U.S. patent application number 12/450223 was filed with the patent office on 2010-06-03 for method of treating malignant mesothelioma.
This patent application is currently assigned to The University of Tokyo. Invention is credited to Teruo Inamoto, Chikao Morimoto, Kei Ohnuma.
Application Number | 20100135993 12/450223 |
Document ID | / |
Family ID | 39765975 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100135993 |
Kind Code |
A1 |
Morimoto; Chikao ; et
al. |
June 3, 2010 |
METHOD OF TREATING MALIGNANT MESOTHELIOMA
Abstract
The present invention relates to a therapeutic agent for
malignant mesothelioma comprising a substance which inhibits
binding of CD26 to extracellular matrix such as an siRNA targeting
CD26 cDNA or an anti-CD26 antibody. The present invention also
relates to a method of treating malignant mesothelioma, which
comprises administering the substance to a patient in need
thereof.
Inventors: |
Morimoto; Chikao; (Tokyo,
JP) ; Ohnuma; Kei; (Tokyo, JP) ; Inamoto;
Teruo; (Tokyo, JP) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
The University of Tokyo
Tokyo
JP
|
Family ID: |
39765975 |
Appl. No.: |
12/450223 |
Filed: |
March 14, 2008 |
PCT Filed: |
March 14, 2008 |
PCT NO: |
PCT/JP2008/055344 |
371 Date: |
September 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60894786 |
Mar 14, 2007 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/146.1; 424/158.1; 435/325; 435/7.21; 514/44A |
Current CPC
Class: |
C07K 16/40 20130101;
A61K 2039/505 20130101; A61P 35/00 20180101; C12N 15/1138 20130101;
A61K 31/7088 20130101; C07K 2317/732 20130101; A61K 35/12 20130101;
A61K 39/3955 20130101; C07K 2317/24 20130101; C12N 2310/14
20130101 |
Class at
Publication: |
424/133.1 ;
514/44.A; 424/158.1; 424/146.1; 435/325; 435/7.21 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7105 20060101 A61K031/7105; C12N 5/071
20100101 C12N005/071; G01N 33/53 20060101 G01N033/53; A61P 35/00
20060101 A61P035/00 |
Claims
1. A pharmaceutical composition for treating malignant
mesothelioma, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression, comprising a
substance which inhibits binding of CD26 to extracellular
matrix.
2. The pharmaceutical composition according to claim 1, wherein the
substance comprises a growth-inhibitory agent for treating
malignant mesothelioma, lung cancer, renal cancer, liver cancer, or
other malignancies associated with CD26 expression.
3. The pharmaceutical composition according to claim 1, wherein the
substance is an siRNA targeting CD26 cDNA.
4. The pharmaceutical composition according to claim 1, wherein the
substance is an anti-CD26 antibody.
5. The pharmaceutical composition according to claim 4, wherein the
anti-CD26 antibody is a monoclonal antibody.
6. The pharmaceutical composition according to claim 4, wherein the
anti-CD26 antibody is 14D10.
7. The pharmaceutical composition according to claim 4, wherein the
anti-CD26 antibody is humanized.
8. The pharmaceutical composition according to claim 7, wherein the
humanized antibody comprises a variable region derived from
14D10.
9. The pharmaceutical composition according to claim 7, wherein the
humanized antibody is produced by a strain designated
s604069YST-pABMC148 (x411) with American Type Culture Collection
(ATCC) accession number PTA-7695.
10. The pharmaceutical composition according to claim 4, further
comprising an effector cell specific to the antibody.
11. A kit for treating malignant mesothelioma, lung cancer, renal
cancer, liver cancer, or other malignancies associated with CD26
expression, comprising an anti-CD26 antibody and an effector cell
specific to the antibody.
12. A method for inhibiting growth of a malignant mesothelioma
cell, lung cancer cell, renal cancer cell, liver cancer cell, or
other malignant cell expressing CD26, which comprises contacting a
malignant mesothelioma cell, lung cancer cell, renal cancer cell,
liver cancer cell, or other malignant cell expressing CD26 with a
substance that inhibits binding of CD26 to extracellular
matrix.
13. The method according to claim 12, wherein the substance is an
anti-CD26 antibody or an siRNA targeting CD26 cDNA.
14. A method for inhibiting growth of a malignant mesothelioma
cell, lung cancer cell, renal cancer cell, liver cancer cell, or
other malignant cell expressing CD26, which comprises contacting
the malignant mesothelioma cell, lung cancer cell, renal cancer
cell, liver cancer cell, or other malignant cell expressing CD26
and an effector cell with an anti-CD26 antibody, wherein the
effector cell is a cell specific to the antibody.
15. The method according to claim 14, wherein the substance is an
anti-CD26 antibody or an siRNA targeting CD26 cDNA.
16. A method for lysing a malignant mesothelioma cell, lung cancer
cell, renal cancer cell, liver cancer cell, or other malignant cell
expressing CD26, which comprises contacting the malignant
mesothelioma cell, lung cancer cell, renal cancer cell, liver
cancer cell, or other malignant cell expressing CD26 and an
effector cell with an anti-CD26 antibody, wherein lysis of the cell
is caused by antibody-dependent cell-mediated cytotoxicity and
wherein the effector cell is a cell specific to the antibody.
17. A method for detecting malignant transformation of
mesothelioma, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression, which comprises
collecting a sample from a patient with mesothelioma, lung cancer,
renal cancer, liver cancer, or other malignancies associated with
CD26 expression, and measuring the expression level of CD26 in the
sample.
18. A diagnostic agent for malignant mesothelioma, lung cancer,
renal cancer, liver cancer, or other malignancies associated with
CD26 expression, comprising an anti-CD26 antibody.
19. A kit for diagnosing malignant mesothelioma, lung cancer, renal
cancer, liver cancer, or other malignancies associated with CD26
expression, comprising an anti-CD26 antibody.
20. A method of screening for a substance for treating malignant
mesothelioma, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression, which comprises
contacting a CD26-positive cell with a test substance, and
measuring the expression level of p27 in the cell.
21. (canceled)
22. A method for the treatment of malignant mesothelioma, lung
cancer, renal cancer, liver cancer, or other malignancies
associated with CD26 expression, comprising administering an
effective amount of a substance according to claim 1 to a subject
in need thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is the U.S. National Phase Application under 35 U.S.C.
.sctn.371 of International Patent Application No. PCT/JP2008/055344
filed Mar. 14, 2008, which claims the priority benefit of U.S.
Provisional Application Ser. No. 60/894,786, filed Mar. 14, 2007,
both of which are hereby incorporated by reference herein in its
entirety. The International Application was published in English on
Sep. 25, 2008 as WO2008/114876 A1 under PCT Article 21(2).
FIELD OF THE INVENTION
[0002] The present invention relates to a therapeutic agent for
malignant mesothelioma and a method of treating malignant
mesothelioma.
BACKGROUND
[0003] Malignant mesothelioma (MM) is an aggressive cancer arising
from the mesothelial cells lining the pleura. MM is usually
associated with history of chronic asbestos exposure (1). Because
of the long latency period between asbestos exposure and tumor
development, the annual incidence of 2500 new cases in US is
expected to increase by more than 50% in the coming decade (2).
Moreover incidence world wide is projected to rise substantially in
the next decades (3). The prognosis is very poor with a medium
survival of 4-12 months despite the therapies currently used,
including surgery, radiotherapy and chemotherapy (4). Because of
the inefficacy of the conventional treatments, development of novel
therapeutic strategies is urgently needed.
[0004] CD26 is 110 kDa surface glycoprotein with dipeptidyl
peptidase IV (DPPIV) activity, able to cleave selected biological
factors to alter their functions (5). CD26/DPPIV is involved in
T-lymphocyte costimulation and signal transduction processes (6,
7), and regulates topoisomerase II alpha levels in hematologic
malignancies, affecting sensitivity to doxorubicin and etoposide
(8). Expressed in various tissues (4, 9), CD26 is involved in the
development of certain human cancers (9-12). CD26 is also known to
serve as a binding motif for ECM in human and rodents (13, 14).
Previously, we reported that CD26 is a collagen binding protein
utilizing a CD26-positive JMN cell line derived from malignant
mesothelioma (15). Moreover, our previous works have shown that
anti-CD26 monoclonal antibody (mAb) inhibits growth of
CD26-positive T-cell malignancies (16, 17) and renal cell carcinoma
(18).
[0005] CD26 structure consists of three regions--an extracellular
region, a 22 residue hydrophobic transmembrane domain, and a 6
amino acid cytoplasmic region, with its extracellular region
containing a membrane-proximal glycosylated domain, a cysteine-rich
domain, and a 260 amino acid C-terminal domain containing DPP IV
enzyme activity. Our previous report shows that the murine
anti-CD26 mAb 14D10, which recognizes the cell membrane-proximal
glycosylated region starting with the 20 amino acid flexible stalk
region of human CD26, has a direct anti-tumor effect by inducing
G1/S arrest while concomitantly blocking the adhesion of cancer
cells to the ECM. However, another murine anti-CD26 mAb termed 5F8,
which detects the cysteine-rich domain of CD26, lacks this
biological activity (18).
[0006] Since human malignant mesothelioma (MM) is a highly
malignant tumor resistant to apparent conventional treatment, the
detection of novel target and development of new treatment
strategies for MM is urgently needed (4,19).
SUMMARY OF THE INVENTION
[0007] CD26 is a 110 kDa cell surface antigen with a role in tumor
development through its association with key intracellular
proteins. In the present invention, we analyzed the expression of
CD26 in the tissues of patients with malignant mesothelioma and
validated the anti-tumor effect of a novel humanized anti-CD26 mAb
(humab) which was constructed from a high affinity Fab clone to the
14D10 variable region, by targeting MM, hence concomitantly showing
the functional role of CD26 in this neoplasm. Specifically, in the
present invention, we show that CD26 is highly expressed on the
cell surface of malignant mesothelioma but not benign mesothelium,
indicating that CD26 may be a marker of malignancy. Importantly,
depletion of CD26 by siRNA oligo transfection results in the loss
of adhesive property, suggesting that CD26 is an extracellular
matrix (ECM) binding protein. We previously showed that binding of
the mouse anti-CD26 monoclonal antibody (mAb) 14D10 induces a
direct anti-tumor effect associated with enhanced p27.sup.kip1
expression, down regulation of CDK2, and dephosphorylation of
retinoblastoma substrate. Given these data, we now analyze the
function of a newly developed humanized anti-CD26 mAb (humAb) which
consists of the high binding affinity Fab clone to the 14D10
variable region. Our in vitro data indicate that humAb induces cell
lysis of malignant mesothelioma cells via antibody-dependent
cell-mediated cytotoxicity (ADCC) in addition to its direct
anti-tumor effect via p27.sup.kip1 accumulation and disruption of
binding to the ECM. In vivo experiments with mouse xenograft models
involving human malignant mesothelioma cells demonstrated that
humAb treatment drastically inhibits tumor growth in tumor-bearing
mice, resulting in enhanced survival. Taken together, our data show
that anti-CD26-humAb treatment may have potential clinical use as a
novel cancer therapeutic agent in CD26-positive malignant
mesothelioma.
[0008] Namely, the present invention is directed to the following.
[0009] (1) A pharmaceutical composition for treating malignant
mesothelioma, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression, comprising a
substance which inhibits binding of CD26 to extracellular matrix.
[0010] (2) The pharmaceutical composition according to (1), which
is a growth-inhibitory agent for malignant mesothelioma, lung
cancer, renal cancer, liver cancer, or other malignancies
associated with CD26 expression. [0011] (3) The pharmaceutical
composition according to (1), wherein the substance is an siRNA
targeting CD26 cDNA. [0012] (4) The pharmaceutical composition
according to (1), wherein the substance is an anti-CD26 antibody.
[0013] (5) The pharmaceutical composition according to (4), wherein
the anti-CD26 antibody is a monoclonal antibody. [0014] (6) The
pharmaceutical composition according to (4), wherein the anti-CD26
antibody is 14D10. [0015] (7) The pharmaceutical composition
according to (4), wherein the anti-CD26 antibody is humanized.
[0016] (8) The pharmaceutical composition according to (7), wherein
the humanized antibody comprises a variable region derived from
14D10. [0017] (9) The pharmaceutical composition according to (7),
wherein the humanized antibody is produced by the strain designated
s604069.YST-pABMC 148 (x411) with American Type Culture Collection
(ATCC) accession number PTA-7695.
[0018] The deposit was made with the ATCC on June 30, 2006, under
the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of a
Patent Procedure, and the deposit was designated with accession
number PTA-7695. The sample deposited was "DH5.alpha. Escherichia
coli with a plasmid having insert of heavy and light chain of a
humanized monoclonal antibody against human CD26 cDNA," having the
strain designation s604069.YST-pABMC148 (x411). [0019] (10) The
pharmaceutical composition according to (4), further comprising an
effector cell specific to the antibody. [0020] (11) A kit for
treating malignant mesothelioma, lung cancer, renal cancer, liver
cancer, or other malignancies associated with CD26 expression,
comprising an anti-CD26 antibody and an effector cell specific to
the antibody. [0021] (12) A method of inhibiting growth of a
malignant mesothelioma cell, lung cancer cell, renal cancer cell,
liver cancer cell or other malignant cell expressing CD26, which
comprises contacting a malignant mesothelioma cell, lung cancer
cell, renal cancer cell, liver cancer cell or other malignant cell
expressing CD26 with a substance which inhibits binding of CD26 to
extracellular matrix. [0022] (13) The method according to (12),
wherein the substance is an anti-CD26 antibody or an siRNA
targeting CD26 cDNA. [0023] (14) A method of inhibiting growth of a
malignant mesothelioma cell, lung cancer cell, renal cancer cell,
liver cancer cell, or other malignant cell that expressing CD26,
which comprises contacting a malignant mesothelioma cell, lung
cancer cell, renal cancer cell, liver cancer cell, or other
malignant cell, and an effector cell with an anti-CD26 antibody,
wherein the effector cell is the cell specific to the antibody.
[0024] (15) The method according to (14), wherein the substance is
an anti-CD26 antibody or an siRNA targeting CD26 cDNA. [0025] (16)
A method of lysing a malignant mesothelioma cell, lung cancer cell,
renal cancer cell, liver cancer cell, or other malignant cell
expressing CD26, which comprises contacting the mesothelioma cell,
lung cancer cell, renal cancer cell, liver cancer cell, or other
malignant cell expressing CD26, and an effector cell with an
anti-CD26 antibody, wherein the lysis of the cell is caused by
antibody-dependent cell-mediated cytotoxicity and wherein the
effector cell is the cell specific to the antibody. [0026] (17) A
method of detecting malignant transformation of mesothelioma, lung
cancer, renal cancer, liver cancer, or other malignancies
associated with CD26 expression, which comprises collecting a
sample from a patient with mesothelioma, lung cancer, renal cancer,
liver cancer, or other malignancies associated with CD26
expression, and measuring the expression level of CD26 in the
sample. [0027] (18) A diagnostic agent for malignant mesothelioma,
lung cancer, renal cancer, liver cancer, or other malignancies
associated with CD26 expression, comprising an anti-CD26 antibody.
[0028] (19) A kit for diagnosing malignant mesothelioma, lung
cancer, renal cancer, liver cancer, or other malignancies
associated with CD26 expression comprising an anti-CD26 antibody.
[0029] (20) A method of screening a substance for treating
malignant mesothelioma, lung cancer, renal cancer, liver cancer, or
other malignancies associated with CD26 expression, which comprises
contacting a CD26-positive cell with a test substance, and
measuring the expression level of p27 in the cell. [0030] (21) Use
of a substance as defined in (1) to (10) for producing a
pharmaceutical composition in a treatment of malignant
mesothelioma, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression. [0031] (22) A
substance as defined in (1) to (10) in a treatment of malignant
mesothelioma, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression. [0032] (23) A method
of treating malignant mesothelioma, lung cancer, renal cancer,
liver cancer, or other malignancies associated with CD26
expression, which comprises administering to a patient an anti-CD26
antibody and an effector cell specific to the antibody. [0033] (24)
A method of diagnosing malignant mesothelioma, which comprises
collecting a sample from a patient, and measuring the expression
level of CD26 in the sample.
[0034] The method according to (24) is useful for differential
diagnosis of malignant mesothelioma, lung cancer, renal cancer,
liver cancer, or other malignancies associated with CD26 expression
from primary lung cancer.
[0035] The adminstered substance may enhance p27.sup.kip1
expression and/or disrupt binding of CD26 to ECM to exhibit
anti-tumor effects on the malignant mesothelioma. When the
administered substance is the anti-CD26 antibody, the antibody may
utilize an effector cell to cause ADCC-induced malignant
mesothelioma cell lysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0037] FIG. 1A. Expression and functional role of CD26 in MM.
[0038] A. Immunohistochemical localization of CD26 in adenomatoid
tumor, reactive mesothelial cells, and MM. a, CD26 in adenomatoid
tumor; b, CD26 in reactive mesothelial cells; c, CD26 in localized
MM; d, CD26 in well-differentiated papillary MM; e and f, H&E
stain in diffuse MM ; g and h, CD26 in diffuse MM. Diffuse MM
specimens showing biphasic features of sarcomatous MM (f, h) and
epithelial MM (g, i). Indicated panels are representative of 12
consecutive specimens. Original magnification.times.100.
[0039] FIG. 1B-D. Expression and functional role of CD26 in MM.
[0040] B. Surface expression of CD26 on mesothelioma cell lines was
analyzed by flow cytometry. Right lines, CD26 histograms were
obtained by staining with mouse anti-CD26mAb (14D10) followed by
staining with rabbit anti-mouse Igb FITC conjugate. Left lines,
control histograms represent background fluorescence obtained by
staining of isotype-matched control mAb (2H4).
[0041] C. Adhesive property of CD26 to ECM. CD26-depleted NCI-H2452
(si), scrambled control oligo-transfected NCI-H2452 (ctl), pEB6
vector-transfected 293T (vec), or pEB6-CD26-transfected 293T (26)
were plated onto 60 mm dishes (2.times.10.sup.6 cells per dish)
coated with fibronectin (FN), collagen I (CL), laminin (LN), or
hyaluronan (HL) and cultured for 18 h. FN and CL are binding
proteins (BP) to the extracellular region of CD26, and HL is a CD44
binding protein. The adhesive ability of cancer cells was expressed
as the mean number of cells that had attached to the bottom surface
of the dish, and the results are presented as mean.+-.SE number of
cells per field of view. Values for adhesion were determined by
calculating the average number of adhesive cells per mm.sup.2 over
three fields per assay and expressed as an average of triplicate
determinations. Adhesive cells (%): adhesive cells/adhesive
cells+nonadhesive cells.
[0042] D. Depletion of CD26 elicits upregulation of p27.sup.kip1.
NCI-H2452 cells and JMN cells were transfected with siRNA-oligo of
CD26 (si), or control-oligo (ctl). At 48 h after transfection,
cells were harvested, lysed, and subjected to SDS-PAGE and probed
with antibodies to p27.sup.kip1, p21.sup.cip1/waf1, CD26, and
CD44.
[0043] FIG. 2. Inhibitory effect of anti-CD26 mAbs on MM
proliferation.
[0044] A. Effect of anti-CD26 mAb on cell adhesion to ECM. JMN
cells treated with isotype matched control mAb (iso), 5F8, 14D10,
or humanized anti-CD26 mAb (humAb) were plated onto 60 mm dishes
(2.times.10.sup.6 cells per dish) coated with fibronectin (FN),
collagen I (CL), laminin (LN), or hyaluronan (HL) and cultured for
18 h. Adhesive cells (%): adhesive cells/adhesive cells+nonadhesive
cells.
[0045] B. 5.times.10.sup.3 cells/well of JMN were incubated in
96-well plates in the presence of either isotype matched control
mAb (iso), 5F8, 14D10, or humanized anti-CD26 mAb (humAb). After
24h of antibody treatment, water soluble formazan dye upon
bioreduction in the presence of an electron carrier,
1-methoxy-5-methylphenazinium, was measured at 450 nm using a
microplate reader as described in Materials and Methods, and growth
inhibitory ratio was calculated as % reduction of OD 450 nm.
