U.S. patent application number 13/827946 was filed with the patent office on 2013-07-25 for human antibodies that bind mesothelin, and uses thereof.
This patent application is currently assigned to BRISTOL-MYERS SQUIBB COMPANY. The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY. Invention is credited to BINGLIANG CHEN, SARAH L. POGUE, CHETANA RAO-NAIK, JONATHAN A. TERRETT, KRISTOPHER TOY, LAN YANG.
Application Number | 20130190481 13/827946 |
Document ID | / |
Family ID | 40526631 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130190481 |
Kind Code |
A1 |
TERRETT; JONATHAN A. ; et
al. |
July 25, 2013 |
HUMAN ANTIBODIES THAT BIND MESOTHELIN, AND USES THEREOF
Abstract
The present disclosure provides isolated monoclonal antibodies
that specifically bind to mesothelin with high affinity,
particularly human monoclonal antibodies. Preferably, the
antibodies bind human mesothelin. In certain embodiments, the
antibodies are capable of internalizing into mesothelin-expressing
cells or are capable of mediating antigen dependent cellular
cytotoxicity. The invention further provides anti-mesothelin
antibodies that can inhibit the binding of mesothelin to the
ovarian cancer antigen CA125. Nucleic acid molecules encoding the
antibodies of this disclosure, expression vectors, host cells and
methods for expressing the antibodies of this disclosure are also
provided. Antibody-partner molecule conjugates, bispecific
molecules and pharmaceutical compositions comprising the antibodies
of this disclosure are also provided. This disclosure also provides
methods for detecting mesothelin, as well as methods for treating
cancers, such as mesotheliomas, pancreatic cancers and ovarian
cancers, using an anti-mesothelin antibody of this disclosure.
Inventors: |
TERRETT; JONATHAN A.;
(SUNNYVALE, CA) ; POGUE; SARAH L.; (FREMONT,
CA) ; TOY; KRISTOPHER; (SAN JOSE, CA) ; YANG;
LAN; (MORGAN HILL, CA) ; RAO-NAIK; CHETANA;
(WALNUT CREEK, CA) ; CHEN; BINGLIANG; (ALAMEDA,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRISTOL-MYERS SQUIBB COMPANY; |
PRINCETON |
NJ |
US |
|
|
Assignee: |
BRISTOL-MYERS SQUIBB
COMPANY
PRINCETON
NJ
|
Family ID: |
40526631 |
Appl. No.: |
13/827946 |
Filed: |
March 14, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13587644 |
Aug 16, 2012 |
8425904 |
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13827946 |
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12681215 |
Apr 1, 2010 |
8268970 |
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PCT/US2008/078123 |
Sep 29, 2008 |
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13587644 |
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60976626 |
Oct 1, 2007 |
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60991692 |
Nov 30, 2007 |
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61077397 |
Jul 1, 2008 |
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Current U.S.
Class: |
530/388.15 |
Current CPC
Class: |
C07K 2317/92 20130101;
A61K 47/6857 20170801; C07K 2317/41 20130101; A61K 47/6869
20170801; A61K 47/6863 20170801; C07K 2317/73 20130101; A61K
39/39558 20130101; C07K 2317/565 20130101; A61K 47/6859 20170801;
C07K 2317/732 20130101; C07K 2317/21 20130101; C07K 2317/80
20130101; A61K 2039/505 20130101; A61K 2300/00 20130101; C07K 16/30
20130101; C07K 2317/76 20130101; C07K 2317/77 20130101; A61K
39/39558 20130101; A61P 35/00 20180101 |
Class at
Publication: |
530/388.15 |
International
Class: |
C07K 16/30 20060101
C07K016/30 |
Claims
1. An isolated monoclonal antibody, or an antigen-binding portion
thereof, comprising a heavy chain variable region that is the
product of or derived from a human V.sub.H 3-33 gene or a human
V.sub.H 3-7 gene, wherein the antibody specifically binds human
mesothelin.
2. An isolated monoclonal antibody, or an antigen-binding portion
thereof, comprising a light chain variable region that is the
product of or derived from a human V.sub.K L6 gene or a human
V.sub.K A27 gene, wherein the antibody specifically binds human
mesothelin.
3. An isolated monoclonal antibody, or an antigen-binding portion
thereof, comprising: (a) a heavy chain variable region that is the
product of or derived from a human V.sub.H 3-33 gene and a light
chain variable region that is the product of or derived from a
human V.sub.K L6 gene; or (b) a heavy chain variable region that is
the product of or derived from a human V.sub.H 3-7 gene and a light
chain variable region that is the product of or derived from a
human V.sub.K A27 gene; wherein the antibody specifically binds
human mesothelin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is continuation of application Ser. No.
13/587,644, filed Aug. 16, 2012, now allowed; which is a divisional
of application Ser. No. 12/681,215, filed Apr. 1, 2010, now U.S.
Pat. No. 8,268,970 B2; which is the National Stage of International
Application No. PCT/US2008/078123, filed Sep. 29, 2008; which
claims the benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Applications Nos. 60/976,626, filed Oct. 1, 2007; 60/991,692, filed
Nov. 30, 2007; and 61/077,397, filed Jul. 1, 2008; the disclosures
of which are incorporated herein by reference.
SEQUENCE LISTING
[0002] Incorporated herein by reference in its entirety is a
Sequence Listing named "SEQT_M00192USCON.txt", comprising SEQ ID
NO:1 through SEQ ID NO:43, which include nucleic acid and/or amino
acid sequences disclosed herein. The Sequence Listing has been
submitted herewith in ASCII text format via EFS-Web, and thus
constitutes both the paper and computer readable form thereof. The
Sequence Listing was first created using PatentIn 3.5 on Mar. 13,
2013, and is 19 KB in size.
BACKGROUND OF THE INVENTION
[0003] Mesothelin is a glycoprotein present on the surface of cells
of the mesothelial lining of the peritoneal, pleural and
pericardial body cavities. It was originally purified from the
human pancreatic cancer cell line HPC-Y5 and was shown to have
megakaryocyte potentiating ability and hence named megakaryocyte
potentiating factor (MPF) (Yamaguchi et al. (1994) J. Biol. Chem.
269:805-808). The mesothelin cDNA was cloned from a library
prepared from the HPC-Y5 cell line (Kojima et al. (1995) J. Biol.
Chem. 270:21984-21990). The cDNA also was cloned using the
monoclonal antibody K1, which recognizes mesotheliomas (Chang and
Pastan (1996) Proc. Natl. Acad. Sci. USA 93:136-40). Structurally,
mesothelin is expressed on the cell surface as a 60 kDa precursor
polypeptide, which is proteolytically processed into a 31 kDa shed
component (corresponding to MPF) and a 40 kDa membrane bound
component (Hassan et al. (2004) Clin. Cancer. Res.
10:3937-3942).
[0004] In addition to being expressed on normal mesothelial cells,
mesothelin is overexpressed in several types of human tumors,
including all mesotheliomas, many ovarian and pancreatic cancers,
and some stomach, lung and endometrial cancers. For example,
mesothelin is expressed on approximately 70% of all ovarian
cancers, approximately 82% of papillary, serous adenocarcinomas,
approximately 83% of all pancreatic adenocarcinomas and
approximately 86% of all ductal pancreatic adenocarcinomas.
[0005] Mutant mice have been prepared in which the mesothelin gene
was disrupted by homologous recombination (Bera, T. K. and Pastan,
I. (2000) Mol. Cell. Biol. 20:2902-2906). No anatomical,
hematologic or reproductive abnormalities were detected, indicating
that mesothelin function is not essential for growth or
reproduction, at least in those mice.
[0006] Mesothelin specifically interacts with CA125 (also known as
MUC-16), a mucin-like glycoprotein present on the surface of tumor
cells that previously had been identified as an ovarian cancer
antigen. Further, binding of CA125 to membrane-bound mesothelin
mediates heterotypic cell adhesion and CA125 and mesothelin are
co-expressed in advanced grade ovarian adenocarcinoma (Rump, A. et
al. (2004) J. Biol. Chem. 279:9190-9198). Expression of mesothelin
in the lining of the peritoneum correlates with the preferred site
of metastasis formation of ovarian cancer and mesothelin-CA125
binding is thought to facilitate peritoneal metastasis of ovarian
tumors (Gubbels, J. A. et al. (2006) Mol. Cancer. 5:50).
[0007] In view of the foregoing, additional agents for modulating
the activity of mesothelin are of interest.
SUMMARY
[0008] The present disclosure provides isolated monoclonal
antibodies, in particular human ones, which specifically bind
mesothelin and have desirable properties such as high affinity
binding to human mesothelin, internalization by cells expressing
mesothelin, inhibition of mesothelin binding to CA125, and/or the
mediation of antibody dependent cellular cytotoxicity (ADCC). The
antibodies of the invention can be used, for example, to detect
mesothelin or to inhibit the growth of cells expressing mesothelin,
such as mesothelin-expressing tumor cells.
[0009] In one aspect, the invention pertains to an isolated human
monoclonal antibody, or an antigen-binding portion thereof, wherein
the antibody binds human mesothelin and exhibits at least one of
the following properties: [0010] (a) binds to human mesothelin with
a K.sub.D of 1.times.10.sup.-8 M or less; [0011] (b) is
internalized by mesothelin-expressing cells; [0012] (c) inhibits
binding of mesothelin to ovarian cancer antigen CA125; [0013] (d)
exhibits ADCC against mesothelin expressing cells; and [0014] (e)
inhibits growth of mesothelin-expressing cells in vivo when
conjugated to a cytotoxin.
[0015] Preferably, the antibody exhibits at least two of properties
(a), (b), (c), (d), and (e). More preferably, the antibody exhibits
at least three of properties (a), (b), (c), (d), and (e). More
preferably, the antibody exhibits four of properties (a), (b), (c),
(d), and (e). Even, more preferably, the antibody exhibits all five
of properties (a), (b), (c), (d), and (e). In another preferred
embodiment, the antibody binds to mesothelin with a K.sub.D of
5.times.10.sup.-9 M or less. In yet another preferred embodiment,
the antibody inhibits growth of mesothelin-expressing tumor cells
in vivo when the antibody is conjugated to a cytotoxin.
[0016] In another aspect, the invention pertains to an isolated
human monoclonal antibody, or antigen binding portion thereof,
wherein the antibody cross-competes for binding to an epitope on
human mesothelin that is recognized by a reference antibody having:
[0017] (a) a heavy chain variable region (V.sub.H) comprising the
amino acid sequence of SEQ ID NO: 19 and a light chain variable
region (V.sub.L) comprising the amino acid sequence of SEQ ID NO:
22; [0018] (b) a V.sub.H comprising the amino acid sequence of SEQ
ID NO: 20 and a V.sub.L comprising the amino acid sequence of SEQ
ID NO: 23; or [0019] (c) a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 21 and a V.sub.L comprising the amino acid
sequence of SEQ ID NO: 24.
[0020] The reference antibodies described above are referred to
hereinbelow as Reference Antibodies (a), (b), and (c),
respectively.
[0021] In a preferred embodiment, the reference antibody comprises
a V.sub.H comprising the amino acid sequence of SEQ ID NO: 19 and a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 22. In
another preferred embodiment, the reference antibody comprises a
V.sub.H comprising the amino acid sequence of SEQ ID NO: 20 and a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 23. In yet
another preferred embodiment, the reference antibody comprises a
V.sub.H comprising the amino acid sequence of SEQ ID NO: 21 and a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 24.
[0022] In another aspect, the invention pertains to an isolated
monoclonal antibody, or an antigen-binding portion thereof,
comprising a V.sub.H that is the product of or derived from a human
V.sub.H 3-33 gene or a human V.sub.H 3-7 gene, wherein the antibody
specifically binds human mesothelin. In another aspect, the
invention pertains to an isolated monoclonal antibody, or an
antigen-binding portion thereof, comprising a V.sub.L that is the
product of or derived from a human V.sub.K L6 gene or a human
V.sub.K A27 gene, wherein the antibody specifically binds human
mesothelin. In a preferred embodiment, the invention provides an
isolated monoclonal antibody, or an antigen-binding portion
thereof, comprising: [0023] (a) a V.sub.H that is the product of or
derived from a human V.sub.H 3-33 gene and a V.sub.L that is the
product of or derived from a human V.sub.K L6 gene; or [0024] (b) a
V.sub.H that is the product of or derived from a human V.sub.H 3-7
gene and a V.sub.L that is the product of or derived from a human
V.sub.K A27 gene; wherein the antibody specifically binds human
mesothelin.
[0025] A particularly preferred antibody, or antigen-binding
portion thereof ("Embodiment A"), comprises: [0026] (a) a heavy
chain variable region CDR1 comprising SEQ ID NO: 1; [0027] (b) a
heavy chain variable region CDR2 comprising SEQ ID NO: 4; [0028]
(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 7;
[0029] (d) a light chain variable region CDR1 comprising SEQ ID NO:
10; [0030] (e) a light chain variable region CDR2 comprising SEQ ID
NO: 13; and [0031] (f) a light chain variable region CDR3
comprising SEQ ID NO: 16.
[0032] Another particularly preferred antibody, or antigen-binding
portion thereof ("Embodiment B"), comprises: [0033] (a) a heavy
chain variable region CDR1 comprising SEQ ID NO: 2; [0034] (b) a
heavy chain variable region CDR2 comprising SEQ ID NO: 5; [0035]
(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 8;
[0036] (d) a light chain variable region CDR1 comprising SEQ ID NO:
11; [0037] (e) a light chain variable region CDR2 comprising SEQ ID
NO: 14; and [0038] (f) a light chain variable region CDR3
comprising SEQ ID NO: 17.
[0039] Another particularly preferred antibody, or antigen-binding
portion thereof ("Embodiment C"), comprises: [0040] (a) a heavy
chain variable region CDR1 comprising SEQ ID NO: 3; [0041] (b) a
heavy chain variable region CDR2 comprising SEQ ID NO: 6; [0042]
(c) a heavy chain variable region CDR3 comprising SEQ ID NO: 9;
[0043] (d) a light chain variable region CDR1 comprising SEQ ID NO:
12; [0044] (e) a light chain variable region CDR2 comprising SEQ ID
NO: 15; and [0045] (f) a light chain variable region CDR3
comprising SEQ ID NO: 18.
[0046] In another aspect, the invention pertains to an isolated
monoclonal antibody, or antigen binding portion thereof,
comprising: (a) a V.sub.H comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 19-21; and (b) a
V.sub.L comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 22-24; wherein the antibody specifically
binds human mesothelin.
[0047] A preferred combination comprises: (a) a V.sub.H comprising
the amino acid sequence of SEQ ID NO: 19; and (b) a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 22.
[0048] Another preferred combination comprises: (a) a V.sub.H
comprising the amino acid sequence of SEQ ID NO: 20; and (b) a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 23.
[0049] Another preferred combination comprises: (a) a V.sub.H
comprising the amino acid sequence of SEQ ID NO: 21; and (b) a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 24.
[0050] The antibodies of this disclosure can be, for example, of an
IgG1 or IgG4 isotype. Alternatively, they can be antibody
fragments, such as Fab, Fab' or Fab'2 fragments, or single chain
antibodies.
[0051] This disclosure also provides an immunoconjugate comprising
an antibody of this disclosure, or antigen-binding portion thereof,
linked to a partner molecule. In a particularly preferred
embodiment, the invention provides an immunoconjugate comprising an
antibody of this disclosure, or antigen-binding portion thereof,
linked to the compound Cytotoxin A, which is identified hereinbelow
and is discussed in WO 2008/083312, the disclosure of which is
incorporated herein by reference. The invention provides the
following preferred immunoconjugates, comprising Cytotoxin A linked
to an antibody of this invention, wherein the antibody (i)
cross-competes with Reference Antibody (a), (b), or (c) for binding
to an epitope of human mesothelin; (ii) is according to Embodiment
A; (iii) is according to Embodiment B; or (iv) is according to
Embodiment C. Certain of such antibody-partner molecule conjugates
are capable of being internalized into mesothelin-expressing cells
and are capable of mediating ADCC.
[0052] In one aspect, such antibody-partner molecule conjugates are
conjugated via chemical linkers. In some embodiments, the linker is
a peptidyl linker, and is depicted herein as
(L.sup.4).sub.p-F-(L.sup.1).sub.m. Other linkers include hydrazine
and disulfide linkers, and is depicted herein as
(L.sup.4).sub.p-H-(L.sup.1), or (L.sup.4).sub.p-J-(L.sup.1).sub.m,
respectively. In addition to the linkers as being attached to the
partner, the present invention also provides cleavable linker arms
that are appropriate for attachment to essentially any molecular
species.
[0053] This disclosure also provides a bispecific molecule
comprising an antibody, or antigen-binding portion thereof, of this
disclosure, linked to a second functional moiety having a different
binding specificity than said antibody, or antigen binding portion
thereof.
[0054] Compositions comprising an antibody, or antigen-binding
portion thereof, or immunoconjugate or bispecific molecule of this
disclosure and a pharmaceutically acceptable carrier are also
provided.
[0055] Nucleic acid molecules encoding the antibodies, or
antigen-binding portions thereof, of this disclosure are also
encompassed by this disclosure, as well as expression vectors
comprising such nucleic acids and host cells comprising such
expression vectors. Methods for preparing anti-mesothelin
antibodies using the host cells comprising such expression vectors
are also provided and may include the steps of (i) expressing the
antibody in the host cell and (ii) isolating the antibody from the
host cell.
[0056] In yet another aspect, the invention pertains to a method
for preparing an anti-mesothelin antibody. The method comprises:
[0057] (a) providing: (i) a V.sub.H antibody sequence comprising a
CDR1 sequence selected from the group consisting of SEQ ID NOs:
1-3, a CDR2 sequence selected from the group consisting of SEQ ID
NOs: 4-6, and/or a CDR3 sequence selected from the group consisting
of SEQ ID NOs: 7-9; and/or (ii) a V.sub.L antibody sequence
comprising a CDR1 sequence selected from the group consisting of
SEQ ID NOs: 10-12, a CDR2 sequence selected from the group
consisting of SEQ ID NOs: 13-15, and/or a CDR3 sequence selected
from the group consisting of SEQ ID NOs: 16-18; [0058] (b) altering
at least one amino acid residue within the V.sub.H antibody
sequence and/or the V.sub.L antibody sequence to create at least
one altered antibody sequence; and [0059] (c) expressing the
altered antibody sequence as a protein.
[0060] In another aspect, the invention pertains to a method of
inhibiting growth of a mesothelin-expressing tumor cell, comprising
contacting the tumor cell with an antibody-partner molecule
conjugate of the disclosure, such that growth of the tumor cell is
inhibited. Preferably, the mesothelin-expressing tumor cell is a
mesothelioma cell, pancreatic tumor cell, ovarian tumor cell,
stomach tumor cell, lung tumor cell or endometrial tumor cell. In
still other embodiments, the mesothelin-expressing tumor cell is
from a cancer selected from the group consisting of mesothelioma,
papillary serous ovarian adenocarcinoma, clear cell ovarian
carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous
ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic
adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma,
extrahepatic bile duct carcinoma, gastric adenocarcinoma,
esophageal adenocarcinoma, colorectal adenocarcinoma and breast
adenocarcinoma.
[0061] In another aspect, the invention pertains to a method of
treating cancer in a subject. The method comprises administering to
the subject an antibody-partner molecule conjugate of the
disclosure such that the cancer is treated in the subject.
Particularly preferred cancers for treatment are mesotheliomas,
pancreatic cancers, ovarian cancers, stomach cancers, lung cancers
and endometrial cancers. In still other embodiments, the cancer to
be treated is selected from the group consisting of mesotheliomas,
papillary serous ovarian adenocarcinomas, clear cell ovarian
carcinomas, mixed Mullerian ovarian carcinomas, endometroid
mucinous ovarian carcinomas, pancreatic adenocarcinomas, ductal
pancreatic adenocarcinomas, uterine serous carcinomas, lung
adenocarcinomas, extrahepatic bile duct carcinomas, gastric
adenocarcinomas, esophageal adenocarcinomas, colorectal
adenocarcinomas and breast adenocarcinomas.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIG. 1A shows the nucleotide (SEQ ID NO: 25) and amino acid
sequences (SEQ ID NO: 19) of the V.sub.H of the 3C10 human
monoclonal antibody. The CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 4)
and CDR3 (SEQ ID NO: 7) regions are delineated.
[0063] FIG. 1B shows the nucleotide (SEQ ID NO: 28) and amino acid
sequences (SEQ ID NO: 22) of the V.sub.L of the 3C10 human
monoclonal antibody. The CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 13)
and CDR3 (SEQ ID NO: 16) regions are delineated.
[0064] FIG. 2A shows the nucleotide (SEQ ID NO: 26) and amino acid
sequences (SEQ ID NO: 20) of the V.sub.H of the 6A4 human
monoclonal antibody. The CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 5)
and CDR3 (SEQ ID NO: 8) regions are delineated.
[0065] FIG. 2B shows the nucleotide (SEQ ID NO: 29) and amino acid
sequences (SEQ ID NO: 23) of the V.sub.L of the 6A4 human
monoclonal antibody. The CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO: 14)
and CDR3 (SEQ ID NO: 17) regions are delineated.
[0066] FIG. 3A shows the nucleotide (SEQ ID NO: 27) and amino acid
sequences (SEQ ID NO: 21) of the V.sub.H of the 7B1 human
monoclonal antibody. The CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 6)
and CDR3 (SEQ ID NO: 9) regions are delineated.
[0067] FIG. 3B shows the nucleotide (SEQ ID NO: 30) and amino acid
sequences (SEQ ID NO: 24) of the V.sub.L of the 7B1 human
monoclonal antibody. The CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO: 15)
and CDR3 (SEQ ID NO: 18) regions are delineated.
[0068] FIG. 4 shows the alignment of the amino acid sequence of the
V.sub.Hs of 3C10 (SEQ ID NO: 19) and 6A4 (SEQ ID NO: 20) with the
human germline V.sub.H 3-33 amino acid sequence (SEQ ID NO:
31).
[0069] FIG. 5 shows the alignment of the amino acid sequence of the
V.sub.Ls of 3C10 (SEQ ID NO: 22) and 6A4 (SEQ ID NO: 23) with the
human germline V.sub.k L6 amino acid sequence (SEQ ID NO: 33).
[0070] FIG. 6 shows the alignment of the amino acid sequence of the
V.sub.H of 7B1 (SEQ ID NO: 21) with the human germline V.sub.H 3-7
amino acid sequence (SEQ ID NO: 32).
[0071] FIG. 7 shows the alignment of the amino acid sequence of the
V.sub.L of 7B 1 (SEQ ID NO: 24) with the human germline V.sub.k A27
amino acid sequence (SEQ ID NO: 34).
[0072] FIG. 8 shows the results of an OVCAR3 ovarian cancer cell
adhesion assay.
[0073] FIGS. 9A and 9B show the results of an in vivo study using
an NCI-H226 (lung mesothelioma) xenograft mouse model.
[0074] FIGS. 10A and 10B show the results of an in vivo study using
an HPAC (human pancreatic carcinoma) xenograft mouse model.
[0075] FIG. 11 is a graph showing that ovarian cancer tumor volumes
were reduced an immunoconjugate of this invention in an ovarian
cancer mouse xenograft model.
[0076] FIG. 12 shows ADCC of ovarian cancer cells (OVCAR3 cells,
FIG. 12a) and NSCLC cells (H226 cells, FIG. 12b) by a
non-fucosylated antibody of this invention.
DETAILED DESCRIPTION OF THIS DISCLOSURE
[0077] The present disclosure relates to isolated monoclonal
antibodies, particularly human ones, which bind to human mesothelin
and have desirable properties. In certain embodiments, the
antibodies of this disclosure are derived from particular heavy and
light chain germline sequences and/or comprise particular
structural features such as CDR regions comprising particular amino
acid sequences. This disclosure provides isolated antibodies,
methods of making such antibodies, antibody-partner molecule
conjugates, and bispecific molecules comprising such antibodies and
pharmaceutical compositions containing such antibodies,
antibody-partner molecule conjugates or bispecific molecules. Also
provided are variants or alternatives such as homologous
antibodies, antibodies with conservative modifications, engineered
and modified antibodies, antibody fragments, and antibody mimetics,
each further described hereinbelow. This disclosure also relates to
methods of using the antibodies, such as to detect mesothelin
protein, as well as to methods of using the anti-mesothelin
antibodies of the invention to inhibit the growth of
mesothelin-expressing cells, such as tumor cells. Accordingly, this
disclosure also provides methods of using the anti-mesothelin
antibodies of this disclosure to treat various types of cancer, for
example, ovarian cancer, pancreatic cancer, stomach cancer, lung
cancer, endometrial cancer and mesotheliomas. Preferably, the
antibody, conjugate, bispecific molecule, alternative or variant
one or more of these properties: binding to human mesothelin,
internalization by mesothelin expressing cells, inhibition of
mesothelin binding to CA125, mediation of ADCC against mesothelin
expressing cells, and inhibition of growth of mesothelin-expressing
tumor cells in vivo. Antibodies can be human (preferably),
humanized, or chimeric.
[0078] In order that the present disclosure may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0079] The term "mesothelin" includes variants, isoforms, homologs,
orthologs and paralogs. For example, antibodies specific for a
human mesothelin protein may, in certain cases, cross-react with a
mesothelin protein from a species other than human. In other
embodiments, the antibodies can be completely specific for the
human mesothelin protein and not exhibit species or other types of
cross-reactivity, or cross-react with mesothelin from certain other
species but not all other species (e.g., cross-react with a primate
mesothelin but not mouse mesothelin). The term "human mesothelin"
refers to human sequence mesothelin, such as the complete amino
acid sequence of human mesothelin having Genbank Accession Number
NP.sub.--005814. The term "mouse mesothelin" refers to mouse
sequence mesothelin, such as the complete amino acid sequence of
mouse mesothelin having Genbank Accession Number NP.sub.--061345.
The N-terminal portion of mesothelin is also known as megakaryocyte
potentiating factor (MPF). The human mesothelin sequence may differ
from human mesothelin of Genbank Accession Number NP.sub.--005814
by having, for example, conserved mutations or mutations in
non-conserved regions and the mesothelin has substantially the same
biological function as the human mesothelin of Genbank Accession
Number NP.sub.--005814, such as binding to CA125.
[0080] A particular human mesothelin sequence will generally be at
least 90% identical in amino acids sequence to human mesothelin of
Genbank Accession Number NP.sub.--005814 and contains amino acid
residues that identify the amino acid sequence as being human when
compared to mesothelin amino acid sequences of other species (e.g.,
murine). In certain cases, a human mesothelin may be at least 95%,
or even at least 96%, 97%, 98%, or 99% identical in amino acid
sequence to mesothelin of Genbank Accession Number NP.sub.--005814.
In certain embodiments, a human mesothelin sequence will display no
more than 10 amino acid differences from the mesothelin sequence of
Genbank Accession Number NP.sub.--005814. In certain embodiments,
the human mesothelin may display no more than 5, or even no more
than 4, 3, 2, or 1 amino acid difference from the mesothelin
sequence of Genbank Accession Number NP.sub.--005814. Percent
identity can be determined as described herein.
[0081] The term "CA125" refers to an ovarian carcinoma antigen, or
ovarian cancer tumor marker, also known as MUC-16. The term "human
CA125" refers to human sequence CA125 such as the complete amino
acid sequence of human CA125 having Genbank Accession Number
NP.sub.--078966. The human CA125 sequence may differ from human
CA125 of Genbank Accession Number NP.sub.--078966 by having, for
example, conserved mutations or mutations in non-conserved regions.
A particular human CA125 sequence will generally be at least 90%
identical in amino acids sequence to human CA125 of Genbank
Accession Number NP.sub.--078966 and contains amino acid residues
that identify the amino acid sequence as being human when compared
to CA125 amino acid sequences of other species (e.g., murine). In
certain cases, a human CA125 may be at least 95%, or even at least
96%, 97%, 98%, or 99% identical in amino acid sequence to CA125 of
Genbank Accession Number NP.sub.--078966. In certain embodiments, a
human CA125 sequence will display no more than 10 amino acid
differences from the CA125 sequence of Genbank Accession Number
NP.sub.--078966. In certain embodiments, the human CA125 may
display no more than 5, or even no more than 4, 3, 2, or 1 amino
acid difference from the CA125 sequence of Genbank Accession Number
NP.sub.--078966. Percent identity can be determined as described
herein.
[0082] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0083] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules
that play a role in the transmission of a signal from one portion
of a cell to another portion of a cell. The phrase "cell surface
receptor" includes molecules and complexes of molecules capable of
receiving a signal and transmitting signal across the plasma
membrane of a cell. An example of a "cell surface receptor" is
human mesothelin.
[0084] The term "antibody" refers to whole antibodies and any
antigen binding fragment ("antigen-binding portion") or single
chains thereof. An "antibody" refers to a glycoprotein comprising
at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds, or an antigen binding portion
thereof. Each heavy chain comprises a heavy chain variable region
(abbreviated herein as V.sub.H) and a heavy chain constant region.
The heavy chain constant region is comprised of three domains,
C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain comprises a light
chain variable region (abbreviated herein as V.sub.L) and a light
chain constant region. The light chain constant region is comprised
of one domain, C.sub.L. V.sub.H and V.sub.L can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies may mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system.
[0085] The term "antibody fragment" and "antigen-binding portion"
of an antibody (or simply "antibody portion"), as used herein,
refer to one or more fragments of an antibody that retain the
ability to specifically bind to an antigen (e.g., mesothelin). It
has been shown that the antigen-binding function of an antibody can
be performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fab' fragment, which is essentially an Fab
with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul
ed., 3rd ed. 1993); (iv) a Fd fragment consisting of the V.sub.H
and C.sub.H1 domains; (v) a Fv fragment consisting of the V.sub.L
and V.sub.H domains of a single arm of an antibody, (vi) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a V.sub.H domain; (vii) an isolated complementarity determining
region (CDR); and (viii) a nanobody, a heavy chain variable region
containing a single variable domain and two constant domains.
Furthermore, although the two domains of the Fv fragment, V.sub.L
and V.sub.H, are coded for by separate genes, they can be joined,
using recombinant methods, by a synthetic linker that enables them
to be made as a single protein chain in which the V.sub.L and
V.sub.H regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. These
antibody fragments are obtained using conventional techniques known
to those with skill in the art, and the fragments are screened for
utility in the same manner as intact antibodies.
