U.S. patent application number 12/919902 was filed with the patent office on 2011-01-06 for selectin ligands useful in the diagnosis and treatment of cancer.
This patent application is currently assigned to John Hopkins University. Invention is credited to Konstantinos Konstantopoulos, Susan Napier Thomas.
Application Number | 20110003710 12/919902 |
Document ID | / |
Family ID | 41016750 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110003710 |
Kind Code |
A1 |
Konstantopoulos; Konstantinos ;
et al. |
January 6, 2011 |
SELECTIN LIGANDS USEFUL IN THE DIAGNOSIS AND TREATMENT OF
CANCER
Abstract
The present invention provides methods and compositions useful
in the diagnosis and treatment of cancer. More specifically, the
present invention provides compositions and methods of use
comprising a targeting composition comprising a solid substrate, an
antibody composition, and optionally a chemotherapeutic agent.
Inventors: |
Konstantopoulos; Konstantinos;
(Baltimore, MD) ; Thomas; Susan Napier;
(Baltimore, MD) |
Correspondence
Address: |
VENABLE LLP
P.O. BOX 34385
WASHINGTON
DC
20043-9998
US
|
Assignee: |
John Hopkins University
Baltimore
MD
|
Family ID: |
41016750 |
Appl. No.: |
12/919902 |
Filed: |
March 2, 2009 |
PCT Filed: |
March 2, 2009 |
PCT NO: |
PCT/US09/35724 |
371 Date: |
August 27, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61072945 |
Apr 3, 2008 |
|
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61067438 |
Feb 28, 2008 |
|
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Current U.S.
Class: |
506/9 ; 424/1.49;
424/178.1; 424/450; 424/9.1; 424/9.34; 424/9.6; 436/501; 506/18;
530/391.3; 530/391.7; 977/774; 977/915 |
Current CPC
Class: |
B82Y 5/00 20130101; A61K
47/6927 20170801; A61K 47/6849 20170801; A61K 47/6853 20170801;
A61K 51/1048 20130101; A61K 39/39558 20130101; C07K 16/18 20130101;
A61K 51/1027 20130101; A61P 35/00 20180101; A61K 39/3955 20130101;
A61K 39/3955 20130101; A61K 39/39558 20130101; C07K 16/3046
20130101; A61P 35/02 20180101; C07K 16/2896 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 51/1093 20130101; A61K
51/1063 20130101 |
Class at
Publication: |
506/9 ; 506/18;
530/391.3; 530/391.7; 424/178.1; 424/450; 424/1.49; 424/9.1;
424/9.6; 424/9.34; 436/501; 977/774; 977/915 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/10 20060101 C40B040/10; C07K 17/00 20060101
C07K017/00; A61K 39/44 20060101 A61K039/44; A61P 35/00 20060101
A61P035/00; A61P 35/02 20060101 A61P035/02; A61K 9/127 20060101
A61K009/127; A61K 51/10 20060101 A61K051/10; A61K 49/00 20060101
A61K049/00; A61K 49/06 20060101 A61K049/06; G01N 33/53 20060101
G01N033/53 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with U.S. government support under
grant no. R01 CA101135. The U.S. government has certain rights in
the invention.
Claims
1. A targeting composition comprising: a. a solid substrate; b. an
antibody composition bound to the substrate, comprising at least
one antibody to one or more E-selectin and L-selectin ligand
antigens, the antigens comprising podocalyxin-like protein (PCLP)
or two or more of PCLP, carcinoembryonic antigen (CEA), and CD44v,
the antibody composition binding specifically to metastatic tumor
cells having E-selectin and L-selectin binding activity, wherein
the substrate is a patterned array and/or the composition further
comprises a therapeutic or imaging agent bound to the substrate or
antibody.
2. The targeting composition of claim 1, wherein the substrate is a
particle suspendable in a biocompatible medium.
3. The targeting composition of claim 1, wherein the particle is
selected from the group consisting of a nanoparticle, a quantum
dot, a liposome, a micelle and a polymersome.
4. The targeting composition of claim 1, wherein the substrate is
an array.
5. The composition of claim 1, wherein the at least one antibody is
selected from the group consisting of a polyclonal antibody, a
monoclonal antibody, a recombinant antibody, a humanized antibody,
a single chain antibody, and a Fab fragment.
6. The composition of claim 1, wherein the antibody composition
comprises an antibody to CEA, an antibody to PCLP, and an antibody
to CD44v.
7. The composition of claim 1, wherein the antibody composition
comprises an antibody to PCLP and an antibody to CEA.
8. The composition of claim 1, wherein the antibody composition
comprises an antibody to PCLP and CD44v.
9. The composition of claim 1, wherein the antibody composition
comprises an antibody to CEA and CD44v.
10. The composition of claim 1, wherein each of the at least one
antibody is conjugated to the substrate via at least one linker
molecule.
11. The composition of claim 1, wherein the substrate is a
nanoparticle.
12. The composition of claim 1, wherein the substrate is a quantum
dot.
13. The composition of claim 11, wherein the nanoparticle is a gold
nanoparticle.
14. A method for treating cancer comprising administering to a
subject with cancer a therapeutically effective amount of a
composition comprising the targeting composition of claim 1,
wherein the substrate is a particle suspendable in biocompatible
medium, and further comprises at least one chemotherapeutic agent
conjugated to the suspendable particle.
15. The method of claim 14, wherein the cancer is a primary
tumor.
16. The method of claim 15, wherein the targeting composition
prevents migration of the cancer from the primary tumor site.
17. The method of claim 14, wherein the cancer is a metastatic
cancer.
18. The method of claim 14, wherein the targeting composition
prevents formation of a secondary tumor.
19. The method of claim 14, wherein the at least one antibody is
selected from the group consisting of a polyclonal antibody, a
monoclonal antibody, a recombinant antibody, a humanized antibody,
a single chain antibody, and a Fab fragment.
20. The method of claim 14, wherein the antibody composition
comprises an antibody to CEA, an antibody to PCLP, and an antibody
to CD44v.
21. The composition of claim 14, wherein the antibody composition
comprises an antibody to PCLP and an antibody to CEA.
22. The composition of claim 14, wherein the antibody composition
comprises an antibody to PCLP and CD44v.
23. The composition of claim 14, wherein the antibody composition
comprises an antibody to CEA and CD44v.
24. The method of claim 14, the particle is a nanoparticle, a
quantum dot, a liposome, a micelle and a polymersome.
25. The method of claim 14, wherein the at least one antibody is
conjugated to the particle via at least one linker molecule.
26. The method of claim 14, wherein the particle is a quantum
dot.
27. The method of claim 14, wherein the particle is a gold
nanoparticle.
28. The method of claim 14, wherein the at least one
chemotherapeutic agent is selected from the group consisting of
paclitaxel, docetaxel, daunorubicin, cisplatin, carboplatin,
oxaliplatin, colchicine, dolastatin 15, nocodazole podophyllotoxin,
rhizoxin, vinblastine, vindesine, vinorelbine (navelbine), the
epothilones, the mitomycins, bleomycin chlorambucil, cannustine,
melphalan, mitoxantrone 5-fluoro-5'-deoxyuridine, camptothecin,
topotecan, irinotecanetoposide, tenoposide, geldanamycin,
methotrexate, adriamycin, actinomycin D, mifepristone, raloxifene,
5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, tamoxifen,
4-hydroxytamoxifen apigenin, rapamycin, angiostatin K1-3,
staurosporine, genistein, fumagillin, endostatin, thalidomide,
analogs thereof and combinations thereof.
29. A method for imaging a cancer cell in a patient comprising: a.
administering to a subject a pharmaceutically acceptable
composition comprising the targeting composition of claim 1,
wherein the substrate is at least one particle suspendable in a
biocompatible medium, and further comprises at least one imaging
agent conjugated to the at least one particle; and b. detecting the
at least one imaging agent.
30. The method of claim 29, wherein the imaging agent is selected
from the group consisting of a radiologic contrast agent,
diatrizoic acid sodium salt dihydrate, an iodine-containing agent,
a barium-containing agent, a fluorescent imaging agent, Lissamine
Rhodamine PE, a stain, a dye, a radioisotope, a metal, a
ferromagnetic compound, a paramagnetic compound, gadolinium, a
superparamagnetic compound, iron oxide, a diamagnetic compound, and
barium sulfate.
31. The method of claim 29, wherein the at least one antibody is
selected from the group consisting of a polyclonal antibody, a
monoclonal antibody, a recombinant antibody, a humanized antibody,
a single chain antibody, and a Fab fragment.
32. The method of claim 29, wherein the antibody composition
comprises an antibody to CEA, an antibody to PCLP, and an antibody
to CD44v.
33. The composition of claim 29, wherein the antibody composition
comprises an antibody to PCLP and an antibody to CEA.
34. The composition of claim 29, wherein the antibody composition
comprises an antibody to PCLP and CD44v.
35. The composition of claim 29, wherein the antibody composition
comprises an antibody to CEA and CD44v.
36. The method of claim 29, the particle is a nanoparticle, a
quantum dot, a liposome, a micelle and a polymersome.
37. The method of claim 29, wherein the at least one antibody is
conjugated to the particle via at least one linker molecule.
38. The method of claim 29, wherein the particle is a quantum
dot.
39. The method of claim 29, wherein the article is a gold
nanoparticle.
40. The method of claim 29, wherein the detecting step comprises
using an imaging device.
41. An array comprising a fixed matrix and an antibody composition
bound to the fixed matrix in a predetermined pattern, comprising at
least one antibody to one or more E-selectin and L-selectin ligand
antigens, the antigens comprising at least one of podocalyxin-like
protein (PCLP), carcinoembryonic antigen (CEA), and CD44v, the
antibody composition binding specifically to metastatic tumor cells
having E-selectin and L-selectin binding activity.
42. A method for diagnosing metastatic cancer comprising obtaining
a biological sample from a patient and contacting the sample with
an array of claim 41, wherein the specific binding by the targeting
composition indicates the presence of metastatic cancer cells.
43. The method of claim 42, wherein the array is a microarray.
44. The method of claim 42, wherein the biological sample is
blood.
45. A method for preparing a targeting composition for metastatic
tumor cells comprising binding an antibody composition to a
substrate, the substrate being selected from a particle suspendable
in a biocompatible medium and an array, and the antibody
composition comprising at least one antibody to an E-selectin and
L-selectin ligand antigen.
46. The method of claim 42, wherein the antigen is selected from
the group consisting of CEA, PCLP and CD44v.
47. A composition comprising an antibody to PCLP, CEA, and/or CD44v
conjugated with an imaging agent.
48. The composition of claim 47, wherein the imaging agent is a
radioactive isotope.
49. The composition of claim 48, wherein the radioactive isotope is
Iodine.sup.131.
50. The composition of claim 47, wherein the antibody is selected
from the group consisting of a polyclonal antibody, a monoclonal
antibody, a recombinant antibody, a humanized antibody, a single
chain antibody, and a Fab fragment.
51. The composition of claim 47, wherein the antibody is to
PCLP.
52. A method for imaging a cancer cell in a patient comprising: a.
administering to a subject the composition of claim 47; and b.
detecting the at least one imaging agent.
53. The method of claim 52, wherein the imaging agent is selected
from the group consisting of a radiologic contrast agent,
diatrizoic acid sodium salt dihydrate, an iodine-containing agent,
a barium-containing agent, a fluorescent imaging agent, Lissamine
Rhodamine PE, a stain, a dye, a radioisotope, a metal, a
ferromagnetic compound, a paramagnetic compound, gadolinium, a
superparamagnetic compound, iron oxide, a diamagnetic compound, and
barium sulfate.
54. The method of claim 52, wherein the antibody is selected from
the group consisting of a polyclonal antibody, a monoclonal
antibody, a recombinant antibody, a humanized antibody, a single
chain antibody, and a Fab fragment.
55. The method of claim 52, wherein the detecting step comprises
using an imaging device.
56. A method of identifying a biomarker specific for metastatic
cancer cells comprising: a. selecting a putative glycopolypeptide
selectin ligand found in a lysate of carcinoma cells; b. detecting
selectin binding activity of the putative glycopolypeptide selectin
ligand in a blot rolling assay under shear flow conditions; c.
identifying the putative glycopolypeptide ligand, and d. measuring
selectin binding activity of the identified glycopolypeptide
selectin ligand in a cell-free flow-based adhesion study.
57. The method of claim 56, further comprising obtaining an
antibody specific to the glycopolypeptide selectin ligand, wherein
the antibody is specific to metastatic cells of the carcinoma.
58. The method of claim 56, wherein the carcinoma cells are from
colon, breast, prostate, squamous, neural blastoma, pancreatic, or
lung cancer.
59. The method of claim 56, wherein the identifying step comprises
immunoaffinity chromatography, sequencing of isolated polypeptide
fragments, and proteomics analysis.
60. The method of claim 56, further comprising preparing a
derivative of the identified glycopolypeptide selectin ligand and
retesting in a cell-free flow-based adhesion study.
61. The method of claim 60, wherein the derivative is a
glycoprotein, a peptide, or a peptidomimetic.
62. The method of claim 60, wherein the identified glycopolypeptide
selectin ligand is selected from PCLP, CEA, and CD44v.
63. The method of claim 57, wherein the antibody is a monoclonal
antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/067,438, filed Feb. 28, 2008, and U.S.
Provisional Application Ser. No. 61/072,945, filed Apr. 3, 2008,
each of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of cancer. More
specifically, the present invention relates to diagnosis and
treatment of cancer.
BACKGROUND OF THE INVENTION
[0004] Blood-borne metastasis is a highly orchestrated and dynamic
process initiated when cancerous cells disassociate from a primary
tumor and migrate across vessel walls into the circulation. During
their passage through the vascular system, tumor cells are exposed
to mechanical and immunological stresses, which affect their
ability to metastasize. Only tumor cells uniquely fit to overcome
or even exploit the detrimental effects of hemodynamic forces and
immunosurveillance will adhere to the vascular endothelium of
distant organs, extravasate and successfully colonize these
sites.
[0005] The adhesive interactions of circulating tumor cells (CTCs)
with host cells including platelets, leukocytes and endothelial
cells may regulate their extravasation from the vasculature.
Platelets, by forming heterotypic complexes with tumor cells, may
mask them from immune-mediated mechanisms of clearance. Palumbo et
al., 105 BLOOD 178-185 (2005); Borsig et al., 99 PROC. NATL. ACAD.
SCI. USA 2193-2198 (2002); Borsig et al., 98 PROC. NATL. ACAD. SCI.
USA 3352-3357 (2001); and Nieswandt et al., 59 CANCER RES.
1295-1300 (1999) Alternatively, platelets may potentiate tumor cell
adhesion to the vessel wall (Burdick et al., 287 J. PHYSIOL. CELL.
PHYSIOL. 539-547 (2004) and release an array of bioactive compounds
such as vascular endothelial growth factor at points of attachment
to endothelium, thereby promoting vascular hyperpermeability and
extravasation (Nash et al., 3 LANCET ONCOL. 425-430 (2002)).
Moreover, polymorphonuclear leukocytes (PMNs) facilitate tumor cell
extravasation in vitro (Slattery et al., 106 INT. J. CANCER 713-722
(2003); Starkey et al., 34 INT. J. CANCER 535-543 (1984)), and
promote the arrest and deposition of tumors in the microvasculature
of target organs in animal models (Starkey et al., 34 INT. J.
CANCER 535-543 (1984)). PMN-facilitated tumor cell extravasation
under dynamic flow conditions involves initial PMN tethering on the
endothelium and subsequent arrest of free-flowing tumor cells by
tethered PMNs. Lian et al., 295 AM. J. PHYSIOL. CELL PHSIOL.
C701-707 (2008).
[0006] Selectins may facilitate cancer metastasis and tumor cell
arrest in the microvasculature by mediating specific interactions
between selectin-expressing host cells and ligands on tumor cells.
The molecular and biochemical underpinnings of selectin-ligand
interactions involved in heterotypic tumor cell-host cell adhesion
events are not well understood. There is a need for new
therapeutics and diagnostics for metastatic cancer.
SUMMARY OF THE INVENTION
[0007] The invention provides therapeutics and diagnostics for
cancer metastasis using selectin ligands as biomarkers for the
tissue specific biochemistry of malignant versus normal tissue.
[0008] The invention provides therapeutics and diagnostics for
cancer metastasis using selectin ligands as biomarkers for the
tissue specific biochemistry of malignant versus normal tissue. In
one embodiment, the present invention provides a targeting
composition comprising (a) a solid substrate; and (b) an antibody
composition bound to the substrate, comprising at least one
antibody to one or more E-selectin and L-selectin ligand antigens,
the antigens comprising podocalyxin-like protein (PCLP) or two or
more of PCLP, carcinoembryonic antigen (CEA), and CD44v, the
antibody composition binding specifically to metastatic tumor cells
having E-selectin and L-selectin binding activity, wherein the
substrate is a patterned array and/or the composition further
comprises a therapeutic or imaging agent bound to the substrate or
antibody.
[0009] In a particular embodiment, the substrate may be a particle
suspendable in a biocompatible medium. The particle may be selected
from the group consisting of a nanoparticle, a quantum dot, a
liposome, a micelle and a polymersome. In a specific embodiment,
the substrate is a quantum dot. In another embodiment, the
nanoparticle is a gold nanoparticle. Alternatively, the substrate
may be an array.
[0010] In another embodiment, the at least one antibody is selected
from the group consisting of a polyclonal antibody, a monoclonal
antibody, a recombinant antibody, a humanized antibody, a single
chain antibody, and a Fab fragment. In a specific embodiment, the
antibody composition comprises an antibody to CEA, an antibody to
PCLP, and an antibody to CD44v. The antibody composition may
comprise an antibody to PCLP and an antibody to CEA. In another
embodiment, the antibody composition may comprise an antibody to
PCLP and CD44v. The antibody composition may also comprise an
antibody to CEA and CD44v. Furthermore, each of the at least one
antibody may be conjugated to the substrate via at least one linker
molecule.
[0011] In another aspect, the present invention provides a method
for treating cancer comprising administering to a subject with
cancer a therapeutically effective amount of a composition
comprising a targeting composition, wherein the substrate is a
particle suspendable in biocompatible medium, and further comprises
at least one chemotherapeutic agent conjugated to the suspendable
particle. In the method, the cancer may be a primary tumor. The
targeting composition may also prevent migration of the cancer from
the primary tumor site. In another embodiment, the cancer is a
metastatic cancer. In a further embodiment, the targeting
composition prevents formation of a secondary tumor.
