U.S. patent application number 13/310840 was filed with the patent office on 2012-06-21 for anti-jam-a antibodies.
This patent application is currently assigned to Pierre Fabre Medicament. Invention is credited to Cedric Bes, Nathalie Corvaia, Liliane Goetsch, Jean-Francois Haeuw.
Application Number | 20120156191 13/310840 |
Document ID | / |
Family ID | 37903981 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120156191 |
Kind Code |
A1 |
Goetsch; Liliane ; et
al. |
June 21, 2012 |
ANTI-JAM-A ANTIBODIES
Abstract
The present invention relates to novel isolated antibodies,
derived compounds, and functional isolated antibody fragments,
capable of inhibiting the proliferation of tumor cells in vitro
and/or in vivo and obtained by functional screening. More
particularly, the present invention relates to the 6F4 antibody,
specific to the JAM-A protein, as well as its use for the treatment
of cancer. Pharmaceutical compositions composed of these antibodies
are also covered.
Inventors: |
Goetsch; Liliane; (Ayze,
FR) ; Corvaia; Nathalie; (Collonges Sous Saleve,
FR) ; Haeuw; Jean-Francois; (Beaumont, FR) ;
Bes; Cedric; (Ambilly, FR) |
Assignee: |
Pierre Fabre Medicament
Boulogne-Billancourt
FR
|
Family ID: |
37903981 |
Appl. No.: |
13/310840 |
Filed: |
December 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12125726 |
May 22, 2008 |
8071730 |
|
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13310840 |
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Current U.S.
Class: |
424/130.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/2803 20130101; A61P 35/04 20180101; C07K 2317/92 20130101;
C07K 2317/73 20130101; A61K 2039/505 20130101; C07K 2317/24
20130101; C07K 2317/54 20130101 |
Class at
Publication: |
424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 24, 2006 |
FR |
FR 0610329 |
Claims
1. A method for preventing or treating a disease related to tumor
cell proliferation in a mammal comprising administering to said
mammal an antibody or a derived compound or functional fragment of
said antibody, wherein said antibody, derived compound, or
functional fragment comprises at least one
complementarity-determining region ("CDR") and wherein said at
least one CDR comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or a combination thereof.
2. The method according to claim 1, wherein said disease related to
tumor cells proliferation is cancer.
3. The method according to claim 2, wherein said cancer is a cancer
selected from the group consisting of prostate cancer,
osteosarcoma, lung cancer, breast cancer, endometrial cancer,
multiple myeloma, ovarian cancer, pancreatic cancer, and colon
cancer.
4. The method according to claim 3, wherein said cancer is a cancer
selected from the group consisting of estrogen-related breast
cancer, non-small cell lung cancer, colon cancer, and pancreatic
cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional Application of U.S. patent
application Ser. No. 12/125,726, filed on May 22, 2008, now U.S.
Pat. No. 8,071,730, which is a nonprovisional application under 35
U.S.C. .sctn.111(a) of International Patent Application
PCT/EP2007/062760, filed on Nov. 23, 2007, and published as WO
2008/062063 on May 29, 2008, which claims priority to French Patent
Application FR 06/10329, filed on Nov. 24, 2006, all of which are
incorporated herein by reference in their entireties for all
purposes.
BACKGROUND OF THE INVENTION
Technical Field of the Invention
[0002] The present invention relates to novel antibodies, in
particular murine monoclonal antibodies, chimeric and humanized,
able to inhibit tumor growth, as well as the amino and nucleic acid
sequences coding for such antibodies. From one aspect, the
invention relates to novel antibodies, derived compounds or
functional fragments, able to inhibit the proliferation of tumor
cells. The invention also comprises the use of such antibodies as a
drug for the preventive and/or therapeutic treatment of cancers, as
well as in the procedures or kits related to cancer diagnosis.
Finally, the invention comprises compositions comprising such
antibodies in combination with other anticancer compounds, such as
antibodies, or conjugated with toxins, and the use of same for the
prevention and/or treatment of certain cancers.
[0003] Generally, the criterion selected for the production of
monoclonal antibodies is the recognition of the immunogen
identified as a potential target of a treatment. In practice, mice
are immunized with a recombinant protein that corresponds to the
immunogen and, after the monoclonal antibodies produced by the
mouse are recovered, they are first screened for their capacity to
recognize the immunogen in a specific manner. In a second stage,
the antibodies thus selected are tested in vivo and in vitro in
order to determine their activity as well as their properties
and/or mechanisms of action.
[0004] This "traditional" approach, even if it makes it possible to
know the working target from the beginning, often generates a large
number of antibodies which are certainly capable of specifically
recognizing a given target but which in vivo do not exhibit
significant biological activity. In the field of cancer, it is
indeed known that, even if an antibody produces good results in
vitro, that does not inevitably mean that such an antibody will
later show genuine anti-tumor activity in vivo.
[0005] The present invention differs from this manner of
proceeding, and goes even against the aforementioned, since it is
based on a "functional" approach, and more particularly on primary
screening based on the function sought for the antibody and not on
the recognized antigen.
[0006] More particularly, the inventors have selected a given
function, namely inhibition of basal proliferation, not induced, of
the cell, as an antibody selection parameter.
[0007] The production method used will be described in more detail
in the examples below.
[0008] In a surprising way, by this functional approach, the
inventors have produced and selected an antibody capable of
inhibiting in vitro and/or in vivo, in a significant manner, the
proliferation of tumor cells.
[0009] According to a first aspect, the invention relates to an
isolated antibody, or a derived compound or functional fragment of
same, capable of inhibiting the proliferation of tumor cells in
vitro and/or in vivo; said antibody, or a derived compound or
functional fragment of same, comprising at least one CDR selected
among the complementarity-determining regions (CDRs) of sequences
SEQ ID No. 1, 2, 3, 4, 5 or 6 or at least one CDR whose sequence
has at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequences SEQ ID No. 1, 2, 3, 4, 5 or 6.
[0010] A "functional fragment" of an antibody means in particular
an antibody fragment, such as fragments Fv, scFv (sc=simple chain),
Fab, F(ab').sub.2, Fab', scFv-Fc or diabodies, or any fragment
whose half-life has been increased. Such functional fragments will
be described in detail later in the present description.
[0011] A "derived compound" of an antibody means in particular a
binding protein composed of a peptide scaffold and at least one of
the CDRs of the original antibody in order to preserve its ability
to be recognized. Such derived compounds, well-known to a person
skilled in the art, will be described in more detail later in the
present description.
[0012] More preferably, the invention comprises the antibodies,
their derived compounds or their functional fragments, according to
the present invention, notably chimeric or humanized, obtained by
genetic recombination or chemical synthesis.
[0013] According to a preferred embodiment, the antibody according
to the invention, or its derived compounds or functional fragments,
is characterized in that it consists of a monoclonal antibody.
[0014] "Monoclonal antibody" is understood to mean an antibody
arising from a nearly homogeneous antibody population. More
particularly, the individual antibodies of a population are
identical except for a few possible naturally-occurring mutations
which can be found in minimal proportions. In other words, a
monoclonal antibody consists of a homogeneous antibody arising from
the growth of a single cell clone (for example a hybridoma, a
eukaryotic host cell transfected with a DNA molecule coding for the
homogeneous antibody, a prokaryotic host cell transfected with a
DNA molecule coding for the homogeneous antibody, etc.) and is
generally characterized by heavy chains of one and only one class
and subclass, and light chains of only one type. Monoclonal
antibodies are highly specific and are directed against a single
antigen. In addition, in contrast with preparations of polyclonal
antibodies which typically include various antibodies directed
against various determinants, or epitopes, each monoclonal antibody
is directed against a single epitope of the antigen.
[0015] It must be understood here that the invention does not
relate to antibodies in natural form, i.e., they are not taken from
their natural environment but are isolated or obtained by
purification from natural sources or obtained by genetic
recombination or chemical synthesis and thus they can carry
unnatural amino acids as will be described below.
[0016] More particularly, according to a preferred embodiment of
the invention, the antibody, or its derived compounds or functional
fragments, is characterized in that it comprises a light chain
comprising at least one CDR selected among the CDRs of amino acid
sequences SEQ ID No. 1, 3 or 5, or at least one CDR whose sequence
has at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequences SEQ ID No. 1, 3 or 5; or it
comprises a heavy chain comprising at least one CDR selected among
the CDRs of amino acid sequences SEQ ID No. 2, 4 or 6, or at least
one CDR whose sequence has at least 80%, preferably 85%, 90%, 95%
and 98% identity after optimal alignment with sequences SEQ ID No.
2, 4 or 6.
[0017] More particularly, the antibodies of the invention, or one
of their derived compounds or functional fragments, are
characterized in that they comprise a heavy chain comprising at
least one of the three CDRs of the sequences SEQ ID Nos. 2, 4 and
6, or at least one sequence with at least 80%, preferably 85%, 90%,
95% and 98% identity after optimal alignment with sequences SEQ ID
Nos. 2,4 or 6.
[0018] Even more preferably, the antibodies of the invention, or
one of their derived compounds or functional fragments, are
characterized in that they comprise a heavy chain comprising the
following three CDRs, respectively CDR-H1, CDR-H2 and CDR-H3,
wherein:
[0019] CDR-H1 comprises the sequence SEQ ID No. 2, 7 or 9, or a
sequence with at least 80% identity after optimal alignment with
sequence SEQ ID No. 2, 7 or 9;
[0020] CDR-H2 comprises the sequences SEQ ID No. 4 or 11, or a
sequence with at least 80% identity after optimal alignment with
sequence SEQ ID No. 4 or 11; and
[0021] CDR-H3 comprises the sequences SEQ ID No. 6 or 12, or a
sequence with at least 80% identity after optimal alignment with
sequence SEQ ID No. 6 or 12.
[0022] According to a particular embodiment, antibodies, or one of
their derived compounds or functional fragments, are characterized
in that they comprise a heavy chain comprising the CDR-H1 of the
sequence SEQ ID No. 7, the CDR-H2 of the sequence SEQ ID No. 4 and
the CDR-H3 of the sequence SEQ ID No. 12.
[0023] According to another particular embodiment, antibodies, or
one of their derived compounds or functional fragments, are
characterized in that they comprise a heavy chain comprising the
CDR-H1 of the sequence SEQ ID No. 9, the CDR-H2 of the sequence SEQ
ID No. 11 and the CDR-H3 of the sequence SEQ ID No. 6.
[0024] According to another embodiment, the antibodies of the
invention, or one of their derived compounds or functional
fragments, are characterized in that they comprise a light chain
comprising at least one of the three CDRs of the sequences SEQ ID
Nos. 1, 3 and 5, or at least one sequence with at least 80%,
preferably 85%, 90%, 95% and 98% identity after optimal alignment
with sequences SEQ ID Nos. 1, 3 or 5.
[0025] In a preferred manner, the antibodies of the invention, or
one of their derived compounds or functional fragments, are
characterized in that they comprise a light chain comprising the
following three CDRs, respectively CDR-L1, CDR-L2 and CDR-L3,
wherein:
[0026] CDR-L1 comprises the sequence SEQ ID No. 1 or 8, or a
sequence with at least 80% identity after optimal alignment with
sequence SEQ ID No. 1 or 8;
[0027] CDR-L2 comprises the sequences SEQ ID No. 3 or 10, or a
sequence with at least 80% identity after optimal alignment with
sequence SEQ ID No. 3 or 10; and
[0028] CDR-L3 comprises the sequence SEQ ID No. 5, or a sequence
with at least 80% identity after optimal alignment with sequence
SEQ ID No. 5.
[0029] According to a particular embodiment, antibodies, or one of
their derived compounds or functional fragments, are characterized
in that they comprise a light chain comprising the CDR-L1 of the
sequence SEQ ID No. 1, the CDR-L2 of the sequence SEQ ID No. 3 and
the CDR-L3 of the sequence SEQ ID No. 5.
[0030] According to another particular embodiment, antibodies, or
one of their derived compounds or functional fragments, are
characterized in that they comprise a light chain comprising the
CDR-L1 of the sequence SEQ ID No. 8, the CDR-L2 of the sequence SEQ
ID No. 10 and the CDR-L3 of the sequence SEQ ID No. 5.
[0031] In the present description, the terms "polypeptides",
"polypeptide sequences", "peptides" and "proteins attached to
antibody compounds or to their sequences" are interchangeable.
[0032] It must be understood here that the invention does not
relate to antibodies in natural form, i.e., they are not taken from
their natural environment but are isolated or obtained by
purification from natural sources or obtained by genetic
recombination or chemical synthesis and thus they can carry
unnatural amino acids as will be described below.
[0033] In a first embodiment, complementarity-determining region,
or CDR, means the hypervariable regions of the heavy and light
chains of immunoglobulins as defined by Kabat et al. (Kabat et al.,
Sequences of proteins of immunological interest, 5.sup.th Ed., U.S.
Department of Health and Human Services, NIH, 1991, and later
editions). There are three heavy-chain CDRs and three light-chain
CDRs. Here, the terms "CDR" and "CDRs" are used to indicate,
depending on the case, one or more, or even all, of the regions
containing the majority of the amino acid residues responsible for
the antibody's binding affinity for the antigen or epitope it
recognizes.
[0034] In a second embodiment, by CDR regions or CDR(s), it is
intended to indicate the hypervariable regions of the heavy and
light chains of the immunoglobulins as defined by IMGT.
[0035] The IMGT unique numbering has been defined to compare the
variable domains whatever the antigen receptor, the chain type, or
the species [Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc
M.-P., The Immunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommie,
C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L.,
Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77
(2003)]. In the IMGT unique numbering, the conserved amino acids
always have the same position, for instance cystein 23 (1st-CYS),
tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cystein
104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP).
The IMGT unique numbering provides a standardized delimitation of
the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to
55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the
complementarity determining regions: CDR1-IMGT: 27 to 38,
CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent
unoccupied positions, the CDR-IMGT lengths (shown from brackets and
separated by dots, e.g. [8.8.13]) become crucial information. The
IMGT unique numbering is used in 2D graphical representations,
designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P.,
Immunogenetics, 53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P.,
Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in
IMGT/3D structure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell
receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210
(2004)].
[0036] Three heavy chain CDRs and 3 light chain CDRs exist. The
term CDR or CDRs is used here in order to indicate, according to
the case, one of these regions or several, or even the whole, of
these regions which contain the majority of the amino acid residues
responsible for the binding by affinity of the antibody for the
antigen or the epitope which it recognizes.
[0037] For more clarity, it must be understood that in the
following description, and more particularly in table 2 and 3, the
CDRs will be defined by IMGT numbering, kabat numbering and by
common numbering.
[0038] Common numbering regroups the residues part of each CDR
which are common to the CDRs as defined by the IMGT and the Kabat
numbering systems.
[0039] IMGT numbering system defines the CDRs according to the IMGT
system as above defined whereas kabat numbering system defines the
CDRs according to the kabat system as above defined.
[0040] More particularly, CDR-L1 consist of SEQ ID No. 1 (QDINNY)
in the common and IMGT numbering systems and of SEQ ID No. 8
(KASQDINNYIA) in the kabat numbering system.
[0041] Concerning the CDR-L2, it consists of SEQ ID No. 3 (YTS) in
the common and IMGT numbering systems and of SEQ ID No. 10
(YTSTLQA) in the kabat numbering system.
[0042] The CDR-L3 consists of SEQ ID No. 5 (LQYDNLWT) for each of
the three numbering systems.
[0043] For the heavy chain, the CDR-H1 consists of the SEQ ID No. 2
(TDYS) in the common numbering system, of SEQ ID No. 7 (GYSFTDYS)
in the IMGT numbering system and of SEQ ID No. 9 (TDYSMY) in the
kabat numbering system.
[0044] The CDR-H2 consists of SEQ ID No. 4 (IDPYNGGT) in the common
and IMGT numbering systems and of SEQ ID No. 11 (YIDPYNGGTRYNQKFKG)
in the kabat numbering system.
[0045] At last, the CDR-H3 consists in the SEQ ID No. 6 (QTDYFDY)
in the common and kabat numbering systems whereas it consists of
SEQ ID No. 12 (ARQTDYFDY) in the IMGT numbering system.
[0046] In the sense of the present invention, the "percentage
identity" from two sequences of nucleic acids or amino acids means
the percentage of identical nucleotides or amino acid residues from
the two sequences to be compared, obtained after optimal alignment,
this percentage being purely statistical and the differences from
the two sequences being distributed randomly along their length.
The comparison of two nucleic acid or amino acid sequences is
traditionally carried out by comparing the sequences after having
optimally aligned them, said comparison being able to be conducted
by segment or by using an "alignment window". Optimal alignment of
the sequences for comparison can be carried out, in addition to
comparison by hand, by means of the local homology algorithm of
Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the
local homology algorithm of Neddleman and Wunsch (1970) [J. Mol.
Biol. 48:443], by means of the similarity search method of Pearson
and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444] or by means
of computer software using these algorithms (GAP, BESTFIT, FASTA
and TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis., or by the
comparison software BLAST NR or BLAST P).
[0047] The percentage identity from two nucleic acid or amino acid
sequences is determined by comparing the two optimally-aligned
sequences in which the nucleic acid or amino acid sequence to
compare can have additions or deletions compared to the reference
sequence for optimal alignment from the two sequences. Percentage
identity is calculated by determining the number of positions at
which the amino acid nucleotide or residue is identical from the
two sequences, dividing the number of identical positions by the
total number of positions in the alignment window and multiplying
the result by 100 to obtain the percentage identity from the two
sequences.
[0048] For example, the BLAST program, "BLAST 2 sequences"
(Tatusova et al., "Blast 2 sequences--a new tool for comparing
protein and nucleotide sequences", FEMS Microbial., 1999, Lett.
174:247-250) can be used with the default parameters (notably for
the parameters "open gap penalty": 5, and "extension gap penalty":
2; the selected matrix being for example the "BLOSUM 62" matrix
proposed by the program); the percentage identity from the two
sequences to compare is calculated directly by the program.
[0049] For the amino acid sequence exhibiting at least 80%,
preferably 85%, 90%, 95% and 98% identity with a reference amino
acid sequence, preferred examples include those containing the
reference sequence, certain modifications, notably a deletion,
addition or substitution of at least one amino acid, truncation or
extension. In the case of substitution of one or more consecutive
or non-consecutive amino acids, substitutions are preferred in
which the substituted amino acids are replaced by "equivalent"
amino acids. Here, the expression "equivalent amino acids" is meant
to indicate any amino acids likely to be substituted for one of the
structural amino acids without however modifying the biological
activities of the corresponding antibodies and of those specific
examples defined below.
[0050] Equivalent amino acids can be determined either on their
structural homology with the amino acids for which they are
substituted or on the results of comparative tests of biological
activity from the various antibodies likely to be generated.
[0051] As a non-limiting example, table 1 below summarizes the
possible substitutions likely to be carried out without resulting
in a significant modification of the biological activity of the
corresponding modified antibody; inverse substitutions are
naturally possible under the same conditions.
TABLE-US-00001 TABLE 1 Original residue Substitution(s) Ala (A)
Val, Gly, Pro Arg (R) Lys, His Asn (N) Gln Asp (D) Glu Cys (C) Ser
Gln (Q) Asn Glu (G) Asp Gly (G) Ala His (H) Arg Ile (I) Leu Leu (L)
Ile, Val, Met Lys (K) Arg Met (M) Leu Phe (F) Tyr Pro (P) Ala Ser
(S) Thr, Cys Thr (T) Ser Trp (W) Tyr Tyr (Y) Phe, Trp Val (V) Leu,
Ala
[0052] It is known by those skilled in the art that in the current
state of the art the greatest variability (length and composition)
from the six CDRs is found at the three heavy-chain CDRs and, more
particularly, at CDR-H3 of this heavy chain. Consequently, it will
be evident that the preferred characteristic CDRs of the antibodies
of the invention, or of one of their derived compounds or
functional fragments, will be the three CDRs of the heavy chain,
i.e., the CDRs coded by sequences SEQ ID Nos. 2, 4 and 6,
respectively, and even more preferentially, the CDR corresponding
to the CDR-H3 coded by sequence SEQ ID No. 6.
[0053] In a specific embodiment, the present invention relates to a
murine antibody, or derived compounds or functional fragments of
same.
[0054] Another embodiment of the invention discloses an antibody,
or its derived compounds or functional fragments, comprising a
light chain comprising the following three CDRs: [0055] CDR-L1 of
the sequence SEQ ID No. 1 or of a sequence with at least 80%,
preferably 85%, 90%, 95% and 98% identity after optimal alignment
with sequence SEQ ID No. 1; [0056] CDR-L2 of the sequence SEQ ID
No. 3 or of a sequence with at least 80%, preferably 85%, 90%, 95%
and 98% identity after optimal alignment with sequence SEQ ID No.
3; and [0057] CDR-L3 of the sequence SEQ ID No. 5 or of a sequence
with at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequence SEQ ID No. 5, and a heavy chain
comprising the following three CDRs: [0058] CDR-H1 of the sequence
SEQ ID No. 7 or of a sequence with at least 80%, preferably 85%,
90%, 95% and 98% identity after optimal alignment with sequence SEQ
ID No. 7; [0059] CDR-H2 of the sequence SEQ ID No. 4 or of a
sequence with at least 80%, preferably 85%, 90%, 95% and 98%
identity after optimal alignment with sequence SEQ ID No. 4; and
[0060] CDR-H3 of the sequence SEQ ID No. 12 or of a sequence with
at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequence SEQ ID No. 12.
[0061] Still another embodiment of the invention discloses an
antibody, or a derived compound or functional fragment of same,
comprising a light chain comprising the following three CDRs:
[0062] CDR-L1 of the sequence SEQ ID No. 8 or of a sequence with at
least 80% identity after optimal alignment with sequence SEQ ID No.
8;
[0063] CDR-L2 of the sequence SEQ ID No. 10 or of a sequence with
at least 80% identity after optimal alignment with sequence SEQ ID
No. 10; and
[0064] CDR-L3 of the sequence SEQ ID No. 5 or of a sequence with at
least 80% identity after optimal alignment with sequence SEQ ID No.
5, and
a heavy chain comprising the following three CDRs:
[0065] CDR-H1 of the sequence SEQ ID No. 9 or of a sequence with at
least 80% identity after optimal alignment with sequence SEQ ID No.
9;
[0066] CDR-H2 of the sequence SEQ ID No. 11 or of a sequence with
at least 80% identity after optimal alignment with sequence SEQ ID
No. 11; and
[0067] CDR-H3 of the sequence SEQ ID No. 6 or of a sequence with at
least 80% identity after optimal alignment with sequence SEQ ID No.
6.