[0086] An "isolated antibody" refers to an antibody that is
substantially free of other antibodies having different antigenic
specificities (e.g., an isolated antibody that specifically binds
mesothelin is substantially free of antibodies that specifically
bind other antigens). An isolated antibody that specifically binds
mesothelin may, however, have cross-reactivity to other antigens,
such as mesothelin molecules from other species. Moreover, an
isolated antibody may be substantially free of other cellular
material and/or chemicals.
[0087] The terms "monoclonal antibody" or "monoclonal antibody
composition" refer to a preparation of antibody molecules of single
molecular composition. A monoclonal antibody composition displays a
single binding specificity and affinity for a particular
epitope.
[0088] The term "human antibody" refers to antibodies having
variable regions in which both the framework and CDR regions are
derived from human germline immunoglobulin sequences. Furthermore,
if the antibody contains a constant region, the constant region
also is derived from human germline immunoglobulin sequences. The
human antibodies of the invention may include amino acid residues
not encoded by human germline immunoglobulin sequences (e.g.,
mutations introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). However, "human antibody" is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
[0089] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable regions
in which both the framework and CDR regions are derived from human
germline immunoglobulin sequences. In one embodiment, the human
monoclonal antibodies are produced by a hybridoma which includes a
B cell obtained from a transgenic nonhuman animal, e.g., a
transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene fused to an immortalized
cell.
[0090] The term "recombinant human antibody," as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom (described further below), (b) antibodies
isolated from a host cell transformed to express the human
antibody, e.g., from a transfectoma, (c) antibodies isolated from a
recombinant, combinatorial human antibody library, and (d)
antibodies prepared, expressed, created or isolated by any other
means that involve splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable regions in which the framework and CDR regions are derived
from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies can be
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the V.sub.H and V.sub.L regions of
the recombinant antibodies are sequences that, while derived from
and related to human germline V.sub.H and V.sub.L sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
[0091] The term "isotype" refers to the antibody class (e.g., IgM
or IgG1) that is encoded by the heavy chain constant region
genes.
[0092] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably with "an
antibody which binds specifically to an antigen."
[0093] The term "human antibody derivatives" refers to any modified
form of the human antibody, e.g., a conjugate of the antibody and
another agent or antibody.
[0094] The term "humanized antibody" refers to antibodies in which
CDR sequences derived from the germline of another mammalian
species, such as a mouse, have been grafted onto human framework
sequences. Additional framework region modifications may be made
within the human framework sequences.
[0095] The term "chimeric antibody" refers to antibodies in which
the variable region sequences are derived from one species and the
constant region sequences are derived from another species, such as
an antibody in which the variable region sequences are derived from
a mouse antibody and the constant region sequences are derived from
a human antibody.
[0096] The term "antibody mimetic" refers to molecules capable of
mimicking an antibody's ability to bind an antigen, but which are
not limited to native antibody structures. Examples of such
antibody mimetics include, but are not limited to, Affibodies,
DARPins, Anticalins, Avimers, and Versabodies, all of which are
further described hereinbelow.
[0097] The term "partner molecule" refers to the entity which is
conjugated to an antibody in an antibody-partner molecule
conjugate. Examples of partner molecules include drugs, toxins,
marker molecules, proteins and therapeutic agents.
[0098] As used herein, an antibody that "specifically binds to
human mesothelin" is intended to refer to an antibody that binds to
human mesothelin with a K.sub.D of 1.times.10.sup.-7 M or less,
more preferably 5.times.10.sup.-8 M or less, more preferably
3.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-8 M or
less, even more preferably 5.times.10.sup.-9 M or less.
[0099] The term "does not substantially bind" to a protein or
cells, as used herein, means does not bind or does not bind with a
high affinity to the protein or cells, i.e. binds to the protein or
cells with a K.sub.D of 1.times.10.sup.-6 M or more, more
preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more.
[0100] The term "K.sub.assoc" or "K.sub.a" refers to the
association rate of a particular antibody-antigen interaction,
whereas the term "K.sub.dis" or "K.sub.d," as used herein, is
intended to refer to the dissociation rate of a particular
antibody-antigen interaction. The term "K.sub.D," as used herein,
refers to the dissociation constant, which is obtained from the
ratio of K.sub.d to K.sub.a (i.e., K.sub.d/K.sub.a) and is
expressed as a molar concentration (M). K.sub.D values for
antibodies can be determined using methods well established in the
art. A preferred method for determining the K.sub.D of an antibody
is by using surface plasmon resonance, preferably using a biosensor
system such as a Biacore.RTM. system.
[0101] The term "high affinity" for an IgG antibody refers to an
antibody having a K.sub.D of 1.times.10.sup.-7 M or less, more
preferably 5.times.10.sup.-8 M or less, even more preferably
1.times.10.sup.-8 M or less, even more preferably 5.times.10.sup.-9
M or less and even more preferably 1.times.10.sup.-9 M or less for
a target antigen. However, "high affinity" binding can vary for
other antibody isotypes. For example, "high affinity" binding for
an IgM isotype refers to an antibody having a K.sub.D of 10.sup.-6
M or less, more preferably 10.sup.-7 M or less, even more
preferably 10.sup.-8 M or less.
[0102] The term "subject" includes any human or nonhuman animal.
The term "nonhuman animal" includes all vertebrates, e.g., mammals
and non-mammals, such as nonhuman primates, sheep, dogs, cats,
horses, cows, chickens, amphibians, reptiles, etc.
[0103] The term "alkyl," by itself or as part of another
substituent, means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
di- and multivalent radicals, having the number of carbon atoms
designated (i.e. C.sub.1-C.sub.10 means one to ten carbons).
Examples of saturated hydrocarbon radicals include, but are not
limited to, groups such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups, which are limited to hydrocarbon
groups are termed "homoalkyl".
[0104] The term "alkylene" by itself or as part of another
substituent means a divalent radical derived from an alkane, as
exemplified, but not limited, by
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and further includes those
groups described below as "heteroalkylene." Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred in the
present invention. A "lower alkyl" or "lower alkylene" is a shorter
chain alkyl or alkylene group, generally having eight or fewer
carbon atoms.
[0105] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or cyclic hydrocarbon radical, or combinations
thereof, consisting of the stated number of carbon atoms and at
least one heteroatom selected from O, N, Si, and S, and wherein the
nitrogen, carbon and sulfur atoms may optionally be oxidized and
the nitrogen heteroatom may optionally be quaternized. The
heteroatom(s) O, N, S, and Si may be placed at any interior
position of the heteroalkyl group or at the position at which the
alkyl group is attached to the remainder of the molecule. Examples
include, but are not limited to, --CH.sub.2CH.sub.2OCH.sub.3,
--CH.sub.2CH.sub.2NHCH.sub.3, --CH.sub.2CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2SCH.sub.2CH.sub.3, --CH.sub.2CH.sub.2S(O)CH.sub.3,
--CH.sub.2CH.sub.2S(O).sub.2CH.sub.3, --CH.dbd.CHOCH.sub.3,
--Si(CH.sub.3).sub.3, --CH.sub.2CH.dbd.NOCH.sub.3, and
--CH.dbd.CHN(CH.sub.3).sub.2. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2NHOCH.sub.3 and
--CH.sub.2OSi(CH.sub.3).sub.3. Similarly, the term "heteroalkylene"
by itself or as part of another substituent means a divalent
radical derived from heteroalkyl, as exemplified, but not limited
by, --CH.sub.2CH.sub.2SCH.sub.2CH.sub.2-- and
--CH.sub.2SCH.sub.2CH.sub.2NHCH.sub.2--. For heteroalkylene groups,
heteroatoms can also occupy either or both of the chain termini
(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,
and the like). The terms "heteroalkyl" and "heteroalkylene"
encompass poly(ethylene glycol) and its derivatives (see, for
example, Shearwater Polymers Catalog, 2001). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0106] The term "lower" in combination with the terms "alkyl" or
"heteroalkyl" refers to a moiety having from 1 to 6 carbon
atoms.
[0107] The terms "alkoxy," "alkylamino," "alkylsulfonyl," and
"alkylthio" (or thioalkoxy) are used in their conventional sense,
and refer to those alkyl groups attached to the remainder of the
molecule via an oxygen atom, an amino group, an SO.sub.2 group or a
sulfur atom, respectively. The term "arylsulfonyl" refers to an
aryl group attached to the remainder of the molecule via an
SO.sub.2 group, and the term "sulfhydryl" refers to an SH
group.
[0108] In general, an "acyl substituent" is also selected from the
group set forth above. As used herein, the term "acyl substituent"
refers to groups attached to, and fulfilling the valence of a
carbonyl carbon that is either directly or indirectly attached to
the polycyclic nucleus of the compounds of the present
invention.
[0109] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of substituted or unsubstituted "alkyl" and
substituted or unsubstituted "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like. The heteroatoms and carbon atoms of
the cyclic structures are optionally oxidized.
[0110] The terms "halo" or "halogen," by themselves or as part of
another substituent, mean, unless otherwise stated, a fluorine,
chlorine, bromine, or iodine atom. Additionally, terms such as
"haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.
For example, the term "halo(C.sub.1-C.sub.4)alkyl" includes, but is
not limited to, trifluoromethyl, 2,2,2-trifluoroethyl,
4-chlorobutyl, 3-bromopropyl, and the like.
[0111] The term "aryl" means, unless otherwise stated, a
substituted or unsubstituted polyunsaturated, aromatic, hydrocarbon
substituent which can be a single ring or multiple rings
(preferably from 1 to 3 rings) which are fused together or linked
covalently. The term "heteroaryl" refers to aryl groups (or rings)
that contain from one to four heteroatoms selected from N, O, and
S, wherein the nitrogen, carbon and sulfur atoms are optionally
oxidized, and the nitrogen atom(s) are optionally quaternized. A
heteroaryl group can be attached to the remainder of the molecule
through a heteroatom. Non-limiting examples of aryl and heteroaryl
groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,
1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,
4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,
2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl,
3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,
2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,
2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.
Substituents for each of the above noted aryl and heteroaryl ring
systems are selected from the group of acceptable substituents
described below. "Aryl" and "heteroaryl" also encompass ring
systems in which one or more non-aromatic ring systems are fused,
or otherwise bound, to an aryl or heteroaryl system.
[0112] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0113] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") include both substituted and unsubstituted
forms of the indicated radical. Preferred substituents for each
type of radical are provided below.
[0114] Substituents for the alkyl, and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generally referred to as "alkyl substituents" and "heteroalkyl
substituents," respectively, and they can be one or more of a
variety of groups selected from, but not limited to: --OR', .dbd.O,
.dbd.NR', .dbd.NOR', --NR'R'', --SR', -halogen, --SiR'R''R''',
--OC(.dbd.O)R', --C(.dbd.O)R', --CO.sub.2R', --CONR'R'',
--OC(.dbd.O)NR'R'', --NR''C(.dbd.O)R', --NR'C(.dbd.O)NR''R''',
--NR''C(.dbd.O).sub.2R', --NRC(NR'R''R''').dbd.NR'''',
--NRC(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2 m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R''''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5, 6, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(.dbd.O)CH.sub.3, --C(.dbd.O)CF.sub.3,
--C(.dbd.O)CH.sub.2OCH.sub.3, and the like).
[0115] Similar to the substituents described for the alkyl radical,
the aryl substituents and heteroaryl substituents are generally
referred to as "aryl substituents" and "heteroaryl substituents,"
respectively and are varied and selected from, for example:
halogen, --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R'', --OC(.dbd.O)R', --C(.dbd.O)R',
--CO.sub.2R', --C(.dbd.O)NR'R'', --OC(.dbd.O)NR'R'',
--NR''C(.dbd.O)R', --NR'C(.dbd.O)NR''R''', --NR''CO.sub.2R',
--NRC(NR'R'').dbd.NR''', --S(.dbd.O)R', --S(.dbd.O).sub.2R',
--S(.dbd.O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2, --R',
--N.sub.3, --CH(Ph).sub.2, fluoro(C.sub.1-C.sub.4)alkoxy, and
fluoro(C.sub.1-C.sub.4)alkyl, in a number ranging from zero to the
total number of open valences on the aromatic ring system; and
where R', R'', R''' and R'''' are preferably independently selected
from hydrogen, (C.sub.1-C.sub.8)alkyl and heteroalkyl,
unsubstituted aryl and heteroaryl, (unsubstituted
aryl)-(C.sub.1-C.sub.4)alkyl, and (unsubstituted
aryl)oxy-(C.sub.1-C.sub.4)alkyl. When a compound of the invention
includes more than one R group, for example, each of the R groups
is independently selected as are each R', R'', R''' and R''''
groups when more than one of these groups is present.
[0116] Two of the aryl substituents on adjacent atoms of the aryl
or heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.sX(CR''R''').sub.d--, where s and d are independently
integers of from 0 to 3, and X is --O--, --NR'--, --S--,
--S(.dbd.O)--, --S(.dbd.O).sub.2--, or --S(.dbd.O).sub.2NR'--. The
substituents R, R', R'' and R' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6) alkyl.
[0117] As used herein, the term "diphosphate" includes but is not
limited to an ester of phosphoric acid containing two phosphate
groups. The term "triphosphate" includes but is not limited to an
ester of phosphoric acid containing three phosphate groups.
[0118] As used herein, the term "heteroatom" includes oxygen (O),
nitrogen (N), sulfur (S) and silicon (Si).
[0119] The symbol "R" is a general abbreviation that represents a
substituent group that is selected from substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, and substituted or unsubstituted heterocyclyl groups,
unless the context indicates otherwise.
[0120] Various aspects of the invention are described in further
detail in the following subsections.
Anti-Mesothelin Antibodies Having Particular Functional
Properties
[0121] The antibodies of this disclosure are characterized by
particular functional features or properties. For example, they
specifically bind to human mesothelin, such as human mesothelin
expressed on the cell surface. Preferably, an antibody of this
disclosure binds to human mesothelin with high affinity, for
example with a K.sub.D of 1.times.10.sup.-7 M or less, more
preferably with a K.sub.D of 5.times.10.sup.-8 M or less and even
more preferably with a K.sub.D of 1.times.10.sup.-8 M or less. An
anti-mesothelin antibody of this disclosure binds to human
mesothelin and preferably exhibits one or more of the following
properties: [0122] (a) binds to human mesothelin with a K.sub.D of
1.times.10.sup.-8 M or less; [0123] (b) is internalized by
mesothelin-expressing cells; [0124] (c) inhibits binding of
mesothelin to ovarian cancer antigen CA125; [0125] (d) exhibits
antibody dependent cellular cytotoxicity (ADCC) against
mesothelin-expressing cells; and [0126] (e) inhibits growth of
mesothelin-expressing cells in vivo when conjugated to a
cytotoxin.
[0127] Preferably, an antibody of this disclosure binds to a
mesothelin protein with a K.sub.D of 5.times.10.sup.-8 M or less,
binds to a mesothelin protein with a K.sub.D of 3.times.10.sup.-8 M
or less, binds to a mesothelin protein with a K.sub.D of
1.times.10.sup.-8 M or less, binds to a mesothelin protein with a
K.sub.D of 7.times.10.sup.-9 M or less, binds to a mesothelin
protein with a K.sub.D of 6.times.10.sup.-9 M or less or binds to a
mesothelin protein with a K.sub.D of 5.times.10.sup.-9 M or less.
The binding affinity of the antibody for mesothelin can be
evaluated, for example, by standard BIACORE analysis. (see e.g.,
Example 3B).
[0128] Preferred antibodies of the invention are human monoclonal
antibodies. Additionally or alternatively, the antibodies can be,
for example, chimeric or humanized monoclonal antibodies.
Monoclonal Antibodies 3C10, 6A4 and 7B1
[0129] Preferred antibodies of this disclosure are the human
monoclonal antibodies 3C10, 6A4 and 7B1, isolated and structurally
characterized as described in Examples 1 and 2. The V.sub.H amino
acid sequences of 3C10, 6A4 and 7B1 are shown in SEQ ID NOs: 19, 20
and 21, respectively. The V.sub.L amino acid sequences of 3C10, 6A4
and 7B1 are shown in SEQ ID NOs: 22, 23 and 24, respectively.
[0130] In another aspect, this disclosure provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of
3C10, 6A4 or 7B1, or combinations thereof. The amino acid sequences
of the V.sub.H CDR1s of 3C10, 6A4 and 7B1 are shown in SEQ ID NOs:
1-3, respectively. The amino acid sequences of the V.sub.H CDR2s of
3C10, 6A4 and 7B1 are shown in SEQ ID NOs: 4-6, respectively. The
amino acid sequences of the V.sub.H CDR3s of 3C10, 6A4 and 7B1 are
shown in SEQ ID NOs: 7-9, respectively. The amino acid sequences of
the V.sub.L CDR1s of 3C10, 6A4 and 7B1 are shown in SEQ ID NOs:
10-12, respectively. The amino acid sequences of the V.sub.L CDR2s
of 3C10, 6A4 and 7B1 are shown in SEQ ID NOs: 13-15. The amino acid
sequences of the V.sub.L CDR3s of 3C10, 6A4 and 7B1 are shown in
SEQ ID NOs: 16-18, respectively. The CDR regions are delineated
using the Kabat system (Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, US Department of
Health and Human Services, NIH Publication No. 91-3242, hereinafter
"Kabat '242").
[0131] Given that each of these antibodies can bind to human
mesothelin and that antigen-binding specificity is provided
primarily by the CDR1, CDR2, and CDR3 regions, the V.sub.H CDR1,
CDR2, and CDR3 sequences and V.sub.L CDR1, CDR2, and CDR3 sequences
can be "mixed and matched" (i.e., CDRs from different antibodies
can be mixed and match, although each antibody must contain a
V.sub.H CDR1, CDR2, and CDR3 and a V.sub.L CDR1, CDR2, and CDR3) to
create other anti-mesothelin binding molecules of this disclosure.
Preferably, when V.sub.H CDR sequences are mixed and matched, the
CDR1, CDR2 and/or CDR3 sequence from a particular V.sub.H sequence
is replaced with a structurally similar CDR sequence(s). Likewise,
when V.sub.L CDR sequences are mixed and matched, the CDR1, CDR2
and/or CDR3 sequence from a particular V.sub.L sequence preferably
is replaced with a structurally similar CDR sequence(s). It will be
readily apparent to the ordinarily skilled artisan that novel
V.sub.H and V.sub.L sequences can be created by substituting one or
more V.sub.H and/or V.sub.L CDR region sequences with structurally
similar sequences from the CDR sequences disclosed herein for
monoclonal antibodies 3C10, 6A4 and 7B1.
[0132] Accordingly, in another aspect, this disclosure provides an
isolated monoclonal antibody, or antigen binding portion thereof
comprising: [0133] (a) a heavy chain variable region CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-3; [0134] (b) a heavy chain variable
region CDR2 comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 4-6; [0135] (c) a heavy chain
variable region CDR3 comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 7-9; [0136] (d) a light
chain variable region CDR1 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 10-12; [0137] (e)
a light chain variable region CDR2 comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 13-15;
and [0138] (f) a light chain variable region CDR3 comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 16-18; wherein the antibody specifically binds human
mesothelin.
[0139] In one preferred embodiment, the antibody is according to
Embodiment A. In another preferred embodiment, the antibody is
according to Embodiment B. In yet another preferred embodiment, the
antibody is according to Embodiment C.
[0140] It is well known in the art that the CDR3 domain,
independently from the CDR1 and/or CDR2 domain(s), alone can
determine the binding specificity of an antibody for a cognate
antigen and that multiple antibodies can predictably be generated
having the same binding specificity based on a common CDR3
sequence.
[0141] Accordingly, the present disclosure provides monoclonal
antibodies comprising one or more heavy and/or light chain CDR3
domains from an antibody derived from a human or non-human animal,
wherein the monoclonal antibody is capable of specifically binding
to human mesothelin. Within certain aspects, the present disclosure
provides monoclonal antibodies comprising one or more heavy and/or
light chain CDR3 domain from a non-human antibody, such as a mouse
or rat antibody, wherein the monoclonal antibody is capable of
specifically binding to mesothelin. Within some embodiments, such
inventive antibodies comprising one or more heavy and/or light
chain CDR3 domain from a non-human antibody (a) are capable of
competing for binding with; (b) retain the functional
characteristics of; (c) bind to the same epitope as; and/or (d)
have a similar binding affinity as the corresponding parental
non-human antibody.
[0142] Within other aspects, the present disclosure provides
monoclonal antibodies comprising one or more heavy and/or light
chain CDR3 domain from a human antibody, such as, for example, a
human antibody obtained from a non-human animal, wherein the human
antibody is capable of specifically binding to human mesothelin.
Within other aspects, the present disclosure provides monoclonal
antibodies comprising one or more heavy and/or light chain CDR3
domain from a first human antibody, such as, for example, a human
antibody obtained from a non-human animal, wherein the first human
antibody is capable of specifically binding to human mesothelin and
wherein the CDR3 domain from the first human antibody replaces a
CDR3 domain in a human antibody that is lacking binding specificity
for mesothelin to generate a second human antibody that is capable
of specifically binding to human mesothelin. Within some
embodiments, such inventive antibodies comprising one or more heavy
and/or light chain CDR3 domain from the first human antibody (a)
are capable of competing for binding with; (b) retain the
functional characteristics; (c) bind to the same epitope; and/or
(d) have a similar binding affinity as the corresponding parental
first human antibody.
Antibodies Having Particular Germline Sequences
[0143] In certain embodiments, an antibody of this disclosure
comprises a V.sub.H from a particular germline heavy chain
immunoglobulin gene and/or a V.sub.L from a particular germline
light chain immunoglobulin gene.
[0144] For example, in a preferred embodiment, this disclosure
provides an isolated monoclonal antibody, or an antigen-binding
portion thereof, comprising a V.sub.H that is the product of or
derived from a human V.sub.H 3-33 gene or a human V.sub.H 3-7 gene,
wherein the antibody specifically binds human mesothelin. In
another preferred embodiment, this disclosure provides an isolated
monoclonal antibody, or an antigen-binding portion thereof;
comprising a V.sub.L that is the product of or derived from a human
V.sub.K L6 gene or a human V.sub.K A27 gene, wherein the antibody
specifically binds human mesothelin. In yet another preferred
embodiment, this disclosure provides an isolated monoclonal
antibody, or antigen-binding portion thereof, wherein the antibody
comprises a V.sub.H that is the product of or derived from a human
V.sub.H 3-33 gene and comprises a V.sub.L that is the product of or
derived from a human V.sub.K L6 gene, wherein the antibody
specifically binds human mesothelin. In yet another preferred
embodiment, this disclosure provides an isolated monoclonal
antibody, or antigen-binding portion thereof, wherein the antibody
comprises a V.sub.H that is the product of or derived from a human
V.sub.H 3-7 gene and comprises a V.sub.L that is the product of or
derived from a human V.sub.K A27 gene, wherein the antibody
specifically binds human mesothelin.
[0145] Such antibodies also may possess one or more of the
functional characteristics described in detail above, such as high
affinity binding to human mesothelin, internalization by
mesothelin-expressing cells, inhibition of binding of mesothelin to
CA125, the ability to mediate ADCC against mesothelin-expressing
cells and/or the ability to inhibit tumor growth of
mesothelin-expressing tumor cells in vivo when conjugated to a
cytotoxin.
[0146] Examples of antibodies having V.sub.H and V.sub.L of V.sub.H
3-33 and V.sub.K L6, respectively, are the 3C10 and 6A4 antibodies.
An example of an antibody having V.sub.H and V.sub.L of V.sub.H 3-7
and V.sub.K A27, respectively, is the 7B1 antibody.
[0147] As used herein, a human antibody comprises heavy or light
chain variable regions that is "the product of" or "derived from" a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic
mouse carrying human immunoglobulin genes with the antigen of
interest or screening a human immunoglobulin gene library displayed
on phage with the antigen of interest. A human antibody that is
"the product of" or "derived from" a human germline immunoglobulin
sequence can be identified as such by comparing the amino acid
sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline
immunoglobulin sequence that is closest in sequence (i.e., greatest
% identity) to the sequence of the human antibody. A human antibody
that is "the product of" or "derived from" a particular human
germline immunoglobulin sequence may contain amino acid differences
as compared to the germline sequence, due to, for example,
naturally-occurring somatic mutations or intentional introduction
of site-directed mutation. However, a selected human antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody
as being human when compared to the germline immunoglobulin amino
acid sequences of other species (e.g., murine germline sequences).
In certain cases, a human antibody may be at least 95%, or even at
least 96%, 97%, 98%, or 99% identical in amino acid sequence to the
amino acid sequence encoded by the germline immunoglobulin gene.
Typically, a human antibody derived from a particular human
germline sequence will display no more than 10 amino acid
differences from the amino acid sequence encoded by the human
germline immunoglobulin gene. In certain cases, the human antibody
may display no more than 5, or even no more than 4, 3, 2, or 1
amino acid difference from the amino acid sequence encoded by the
germline immunoglobulin gene.
Homologous Antibodies
[0148] In yet another embodiment, an antibody of this disclosure
comprises heavy and light chain variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the preferred antibodies described herein, and wherein the
antibodies retain the desired functional properties of the
anti-mesothelin antibodies of this disclosure.
[0149] For example, this disclosure provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising a V.sub.H
and a V.sub.L, wherein: (a) the V.sub.H comprises an amino acid
sequence that is at least 80% homologous to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 19-21; (b) the
V.sub.L comprises an amino acid sequence that is at least 80%
homologous to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 22-24; and (c) the antibody specifically
binds to human mesothelin.
[0150] In other embodiments, the V.sub.H and/or V.sub.L amino acid
sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to
the sequences set forth above. An antibody having V.sub.H and
V.sub.L regions having high (i.e., 80% or greater) homology to the
V.sub.H and V.sub.L regions of the sequences set forth above, can
be obtained by mutagenesis (e.g., site-directed or PCR-mediated
mutagenesis) of nucleic acid molecules encoding SEQ ID NOs: 25-27
or 28-30, followed by testing of the encoded altered antibody for
retained function (i.e., the functions set forth above) using the
functional assays described herein.
[0151] The percent homology between two amino acid sequences is
equivalent to the percent identity between the two sequences. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions.times.100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, as described in the non-limiting examples
below.
[0152] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package, using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0153] The protein sequences of the present disclosure also can be
used as a "query sequence" to search against public databases to,
for example, to identify related sequences. Such searches can be
performed using the XBLAST program (version 2.0) of Altschul, et
al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain
amino acid sequences homologous to the antibody molecules of this
disclosure. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., XBLAST and NBLAST) are useful.
Antibodies with Conservative Modifications
[0154] In certain embodiments, an antibody of this disclosure
comprises a V.sub.H comprising CDR1, CDR2 and CDR3 sequences and a
V.sub.L comprising CDR1, CDR2 and CDR3 sequences, wherein one or
more of these CDR sequences comprise specified amino acid sequences
based on known anti-mesothelin antibodies, or conservative
modifications thereof, and wherein the antibodies retain the
desired functional properties of the anti-mesothelin antibodies of
this disclosure. It is understood in the art that certain
conservative sequence modification can be made which do not remove
antigen binding. Accordingly, this disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof, comprising
a V.sub.H comprising CDR1, CDR2, and CDR3 sequences and a V.sub.L
comprising CDR1, CDR2, and CDR3 sequences, wherein: (a) the V.sub.H
CDR3 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs: 7-9, and
conservative modifications thereof; (b) the V.sub.L CDR3 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequence of SEQ ID NOs: 16-18, and conservative
modifications thereof; and (c) the antibody specifically binds
human mesothelin.
[0155] In a preferred embodiment, the heavy chain variable region
CDR2 sequence comprises an amino acid sequence selected from the
group consisting of amino acid sequences of SEQ ID NOs: 4-6, and
conservative modifications thereof; and the light chain variable
region CDR2 sequence comprises an amino acid sequence selected from
the group consisting of amino acid sequences of SEQ ID NOs: 13-15,
and conservative modifications thereof. In another preferred
embodiment, the heavy chain variable region CDR1 sequence comprises
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOs: 1-3, and conservative modifications
thereof; and the light chain variable region CDR1 sequence
comprises an amino acid sequence selected from the group consisting
of amino acid sequences of SEQ ID NOs: 10-12, and conservative
modifications thereof.
[0156] The term "conservative sequence modifications" refers to
amino acid modifications that do not significantly affect or alter
the binding characteristics of the antibody containing the amino
acid sequence. Such conservative modifications include amino acid
substitutions, additions and deletions. Modifications can be
introduced into an antibody by standard techniques known in the
art, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Conservative amino acid substitutions are ones in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of this disclosure can be replaced with other amino acid residues
from the same side chain family and the altered antibody can be
tested for retained function using the functional assays described
herein.
Antibodies that Bind to the Same Epitope as Anti-Mesothelin
Antibodies
[0157] In another embodiment, this disclosure provides antibodies
that bind an epitope on mesothelin recognized by any of the
anti-mesothelin monoclonal antibodies of this disclosure (i.e.,
antibodies that have the ability to cross-compete for binding to
human mesothelin with any of the monoclonal antibodies of this
disclosure). In preferred embodiments, the reference antibody for
cross-competition studies can be the monoclonal antibody 3C10
(having V.sub.H and V.sub.L sequences as shown in SEQ ID NOs: 19
and 22, respectively), or the monoclonal antibody 6A4 (having
V.sub.H and V.sub.L sequences as shown in SEQ ID NOs: 20 and 23,
respectively), or the monoclonal antibody 7B1 (having V.sub.H and
V.sub.L sequences as shown in SEQ ID NOs: 21 and 24,
respectively).
[0158] Such cross-competing antibodies can be identified based on
their ability to cross-compete with 3C10, 6A4 or 7B1 in standard
mesothelin binding assays, such as ELISA or BIAcore analysis. In a
preferred embodiment, the antibody that binds to the same epitope
on human mesothelin as is recognized by 3C10, 6A4 or 7B 1 is a
human monoclonal antibody. Such human monoclonal antibodies can be
prepared and isolated as described in the Examples.