[0012] The chemotherapeutic agent may be selected from the group
consisting of paclitaxel, docetaxel, daunorubicin, cisplatin,
carboplatin, oxaliplatin, colchicine, dolastatin 15, nocodazole
podophyllotoxin, rhizoxin, vinblastine, vindesine, vinorelbine
(navelbine), the epothilones, the mitomycins, bleomycin
chlorambucil, cannustine, melphalan, mitoxantrone
5-fluoro-5'-deoxyuridine, camptothecin, topotecan,
irinotecanetoposide, tenoposide, geldanamycin, methotrexate,
adriamycin, actinomycin D, mifepristone, raloxifene, 5-azacytidine,
5-aza-T-deoxycytidine, zebularine, tamoxifen, 4-hydroxytamoxifen
apigenin, rapamycin, angiostatin K1-3, staurosporine, genistein,
fumagillin, endostatin, thalidomide, analogs thereof and
combinations thereof.
[0013] The present invention also provides a method for imaging a
cancer cell in a patient comprising administering to a subject a
pharmaceutically acceptable composition comprising a targeting
composition, wherein the substrate is at least one particle
suspendable in a biocompatible medium, and further comprises at
least one imaging agent conjugated to the at least one particle;
and detecting the at least one imaging agent. In one embodiment,
the detecting step comprises using an imaging device. In a
particular embodiment, the imaging agent may be selected from the
group consisting of a radiologic contrast agent, diatrizoic acid
sodium salt dihydrate, an iodine-containing agent, a
barium-containing agent, a fluorescent imaging agent, Lissamine
Rhodamine PE, a stain, a dye, a radioisotope, a metal, a
ferromagnetic compound, a paramagnetic compound, gadolinium, a
superparamagnetic compound, iron oxide, a diamagnetic compound, and
barium sulfate.
[0014] In the method, the at least one antibody may be selected
from the group consisting of a polyclonal antibody, a monoclonal
antibody, a recombinant antibody, a humanized antibody, a single
chain antibody, and a Fab fragment. More particularly, the antibody
composition comprises an antibody to CEA, an antibody to PCLP, and
an antibody to CD44v. In a specific embodiment, the antibody
composition comprises an antibody to PCLP and an antibody to CEA.
The antibody composition may also comprise an antibody to PCLP and
CD44v. In an alternative embodiment, the antibody composition
comprises an antibody to CEA and CD44v. Furthermore, the at least
one antibody may be conjugated to the particle via at least one
linker molecule.
[0015] With regard to the methods, the particle may be a
nanoparticle, a quantum dot, a liposome, a micelle and a
polymersome. In one embodiment, the particle is a quantum dot. In
another embodiment, the particle is a gold nanoparticle.
[0016] The present invention further provides an array comprising a
fixed matrix and an antibody composition bound to the fixed matrix
in a predetermined pattern, comprising at least one antibody to one
or more E-selectin and L-selectin ligand antigens, the antigens
comprising podocalyxin-like protein (PCLP) or two or more of PCLP,
carcinoembryonic antigen (CEA), and CD44v, the antibody composition
binding specifically to metastatic tumor cells having E-selectin
and L-selectin binding activity.
[0017] In one embodiment, the present invention provides a method
for diagnosing metastatic cancer comprising obtaining a biological
sample from a patient and contacting the sample with an array,
wherein the specific binding by the targeting composition indicates
the presence of metastatic cancer cells. In a specific embodiment,
the array is a microarray. In another embodiment, the biological
sample is blood.
[0018] The present invention further provides a method for
preparing a targeting composition for metastatic tumor cells
comprising binding an antibody composition to a substrate, the
substrate being selected from a particle suspendable in a
biocompatible medium and an array, and the antibody composition
comprising at least one antibody to an E-selectin and L-selectin
ligand antigen. The antigen may be selected from the group
consisting of CEA, PCLP and CD44v.
[0019] The present invention also provides a composition comprising
an antibody to PCLP, CEA, and/or CD44v conjugated with an imaging
agent. The imaging agent may be a radioactive isotope. In a
specific embodiment, the radioactive isotope is Iodine.sup.131. The
antibody may be selected from the group consisting of a polyclonal
antibody, a monoclonal antibody, a recombinant antibody, a
humanized antibody, a single chain antibody, and a Fab fragment. IN
a particular embodiment, the antibody is to PCLP.
[0020] In yet another embodiment, a method for imaging a cancer
cell in a patient comprises administering to a subject a
composition; and detecting the at least one imaging agent. The
imaging agent may be selected from the group consisting of a
radiologic contrast agent, diatrizoic acid sodium salt dihydrate,
an iodine-containing agent, a barium-containing agent, a
fluorescent imaging agent, Lissamine Rhodamine PE, a stain, a dye,
a radioisotope, a metal, a ferromagnetic compound, a paramagnetic
compound, gadolinium, a superparamagnetic compound, iron oxide, a
diamagnetic compound, and barium sulfate.
[0021] Moreover, the antibody may be selected from the group
consisting of a polyclonal antibody, a monoclonal antibody, a
recombinant antibody, a humanized antibody, a single chain
antibody, and a Fab fragment. In another embodiment, the detecting
step comprises using an imaging device.
[0022] The present invention also provides a method of identifying
a biomarker specific for metastatic cancer cells comprising
selecting a putative glycopolypeptide selectin ligand found in a
lysate of carcinoma cells; detecting selectin binding activity of
the putative ligand in a blot rolling assay under shear flow
conditions; identifying the putative glycopolypeptide selectin
ligand, and measuring selectin binding activity of the identified
glycopolypeptide selectin ligand in a cell-free flow-based adhesion
study. The method may further comprise the step of obtaining an
antibody specific to the glycopolypeptide selectin ligand, wherein
the antibody is specific to metastatic cells of the carcinoma.
[0023] The carcinoma cells may be from colon, breast, prostate,
squamous, neural blastoma, pancreatic, or lung cancer. In another
embodiment, the identifying step comprises immunoaffinity
chromatography, sequencing of isolated polypeptide fragments, and
proteomics analysis.
[0024] The method may further comprise the step of preparing a
derivative of the identified glycopolypeptide selectin ligand and
retesting in a cell-free flow-based adhesion study. The derivative
may be a glycoprotein, a peptide, or a peptidomimetic. In a
particular embodiment, the identified glycopolypeptide selectin
ligand is selected from PCLP, CEA, and CD44v. In another
embodiment, the antibody is a monoclonal antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 is a schematic diagram outlining a methodology used
to isolate, identify and verify carcinoma-expressed selectin
ligands. A combination of biochemistry, flow-based adhesion assays
and tandem mass spectrometry in conjunction with bioinformatics
techniques was used to identify and subsequently verify PCLP,
CD66e, and CD44v to be carcinoma-expressed selectin ligands. Solid
lines denote chronological order of methods used to purify and
identify targets. Dotted lines denote chronological order of
methods used to verify targets as selectin ligands.
[0026] FIG. 2 is a schematic diagram of colon carcinoma
cell-expressed selectin ligands PCLP, CD66e, and CD44v.
Diamond-enclosed Os represent putative sites for O-linked
glycosylation. Circled Ns represent putative sites for N-linked
glycosylation, SS represents disulfide bonds, P represents serine
phophatases, PKC represent Protein Kinase C site, CK2 represents
Casein Kinase 2 site. CD663 is GPI-linked to the cell membrane
whereas CD44v and PCLP are type 1 transmembrane glycoproteins.
Membrane proximal stem domain of CD44v and mucin-like domain of
PCLP do not depict all putative sites for posttranslational
modifications in this diagram.
[0027] FIG. 3 provides Western blot results from PCLP experiments.
Panel A: Western blots of HECA-452 immunoaffinity product from
CD44-knockdown LS174T colon carcinoma cell lysate stained with the
anti-PCLP (3D3) (lane 1) or HECA-452 (lane 2) mAbs. Western blots
reveal that PCLP is a 180-kDa molecule recovered after HECA-452
immunoaffinity chromatography of CD44-knockdown LS174T whole cell
lysate. Panel B: Western blots of whole cell lysate from wild-type
(lane 1) and CD44-knockdown (lane 2) LS174T colon carcinoma cells
stained with the anti-PCLP mAB 3D3. Panel C: Western blots of
immunoprecipitated PCLP from CD44-knockdown LS174T whole cell
lysate stained with the anti-PCLP (3D3) (lane 1) or HECA-452 (lane
2) mAbs demonstrating that PCLP is a 180 kDa sialofucosylated
glycoprotein.
[0028] FIG. 4 presents results from flow-based adhesion assays
using PCLP. Panels A and B: Representative flow cytometric
histograms of (A) PCLP and (B) HECA-452 expression on microbeads
coated with PCLP immunopurified from wild-type (black line) and
CD44-knockdown (gray line) LS174T cells. Microspheres were stained
with the FITC-conjugated anti-PCLP mAb 53D11 (A), FITC-conjugated
HECA-452 mAb (B), or FITC-conjugated isotype control antibody
(dashed lines). Panel C: Extent of adhesion of microbeads
(10.sup.6/ml) coated with PCLP immunopurified from wild-type or
CD44-knockdown LS174T cells over 10 .mu.g/ml E-(black bars), L-
(white bars), or P-selectin at a wall shear stress level of 1
dyn/cm.sup.2 for 2 min. Data represent the mean.+-.SEM. *p<0.05
with respect to microbeads coated with PCLP immunopurified from
wild-type LS174T cells. Panel D and E: Average rolling velocities
of microspheres (10.sup.6/ml) coated with PCLP immunopurified from
wild-type or CD44-knockdown LS174T cells over 10 .mu.g/ml (D)
E-selectin or (E) L-selectin over a range of shear stresses. Data
represent the mean.+-.SEM. *p<0.05 with respect to microbeads
coated with PCLP immunopurified from wild-type LS174T cells.
[0029] FIG. 5 shows results from PCLP-coated microsphere and flow
cytometry experiments. Panels A and B: Site densities of (A) PCLP
and (B) sLe.sup.x on wild-type LS174T PCLP-bearing polystyrene
microbeads, pretreated with enzymes or metabolic inhibitors,
determined by flow cytometry. Wild-type LS174T PCLP-absorbed
microspheres were treated with Vibrio cholerae sialidase (0.1 U/ml)
or OSGE (120 .mu.g/ml). In other experiments, microbeads were
coated with PCLP immunoprecipitated from wild-type LS174T whole
cell lysate pretreated with DMJ or benzyl-GalNAc. Site densities of
adsorbed PCLP were quantified using the FITC-conjugated anti-PCLP
mAb 53D11. Site densities of HECA-452-reactive epitopes on
untreated (control) and treated wild-type LS174T PCLP-coated
microbeads were determined by flow cytometry using a HECA-452 mAb.
Panels C and D: Extent of adhesion of wild-type LS174T PCLP-coated
polystyrene microbeads pretreated with sialidase or microbeads
coated with PCLP immunopurified from the whole cell lysate of
wild-type LS174T cells cultured in benzyl-Ga1NAc or DMJ-containing
media to 10 .mu.g/ml (C) E-selectin or (D) L-selectin at a wall
shear stress level of 1 dyn/cm.sup.2 for 2 min. Data represent the
mean.+-.SEM. *p<0.05 by ANOVA.
[0030] FIG. 6 provides the Western blot results from CD66e
experiments. Panel A: Western blots of whole cell lysate or
immunopurified HECA-452-reactive epitopes or immunopurified CD66e
from CD44-knockdown LS174T colon carcinoma cells. Anti-CD66de
(Col-1) (lanes 1, 3 and 5) or HECA-452 (lanes 2, 4 and 6) mAbs were
used to stain Western blots of CD44-knockdown LS174T whole cell
lysate (lanes 1 and 2), immunoprecipitated HECA-452-reactive
epitopes (lanes 3 and 4), and immunoprecipitated CD66e (lanes 5 and
6) from CD44-knockdown LS174T cells. Panel B: Selectin-dependent
adhesion to SDS-PAGE resolved and blotted CEA immunoprecipitated
from CD44-knockdown LS174T whole cell lysate. CHO-E cells,
lymphocytes or CHO-P cells were perfused at the wall shear stress
level of 0.5 dyn/cm.sup.2 over SDS-PAGE immunoblots of
immunopurified CD66e from whole cell lysate of CD44-knockdown
LS174T cells. In select experiments, CHO-E cells and lymphocytes
were pre-treated with an anti-E-selectin or an anti-L-selectin
function blocking mAb (20 .mu.g/ml), respectively, before use in
blot rolling assays. The saturating concentration of the mAb (20
.mu.g/ml) was maintained in the perfusion assays. Data represent
the mean.+-.S.E. of n>3 experiments.
[0031] FIG. 7 presents results from flow-based adhesions assays
using CD66e. Panel A: Representative flow cytometric histograms of
CEACAM expression by wildtype LS174T cells. CEACAM expression on
colon carcinoma cells was investigated by using primary anti-CD66
mAbs (CD66a, GM8G5; CD66b, 80H3; CD66c, 9A6; CD66de, Col-1; CD66f,
BAP3; CEACAM7, BAC2) in conjunction with appropriate PE-conjugated
secondary and isotype control antibodies. Panel B: Western blots of
whole cell lysate or immunoprecipitated CEA from wildtype LS174T
colon carcinoma cells. Anti-CD66de (Col-1) (lanes 1 and 3) or
HECA-452 (lanes 2 and 4) mAbs were used to stain Western blots of
whole cell lysate (lanes 1 and 2) and immunoprecipitated CEA (lanes
3 and 4) from wildtype LS174T colon carcinoma cells. Panel C:
Western blots of whole cell lysate or immunoprecipitated CD66c from
wildtype LS174T colon carcinoma cells. Anti-CD66c (B6.2) (lanes 1
and 3) or HECA-452 (lanes 2 and 4) mAbs were used to stain Western
blots of whole cell lysate (lanes 1 and 2) and immunoprecipitated
CD66c (lanes 3 and 4) from wildtype LS174T cells.
[0032] FIG. 8 show results from CD66e and blot-rolling assays.
Panels A and B: Site densities of CD66e (A) and HECA-452-reactive
epitopes (B) on polystyrene microspheres coated with CD66e
immunopurified from either wildtype (bold line) or CD44-knockdown
(thin line) LS174T cells. Microspheres were stained with
PE-conjugated anti-CD66 B1.1 (A), FITC-conjugated HECA-452 (B), or
PE- or FITC-conjugated isotype control antibodies (dashed lines).
Panel C: Extent of adhesion of microspheres (10.sup.6/ml) coated
with CD66e immunopurified from wildtype (black bars) or
CD44-knockdown (white bars) LS174T colon carcinoma cells to 10
.mu.g/ml E-, L-, or P-selectin at a wall shear stress level of 1
dyn/cm.sup.2 for 2 min. Data represent the mean.+-.S.E. *p<0.05
with respect to microspheres coated with CD66e immunopurified from
wildtype LS174T cells. ND: not done. Average rolling velocities of
microspheres (10.sup.6/ml) coated with CD66e immunopurified from
wildtype or CD44-knockdown LS174T cells on 10 .mu.g/ml E-selectin
(Panel D) or L-selectin (Panel E) at prescribed wall shear
stresses. Data represent the mean.+-.S.E. *p<0.05 with respect
to microspheres coated with CEA immunopurified from wildtype LS174T
cells.
[0033] FIG. 9 provides results from CD66e-coated microsphere and
flow cytometry experiments. Panel A: Site densities of wildtype
LS174T CD66e-coated polystyrene microspheres, pretreated with
enzymes or metabolic inhibitors, determined by flow cytometry.
Wildtype LS174T CD66e-absorbed microspheres were treated with
Vibrio cholerae sialidase (0.1 U/ml). Alternatively, CD66e
immunoprecipitated from N-glycosidase F-treated wildtype LS174T
whole cell lysate was used to coat microspheres. In other
experiments, microspheres were coated with CD66e immunoprecipitated
from wildtype LS174T whole cell lysate pretreated with DMJ or
benzyl-GalNAc. Site densities of adsorbed CD66e were quantified
using a PE-conjugated anti-CD66 mAb (B1.1) after treatments. Panel
B: Site densities of HECA-452-reactive epitopes on treated wildtype
LS174T CD66e-coated microspheres determined by flow cytometry.
Panel C: Extent of adhesion of wildtype LS174T CD66e-coated
polystyrene microspheres, pretreated with highly specific enzymes
and perfused over 10 .mu.g/ml E- (white bars) or L-selectin (black
bars) at a wall shear stress level of 1 dyn/cm2 for 2 min. Data
represent the mean.+-.S.E. *p<0.05 with respect to untreated
control microspheres. Panel D: Extent of adhesion of CD66e-coated
microspheres generated using CD66e immunoprecipitated from wildtype
LS174T cells cultured in the presence and absence (control) of
metabolic inhibitors, and perfused over 10 .mu.g/ml E- (white bars)
or L-selectin (black bars) at a wall shear stress level of 1
dyn/cm2 for 2 min. Data represent the mean S.E. *p<0.05 with
respect to control microspheres.
[0034] FIG. 10 provides results from additional CD66e, flow
cytometry, and flow-based adhesion assays. Panel A: Representative
flow cytometric histograms of CD66, CD44 and CD29 expression by
wildtype, CD44-knockdown, CD66e-knockdown, and CD66e/CD44-double
knockdown LS174T cells. Cells were stained by indirect single-color
immunofluorescence using the anti-CD66de mAb Col-1 (solid line) or
an isotype control antibody (dashed line). Alternatively, cells
were stained with the PE-conjugated anti-CD44 mAb 515 (solid line)
or PE-conjugated isotype control antibody (dashed line). In other
experiments, cells were stained with the PE-conjugated anti-CD29
mAb MAR4 (solid line) or PE-conjugated isotype control antibody
(dashed line). Panel B: Extent of adhesion of wildtype,
CD44-knockdown, CD66e-knockdown, and two distinct CD66e/CD44-double
knockdown LS174T cell lines (10.sup.6/ml) to E-selectin (0.75
.mu.g/ml) under physiological flow conditions. The average number
of wildtype LS174T cells per mm.sup.2 that tethered and rolled on
E-selectin at 1.0 and 2.0 dyn/cm.sup.2 was 380.+-.60 and 290.+-.30,
respectively. Data represent the mean.+-.S.E. of n=3 experiments.