[0068] According to still another embodiment, the antibody of the
invention, or its derived compounds or functional fragments, is
characterized in that it comprises a light-chain sequence
comprising the amino acid sequence SEQ ID No. 13 or a sequence with
at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequence SEQ ID No. 13; and in that it
comprises a heavy-chain sequence comprising the amino acid sequence
SEQ ID No. 14 or a sequence with at least 80%, preferably 85%, 90%,
95% and 98% identity after optimal alignment with sequence SEQ ID
No. 14.
[0069] It is also disclosed a humanized antibody, or a derived
compound or functional fragment of same, which is characterized in
that it comprises a light chain sequence comprising the amino acid
sequence SEQ ID No. 17 or a sequence with at least 80% identity
after optimal alignment with sequence SEQ ID No. 17, and in that it
comprises a heavy chain sequence comprising the amino acid sequence
SEQ ID No. 18 or 19 or a sequence with at least 80% identity after
optimal alignment with sequence SEQ ID No. 18 or 19.
[0070] As seen above, the invention also relates to any compound
derived from an antibody as described in the invention.
[0071] More particularly, the antibody of the invention, or its
derived compounds or functional fragments, is characterized in that
said derived compound consists of a binding protein comprising a
peptide scaffold on which is grafted at least one CDR in such a way
as to preserve all or part of the paratope recognition properties
of the initial antibody.
[0072] One or more sequences among the six CDR sequences described
in the present invention can also be present on the various
immunoglobulin protein scaffolding. In this case, the protein
sequence makes it possible to recreate a peptide skeleton favorable
to the folding of the grafted CDRs, enabling them to preserve their
paratope antigen-recognition properties.
[0073] Generally, a person skilled in the art knows how to
determine the type of protein scaffold on which to graft at least
one of the CDRs arising from the original antibody. More
particularly, it is known that to be selected such scaffolds must
meet the greatest number of criteria as follows (Skerra A., J. Mol.
Recogn., 2000, 13:167-187): [0074] good phylogenetic conservation;
[0075] known three-dimensional structure (as, for example, by
crystallography, NMR spectroscopy or any other technique known to a
person skilled in the art); [0076] small size; [0077] few or no
post-transcriptional modifications; and/or [0078] easy to produce,
express and purify.
[0079] The origin of such protein scaffolds can be, but is not
limited to, the structures selected among: fibronectin and
preferentially fibronectin type III domain 10, lipocalin, anticalin
(Skerra A., J. Biotechnol., 2001, 74(4):257-75), protein Z arising
from domain B of protein A of Staphylococcus aureus, thioredoxin A
or proteins with a repeated motif such as the "ankyrin repeat"
(Kohl et al., PNAS, 2003, vol. 100, No. 4, 1700-1705), the
"armadillo repeat", the "leucine-rich repeat" and the
"tetratricopeptide repeat".
[0080] Scaffolds derived from toxins such as, for example, toxins
from scorpions, insects, plants, mollusks, etc., and the protein
inhibiters of neuronal NO synthase (PIN) should also be
mentioned.
[0081] An example, in no way limiting, of such hybrid
constructions, is the insertion of the CDR-H1 (heavy chain) of an
anti-CD4 antibody, namely 13B8.2, in one of the loops in the PIN,
the new binding protein thus obtained preserving the same binding
properties as the original antibody (Bes et al., Biochem. Biophys.
Res. Commun., 2006, 343(1), 334-344). On a purely illustrative
basis, grafting the CDR-H3 (heavy chain) of an anti-lysozyme VHH
antibody on one of the loops of neocarzinostatin (Nicaise et al.,
Protein Science, 2004, 13(7):1882-1891) can also be mentioned.
[0082] Lastly, as described above, such peptide scaffolds can
comprise from one to six CDRs arising from the original antibody.
Preferably, but not being a requirement, a person skilled in the
art will select at least one CDR from the heavy chain, the latter
being known to be primarily responsible for the specificity of the
antibody. The selection of one or more relevant CDRs is obvious to
a person skilled in the art, who will then choose suitable known
techniques (Bes et al., FEBS letters 508, 2001, 67-74).
[0083] A specific aspect of the present invention relates to a
method for selecting a compound derived from an antibody according
to the invention, said derived compound being capable of inhibiting
in vitro and/or in vivo the growth of tumor cells and said derived
compound comprising a peptide scaffold on which is grafted at least
one antibody CDR, characterized in that it comprises the following
steps: [0084] a) the placing in contact in vitro of a compound
composed of a peptide scaffold on which is grafted at least one
antibody CDR with a biological sample containing tumor cells able
to grow and under conditions allowing these cells to grow; and
[0085] b) selection of said compound if said compound is capable of
inhibiting the growth of these tumor cells, and characterized in
that said at least one grafted CDR is selected among the following
CDRs: [0086] the CDR of sequence SEQ ID No. 1, 8 or a sequence with
at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequence SEQ ID No. 1, 8; [0087] the CDR of
sequence SEQ ID No. 3, 10 or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98% identity after optimal alignment
with sequence SEQ ID No. 3, 10; [0088] the CDR of sequence SEQ ID
No. 5 or a sequence with at least 80%, preferably 85%, 90%, 95% and
98% identity after optimal alignment with sequence SEQ ID No. 5;
[0089] the CDR of sequence SEQ ID No. 2, 7, 9 or a sequence with at
least 80%, preferably 85%, 90%, 95% and 98% identity after optimal
alignment with sequence SEQ ID No. 2, 7, 9; [0090] the CDR of
sequence SEQ ID No. 4, 11 or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98% identity after optimal alignment
with sequence SEQ ID No. 4, 11; and [0091] the CDR of sequence SEQ
ID No. 6, 12 or a sequence with at least 80%, preferably 85%, 90%,
95% and 98% identity after optimal alignment with sequence SEQ ID
No. 6, 12.
[0092] According to a preferred mode, the method can include in
step a) the placing in contact in vitro of a compound comprising a
peptide scaffold on which is grafted at least two or three antibody
CDRs.
[0093] According to an even more preferred mode of this method, the
peptide scaffold is selected among the scaffolds or binding
proteins whose structures were mentioned above.
[0094] Obviously, these examples are in no way limiting, and any
other structure known or obvious to a person skilled in the art
should be considered as being covered by the protection conferred
by the present patent application.
[0095] The present invention thus relates to an antibody, or its
derived compounds or functional fragments, characterized in that
the peptide scaffold is selected among proteins that are a)
phylogenetically well preserved, b) of robust architecture, c) with
a well-known 3-D molecular organization, d) of small size and/or e)
comprising regions that can be modified by deletion and/or
insertion without modifying stability properties.
[0096] According to a preferred embodiment, the antibody of the
invention, or its derived compounds or functional fragments, is
characterized in that said peptide scaffold is selected among i)
scaffolds arising from fibronectin, preferentially fibronectin type
3 domain 10, lipocalin, anticalin, protein Z arising from domain B
of protein A of Staphylococcus aureus, thioredoxin A or proteins
with a repeated motif such as the "ankyrin repeat" (Kohl et al.,
PNAS, 2003, vol. 100, No. 4, 1700-1705), the "armadillo repeat",
the "leucine-rich repeat" and the "tetratricopeptide repeat" or
iii) protein inhibiters of neuronal NO synthase (PIN).
[0097] Another aspect of the invention relates to the functional
fragments of the antibody described above.
[0098] More particularly, the invention targets an antibody, or its
derived compounds or functional fragments, characterized in that
said functional fragment is selected among the fragments Fv, Fab,
(Fab').sub.2, Fab', scFv, scFv-Fc and diabodies, or any fragment
whose half-life has been increased such as PEGylated fragments.
[0099] Such functional fragments of the antibody according to the
invention consist, for example, of the fragments Fv, scFv
(sc=simple chain), Fab, F(ab').sub.2, Fab', scFv-Fc or diabodies,
or any fragment whose half-life has been increased by chemical
modification, such as the addition of polyalkylene glycol such as
polyethylene glycol (PEGylation) (PEGylated fragments are referred
to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab').sub.2-PEG and Fab'-PEG), or
by incorporation in a liposome, microspheres or PLGA, said
fragments possessing at least one of the characteristic CDRs of the
invention which is notably capable of exerting in a general manner
activity, even partial, of the antibody from which it arises.
[0100] Preferably, said functional fragments will comprise or
include a partial sequence of the variable heavy or light chain of
the antibody from which they are derived, said partial sequence
being sufficient to retain the same binding specificity as the
antibody from which it arises and sufficient affinity, preferably
at least equal to 1/100, more preferably at least 1/10 of that of
the antibody from which it arises.
[0101] Such a functional fragment will contain at least five amino
acids, preferably 6, 7, 8, 10, 15, 25, 50 or 100 consecutive amino
acids of the sequence of the antibody from which it arises.
[0102] Preferably, these functional fragments will be of the types
Fv, scFv, Fab, F(ab').sub.2, F(ab'), scFv-Fc or diabodies, which
generally have the same binding specificity as the antibody from
which they result. According to the present invention, fragments of
the antibody of the invention can be obtained from the antibodies
described above by methods such as enzyme digestion, including
pepsin or papain, and/or by cleavage of the disulfide bridges by
chemical reduction. The antibody fragments can be also obtained by
recombinant genetics techniques also known to a person skilled in
the art or by peptide synthesis by means, for example, of automatic
peptide synthesizers such as those marketed by Applied BioSystems,
etc.
[0103] The invention also targets the original murine antibody,
namely an antibody according to the invention, or its derived
compounds or functional fragments, characterized in that said
antibody is a murine antibody and in that it comprises a
light-chain of amino acid sequence SEQ ID No. 15, or a sequence
with at least 80%, preferably 85%, 90%, 95% and 98% identity after
optimal alignment with sequence SEQ ID No. 15, and a heavy-chain of
amino acid sequence SEQ ID No. 16, or a sequence with at least 80%,
preferably 85%, 90%, 95% and 98% identity after optimal alignment
with sequence SEQ ID No. 16.
[0104] For more clarity, table 2 below summarizes the various amino
acid sequences corresponding to the antibody of the invention.
TABLE-US-00002 TABLE 2 (wherein Mu. = murine and Hu. = humanized):
CDR SEQ ID Antibody numbering Heavy chain Light chain NO. 6F4
Common CDR-L1 1 CDR-L2 3 CDR-L3 5 CDR-H1 2 CDR-H2 4 CDR-H3 6 IMGT
CDR-L1 1 CDR-L2 3 CDR-L3 5 CDR-H1 7 CDR-H2 4 CDR-H3 12 Kabat CDR-L1
8 CDR-L2 10 CDR-L3 5 CDR-H1 9 CDR-H2 11 CDR-H3 6 Mu. variable 13
domain Mu. variable 14 domain Mu. entire 15 Mu. entire 16 Hu.
variable 17 domain Hu. variable 18 domain (V1) Hu. variable 19
domain (V2)
[0105] Another specific aspect of the present invention relates to
a chimeric antibody, or its derived compounds or functional
fragments, characterized in that said antibody also comprises
light-chain and heavy-chain constant regions derived from an
antibody of a species heterologous with the mouse, notably man.
[0106] Yet another specific aspect of the present invention relates
to a humanized antibody, or its derived compounds or functional
fragments, characterized in that the constant regions of the
light-chain and the heavy-chain derived from human antibody are,
respectively, the lambda or kappa region and the gamma-1, gamma-2
or gamma-4 region.
[0107] According to another aspect, the invention relates to a
murine hybridoma capable of secreting a monoclonal antibody
according to the invention, notably the hybridoma of murine origin
filed with the French center for microorganism cultures (CNCM,
Pasteur Institute, Paris, France) on Jul. 6, 2006, under number
1-3646. Said hybridoma was obtained by the fusion of Balb/C
immunized mice splenocytes and cells of the myeloma Sp 2/O-Ag 14
lines.
[0108] The monoclonal antibody, here referred to as 6F4, or its
derived compounds or functional fragments, characterized in that
said antibody is secreted by the hybridoma filed with the CNCM on
Jul. 4, 2006, under number I-3646, obviously forms part of the
present invention.
[0109] The antibody of the invention also comprises chimeric or
humanized antibodies.
[0110] A chimeric antibody is one containing a natural variable
region (light chain and heavy chain) derived from an antibody of a
given species in combination with constant regions of the light
chain and the heavy chain of an antibody of a species heterologous
to said given species.
[0111] The antibodies, or chimeric fragments of same, can be
prepared by using the techniques of recombinant genetics. For
example, the chimeric antibody could be produced by cloning
recombinant DNA containing a promoter and a sequence coding for the
variable region of a nonhuman monoclonal antibody of the invention,
notably murine, and a sequence coding for the human antibody
constant region. A chimeric antibody according to the invention
coded by one such recombinant gene could be, for example, a
mouse-human chimera, the specificity of this antibody being
determined by the variable region derived from the murine DNA and
its isotype determined by the constant region derived from human
DNA. Refer to Verhoeyn et al. (BioEssays, 8:74, 1988) for methods
for preparing chimeric antibodies.
[0112] "Humanized antibodies" means an antibody that contains CDR
regions derived from an antibody of nonhuman origin, the other
parts of the antibody molecule being derived from one (or several)
human antibodies. In addition, some of the skeleton segment
residues (called FR) can be modified to preserve binding affinity
(Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al.,
Science, 239:1534-1536, 1988; Riechmann et al., Nature,
332:323-327, 1988).
[0113] The humanized antibodies of the invention or fragments of
same can be prepared by techniques known to a person skilled in the
art (such as, for example, those described in the documents Singer
et al., J. Immun., 150:2844-2857, 1992; Mountain et al.,
Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; and Bebbington et
al., Bio/Technology, 10:169-175, 1992). Such humanized antibodies
are preferred for their use in methods involving in vitro diagnoses
or preventive and/or therapeutic treatment in vivo. Other
humanization techniques, also known to a person skilled in the art,
such as, for example, the "CDR grafting" technique described by PDL
in patents EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647
or US 5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761. U.S.
Pat. Nos. 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also
be cited.
[0114] In addition, the invention also relates to humanized
antibodies arising from the murine antibodies described above.
[0115] More particularly, the humanization method for the 6F4
antibody is described in detail in examples 2 and 3 for the light
and heavy chains, respectively.
[0116] In a preferred manner, constant regions of the light-chain
and the heavy-chain derived from human antibody are, respectively,
the lambda or kappa and the gamma-1, gamma-2 or gamma-4 region.
[0117] In the embodiment corresponding to IgG1 isotype IgG1, an
additional characteristic of the antibody is to exhibit effector
functions, such as antibody-dependant cellular cytotoxicity (ADCC)
and/or complement-dependant cytotoxicity (CDC).
[0118] In the field of the present invention, several backup
humanized antibodies have been developed. More particularly, the
invention concerns two variants heavy chains and two variants light
chains, both derived from the 6F4 antibody.
[0119] In a first aspect, the invention relates to a humanized
antibody, or a derived compound or functional fragment thereof,
said antibody being characterized in that it comprises a heavy
chain variable domain, called BU-H1, comprising the amino acid
sequence SEQ ID No. 65.
TABLE-US-00003 SEQ ID No. 65:
(X01)VQL(X02)QSGAEVKKPGASVKVSCKASGYSFTDYSMHWVRQAPG
Q(X03)LEWMG(X04)IDPYNGGT(X05)YSQKFQGR(X06)T(X07)T (X08)
DTSASTAYM(X09)LSSLRSEDTAVYYCARQTDYFDYWGQGTLV TVSS
wherein bolded residues correspond to CDR-IMGT, [0120] and X01 is E
or Q; X02 is V or Q; X03 is S or R; X04 is Y or W; X05 is R or K;
X06 is A or V; X07 is L or I; X08 is V or R and X09 is H or E.
[0121] In a second aspect, the invention relates to a humanized
antibody, or a derived compound or functional fragment thereof,
said antibody being characterized in that it comprises a heavy
chain variable domain, called BU-H2, comprising the amino acid
sequence SEQ ID No. 66.
TABLE-US-00004 SEQ ID No. 66:
(X01)VQL(X02)QSGAEVKKPGASVKVSCKASGYSFTDYSMHVVVRQAP
GQ(X03)LEWMG(X04)IDPYNGGT(X05)YAQKFQGR(X06)T(X07)T
(X08)DTSTSTVYM(X09)LSSLRSEDTAVYYCARQTDYFDYWGQGTLVT VSS
wherein bolded residues correspond to CDR-IMGT, [0122] and X01 is E
or Q; X02 is V or Q; X03 is S or G; X04 is Y or I; X05 is R or S;
X06 is A or V; X07 is L or M; X08 is V or R and X09 is H or E.
[0123] In a third aspect, the invention relates to a humanized
antibody, or a derived compound or functional fragment thereof,
said antibody being characterized in that it comprises a light
chain variable domain, called BU-L1, comprising the amino acid
sequence SEQ ID No. 67.
TABLE-US-00005 SEQ ID No. 67:
DIQMTQSPSSLSASVGDRVTITC(X01)ASQDINNY(X02)AWYQQKPGK
VPKLLI(X03)YTSTLQSGVPSRFSGSGSGTD(X04)TLTISSLQPEDVA
TYYCLQYDNLWTFGQGTKVEIK
wherein bolded residues correspond to CDR-IMGT, [0124] and X01 is K
or R; X02 is L or I; X03 is H or Y and X04 is Y or F.
[0125] In a fourth aspect, the invention relates to a humanized
antibody, or a derived compound or functional fragment thereof,
said antibody being characterized in that it comprises a light
chain variable domain, called BU-L2, comprising the amino acid
sequence SEQ ID No. 68.
TABLE-US-00006 SEQ ID No. 68:
(X01)I(X02)MTQSPFSLSASVGDRVTITC(X03)ASQDINNY(X04)A
WYQQKPAKAPKLFI(X05)YTS(X06)LQSGVPSRFSGSGSGTDYTLTIS
SLQPEDFATYYCLQYDNLWTFGQGTKVEIK
wherein bolded residues correspond to CDR-IMGT, [0126] and X01 is D
or A; X02 is R or Q; X03 is K or W; X04 is L or I; X05 is H or Y
and X06 is S or T.
[0127] In a preferred embodiment, the invention relates to a
humanized antibody, or a derived compound or functional fragment
thereof, characterized in that it comprises a heavy chain variable
domain comprising the amino acid sequences SEQ ID No. 65 and a
light chain variable domain comprising the amino acid sequence SEQ
ID No. 67.
[0128] In another preferred embodiment, the invention relates to a
humanized antibody, or a derived compound or functional fragment
thereof, characterized in that it comprises a heavy chain variable
domain comprising the amino acid sequences SEQ ID No. 65 and a
light chain variable domain comprising the amino acid SEQ ID No.
68.
[0129] In yet another preferred embodiment, the invention relates
to a humanized antibody, or a derived compound or functional
fragment thereof, characterized in that it comprises a heavy chain
variable domain comprising the amino acid sequences SEQ ID No. 66
and a light chain variable domain comprising the amino acid
sequence SEQ ID No. 67.
[0130] Still in another preferred embodiment, the invention relates
to a humanized antibody, or a derived compound or functional
fragment thereof, characterized in that it comprises a heavy chain
variable domain comprising the amino acid sequences SEQ ID No. 66
and a light chain variable domain comprising the amino acid
sequence SEQ ID No. 68.
[0131] For more clarity, table 3 below summarizes the various amino
acid sequences corresponding to the back-up antibodies.
TABLE-US-00007 TABLE 3 (wherein Mu. = murine and Hu. = humanized):
CDR SEQ ID Antibody numbering Heavy chain Light chain NO. 6F4 BU
Hu. variable 65 domain (H1) Hu. variable 66 domain (H2) Hu.
variable 67 domain (L1) Hu. variable 68 domain (L2)
[0132] In another aspect of the invention, the applicant has also
identified the antigen recognized by the antibody according to the
invention.
[0133] The method used to accomplish this is described in detail in
example 4 below.
[0134] JAM-A is a membrane protein belonging to the immunoglobulin
superfamily (IgSF), in which it belongs to the junctional adhesion
molecule (JAM) family. In man, the JAM family comprises several
members, including the JAM-A, JAM-B, JAM-C, A33 and A34 proteins.
Among the members of the JAM family, JAM-A has the highest homology
with JAM-B and JAM-C, approximately 35% sequence identity in amino
acids and 45% similarity with these two proteins. JAM-A protein is
also called JAM A, F11R, F11 receptor, JAM-1, JAM 1, PAM-1 or
CD321.
[0135] Two isoforms of the JAM-A precursor differing by the length
of the extracellular region were identified: [0136] isoform a: 299
amino acids (SEQ ID No. 61) [0137] isoform b: 259 amino acids (SEQ
ID No. 63).
[0138] The nucleotide sequences of the two isoforms are represented
with SEQ ID No. 62 for isoform a and SEQ ID No. 64 for isoform
b.
[0139] The protein expressed on the surface of the human cells has
a single polypeptide chain with an intracellular C-terminal domain,
a single transmembrane domain (21 amino acids) and an N-terminal
extracellular region containing two "Ig-like" domains.
[0140] JAM-A has an N-glycosylation site, an Asn residue in
position 185 for isoform a and 145 for isoform b, and two disulfide
bridges, one from Cys residues 50 and 109 in the Ig N-terminal
domain and one from residues Cys 153 and 212 in the second Ig
domain.
[0141] The presence of the two extracellular Ig-like domains was
confirmed by crystallography (Kostrewa et al., 2001, EMBO J.
16:4391-4398; Prota et al., 2003, Proc. Natl. Acad. Sci. USA,
100:5366-5371). These two domains are connected by a tripeptide
linker (sequence VLV [127-129], isoform A). These structural
studies also confirmed the implication of JAM-A in homophilic
interactions on the cell surface involving the extracellular
region; this region, produced in recombinant form and capable of
forming homodimers in solution (Bazzoni et al., 2000, J. Biol.
Chem. 275:30970-30976) also made it possible to identify the amino
acids involved in these interactions: Arg 59, Glu 61, Lys 63, Leu
72, Tyr 75, Met 110, Glu 114, Tyr 119 and Glu 121. The tripeptide
RVE [59-61] is relatively conserved within the JAM family (RLE for
JAM-B, RIE for JAM-C) and constitutes the minimal motif for
homodimer formation (Kostrewa et al., 2001, EMBO J.
16:4391-4398).
[0142] In epithelial and endothelial cells, JAM-A is mainly found
in the tight junctions (Liu et al., 2000, J. Cell Sci.,
113:2363-2374). The cytoplasmic region contains a type II PDZ
domain in the C-terminal position (sequence FLV [298-300], isoform
a, which is responsible for the interaction of JAM-A with various
cytosolic proteins associated with the tight junction, also
containing a PDZ domain, such as ZO-1, AF-6, MUPP-1 and PAR-3
(Ebnet et al., 2000, J. Biol. Chem., 275:27979-27988; Itoh et al.,
2001, J. Cell Biol., 154:491-498; Hamazaki et al., 2002, J. Biol.