Engineered and Modified Antibodies
[0159] An antibody of the invention further can be prepared using
an antibody having one or more known anti-mesothelin antibody
V.sub.H and/or V.sub.L sequences as starting material to engineer a
modified antibody having altered properties as compared to the
starting antibody. One or more amino acids within one or both of
V.sub.H and V.sub.L, within one or more CDR regions and/or within
one or more framework regions can be modified. Additionally or
alternatively, residues within the constant region(s) can be
modified to alter the effector function(s) of the antibody.
[0160] In certain embodiments, CDR grafting can be used to engineer
variable regions of antibodies. Antibodies interact with target
antigens predominantly through amino acid residues in their CDRs.
For this reason, the amino acid sequences within CDRs are more
diverse between individual antibodies than sequences outside of
CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann, L. et al. (1998)
Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525;
Queen, C. et al. (1989) Proc. Natl. Acad. See. USA. 86:10029-10033;
U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,762 and 6,180,370 to Queen et al.)
[0161] Accordingly, another embodiment of this disclosure pertains
to an isolated monoclonal antibody, or antigen binding portion
thereof, comprising a V.sub.H comprising CDR1, CDR2, and CDR3
sequences comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-3, SEQ ID NOs: 4-6, and SEQ ID NOs:
7-9, respectively, and a V.sub.L comprising CDR1, CDR2, and CDR3
sequences comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 10-12, SEQ ID NOs: 13-15, and SEQ ID NOs:
16-18, respectively. Thus, such antibodies contain the V.sub.H and
V.sub.L CDR sequences of monoclonal antibodies 3C10, 6A4 or 7B 1
yet may contain different framework sequences from these
antibodies.
[0162] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human
V.sub.H and V.sub.L genes can be found in the "VBase" human
germline sequence database, as well as in Kabat '242; Tomlinson, I.
M., et al. (1992) J. Mol. Biol. 227:776-798; and Cox, J. P. L. et
al. (1994) Eur. J. Immunol. 24:827-836; each of which is
incorporated herein by reference. As another example, the germline
DNA sequences for human V.sub.H and V.sub.L genes can be found in
the Genbank database. For example, the following heavy chain
germline sequences found in the HCo7 HuMAb mouse are available in
the accompanying Genbank accession numbers: 1-69 (NG.sub.--0010109,
NT.sub.--024637 and BC070333), 3-33 (NG.sub.--0010109 and
NT.sub.--024637) and 3-7 (NG.sub.--0010109 and NT.sub.--024637). As
another example, the following heavy chain germline sequences found
in the HCo12 mouse of the HuMAb Mouse type are available in the
accompanying Genbank accession numbers: 1-69 (NG.sub.--0010109,
NT.sub.--024637 and BC070333), 5-51 (NG.sub.--0010109 and
NT.sub.--024637), 4-34 (NG.sub.--0010109 and NT.sub.--024637),
3-30.3 (CAJ556644) and (AJ406678). Yet another source of human
heavy and light chain germline sequences is the database of human
immunoglobulin genes available from IMGT.
[0163] Antibody protein sequences are compared against a compiled
protein sequence database using one of the sequence similarity
searching methods called the Gapped BLAST (Altschul et al. (1997)
Nucleic Acids Research 25:3389-3402), which is well known to those
skilled in the art. BLAST is a heuristic algorithm in that a
statistically significant alignment between the antibody sequence
and the database sequence is likely to contain high-scoring segment
pairs (HSP) of aligned words. Segment pairs whose scores cannot be
improved by extension or trimming is called a hit. Briefly, the
nucleotide sequences of VBASE origin are translated and the region
between and including FR1 through FR3 framework region is retained.
The database sequences have an average length of 98 residues.
Duplicate sequences which are exact matches over the entire length
of the protein are removed. A BLAST search for proteins using the
program blastp with default, standard parameters except the low
complexity filter, which is turned off, and the substitution matrix
of BLOSUM62, filters for top 5 hits yielding sequence matches. The
nucleotide sequences are translated in all six frames and the frame
with no stop codons in the matching segment of the database
sequence is considered the potential hit. This is in turn confirmed
using the BLAST program tblastx, which translates the antibody
sequence in all six frames and compares those translations to the
VBASE nucleotide sequences dynamically translated in all six
frames. Other human germline sequence databases, such as that
available from IMGT, can be searched similarly to VBASE as
described above.
[0164] The identities are exact amino acid matches between the
antibody sequence and the protein database over the entire length
of the sequence. The positives (identities+substitution match) are
not identical but amino acid substitutions guided by the BLOSUM62
substitution matrix. If the antibody sequence matches two of the
database sequences with same identity, the hit with most positives
would be decided to be the matching sequence hit.
[0165] Preferred framework sequences for use in the antibodies of
this disclosure are those structurally similar to the framework
sequences used by selected antibodies of this disclosure, e.g.,
similar to the V.sub.H 3-33 (SEQ ID NO: 31) or V.sub.H 3-7 (SEQ ID
NO: 32) framework sequences and/or the V.sub.K L6 (SEQ ID NO: 33)
or V.sub.K A27 (SEQ ID NO: 34) framework sequences. The V.sub.H
CDR1, CDR2, and CDR3 sequences and the V.sub.K CDR1, CDR2, and CDR3
sequences can be grafted onto framework regions that have the
identical sequence as that found in the germline immunoglobulin
gene from which the framework sequence derives, or the CDR
sequences can be grafted onto framework regions that contain one or
more mutations as compared to the germline sequences. For example,
it has been found that in certain instances it is beneficial to
mutate residues within the framework regions to maintain or enhance
the antigen binding ability (see e.g., U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
[0166] Another type of variable region modification is the mutation
of amino acid residues within the V.sub.H and/or V.sub.K CDR1, CDR2
and/or CDR3 regions to improve one or more binding properties
(e.g., affinity). Site-directed mutagenesis or PCR-mediated
mutagenesis can be performed to introduce the mutation(s) and the
effect on antibody binding, or other functional property of
interest, can be evaluated in in vitro or in vivo assays as
described herein. Preferably conservative modifications are
introduced. The mutations may be amino acid substitutions,
additions or deletions, but are preferably substitutions. Moreover,
typically no more than one, two, three, four or five residues
within a CDR region are altered.
[0167] Accordingly, in another embodiment, the instant disclosure
provides isolated anti-mesothelin monoclonal antibodies, or antigen
binding portions thereof, comprising: (a) a V.sub.H CDR1 region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-3, or an amino acid sequence having
one, two, three, four or five amino acid substitutions, deletions
or additions as compared to SEQ ID NOs: 1-3; (b) a V.sub.H CDR2
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 4-6, or an amino acid sequence having
one, two, three, four or five amino acid substitutions, deletions
or additions as compared to SEQ ID NOs: 4-6; (c) a V.sub.H CDR3
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 7-9, or an amino acid sequence having
one, two, three, four or five amino acid substitutions, deletions
or additions as compared to SEQ ID NOs: 7-9; (d) a V.sub.L CDR1
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 10-12, or an amino acid sequence having
one, two, three, four or five amino acid substitutions, deletions
or additions as compared to SEQ ID NOs: 10-12; (e) a V.sub.L CDR2
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 13-15, or an amino acid sequence having
one, two, three, four or five amino acid substitutions, deletions
or additions as compared to SEQ ID NOs: 13-15; and (f) a V.sub.L
CDR3 region comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 16-18, or an amino acid sequence
having one, two, three, four or five amino acid substitutions,
deletions or additions as compared to SEQ ID NOs: 16-18.
[0168] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. One approach is to
"backmutate" one or more framework residues to the corresponding
germline sequence. More specifically, an antibody that has
undergone somatic mutation may contain framework residues that
differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived.
[0169] For example, Table A shows regions where a framework region
amino acid position (using the Kabat numbering system) differs from
the germline and how this position can be backmutated to the
germline by the indicated substitutions:
TABLE-US-00001 TABLE A Exemplary Backmutations Region Framework
Amino Acid Position Backmutation 3C10 V.sub.H 3 Y3Q 3C10 V.sub.H 27
I27F 3C10 V.sub.H 82 L82Q 6A4 V.sub.H 3 H3Q 6A4 V.sub.H 23 V23A 6A4
V.sub.H 27 I27F 6A4 V.sub.H 30 R30S 6A4 V.sub.H 93 I93V 7B1 V.sub.H
3 H3Q 7B1 V.sub.H 41 Q41P 7B1 V.sub.H 80 S80Y
[0170] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in US 2003/0153043 by Carr et al.
[0171] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties, such as serum
half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties. Each of these embodiments is described in further
detail below. The numbering of residues in the Fc region is that of
the EU index of Kabat.
[0172] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is altered
(i.e., increased or decreased), as described in U.S. Pat. No.
5,677,425 by Bodmer et al. Such alteration can facilitate assembly
of the light and heavy chains or increase or decrease the stability
of the antibody.
[0173] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding, as
described in U.S. Pat. No. 6,165,745 by Ward et al.
[0174] In another embodiment, the antibody is modified to increase
its biological half life. For example, one or more of the following
mutations can be introduced: T252L, T254S, T256F, as described in
U.S. Pat. No. 6,277,375 to Ward. Alternatively, the antibody can be
altered within the CH1 or C.sub.L region to contain a salvage
receptor binding epitope taken from two loops of a CH2 domain of an
Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and
6,121,022 by Presta et al.
[0175] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter its effector function(s). For example, one or
more of amino acid residues 234, 235, 236, 237, 297, 318, 320 and
322 can be replaced with a different amino acid residue such that
the antibody has an altered affinity for an effector ligand--e.g.,
an Fc receptor or the C1 component of complement--but retains the
antigen-binding ability of the parent antibody, as described in
U.S. Pat. Nos. 5,624,821 and 5,648,260, both to Winter et al.
[0176] In another example, one or more of amino acid residues 329,
331 and 322 can be replaced such that the antibody has altered C1q
binding and/or reduced or abolished complement dependent
cytotoxicity (CDC), as described in U.S. Pat. No. 6,194,551 by
Idusogie et al.
[0177] In another example, one or more amino acid residues within
amino acid positions 231 and 239 are altered to thereby alter the
ability of the antibody to fix complement. This approach is
described further in WO 94/29351 by Bodmer et al.
[0178] In yet another example, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids at the following positions: 238, 239, 248, 249, 252, 254,
255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283,
285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,
307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,
334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,
414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is
described further in WO 00/42072 by Presta. Moreover, the binding
sites on human IgG1 for Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII
and FcRn have been mapped and variants with improved binding have
been described (see Shields, R. L. et al. (2001) J. Biol. Chem.
276:6591-6604). Specific mutations at positions 256, 290, 298, 333,
334 and 339 were shown to improve binding to Fc.gamma.RIII.
Additionally, the following combination mutants were shown to
improve Fc.gamma.RIII binding: T256A/S298A, S298A/E333A,
S298A/K224A and S298A/E333A/K334A.
[0179] In still another embodiment, the C-terminal end of an
antibody of the present invention is modified by the introduction
of a cysteine residue, as described in PCT/US2008/073569, which is
incorporated by reference in its entirety. Such modifications
include, but are not limited to, the replacement of an existing
amino acid residue at or near the C-terminus of a full-length heavy
chain sequence, as well as the introduction of a
cysteine-containing extension to the c-terminus of a full-length
heavy chain sequence. In preferred embodiments, the
cysteine-containing extension comprises the sequence
cysteine-alanine-alanine (from N-terminal to C-terminal).
[0180] In preferred embodiments the presence of such C-terminal
cysteine modifications provide a location for conjugation of a
partner molecule, such as a therapeutic agent or a marker molecule.
In particular, the presence of a reactive thiol group, due to the
C-terminal cysteine modification, can be used to conjugate a
partner molecule employing the disulfide linkers or a
sulfhydryl-reactive maleimide group. Conjugation of the antibody to
a partner molecule in this manner allows for increased control over
the specific site of attachment. Furthermore, by introducing the
site of attachment at or near the C-terminus, conjugation can be
optimized such that it reduces or eliminates interference with the
antibody's functional properties, and allows for simplified
analysis and quality control of conjugate preparations.
[0181] In still another embodiment, the glycosylation of an
antibody is modified by eliminating glycosylation (aglycosylation)
or altering the site(s) of glycosylation, to increase its affinity
for its antigen. For example, one or more amino acid substitutions
can be made that result in elimination of one or more variable
region framework glycosylation sites. Such aglycosylation may
increase the affinity of the antibody for antigen. See U.S. Pat.
Nos. 5,714,350 and 6,350,861 to Co et al. Additional approaches for
altering glycosylation are described in U.S. Pat. No. 7,214,775 to
Hanai et al.; U.S. Pat. No. 6,737,056 to Presta; US 20070020260 to
Presta; WO 2007/084926 to Dickey et al.; WO 2006/089294 to Zhu et
al.; and WO 2007/055916 to Ravetch et al.; each of which is
incorporated by reference in its entirety.
[0182] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. For example, the cell
lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene,
FUT8 (alpha (1,6) fucosyltransferase), such that antibodies
expressed in these cell lines lack fucose on their carbohydrates.
See US 2004/0110704 by Yamane et al. and Yamane-Ohnuki et al.
(2004) Biotechnol Bioeng 87:614-22. As another example, EP
1,176,195 by Hanai et al. describes a cell line with a functionally
disrupted FUT8 gene, which encodes a fucosyl transferase, such that
antibodies expressed in such a cell line exhibit hypofucosylation.
Hanai et al. also describe cell lines which have a low enzyme
activity for adding fucose to the N-acetylglucosamine that binds to
the Fc region of the antibody. WO 03/035835 by Presta describes a
variant CHO cell line, Lec13 cells, with reduced ability to attach
fucose to Asn(297)-linked carbohydrates, also resulting in
hypofucosylation (see also Shields, R. L. et al. (2002) J. Biol.
Chem. 277:26733-26740). WO 99/54342 by Umana et al. describes cell
lines engineered to express glycoprotein-modifying glycosyl
transferases, such that antibodies expressed by these cell lines
exhibit increased bisecting GlcNac structures and increased ADCC
activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech. 17:176-180). Alternatively, the fucose residues of the
antibody may be cleaved off using a fucosidase enzyme (Tarentino,
A. L. et al. (1975) Biochem. 14:5516-23).
[0183] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, wherein that alteration
relates to the level of sialyation of the antibody. Such
alterations are described in WO 2007/084926 to Dickey et al, and WO
2007/055916 to Ravetch et al., both of which are incoporated by
reference. For example, one may employ an enzymatic reaction with
sialidase, such as described in the U.S. Pat. No. 5,831,077, which
is hereby incorporated by reference. Other examples of suitable
enzymes are neuraminidase and N-Glycosidase F, as described in
Schloemer et al., J. Virology, 15(4), 882-893 (1975) and in
Leibiger et al., Biochem J., 338, 529-538 (1999), respectively.
Alternatively, one may employ methods to increase the level of
sialyation, such as by employing sialytransferase enzymes, as
described in Basset et al., Scandinavian Journal of Immunology,
51(3), 307-311 (2000).
[0184] Another modification of the antibodies herein that is
contemplated by the invention is pegylation. An antibody can be
pegylated to, for example, increase its biological (e.g., serum)
half life. To pegylate an antibody, the antibody, or fragment
thereof, typically is reacted with a polyethylene glycol (PEG)
derivative, such as a reactive ester or aldehyde derivative of PEG.
Or, the pegylation is carried out via an acylation reaction or an
alkylation reaction with a reactive PEG molecule (or an analogous
reactive water-soluble polymer). As used herein, the term
"polyethylene glycol" encompasses any of the forms of PEG that have
been used to derivatize other proteins, such as mono (C1-C10)
alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. The antibody to be pegylated can be an
aglycosylated antibody. Methods for pegylating proteins are known
in the art and can be applied to the antibodies of the invention.
See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384
by Ishikawa et al.
Antibody Fragments and Antibody Mimetics
[0185] The instant invention is not limited to traditional
antibodies and may be practiced through the use of antibody
fragments and antibody mimetics. A wide variety of antibody
fragment and antibody mimetic technologies have now been developed
and are widely known in the art.
[0186] Domain Antibodies (dAbs) are the smallest functional binding
units of antibodies, corresponding to the variable regions of
either the heavy (VH) or light (VL) chains of human antibodies.
Domain Antibodies have a molecular weight of approximately 13 kDa.
Domantis has developed a series of large and highly functional
libraries of fully human VH and VL dAbs (more than ten billion
different sequences in each library), and uses these libraries to
select dAbs that are specific to therapeutic targets. In contrast
to many conventional antibodies, Domain Antibodies are well
expressed in bacterial, yeast, and mammalian cell systems. Further
details of domain antibodies and methods of production thereof may
be obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915;
6,593,081; 6,172,197; and 6,696,245; US 2004/0110941; EP 1433846,
0368684 and 0616640; WO 2005/035572, 2004/101790, 2004/081026,
2004/058821, 2004/003019 and 2003/002609, each of which is herein
incorporated by reference in its entirety.
[0187] Nanobodies are antibody-derived therapeutic proteins that
contain the unique structural and functional properties of
naturally-occurring heavy-chain antibodies. These heavy-chain
antibodies contain a single variable domain (VHH) and two constant
domains (CH2 and CH3). Importantly, the cloned and isolated VHH
domain is a perfectly stable polypeptide harbouring the full
antigen-binding capacity of the original heavy-chain antibody.
Nanobodies have a high homology with the VH domains of human
antibodies and can be further humanized without any loss of
activity. Importantly, Nanobodies have a low immunogenic potential,
which has been confirmed in primate studies with Nanobody lead
compounds.
[0188] Nanobodies combine the advantages of conventional antibodies
with important features of small molecule drugs. Like conventional
antibodies, Nanobodies show high target specificity and affinity
and low inherent toxicity. However, like small molecule drugs they
can inhibit enzymes and readily access receptor clefts.
Furthermore, Nanobodies are extremely stable, can be administered
by means other than injection (see, e.g., WO 2004/041867,
incorporated herein by reference in its entirety) and are easy to
manufacture. Other advantages of Nanobodies include recognizing
uncommon or hidden epitopes as a result of their small size,
binding into cavities or active sites of protein targets with high
affinity and selectivity due to their unique 3-dimensional, drug
format flexibility, tailoring of half-life and ease and speed of
drug discovery.
[0189] Nanobodies are encoded by single genes and are efficiently
produced in almost all prokaryotic and eukaryotic hosts, e.g., E.
coli (see, e.g., U.S. Pat. No. 6,765,087, which is herein
incorporated by reference in its entirety), molds (for example
Aspergillus or Trichoderma) and yeast (for example Saccharomyces,
Kluyveromyces, Hansenula or Pichia) (see, e.g., U.S. Pat. No.
6,838,254, which is herein incorporated by reference in its
entirety).
[0190] The Nanoclone method (see, e.g., WO 06/079372, which is
herein incorporated by reference in its entirety) generates
Nanobodies against a desired target, based on automated
high-throughout selection of B-cells and could be used in the
context of the instant invention.
[0191] UniBodies are another antibody fragment technology, however
based upon the removal of the hinge region of IgG4 antibodies. The
deletion of the hinge region results in a molecule that is
essentially half the size of a traditional IgG4 antibody and has a
univalent binding region rather than the bivalent binding region of
an IgG4 antibody. Like IgG4 antibodies, Unibodies are inert and do
not interact with the immune system, which may be advantageous for
the treatment of diseases where an immune response is not desired.
UniBodies may function to inhibit or silence, but not kill, the
cells to which they are bound. Additionally, UniBody binding to
cancer cells does not stimulate them to proliferate. Furthermore,
because UniBodies are about smaller, they may show better
distribution over larger solid tumors with potentially advantageous
efficacy. UniBodies are cleared from the body at a similar rate to
whole IgG4 antibodies and are able to bind with a similar affinity
for their antigens. Further details of UniBodies may be obtained by
reference to WO 2007/059782, which is incorporated by reference in
its entirety.
[0192] Affibody molecules are affinity proteins based on a 58-amino
acid residue protein domain derived from a three helix bundle
IgG-binding domain of staphylococcal protein A. This domain has
been used as a scaffold for the construction of combinatorial
phagemid libraries, from which Affibody variants targeting the
desired molecules can be selected using phage display technology
(Nord et al., Nat Biotechnol 1997; 15:772-7; Ronmark et al., Eur J
Biochem 2002; 269:2647-55). The simple, robust structure and low
molecular weight (6 kDa) of Affibody molecules makes them suitable
for a wide variety of applications, for instance, as detection
reagents and inhibitors of receptor interactions. Further details
on Affibodies and their production are found in U.S. Pat. No.
5,831,012 which is incorporated by reference in its entirety.
Labelled Affibodies may also be useful in imaging applications for
determining abundance of isoforms.
[0193] DARPins (Designed Ankyrin Repeat Proteins) embody DRP
(Designed Repeat Protein) antibody mimetic technology developed to
exploit the binding abilities of non-antibody polypeptides. Repeat
proteins, such as ankyrin and leucine-rich repeat proteins, are
ubiquitous binding molecules that, unlike antibodies, occur intra-
and extracellularly. Their unique modular architecture features
repeating structural units (repeats) that stack together to form
elongated repeat domains displaying variable and modular
target-binding surfaces. Based on this modularity, combinatorial
libraries of polypeptides with highly diversified binding
specificities can be generated. This strategy includes the
consensus design of self-compatible repeats displaying variable
surface residues and their random assembly into repeat domains.
[0194] DARPins can be produced in bacterial expression systems at
very high yields and are among the most stable proteins known.
Highly specific, high-affinity DARPins that bind to a broad range
of target proteins, including human receptors, cytokines, kinases,
human proteases, viruses and membrane proteins, have been made,
including some having affinities in the single-digit nanomolar to
picomolar range.
[0195] DARPins have been used in a wide range of applications,
including ELISA, sandwich ELISA, flow cytometric analysis (FACS),
immunohistochemistry (IHC), chip applications, affinity
purification or Western blotting. DARPins are not only ideal to
block protein-protein interactions, but also to inhibit enzymes.
Very fast and specific enrichments on the tumor and very favorable
tumor to blood ratios make DARPins well suited for in vivo
diagnostics or therapeutic approaches. Additional information
regarding DARPins and other DRP technologies can be found in US
2004/0132028 and WO 02/20565, both of which are incorporated by
reference.
[0196] Anticalins are another antibody mimetic technology. However,
in this case the binding specificity is derived from lipocalins, a
family of low molecular weight proteins that are naturally and
abundantly expressed in human tissues and body fluids. Lipocalins
have evolved to perform a range of functions in vivo associated
with the physiological transport and storage of chemically
sensitive or insoluble compounds. Lipocalins have a robust
intrinsic structure comprising a highly conserved .beta.-barrel
which supports four loops at one terminus of the protein. These
loops form the entrance to a binding pocket and conformational
differences in this part of the molecule account for the variation
in binding specificity between individual lipocalins.
[0197] While the overall structure of hypervariable loops supported
by a conserved .beta.-sheet framework is reminiscent of
immunoglobulins, lipocalins differ considerably from antibodies in
terms of size, being composed of a single polypeptide chain of
160-180 amino acids, which is marginally larger than a single
immunoglobulin domain.
[0198] Lipocalins can be cloned and their loops subjected to
engineering in order to create Anticalins. Libraries of
structurally diverse Anticalins have been generated and Anticalin
display allows the selection and screening of binding function,
followed by the expression and production of soluble protein for
further analysis in prokaryotic or eukaryotic systems. Studies have
demonstrated that Anticalins can be developed that are specific for
virtually any human target protein and binding affinities in the
nanomolar or higher range can be obtained.
[0199] Anticalins can also be formatted as dual targeting proteins
(Duocalins). A Duocalin binds two separate therapeutic targets in
one easily produced monomeric protein using standard manufacturing
processes while retaining target specificity and affinity
regardless of the structural orientation of its two binding
domains.
[0200] Modulation of multiple targets through a single molecule is
particularly advantageous in diseases known to involve more than a
single causative factor. Moreover, bi- or multivalent binding
formats such as Duocalins have significant potential in targeting
cell surface molecules in disease, mediating agonistic effects on
signal transduction pathways or inducing enhanced internalization
effects via binding and clustering of cell surface receptors.
Furthermore, the high intrinsic stability of Duocalins is
comparable to monomeric Anticalins.
[0201] Additional information regarding Anticalins can be found in
U.S. Pat. No. 7,250,297 and WO 99/16873, both of which are hereby
incorporated by reference in their entirety.
[0202] Avimers are another type of antibody mimetic technology
useful in the context of the instant invention. Avimers are evolved
from a large family of human extracellular receptor domains by in
vitro exon shuffling and phage display, generating multidomain
proteins with binding and inhibitory properties. Linking multiple
independent binding domains has been shown to create avidity and
results in improved affinity and specificity compared to
conventional single-epitope binding proteins. Other potential
advantages include simple and efficient production of
multi-target-specific molecules in Escherichia coli, improved
thermostability and resistance to proteases. Avimers with
sub-nanomolar affinities have been obtained against a variety of
targets. Additional information regarding Avimers can be found in
US 2006/0286603, 2006/0234299, 2006/0223114, 2006/0177831,
2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932,
2005/0053973, 2005/0048512, 2004/0175756, all of which are hereby
incorporated by reference in their entirety.
[0203] Versabodies are another antibody mimetic technology that can
be used in the context of the instant invention. Versabodies are
small proteins of 3-5 kDa with >15% cysteines, which form a high
disulfide density scaffold replacing the hydrophobic core that
typical proteins have. This replacement results in a protein that
is smaller, is more hydrophilic (i.e., less prone to aggregation
and non-specific binding), is more resistant to proteases and heat,
and has a lower density of T-cell epitopes, because the residues
that contribute most to MHC presentation are hydrophobic. All four
of these properties are well-known to affect immunogenicity, and
together they are expected to cause a large decrease in
immunogenicity.
[0204] Given the structure of Versabodies, these antibody mimetics
offer a versatile format that includes multi-valency,
multi-specificity, a diversity of half-life mechanisms, tissue
targeting modules and the absence of the antibody Fc region.
Furthermore, Versabodies are manufactured in E. coli at high
yields, and because of their hydrophilicity and small size,
Versabodies are highly soluble and can be formulated to high
concentrations. Versabodies are exceptionally heat stable and offer
extended shelf-life. Additional information regarding Versabodies
can be found in US 2007/0191272, which is hereby incorporated by
reference in its entirety.
[0205] The above descriptions of antibody fragment and mimetic
technologies is not intended to be comprehensive. A variety of
additional technologies including alternative polypeptide-based
technologies, such as fusions of complementarity determining
regions as outlined in Qui et al., Nature Biotechnology, 25(8)
921-929 (2007), as well as nucleic acid-based technologies, such as
the RNA aptamer technologies described in U.S. Pat. Nos. 5,789,157;
5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526;
6,114,120; 6,261,774; and 6,387,620; all of which are hereby
incorporated by reference, could be used in the context of the
instant invention.
Antibody Physical Properties
[0206] Antibodies of this disclosure can be characterized by their
various physical properties, to detect and/or differentiate
different classes thereof.
[0207] Antibodies of the present disclosure may contain one or more
glycosylation sites in either the light or heavy chain variable
region. Such glycosylation sites may result in increased
immunogenicity of the antibody or an alteration of the pK of the
antibody due to altered antigen binding (Marshall et al (1972) Annu
Rev Biochem. 41:673-702; Gala and Morrison (2004) J Immunol
172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro
(2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature
316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).
Glycosylation has been known to occur at motifs containing an
N--X--S/T sequence. In some instances, it is preferred to have an
anti-mesothelin antibody that does not contain variable region
glycosylation. This can be achieved either by selecting antibodies
that do not contain the glycosylation motif in the variable region
or by mutating residues within the glycosylation region.
[0208] In a preferred embodiment, the antibodies of the present
disclosure do not contain asparagine isomerism sites. The
deamidation of asparagine may occur on N-G or D-G sequences and
result in the creation of an isoaspartic acid residue that
introduces a kink into the polypeptide chain and decreases its
stability (isoaspartic acid effect).
[0209] Each antibody will have a unique isoelectric point (pI),
which generally falls in the pH range between 6 and 9.5. The pI for
an IgG1 antibody typically falls within the pH range of 7-9.5 and
the pI for an IgG4 antibody typically falls within the pH range of
6-8. There is speculation that antibodies with a pI outside the
normal range may have some unfolding and instability under in vivo
conditions. Thus, it is preferred to have an anti-mesothelin
antibody that contains a pI value that falls in the normal range.
This can be achieved either by selecting antibodies with a pI in
the normal range or by mutating charged surface residues.
[0210] Each antibody will have a characteristic melting
temperature, with a higher melting temperature indicating greater
overall stability in vivo (Krishnamurthy R and Manning M C (2002)
Curr Pharm Biotechnol 3:361-71). Generally, it is preferred that
the T.sub.M1 (the temperature of initial unfolding) be greater than
60.degree. C., preferably greater than 65.degree. C., even more
preferably greater than 70.degree. C. The melting point of an
antibody can be measured using differential scanning calorimetry
(Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999)
Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002)
J. Chromatogr Sci 40:343-9).
[0211] In a preferred embodiment, antibodies are selected that do
not degrade rapidly. Degradation of an antibody can be measured
using capillary electrophoresis (CE) and MALDI-MS (Alexander A J
and Hughes D E (1995) Anal Chem 67:3626-32).
[0212] In another preferred embodiment, antibodies are selected
that have minimal aggregation effects, which can lead to the
triggering of an unwanted immune response and/or altered or
unfavorable pharmacokinetic properties. Generally, antibodies are
acceptable with aggregation of 25% or less, preferably 20% or less,
even more preferably 15% or less, even more preferably 10% or less
and even more preferably 5% or less. Aggregation can be measured by
several techniques, including size-exclusion column (SEC), high
performance liquid chromatography (HPLC), and light scattering.
Methods of Engineering Antibodies
[0213] As discussed above, the anti-mesothelin antibodies having
known V.sub.H and V.sub.L sequences can be used to create new
anti-mesothelin antibodies by modifying them or the constant
region(s) attached thereto. Thus, in another aspect of this
disclosure, the structural features of a known anti-mesothelin
antibody, e.g., 3C10, 6A4 or 7B 1, are used to create structurally
related anti-mesothelin antibodies that retain at least one
functional property of the antibodies of this disclosure, such as
binding to human mesothelin. For example, one or more CDR regions
of known anti-mesothelin antibodies or mutations thereof, can be
combined recombinantly with known framework regions and/or other
CDRs to create additional, recombinantly-engineered,
anti-mesothelin antibodies of this disclosure, as discussed above.