[0046] C. JMN cells were treated with isotype matched control mAb
(iso), 5F8, 14D10, or humanized anti-CD26 mAb (humAb). At 18 h
after antibody administration, cells were harvested, lysed, and
subjected to SDS-PAGE and probed by antibodies to p27.sup.kip1,
p21.sup.cip1/waf1, CDK2, CDK4, CDK6, cyclinD1, cyclinE, and
.beta.-actin.
[0047] FIG. 3. Antibody-dependent cell-mediated cytotoxicity (ADCC)
specific lysis of JMN cells by humanized anti-CD26 mAb.
[0048] A. Left panel; ADCC of humanized anti-CD26 mAb (humAb) and
14D10 at the indicated concentrations on the X-axis were examined.
Effector/Target (E/T) ratio was held constant at 50. Right panel;
ADCC of humanized anti-CD26 mAb (humAb) and 14D10 in the presence
of varying E/T ratios were examined. Concentrations of mAbs were
held constant at 5 .mu.g/ml. NK cells from a healthy donor were
used as effector cells.
[0049] B. To mimic effector cells in ADCC effects, cross-linking
(XL) method of humanized anti-CD26 mAb (humAb) and 14D10 was
utilized. Upper three panels indicate the cross-linked 14D10,
intact humAb, cross-linked humAb, respectively. To examine the
complementary-dependent cytotoxicity (CDC), human-serum was
utilized. Lower three panels indicate 14D10 with serum, humAb with
serum, and humAb with heat-inactivated serum. X-axis indicates the
annexinV. Y-axis indicates the propidium iodide (PI).
[0050] C. Activated caspase 3 was evaluated in JMN cells
pre-treated with the cross-linked 14D10, intact humAb, cross-linked
humAb, respectively (upper three panels), or in JMN cells
pre-treated with the 14D10 plus serum, humAb plus serum, and humAb
plus heat-inactivated serum, respectively (lower three panels).
[0051] FIG. 4. In vivo direct effect of humanized anti-CD26 mAb:
ADCC depletion model. 6 week old female NOD SCID mice were
pre-treated with anti-asialo-GM1 polyclonal antisera 1 day before
treatment.
[0052] A. Effect of humanized anti-CD26 mAb in subcutaneous
tumorigenicity was evaluated. JMN cells (1.times.10.sup.6) were
inoculated subcutaneously into the left flank of mice. Mice were
treated with intra-tumoral injection of isotype matched control mAb
(iso, n=4), 5F8 (n=4), 14D10 (n=4), or humanized anti-CD26 mAb
(humAb, n=4) on the 14th day after cancer cell inoculation when the
tumor mass became visible (5 mm in size). Each mAb was administered
at 10 .mu.g/injection three times per week.
[0053] B. Representative resected specimens in subcutaneous
tumorigenicity model on 35th day after first mAb treatment.
[0054] C. Effect of humanized anti-CD26 mAb in tumor dissemination
model was evaluated. JMN cells (1.times.10.sup.5) were injected
intra-venously into mice in each group. Mice were treated with
intra-venous injection of isotype matched control mAb (iso, n=4),
5F8 (n=4), 14D10 (n=4), or humanized anti-CD26 mAb (humAb, n=4) on
the day of cancer cell injection. Each mAb was administered at 10
.mu.g/injection three times per week.
[0055] FIG. 5A-B. In vivo direct and indirect effects of humanized
anti-CD26 mAb: mouse ADCC presence model.
[0056] 6 week old female Balb mice were used in this
experiment.
[0057] A. Effect of humanized anti-CD26 mAb in subcutaneous
tumorigenicity was evaluated. JMN cells (1.times.10.sup.6) were
inoculated subcutaneously into the left flank of mice. Mice were
treated with intra-tumoral injection of isotype matched control mAb
(iso, n=4), 5F8 (n=4), 14D10 (n=4), or humanized anti-CD26 mAb
(humAb, n=4) on the 14th day after cancer cell inoculation when the
tumor mass became visible (5 mm in size). Each mAb was administered
at 10 .mu.g/injection three times per week.
[0058] B. Representative H&E stain of resected specimens in
subcutaneous tumorigenicity model on 35th day after first mAb
treatment. a, isotype matched control mAb (.times.100); b, isotype
matched control mAb (.times.600); c, 5F8 (.times.100); d, 5F8
(.times.600); e, 14D10 (.times.100); f, 14D10 (.times.600); g,
humanized anti-CD26 mAb (.times.100) ; and h, humanized anti-CD26
mAb (.times.600). White broken line indicates the boundary between
tumor (T) and dead tissue (D).
[0059] FIG. 5C-D. In vivo direct and indirect effect of humanized
anti-CD26 mAb: mouse ADCC presence model.
[0060] C. Effect of humanized anti-CD26 mAb in tumor dissemination
model was evaluated. JMN cells (1.times.10.sup.5) were injected
intra-venously into mice in each group. Mice were treated with
intra-venous injection of isotype matched control mAb (iso, n=5),
or humanized anti-CD26 mAb (humAb, n=5) on the day of cancer cell
injection. Each mAb was administered at 10 .mu.g/injection three
times per week.
[0061] D. Effect of humanized anti-CD26 mAb on distant metastasis
formation in the tumor dissemination model was evaluated. JMN cells
(1.times.10.sup.5) were injected intra-venously into mice in each
group. Mice were treated with intra-venous injection of isotype
matched control mAb (lane 1, n=4), 5F8 (lane 2, n=4), 14D10 (lane
3, n=4), or humanized anti-CD26 mAb (lane 4, n=4) on the day of
cancer cell injection. Each mAb was administered at 10
.mu.g/injection three times per week. On 35th day after cancer cell
injection, mice were euthanized and multiple metastasis formation
in the lung and liver was calculated.
[0062] FIG. 6. In vivo direct and indirect effect of humanized
anti-CD26 mAb: human ADCC presence model.
[0063] 6 week old female NOG SCID mice were used in this
experiment. Mice were divided into two groups, human effector cells
(HuEC)-implanted group and HuEC-negative group, respectively. All
mice were pre-treated with anti-asialo-GM1 polyclonal antisera
intra-peritoneally 2 days before HuEC implantation. HuEC were
implanted intra-peritoneally with effector:target ratio=10:1. JMN
cells (1.times.10.sup.6) were implanted 1 day after HuEC
implantation into the peritoneal cavity of mice. The latter group
was left untreated. All mice were treated with human normal
IgG+HuEC (H-IgG+HuEC, n=4), 14D10 (n=4), 14D10+HuEC (n=4),
humanized anti-CD26 mAb (humAb, n=4), or humanized anti-CD26
mAb+HuEC (humAb+HuEC, n=4). Each mAb was i.p. administered at 10
.mu.g/injection, 1, 3, and 5 days after cancer cells
implantation.
[0064] All publications, patents, patent applications, internet
sites, and accession numbers/database sequences (including both
polynucleotide and polypeptide sequences) cited herein are hereby
incorporated by reference herein in their entirety for all purposes
to the same extent as if each individual publication, patent,
patent application, internet site, or accession number/database
sequence were specifically and individually indicated to be so
incorporated by reference.
DETAILED DESCRIPTION
[0065] The present invention provides a pharmaceutical composition
for treating malignant mesothelioma comprising a substance which
inhibits binding of CD26 to extracellular matrix. Examples of the
substance include novel polypeptides such as anti-CD26 antibodies
and an siRNA targeting CD26 mRNA and/or cDNA.
[0066] The present invention provides novel polypeptides such as
anti-CD26 antibodies, fragments of anti-CD26 antibodies, and other
polypeptides related to anti-CD26 antibodies. In some embodiments,
the anti-CD26 antibodies are humanized anti-CD26 antibodies.
Polynucleotides comprising nucleic acid sequences encoding the
polypeptides are also provided. Vectors and host cells comprising
the polynucleotides are also provided. Compositions, such as
pharmaceutical compositions, comprising the polypeptides of the
invention are also provided. Methods of making the polypeptides are
also provided. In addition, methods of using the polypeptides or
compositions comprising the polypeptides to inhibit proliferation
of cells expressing CD26 or in the treatment or diagnosis of
conditions associated with CD26 expression are further
provided.
[0067] It is understood that wherever embodiments are described
herein with the language "comprising," otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0068] General Techniques
[0069] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature, such
as, Molecular Cloning: A Laboratory Manual, second edition
(Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide
Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology,
Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis,
ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,
ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather
and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:
Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,
eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
[0070] Definitions
[0071] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also fragments
thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv),
mutants thereof, fusion proteins comprising an antibody portion,
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site. An antibody includes an
antibody of any class, such as IgG, IgA, or IgM (or sub-class
thereof), and the antibody need not be of any particular class.
Depending on the antibody amino acid sequence of the constant
domain of its heavy chains, immunoglobulins can be assigned to
different classes. There are five major classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, and several of these may be further
divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2. The heavy-chain constant domains that correspond to
the different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0072] As used herein, "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible naturally-occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by
recombinant DNA methods such as described in U.S. Pat. No.
4,816,567. The monoclonal antibodies may also be isolated from
phage libraries generated using the techniques described in
McCafferty et al., 1990, Nature, 348:552-554, for example.
[0073] As used herein, "humanized" antibodies refers to forms of
non-human (e.g. murine) antibodies that are specific chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2, or other antigen-binding
subsequences of antibodies) that contain minimal sequence derived
from non-human immunoglobulin. Some humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Some humanized
antibodies comprise at least one, and typically two, variable
domains that are generally derived from a non-human species (donor
antibody) such as a mouse, rat, or rabbit having the desired
specificity, affinity, and/or capacity, but in which one or more Fv
framework region residues and/or one or more Fv CDR residues have
been replaced by a corresponding human residue (i.e., a residue
derived from a human antibody sequence). Most typically, at least a
plurality of Fv framework region residues will have been replaced
in one or more of the variable domains of the humanized antibody.
Furthermore, the humanized antibody may comprise residues that are
found neither in the recipient antibody nor in the imported CDR or
framework sequences, but are included to further refine and
optimize antibody performance. Some humanized antibodies will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDRs
correspond to those of a non-human immunoglobulin and all or
substantially all of the FRs are those of a human immunoglobulin
consensus sequence. Some humanized antibodies will comprise
substantially all of at least one, and typically two, variable
domains, in which the majority of the amino acid residues of the
CDRs correspond to those of a non-human immunoglobulin and one or
more of the amino acid residues of the FRs are those of a human
immunoglobulin consensus sequence. A humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Some humanized antibodies have Fc regions modified as described in
WO 99/58572. Some forms of humanized antibodies have one or more
(e.g., one, two, three, four, five, six) CDRs which are altered
with respect to the original antibody, which are also termed one or
more CDRs "derived from" one or more CDRs from the original
antibody.
[0074] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody produced
by a human and/or has been made using any of the techniques for
making human antibodies known in the art or disclosed herein. This
definition of a human antibody includes antibodies comprising at
least one human heavy chain polypeptide or at least one human light
chain polypeptide. One such example is an antibody comprising
murine light chain and human heavy chain polypeptides. Human
antibodies can be produced using various techniques known in the
art. In one embodiment, the human antibody is selected from a phage
library, where that phage library expresses human antibodies
(Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et
al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991,
J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,
222:581). Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016. Alternatively, the human antibody may be prepared by
immortalizing human B lymphocytes that produce an antibody directed
against a target antigen (such B lymphocytes may be recovered from
an individual or may have been immunized in vitro). See, e.g., Cole
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77 (1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and
U.S. Pat. No. 5,750,373. In some embodiments, a human antibody is
"fully human," meaning the antibody contains human heavy chain and
light chain polypeptides.
[0075] The terms "polypeptide", "oligopeptide", "peptide", and
"protein" are used interchangeably herein to refer to polymers of
amino acids of any length. The polymer may be linear or branched,
it may comprise modified amino acids, and it may be interrupted by
non-amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that, because the polypeptides of this invention are
based upon an antibody, the polypeptides can occur as single chains
or associated chains.
[0076] "Polynucleotide," or "nucleic acid," as used interchangeably
herein, refer to polymers of nucleotides of any length, and include
DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their
analogs, or any substrate that can be incorporated into a polymer
by DNA or RNA polymerase. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and their analogs. If
present, modification to the nucleotide structure may be imparted
before or after assembly of the polymer. The sequence of
nucleotides may be interrupted by non-nucleotide components. A
polynucleotide may be further modified after polymerization, such
as by conjugation with a labeling component. Other types of
modifications include, for example, "caps", substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, cabamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing
pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, ply-L-lysine, etc.), those
with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron,
oxidative metals, etc.), those containing alkylators, those with
modified linkages (e.g., alpha anomeric nucleic acids, etc.), as
well as unmodified forms of the polynucleotide(s). Further, any of
the hydroxyl groups ordinarily present in the sugars may be
replaced, for example, by phosphonate groups, phosphate groups,
protected by standard protecting groups, or activated to prepare
additional linkages to additional nucleotides, or may be conjugated
to solid supports. The 5' and 3' terminal OH can be phosphorylated
or substituted with amines or organic capping group moieties of
from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized
to standard protecting groups. Polynucleotides can also contain
analogous forms of ribose or deoxyribose sugars that are generally
known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl,
2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs,
.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses
or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,
acyclic analogs and abasic nucleoside analogs such as methyl
riboside. One or more phosphodiester linkages may be replaced by
alternative linking groups. These alternative linking groups
include, but are not limited to, embodiments wherein phosphate is
replaced by P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2
("amidate"), P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in
which each R or R' is independently H or substituted or
unsubstituted alkyl (1-20 C) optionally containing an ether (--O--)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not
all linkages in a polynucleotide need be identical. The preceding
description applies to all polynucleotides referred to herein,
including RNA and DNA.
[0077] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FR) connected by three complementarity determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FRs and, with the CDRs
from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al (1997) J. Molec. Biol. 273:927-948)). In addition,
combinations of these two approaches are sometimes used in the art
to determine CDRs.
[0078] A "constant region" of an antibody refers to the constant
region of the antibody light chain or the constant region of the
antibody heavy chain, either alone or in combination.
[0079] An epitope that "preferentially binds" or "specifically
binds" (used interchangeably herein) to an antibody or a
polypeptide is a term well understood in the art, and methods to
determine such specific or preferential binding are also well known
in the art. A molecule is said to exhibit "specific binding" or
"preferential binding" if it reacts or associates more frequently,
more rapidly, with greater duration and/or with greater affinity
with a particular cell or substance than it does with alternative
cells or substances. An antibody "specifically binds" or
"preferentially binds" to a target if it binds with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other substances. For example, an antibody that
specifically or preferentially binds to a CD26 epitope is an
antibody that binds this CD26 epitope with greater affinity,
avidity, more readily, and/or with greater duration than it binds
to other CD26 epitopes or non-CD26 epitopes. It is also understood
by reading this definition that, for example, an antibody (or
moiety or epitope) that specifically or preferentially binds to a
first target may or may not specifically or preferentially bind to
a second target. As such, "specific binding" or "preferential
binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
binding means preferential binding.
[0080] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected in vivo with a
polynucleotide(s) of this invention.
[0081] A polypeptide, antibody, polynucleotide, vector, cell, or
composition which is "isolated" is a polypeptide, antibody,
polynucleotide, vector, cell, or composition which is in a form not
found in nature. Isolated polypeptides, antibodies,
polynucleotides, vectors, cell, or compositions include those which
have been purified to a degree that they are no longer in a form in
which they are found in nature. In some embodiments, an antibody,
polynucleotide, vector, cell, or composition which is isolated is
substantially pure.
[0082] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of one or more symptoms, diminishment
of extent of disease, stabilized (i.e., not worsening) state of
disease, delay or slowing of disease progression, amelioration or
palliation of the disease state, and remission (whether partial or
total), whether detectable or undetectable. "Treatment" can also
mean prolonging survival as compared to expected survival if not
receiving treatment.
[0083] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results including clinical results.
An effective amount can be administered in one or more
administrations. For purposes of this invention, an effective
amount of a polypeptide, such as an anti-CD26 antibody, described
herein is an amount sufficient to ameliorate, stabilize, reverse,
slow and/or delay progression of a condition associated with CD26
expression. As is understood in the art, an effective amount of,
for example, an anti-CD26 antibody may vary, depending on, inter
alia, patient history as well as other factors such as the type
(and/or dosage) of an anti-CD26 antibody used. As evident by this
disclosure to one skilled in the art, these principles apply to
polypeptide embodiments.
[0084] An "individual," also referred to herein as a "subject," is
a vertebrate, preferably a mammal, more preferably a human. Mammals
include, but are not limited to, farm animals (such as cows), sport
animals, pets (such as cats, dogs, and horses), primates, mice and
rats.
[0085] As used herein, "vector" means a construct, which is capable
of delivering, and preferably expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0086] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable excipient" includes any material
which, when combined with an active ingredient, allows the
ingredient to retain biological activity and is non-reactive with
the subject's immune system and non-toxic to the subject when
delivered. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline or normal
(0.9%) saline. Compositions comprising such carriers are formulated
by well known conventional methods (see, for example, Remington's
Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990; and Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
[0087] Anti-CD26 Antibody
[0088] In some embodiments, the polypeptides described herein
preferentially bind to the one or more peptides. These peptides are
regions of human CD26. In some embodiments, the polypeptides
described herein bind to the same epitope as the mouse monoclonal
antibody 14D10. In some embodiments, the polypeptides described
herein are capable of blocking the binding of mouse monoclonal
antibody 14D10 to human CD26 in a competition assay. In some
embodiments, the polypeptides described herein are capable of
blocking the binding of mouse monoclonal antibody 1F7 to human CD26
in a competition assay.
[0089] Methods of determining affinity are known in the art. For
instance, binding affinity may be determined using a BIAcore
biosensor, a KinExA biosensor, scintillation proximity assays,
ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching,
fluorescence transfer, and/or yeast display. Binding affinity may
also be screened using a suitable bioassay.
[0090] One way of determining binding affinity of antibodies to
CD26 is by measuring affinity of monofunctional Fab fragments of
the antibodies. To obtain monofunctional Fab fragments, antibodies,
for example, IgGs can be cleaved with papain or expressed
recombinantly. Affinities of anti-CD26 Fab fragments of monoclonal
antibodies can be determined by Surface Plasmon Resonance (SPR)
system (BIAcore 3000.TM., BIAcore, Inc., Piscaway, N.J.). SA chips
(streptavidin) are used according to the supplier's instructions.
Biotinylated CD26 can be diluted into HBS-EP (100 mM HEPES pH 7.4,
150 mM NaCl, 3 mM EDTA, 0.005% P20) and injected over the chip at a
concentration of 0.005 mg/mL. Using variable flow time across the
individual chip channels, two ranges of antigen density are
achieved: 10-20 response units (RU) for detailed kinetic studies
and 500-600 RU for concentration. A mixture of Pierce elution
buffer and 4 M NaCl (2:1) effectively removes the bound Fab while
keeping the activity of CD26 on the chip for over 200 injections.
HBS-EP buffer can be used as running buffer for all the BIAcore
assays. Serial dilutions (0.1-10.times. estimated K.sub.D) of
purified Fab samples are injected for 2 min at 100 .mu.L/min and
dissociation times of up to 30 min are generally allowed. The
concentrations of the Fab proteins can be determined by ELISA
and/or SDS-PAGE electrophoresis using a standard Fab of known
concentration (determined by amino acid analysis). Kinetic
association rates (k.sub.on) and dissociation rates (k.sub.off) are
obtained simultaneously by fitting the data to a 1:1 Langmuir
binding model (Lofas & Johnsson, 1990) using the BIAevaluation
program. Equilibrium dissociation constant (K.sub.D) values are
calculated as k.sub.off/k.sub.on.
[0091] In some embodiments, the invention encompasses polypeptides,
such as antibodies, which inhibit proliferation of cells expressing
CD26. The invention also encompasses embodiments where the
polypeptides are useful in the treatment of a condition (such as a
disease or disorder) associated with CD26 expression (e.g., a
malignant mesothelioma). In some embodiments, the polypeptides
(e.g., antibodies) of the invention may have one or more of the
following characteristics: (a) bind CD26; (b) modulate CD26
activity, (c) cause cell cycle arrest of CD26+ cells at the G1/S
checkpoint; (d) inhibit proliferation of cells expressing CD26
(e.g., malignant mesothelioma), (e) inhibit binding of CD26 to
extracellular matrix, and/or (f) are useful in the treatment of a
condition associated with CD26 expression. In some embodiments, the
condition associated with CD26 expression is a disease or disorder
associated with CD26 overexpression. In some embodiments, the
condition associated with CD26 expression is mediated, at least in
part, by CD26. In some embodiments, the condition associated with
CD26 expression is a condition associated with the proliferation of
cells expressing CD26. In some embodiments, the disease or disorder
is a cancer (e.g., malignant mesothelioma, lung cancer, renal
cancer; liver cancer, or other malignancies associated with CD26
expression), an autoimmune disease or disorder, graft versus host
disease (GVHD), or an inflammatory disease or disorder.