Black bars represent data acquired at the wall shear stress level
of 1.0 dyn/cm.sup.2, whereas white bars represent data at 2.0
dyn/cm.sup.2. *p<0.05 with respect to wildtype, CD44-knockdown
and CD66e-knockdown LS174T cells. Panel C: Extent of adhesion of
wildtype, CD44-knockdown, CD66e-knockdown, and CD66e/CD44-double
knockdown LS174T cells (10.sup.6/ml) to L-selectin (1.5 .mu.g/ml)
under physiological flow conditions. The average number of wildtype
LS174T cells per mm.sup.2 that tethered and rolled on L-selectin at
1.0 and 2.0 dyn/cm.sup.2 was 700.+-.100 and 600.+-.100,
respectively. Data are normalized with respect to wildtype LS174T
cells, and represent the mean.+-.S.E. of n=3-4 experiments. Black
bars represent data acquired at the wall shear stress level of 1.0
dyn/cm.sup.2, whereas white bars represent data at 2.0
dyn/cm.sup.2. *p<0.05 with respect to wildtype, CD44-knockdown
and CD66e-knockdown LS174T cells. Panel D: Extent of adhesion of
wildtype, CD44-knockdown, CD66e-knockdown, and CD66e/CD44-double
knockdown LS174T cells (10.sup.6/ml) to P-selectin (1.5 .mu.g/ml)
under physiological flow conditions. The average number of wildtype
LS174T cells per mm.sup.2 that tethered and rolled on P-selectin at
1.0 and 2.0 dyn/cm.sup.2 was 1200.+-.200 and 610.+-.90,
respectively. Data are normalized with respect to wildtype LS174T
cells, and represent the mean.+-.S.E. of n=3 experiments. Black
bars represent data acquired at the wall shear stress level of 1.0
dyn/cm.sup.2, whereas white bars represent data at 2.0
dyn/cm.sup.2. *p<0.05 with respect to wildtype and CEA-knockdown
LS174T cells.
[0035] FIG. 11 is a schematic illustration showing the creation of
multifunctional surfaces by serial use of micro contact printing
(.mu.CP) (left panel) and microfluidics (right panel). A PDMS stamp
(a1, a2) inked with an active species that promotes adsorption (b)
is contacted with the substrate (c), rinsed (d), and backfilled
with a passive species that prevents adsorption (e, f). The
substrate is affixed as a lid to a microfluidics (MF) network with
n channels (g) through which adsorbing species are introduced. (h)
shows MF network of two channels. Images of the indicated areas of
the actual MF network with flow directions within the network are
shown in (1) and (2). By this method, surfaces presenting n species
on distinct patches surrounded by continuous passivated regions are
created. The pictures are not drawn to scale.
[0036] FIG. 12 shows results of an experiment described herein and
in particular, FIG. 3. Multifunctional surfaces with discrete
functionalized regions were created to capture normal versus
malignant cells from mixed cell suspensions. Sorting of a mixture
of CMFDA-labeled LS174T colon carcinoma cells (green; also referred
to by indicator number 20) and SNARF-stained polymorphonuclear
leukocytes (PMNs) (red; also referred to by indicator number 10) on
anti-PSGL1 and anti-CEA mAb micropatterned glass slides surrounded
by PEG-functionalized regions. (a) The cell mixture was incubated
for 30 min on the slide. (b) The cell mixture was perfused over the
micropatterned glass slide through a parallel plate flow chamber
assay at 0.35 dyn/cm.sup.2. For the multicolored images (not
shown), two separate gray scale images of the fluorophores are
taken, color labeled, and merged with Image J. The scale bars
represent 100 .mu.m. LS174T colon carcinoma cells 20 were
effectively sorted from PMNs 10 into prescribed patches under both
static and physiological flow conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0037] It is understood that the present invention is not limited
to the particular methods and components, etc., described herein,
as these may vary. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention. It must be noted that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
the plural reference unless the context clearly dictates otherwise.
Thus, for example, a reference to a "protein" is a reference to one
or more proteins, and includes equivalents thereof known to those
skilled in the art and so forth.
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Specific
methods, devices, and materials are described, although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention.
[0039] All publications cited herein are hereby incorporated by
reference including all journal articles, books, manuals, published
patent applications, and issued patents. In addition, the meaning
of certain terms and phrases employed in the specification,
examples, and appended claims are provided. The definitions are not
meant to be limiting in nature and serve to provide a clearer
understanding of certain aspects of the present invention.
Selectin Ligands
[0040] The targeting compositions and related methods of the
present invention are useful in diagnosing, detecting, imaging,
treating, and/or preventing cancer. In particular, the targeting
compositions are designed to bind to ligands expressed on cancer
cells, which ligands typically bind selectins expressed on
epithelial cells.
[0041] Furthermore, the inventive compositions and methods involve
targeting antibodies to antigens that are specific to metastatic
tumor cells, for example sialomucin markers for metastatic
carcinoma such as sialofucosylated glycoprotein antigens that
include a selectin binding determinant. A cell-free flow-based
adhesion assay as described herein has been used to identify three
such antigens (PCLP, CEA, and C44v) and may be used to identify
peptides, peptidomimetics, or other suitable variants of these
proteins, or other proteins or variants.
[0042] In one embodiment, the targeting composition comprises at
least one antibody to one or more E-selectin and L-selectin ligand
antigens. In a specific embodiment, the selectin ligand comprises
podocalyxin-like protein (PCLP). PCLP is a type I transmembrane
sialoprotein and a member of the CD34 subfamily of sialomucins,
which includes CD34 and endoglycan with whom PCLP shares a
considerable degree of structural and functional homology. See
Furnessand et al., 34 IMMUNOL. RES. 13-32 (2006); Takeda et al., 11
MOL. BIOL. CELL 3219-3232 (2000). Structural similarities include
an N-terminus-proximal mucin-like domain with numerous sites for
putative N- and O-linked glycosylation, four cysteines available
for disulfide formation in the globular stalk domain, and a
one-pass transmembrane domain. See FIG. 2; Kershaw et al., 272 J.
BIOL. CHEM. 15708-15714 (1997). At the cytoplasmic tail, PCLP
possesses consensus phosphorylation sites as well as a C-terminal
PDZ docking motif. Furnessand et al., 34 IMMUNOL. RES. 13-32
(2006). The human PCLP gene (PODXL) is located at chromosome
7q32-q33. Kershaw et al., 272 J. BIOL. CHEM. 15708-15714
(1997).
[0043] PCLP was first described as a kidney podocyte
surface-expressed molecule, which maintains the integrity of the
filtration slits in the foot processes in the kidney glomerulus via
its anti-adhesive properties due to a high net negative charge.
Furnessand et al., 34 IMMUNOL. RES. 13-32 (2006). Mice lacking
podocalyxin die soon after birth with defective kidney development
and anuria. Doyonnas et al., 194 J. EXP. MED. 13-27 (2001). PCLP is
also expressed in other cell types including early hematopoietic
precursors, developing but not mature hematopoietic stem cells, and
in high endothelial venules (HEVs). Furnessand et al., 34 IMMUNOL.
RES. 13-32 (2006). As opposed to its anti-adhesive function in
kidney podocytes, HEV-expressed PCLP mediates L-selectin-dependent
lymphocyte recruitment to secondary lymphoid tissues. Sassetti et
al., 187 J. EXP. MED. 1965-1975 (1998). The nucleotide and amino
acid sequences encoding PCLP are known in the art and are easily
retrievable by known methods. The database accession number for
PCLP variant1 is NM.sub.--001018111.2 (SEQ ID NO:1), and PCLP
variant2 is NM.sub.--005397.3 (SEQ ID NO:2), the entire sequences
of which are incorporated herein by reference.
[0044] In another embodiment, the selectin ligand comprises
carcinoembryonic antigen (CEA). The CEA gene family is tandemly
arranged on chromosome 19q13.2 and is divided into major
subfamilies, including CEA cell adhesion molecule (CEACAM) and
pregnancy-specific glycoprotein, based on corresponding domain
sequence homologies. Beauchemin et al., 252 EXP. CELL RES. 243-249
(1999); and Obrink, 9 CURR. OPIN. CELL. BIOL. 616-626 (1997). As
immunoglobulin (Ig) family members, every CEACAM molecule contains
an N-terminal Ig variable-like domain, which lacks an intra-chain
disulfide link followed by a variable number (zero-six) of Ig
C2-type constant-like domains. Hammarstrom, 9 SEMIN. CANCER BIOL.
67-81 (1999). The largest member of the CEACAM family, CEA (used
interchangeably with CEACAM5, CD66, and CD66e), is a glycosyl
phosphatidyl inositol (GPI)-cell surface anchored glycoprotein that
possesses six Ig C2-type domains. See FIG. 2. Sequence analysis has
revealed multiple sites for putative N-glycosylation by
multi-antennary complex type carbohydrate chains, which may
constitute up to 50% of its total mass. Hammarstrom, 9 SEMIN.
CANCER BIOL. 67-81 (1999); Paxton et al., 84 PROC. NATL. ACAD. SCI.
USA 920-924 (1987).
[0045] The nucleotide and amino acid sequences encoding CEA are
known in the art and are easily retrievable by known methods. The
database accession number for CEA is NM.sub.--004363.2 (SEQ ID
NO:3), the entire sequence of which is incorporated herein by
reference.
[0046] In yet another embodiment, the selectin ligand comprises
CD44v. CD44 proteins comprise a group of type I transmembrane
molecules encoded by a single gene, which consists of at least 20
exons. Exons 1-5, 16-18 and 20 are spliced together to form the
smallest CD44 transcript known as standard isoform (CD44s). At
least ten exons (6-15; typically identified as v1-v10) encoding a
membrane proximal portion of the extracellular domain of CD44 can
be alternatively spliced and inserted at a single site between
exons 5 and 16 to generate CD44v. See FIG. 2; and Ponta et al., 4
NAT. REV. MOL. CELL. BIOL. 33-45 (2003). See also Hanley et al., 20
FASEB J. 337-339 (2006). The CD44 protein family can function as:
(i) a ligand binding receptor for various components of the
extracellular matrix such as hyaluronan (HA), collagen, laminin and
fibronectin; (ii) a platform for growth factors and other molecules
such as metalloproteinases; (iii) a co-receptor which mediates the
signaling of receptor tyrosine kinases; and (iv) an organizer of
the cortical actin cytoskeleton. Id.
[0047] The nucleotide and amino acid sequences encoding CD44 are
known in the art and are easily retrievable by known methods. The
database accession number for CD44v is NM.sub.--000610 (SEQ ID
NO:4), the entire sequence of which is incorporated herein by
reference.
[0048] Variants of the selectin ligands including PCLP, CEA, and
CD44v may be used to generate other antibodies with increased or
alternative functionality compared to commercially available
antibodies. In particular, the nucleotide and amino acid sequences
of the ligands may be analyzed using known methods to identify
particular and potential binding sites for antibodies. In addition,
comparative analysis of the ligands expressed on normal versus
cancer cells will result in the identification of particular
conformational differences (including glycosylation patterns) that
may be used to generate antibodies that will recognize ligands
expressed only on cancer cells, thereby reducing or eliminating any
cross-reactivity.
[0049] When referring to a ligand or a ligand expressed on the
surface of a cell including a metastatic tumor cell, the phrase
"E-selectin and L-selectin binding activity", means that the ligand
has a higher affinity for E-selectin and L-selectin than for other
molecular structures typically found on cell surfaces. In a more
narrow sense, E-selectin and L-selectin binding activity or binding
selectivity refers to those ligands which have a higher affinity
for E-selectin and L-selectin than for other cell adhesion
molecules which are related to E-selectin and L-selectin, such as
P-selectin.
[0050] "E-selectin and L-selectin ligand antigens" refers to a
ligand that specifically binds to E-selectin and L-selectin and
includes, for example, CEA, PCLP, and CD44v. The term also includes
other sialofucosylated glycoproteins that specifically bind
E-selectin and L-selectin. The term further includes all
derivatives, analogs, peptidomimetics, and fragments of the
foregoing that exhibit E-selectin and L-selectin binding
activity.
[0051] Table 1 below summarizes the biochemical differences in the
recently identified selectin ligands expressed by normal versus
malignant cells
TABLE-US-00001 TABLE 1 Biochemical and known selectin binding
characteristics of normal cell- versus malignant cell-expressed
selectin ligands. ##STR00001## White columns represent selectin
ligands expressed in normal (host) cells, gray shaded columns
represent selectin ligands expressed by malignant cells. LS174T,
T84 and Colo 205: colon carcinoma cell lines; KS: breast carcinoma
cell line; NCI-H128 and SW2: small cell lung cancer cell line;
SK-N-SH: neuroblastoma cell line; HL-60: myeloid cell line; KG1a:
hematopoetic progenitor cell line (HPC). *Sialidase-treated LS174T
PCLP retained <20% tethering to E-selectin.
.dagger.Sialidase-treated LS174T CD44 retained >60% tethering to
E-selectin compared with the control.
.dagger-dbl.Selectin-dependent adhesion was shown to be
sulfation-independent by chlorate treatment. ND: no data.
Antibodies to Selectin Ligands
[0052] The present invention contemplates the use of antibodies
specific for selectin ligands including PCLP, CEA, and CD44v. The
phrases "binding specificity," "binding specifically to, "specific
binding" or otherwise any reference to an antibody to PCLP, CEA,
and/or CD44v, refers to a binding reaction that is determinative of
the presence of the corresponding antigen to the antibody in a
heterogeneous population of antigens and other biologics. Thus, the
specified antibodies bind selectively to an antigen present on a
target cell and do not substantially bind in a statistically
significant amount to other non-target cells. The parameters
required to achieve such specificity can be determined routinely,
using conventional methods in the art including, but not limited
to, competitive binding studies. For example, an antibody of the
invention may bind at least about 25% to 100 fold, or more, as
efficiently to an antigen expressed on a target cell than it binds
to other antigens expressed on non-target cells in a sample.
[0053] "Immunoglobulins" (Igs) and "antibodies" are glycoproteins
having the same structural characteristics. These terms are used
interchangeably herein.
[0054] Native antibodies are usually heterotetrameric glycoproteins
of about 150,000 daltons, composed of two identical light (L)
chains and two identical heavy (H) chains. Each light chain is
linked to a heavy chain by one covalent disulfide bond, while the
number of disulfide linkages varies among the heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also
has regularly spaced intrachain disulfide bridges. Each heavy chain
has at one end a variable domain (VH) followed by a number of
constant domains. Each light chain has a variable domain at one end
(VL) and a constant domain at its other end. The constant domain of
the light chain is aligned with the first constant domain of the
heavy chain, and the light-chain variable domain is aligned with
the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light chain
and heavy chain variable domains.
[0055] The terms "antibody" and "immunoglobulin" cover fully
assembled antibodies and antibody fragments that can bind antigen
(e.g., Fab', F'(ab).sup.2, Fv, single chain antibodies, diabodies),
including recombinant antibodies and antibody fragments. In certain
embodiment, the immunoglobulins or antibodies are chimeric, human,
or humanized.
[0056] The variable domains of the heavy and light chain recognize
or bind to a particular epitope of a cognate antigen. The term
"epitope" is used to refer to the specific binding sites or
antigenic determinant on an antigen that the variable end of the
immunoglobulin binds. Epitopes can be linear, i.e., be composed of
a sequence of amino acid residues found in the primary selectin
ligand sequence. Epitopes also can be conformational, such that an
immunoglobulin recognizes a 3-D structure found on a folded
selectin ligand as expressed on the surface of a cancer cell, such
that the amino acids recognized are not necessarily contiguous in
the primary sequence. Epitopes can also be a combination of linear
and conformational elements. Further, carbohydrate portions of a
molecule, as expressed by the target bearing tumor cells can also
be epitopes.
[0057] Antibodies are said to be "specifically binding" if: 1) they
exhibit a threshold level of binding activity, and/or 2) they do
not significantly cross-react with known related polypeptide
molecules. The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, ANN. NY ACAD. SCI. 51: 660-672,
1949). In some embodiments, the immunoglobulins of the present
invention bind to a selectin ligand, including PCLP, CEA, and CD44v
at least about 5, at least about 10, at least about 100, at least
about 10.sup.3, at least about 10.sup.4, at least 10.sup.5, and at
least 10.sup.6 fold higher than to other proteins.
[0058] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a P-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the P-sheet structure. The hypervariable
regions in each chain are held together in close proximity by the
FRs and, with the hypervariable regions from the other chain,
contribute to the formation of the antigen-binding site of
immunoglobulins. Kabat et al., SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST (1991). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody
in antibody dependent cellular cytotoxicity (ADCC).
[0059] The term "hypervariable region" refers to the amino acid
residues of an immunoglobulin that are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR"
and/or those residues from a "hypervariable loop." "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined. An antibody "which
binds" an antigen of interest, e.g., PCLP, CEA, or CD44v, is one
capable of binding that antigen with sufficient affinity and/or
avidity such that the antibody is useful as a diagnostic or
therapeutic agent for targeting a cell expressing the antigen.
[0060] The present invention contemplates the use of selectin
ligand antibodies or functional equivalents thereof, in the
treatment and prevention of cancer. In one embodiment, the selectin
ligand antibodies or functional equivalents thereof specifically
bind to the extracellular domain of PCLP (amino acids 23-431 of the
528 amino acid protein), CEA (amino acids 35-702 of the 702 amino
acid GPI-linked protein), and/or CD44v (amino acids 21-649 of the
742 amino acid protein). In a specific embodiment, an antibody or
functional equivalent thereof specifically binds an epitope of the
extracellular domain of PCLP, CEA, or CD44v as expressed in a
cancer cell. In a more specific embodiment, an antibody or
functional equivalent thereof specifically binds a carbohydrate
epitope of the extracellular domain of PCLP, CEA, or CD44v as
expressed in a cancer cell. General methods for the production of
antibodies that specifically bind to particular epitopes of the
extracellular domain of transmembrane proteins are known in the
art. See, e.g., U.S. Pat. Nos. 6,344,339; 6,218,516; and
6,150,508.