Other types of modifications include those described in the
previous section. The starting material for the engineering method
is one or more of the V.sub.H and/or V.sub.L sequences provided
herein, or one or more CDR regions thereof. To create the
engineered antibody, it is not necessary to actually prepare (i.e.,
express as a protein) an antibody having one or more of the V.sub.H
and/or V.sub.L sequences provided herein, or one or more CDR
regions thereof. Rather, the information contained in the
sequence(s) is used as the starting material to create a "second
generation" sequence(s) derived from the original sequence(s) and
then the "second generation" sequence(s) is prepared and expressed
as a protein.
[0214] Accordingly, in another embodiment, this disclosure provides
a method for preparing an anti-mesothelin antibody comprising:
[0215] (a) providing: (i) a V.sub.H antibody sequence comprising a
CDR1 sequence selected from the group consisting of SEQ ID NOs:
1-3, a CDR2 sequence selected from the group consisting of SEQ ID
NOs: 4-6, and/or a CDR3 sequence selected from the group consisting
of SEQ ID NOs: 7-9; and/or (ii) a V.sub.L antibody sequence
comprising a CDR1 sequence selected from the group consisting of
SEQ ID NOs: 10-12, a CDR2 sequence selected from the group
consisting of SEQ ID NOs: 13-15, and/or a CDR3 sequence selected
from the group consisting of SEQ ID NOs: 16-18; [0216] (b) altering
at least one amino acid residue within the V.sub.L antibody
sequence and/or the V.sub.L antibody sequence to create at least
one altered antibody sequence; and [0217] (c) expressing the
altered antibody sequence as a protein.
[0218] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence.
[0219] In certain embodiments, mutations can be introduced randomly
or selectively along all or part of an anti-mesothelin antibody
coding sequence and the resulting modified anti-mesothelin
antibodies can be screened for binding activity and/or other
functional properties as described herein. Mutational methods have
been described in the art. For example, WO 02/092780 by Short
describes methods for creating and screening antibody mutations
using saturation mutagenesis, synthetic ligation assembly, or a
combination thereof. Alternatively, WO 03/074679 by Lazar et al.,
describes methods of using computational screening methods to
optimize physiochemical properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of the Invention
[0220] Another aspect of this disclosure pertains to nucleic acid
molecules that encode the antibodies of this disclosure. The
nucleic acids may be present in whole cells, in a cell lysate, or
in a partially purified or substantially pure form. A nucleic acid
is "isolated" or "rendered substantially pure" when purified away
from other cellular components or other contaminants, e.g., other
cellular nucleic acids or proteins, by standard techniques,
including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols
in Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of this disclosure can be, for example, DNA or
RNA and may or may not contain intronic sequences. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0221] Nucleic acids of this disclosure can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), a nucleic acid
encoding such antibodies can be recovered from the gene
library.
[0222] Preferred nucleic acids molecules are those encoding the
V.sub.H and V.sub.L sequences of the 3C10, 6A4 or 7B1 monoclonal
antibodies. DNA sequences encoding the V.sub.H sequences of 3C10,
6A4 and 7B1 are shown in SEQ ID NOs: 25-27, respectively. DNA
sequences encoding the V.sub.L sequences of 3C10, 6A4 and 7B1 are
shown in SEQ ID NOs: 28-30, respectively.
[0223] Once DNA fragments encoding V.sub.H and V.sub.L segments are
obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes, or to scFv genes. In these manipulations, a
V.sub.L- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA fragment encoding another protein, such as an antibody
constant region or a flexible linker. The term "operatively
linked", as used in this context, means that the two DNA fragments
are joined such that the amino acid sequences encoded by them
remain in-frame.
[0224] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to a DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat
'242) and DNA fragments encompassing these regions can be obtained
by standard PCR amplification. The heavy chain constant region can
be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region,
but most preferably is an IgG1 or IgG4 constant region. For a Fab
fragment heavy chain gene, the V.sub.H-encoding DNA can be
operatively linked to a DNA molecule encoding only the heavy chain
CH1 constant region.
[0225] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as a Fab light
chain gene) by operatively linking the V.sub.L-encoding DNA to
another DNA molecule encoding the light chain constant region, CL.
The sequences of human light chain constant region genes are known
in the art (see e.g., Kabat '242) and DNA fragments encompassing
these regions can be obtained by standard PCR amplification. In
preferred embodiments, the light chain constant region can be a
kappa or lambda constant region.
[0226] To create a scFv gene, the V.sub.H- and V.sub.L-encoding DNA
fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences
can be expressed as a contiguous single-chain protein, with the
V.sub.L and V.sub.H regions joined by the flexible linker (see
e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990)
Nature 348:552-554).
Production of Monoclonal Antibodies of this Disclosure
[0227] Monoclonal antibodies (mAbs) of the present disclosure can
be produced by a variety of techniques, including the somatic cell
hybridization technique of Kohler and Milstein (1975) Nature 256:
495. Although somatic cell hybridization procedures are preferred,
in principle other techniques can be employed e.g., viral or
oncogenic transformation of B lymphocytes.
[0228] The preferred animal system for preparing hybridomas is the
murine system. Hybridoma production in the mouse is a very
well-established procedure. Immunization protocols and techniques
for isolation of immunized splenocytes for fusion are known in the
art. Fusion partners (e.g., murine myeloma cells) and fusion
procedures are also known.
[0229] Chimeric or humanized antibodies of the present disclosure
can be prepared based on the sequence of a non-human monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the non-human
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, murine
variable regions can be linked to human constant regions (see e.g.,
U.S. Pat. No. 4,816,567 to Cabilly et al.). To create a humanized
antibody, murine CDR regions can be inserted into a human framework
(see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat. Nos.
5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
[0230] Preferably, the antibodies are human monoclonal antibodies.
They can be generated using transgenic or transchromosomic mice
carrying parts of the human immune system rather than the mouse
system. These transgenic and transchromosomic mice include those of
the HuMAb Mouse.RTM. and KM Mouse.RTM. types, respectively, and are
collectively referred to herein as "human Ig mice."
[0231] The HuMAb Mouse.RTM. type of mouse (Medarex.RTM., Inc.)
contains human immunoglobulin gene miniloci that encode
unrearranged human heavy (.beta. and .gamma.) and light chain
immunoglobulin sequences, together with targeted mutations that
inactivate the endogenous .mu. and .kappa. chain loci (see e.g.,
Lonberg et al. (1994) Nature 368(6474): 856-859). Accordingly, the
mice exhibit reduced expression of mouse IgM or .kappa., and in
response to immunization, the introduced human heavy and light
chain transgenes undergo class switching and somatic mutation to
generate high affinity human IgG.kappa. monoclonal antibodies
(Lonberg et al. (1994), supra; reviewed in Lonberg (1994) Handbook
of Experimental Pharmacology 113:49-101; Lonberg and Huszar (1995)
Intern. Rev. Immunol. 13: 65-93, and Harding and Lonberg (1995)
Ann. N.Y. Acad. Sci. 764:536-546). Preparation and use of the HuMAb
Mouse.RTM. type of mice, and the genomic modifications carried by
such mice, is further described in Taylor et al. (1992) Nucleic
Acids Research 20:6287-6295; Chen et al. (1993) International
Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad.
Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics
4:117-123; Chen et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.
(1994) J. Immunol. 152:2912-2920; Taylor. et al. (1994)
International Immunology 6: 579-591; and Fishwild et al. (1996)
Nature Biotechnology 14: 845-851, the contents of all of which are
hereby specifically incorporated by reference in their entirety.
See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299;
and 5,770,429; all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to
Surani et al.; WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852,
WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and WO
01/14424 to Korman et al. Transgenic mice carrying human lambda
light chain genes also can be used, such as those described in WO
00/26373 by Bruggemann. For example, a mouse carrying a human
lambda light chain transgene can be crossbred with a mouse carrying
a human heavy chain transgene (e.g., HCo7), and optionally also
carrying a human kappa light chain transgene (e.g., KCo5) to create
a mouse carrying both human heavy and light chain transgenes (see
e.g., Example 1).
[0232] In another embodiment, human antibodies of this disclosure
can be generated using a mouse that carries human immunoglobulin
sequences on transgenes and transchomosomes, such as one carrying a
human heavy chain transgene and a human light chain
transchromosome. This mouse is referred to herein as being of the
KM Mouse.RTM. strain or type and is described in detail in WO
02/43478 to Ishida et al.
[0233] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-mesothelin antibodies of this disclosure.
For example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;
6,150,584 and 6,162,963 to Kucherlapati et al.
[0234] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to generate anti-mesothelin antibodies of this
disclosure. For example, mice carrying both a human heavy chain
transchromosome and a human light chain tranchromosome, referred to
as "TC mice" can be used; such mice are described in Tomizuka et
al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Furthermore, cows
carrying human heavy and light chain transchromosomes have been
disclosed (e.g., Kuroiwa et al. (2002) Nature Biotechnology
20:889-894 and WO 2002/092812).
[0235] Human monoclonal antibodies of this disclosure can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. See for example: U.S. Pat. Nos.
5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat.
Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos.
5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.
5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081
to Griffiths et al.
[0236] Human monoclonal antibodies of this disclosure can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in U.S. Pat. Nos.
5,476,996 and 5,698,767 to Wilson et al.
[0237] In another embodiment, human anti-mesothelin antibodies are
prepared using a combination of human Ig mouse and phage display
techniques, as described in U.S. Pat. No. 6,794,132 by Buechler et
al. More specifically, the method first involves raising an
anti-mesothelin antibody response in a human Ig mouse (such as
those of the HuMab Mouse.RTM. or KM Mouse.RTM. strains) by
immunizing the mouse with one or more mesothelin antigens, followed
by isolating nucleic acids encoding human antibody chains from
lymphatic cells of the mouse and introducing these nucleic acids
into a display vector (e.g., phage) to provide a library of display
packages. Thus, each library member comprises a nucleic acid
encoding a human antibody chain and each antibody chain is
displayed from the display package. The library then is screened
with mesothelin protein to isolate library members that
specifically bind to mesothelin. Nucleic acid inserts of the
selected library members then are isolated and sequenced by
standard methods to determine the light and heavy chain variable
sequences of the selected mesothelin binders. The variable regions
can be converted to full-length antibody chains by standard
recombinant DNA techniques, such as cloning of the variable regions
into an expression vector that carries the human heavy and light
chain constant regions such that the V.sub.H region is operatively
linked to the C.sub.H region and the V.sub.L region is operatively
linked to the C.sub.L region.
Immunization of Human Ig Mice
[0238] When human Ig mice are used to generate human antibodies of
this disclosure, such mice can be immunized with a purified or
enriched preparation of a mesothelin antigen and/or recombinant
mesothelin protein, cells expressing a mesothelin protein, or a
mesothelin fusion protein, as described by Lonberg, N. et al.
(1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature
Biotechn. 14: 845-851; and WO 98/24884 and WO 01/14424. Preferably,
the mice are 6-16 weeks of age upon the first infusion. For
example, a purified or recombinant preparation (5-50 .mu.g) of
mesothelin antigen is used to immunize the human Ig mice
intraperitoneally and/or subcutaneously. Most preferably, the
immunogen used to raise the antibodies of this disclosure is a
mesothelin fusion protein comprising the extracellular domain of a
mesothelin protein, fused at its N-terminus to a non-mesothelin
polypeptide (e.g., a His tag) (see, e.g., Example 1).
[0239] Detailed procedures to generate fully human monoclonal
antibodies that bind human mesothelin are described in Example 1.
Cumulative experience with various antigens has shown that
transgenic mice of the HuMAb Mouse type respond when initially
immunized intraperitoneally (IP) with antigen in complete Freund's
adjuvant, followed by every other week IP immunizations (up to a
total of 6) with antigen in incomplete Freund's adjuvant. Adjuvants
other than Freund's (e.g., RIBI adjuvant) are also effective. In
addition, whole cells in the absence of adjuvant are found to be
highly immunogenic. The immune response can be monitored over the
course of the immunization protocol with plasma samples obtained by
retroorbital bleeds. The plasma can be screened by ELISA, and mice
with sufficient titers of anti-mesothelin human immunoglobulin can
be used for fusions. Mice can be boosted intravenously with
antigen, for example 3 days before sacrifice and removal of the
spleen. It is expected that 2-3 fusions for each immunization may
be needed. Between 6 and 24 mice are typically immunized for each
antigen. Usually both HCo7 and HCo12 strains are used. In addition,
both HCo7 and HCo12 transgene can be bred together into a single
mouse having two different human heavy chain transgenes
(HCo7/HCo12). Alternatively or additionally, the KM Mouse.RTM.
strain can be used.
Generation of Hybridomas Producing Human Monoclonal Antibodies of
the Invention
[0240] To generate hybridomas producing human monoclonal antibodies
of this disclosure, splenocytes and/or lymph node cells from
immunized mice are isolated and fused to an appropriate
immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas are screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice are fused to one-sixth
the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC,
CRL 1580) with 50% PEG. Alternatively, the single cell suspension
of splenic lymphocytes from immunized mice can be fused by
electrofusion, using a CytoPulse large chamber cell fusion
electroporator (CytoPulse Sciences, Inc., Glen Burnie, Md.). Cells
are plated at approximately 2.times.10.sup.5 in flat bottom
microtiter plate, followed by a two week incubation in selective
medium containing 20% fetal Clone Serum, 18% "653" conditioned
media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5
mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/mL penicillin, 50
mg/mL streptomycin, 50 mg/mL gentamycin and 1.times.HAT (Sigma; the
HAT is added 24 hours after the fusion). After approximately two
weeks, cells are cultured in medium in which the HAT is replaced
with HT. Individual wells are then screened by ELISA for human
monoclonal IgM and IgG antibodies. Once extensive hybridoma growth
occurs, medium is observed, usually after 10-14 days. The antibody
secreting hybridomas are replated, screened again, and if still
positive for human IgG, the monoclonal antibodies are subcloned at
least twice by limiting dilution. The stable subclones are then
cultured in vitro to generate small amounts of antibody in tissue
culture medium for characterization.
[0241] To purify human monoclonal antibodies, selected hybridomas
are grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG fractions are checked by gel
electrophoresis and HPLC to ensure purity. The buffer solution is
exchanged into PBS, and the concentration is determined by OD280
using extinction coefficient of 1.43. The monoclonal antibodies are
aliquoted and stored at -80.degree. C.
Generation of Transfectomas Producing Monoclonal Antibodies of the
Invention
[0242] Antibodies of this disclosure also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (e.g., Morrison, S. (1985) Science 229:1202).
[0243] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, are obtained by standard molecular biology techniques
(e.g., PCR amplification or cDNA cloning using a hybridoma that
expresses the antibody of interest) and the DNAs can be inserted
into expression vectors such that the genes are operatively linked
to transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene are inserted into separate vectors or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). The light and heavy chain variable regions of
the antibodies described herein are used to create full-length
antibody genes of any antibody isotype by inserting them into
expression vectors already encoding heavy chain constant and light
chain constant regions of the desired isotype such that the V.sub.H
segment is operatively linked to the C.sub.H segment(s) within the
vector and the V.sub.L segment is operatively linked to the C.sub.L
segment within the vector. Additionally or alternatively, the
recombinant expression vector can encode a signal peptide that
facilitates secretion of the antibody chain from a host cell. The
antibody chain gene can be cloned into the vector such that the
signal peptide is linked in-frame to the amino terminus of the
antibody chain gene. The signal peptide can be an immunoglobulin
signal peptide or a heterologous signal peptide (i.e., a signal
peptide from a non-immunoglobulin protein).
[0244] In addition to the antibody chain genes, the recombinant
expression vectors of this disclosure carry regulatory sequences
that control the expression of the antibody chain genes in a host
cell. The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990)). It will be appreciated by those skilled in the art that
the design of the expression vector, including the selection of
regulatory sequences, may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as those derived from
cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g.,
the adenovirus major late promoter (AdMLP) and polyoma.
Alternatively, nonviral regulatory sequences may be used, such as
the ubiquitin promoter or .beta.-globin promoter. Still further,
regulatory elements composed of sequences from different sources,
such as the SR.alpha. promoter system, which contains sequences
from the SV40 early promoter and the long terminal repeat of human
T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell.
Biol. 8:466-472).
[0245] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of this disclosure
may carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0246] For expression of the light and heavy chains, the expression
vector(s) encoding them are transfected into a host cell. The
various forms of the term "transfection" are intended to encompass
a wide variety of techniques commonly used for the introduction of
exogenous DNA into a prokaryotic or eukaryotic host cell, e.g.,
electroporation, calcium-phosphate precipitation, DEAE-dextran
transfection and the like. Although it is theoretically possible to
express the antibodies of this disclosure in either prokaryotic or
eukaryotic host cells, expression of antibodies in eukaryotic
cells, and most preferably mammalian host cells, is the most
preferred because they are more likely than prokaryotic cells to
assemble and secrete a properly folded and immunologically active
antibody.
[0247] Preferred mammalian host cells for expressing the
recombinant antibodies of this disclosure include Chinese Hamster
Ovary (CHO cells) (including dhfr.sup.- CHO cells, described in
Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,
used with a DHFR selectable marker, e.g., as described in R. J.
Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO
myeloma cells, COS cells and SP2 cells. In particular, for use with
NSO myeloma cells, another preferred expression system is the GS
gene expression system disclosed in WO 87/04462 (to Wilson), WO
89/01036 (to Bebbington) and EP 338,841 (to Bebbington). When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in
which the host cells are grown. Antibodies can be recovered from
the culture medium using standard protein purification methods.
Characterization of Antibody Binding to Antigen
[0248] Antibodies of the invention can be tested for binding to
human mesothelin by, for example, standard ELISA. Briefly,
microtiter plates are coated with purified and/or recombinant
mesothelin protein (see, e.g., Example 1) at 1 .mu.g/mL in PBS, and
then blocked with 5% bovine serum albumin in PBS. Dilutions of
antibody (e.g., dilutions of plasma from mesothelin-immunized mice)
are added to each well and incubated for 1-2 hours at 37.degree. C.
The plates are washed with PBS/Tween and then incubated with
secondary reagent (e.g., for human antibodies, a goat-anti-human
IgG Fc-specific polyclonal reagent) conjugated to alkaline
phosphatase for 1 hour at 37.degree. C. After washing, the plates
are developed with pNPP substrate (1 mg/mL), and analyzed at OD of
405-650. Preferably, mice that develop the highest titers will be
used for fusions.
[0249] An ELISA assay as described above can also be used to screen
for hybridomas that show positive reactivity with a mesothelin
protein. Hybridomas that bind with high avidity and/or affinity to
a mesothelin protein are subcloned and further characterized. One
clone from each hybridoma, which retains the reactivity of the
parent cells (by ELISA), can be chosen for making a 5-10 vial cell
bank stored at -140.degree. C., and for antibody purification.
[0250] To purify anti-mesothelin antibodies, selected hybridomas
are grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants are filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.). Eluted IgG fractions are checked by gel
electrophoresis and HPLC to ensure purity. The buffer solution is
exchanged into PBS, and the concentration is determined by OD280
using an extinction coefficient of 1.43. The monoclonal antibodies
are aliquoted and stored at -80.degree. C.
[0251] To determine if the selected anti-mesothelin monoclonal
antibodies bind to unique epitopes, each antibody is biotinylated
using commercially available reagents (Pierce, Rockford, Ill.).
Competition studies using unlabeled monoclonal antibodies and
biotinylated monoclonal antibodies are performed using mesothelin
protein coated-ELISA plates as described above. Biotinylated mAb
binding can be detected with a strep-avidin-alkaline phosphatase
probe.
[0252] To determine the isotype of purified antibodies, isotype
ELISAs can be performed using reagents specific for a particular
isotype. For example, to determine the isotype of a human
monoclonal antibody, wells of microtiter plates can be coated with
1 .mu.g/mL of anti-human immunoglobulin overnight at 4.degree. C.
After blocking with 1% BSA, the plates are reacted with 1 .mu.g/mL
or less of test monoclonal antibodies or purified isotype controls,
at ambient temperature for one to two hours. The wells are then
reacted with either human IgG1 or human IgM-specific alkaline
phosphatase-conjugated probes. Plates are developed and analyzed as
described above.
[0253] Anti-mesothelin human IgGs also can be tested for reactivity
with a mesothelin antigen by Western blotting. Briefly, a
mesothelin antigen is prepared and subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis. The separated antigens
are transferred to nitrocellulose membranes, blocked with 10% fetal
calf serum, and probed with the monoclonal antibodies to be tested.
Human IgG binding is detected using anti-human IgG alkaline
phosphatase and developed with BCIP/NBT substrate tablets (Sigma
Chem. Co., St. Louis, Mo.).
[0254] The binding specificity of an antibody of this disclosure
can also be determined by monitoring binding of the antibody to
cells expressing a mesothelin protein, for example by flow
cytometry. Cells or cell lines that naturally expresses mesothelin
protein, such OVCAR3, NC1-H226, CFPAC-1 or KB cells, can be used,
or a cell line such as a CHO cell line can be transfected with an
expression vector encoding mesothelin such that mesothelin is
expressed on the cell surface. The transfected protein may comprise
a tag, such as a myc-tag or a his-tag, preferably at the
N-terminus, for detection using an antibody to the tag. Binding of
an antibody of this disclosure to a mesothelin protein can be
determined by incubating the transfected cells with the antibody,
and detecting bound antibody. Binding of an antibody to the tag on
the transfected protein may can used as a positive control.
Bispecific Molecules
[0255] In another aspect, the present disclosure features
bispecific molecules comprising an anti-mesothelin antibody, or a
fragment thereof, of this disclosure linked to another functional
molecule, e.g., another peptide or protein such as another
antibody, a binding mimetic, or a ligand for a receptor, which bind
to at least two different binding sites or target molecules. The
antibody of this disclosure may in fact be linked to more than one
other functional molecule to generate multispecific molecules that
bind to more than two different binding sites and/or target
molecules. Such multispecific molecules are also intended to be
encompassed by the term "bispecific molecule" as used herein. The
linkage can be by chemical coupling, genetic fusion, noncovalent
association or otherwise.
[0256] Accordingly, the present disclosure includes bispecific
molecules comprising at least one first binding specificity for
mesothelin and a second binding specificity for a second target
epitope. In a particular embodiment of this disclosure, the second
target epitope is an Fc receptor, e.g., human Fc.gamma.RI (CD64) or
a human Fc.alpha. receptor (CD89). Therefore, this disclosure
includes bispecific molecules capable of binding both to Fc.gamma.R
or Fc.alpha.R expressing effector cells (e.g., monocytes,
macrophages or polymorphonuclear cells (PMNs)), and to target cells
expressing mesothelin protein. These bispecific molecules target
mesothelin expressing cells to effector cell and trigger Fc
receptor-mediated effector cell activities, such as phagocytosis of
mesothelin expressing cells, ADCC, cytokine release, or generation
of superoxide anion.
[0257] Where the bispecific molecule is multispecific, the molecule
can further include a third binding specificity, in addition to an
anti-Fc binding specificity and an anti-mesothelin binding
specificity. In one embodiment, the third binding specificity is an
anti-enhancement factor (EF) portion, e.g., a molecule which binds
to a surface protein involved in cytotoxic activity and thereby
increases the immune response against the target cell. The
"anti-enhancement factor portion" can be an antibody, functional
antibody fragment or a ligand that binds to a given molecule, e.g.,
an antigen or a receptor, and thereby results in an enhancement of
the effect of the binding determinants for the Fc receptor or
target cell antigen. The "anti-enhancement factor portion" can bind
an Fc receptor or a target cell antigen. Alternatively, the
anti-enhancement factor portion can bind to an entity that is
different from the entity to which the first and second binding
specificities bind. For example, the anti-enhancement factor
portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28,
CD4, CD40, ICAM-1) or other immune cell that results in an
increased immune response against the target cell).
[0258] In one embodiment, the bispecific molecules of this
disclosure comprise as a binding specificity at least one antibody,
or an antibody fragment thereof, including, e.g., an Fab, Fab',
F(ab').sub.2, Fv, Fd, dAb or a single chain Fv. The antibody may
also be a light chain or heavy chain dimer, or any minimal fragment
thereof such as a Fv or a single chain construct as described in
U.S. Pat. No. 4,946,778 to Ladner et al., the contents of which is
expressly incorporated by reference.
[0259] In one embodiment, the binding specificity for an Fc.gamma.
receptor is provided by a monoclonal antibody, the binding of which
is not blocked by human immunoglobulin G (IgG). As used herein, the
term "IgG receptor" refers to any of the eight .gamma.-chain genes
located on chromosome 1. These genes encode a total of twelve
transmembrane or soluble receptor isoforms which are grouped into
three Fc.gamma. receptor classes: Fc.gamma.RI (CD64), Fc.gamma.RII
(CD32), and Fc.gamma.RIII (CD16). In one preferred embodiment, the
Fc.gamma. receptor a human high affinity Fc.gamma.RI. The human
Fc.gamma.RI is a 72 kDa molecule, which shows high affinity for
monomeric IgG (10.sup.8-10.sup.9 M.sup.-1).
[0260] The production and characterization of certain preferred
anti-Fc.gamma. monoclonal antibodies are described in WO 88/00052
and in U.S. Pat. No. 4,954,617 to Fanger et al., which are fully
incorporated by reference herein. These antibodies bind to an
epitope of Fc.gamma.RI, Fc.gamma.RII or Fc.gamma.RIII at a site
which is distinct from the Fc.gamma. binding site of the receptor
and, thus, their binding is not blocked substantially by
physiological levels of IgG. Specific anti-Fc.gamma.RI antibodies
useful in this disclosure are mAb 22, mAb 32, mAb 44, mAb 62 and
mAb 197. The hybridoma producing mAb 32 is available from the
American Type Culture Collection, ATCC Accession No. HB9469. In
other embodiments, the anti-Fc.gamma. receptor antibody is a
humanized form of monoclonal antibody 22 (H22). The production and
characterization of the H22 antibody is described in Graziano, R.
F. et al. (1995) J. Immunol 155 (10): 4996-5002 and WO 94/10332 to
Tempest et al. The H22 antibody producing cell line was deposited
at the American Type Culture Collection under the designation
HA022CL1 and has the accession no. CRL 11177.
[0261] In still other preferred embodiments, the binding
specificity for an Fc receptor is provided by an antibody that
binds to a human IgA receptor, e.g., an Fc-alpha receptor
(Fc.alpha.RI (CD89)), the binding of which is preferably not
blocked by human immunoglobulin A (IgA). The term "IgA receptor" is
intended to include the gene product of one .alpha.-gene
(Fc.alpha.RI) located on chromosome 19. This gene is known to
encode several alternatively spliced transmembrane isoforms of 55
to 110 kDa. Fc.alpha.RI (CD89) is constitutively expressed on
monocytes/macrophages, eosinophilic and neutrophilic granulocytes,
but not on non-effector cell populations. Fc.alpha.RI has medium
affinity 5.times.10.sup.7 M.sup.-1) for both IgA1 and IgA2, which
is increased upon exposure to cytokines such as G-CSF or GM-CSF
(Morton, H. C. et al. (1996) Critical Reviews in Immunology
16:423-440). Four Fc.alpha.RI-specific monoclonal antibodies,
identified as A3, A59, A62 and A77, which bind Fc.alpha.RI outside
the IgA ligand binding domain, have been described (Monteiro, R. C.
et al. (1992) J. Immunol. 148:1764).
[0262] Fc.alpha.RI and Fc.gamma.RI are preferred trigger receptors
for use in the bispecific molecules of this disclosure because they
are (1) expressed primarily on immune effector cells, e.g.,
monocytes, PMNs, macrophages and dendritic cells; (2) expressed at
high levels (e.g., 5,000-100,000 per cell); (3) mediators of
cytotoxic activities (e.g., ADCC, phagocytosis); and (4) mediate
enhanced antigen presentation of antigens, including self-antigens,
targeted to them.
[0263] While human monoclonal antibodies are preferred, other
antibodies which can be employed in the bispecific molecules of
this disclosure are murine, chimeric and humanized monoclonal
antibodies.
[0264] The bispecific molecules of the present disclosure can be
prepared by conjugating the constituent binding specificities,
e.g., the anti-FcR and anti-mesothelin binding specificities, using
methods known in the art. For example, each binding specificity of
the bispecific molecule can be generated separately and then
conjugated to one another. When the binding specificities are
proteins or peptides, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med.
160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus (1985)
Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science
229:81-83, and Glennie et al. (1987) J. Immunol. 139: 2367-2375).
Preferred conjugating agents are SATA and sulfo-SMCC, both
available from Pierce Chemical Co. (Rockford, Ill.).
[0265] When the binding specificities are antibodies, they can be
conjugated via sulfhydryl bonding of the C-terminus hinge regions
of the two heavy chains. In a particularly preferred embodiment,
the hinge region is modified to contain an odd number of sulfhydryl
residues, preferably one, prior to conjugation.
[0266] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2 or
ligand.times.Fab fusion protein. A bispecific molecule of this
disclosure can be a single chain molecule comprising one single
chain antibody and a binding determinant, or a single chain
bispecific molecule comprising two binding determinants. Bispecific
molecules may comprise at least two single chain molecules. Methods
for preparing bispecific molecules are described for example in
U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405;
5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858, all of
which are expressly incorporated herein by reference.
[0267] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, ELISA, radioimmunoassay
(RIA), FACS analysis, bioassay (e.g., growth inhibition), or
Western Blot assay. Each of these assays generally detects the
presence of protein-antibody complexes of interest by employing a
labeled reagent (e.g., an antibody) specific for the complex of
interest. For example, the FcR-antibody complexes can be detected
using e.g., an enzyme-linked antibody or antibody fragment which
recognizes and specifically binds to the antibody-FcR complexes.
Alternatively, the complexes can be detected using any of a variety
of other immunoassays. For example, the antibody can be
radioactively labeled and used in a radioimmunoassay (RIA) (see,
for example, Weintraub, B., Principles of Radioimmunoassays,
Seventh Training Course on Radioligand Assay Techniques, The
Endocrine Society, March, 1986, which is incorporated by reference
herein). The radioactive isotope can be detected by such means as
the use of a .gamma. or scintillation counter or by
autoradiography.