[0092] In some embodiments, the antibody comprises both a heavy
chain variable region comprising an amino acid sequence having at
least about 80% identity to an amino acid sequence selected from
the group consisting of SEQ ID NOS:8-14 and a light chain variable
region comprising an amino acid sequence having at least about 80%
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-7. In some embodiments, the antibody
comprises a light chain variable region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOS:1-7 and a
heavy chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS:8-14.
[0093] In some embodiments, the polypeptide comprises at least 5
contiguous amino acids, at least 8 contiguous amino acids, at least
about 10 contiguous amino acids, at least about 15 contiguous amino
acids, at least about 20 contiguous amino acids, at least about 30
contiguous amino acids, or at least about 50 contiguous amino acids
of an amino acid sequence of any one of SEQ ID NOS:1-14.
[0094] The invention further provides polypeptides comprising
fragments of the polypeptide sequences described herein (e.g., any
one of SEQ ID NOS:1-7, SEQ IDS NOS:8-14, SEQ IDS NO:15, or SEQ IDS
NO:16). In some embodiments, the polypeptide comprises a fragment
of a polypeptide sequence described herein, wherein the fragment is
at least about 10 amino acids in length, at least about 25 amino
acids in length, at least about 50 amino acids in length, at least
about 75 amino acids in length, or at least about 100 amino acids
in length.
SEQ ID NO:15
[0095]
EVQLVX.sub.1SGX.sub.2X.sub.3X.sub.4X.sub.5QPGX.sub.6X.sub.7LRLX.sub-
.8CX.sub.9ASGX.sub.10X.sub.11LX.sub.12TYGVHWVRQAPGKGLE
WX.sub.13GVIWGX.sub.14GRTDYDX.sub.15X.sub.16FMSRVTISX.sub.17DX.sub.18SKX.-
sub.19TX.sub.20YLQX.sub.21NSLRAEDTAV
YYCX.sub.22RX.sub.23RHDWFDYWGQGTTVTVSS, wherein X.sub.1 is E or Q,
X.sub.2 is A or G, X.sub.3 is G or E, X.sub.4 is L or V, X.sub.5 is
V, K, or E, X.sub.6 is G or E, X.sub.7 is T or S, X.sub.8 is T or
S, X.sub.9 is T or K, X.sub.10 is F or Y, X.sub.11 is S or T,
X.sub.12 is T, N, or S, X.sub.13 is V or M, X.sub.14 is G or D,
X.sub.15 is A or S, X.sub.16 is A or S, X.sub.17 is K or R,
X.sub.18 is N or T, X.sub.19 is S or N, X.sub.20 is V or A,
X.sub.21 is M or L, X.sub.22 is V, M, or T, and X.sub.23 is N or
S.
SEQ ID NO:16
[0096]
X.sub.1IX.sub.2X.sub.3TQSPSSLSX.sub.4X.sub.5X.sub.6GX.sub.7RX.sub.8-
TIX.sub.9CX.sub.10ASQX.sub.11 IRNX.sub.12LNWYQQKPGQAPRLLIY
YSSNLX.sub.13X.sub.14GVPX.sub.15RFSGSGSGTDFTLTISRLX.sub.16X.sub.17EDX.sub-
.18AX.sub.19YYCQQSX.sub.20KLPX.sub.21 TFGSGTKVEIK, wherein X.sub.1
is D or E, X.sub.2 is L or E, X.sub.3 is M or L, X.sub.4 is A or V,
X.sub.5 is S or T, X.sub.6 is L, P, or A, X.sub.7 is D or E,
X.sub.8 is V or A, X.sub.9 is T or S, X.sub.10 is S or R, X.sub.11
is G or D, X.sub.12 is S or N, X.sub.13 is H or Q, X.sub.14 is S or
T, X.sub.15 is S, D, or A, X.sub.16 is E or Q, X.sub.17 is P or A,
X.sub.18 is F or V, X.sub.19 is T, A, or I, X.sub.20 is I or N and
X.sub.21 is F or L.
[0097] Table 1 shows the amino acid sequences of humanized VL
variants X376 (SEQ ID NO:1), X377 (SEQ ID NO:2), X378 (SEQ ID
NO:3), X379 (SEQ ID NO:4), X380 (SEQ ID NO:5), X381 (SEQ ID NO:6),
and X394 (SEQ ID NO:7). Kabat and sequential numbering schemes are
identical for the light chain variable regions.
[0098] Table 2 shows the amino acid sequences of humanized VH
variants X384 (SEQ ID NO:8), X385 (SEQ ID NO:9), X386 (SEQ ID
NO:10), X387 (SEQ ID NO:11) and X388 (SEQ ID NO:12), X399 (SEQ ID
NO:13) and X420 (SEQ ID NO:14). Both the sequential and Kabat
numbering schemes are shown. The Kabat numbering scheme includes
82a, 82b, and 82c.
TABLE-US-00001 TABLE 1 Sequential 10 20 30 40 50 Numbering
123456789012345678901234567890123456789012345678901234
<---------FR1---------X--CDR1---X-----FR2-----XCDR2-X- CM03 VL
DIQMTQSPSSLSASLGDRVTITCSASQGIRNSLNWYQQKPDGAVKLLIYYSSNL X376
DILMTQSPSSLSASPGDRVTISCRASQDIRNNLNWYQQKPGQAPRLLIYYSSNL X377
EIELTQSPSSLSVSLGDRVTISCSASQDIRNNLNWYQQKPGQAPRLLIYYSSNL X378
DIEMTQSPSSLSASAGERVTISCRASQGIRNSLNWYQQKPGQAPRLLIYYSSNL X379
DILLTQSPSSLSATPGERATITCRASQGIRNNLNWYQQKPGQAPRLLIYYSSNL X380
EIEMTQSPSSLSVSAGERATISCSASQDIRNSLNWYQQKPGQAPRLLIYYSSNL X381
EIELTQSPSSLSVSPGDRVTISCSASQGIRNSLNWYQQKPGQAPRLLIYYSSNL X394
DILMTQSPSSLSASPGDRVTISCRASQDIRNNLNWYQQKPGQAPRLLIYYSSNL Kabat
Numbering 24 34 50 (same as sequential numbering; no insertion)
Sequential 60 70 80 90 100 Numbering
56789012345678901234567890123456789012345678901234567
------------FR3--------------X-CDR3--X--FR4---> CM03 VL
HSGVPSRFSGSGSGTDFSLTISNLEPEDIATYYCQQSIKLPFTFGSGTKLEIK X376
HSGVPDRFSGSGSGTDFTLTISRLEPEDFAAYYCQQSIKLPLTFGSGTKVEIK X377
QTGVPARFSGSGSGTDFTLTISRLEPEDVAAYYCQQSIKLPFTFGSGTKVEIK X378
QTGVPSRFSGSGSGTDFTLTISRLQAEDFATYYCQQSNKLPFTFGSGTKVEIK X379
QSGVPSRFSGSGSGTDFTLTISRLQPEDVAAYYCQQSIKLPFTFGSGTKVEIK X380
HTGVPARFSGSGSGTDFTLTISRLEPEDVAIYYCQQSNKLPLTFGSGTKVEIK X381
HTGVPARFSGSGSGTDFTLTISRLQAEDFATYYCQQSIKLPLTFGSGTKVEIK X394
QTGVPARFSGSGSGTDFTLTISRLEPEDFAAYYCQQSIKLPLTFGSGTKVEIK Kabat
Numbering 56 89 97 (same as sequential numbering; no insertion)
TABLE-US-00002 TABLE 2 Sequential 10 20 30 40 50 Numbering
1234567890123456789012345678901234567890123456789012345678
<----------FR1----------X--CDR1--X----FR2-----X----CDR2--- CM03
VH QVKLQESGPGLVQPSQTLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWGGGRTD X384
EVQLVESGAGVKQPGGTLRLTCTASGFSLTTYGVHWVRQAPGKGLEWVGVIWGDGRTD X385
EVQLVQSGGGVKQPGETLRLTCTASGFSLTTYGVHWVRQAPGKGLEWVGVIWGDGRTD X386
EVQLVESGAGVEQPGGTLRLTCTASGFSLTTYGVHWVRQAPGKGLEWMGVIWGDGRTD X387
EVQLVESGAELVQPGGSLRLTCKASGFTLNTYGVHWVRQAPGKGLEWMGVIWGGGRTD X388
EVQLVQSGGGLKQPGETLRLSCTASGYSLTTYGVHWVRQAPGKGLEWMGVIWDGDRTD X399
EVQLVQSGGGVKQPGETLRLTCTASGFSLSTYGVHWVRQAPGKGLEWVGVIWGDGRTD X420
EVQLVESGGGVKQPGETLRLTCTASGFSLSTYGVHWVRQAPGKGLEWVGVIWGDGRTD Kabat
Numbering 26 35 50 Sequential 60 70 80 90 100 110 Numbering
9012345678901234567890123456789012345678901234567890123456
---X-------------FR3--------------X-CDR3-X--FR4----> CM03 VH
YDAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCVRNRHDWFDYWGQGTTVTVSS X384
YDAAFMSRVTISKDTSKSTVYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVTVSS X385
YDAAFMSRVTISKDTSKSTAYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVTVSS X386
YDAAFMSRVTISRDTSKSTAYLQLNSLRAEDTAVYYCVRNRHDWFDYWGQGTTVTVSS X387
YDASFMSRVTISKDNSKNTAYLQLNSLRAEDTAVYYCTRSRHDWFDYWGQGTTVTVSS X388
YDSSFMSRVTISKDTSKSTAYLQLNSLRAEDTAVYYCTRNRHDWFDYWGQGTTVTVSS X399
YDAAFMSRVTISKDTSKSTVYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVTVSS X420
YDAAFMSRVTISKDTSKSTVYLQMNSLRAEDTAVYYCMRNRHDWFDYWGQGTTVTVSS Kabat
Numbering 65 abc3456789012345678901234567890123 82 90 100 110 95
102
[0099] The invention further provides a polypeptide (e.g., an
antibody) comprising SEQ ID NO:17, or a fragment or variant
thereof. In some embodiments, the polypeptide comprises SEQ ID NO:
17. In some embodiments, the polypeptide comprises SEQ ID NO:17
except for the signal sequence. (One of ordinary skill in the art
will readily appreciate that in some embodiments, the signal
sequence of a polypeptide is cleaved off of the polypeptide.) In
some embodiments, the polypeptide comprises the variable region of
SEQ ID NO:17. In some embodiments, the polypeptide comprises a
polypeptide having at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or at least about 98% identity to
SEQ ID NO:17 (or a fragment thereof). In some embodiments, the
polypeptide comprises a fragment of SEQ ID NO:17, wherein the
fragment is at least about 10 amino acids in length, at least about
25 amino acids in length, at least about 50 amino acids in length,
at least about 75 amino acids in length, or at least about 100
amino acids in length. In some embodiments, the polypeptide binds
human CD26.
TABLE-US-00003 Heavy chain (SEQ ID NO: 17): MEWSWVFLFFLSVTTGVHS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKITVDKSRW
QQGNVFSGSVMHEALHNHYTQKSLSLSPGK
[0100] The invention further provides a polypeptide (e.g., an
antibody) comprising SEQ ID NO:18, or a fragment or variant
thereof. In some embodiments, the polypeptide comprises SEQ ID
NO:18. In some embodiments, the polypeptide comprises SEQ ID NO:18
except for the signal sequence. (One of ordinary skill in the art
will readily appreciate that in some embodiments, the signal
sequence of a polypeptide is cleaved off of the polypeptide.) In
some embodiments, the polypeptide comprises the variable region of
SEQ ID NO:18. In some embodiments, the polypeptide comprises a
polypeptide having at least about 80%, at least about 85%, at least
about 90%, at least about 95%, or at least about 98% identity to
SEQ ID NO:18 (or a fragment thereof). In some embodiments, the
polypeptide comprises a fragment of SEQ ID NO:18, wherein the
fragment is at least about 10 amino acids in length, at least about
25 amino acids in length, at least about 50 amino acids in length,
at least about 75 amino acids in length, or at least about 100
amino acids in length. In some embodiments, the polypeptide further
comprises SEQ ID NO:18, or a fragment or variant thereof. In some
embodiments, the polypeptide binds human CD26. For instance, in
some embodiments, the polypeptide is an antibody comprising at
least one heavy chain (e.g., two heavy chains), each of which
comprises SEQ ID NO:17 without the signal sequence, and at least
one light chain (e.g., two light chains), each of which comprises
SEQ ID NO:18 without the signal sequence.
TABLE-US-00004 Light chain (SEQ ID NO: 18): MSVPTQVLGLLLLWLTDARC
RTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT
EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0101] In another aspect, the invention provides a polypeptide,
such as an antibody, that binds to one or more peptides selected
from the group consisting of YSLRWISDHEYLY (SEQ ID NO:19; peptide
6), LEYNYVKQWRHSY (SEQ ID NO:20;,peptide 35), TWSPVGHKLAYVW (SEQ ID
NO:21; peptide 55), LWWSPNGTFLAYA (SEQ ID NO:22; peptide 84),
RISLQWLRRIQNY (SEQ ID NO:23; peptide 132), YVKQWRHSYTASY (SEQ ID
NO:24; peptide 37), EEEVFSAYSALWW (SEQ ID NO:25; peptide 79),
DYSISPDGQFILL (SEQ ID NO:26; peptide 29), SISPDGQFILLEY (SEQ ID
NO:27; peptide 30), and IYVKIEPNLPSYR (SEQ ID NO:28; peptide 63).
In some embodiments, the polypeptide preferentially binds to the
one or more peptides. These peptides are regions of human CD26. In
some embodiments, the polypeptide preferentially binds to one or
more peptides selected from the group consisting of YSLRWISDHEYLY
(SEQ ID NO:19; peptide 6), LEYNYVKQWRHSY (SEQ ID NO:20; peptide
35), TWSPVGHKLAYVW (SEQ ID NO:21; peptide 55), LWWSPNGTFLAYA (SEQ
ID NO:22; peptide 84), RISLQWLRRIQNY (SEQ ID NO:23; peptide 132),
YVKQWRHSYTASY (SEQ ID NO:24; peptide 37), EEEVFSAYSALWW (SEQ ID
NO:25; peptide 79), DYSISPDGQFILL (SEQ ID NO:26; peptide 29),
SISPDGQFILLEY (SEQ ID NO:27; peptide 30), and IYVKIEPNLPSYR (SEQ ID
NO:28; peptide 63), relative to one or more peptides corresponding
to other regions of human CD26.
[0102] In some embodiments, the polypeptide (e.g., antibody) binds
to each of the following peptides: YSLRWISDHEYLY (SEQ ID NO:19;
peptide 6); LEYNYVKQWRHSY (SEQ ID NO:20; peptide 35); TWSPVGHKLAYVW
(SEQ ID NO:21; peptide 55); LWWSPNGTFLAYA (SEQ ID NO:22; peptide
84); and RISLQWLRRIQNY (SEQ ID NO:23; peptide 132). In some other
embodiments, the polypeptide binds to each of the following
peptides: YSLRWISDHEYLY (SEQ ID NO:19; peptide 6); TWSPVGHKLAYVW
(SEQ ID NO:21; peptide 55); RISLQWLRRIQNY (SEQ ID NO:23; peptide
132); YVKQWRHSYTASY (SEQ ID NO:24; peptide 37); and EEEVFSAYSALWW
(SEQ ID NO:25; peptide 79). In some embodiments, the polypeptide
binds to each of the following peptides: DYSISPDGQFILL (SEQ ID
NO:26; peptide 29); SISPDGQFILLEY (SEQ ID NO:27; peptide 30); and
TWSPVGHKLAYVW (SEQ ID NO:21; peptide 55). In some other
embodiments, the polypeptide binds to each of the following
peptides: DYSISPDGQFILL (SEQ ID NO:26; peptide 29); SISPDGQFILLEY
(SEQ ID NO:27; peptide 30); TWSPVGHKLAYVW (SEQ ID NO:21; peptide
55); and IYVKIEPNLPSYR (SEQ ID NO:28; peptide 63). In some
embodiments, the polypeptides preferentially bind to the specified
peptides.
[0103] Competition assays can be used to determine whether two
antibodies bind the same epitope by recognizing identical or
sterically overlapping epitopes. Typically, antigen is immobilized
on a multi-well plate and the ability of unlabeled antibodies to
block the binding of labeled antibodies is measured. Common labels
for such competition assays are radioactive labels or enzyme
labels. In addition, epitope mapping techniques known to those in
the art can be used to determine the epitopes to which antibodies
bind.
[0104] In some embodiments, a polypeptide described herein
comprises one or more constant regions. In some embodiments, a
polypeptide described herein comprises a human constant region. In
some embodiments, the constant region is a constant region of the
heavy chain. In other embodiments, the constant region is a
constant region of the light chain. In some embodiments, the
polypeptide comprises a constant region which has at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 98%, or 100% identity to a human constant region. In
some embodiments, a polypeptide (e.g., an antibody) described
herein comprises an Fc region. In some embodiments, the polypeptide
comprises a human Fc region. In some embodiments, a polypeptide
described herein comprises an Fc region which has at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 98%, or 100% identity to a human Fc region.
[0105] In some embodiments, an antibody described herein is an IgG
antibody. In some embodiments, the antibody is an IgG1 antibody. In
some other embodiments, the antibody is an IgG2 antibody. In some
embodiments, the antibody is a human IgG antibody.
[0106] The invention provides antibodies in monomeric, dimeric, and
multivalent forms. For example, bispecific antibodies, monoclonal
antibodies that have binding specificities for at least two
different antigens, can be prepared using the antibodies disclosed
herein. Methods for making bispecific antibodies are known in the
art (see, e.g., Suresh et al., 1986, Methods in Enzymology
121:210). Traditionally, the recombinant production of bispecific
antibodies was based on the coexpression of two immunoglobulin
heavy chain-light chain pairs, with the two heavy chains having
different specificities (Millstein and Cuello, 1983, Nature 305,
537-539).
[0107] According to one approach to making bispecific antibodies,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1), containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0108] In one approach, the bispecific antibodies are composed of a
hybrid immunoglobulin heavy chain with a first binding specificity
in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair (providing a second binding specificity) in the other arm.
This asymmetric structure, with an immunoglobulin light chain in
only one half of the bispecific molecule, facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations. This approach is described in
PCT Publication No. WO 94/04690, published Mar. 3, 1994.
[0109] Heteroconjugate antibodies, comprising two covalently joined
antibodies, are also within the scope of the invention. Such
antibodies have been used to target immune system cells to unwanted
cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(PCT application publication Nos. WO 91/00360 and WO 92/200373; EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents and techniques
are well known in the art, and are described in U.S. Pat. No.
4,676,980.
[0110] In certain embodiments, an antibody described herein is an
antibody fragment. For instance, in some embodiments, the antibody
is selected from the group consisting of Fab, Fab', Fab'-SH, Fv,
scFv, and F(ab').sub.2. In some embodiments, the antibody is a Fab.
Various techniques have been developed for the production of
antibody fragments. These fragments can be derived via proteolytic
digestion of intact antibodies (see, e.g., Morimoto et al., 1992,
J. Biochem. Biophys. Methods 24:107-117 and Brennan et al., 1985,
Science 229:81), or produced directly by recombinant host cells.
For example, Fab'-SH fragments can be directly recovered from E.
coli and chemically coupled to form F(ab').sub.2 fragments (Carter
et al., 1992, Bio/Technology 10:163-167). In another embodiment,
the F(ab').sub.2 is formed using the leucine zipper of GCN4 to
promote assembly of the F(ab').sub.2 molecule. According to another
approach, Fv, Fab, or F(ab').sub.2 fragments are isolated directly
from recombinant host cell culture.