Chem., 277:455-461). Murine antibodies directed against the region
[111-123] involved in dimer formation, so-called J3F.1 and J10.4
antibodies, are capable of inhibiting the homodimerization of JAM-A
and the reconstruction of the epithelial barrier in vitro (Mandell
et al., 2004, J. Biol. Chem., 279:16254-16262).
[0143] JAM-A interacts with integrin .alpha..sub.V.beta..sub.3 and
is involved in the migration of endothelial cells on vitronectin,
ligand of integrin .alpha..sub.V.beta..sub.3 (Naik and Naik, 2005,
J. Cell Sci. 119:490-499). AntiJAM-A antibody J3F.1, in the same
manner as an anti-.alpha..sub.V.beta..sub.3 antibody, inhibits the
migration of endothelial cells and the angiogenesis induced by bFGF
in vitro (Naik et al., 2003, Blood, 102:2108-2114). Various
signaling pathways were demonstrated in endothelial cells: MAP
kinases, PI3-kinase and PKC (Naik et Naik, 2005, J. Cell Sci.,
119:490-499; Naik et al., 2003, Blood, 102:2108-2114; Naik et al.,
2003, Artherioscler. Thromb. Vasc. Biol., 23:2165-2171).
[0144] JAM-A is also expressed in monocytes, lymphocytes,
neutrophils and platelets (Williams et al., 1999, Mol. Immunol.,
36:1175-1188). JAM-A protein was however initially identified as a
receptor of the F11 antibody, an antibody capable of activating
platelets and inducing their aggregation (Naik et al., 1995,
Biochem. J., 310:155-162; Sobocka et al., 2000, Blood,
95:2600-2609). Peptides [28-60] and [97-109] belong to the F11
antibody epitope and are involved in platelet activation and
aggregation phenomena and in homodimerization (Babinska et al.,
2002, Thromb. Haemost., 87:712-721).
[0145] Rat antibody BV11, directed against the murine form of
JAM-A, inhibits the trans-endothelial migration of monocytes in
vitro and in vivo (Del Maschio et al., 1999, J. Exp. Med.,
190:1351-1356). Ostermann and colleagues (2002, Nature Immunol.,
3:151-158) showed that JAM-A was a ligand of
.alpha..sub.L.beta..sub.2 or LFA-1 (lymphocyte function-associated
antigen 1) integrin, which is overexpressed in response to certain
chemokines during the development of an anti-inflammatory response
and is required for the diapedesis or migration of leukocytes to
the site of inflammation. JAM-A, via the second Ig-like domain,
contributes to the adhesion and trans-endothelial migration of T
lymphocytes and neutrophils (Ostermann et al., 2002, Nature
Immunol., 3:151-158), and thus plays an important role in the
recruitment of leukocytes to the site of inflammation.
[0146] JAM-A protein is also implicated in viral infection
phenomena. JAM-A is indeed a receptor of reovirus, viruses
responsible for certain types of encephalitis by means of
interacting with attachment protein .sigma.1 (Barton et al., 2001,
Cell 104:441-451). AntiJAM-A antibody J10.4 inhibits the binding of
reovirus to JAM-A (Forrest et al., 2003, J. Biol. Chem.,
278:48434-48444).
[0147] To date, none of the antibodies mentioned above directed
against the human form of JAM-A exhibit activity in vivo, much less
anti-tumor activity. Such antibodies are used only as research
tools. Thus, in the former art, there is a genuine lack of an
anti-tumor antibody active in vitro and in vivo.
[0148] According to a specific aspect, the antibody of the
invention, or its derived compounds or functional fragments, is
characterized in that it is capable of specifically binding to
JAM-A protein (according to the English nomenclature "Junctional
Adhesion Molecules").
[0149] According to still another aspect, the antibody of the
invention, or its derived compounds or functional fragments, is
characterized in that it exhibits a K.sub.D for JAM-A from roughly
1 nM and roughly 1 pM. More preferably, said K.sub.D for JAM-A is
from roughly 10 pM and roughly 40 pM.
[0150] The expression "K.sub.D" refers to the dissociation constant
of a given antibody-antigen complex. K.sub.D=K.sub.off/K.sub.on
with K.sub.off consisting of the "off rate" constant for the
dissociation of the antibody from the antibody-antigen complex and
K.sub.on consisting of the level at which the antibody binds the
antigen (Chen Y. et al., 1999, J. Mol. Biol., 293:865-881).
[0151] A novel aspect of the present invention relates to an
isolated nucleic acid characterized in that it is selected among
the following nucleic acids (including any degenerate genetic
code):
[0152] a) a nucleic acid, DNA or RNA, coding for an antibody
according to the invention, or one of its derived compounds or
functional fragments;
[0153] b) a nucleic acid complementary to a nucleic acid as defined
in a);
[0154] c) a nucleic acid of at least 18 nucleotides capable of
hybridizing under highly stringent conditions with at least one of
the CDRs of nucleic acid sequences SEQ ID Nos. 20 to 31 or with a
sequence with at least 80%, preferably 85%, 90%, 95% and 98%,
identity after optimal alignment with sequence SEQ ID Nos. 20 to
31; and
[0155] d) a nucleic acid of at least 18 nucleotides capable of
hybridizing under highly stringent conditions with at least the
light chain of nucleic acid sequence SEQ ID No. 32 or 36 and/or the
heavy chain of nucleic acid sequence SEQ ID No. 33, 37 or 38, or
with a sequence with at least 80% identity after optimal alignment
with sequence SEQ ID No. 32 or 36 and/or 33, 37 or 38.
[0156] Table 4 below summarizes the various nucleotide sequences
concerning the antibody of the invention.
TABLE-US-00008 TABLE 4 CDR SEQ ID Antibody numbering Heavy chain
Light chain NO. 6F4 Common CDR-L1 20 CDR-L2 22 CDR-L3 24 CDR-H1 21
CDR-H2 23 CDR-H3 25 IMGT CDR-L1 20 CDR-L2 22 CDR-L3 24 CDR-H1 26
CDR-H2 23 CDR-H3 27 Kabat CDR-L1 28 CDR-L2 29 CDR-L3 24 CDR-H1 30
CDR-H2 31 CDR-H3 25 Mu. variable 32 domain Mu. variable 33 domain
Mu. entire 34 Mu. entire 35 Hu. variable 36 domain Hu. variable 37
domain (V1) Hu. variable 38 domain (V2)
[0157] The terms "nucleic acid", "nucleic sequence", "nucleic acid
sequence", "polynucleotide", "oligonucleotide", "polynucleotide
sequence" and "nucleotide sequence", used interchangeably in the
present description, mean a precise sequence of nucleotides,
modified or not, defining a fragment or a region of a nucleic acid,
containing unnatural nucleotides or not, and being either a
double-strand DNA, a single-strand DNA or transcription products of
said DNAs.
[0158] It should also be included here that the present invention
does not relate to nucleotide sequences in their natural
chromosomal environment, i.e., in a natural state. The sequences of
the present invention have been isolated and/or purified, i.e.,
they were sampled directly or indirectly, for example by a copy,
their environment having been at least partially modified. Isolated
nucleic acids obtained by recombinant genetics, by means, for
example, of host cells, or obtained by chemical synthesis should
also be mentioned here.
[0159] "Nucleic sequences exhibiting a percentage identity of at
least 80%, preferably 85%, 90%, 95% and 98%, after optimal
alignment with a preferred sequence" means nucleic sequences
exhibiting, with respect to the reference nucleic sequence, certain
modifications such as, in particular, a deletion, a truncation, an
extension, a chimeric fusion and/or a substitution, notably
punctual. Preferably, these are sequences which code for the same
amino acid sequences as the reference sequence, this being related
to the degeneration of the genetic code, or complementarity
sequences that are likely to hybridize specifically with the
reference sequences, preferably under highly stringent conditions,
notably those defined below.
[0160] Hybridization under highly stringent conditions means that
conditions related to temperature and ionic strength are selected
in such a way that they allow hybridization to be maintained from
two complementarity DNA fragments. On a purely illustrative basis,
the highly stringent conditions of the hybridization step for the
purpose of defining the polynucleotide fragments described above
are advantageously as follows.
[0161] DNA-DNA or DNA-RNA hybridization is carried out in two
steps: (1) prehybridization at 42.degree. C. for three hours in
phosphate buffer (20 mM, pH 7.5) containing 5.times.SSC
(1.times.SSC corresponds to a solution of 0.15 M NaCl+0.015 M
sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS),
10.times. Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA;
(2) primary hybridization for 20 hours at a temperature depending
on the length of the probe (i.e.: 42.degree. C. for a probe>100
nucleotides in length) followed by two 20-minute washings at
20.degree. C. in 2.times.SSC+2% SDS, one 20-minute washing at
20.degree. C. in 0.1.times.SSC+0.1% SDS. The last washing is
carried out in 0.1.times.SSC+0.1% SDS for 30 minutes at 60.degree.
C. for a probe>100 nucleotides in length. The highly stringent
hybridization conditions described above for a polynucleotide of
defined size can be adapted by a person skilled in the art for
longer or shorter oligonucleotides, according to the procedures
described in Sambrook, et al. (Molecular cloning: a laboratory
manual, Cold Spring Harbor Laboratory; 3rd edition, 2001).
[0162] The invention also relates to a vector comprising a nucleic
acid as described in the invention.
[0163] The invention notably targets cloning and/or expression
vectors that contain such a nucleotide sequence.
[0164] The vectors of the invention preferably contain elements
which allow the expression and/or the secretion of nucleotide
sequences in a given host cell. The vector thus must contain a
promoter, translation initiation and termination signals, as well
as suitable transcription regulation regions. It must be able to be
maintained in a stable manner in the host cell and may optionally
have specific signals which specify secretion of the translated
protein. These various elements are selected and optimized by a
person skilled in the art according to the host cell used. For this
purpose, the nucleotide sequences can be inserted in
self-replicating vectors within the selected host or be integrative
vectors of the selected host.
[0165] Such vectors are prepared by methods typically used by a
person skilled in the art and the resulting clones can be
introduced into a suitable host by standard methods such as
lipofection, electroporation, heat shock or chemical methods.
[0166] The vectors are, for example, vectors of plasmid or viral
origin. They are used to transform host cells in order to clone or
express the nucleotide sequences of the invention.
[0167] The invention also comprises host cells transformed by or
comprising a vector as described in the present invention.
[0168] The host cell can be selected among prokaryotic or
eukaryotic systems such as bacterial cells, for example, but also
yeast cells or animal cells, notably mammal cells. Insect or plant
cells can also be used.
[0169] The invention also relates to animals, other than man, that
have a transformed cell according to the invention.
[0170] Another aspect of the invention relates to a method for the
production of an antibody according to the invention, or one of its
functional fragments, characterized in that said method comprises
the following steps:
[0171] a) the culture in a medium of and the suitable culture
conditions for a host cell according to the invention; and
[0172] b) the recovery of said antibody, or one of its functional
fragments, thus produced from the culture medium or from said
cultured cells.
[0173] The transformed cells according to the invention are of use
in methods for the preparation of recombinant polypeptides
according to the invention. Methods for the preparation of
polypeptide according to the invention in recombinant form,
characterized in that said methods use a vector and/or a cell
transformed by a vector according to the invention, are also
comprised in the present invention. Preferably, a cell transformed
by an vector according to the invention is cultured under
conditions that allow the expression of the aforesaid polypeptide
and recovery of said recombinant peptide.
[0174] As already mentioned, the host cell can be selected among
prokaryotic or eukaryotic systems. In particular, it is possible to
identify the nucleotide sequences of the invention that facilitate
secretion in such a prokaryotic or eukaryotic system. An vector
according to the invention carrying such a sequence can thus be
used advantageously for the production of recombinant proteins to
be secreted. Indeed, the purification of these recombinant proteins
of interest will be facilitated by the fact that they are present
in the supernatant of the cellular culture rather than inside host
cells.
[0175] The polypeptides of the invention can also be prepared by
chemical synthesis. One such method of preparation is also an
object of the invention. A person skilled in the art knows methods
for chemical synthesis, such as solid-phase techniques (see notably
Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem.
Company, Rockford, 111, 2nd ed.) or partial solid-phase techniques,
by condensation of fragments or by conventional synthesis in
solution. Polypeptides obtained by chemical synthesis and capable
of containing corresponding unnatural amino acids are also
comprised in the invention.
[0176] The antibodies, or the derived compounds or functional
fragments of same, likely to be obtained by the method of the
invention are also comprised in the present invention.
[0177] According to still another aspect, the present invention
relates to an antibody as described above, characterized in that it
is, in addition, capable of specifically binding to a human
tyrosine kinase family receptor and/or capable of specifically
inhibiting the tyrosine kinase activity of such a receptor.
[0178] According to a novel embodiment, the invention relates to an
antibody, or its derived compounds or functional fragments,
consisting of an antibody that is bispecific in the sense that it
comprises a second motif capable of interacting with any receptor
implicated in the development of tumors, such as, for example,
VEGFR, VEGF, EGFR, IGF-1R, HER2neu, HGF, cMET, FGF, tetraspanins,
integrins, CXCR4 or CXCR2.
[0179] According to a first embodiment, one such antibody consists
of a bispecific antibody and comprises a second motif that
specifically inhibits the binding of EGF with human epidermal
growth factor receptor (EGFR) and/or specifically inhibiting the
tyrosine kinase activity of said EGFR. According to an even more
preferred aspect of the invention, said second anti-EGFR motif
arises from the monoclonal antibody cetuximab (C225 or erbitux),
matuzumab, huR3, HuMax-EGFR or panitumab.
[0180] According to a second embodiment, the antibody according to
the invention consists of a bispecific antibody and comprises a
second motif specifically inhibiting the activity modulated by the
HER2/neu receptor and/or specifically inhibiting the tyrosine
kinase activity of said HER2/neu receptor. More particularly, said
second antiHER2/neu motif arises from the mouse monoclonal antibody
4D5 or 2C4 or from the humanized antibody trastuzumab or
pertuzumab.
[0181] According to a third embodiment, the antibody according to
the invention consists of a bispecific antibody and comprises a
second motif specifically inhibiting the binding of hepatocyte
growth factor (HGF) with the cMET receptor and/or specifically
inhibiting the tyrosine kinase activity of said cMET receptor.
[0182] According to a fourth embodiment, the antibody according to
the invention consists of a bispecific antibody and comprises a
second motif specifically inhibiting the activity modulated by the
IGF-1R receptor and/or specifically inhibiting the tyrosine kinase
activity of said IGF-1R receptor. More particularly, said second
antiIGF-1R motif arises from mouse monoclonal antibody 7C10, from
corresponding humanized antibody h7C10 (Goetsch et al.,
international patent application WO 03/059951), from hEM164
antibodies (Maloney et al., Cancer Res., 2003, 63 (16):5073-5083),
from the antiIGF-1R antibodies developed by Abgenix (see US patent
application 2005/281812) or from Mab 39, 1H7 (Li et al., Cancer
Immunol. Immunother., 2000, 49(4-5):243-252) or 4G11 (Jackson-Booth
et al., Horm. Metab. Res., 2003, 35(11-12):850-856).
[0183] Lastly, according to a final embodiment, the antibody of the
invention consists in a bispecific antibody and comprises a second
motif capable of interacting with any type of receptor implicated
in tumor development, such as, as non-limiting examples, VEGFR,
VEGF, FGF (fibroblast growth factor) or any member of the CXCR
(chemokine receptor) family, such as CXCR2 or CXCR4.
[0184] Also suitable for mention are antiCD20 antibodies such as a
rituximab, ibritumomab or tositumomab; antiCD33 antibodies such as
gemtuzumab or lintuzumab; antiCD22 antibodies such as epratuzumab;
antiCD52 antibodies such as alemtuzumab; antiEpCAM antibodies such
as edrecolomab, Ch 17-1A or IGN-101; antiCTP21 or 16 antibodies
such as Xactin; antiDNA-Ag antibodies such as .sup.131I-Cotara
TNT-1; antiMUC1 antibodies such as pemtumomab or R1150; antiMUC18
antibodies such as ABX-MA1; antiGD3 antibodies such as mitumomab;
antiECA antibodies such as CeaVac or labetuzumab; antiCA125
antibodies such as OvaRex; antiHLA-DR antibodies such as
apolizumab; antiCTLA4 antibodies such as MDX-010; antiPSMA
antibodies such as MDX-070, .sup.111In & .sup.90Y-J591,
.sup.177Lu J591, J591-DM1; antiLewis Y antibodies such as IGN311;
antiangiogenesis antibodies such as AS1405 and 90YmuBC1;
antiTrail-R1 antibodies such as TRAIL R1mAb or TRAIL R2mAb.
[0185] The bispecific or bifunctional antibodies constitute a
second generation of monoclonal antibodies in which two different
variable regions are combined in the same molecule (Hollinger and
Bohlen, 1999, Cancer and metastasis, rev. 18:411-419). Their
utility was demonstrated in both diagnostic and therapeutic domains
relative to their capacity to recruit new effector functions or to
target several molecules on the surface of tumor cells; such
antibodies can be obtained by chemical methods (Glennie M J et al.,
1987, J. Immunol. 139, 2367-2375; Repp R. et al., 1995, J. Hemat.,
377-382) or somatic methods (Staerz U. D. and Bevan M. J., 1986,
PNAS 83, 1453-1457; Suresh M. R. et al., 1986, Method Enzymol.,
121:210-228) but also, preferentially, by genetic engineering
techniques that make it possible to force heterodimerization and
thus facilitate the purification of the antibody sought (Merchand
et al., 1998, Nature Biotech., 16:677-681).
[0186] These bispecific antibodies can be constructed as whole IgG,
bispecific Fab'2, Fab'PEG, diabodies or bispecific scFv, but also
as a tetravalent bispecific antibody in which two binding sites are
present for each antigen targeted (Park et al., 2000, Mol.
Immunol., 37(18):1123-30) or the fragments of same as described
above.
[0187] In addition to an economic advantage given that the
production and administration of a bispecific antibody are cheaper
than the production of two specific antibodies, the use of such
bispecific antibodies has the advantage of reducing the treatment's
toxicity. Indeed, the use of a bispecific antibody makes it
possible to decrease the overall quantity of circulating antibodies
and, consequently, possible toxicity.
[0188] In a preferred embodiment of the invention, the bispecific
antibody is a bivalent or tetravalent antibody.
[0189] Lastly, the present invention relates to the antibody
described above, or its derived compounds or functional fragments,
for use as a drug.
[0190] The invention also relates to a pharmaceutical composition
comprising as an active ingredient a compound consisting of an
antibody of the invention, or one of its derived compounds or
functional fragments. Preferably, said antibody is supplemented by
an excipient and/or a pharmaceutically acceptable carrier.
[0191] According to still another embodiment, the present invention
also relates to a pharmaceutical composition as described above
that comprises at least a second anti-tumor compound selected among
the compounds capable of specifically inhibiting the tyrosine
kinase activity of receptors such as IGF-IR, EGFR, HER2/neu, cMET,
VEGFR or VEGF, or any other anti-tumor compound known to a person
skilled in the art. In a second preferred aspect of the invention,
said second compound can be selected among the antibodies antiEGFR,
antiIGF-IR, antiHER2/neu, anticMET, VEGFR, VEGF, etc., isolated, or
their functional fragments and derived compounds, capable of
inhibiting the proliferative and/or anti-apoptotic and/or
angiogenic and/or inductive activity of metastatic dissemination
promoted by said receptors.
[0192] According to still another embodiment of the invention, the
composition comprises, in addition, as a combination product for
use in a simultaneous, separated or extended fashion, at least one
inhibiter of the tyrosine kinase activity of receptors such as
IGF-IR, EGFR, HER2/neu, cMET and VEGFR.
[0193] In another preferred embodiment, said inhibiter of the
tyrosine kinase activity of these receptors is selected from the
group comprising derived natural agents, dianilinophthalimides,
pyrazolo- or pyrrolo-pyridopyrimidines or quinazolines. Such
inhibiting agents, well-known to a person skilled in the art, are
described in the literature (Ciardiello F., Drugs 2000, Suppl. 1,
25-32).
[0194] Another embodiment complementary to the invention consists
of a composition as described above comprised of, in addition, as a
combination product for simultaneous, separated or extended use, a
cytotoxic/cytostatic agent.
[0195] "Simultaneous use" means the administration of both
compounds of the composition comprised in a single dosage form.
[0196] "Separated use" means administration, at the same time, of
both compounds of the composition, comprised in distinct dosage
forms.
[0197] "Extended use" means the successive administration of both
compounds of the composition, each comprised in a distinct dosage
form.
[0198] Generally, the composition according to the invention
considerably increases cancer treatment effectiveness. In other
words, the therapeutic effect of the antibody of the invention is
enhanced in an unexpected way by the administration of a cytotoxic
agent. Another major subsequent advantage produced by a composition
of the invention relates to the possibility of using lower
effective doses of the active ingredient, thus making it possible
to avoid or reduce the risks of the appearance of side effects, in
particular the effect of the cytotoxic agent. Moreover, this
composition makes it possible to achieve the expected therapeutic
effect more quickly.
[0199] "Therapeutic anticancer agent" or "cytotoxic agent" means a
substance which, when it is administered to a patient, treats or
prevents the development of cancer in the patient. Non-limiting
examples of such agents include "alkylating" agents,
antimetabolites, antitumor antibiotics, mitotic inhibitors,
inhibitors of chromatin functioning, antiangiogenics,
antiestrogens, antiandrogens and immunomodulators.
[0200] Such agents, for example, are cited in VIDAL, on the page
devoted to compounds related to oncology and hematology under the
heading "Cytotoxic"; the cytotoxic compounds cited by reference to
this document are cited herein as preferred cytotoxic agents.
[0201] "Alkylating agent" refers to any substance that can bind
covalently with or can alkylate any molecule, preferentially a
nucleic acid (e.g., DNA), within a cell. Examples of such
alkylating agents include nitrogen mustards such as
mechlorethamine, chlorambucil, melphalan, chlorhydrate, pipobroman,
prednimustine, disodium phosphate or estramustine;
oxazaphosphorines such as cyclophosphamide, altretamine,
trofosfamide, sulfofosfamide or ifosfamide; aziridines or
ethylene-imines such as thiotepa, triethyleneamine or altetramine;
nitrosoureas such as carmustine, streptozocine, fotemustine or
lomustine; alkyl sulfonates such as busulfan, treosulfan or
improsulfan; triazenes such as dacarbazine; or platinum complexes
such as cisplatine, oxaliplatine or carboplatine.
[0202] "Antimetabolite" refers to a substance that blocks growth
and/or cellular metabolism by interfering with certain activities,
generally DNA synthesis. Examples of antimetabolites include
methotrexate, 5-fluorouracile, floxuridine, 5-fluorodeoxyuridine,
capecitabine, cytarabine, fludarabine, cytosine arabinoside,
6-mercaptopurine (6-MP), 6-thioguanine (6-TG),
chlorodesoxyadenosine, 5-azacytidine, gemcitabine, cladribine,
deoxycoformycin and pentostatin.