Conjugates
[0268] In another aspect, there is provided an antibody-partner
molecule conjugate, wherein a partner molecule is conjugated to an
antibody of this invention by a chemical linker (sometimes referred
to herein simply as "linker"). The partner molecule can be a
therapeutic agent or a marker. The therapeutic agent can be, for
example, a cytotoxin, a non-cytotoxic drug (e.g., an
immunosuppressant), a radioactive agent, another antibody, or an
enzyme. Preferably, the partner molecule is a cytotoxin. The marker
can be any label that generates a detectable signal, such as a
radiolabel, a fluorescent label, or an enzyme that catalyzes a
detectable modification to a substrate. The antibody serves a
targeting function: by binding to a target tissue or cell where its
antigen is found, the antibody steers the conjugate to the target
tissue or cell. There, the linker is cleaved, releasing the partner
molecule to perform its desired biological function. In some
instances, the conjugate is internalized within a target cell and
the cleavage occurs therewithin.
Linkers
[0269] In some embodiments, the linker is a peptidyl linker,
depicted herein as (L.sup.4).sub.p-F-(L.sup.1).sub.m. Other linkers
include hydrazine and disulfide linkers, depicted herein as
(L.sup.4).sub.p-H-(L.sup.1), and (L.sup.4).sub.p-J-(L.sup.1).sub.m,
respectively. F, H, and J are peptidyl, hydrazine, and disulfide
moieties, respectively, that are cleavable to release the partner
molecule from the antibody, while L.sup.1 and L.sup.4 are linker
groups. F, H, J, L.sup.1, and L.sup.4 are more fully defined
hereinbelow, along with the subscripts p and m. The preparation and
use of these and other linkers is described in WO 2005/112919, the
disclosure of which is incorporated herein by reference.
[0270] The use of peptidyl and other linkers in antibody-partner
conjugates is described in US 2006/0004081; 2006/0024317;
2006/0247295; U.S. Pat. Nos. 6,989,452; 7,087,600; and 7,129,261;
WO 2007/051081; 2007/038658; 2007/059404; and 2007/089100; all of
which are incorporated herein by reference.
[0271] Additional linkers are described in U.S. Pat. No. 6,214,345;
2003/0096743; and 2003/0130189; de Groot et al., J. Med. Chem. 42,
5277 (1999); de Groot et al. J. Org. Chem. 43, 3093 (2000); de
Groot et al., J. Med. Chem. 66, 8815, (2001); WO 02/083180; Carl et
al., J. Med. Chem. Lett. 24, 479, (1981); Dubowchik et al., Bioorg
& Med. Chem. Lett. 8, 3347 (1998), the disclosures of which are
incorporated herein by reference.
[0272] In addition to connecting the antibody and the partner
molecule, a linker can impart stability to the partner molecule,
reduce its in vivo toxicity, or otherwise favorably affect its
pharmacokinetics, bioavailability and/or pharmacodynamics. It is
generally preferred that the linker is cleaved, releasing the
partner molecule, once the conjugate is delivered to its site of
action. Also preferably, the linkers are traceless, such that once
removed from the partner molecule (such as during activation), no
trace of the linker's presence remains.
[0273] In another embodiment, the linkers are characterized by
their ability to be cleaved at a site in or near a target cell such
as at the site of therapeutic action or marker activity of the
partner molecule. Such cleavage can be enzymatic in nature. This
feature aids in reducing systemic activation of the partner
molecule, reducing toxicity and systemic side effects. Preferred
cleavable groups for enzymatic cleavage include peptide bonds,
ester linkages, and disulfide linkages, such as the aforementioned
F, H, and J moieties. In other embodiments, the linkers are
sensitive to pH and are cleaved through changes in pH.
[0274] An important aspect is the ability to control the speed with
which the linkers cleave. Often a linker that cleaves quickly is
desired. In some embodiments, however, a linker that cleaves more
slowly may be preferred. For example, in a sustained release
formulation or in a formulation with both a quick release and a
slow release component, it may be useful to provide a linker which
cleaves more slowly. The aforecited WO 2005/112919 discloses
hydrazine linkers that can be designed to cleave at a range of
speeds, from very fast to very slow.
[0275] The linkers can also serve to stabilize the partner molecule
against degradation while the conjugate is in circulation, that is,
before it reaches the target tissue or cell. This feature provides
a significant benefit since it prolongates the circulation
half-life of the partner molecule. The linker also serves to
attenuate the activity of the partner molecule so that the
conjugate is relatively benign while in circulation but the partner
molecule has the desired effect--for example is cytotoxic--after
activation at the desired site of action. For therapeutic agent
conjugates, this feature of the linker serves to improve the
therapeutic index of the agent.
[0276] In addition to the cleavable peptide, hydrazine, or
disulfide groups F, H, or J, respectively, one or more linker
groups L.sup.1 are optionally introduced between the partner
molecule and F, H, or J, as the case may be. These linker groups
L.sup.1 may also be described as spacer groups and contain at least
two functional groups. Depending on the value of the subscript m
(i.e., the number of L.sup.1 groups present) and the location of a
particular group L.sup.1, a chemical functionality of a group
L.sup.1 can bond to a chemical functionality of the partner
molecule, of F, H or J, as the case may be, or of another linker
group L.sup.1 (if more than one L.sup.1 is present). Examples of
suitable chemical functionalities for spacer groups L.sup.1 include
hydroxy, mercapto, carbonyl, carboxy, amino, ketone, aldehyde, and
mercapto groups.
[0277] The linkers L.sup.1 can be a substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl or substituted or unsubstituted
heteroalkyl group. In one embodiment, the alkyl or aryl groups may
comprise between 1 and 20 carbon atoms. They may also comprise a
polyethylene glycol moiety.
[0278] Exemplary groups L.sup.1 include, for example,
6-aminohexanol, 6-mercaptohexanol, 10-hydroxydecanoic acid, glycine
and other amino acids, 1,6-hexanediol, .beta.-alanine,
2-aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic
acid, 6-aminohexanoic acid, 3-maleimidobenzoic acid, phthalide,
.alpha.-substituted phthalides, the carbonyl group, aminal esters,
nucleic acids, peptides and the like.
[0279] One function of the groups L.sup.1 is to provide spatial
separation between F, H or J, as the case may be, and the partner
molecule, lest the latter interfere (e.g., via steric or electronic
effects) with cleavage chemistry at F, H, or J. The groups L.sup.1
also can serve to introduce additional molecular mass and chemical
functionality into conjugate. Generally, the additional mass and
functionality affects the serum half-life and other properties of
the conjugate. Thus, through careful selection of spacer groups,
conjugates with a range of serum half-lives can be produced.
Optionally, one or more linkers L.sup.1 can be a self-immolative
group, as described hereinbelow.
[0280] The subscript m is an integer selected from 0, 1, 2, 3, 4,
5, and 6. When multiple L.sup.1 groups are present, they can be the
same or different.
[0281] L.sup.4 is a linker moiety that provides spatial separation
between F, H, or J, as the case may be, and the antibody, lest F,
H, or J interfere with the antigen binding by the antibody or the
antibody interfere with the cleavage chemistry at F, H, or J.
Preferably, L.sup.4 imparts increased solubility or decreased
aggregation properties to conjugates utilizing a linker that
contains the moiety or modifies the hydrolysis rate of the
conjugate. As in the case of L.sup.1, L.sup.4 optionally is a self
immolative group. In one embodiment, the L.sup.4 moiety is
substituted alkyl, unsubstituted alkyl, substituted aryl,
unsubstituted aryl, substituted heteroalkyl, or unsubstituted
heteroalkyl, any of which may be straight, branched, or cyclic. The
substitutions can be, for example, a lower (C.sub.1-C.sub.6) alkyl,
alkoxy, alkylthio, alkylamino, or dialkylamino. In certain
embodiments, L.sup.4 comprises a non-cyclic moiety. In another
embodiment, L.sup.4 comprises a positively or negatively charged
amino acid polymer, such as polylysine or polyarginine. L.sup.4 can
comprise a polymer such as a polyethylene glycol moiety.
Additionally the L.sup.4 linker can comprise, for example, both a
polymer component and a small molecule moiety.
[0282] In a preferred embodiment, L.sup.4 comprises a polyethylene
glycol (PEG) moiety. The PEG portion of L.sup.4 may be between 1
and 50 units long. Preferably, the PEG will have 1-12 repeat units,
more preferably 3-12 repeat units, more preferably 2-6 repeat
units, or even more preferably 3-5 repeat units and most preferably
4 repeat units. L.sup.4 may consist solely of the PEG moiety, or it
may also contain an additional substituted or unsubstituted alkyl
or heteroalkyl. It is useful to combine PEG as part of the L.sup.4
moiety to enhance the water solubility of the complex.
Additionally, the PEG moiety reduces the degree of aggregation that
may occur during the conjugation of the drug to the antibody.
[0283] The subscript p is 0 or 1; that is, the presence of L.sup.4
is optional. Where present, L.sup.4 has at least two functional
groups, with one functional group binding to a chemical
functionality in F, H, or J, as the case may be, and the other
functional group binding to the antibody. Examples of suitable
chemical functionalities of groups L.sup.4 include hydroxy,
mercapto, carbonyl, carboxy, amino, ketone, aldehyde, and mercapto
groups. As antibodies typically are conjugated via sulfhydryl
groups (e.g., from unoxidized cysteine residues, the addition of
sulfhydryl-containing extensions to lysine residues with
iminothiolane, or the reduction of disulfide bridges), amino groups
(e.g., from lysine residues), aldehyde groups (e.g., from oxidation
of glycoside side chains), or hydroxyl groups (e.g., from serine
residues), preferred chemical functionalities for attachment to the
antibody are those reactive with the foregoing groups, examples
being maleimide, sulfhydryl, aldehyde, hydrazine, semicarbazide,
and carboxyl groups. The combination of a sulfhydryl group on the
antibody and a maleimide group on L.sup.4 is preferred.
[0284] In some embodiments, L.sup.4 comprises
##STR00001##
directly attached to the N-terminus of (AA.sup.1).sub.c. R.sup.20
is a member selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, and acyl. Each R.sup.25,
R.sup.25', R.sup.26, and R.sup.26' is independently selected from
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, and substituted or unsubstituted
heterocycloalkyl; and s and t are independently integers from 1 to
6. Preferably, R.sup.20, R.sup.25, R.sup.25', R.sup.26 and
R.sup.26' are hydrophobic. In some embodiments, R.sup.20 is H or
alkyl (preferably, unsubstituted lower alkyl). In some embodiments,
R.sup.25, R.sup.25', R.sup.26 and R.sup.26' are independently H or
alkyl (preferably, unsubstituted C.sup.1 to C.sup.4 alkyl). In some
embodiments, R.sup.25, R.sup.25', R.sup.26 and R.sup.26' are all H.
In some embodiments, t is 1 and s is 1 or 2.
Peptide Linkers (F)
[0285] As discussed above, the peptidyl linkers of the invention
can be represented by the general formula:
(L.sup.4).sub.p-F-(L.sup.1).sub.m, wherein F represents the portion
comprising the peptidyl moiety. In one embodiment, the F portion
comprises an optional additional self-immolative linker L.sup.2 and
a carbonyl group, corresponding to a conjugate of formula (a):
##STR00002##
[0286] In this embodiment, L.sup.1, L.sup.4, p, and m are as
defined above. X.sup.4 is an antibody and D is a partner molecule.
The subscript o is 0 or 1 and L.sup.2, if present, represents a
self-immolative linker AA.sup.1 represents one or more natural
amino acids, and/or unnatural .alpha.-amino acids; c is an integer
from 1 and 20. In some embodiments, c is in the range of 2 to 5 or
c is 2 or 3.
[0287] In formula (a), AA.sup.1 is linked, at its amino terminus,
either directly to L.sup.4 or, when L.sup.4 is absent, directly to
X.sup.4. In some embodiments, when L.sup.4 is present, L.sup.4 does
not comprise a carboxylic acyl group directly attached to the
N-terminus of (AA.sup.1).sub.c.
[0288] In another embodiment, the F portion comprises an amino
group and an optional spacer group L.sup.3 and L.sup.1 is absent
(i.e., m is 0), corresponding to a conjugate of formula (b):
##STR00003##
[0289] In this embodiment, X.sup.4, D, L.sup.4, AA.sup.1, c, and p
are as defined above. The subscript o is 0 or 1. L.sup.3, if
present, is a spacer group comprising a primary or secondary amine
or a carboxyl functional group, and either the amine of L.sup.3
forms an amide bond with a pendant carboxyl functional group of D
or the carboxyl of L.sup.3 forms an amide bond with a pendant amine
functional group of D.
Self-Immolative Linkers
[0290] A self-immolative linker is a bifunctional chemical moiety
which is capable of covalently linking together two spaced chemical
moieties into a normally stable tripartate molecule, releasing one
of said spaced chemical moieties from the tripartate molecule by
means of enzymatic cleavage; and following said enzymatic cleavage,
spontaneously cleaving from the remainder of the molecule to
release the other of said spaced chemical moieties. In accordance
with the present invention, the self-immolative spacer is
covalently linked at one of its ends to the peptide moiety and
covalently linked at its other end to the chemically reactive site
of the drug moiety whose derivatization inhibits pharmacological
activity, so as to space and covalently link together the peptide
moiety and the drug moiety into a tripartate molecule which is
stable and pharmacologically inactive in the absence of the target
enzyme, but which is enzymatically cleavable by such target enzyme
at the bond covalently linking the spacer moiety and the peptide
moiety to thereby effect release of the peptide moiety from the
tripartate molecule. Such enzymatic cleavage, in turn, will
activate the self-immolating character of the spacer moiety and
initiate spontaneous cleavage of the bond covalently linking the
spacer moiety to the drug moiety, to thereby effect release of the
drug in pharmacologically active form. See, for example, Carl et
al., J. Med. Chem., 24 (3), 479-480 (1981); Carl et al., WO
81/01145 (1981); Toki et al., J. Org. Chem. 67, 1866-1872 (2002);
Boyd et al., WO 2005/112919; and Boyd et al., WO 2007/038658, the
disclosures of which are incorporated herein by reference.
[0291] One particularly preferred self-immolative spacer may be
represented by the formula (c):
##STR00004##
[0292] The aromatic ring of the aminobenzyl group may be
substituted with one or more "K" groups. A "K" group is a
substituent on the aromatic ring that replaces a hydrogen otherwise
attached to one of the four non-substituted carbons that are part
of the ring structure. The "K" group may be a single atom, such as
a halogen, or may be a multi-atom group, such as alkyl,
heteroalkyl, amino, nitro, hydroxy, alkoxy, haloalkyl, and cyano.
Each K is independently selected from the group consisting of
substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,
unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.21R.sup.22, NR.sup.21COR.sup.22,
OCONR.sup.21R.sup.22, OCOR.sup.21R.sup.22, and OR.sup.21, wherein
R.sup.21 and R.sup.22 are independently selected from the group
consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, unsubstituted heteroalkyl, substituted
aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted
heteroaryl, substituted heterocycloalkyl and unsubstituted
heterocycloalkyl. Exemplary K substituents include, but are not
limited to, F, Cl, Br, I, NO.sub.2, OH, OCH.sub.3, NHCOCH.sub.3,
N(CH.sub.3).sub.2, NHCOCF.sub.3 and methyl. For "K.sub.i", i is an
integer of 0, 1, 2, 3, or 4. In one preferred embodiment, i is
0.
[0293] The ether oxygen atom of the structure shown above is
connected to a carbonyl group. The line from the NR.sup.24
functionality into the aromatic ring indicates that the amine
functionality may be bonded to any of the five carbons that both
form the ring and are not substituted by the --CH.sub.2--O-- group.
Preferably, the NR.sup.24 functionality of X is covalently bound to
the aromatic ring at the para position relative to the
--CH.sub.2--O-- group. R.sup.24 is a member selected from the group
consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl. In a
specific embodiment, R.sup.24 is hydrogen.
[0294] In one embodiment, the invention provides a peptide linker
of formula (a) above, wherein F comprises the structure:
##STR00005##
where R.sup.24, AA.sup.1, K, i, and c are as defined above.
[0295] In another embodiment, the peptide linker of formula (a)
above comprises a --F-(L.sup.1).sub.m- that comprises the
structure:
##STR00006##
where R.sup.24, AA.sup.1, K, i, and c are as defined above.
[0296] In some embodiments, a self-immolative spacer L.sup.1 or
L.sup.2 includes
##STR00007##
where each R.sup.17, R.sup.18, and R.sup.19 is independently
selected from H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
and w is an integer from 0 to 4. In some embodiments, R.sup.17 and
R.sup.18 are independently H or alkyl (preferably, unsubstituted
C.sub.1-C.sub.4 alkyl). Preferably, R.sup.17 and R.sup.18 are
C.sub.1-4 alkyl, such as methyl or ethyl. In some embodiments, w is
0. It has been found experimentally that this particular
self-immolative spacer cyclizes relatively quickly.
[0297] In some embodiments, L.sup.1 or L.sup.2 includes
##STR00008##
where R.sup.17, R.sup.18, R.sup.19, R.sup.24, and K are as defined
above.
Spacer Groups
[0298] The spacer group L.sup.3 is characterized in that it
comprises a primary or secondary amine or a carboxyl functional
group, and either the amine of the L.sup.3 group forms an amide
bond with a pendant carboxyl functional group of D or the carboxyl
of L.sup.3 forms an amide bond with a pendant amine functional
group of D. L.sup.3 can be selected from the group consisting of
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, or substituted or unsubstituted
heterocycloalkyl. In a preferred embodiment, L.sup.3 comprises an
aromatic group. More preferably, L.sup.3 comprises a benzoic acid
group, an aniline group or indole group. Non-limiting examples of
structures that can serve as an -L.sup.3-NH-- spacer include the
following structures:
##STR00009##
where Z is a member selected from O, S and NR.sup.23, and where
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl.
[0299] Upon cleavage of the linker of the invention containing
L.sup.3, the L.sup.3 moiety remains attached to the drug, D.
Accordingly, the L.sup.3 moiety is chosen such that its attachment
to D does not significantly alter the activity of D. In another
embodiment, a portion of the drug D itself functions as the L.sup.3
spacer. For example, in one embodiment, the drug, D, is a
duocarmycin derivative in which a portion of the drug functions as
the L.sup.3 spacer. Non-limiting examples of such embodiments
include those in which NH.sub.2-(L.sup.3)-D has a structure
selected from the group consisting of:
##STR00010## ##STR00011##
where Z is O, S or NR.sup.23, where R.sup.23 is H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or
acyl; and the NH.sub.2 group on each structure reacts with
(AA.sup.1).sub.e to form -(AA.sup.1).sub.n-NH--.
Peptide Sequence (AA.sup.1).sub.c
[0300] The group AA.sup.1 represents a single amino acid or a
plurality of amino acids that are joined together by amide bonds.
The amino acids may be natural amino acids and/or unnatural
.alpha.-amino acids. They may be in the L or the D configuration.
In one embodiment, at least three different amino acids are used.
In another embodiment, only two amino acids are used.
[0301] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate,
citrulline, and O-phosphoserine. Amino acid analogs refers to
compounds that have the same basic chemical structure as a
naturally occurring amino acid, i.e., an .alpha. carbon that is
bound to a hydrogen, a carboxyl group, an amino group, and an R
group, e.g., homoserine, norleucine, methionine sulfoxide,
methionine methyl sulfonium. Such analogs have modified R groups
(e.g., norleucine) or modified peptide backbones, but retain the
same basic chemical structure as a naturally occurring amino acid.
One amino acid that may be used in particular is citrulline, which
is a precursor to arginine and is involved in the formation of urea
in the liver. Amino acid mimetics refers to chemical compounds that
have a structure that is different from the general chemical
structure of an amino acid, but functions in a manner similar to a
naturally occurring amino acid. The term "unnatural amino acid" is
intended to represent the "D" stereochemical form of the twenty
naturally occurring amino acids described above. It is further
understood that the term unnatural amino acid includes homologues
of the natural amino acids, and synthetically modified forms of the
natural amino acids. The synthetically modified forms include, but
are not limited to, amino acids having alkylene chains shortened or
lengthened by up to two carbon atoms, amino acids comprising
optionally substituted aryl groups, and amino acids comprised
halogenated groups, preferably halogenated alkyl and aryl groups.
When attached to a linker or conjugate of the invention, the amino
acid is in the form of an "amino acid side chain", where the
carboxylic acid group of the amino acid has been replaced with a
keto (C(O)) group. Thus, for example, an alanine side chain is
--C(O)--CH(NH.sub.2)--CH.sub.3, and so forth.
[0302] The peptide sequence (AA.sup.1).sub.c is functionally the
amidification residue of a single amino acid (when c=1) or a
plurality of amino acids joined together by amide bonds. The
peptide sequence (AA.sup.1).sub.c preferably is selected for
enzyme-catalyzed cleavage by an enzyme in a location of interest in
a biological system. For example, for conjugates that are targeted
to but not internalized by a cell, a peptide is chosen that is
cleaved by a protease that in the extracellular matrix, e.g., a
protease released by nearby dying cells or a tumor-associated
protease, such that the peptide is cleaved extracellularly. For
conjugates that are designed for internalization by a cell, the
sequence (AA.sup.1), preferably is selected for cleavage by an
endosomal or lysosomal protease. The number of amino acids within
the peptide can range from 1 to 20; but more preferably there will
be 1-8 amino acids, 1-6 amino acids or 1, 2, 3 or 4 amino acids
comprising (AA.sup.1).sub.c. Peptide sequences that are susceptible
to cleavage by specific enzymes or classes of enzymes are well
known in the art.
[0303] Preferably, (AA.sup.1), contains an amino acid sequence
("cleavage recognition sequence") that is a cleavage site by the
protease. Many protease cleavage sequences are known in the art.
See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al.
Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244:
175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et
al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol.
244: 412 (1994); Bouvier et al. Meth. Enzymol. 248: 614 (1995),
Hardy et al., in Amyloid Protein Precursor in Development, Aging,
and Alzheimer's Disease, ed. Masters et al. pp. 190-198 (1994).
[0304] In a preferred embodiment, the peptide sequence
(AA.sup.1).sub.c is chosen based on its ability to be cleaved by a
lysosomal proteases, non-limiting examples of which include
cathepsins B, C, D, H, L and S. Preferably, the peptide sequence
(AA.sup.1).sub.c is capable of being cleaved by cathepsin B in
vitro.
[0305] In another embodiment, the peptide sequence (AA.sup.1).sub.c
is chosen based on its ability to be cleaved by a tumor-associated
protease, such as a protease found extracellularly in the vicinity
of tumor cells, examples of which include thimet oligopeptidase
(TOP) and CD10. In other embodiments, the sequence (AA.sup.1).sub.c
is designed for selective cleavage by urokinase or tryptase.
[0306] Suitable, but non-limiting, examples of peptide sequences
suitable for use in the conjugates of the invention include
Val-Cit, Cit-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit,
Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N.sup.9-tosyl-Arg,
Phe-N.sup.9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys,
Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ. ID NO:
40), .beta.-Ala-Leu-Ala-Leu (SEQ. ID NO: 41) and Gly-Phe-Leu-Gly
(SEQ. ID NO: 42) Val-Ala, Leu-Leu-Gly-Leu (SEQ. ID NO: 43),
Leu-Asn-Ala, and Lys-Leu-Val. Preferred peptides sequences are
Val-Cit and Val-Lys.
[0307] In another embodiment, the amino acid located the closest to
the drug moiety is selected from the group consisting of: Ala, Asn,
Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, and Val. In yet another embodiment, the amino acid
located the closest to the drug moiety is selected from the group
consisting of: Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
[0308] One of skill in the art can readily evaluate an array of
peptide sequences to determine their utility in the present
invention without resort to undue experimentation. See, for
example, Zimmerman, M., et al., (1977) Analytical Biochemistry
78:47-51; Lee, D., et al., (1999) Bioorganic and Medicinal
Chemistry Letters 9:1667-72; and Rano, T. A., et al., (1997)
Chemistry and Biology 4:149-55.
[0309] A conjugate of this invention may optionally contain two or
more linkers. These linkers may be the same or different. For
example, a peptidyl linker may be used to connect the drug to the
ligand and a second peptidyl linker may attach a diagnostic agent
to the complex. Other uses for additional linkers include linking
analytical agents, biomolecules, targeting agents, and detectable
labels to the antibody-partner complex.
Hydrazine Linkers (H)
[0310] In another embodiment, the conjugate of the invention
comprises a hydrazine self-immolative linker, wherein the conjugate
has the structure:
X.sup.4-(L.sup.4).sub.p-H-(L.sup.1).sub.m-D
wherein D, L.sup.1, L.sup.4, p, m, and X.sup.4 are as defined above
and described further herein, and H is a linker comprising the
structure:
##STR00012##
wherein n.sub.1 is an integer from 1-10; n.sub.2 is 0, 1, or 2;
each R.sup.24 is a member independently selected from the group
consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl; and I is
either a bond (i.e., the bond between the carbon of the backbone
and the adjacent nitrogen) or:
##STR00013##
wherein n.sub.3 is 0 or 1, with the proviso that when n.sub.3 is 0,
n.sub.2 is not 0; and n.sub.4 is 1, 2, or 3.
[0311] In one embodiment, the substitution on the phenyl ring is a
para substitution. In preferred embodiments, n.sub.1 is 2, 3, or 4
or n.sub.1 is 3. In preferred embodiments, n.sub.2 is 1. In
preferred embodiments, I is a bond (i.e., the bond between the
carbon of the backbone and the adjacent nitrogen). In one aspect,
the hydrazine linker, H, can form a 6-membered self immolative
linker upon cleavage, for example, when n.sub.3 is 0 and n.sub.4 is
2. In another aspect, the hydrazine linker, H, can form two
5-membered self immolative linkers upon cleavage. In yet other
aspects, H forms a 5-membered self immolative linker, H forms a
7-membered self immolative linker, or H forms a 5-membered self
immolative linker and a 6-membered self immolative linker, upon
cleavage. The rate of cleavage is affected by the size of the ring
formed upon cleavage. Thus, depending upon the rate of cleavage
desired, an appropriate size ring to be formed upon cleavage can be
selected.
[0312] Another hydrazine structure, H, has the formula:
##STR00014##
where q is 0, 1, 2, 3, 4, 5, or 6; and each R.sup.24 is a member
independently selected from the group consisting of H, substituted
alkyl, unsubstituted alkyl, substituted heteroalkyl, and
unsubstituted heteroalkyl. This hydrazine structure can also form
five-, six-, or seven-membered rings and additional components can
be added to form multiple rings.
[0313] The preparation, cleavage chemistry and cyclization kinetics
of the various hydrazine linkers is disclosed in WO 2005/112919,
the disclosure of which is incorporated herein by reference.
Disulfide Linkers (J)
[0314] In yet another embodiment, the linker comprises an
enzymatically cleavable disulfide group. In one embodiment, the
invention provides a cytotoxic antibody-partner compound having a
structure according to Formula (d):
##STR00015##
wherein D, L.sup.1, L.sup.4, p, m, and X.sup.4 are as defined above
and described further herein, and J is a disulfide linker
comprising a group having the structure:
##STR00016##
wherein each R.sup.24 is a member independently selected from the
group consisting of H, substituted alkyl, unsubstituted alkyl,
substituted heteroalkyl, and unsubstituted heteroalkyl; each K is a
member independently selected from the group consisting of
substituted alkyl, unsubstituted alkyl, substituted heteroalkyl,
unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,
substituted heteroaryl, unsubstituted heteroaryl, substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.21R.sup.22, NR.sup.21COR.sup.22,
OCONR.sup.21R.sup.22, OCOR.sup.21, and OR.sup.21 wherein R.sup.21
and R.sup.22 are independently selected from the group consisting
of H, substituted alkyl, unsubstituted alkyl, substituted
heteroalkyl, unsubstituted heteroalkyl, substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted
heteroaryl, substituted heterocycloalkyl and unsubstituted
heterocycloalkyl; i is an integer of 0, 1, 2, 3, or 4; and d is an
integer of 0, 1, 2, 3, 4, 5, or 6.
[0315] The aromatic ring of a disulfides linker can be substituted
with one or more "K" groups. A "K" group is a substituent on the
aromatic ring that replaces a hydrogen otherwise attached to one of
the four non-substituted carbons that are part of the ring
structure. The "K" group may be a single atom, such as a halogen,
or may be a multi-atom group, such as alkyl, heteroalkyl, amino,
nitro, hydroxy, alkoxy, haloalkyl, and cyano. Exemplary K
substituents independently include, but are not limited to, F, Cl,
Br, I, NO.sub.2, OH, OCH.sub.3, NHCOCH.sub.3, N(CH.sub.3).sub.2,
NHCOCF.sub.3 and methyl. For "K.sub.i", i is an integer of 0, 1, 2,
3, or 4. In a specific embodiment, i is 0.
[0316] In a preferred embodiment, the linker comprises an
enzymatically cleavable disulfide group of the following
formula:
##STR00017##
wherein L.sup.4, X.sup.4, p, and R.sup.24 are as described above,
and d is 0, 1, 2, 3, 4, 5, or 6. In a particular embodiment, d is 1
or 2.
[0317] A more specific disulfide linker is shown in the formula
below:
##STR00018##
[0318] Preferably, d is 1 or 2 and each K is H.
[0319] Another disulfide linker is shown in the formula below:
##STR00019##
[0320] Preferably, d is 1 or 2 and each K is H.
[0321] In various embodiments, the disulfides are ortho to the
amine. In another specific embodiment, a is 0. In preferred
embodiments, R.sup.24 is independently selected from H and
CH.sub.3.
[0322] The preparation and chemistry of disulfide linkers such as
those described above is disclosed in WO 2005/112919, the
disclosure of which is incorporated herein by reference.
[0323] For further discussion of types of cytotoxins, linkers and
other methods for conjugating therapeutic agents to antibodies, see
also U.S. Pat. No. 7,087,600; U.S. Pat. No. 6,989,452; U.S. Pat.