[0111] In some embodiments, the antibodies of the invention are
single chain (ScFv), mutants thereof, fusion proteins comprising an
antibody portion, humanized antibodies, chimeric antibodies,
diabodies linear antibodies, single chain antibodies, and any other
modified configuration of the immunoglobulin molecule.
[0112] Single chain variable region fragments are made by linking
light and/or heavy chain variable regions by using a short linking
peptide. Bird et al. (1988) Science 242:423-426. An example of a
linking peptide is (GGGGS).sub.3 (SEQ ID NO:29), which bridges
approximately 3.5 nm between the carboxy terminus of one variable
region and the amino terminus of the other variable region. Linkers
of other sequences have been designed and used. Bird et al. (1988).
Linkers can in turn be modified for additional functions, such as
attachment of drugs or attachment to solid supports. The single
chain variants can be produced either recombinantly or
synthetically. For synthetic production of scFv, an automated
synthesizer can be used. For recombinant production of scFv, a
suitable plasmid containing polynucleotide that encodes the scFv
can be introduced into a suitable host cell, either eukaryotic,
such as yeast, plant, insect or mammalian cells, or prokaryotic,
such as E. coli. Polynucleotides encoding the scFv of interest can
be made by routine manipulations such as ligation of
polynucleotides. The resultant scFv can be isolated using standard
protein purification techniques known in the art.
[0113] Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see e.g., Holliger, P., et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et
al. (1994) Structure 2:1121-1123).
[0114] The invention encompasses modifications to antibodies or
other polypeptides described herein, including functionally
equivalent antibodies which do not significantly affect their
properties and variants which have enhanced or decreased activity.
It is understood that the principles of modification apply to
polypeptides as well as antibodies. Modification of polypeptides is
routine practice in the art and need not be described in detail
herein. Examples of modified polypeptides include polypeptides with
conservative substitutions of amino acid residues, one or more
deletions or additions of amino acids which do not significantly
deleteriously change the functional activity, or use of chemical
analogs.
[0115] Amino acid sequence insertions or additions include amino-
and/or carboxyl-terminal fusions ranging in length from one residue
to polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the antibody molecule include
the fusion to the N- or C-terminus of the antibody of an enzyme or
a polypeptide which increases the serum half-life of the
antibody.
[0116] Substitution variants have at least one amino acid residue
in the antibody or other polypeptide sequence removed and a
different residue inserted in its place. The sites of greatest
interest for substitutional mutagenesis include the CDRs, but FR
alterations are also contemplated. Conservative substitutions are
shown in Table 3 under the heading of "conservative substitutions".
If such substitutions result in a change in biological activity,
then more substantial changes, denominated "exemplary
substitutions" in Table 3, or as further described below in
reference to amino acid classes, may be introduced and the products
screened.
TABLE-US-00005 TABLE 3 Amino Acid Substitutions Original Residue
Conservative Substitutions Exemplary Substitutions Ala (A) Val Val;
Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys;
Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu
Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg
Ile (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile
Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met
(M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P)
Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr
(Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;
Norleucine
[0117] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties: [0118]
(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; [0119] (2)
Neutral hydrophilic: Cys, Ser, Thr; [0120] (3) Acidic: Asp, Glu;
[0121] (4) Basic: Asn, Gln, His, Lys, Arg; [0122] (5) Residues that
influence chain orientation: Gly, Pro; and [0123] (6) Aromatic:
Trp, Tyr, Phe.
[0124] Non-conservative substitutions are made by exchanging a
member of one of these classes for another class. More conservative
substitutions involve exchanging one member of a class for another
member of the same class.
[0125] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant cross-linking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an antibody fragment such as an Fv fragment.
[0126] Amino acid modifications can range from changing or
modifying one or more amino acids to complete redesign of a region,
such as the variable region. Changes in the variable region can
alter binding affinity and/or specificity. In some embodiments, no
more than one to five conservative amino acid substitutions are
made within a CDR domain. In other embodiments, no more than one to
three conservative amino acid substitutions are made within a CDR3
domain. In still other embodiments, the CDR domain is CDRH3 and/or
CDR L3.
[0127] Modifications also include glycosylated and nonglycosylated
polypeptides, as well as polypeptides with other post-translational
modifications, such as, for example, glycosylation with different
sugars, acetylation, and phosphorylation. Antibodies are
glycosylated at conserved positions in their constant regions
(Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and
Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains
of the immunoglobulins affect the protein's function (Boyd et al.,
1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem.
29:4175-4180) and the intramolecular interaction between portions
of the glycoprotein, which can affect the conformation and
presented three-dimensional surface of the glycoprotein (Hefferis
and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.
7:409-416). Oligosaccharides may also serve to target a given
glycoprotein to certain molecules based upon specific recognition
structures. Glycosylation of antibodies has also been reported to
affect antibody-dependent cellular cytotoxicity (ADCC). In
particular, CHO cells with tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al., 1999, Nature
Biotech. 17:176-180).
[0128] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine, asparagine-X-threonine, and
asparagine-X-cysteine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0129] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0130] The glycosylation pattern of antibodies may also be altered
without altering the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g. antibodies, as potential therapeutics is rarely
the native cell, variations in the glycosylation pattern of the
antibodies can be expected (see, e.g. Hse et al., 1997, J. Biol.
Chem. 272:9062-9070).
[0131] In addition to the choice of host cells, factors that affect
glycosylation during recombinant production of antibodies include
growth mode, media formulation, culture density, oxygenation, pH,
purification schemes, and the like. Various methods have been
proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example using endoglycosidase H (Endo H). In
addition, the recombinant host cell can be genetically engineered
to be defective in processing certain types of polysaccharides.
These and similar techniques are well known in the art.
[0132] Other methods of modification include using coupling
techniques known in the art, including, but not limited to,
enzymatic means, oxidative substitution and chelation.
Modifications can be used, for example, for attachment of labels
for immunoassay. Modified polypeptides are made using established
procedures in the art and can be screened using standard assays
known in the art, some of which are described below and in the
Examples.
[0133] Other antibody modifications include antibodies that have
been modified as described in PCT Publication No. WO 99/58572,
published Nov. 18, 1999. These antibodies comprise, in addition to
a binding domain directed at the target molecule, an effector
domain having an amino acid sequence substantially homologous to
all or part of a constant domain of a human immunoglobulin heavy
chain. These antibodies are capable of binding the target molecule
without triggering significant complement dependent lysis, or
cell-mediated destruction of the target. In some embodiments, the
effector domain is capable of specifically binding FcRn and/or
Fc.gamma.RIIb. These are typically based on chimeric domains
derived from two or more human immunoglobulin heavy chain C.sub.H2
domains. Antibodies modified in this manner are particularly
suitable for use in chronic antibody therapy, to avoid inflammatory
and other adverse reactions to conventional antibody therapy.
[0134] In some embodiments, the polypeptides of the invention are
conjugates. For instance, in some embodiments, the polypeptide is
conjugated to another agent such as a chemotherapeutic agent, a
radionuclide, an immunotherapeutic agent, a cytokine, a chemokine,
an imaging agent, a toxin, a biological agent, an enzyme inhibitor,
or an antibody.
[0135] In some embodiments the polypeptides, such as antibodies,
are conjugated to water-soluble polymer moieties. The polypeptides
may be conjugated to polyethylene glycol (PEG), monomethoxy-PEG,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol or the like. The
polypeptides may be modified at random positions with the molecule,
or at predetermined positions with the molecule and may include
one, two, three or more attached moieties. The polymer may be of
any molecular weight, and may be branched or unbranched. In some
embodiments, the moiety is attached to the polypeptide via a
linker. In some embodiments, the attached moiety increases the
circulating half-life of the polypeptide in an animal. Methods of
attaching polymers such as PEG to polypeptides including antibodies
are well known in the art. In some embodiments, the polypeptides
are PEGylated polypeptides, such as PEGylated antibodies.
[0136] The invention includes affinity matured embodiments. For
example, affinity matured antibodies can be produced by procedures
known in the art (Marks et al., 1992, Bio/Technology, 10:779-783;
Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier
et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol.,
155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9;
Hawkins et al, 1992, J. Mol. Biol., 226:889-896; and
WO2004/058184). Candidate affinity matured antibodies may be
screened or selected for improved and/or altered binding affinity
using any method known in the art, including screening using
BIAcore surface plasmon resonance analysis, and selection using any
method known in the art for selection, including phage display,
yeast display, and ribosome display.
[0137] Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, 1975,
Nature 256:495. In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0138] The monoclonal antibodies (as well as other polypeptides) of
the invention may also be made by recombinant DNA methods, such as
those described in U.S. Pat. No. 4,816,567. DNA encoding the
monoclonal antibodies is isolated and sequenced using conventional
procedures, such as by using oligonucleotide probes that are
capable of binding specifically to genes encoding the heavy and
light chains of the monoclonal antibodies. Once isolated, the DNA
may be placed into expression vectors, which are then transfected
into host cells such as E. coli cells, simian COS cells, Chinese
hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells.
[0139] In some embodiments, the polypeptides of the invention
(e.g., antibodies) are expressed in any organism, or cells derived
from any organism, including, but not limited to bacteria, yeast,
plant, insect, and mammal. Particular types of cells include, but
are not limited to, Drosophila melanogaster cells, Saccharomyces
cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells,
HEK-293 cells, Neurospora, BHK cells, CHO cells, COS cells, HeLa
cells, fibroblasts, Schwannoma cell lines, immortalized mammalian
myeloid and lymphoid cell lines, Jurkat cells, mast cells and other
endocrine and exocrine cells, and neuronal cells.
[0140] A variety of protein expression systems, vectors, and cell
media useful in the production of polypeptides are known to those
of ordinary skill in the art. See, e.g., international patent
publications WO 03/054172, WO 04/009823, and WO 03/064630, each of
which is incorporated herein by reference in its entirety. In some
embodiments, a glutamine synthetase (GS) expression system is used
for expression of the polypeptides (e.g., antibodies).
[0141] Preferably, the polypeptide is purified or isolated after
expression according to methods known to those skilled in the art.
Examples of purification methods include electrophoretic,
molecular, immunological, and chromatographic techniques, including
ion exchange, hydrophobic affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. The degree of purification
necessary will vary depending on the use of the polypeptide. In
some instances, no purification will be necessary.
[0142] The DNA can be modified, for example, by covalently joining
to the immunoglobulin coding sequence all or part of the coding
sequence for a non-immunoglobulin polypeptide. In that manner,
"chimeric" or "hybrid" antibodies are prepared that have the
binding specificity of a monoclonal antibody disclosed herein.
Typically such non-immunoglobulin polypeptides are substituted for
the constant domains of an antibody of the invention, or they are
substituted for the variable domains of one antigen-combining site
of an antibody of the invention to create a chimeric bivalent
antibody comprising one antigen-combining site having specificity
for one surface epitope CD26 and another antigen-combining site
having specificity for a different antigen or CD26 epitope.
[0143] The invention also encompasses humanized antibodies.
Therapeutic antibodies often elicit adverse effects, in part due to
triggering of an immune response directed against the administered
antibody. This can result in reduced drug efficacy, depletion of
cells bearing the target antigen, and an undesirable inflammatory
response. To circumvent the above, recombinant anti-CD26 humanized
antibodies may be generated. The general principle in humanizing an
antibody involves retaining the basic sequence of the
antigen-binding portion of the antibody, while swapping at least
portions of the non-human remainder of the antibody with human
antibody sequences. Four traditional, but non-limiting, general
steps to humanize a monoclonal antibody include: (1) determining
the nucleotide and predicted amino acid sequence of the starting
antibody light and heavy variable domains, (2) designing the
humanized antibody, i.e., deciding which antibody framework region
or residues and/or CDR residues to use during the humanizing
process, (3) the actual humanizing methodologies/techniques, and
(4) the transfection and expression of the humanized antibody. The
constant region may also be engineered to more resemble human
constant regions to avoid immune response if the antibody is used
in clinical trials and treatments in humans. See, for example, U.S.
Pat. Nos. 5,997,867 and 5,866,692.
[0144] In the recombinant humanized antibodies, the Fc.gamma.
portion can be modified to avoid interaction with Fc.gamma.
receptor and the complement immune system. Techniques for
preparation of such antibodies are described in WO 99/58572.
[0145] A number of "humanized" antibody molecules comprising an
antigen-binding site derived from a non-human immunoglobulin have
been described, including chimeric antibodies having rodent V
regions and their associated complementarity determining regions
(CDRs) fused to human constant domains. See, for example, Winter et
al. Nature 349:293-299 (1991); Lobuglio et al. Proc. Nat. Acad.
Sci. USA 86:4220-4224 (1989); Shaw et al. J Immunol. 138:4534-4538
(1987); and Brown et al. Cancer Res. 47:3577-3583 (1987). Other
references describe rodent CDRs grafted into a human supporting
framework region (FR) prior to fusion with an appropriate human
antibody constant domain. See, for example, Riechmann et al. Nature
332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536 (1988);
and Jones et al. Nature 321:522-525 (1986). Another reference
describes rodent CDRs supported by recombinantly veneered rodent
framework regions. See, for example, European Patent Publication
No. 519,596. These types of "humanized" molecules are designed to
minimize unwanted immunological response toward rodent antihuman
antibody molecules which limits the duration and effectiveness of
therapeutic applications of those moieties in human recipients.
Other methods of humanizing antibodies that may also be utilized
are disclosed by Daugherty et al., Nucl. Acids Res., 19:2471-2476
(1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867;
5,866,692; 6,210,671; 6,350,861; and PCT WO 01/27160.
[0146] Additional exemplary methods of humanizing antibodies are
described in International Publication No. WO 02/084277 and U.S.
Publication No. US 2004/0133357, both of which are incorporated by
reference herein in their entirety.
[0147] In yet another alternative, fully human antibodies may be
obtained by using commercially available mice which have been
engineered to express specific human immunoglobulin proteins.
Transgenic animals which are designed to produce a more desirable
(e.g., fully human antibodies) or more robust immune response may
also be used for generation of humanized or human antibodies.
Examples of such technology are Xenomouse.TM. from Abgenix, Inc.
(Fremont, Calif.) and HuMAb-Mouse.RTM. and TC Mouse.TM. from
Medarex, Inc. (Princeton, N.J.).
[0148] In another alternative, antibodies may be made recombinantly
by phage display technology. See, for example, U.S. Pat. Nos.
5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter et al.,
Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, the phage
display technology (McCafferty et al., Nature 348:552-553 (1990))
can be used to produce human antibodies and antibody fragments in
vitro, from immunoglobulin variable (V) domain gene repertoires
from unimmunized donors. According to this technique, antibody V
domain genes are cloned in-frame into either a major or minor coat
protein gene of a filamentous bacteriophage, such as M13 or fd, and
displayed as functional antibody fragments on the surface of the
phage particle. Because the filamentous particle contains a
single-stranded DNA copy of the phage genome, selections based on
the functional properties of the antibody also result in selection
of the gene encoding the antibody exhibiting those properties.
Thus, the phage mimics some of the properties of the B cell. Phage
display can be performed in a variety of formats; for review see,
e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in
Structural Biology 3, 564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature
352:624-628 (1991), isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Mark et
al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.
12:725-734 (1993). In a natural immune response, antibody genes
accumulate mutations at a high rate (somatic hypermutation). Some
of the changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling." Marks, et al., Bio/Technol. 10:779-783
(1992)). In this method, the affinity of "primary" human antibodies
obtained by phage display can be improved by sequentially replacing
the heavy and light chain V region genes with repertoires of
naturally occurring variants (repertoires) of V domain genes
obtained from unimmunized donors. This technique allows the
production of antibodies and antibody fragments with affinities in
the pM-nM range. A strategy for making very large phage antibody
repertoires (also known as "the mother-of-all libraries") has been
described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266
(1993). Gene shuffling can also be used to derive human antibodies
from rodent antibodies, where the human antibody has similar
affinities and specificities to the starting rodent antibody.
According to this method, which is also referred to as "epitope
imprinting", the heavy or light chain V domain gene of rodent
antibodies obtained by phage display technique is replaced with a
repertoire of human V domain genes, creating rodent-human chimeras.
Selection on antigen results in isolation of human variable regions
capable of restoring a functional antigen-binding site, i.e., the
epitope governs (imprints) the choice of partner. When the process
is repeated in order to replace the remaining rodent V domain, a
human antibody is obtained (see PCT patent application PCT WO
9306213, published Apr. 1, 1993). Unlike traditional humanization
of rodent antibodies by CDR grafting, this technique provides
completely human antibodies, which have no framework or CDR
residues of rodent origin. It is apparent that although the above
discussion pertains to humanized antibodies, the general principles
discussed are applicable to customizing antibodies for use, for
example, in dogs, cats, primates, equines, and bovines.
[0149] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods of synthetic protein chemistry, including those
involving cross-linking agents. For example, immunotoxins may be
constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0150] Single chain Fv fragments may also be produced, such as
described in Iliades et al., 1997, FEBS Letters, 409:437-441.
Coupling of such single chain fragments using various linkers is
described in Kortt et al., 1997, Protein Engineering, 10:423-433. A
variety of techniques for the recombinant production and
manipulation of antibodies are well known in the art.
[0151] In one aspect, the invention provides methods of producing
the polypeptides described herein. In some embodiments, the method
comprises expressing a polynucleotide in a host cell, wherein the
polynucleotide encodes the polypeptide. In some embodiments, the
method is a method of producing an antibody and comprises
expressing one or more polynucleotides in a host cell (e.g., in
cell culture), wherein each chain of the antibody is encoded by at
least one of the polynucleotides. In some embodiments, the one or
more polynucleotides are on the same vector. In other embodiments,
the one or more polynucleotides are located on separate vectors. In
some embodiments, the methods of producing polypeptides described
herein further comprise the step of isolating the polypeptides from
the host cells in which they are expressed (e.g., isolated from the
cell culture in which the host cells are grown).
[0152] siRNA Targeting CD26 mRNA and/or CD26 cDNA
[0153] Compositions and methods comprising siRNA targeted to CD26
are advantageously used to inhibit binding of CD26 to extracellular
matrix, in particular for the treatment of tumorigenesis disease.
The siRNA of the invention are believed to cause the RNAi-mediated
degradation of these mRNAs, so that the protein product of the CD26
genes is not produced or is produced in reduced amounts.
[0154] The invention therefore provides isolated siRNA comprising
short double-stranded RNA from about 17 nucleotides to about 29
nucleotides in length, preferably from about 19 to about 25
nucleotides in length, that are targeted to the target mRNA and/or
target cDNA. The siRNA comprise a sense RNA strand and a
complementary antisense RNA strand annealed together by standard
Watson-Crick base-pairing interactions (hereinafter "base-paired").
As is described in more detail below, the sense strand comprises a
nucleic acid sequence which is identical to a target sequence
contained within the target mRNA and/or target cDNA.
[0155] The sense and antisense strands of the present siRNA can
comprise two complementary, single-stranded RNA molecules or can
comprise a single molecule in which two complementary portions are
base-paired and are covalently linked by a single-stranded
"hairpin" area. Without wishing to be bound by any theory, it is
believed that the hairpin area of the latter type of siRNA molecule
is cleaved intracellularly by the "Dicer" protein (or its
equivalent) to form an siRNA of two individual base-paired RNA
molecules.
[0156] As used herein, "isolated" means altered or removed from the
natural state through human intervention. For example, an siRNA
naturally present in a living animal is not "isolated," but a
synthetic siRNA, or an siRNA partially or completely separated from
the coexisting materials of its natural state is "isolated." An
isolated siRNA can exist in substantially purified form, or can
exist in a non-native environment such as, for example, a cell into
which the siRNA has been delivered.
[0157] As used herein, "target mRNA" and "target cDNA" means human
CD26 mRNA and human CD26 cDNA, respectively, mutant or alternative
splice forms thereof.
[0158] RT-PCR can also be used to identify alternatively spliced
CD26 mRNAs. In RT-PCR, mRNA from the diseased tissue is converted
into cDNA by the enzyme reverse transcriptase, using methods
well-known to those of ordinary skill in the art. The entire coding
sequence of the cDNA is then amplified via PCR using a forward
primer located in the 3' untranslated region, and a reverse primer
located in the 5' untranslated region. The amplified products can
be analyzed for alternative splice forms, for example by comparing
the size of the amplified products with the size of the expected
product from normally spliced mRNA, e.g., by agarose gel
electrophoresis. Any change in the size of the amplified product
can indicate alternative splicing.