[0203] "Antitumor antibiotic" refers to a compound that can prevent
or inhibit the synthesis of DNA, RNA and/or proteins. Examples of
such antitumor antibiotics include doxorubicin, daunorubicin,
idarubicin valrubicin, mitoxantrone, dactinomycin, mithramycin,
plicamycin, mitomycin C, bleomycin and procarbazine.
[0204] "Mitotic inhibiters" prevent the normal progression of the
cell cycle and mitosis. In general, microtubule inhibiters or
"taxoids" such as paclitaxel and docetaxel are capable of
inhibiting mitosis. The vinca alkaloids, such as vinblastine,
vincristine, vindesine and vinorelbine, are also capable of
inhibiting mitosis.
[0205] "Chromatin inhibiters" or "topoisomerase inhibiters" are
substances that inhibit the normal functioning of proteins that
shape chromatin, such as topoisomerases I and II. Examples of such
inhibiters include, for topoisomerase I, camptothecine and its
derivatives, such as irinotecan or topotecan; for topoisomerase II,
etoposide, etiposide phosphate and teniposide.
[0206] An "antiangiogenic" is any drug, compound, substance or
agent that inhibits the growth of the blood vessels. Examples of
antiangiogenics include, without being limiting, razoxin,
marimastat, batimastat, prinomastat, tanomastat, ilomastat,
CGS-27023A, halofuginone, COL-3, neovastat, BMS-275291,
thalidomide, CDC 501, DMXAA, L-651582, squalamine, endostatine,
SU5416, SU6668, interferon-alpha, EMD121974, interleukin-12, IM862,
angiostatin and vitaxin.
[0207] "Antiestrogen" or "estrogen antagonist" refers to any
substance that decreases, antagonizes or inhibits estrogen action.
Examples of such agents are tamoxifene, toremifene, raloxifene,
droloxifene, iodoxyfene, anastrozole, letrozole and exemestane.
[0208] "Antiandrogen" or "androgen antagonist" refers to any
substance that reduces, antagonizes or inhibits androgen action.
Examples of antiandrogens include flutamide, nilutamide,
bicalutamide, sprironolactone, cyproterone acetate, finasteride and
cimitidine.
[0209] Immunomodulators are substances that stimulate the immune
system. Examples of immunomodulators include interferon,
interleukins such as aldesleukin, OCT-43, denileukin diftitox or
interleukine-2, tumor necrosis factors such as tasonermine, or
other types of immunomodulators such as lentinan, sizofiran,
roquinimex, pidotimod, pegademase, thymopentine, poly I:C or
levamisole in combination with 5-fluorouracil.
[0210] For further details, a person skilled in the art can refer
to the manual published by the French Association of Therapeutic
Chemistry Teachers titled "Therapeutic chemistry, vol. 6, Antitumor
drugs and perspectives in the treatment of cancer, TEC and DOC
edition, 2003 [in French]".
[0211] In a particularly preferred embodiment, said composition of
the invention as a combination product is characterized in that
said cytotoxic agent is bound chemically to said antibody for use
simultaneously.
[0212] In a particularly preferred embodiment, said composition is
characterized in that said cytotoxic/cytostatic agent is selected
among the spindle inhibitors or stabilizers, preferably vinorelbine
and/or vinflunine and/or vincristine.
[0213] In order to facilitate binding from said cytotoxic agent and
the antibody according to the invention, spacer molecules can be
introduced from the two compounds to bind, such as the
poly(alkylene)glycol polyethyleneglycol or the amino acids; or, in
another embodiment, said cytotoxic agents' active derivatives, into
which have been introduced functions capable of reacting with said
antibody, can be used. These binding techniques are well-known to a
person skilled in the art and will not be discussed in more detail
in the present description.
[0214] Other EGFR inhibiters include, without being limiting,
monoclonal antibodies C225 and antiEGFR 22Mab (ImClone Systems
Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck
KgaA) or compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca),
PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787
(Novartis), CP 701 (Cephalon), flunomide (Pharmacia/Sugen), CI-1033
(Warner Lambert Parke Davis), CI-1033/PD 183, 805 (Warner Lambert
Parke Davis), CL-387, 785 (Wyeth-Ayerst), BBR-1611 (Boehringer
Mannheim GMBH/Roche), Naamidine A (Bristol-board Myers Squibb),
RC-3940-II (Pharmacia), BIBX-1382 (Boehringer Ingelheim), OLX-103
(Merck & Co), VRCTC-310 (Ventech Research), EGF fusion toxin
(Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808 (Imperial
Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (Parker Hughes
Center Cancer), WHI-P97 (Parker Hughes Center Cancer), GW-282974
(Glaxo), KT-8391 (Kyowa Hakko) or the "EGFR vaccine" (York
Medical/Centro of Immunologia Molecular).
[0215] Another aspect of the invention relates to a composition
characterized in that at least one of said antibodies, or of the
derived compounds or functional fragments of same, is conjugated
with a cellular toxin and/or a radioisotope.
[0216] Preferably, said toxin or said radioisotope is capable of
preventing the growth or proliferation of the tumor cell, notably
of completely inactivating said tumor cell.
[0217] Also preferably, said toxin is an enterobacteria toxin,
notably Pseudomonas exotoxin A.
[0218] The radioisotopes preferentially combined with therapeutic
antibodies are radioisotopes that emit gamma rays, preferentially
iodine.sup.131, yttrium.sup.90, gold.sup.199, palladium.sup.100,
copper.sup.67, bismuth.sup.217 and antimony.sup.211. Radioisotopes
that emit alpha and beta rays can also be used in therapy.
[0219] "Toxin or radioisotope combined with at least one antibody
of the invention, or a functional fragment of same" refers to any
means that makes it possible to bind said toxin or said
radioisotope to that at least one antibody, notably by covalent
binding from the two compounds, with or without the introduction of
the binding molecule.
[0220] Examples of agents that allow chemical (covalent),
electrostatic, or non-covalent bonding of all or part of the
conjugate's elements include, in particular, benzoquinone,
carbodiimide and more particularly EDC
(1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide-hydrochloride),
dimaleimide, dithiobis-nitrobenzoic (DTNB) acid, N-succinimidyl
S-acetyl thio-acetate (SATA), bridging agents with one or more
groups, with one or more phenylaside groups, reacting with
ultraviolet (UV) rays, most preferentially
N-[-4(azidosalicylamino)butyl]-3'-(2'-pyridyldithio)-propionamide
(APDP), N-succinimid-yl 3(2-pyridyldithio)propionate (SPDP) and
6-hydrazino-nicotinamide (HYNIC).
[0221] Another form of binding, notably for radioisotopes, can
consist of the use of bifunctional ion chelating agents.
[0222] Examples of such chelators include the chelators derived
from EDTA (ethylenediaminetetraacetic acid) or DTPA (d
iethylenetriaminepentaacetic acid) which were developed to bind
metals, particularly radioactive metals, with immunoglobulins.
Thus, DTPA and its derivatives can be substituted on the carbon
chain by various groups in such a way as to increase the stability
and the rigidity of the ligand-metal complex (Krejcarek et al.,
1977; Brechbiel et al., 1991; Gansow, 1991; U.S. Pat. No.
4,831,175).
[0223] For example, DTPA (diethylenetriaminepentaacetic acid) and
its derivatives, which long have been widely used in drug and
biology either in its free form or in a complex with a metal ion,
exhibit the remarkable characteristic of forming stable chelates
with metal ions which can be coupled with proteins of therapeutic
or diagnostic interest, such as antibodies, for the development of
radio-immuno conjugates for cancer therapy (Meases et al., 1984;
Gansow et al., 1990).
[0224] Also preferably, said at least one antibody of the invention
forming said conjugate is selected among its functional fragments,
notably fragments that have lost their Fc component, such as scFv
fragments.
[0225] The present invention also comprises the use of the
composition for the preparation of a drug intended for the
prevention or the treatment of cancer.
[0226] The present invention also relates to the use of an
antibody, or a derived compound or functional fragment of same,
preferably humanized, and/or of a composition according to the
invention for the preparation of a drug for inhibiting the growth
of tumor cells. Generally, the present invention relates to the use
of an antibody, or a derived compound or functional fragment of
same, preferably humanized, and/or of a composition, for the
preparation of a drug for cancer prevention or treatment.
[0227] Preferred cancers that can be prevented and/or treated
include prostate cancer, osteosarcoma, lung cancer, breast cancer,
endometrial cancer, colon cancer, multiple myeloma, ovarian cancer,
pancreatic cancer or any other cancer.
[0228] In a preferred manner, said cancer is a cancer selected
among estrogen-related breast cancer, non-small cell lung cancer,
colon cancer and/or pancreatic cancer.
[0229] Another aspect of the present invention relates to the use
of the antibody as described in a diagnostic method, preferably in
vitro, of diseases related to JAM-A expression level. Preferably,
said JAM-A protein related diseases in said diagnostic method will
be cancers.
[0230] Thus, the antibodies of the invention, or the derived
compounds or functional fragments of same, can be employed in a
method for the detection and/or quantification of JAM-A protein in
a biological sample in vitro, notably for the diagnosis of diseases
associated with an abnormal expression with this protein, such as
cancers, wherein said method comprises the following steps:
[0231] a) placing the biological sample in contact with an antibody
according to the invention, or a derived compound or functional
fragment of same;
[0232] b) demonstrating the antigen-antibody complex possibly
formed.
[0233] Thus, the present invention also comprises the kits or
accessories for the implementation of a method as described (for
detecting the expression of a gene from Legionella pneumophila
Paris or from an associated organism, or for detecting and/or
identifying Legionella pneumophila Paris bacteria or associated
microorganisms), comprising the following elements:
[0234] a) a polyclonal or monoclonal antibody of the invention;
[0235] b) optionally, reagents for constituting the medium
favorable to immunological reactions;
[0236] c) optionally, reagents that reveal the antigen-antibodies
complexes produced by the immunological reaction.
[0237] Advantageously, the antibodies or functional fragments of
same can be immobilized on a support, notably a protein chip. One
such protein chip is an object of the invention.
[0238] Advantageously, the protein chips can be used in the kits or
accessories required for detecting and/or quantifying JAM-A protein
in a biological sample.
[0239] It must be stated that the term "biological sample" relates
herein to samples taken from a living organism (notably blood,
tissue, organ or other samples taken from a mammal, notably man) or
any sample likely to contain one such JAM-A protein (such as a
sample of cells, transformed if needed).
[0240] Said antibody, or a functional fragment of same, can be in
the form of an immunoconjugate or of a labeled antibody in order to
obtain a detectable and/or quantifiable signal.
[0241] The labeled antibodies of the invention, or the functional
or fragments of same, include, for example, antibody conjugates
(immunoconjugates), which can be combined, for example, with
enzymes such as peroxidase, alkaline phosphatase,
.alpha.-D-galactosidase, glucose oxidase, glucose amylase, carbonic
anhydrase, acetyl-cholinesterase, lysozyme, malate dehydrogenase or
glucose-6 phosphate dehydrogenase or by a molecule such as biotin,
digoxigenin or 5-bromo-desoxyuridine. Fluorescent labels can be
also combined with the antibodies of the invention or functional
fragments of same, including notably fluorescein and its
derivatives, fluorochrome, rhodamine and its derivatives, green
fluorescent protein (GFP), dansyl, umbelliferone, etc. In such
conjugates, the antibodies of the invention or functional fragments
of same can be prepared by methods known to a person skilled in the
art. They can be bound with enzymes or fluorescent labels directly;
via a spacer group or a linkage group such as polyaldehyde,
glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) or
diethylenetriaminepentaacetic acid (DPTA); or in the presence of
binding agents such as those mentioned above for therapeutic
conjugates. Conjugates carrying fluorescein labels can be prepared
by reaction with an isothiocyanate.
[0242] Others conjugates can also include chemiluminescent labels
such as luminol and dioxetane, bioluminescent labels such as
luciferase and luciferin, or radioactive labels such as
iodine.sup.123, iodine.sup.125, iodine.sup.126, iodine.sup.133,
bromine.sup.77, technetium99m, indium.sup.111, indium.sup.113m,
gallium.sup.67, gallium.sup.68, ruthenium.sup.95, ruthenium.sup.97,
ruthenium.sup.103, ruthenium.sup.105, mercury.sup.107,
mercury.sup.203, rhenium.sup.99m, rhenium.sup.101, rhenium.sup.105,
scandium.sup.47, tellurium.sup.121m, tellurium.sup.122m,
tellurium.sup.125m, thulium.sup.165, thulium.sup.187,
thulium.sup.168, fluorine.sup.18, yttrium.sup.199 and
iodine.sup.131. Existing methods known to a person skilled in the
art for binding radioisotopes with antibodies, either directly or
via a chelating agent such as the EDTA or DTPA mentioned above, can
be used for as diagnostic radioisotopes. Thus should be mentioned
labeling with [I.sup.125]Na by the chloramine-T technique [Hunter
W. M. and Greenwood F. C. (1962) Nature 194:495]; labeling with
technetium.sup.99m as described by Crockford et al. (U.S. Pat. No.
4,424,200) or bound via DTPA as described by Hnatowich (U.S. Pat.
No. 4,479,930).
[0243] The invention also relates to the use of an antibody
according to the invention for the preparation of a drug for the
specific targeting of a compound that is biologically active toward
cells expressing or overexpressing JAM-A protein.
[0244] In the sense of the present description, a "biologically
active compound" is any compound capable of modulating, notably
inhibiting, cellular activity, notably growth, proliferation,
transcription and gene translation.
[0245] The invention also relates to an in vivo diagnostic reagent
composed of an antibody according to the invention, or a functional
fragment of same, preferably labeled, notably radiolabeled, and its
use in medical imaging, notably for the detection of cancer related
to the cellular expression or overexpression of JAM-A protein.
[0246] The invention also relates to a composition as a combination
product or to an anti-JAM-A/toxin conjugate or radioisotope,
according to the invention, used as drug.
[0247] Preferably, said composition as a combination product or
said conjugate will be supplemented by an excipient and/or a
pharmaceutical vehicle.
[0248] In the present description, "pharmaceutical vehicle" means a
compound, or a combination of compounds, entering a pharmaceutical
composition that does not cause secondary reactions and that, for
example, facilitates administration of the active compounds,
increases its lifespan and/or effectiveness in the organism,
increases its solubility in solution or improves its storage. Such
pharmaceutical carriers are well-known and will be adapted by a
person skilled in the art according to the nature and the
administration route of the active compounds selected.
[0249] Preferably, such compounds will be administered by systemic
route, notably by intravenous, intramuscular, intradermal,
intraperitoneal, subcutaneous or oral route. More preferably, the
composition composed of the antibody according to the invention
will be administered in several doses spaced equally over time.
[0250] Their administration routes, dosing schedules and optimal
galenic forms can be determined according to the criteria generally
taken into account when establishing a treatment suited to a
patient such as, for example, the patient's age or body weight, the
seriousness of his general state, his tolerance for the treatment
and the side effects experienced.
[0251] Thus, the invention relates to the use of an antibody, or
one of its functional fragments, for the preparation of a drug for
the specific targeting of a compound that is biologically active
toward cells expressing or overexpressing JAM-A.
[0252] Other characteristics and advantages of the invention appear
further in the description with the examples and figures whose
legends are presented below.
BRIEF DESCRIPTION OF THE FIGURES
[0253] FIG. 1 shows the respective sequences of the heavy (DNA
sequence disclosed as SEQ ID NO: 33; protein sequence disclosed as
SEQ ID NO: 14) and light (DNA sequence disclosed as SEQ ID NO: 32;
protein sequence disclosed as SEQ ID NO: 13) chains of the murine
6F4 antibody. CDRs are underlined and in bold (according to the
Kabat numbering).
[0254] FIGS. 2A and 2B represent the respective alignments of the V
(FIG. 2A) and J (FIG. 2B) regions of murine 6F4 antibody and the
murine cell lines selected, namely IGKV19-93*01 (SEQ ID No. 39) for
the V region and IGKJ1*01 (SEQ ID No. 40) for the J region. FIG. 2A
discloses SEQ ID NOS 13 and 69-70, respectively, in order of
appearance. FIG. 2B discloses SEQ ID NOS 40, 71 and 71,
respectively, in order of appearance.
[0255] FIGS. 3A and 3B represent the respective alignments of the V
(FIG. 3A) and J (FIG. 3B) regions of murine 6F4 antibody and the
human cell lines selected, namely IGKV1-33*01 (SEQ ID No. 41) for
the V region and IGKJ1*01 (SEQ ID No. 42) for the J region. FIG. 3A
discloses SEQ ID NOS 72-75, respectively, in order of appearance.
FIG. 3B discloses SEQ ID NOS 76, 40 and 77-78, respectively, in
order of appearance.
[0256] FIG. 4 is the protein sequence of the light chain of the 6F4
antibody (SEQ ID NO: 13) with reference to the respective KABAT and
IMGT numbering systems.
[0257] FIGS. 5A, 5B and 5C represent the respective alignments of
the V (FIG. 5A), D (FIG. 5B) and J (FIG. 5C) regions of the murine
6F4 antibody and the murine cell lines selected, namely
IGHV1S135*01 (SEQ ID No. 43) for the V region, IgHD-ST4*01 (SEQ ID
No. 44) for the D region and IgHJ2*01 (SEQ ID No. 45) for the J
region. FIG. 5A discloses SEQ ID NOS 14, 79 and 54, respectively,
in order of appearance. FIG. 5B discloses SEQ ID NOS 80-81,
respectively, in order of appearance. FIG. 5C discloses SEQ ID NOS
82-84, respectively, in order of appearance.
[0258] FIGS. 6A, 6B and 6C represent the respective alignments of
the V (FIG. 6A), D (FIG. 6B) and J (FIG. 6C) regions of the murine
6F4 antibody and the human cell lines selected, namely IGHV1-f*01
(SEQ ID No. 46) for the V region, IGHD1-1*01 (SEQ ID No. 47) for
the D region and IGHJ4*01 (SEQ ID No. 48) for the J region. FIG. 6A
discloses SEQ ID NOS 85-86, 57 and 46, respectively, in order of
appearance. FIG. 6B discloses SEQ ID NOS 87-88, respectively, in
order of appearance. FIG. 6C discloses SEQ ID NOS 89, 45, 90 and
48, respectively, in order of appearance.
[0259] FIG. 7 is the protein sequence of the heavy chain of the 6F4
antibody (SEQ ID NO: 14) with reference to the respective KABAT and
IMGT numbering systems.
[0260] FIGS. 8A and 8B represent the 6F4-sepharose
immunopurification of 6F4 antigen from HT-29 cell membranes.
Analyses of fractions collected by SDS-PAGE electrophoresis (FIG.
8A) and western blot (FIG. 8B) are presented as well.
[0261] FIGS. 9A and 9B present an analysis by SDS-PAGE
electrophoresis (FIG. 9A) and western blot (FIG. 9B) of
immunopurified protein. Two purifications (#1 and #2) were
performed and analyzed under reducing and in non-reducing
conditions.
[0262] FIG. 10 presents an analysis by MALDI-TOF mass spectrometry
of the mixture of peptides extracted after tryptic hydrolysis.
[0263] FIGS. 11A and 11B consist of the confirmation of a protein
identified by western blot (non-reducing conditions): revealed
using 6F4 antibody (FIG. 11A) and anti-human JAM-A polyclonal
antibody (FIG. 11B).
[0264] FIG. 12 shows the specificity of the 6F4 antibody for human
JAM-A protein. The quantities deposited for each protein are 250
ng, 25 ng and 10 ng.
[0265] FIG. 13 is sensorgrams obtained after 2 minutes of injection
(double arrow) of the 6F4 antibody at 100 nm in HBS-EP buffer on
murine JAM1 Fc protein (Flow cell #1, bottom graph) and on murine
JAM1 Fc protein (Flow cell #2, top graph) with a dissociation time
at 25.degree. C. of 5 minutes and a flow rate of 30 .mu.l/min (CM4:
m-JAM1-Fc 501.6 RU (Fc1) and 511.5 RU (Fc2)).
[0266] FIG. 14 is sensorgrams obtained with a double reference,
(Fc2-Fc1)6F4(Fc2-Fc1)HBS-EP. The curve is fitted using a Langmuir
A+B binding model. The calculated kinetic parameters (black curve)
are as follows: ka=(1.38.+-.0.001)*.sup.10.sup.5 M.sup.-1s.sup.-1;
kd=(0.25.+-.1.58)10.sup.-6 s.sup.-1; Rmax (global fitting)=371 RU;
.quadrature..sup.2=0.853.
[0267] FIG. 15 illustrates the antitumor activity of the 6F4
antibody in a xenograft model of MCF-7 cells in the Swiss nude
mouse. The 6F4 antibody was tested by IP route in unpurified form
(peritoneal cavity fluid), at the theoretical dose of 250
.mu.g/mouse, twice per week. The 9G4 antibody is an antibody of the
same isotype (IgG1), non-relevant with respect to the activity
measured.
[0268] FIG. 16 illustrates JAM-A protein expression recognized by
Mab 6F4 on the surface of various tumor lines.
[0269] FIG. 17 is the sequence of the humanized 6F4 VL domain (SEQ
ID NO: 91) wherein: * correspond to amino acids changed de facto to
their human counterparts, 1 correspond to amino acids analyzed for
their abilities to be humanized, the human residue being indicated
below the sign, and 2 correspond to amino acids that remain murin
in the humanized 6F4 VL domain.
[0270] FIG. 18 is the sequence of the humanized 6F4 VH domain (SEQ
ID NO: 92) wherein: * correspond to amino acids changed de facto to
their human counterparts, 1 correspond to amino acids analysed for
their abilities to be humanized, the human residue being indicated
below the sign, and 2 correspond to amino acids that remain murin
in the humanized 6F4 VH domain.
[0271] FIG. 19 illustrates the in vitro JAM-A down-regulation
induced by the 6F4 MAb.
[0272] FIG. 20 illustrates the in vivo inhibition of tumor cell
proliferation induced by the 6F4 MAb.
[0273] FIG. 21 is the in vivo down-regulation of JAM-A by the 6F4
Mab.
[0274] FIG. 22 is curves of the Comparison of 6F4 and its
F(ab').sub.2 fragment on the MCF-7 in vivo model.
[0275] FIG. 23 illustrates the comparison of normal versus tumoral
expression of JAM-A on thyroid tissues.
[0276] FIG. 24 illustrates the comparison of normal versus tumoral
expression of JAM-A on lung tissues.
[0277] FIG. 25 illustrates the comparison of normal versus tumoral
expression of JAM-A on Breast tissues.
[0278] FIG. 26 is curves illustrating the in vivo activity of 6F4
on A431 epidermoid carcinoma xenograft in nude mice.