[0061] Commercially Available Antibodies
[0062] Commercially available antibodies specific for PCLP, CEA,
and CD44v may be used in a targeting composition of the present
invention. For example, anti-PCLP antibody is available from Santa
Cruz Biotechnology, Inc. (Santa Cruz, Calif.) (Clone 3D3, Catalog
No. sc-23904). Anti-CEA antibodies are available from BD
Biosciences (San Jose, Calif.) (Clone Col-1, Catalog No. 551477)
and Abcam, Inc. (Cambridge, Mass. (Catalog No. ab17254). Anti-CD44v
antibodies are available from AbD Serotec (Oxford, United Kingdom)
(CD44v3, Clone VFF-327, Catalog No. MCA1734), (CD44v4, Clone 10D1,
Catalog No. MCA1970), (CD44v5, Clone VFF-8, Catalog No MCA1729),
(CD44v6, Clone VFF-7, Catalog #MCA1730), (CD44v7, Clone VFF-9,
Catalog No MCA 1731), (CD44v7/8, Clone VFF-17 Catalog No. MCA1732),
and (CD44c10, Clone VFF-14, Catalog No. MCA1733)
[0063] Polyclonal Antibodies
[0064] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc), intraperitoneal (ip) or intramuscular
(im) injections of the relevant antigen and an adjuvant. It may be
useful to conjugate the relevant antigen to a protein that is
immunogenic in the species to be immunized, e.g., keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin
inhibitor using a bifunctional or derivatizing agent, for example,
maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl2, or RIN.dbd.C--NR, where
R and RI are different alkyl groups. Animals are immunized against
the antigen, immunogenic conjugates, or derivatives by combining,
e.g., 100 pg of the protein or conjugate (for rabbits or mice,
respectively) with 3 volumes of Freund's complete adjuvant and
injecting the solution intradermally at multiple sites. One month
later the animals are boosted with 1/5 to 1/10 the original amount
of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later,
the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with the conjugate of the same antigen, but
conjugated to a different protein and/or through a different
cross-linking reagent. Conjugates also can be made in recombinant
cell culture as protein fusions. In addition, aggregating agents
such as alum are suitably used to enhance the immune response.
[0065] Monoclonal Antibodies
[0066] The term "monoclonal antibody" as used herein 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 conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants, each monoclonal
antibody is directed against a single determinant on the
antigen.
[0067] In addition to their specificity, monoclonal antibodies are
advantageous in that they may be synthesized while uncontaminated
by other immunoglobulins. For example, the monoclonal antibodies to
be used in accordance with the invention may be produced by the
hybridoma method first described by Kohler et al., 256 NATURE 495
(1975). Alternatively, monoclonal antibodies may be produced by
recombinant DNA methods. See, e.g., U.S. Pat. No. 4,816,567. In
another embodiment, monoclonal antibodies may be isolated from
phage antibody libraries using the techniques described, for
example, in Clackson et al., 352 NATURE 624-28 (1991) and in Marks
et al., 222 J. MOL. BIOL. 581-97 (1991).
[0068] Human Antibodies
[0069] As an alternative to humanization, human antibodies may be
generated. For example, transgenic animals (e.g., mice) may be
produced that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. It has been described that the
homozygous deletion of the antibody heavy-chain joining region (JH)
gene in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene in such germ-line mutant mice will
result in the production of human antibodies upon antigen
challenge. See, e.g., Jakobovits et al., 90 PROC. NATL. ACAD. SCI.
USA 2551 (1993); Jakobovits et al., 362 NATURE 255-58 (1993);
Bruggermann et al., 7 YEAR IN IMMUNOL. 33 (1993); U.S. Pat. Nos.
5,591,669; 5,589,369; 5,545,807.
[0070] Alternatively, phage display technology may be used to
produce human antibodies and antibody fragments in vitro, from
immunoglobulin variable (V) domain gene repertoires from
unimmunized donors. See, e.g., Johnson and Chiswell, 3 CURRENT
OPIN. IN STRUCT. BIOL. 564-71 (1993); McCafferty et al., 348 NATURE
552-53 (1990). 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, 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 may be
performed in a variety of formats. Human antibodies may also be
generated by in vitro activated B cells. See U.S. Pat. Nos.
5,567,610 and 5,229,275.
[0071] Humanized Antibodies
[0072] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody.
[0073] In general, the humanized antibody may comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the hypervariable
loops correspond to those of a non-human immunoglobulin and all or
substantially all of the FRs are those of a human immunoglobulin
sequence. In one embodiment, humanized antibodies comprise a
humanized FR that exhibits at least 65% sequence identity with an
acceptor (non-human) FR, e.g., murine FR. The humanized antibody
also may comprise at least a portion of an immunoglobulin constant
region (Fc), particularly a human immunoglobulin. For further
details, see Jones et al., 321 NATURE 522-25 (1986); Riechmann et
al., 332 NATURE 323-29 (1988); Presta, 2 CURR. OPIN. STRUCT. BIOL.
593-96 (1992); WO 01/27160.
[0074] Methods for humanizing non-human antibodies have been
described in the art. A humanized antibody may have one or more
amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization may be essentially performed
following the method of Winter and co-workers (Jones et al., 321
NATURE 522-25 (1986); Riechmann et al., 332 NATURE 323-27 (1988);
Verhoeyen et al., 239 SCIENCE 1534-36 (1988)), by substituting
hypervariable region sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0075] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence that is closest to that of the rodent
is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., 151 J. IMMUNOL. 2296 (1993);
Chothia et al., 196 J. MOL. BIOL. 901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., 89 PROC. NATL. ACAD. SCI. USA
4285 (1992); Presta et al., 151 J. IMMUNOL. 2623 (1993)).
[0076] Importantly, antibodies may be humanized to retain and/or
enhance affinity for the antigen and other favorable biological
properties. In one embodiment, humanized antibodies maybe designed
by analyzing the parental sequences and various conceptual
humanized products using three dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available that illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this regard, FR residues may
be selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved.
[0077] Other methods generally involve conferring donor CDR binding
affinity onto an antibody acceptor variable region framework. One
method involves simultaneously grafting and optimizing the binding
affinity of a variable region binding fragment. Another method
relates to optimizing the binding affinity of an antibody variable
region. See generally WO 01/27160.
[0078] The proteins discussed herein, particularly immunoglobulins
and more particularly, humanized antibodies, may be rendered
non-immunogenic, or less immunogenic, to a given species by
identifying in the amino acid sequences one or more potential
epitopes for T-cells of the given species and modifying the amino
acid sequence to eliminate at least one of the T-cell epitopes.
This procedure eliminates or reduces the immunogenicity of the
protein when exposed to the immune system of the given species.
Indeed, monoclonal antibodies and other immunoglobulin-like
molecules can particularly benefit from being de-immunized in this
way, for example, mouse-derived immunoglobulins can be de-immunized
for human therapeutic use in treating cancers. See WO 98/52976.
[0079] Furthermore, certain epitopes may be retained in a protein
sequence if the peptides constituting such epitopes are present in
endogenous human protein, because they would be recognized as
"self". It has now been found, however, that even self epitopes may
give rise to immune reactions. Thus, one aspect of the invention
provides for the elimination of self epitopes, for example, by
recombinant DNA technology, to render them more useful for
administration to humans, for example for therapeutic or diagnostic
purposes relating to cancer. See WO 00/34317.
[0080] Therapeutic selectin ligand-binding immunoglobulins or
antibodies can also be "chimeric" in the sense that a variable
region can come from a one species, such as a rodent, and the
constant region can be from a second species, such as a human. See
U.S. Pat. No. 6,331,415.
[0081] Human, humanized, chimeric, or non-human antibodies can also
be subject to affinity maturation. A library of mutant antibody
chains based on a previously identified PCLP, CEA, and/or CD44v
antibody heavy and light chains is generated, and then screened for
changes in activity. Screens can be designed to identify mutants
with enhanced activity, including higher affinity, lower
dissociation constant, less cross reactivity with other proteins,
or stronger effector functions. Examples of a process of generating
such a library and identifying antibodies with high affinity are
disclosed in WO 01/27160.
[0082] Antibody Fragments
[0083] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen-binding or variable region
thereof. Examples of antibody fragments include Fab, Fab',
F(ab').sup.2, Fv fragments, diabodies, linear antibodies,
single-chain antibody molecules, and multispecific antibodies
formed from antibody fragments.
[0084] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment. The Fab
fragments also contain the constant domain of the light chain and
the first constant domain (CHI) of the heavy chain.
[0085] Pepsin treatment yields an F(ab').sup.2 fragment that has
two antigen-binding sites and is still capable of crosslinking
antigen. Fab' fragments differ from Fab fragments by the addition
of a few residues at the carboxy terminus of the heavy chain CHI
domain including one or more cysteines from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear at least one free
thiol group. F(ab').sup.2 antibody fragments originally were
produced as pairs of Fab' fragments which have hinge cysteines
between them. Other chemical couplings of antibody fragments are
well known in the art.
[0086] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six hypervariable regions confer
antigen binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three
hypervariable regions specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
[0087] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. The Fv polypeptide may further comprise
a polypeptide linker between the VH and VL domains that enables the
scFv to form the desired structure for antigen binding. See
Pluckthun, 113 THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES 269-315
(Rosenburg and Moore eds. 1994). See also WO 93/16185; U.S. Pat.
Nos. 5,587,458 and 5,571,894.
[0088] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies. See, e.g., Morimoto et
al., 24 J. BIOCHEM. BIOPHYS. METH. 107-17 (1992); Brennan et al.,
229 SCIENCE 81 (1985). However, these fragments may now be produced
directly by recombinant host cells. For example, the antibody
fragments may be isolated from the antibody phage libraries
discussed above. Alternatively, Fab'-SH fragments may be directly
recovered from E. coli and chemically coupled to form F(ab').sup.2
fragments. Carter et al., 10 BIO/TECHNOLOGY 163-67 (1992)). In
another approach, F(ab').sup.2 fragments may be isolated directly
from recombinant host cell culture. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner.
[0089] Bispecific Antibodies
[0090] The term "bispecific antibody" refers to small antibody
fragments with two antigen-binding sites. Each fragment comprises a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) in the same polypeptide chain (VH-VL). By
using a linker that is too short to allow pairing between the two
domains on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two antigen
binding sites. EP 0 404 097; WO 93/11161; Hollinger et al., 90
PROC. NATL. ACAD. SCI. USA 6444-48 (1993).
[0091] Methods for making bispecific antibodies are well known in
the art. Traditional production of full length bispecific
antibodies is based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, where the two chains have different
specificities. Millstein et al., 305 NATURE 537-39 (1983). Because
of the random assortment of immunoglobulin heavy and light chains,
these hybridomas (quadromas) produce a potential mixture of 10
different antibody molecules, of which only one has the correct
bispecific structure. Purification of the correct molecule, which
is usually accomplished by affinity chromatography steps, is rather
cumbersome, and the product yields are low. See also WO 93/08829;
Traunecker et al., 10 EMBO J. 3655-59 (1991).
[0092] In another approach, antibody variable domains with the
desired binding specificities (antibody-antigen combining sites)
may be fused to immunoglobulin constant domain sequences.
Specifically, the variable domains are fused with an immunoglobulin
heavy chain constant domain, comprising at least part of the hinge,
CH2, and CH3 regions. In one embodiment, the fusion protein
comprises the first heavy-chain constant region (CHI) because it
contains the site necessary for light chain binding.
Polynucleotides encoding the immunoglobulin heavy chain fusions
and, if desired, the immunoglobulin light chain, may be inserted
into separate expression vectors and co-transfected 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.
[0093] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which may be chemically coupled to
form bispecific antibodies. Shalaby et al., 175 J. EXP. MED.
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sup.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0094] Bispecific antibodies have been produced using leucine
zippers. Kostelny et al., 148(5) J. IMMUNOL. 1547-53 (1992). The
leucine zipper peptides from the Fos and Jun proteins are linked to
the Fab' portions of two different antibodies by gene fusion. The
antibody homodimers are then reduced at the hinge region to form
monomers and then re-oxidized to form the antibody heterodimers.
This method may also be utilized for the production of antibody
homodimers.
[0095] Another strategy for making bispecific antibody fragments by
the use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., 152 J. IMMUNOL. 5368 (1994). Furthermore, the
invention contemplates antibodies with more than two valencies,
such as trispecific antibodies. See Tutt et al., 147 J. IMMUNOL. 60
(1991).
[0096] Modifications of Antibodies to Selectin Ligands
[0097] Peptide Mimetics
[0098] Another embodiment for the preparation of antibodies
according to the invention is the use of peptide mimetics. Mimetics
are peptide-containing molecules that mimic elements of protein
secondary structure. See, for example, Johnson et al., "Peptide
Turn Mimetics" in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds.,
Chapman and Hall, New York (1993). The underlying rationale behind
the use of peptide mimetics in rational design is that the peptide
backbone of proteins exists chiefly to orient amino acid side
chains in such a way as to facilitate molecular interactions, such
as those of antibody and antigen. A peptide mimetic is expected to
permit molecular interactions similar to the natural molecule.
These principles may be used to engineer second generation
molecules having many of the natural properties of the targeting
antibodies disclosed herein, but with altered and even improved
characteristics. More specifically, under this rational design
approach, peptide mapping may be used to determine "active" antigen
recognition residues, and along with molecular modeling and
molecular dynamics trajectory analysis, peptide mimic of the
antibodies containing antigen contact residues from multiple CDRs
may be prepared.
[0099] Epitopes of Selectin Ligands
[0100] In some embodiments, the anti-PCLP antibody, anti-CEA
antibody, and the anti-CD44v antibody specifically binds an epitope
of the respective selectin ligand that maps to a peptide region
from the extracellular domain. It is to be understood that the
peptide regions may not necessarily precisely map one epitope, but
may also contain selectin ligand sequence that is not immunogenic.
Methods of predicting other potential epitopes to which an
immunoglobulin of the invention can bind are well-known to those of
skill in the art and include, without limitation, Kyte-Doolittle
Analysis (Kyte, J. and Dolittle, R. F., J. Mol. Biol. (1982)
157:105-132), Hopp and Woods Analysis (Hopp, T. P. and Woods, K.
R., Proc. Natl. Acad. Sci. USA (1981) 78:3824-3828; Hopp, T. J. and
Woods, K. R., Mol. Immunol. (1983) 20:483-489.; Hopp, T. J., J.
Immunol. Methods (1986) 88:1-18), Jameson-Wolf Analysis (Jameson,
B. A. and Wolf, H., Comput. Appl. Biosci. (1988) 4:181-186), and
Emini Analysis (Emini, E. A., Schlief, W. A., Colonno, R. J. and
Wimmer, E., Virology (1985) 140:13-20).
[0101] Other Modifications
[0102] Amino acid sequence variants of the antibodies of the
present invention may be prepared by introducing appropriate
nucleotide changes into the polynucleotide that encodes the
antibody or by peptide synthesis. Such modifications include, for
example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletions, insertions, and
substitutions may be made to arrive at the final construct.
[0103] Amino acid sequence insertions include amino-terminal 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 a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody of a
polypeptide that increases the serum half-life of the antibody.
[0104] Another type of antibody variant is an amino acid
substitution variant. These variants have at least one amino acid
residue in the antibody molecule replaced by a different residue.
For example, the sites of greatest interest for substitutional
mutagenesis of antibodies include the hypervariable regions, but
framework region (FR) alterations are also contemplated.
[0105] A useful method for the identification of certain residues
or regions of the anti-selectin ligand antibodies (including
antibodies specific for PCLP, CEA, and CD44v) that are preferred
locations for substitution, i.e., mutagenesis, is alanine scanning
mutagenesis. See Cunningham & Wells, 244 SCIENCE 1081-85
(1989). Briefly, a residue or group of target residues are
identified (e.g., charged residues such as arg, asp, his, lys, and
glu) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction
of the amino acids with antigen. The amino acid locations
demonstrating functional sensitivity to the substitutions are
refined by introducing further or other variants at, or for, the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to analyze
the performance of a mutation at a given site, alanine scanning or
random mutagenesis may be conducted at the target codon or region
and the expressed antibody variants screened for the desired
activity.
[0106] Substantial modifications in the biological properties of
the antibody can be accomplished by selecting substitutions that
differ significantly in their effect on, maintaining (i) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (ii)
the charge or hydrophobicity of the molecule at the target site, or
(iii) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0107] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0108] (2) neutral hydrophilic: cys, ser, thr;
[0109] (3) acidic: asp, glu;
[0110] (4) basic: asn, gln, his, lys, arg;
[0111] (5) residues that influence chain orientation: gly, pro;
and
[0112] (6) aromatic: trp, tyr, phe.
[0113] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Conservative
substitutions involve exchanging of amino acids within the same
class.
[0114] 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 crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability, particularly where
the antibody is an immunoglobulin fragment such as an Fv
fragment.
[0115] Another type of substitutional variant involves substituting
one or more hypervariable region residues of a parent antibody.
Generally, the resulting variant(s), i.e., functional equivalents
as defined above, selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants is by affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed.
[0116] In order to identify candidate hypervariable region sites
for modification, alanine-scanning mutagenesis may be performed to
identify hypervariable region residues contributing significantly
to antigen binding. Alternatively, or additionally, it may be
beneficial to analyze a crystal structure of the antibody-antigen
complex to identify contact points between the antibody and
antigen. Such contact residues and neighboring residues are
candidates for substitution according to the techniques elaborated
herein. Once generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0117] It may be desirable to modify the antibodies of the present
invention, i.e., create functional equivalents, with respect to
effector function, e.g., so as to enhance antigen-dependent
cell-mediated cyotoxicity (ADCC) and/or complement dependent
cytotoxicity (CDC) of the antibody. This may be achieved by
introducing one or more amino acid substitutions in an Fc region of
an antibody. Alternatively or additionally, cysteine residue(s) may
be introduced in the Fc region, thereby allowing interchain
disulfide bond formation in this region. The homodimeric antibody
thus generated may have improved internalization capability and/or
increased complement-mediated cell killing and antibody-dependent
cellular cytotoxicity (ADCC). Caron et al., 176 J. EXP MED. 1191-95
(1992); Shopes, 148 J. IMMUNOL. 2918-22 (1992). Homodimeric
antibodies with enhanced anti-tumor activity may also be prepared
using heterobifunctional cross-linkers as described in Wolff et
al., 53 CANCER RESEARCH 2560-65 (1993). Alternatively, an antibody
can be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. Stevenson et al.,
3 ANTI-CANCER DRUG DESIGN 219-30 (1989).
[0118] To increase the serum half life of an antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an immunoglobulin fragment) as described in, for
example, U.S. Pat. No. 5,739,277. As used herein, the term "salvage
receptor binding epitope" refers to an epitope of the Fc region of
an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is
responsible for increasing the in vivo serum half-life of the IgG
molecule.
[0119] Polynucleotide molecules encoding amino acid sequence
variants of the antibody are prepared by a variety of methods known
in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-selectin ligand
antibodies of the present invention.