[0159] The siRNA of the invention can comprise partially purified
RNA, substantially pure RNA, synthetic RNA, or recombinantly
produced RNA, as well as altered RNA that differs from
naturally-occurring RNA by the addition, deletion, substitution
and/or alteration of one or more nucleotides. Such alterations can
include addition of non-nucleotide material, such as to the end(s)
of the siRNA or to one or more internal nucleotides of the siRNA,
including modifications that make the siRNA resistant to nuclease
digestion.
[0160] One or both strands of the siRNA of the invention can also
comprise a 3' overhang. As used herein, a "3' overhang" refers to
at least one unpaired nucleotide extending from the 3'-end of an
RNA strand.
[0161] Thus in one embodiment, the siRNA of the invention comprises
at least one 3' overhang of from 1 to about 6 nucleotides (which
includes ribonucleotides or deoxynucleotides) in length, preferably
from 1 to about 5 nucleotides in length, more preferably from 1 to
about 4 nucleotides in length, and particularly preferably from
about 2 to about 4 nucleotides in length.
[0162] In the embodiment in which both strands of the siRNA
molecule comprise a 3' overhang, the length of the overhangs can be
the same or different for each strand. In a most preferred
embodiment, the 3' overhang is present on both strands of the
siRNA, and is 2 nucleotides in length. For example, each strand of
the siRNA of the invention can comprise 3' overhangs of
dithymidylic acid ("TT") or diuridylic acid ("uu").
[0163] In order to enhance the stability of the present siRNA, the
3' overhangs can be also stabilized against degradation. In one
embodiment, the overhangs are stabilized by including purine
nucleotides, such as adenosine or guanosine nucleotides.
Alternatively, substitution of pyrimidine nucleotides by modified
analogues, e.g., substitution of uridine nucleotides in the 3'
overhangs with 2'-deoxythymidine, is tolerated and does not affect
the efficiency of RNAi degradation.
[0164] In certain embodiments, the siRNA of the invention comprises
the sequence AA(N19)TT or NA(N21), where N is any nucleotide. These
siRNA comprise approximately 30-70% GC, and preferably comprise
approximately 50% G/C. The sequence of the sense siRNA strand
corresponds to (N19)TT or N21 (i.e., positions 3 to 23),
respectively. In the latter case, the 3' end of the sense siRNA is
converted to TT. The rationale for this sequence conversion is to
generate a symmetric duplex with respect to the sequence
composition of the sense and antisense strand 3' overhangs. The
antisense RNA strand is then synthesized as the complement to
positions 1 to 21 of the sense strand.
[0165] Because position 1 of the 23-nt sense strand in these
embodiments is not recognized in a sequence-specific manner by the
antisense strand, the 3'-most nucleotide residue of the antisense
strand can be chosen deliberately. However, the penultimate
nucleotide of the antisense strand (complementary to position 2 of
the 23-nt sense strand in either embodiment) is generally
complementary to the targeted sequence.
[0166] The siRNA of the invention can be targeted to any stretch of
approximately 19-25 contiguous nucleotides in any of the target
mRNA and/or target cDNA sequences (the "target sequence").
Techniques for selecting target sequences for siRNA are given, for
example, in Tuschl T et al., "The siRNA User Guide," revised Oct.
11, 2002, the entire disclosure of which is herein incorporated by
reference. "The siRNA User Guide" is available on the world wide
web at a website maintained by Dr. Thomas Tuschl, Department of
Cellular Biochemistry, AG105, Max-Planck-Institute for Biophysical
Chemistry, 37077 Gottingen, Germany, and can be found by accessing
the website of the Max Planck Institute and searching with the
keyword "siRNA." Thus, the sense strand of the present siRNA
comprises a nucleotide sequence identical to any contiguous stretch
of about 19 to about 25 nucleotides in the target mRNA.
[0167] Generally, a target sequence on the target mRNA can be
selected from a given cDNA sequence corresponding to the target
mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the 3'
direction) from the start codon. The target sequence can, however,
be located in the 5' or 3' untranslated regions, or in the region
nearby the start codon.
[0168] For example, a suitable target sequence in the CD26 cDNA
sequence can be obtained from accession No. NM 001935
[0169] Thus, an siRNA of the invention targeting this sequence, and
which has 3' uu overhangs on each strand (overhangs shown in bold),
is:
TABLE-US-00006 (SEQ ID NO: 30) sense: 5'-GAAAGGUGUCAGUACUAUU TT-3',
(SEQ ID NO: 31) antisense: 3'-TT CUUUCCACAGUCAUGAUAA-5'
[0170] The siRNA of the invention can be obtained using a number of
techniques known to those of skill in the art. For example, the
siRNA can be chemically synthesized or recombinantly produced using
methods known in the art.
[0171] Preferably, the siRNA of the invention are chemically
synthesized using appropriately protected ribonucleoside
phosphoramidites and a conventional DNA/RNA synthesizer. The siRNA
can be synthesized as two separate, complementary RNA molecules, or
as a single RNA molecule with two complementary regions. Commercial
suppliers of synthetic RNA molecules or synthesis reagents include
Proligo (Hamburg, Germany), Pierce Chemical (part of Perbio
Science, Rockford, Ill., USA), etc.
[0172] Alternatively, siRNA can also be expressed from recombinant
circular or linear DNA plasmids using any suitable promoter.
Suitable promoters for expressing siRNA of the invention from a
plasmid include, for example, the U6 or H1 RNA pol III promoter
sequences and the cytomegalovirus promoter. Selection of other
suitable promoters is within the skill in the art. The recombinant
plasmids of the invention can also comprise inducible or
regulatable promoters for expression of the siRNA in a particular
tissue or in a particular intracellular environment.
[0173] The siRNA expressed from recombinant plasmids can either be
isolated from cultured cell expression systems by standard
techniques, or can be expressed intracellularly at or near the area
of neovascularization in vivo. The use of recombinant plasmids to
deliver siRNA of the invention to cells in vivo is discussed in
more detail below.
[0174] siRNA of the invention can be expressed from a recombinant
plasmid either as two separate, complementary RNA molecules, or as
a single RNA molecule with two complementary regions.
[0175] Selection of plasmids suitable for expressing siRNA of the
invention, methods for inserting nucleic acid sequences for
expressing the siRNA into the plasmid, and methods of delivering
the recombinant plasmid to the cells of interest are within the
skill in the art. See, for example Tuschl, T. (2002), Nat.
Biotechnol, 20: 446 448; Brummelkamp T R et al. (2002), Science
296: 550 553; Miyagishi M et al. (2002), Nat. Biotechnol. 20: 497
500; Paddison P J et al. (2002), Genes Dev. 16: 948 958; Lee N S et
al. (2002), Nat. Biotechnol. 20: 500 505; and Paul C P et al.
(2002), Nat. Biotechnol. 20: 505 508, the entire disclosures of
which are herein incorporated by reference.
[0176] The siRNA of the invention can also be expressed from
recombinant viral vectors intracellularly at or near the area of
neovascularization in vivo. The recombinant viral vectors of the
invention comprise sequences encoding the siRNA of the invention
and any suitable promoter for expressing the siRNA sequences.
Suitable promoters include, for example, the U6 or H1 RNA pol III
promoter sequences and the cytomegalovirus promoter. Selection of
other suitable promoters is within the skill in the art. The
recombinant viral vectors of the invention can also comprise
inducible or regulatable promoters for expression of the siRNA in a
particular tissue or in a particular intracellular environment. The
use of recombinant viral vectors to deliver siRNA of the invention
to cells in vivo is discussed in more detail below.
[0177] siRNA of the invention can be expressed from a recombinant
viral vector either as two separate, complementary RNA molecules,
or as a single RNA molecule with two complementary regions.
[0178] Any viral vector capable of accepting the coding sequences
for the siRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g. lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of the
viral vectors can also be modified by pseudotyping the vectors with
envelope proteins or other surface antigens from other viruses. For
example, an AAV vector of the invention can be pseudotyped with
surface proteins from vesicular stomatitis virus (VSV), rabies,
Ebola, Mokola, and the like.
[0179] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing the siRNA into the vector, and methods of delivering the
viral vector to the cells of interest are within the skill in the
art. See, for example, Domburg R (1995), Gene Therap. 2: 301 310;
Eglitis M A (1988), Biotechniques 6: 608 614; Miller A D (1990),
Hum Gene Therap. 1: 5 14; and Anderson W F (1998), Nature 392: 25
30, the entire disclosures of which are herein incorporated by
reference.
[0180] The ability of an siRNA containing a given target sequence
to cause RNAi-mediated degradation of the target mRNA can be
evaluated using standard techniques for measuring the levels of RNA
or protein in cells. For example, siRNA of the invention can be
delivered to cultured cells, and the levels of target mRNA can be
measured by Northern blot or dot blotting techniques, or by
quantitative RT-PCR. Alternatively, the levels of CD26 protein in
the cultured cells can be measured by ELISA or Western blot.
[0181] RNAi-mediated degradation of target mRNA by an siRNA
containing a given target sequence can also be evaluated with
animal models of neovascularization, such as the ROP or CNV mouse
models. For example, areas of neovascularization in an ROP or CNV
mouse can be measured before and after administration of an
siRNA.
[0182] As discussed above, the siRNA of the invention target and
cause the RNAi-mediated degradation of CD26 mRNA and/or CD26 cDNA,
or alternative splice forms, mutants or cognates thereof.
Degradation of the target mRNA by the present siRNA reduces the
production of a functional gene product from the CD26 genes. Thus,
the invention provides a method of inhibiting expression of CD26 in
a subject, comprising administering an effective amount of an siRNA
of the invention to the subject, such that the target mRNA and/or
target CD26 cDNA is degraded. As the products of the CD26 genes are
required for initiating and maintaining angiogenesis, the invention
also provides a method of inhibiting angiogenesis in a subject by
the RNAi-mediated degradation of the target mRNA and/or target CD26
cDNA by the present siRNA.
[0183] As used herein, a "subject" includes a human being or
non-human animal. Preferably, the subject is a human being.
[0184] As used herein, an "effective amount" of the siRNA is an
amount sufficient to cause RNAi-mediated degradation of the target
mRNA, or an amount sufficient to inhibit the progression of
angiogenesis in a subject.
[0185] RNAi-mediated degradation of the target mRNA and/or target
CD26 cDNA can be detected by measuring levels of the target mRNA or
protein in the cells of a subject, using standard techniques for
isolating and quantifying mRNA and/or cDNA or protein as described
above.
[0186] Inhibition of tumorigenesis can be evaluated by directly
measuring the progress of pathogenic or nonpathogenic tumorigenesis
in a subject; for example, by observing the size of a tumor before
and after treatment with the siRNA of the invention. An inhibition
of tumorigenesis is indicated if the tumor size stays the same or
is reduced. Techniques for observing and measuring the tumor size
in a subject are within the skill in the art.
[0187] It is understood that the siRNA of the invention can degrade
the target mRNA and/or target cDNA (and thus inhibit tumorigenesis)
in substoichiometric amounts. Without wishing to be bound by any
theory, it is believed that the siRNA of the invention causes
degradation of the target mRNA in a catalytic manner. Thus,
compared to standard anti-tumor therapies, significantly less siRNA
needs to be delivered at or near the tumor site to have a
therapeutic effect.
[0188] One skilled in the art can readily determine an effective
amount of the siRNA of the invention to be administered to a given
subject, by taking into account factors such as the size and weight
of the subject; the extent of the tumorigenesis or disease
penetration; the age, health, and sex of the subject; the route of
administration; and whether the administration is regional or
systemic. Generally, an effective amount of the siRNA of the
invention comprises an intercellular concentration at or near the
tumorigenesis site of from about 1 nanomolar (nM) to about 100 nM,
preferably from about 2 nM to about 50 nM, more preferably from
about 2.5 nM to about 10 nM. It is contemplated that greater or
lesser amounts of siRNA can be administered.
[0189] The present methods can also inhibit angiogenesis which is
associated with an angiogenic disease; i.e., a disease in which
pathogenicity is associated with inappropriate or uncontrolled
angiogenesis. For example, most cancerous solid tumors generate an
adequate blood supply for themselves by inducing angiogenesis in
and around the tumor site. This tumor-induced angiogenesis is often
required for tumor growth, and also allows metastatic cells to
enter the bloodstream.
[0190] Preferably, an siRNA of the invention is used to inhibit the
growth or metastasis of solid tumors associated with malignant
mesothelioma; for example breast cancer, lung cancer, head and neck
cancer, brain cancer, abdominal cancer, colon cancer, colorectal
cancer, esophagus cancer, gastrointestinal cancer, glioma, liver
cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer,
pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor,
multiple myeloma; skin cancer (e.g., melanoma), lymphomas, and
blood cancer.
[0191] For treating angiogenic diseases, the siRNA of the invention
can administered to a subject in combination with a pharmaceutical
agent which is different from the present siRNA. Alternatively, the
siRNA of the invention can be administered to a subject in
combination with another therapeutic method designed to treat the
angiogenic disease. For example, the siRNA of the invention can be
administered in combination with therapeutic methods currently
employed for treating mesothelioma or preventing tumor metastasis
(e.g., radiation therapy, chemotherapy, and surgery). For treating
tumors, the siRNA of the invention is preferably administered to a
subject in combination with radiation therapy, or in combination
with chemotherapeutic agents such as cisplatin, carboplatin,
cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin, or
tamoxifen.
[0192] In the present methods, the present siRNA can be
administered to the subject either as naked siRNA, in conjunction
with a delivery reagent, or as a recombinant plasmid or viral
vector which expresses the siRNA.
[0193] Suitable delivery reagents for administration in conjunction
with the present siRNA include the Minis Transit TKO lipophilic
reagent; lipofectin; lipofectamine; cellfectin; or polycations
(e.g., polylysine), or liposomes. A preferred delivery reagent is a
liposome.
[0194] Liposomes can aid in the delivery of the siRNA to a
particular tissue, such as tumor tissue, and can also increase the
blood half-life of the siRNA. Liposomes suitable for use in the
invention are formed from standard vesicle-forming lipids, which
generally include neutral or negatively charged phospholipids and a
sterol, such as cholesterol. The selection of lipids is generally
guided by consideration of factors such as the desired liposome
size and half-life of the liposomes in the blood stream. A variety
of methods are known for preparing liposomes, for example as
described in Szoka et al. (1980), Ann Rev. Biophys. Bioeng. 9: 467;
and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369,
the entire disclosures of which are herein incorporated by
reference.
[0195] Preferably, the liposomes encapsulating the present siRNA
comprises a ligand molecule that can target the liposome to a
malignant mesothelioma cell or tissue. Ligands which bind to
receptors prevalent in tumor cells, such as monoclonal antibodies
that bind to CD26 are preferred.
[0196] Particularly preferably, the liposomes encapsulating the
present siRNA are modified so as to avoid clearance by the
mononuclear macrophage and reticuloendothelial systems, for example
by having opsonization-inhibition moieties bound to the surface of
the structure. In one embodiment, a liposome of the invention can
comprise both opsonization-inhibition moieties and a ligand.
[0197] Opsonization-inhibiting moieties for use in preparing the
liposomes of the invention are typically large hydrophilic polymers
that are bound to the liposome membrane. As used herein, an
opsonization inhibiting moiety is "bound" to a liposome membrane
when it is chemically or physically attached to the membrane, e.g.,
by the intercalation of a lipid-soluble anchor into the membrane
itself, or by binding directly to active groups of membrane lipids.
These opsonization-inhibiting hydrophilic polymers form a
protective surface layer which significantly decreases the uptake
of the liposomes by the macrophage-monocyte system ("MMS") and
reticuloendothelial system ("RES"); e.g., as described in U.S. Pat.
No. 4,920,016, the entire disclosure of which is herein
incorporated by reference. Liposomes modified with
opsonization-inhibition moieties thus remain in the circulation
much longer than unmodified liposomes. For this reason, such
liposomes are sometimes called "stealth" liposomes.
[0198] Stealth liposomes are known to accumulate in tissues fed by
porous or "leaky" microvasculature. Thus, target tissue
characterized by such microvasculature defects, for example solid
tumors, will efficiently accumulate these liposomes; see Gabizon,
et al. (1988), P.N.A.S., USA, 18: 6949 53. In addition, the reduced
uptake by the RES lowers the toxicity of stealth liposomes by
preventing significant accumulation in the liver and spleen. Thus,
liposomes of the invention that are modified with
opsonization-inhibition moieties can deliver the present siRNA to
tumor cells.
[0199] Opsonization inhibiting moieties suitable for modifying
liposomes are preferably water-soluble polymers with a molecular
weight from about 500 to about 40,000 daltons, and more preferably
from about 2,000 to about 20,000 daltons. Such polymers include
polyethylene glycol (PEG) or polypropylene glycol (PPG)
derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;
synthetic polymers such as polyacrylamide or poly N-vinyl
pyrrolidone; linear, branched, or dendrimeric polyamidoamines;
polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and
polyxylitol to which carboxylic or amino groups are chemically
linked, as well as gangliosides, such as ganglioside GM.sub.1.
Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives
thereof, are also suitable. In addition, the opsonization
inhibiting polymer can be a block copolymer of PEG and either a
polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine,
or polynucleotide. The opsonization inhibiting polymers can also be
natural polysaccharides containing amino acids or carboxylic acids,
e.g., galacturonic acid, glucuronic acid, mannuronic acid,
hyaluronic acid, pectic acid, neuraminic acid, alginic acid,
carrageenan; aminated polysaccharides or oligosaccharides (linear
or branched); or carboxylated polysaccharides or oligosaccharides,
e.g., reacted with derivatives of carbonic acids with resultant
linking of carboxylic groups.
[0200] Preferably, the opsonization-inhibiting moiety is a PEG,
PPG, or derivatives thereof Liposomes modified with PEG or
PEG-derivatives are sometimes called "PEGylated liposomes."
[0201] The opsonization inhibiting moiety can be bound to the
liposome membrane by any one of numerous well-known techniques. For
example, an N-hydroxysuccinimide ester of PEG can be bound to a
phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a
membrane. Similarly, a dextran polymer can be derivatized with a
stearylamine lipid-soluble anchor via reductive amination using
Na(CN)BH.sub.3 and a solvent mixture such as tetrahydrofuran and
water in a 30:12 ratio at 60.degree. C.
[0202] Pharmaceutical Compositions and Kits
[0203] The present invention further provides compositions
comprising the polypeptides (e.g., antibodies) and/or
polynucleotides described herein. For instance, the invention
provides pharmaceutical compositions comprising the polypeptides
described herein. Kits comprising the polypeptides are also
provided.
[0204] In some embodiments, the pharmaceutical composition
comprises a polypeptide described herein and a pharmaceutically
acceptable excipient (also referred to herein as a
"pharmaceutically acceptable carrier"). In some embodiments, the
polypeptide in the pharmaceutical composition is an antibody. In
some embodiments, the polypeptide in the pharmaceutical composition
is a humanized antibody.
[0205] Pharmaceutical compositions within the scope of the present
invention may also contain other compounds that may be biologically
active or inactive.
[0206] A pharmaceutical composition can contain DNA encoding one or
more of the polypeptides as described above, such that the
polypeptide is generated in situ. As noted above, the DNA may be
present within any of a variety of delivery systems known to those
of ordinary skill in the art, including nucleic acid expression
systems, bacteria, and viral expression systems. Numerous gene
delivery techniques are well known in the art, such as those
described by Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems
15:143-198, and references cited therein. Appropriate nucleic acid
expression systems contain the necessary DNA sequences for
expression in the patient (such as a suitable promoter and
terminating signal).
[0207] In a preferred embodiment, the DNA may be introduced using a
viral expression system (e.g., vaccinia or other pox virus,
retrovirus, or adenovirus), which may involve the use of a
non-pathogenic (defective), replication competent virus. Suitable
systems are disclosed, for example, in Fisher-Hoch et al., 1989,
Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann.
N. Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21;
U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973;
U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;
Berkner-Biotechniques 6:616-627, 1988; Rosenfeld et al., 1991,
Science 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA
91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA
90:11498-11502; Guzman et al., 1993, Circulation 88:2838-2848; and
Guzman et al., 1993, Cir. Res. 73:1202-1207. Techniques for
incorporating DNA into such expression systems are well known to
those of ordinary skill in the art. The DNA may also be "naked," as
described, for example, in Ulmer et al., 1993, Science
259:1745-1749, and reviewed by Cohen, 1993, Science 259:1691-1692.