[0279] FIG. 27 illustrates the effect of the 6F4 antibody on A. non
specific lyphoproliferation induced with PHA and B. antigen
presentation process. First experiment with 2 independent
donors.
[0280] FIG. 28 illustrates the effect of the 6F4 antibody on A. non
specific lyphoproliferation induced with PHA and B. antigen
presentation process. Second experiment with 2 independent
donors.
[0281] FIG. 29 illustrates the platelet aggregation on 10 human
normal donors. Results are expected as mean+/-sd.
[0282] FIG. 30 is the serotonine release on 10 human normal donors.
Results are expected as mean+/-sd.
[0283] FIG. 31 is the alignment of the 6F4 VH domain and
IGHV1-03*01 germline gene (SEQ ID No. 49). Figure discloses SEQ ID
NOS 93-95, respectively, in order of appearance.
[0284] FIG. 32 is the IGKV1-27*01 based humanized version of 6F4 VL
with mentioned mutations (BU-L1). Figure discloses SEQ ID NOS 13
and 96-97, respectively, in order of appearance.
[0285] FIG. 33 is the IGKV1D-43*01 based humanized version of 6F4
VL with mentioned mutations (BU-L2). Figure discloses SEQ ID NOS 13
and 98-99, respectively, in order of appearance.
[0286] FIG. 34 is the IGHV1-3*01 based humanized version of 6F4 VH
with mentioned mutations (BU-H1). Figure discloses SEQ ID NOS 14
and 100-101, respectively, in order of appearance.
[0287] FIG. 35 is the IGHV1-46*01 based humanized version of 6F4 VH
with mentioned mutations (BU-H2). Figure discloses SEQ ID NOS 14,
102 and 101, respectively, in order of appearance.
[0288] In order to further illustrate the present invention and the
advantages thereof, the following specific examples are given, it
being understood that same are intended only as illustrative and in
nowise limitative.
EXAMPLES
Example 1
Generation of the 6F4 Antibody
[0289] To generate the murine monoclonal antibody (Mab), BALB/C
mice were immunized using 5.times.10.sup.6 MCF-7 cells from ATCC.
After a final booster injection of 10.sup.7 MCF-7 cells, cells from
lymph nods of mice are fused with Sp2/O-Ag14 myeloma cells using
the techniques classically described by Kohler and Milstein. The
supernatants of the hybridomas arising from the fusion were then
screened for functional activity, namely the inhibition of the
proliferation of MCF-7 cells in vitro.
[0290] For this screening, MCF-7 cells are cultured in 96-well
culture dishes at 5.times.10.sup.3 cells/well in 100 .mu.l of
hybridoma medium without fetal calf serum. The plates are incubated
for 24 hours at 37.degree. C. under an atmosphere of 5% CO.sub.2.
After 24 hours, 50 .mu.l of the supernatant of the hybridomas to be
screened are added to each well. The last line on the plate is
reserved for the controls: [0291] three wells are supplemented by
50 .mu.l of a hybridoma supernatant that is non-relevant with
respect to the activity sought and that is cultured in the same
culture medium as that used for the fused cells. These wells will
be used to calibrate the impact of inactive supernatant on the
incorporation of tritiated thymidine; [0292] three wells will
receive 50 .mu.l of hybridoma culture medium.
[0293] After roughly 52 hours of culture, each well is supplemented
by 0.25 .mu.Ci of [.sup.3H]thymidine and incubated again for 20
hours at 37.degree. C. The incorporation of [.sup.3H]thymidine in
the DNA, indicating cell proliferation, is quantified by measuring
liquid scintillation. Background noise and thresholds are
determined for each plate as a function of the control wells
containing the medium alone and the non-relevant hybridoma
supernatant.
[0294] By this method, 43 hybridomas secreting antibodies
inhibiting the growth of MCF-7 cells were selected after a first
screening. Eleven of these 43 hybridomas had weak or non-existent
growth and were abandoned. During proliferation tests performed
following the expansion and cloning of the hybridomas, only the
hybridomas whose supernatant had a .gtoreq.20% inhibiting activity
on the proliferation of MCF-7 cells were selected. At the end of
the cloning/selection process, only one clone proved to have the
required properties, the 6F4 clone.
Example 2
Process of Humanization by CDR-Grafting of the Variable Region of
the Light Chain of the 6F4 Antibody (6F4 VL)
[0295] a) Comparison of the 6F4 VL Nucleotide Sequence with all
Known Murine Cell Line Sequences
[0296] As a preliminary step in humanization by CDR-grafting, the
6F4 VL nucleotide sequence initially was compared with all of the
murine cell line sequences present in the IMGT data bank.
[0297] Regions V and J of mouse cell lines having a sequence
identity of 98.56% for the V region and 100% for the J region were
identified, respectively IGKV19-93*01 (SEQ ID No. 39, EMBL
nomenclature: AJ235935) and IGKJ1*01 (SEQ ID No. 40, EMBL
nomenclature: V00777).
[0298] Considering these identity percentages, it was decided to
use the 6F4 VL sequence directly.
[0299] These alignments are represented in FIG. 2A for the V region
and in FIG. 2B for the J region.
[0300] b) Comparison of the Nucleotide Sequence of 6F4 VL with all
Known Human Cell Line Sequences
[0301] In order to identify the best human candidate for
CDR-grafting, the human-origin germline having the greatest
possible identity with 6F4 VL was sought. For this purpose, the
nucleotide sequence of mouse 6F4 VL was compared with all of the
human cell line sequences present in the IMGT data base.
[0302] Regions V and J of human-origin cell lines were identified
with a sequence identity of 81.36% for the V region, namely
IGKV1-33*01 (SEQ ID No. 41, EMBL nomenclature: M64856) and 86.84%
for the J region, namely IGKJ1*01 (SEQ ID No. 42, EMBL
nomenclature: J00242).
[0303] Cell lines IGKV1-33*01 for the V region and IGKJ1*01 for the
J region were thus selected as human receptor sequences for mouse
6F4 VL CDRs.
[0304] These alignments are presented in FIG. 3A for the V region
and in FIG. 3B for the J region.
[0305] c) Humanized Versions of 6F4 VL
[0306] The following step in the humanization process consists of
joining together the IGKV1-33*01 and IGKJ1*01 cell line sequences
and then joining the mouse 6F4 VL CDRs to the scaffold regions of
these same germlines.
[0307] This stage of the process the molecular model of the mouse
6F4 Fv regions will be particularly useful in the choice of the
mouse residues to preserve because they may play a role either in
maintaining the molecule's three-dimensional structure (canonical
structure of CDRs, VH/VL interfaces, etc.) or in binding the
antigen. In the scaffold regions, each difference from mouse (6F4
VL) and human (IGKV1-33*01/IGKJ1*01) nucleotides will be examined
very carefully.
[0308] For more clarity in the following, FIG. 4 presents the 6F4VL
sequence with reference to KABAT and IMGT classifications.
[0309] Three murine residues were identified which must be
preserved.
[0310] Residue 33 (Ile) takes part in CDR1 anchoring according to
IMGT and is part of CDR1 according to Kabat.
[0311] Residue 49 (His) takes part in CDR2 anchoring according to
IMGT, takes part in the VH/VL interface and belongs to the Vernier
zone.
[0312] Residue 53 (Thr) takes part in CDR2 anchoring according to
IMGT and is part of CDR2 according to Kabat.
[0313] Initially, three changes in the scaffold regions of
IGKV1-33*01 and IGKJ1*01 will be studied. These changes relate to
residues 24, 69 and 71 (IMGT nomenclature). It should be
understood, of course, that these three changes will be studied
independently of each other and also in various combinations. The
aim is to have available all possible mutants in order to test them
and to select the mutant that has preserved the best binding
properties. ELISA/Biacore binding tests will thus be performed on
each mutant.
[0314] Residue 24 (Lys/Gln) is near CDR1 and could as a result be
critical for maintaining a conformation that enables proper CDR1
presentation. More particularly, this residue is likely to interact
with residues 69-70 within the Vernier zone. Lys is only slightly
represented in human VLs but is part of CDR1 according to
Kabat.
[0315] Although residue 69 (Arg/Thr) is in the Vernier zone and
thus directly takes part in CDR1's canonical structure, this
residue is always Thr in the human VL.
[0316] Although residue 71 (Tyr/Phe) directly takes part in CDR1's
canonical structure, it is systematically Phe in the human VL.
[0317] Secondly, a modification of residue 56 (Ala) into Thr can be
considered. This residue, although outside of CDRs according to
IMGT, belongs to CDR2 according to Kabat.
[0318] Third and last, two additional changes could be made at
residues 34 and 55 (IMGT nomenclature). The two residues, outside
of the CDRs defined IMGT, are included in the CDRs defined by
Kabat.
[0319] Residue 34 (Ala/Asn) belongs to CDR1 according to Kabat and
takes part in the VH/VL interface. Such a mutation remains relevant
in spite of the strong representation of Ala in man.
[0320] Residue 55 (Gln/Glu) is part of CDR2 according to Kabat and
also takes part in the VH/VL interface. Such a mutation also
remains relevant in spite of the strong representation of Gln in
man.
[0321] As was described above, these three mutations could be
tested independently or in various combinations.
Example 3
Process of Humanization by CDR-Grafting of the Variable Region of
the Heavy Chain of the 6F4 Antibody (6F4 VH)
[0322] a) Comparison of the 6F4 VH Nucleotide Sequence with all
Known Murine Cell Line Sequences
[0323] As a preliminary step in humanization by CDR-grafting, the
6F4 VH nucleotide sequence initially was compared with all of the
murine cell line sequences present in the IMGT data bank.
[0324] Regions V, D and J of murine cell lines having a sequence
identity of 99.30% for the V region (IGHV1S135*01; SEQ ID No. 43;
EMBL nomenclature: AF304556), of 80% for the D region (IgHD-ST4*01;
SEQ ID No. 44; EMBL nomenclature: M23243) and of 100% for the J
region (IgHJ2*01; SEQ ID No. 45; EMBL nomenclature: V00770).
[0325] These alignments are represented in FIG. 5A for the V
region, FIG. 5B for the D region and FIG. 5C for the J region.
[0326] Considering these identity percentages, it was decided to
use the 6F4 VH sequence directly, as was the case for 6F4 VL.
[0327] b) Comparison of the Nucleotide Sequence of 6F4 VH with all
Known Human Cell Line Sequences
[0328] In order to identify the best human candidate for
CDR-grafting, the human-origin germline having the greatest
possible identity with each of the three regions V, D and J of 6F4
VH was sought. For this purpose, the nucleotide sequence of mouse
6F4 VH was compared with all of the human cell line sequences
present in the IMGT data base.
[0329] Human-origin germlines were identified having an sequence
identity of 75.34% for the V region (IGHV1-f*01; SEQ ID No. 46;
EMBL nomenclature: Z12305), of 71.42% for the D region (IGHD1-1*01;
SEQ ID No. 47; EMBL nomenclature: X97051) and of 87.51% for the J
region (IGHJ4*01; SEQ ID No. 48, EMBL nomenclature: J00256).
[0330] For each of the regions V, D and J, the germinal lines above
were selected and rearranged from them.
[0331] These alignments are presented in FIG. 6A for the V region,
FIG. 6B for the D region and FIG. 6C for the J region.
[0332] c) Humanized Versions of 6F4 VH
[0333] The following step in the humanization process consists of
joining together the IGHV1-f*01, IGHD1-1*01 and IGHJ4*01 cell line
sequences and then joining the mouse 6F4 VH CDRs to the scaffold
regions of these same germlines.
[0334] This stage of the process the molecular model of the mouse
6F4 Fv regions will be particularly useful in the choice of the
mouse residues to preserve because they may play a role either in
maintaining the molecule's three-dimensional structure (canonical
structure of CDRs, VH/VL interfaces, etc.) or in binding the
antigen. In the scaffold regions, each difference from mouse (6F4
VH) and human (IGHV1-f*01, IGHD1-1*01 and IGHJ4*01) nucleotides
will be examined very carefully.
[0335] For more clarity in the following, FIG. 7 presents the 6F4VH
sequence with reference to KABAT and IMGT classifications.
[0336] As was the case with the light chain, four residues that
must remain unchanged were identified.
[0337] Residue 2 (Ile) is part of Vernier zone and takes part in
CDR3 structuring.
[0338] Residue 35 (Tyr) takes part in CDR1 anchoring according to
IMGT, is part of CDR1 according to Kabat, and also takes part in
the VH/VL interface and interacts with CDR3.
[0339] Residue 50 (Tyr) takes part in CDR2 anchoring according to
IMGT, is part of CDR2 according to Kabat, is also part of the
Vernier zone and also takes part in the VH/VL interface.
[0340] Residue 59 (Arg) takes part in CDR2 anchoring according to
IMGT, is part of CDR2 according to Kabat and takes part in the
VH/VL interface.
[0341] A first humanized version will be able to include three
mutations at residues 61, 62 and 65, respectively (IMGT
classification).
[0342] These three residues are located in CDR2 according to Kabat
and take part in the VH/VL interface.
[0343] Residue 61 (Asn/Ala) is not directly implicated in antigen
recognition. Its mutation can thus be considered.
[0344] Residue 62 (Gln/Glu) and residue 65 (Lys/Gln).
[0345] Secondly, two additional changes will be evaluated. The two
changes relate to residues 48 and 74 (IMGT nomenclature).
[0346] Residue 48 (Ile/Met), belonging to the scaffold region,
takes part in the VH/VL interface.
[0347] Residue 74 (Lys/Thr) is part of the Vernier zone and may be
implicated in CDR2 structuring.
[0348] Third and last, a third series of mutations could be
considered, namely a change of residues 9 (Pro/Ala) and 41
(His/Pro). The aim is thus, in a way similar to the mutations
planned for 6F4 VL, to approach the human germline as closely as
possible without modifying CDR anchoring.
[0349] For summary purpose only, tables 5 and 6 below list the cell
lines used as well as, respectively, their amino acid and
nucleotide sequence numbers.
TABLE-US-00009 TABLE 5 GERMLINES (EMBL ref.) SEQ ID No.
IGKV19-93*01 (AJ235935) 39 IGKJ1*01 (V00777) 40 IGKV1-33*01
(M64856) 41 IGKJ1*01 (J00242) 42 IGHV1S135*01 (AF304556) 43
IGHD-ST4*01 (M23243) 44 IGHJ2*01 (V00770) 45 IGHV1-f*01 (Z12305) 46
IGHD1-1*01 (X97051) 47 IGHJ4*01 (J00256) 48 IGHV1-03*01 (X62109)
49
TABLE-US-00010 TABLE 6 GERMLINES (EMBL ref.) SEQ ID No.
IGKV19-93*01 (AJ235935) 50 IGKJ1*01 (V00777) 51 IGKV1-33*01
(M64856) 52 IGKJ1*01 (J00242) 53 IGHV1S135*01 (AF304556) 54
IGHD-ST4*01 (M23243) 55 IGHJ2*01 (V00770) 56 IGHV1-f*01 (Z12305) 57
IGHD1-1*01 (X97051) 58 IGHJ4*01 (J00256) 59 IGHV1-03*01 (X62109)
60
Example 4
Purification and Identification of the 6F4 Antibody Antigen
Target
Purification by Immunoaffinity:
[0350] The antigen target of the 6F4 antibody is purified from a
membrane fraction enriched by HT-29 cells. After solubilization in
a 50 mM Tris/HCl buffer, pH 7.4, containing 150 mM NaCl, Triton
X-100 and IGEPAL, membrane proteins are incubated in the presence
of the 6F4 antibody immobilized on sepharose beads overnight at
+4.degree. C. under gentle mixing. The 6F4-Ag complex formed on the
beads is then washed with various solutions containing detergents
in order to eliminate proteins adsorbed nonspecifically. The 6F4
antigen target is eluted from the 6F4-sepharose support using a 0.1
M Gly/HCl buffer, pH 2.7. The fractions collected are analyzed by
SDS-PAGE electrophoresis (10% gel, non-reducing conditions) and
western blot after transfer to nitrocellulose membrane (primary 6F4
antibody used at 0.5 .mu.g/ml, detection by chemiluminescence) in
order to select the fractions enriched in the antigen of interest
(FIGS. 8A and 8B). The analysis by western blot confirms the
absence of the protein of interest in the un-selected fractions and
washings, and a specific elution of the latter at acid pH.
[0351] The enriched fractions arising from two purifications were
then analyzed by SDS-PAGE electrophoresis (10% gel) and western
blot under the conditions described previously. The antigen
recognized by the 6F4 antibody in the western blot had an apparent
molecular weight of 35 kDa after analysis in reducing conditions
(FIGS. 9A and 9B). A difference in apparent molecular weight can be
noted when electrophoresis is performed in non-reducing conditions:
under these conditions, the apparent molecular weight is indeed
slightly lower than that observed in reducing conditions.
Identification of the Antigen Target:
[0352] After SDS-PAGE electrophoresis (10% gel), the proteins are
stained with colloidal blue using a method compatible with mass
spectrometry analysis (FIG. 10). The band of interest corresponding
to the protein detected by western blot is cut out using a scalpel
and then de-stained by incubation in a 25 mM ammonium bicarbonate
solution. After reduction (DTT)/alkylation (iodoacetamide) and "in
gel" hydrolysis (overnight at 37.degree. C.) of the protein by
trypsin (Promega), a proteolytic enzyme that hydrolyzes proteins at
the Lysine and Arginine residues and thus releases peptides having
a Lysine or Arginine residue in the C-terminal position, the
peptides generated are extracted using an acetonitrile/water
mixture (70/30, v/v) in the presence of formic acid. These are then
deposited on the MALDI target in a mixture with a matrix
(alpha-cyano-4-hydroxycinnamic acid, Bruker Daltonics) and in the
presence of ATFA, and then analyzed by MALDI-TOF mass spectrometry
(Autoflex, Bruker Daltonics). The mass spectrum obtained is
presented in FIG. 10. The list of the peptides deduced from this
analysis is used to identify the protein by searching data banks
using the Mascot search engine (Matrix Sciences).
[0353] The NCBInr data bank search results, restricted to proteins
of human origin, indicate that three proteins have a significant
score (score>64):
[0354] 1. Crystal structure of human junctional adhesion molecule
type 1 [0355] Score=116 [0356] This protein corresponds to the
extracellular domain of the F11R/JAM-A protein used for structural
studies.
[0357] 2. F11 receptor (Homo sapiens) [0358] Score=116 [0359] This
protein corresponds to the precursor of protein F11R/isoform a.
[0360] 3. F11 receptor isoform b (Homo sapiens) [0361] Score=65
[0362] This is the precursor of the isoform b of protein F11R, with
two deletions of 20 amino acids with respect to isoform a.
[0363] The identified protein, by this approach, is thus called
F11R or F11 receptor. This is in fact the official designation of
the protein adopted when it was first described as a receptor of a
so-called F11 antibody (Naik et al., 1995, Biochem. J., 310,
155-162). This protein is better known today under the name of
JAM-A or "junctional adhesion molecule A", and is also called JAM1,
PAM-1, CD321 or antigen 106.
[0364] Among the peptides released by tryptic hydrolysis and
analyzed by mass spectrometry, nine peptides have an experimental
molecular weight corresponding, within 0.1 Da, to that of peptides
arising from the theoretical hydrolysis of the human form of
JAM-A/isoform a. These nine peptides cover 37% of the protein's
primary sequence. Moreover, the theoretical molecular weight of the
JAM-A precursor (.about.32.9 kDa) is in agreement with the apparent
molecular weight determined experimentally by SDS-PAGE.
Confirmation of the Target Identified by Western Blot:
[0365] The identification of JAM-A by a proteomic approach was then
confirmed by western blot (10% SDS-PAGE gel in non-reducing
conditions, 6F4 antibody at 0.5 .mu.g/ml, detection by
chemiluminescence).
[0366] As shown in FIG. 11A, the 6F4 antibody recognizes natural
JAM-A protein in the HT-29 membrane extract and in the fraction
enriched by immunopurification (apparent MW=35 kDa), as well as the
dimeric recombinant protein JAM-A/Fc (R&D Systems ref. 1103-JM,
apparent MW .about.120 kDa). This recognition is equivalent to that
of a commercial anti-human JAM-A goat polyclonal antibody (R&D
Systems, ref. AF1103) diluted to 1/1000 (FIG. 11B).
Example 5
Specificity of the 6F4 Antibody for Human JAM-A
[0367] The specificity of the 6F4 antibody was determined by
western blot under the conditions described above.
[0368] FIG. 12 shows that the 6F4 antibody is specific for the
human form of JAM-A since it recognizes the recombinant protein
hJAM-A/Fc (R&D Systems ref. 1103-JM), but recognizes neither
the human forms of JAM-B and JAM-C (recombinant proteins hJAM-B/Fc
and hJAM-C/Fc, R&D Systems ref. 1074-VJ and 1189-J3) nor the
murine form of JAM-A (recombinant protein mJAM-A/Fc, R&D
Systems ref. 1077-JM).
Example 6
Measurement of the Affinity of the 6F4 Antibody by BIAcore (Surface
Plasmon Resonance):
Principle:
[0369] Using BIAcore, the affinity constant K.sub.D (M) of the 6F4
antibody for the soluble protein JAM-1-Fc (extracellular domain
fused with a Fc fragment of the antibody and produced in
recombinant form in NSO cells) can be calculated from the
determination of the association kinetics (k.sub.a) (1/m.s) and the
dissociation kinetics (k.sub.d) (1/s) according to the formula
K.sub.D=k.sub.d/k.sub.a (Rich and Myszka, J. Mol. Recog., 2005, 18,
431).
Materials and Methods:
[0370] Instrument used: BIAcore X and BIAevaluation 3.1.times.
software (Uppsala, SW) [0371] Reagents: [0372] Murine monoclonal
6F4 antibody: 1.3 mg/ml [0373] Human JAM-1-Fc (ref. 1103-JM R&D
Systems): 50 .mu.g carrier-free) [0374] Mouse JAM-1-Fc (ref.
1077-JM R&D Systems): 50 .mu.g carrier-free [0375] Running
buffer: HBS-EP (BIAcore) [0376] Binding kit: "Amine" (BIAcore)
[0377] Binding buffer: Acetate pH 5.0 (BIAcore) [0378] Capturing
antibody: goat IgG Fc anti-human (=GAH, goat anti-human)
(Bioscience) [0379] Regeneration buffer: Glycine, HCl pH 1.5 for 30
seconds (BIAcore).
Discussion and Conclusions:
[0380] The data in FIG. 13 show that the murine 6F4 antibody is
bound to the extracellular part of the human JAM-1 protein but not
to the extracellular part of the murine JAM-1 protein.
[0381] The data in FIG. 14 make it possible to calculate a K.sub.D
of 22 pM of the 6F4 antibody for the human JAM-1 protein under
these experimental conditions.
[0382] The slow dissociation kinetics indicates the involvement of
a phenomenon of antibody avidity for the antigen (divalent
analytical model).