No. 7,129,261; US 2006/0004081; US 2006/0247295; WO 02/096910; WO
2007/051081; WO 2005/112919; WO 2007/059404; WO 2008/083312; PCT
application no. PCT/US2008/054362; Saito et al. (2003) Adv. Drug
Deliv. Rev. 55:199-215; Trail et al. (2003) Cancer Immunol.
Immunother. 52:328-337; Payne. (2003) Cancer Cell 3:207-212; Allen
(2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman (2002) Curr.
Opin. Investig. Drugs 3:1089-1091; Senter and Springer (2001) Adv.
Drug Deliv. Rev. 53:247-264, each of which is hereby incorporated
by reference in its entirety.
Cytotoxins as Partner Molecules
[0324] In one aspect, the present invention features an antibody
conjugated to a partner molecule, such as a cytotoxin, a drug
(e.g., an immunosuppressant) or a radiotoxin. Such conjugates are
also referred to as "immunotoxins." A cytotoxin or cytotoxic agent
includes any agent that is detrimental to (e.g., kills) cells.
[0325] Examples of partner molecules of the present invention
include taxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Examples of partner molecules also include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan,
tubulysin, dibromomannitol, streptozotocin, mitomycin C, cisplatin,
anthracyclines (e.g., daunorubicin (formerly daunomycin) and
doxorubicin), antibiotics (e.g., dactinomycin (formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and
anti-mitotic agents (e.g., vincristine and vinblastine). Other
preferred examples of partner molecules that can be conjugated to
an antibody of the invention include calicheamicins, maytansines
and auristatins, and derivatives thereof.
[0326] Preferred examples of partner molecule are analogs and
derivatives of CC-1065 and the structurally related duocarmycins.
Despite its potent and broad antitumor activity, CC-1065 cannot be
used in humans because it causes delayed death in experimental
animals, prompting a search for analogs or derivatives with a
better therapeutic index.
[0327] Many analogues and derivatives of CC-1065 and the
duocarmycins are known in the art. The research into the structure,
synthesis and properties of many of the compounds has been
reviewed. See, for example, Boger et al., Angew. Chem. Int. Ed.
Engl. 35: 1438 (1996); and Boger et al., Chem. Rev. 97: 787 (1997).
Other disclosures relating to CC-1065 analogs or derivatives
include: U.S. Pat. No. 5,101,038; U.S. Pat. No. 5,641,780; U.S.
Pat. No. 5,187,186; U.S. Pat. No. 5,070,092; U.S. Pat. No.
5,703,080; U.S. Pat. No. 5,070,092; U.S. Pat. No. 5,641,780; U.S.
Pat. No. 5,101,038; U.S. Pat. No. 5,084,468; U.S. Pat. No.
5,739,350; U.S. Pat. No. 4,978,757, U.S. Pat. No. 5,332,837 and
U.S. Pat. No. 4,912,227; WO 96/10405; and EP 0,537,575 A1
[0328] In a particularly preferred aspect, the partner molecule is
a CC-1065/duocarmycin analog having a structure according to the
following formula (e):
##STR00020##
in which ring system A is a member selected from substituted or
unsubstituted aryl substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl groups. Exemplary
ring systems A include phenyl and pyrrole.
[0329] The symbols E and G are independently selected from H,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, a heteroatom, a single bond or E and G are optionally
joined to form a ring system selected from substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl and
substituted or unsubstituted heterocycloalkyl.
[0330] The symbol X represents a member selected from O, S and
NR.sup.23. R.sup.23 is a member selected from H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and
acyl.
[0331] The symbol R.sup.3 represents a member selected from
(.dbd.O), SR.sup.11, NHR.sup.11 and OR.sup.11, in which R.sup.11 is
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, monophosphates, diphosphates, triphosphates,
sulfonates, acyl, C(O)R.sup.12R.sup.13, C(O)OR.sup.12,
C(O)NR.sup.12R.sup.13, P(O)(OR.sup.12).sub.2,
C(O)CHR.sup.12R.sup.13, SR.sup.12 or SiR.sup.12R.sup.13R.sup.14.
The symbols R.sup.12, R.sup.13, and R.sup.14 independently
represent H, substituted or unsubstituted alkyl, substituted or
unsubstituted heteroalkyl and substituted or unsubstituted aryl,
where R.sup.12 and R.sup.13 together with the nitrogen or carbon
atom to which they are attached are optionally joined to form a
substituted or unsubstituted heterocycloalkyl ring system having
from 4 to 6 members, optionally containing two or more
heteroatoms.
[0332] R.sup.4, R.sup.4', R.sup.5 and R.sup.5' are members
independently selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen,
NO.sub.2, NR.sup.15R.sup.16, NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16,
OC(O)OR.sup.15, C(O)R.sup.15, SR.sup.15, OR.sup.15,
CR.sup.15.dbd.NR.sup.16, and O(CH.sub.2).sub.nN(CH.sub.3).sub.2,
where n is an integer from 1 to 20, or any adjacent pair of
R.sup.4, R.sup.4', R.sup.5 and R.sup.5', together with the carbon
atoms to which they are attached, are joined to form a substituted
or unsubstituted cycloalkyl or heterocycloalkyl ring system having
from 4 to 6 members. R.sup.15 and R.sup.16 independently represent
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl and substituted or unsubstituted peptidyl, where
R.sup.15 and R.sup.16 together with the nitrogen atom to which they
are attached are optionally joined to form a substituted or
unsubstituted heterocycloalkyl ring system having from 4 to 6
members, optionally containing two or more heteroatoms. One
exemplary structure is aniline.
[0333] One of R.sup.3, R.sup.4, R.sup.4', R.sup.5, and R.sup.5'
joins the cytotoxin to a linker or enzyme cleavable substrate of
the present invention, as described herein, for example to L.sup.1
or L.sup.3, if present or to F, H, or J.
[0334] R.sup.6 is a single bond which is either present or absent.
When R.sup.6 is present, R.sup.6 and R.sup.7 are joined to form a
cyclopropyl ring. R.sup.7 is CH.sub.2--X.sup.1 or --CH.sub.2--.
When R.sup.7 is --CH.sub.2-- it is a component of the cyclopropane
ring. The symbol X.sup.1 represents a leaving group such as a
halogen, for example Cl, Br or F. The combinations of R.sup.6 and
R.sup.7 are interpreted in a manner that does not violate the
principles of chemical valence.
[0335] X.sup.1 may be any leaving group. Useful leaving groups
include, but are not limited to, halogens, azides, sulfonic esters
(e.g., alkylsulfonyl, arylsulfonyl), oxonium ions, alkyl
perchlorates, ammonioalkanesulfonate esters, alkylfluorosulfonates
and fluorinated compounds (e.g., triflates, nonaflates, tresylates)
and the like. Particular halogens useful as leaving groups are F,
Cl and Br.
[0336] The curved line within the six-membered ring indicates that
the ring may have one or more degrees of unsaturation, and it may
be aromatic. Thus, ring structures such as those set forth below,
and related structures, are within the scope of Formula (f):
##STR00021##
[0337] In one embodiment, R.sup.11 includes a moiety, X.sup.5, that
does not self-cyclize and links the drug to L.sup.1 or L.sup.3, if
present, or to F, H, or J. The moiety, X.sup.5, is preferably
cleavable using an enzyme and, when cleaved, provides the active
drug. As an example, R.sup.11 can have the following structure
(with the right side coupling to the remainder of the drug):
##STR00022##
[0338] In some embodiments, at least one of R.sup.4, R.sup.4',
R.sup.5, and R.sup.5' links said drug to L.sup.1, if present, or to
F, H, J, or X.sup.2, and R.sup.3 is selected from SR.sup.11,
NHR.sup.11 and OR.sup.11. R.sup.11 is selected from --SO(OH).sub.2,
--PO(OH).sub.2, -AA.sub.n, --Si(CH.sub.3).sub.2C(CH.sub.3).sub.3,
--C(O)OPhNH(AA).sub.m,
##STR00023##
or any other sugar or combination of sugars
##STR00024##
and pharmaceutically acceptable salts thereof, where n is any
integer in the range of 1 to 10, m is any integer in the range of 1
to 4, p is any integer in the range of 1 to 6, and AA is any
natural or non-natural amino acid. Where the compound of formula
(e) is conjugated via R.sup.4, R.sup.4', R.sup.5, or R.sup.6,
R.sup.3 preferably comprises a cleavable blocking group whose
presence blocks the cytotoxic activity of the compound but is
cleavable under conditions found at the intended site of action by
a mechanism different from that for cleavage of the linker
conjugating the cytotoxin to the antibody. In this way, if there is
adventitiouis cleavage of the conjugate in the plasma, the blocking
group attenuates the cytotoxicity of the released cytotoxin. For
instance, if the conjugate has a hydrazone or disulfide linker, the
blocking group can be an enzymatically cleavable amide. Or, if the
linker is a peptidyl one cleavable by a protease, the blocking
group can be an ester or carbamate cleavable by a
carboxyesterase.
[0339] For example, in a preferred embodiment, D is a cytotoxin
having a structure (j):
##STR00025##
[0340] In this structure, R.sup.3, R.sup.6, R.sup.7, R.sup.4,
R.sup.4', R.sup.5, R.sup.5' and X are as described above for
Formula (e). Z is a member selected from O, S and NR.sup.23, where
R.sup.23 is a member selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted heteroalkyl, and acyl.
[0341] R.sup.1 is H, substituted or unsubstituted lower alkyl,
C(O)R.sup.8, or CO.sub.2R.sup.8, wherein R.sup.8 is a member
selected from NR.sup.9R.sup.10 and OR.sup.9, in which R.sup.9 and
R.sup.10 are members independently selected from H, substituted or
unsubstituted alkyl and substituted or unsubstituted
heteroalkyl.
[0342] R.sup.1' is H, substituted or unsubstituted lower alkyl, or
C(O)R.sup.8, wherein R.sup.8 is a member selected from
NR.sup.9R.sup.10 and OR.sup.9, in which R.sup.9 and R.sup.10 are
members independently selected from H, substituted or unsubstituted
alkyl and substituted or unsubstituted heteroalkyl.
[0343] R.sup.2 is H, or substituted or unsubstituted lower alkyl or
unsubstituted heteroalkyl or cyano or alkoxy; and R.sup.2' is H, or
substituted or unsubstituted lower alkyl or unsubstituted
heteroalkyl.
[0344] One of R.sup.3, R.sup.4, R.sup.4', R.sup.5, or R.sup.5'
links the cytotoxin to L.sup.1 or L.sup.3, if present, or to F, H,
or.
[0345] A further embodiment has the formula:
##STR00026##
[0346] In this structure, A, R.sup.6, R.sup.7, X, R.sup.4,
R.sup.4', R.sup.5, and R.sup.5' are as described above for Formula
(e). Z is a member selected from O, S and NR.sup.23, where R.sup.23
is a member selected from H, substituted or unsubstituted alkyl,
substituted or unsubstituted heteroalkyl, and acyl;
[0347] R.sup.34 is C(.dbd.O)R.sup.33 or C.sub.1-C.sub.6 alkyl,
where R.sup.33 is selected from H, substituted or unsubstituted
alkyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl, halogen, NO.sub.2, NR.sup.15R.sup.16,
NC(O)R.sup.15, OC(O)NR.sup.15R.sup.16, OC(O)OR.sup.15,
C(O)R.sup.15, SR.sup.15, OR.sup.15, CR.sup.15.dbd.NR.sup.16, and
O(CH.sub.2).sub.nN(CH.sub.3).sub.2, where n is an integer from 1 to
20. R.sup.15 and R.sup.16 independently represent H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heterocycloalkyl and
substituted or unsubstituted peptidyl, where R.sup.15 and R.sup.16
together with the nitrogen atom to which they are attached are
optionally joined to form a substituted or unsubstituted
heterocycloalkyl ring system having from 4 to 6 members, optionally
containing two or more heteroatoms.
[0348] Preferably, A is substituted or unsubstituted phenyl or
substituted or unsubstituted pyrrole. Moreover, any selection of
substituents described herein for R.sup.11 is also applicable to
R.sup.33.
Markers as Partner Molecules
[0349] Where the partner molecule is a marker, it can be any moiety
having or generating a detectable physical or chemical property,
thereby indicating its presence in a particular tissue or cell.
Markers (sometimes also called reporter groups) have been well
developed in the area of immunoassays, biomedical research, and
medical diagnosis. A marker may be detected by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Examples include magnetic beads (e.g.,
DYNABEADS.TM.), fluorescent dyes (e.g., fluorescein isothiocyanate,
Texas red, rhodamine, and the like), radiolabels (e.g., .sup.3H,
.sup.125I, .sup.35S, .sup.4C, or .sup.32P), enzymes (e.g., horse
radish peroxidase, alkaline phosphatase and others commonly used in
an ELISA), and colorimetric labels such as colloidal gold or
colored glass or plastic beads (e.g., polystyrene, polypropylene,
latex, etc.).
[0350] The marker is preferably a member selected from the group
consisting of radioactive isotopes, fluorescent agents, fluorescent
agent precursors, chromophores, enzymes and combinations thereof.
Examples of suitable enzymes are horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, and glucose oxidase. Fluorescent
agents include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds
include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
For a review of various labeling or signal producing systems that
may be used, see, U.S. Pat. No. 4,391,904.
[0351] Markers can be attached by indirect means: a ligand molecule
(e.g., biotin) is covalently bound to an antibody. The ligand then
binds to another molecule (e.g., streptavidin), which is either
inherently detectable or covalently bound to a signal system, such
as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound.
Examples of Conjugates
[0352] Specific examples of partner molecule-linker combinations
suitable for conjugation to an antibody of this invention are shown
following:
##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031##
##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036##
[0353] In the foregoing compounds, where the subscript r is present
in a formula, it is an integer in the range of 0 to 24. R, wherever
it occurs, is
##STR00037##
[0354] Each of the foregoing compounds has a maleimide group and is
ready for conjugation to an antibody via a sulfhydryl group
thereon.
[0355] Conjugation to the antibodies of this invention can also be
effected by other types of chemical functionalities, as described
hereinabove in the context of bispecific molecules.
Pharmaceutical Compositions
[0356] In another aspect, the present disclosure provides a
pharmaceutical composition, comprising one or a combination of
monoclonal antibodies, or antigen-binding portion(s) thereof, of
the present disclosure, formulated together with a pharmaceutically
acceptable carrier.
[0357] Pharmaceutical compositions of this disclosure also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-mesothelin antibody of the present disclosure combined with at
least one other anti-cancer agent. Examples of therapeutic agents
that can be used in combination therapy are described in greater
detail below.
[0358] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody,
immunoconjugate, or bispecific molecule, may be coated in a
material to protect the compound from the action of acids and other
natural conditions that may inactivate it.
[0359] The pharmaceutical compounds of this disclosure may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects. Examples of such salts include
acid addition salts and base addition salts. Acid addition salts
include those derived from nontoxic inorganic acids, such as
hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0360] A pharmaceutical composition of this disclosure also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0361] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of this
disclosure include water, ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable
organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants.
[0362] The compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol, sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents that delay absorption such as aluminum mono
stearate and gelatin.
[0363] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of this disclosure is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0364] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride.
[0365] Sterile injectable solutions can be prepared by
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) to yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0366] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0367] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of this disclosure are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0368] For administration of an antibody of this invention, the
dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01
to 5 mg/kg, of the host body weight. For example dosages can be 0.3
mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5
mg/kg body weight or 10 mg/kg body weight or within the range of
1-10 mg/kg. An exemplary treatment regime entails administration
once per week, once every two weeks, once every three weeks, once
every four weeks, once a month, once every 3 months or once every
three to 6 months. Preferred dosage regimens include 1 mg/kg body
weight or 3 mg/kg body weight via intravenous administration, with
the antibody being given using one of the following dosing
schedules: (i) every four weeks for six dosages, then every three
months; (ii) every three weeks; (iii) 3 mg/kg body weight once
followed by 1 mg/kg body weight every three weeks.
[0369] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/mL and in some methods about 25-300 .mu.g/mL.
[0370] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated, and preferably until the patient shows
partial or complete amelioration of symptoms of disease.
Thereafter, the patient can be administered a prophylactic
regime.
[0371] For use in the prophylaxis and/or treatment of diseases
related to abnormal cellular proliferation, a circulating
concentration of administered compound of about 0.001 .mu.M to 20
.mu.M is preferred, with about 0.01 .mu.M to 5 .mu.M being
preferred.
[0372] Patient doses for oral administration of the compounds
described herein, typically range from about 1 mg/day to about
10,000 mg/day, more typically from about 10 mg/day to about 1,000
mg/day, and most typically from about 50 mg/day to about 500
mg/day. Stated in terms of patient body weight, typical dosages
range from about 0.01 to about 150 mg/kg/day, more typically from
about 0.1 to about 15 mg/kg/day, and most typically from about 1 to
about 10 mg/kg/day, for example 5 mg/kg/day or 3 mg/kg/day.
[0373] In at least some embodiments, patient doses that retard or
inhibit tumor growth can be 1 mmol/kg/day or less. For example, the
patient doses can be 0.9, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, or 0.1
mmol/kg/day or less (referring to moles of the drug). Preferably,
the antibody-drug conjugate retards growth of the tumor when
administered in the daily dosage amount over a period of at least
five days.
[0374] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present disclosure may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions, or the ester, salt or
amide thereof, the route of administration, the time of
administration, the rate of excretion of the particular compound
being employed, the duration of the treatment, other drugs,
compounds and/or materials used in combination with the particular
compositions employed, the age, sex, weight, condition, general
health and prior medical history of the patient, and like factors
well known in the medical arts.
[0375] A "therapeutically effective dosage" of an anti-mesothelin
antibody of this disclosure preferably results in a decrease in
severity of disease symptoms, an increase in frequency and duration
of disease symptom-free periods, or a prevention of impairment or
disability due to the disease affliction. For example, for the
treatment of tumor-bearing subjects, a "therapeutically effective
dosage" preferably inhibits tumor growth by at least about 20%,
more preferably by at least about 40%, even more preferably by at
least about 60%, and still more preferably by at least about 80%
relative to untreated subjects. The ability of a compound to
inhibit tumor growth can be evaluated in an animal model system
predictive of efficacy in human tumors. Alternatively, this
property of a composition can be evaluated by examining the ability
of the compound to inhibit cell growth, such inhibition can be
measured in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
[0376] A composition of the present disclosure can be administered
via one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Preferred routes of
administration for antibodies of this disclosure include
intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration,
for example by injection or infusion. The phrase "parenteral
administration" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0377] Alternatively, an antibody of this disclosure can be
administered via a non-parenteral route, such as a topical,
epidermal or mucosal route of administration, for example,
intranasally, orally, vaginally, rectally, sublingually or
topically.
[0378] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0379] Therapeutic compositions can be administered with medical
devices known in the art. For example, a therapeutic composition of
this disclosure can be administered with a needleless hypodermic
injection device, such as those disclosed in U.S. Pat. Nos.
5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;
or 4,596,556. Examples of implants and modules useful in the
present disclosure include: U.S. Pat. No. 4,487,603 (implantable
micro-infusion pump for dispensing medication at a controlled
rate); U.S. Pat. No. 4,486,194 (therapeutic device for
administering medicants through the skin); U.S. Pat. No. 4,447,233
(infusion pump for delivering medication at a precise infusion
rate); U.S. Pat. No. 4,447,224 (variable flow implantable infusion
apparatus for continuous drug delivery); U.S. Pat. No. 4,439,196
(osmotic drug delivery system having multi-chamber compartments);
and U.S. Pat. No. 4,475,196 (osmotic drug delivery system). These
patents are incorporated herein by reference. Many other implants,
delivery systems, and modules are known to those skilled in the
art.
[0380] The antibodies of this disclosure can be formulated to
ensure proper distribution in vivo. For example, the blood-brain
barrier (BBB) excludes many highly hydrophilic compounds. To ensure
that the therapeutic compounds of this disclosure cross the BBB (if
desired), they can be formulated, for example, in liposomes. For
methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos.
4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one
or more moieties which are selectively transported into specific
cells or organs, thus enhance targeted drug delivery (see, e.g.,
Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting
moieties include folate or biotin (see, e.g., U.S. Pat. No.
5,416,016 to Low et al.); mannosides (Umezawa et al., (1988)
Biochem. Biophys. Res. Commun. 153:1038); antibodies (Bloeman et
al. (1995) FEBS Lett. 357:140; Owais et al. (1995) Antimicrob.
Agents Chemother. 39:180); surfactant protein A receptor (Briscoe
et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al.
(1994) J. Biol. Chem. 269:9090); see also Keinanen and Laukkanen
(1994) FEBS Lett. 346:123; Killion and Fidler (1994) Immunomethods
4:273.
Uses and Methods of the Invention
[0381] The antibodies, particularly the human antibodies, antibody
compositions, antibody-partner molecule conjugate compositions and
methods of the present invention have numerous in vitro and in vivo
diagnostic and therapeutic utilities involving the diagnosis and
treatment of mesothelin-mediated disorders. For example, these
molecules can be administered to cells in culture, in vitro or ex
vivo, or to human subjects, to treat, prevent and to diagnose a
variety of disorders. As used herein, the term "subject" is
intended to include human and non-human animals. Non-human animals
include all vertebrates, e.g., mammals and non-mammals, such as
non-human primates, sheep, dogs, cats, cows, horses, chickens,
amphibians, and reptiles. Preferred subjects include human patients
having disorders mediated by mesothelin activity, particularly
human patients having a disorder associated with aberrant
mesothelin expression. When antibody-partner molecule conjugates to
mesothelin are administered together with another agent, the two
can be administered in either order or simultaneously.
[0382] Given the specific binding of the antibodies of the
invention for mesothelin, the antibodies of the invention can be
used to specifically detect mesothelin expression and, moreover,
can be used to purify mesothelin via immunoaffinity
purification.
[0383] In one embodiment, the compositions of the invention can be
used to detect levels of mesothelin, which levels can then be
linked to certain disease symptoms. Alternatively, the compositions
can be used to inhibit or block mesothelin function which, in turn,
can be linked to the prevention or amelioration of certain disease
symptoms, thereby implicating mesothelin as a mediator of the
disease. This can be achieved by contacting a sample and a control
sample with the anti-mesothelin antibody under conditions that
allow for the formation of a complex between the antibody and
mesothelin. Any complexes formed between the antibody and
mesothelin are detected and compared in the sample and the
control.
[0384] In another embodiment, the compositions of the invention can
be initially tested for binding activity associated with
therapeutic or diagnostic use in vitro. For example, compositions
of the invention can be tested using flow cytometric assays known
in the art.
[0385] The compositions of the invention have additional utility in
therapy and diagnosis of mesothelin-related diseases. For example,
the immunoconjugates can be used to elicit in vivo or in vitro one
or more of the following biological activities: to inhibit the
growth of and/or kill a cell expressing mesothelin; to mediate
phagocytosis or ADCC of a cell expressing mesothelin in the
presence of human effector cells, or to block mesothelin ligand
binding to mesothelin.
[0386] In a particular embodiment, the compositions are used in
vivo to treat, prevent or diagnose a variety of mesothelin-related
diseases. Examples of mesothelin-related diseases include, among
others, ovarian, pancreatic, stomach, lung, uterine, endometrial,
bile duct, gastric/esophageal, colorectal, and breast cancers.
[0387] The compositions of the invention can comprise agents
including, among others, anti-neoplastic agents such as doxorubicin
(adriamycin), cisplatin, bleomycin sulfate, carmustine,
chlorambucil, cyclophosphamide and hydroxyurea which, by
themselves, are only effective at levels which are toxic or
subtoxic to a patient. Cisplatin is intravenously administered as a
100 mg/kg dose once every four weeks and adriamycin is
intravenously administered as a 60-75 mg/mL dose once every 21
days. Co-administration of the human anti-mesothelin antibodies, or
antigen binding fragments thereof, of the present invention with
chemotherapeutic agents provides two anti-cancer agents which
operate via different mechanisms and yield a cytotoxic effect to
human tumor cells. Such co-administration can solve problems due to
development of resistance to drugs or a change in the antigenicity
of the tumor cells, which would render them unreactive with the
antibody.
[0388] Bispecific and multispecific molecules of the invention can
also be used to modulate Fc.gamma.R or Fc.gamma.R levels on
effector cells, such as by capping and elimination of receptors on
the cell surface. Mixtures of anti-Fc receptors can also be used
for this purpose.
[0389] The compositions of the invention which have complement
binding sites, such as portions from IgG1, -2, or -3 or IgM which
bind complement, can also be used in the presence of complement. In
one embodiment, ex vivo treatment of a population of cells
comprising target cells with a binding agent of the invention and
appropriate effector cells can be supplemented by the addition of
complement or serum containing complement. Phagocytosis of target
cells coated with a binding agent of the invention can be improved
by binding of complement proteins. In another embodiment target
cells coated with the compositions (e.g., human antibodies,
multispecific and bispecific molecules) of the invention can also
be lysed by complement. In yet another embodiment, the compositions
of the invention do not activate complement.
[0390] The compositions of the invention can also be administered
together with complement. Thus, the instant disclosure provides
compositions comprising human antibodies, multispecific or
bispecific molecules and serum or complement. These compositions
can be advantageous when the complement is located in close
proximity to the human antibodies, multispecific or bispecific
molecules. Alternatively, the human antibodies, multispecific or
bispecific molecules of the invention and the complement or serum
can be administered separately.
[0391] Also within the scope of the present invention are kits
comprising the antibody compositions of the invention and
instructions for use. The kit can further contain one ore more
additional reagents, such as an immunosuppressive reagent, a
cytotoxic agent or a radiotoxic agent, or one or more additional
human antibodies of the invention (e.g., a human antibody having a
complementary activity which binds to an epitope in the mesothelin
antigen distinct from the first human antibody).
[0392] Accordingly, patients treated with antibody compositions of
the invention can be additionally administered (prior to,
simultaneously with, or following administration of a composition
of the invention) with another therapeutic agent, such as a
cytotoxic or radiotoxic agent, which enhances or augments the
therapeutic effect of the composition.
[0393] In other embodiments, the subject can be additionally
treated with an agent that modulates, e.g., enhances or inhibits,
the expression or activity of Fc.gamma. or Fc.gamma. receptors by,
for example, treating the subject with a cytokine. Preferred
cytokines for administration during treatment with the
multispecific molecule include of granulocyte colony-stimulating
factor (G-CSF), granulocyte-macrophage colony-stimulating factor
(GM-CSF), interferon-.gamma. (IFN-.gamma.), and tumor necrosis
factor (TNF).
[0394] In a particular embodiment, the invention provides methods
for detecting the presence of mesothelin antigen in a sample, or
measuring the amount of mesothelin antigen, comprising contacting
the sample, and a control sample, with a human monoclonal antibody,
or an antigen binding portion thereof, which specifically binds to
mesothelin, under conditions that allow for formation of a complex
between the antibody or portion thereof and mesothelin. The
formation of a complex is then detected, wherein a difference in
complex formation between the sample compared to the control sample
is indicative the presence of mesothelin antigen in the sample.
[0395] In other embodiments, the invention provides methods for
treating a mesothelin-mediated disorder in a subject, e.g.,
ovarian, pancreatic, stomach, lung, uterine, endometrial, bile
duct, gastric/esophageal, colorectal, and breast cancers.
[0396] In yet another embodiment, immunoconjugates of the invention
can be used to partner molecules to cells that express mesothelin
by linking such partner molecules to the antibody. For example, an
anti-mesothelin antibody can be conjugated to any of the toxin
compounds described in U.S. Pat. Nos. 6,281,354; 6,548,530;
2003/0050331; 2003/0064984; 2003/0073852; and 2004/0087497; or WO
03/022806. Thus, the invention also provides methods for localizing
ex vivo or in vivo cells expressing mesothelin (e.g., with a
detectable label, such as a radioisotope, a fluorescent compound,
an enzyme, or an enzyme co-factor). Alternatively, the
immunoconjugates can be used to kill cells that have mesothelin
cell surface receptors by targeting cytotoxins or radiotoxins
conjugated to mesothelin-binding antibodies
[0397] In view of the association of mesothelin with certain
tumors, the invention provides a method of inhibiting the growth of
a mesothelin-expressing tumor cell, comprising contacting the tumor
cell with an anti-mesothelin antibody of the disclosure such that
growth of the tumor cells is inhibited. In a preferred embodiment,
the antibody is conjugated to a therapeutic agent, such as a
cytotoxin. In particularly preferred embodiments, the
mesothelin-expressing tumor cell is a mesothelioma cell, or a tumor
cell associated with ovarian, pancreatic, stomach, lung, uterine,
endometrial, bile duct, gastric/esophageal, colorectal, and breast
cancers. In other preferred embodiments, the mesothelin-expressing
tumor cell is a mesothelioma cell, a pancreatic tumor cell, an
ovarian tumor cell, a stomach tumor cell, a lung tumor cell or an
endometrial tumor cell. In still other embodiments, the tumor cell
is from a cancer selected from the group consisting of
mesotheliomas, papillary serous ovarian adenocarcinomas, clear cell
ovarian carcinomas, mixed Mullerian ovarian carcinomas, endometroid
mucinous ovarian carcinomas, pancreatic adenocarcinomas, ductal
pancreatic adenocarcinomas, uterine serous carcinomas, lung
adenocarcinomas, extrahepatic bile duct carcinomas, gastric
adenocarcinomas, esophageal adenocarcinomas, colorectal
adenocarcinomas and breast adenocarcinomas.
[0398] Furthermore, in another aspect, the anti-mesothelin
antibodies and antibody drug conjugates thereof can be used in the
treatment of cancer in a subject. Accordingly, the invention
provides a method of treating cancer in a subject comprising
administering to the subject an antibody of the disclosure such
that the cancer is treated in the subject. For example, in a
particularly preferred embodiment, the antibodies can be used in
the treatment of mesotheliomas, ovarian cancers or pancreatic
cancers. In other preferred embodiments, the antibodies are used in
the treatment of mesotheliomas, pancreatic cancers, ovarian
cancers, stomach cancers, lung cancers or endometrial cancers. In
still other embodiments, the antibodies are used in the treatment
of a cancer selected from the group consisting of mesotheliomas,
papillary serous ovarian adenocarcinomas, clear cell ovarian
carcinomas, mixed Mullerian ovarian carcinomas, endometroid
mucinous ovarian carcinomas, pancreatic adenocarcinomas, ductal
pancreatic adenocarcinomas, uterine serous carcinomas, lung
adenocarcinomas, extrahepatic bile duct carcinomas, gastric
adenocarcinomas, esophageal adenocarcinomas, colorectal
adenocarcinomas and breast adenocarcinomas.