The uptake of naked DNA may be increased by coating the DNA onto
biodegradable beads, which are efficiently transported into the
cells.
[0208] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration. Compositions of the present invention may be
formulated for any appropriate manner of administration, including
for example, topical, oral, nasal, intravenous, intracranial,
intraperitoneal, subcutaneous, or intramuscular administration. For
parenteral administration, such as subcutaneous injection, the
carrier preferably comprises water, saline, alcohol, a fat, a wax,
or a buffer. For oral administration, any of the above carriers or
a solid carrier, such as mannitol, lactose, starch, magnesium
stearate, sodium saccharine, talcum, cellulose, glucose, sucrose,
and magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactate polyglycolate) may also be employed
as carriers for the pharmaceutical compositions of this invention.
Suitable biodegradable microspheres are disclosed, for example, in
U.S. Pat. Nos. 4,897,268 and 5,075,109.
[0209] Such compositions may also comprise buffers (e.g., neutral
buffered saline or phosphate buffered saline), carbohydrates (e.g.,
glucose, mannose, sucrose or dextran), mannitol, proteins,
polypeptides or amino acids such as glycine, antioxidants,
chelating agents such as EDTA or glutathione, adjuvants (e.g.,
aluminum hydroxide), and/or preservatives. Alternatively,
compositions of the present invention may be formulated as a
lyophilizate. Compounds may also be encapsulated within liposomes
using well known technology.
[0210] The compositions described herein may be administered as
part of a sustained release formulation (i.e., a formulation such
as a capsule or sponge that effects a slow release of compound
following administration). Such formulations may generally be
prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain a polypeptide, polynucleotide or antibody
dispersed in a carrier matrix and/or contained within a reservoir
surrounded by a rate controlling membrane. Carriers for use within
such formulations are biocompatible, and may also be biodegradable;
preferably the formulation provides a relatively constant level of
active component release. The amount of active compound contained
within a sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated.
[0211] In some embodiments, the polypeptide also may be entrapped
in microcapsules prepared, for example, by coacervation techniques
or by interfacial polymerization (for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively), in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles, and nanocapsules), or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990; and Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing, 2000. To increase
the serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0212] The antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al., 1980, Proc.
Natl Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed
in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be
generated by the reverse phase evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol and
PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through filters of defined pore size to yield liposomes
with the desired diameter. In addition, Fab' fragments of the
antibody of the present invention can be conjugated to the
liposomes as described in Martin et al., 1982, J. Biol. Chem.
257:286-288, via a disulfide interchange reaction.
[0213] Kits and articles of manufacture comprising the
polypeptides, polynucleotides, vectors, or host cells described
herein are also provided.
[0214] In some embodiments, the article of manufacture comprises a
container with a label. Suitable containers include, for example,
bottles, vials, and test tubes. The containers may be formed from a
variety of materials such as glass or plastic. In some embodiments,
the container holds a composition useful in the identification or
quantitation of cells expressing CD26, the inhibition of
proliferation of cells expressing CD26, or the treatment of a
disease associated with expression of CD26. In some embodiments,
the label on the container indicates the composition is useful for
the identification or quantitation of cells expressing CD26, the
inhibition of proliferation of cells expressing CD26, or the
treatment of a disease associated with expression of CD26.
[0215] In some embodiments, the kit of the invention comprises the
container described above. In other embodiments, the kit of the
invention comprises the container described above and a second
container comprising a buffer. In some embodiments, the kit
comprises a package insert with instructions for use of a
polypeptide, polynucleotide, vector or host cell contained
therein.
[0216] Recombinant plasmids which express siRNA of the invention
are discussed above. Such recombinant plasmids can also be
administered directly or in conjunction with a suitable delivery
reagent, including the Minis Transit LT1 lipophilic reagent;
lipofectin; lipofectamine; cellfectin; polycations (e.g.,
polylysine) or liposomes. Recombinant viral vectors which express
siRNA of the invention are also discussed above, and methods for
delivering such vectors to a tumor area in a patient are within the
skill in the art.
[0217] The siRNA of the invention can be administered to the
subject by any means suitable for delivering the siRNA to the cells
of the tissue at or near the tumor area. For example, the siRNA can
be administered by gene gun, electroporation, or by other suitable
parenteral or enteral administration routes.
[0218] Suitable enteral administration routes include oral, rectal,
or intranasal delivery.
[0219] Suitable parenteral administration routes include
intravascular administration (e.g. intravenous bolus injection,
intravenous infusion, intra-arterial bolus injection,
intra-arterial infusion, and catheter instillation into the
vasculature); peri- and intra-tissue injection (e.g., peri-tumoral
and intra-tumoral injection, intra-retinal injection, or subretinal
injection); subcutaneous injection or deposition including
subcutaneous infusion (such as by osmotic pumps); direct
application to the area at or near the site of neovascularization,
for example by a catheter or other placement device (e.g., a
suppository or an implant comprising a porous, non-porous, or
gelatinous material); and inhalation. It is preferred that
injections or infusions of the siRNA be given at or near the tumor
site.
[0220] The siRNA of the invention can be administered in a single
dose or in multiple doses. Where the administration of the siRNA of
the invention is by infusion, the infusion can be a single
sustained dose or can be delivered by multiple infusions. Injection
of the agent directly into the tissue is at or near the tumor site.
Multiple injections of the agent into the tissue at or near the
tumor site are particularly preferred.
[0221] One skilled in the art can also readily determine an
appropriate dosage regimen for administering the siRNA of the
invention to a given subject. For example, the siRNA can be
administered to the subject once, for example as a single injection
or deposition at or near the tumor site. Alternatively, the siRNA
can be administered once or twice daily to a subject for a period
of from about three to about twenty-eight days, more preferably
from about seven to about ten days. In a preferred dosage regimen,
the siRNA is injected at or near the site of tumor once a day for
seven days. Where a dosage regimen comprises multiple
administrations, it is understood that the effective amount of
siRNA administered to the subject can comprise the total amount of
siRNA administered over the entire dosage regimen.
[0222] The siRNA of the invention are preferably formulated as
pharmaceutical compositions prior to administering to a subject,
according to techniques known in the art. Pharmaceutical
compositions of the present invention are characterized as being at
least sterile and pyrogen-free. As used herein, "pharmaceutical
formulations" include formulations for human and veterinary use.
Methods for preparing pharmaceutical compositions of the invention
are within the skill in the art, for example as described in
Remington's Pharmaceutical Science, 17th ed., Mack Publishing
Company, Easton, Pa. (1985), the entire disclosure of which is
herein incorporated by reference.
[0223] The present pharmaceutical formulations comprise an siRNA of
the invention (e.g., 0.1 to 90% by weight), or a physiologically
acceptable salt thereof, mixed with a physiologically acceptable
carrier medium. Preferred physiologically acceptable carrier media
are water, buffered water, normal saline, 0.4% saline, 0.3%
glycine, hyaluronic acid, and the like.
[0224] Pharmaceutical compositions of the invention can also
comprise conventional pharmaceutical excipients and/or additives.
Suitable pharmaceutical excipients include stabilizers,
antioxidants, osmolality adjusting agents, buffers, and pH
adjusting agents. Suitable additives include physiologically
biocompatible buffers (e.g., tromethamine hydrochloride), additions
of chelants (such as, for example, DTPA or DTPA-bisamide) or
calcium chelate complexes (as for example calcium DTPa.,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium
salts (for example, calcium chloride, calcium ascorbate, calcium
gluconate or calcium lactate). Pharmaceutical compositions of the
invention can be packaged for use in liquid form, or can be
lyophilized.
[0225] For solid compositions, conventional nontoxic solid carriers
can be used; for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like.
[0226] For example, a solid pharmaceutical composition for oral
administration can comprise any of the carriers and excipients
listed above and 10-95%, preferably 25% -75%, of one or more siRNA
of the invention. A pharmaceutical composition for aerosol
(inhalational) administration can comprise 0.01-20% by weight,
preferably 1% -10% by weight, of one or more siRNA of the invention
encapsulated in a liposome as described above, and propellant. A
carrier can also be included as desired; e.g., lecithin for
intranasal delivery.
[0227] Methods of Using the Polypeptides
[0228] The polypeptides (such as antibodies) of the present
invention are useful in a variety of applications including, but
not limited to, diagnostic methods and therapeutic treatment
methods, diagnosing methods, a method of lysing malignant cell, and
a method of screening a substance for treating malignant disorders.
Methods of inhibiting proliferation of cells expressing CD26 are
also provided.
[0229] Antibodies and polypeptides of the invention can be used in
the detection, diagnosis and/or monitoring of a condition (such as
a disease or disorder) associated with CD26 expression. In some
embodiments, the condition associated with CD26 expression is a
condition associated with abnormal CD26 expression. For instance,
in some embodiments, the condition associated with CD26 expression
is a condition associated with altered or aberrant CD26 expression
(in some embodiments, increased or decreased CD26 expression
(relative to a normal sample), and/or inappropriate expression,
such as presence of expression in tissue(s) and/or cell(s) that
normally lack CD26 expression, or absence of CD26 expression in
tissue(s) or cell(s) that normally possess CD26 expression). In
some embodiments, the condition associated with CD26 expression is
a condition associated with CD26 overexpression. Overexpression of
CD26 is understood to include both an increase in expression of
CD26 in cell(s) or tissue(s) which normally expresses CD26 relative
to the normal level of expression of CD26 in those cell(s) or
tissue(s), as well as the presence of expression of CD26 in
tissue(s) or cell(s) that normally lack CD26 expression. In some
embodiments, the condition associated with CD26 expression is
mediated, at least in part, by CD26. In some embodiments, the
condition associated with CD26 expression is a condition associated
with cells expressing CD26. In some embodiments, the condition
associated with CD26 expression is a condition associated with the
proliferation of cells expressing CD26. In some embodiments, the
proliferation of the cells expressing CD26 is an abnormal
proliferation. The diagnostic method may be in vitro or in
vivo.
[0230] Exemplary reference is made in this discussion to
antibodies, with the understanding that this discussion also
pertains to the polypeptides of the invention.
[0231] For diagnostic applications, the antibody typically will be
labeled with a detectable moiety including but not limited to
radioisotopes, fluorescent labels, and various enzyme-substrate
labels. Methods of conjugating labels to an antibody are known in
the art. In other embodiment of the invention, antibodies of the
invention need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the antibodies of
the invention.
[0232] The antibodies of the present invention may be employed in
any known assay method, such competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0233] The antibodies may also be used for in vivo diagnostic
assays, such as in vivo imaging. Generally, the antibody is labeled
with a radionuclide (such as .sup.111In, .sup.99Tc, 14C, .sup.131I,
.sup.125I, or .sup.3H) so that the cells or tissue of interest can
be localized using immunoscintiography.
[0234] The antibody may also be used as staining reagent in
pathology, following techniques well known in the art.
[0235] In another aspect, the invention provides a method of
inhibiting proliferation of a cell expressing CD26. Inhibition of
proliferation of a cell expressing CD26 encompasses any observable
level of inhibition, including partial to complete inhibition of
proliferation. In some embodiments, proliferation of the cells is
inhibited at least about 10%, at least about 25%, at least about
50%, at least about 75%, at least about 90%, or at least about 95%,
or least about 98%, or about 100%. The method may be in vitro or in
vivo. In some embodiments, the method comprises contacting the cell
with a polypeptide (e.g., antibody) described herein. Generally,
the cell will be contacted with an amount of the polypeptide
sufficient to inhibit proliferation of the cell.
[0236] In some embodiments of the aspects described herein, a cell
expressing CD26 is a human cell. In some embodiments, a cell
expressing CD26 is a cancer cell. In some embodiments, a cell
expressing CD26 is a malignant mesothelioma, lung cancer, renal
cancer, liver cancer, or other malignancies associated with CD26
expression. In some embodiments, the tumor cell is malignant or
benign.
[0237] The antibody of the present invention may be used in
combination with an effector cells specific to the antibody for
treating, diagnosing, or inhibiting malignant mesothelioma, lung
cancer, renal cancer, liver cancer, or other malignancies
associated with CD26 expression.
[0238] Methods of assessing the inhibition of proliferation of a
cell are known in the art and include MTT assays. (See, e.g., Aytac
et al. (2003) British Journal of Cancer 88:455-462, Ho et al.
(2001) Clinical Cancer Research 7:2031-2040, Hansen et al. (1989)
J. Immunol. Methods, 119:203-210, and Aytac et al. (2001) Cancer
Res. 61:7204-7210.) Examples of MTT assays are also provided in the
specific examples Examples 2(G), 3(D), and 5 below.
[0239] The invention further provides methods for treating a
condition associated with CD26 expression in a subject. In some
embodiments, the method of treating a condition associated with
CD26 expression in a subject, comprises administering an effective
amount of a composition comprising a polypeptide, such as an
antibody, described herein to the subject. In some embodiments, the
condition associated with CD26 expression is associated with
abnormal expression of CD26. In some embodiments, the condition
associated with CD26 expression is a condition associated with
altered or aberrant CD26 expression (in some embodiments, increased
or decreased CD26 expression (relative to a normal sample), and/or
inappropriate expression, such as presence of expression in
tissue(s) and/or cell(s) that normally lack CD26 expression, or
absence of CD26 expression in tissue(s) or cell(s) that normally
possess CD26 expression). In some embodiments, the condition
associated with CD26 expression is a condition associated with CD26
overexpression. In some embodiments, the condition associated with
CD26 expression is mediated, at least in part, by CD26. In some
embodiments, the condition associated with CD26 expression is a
condition associated with cells expressing CD26. In some
embodiments, the condition associated with CD26 expression is a
condition associated with the proliferation of cells expressing
CD26. In some embodiments, the proliferation of CD26-expressing
cells is abnormal. In some embodiments, the cell expressing CD26 is
a T-cell. In some embodiments, the cell expressing CD26 is a tumor
cell, which may be malignant or benign. (Additional cells
expressing CD26 are described above.)
[0240] In some embodiments, the condition associated with CD26
expression in a subject is a proliferative disorder. In some
embodiments, the condition associated with CD26 expression in a
subject is a cancer. In some embodiments, the cancer is a malignant
mesothelium, lung cancer, renal cancer, liver cancer, or other
malignancies associated with CD26 expression.
[0241] The methods described herein (including therapeutic methods)
can be accomplished by a single direct injection at a single time
point or multiple time points to a single or multiple sites.
Administration can also be nearly simultaneous to multiple sites.
Frequency of administration may be determined and adjusted over the
course of therapy, and is based on accomplishing desired results.
In some cases, sustained continuous release formulations of
polypeptides (including antibodies), polynucleotides, and
pharmaceutical compositions of the invention may be appropriate.
Various formulations and devices for achieving sustained release
are known in the art.
[0242] Patients, subjects, or individuals include mammals, such as
human, bovine, equine, canine, feline, porcine, and ovine animals.
The subject is preferably a human, and may or may not be afflicted
with disease or presently show symptoms.
[0243] The antibody is preferably administered to the mammal in a
carrier; preferably a pharmaceutically-acceptable carrier. Suitable
carriers and their formulations are described in Remington's
Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990; and Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing, 2000. Typically, an
appropriate amount of a pharmaceutically-acceptable salt is used in
the formulation to render the formulation isotonic. Examples of the
carrier include saline, Ringer's solution and dextrose solution.
The pH of the solution is preferably from about 5 to about 8, and
more preferably from about 7 to about 7.5. Further carriers include
sustained release preparations such as semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, liposomes or
microparticles. It will be apparent to those persons skilled in the
art that certain carriers may be more preferable depending upon,
for instance, the route of administration and concentration of
antibody being administered.
[0244] The antibody can be administered to the mammal by injection
(e.g., systemic, intravenous, intraperitoneal, subcutaneous,
intramuscular, intraportal), or by other methods, such as infusion,
which ensure its delivery to the bloodstream in an effective form.
The antibody may also be administered by isolated perfusion
techniques, such as isolated tissue perfusion, to exert local
therapeutic effects. Intravenous injection is preferred.
[0245] Effective dosages and schedules for administering the
polypeptide (e.g., antibody) may be determined empirically, and
making such determinations is within the skill in the art. Those
skilled in the art will understand that the dosage of antibody that
must be administered will vary depending on, for example, the
mammal that will receive the antibody, the route of administration,
the particular type of antibody used, and other drugs being
administered to the mammal. Guidance in selecting appropriate doses
for antibody is found in the literature on therapeutic uses of
antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et
al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and
pp. 303-357; Smith et al., Antibodies in Human Diagnosis and
Therapy, Haber et al., eds., Raven Press, New York, 1977, pp.
365-389. A typical daily dosage of the antibody used alone might
range from about 1 .mu.g/kg to up to 100 mg/kg of body weight or
more per day, depending on the factors mentioned above. Generally,
any of the following doses may be used: a dose of at least about 50
mg/kg body weight; at least about 10 mg/kg body weight; at least
about 3 mg/kg body weight; at least about 1 mg/kg body weight; at
least about 750 .mu.g/kg body weight; at least about 500 .mu.g/kg
body weight; at least about 250 .mu.g/kg body weight; at least
about 100 .mu.g/kg body weight; at least about 50 .mu.g/kg body
weight; at least about 10 .mu.g/kg body weight; at least about 1
.mu.g/kg body weight, or more, is administered.
[0246] In some embodiments, more than one antibody may be present.
Such compositions may contain at least one, at least two, at least
three, at least four, or at least five different antibodies
(including polypeptides) of the invention.
[0247] The polypeptide may also be administered to the mammal in
combination with effective amounts of one or more other therapeutic
agents. The polypeptide may be administered sequentially or
concurrently with the one or more other therapeutic agents. The
amounts of polypeptide and therapeutic agent depend, for example,
on what type of drugs are used, the pathological condition being
treated, and the scheduling and routes of administration but would
generally be less than if each were used individually.
[0248] Following administration of antibody to the mammal, the
mammal's physiological condition can be monitored in various ways
well known to the skilled practitioner.
[0249] The above principles of administration and dosage can be
adapted for polypeptides described herein.
[0250] A polynucleotide encoding a polypeptide (including an
antibody) of the invention may also be used for delivery and
expression of the antibody or the polypeptide in a desired cell. It
is apparent that an expression vector can be used to direct
expression of the antibody. The expression vector can be
administered systemically, intraperitoneally, intravenously,
intramuscularly, subcutaneously, intrathecally, intraventricularly,
orally, enterally, parenterally, intranasally, dermally, or by
inhalation. For example, administration of expression vectors
includes local or systemic administration, including injection,
oral administration, particle gun or catheterized administration,
and topical administration. One skilled in the art is familiar with
administration of expression vectors to obtain expression of an
exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;
6,413,942; and 6,376,471.
[0251] Targeted delivery of therapeutic compositions comprising a
polynucleotide encoding a polypeptide or antibody of the invention
can also be used. Receptor-mediated DNA delivery techniques are
described in, for example, Findeis et al., Trends Biotechnol.
(1993) 11:202; Chiou et al., Gene Therapeutics: Methods And
Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu
et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem.
(1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990)
87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic
compositions containing a polynucleotide are administered in a
range of about 100 ng to about 200 mg of DNA for local
administration in a gene therapy protocol. Concentration ranges of
about 500 ng to about 50 mg, about 1 .mu.g to about 2 mg, about 5
.mu.g to about 500 .mu.g, and about 20 .mu.g to about 100 .mu.g of
DNA can also be used during a gene therapy protocol. The
therapeutic polynucleotides and polypeptides of the present
invention can be delivered using gene delivery vehicles. The gene
delivery vehicle can be of viral or non-viral origin (see
generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human
Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995)
1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of
such coding sequences can be induced using endogenous mammalian or
heterologous promoters. Expression of the coding sequence can be
either constitutive or regulated.
[0252] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5, 219,740; 4,777,127; GB
Patent No. 2,200,651; and EP 0 345 242), alphavirus-based vectors
(e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67;
ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and
Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250;
ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV)
vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769;
WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).
Administration of DNA linked to killed adenovirus as described in
Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
[0253] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther. (1992) 3:147); ligand-linked DNA(see, e.g., Wu, J.
Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338), and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP 0 524 968. Additional
approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411,
and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
[0254] The invention will now be illustrated with the following
non-limiting examples.
Examples
Materials and Methods
Reagents and Antibodies
[0255] Anti-CD26 mouse mAb (IgG1)14D10, 5F8, and anti-CD45 RA mouse
mAb (IgG1) 2H4 were developed in our laboratory as described
previously (20, 21), with the last one being used as control.
Normal human IgG (Sigma Aldrich, St. Louis, Mo.) was also used as a
control. Humanized anti-CD26 mAb was generated from 14D10 coding
sequence. In brief, several Fab clones which posses high binding
affinity to the epitope were selected. They were tested for
biological efficacies using in vitro proliferation assay. One was
selected for generation of a humanized anti-CD26 mAb (humAb). Mouse
mAb to PKBa/Akt, CDK2, CDK4, CDK6, cyclinE, and .beta.-actin were
from Cell Signaling Technology Inc. (Beverly, Mass.), and mouse mAb
to p27.sup.kip1, p21.sup.cip1/waf1, cyclinD1, and activated caspase
3 were from BD PharMingen.TM. (Lexington, Ky.). Anti-human IgG,
Fc.gamma. fragment specific F(ab')2 fragment of goat, anti-mouse
IgG, and Fc.gamma. fragment specific F(ab')2 fragment of goat were
from Jackson ImmunoResearch (West Grove, Pa.).
Cell Culture and Transfection
[0256] JMN cells were a kind gift from Dr. Brenda Gerwin
(Laboratory of Human Carcinogenesis, National Institutes of Health,
Bethesda, Md.). NCI-H2452 cells and 293T cells were obtained from
the American Type Culture Collection (Rockville, Md.). JMN cell
line and NCI-H2452 cell line were derived from patients with
malignant mesothelioma. All cells were grown in RPMI medium (Life
Technologies Inc., Grand Island, N.Y.) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), penicillin (100
units/ml), and streptomycin (100 .mu.g/m1) (Life Technologies Inc.,
Gaithersburg, Md.), or G418 (500 .mu.g/ml) (Sigma-Aldrich, St.
Louis, Mo.). 293T cells were transfected with full length CD26
subcloned into a pEB6 vector (22) using FuGENE6 reagent (Roche
Diagnostics, Indianapolis, Ind.). pEB6 vector was a kind gift from
Dr Y. Miwa (University of Tsukuba, Ibaraki, Japan).
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tet-
razolium assay
[0257] Cells were incubated in 96-well plates in media alone or in
the presence of humAb (0.1, 1.0, or 10 .mu.g/ml), or 2H4 (0.1, 1.0,
or 10 .mu.g/ml) in a total volume of 100 .mu.L (5.times.10.sup.3
cells per well). After 24 h of incubation in 37.degree. C.,
2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-te-
trazolium (Seikagaku, Tokyo, Japan) was added to each well. After
another 2 h of incubation, water soluble formazan dye upon
bioreduction in the presence of an electron carrier,
1-methoxy-5-methylphenazinium, was measured at 450 nm using a
microplate reader (BioRad, Hercules, Calif.). All samples were
tested in triplicate. Values reported represent the means of
triplicate wells, and the standard error (SE) of mean was within
15.
Immunohistochemistry
[0258] For immunohistochemistry, surgical specimens from twelve
patients consisting of seven MM, three reactive mesothelial cells,
and two adenomatoid tumors were evaluated. For each, 10%
formalin-fixed, paraffin-embedded specimens containing both the
carcinoma and its adjacent non-neoplastic tissue were prepared.
Paraffin-embedded tissue were dewaxed and rehydrated using xylene
and ethanol, respectively. Slides were deparaffinized, then heated
in a microwave processor for antigen retrieval in 10 mM citrate
buffer (pH 6.0) for 10 min. After blocking in 3% (vol/vol) BSA,
slides were incubated at 4.degree. C. overnight with the primary
antibody (anti-CD26 mAb), washed with PBS, and the secondary
antibody was labeled with biotin and applied for 30 min.
Streptavidin-LSA amplification method was carried out for 30 min
followed by peroxidase/diaminobenzidine substrate/chromagen. The
slides were counterstained with hematoxylin. Two different
pathologists checked the validity of the obtained results. All
human specimens were obtained from Department of Pathology, Keio
University (Tokyo, Japan), and informed consent was obtained from
all patients in accordance with requirements of the institutional
review board (IRB).
Depletion of Endogenous CD26
[0259] To deplete endogenous CD26, siRNA-oligo targeting CD26 cDNA
(accession no. NM.sub.--001935) was made according to the design
site of TAKARA BIO (http://www.takara-bio.co.jp/RNAi.htm); sense:
5'- GAAAGGUGUCAGUACUAUU TT-3' (SEQ ID NO: 30), antisense: 3'-TT
CUUUCCACAGUCAUGAUAA-5' (SEQ ID NO: 31), with scrambled control
siRNA-oligo targeting human Cas-L; sense: 5'-UAAUUAGGGUCGGGUAAAC
TT-3' (SEQ ID NO: 32), antisense: 3'-TT AUUAAUCCCAGCCCAUUUG -5'
(SEQ ID NO: 33), being used as control. CD26 siRNA oligo (siCD26)
was transfected using TranslT-TKO.RTM. transfection reagent (Mirus
Bio Corporation, Madison, Wis.) according to the manufacturer's
protocol.
SDS-PAGE and Immuno-Blotting
[0260] Preparation of whole cell lysates and cell fractionations
were performed as described elsewhere (23). The protein samples
were subjected to SDS-PAGE and transferred to polyvinylidene
difluoride membrane (Immobilon-P; Millipore, Bedford, Mass.).
Specific antigens were probed using the corresponding mAbs,
followed by HRP-conjugated secondary Ig (Amersham Pharmacia
Biotech, Piscataway, N.J.). Western blots were visualized by the
enhanced chemiluminescence technique (NEN, Boston, Mass.).
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
[0261] The capacity of mAb to induce effector cell-dependent lysis
of tumor cells was evaluated with the Calcein-AM-release assay.
Healthy donor NK cells were isolated from PBMCs with the NK Cell
Isolation Kit II Miltenyi Biotec (Bergisch Gladbach, Germany), and
used as effector cells. Target cells (1.times.10.sup.6 cells) were
labeled with 10 .mu.M Calcein-AM (Dojindo, Kumamoto, Japan) under
shaking conditions at 37.degree. C. for 1 h. Cells were washed
three times with PBS and resuspended in culture medium
(1.times.10.sup.5 cells/ml). Labeled cells were dispensed in
96-well U-bottom plates (5.times.10.sup.3, in 50 .mu.l/well) and
preincubated (37.degree. C., 30 min) with 50 .mu.l of 7-fold serial
dilutions of humAb or 14D10, in culture medium, ranging from 0.1
pg/ml to 0.1 mg/ml (final concentrations). Culture medium was added
instead of mAb to determine spontaneous Calcein-AM release, with
Triton X-100 (1% final concentration) being added to determine
maximal Calcein-AM release. Thereafter, human effector cells were
added to the wells (5.times.10.sup.5 cells/well) and cells were
incubated at 37.degree. C. overnight. Supernatants were then
collected for measurement of the Calcein-AM release. Percentage of
specific lysis was calculated using the following formula: %
specific lysis=(experimental release-spontaneous release)/(maximal
release-spontaneous release).times.100; where maximal release was
determined by adding Triton X-100 to target cells, and spontaneous
release was measured in the absence of sensitizing Abs and effector
cells.
Complement-Dependent Cytotoxicity (CDC)
[0262] CDC assay was performed as described previously (24). Target
cells were dispensed in 96-well U-bottom plates at 1.times.10.sup.5
cells/well, and incubated with various concentrations of mAbs at
4.degree. C. for 30 min. Subsequently, human serum was added and
cells were incubated at 37.degree. C. for 2 h. Evaluation of CDC
specific cell death along with ADCC specific cell death was
assessed with the Annexin V-FITC Apoptosis Detection Kit
(BioVision, Mountain View, Calif.) and detection of activated
caspase 3.
Assessment of Antitumor Activity of Humanized Anti-CD26 mAb in
Effector-Depleted SCID Mice
[0263] All in vivo studies were approved by the Institute Animal
Care and Use Committee. 6 week old female NOD-SCID mice were
purchased from Charles River (Kanagawa, Japan), and were
pre-treated with anti-asialo-GM1 polyclonal antisera 25% (v/v,
WAKO, Osaka, Japan) i.p. 1 day before mAb treatment.
[0264] To assess the effect of humAb against tumorigenicity, JMN
cells (1.times.10.sup.6) were inoculated subcutaneously into the
left flank of mice. Mice were treated with intratumoral injection
of isotype matched control mAb, 5F8, 14D10, or humanized anti-CD26
mAb (10 .mu.g/each injection) on the 14.sup.th day after cancer
cell inoculation when the tumor mass became visible (5 mm in size).
Each mAb was admnistered three times per week. Tumor-bearing mice
were then monitored for tumor development and progression. Tumor
size was determined by caliper measurement of the largest (x) and
smallest (y) perpendicular diameters, and was calculated according
to the formula V=.pi./6.times.xy.sup.2.
[0265] To assess the effect of humAb against tumor dissemination,
JMN cells (1.times.10.sup.5) were injected intravenously via tail
vein. Thereafter, mice were treated with intra-venous injection of
isotype matched control mAb, 5F8, 14D10, or humanized anti-CD26 mAb
(10 .mu.g/each injection), starting on the day of cancer cell
injection. Each mAb was administered three times per week.
Cumulative proportion survival was assessed by Kaplan-Meier.
Assessment of Antitumor Activity of Humanized Anti-CD26 mAb in
Effector-Present Balb Mice
[0266] 6 week old female Balb mice were purchased from Charles
River (Kanagawa, Japan), and treatment with anti-asialo-GM1
polyclonal antisera was not introduced to preserve the binding of
the mouse effector system.
[0267] To assess the effect of humAb against tumorigenicity, JMN
cells (1.times.10.sup.6) were inoculated subcutaneously into the
left flank of mice. Mice were treated with intratumoral injection
of isotype matched control mAb, 5F8, 14D10, or humanized anti-CD26
mAb (10 .mu.g/each injection) on the 14th day after cancer cell
inoculation when the tumor mass became visible (5 mm in size). Each
mAb was administered three times per week. Tumor-bearing mice were
then monitored for tumor development and progression. Tumor size
was determined by caliper measurement of the largest (x) and
smallest (y) perpendicular diameters, and was calculated according
to the formula V=.pi./6.times.xy.sup.2. On the 35th day after the
first mAb treatment, all mice were euthanized to assess the
microscopic feature of resected specimens in subcutaneous
tumorigenicity model.
[0268] To assess the effect of humAb against tumor dissemination,
JMN cells (1.times.10.sup.5) were intravenously injected via tail
vein. Thereafter, mice were treated with intra-venous injection of
isotype matched control mAb, or humanized anti-CD26 mAb (10
.mu.g/each injection) starting on the day of cancer cell injection.
Each mAb was administered three times per week. Cumulative
proportion of survival was assessed by Kaplan-Meier. To further
assess the effect of humanized anti-CD26 mAb on distant metastasis
formation, treated mice were euthanized and multiple metastasis
formation in the lung and liver was calculated in another tumor
dissemination model. JMN cells (1.times.10.sup.5) were injected
intravenously into mice in each group. Mice were treated with
intra-venous injection of isotype matched control mAb (lane 1,
n=4), 5F8 (lane 2, n=4), 14D10 (lane 3, n=4), or humanized
anti-CD26 mAb (lane 4, n=4) on the day of cancer cell injection.
Each mAb was administered three times per week. On the 35th day
after cancer cell injection, mice were euthanized and multiple
metastasis formation in the lung and liver was calculated.
Construction of Human Effector Cell (HuEC)-Engrafted Mice and
Assessment of Antitumor Activity in NOD/Shi-scid,
IL-R.gamma..sup.null Mice
[0269] NOD/Shi-scid, IL-R.gamma..sup.null (NOG mice) were obtained
from Central Institute for Experimental Animals (CIEA) (Kanagawa,
Japan). Human PBMCs were isolated from the peripheral blood of a
healthy donor using Lymphoprep (AXIS-SHIELD, Oslo, Norway) and used
as HuEC. Thereafter, HuEC (5.times.10.sup.6) were injected
intraperitoneally (i.p.) in a volume of 0.2 ml suspended in PBS
into NOG-SCID mice under sterile conditions. The mice were
pretreated with a 0.2 mL anti-asialo-GM1 polyclonal antisera 25%
(v/v, WAKO, Osaka, Japan) given i.p. 1 day before HuEC injection.
NCI-H2452 cells (5.times.10.sup.4) were injected i.p. into SCID
mice engrafted with human HuEC, 1 days after HuEC injection. One,
3, and 5 days later, humAb were injected i.p. Mice were observed
daily to monitor for death due to ascites tumor development.
Cumulative proportion of survival was assessed by Kaplan-Meier.
Results
Cell Surface CD26 is Highly Expressed on Human MM Tissue.
[0270] We first evaluated the level of CD26 expression on
surgically resected human MM tissues from patients. Twelve
consecutive surgically resected specimens from the primary sites
were examined for cell surface CD26 expression. CD26 was highly
expressed on all MM tissues (FIG. 1A). In adenomatoid tumor, or
reactive mesothelial cells, CD26 expression was very weak (FIG.
1A-a,b). In contrast, CD26 was highly expressed in various
pathological types of MM, including localized MM,
well-differentiated papillary MM, and diffuse MM (FIG. 1A-c to h).
These results suggested that CD26 is highly expressed in MM, but
not in benign mesothelial tissues.
CD26 Plays a Role in Cell Adhesion to Extracellular Matrix.
[0271] MM cell lines, JMN and NCI-H2452, exhibited high levels of
surface CD26 expression (FIG. 1B).
[0272] Since CD26 has been described previously to play a role in
cell adhesion to extracellular matrix (ECM) proteins (13, 25), we
examined whether CD26 plays a role in cellular interaction with the
ECM. As seen in FIG. 1C, NCI-H2452 depleted of endogenous CD26
using siRNA oligo showed significant loss of CD26 binding to
extracellular matrix proteins including fibronectin and collagen I.
In contrast to these results, depletion of CD26 did not alter
binding to laminin (an ECM protein lacking binding ability to
CD26), or hyaluronan (a ligand for CD44) (FIG. 1C). In further
support of these findings, 293T cells transfected with full length
CD26 cDNA subcloned into pEB6 vector showed higher binding ability
to fibronectin and collagen I than control pEB6 transfected 293T
cells (FIG. 1C). Moreover, depletion of CD26 was associated with
the upregulation of p27.sup.kip1 (FIG. 1D). These findings thus
suggested that CD26 serves as a binding molecule to distinct ECM
proteins and that contact inhibition may play a contributing role
to the observed CD26-depletion-mediated upregulation of
p27.sup.kip1 associated with CD26 depletion (26, 27).
Anti-CD26 mAb Perturbs Cellular Binding to ECM.
[0273] Since CD26 proved to be an ECM binding protein, we further
evaluated whether anti-CD26 mAbs disrupt cellular adhesion to ECM.
For this purpose, isotype matched control mAb, 5F8, 14D10, and
humanized anti-CD26 mAb (humAb) were evaluated for potential
disruption to cellular adhesion to ECM. As seen in FIG. 2A, JMN
cells treated with 14D10 and humAb had decreased binding to
fibronectin and collagen I, while control mAb and 5F8 (anti-CD26
mAb without biological function) did not influence binding to
fibronectin and collagen I. Moreover, 14D10 and humAb conferred
direct growth inhibition to JMN cells by in vitro proliferation
assay in a dose dependent manner, with humAb having a stronger
antiproliferative effect than 14D10 (FIG. 2B). Importantly, 14D10
and humAb induced upregulation of p27.sup.kip1 and downregulation
of CDK2. These results suggested that both 14D10 and humAb
dynamically confer contact inhibition-related growth inhibition via
upregulation of p27.sup.kip1 and downregulation of CDK2.
Humanization of Anti-CD26 mAb Results in Antibody-Dependent
Cell-Mediated Cytotoxicity.
[0274] While both 14D10 and humAb had similar direct effects on
cancer cells, our present studies revealed different biological
effects of humAb as compared to 14D10, through the use of ADCC
assay with human effector cells. When effector/target (E/T) ratio
was held constant at 50, JMN cells treated with humAb showed
specific lysis via ADCC in an antibody-dose dependent manner (FIG.
3A, left panel). Importantly, JMN cells treated with 14D10 did not
show ADCC specific lysis (FIG. 3A, left panel), suggesting that
humanization of 14D10 to humAb results in the induction of potent
ADCC activity via engagement of the human effector system.
Moreover, as seen in FIG. 3A, right panel, humAb provoked ADCC
specific lysis in an effector-dose dependent manner. These results
were also observed when other CD26-positive MM lines besides JMN
(NCI-H2452) were used as target cells. These data suggested that
humAb possesses a novel biological function other than the direct
effect on target cells seen with 14D10, namely ADCC specific lysis.
To better characterize the humAb-mediated ADCC, apoptosis assays
using PI-annexinV staining and detection of cleaved caspase 3 were
employed. In these assays, a cross-linking method using anti-human
IgG, Fc.gamma. fragment specific F(ab')2 fragment of goat,
anti-mouse IgG, and Fc.gamma. fragment specific F(ab')2 fragment of
goat were used to mimic human effectors to humAb and 14D10,
respectively. As seen FIG. 3B, upper three panels, cross-linked
humAb induced late apoptosis, while cross-linked 14D10 did not
induce late or early apoptosis. Importantly, neither humAb nor
14D10 induced CDC using human complement (FIG. 3B, lower three
panels; Supplementary FIG. 1). To furher support these findings,
only cross-linked humAb induced activation of caspase 3 in JMN
cells, while cross-linked 14D10, humAb plus human complement, and
14D10 plus human complement did not (FIG. 3C). These results
therefore indicated that humAb elicits ADCC specific lysis, but not
CDC specific lysis.
Humanized Anti-CD26 mAb Exhibits a Direct In Vivo Anti-Tumor Effect
on MM Cells.
[0275] Since we recently demonstrated that 14D10 exhibits direct in
vivo anti-tumor effect on solid tumors (24), we further examined
whether humAb has a similar in vivo anti-tumor effect. For this
purpose, we utilized NOD-SCID mice, which lack functional B and T
cells as well as most natural killer (NK) cell activity (28). To
minimize the effect of mouse effector cells, NOD-SCID mice were
pre-treated by anti-asialo-GM1 polyclonal antisera, prior to being
subjected to humAb functional evaluation. As seen in FIGS. 4A and
B, humAb and 14D10 reduced the tumorigenicity of subcutaneously
inoculated JMN, with humAb being more potent in reducing tumor
formation. These observed results suggested that humAb possesses a
stronger direct anti-tumor effect than 14D10. To further examine
the direct anti-tumor activity of humAb on tumor dissemination, we
examined the effect of intravenously administered antibodies in a
JMN xenograft model. As seen in FIG. 4C, humAb and 14D10 enhanced
mouse survival when both antibodies were administered
intravenously, with humAb being more efficient in promoting
survival. All together, these observed results suggested that humAb
is more potent than 14D10 in its direct anti-tumor activity.
Mouse Effector System May Potentiate the Anti-Tumor Effect of
Anti-CD26 mAb.
[0276] While both humAb and 14D10 showed a direct in vivo
anti-tumor effect, we next examined the potential involvement of
mouse effector system in the anti-CD26 mAb activity induced
anti-tumor effect. For this purpose, we utilized Balb mice, which
possesses robust NK cell activity. As seen in FIG. 5A, humAb and
14D10 reduced the tumorigenicity of subcutaneously inoculated JMN.
It should be noted that both 14D10 and humAb reduced tumor
formation in the presence of the mouse effector system (FIG. 5A).
As seen in FIG. 5B, both humAb and 14D10-treated tumors showed
resultant dead tissues upon microscopic analyses. These results
suggested that both humAb and 14D10 utilized the mouse effector
system, in marked contrast to the observed differences between
humAb and 14D10 in the mouse effector-depleted xenograft model.