Solid Substrates
[0120] The targeting compositions of the present invention may
comprise a solid substrate. The substrates described herein may be
present in a dry formulation (such as lyophilized composition) or
the substrate may comprise a particle suspended in a biocompatible
medium. Suitable biocompatible media include, but are not limited
to, water, deionized water, buffered aqueous media, saline,
buffered saline, optionally buffered solutions of amino acids,
optionally buffered solutions of proteins, optionally buffered
solutions of sugars, optionally buffered solutions of vitamins,
optionally buffered solutions of synthetic polymers,
lipid-containing emulsions, and the like.
[0121] In other embodiments, the substrate particle may comprise a
nanoparticle, a quantum dot, a liposome, a micelle, a polymersome,
or other delivery compositions known in the art.
[0122] According to the present invention, in one embodiment, a
substrate particle may be conjugated to at least one antibody. In a
specific embodiment, a substrate particle may be conjugated to at
least one anti-PCLP antibody. In another embodiment, a substrate
particle may be conjugated to at least one anti-CEA antibody. In
yet another embodiment, a substrate particle may be conjugated to
at least one anti-CD44v antibody. In a further embodiment, a
substrate particle may be conjugated to at least one anti-PCLP
antibody and at least one anti-CEA antibody. In an alternative
embodiment, a substrate particle may be conjugated to at least one
anti-PCLP antibody and at least one anti-CD44v antibody. The
substrate particle may also be conjugated to at least one anti-CEA
antibody and at least one anti-CD44v antibody. In a further
embodiment, the substrate particle may be conjugated to at least
one anti-PCLP antibody, at least one anti-CEA antibody, and at
least one anti-CD44v antibody.
[0123] Nanoparticles
[0124] The nanoparticle substrates of the present invention may be
prepared using conventional methods known to those of ordinary
skill in the art. For example, in certain embodiments, the
nanoparticle substrate may comprise a polymeric matrix or
"nanospheres" where the antibody composition is attached, directly
or indirectly, to the surface of the nanoparticle substrate. In
other embodiments, the nanoparticle substrate may comprise a
reservoir system comprising an oily core surrounded by a thin
polymeric wall ("nanocapsules"), where the core comprises the
antibody composition which may protrude through the wall or be
released and function as intended. Polymers suitable for the
preparation of nanoparticles include, but are not limited to,
poly(alkylcyano-acrylates), and polyesters such as poly(lactic
acid) (PLA), poly(glycolic acid), poly(-capro-lactone) and their
copolymers.
[0125] Nanoparticles may be fabricated using biodegradable
polyesters, e.g., polymers of poly(lactic acid) (PLA) and
copolymers that are manufactured with varying quantities of
glycolic acid (PLGA). PLA is more hydrophobic in comparison to
PLGA; therefore, PLA offers a relatively extended release profile.
Similarly, the ratio of glycolic acid to lactic acid in the
copolymerization process affects the degradative properties of the
resultant copolymer. In one embodiment, low molecular weight (14
kDa) PLGA may be copolymerized with a high (50%) glycolide content
(PLGA 50:50). These particles will degrade comparatively rapidly
due to the low molecular weight and high glycolide content of the
PLGA used. To obtain nanoparticles with an intermediate or long
degradation profile, the formulation may comprise a higher
molecular weight copolymer (e.g., 60-100 kDa), with or without a
lower glycolide content (PLGA 65:35 or 75:25). In short, a
comprehensive range of PLA and PLGA polymer molecular weight,
lactic/glycolic acid ratios, and PLA-PLGA blends may be used to
optimize loading and release profiles.
[0126] Furthermore, nanoparticles (NPs) may comprise a metal, a
semiconductor, and an insulator particle compositions, and a
dendrimer (organic or inorganic). Thus, nanoparticles are
contemplated for use in the methods which comprise a variety of
inorganic materials including, but not limited to, metals,
semi-conductor materials or ceramics as described in U.S. Patent
Publication No 20030147966. Ceramic nanoparticle materials include,
but are not limited to, brushite, tricalcium phosphate, alumina,
silica, and zirconia. Organic materials from which nanoparticles
are produced include carbon. Nanoparticle polymers include
polystyrene, silicone rubber, polycarbonate, polyurethanes,
polypropylenes, polymethylmethacrylate, polyvinyl chloride,
polyesters, polyethers, and polyethylene. Biodegradable, biopolymer
(e.g. polypeptides such as BSA, polysaccharides, etc.), other
biological materials (e.g. carbohydrates), and/or polymeric
compounds are also contemplated for use in producing
nanoparticles.
[0127] In one embodiment, the nanoparticle is metallic, and in
various aspects, the nanoparticle is a colloidal metal. Thus, in
various embodiments, nanoparticles useful in the practice of the
methods include metal (including for example and without
limitation, gold, silver, platinum, aluminum, palladium, copper,
cobalt, indium, nickel, or any other metal amenable to nanoparticle
formation), semiconductor (including for example and without
limitation, CdSe, CdS, and CdS or CdSe coated with ZnS) and
magnetic (for example., ferromagnetite) colloidal materials. Other
nanoparticles useful in the practice of the invention include, also
without limitation, ZnS, ZnO, Ti, TiO.sub.2, Sn, SnO.sub.2, Si,
SiO.sub.2, Fe, Fe.sup.+4, Ag, Cu, Ni, Al, steel, cobalt-chrome
alloys, Cd, titanium alloys, AgI, AgBr, HgI.sub.2, PbS, PbSe, ZnTe,
CdTe, In.sub.2S.sub.3, In.sub.2Se.sub.3, Cd.sub.3P.sub.2,
Cd.sub.33As.sub.2, InAs, and GaAs. Methods of making ZnS, ZnO,
TiO.sub.2, AgI, AgBr, HgI.sub.2, PbS, PbSe, ZnTe, CdTe,
In.sub.2S.sub.3, In.sub.2Se.sub.3, Cd.sub.3P.sub.2,
Cd.sub.3As.sub.2, InAs, and GaAs nanoparticles are also known in
the art. See, e.g., Weller, 32 CHEM. INT. ED. ENGL. 41 (1993);
Henglein, 143 TOP. CURR. CHEM 113 (1988); Henglein, 89 CHEM. REV.
1861 (1989); 53 BRUS, APPL. PHYS. A. 465 (1991); Bahncmann, in
Photochemical Conversion and Storage of Solar Energy (eds.
Pelizetti and Schiavello 1991), page 251; Wang and Herron, 95 J.
PHYS. CHEM. 525 (1991); Olshaysky, et al., 112J. AM. CHEM. SOC.
9438 (1990); Ushida et al., 95 J. PHYS. CHEM. 5382 (1992).
[0128] Methods of making metal, semiconductor and magnetic
nanoparticles are well-known in the art. See, for example, Schmid,
G. (ed.) Clusters and Colloids (VCH, Weinheim, 1994); Hayat, M. A.
(ed.) Colloidal Gold: Principles, Methods, and Applications
(Academic Press, San Diego, 1991); Massart, R., IEEE Transactions
On Magnetics, 17, 1247 (1981); Ahmadi, T. S. et al., Science, 272,
1924 (1996); Henglein, A. et al., J. Phys. Chem., 99, 14129 (1995);
Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27, 1530
(1988). Preparation of polyalkylcyanoacrylate nanoparticles
prepared is described in Fattal, et al., J. Controlled Release
(1998) 53: 137-143 and U.S. Pat. No. 4,489,055. Methods for making
nanoparticles comprising poly(D-glucaramidoamine)s are described in
Liu, et al., J. Am. Chem. Soc. (2004) 126:7422-7423.
[0129] Preaparation of nanoparticles comprising polymerized
methylmethacrylate (MMA) is described in Tondelli, et al., Nucl.
Acids Res. (1998) 26:5425-5431, and preparation of dendrimer
nanoparticles is described in, for example Kukowska-Latallo, et
al., Proc. Natl. Acad. Sci. USA (1996) 93:4897-4902 (Starburst
polyamidoamine dendrimers). Suitable nanoparticles are also
commercially available from, for example, Ted Pella, Inc. (gold),
Amersham Corporation (gold) and Nanoprobes, Inc. (gold). Also as
described in US Patent Publication No. 20030147966, nanoparticles
comprising materials described herein are available commercially or
they can be produced from progressive nucleation in solution (e.g.,
by colloid reaction), or by various physical and chemical vapor
deposition processes, such as sputter deposition. See, e.g.,
HaVashi, (1987) Vac. Sci. Technol. July/August 1987, A5(4):1375-84;
Hayashi, (1987) Physics Today, December 1987, pp. 44-60; MRS
Bulletin, January 1990, pp. 16-47.
[0130] As further described in US Patent Publication No.
20030147966, nanoparticles contemplated are produced using
HAuCl.sub.4 and a citrate-reducing agent, using methods known in
the art. See, e.g., Marinakos et al., (1999) Adv. Mater. 11: 34-37;
Marinakos et al., (1998) Chem. Mater. 10: 1214-19; Enustun &
Turkevich, (1963) J. Am. Chem. Soc. 85: 3317. Tin oxide
nanoparticles having a dispersed aggregate particle size of about
140 nm are available commercially from Vacuum Metallurgical Co.,
Ltd. of Chiba, Japan. Other commercially available nanoparticles of
various compositions and size ranges are available, for example,
from Vector Laboratories, Inc. of Burlingame, Calif.
[0131] Nanoparticles may be prepared using an emulsification and
solvent evaporation process, or so-called double emulsion process,
for example. Other procedures comprise adding additional polymers
through covalent modification of existing nanoparticles. To form a
primary emulsion, an internal aqueous phase that contains a
stabilizing emulsifier and an antibody composition is added to an
ice-cold organic phase. The stabilizing emulsifier may be 10% w/v
polyvinyl alcohol (PVA), and the organic phase may comprise a
polymer dissolved in dichloromethane (DCM). The polymer content is
modified according to the particle size required.
[0132] The surface of hydrophobic nanoparticles may be modified to
minimize phagocytosis, allowing sustained systemic circulation of
nanoparticles. Following intravenous administration, hydrophobic
nanoparticles are rapidly cleared from systemic circulation by the
mononuclear phagocytic system (MPS), resulting in rapid deposition
of nanoparticles in the liver or spleen. When the liver, spleen or
MPS itself are not targets of choice, various modifications of the
nanoparticle surface are possible to minimize phagocytosis,
including modification with poly(ethylene glycol) (PEG). PEG is a
hydrophilic, nonionic polymer that exhibits excellent
biocompatibility. PEG molecules, like other polymers, can be added
to the nanoparticles by a number of different methods, including
covalent bonding, blending during nanoparticle preparation, or
surface adsorption. The presence of PEG on the nanoparticle surface
serves other functions besides increasing residence time in
systemic circulation. PEG has been shown to reduce protein and
enzyme adsorption to the nanoparticle, retarding degradation of
PLGA-based nanoparticles. The density and molecular weight of PEG
on the surface can be adjusted to minimize protein adsorption.
Poloxamer and poloxamines also have been shown to reduce
nanoparticle capture by macrophages and increase nanoparticle
residence time in systemic circulation. PLGA particles also may be
coated with poloxamer 407 and poloxamine 908 to extend the
half-life of the nanoparticles. Poly(ethylene glycol) can be
introduced at the surface either (a) by adsorption of surfactants
(e.g., poloxamer 188) or (b) as block or branched co-polymers,
usually based on polyesters, such as poly(lactic acid) (PLA).
[0133] Quantum Dots
[0134] In certain embodiments of the present invention, a particle
may comprise a quantum dot. Quantum dots are a semiconductor
nanocrystal with size-dependent optical and electronic properties.
Many semiconductors that are constructed of elements from groups
II-VI, III-V and IV of the periodic table have been prepared as
quantum sized particles, exhibit quantum confinement effects in
their physical properties, and can be used in the composition of
the present invention. Exemplary materials suitable for use as
quantum dots include ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP,
GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge,
and Si and ternary and quaternary mixtures thereof. The quantum
dots may further include an overcoating layer of a semiconductor
having a greater band gap.
[0135] The quantum dots are characterized by their uniform
nanometer size. By "nanometer" size, it is meant less than about
150 angstroms, including in the range of 12-150 angstroms. The
quantum dots also may be substantially monodisperse within the
broad range given above. By monodisperse, as that term is used
herein, it is meant a colloidal system in which the suspended
particles may have substantially identical size and shape. For the
purposes of the present invention, monodisperse particles mean that
at least about 50% of the particles fall within a specified
particle size range. Monodisperse particles may deviate less than
about 10% in tins diameter, and in some embodiments, less than
about 5% in rms diameter.
[0136] Liposomes
[0137] In one aspect, the particles of the present invention may
comprise a liposome. The liposomes in the composition can be
comprised primarily of one or more vesicle-forming lipids. Such a
vesicle-forming lipid is one which can form spontaneously into
bilayer vesicles in water, as exemplified by the phospholipids,
with its hydrophobic moiety in contact with the interior,
hydrophobic region of the bilayer membrane, and its head group
moiety oriented toward the exterior, polar surface of the
membrane.
[0138] Liposomes can be categorized into multilamellar vesicles,
multivesicular liposomes, unilamellar vesicles and giant liposomes.
Multilamellar liposomes (also known as multilamellar vesicles or
"MLV") contain multiple concentric bilayers within each liposome
particle, resembling the "layers of an onion". Multivesicular
liposomes consist of lipid membranes enclosing multiple
non-concentric aqueous chambers. Unilamellar liposomes enclose a
single internal aqueous compartment. Single bilayer (or
substantially single bilayer) liposomes include small unilamellar
vesicles (SUV) and large unilamellar vesicles (LUV). LUVs and SUVs
range in size from about 50 to 500 nm and 20 to 50 nm respectively.
Giant liposomes typically range in size from 5000 nm to 50,000 nm
and are used mainly for studying mechanochemical and interactive
features of lipid bilayer vesicles in vitro. Needham et al., 18
COLLOIDS AND SURFACES B: BIOINTERFACES 183-195 (2000).
[0139] Any suitable vesicle-forming lipid may be utilized in the
practice of this invention as judged by one of skill in the art.
This includes phospholipids such as phosphatidylcholine (PC),
phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic
acid (PA), phosphatidyethanolamine (PE) and phosphatidylserine
(PS); sterols such as cholesterol; glycolipids; sphingolipids such
as sphingosine, ceramides, sphingomyelin, and glycosphingolipids
(such as cerebrosides and gangliosides). Suitable phospholipids may
include one or two acyl chains having any number of carbon atoms,
between about 6 to about 24 carbon atoms, selected independently of
one another and with varying degrees of unsaturation. Thus,
combinations of phospholipid of different species and different
chain lengths in varying ratios may be selected. Mixtures of lipids
in suitable ratios, as judged by one of skill in the art, may also
be used.
[0140] Liposomes for use in the present invention may be generated
using a variety of conventional techniques. These techniques
include: the ether injection method (Deamer et al., 308 ACAD. SCl.
250 (1978); the surfactant method (Brunner et al., 455 BIOCHIM.
BIOPHYS. ACTA 322 (1978); the Ca.sup.2+ fusion method
(Paphadjopoulos et al., 394 BIOCHIM. BIOPHYS. ACTA 483 (1978); the
freeze-thaw method (Pick et al., 212 ARACH. BIOCHIM. BIOPHYS. 186
(1981); the reverse-phase evaporation method (Szoka et al., 601
BIOCHIM. BIOPHYS. ACTA 559 (1981); the ultrasonic treatment method
(Huang et al., 8 BIOCHEMISTRY 344 (1969); the ethanol injection
method (Kremer et al., 16 BIOCHEMISTRY 3932 (1977); the extrusion
method (Hope et al., 812 BIOCHIM. BIOPHYSICA ACTA 55 (1985); the
French press method (Barenholz et al., 99 FEBS LETT. 210 (1979); or
any other technique described herein or known in the art.
[0141] Liposomes may range from any value between about 1 nm to
about 100 um in diameter. For example, liposomes in a liposomal
composition according to the invention may range from any value
between about 10 to about 200 nm in diameter. In some embodiments,
liposomes in a liposomal composition according to the invention may
be less than about 200 nm in diameter, or less than about 160 nm in
diameter, or less than about 140 nm in diameter. In some
embodiments, liposomes in a liposomal composition according to the
invention may be substantially uniform in size, for example, 10% to
100%, or more generally at least 10%, 20%, 30%, 40%, 50, 55% or
60%, or at least 65%, 75%, 80%, 85%, 90%, or 95%, or as much as
96%, 97%, 98%, 99%, or 100% of the liposomes in the liposomal
composition may be between the size values indicated herein.
Liposomes may be sized by extrusion through a filter (e.g., a
polycarbonate filter) having pores or passages of the desired
diameter.
[0142] The liposomes can also include a lipopolymer, i.e., a lipid
covalently attached to a hydrophilic polymer. Lipopolymers, in
particular mPEG-DSPE conjugates, have been used extensively in
various liposomal delivery systems. See Woodle, M. C. in
POLY(ETHYLENE GLYCOL) CHEMISTRY AND BIOLOGICAL APPLICATIONS; and J.
M. Harris and S. Zalipsky, Eds., ACS Symp. Series 680, pp. 60-81,
American Chemical Soc., Washington, D.C. (1997). As has been
described, for example in U.S. Pat. No. 5,013,556, including such a
polymer-derivatized lipid in the liposome composition forms a
surface coating of hydrophilic polymer chains around the liposome.
The surface coating of hydrophilic polymer chains is effective to
increase the in vivo blood circulation lifetime of the liposomes
when compared to liposomes lacking such a coating.
Polymer-derivatized lipids comprised of methoxy(polyethylene
glycol) (mPEG) and a phosphatidylethanolamine (e.g., dimyristoyl
phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine,
distearoyl phosphatidylethanolamine (DSPE), or dioleoyl
phosphatidylethanolamine) can be obtained from Avanti Polar Lipids,
Inc. (Alabaster, Ala.) at various mPEG molecular weights (350, 550,
750, 1000, 2000, 3000, 5000 Daltons). Lipopolymers of mPEG-ceramide
can also be purchased from Avanti Polar Lipids, Inc. Preparation of
Lipid-Polymer Conjugates is Also Described in the Literature, See
U.S. Pat. Nos. 5,631,018, 6,586,001, and 5,013,556; Zalipsky, S.,
et al., 8 BIOCONJUGATE CHEM. 111 (1997); Zalipsky, S., et al., 387
METH. ENZYMOL. 50 (2004). These lipopolymers can be prepared as
well-defined, homogeneous materials of high purity, with minimal
molecular weight dispersity (Zalipsky, S., et al., 8 BIOCONJUGATE
CHEM. 111 (1997); Wong, J., et al., 275 SCIENCE 820 (1997)). The
lipopolymer can also be a "neutral" lipopolymer, such as a
polymer-distearoyl conjugate, as described in U.S. Pat. No.