Example 7
In vivo Activity of the 6F4 Antibody in the MCF-7 Xenograft
Model
[0383] A test of the 6F4 antibody, unpurified and injected by IP
route at a dose of 250 .mu.g/mouse, demonstrates that this antibody
significantly inhibits the growth of MCF-7 cells in vivo with
inhibition percentages reaching 56% compared to mice injected with
PBS (FIG. 15). The non-relevant 9G4 antibody used as an IgG1
control isotype is, as expected, without antitumor activity.
Example 8
Study of the Distribution of the Antigen Recognized by 6F4 on a
Panel of Tumor Cells
[0384] In order to determine the potential indications for the 6F4
antibody, four types of tumors were studied by flow cytometry in
terms of a membrane expression profile. The selected cell lines are
MCF-7 (estrogen-related breast cancer), A549 (non-small cell lung
cancer), HT29 and Colo 205 (colon cancer) and BxPC3 (pancreatic
cancer). For labeling cells, a range of doses (10 .mu.g/ml, 5
.mu.g/ml, 1 .mu.g/ml, 0.5 .mu.g/ml, 0.25 .mu.g/ml and 0.125
.mu.g/ml) was tested.
[0385] The results presented in FIG. 16 show that the 6F4 antibody
recognizes an antigen significantly expressed on the surface of all
cells tested. The labeling obtained is saturable, which attests to
its specificity. Saturation of the sites is obtained from a
concentration of 1 .mu.g/ml of antibody, which is evidence that the
6F4 antibody's affinity for the JAM-A antigen is high.
Example 9
Humanization by CDR-Grafting of the Variable Region of the Light
Chain of the 6F4 Antibody (6F4 VL)
[0386] Summary of the Immunogenetic Analysis:
TABLE-US-00011 Result Productive IGK rearranged sequence summary:
(no stop codon and in frame junction) V-GENE and IGKV1-33*01 score
= identity = 81, 36% allele 922 (227/279 nt) J-GENE and IGKJ1*01
score = identity = 86, 49% allele 140 (32/37 nt) CDR-IMGT [6, 3, 8]
CLQYDNLWTF (SEQ ID NO: 103) lengths and AA JUNCTION
Detailed Data for Closest Human V-Gene Identification:
[0387] Closest V-REGIONs (evaluated from the V-REGION first
nucleotide to the 2nd-CYS codon plus 15 nt of the CDR3-IMGT)
TABLE-US-00012 [0387] Score Identity M64856 IGKV1-33*01 922 81.36%
(227/279 nt) M64855 IGKV1D-33*01 922 81.36% (227/279 nt) X63398
IGKV1-27*01 868 79.21% (221/279 nt) Y14865 IGKV1-NL1*01 841 78.14%
(218/279 nt) X72817 IGKV1D-43*01 841 78.14% (218/279 nt)
Detailed Data for Closest Human J-Gene Identification:
[0388] Closest J-REGIONs:
TABLE-US-00013 [0388] Score Identity J00242 IGKJ1*01 140 86.49%
(32/37 nt) AF103571 IGKJ4*02 122 81.08% (30/37 nt) J00242 IGKJ4*01
113 78.38% (29/37 nt) Z70260 IGKJ2*02 104 75.68% (28/37 nt) Z46620
IGKJ2*04 95 72.97% (27/37 nt)
Identification of Critical Residues:
[0389] Several criteria are involved in the definition and ranking
of outside CDR critical residues. These include at least, known
participation of the residue in VH/VL interface, in antigen binding
or in CDR structure, the amino acid class changes from murine and
human residues, localization of the residue in the 3D structure of
a variable domain etc.
[0390] 21 amino acids are found different from 6F4 VL domain and
the closest IGKV1-33*01 human germline V gene, all of them being
outside CDR residues. Out of these 21 residues, analysis of the
above cited parameters lead to the identification of 9 most
potentially contributing residues. These murine residues are K24,
I39, A40, H55, T66, Q68, A69, R85 and Y87. Out of these 9 residues,
3 of them are supposed to be even more important so that they will
keep their murine origin in the humanized form. These are residues
I39 and H55 and T66, located at the CDR1 and CDR2 anchors,
respectively. Finally, 6 amino acids will be analysed individually
and/or in combination to determine whether they can be humanized or
if they have to keep their murine origin.
[0391] Looking to the non-involvement of the J-region in antigen
binding and structuration of the V-region, it was decided to use
the native human IGKJ1*01 germline gene.
[0392] In the designed sequence of the humanized 6F4 VL domain
depicted in FIG. 17:
[0393] *, correspond to amino acids changed de facto to their human
counterparts
[0394] 1, correspond to amino acids analysed for their abilities to
be humanized, the human residue being indicated below the sign
[0395] 2, correspond to amino acids that remain murin in the
humanized 6F4 VH domain
Example 10
First Version of Humanization by CDR-Grafting of the Variable
Region of the Heavy Chain of the 6F4 Antibody (6F4 VH)
Summary of the Immunogenetic Analysis:
TABLE-US-00014 [0396] Result Productive IGH rearranged sequence
summary: (no stop codon and in frame junction) V-GENE and
IGHV1-f*01 score = identity = 75, 35% allele 796 (217/288 nt)
J-GENE and IGHJ4*01 score = identity = 87, 23% allele 181 (41/47
nt) CDR-IMGT [8, 8, 9] CARQTDYFDYW (SEQ ID NO 104) lengths and AA
JUNCTION
[0397] D-gene strictly belongs to the CDR3 region in the VH domain.
The humanization process is based on a <<CDR-grafting>>
approach. Analysis of the closest human D-genes is not useful in
this strategy.
Detailed Data for Closest Human V-Gene Identification:
[0398] Closest V-REGIONs (evaluated from the V-REGION first
nucleotide to the 2nd-CYS codon)
TABLE-US-00015 [0398] Score Identity Z12305 IGHV1-f*01 796 75.35%
(217/288 nt) X62106 IGHV1-2*02 787 75.00% (216/288 nt) X92208
IGHV1-2*03 782 74.65% (215/288 nt) Z12310 IGHV1-2*04 778 74.65%
(215/288 nt) M99642 IGHV1-24*01 760 73.96% (213/288 nt)
Detailed Data for Closest Human J-Gene Identification:
[0399] Closest J-REGIONs:
TABLE-US-00016 [0399] Score Identity J00256 IGHJ4*01 181 87.23%
(41/47 nt) X86355 IGHJ4*02 172 85.11% (40/47 nt) M25625 IGHJ4*03
172 85.11% (40/47 nt) J00256 IGHJ1*01 138 74.51% (38/51 nt) J00256
IGHJ5*01 133 74.00% (37/50 nt)
Identification of Critical Residues:
[0400] Several criteria are involved in the definition and ranking
of outside CDR critical residues. These include at least, known
participation of the residue in VH/VL interface, in antigen binding
or in CDR structure, the amino acid class changes from murine and
human residues, localization of the residue in the 3D structure of
a variable domain etc.
[0401] 31 amino acids are found different from 6F4 VH domain and
the closest IGHV1-f*01 human germline V gene, all of them being
outside CDR residues. Out of these 31 residues, analysis of the
above cited parameters lead to the identification of 9 most
potentially contributing residues. These murine residues are I2,
Y40, I53, Y55, R66, N68, Q69, K72 and K82. Out of these 9 residues,
2 of them are supposed to be even more important so that they will
keep their murine origin in the humanized form. These are residues
Y55 and R66, located at the CDR2 anchors. Finally, 7 amino acids
will be analysed individually and/or in combination to determine
whether they can be humanized or if they have to keep their murine
origin.
[0402] Looking to the non-involvement of the J-region in antigen
binding and structuration of the V-region, it was decided to use
the native human IGHJ4*01 germline gene.
[0403] In the designed sequence of the humanized 6F4 VH domain
depicted in FIG. 18:
[0404] *, correspond to amino acids changed de facto to their human
counterparts
[0405] 1, correspond to amino acids analysed for their abilities to
be humanized, the human residue being indicated below the sign
[0406] 2, correspond to amino acids that remain murin in the
humanized 6F4 VH domain
Example 11
Second Version of Humanization by CDR-Grafting of the Variable
Region of the Heavy Chain of the 6F4 Antibody (6F4 VH)
[0407] An other way to identify human V-gene candidates for
CDR-grafting was to look for human homologies at the amino acid
level using IMGT/DomainGapAlign tool.
Results of the IMGT/DomainGapAlign Immunogenetic Analysis are
Summarized Hereinafter:
TABLE-US-00017 [0408] Smith- Waterman Identity Allele Species
Domain Score percentage Overlap IGHV1-3*01 Homo sapiens 1 451 64.3
98
[0409] Identification of critical residues in IGHV1-03*01 germline
gene (SEQ ID No. 49, EMBL nomenclature: X62109).
[0410] The alignment of 6F4 VH domain and IGHV1-3*01 proteic
sequences is represented in FIG. 31.
[0411] The selection and ranking of those residues is based on
differential criteria based on the relative importance of each
single position according to their structural relevance, their
known structure-function relationship, the relevance of the amino
acid class change if it happen and it also take advantage of the
results obtained during the first humanization process.
[0412] In a first intention, all the different "out-side CDRs"
amino acids have been changes for their human counterparts, except
residues Y55 and R66 which both are strongly supposed to be
involved in binding as CDR2-anchors assigned residues.
Humanizability of those two residues will be explored at the end of
the process, when all the other analyses described after will be
performed. Indeed, recovery of the fully activity of the parental
antibody, the 6F4 Hz2 re-humanized VH domain would have to be
improved as follow; a "de-humanization" process would consist in
back mutating, if necessary, these amino acids in their murine
counterpart:
[0413] The first group residues, namely E1Q, K43R and K75R present
a strong combination of criteria and correspond to the first
positions that "de-humanization" will be assessed if looking for a
benefit.
[0414] Then, residues from group 2, namely K48Q, S49R, F88Y and
H90R, are chemically relevant mutations but structurally a little
less supposed key residues and will be tested in a second round of
experiment.
[0415] The six residues from the third group, are presumably more
involved in an overall and/or core-oriented residues and thus
supposed to be less involved in binding and thus be explored in a
third round of improving, whenever necessary.
[0416] Residues from the group 4, are supposed to be the less
structurally and/or amino acid class change relevant and for who
"de-humanization" would be explored lately.
[0417] Finally, the following six residues, I2V, Y40H, I53M, N68S,
K72Q and K82T, correspond to amino acids that humanization did not,
at least in this initial combination, alter binding activity of the
firstly humanized VH domain. "De-humanization" of these residues
will be performed in a last round of improving.
[0418] D-gene strictly belongs to the CDR3 region in the VH domain.
The humanization process is based on a <<CDR-grafting>>
approach. Analysis of the closest human D-genes is not useful in
this strategy.
[0419] Looking to the non-involvement of the J-region in antigen
binding and structuration of the V-region, it was decided to use
the native human IGHJ4*01 germline gene.
Experimental Data Obtained for the Re-Humanized 6F4 Antibody:
[0420] In the following experiments, the re-humanization only
concern the heavy chain, the light chain always corresponding to
the QTY/AET humanized 6F4 VL domain as exemplified in example 9
this finally selected humanized VL domain exhibits an anti-JAM-a
binding activity similar to that of the recombinant chimeric 6F4
antibody. Similarly, the re-humanized version improvement assays
were performed with reference to recombinant chimeric 6F4 antibody
anti-JAM-a binding activity as defined by an ELISA assay (data not
showed).
Example 12
In vitro Down-Regulation of JAM-A Expression by the 6F4 MAb
[0421] MCF-7, HT29 and A549 cell lines were selected to determine
the effect of the 6F4 MAb on JAMA expression. Briefly cells were
plated in 75 cm.sup.2 flasks and incubated at 37.degree. C., in 5%
CO.sub.2 atmosphere, for 24 hours, in medium supplemented with 10%
Fecal Calf Serum (FCS). Then cells were washed 3 times with PBS and
incubated for an additional day in serum-free medium. After this
second incubation, the serum-free medium was removed and replaced
by fresh serum-free medium alone or fresh serum-free medium
containing either 6F4 or an IgG1 isotype control described as 9G4.
After either 5 or 16 hours of incubation, cold lysis buffer (10 mM
Tris HCl buffer, pH 7.5, 15% NaCl 1 M (Sigma Chemical Co.), 10%
detergent mix (10 mM Tris-HCl, 10% Igepal lysis buffer) (Sigma
Chemical Co.), 5% sodium deoxycholate (Sigma Chemical Co.), 1
protease inhibitor cocktail complete TM tablet (Roche) and 1%
phosphatase inhibitor Cocktail Set II (Calbiochem), pH 7.5) was
added and cells were scrapped on ice. The lysates were clarified by
centrifugation at 4.degree. C. Protein was quantified by BCA
protein assay and 25pg of protein were loaded in each lane of a
Biorad 4-12% Bis-Tris gel. Samples were heated for 5 minutes at
100.degree. C. and kept at -20.degree. C. or loaded directly on
4-12% SDS-PAGE gels and transferred to nitrocellulose membrane.
Blots were first blocked with 5% BSA for all antibodies. Incubation
of specific anti-JAMA primary antibody was performed for 2 hours at
room temperature. Filters were washed in TBST and incubated with
appropriate HRP-linked secondary antibodies for 1 hour at room
temperature. Membranes were washed in TBST prior visualization of
proteins with ECL (Amersham).
[0422] As shown in FIG. 19, a significant down-regulation of JAM-A
was observed for the 3 cell lines treated with the 6F4 MAb. MCF-7
seemed to be the most sensitive one with a complete and stable
down-regulation observed as early as 5 hours post 6F4 incubation.
For HT29 cells a partial but sustained down-regulation of JAM-A was
also noticed. The kinetic of down-regulation was different for A549
cells as no significant effect was observed at the early incubation
time while a complete inhibition occurred after 16 hours of
incubation with the 6F4 MAb. As expected no significant differences
were observed from untreated cells and cells incubated with the 9G4
isotype control.
Example 13
Effect of a Single Injection of 6F4 on in vivo Tumor
Proliferation
[0423] To determine the in vivo mechanism of action of the 6F4 MAb,
7 weeks old female mice bearing estrogen pellets have been injected
with MCF-7 cells. When tumors reached a volume of 80 to 100
mm.sup.3, 3 groups of mice with comparable tumors were generated.
Before any injection, tumors were removed from one of these groups
to check the basal proliferation of tumor cells within an untreated
tumor. Mice from the 2 other groups were injected either with 1 mg
of 6F4 or with the same dose of an IgG1 isotype control described
as 9G4.
[0424] Six hours post injection, tumors were removed, fixed in
formalin, paraffin embedded, cut into 5 .mu.m sections and stained
with an anti-Ki67 antibody to determine the level of proliferation
in treated versus control tumors.
[0425] As shown in FIG. 20 no difference was observed from tumors
removed before injection (described as T0 for time 0) and tumors
treated with the isotype control 9G4. On the other hand, a
significant inhibition of tumor cell proliferation was observed
after a single injection, 6F4.
Example 14
Effect of a Single Injection of 6F4 on in vivo JAM-A Expression
[0426] For this study the in vivo protocol is the same as the one
described in in vivo proliferation experiments except that removed
tumors were quickly frozen in liquid nitrogen for Western blot
analysis. The Western blot was performed as described in the
Example 13 above.
[0427] FIG. 21 demonstrate that no difference in JAM-A expression
was observed from untreated mice (described as T0 for Time 0) and
mice injected once with the 9G4 isotype control. A significant
down-regulation was noticed when mice were treated with the 6F4 MAb
indicating that a potential mechanism of action involved in the in
vivo antitumor activity of this antibody could be the
down-regulation of the receptor. These results were in agreement
with the one observed in vitro and described below in example
13.
Example 15
Comparison of the Anti-Tumoral Activity of 6F4 and its F(ab').sub.2
Fragment
[0428] As JAM-A is highly expressed by MCF-7 cells and despite the
fact that 6F4 is an IgG1 (isotype known to be poorly involved in
effector functions in mice), an in vivo comparison from 6F4 and its
F(ab')2 fragment has been set up in the MCF-7 model to determine a
potential involvement of effector functions in the in vivo
activity.
[0429] For that purpose, Five millions MCF7 cells were engrafted
into 7 weeks old mice female bearing estrogens pellet. Five days
after cells implantation, mice were treated either with 300 .mu.g
of 6F4 or with 200 .mu.g of 6F4 F(ab').sub.2 three times per week.
For the first injection, 600 .mu.g of antibody and 400 .mu.g of 6F4
F(ab').sub.2 were injected. Tumor volume was measured twice a week
for 4 weeks.
[0430] FIG. 22 showed that tumor growth in mice treated with 6F4
and 6F4 F(ab').sub.2 was significantly different from tumor growth
of control mice from D3 to D27 (p.ltoreq.0.03 for 6F4 and
p.ltoreq.0.015 for 6F4 F(ab').sub.2). No difference was observed
from 6F4 and 6F4 F(ab').sub.2 groups of mice showing that effector
functions are not involved in the 6F4 activity.
Example 16
Evaluation of the Expression of JAM-A on Human Tissue
[0431] A comparison of JAM-A expression on tumoral versus normal
patient tissues has been performed to select tumor types
overexpressing JAMA. Pairs of normal versus tumoral tissues from
the same patient were selected for this study. In these patients
normal tissues was taken near to the tumor. JAM-A expression was
determined by ImmunoHistoChemistry (IHC) using tissue arrays from
Superships. Briefly, Slides were dewaxed and antigen retrieval was
performed using the Dakocytomation solution S1699, at 98.degree. C.
for 20 minutes. After quenching endogenous peroxidase (0.3%
H.sub.2O.sub.2 solution for 5 minutes) and blocking non specific
sites (Ultra-V-Block; Labvision, ref. TA-125-UB), the primary
antibody (anti-hJAM-A, AF1103 from R&Dsytem or goat IgG isotype
control from Zymed) was incubated for 1 hour at room temperature.
After washes in TBS-tween, the binding of the anti-hJAM-A was
revealed using the LSAB+ kit from dakocytomation. Visualization of
the complex primary Ab and LSAB+ was performed by the chromogenic
reaction HRP-DAB. Slides were then counterstained by
hematoxylin.
[0432] Samples of thyroid, lung and breast cancer were analysed.
For thyroid samples (FIG. 23), no expression was observed on normal
thyroid tissue while JAM-A appeared to be strongly expressed in
tumoral sections (membrane staining) from the same patient. In lung
normal tissue JAM-A was expressed by pneumocytes. However, a strong
membrane expression was observed in all tumoral samples (FIG. 24).
For breast cancer, a weak JAM-A expression, located on lobular
ducts, was observed on normal breast tissue. In cancer sections,
the 3 examples of carcinoma shown in FIG. 25 (infiltrating duct,
atypically medullary and infiltrating papillary) demonstrate that
JAM-A is over expresses on breast cancer tissues.
[0433] These data suggested that thyroid, breast and lung cancers
could be good targets for a JAMA therapy.
Example 17
In vivo Activity of 6F4 on A431 Epidermoid Carcinoma Xenograft in
Nude Mice
[0434] A-431 cells were routinely cultured in DMEM (Lonza)
supplemented with 10% heat inactivated Fetal Calf Serum (Sigma).
Cells were split two days before engraftment so that they were in
exponential phase of growth. Ten million A-431 cells were engrafted
on 7 weeks old Athymic Nude mice. Five days after engraftment (D5)
mice were randomized and treated i.p. with the following schemes:
The control group received twice a week injections of PBS and the
6F4 treated group was injected i.p. with a loading dose of 2 mg
followed by twice a week injections of 1 mg dose of antibody. Tumor
were measured twice a week and tumor volumes were calculated using
the formula: .quadrature./6.length.width.height. Statistical
analysis were performed for each time point using a Mann-Whitney
Test and SigmaStat software. FIG. 26 showed that the 6F4 MAb is
capable of significantly inhibiting the in vivo growth of A431 cell
line (p<0.009 from day 38 to day 56).
Example 18
Evaluation of 6F4 Activity on Antigen Presentation by Antigen
Presenting Cells (APC)
[0435] JAM proteins are expressed in a variety of tissues
throughout the human body as well as on the surface of platelets,
leukocytes, and erythrocytes [Naik 1995; Malergue 1998; Korneki
1990; Williams 1999; Gupta 2000]. JAM-A appears to be expressed in
platelets, neutrophils, monocytes, lymphocytes, and erythrocytes
[For review see Mandell 2005].
[0436] To determine whether a treatment with 6F4 could impair
antigen presentation in patients an evaluation of a potential
interference with Antigen Presenting Cells (APC) including
macrophages and dendritic cells has been performed. In the
presentation process, APC internalize antigens and degrade them to
generate peptides which are associated within the cytoplasm with
CMH class II molecules. Then the complex is expressed on APC
membranes and presented to specific T lymphocytes responding to
that stimulation by proliferation.
[0437] In the study presented below, the potential effect of 6F4 on
Tetanus Toxoid presentation by human PBMC was evaluated. For that
purpose, PBMC were isolated by Ficoll gradient centrifugation from
blood. Cells were washed in PBS, counted and suspended in RPMI 1640
medium supplemented with 10% heat-inactivated foetal calf serum
(FCS), glutamine and antibiotics at the concentration of
0.25.10.sup.6 cells/ml. 100 .mu.l of PBMC were seeded in each well
of a 96 well plate previously filled with the antigen and the
antibody to be tested 10 .mu.g/ml final concentration). The 9G4 Mab
was used as an IgG1 isotype control and phytohemagglutinin PHA (2.5
.mu.g/ml final concentration), a polyclonal activator of
lymphocytes, was introduced as a positive control.
[0438] Specific antigen activator Tetanus Toxoid (TT) was selected
and added to PBMC at a final concentration of 100 .mu.g/ml. Plates
were then incubated at 37.degree. C. in an atmosphere containing 5%
CO.sub.2 for 96 h. Then, 0.25 .mu.Ci of [.sup.3H]-Thymidine is
added to the wells and incubated for 24 h. After incubation the
cells were harvested, the filter membrane was dried and the amount
of radioactivity was counted in a scintillation counter.
[0439] Regarding to FIGS. 27A and 28A that display the values of
two independent experiments, the polyclonal activator, PHA used as
a positive control of PBMC preparation is a potent inducer of
lymphoproliferation, with indexes ranging from 30 and 70 depending
on the donors and the experiment. In these conditions, the
lymphoproliferation index was not modified whatever the antibody
incubated, and 6F4 did not display any significant agonist or
antagonist activity. FIGS. 27B and 28B that display the values of
two independent experiments, showed that significant variations
could occur from donors towards TT activation of
lymphoproliferation. In these experiments, indexes ranged from 2
and 5 depending on the donors and the experiment. However, no
interference on the antigen presentation was observed in presence
of 6F4.