Additional studies using intravenous administration of JMN cells
showed that intravenous injection of humAb effectively enhanced
mouse survival in the presence of the mouse effector system (FIG.
5C). Importantly, formation of distant JMN was similarly inhibited
by both humAb and 14D10 (FIG. 5D). These data indicated that the
mouse effector system potentiates the anti-CD26 mAb-mediated direct
anti-tumor effect.
Human Effector System May Potentiate Anti-Tumor Effect of Humanized
Anti-CD26 mAb.
[0277] We next evaluated the potential involvement of human
effector system in the anti-CD26 mAb-induced anti-tumor effect. For
this purpose, NOD/Shi-scid, IL-R.gamma..sup.null (NOG mice) which
have significant defects in T, B, and NK cell activity, were
employed in a NCI-H2452 xenograft model construction. Human PBMCs
were used as human effector cells (HuEC) in this in vivo model. To
completely deplete the mouse effector system, NOG mice were
pretreated with anti-asialo-GM1 antisera one day prior to
intraperitoneal HuEC implantation. As seen in FIG. 6,
intraperitoneal administration of humAb drastically enhanced
NCI-H2452 xenograft mouse survival in the absence of HuEC. It
should be noted that while 14D10 also enhanced mouse survival, its
effect was much weaker than humAb in the absence of HuEC (FIG. 6).
These results suggested that humAb possesses a stronger direct
anti-tumor effect. Importantly, in the presence of HuEC, the
anti-tumor effect of humAb was exaggerated, while the anti-tumor
effect of 14D10 was not altered significantly (FIG. 6). All
together these observed results suggested that CD26 is an
appropriate molecular target for mesothelioma therapy and humAb
regulates tumor growth by at least two distinct mechanisms of
action, through its direct anti-tumor activity as well as its
ability to engaze human effector system.
Discussion
[0278] In this study, we demonstrate the anti-tumor effect of
anti-CD26 mAb in an in vitro and in vivo model. Importantly, our
study suggests that humanization of anti-CD26 mAb yields additive
anti-tumor effect to contact inhibition associated with
p27.sup.kip1 induction. Our study also indicates the functional
role of CD26 as a binding protein to ECM in human MM.
[0279] Immunohistological analysis indicated that human MM cells
express high levels of surface CD26 than non-malignant tissue,
suggesting that CD26 may play a role in cancer growth and
progression. It should be noted that depletion of endogenous CD26
in NCI-H2452 using siRNA oligo results in significant loss of
binding to ECM, including fibronectin and collagen I. Moreover,
293T cells transfected with full length CD26 cDNA exhibit higher
binding affinity to fibronectin and collagen I than control mock
transfected 293T cells. Moreover, depletion of CD26 leads to the
upregulation of p27.sup.kip1. These findings thus suggest that CD26
is involved in cancer cell adhesion to ECM and that contact
inhibition may play a contributing role to the observed
CD26-depletion-mediated upregulation of p27.sup.kip1. It has been
previously reported that p27.sup.kip1 is up-regulated during
contact inhibition (26).
[0280] Both humAb and 14D10 display direct inhibition of MM growth
via p27.sup.kip1 upregulation and disruption of binding to ECM.
Hence, our results with these ani-CD26 monoclonal antibodies are
consistant with those obtained from above siRNA study, showing that
both humAb and 14D10 have an antagonistic effect on the adhesive
property of MM.
[0281] Further examination of their effector functions associated
with anti-CD26 mAb mediated anti-tumor effect indicates that humAb,
but not 14D10, elicits ADCC induced cell lysis. Cross-linking of
humAb results in an accumulation of annexinV-positive and
PI-positive population, and cleavage of activated caspase 3. These
data suggest that humanization of anti-CD26 mAb elicits greater
contribution from ADCC in addition to a direct anti-tumor effect.
Meanwhile, the reason why humAb does not induce CDC activity is not
clear at the moment. One potential reason is the high surface
expression of DAF and CD59, which are antagonistic to human
complement proteins (data not shown). Alternatively, our in vitro
system may not be appropriate for the induction of CDC
activation.
[0282] In vivo study with NOD-SCID mice showed that humAb and 14D10
reduce the tumorigenicity of subcutaneously inoculated JMN cells,
suggesting that humAb possesses a direct anti-tumor effect as well.
Our results also suggest that humAb is more potent in reducing
tumor formation, possibly due to its higher binding affinity to
CD26 than 14D10.
[0283] Meanwhile, in vivo study with Balb mice showed that humAb
and 14D10 are equally effective in reducing the tumorigenicity of
subcutaneously inoculated JMN cells. These data suggest that the
mouse effector system may potentiate the anti-tumor effect of 14D10
more than humAb. In fact, not only humAb but also 14D10-treated
tumor specimens from these mice exhibit a reduction of viable cells
in tumor mass. It is also noteworthy that both humAb and 14D10
reduce the formation of distant metastasis, findings which may be
partly explained by our in vitro results that CD26 serves as a
binding protein to distinct ECM proteins.
[0284] In vivo study with NOG-SCID mice which lack functional mice
effectors showed that dual-xenograft of human effector cells plus
target cells results in greater mouse survival than single
xenograft of target when combined with humAb. These data clearly
corroborate the in vitro data suggesting that humAb induces a
biphasic anti-tumor action with a human effector system.
[0285] CD26 status may be altered in cancer and may have an effect
on the growth and metastatic potential of various tumors. CD26
absence is associated with the development of some cancers while
CD26 presence is associated with a more aggressive phenotype in
other neoplasms. For example, in non small cell lung cancer cell
lines, cells transfected with CD26 develop morphologic changes,
altered contact inhibition, and reduced ability for
anchorage-independent growth (29). CD26 reexpression also
correlates with increased p21 expression, leading to induction of
apoptosis and cell cycle arrest in G1 stage. Wesley et al reported
that CD26/DPPIV up-regulates the expression of CDKI p27.sup.kip1 by
4 to 6 fold in CD26 transfected DU-145 metastatic prostate cancer
cells compared with the parent and vector transfected DU-145 cells
(30). It is also reported that overexpression of CD26 in ovarian
cancer leads to increased E-cadherin and tissue inhibitors of MMPs
resulting in decreased invasive potential (31). CD26/DPPIV thus
functions as a tumor suppressor in the cases described above and
its downregulation may contribute to the loss of growth control. In
contrast, CD26 expression is associated with a more aggressive
clinical course in T-cell large granular lymphocyte leukemia
(T-LGLL) (32).
[0286] In non-Hodgkin Lymphoma (NHL), CD26 expression is found
mainly in aggressive subtypes such as T-lymphoblastic lymphoma
(LBL)/T-acute lymphoblastic leukemia (ALL) and T-cell CD30 +
anaplastic large cell lymphoma (ALCL). An earlier report indicated
that CD26 and CD40L expression is mutually exclusive, with CD40L
expressed on cells from more indolent diseases. Of note is that
CD26 expression on T-cell LBL/ALL is associated with a worse
survival (33). In renal cell carcinoma, CD26 is highly expressed on
the cell surface of RCC tissues and many cell lines derived from
RCC such as Caki-2, VMRC-RCW, and Caki-1. Moreover, anti-CD26
mAb-mediated growth inhibition of the Caki-2 cell line is
associated with G1/S cell cycle arrest, enhanced p27.sup.kip1
expression, and down regulation of CDK2 (18). We now show that CD26
is highly expressed in MM tissues, and ani-CD26 mAb treatment and
CD26 downregulaton by RNAi in CD26 positive MM cell lines lead to
contact inhibition and p27.sup.kip1 upregulation.
[0287] Therefore in case of malignant tumors such as T-cell
lymphoma, renal cell carcinoma, and malignant mesothelioma, CD26
plays a role in tumor growth and may be involved in invasion and
metastasis. In view of its complex historical effect, the role of
CD26 in cancers needs to be evaluated individually for each tumor
type.
[0288] Malignant mesothelioma is an aggressive neoplasm with a
dismal prognosis and is relatively unpresponsive to chemotherapy.
One study systematically reviewed evidence for chemotherapy effect
from 1965 through June 2001, and found 83 studies with 88 treatment
arms (34). Cisplatin was the most active single drug, and cisplatin
with doxorubicin had the highest response rate (28.5% response
rate, confidence interval 21.3-35.7%). Since this report, results
of a phase III randomized trial (using 448 chemotherapy naive
patients with unresectable mesothelioma) involving the combination
cisplatis/pemetrexed (an antimetabolite) or cisplatin alone have
demonstrated that medium survival is extended from 9.3 months in
those treated with cisplatin to 12.1 months in those treated with
both agents (35). However standard treatments for MM are still not
satisfactory in term of survival, hence there is an urgent need for
novel therapeutic approaches for MM.
[0289] Our data therefore indicate that the novel humanized
anti-CD26 mAb humAb is an effective therapeutic tool for cancer
treatment including MM, as it can utilize the human effector system
to target cancer cells in addition to its direct anti-tumor
effect.
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Sequence CWU 1
1
331107PRTArtificialSynthetic Peptide 1Asp Ile Leu Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Pro Gly1 5 10 15Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Gln Asp Ile Arg Asn Asn 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu His Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu
Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Ser Ile Lys Leu Pro Leu 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1052107PRTArtificialSynthetic Peptide 2Glu Ile Glu Leu Thr Gln Ser
Pro Ser Ser Leu Ser Val Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser
Cys Ser Ala Ser Gln Asp Ile Arg Asn Asn 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu Gln Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu
Asp Val Ala Ala Tyr Tyr Cys Gln Gln Ser Ile Lys Leu Pro Phe 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1053107PRTArtificialSynthetic Peptide 3Asp Ile Glu Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Ala Gly1 5 10 15Glu Arg Val Thr Ile Ser
Cys Arg Ala Ser Gln Gly Ile Arg Asn Ser 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Ala65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Lys Leu Pro Phe 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1054107PRTArtificialSynthetic Peptide 4Asp Ile Leu Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Thr Pro Gly1 5 10 15Glu Arg Ala Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Arg Asn Asn 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro65 70 75 80Glu
Asp Val Ala Ala Tyr Tyr Cys Gln Gln Ser Ile Lys Leu Pro Phe 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1055107PRTArtificialSynthetic Peptide 5Glu Ile Glu Met Thr Gln Ser
Pro Ser Ser Leu Ser Val Ser Ala Gly1 5 10 15Glu Arg Ala Thr Ile Ser
Cys Ser Ala Ser Gln Asp Ile Arg Asn Ser 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu His Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu
Asp Val Ala Ile Tyr Tyr Cys Gln Gln Ser Asn Lys Leu Pro Leu 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1056107PRTArtificialSynthetic Peptide 6Glu Ile Glu Leu Thr Gln Ser
Pro Ser Ser Leu Ser Val Ser Pro Gly1 5 10 15Asp Arg Val Thr Ile Ser
Cys Ser Ala Ser Gln Gly Ile Arg Asn Ser 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu His Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Ala65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Ile Lys Leu Pro Leu 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1057107PRTArtificialSynthetic Peptide 7Asp Ile Leu Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Pro Gly1 5 10 15Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Gln Asp Ile Arg Asn Asn 20 25 30Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser Ser
Asn Leu Gln Thr Gly Val Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro65 70 75 80Glu
Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Ser Ile Lys Leu Pro Leu 85 90
95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
1058116PRTArtificialSynthetic Peptide 8Glu Val Gln Leu Val Glu Ser
Gly Ala Gly Val Lys Gln Pro Gly Gly1 5 10 15Thr Leu Arg Leu Thr Cys
Thr Ala Ser Gly Phe Ser Leu Thr Thr Tyr 20 25 30Gly Val His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Val Ile Trp
Gly Asp Gly Arg Thr Asp Tyr Asp Ala Ala Phe Met 50 55 60Ser Arg Val
Thr Ile Ser Lys Asp Thr Ser Lys Ser Thr Val Tyr Leu65 70 75 80Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Met 85 90
95Arg Asn Arg His Asp Trp Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val
100 105 110Thr Val Ser Ser 1159116PRTArtificialSynthetic Peptide
9Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Lys Gln Pro Gly Glu1 5
10 15Thr Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Thr Thr
Tyr 20 25 30Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Val Ile Trp Gly Asp Gly Arg Thr Asp Tyr Asp Ala
Ala Phe Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Ser
Thr Ala Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Met 85 90 95Arg Asn Arg His Asp Trp Phe Asp Tyr
Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val Ser Ser
11510116PRTArtificialSynthetic Peptide 10Glu Val Gln Leu Val Glu
Ser Gly Ala Gly Val Glu Gln Pro Gly Gly1 5 10 15Thr Leu Arg Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Thr Thr Tyr 20 25 30Gly Val His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Val Ile
Trp Gly Asp Gly Arg Thr Asp Tyr Asp Ala Ala Phe Met 50 55 60Ser Arg
Val Thr Ile Ser Arg Asp Thr Ser Lys Ser Thr Ala Tyr Leu65 70 75
80Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95Arg Asn Arg His Asp Trp Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 11511116PRTArtificialSynthetic
Peptide 11Glu Val Gln Leu Val Glu Ser Gly Ala Glu Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Lys Ala Ser Gly Phe Thr Leu
Asn Thr Tyr 20 25 30Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Met 35 40 45Gly Val Ile Trp Gly Gly Gly Arg Thr Asp Tyr
Asp Ala Ser Phe Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Asn Ser
Lys Asn Thr Ala Tyr Leu65 70 75 80Gln Leu Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95Arg Ser Arg His Asp Trp Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val Ser Ser
11512116PRTArtificialSynthetic Peptide 12Glu Val Gln Leu Val Gln
Ser Gly Gly Gly Leu Lys Gln Pro Gly Glu1 5 10 15Thr Leu Arg Leu Ser
Cys Thr Ala Ser Gly Tyr Ser Leu Thr Thr Tyr 20 25 30Gly Val His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Val Ile
Trp Gly Asp Gly Arg Thr Asp Tyr Asp Ser Ser Phe Met 50 55 60Ser Arg
Val Thr Ile Ser Lys Asp Thr Ser Lys Ser Thr Ala Tyr Leu65 70 75
80Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr
85 90 95Arg Asn Arg His Asp Trp Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 11513116PRTArtificialSynthetic
Peptide 13Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Lys Gln Pro
Gly Glu1 5 10 15Thr Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
Ser Thr Tyr 20 25 30Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Gly Val Ile Trp Gly Asp Gly Arg Thr Asp Tyr
Asp Ala Ala Phe Met 50 55 60Ser Arg Val Thr Ile Ser Lys Asp Thr Ser
Lys Ser Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Met 85 90 95Arg Asn Arg His Asp Trp Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val Ser Ser
11514116PRTArtificialSynthetic Peptide 14Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Lys Gln Pro Gly Glu1 5 10 15Thr Leu Arg Leu Thr
Cys Thr Ala Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30Gly Val His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Val Ile
Trp Gly Asp Gly Arg Thr Asp Tyr Asp Ala Ala Phe Met 50 55 60Ser Arg
Val Thr Ile Ser Lys Asp Thr Ser Lys Ser Thr Val Tyr Leu65 70 75
80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Met
85 90 95Arg Asn Arg His Asp Trp Phe Asp Tyr Trp Gly Gln Gly Thr Thr
Val 100 105 110Thr Val Ser Ser 11515116PRTArtificialSynthetic
Peptide 15Glu Val Gln Leu Val Xaa Ser Gly Xaa Xaa Xaa Xaa Gln Pro
Gly Xaa1 5 10 15Xaa Leu Arg Leu Xaa Cys Xaa Ala Ser Gly Xaa Xaa Leu
Xaa Thr Tyr 20 25 30Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Xaa 35 40 45Gly Val Ile Trp Gly Xaa Gly Arg Thr Asp Tyr
Asp Xaa Xaa Phe Met 50 55 60Ser Arg Val Thr Ile Ser Xaa Asp Xaa Ser
Lys Xaa Thr Xaa Tyr Leu65 70 75 80Gln Xaa Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Xaa 85 90 95Arg Xaa Arg His Asp Trp Phe
Asp Tyr Trp Gly Gln Gly Thr Thr Val 100 105 110Thr Val Ser Ser
11516107PRTArtificialSynthetic Peptide 16Xaa Ile Xaa Xaa Thr Gln
Ser Pro Ser Ser Leu Ser Xaa Xaa Xaa Gly1 5 10 15Xaa Arg Xaa Thr Ile
Xaa Cys Xaa Ala Ser Gln Xaa Ile Arg Asn Xaa 20 25 30Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ser
Ser Asn Leu Xaa Xaa Gly Val Pro Xaa Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Xaa Xaa65 70 75
80Glu Asp Xaa Ala Xaa Tyr Tyr Cys Gln Gln Ser Xaa Lys Leu Pro Xaa
85 90 95Thr Phe Gly Ser Gly Thr Lys Val Glu Ile Lys 100
10517465PRTArtificialSynthetic Peptide 17Met Glu Trp Ser Trp Val
Phe Leu Phe Phe Leu Ser Val Thr Thr Gly1 5 10 15Val His Ser Glu Val
Gln Leu Val Glu Ser Gly Ala Gly Val Lys Gln 20 25 30Pro Gly Gly Thr
Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu 35 40 45Thr Thr Tyr
Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp
Val Gly Val Ile Trp Gly Asp Gly Arg Thr Asp Tyr Asp Ala65 70 75
80Ala Phe Met Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Ser Thr
85 90 95Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 100 105 110Tyr Cys Met Arg Asn Arg His Asp Trp Phe Asp Tyr Trp
Gly Gln Gly 115 120 125Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 130 135 140Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu145 150 155 160Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 180 185 190Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 195 200
205Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
210 215 220Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys225 230 235 240Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro 245 250 255Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 260 265 270Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 275 280 285Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val305 310 315
320Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr 355 360 365Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr 370 375 380Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu385 390 395 400Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 405 410 415Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 420 425 430Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 435 440
445Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
450 455 460Lys46518234PRTArtificialSynthetic Peptide 18Met Ser Val
Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Asp Ala
Arg Cys Asp Ile Leu Leu Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala
Thr Pro Gly Glu Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Gly 35 40
45Ile Arg Asn Asn Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60Arg Leu Leu Ile Tyr Tyr Ser Ser Asn Leu Gln Ser Gly Val Pro
Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 85 90 95Arg Leu Gln Pro Glu Asp Val Ala Ala Tyr Tyr Cys
Gln Gln Ser Ile 100 105 110Lys Leu Pro Phe Thr Phe Gly Ser Gly Thr
Lys Val Glu Ile Lys Arg 115 120 125 Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 130
135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys225 2301913PRTArtificialSynthetic Peptide
19Tyr Ser Leu Arg Trp Ile Ser Asp His Glu Tyr Leu Tyr1 5
102013PRTArtificialSynthetic Peptide 20Leu Glu Tyr Asn Tyr Val Lys
Gln Trp Arg His Ser Tyr1 5 102113PRTArtificialSynthetic Peptide
21Thr Trp Ser Pro Val Gly His Lys Leu Ala Tyr Val Trp1 5
102213PRTArtificialSynthetic Peptide 22Leu Trp Trp Ser Pro Asn Gly
Thr Phe Leu Ala Tyr Ala1 5 102313PRTArtificialSynthetic Peptide
23Arg Ile Ser Leu Gln Trp Leu Arg Arg Ile Gln Asn Tyr1 5
102413PRTArtificialSynthetic Peptide 24Tyr Val Lys Gln Trp Arg His
Ser Tyr Thr Ala Ser Tyr1 5 102513PRTArtificialSynthetic Peptide
25Glu Glu Glu Val Phe Ser Ala Tyr Ser Ala Leu Trp Trp1 5
102613PRTArtificialSynthetic Peptide 26Asp Tyr Ser Ile Ser Pro Asp
Gly Gln Phe Ile Leu Leu1 5 102713PRTArtificialSynthetic Peptide
27Ser Ile Ser Pro Asp Gly Gln Phe Ile Leu Leu Glu Tyr1 5
102813PRTArtificialSynthetic Peptide 28Ile Tyr Val Lys Ile Glu Pro
Asn Leu Pro Ser Tyr Arg1 5 102915PRTArtificialSynthetic Peptide
29Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
153021DNAArtificialSynthetic RNA 30gaaagguguc aguacuauut t
213121DNAArtificialSynthetic RNA 31ttcuuuccac agucaugaua a
213221DNAArtificialSynthetic RNA 32uaauuagggu cggguaaact t
213321DNAArtificialSynthetic RNA 33ttauuaaucc cagcccauuu g 21
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References