6,586,001.
[0143] Methods for attaching other molecules to liposomes are
known, where the liposome can be functionalized for subsequent
reaction with a selected molecule. See U.S. Pat. No. 6,180,134;
Zalipsky et al., 353 FEBS LETT. 71 (1994); Zalipsky et. al., 4
BIOCONJUGATE CHEM. 296 (1993); Zalipsky et al., 39 J. CONTROL. REL.
153 (1996); Zalipsky et al., 8(2) BIOCONJUGATE CHEM. 111 (1997);
Zalipsky et al., 387 METH. ENZYMOL. 50 (2004). Functionalized
polymer-lipid conjugates can also be obtained commercially, such as
end-functionalized PEG-lipid conjugates (Avanti Polar Lipids,
Inc.). The linkage between the molecule and the liposome polymer
can be a stable covalent linkage or a releasable linkage that is
cleaved in response to a stimulus, such as a change in pH or
presence of a reducing agent.
[0144] The liposome composition can also include a cyclodextrin.
Cyclodextrins are cyclic oligosaccharides of
.alpha.-D-gluco-pyranose and can be formed by the catalytic
cyclization of starch. Due to a lack of free rotation about the
bonds connecting the glycopyranose units, cyclodextrins are
toroidal or cone shaped, rather than cylindrical. The cyclodextrins
have a relatively hydrophobic central cavity and a hydrophilic
outer surface. The hydrophobic cage-like structure of cyclodextrins
has the ability to entrap a variety of guest compounds to form
host-guest complexes in the solid state and in solution. These
complexes are often termed inclusion complexes and the guest
compounds are released from the inclusion site. Common
cyclodextrins are alpha-, beta-, and gamma-cyclodextrin, which
consist of six, seven, or eight glucopyranose units, respectively.
Cyclodextrins containing nine, ten, eleven, twelve, and thirteen
glucopyranose units are designated delta-, epsilon-, xi-, eta-, and
theta-cyclodextrin, respectively.
[0145] Micelles
[0146] In yet another aspect of the present invention, the
substrate particle may comprise a micelle. As used herein, the term
"micelle" means a vesicle including a single lipid monolayer
encapsulating an aqueous phase. Micelles may be spherical or
tubular or wormlike and form spontaneously about the critical
micelle concentration (CMC). In general, micelles are in
equilibrium with the monomers under a given set of physical
conditions such as temperature, ionic environment, concentration,
etc.
[0147] Formation of a micelle requires the presence of
"micelle-forming compounds," which include amphipathic lipids
(e.g., a vesicle-forming lipid as described herein or known in the
art), lipoproteins, detergents, non-lipid polymers, or any other
compound capable of either forming or being incorporated into a
monolayer vesicle structure. Thus, a micelle-forming compound
includes compounds that are capable of forming a monolayer by
themselves or when in combination with another compound, and may be
polymer micelles, block co-polymer micelles, polymer-lipid mixed
micelles, or lipid micelles. A micelle-forming compound, in an
aqueous environment, generally has a hydrophobic moiety in contact
with the interior of the vesicle, and a polar head moiety oriented
outwards into the aqueous environment. Hydrophilicity generally
arises from the presence of functional groups such as hydroxyl,
phosphate, carboxyl, sulfate, amino or sulfhydryl groups.
Hydrophobicity generally results from the presence of a long chain
of aliphatic hydrocarbon groups.
[0148] A micelle may be prepared from lipoproteins or artificial
lipoproteins including low density lipoproteins, chylomicrons and
high density lipoproteins. Artificial lipoproteins may also
comprise lipidized protein with targeting capabilities. Uptake of
lipoproteins into cell populations may be facilitated by receptors
present on the target cells. For instance, uptake of low density
lipoproteins into cancerous cells may be facilitated by LDL
receptors present on such cells and uptake of chylomicrons and
lactosylated high density lipoproteins into hepatocytes may be
facilitated by the remnant receptor and the lactosylated receptor
respectively.
[0149] Micelles for use in the present invention may be generated
using a variety of conventional techniques. These techniques
include: simple dispersion by mixing in aqueous or hydroalcoholic
media or media containing surfactants or ionic substances;
sonication, solvent dispersion or any other technique described
herein or known in the art. Different techniques may be appropriate
depending on the type of micelle desired and the physicochemical
properties of the micelle-forming components, such as solubility,
hydrophobicity and behaviour in ionic or surfactant-containing
solutions.
[0150] Micelles for use in the present invention may range from any
value between about 1 nm to about 1000 .mu.m in diameter. In some
embodiments, micelles may be less than about 50 nm in diameter, or
less than about 30 nm in diameter, or less than about 20 nm in
diameter.
[0151] In some embodiments, micelles for use in the present
invention may include a hydrophilic polymer-lipid conjugate, as
described herein or known in the art. As indicated herein, the term
"hydrophilic polymer-lipid conjugate" refers to a lipid, e.g., a
vesicle-forming lipid, covalently joined at its polar head moiety
to a hydrophilic polymer, and is typically made from a lipid that
has a reactive functional group at the polar head moiety in order
to attach the polymer. The covalent linkage may be releasable such
that the polymer may dissociate from the lipid at for example
physiological pH after a variable length of time, such as over
several to many hours. Adlakha-Hutcheon et al., 17(8) NAT.
BIOTECHNOL. 775-779 (1999). Such conjugates may include any
compounds known and routinely utilized in the art of sterically
stabilized liposome technology and technologies which are useful
for increasing circulatory half-life for proteins, including for
example polyethylene glycol (PEG), polyvinyl alcohol, polylactic
acid, polyglycolic acid, polyvinylpyrrolidone, polyacrylamide,
polyglycerol, or synthetic lipids with polymeric head groups. For
example, a distearoyl-phosphatidylethanolamine covalently bonded to
a PEG alone, or in further combination with phosphatidylcholine
(PC), may be used to produce a micelle according to the invention.
The molecular weight of the PEG may be any value between about 500
Daltons to about 10,000 Daltons, inclusive, for example, 1000,
2000, 4000, 6000, 8000, etc. The CMC of the hydrophilic
polymer-lipid conjugate will be dependent on the molecular weight
of the PEG as well as the lipid anchor and the added components
used when preparing mixed micelles (e.g. PEG modified
distearoyl-phosphatidylethanolamine and PC).
[0152] Polymersomes
[0153] In another aspect, the substrate particle of the present
invention may comprise a polymersome. A "polymersome" generally
refers to a vesicle which is assembled from polymers or copolymers
in aqueous solutions. Polymersomes are composed substantially of
synthetic polymers and/or copolymers. Unlike liposomes, a
polymersome does not include lipids or phospholipids as its
majority component. Consequently, polymersomes can be acoustically,
thermally, mechanically, and chemically distinct and, in
particular, more durable and resilient than the most stable of
lipid vesicles. Polymersomes assemble during processes of lamellar
swelling (e.g., by film or bulk rehydration), through an additional
phoresis step, or by other known methods. Like liposomes,
polymersomes form by "self-assembly," a spontaneous, entropy-driven
process of preparing a closed semi-permeable membrane. The choice
of synthetic polymers, as well as the choice of molecular weight of
the polymer, are important. The term "substantially" means that
greater than 50 mole percent (%) of the vesicle components are
composed of synthetic polymers. If desired, greater than
approximately 60%, 70%, 80%, 90%, 95%, or even 100 mole % of the
polymersome components are composed of synthetic polymers.
Polymersomes may be, for example, supramolecular complexes,
stabilized, or otherwise cross-linked.
[0154] The polymersomes of the present invention are composed of a
class of molecules represented by, but not limited to, block
copolymers. For example, one such species is the hydrophilic
polyethyleneoxide (EO) linked to hydrophobic polyethylethylene
(EE). The synthetic diversity of block copolymers provides the
opportunity to make a wide variety of vesicles, of which some
embodiments form bilayer membranes with material properties that
greatly expand what is currently available from the spectrum of
naturally occurring phospholipids.
[0155] Because of the self-assembled bilayer membrane's
preselectivity, materials, (e.g., therapeutic macromolecules) may
be "encapsulated" in the aqueous interior or intercalated into the
hydrophobic membrane core of the polymersomc vesicle. Numerous drug
delivery technologies can be developed from such vesicles, owing to
the numerous unique features of the bilayer membrane and the broad
availability of amphiphiles (e.g., block copolymers).
Alternative Substrates: Antibody Arrays
[0156] In another aspect, the substrate of the present invention
may comprise a fixed matrix. In a particular embodiment, the fixed
matrix may comprise an array or a chip. As used herein, an "array"
is a linear or two-dimensional (or three-dimensional) array of
preferably discrete regions, each having a defined area, formed on
the surface of a solid support. As used herein, an antibody array
is an array of antibodies placed on a chip or other surfaces.
Because the position of each particular group of antibodies on the
array is known, the identities of the target cells and non-target
cells can be determined based on their binding to a particular
position on the array.
[0157] Generally, the array comprises a suitable solid support. By
"solid support" or "solid phase support" is meant any material that
can be modified to contain discrete individual sites appropriate
for the attachment or association of the antibodies and is amenable
to at least one detection method. The solid phase support of the
present invention can be of any solid materials and structures
suitable for supporting antibody-antigen binding. In one
embodiment, the solid phase support comprises at least one
substantially rigid surface on which the antibodies can be
immobilized and a biological containing target and non-target cells
can be probed thereon. The solid supports with which the antibody
array elements are stably associated may be fabricated from a
variety of materials including, but not limited to, plastics,
ceramics, metals, acrylamide, cellulose, nitrocellulose, glass,
polystyrene, polyethylene vinyl acetate, polypropylene,
polymethacrylate, polyethylene, polyethylene oxide, polysilicates,
polycarbonates, Teflon.RTM., fluorocarbons, nylon, silicon rubber,
polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,
and polyamino acids. Solid supports may be two-dimensional or
three-dimensional in form, such as gels, membranes, thin films,
glasses, slides, plates, cylinders, beads, magnetic beads, optical
fibers, woven fibers, etc. In particular embodiments, the array is
a three-dimensional array. A three-dimensional array may comprise a
collection of tagged beads. Each tagged bead has different
antibodies attached to it. Tags are detectable by signaling means
such as color (Luminex, Illumina) and electromagnetic field
(Pharmaseq) and signals on tagged beads can even be remotely
detected (e.g., using optical fibers). The size of the solid
support can be any of the standard array sizes, useful for protein
array technology, and the size may be tailored to fit the
particular machine being used to conduct a diagnosis and/or
detection method of the present invention.
[0158] In a specific embodiment, the solid support and the antibody
may be derivatized with chemical functional groups for subsequent
attachment of the two. Thus, for example, the array can be
derivatized with a chemical functional group including, but not
limited to, amino groups, carboxy groups, oxo groups and thiol
groups. Using these functional groups, the antibodies can be
attached to the solid support using linkers known in the art
including, for example, homo- or hetero-bifunctional linkers. In
addition, in some cases, additional linkers, such as alkyl groups
(including substituted and heteroalkyl groups) may be used.
[0159] Arrays comprising discrete regions or spots can by prepared
using conventional methods including, but not limited to,
microfluidics printing (Rowe et al., 71 ANAL. CHEM. 433-439 (1999)
and Bernard et al., 73 Anal. Chem. 8-12 (2001)), microstamping
(U.S. Pat. Nos. 5,512,131 and 5,731,152, Martin et al., 14 LANGMUIR
3971-3975 (1998), and MacBeath et al., 289 SCIENCE 1760-1763
(2000)), microcontact printing (PCT Publication WO 96/29629), and
electrospray deposition (Morozov et al., 71 Anal. Chem. 1415-1420
(1999) and Moerman et al., 73 ANAL. CHEM. 2183-2189 (2001)). Inkjet
printer heads provide another option for patterning molecules, or
components thereof, to nanometer or micrometer scale sites on the
surface of the substrate or coating formed thereon. See Lemmo et
al., 60 ANAL. CHEM. 543-551 (1997); Roda et al., 28 BIOTECHNIQUES
492-496 (2000), and Silzel et al., 44 CLIN. CHEM. 2036-2043
(1998)); and U.S. Pat. Nos. 5,843,767 and 5,837,860. In some cases,
commercially available arrayers based on capillary dispensing
(OmniGrid.TM. (Genemachines, Inc. (San Carlos, Calif.)) and
High-Throughput Microarrayer from (Intelligent Bio-Instruments
(Cambridge, Mass.)) may also be used.
[0160] The boundaries between the patches of proteins immobilized
on substrate arrays may be integrated as topographic patterns
(physical barriers) or surface functionalities with orthogonal
wetting behavior (chemical barriers). For instance, walls of
substrate material or photoresist may be used to separate some of
the patches from some of the others or all of the patches from each
other. Alternatively, non-bioreactive organic thinfilms, such as
monolayers, with different wettability may be used to separate
patches from one another.
[0161] Generally, the dispensing device includes calibrating means
for controlling the amount of sample deposition, and may also
include a structure for moving and positioning the sample in
relation to the support surface. The volume of fluid to be
dispensed per antibody in an array varies with the intended use of
the array, and available equipment. In particular embodiments, a
volume formed by one dispensation is less than about 100 mL, less
than about 10 mL, and about 1 mL. Solutions of blocking agents may
be applied to the arrays to prevent non-specific binding by
non-target cells and other molecules that have not bound to an
antibody. Solutions of bovine serum albumin (BSA), polyethylene
glycol (PEG), casein, or nonfat milk, for example, may be used as
blocking agents to reduce background binding in subsequent
assays.
[0162] The fixed matrix substrates of the present invention may
comprise a plurality of spots or discrete regions of antibodies
conjugated to such substrate. The size of the spot or discrete
region can be varied and may include, for example, at least about a
10 .mu.m.times.10 .mu.m square. In certain embodiments, an antibody
region may be arranged on an area ranging from about 5
.mu.m.times.about 5 .mu.m to about 500 .mu.m to about 500 .mu.m. In
other embodiments, the area may be greater. More specifically, an
antibody region may be arranged on an area of about 15
.mu.m.times.about 15 .mu.m, about 20 .mu.m.times.about 20 .mu.m,
about 25 .mu.m.times.about 25 .mu.m, about 35 .mu.m.times.about 35
.mu.m, about 50 .mu.m.times.about 50 .mu.m, or more.
[0163] The antibody spot may take any shape including, for example,
a square, a rectangle, a circle, etc. Any suitable pattern of
antibody spots may be used, and the distance between antibody spots
may be varied and/or optimized by one of ordinary skill in the
art.
[0164] Furthermore, the number of antibody molecules bound to a
particular spot or region (the antibody density) may range from
about 1 molecule per site to about 10,000 antibody molecule per
site. In fact, the antibody density may range from about 1 molecule
per .mu.m.sup.2 to about 10,000 molecules per .mu.m.sup.2. More
specifically, the antibody density may range from about 100
molecules per .mu.m.sup.2 to about 5000 molecules per .mu.m.sup.2,
from about 200 molecules per .mu.m.sup.2 to about 1000 molecules
per .mu.m.sup.2. In one specific embodiment, the antibody density
may comprise about 200 molecules per .mu.m.sup.2.
[0165] Each spot or site on the substrate array may comprise one
type of antibody, for example, a spot comprising only anti-PCLP
antibody. Multiple spots of single antibodies may comprise
different concentrations or densities of that particular antibody.
Such an approach may be utilized as a type of titration to reduce
cross-reactivity for selectin ligands that are expressed on normal
and cancer target cells.
[0166] Alternatively, a region of the array may comprise more than
one type of antibody. In a specific embodiment, a spot may comprise
anti-PCLP antibody and anti-CEA antibody. In another embodiment,
the spot may comprise anti-PCLP antibody and anti-CD44v antibody.
In yet another embodiment, the spot may comprise anti-CEA antibody
and anti-CD44v antibody. In another specific embodiment, a spot on
the array may comprise anti-PCLP antibody, anti-CEA antibody, and
anti-CD44v antibody. The multi-antibody spots may be present in
different concentrations or densities of the antibodies as well.
The methods for creating these types of array are known in the art.
See, e.g., Ghosh et al., 24 LANGMUIR 8134-8142 (2008).
Substrate Conjugates
[0167] In another aspect, the present invention contemplates the
conjugation of several molecules to the solid substrate of the
targeting composition. Antibodies, chemotherapeutic agents, imaging
agents, etc. may be conjugated to a substrate using conventional
methods known to those of ordinary skill in the art.
[0168] Antibodies
[0169] The antibody compositions of the present invention may be
bound to the substrate using known techniques including, but not
limited to, by chemical coupling means or by genetic engineering.
Covalent conjugates of an antibody and a substrate can be prepared
by linking chemical moieties of a substrate to functional groups on
amino acid sidechains or at the N-terminus or at the C-terminus of
the antibody. The antibody may also be chemically modified with
other chemical moieties, such as glycosyl groups, lipids,
phosphate, acetyl groups and the lice, to facilitate chemical
coupling.
[0170] To illustrate, there are a large number of chemical
cross-linking agents that are known to those skilled in the art. In
particular embodiments, the cross-linking agents may comprise
heterobifunctional cross-linkers, which can be used to link an
antibody and a substrate in a stepwise manner. Heterobifunctional
cross-linkers provide the ability to design more specific coupling
methods for conjugating to proteins, thereby reducing the
occurrences of unwanted side reactions such as homo-protein
polymers. A wide variety of heterobifunctional cross-linkers are
known in the art. These include, but are not limited to,
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl
(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl
4-(p-maleimidophenyl) butyrate (SMPB),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC);
4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene
(SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP),
succinimidyl 6-[3-(2-pyridyldithio) propionate] hexanoate
(LC-SPDP). Those cross-linking agents having N-hydroxysuccinimide
moieties can be obtained as the N-hydroxysulfosuccinimide analogs,
which generally have greater water solubility. In addition, those
cross-linking agents having disulfide bridges within the linking
chain can be synthesized instead as the alkyl derivatives so as to
reduce the amount of linker cleavage in vivo.
[0171] In addition to the heterobifunctional cross-linkers, there
exist a number of other cross-linking agents including, but not
limited to, homobifunctional and photoreactive cross-linkers.
Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH) and
dimethylpimelimidate-2 HCl (DMP) are examples of useful
homobifunctional cross-linking agents, and
bis-[.beta.-(4-azidosalicylamido)ethyl]disulfide (BASED) and
N-succinimidyl-6(4'-azido-2'-nitrophenyl-amino)hexanoate (SANPAH)
are examples of useful photoreactive cross-linkers for use in the
present invention. For a review of protein coupling techniques, see
Means et al., 1 BIOCONJUGATE CHEMISTRY 2-12 (1990).