[0440] In conclusion, despite the significant expression of JAM-A
on APC and lymphocyte, the use of an antibody directed against this
target does not impair neither the non specific proliferation of
lymphocyte nor the antigen presentation process.
Example 19
Evaluation of Platelet Aggregation and Activation After 6F4
Incubation
[0441] In order to investigate whether 6F4, which binds to human
platelets, could have any biological function, two parameters were
measured: platelet aggregation and serotonine release.
[0442] For this purpose, human platelets from 10 normal donors were
incubated with 5 .mu.g/ml of several antibodies to be tested.
[0443] PM6/248 (an anti-.quadrature..sub.IIb.quadrature..sub.3)
have been reported to induce platelet aggregation. 9G4 was used as
negative isotype control.
[0444] As expected when tested on human platelets, thrombine and
ADP induced aggregation. PM6/248 also induced platelet
aggregation.
[0445] No platelet aggregation was measured after incubation with
6F4. The effect was comparable to the one observed with 9G4, used
as positive control (FIG. 29).
[0446] In a similar way, 6F4 was not able to induce serotonine
release (FIG. 30) whereas thrombine induced, as expected, 5-HT
release.
[0447] All together, these results indicate that whereas JAM-A is
expressed, no biological function is triggered on human platelet
after 6F4 activation.
Example 20
Humanization by CDR-Grafting of the Variable Region of the Light
Chain of the 6F4-Back Up Antibody (6F4-BU)
Detailed Data for Closest Human Gene Identification:
[0448] In order to identify alternative human candidates for the
CDR grafting, human germline genes displaying the best identity
with the 6F4 VL have been searched. To this end, the nucleotidic
sequence of 6F4 VL has been aligned with the human germline genes
sequences part of the IMGT database. For optimization of the
selection, alignments from the proteic sequences were also made to
search for better homologies.
[0449] For the J region, the best homology score was obtained with
the human IGKJ1*01 showing a nucleotidic sequence identity of
86,49%. Thus the IGKJ1*01 germline gene was selected as receiving
human J region for the murine 6F4 VL CDRs. [0450] IGKV1-27*01 and
IGKV1D-43*01 human V genes were selected for further use as human
framework sequence for CDR-grafting.
TABLE-US-00018 [0450] % Identity IGKV1-27*01 79.21% (221/279 nt)
IGKV1D-43*01 78.14% (218/279 nt)
Humanization of the 6F4 VL Domain by CDR-Grafting:
[0451] Given the possibility of two receiving human V regions for
the murine 6F4 VL CDRs, 2 humanized versions of the 6F4 VL domain
will be described.
a) IGKV1-27*01 Based Humanized Version of 6F4 VL (BU-L1)
[0452] The following steps in the humanization process consist in
linking the selected germline genes sequences IGKV1-27*01 and
IGKJ1*01 and also the CDRs of the murine 6F4 VL to the frameworks
of these germline genes sequences.
[0453] As depicted in FIG. 32, the bolded residues in the 6F4 VL
sequence corresponds to the twenty-one amino acids that were found
different from 6F4 VL domain and the selected human frameworks
(Human FR, i.e. IGKV1-27*01 and IGKJ1*01).
[0454] FIG. 32 is the implemented IGKV1-27*01 based humanized 6F4
VL with the described mutations clearly identified. The number
under each proposed mutation corresponds to the rank at which said
mutation will be done.
[0455] The numbering of amino acids and subsequent mutations
correspond to the IMGT numbering system in FIG. 32. For example,
residue 33 in the sequence listing (linear numbering) corresponds
to residue 39 in the FIG. 32 (IMGT numbering).
[0456] Regarding to several criteria such as their known
participation in VH/VL interface, in antigen binding or in CDR
structure, the amino acid class changes from murine and human
residues, localization of the residue in the 3D structure of the
variable domain, four out of the twenty-one different residues have
been identified to be eventually mutated. These four most important
defined residues and mutations into their human counterparts being
murine K24 into human R, I39 into L, H54 into Y and Y87 into F.
These ranked one residues are shown in FIG. 32 as bolded residues
in the 6F4 BU-L1 humanized VL sequence where they remained
murine.
[0457] Of course, the above mentioned residues to be tested are not
limited but must be considered as preferential mutations.
[0458] With the help of a molecular model, other mutations could be
identified. Can be mentioned the following ranked two residues,
i.e. residues 17 (G/D), 44 (H/Q), 69 (A/S), 85 (R/T), 89 (F/L), 93
(N/S) and 115 (G/Q) on which mutations could also be envisaged in
another preferred embodiment.
[0459] The above mentioned residues to be eventually tested are not
limited but must be considered as preferential mutations. In
another preferred embodiment, all the ten others ranked three
residues among the twenty-one different amino acids could be
reconsidered.
[0460] All the above mentioned mutations will be tested
individually or according various combinations.
b) IGKV1D-43*01 Based Humanized Version of 224G11 VL (BU-L2)
[0461] The following steps in the humanization process consist in
linking the selected germline genes sequences IGKV1D-43*01 and
IGKJ1*01 and also the CDRs of the murine 6F4 VL to the frameworks
of these germline genes sequences.
[0462] As depicted in FIG. 33, the bolded residues in the 6F4 VL
sequence correspond to the twenty-seven amino acids that were found
different from 6F4 VL domain and the selected human frameworks
(Human FR, i.e. IGKV1D-43*01 and IGKJ1*01). FIG. 33 is the
implemented IGKV1D-43*01 based humanized 6F4 VL with the described
mutations clearly identified. The number under each proposed
mutation corresponds to the rank at which said mutation will be
done.
[0463] The numbering of amino acids and subsequent mutations
correspond to the IMGT numbering system in FIG. 33. For example,
residue 33 in the sequence listing (linear numbering) corresponds
to residue 39 in the FIG. 33 (IMGT numbering).
[0464] Regarding to several criteria such as their known
participation in VH/VL interface, in antigen binding or in CDR
structure, the amino acid class changes from murine and human
residues, localization of the residue in the 3D structure of the
variable domain, six out of the twenty-seven different residues
have been identified to be eventually mutated. These six most
important defined residues and mutations into their human
counterparts being murine D1 into human A, Q3 into R, K24 into W,
I39 into L, H55 into Y and T66 into S. These ranked one residues
are shown in FIG. 33 as bolded residues in the 6F4 BU-L2 humanized
VL sequence where they remained murine. Of course, the above
mentioned residues to be tested are not limited but must be
considered as preferential mutations.
[0465] With the help of a molecular model, other mutations could be
identified. Can be mentioned the following ranked two residues,
i.e. residues 9 (S/F), 17 (G/D), 44 (H/Q), 53 (L/F), 69 (A/S), 85
(R/T), 89 (F/L), 93 (N/S) and 115 (G/Q) on which mutations could
also be envisaged in another preferred embodiment.
[0466] The above mentioned residues to be eventually tested are not
limited but must be considered as preferential mutations. In
another preferred embodiment, all the twelve others ranked three
residues among the twenty-seven different amino acids could be
reconsidered.
[0467] All the above mentioned mutations will be tested
individually or according various combinations.
Example 21
Humanization by CDR-Grafting of the Variable Region of the Heavy
Chain of the 6F4-Back Up Antibody (6F4-BU)
Detailed Data for Closest Human Gene Identification:
[0468] In order to identify alternative human candidates for the
CDR grafting, human germline genes displaying the best identity
with the 6F4 VH have been searched. To this end, the nucleotidic
sequence of 6F4 VH has been aligned with the human germline genes
sequences part of the IMGT database. For optimization of the
selection, alignments from the proteic sequences were also made to
search for better homologies.
[0469] For the J region, the best homology score was obtained with
the human IGHJ4*01 showing a nucleotidic sequence identity of
87.23%. Thus the IGHJ4*01 germline gene was selected as receiving
human J region for the murine 6F4 VH CDRs. IGHV1-3*01 and
IGHV1-46*01 human V genes were selected for further use as human
framework sequence for CDR-grafting.
TABLE-US-00019 % Identity IGHV1-46*01 73.61% (212/288 nt)
IGHV1-3*01 64.30% (63/98 aa)
Humanization of the 6F4 VH Domain by CDR-Grafting:
[0470] Given the possibility of two receiving human V regions for
the murine 6F4 VH CDRs, 2 humanized versions of the 6F4 VH domain
will be described.
a) IGHV1-3*01 Based Humanized Version of 6F4 VH (BU-H1)
[0471] The following steps in the humanization process consist in
linking the selected germline genes sequences IGHV1-3*01 and
IGHJ4*01 and also the CDRs of the murine 6F4 VH to the frameworks
of these germline genes sequences.
[0472] As depicted in FIG. 34, the bolded residues in the 6F4 VH
sequence corresponds to the thirty amino acids that were found
different from 6F4 VH domain and the selected human frameworks
(Human FR, i.e. IGHV1-3*01 and IGHJ4*01). FIG. 34 is the
implemented IGHV1-3*01 based humanized 6F4 VL with the described
mutations clearly identified. The number under each proposed
mutation corresponds to the rank at which said mutation will be
done.
[0473] The numbering of amino acids and subsequent mutations
correspond to the IMGT numbering system in FIG. 34. For example,
residue 44 in the sequence listing (linear numbering) corresponds
to residue 49 in the FIG. 34 (IMGT numbering).
[0474] Regarding to several criteria such as their known
participation in VH/VL interface, in antigen binding or in CDR
structure, the amino acid class changes from murine and human
residues, localization of the residue in the 3D structure of the
variable domain, nine out of the thirty different residues have
been identified to be eventually mutated. These nine most important
defined residues and mutations into their human counterparts being
murine E1 into human Q, Q5 into V, S49 into R, Y55 into W, R66 into
K, A76 into V, L78 into I, V80 into R and H90 into R. These ranked
one residues are shown in FIG. 34 as bolded residues in the 6F4
BU-H1 humanized VH sequence where they remained murine. It must be
noted that the above mentioned residues to be tested are not
limited but must be considered as preferential mutations.
[0475] With the help of a molecular model, other mutations could be
identified. Can be mentioned the following ranked two residues,
i.e. residues 2 (I/V), 9 (A/P), 40 (Y/H), 46 (H/P), 53 (I/M) and 84
(S/A) on which mutations could also be envisaged in another
preferred embodiment.
[0476] Of course, the above mentioned residues to be eventually
tested are not limited but must be considered as preferential
mutations. In another preferred embodiment, all the fifteen others
ranked three residues among the thirty different amino acids could
be reconsidered.
[0477] All the above mentioned mutations will be tested
individually or according various combinations.
b) IGHV1-46*01 Based Humanized Version of 6F4 VH (BU-H2)
[0478] The following steps in the humanization process consist in
linking the selected germline genes sequences IGHV1-46*01 and
IGHJ4*01 and also the CDRs of the murine 6F4 VH to the frameworks
of these germline genes sequences.
[0479] As depicted in FIG. 35, the bolded residues in the 6F4 VH
sequence correspond to the thirty-one amino acids that were found
different from 6F4 VH domain and the selected human frameworks
(Human FR, i.e. IGHV1-46*01 and IGHJ4*01). FIG. 35 is the
implemented IGHV1-46*01 based humanized 6F4 VL with the described
mutations clearly identified. The number under each proposed
mutation corresponds to the rank at which said mutation will be
done.
[0480] The numbering of amino acids and subsequent mutations
correspond to the IMGT numbering system in FIG. 35. For example,
residue 44 in the sequence listing (linear numbering) corresponds
to residue 49 in the FIG. 35 (IMGT numbering).
[0481] Regarding to several such as their known participation in
VH/VL interface, in antigen binding or in CDR structure, the amino
acid class changes from murine and human residues, localization of
the residue in the 3D structure of the variable domain, nine out of
the thirty different residues have been identified to be eventually
mutated. These nine most important defined residues and mutations
into their human counterparts being murine El into human Q, Q5 into
V, S49 into G, Y55 into I, R66 into S, A76 into V, L78 into M, V80
into R and H90 into E. These ranked one residues are shown in FIG.
35 as bolded residues in the 6F4 BU-H2 humanized VH sequence where
they remained murine. Of course, the above mentioned residues to be
tested are not limited but must be considered as preferential
mutations.
[0482] With the help of a molecular model, other mutations could be
identified. Can be mentioned the following ranked two residues,
i.e. residues 2 (IN), 9 (A/P), 40 (Y/H), 46 (H/P) and 53 (I/M) on
which mutations could also be envisaged in another preferred
embodiment. It must be notice that the above mentioned residues to
be eventually tested are not limited but must be considered as
preferential mutations. In another preferred embodiment, all the
sixteen others ranked three residues among the thirty-one different
amino acids could be reconsidered.
[0483] All the above mentioned mutations will be tested
individually or according various combinations.
[0484] Each patent, patent application, publication, text and
literature article/report cited or indicated herein is hereby
expressly incorporated by reference in its entirety.
[0485] While the invention has been described in terms of various
specific and preferred embodiments, the skilled artisan will
appreciate that various modifications, substitutions, omissions,
and changes may be made without departing from the spirit thereof.
Accordingly, it is intended that the scope of the present invention
be limited solely by the scope of the following claims, including
equivalents thereof.
Sequence CWU 1
1
10416PRTMus musculus 1Gln Asp Ile Asn Asn Tyr1 524PRTMus musculus
2Thr Asp Tyr Ser133PRTMus musculus 3Tyr Thr Ser148PRTMus musculus
4Ile Asp Pro Tyr Asn Gly Gly Thr1 558PRTMus musculus 5Leu Gln Tyr
Asp Asn Leu Trp Thr1 567PRTMus musculus 6Gln Thr Asp Tyr Phe Asp
Tyr1 578PRTMus musculus 7Gly Tyr Ser Phe Thr Asp Tyr Ser1
5811PRTMus musculus 8Lys Ala Ser Gln Asp Ile Asn Asn Tyr Ile Ala1 5
1096PRTMus musculus 9Thr Asp Tyr Ser Met Tyr1 5107PRTMus musculus
10Tyr Thr Ser Thr Leu Gln Ala1 51117PRTMus musculus 11Tyr Ile Asp
Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe Lys1 5 10
15Gly129PRTMus musculus 12Ala Arg Gln Thr Asp Tyr Phe Asp Tyr1
513106PRTMus musculus 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Leu Gly1 5 10 15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Ile Asn Asn Tyr 20 25 30Ile Ala Trp Tyr Gln His Lys Pro Gly
Lys Gly Pro Arg Leu Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ala
Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr
Ser Phe Ser Ile Ser Asn Leu Glu Pro65 70 75 80Glu Asp Ile Gly Thr
Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 10514116PRTMus musculus 14Glu Ile Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser
Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Phe65 70 75 80Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Thr Leu 100 105 110Thr Val Ser Ser 11515215PRTMus musculus
15Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu
Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ala Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
Asn Leu Glu Pro65 70 75 80Glu Asp Ile Gly Thr Tyr Tyr Cys Leu Gln
Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Ala Asp Ala Ala Pro 100 105 110Thr Val Ser Ile Phe Pro Pro
Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120 125Ala Ser Val Val Cys
Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 130 135 140Val Lys Trp
Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn145 150 155
160Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser
Tyr Thr 180 185 190Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile
Val Lys Ser Phe 195 200 205Asn Arg Asn Glu Cys Asn His 210
21516440PRTMus musculus 16Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met Tyr Trp Val Lys Gln Ser
His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Tyr Asn
Gly Gly Thr Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Phe65 70 75 80Met His Leu Asn
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln
Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110Thr
Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala 115 120
125Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu
130 135 140Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn
Ser Gly145 150 155 160Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Asp 165 170 175Leu Tyr Thr Leu Ser Ser Ser Val Thr
Val Pro Ser Ser Thr Trp Pro 180 185 190Ser Glu Thr Val Thr Cys Asn
Val Ala His Pro Ala Ser Ser Thr Lys 195 200 205Val Asp Lys Lys Ile
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile 210 215 220Cys Thr Val
Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro225 230 235
240Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val
245 250 255Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp
Phe Val 260 265 270Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
Arg Glu Glu Gln 275 280 285Phe Asn Ser Thr Phe Arg Ser Val Ser Glu
Leu Pro Ile Met His Gln 290 295 300Asp Trp Leu Asn Gly Lys Glu Phe
Lys Cys Arg Val Asn Ser Ala Ala305 310 315 320Phe Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro 325 330 335Lys Ala Pro
Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala 340 345 350Lys
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu 355 360
365Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr
370 375 380Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe
Val Tyr385 390 395 400Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu
Ala Gly Asn Thr Phe 405 410 415Thr Cys Ser Val Leu His Glu Gly Leu
His Asn His His Thr Glu Lys 420 425 430Ser Leu Ser His Ser Pro Gly
Lys 435 44017106PRTMus musculus 17Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Gln Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30Ile Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45His Tyr Thr Ser Thr
Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10518116PRTMus musculus
18Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Asp
Tyr 20 25 30Ser Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Ala
Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln Thr Asp Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
11519116PRTMus musculus 19Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg Gln Ala
Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Asp Pro Tyr Asn
Gly Gly Thr Arg Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln
Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 1152018DNAMus musculus 20caagacatta acaattat
182112DNAMus musculus 21actgactaca gc 12229DNAMus musculus
22tacacatct 92324DNAMus musculus 23attgatcctt acaatggtgg tact
242424DNAMus musculus 24ctacagtatg ataatctgtg gacg 242521DNAMus
musculus 25cagacggact actttgacta c 212624DNAMus musculus
26ggttactcat tcactgacta cagc 242727DNAMus musculus 27gcaagacaga
cggactactt tgactac 272833DNAMus musculus 28aaggcaagcc aagacattaa
caattatata gct 332921DNAMus musculus 29tacacatcta cattacaagc a
213018DNAMus musculus 30actgactaca gcatgtac 183151DNAMus musculus
31tatattgatc cttacaatgg tggtactagg tacaaccaga agttcaaggg c
5132318DNAMus musculus 32gacatccaga tgacacagtc tccatcctca
ctgtctgcat ctctgggagg caaagtcacc 60atcacttgca aggcaagcca agacattaac
aattatatag cttggtacca acacaagcct 120ggaaaaggtc ctaggctgct
catacattac acatctacat tacaagcagg catcccatca 180aggttcagtg
gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct
240gaagatattg gaacttatta ttgtctacag tatgataatc tgtggacgtt
cggtggaggc 300accaagctgg aaatcaaa 31833348DNAMus musculus
33gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
60tcctgcaagg cttctggtta ctcattcact gactacagca tgtactgggt gaagcagagc
120catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg
tactaggtac 180aaccagaagt tcaagggcaa ggccacattg actgttgaca
agtcctccag cacagccttc 240atgcatctca acagcctgac atctgaggac
tctgcagtct attactgtgc aagacagacg 300gactactttg actactgggg
ccaaggcacc actctcacag tctcctca 34834639DNAMus musculus 34gacatccaga
tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 60atcacttgca
aggcaagcca agacattaac aattatatag cttggtacca acacaagcct
120ggaaaaggtc ctaggctgct catacattac acatctacat tacaagcagg
catcccatca 180aggttcagtg gaagtgggtc tgggagagat tattccttca
gcatcagcaa cctggagcct 240gaagatattg gaacttatta ttgtctacag
tatgataatc tgtggacgtt cggtggaggc 300accaagctgg aaatcaaacg
ggctgatgct gcaccaactg tatccatctt cccaccatcc 360agtgagcagt
taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc
420aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg
cgtcctgaac 480agttggactg atcaggacag caaagacagc acctacagca
tgagcagcac cctcacgttg 540accaaggacg agtatgaacg acataacagc
tatacctgtg aggccactca caagacatca 600acttcaccca ttgtcaagag
cttcaacagg aatgagtgt 639351320DNAMus musculus 35gagatccagc
tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60tcctgcaagg
cttctggtta ctcattcact gactacagca tgtactgggt gaagcagagc
120catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg
tactaggtac 180aaccagaagt tcaagggcaa ggccacattg actgttgaca
agtcctccag cacagccttc 240atgcatctca acagcctgac atctgaggac
tctgcagtct attactgtgc aagacagacg 300gactactttg actactgggg
ccaaggcacc actctcacag tctcctcagc caaaacaaca 360gccccatcgg
tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc
420ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg
gaactctgga 480tccctgtcca gcggtgtgca caccttccca gctgtcctgc
agtctgacct ctacactctg 540agcagctcag tgactgtccc ctccagcacc
tggcccagcg agaccgtcac ctgcaacgtt 600gcccacccgg ccagcagcac
caaggtggac aagaaaattg tgcccaggga ttgtggttgt 660aagccttgca
tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc
720aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt
agacatcagc 780aaggatgatc ccgaggtcca gttcagctgg tttgtagatg
atgtggaggt gcacacagct 840cagacgcaac cccgggagga gcagttcaac
agcactttcc gctcagtcag tgaacttccc 900atcatgcacc aggactggct
caatggcaag gagttcaaat gcagggtcaa cagtgcagct 960ttccctgccc
ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag
1020gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag
tctgacctgc 1080atgataacag acttcttccc tgaagacatt actgtggagt
ggcagtggaa tgggcagcca 1140gcggagaact acaagaacac tcagcccatc
atggacacag atggctctta cttcgtctac 1200agcaagctca atgtgcagaa
gagcaactgg gaggcaggaa atactttcac ctgctctgtg 1260ttacatgagg
gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa
132036318DNAMus musculus 36gacatacaga tgactcagag cccatcatca
ttgagcgcgt ctgtcggcga tcgggttacc 60attacctgcc aggcaagtca agatatcaac
aactatattg cttggtatca acagaagccc 120ggtaaagccc caaagctgct
gatacactac acctccaccc tggagaccgg cgtgccttct 180agattttctg
gaagcgggtc cggaaccgat tatacgttca caatctccag ccttcagccc
240gaagacatcg ccacatacta ctgtctgcaa tacgacaatc tgtggacatt
tggccagggg 300actaaggtgg agatcaaa 31837345DNAMus musculus
37gaagtgcagc tggttcagag cggcgccgag gtaaaaccag gggcgacggt gaagataagc
60tgcaaggtga gtgggtactc attcaccgac tattcaatgc actgggtcca acaggcccct
120ggtaaaggac tggagtggat gggatacatc gatccctaca atggaggcac
taggtacgcc 180gagaagttcc aggggagagt cactattacc gcagatactt
ctaccgatac tgcctacatg 240gaactcagca gtctgcggtc cgaggacaca
gcagtctact attgtgctcg ccaaacagac 300tattttgact attggggcca
gggaaccttg gtgacagtgt cctct 34538348DNAMus musculus 38caggtgcaat