[0399] The antibody can be used alone or in combination other
cancer treatments, such as surgery and/or radiation, and/or with
other anti-neoplastic agents, such as the anti-neoplastic agents
discussed and set forth above, including chemotherapeutic drugs and
other anti-tumor antigen antibodies, such as those that bind CD20,
Her2, PSMA, Campath-1, EGFR and the like.
[0400] Optionally, antibodies to mesothelin can be combined with an
immunogenic agent, such as a vaccine comprising cancerous cells,
purified tumor antigens (including recombinant proteins, peptides,
and carbohydrate molecules), cells, and cells transfected with
genes encoding immune stimulating cytokines (He et al (2004) J.
Immunol. 173:4919-28). Many experimental strategies for vaccination
against tumors have been devised (see Rosenberg, S., 2000,
Development of Cancer Vaccines, ASCO Educational Book Spring:
60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302;
Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K.
2000, ASCO Educational Book Spring: 730-738; see also Restifo, N.
and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita, V.
et al. (eds.), 1997, Cancer: Principles and Practice of Oncology.
Fifth Edition). In one of these strategies, a vaccine is prepared
using autologous or allogeneic tumor cells. These cellular vaccines
have been shown to be most effective when the tumor cells are
transduced to express GM-CSF. GM-CSF has been shown to be a potent
activator of antigen presentation for tumor vaccination (Dranoff et
al. (1993) Proc. Natl. Acad. Sci. USA. 90: 3539-43).
[0401] Other tumor vaccines may include the proteins from viruses
implicated in human cancers such a Human Papilloma Viruses (HPV),
Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus
(KHSV). Another form of tumor specific antigen which may be used in
as a tumor vaccine is purified heat shock proteins (HSP) isolated
from the tumor tissue itself. These heat shock proteins contain
fragments of proteins from the tumor cells and these HSPs are
highly efficient at delivery to antigen presenting cells for
eliciting tumor immunity (Suot, R & Srivastava, P (1995)
Science 269:1585-1588; Tamura, Y. et al. (1997) Science
278:117-120).
[0402] Dendritic cells (DC) are potent antigen presenting cells
that can be used to prime antigen-specific responses. DC's can be
produced ex vivo and loaded with various protein and peptide
antigens as well as tumor cell extracts (Nestle, F. et al. (1998)
Nature Medicine 4: 328-332). DCs may also be transduced by genetic
means to express these tumor antigens as well. DCs have also been
fused directly to tumor cells for the purposes of immunization
(Kugler, A. et al. (2000) Nature Medicine 6:332-336). As a method
of vaccination, DC immunization may be effectively combined with
anti-mesothelin antibody treatment to activate anti-tumor
responses.
[0403] The use of anti-mesothelin antibodies may also be combined
with standard cancer treatments. Anti-mesothelin antibody treatment
may be effectively combined with chemotherapeutic regimes. In these
instances, it may be possible to reduce the dose of
chemotherapeutic reagent administered (Mokyr, M. et al. (1998)
Cancer Research 58: 5301-5304). Other combination therapies that
may result in synergy with anti-mesothelin antibody treatment are
radiation, surgery, and hormone deprivation. Angiogenesis
inhibitors may also be combined with anti-mesothelin antibody
treatment.
[0404] Anti-mesothelin antibody treatment can also be used in
combination with bispecific antibodies that target Fc alpha or Fc
gamma receptor-expressing effectors cells to tumor cells (see,
e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243). For example anti-Fc
receptor/anti tumor antigen (e.g., Her-2/neu) bispecific antibodies
have been used to target macrophages to sites of tumor. This
targeting may more effectively activate tumor specific responses.
Alternatively, antigen may be delivered directly to DCs by the use
of bispecific antibodies which bind to tumor antigen and a
dendritic cell specific cell surface marker.
[0405] Tumors evade host immune surveillance by a large variety of
mechanisms. Many of these mechanisms may be overcome by the
inactivation of immunosuppressive proteins expressed by the tumors.
These include TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163:
1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology
Today 13: 198-200), and Fas ligand (Hahne, M. et al. (1996) Science
274: 1363-1365). Antibodies to each of these entities may be used
in combination with anti-mesothelin to counteract the effects of
the immunosuppressive agent and favor tumor immune responses by the
host.
[0406] Other antibodies which may be used to activate host immune
responsiveness can be used in combination with anti-mesothelin.
These include molecules on the surface of dendritic cells which
activate DC function and antigen presentation. Anti-CD40 antibodies
are able to substitute effectively for T cell helper activity
(Ridge, J. et al. (1998) Nature 393: 474-478) and can be used in
conjunction with anti-mesothelin antibodies (Ito, N. et al. (2000)
Immunobiology 201 (5) 527-40). Antibodies to T cell costimulatory
molecules such as CTLA-4 (e.g., U.S. Pat. Nos. 5,811,097, and
6,984,720), OX-40 (Weinberg, A. et al. (2000) Immunol 164:
2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine 3:
682-685 (1997), ICOS (Hutloff, A. et al. (1999) Nature 397:
262-266), PD-1 and/or PD-L1 may also provide for increased levels
of T cell activation and thus, such T cell activating antibodies
can be used in combination with the anti-mesothelin antibodies of
the invention.
[0407] Bone marrow transplantation is currently being used to treat
a variety of tumors of hematopoietic origin. While graft versus
host disease is a consequence of this treatment, therapeutic
benefit may be obtained from graft vs. tumor responses. Thus,
anti-mesothelin antibody treatment can be used in combination with
bone marrow transplantation as part of a tumor treatment
regimen.
[0408] The present disclosure is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of all figures and all references, Genbank
sequences, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference in their entirety.
Example 1
Generation of Human Monoclonal Antibodies Against Mesothelin
Protein
[0409] Anti-mesothelin human monoclonal antibodies were generated
using transgenic mice that express human antibody genes, as
follows.
Antigen
[0410] A mesothelin soluble fusion protein was used as the antigen
for immunization. The soluble fusion protein was composed of the 40
kD portion of mesothelin (which is membrane-associated via a
GPI-linkage in native mesothelin) linked to a His tag at its
C-terminus. This fusion protein is referred to herein as "hu
mesothelin-his fusion protein". The fusion protein was generated by
standard recombinant DNA methods and expressed in transfected CHO
cells, which secreted the soluble fusion protein into the culture
supernatant. The CHO host cells used for transfection were obtained
from Invitrogen (Cat #11619-012). The secreted soluble fusion
protein was purified for use as immunogen. Furthermore, supernatant
from the transfected CHO cells secreting hu mesothelin-his fusion
protein was used in the ELISA assays for screening HuMAbs
(described further below).
Transgenic Mice
[0411] Fully human monoclonal antibodies to human mesothelin were
prepared using mice from the HCo7.times.hu Lamda strain of a
transgenic mouse of the HuMab Mouse.TM. type ("Hco7.times.hu
mouse"). In this strain, the endogenous mouse kappa light chain
gene has been homozygously disrupted as described in Chen et al.
(1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene
has been homozygously disrupted as described in Example 1 of WO
2001/09187. Furthermore, this mouse strain carries a human kappa
light chain transgene, KCo5, as described in Fishwild et al. (1996)
Nature Biotechnology 14:845-851, a yeast artificial chromosome
(YAC) carrying most of the human lambda light chain locus, as
described in WO 2000/026373, and a human heavy chain transgene,
HCo7, as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and
5,545,807.
Immunization of Mice
[0412] To generate fully human monoclonal antibodies to human
mesothelin, HCo7.times.hu mice were immunized with purified hu
mesothelin-his fusion protein. General immunization schemes are
described in Lonberg, N. et al (1994) Nature 368(6474): 856-859;
Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO
98/24884. The mice were 3-4 months of age upon the first infusion
of antigen. A purified recombinant human mesothelin-his antigen
preparation (10 .mu.g purified from transfected mammalian cells
expressing the fusion protein) was used to immunize the mice
intraperitonealy and subcutaneously. The antigen was mixed 1:1 with
RIBI adjuvant (Sigma Cat#M6536).
[0413] The mice were immunized 9 times at weekly intervals. The
immune response was monitored by retro orbital bleeds. The plasma
was screened by an indirect ELISA analysis (as described below),
and mice with highest titers for anti-human mesothelin human IgG
were used for fusions. Mice received a final boost by intravenous
(IV) and intraperitoneal (IP) injection of soluble antigen 2 and 3
days before sacrifice and removal of the spleen.
Selection of Mice Producing Anti-Human Mesothelin Human Monoclonal
Antibodies
[0414] To select the Hco7.times.hu mice producing high titers of
anti-mesothelin antibodies, an indirect ELISA assay was used to
monitor serum antibody titers, as follows. His-tag antibody coated
microtiter plates (Novagen Cat. #71184) were hydrated and blocked
with 300 .mu.L/well of 1% BSA/DPBS for 10 to 15 minutes. After
several washes, supernatant from CHO cells secreting hu
mesothelin-his fusion protein, titered for maximum signal, was
added (50 .mu.l/well) and incubated for 1 hour. After washing
plates with DPBS/Tween, dilutions of plasma from mesothelin
immunized mice (starting at 1:50 dilution and serially diluted 1:2
11 times) were added to each well and incubated for 1 hour. The
plates were washed with DPBS/Tween and incubated for 1 hour with
goat-anti-human IgG polyclonal antibodies conjugated with
horseradish peroxidase (Jackson ImmunoResearch Labs Cat.
#115-036-098). ABTS (2,2'-azino-bis[3-ethylbenthiazolin 6-sulfonic
acid]) substrate (Sigma Cat. #S9941) was then added and plates were
analyzed spectrophotometrically at OD 415 nm. Titer was defined as
the dilution of serum that produced an OD two times the background
OD. Titer was calculated using an Excel template. Mice that
developed the highest titers of anti-human mesothelin antibodies
were used for fusions. Fusions were performed as described below
and hybridoma supernatants were tested for anti-human mesothelin
activity using two different ELISA assay formats, also described
below.
Generation of Hybridomas Producing Human Monoclonal Antibodies to
Mesothelin Protein
[0415] Mouse splenocytes isolated from high titer HCo7.times.hu
mice and a mouse myeloma fusion partner were fused with an electric
field based electrofusion using a Cyto Pulse large chamber cell
fusion electroporator (Cyto Pulse Sciences, Inc., Glen Burnie,
Md.). Single cell suspensions of splenic lymphocytes from immunized
mice were fused to an equal number of P3X63 Ag8.6.53 (ATCC CRL
1580) nonsecreting mouse myeloma cells. Resulting cells were plated
at 2.0.times.10.sup.4 cells/well in flat bottom microtiter plates
in selective DMEM medium containing high glucose (Cellgro
#10-013-CM) and 10% fetal calf serum (Hyclone #SH30071.03), and
supplemented with beta-mercaptoethanol (1000.times., Gibco
#21985-023), 7 mM HEPES (Cellgro 25-060-C1), additional 2 mM
L-glutamine (Cellgro 25-005-C1), HAT (50.times., Sigma #H-0262), 5%
Hybridoma Cloning Factor (BioVeris #210001), 10% P388DI (ATCC #CRL
TIB-63) conditioned medium and Penicillin-Streptomycin (100.times.,
Cellgro #30-002-CI). After about 7 days, some of the medium
containing HAT was replaced with medium containing HT (Cellgro
#25-047-CI).
[0416] After 10 to 12 days, individual wells were screened in a
direct ELISA assay for wells containing human IgG/human kappa light
chain or human IgG/human lambda light chain antibodies. In the
direct ELISA format, microtiter plates were coated with goat
anti-human IgG F(ab').sub.2 Fc fragment specific (Jackson
ImmunoResearch Labs, Cat. #109-006-098), 2.5 .mu.g/mL, 50
.mu.l/wells, incubated for 1 hour and then blocked with 300
.mu.L/well of 1% BSA/DPBS for 15 minutes. Blocking buffer was
removed and supernatant from fusion plates was added (50
.mu.L/well) and incubated for 1 hour. The plates were washed with
DPBS/Tween and then incubated for 1 hour with goat-anti-human kappa
light chain polyclonal antibodies conjugated with horseradish
peroxidase (Bethyl Cat. #A80-115P) or goat-anti-human lambda light
chain polyclonal antibodies conjugated with horseradish peroxidase
(Biosource Cat. #10904). After washing, the plates were developed
with ABTS substrate and analyzed spectrophotometrically at OD 415
nm. Wells with an elevated OD>0.75, were selected for further
analysis.
[0417] Hybridoma cells from wells positive for human IgG/human
kappa light chain or human IgG/human lambda light chain antibodies
were transferred to 24-well cultures. A few days later, cell
supernatant from individual wells was rescreened for specificity
using an indirect ELISA to identify human IgG antibody producing
hybridomas specific to human mesothelin. The protocol for the
indirect ELISA used for the secondary screen of the hybridomas was
the same protocol as described above for testing serum antibody
titers in the Hco7.times.hu mice, except that the sample
supernatant was from hu IgG/hu kappa or hu IgG/hu lambda positive
cultures.
[0418] The hybridomas were cloned by serial dilution and screened
two times. Multiple characterization assays (described in the
following examples) were performed using supernatants from
overgrown 24-well cultures prior to cloning. Ten antibodies were
selected and their characteristics were confirmed using clonal in
vitro produced and purified antibody. Three antibodies, 3C10
(original clone name: 1382.210.3C10.1.6), 6A4 (original clone name:
1382.210.6A4.1.6) and 7B1 (original clone name: 1382.210.7B1.2.18),
were selected for sequencing and further analysis.
Example 2
Structural Characterization of Antibodies 3C10, 6A4 and 7B1
[0419] The cDNA sequences encoding the heavy and light chain
variable regions of the mAbs expressed by the 3C10, 6A4 and 7B1
clones, as described in Example 1, were sequenced using standard
DNA sequencing techniques and the expressed proteins were
characterized by standard protein chemistry analysis. All three
clones were found to express an antibody comprising an IgG1 heavy
chain and a kappa light chain.
[0420] The nucleotide and amino acid sequences of the V.sub.H of
3C10 are shown in FIG. 1A and in SEQ ID NO: 25 and 19,
respectively. The nucleotide and amino acid sequences of the kappa
V.sub.L of 3C10 are shown in FIG. 1B and in SEQ ID NO: 28 and 22,
respectively.
[0421] Comparison of the 3C10 heavy chain immunoglobulin sequence
to the known human germline immunoglobulin heavy chain sequences
demonstrated that the 3C10 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 3-33, a D segment from human germline
D3-10 and a JH segment from human germline JH 4B. Further analysis
of the 3C10 V.sub.H sequence using the Kabat system of CDR region
determination led to the delineation of the heavy chain CDR1, CDR2
and CD3 regions as shown in FIG. 1A and in SEQ ID NOs: 1, 4 and 7,
respectively.
[0422] Comparison of the 3C10 light chain immunoglobulin sequence
to the known human germline immunoglobulin light chain sequences
demonstrated that the 3C10 kappa light chain utilizes a V.sub.K
segment from human germline V.sub.K L6 and a J.sub.K segment from
human germline JK 4. Further analysis of the 3C10 V.sub.K sequence
using the Kabat system of CDR region determination led to the
delineation of the light chain CDR1, CDR2 and CD3 regions as shown
in FIG. 1B and in SEQ ID NOs: 10, 13 and 16, respectively.
[0423] The nucleotide and amino acid sequences of the V.sub.H of
6A4 are shown in FIG. 2A and in SEQ ID NO: 26 and 20, respectively.
The nucleotide and amino acid sequences of the V.sub.L of 6A4 are
shown in FIG. 2B and in SEQ ID NO: 29 and 23, respectively.
[0424] Comparison of the 6A4 heavy chain immunoglobulin sequence to
the known human germline immunoglobulin heavy chain sequences
demonstrated that the 6A4 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 3-33, a D segment from the human
germline D3-10, and a JH segment from human germline JH 4B. Further
analysis of the 6A4 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CD3 regions as shown in FIG. 2A and in SEQ ID NOs:
2, 5 and 8, respectively.
[0425] Comparison of the 6A4 light chain immunoglobulin sequence to
the known human germline immunoglobulin light chain sequences
demonstrated that the 6A4 kappa light chain utilizes a V.sub.k
segment from human germline V.sub.K L6 and a J.sub.K segment from
human germline JK 4. Further analysis of the 6A4 V.sub.k sequence
using the Kabat system of CDR region determination led to the
delineation of the light chain CDR1, CDR2 and CD3 regions as shown
in FIG. 2B and in SEQ ID NOs: 11, 14 and 17, respectively.
[0426] The nucleotide and amino acid sequences of the V.sub.H of 7B
1 are shown in FIG. 3A and in SEQ ID NO: 27 and 21, respectively.
The nucleotide and amino acid sequences of the V.sub.L of 7B1 are
shown in FIG. 3B and in SEQ ID NO: 30 and 24, respectively.
[0427] Comparison of the 7B1 heavy chain immunoglobulin sequence to
the known human germline immunoglobulin heavy chain sequences
demonstrated that the 7B 1 heavy chain utilizes a V.sub.H segment
from human germline V.sub.H 3-7, a D segment from the human
germline D3-10, and a JH segment from human germline JH 6B. Further
analysis of the 7B 1 V.sub.H sequence using the Kabat system of CDR
region determination led to the delineation of the heavy chain
CDR1, CDR2 and CD3 regions as shown in FIG. 3A and in SEQ ID NOs:
3, 6 and 9, respectively.
[0428] Comparison of the 7B 1 light chain immunoglobulin sequence
to the known human germline immunoglobulin light chain sequences
demonstrated that the 7B 1 kappa light chain utilizes a V.sub.k
segment from human germline V.sub.K A27 and a J.sub.K segment from
human germline JK 2. Further analysis of the 7B 1 V.sub.k sequence
using the Kabat system of CDR region determination led to the
delineation of the light chain CDR1, CDR2 and CD3 regions as shown
in FIG. 3B and in SEQ ID NOs: 12, 15 and 18, respectively.
[0429] FIG. 4 shows the alignment of the 3C10 and 6A4 heavy chain
variable amino acid sequences (SEQ ID NOs: 19 and 20, respectively)
with the germline V.sub.H 3-33 encoded amino acid sequence (SEQ ID
NO: 31). The CDR1, CDR2 and CDR3 regions are delineated.
[0430] FIG. 5 shows the alignment of the 3C10 and 6A4 kappa light
chain variable amino acid sequences (SEQ ID NOs: 22 and 23,
respectively) with the germline V.sub.K L6 encoded amino acid
sequence (SEQ ID NO: 33). The CDR1, CDR2 and CDR3 regions are
delineated.
[0431] FIG. 6 shows the alignment of the 7B 1 heavy chain variable
amino acid sequence (SEQ ID NO: 21) with the germline V.sub.H 3-7
encoded amino acid sequence (SEQ ID NO: 32). The CDR1, CDR2 and
CDR3 regions are delineated.
[0432] FIG. 7 shows the alignment of the 7B 1 kappa light chain
variable amino acid sequence (SEQ ID NO: 24) with the germline
V.sub.K A27 encoded amino acid sequence (SEQ ID NO: 34). The CDR1,
CDR2 and CDR3 regions are delineated.
[0433] The 3C10, 6A4 and 7B1 variable regions can be converted to
full-length antibodies of any desired isotype using standard
recombinant DNA techniques. For example, DNA encoding the V.sub.H
and V.sub.L regions can be cloned into an expression vector that
carries the heavy and light chain constant regions such that the
variable regions are operatively linked to the constant regions.
Alternatively, separate vectors can be used for expression of the
full-length heavy chain and the full-length light chain.
Non-limiting examples of expression vectors suitable for use in
creating full-length antibodies include the pIE vectors described
in US 2005/0153394 by Black.
Example 3
Characterization of Binding Properties of Mesothelin Monoclonal
Antibodies
[0434] In this example, the binding of mAbs 3C10, 6A4 and 7B1 to
cell surface mesothelin was examined by flow cytometry.
Furthermore, binding kinetics to mesothelin were analyzed by
BIACORE analysis.
A. Flow Cytometry Studies
[0435] To test the ability of the antibodies to bind to cell
surface mesothelin protein, the antibodies were incubated with four
different mesothelin-expressing cells: OVCAR3 (ATCC Designation
HTB-161, a human ovarian cancer cell line), NCI-H226 (ATCC
Designation CRL-5826, a human lung-derived mesothelioma), CFPAC-1
(ATCC Designation CRL-1918, a human pancreatic cancer cell line),
and KB (ATCC Designation CCL-17, a human oral carcinoma cell line).
For the flow cytometry studies, the 3C10, 6A4 and 7B1 monoclonal
antibodies were diluted with cold 1.times.PBS+0.1% BSA to 40
.mu.g/mL. For the binding reaction, 50 .mu.l of diluted antibody
solution was added to a 50 .mu.l cell suspension containing
4.times.10.sup.5 cells and the mixture was incubated on ice for
30-60 minutes. The cells were then washed three times with
1.times.PBS+0.1% BSA. A 1:50 dilution of R-phycoerythrin-labeled
goat anti-human IgG F.gamma. F(ab).sub.2 fragment (Jackson
ImmunoResearch Labs, Cat. #109-116-098) was added and the mixture
was incubated on ice for 1 hour, followed by washing twice with
cold 1.times.PBS+0.1% BSA. After the final wash, 200 .mu.l of cold
1.times.PBS+0.1% BSA was added to each solution and analysis of
antibody binding was carried out by FACS.
[0436] The results of the flow cytometry analysis are summarized
below in Table 1, which shows EC.sub.50 for binding to the four
different cell lines. The results demonstrate that all three
monoclonal antibodies bind effectively to cell-surface human
mesothelin.
TABLE-US-00002 TABLE 1 Binding of Anti-Mesothelin Antibodies to
Cells Expressing Human Mesothelin NCI-H226 CFPAC-1 OVCAR3 Cells
Cells Cells KB Cells Antibody EC.sub.50 (nM) EC.sub.50 (nM)
EC.sub.50 (nM) EC.sub.50 (nM) 3C10 0.2126 0.1968 0.1933 0.1441 6A4
0.1697 0.1556 0.1932 0.1815 7B1 0.1103 0.6642 0.0643 0.06793
B. BIACORE Analysis
[0437] The binding of the 3C10, 6A4 and 7B1 antibodies to
recombinant soluble mesothelin his-fusion protein was examined by
BIAcore.TM. using a capture method. The 3C10, 6A4 and 7B1
antibodies each were separately captured on a protein G (Pierce,
Rockford, Ill.) coated CM5 chip at high density (4000 RUs). The
coating was performed based on the standard immobilization
procedure recommended by the manufacturer. After the 3C10, 6A4 or
7B1 purified antibody was captured on protein G, a concentration
series of recombinant human mesothelin His-fusion protein (450,
225, 112.5, 56, and 28 nM) was injected over the captured antibody
for 2 minutes at a flow rate of 25 .mu.L/mL. The antigen was
allowed to dissociate for 8 minutes. Isotype controls were run on
the chip, and the data used to subtract non-specific binding. All
the analyses were carried out on a Biacore 2000 or 3000 surface
plasmon resonance instrument, using BIAcore Control software v
4.01. Data was analyzed using BiaEvaluation v4.01 software.
[0438] The results are shown in Table 2 below. The BIAcore results
for 3C10, 6A4 and 7B 1 confirm the flow cytometry results that all
three antibodies are capable of binding with high affinity to human
mesothelin.
TABLE-US-00003 TABLE 2 Binding Affinity and Kinetics of
Anti-Mesothelin Antibodies to Recombinant Human Mesothelin Antibody
K.sub.D .times. 10.sup.-9 (M) k.sub.on (1/Ms) .times. 10.sup.4
k.sub.off (1/s) .times. 10.sup.-4 3C10 5.8 6.30 3.62 6A4 5.9 6.39
3.77 7B1 2.4 7.15 1.71
[0439] To investigate the epitopes bound by the 3C10, 6A4 and 7B1
mAbs, epitope binning analyses were performed with BIAcore. 3C10
and 7B1 mAbs were covalently linked to a CM5 chip (carboxy methyl
dextran coated chip) via primary amines, using the standard amine
coupling chemistry and kit provided by Biacore. Mixtures of
antibody-antigen complex were flowed across the immobilized
antibodies. The antibody concentrations were a two-fold dilution
series starting at between 50 and 500 nM. The mesothelin
concentration was between 22 and 44 nM. Antibodies and mesothelin
were pre-incubated for at least 1 hour prior to injection.
Antibody-antigen mixtures (15 uL) were injected at a flow rate of 6
.mu.l/min. Antibodies that have overlapping epitopes will compete
out (decreasing response with increasing antibody concentration)
whereas those with distinct epitopes will simultaneously bind to
the antigen (increasing response with increasing antibody
concentration). The results were that 3C10 mAb bound to the same
(or very similar epitope) as the previously described mouse
anti-mesothelin mAb K1. The 7B 1 mAb bound to a distinct epitope
from 3C10 and K1. The 6A4 mAb was able to block both 3C10 and 6A4
binding to recombinant mesothelin.
Example 4
Characterization of Stability of Mesothelin Monoclonal
Antibodies
[0440] In this example, the stability of mAbs 3C10, 6A4 and 7B1 was
examined in chemical unfolding and thermal stability analyses, as
follows.
[0441] The stability of the anti-mesothelin monoclonal antibodies
were compared by measuring the midpoint of chemical denaturation by
fluorescence spectroscopy. Fluorescence measurements of chemical
denaturation were performed on a SPEX Fluorolog 3.22 equipped with
a Micromax plate reader (SPEX, Edison, N.J.). The measurements were
performed on antibody samples that had been left to equilibrate for
20 hours in 16 different concentrations of guanidinium
hydrochloride (GdHCL) in PBS buffer ranging from 0 to 6 Molar. The
measurements were made in black, low volume, non-binding surface
384-well plates (Corning, Acton, Mass.) and required 1 .mu.M of
antibody in a well volume of 12 .mu.L. Fluorescence was excited at
280 nm and the emission spectra were measured between 320 and 400
nm. The scan speed was 1 second per nm and slits were set to 5 nm
bandpass. A buffer blank was performed using PBS and automatically
subtracted from the data. Data was fitted to a two-state
denaturation model using the GraphPad Prism software. The results,
expressed as the molar concentration of GdHCl that was the
unfolding midpoint, are shown below in Table 3.
TABLE-US-00004 TABLE 3 Chemical Stability of anti-Mesothelin
Antibodies Antibody Unfolding Midpoint (GdHCl, M) 3C10 2.50 6A4
2.67 7B1 2.21
[0442] The thermal stability of the anti-mesothelin monoclonal
antibodies was determined by calorimetric analysis of the melting
temperature of the antibodies.
[0443] Calorimetric measurements of melting temperatures (T.sub.m)
were performed on a VP-Capillary DSC differential scanning micro
calorimeter platform that is combined with an autosampler (MicroCal
LLC, Northampton, Mass., USA). Denaturation data on the antibodies
was obtained by heating the samples, at a concentration of 0.25
mg/mL, from 30 to 95.degree. C. at a rate of 1.degree. C./min. The
antibody samples were present in phosphate-buffered saline (PBS) at
pH 7.4. The same buffer was used in the reference cell to obtain
the molar heat capacity by comparison. The observed thermograms
were baseline corrected and normalized data analyzed based on a
non-2-state model, using the software Origin v7.0. The results,
expressed as temperature (in .degree. C.) at which peaks indicative
of antibody melting were observed, are shown in Table 4 below.
TABLE-US-00005 TABLE 4 Thermal Stability of Mesothelin Antibodies
Antibody Peak 1 (.degree. C.) Peak 2 (.degree. C.) Peak 3 (.degree.
C.) 3C10 67 81 6A4 69 81 7B1 69.8 72.7 82
[0444] The results above indicate that the 3C10, 6A4 and 7B 1
antibodies have desirable chemical and thermal stability
properties.
Example 5
Internalization of Mesothelin Antibodies
[0445] In this example, the ability of the 3C10, 6A4 and 7B1
antibodies to be internalized by mesothelin-expressing cells was
examined, as follows.
[0446] In the first series of analyses, a Hum-ZAP assay was used to
examine antibody internalization. In the Hum-ZAP assay (kits for
which are commercially available from Advanced Targeting Systems,
San Diego, Calif.), the anti-mesothelin antibodies were reacted
with mesothelin-expressing cells and with a goat anti-human IgG
antibody conjugated to the toxin saporin (a ribosome inactivating
protein). Upon internalization of the primary anti-mesothelin
antibody, the secondary saporin-conjugated antibody is also taken
inside the cell, resulting in protein synthesis inhibition and
eventual cell death after 2-4 days.
[0447] More specifically, cells were diluted to 5.times.10.sup.4
cells/mL in cell culture medium. 50 .mu.l of diluted cells were
added to each well of a flat-bottomed 96 well tissue culture plate
and incubated overnight in a 37.degree. C., 5% CO.sub.2 incubator.
Antibodies were diluted with culture medium to a concentration of 4
.mu.g/mL and 0.4 .mu.g/mL. 25 .mu.l of diluted antibody was added
to the cultured cells so the final antibody concentrations were 1
.mu.g/mL and 0.1 .mu.g/mL. Cell and antibody were incubated for 10
minutes before addition of 25 .mu.l of 8 .mu.g/mL Hum-zap reagent.
The mixture was incubated at 37.degree. C., 5% CO.sub.2 for 72
hours. 100 .mu.l of Celltiter-Glo (a viability dye) was added to
each well and gently swirled for 2 minutes. After 10 minutes at
room temperature, plates were read on a Promega Veritas Microplate
Luminator.
[0448] The Hum-ZAP assay was conducted using the
mesothelin-expressing cell lines NCI-H226, CFPAC-1 and KB
(described in Example 3) as target cells, as well as CHO cells
transfected to express mesothelin on the cell surface via a
GPI-linkage (CHO-mesothelin). The results are expressed
qualitatively below in Table 5:
TABLE-US-00006 TABLE 5 Internalization of Mesothelin Antibodies
Using Hum-ZAP Assay CHO- Antibody Mesothelin NCI-H226 CFPAC-1 KB
3C10 + N.D N.D + 6A4 + + + + 7B1 + N.D N.D +/- N.D. = Not
determined
[0449] The Hum-ZAP assay results demonstrate that the 6A4 antibody
was internalized by all four cell lines. The 3C10 and 7B1
antibodies also were internalized by the CHO-mesothelin cells.