[0172] One particularly useful class of heterobifunctional
cross-linkers, included above, contain the primary amine reactive
group, N-hydroxysuccinimide (NHS), or its water soluble analog
N-hydroxysulfosuccinimide (sulfo-NHS). Primary amines (lysine
epsilon groups) at alkaline pHs are unprotonated and react by
nucleophilic attack on NHS or sulfo-NHS esters. This reaction
results in the formation of an amide bond, and release of NHS or
sulfo-NHS as a by-product.
[0173] Another reactive group useful as part of a
heterobifunctional cross-linker is a thiol reactive group. Common
thiol reactive groups include maleimides, halogens, and pyridyl
disulfides. Maleimides react specifically with free sulfhydryls
(cysteine residues) in minutes, under slightly acidic to neutral
(pH 6.5-7.5) conditions. Halogens (iodoacetyl functions) react with
--SH groups at physiological pHs. Both of these reactive groups
result in the formation of stable thioether bonds.
[0174] A heterobifunctional cross-linker may further comprise a
spacer arm or bridge. The bridge is the structure that connects the
two reactive ends. The most apparent attribute of the bridge is its
effect on steric hindrance. In some instances, a longer bridge can
more easily span the distance necessary to link two complex
molecules. For instance, SMPB has a span of 14.5 angstroms.
[0175] In another aspect of the present invention, antibodies may
be used in a "free" state, i.e., without conjugation to a
substrate. Such antibodies may be conjugated with a
chemotherapeutic sagent or an imaging agent including a radioactive
isotope. In one embodiment, at least one anti-PCLP antibody may be
conjugated to I.sup.131. In another embodiment, at least one
anti-CEA antibody may be conjugated to I.sup.131. In yet another
embodiment, at least one anti-CD44v antibody may be conjugated to
I.sup.131.
[0176] Chemotherapeutic Agents
[0177] In particular embodiments, the targeting composition of the
present invention further comprises at least one chemotherapeutic
agent conjugated to a suspendable particle. A "chemotherapeutic
agent" is a compound useful in the treatment of cancer. Examples of
chemotherapeutic agents include, but are not limited to, alkylating
agents such as thiotepa and cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide and
trimethylolomelamine; nitrogen mustards such as chlorambucil,
chlomaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembiehin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitroureas such as cannustine, chlorozotocin, fotemustine,
lomustine, nimustine, ranimustine; antibiotics such as
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,
chromoinycins, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,
idambicin, marcellomycin, mitomycins, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, 5-FU; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;
mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin;
phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide;
procarbazine; PSK.RTM.; razoxane; sizofrran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer, Antony, France); chlorambucil;
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;
vincristine; vinorelbine; navelbine; novantrone; teniposide;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid;
esperamicins; capecitabine; and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene,
onapristone, and toremifene (Fareston); and anti-androgens such as
flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0178] Other Conjugates
[0179] The present invention further contemplates the conjugation
of therapeutic agents that have nucleolytic activity such as a
ribonuclease and a deoxyribonuclease. In addition, a variety of
radioactive isotopes are available for the production of targeting
compositions. Examples include At.sup.222, Ret.sup.86, Sm.sup.153,
Bi.sup.212, .sup.32P and radioactive isotopes of Lu. Suitable
radioactive isotopes may further include, but are not limited to,
astatine.sup.211, .sup.14carbon, .sup.51chromium, .sup.36chlorine,
.sup.57iron, .sup.58cobalt, copper.sup.67, .sup.152Eu,
gallium.sup.67, .sup.3hydrogen, iodine.sup.123, indium.sup.111,
.sup.59ion, .sup.32phosphorus, rhenium.sup.186, .sup.75selenium,
.sup.35sulphur, technicium.sup.99m, and/or yttrium.sup.90.
[0180] In yet another aspect of the present invention, the
particles of the targeting composition may also be conjugated to a
receptor, such as streptavidin, for utilization in tumor
pretargeting. Briefly, the composition-receptor conjugate is
administered to the patient and unbound conjugate is removed from
circulation with a clearing agent. A ligand, such as biotin, which
is conjugated to a cytotoxic agent is then administered.
Imaging Agents and Detection
[0181] The targeting compositions of the present invention may
further comprise an imaging agent. In other embodiments, antibodies
and imaging agents may be conjugated together and used as a
targeting composition without a solid substrate. Examples of
imaging agents include, but are not limited to a radiologic
contrast agent, diatrizoic acid sodium salt dihydrate, an
iodine-containing agent, a barium-containing agent, a fluorescent
imaging agent, Lissamine Rhodamine PE, a stain, a dye, a
radioisotope, a metal, a ferromagnetic compound, a paramagnetic
compound, gadolinium, a superparamagnetic compound, iron oxide, a
diamagnetic compound, and barium sulfate.
[0182] More specifically, suitable imaging agents, according to the
invention can include, but are not limited to those described by
Molecular Probes (Handbook of fluorescent probes and research
products), such as Rhodamine, fluorescein, Texas red, Acridine
Orange, Alexa Fluor (various), Allophycocyanin, 7-aminoactinomycin
D, BOBO-1, BODIPY (various), Calcien, Calcium Crimson, Calcium
green, Calcium Orange, 6-carboxyrhodamine 6G, Cascade blue, Cascade
yellow, DAPI, DiA, DiD, Dil, DiO, DiR, ELF 97, Eosin, ER Tracker
Blue-White, EthD-1, Ethidium bromide, Fluo-3, Fluo4, FM1-43,
FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342,
7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissamine
rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensor
Green, LysoSensor Yellow/Blu, Lysotracker Green FM, Magnesium
Green, Marina Blue, Mitotracker Green FM, Mitotracker Orange
CMTMRos, MitoTracker Red CMXRos, Monobromobimane, NBD amines,
NeruoTrace 500/525 green, Nile red, Oregon Green, Pacific Blue.
POP-1, Propidium iodide, Rhodamine 110, Rhodamine Red,
R-Phycoerythrin, Resorfin, RH414, Rhod-2, Rhodamine Green,
Rhodamine 123, ROX dye, Sodium Green, SYTO blue (various), SYTO
green (Various), SYTO orange (various), SYTOX blue, SYTOX green,
SYTOX orange, Tetramethylrhodamine B, TOT-1, TOT-3, X-rhod-1,
YOYO-1, and YOYO-3.
[0183] Also included within the scope of the present invention are
metal ions generally used for chelation in paramagnetic T1-type MIR
contrast agents, and include di- and tri-valent cations selected
from the group consisting of copper, chromium, iron, gadolinium,
manganese, erbium, europium, dysprosium and holmium. Metal ions
that can be chelated and used for radionuclide imaging according to
the invention include, but are not limited to, metals selected from
the group consisting of gallium, germanium, cobalt, calcium,
indium, iridium, rubidium, yttrium, ruthenium, yttrium, technetium,
rhenium, platinum, thallium and samarium. Additionally metal ions
known to be useful in neutron-capture radiation therapy include
boron and other metals with large nuclear cross-sections. Also
included are metal ions useful in ultrasound contrast, and X-ray
contrast compositions.
[0184] The imaging agent may be conjugated to each of the various
substrate particles described herein and within the scope of the
present invention using methods known to those of ordinary skill in
the art. More specifically, radioactively labeled antibodies of the
present invention may be produced according to well-known methods
in the art. For instance, they can be iodinated by contact with
sodium or potassium iodide and a chemical oxidizing agent such as
sodium hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase. Antibodies may also be labeled with
technetium.sup.99 by ligand exchange process, for example, by
reducing pertechnate with stannous solution, chelating the reduced
technetium onto a Sephadex column and applying the peptide to this
column or by direct labeling techniques, e.g., by incubating
pertechnate, a reducing agent such as SNCl.sub.2, a buffer solution
such as sodium-potassium phthalate solution, and the peptide.
Intermediary functional groups which are often used to bind
radioisotopes which exist as metallic ions to peptides are
diethylenetriaminepentaacetic acid (DTPA) and ethylene
diaminetetracetic acid (EDTA).
[0185] In certain embodiments, the antibodies may be linked to a
secondary binding ligand or to an enzyme (an enzyme tag) that will
generate a colored product upon contact with a chromogenic
substrate. Examples of suitable enzymes include urease, alkaline
phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
Preferred secondary binding ligands are biotin and avidin or
streptavidin compounds. The use of such labels is well known to
those of skill in the art in light and is described, for example,
in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241.
[0186] Furthermore, in a specific embodiment, quantum dots may be
used as an imaging agent itself because of their luminescent
properties. In particular, a quantum dot may emit at visible light
wavelengths, far-red, near-infrared, and infrared wavelengths, and
at other wavelengths, typically in response to absorption below
their emission wavelength.
[0187] Generally, quantum dots (which may also be referred to
interchangeably as a "semiconductor nanocrystal" or a "fluorescent
semiconductor nanocrystals") demonstrate quantum confinement
effects in their luminescent properties. When quantum dots are
illuminated with a primary energy source, a secondary emission of
energy occurs of a frequency that corresponds to the band gap of
the semiconductor material used in the quantum dot. In quantum
confined particles, the band gap is a function of the size of the
nanocrystal.
[0188] The narrow size distribution of the quantum dots allows the
possibility of light emission in narrow spectral widths.
Monodisperse quantum dots have been described in detail in Murray
et al. (J. Am. Chem. Soc., 115:8706 (1993)); in the thesis of
Christopher Murray, "Synthesis and Characterization of II-VI
Quantum Dots and Their Assembly into 3-D Quantum Dot
Superlattices", Massachusetts Institute of Technology, September
1995; and in U.S. patent application Ser. No. 08/969,302 entitled
"Highly Luminescent Color-selective Materials".
[0189] The fluorescence of semiconductor nanocrystals results from
confinement of electronic excitations to the physical dimensions of
the nanocrystals. Quantum dots may have discrete optical
transitions, which are tunable with size (U.S. patent application
Ser. No. 08/969,302 entitled "Highly Luminescent Color-selective
Materials"). Current technology allows good control of their sizes
(for example, between about 12 to about 150 angstroms), and thus,
enables construction of quantum dots that emit light at a desired
wavelength throughout the UV-visible-IR spectrum with a quantum
yield ranging from 30-50% at room temperature in organic solvents
and 10-30% at room temperature in water.
[0190] Quantum dots are capable of fluorescence when excited by
light. The ability to control the size of quantum dots enables one
to construct quantum dots with fluorescent emissions at any
wavelength in the UV-visible-IR region. Therefore, the emissions of
quantum dots are tunable to any desired spectral wavelength.
Furthermore, the emission spectra of monodisperse quantum dots have
linewidths as narrow as 25-30 nm. The linewidths are dependent on
the size heterogeneity of quantum dots in each preparation.
[0191] The imaging agents of the present invention may be detected
using imaging methods and devices known to those of ordinary skill
in the art. The particular imaging method and device depends on the
type of imaging agent used to image a cancer cell. Imaging methods
and devices include, but are not limited to, microscopy, position
emission tomography, single photon emission computed tomography
(SPECT), radioimaing, fluorescence imaging, color imaging,
biophotonic imaging, magnetic resonance imaging (MRI), X-ray, and
computer-assisted tomography.
Methods and Composition for Treatment of Cancer
[0192] The compositions of the present invention are useful in
treating and/or preventing cancer including, but not limited to,
lung, colon, liver, prostate, ovarian, breast, brain, thyroid,
bone, kidney and skin (e.g., melanoma) cancers, as well as cancers
such as leukemia and lymphoma. Further, more specific examples of
cancer include, but are not limited to, malignant and non-malignant
cell growth, leukemia, acute leukemia, acute lymphoblastic leukemia
(ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML),
chromic myelocytic leukemia (CML), chronic lymphocytic leukemia
(CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a
lymphoma, Hodgkin's disease, a malignant lymphoma, non-hodgkin's
lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma,
colorectal carcinoma, pancreatic carcinoma, nasopharyngeal
carcinoma, neural blastoma, malignant histiocytosis, paraneoplastic
syndrome/hypercalcemia of malignancy, solid tumors,
adenocarcinomas, sarcomas, malignant melanoma, hemangioma,
metastatic disease, cancer related bone resorption, cancer related
bone pain, and the like.
[0193] In a specific embodiment, the methods and compositions of
the present invention may be used to treat a primary tumor. In
another embodiment, the methods and compositions of the present
invention may be used to treat or prevent metastasis. In yet
another embodiment, the methods and compositions of the present
invention may be used to treat a secondary tumor. In an alternative
embodiment, the methods and compositions of the present invention
may be used to treat or prevent colon cancer. In a particular
embodiment, the methods and compositions of the present invention
may be used to treat or prevent pancreatic cancer. In a further
embodiment, the methods and compositions of the present invention
may be used to treat or prevent neural blastoma. In a specific
embodiment, the methods and compositions of the present invention
may be used to treat or prevent prostate cancer. In another
embodiment, the methods and compositions of the present invention
may be used to treat or prevent breast cancer. In several
embodiments, the methods and compositions of the present invention
may be used to treat or prevent any cancer in which any one or more
of PCLP, CEA, or CD44v is expressed on tumor cells.
Routes of Administration
[0194] The compositions of the present invention may be
administered by any particular route of administration including,
but not limited to oral, parenteral, subcutaneous, intramuscular,
intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracelebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, iontophoretic means, or transdermal
means.
[0195] Pulmonary/Nasal Administration
[0196] There are a several desirable features of an inhalation
device for administering a composition of the present invention.
For example, delivery by the inhalation device is reliable,
reproducible, and accurate. For pulmonary administration, at least
one composition may be delivered in a particle size effective for
reaching the lower airways of the lung or sinuses. The inhalation
device can optionally deliver small dry particles, e.g. less than
about 10 .mu.m, including about 1-5 .mu.m, for good
respirability.
[0197] According to the invention, at least one composition can be
delivered by any of a variety of inhalation or nasal devices known
in the art for administration of a therapeutic agent by inhalation.
Devices capable of depositing aerosolized formulations in the sinus
cavity or alveoli of a patient include metered dose inhalers,
nebulizers, dry powder generators, sprayers, and the like. Other
devices suitable for directing pulmonary or nasal administration
are also known in the art.
[0198] All such devices can be used for the administration of a
composition in an aerosol. Such aerosols may comprise either
solutions (both aqueous and non aqueous) or solid particles.
Metered dose inhalers like the Ventolin.RTM. metered dose inhaler,
typically use a propellent gas and require actuation during
inspiration. See, e.g., WO 98/35888; WO 94/16970. Dry powder
inhalers like Turbuhaler.RTM. (Astra), Rotahaler.RTM. (Glaxo),
Diskus.RTM. (Glaxo), Spiros.RTM. inhaler (Dura), devices marketed
by Inhale Therapeutics, and the Spinhaler.RTM. powder inhaler
(Fisons), use breath-actuation of a mixed powder. See U.S. Pat.
Nos. 5,458,135; 4,668,218; WO 97/25086; WO 94/08552; WO 94/06498;
and EP 0 237 507, each entirely expressly incorporated herein by
reference. Nebulizers like AERx.RTM., Aradigm, the Ultravent.RTM.
nebulizer (Mallinckrodt), and the Acorn II.RTM. nebulizer (Marquest
Medical Products), produce aerosols from solutions, while metered
dose inhalers, dry powder inhalers, etc., generate small particle
aerosols. These specific examples of commercially available
inhalation devices are intended to be a representative of specific
devices suitable for the practice of the invention, and are not
intended as limiting the scope of the invention.
[0199] Formulations suitable for nasal administration, wherein the
carrier is a solid, include a coarse powder having a particle size,
for example, in the range of 0.001 to 5000 .mu.m which is
administered in the manner in which snuff is administered, i.e., by
rapid inhalation through the nasal passage from a container of the
powder held close up to the nose. Suitable formulations, wherein
the carrier is a liquid, for administration, as for example, a
nasal spray or as nasal drops, include aqueous or oily solutions of
the composition.
[0200] A spray comprising a composition of the present invention
can be produced by forcing a suspension or solution of a
composition disclosed herein through a nozzle under pressure. The
nozzle size and configuration, the applied pressure, and the liquid
feed rate can be chosen to achieve the desired output and particle
size. An electrospray can be produced, for example, by an electric
field in connection with a capillary or nozzle feed.
Advantageously, particles of at least one composition delivered by
a sprayer have a particle size in a range of about less than 1 nm
to less than about 200 .mu.m.
[0201] Compositions of the present invention suitable for use with
a sprayer typically include a composition disclosed herein in an
aqueous solution at a concentration of about 0.0001 .mu.g to about
100 mg of a composition disclosed herein per ml of solution, or any
range or value therein, including, but not limited to, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40,
45, 50, 60, 70, 80, 90 or 100 pg/ml or mg/ml. The composition can
include agents such as an excipient, a buffer, an isotonicity
agent, a preservative, a surfactant, or other known agents of
pharmaceutical compositions.
[0202] A composition of the present invention can also be
administered by a nebulizer such as a jet nebulizer or an
ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed
air source is used to create a high-velocity air jet through an
orifice. As the gas expands beyond the nozzle, a low-pressure
region is created, which draws a solution of composition protein
through a capillary tube connected to a liquid reservoir. The
liquid stream from the capillary tube is sheared into unstable
filaments and droplets as it exits the tube, creating the aerosol.
A range of configurations, flow rates, and baffle types can be
employed to achieve the desired performance characteristics from a
given jet nebulizer. In an ultrasonic nebulizer, high-frequency
electrical energy is used to create vibrational, mechanical energy,
typically employing a piezoelectric transducer. This energy is
transmitted to the formulation either directly or through a
coupling fluid, creating an aerosol including the composition.
Advantageously, the pharmaceutical composition delivered by a
nebulizer have a particle size range of from about less than 1 nm
to less than about 2000 .mu.m.
[0203] Compositions of the present invention suitable for use with
a nebulizer, either jet or ultrasonic, typically include a
concentration of about 0.1 ng to about 100 mg of a composition
disclosed herein per ml of solution, or any range or value therein
including, but not limited to, the individual amounts disclosed for
spray compositions. The composition can include other
pharmaceutical agents such as an excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and those known in
the art for use in nebulizer administration.
[0204] In a metered dose inhaler (MDI), a propellant, a composition
of the present invention, and any excipients or other additives are
contained in a canister as a mixture including a liquefied,
compressed gas. Actuation of the metering valve releases the
mixture as an aerosol, preferably containing a particle size range
of from about less than 1 nm to less than about 2000 .mu.m.