tggtacagtc aggcgcggag gtgaagaagc ctggggctag tgttaaagtc 60tcctgtaaag
cctccggata ttccttcact gactactcta tgcattgggt tcgccaggca
120ccagggcagc ggctggaatg gatggggtac attgatccct acaacggagg
cacgcgatat 180agtcagaagt tccagggtcg ggtgacaatc acagccgata
cgtccaccag caccgcctac 240atggagttga gcagtctcag gtcagaagac
acagccgtgt actattgcgc aagacagacc 300gattatttcg actactgggg
ccaaggcact ctcgtgaccg tctctagc 34839115PRTMus musculus 39Met Arg
Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His1 5 10 15Gly
Ala Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25
30Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp
35 40 45Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly
Pro 50 55 60Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile
Pro Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser
Phe Ser Ile Ser 85 90 95Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr
Cys Leu Gln Tyr Asp 100 105 110Asn Leu Leu 1154012PRTMus musculus
40Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys1 5 1041117PRTHomo
sapiens 41Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Gln
Leu Trp1 5 10 15Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Gln Ala Ser 35 40 45Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser
Asn Leu Glu Thr Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Tyr Asp Asn Leu Pro
1154212PRTHomo sapiens 42Trp Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys1 5 104398PRTMus musculus 43Glu Ile Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Asn Met Tyr Trp Val Lys
Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro
Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys
Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Phe65 70 75 80Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95Ala Arg442PRTMus musculus 44Gln
Thr14516PRTMus musculus 45Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser1 5 10 154698PRTHomo sapiens 46Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr
Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Thr47121PRTHomo sapiens 47Met Ser Val Ser
Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp1 5 10 15Gly Val Leu
Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val 20 25 30Lys Pro
Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser 35 40 45Val
Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser 50 55
60Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr65
70 75 80Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro
Asp 85 90 95Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu 100 105 110Asp Thr Ala Val Tyr Tyr Cys Ala Arg 115
1204815PRTHomo sapiens 48Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser1 5 10 154997PRTHomo sapiens 49Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Ala Met
His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala50643DNAMus musculus 50cagatgaagc tgatttgcat
gtgctgagat catattctac tgccccagag atttaataat 60ctgatcatac acactccaac
agtcattctt ggtcaggaga cgttgtagaa atgagaccgt 120ctattcagtt
cctggggctc ttgttgttct ggcttcatgg taaggagttt aacattgaat
180atgctaaaaa gagtatgtga tcaggaattt ctggtccttc agaaaaatct
tctttgaata 240taattaattt catagggatt tgtgttcttt ttaattatag
gtgctcagtg tgacatccag 300atgacacagt ctccatcctc actgtctgca
tctctgggag gcaaagtcac catcacttgc 360aaggcaagcc aagacattaa
caagtatata gcttggtacc aacacaagcc tggaaaaggt 420cctaggctgc
tcatacatta cacatctaca ttacagccag gcatcccatc aaggttcagt
480ggaagtgggt ctgggagaga ttattccttc agcatcagca acctggagcc
tgaagatatt 540gcaacttatt attgtctaca gtatgataat cttctaccca
cagtgataca aatcataaca 600aaaaccaccc agggaagcag aagtgagagg
ctaggttgcc cac 6435139DNAMus musculus 51tggacgttcg gtggaggcac
caagctggaa atcaaacgt 3952667DNAHomo sapiens 52ctgcagctgt gcccagcctg
ccctatcccc tgctgatttg catgttcgca gagcacagcc 60ccctgccctg aagacttatt
aataggctgg tcgcaccctg tgcaggagtc agtcccaacc 120aggacacagc
atggacatga gggtccctgc tcagctcctg gggctcctgc agctctggct
180ctcaggtaag gaaggataac actaggaatt ttctcagcca gtgtgctcag
tacagcctgg 240ctcttgatgg aagccttcct ataatatgac taatagtatg
aatatttgtg tttatgtttc 300taatcgcagg tgccagatgt gacatccaga
tgacccagtc tccatcctcc ctgtctgcat 360ctgtaggaga cagagtcacc
atcacttgcc aggcgagtca ggacattagc aactatttaa 420attggtatca
gcagaaacca gggaaagccc ctaagctcct gatctacgat gcatccaatt
480tggaaacagg ggtcccatca aggttcagtg gaagtggatc tgggacagat
tttactttca 540ccatcagcag cctgcagcct gaagatattg caacatatta
ctgtcaacag tatgataatc 600tccctcccac agtgtaacaa gtcataacat
aaatcaccca ggggagcaga tgcgtgaggc 660tcagctg 6675337DNAHomo sapiens
53tggacgttcg gccaagggac caaggtggaa atcaaac 3754294DNAMus musculus
54gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta
60tcctgcaagg cttctggtta ctcattcact gactacaaca tgtactgggt gaagcagagc
120catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg
tactagctac 180aaccagaagt tcaagggcaa ggccacattg actgttgaca
agtcctccag cacagccttc 240atgcatctca acagcctgac atctgaggac
tctgcagtct attactgtgc aaga 29455163DNAMus musculus 55aagcttgccc
aggaaccact agtgctcaca cagctctgcc cacaggggaa acctaaccat 60gcctgccccc
tactcagcag gaaggctctg aagctctgag aggattttga acaagttact
120gtcacagtga gacagctcgg gctaccatgt aagaaaagct caa 1635645DNAMus
musculus 56tactttgact actggggcca aggcaccact ctcacagtct cctca
4557294DNAHomo sapiensCDS(1)..(294) 57gag gtc cag ctg gta cag tct
ggg gct gag gtg aag aag cct ggg gct 48Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15aca gtg aaa atc tcc tgc
aag gtt tct gga tac acc ttc acc gac tac 96Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30tac atg cac tgg gtg
caa cag gcc cct gga aaa ggg ctt gag tgg atg 144Tyr Met His Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45gga ctt gtt gat
cct gaa gat ggt gaa aca ata tac gca gag aag ttc 192Gly Leu Val Asp
Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe 50 55 60cag ggc aga
gtc acc ata acc gcg gac acg tct aca gac aca gcc tac 240Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80atg
gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat tac tgt 288Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95gca aca 294Ala Thr5817DNAHomo sapiens 58ggtacaactg gaacgac
175946DNAHomo sapiens 59tactttgact actggggcca aggaaccctg gtcaccgtct
cctcag 4660291DNAHomo sapiens 60ggggcctcag tgaaggtttc ctgcaaggct
tctggataca ccttccaggt ccagcttgtg 60cagtctgggg ctgaggtgaa gaagcctact
agctatgcta tgcattgggt gcgccaggcc 120cccggacaaa ggcttgagtg
gatgggatgg atcaacgctg gcaatggtaa cacaaaatat 180tcacagaagt
tccagggcag agtcaccatt accagggaca catccgcgag cacagcctac
240atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc g
29161299PRTHomo sapiens 61Met Gly Thr Lys Ala Gln Val Glu Arg Lys
Leu Leu Cys Leu Phe Ile1 5 10 15Leu Ala Ile Leu Leu Cys Ser Leu Ala
Leu Gly Ser Val Thr Val His 20 25 30Ser Ser Glu Pro Glu Val Arg Ile
Pro Glu Asn Asn Pro Val Lys Leu 35 40 45Ser Cys Ala Tyr Ser Gly Phe
Ser Ser Pro Arg Val Glu Trp Lys Phe 50 55 60Asp Gln Gly Asp Thr Thr
Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr65 70 75 80Ala Ser Tyr Glu
Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe 85 90 95Lys Ser Val
Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser 100 105 110Glu
Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu Ile Val 115 120
125Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile Pro Ser Ser Ala Thr
130 135 140Ile Gly Asn Arg Ala Val Leu Thr Cys Ser Glu Gln Asp Gly
Ser Pro145 150 155 160Pro Ser Glu Tyr Thr Trp Phe Lys Asp Gly Ile
Val Met Pro Thr Asn 165 170 175Pro Lys Ser Thr Arg Ala Phe Ser Asn
Ser Ser Tyr Val Leu Asn Pro 180 185 190Thr Thr Gly Glu Leu Val Phe
Asp Pro Leu Ser Ala Ser Asp Thr Gly 195 200 205Glu Tyr Ser Cys Glu
Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser 210 215 220Asn Ala Val
Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val225 230 235
240Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu Val Phe Gly
245 250 255Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp Arg Thr Lys
Lys Gly 260 265 270Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser
Ala Arg Ser Glu 275 280 285Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu
Val 290 29562897DNAHomo sapiens 62atggggacaa aggcgcaagt cgagaggaaa
ctgttgtgcc tcttcatatt ggcgatcctg 60ttgtgctccc tggcattggg cagtgttaca
gtgcactctt ctgaacctga agtcagaatt 120cctgagaata atcctgtgaa
gttgtcctgt gcctactcgg gcttttcttc tccccgtgtg 180gagtggaagt
ttgaccaagg agacaccacc agactcgttt gctataataa caagatcaca
240gcttcctatg aggaccgggt gaccttcttg ccaactggta tcaccttcaa
gtccgtgaca 300cgggaagaca ctgggacata cacttgtatg gtctctgagg
aaggcggcaa cagctatggg 360gaggtcaagg tcaagctcat cgtgcttgtg
cctccatcca agcctacagt taacatcccc 420tcctctgcca ccattgggaa
ccgggcagtg ctgacatgct cagaacaaga tggttcccca 480ccttctgaat
acacctggtt caaagatggg atagtgatgc ctacgaatcc caaaagcacc
540cgtgccttca gcaactcttc ctatgtcctg aatcccacaa caggagagct
ggtctttgat 600cccctgtcag cctctgatac tggagaatac agctgtgagg
cacggaatgg gtatgggaca 660cccatgactt caaatgctgt gcgcatggaa
gctgtggagc ggaatgtggg ggtcatcgtg 720gcagccgtcc ttgtaaccct
gattctcctg ggaatcttgg tttttggcat ctggtttgcc 780tatagccgag
gccactttga cagaacaaag aaagggactt cgagtaagaa ggtgatttac
840agccagccta gtgcccgaag tgaaggagaa ttcaaacaga cctcgtcatt cctggtg
89763259PRTHomo sapiens 63Met Gly Thr Lys Ala Gln Val Glu Arg Lys
Leu Leu Cys Leu Phe Ile1 5 10 15Leu Ala Ile Leu Pro Glu Asn Asn Pro
Val Lys Leu Ser Cys Ala Tyr 20 25 30Ser Gly Phe Ser Ser Pro Arg Ala
Ala Ser Tyr Glu Asp Arg Val Thr 35 40 45Phe Leu Pro Thr Gly Ile Thr
Phe Lys Ser Val Thr Arg Glu Asp Thr 50 55 60Gly Thr Tyr Thr Cys Met
Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly65 70 75 80Glu Val Lys Val
Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr 85 90 95Val Asn Ile
Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val Leu Thr 100 105 110Cys
Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp Phe Lys 115 120
125Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr Arg Ala Phe Ser
130 135 140Asn Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly Glu Leu Val
Phe Asp145 150 155 160Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser
Cys Glu Ala Arg Asn 165 170 175Gly Tyr Gly Thr Pro Met Thr Ser Asn
Ala Val Arg Met Glu Ala Val 180 185 190Glu Arg Asn Val Gly Val Ile
Val Ala Ala Val Leu Val Thr Leu Ile 195 200 205Leu Leu Gly Ile Leu
Val Phe Gly Ile Trp Phe Ala Tyr Ser Arg Gly 210 215 220His Phe Asp
Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr225 230 235
240Ser Gln Pro Ser Ala Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser
245 250 255Phe Leu Val64777DNAHomo sapiens 64atggggacaa aggcgcaagt
cgagaggaaa ctgttgtgcc tcttcatatt ggcgatcctt 60cctgagaata atcctgtgaa
gttgtcctgt gcctactcgg gcttttcttc tccccgtgca 120gcttcctatg
aggaccgggt gaccttcttg ccaactggta tcaccttcaa gtccgtgaca
180cgggaagaca ctgggacata cacttgtatg gtctctgagg aaggcggcaa
cagctatggg 240gaggtcaagg tcaagctcat cgtgcttgtg cctccatcca
agcctacagt taacatcccc 300tcctctgcca ccattgggaa ccgggcagtg
ctgacatgct cagaacaaga tggttcccca 360ccttctgaat acacctggtt
caaagatggg atagtgatgc ctacgaatcc caaaagcacc 420cgtgccttca
gcaactcttc ctatgtcctg aatcccacaa caggagagct ggtctttgat
480cccctgtcag cctctgatac tggagaatac agctgtgagg cacggaatgg
gtatgggaca 540cccatgactt caaatgctgt gcgcatggaa gctgtggagc
ggaatgtggg ggtcatcgtg 600gcagccgtcc ttgtaaccct gattctcctg
ggaatcttgg tttttggcat ctggtttgcc 660tatagccgag gccactttga
cagaacaaag aaagggactt cgagtaagaa ggtgatttac 720agccagccta
gtgcccgaag tgaaggagaa ttcaaacaga cctcgtcatt cctggtg
77765116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 65Xaa Val Gln Leu Xaa Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg Gln Ala Pro
Gly Gln Xaa Leu Glu Trp Met 35 40 45Gly Xaa Ile Asp Pro Tyr Asn Gly
Gly Thr Xaa Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Xaa Thr Xaa Thr
Xaa Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Xaa Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln Thr
Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val
Ser Ser 11566116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 66Xaa Val Gln Leu Xaa Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg
Gln Ala Pro Gly Gln Xaa Leu Glu Trp Met 35 40 45Gly Xaa Ile Asp Pro
Tyr Asn Gly Gly Thr Xaa Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Xaa
Thr Xaa Thr Xaa Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Xaa
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110Thr Val Ser Ser 11567106PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 67Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Xaa Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30Xaa Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Xaa Tyr Thr
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Xaa Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr
85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10568106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 68Xaa Ile Xaa Met Thr Gln Ser Pro Phe Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Xaa Ala Ser
Gln Asp Ile Asn Asn Tyr 20 25 30Xaa Ala Trp Tyr Gln Gln Lys Pro Ala
Lys Ala Pro Lys Leu Phe Ile 35 40 45Xaa Tyr Thr Ser Xaa Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 10569319DNAMus musculusCDS(1)..(318)
69gac atc cag atg aca cag tct cca tcc tca ctg tct gca tct ctg gga
48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15ggc aaa gtc acc atc act tgc aag gca agc caa gac att aac aat
tat 96Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30ata gct tgg tac caa cac aag cct gga aaa ggt cct agg ctg
ctc ata 144Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu
Leu Ile 35 40 45cat tac aca tct aca tta caa gca ggc atc cca tca agg
ttc agt gga 192His Tyr Thr Ser Thr Leu Gln Ala Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60agt ggg tct ggg aga gat tat tcc ttc agc atc agc
aac ctg gag cct 240Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
Asn Leu
Glu Pro65 70 75 80gaa gat att gga act tat tat tgt cta cag tat gat
aat ctg tgg acg 288Glu Asp Ile Gly Thr Tyr Tyr Cys Leu Gln Tyr Asp
Asn Leu Trp Thr 85 90 95ttc ggt gga ggc acc aag ctg gaa atc aaa c
319Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10570287DNAMus
musculus 70gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg
caaagtcacc 60atcacttgca aggcaagcca agacattaac aagtatatag cttggtacca
acacaagcct 120ggaaaaggtc ctaggctgct catacattac acatctacat
tacagccagg catcccatca 180aggttcagtg gaagtgggtc tgggagagat
tattccttca gcatcagcaa cctggagcct 240gaagatattg caacttatta
ttgtctacag tatgataatc ttctacc 2877138DNAMus musculusCDS(2)..(37)
71g tgg acg ttc ggt gga ggc acc aag ctg gaa atc aaa c 38 Trp Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 1072312DNAMus
musculusCDS(1)..(312) 72gac atc cag atg aca cag tct cca tcc tca ctg
tct gca tct ctg gga 48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Leu Gly1 5 10 15ggc aaa gtc acc atc act tgc aag gca agc
caa gac att aac aat tat 96Gly Lys Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Ile Asn Asn Tyr 20 25 30ata gct tgg tac caa cac aag cct gga
aaa ggt cct agg ctg ctc ata 144Ile Ala Trp Tyr Gln His Lys Pro Gly
Lys Gly Pro Arg Leu Leu Ile 35 40 45cat tac aca tct aca tta caa gca
ggc atc cca tca agg ttc agt gga 192His Tyr Thr Ser Thr Leu Gln Ala
Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60agt ggg tct ggg aga gat tat
tcc ttc agc atc agc aac ctg gag cct 240Ser Gly Ser Gly Arg Asp Tyr
Ser Phe Ser Ile Ser Asn Leu Glu Pro65 70 75 80gaa gat att gga act
tat tat tgt cta cag tat gat aat ctg tgg acg 288Glu Asp Ile Gly Thr
Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95ttc ggt gga ggc
acc aag ctg gaa 312Phe Gly Gly Gly Thr Lys Leu Glu 10073104PRTMus
musculus 73Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Leu Gly1 5 10 15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile
Asn Asn Tyr 20 25 30Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro
Arg Leu Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ala Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser
Ile Ser Asn Leu Glu Pro65 70 75 80Glu Asp Ile Gly Thr Tyr Tyr Cys
Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu 10074287DNAHomo sapiensCDS(1)..(285) 74gac atc cag atg acc cag
tct cca tcc tcc ctg tct gca tct gta gga 48Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15gac aga gtc acc atc
act tgc cag gcg agt cag gac att agc aac tat 96Asp Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30tta aat tgg tat
cag cag aaa cca ggg aaa gcc cct aag ctc ctg atc 144Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45tac gat gca
tcc aat ttg gaa aca ggg gtc cca tca agg ttc agt gga 192Tyr Asp Ala
Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60agt gga
tct ggg aca gat ttt act ttc acc atc agc agc ctg cag cct 240Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80gaa gat att gca aca tat tac tgt caa cag tat gat aat ctc cct cc
287Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro 85
90 957595PRTHomo sapiens 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro 85 90 957637DNAMus
musculusCDS(2)..(37) 76g tgg acg ttc ggt gga ggc acc aag ctg gaa
atc aaa 37 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5
107737DNAHomo sapiensCDS(2)..(37) 77g tgg acg ttc ggc caa ggg acc
aag gtg gaa atc aaa 37 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 1 5 107812PRTHomo sapiens 78Trp Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys1 5 1079349DNAMus musculusCDS(1)..(348) 79gag atc cag
ctg cag cag tct gga cct gag ctg gtg aag cct ggg gct 48Glu Ile Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15tca gtg
aag gta tcc tgc aag gct tct ggt tac tca ttc act gac tac 96Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30agc
atg tac tgg gtg aag cag agc cat gga aag agc ctt gag tgg att 144Ser
Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45gga tat att gat cct tac aat ggt ggt act agg tac aac cag aag ttc
192Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe
50 55 60aag ggc aag gcc aca ttg act gtt gac aag tcc tcc agc aca gcc
ttc 240Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Phe65 70 75 80atg cat ctc aac agc ctg aca tct gag gac tct gca gtc
tat tac tgt 288Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95gca aga cag acg gac tac ttt gac tac tgg ggc caa
ggc acc act ctc 336Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Thr Leu 100 105 110aca gtc tcc tca g 349Thr Val Ser Ser
115806PRTMus musculus 80Ala Arg Gln Thr Asp Tyr1 58116DNAMus
musculus 81agacagctc gggctac 168218PRTMus musculus 82Gln Thr Asp
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val1 5 10 15Ser
Ser8354DNAMus musculusCDS(1)..(54) 83cag acg gac tac ttt gac tac
tgg ggc caa ggc acc act ctc aca gtc 48Gln Thr Asp Tyr Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Leu Thr Val1 5 10 15tcc tca 54Ser
Ser8447DNAMus musculus 84actactttga ctactggggc caaggcacca
ctctcacagt ctcctca 4785336DNAMus musculusCDS(1)..(336) 85gag atc
cag ctg cag cag tct gga cct gag ctg gtg aag cct ggg gct 48Glu Ile
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15tca
gtg aag gta tcc tgc aag gct tct ggt tac tca ttc act gac tac 96Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25
30agc atg tac tgg gtg aag cag agc cat gga aag agc ctt gag tgg att
144Ser Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45gga tat att gat cct tac aat ggt ggt act agg tac aac cag aag
ttc 192Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys
Phe 50 55 60aag ggc aag gcc aca ttg act gtt gac aag tcc tcc agc aca
gcc ttc 240Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Phe65 70 75 80atg cat ctc aac agc ctg aca tct gag gac tct gca
gtc tat tac tgt 288Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95gca aga cag acg gac tac ttt gac tac tgg ggc
caa ggc acc act ctc 336Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Thr Leu 100 105 11086112PRTMus musculus 86Glu Ile Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser
Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40
45Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Phe65 70 75 80Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Thr Leu 100 105 1108715DNAMus musculusCDS(1)..(15) 87cag
acg gac tac ttt 15Gln Thr Asp Tyr Phe1 5885PRTMus musculus 88Gln
Thr Asp Tyr Phe1 58956DNAMus musculusCDS(9)..(56) 89gacagacg gac
tac ttt gac tac tgg ggc caa ggc acc act ctc aca gtc 50 Asp Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val 1 5 10tcc tca 56Ser
Ser159047DNAHomo sapiensCDS(3)..(47) 90ac tac ttt gac tac tgg ggc
caa gga acc ctg gtc acc gtc tcc tca 47 Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 1 5 10 1591106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
91Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ala Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Thr Phe Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln
Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10592116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 92Glu Ile Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys
Val Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met Tyr Trp Val Gln
Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro
Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110Thr Val Ser Ser 1159397PRTMus musculus 93Glu Ile Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met Tyr Trp
Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Tyr Ile
Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe65 70 75
80Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala9497PRTMus musculus 94Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Tyr Asn Gly Gly Thr Lys Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala9597PRTMus musculus 95Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg Gln Ala
Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Tyr Ile Asp Pro Tyr Asn
Gly Gly Thr Arg Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala96106PRTHomo sapiens 96Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Asn Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Val Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Thr Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Val Gly
Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 10597106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
97Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu
Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Val Gly Thr Tyr Tyr Cys Leu Gln
Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 10598106PRTHomo sapiens 98Ala Ile Arg Met Thr Gln Ser Pro
Phe Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Trp Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Ala Lys Ala Pro Lys Leu Phe Ile 35 40 45Tyr Tyr Thr Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10599106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
99Asp Ile Gln Met Thr Gln Ser Pro Phe Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30Ile Ala Trp Tyr Gln Gln Lys Pro Ala Lys Ala Pro Lys Leu
Phe Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
Tyr Asp Asn Leu Trp Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105100116PRTHomo sapiens 100Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45Gly Trp Ile Asp Pro Tyr Asn Gly Gly Thr Lys Tyr Ser Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115101116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 101Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Trp Met 35 40 45Gly Tyr Ile Asp Pro Tyr Asn
Gly Gly Thr Arg Tyr Ser Gln Lys Phe 50 55 60Gln Gly Arg Ala Thr Leu
Thr Val Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met His Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gln
Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser 115102116PRTHomo sapiens 102Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Ser Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Asp
Pro Tyr Asn Gly Gly Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110Thr Val Ser Ser 11510310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Cys
Leu Gln Tyr Asp Asn Leu Trp Thr Phe1 5 1010411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Cys
Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp1 5 10
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