Still further, the 3C10 antibody was internalized by the KB cells,
whereas the 7B1 antibody was internalized less well by the KB cells
than the other two antibodies.
[0450] In the second series of analyses, the internalization of the
6A4 antibody by the NCI-H226 cell line was further examined using
an immunofluoresence assay. In this assay, NCI-H226 were grown to
80% confluency. The adherent cells were lifted with a cell
dissociation buffer (Cell Stripper) and 1.35.times.10.sup.6 cells
were transferred to a 15 ml conical tube and washed once with FACS
buffer (PBS+2% fetal bovine serum). The cells were incubated with
6A4 antibody at 20 .mu.g/mL in a volume of 450 .mu.L FACS buffer
(PBS+2% fetal bovine serum) at 4.degree. C. for 30 min followed by
washing with cold FACS buffer. Cells were then stained with a
detection antibody, a goat anti-human IgG antibody conjugated to
Rhodamine at 20 .mu.g/mL in FACS Buffer at 4.degree. C. for 30
minutes, followed by washing with cold FACS buffer. This
receptor/antibody complex was further resuspended in 450 .mu.L FACS
buffer and incubated with the cells at 37.degree. C./5% CO.sub.2
for specified times (zero, 5, 10, 15, 30, 60, 120 minutes, and
overnight) to allow for internalization. At specified time points,
50 .mu.L aliquots, corresponding to 150,000 cells, were transferred
into 96-well plates and stained with a wheat germ agglutinin-Alexa
Fluor 488 conjugate (Invitrogen, Cat. # W11261), which stains the
cell surface. The cell aliquots were incubated on ice for 30
minutes with 10 .mu.g/mL WGA in FACS Buffer, followed by washing
with FACS Buffer. Finally, the cells were washed with FACS buffer
and fixed by addition of 2% paraformaldehyde and analyzed by
fluorescence microscopy for associated immunofluorescence using
excitation/emission wavelengths 495/519 and 554/570 for Alexa488
and Rhodamine respectively. The results confirmed that the 6A4 mAb
is effectively internalized by the NCI-H226 cells.
Example 6
Inhibition of Binding of Mesothelin to CA125 by Anti-Mesothelin
mAbs
[0451] To test the ability of the anti-mesothelin antibodies to
inhibit binding of mesothelin to CA125, an in vitro heterotypic
cell adhesion assay was performed. In this assay, the ability of
the antibodies to inhibit adhesion of OVCAR3 cells (an ovarian
cancer cell line that expresses CA125) to CHO cells transfected to
express mesothelin on their cell surface (CHO-mesothelin) was
examined. Adhesion of OVCAR3 cells (described further in Example 3)
to CHO-mesothelin cells is mediated by the interaction of
mesothelin with CA125.
[0452] To test antibody inhibition of the mesothelin/CA125
interaction, OVCAR cells were plated at a density of
4.times.10.sup.4 cells/200 .mu.l per well in a 96-well plate 24
hours prior to the assay to obtain a subconfluent culture of cells.
After 24 hours, medium was removed and fresh medium containing 1%
bovine serum albumin (BSA) plus 10 .mu.g/mL of antibody (3C10, 7B1,
6A4, isotype control or no antibody) was added to the wells. Cells
were incubated at 37.degree. C. with 5% CO.sub.2 for 30 minutes.
CHO-S (parental cells) and CHO-Mesothelin cells were incubated 30
minutes with Calcein AM (5 .mu.M for 6.times.10.sup.6 cells/mL),
washed with medium and added to the OVCAR cultures and incubated
for 60 minutes at 37.degree. C.
[0453] Following the incubation, samples were washed 4 times with
PBS to remove unattached cells. Attached cells were measured by
adding 100 .mu.l/well PBS and reading plates with the Synergy-HT
Fluorescence reader at 494 nm/517 nm.
[0454] The results are illustrated in FIG. 8. The results show that
OVCAR3 cells adhered to CHO-mesothelin cells in the absence of
antibodies, but did not adhere to control CHO-S cells that did not
express mesothelin on their cell surface. Furthermore, the presence
of the 3C10 mAb or the 6A4 mAb resulted in approximately 68.7%
inhibition or 84.8% inhibition, respectively, in cell adhesion, as
compared to a control antibody (DT). In contrast, the 7B 1 mAb did
not exhibit the ability to inhibit the interaction of mesothelin
with CA125, indicating that the epitope to which 7B 1 binds on
mesothelin (which differs from the 3C10 and 6A4 epitopes) does not
block the mesothelin-CA125 interaction region.
Example 7
Antibody-Dependent Cellular Cytotoxicity
[0455] To determine the ability of antibody 6A4 to kill
mesothelin-expressing cells in the presence of effector cells via
antibody dependent cellular cytotoxicity (ADCC), a fluorescence
cytotoxicity assay was used. CHO cells transfected to express the
40 kD membrane-associated portion of mesothelin on their cell
surface (CHO-mesothelin cells) were used as the target cells.
[0456] Human effector cells were prepared from whole blood as
follows. Human peripheral blood mononuclear cells were purified
from heparinized whole blood by standard Ficoll-paque separation.
The cells were resuspended in RPMI1640 media containing 10% FBS
(heat-inactivated) and 200 U/mL of human IL-2 and incubated
overnight at 37.degree. C. The following day, the cells were
collected and washed four times in culture media and resuspended at
2.times.10.sup.7 cells/mL. Target CHO-mesothelin cells were
prepared by incubating with BATDA reagent (Perkin Elmer, Wellesley,
Mass.) at 2.5 .mu.l BATDA per 1.times.10.sup.6 target cells/mL for
20 minutes at 37.degree. C. The target cells were washed four
times, spun down and brought to a final volume of 1.times.10.sup.5
cells/mL.
[0457] The CHO-mesothelin cells were tested for antibody specific
ADCC to the human anti-mesothelin 6A4 monoclonal antibody using the
Delfia fluorescence emission analysis as follows. CHO-mesothelin
cells (100 .mu.l of labeled target cells, 10.sup.4 cells/well) were
incubated with 50 .mu.l of effector cells (10.sup.6 cells/well) and
50 .mu.l of antibody (10 ug/mL final concentration). A target to
effector ratio of 1:100 was used throughout the analyses. In all
studies, a human IgG1 isotype control was used as a negative
control. Cells were spun down at 2000 rpm and incubated for one
hour incubation at 37.degree. C. The supernatants were then
collected, submitted to centrifugation and 20 .mu.l of supernatant
was transferred to a flat bottom plate, to which 180 .mu.l of Eu
solution (Perkin Elmer, Wellesley, Mass.) was added and read in a
RubyStar reader (BMG Labtech). The % lysis was calculated as
follows: (sample release--spontaneous release*100)/(maximum
release--spontaneous release), where the spontaneous release is the
fluorescence from wells which contain target cells plus effector
cells and maximum release is the fluorescence from wells containing
target cells and have been treated with 2% Triton-X.
[0458] The results are summarized below in Table 6, which shows the
EC.sub.50 in nM.
TABLE-US-00007 TABLE 6 Antibody Dependent Cellular Cytotoxicity
Mediated by 6A4 Antibody Antibody EC.sub.50(nM) 6A4 63.10 Isotype
Control 682.7
[0459] The results demonstrate that the 6A4 monoclonal antibody
exhibits ADCC activity against a cell line expressing mesothelin on
its cell surface.
Example 8
Tumor Growth Inhibition In Vivo by Anti-Mesothelin mAb 6A4
[0460] In this example, the ability of the 6A4 mAb, either as a
naked antibody or as a cytotoxin conjugate, to inhibit tumor growth
in mouse models in vivo, was examined. The cytotoxin conjugate of
6A4 is referred to herein as 6A4-Cytotoxin A, which is composed of
the 6A4 antibody linked to Cytotoxin A. The Cytotoxin A cytotoxin,
and preparation thereof, is described further in WO 2008/083312,
the entire content of which is specifically incorporated herein by
reference. The 6A4-Cytotoxin A conjugate was prepared as
follows:
[0461] Antibody 6A4 was concentrated to approximately 5 mg/mL,
buffer exchanged into 100 mM phosphate buffer, 50 mM NaCl, 2 mM
DTPA pH 8.0 and thiolated by addition of an 8 to 10-fold molar
excess of 2-Imminothiolane for 60 minutes at room temperature.
Following thiolation, the antibody was buffer exchanged into 50 mM
HEPES buffer, containing 5 mM glycine, 2 mM DTPA, and 0.5% Povidone
(10 K) pH 5.5. Thiolation was quantified with
4,4''-dithiodipyridine by measuring thiopyridine release at 324 nM.
Conjugation was achieved by addition of maleimide containing drug
Cytotoxin A at a 3:1 molar ratio of drug to thiols.
[0462] Incubation was at room temperature for 60 minutes followed
by the addition of 10:1 molar ratio of N-ethylmaleimide (NEM) to
thiols to the reaction mix. After incubation in the presence of
NEM, the resulting conjugates were purified by size-exclusion
chromatography on a Sephacryl-200 column run in 50 mM HEPES, 5 mM
glycine, 100 mM NaCl, pH 6.0. Fractions containing monomeric
antibody conjugate were pooled and concentrated by ultrafiltration.
Antibody conjugate concentration and substitution ratios were
determined by measuring absorbance at 280 and 340 nm.
[0463] Cytotoxin A has the structure shown below:
##STR00038##
[0464] Those skilled in the art will appreciate that although the
term "cytotoxin" is employed, the structure shown actually
encompasses both the cytotoxin proper (partner molecule) and a
linker moiety for joining the cytotoxin to the antibody and that,
after cleavage of the conjugate, the toxin is released in a prodrug
form that is then hydrolyzed to the toxin proper. Thus, strictly
speaking, the partner molecule in a conjugate of this invention can
be viewed either as the prodrug form or the cytotoxin proper.
##STR00039##
[0465] In a first series of analyses, the effect of 6A4 and
6A4-Cytotoxin A on the growth of NCI-H226 cells (a lung-derived
mesothelioma; ATCC Designation CRL-5826) in a mouse xenograft model
was examined. In this xenograft model, SCID mice (CB 17, from
Taconic, Germantown, N.Y.) were implanted with 9.times.10.sup.6
NCI-H226 cells/mouse and the NCI-H226 cells were allowed to grow
for 36 days. On day 40, at which time the mean tumor volume was 87
mm.sup.3, the mice were randomized and treated intraperitoneally
(i.p.) with 6A4 (10 mg/kg or 30 mg/kg) or with 6A4-Cytotoxin A
conjugate (0.1 .mu.mole/kg), along with controls: vehicle alone
(PBS, Q3Dx4), an isotype-matched human IgG1 antibody (4A3) or an
isotype-matched human IgG1 antibody linked to the Cytotoxin A
cytotoxin (iso-Cytotoxin A).
[0466] The 6A4 antibody, and its controls, were administered on
days 40, 43, 47 and 51. The 6A4-Cytotoxin A conjugate, and its
controls, were administered on days 40 and 64. Tumor volume was
measured at regular intervals until day 105. FIGS. 9A and 9B show
the mean tumor volume and median tumor volume, respectively, in
mice treated with vehicle alone, 6A4-Cytotoxin A (0.1 mole/kg),
iso-Cytotoxin A (0.1 .mu.mole/kg), unconjugated 6A4 and
unconjugated isotype control (4A3). As would be expected, given
that the mouse xenograft model employed in the instant study is
immuno-compromised and therefore would not be expected to mount a
robust ADCC response, treatment with the naked 6A4 antibody did not
show an effect on NCI-H226 tumor cell volume (i.e., did not inhibit
tumor growth). In contrast, treatment with the 6A4-Cytotoxin A
conjugate significantly inhibited tumor growth, as illustrated in
the graphs of FIGS. 9A and 9B.
[0467] In a second series of analyses, the effect of 6A4 and
6A4-Cytotoxin A on the growth of HPAC cells (human pancreatic
adenocarcinoma; ATCC Designation CRL-2119) in a mouse xenograft
model was examined. HPAC cells (5.times.10.sup.6 cells/mouse) were
implanted into CB 17 SCID mice and allowed to grow for 8 days. On
day 8, at which time the mean tumor volume was 96 mm.sup.3, the
mice were randomized and treated intraperitoneally (i.p.) with 6A4
(10 mg/kg or 30 mg/kg) or with 6A4-Cytotoxin A conjugate (0.15
mole/kg), along with controls: vehicle alone (PBS, Q3Dx4), an
isotype-matched human IgG1 antibody (4A3; 30 mg/kg) or an
isotype-matched human IgG1 antibody linked to the Cytotoxin A
cytotoxin (2A10-Cytotoxin A; 0.15 mole/kg) and unconjugated 6A4 (8
mg/kg) or unconjugated isotype control (2A10; 8.7 mg/kg, SD) at
equivalent protein dose. The 6A4 antibody, and its controls, were
administered with a dose schedule of Q3Dx4 (every three days for
four times). The 6A4-Cytotoxin A conjugate, and its controls, were
administered with a dose schedule of a single dose (SD). Tumor
volume was measured at regular intervals until day 57.
[0468] The results are shown in the graphs of FIGS. 10A and 10B.
FIGS. 10A and 10B show the mean tumor volume and median tumor
volume, respectively, in mice treated with vehicle alone,
6A4-Cytotoxin A (0.15 mmole/kg), isotype-matched conjugate
(2A10-Cytotoxin A; 0.15 .mu.mole/kg), weight-equivalent
unconjugated 6A4 (8 mg/kg), unconjugated isotype control (2A10; 8.7
mg/kg) at equivalent protein dose, naked 6A4 (10 mg/kg or 30 mg/kg)
or isotype-matched IgG1 naked mAb (4A3; 30 mg/kg). The data
indicate that treatment with the naked 6A4 antibody resulted in an
inhibition of the growth of the HPAC tumor cells, even though the
mouse xenograft model would not be expected to mount a robust ADCC
response. Again, as was the case with the previous example,
treatment with the 6A4-Cytotoxin A conjugate was shown to be very
effective in inhibiting the growth of the HPAC tumor cells.
[0469] In a third series of analyses, the effect of 6A4-Cytotoxin A
on the growth of ovarian cancer derived OVCAR3 cells (ATCC
Designation HTB-161) in a mouse xenograft model was examined. In
this xenograft model, CB17 SCID mice were implanted with
5.times.106 OVCAR3 cells per mouse and the OVCAR3 cells were
allowed to grow for 42 days. On day 42, the mean tumor volume was
approximately 100 mm3. The mice were treated intraperitoneally
(i.p.) with 6A4-Cytotoxin A conjugate at a 0.1 or 0.3 .mu.mole/kg
dose. As controls, mice were treated with vehicle alone (PBS,
Q3Dx4) or 0.1 or 0.3 .mu.mole/kg of an isotype-matched human IgG1
antibody linked to Cytotoxin A.
[0470] The 6A4-Cytotoxin A conjugate and its controls were
administered on days 42, 96, 132 and 176. As shown in FIG. 11,
treatment with the 6A4-Cytotoxin A conjugate significantly
inhibited tumor growth. Tumor growth was controlled throughout this
example, including through the final dosing at day 176, for the 0.3
.mu.mol/kg dose of 6A4-Cytotoxin A. Tumor growth was controlled
until day 150 with the 0.1 .mu.mol/kg dose of 6A4-Cytotoxin A. The
isotype controls were not effective in controlling the tumors (data
not shown).
[0471] In summary, a cytotoxin conjugate of 6A4 antibody (at a dose
of 0.1-0.15 .mu.mole/kg) significantly inhibited tumor growth in
vivo in three different xenograft mouse models.
Example 9
ADCC Mediated by Non-Fucosylated 6A4
[0472] In this example, non-fucosylated 6A4 (6A4nf) antibodies were
shown to be efficient mediators of ADCC in lung and ovarian
cancers. A Delfia fluorescence emission analysis showed that
non-fucosylated 6A4 killed mesothelin-expressing H226 (NSCLC) and
OVCAR3 (ovarian cancer) cells in the presence of effector cells via
ADCC. First, human effector cells were prepared from whole blood as
follows: Human peripheral blood mononuclear cells were purified
from heparinized whole blood by standard Ficoll-paque separation.
The cells were resuspended in RPMI1640 media containing 10% FBS
(heat-inactivated) and 200 U/mL of human IL-2 and incubated
overnight at 37.degree. C. The following day, the cells were
collected and washed four times in culture media and resuspended at
2.times.10.sup.7 cells/mL.
[0473] Second, labeled H226 or OVCAR3 cells were prepared by
incubating them with BATDA reagent (Perkin Elmer, Wellesley,
Mass.). Third, the target cells were mixed with effector cells and
antibody. 100 .mu.l of labeled target cells (10.sup.4 cells/well)
were incubated with 50 .mu.l of effector cells (10.sup.6
cells/well) and 50 .mu.l of antibody (10 .mu.g/mL final
concentration). A target to effector ratio of 1:100 was used
throughout the analyses. A human IgG1 (HuIgG1) isotype control was
used as a negative control. Fourth, cell lysis was quantified.
Cells were spun down at 2000 rpm and incubated for one hour
incubation at 37.degree. C. The supernatants were then collected,
submitted to centrifugation and 20 .mu.l of supernatant was
transferred to a flat bottom plate. 180 .mu.l of Eu solution
(Perkin Elmer, Wellesley, Mass.) was added and read in a RubyStar
reader (BMG Labtech). The percent lysis was calculated as follows:
((sample release--spontaneous release).times.100) divided by
(maximum release--spontaneous release) where the spontaneous
release is the fluorescence from wells which contain target cells
plus effector cells and maximum release is the fluorescence from
wells containing target cells and have been treated with 2%
Triton-X. Significant increases in ADCC activity was observed using
non-fucosylated 6A4 on OVCAR3 cells (FIG. 12a) and H226 cells (FIG.
12b).
Example 10
Non-Fucosylated 6A4 Inhibited Tumor Growth in vivo
[0474] In another example, 6A4nf, in combination with Gemcitabine,
significantly slowed the growth of H226 cells in a mouse xenograft
model. CB17 SCID mice were implanted with 9.times.10.sup.6 H226
cells per mouse and the allowed to grow. On day 23, the mean tumor
volume was approximately 100 mm3. The mice were randomized and
treated intraperitoneally with 6A4nf, 80 mg/kg of Gemcitabine, the
combination 6A4nf plus Gemcitabine, a vehicle control, or an IgG1
isotype control. 6A4nf was administered at either 10 mg/kg or 30
mg/kg. H226 growth was inhibited by all combinations of 6A4nf plus
Gemcitabine. Maximal inhibition was achieved when 6A4nf was
administered at 30 mg/kg in combination with Gemcitabine and
suggested a dose-dependent effect.
Example 11
Mesothelin Expression on Tumor Cells
[0475] Immunohistochemistry using antibody 6A4 was used to show its
ability to bind to human tumors and determine the range of
expression of mesothelin in tumors. 6A4 was pre-complexed with a
FITC-labeled Goat anti Human Fab (Jackson #109-097-003) so that the
final concentration of 6A4 was 6 ug/mL. This complex was then used
with standard IHC methods to determine binding. 6A4 was shown to
bind to ovarian cancers, pancreatic cancers, lung cancers,
mesotheliomas, and cancers of the head and neck. Additional
examples of 6A4 binding to head and neck cancers include cancers
from epiglottis, larynx, salivary gland, tongue, and tonsil
tissues.
Example 12
6A4-Cytotoxin A is Not Toxic in Cynomologus Monkeys
[0476] 6A4-Cytotoxin A was shown to not be toxic in Cynomolgus
monkeys. These animals show similar mesothelin expression patterns
to those in humans. Also, the 6A4 antibody binds to the cynomolgus
mesothelin protein with the same affinity as it does to the human
mesothelin protein. Thus, cynomolgus monkeys were suitable to
assess the on target toxicities of 6A4-Cytotoxin A.
[0477] 6A4-Cytotoxin A was dosed intravenously to two male and two
female cynomolgus monkeys. Two doses of 0.4 .mu.mol/kg were given
on days 1 and 15. The animals were observed for behavioral changes,
signs of toxicity, and blood was drawn for analysis. The animals
did not show signs of toxic effects.
[0478] On days 4, 3, 7, 14, 21, and 28, blood was drawn and
multiple parameters were measured. No significant changes were
observed in white blood cells, red blood cells, platelets,
reticulocytes, neutrophil percentages, lymphocyte percentages,
monocytes percentages, eosinophils percentages, basophile
percentages, unstained cell percentages, hemoglobin, hematocrit,
mcv, mch, mchc, protime, apt, albumin, total-protein, globulin,
bilirubin, urea-nitrogen, creatinine, alkaline phosphatase, alanine
transferase, aspartate transferase, glutamyl transferase, glucose,
cholesterol, calcium, chloride, phosphorus, potassium, sodium, and
triglycerides.
[0479] Following necropsy at day 28, no drug related changes were
observed in the following organs: adrenal glands, aorta, sternum,
brain, cecum, colon, duodenum, esophagus, eyes, optic nerve, femur,
gallbladder, heart, ileum, jejunum, kidneys, lymph node, liver,
lymph node, mes, lungs, mammary gland, sciatic nerve ovaries,
pancreas, pituitary, parathyroid, rectum salivary gland, skin,
skeletal muscle, spleen, stomach, thyroid glands, thymus, spinal
cord, trachea, urinary bladder, uterus, vagina, cervix, injection
site, oviducts, ureters, and peyer's patches.
TABLE-US-00008 SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE 1
V.sub.H CDR1 a.a. 3C10 2 V.sub.H CDR1 a.a. 6A4 3 V.sub.H CDR1 a.a.
7B1 4 V.sub.H CDR2 a.a. 3C10 5 V.sub.H CDR2 a.a. 6A4 6 V.sub.H CDR2
a.a. 7B1 7 V.sub.H CDR3 a.a. 3C10 8 V.sub.H CDR3 a.a. 6A4 9 V.sub.H
CDR3 a.a. 7B1 10 V.sub.K CDR1 a.a. 3C10 11 V.sub.K CDR1 a.a. 6A4 12
V.sub.K CDR1 a.a. 7B1 13 V.sub.K CDR2 a.a. 3C10 14 V.sub.K CDR2
a.a. 6A4 15 V.sub.K CDR2 a.a. 7B1 16 V.sub.K CDR3 a.a. 3C10 17
V.sub.K CDR3 a.a. 6A4 18 V.sub.K CDR3 a.a. 7B1 19 V.sub.H a.a. 3C10
20 V.sub.H a.a. 6A4 21 V.sub.H a.a. 7B1 22 V.sub.K a.a. 3C10 23
V.sub.K a.a. 6A4 24 V.sub.K a.a. 7B1 25 V.sub.H n.t. 3C10 26
V.sub.H n.t. 6A4 27 V.sub.H n.t. 7B1 28 V.sub.K n.t. 3C10 29
V.sub.K n.t. 6A4 30 V.sub.K n.t. 7B1 31 V.sub.H 3-33 germaline a.a.
32 V.sub.H 3-7 germline a.a. 33 V.sub.k L6 germline a.a. 34 V.sub.k
A27 germline a.a. 35 3C10 VH fragment presented in Fig. 4 36 6A4 VH
fragment presented in Fig. 4 37 JK4 fragment presented in Fig. 5 38
JH6b fragment presented in Fig. 6 39 JK2 fragment presented in Fig.
7 40 Peptide conjugate sequence 41 Peptide conjugate sequence 42
Peptide conjugate sequence 43 Peptide conjugate sequence
Sequence CWU 1
1
4315PRTHomo sapiens 1Ile Tyr Gly Met His 1 5 25PRTHomo sapiens 2Ile
Tyr Gly Met His 1 5 35PRTHomo sapiens 3Arg Tyr Trp Met Ser 1 5
417PRTHomo sapiens 4Val Ile Trp Tyr Asp Gly Ser His Glu Tyr Tyr Ala
Asp Ser Val Lys 1 5 10 15 Gly 517PRTHomo sapiens 5Val Leu Trp Tyr
Asp Gly Ser His Glu Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
617PRTHomo sapiens 6Ser Ile Lys Gln Ala Gly Ser Glu Lys Thr Tyr Val
Asp Ser Val Lys 1 5 10 15 Gly 713PRTHomo sapiens 7Asp Gly Asp Tyr
Tyr Asp Ser Gly Ser Pro Leu Asp Tyr 1 5 10 813PRTHomo sapiens 8Asp
Gly Asp Tyr Tyr Asp Ser Gly Ser Pro Leu Asp Tyr 1 5 10 922PRTHomo
sapiens 9Glu Gly Ala Tyr Tyr Tyr Asp Ser Ala Ser Tyr Tyr Pro Tyr
Tyr Tyr 1 5 10 15 Tyr Tyr Ser Met Asp Val 20 1011PRTHomo sapiens
10Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 1111PRTHomo
sapiens 11Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10
1212PRTHomo sapiens 12Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu
Ala 1 5 10 137PRTHomo sapiens 13Asp Ala Ser Asn Arg Ala Thr 1 5
147PRTHomo sapiens 14Asp Ala Ser Asn Arg Ala Thr 1 5 157PRTHomo
sapiens 15Gly Ala Ser Ser Arg Ala Thr 1 5 169PRTHomo sapiens 16Gln
Gln Arg Ser Asn Trp Pro Leu Thr 1 5 179PRTHomo sapiens 17Gln Gln
Arg Ser Asn Trp Pro Leu Thr 1 5 189PRTHomo sapiens 18Gln Gln Tyr
Gly Ser Ser Gln Tyr Thr 1 5 19122PRTHomo sapiens 19Gln Val Tyr Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Tyr 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Val Ile Trp Tyr Asp Gly Ser His Glu Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Leu Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asp Tyr Tyr Asp Ser
Gly Ser Pro Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 20122PRTHomo sapiens 20Gln Val His Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu
Ser Cys Val Ala Ser Gly Ile Thr Phe Arg Ile Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Leu Trp Tyr Asp Gly Ser His Glu Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile
Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Asp Tyr Tyr Asp Ser Gly Ser
Pro Leu Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 21131PRTHomo sapiens 21Glu Val His Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser
Ile Lys Gln Ala Gly Ser Glu Lys Thr Tyr Val Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Tyr Tyr Asp Ser Ala Ser
Tyr Tyr Pro Tyr 100 105 110 Tyr Tyr Tyr Tyr Ser Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr 115 120 125 Val Ser Ser 130 22107PRTHomo
sapiens 22Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 23107PRTHomo sapiens
23Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 24108PRTHomo sapiens 24Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Gln 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 25366DNAHomo sapiens 25caggtgtacc
tggtggagtc tgggggaggc gtggtccagc ccgggaggtc cctgagactc 60tcctgtgcag
cgtctggaat caccttcagt atctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtca
tgaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgctaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagatggc 300gattattatg attcggggag
tcctcttgac tactggggcc agggaaccct ggtcaccgtc 360tcctca
36626366DNAHomo sapiens 26caggtgcacc tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgtag cgtctggaat caccttcagg
atctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt ttatggtatg atggaagtca tgaatactat 180gcagactccg
tgaagggccg attcaccatc tccagagaca attccaagaa cacgctatat
240ctgcaaatga acagcctgag agccgaggac acggctatat attactgtgc
gagagatggc 300gattactatg attcggggag tcctcttgac tactggggcc
agggaaccct ggtcaccgtc 360tcctca 36627393DNAHomo sapiens
27gaggttcacc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagt agatactgga tgagctgggt ccgccaggct
120caagggaaag ggctggagtg ggtggccagc ataaagcaag ctggaagtga
gaaaacctat 180gtggactctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcactgtct 240ctgcaaatga acagcctgag agccgaggac
acggctgttt attactgtgc gagggagggg 300gcatattact atgattcggc
gagttattac ccttactact actactacag tatggacgtc 360tggggccaag
ggaccacggt caccgtctcc tca 39328321DNAHomo sapiens 28gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa a
32129321DNAHomo sapiens 29gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agctacttag cctggtacca acagaaacct 120ggccaggctc ccaggctcct
catctatgat gcatccaaca gggccactgg catcccagcc 180aggttcagtg
gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct
240gaagattttg cagtttatta ctgtcagcag cgtagcaact ggccgctcac
tttcggcgga 300gggaccaagg tggagatcaa a 32130318DNAHomo sapiens
30gtgttgacgc agtctccagg caccctgtct ttgtctccag gggaaagagc caccctctcc
60tgcagggcca gtcagagtgt tagcagcagc tacttagcct ggtaccagca gaaacctggc
120caggctccca ggctcctcat ctatggtgca tccagcaggg ccactggcat
cccagacagg 180ttcagtggca gtgggtctgg gacagacttc actctcacca
tcagcagact ggagcctgaa 240gattttgcag tgtattactg tcagcagtat
ggtagctcac agtacacttt tggccagggg 300accaagctgg agatcaaa
3183198PRTHomo sapiens 31Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg 3298PRTHomo sapiens 32Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn
Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg 3395PRTHomo sapiens 33Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro 85 90 95 3495PRTHomo sapiens 34Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly
Ser Ser 85 90 95 3511PRTHomo sapiens 35Asp Gly Asp Tyr Tyr Asp Ser
Gly Ser Pro Leu 1 5 10 3611PRTHomo sapiens 36Asp Gly Asp Tyr Tyr
Asp Ser Gly Ser Pro Leu 1 5 10 3712PRTHomo sapiens 37Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 3813PRTHomo sapiens
38Glu Gly Ala Tyr Tyr Tyr Asp Ser Ala Ser Tyr Tyr Pro 1 5 10
3912PRTHomo sapiens 39Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 1 5 10 404PRTArtificial SequencePeptide conjugate sequence
40Ala Leu Ala Leu 1 414PRTArtificial SequencePeptide conjugate
sequence 41Xaa Leu Ala Leu 1 424PRTArtificial SequencePeptide
conjugate sequence 42Gly Phe Leu Gly 1 434PRTArtificial
SequencePeptide conjugate sequence 43Leu Leu Gly Leu 1
* * * * *