[0205] The desired aerosol particle size can be obtained by
employing a formulation of a composition of the present invention
produced by various methods known to those of skill in the art
including, but not limited to, jet-milling, spray drying, critical
point condensation, and the like. Suitable metered dose inhalers
include those manufactured by 3M or Glaxo and employing a
hydrofluorocarbon propellant.
[0206] Compositions for use with a metered-dose inhaler device will
generally include a finely divided powder containing a composition
disclosed herein as a suspension in a non-aqueous medium, for
example, suspended in a propellant with the aid of a surfactant.
The propellant can be any conventional material employed for this
purpose such as chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon including
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a
(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the
like. In one embodiment, the propellant is a hydrofluorocarbon. The
surfactant can be chosen to stabilize the composition of the
present invention as a suspension in the propellant, to protect the
active agent against chemical degradation, and the like. Suitable
surfactants include sorbitan trioleate, soya lecithin, oleic acid,
or the like. In some cases solution aerosols are preferred using
solvents such as ethanol. One of ordinary skill in the art will
recognize that the methods of the present invention can be achieved
by pulmonary administration of a composition disclosed herein via
devices not described herein.
[0207] For absorption through mucosal surfaces, the compositions
and methods of the present invention for administering a
composition disclosed herein include an emulsion comprising a
plurality of submicron particles, a mucoadhesive macromolecule, a
bioactive peptide, and an aqueous continuous phase, which promotes
absorption through mucosal surfaces by achieving mucoadhesion of
the emulsion particles. See, e.g., U.S. Pat. No. 5,514,670. Mucous
surfaces suitable for application of the emulsions of the present
invention can include corneal, conjunctival, buccal, sublingual,
nasal, vaginal, pulmonary, abdominal, intestinal, and rectal routes
of administration. Compositions for vaginal or rectal
administration such as suppositories, can contain as excipients,
for example, polyalkyleneglycols, vaseline, cocoa butter, and the
like. Compositions for intranasal administration can be solid and
contain excipients, for example, lactose or can be aqueous or oily
solutions of nasal drops. For buccal administration, excipients
include sugars, calcium stearate, magnesium stearate,
pregelinatined starch, and the like. See, e.g., U.S. Pat. No.
5,849,695.
[0208] Transdermal Administration
[0209] In another embodiment, the pharmaceutical compositions of
the present invention may be administered via transdermal routes
using forms of transdermal skin patches well known to those of
ordinary skill in that art. For transdermal administration, a
composition of the present invention is encapsulated in a delivery
device such as a liposome or polymeric nanoparticles,
microparticle, microcapsule, or microspheres (referred to
collectively as particles unless otherwise stated). A number of
suitable devices are known, including particles made of synthetic
polymers such as polyhydroxy acids such as polylactic acid,
polyglycolic acid and copolymers thereof, polyorthoesters,
polyanhydrides, and polyphosphazenes, and natural polymers such as
collagen, polyamino acids, albumin and other proteins, alginate and
other polysaccharides, and combinations thereof. See, e.g., U.S.
Pat. No. 5,814,599. To be administered in the form of a transdermal
delivery system, the dosage administration may be, for example,
continuous rather than intermittent throughout the dosage
regimen.
[0210] Formulations suitable for topical administration to the skin
may be presented as ointments, creams, gels and pastes comprising
the ingredient to be administered in a pharmaceutical acceptable
carrier. A preferred topical delivery system is a transdermal patch
comprising a composition of the present invention.
[0211] Topical compositions containing a composition of the present
invention may be admixed with a variety of carrier materials well
known in the art including alcohols, aloe vera gel, allantoin,
glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl
propionate and the like to form, for example, alcoholic solutions,
topical cleansers, cleansing creams, skin gels, skin lotions, and
shampoos in cream or gel formulations. Examples of such carriers
and methods of formulation may be found in REMINGTON'S
PHARMACEUTICAL SCIENCES (1990). Formulations may contain from about
0.001% to about 40% by weight of the active ingredient. In one
embodiment, the formulations contain from about 0.001% to 10% by
weight of the composition of the present invention.
[0212] Compounds of the present invention may be administered by
bioactive agent delivery systems containing particles suspended in
a polymer matrix. The particles may be microcapsules, microspheres,
nanoparticles, nanospheres, etc., currently known in the art. The
particles should be capable of being entrained intact within a
polymer that is or becomes a gel once inside a biological
environment. The particles can be biodegradable or
non-biodegradable. Many microencapsulation techniques used to
incorporate a bioactive agent into a particle carrier are taught in
the art. See, e.g., U.S. Pat. Nos. 4,652,441; 5,100,669; 4,438,253;
and 5,665,428.
[0213] A polymeric matrix will be biodegradable and exhibit water
solubility at low temperature and will undergo reversible thermal
gelation at physiological mammalian body temperatures. The
polymeric matrix is capable of releasing the composition entrained
within its matrix over time and in a controlled manner. The
polymers are gradually degraded by enzymatic or non-enzymatic
hydrolysis in aqueous or physiological environments. See, e.g.,
U.S. Pat. No. 6,287,588.
[0214] Compounds of the present invention may be administered by a
drug delivery composition comprising particles comprising at least
one chemotherapeutic agent and an antibody composition suspended in
a polymer matrix. The particles may be microcapsules, microspheres,
nanoparticles, or nanospheres currently known in the art. The
particles should be biodegradable and stable in physiological
environments. The particles also permit diffusion of the
chemotherapeutic agent from the core through the matrix at a
predetermined release rate. Ionic chemotherapeutic agents are
suitable for use in the delivery composition of the invention. The
drug delivery compositions may be delivered to a target site
through a variety of known routes of administration. Dosages of the
chemotherapeutic agent incorporated in the targeting composition
will depend on individual needs, the desired effect and on the
chosen route of administration. See, e.g., WO 98/50018.
Dosage Determinations
[0215] In general, the compositions disclosed herein may be used
alone or in concert with other therapeutic agents at appropriate
dosages defined by routine testing in order to obtain optimal
efficacy while minimizing any potential toxicity. The dosage
regimen utilizing a composition of the present invention may be
selected in accordance with a variety of factors including type,
species, age, weight, sex, medical condition of the patient; the
severity of the condition to be treated; the route of
administration; the renal and hepatic function of the patient; and
the particular composition employed. A physician of ordinary skill
can readily determine and prescribe the effective amount of the
drug required to prevent, counter, or arrest the progress of the
condition.
[0216] Optimal precision in achieving concentrations of drug within
the range that yields maximum efficacy with minimal toxicity may
require a regimen based on the kinetics of the composition's
availability to one or more target sites. Distribution,
equilibrium, and elimination of a drug may be considered when
determining the optimal concentration for a treatment regimen. The
dosages of a composition disclosed herein may be adjusted when
combined to achieve desired effects. On the other hand, dosages of
these various therapeutic agents may be independently optimized and
combined to achieve a synergistic result wherein the pathology is
reduced more than it would be if either agent were used alone.
[0217] In particular, toxicity and therapeutic efficacy of a
composition disclosed herein may be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index and it may be expressed
as the ratio LD.sub.50/ED.sub.50. Compositions exhibiting large
therapeutic indices are preferred except when cytotoxicity of the
composition is the activity or therapeutic outcome that is desired.
Although compositions that exhibit toxic side effects may be used,
a delivery system can target such compositions to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects. Generally, the
compositions of the present invention may be administered in a
manner that maximizes efficacy and minimizes toxicity.
[0218] Data obtained from cell culture assays and animal studies
may be used in formulating a range of dosages for use in humans.
The dosages of such compositions lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage may vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any composition used in the methods of the invention,
the therapeutically effective dose may be estimated initially from
cell culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (the concentration of the test composition that achieves
a half-maximal inhibition of symptoms) as determined in cell
culture. Such information may be used to accurately determine
useful doses in humans. Levels in plasma may be measured, for
example, by high performance liquid chromatography.
[0219] Moreover, the dosage administration of the compositions of
the present invention may be optimized using a
pharmacokinetic/pharmacodynamic modeling system. For example, one
or more dosage regimens may be chosen and a
pharmacokinetic/pharmacodynamic model may be used to determine the
pharmacokinetic/pharmacodynamic profile of one or more dosage
regimens. Next, one of the dosage regimens for administration may
be selected which achieves the desired
pharmacokinetic/pharmacodynamic response based on the particular
pharmacokinetic/pharmacodynamic profile. See WO 00/67776, which is
entirely expressly incorporated herein by reference.
Dosages
[0220] More specifically, the compositions may be administered in a
single daily dose, or the total daily dosage may be administered in
divided doses of two, three, or four times daily. In the case of
oral administration, the daily dosage of the compositions may be
varied over a wide range from about 0.1 ng to about 1,000 mg per
patient, per day. The range may more particularly be from about
0.001 ng/kg to 10 mg/kg of body weight per day, about 0.1-100 mg,
about 1.0-50 .mu.g or about 1.0-20 mg per day for adults (at about
60 kg).
[0221] The daily dosage of the pharmaceutical compositions may be
varied over a wide range from about 0.1 ng to about 1000 mg per
adult human per day. For oral administration, the compositions may
be provided in the form of tablets containing from about 0.1 ng to
about 1000 mg of the composition or 0.1, 0.2, 0.5, 1.0, 2.0, 5.0,
10.0, 15.0, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 800, 900, or 1000 milligrams of the composition for the
symptomatic adjustment of the dosage to the patient to be treated.
An effective amount of the composition is ordinarily supplied at a
dosage level of from about 0.1 ng/kg to about 20 mg/kg of body
weight per day. In one embodiment, the range is from about 0.2
ng/kg to about 10 mg/kg of body weight per day. In another
embodiment, the range is from about 0.5 ng/kg to about 10 mg/kg of
body weight per day. The compositions may be administered on a
regimen of about 1 to about 10 times per day.
[0222] In the case of injections, it is usually convenient to give
by an intravenous route in an amount of about 0.0001 .mu.g-30 mg,
about 0.01 .mu.g-20 mg or about 0.01-10 mg per day to adults (at
about 60 kg). In the case of other animals, the dose calculated for
60 kg may be administered as well.
[0223] Doses of a composition of the present invention can
optionally include 0.0001 .mu.g to 1,000 mg/kg/administration, or
0.001 .mu.g to 100.0 mg/kg/administration, from 0.01 .mu.g to 10
mg/kg/administration, from 0.1 .mu.g to 10 mg/kg/administration,
including, but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or
100-500 mg/kg/administration or any range, value or fraction
thereof, or to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0,
1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9,
5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0,
9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0,
13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0,
7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5,
11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9,
16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20,
20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
and/or 5000 .mu.g/ml serum concentration per single or multiple
administration or any range, value or fraction thereof.
[0224] As a non-limiting example, treatment of humans or animals
can be provided as a one-time or periodic dosage of a composition
of the present invention 0.1 ng to 100 mg/kg such as 0.0001, 0.001,
0.01, 0.1 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at
least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or
additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, or 52, or alternatively or
additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20 years, or any combination
thereof, using single, infusion or repeated doses.
[0225] Specifically, the compositions of the present invention may
be administered at least once a week over the course of several
weeks. In one embodiment, the pharmaceutical compositions are
administered at least once a week over several weeks to several
months. In another embodiment, the pharmaceutical compositions are
administered once a week over four to eight weeks. In yet another
embodiment, the pharmaceutical compositions are administered once a
week over four weeks.
[0226] More specifically, the compositions may be administered at
least once a day for about 2 days, at least once a day for about 3
days, at least once a day for about 4 days, at least once a day for
about 5 days, at least once a day for about 6 days, at least once a
day for about 7 days, at least once a day for about 8 days, at
least once a day for about 9 days, at least once a day for about 10
days, at least once a day for about 11 days, at least once a day
for about 12 days, at least once a day for about 13 days, at least
once a day for about 14 days, at least once a day for about 15
days, at least once a day for about 16 days, at least once a day
for about 17 days, at least once a day for about 18 days, at least
once a day for about 19 days, at least once a day for about 20
days, at least once a day for about 21 days, at least once a day
for about 22 days, at least once a day for about 23 days, at least
once a day for about 24 days, at least once a day for about 25
days, at least once a day for about 26 days, at least once a day
for about 27 days, at least once a day for about 28 days, at least
once a day for about 29 days, at least once a day for about 30
days, or at least once a day for about 31 days.
[0227] Alternatively, the compositions may be administered about
once every day, about once every 2 days, about once every 3 days,
about once every 4 days, about once every 5 days, about once every
6 days, about once every 7 days, about once every 8 days, about
once every 9 days, about once every 10 days, about once every 11
days, about once every 12 days, about once every 13 days, about
once every 14 days, about once every 15 days, about once every 16
days, about once every 17 days, about once every 18 days, about
once every 19 days, about once every 20 days, about once every 21
days, about once every 22 days, about once every 23 days, about
once every 24 days, about once every 25 days, about once every 26
days, about once every 27 days, about once every 28 days, about
once every 29 days, about once every 30 days, or about once every
31 days.
[0228] The compositions of the present invention may alternatively
be administered about once every week, about once every 2 weeks,
about once every 3 weeks, about once every 4 weeks, about once
every 5 weeks, about once every 6 weeks, about once every 7 weeks,
about once every 8 weeks, about once every 9 weeks, about once
every 10 weeks, about once every 11 weeks, about once every 12
weeks, about once every 13 weeks, about once every 14 weeks, about
once every 15 weeks, about once every 16 weeks, about once every 17
weeks, about once every 18 weeks, about once every 19 weeks, about
once every 20 weeks.
[0229] Alternatively, the compositions of the present invention may
be administered about once every month, about once every 2 months,
about once every 3 months, about once every 4 months, about once
every 5 months, about once every 6 months, about once every 7
months, about once every 8 months, about once every 9 months, about
once every 10 months, about once every 11 months, or about once
every 12 months.
[0230] Alternatively, the compositions may be administered at least
once a week for about 2 weeks, at least once a week for about 3
weeks, at least once a week for about 4 weeks, at least once a week
for about 5 weeks, at least once a week for about 6 weeks, at least
once a week for about 7 weeks, at least once a week for about 8
weeks, at least once a week for about 9 weeks, at least once a week
for about 10 weeks, at least once a week for about 11 weeks, at
least once a week for about 12 weeks, at least once a week for
about 13 weeks, at least once a week for about 14 weeks, at least
once a week for about 15 weeks, at least once a week for about 16
weeks, at least once a week for about 17 weeks, at least once a
week for about 18 weeks, at least once a week for about 19 weeks,
or at least once a week for about 20 weeks.
[0231] Alternatively the compositions may be administered at least
once a week for about 1 month, at least once a week for about 2
months, at least once a week for about 3 months, at least once a
week for about 4 months, at least once a week for about 5 months,
at least once a week for about 6 months, at least once a week for
about 7 months, at least once a week for about 8 months, at least
once a week for about 9 months, at least once a week for about 10
months, at least once a week for about 11 months, or at least once
a week for about 12 months.
Kits
[0232] The present invention also provides kits for use in treating
and/or diagnosing cancer. The kits of the present invention include
one or more containers comprising targeting compositions (or unit
dosage forms and/or articles of manufacture), and in some
embodiments, further comprise instructions for use in accordance
with any of the methods described herein. The kit may further
comprise a description of selecting an individual suitable or
treatment. Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0233] In some embodiments, the kit comprises a) a targeting
composition comprising a solid substrate, an antibody composition,
and optionally a chemotherapeutic agent, and b) instructions for
administering the targeting composition for treatment of a
proliferative disease such as cancer.
[0234] The kits of the present invention are in suitable packaging.
Suitable packaging include, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Kits may optionally provide additional components such as
buffers and interpretative information.
[0235] The instructions relating to the use of the compositions
generally include information as to dosage, dosing schedule, and
route of administration for the intended treatment. The containers
may be unit doses, bulk packages (e.g., multi-dose packages) or
sub-unit doses. For example, kits may be provided that contain
sufficient dosages of the composition as disclosed herein to
provide effective treatment of an individual for an extended
period, such as any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks,
8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9
months, or more. Kits may also include multiple unit doses of the
compositions and instructions for use and packaged in quantities
sufficient for storage and use in pharmacies, for example, hospital
pharmacies and compounding pharmacies.
[0236] Without further elaboration, it is believed that one skilled
in the art, using the preceding description, can utilize the
present invention to the fullest extent. The following examples are
illustrative only, and not limiting of the remainder of the
disclosure in any way whatsoever.
EXAMPLES
Materials and Methods
[0237] Adhesion Molecules, Antibodies and Reagents. The chimeric
form of E- and P-selectin-IgG Fc (E-selectin; P-selectin)
consisting of the lectin, epidermal growth factor and consensus
repeat domains for human E-selectin and P-selectin, respectively,
linked to each arm of human IgG.sub.i, were obtained from Wyeth
External Research (Cambridge, Mass.). Somers et al., 103 CELL
467-479 (2000). L-selectin-IgG Fc (L-selectin) was purchased from
R&D Systems (Minneapolis, Minn.). Anti-PCLP mAb 3D3 was from
Santa Cruz Biotechonology Inc. (Santa Cruz, Calif.). Fluorescein
isothiocyanate (FITC)-conjugated anti-PCLP antibody 53D11 and
isotype control were from MBL International (Woburn, Mass.).
Alkaline phosphatase (AP)- and Horseradish peroxidase
(HRP)-conjugated anti-mouse IgG and AP-conjugated anti-rat IgM were
from Southern Biotech (Birmingham, Ala.). All other unlabeled and
phycoerythrin (PE)- or FITC-conjugated antibodies were obtained
from BD Biosciences Pharmingen (San Jose, Calif.) unless otherwise
specified. All other reagents were from Sigma (St. Louis, Mo.)
unless otherwise stated.
[0238] Cell Culture. The human colorectal carcinoma cell line
LS174T was obtained from the American Type Culture Collection
(Manassas, Va.), and cultured in the recommended medium. Prior to
cell lysis, LS174T carcinoma cells were detached from culture
flasks using Enzyme Free Cell Dissociation Media (15 min at
37.degree. C.; Chemicon, Phillipsburg, N.J.). Napier et al., 282 J.
BIOL. CHEM. 3433-3441 (2007). For flow cytometric and flow-based
adhesion assays, LS174T cells were harvested by mild trypsinization
(0.25% trypsin/EDTA for 5 min at 37.degree. C.), and subsequently
incubated (10.sup.7 cells/ml) at 37.degree. C. for 2 h to allow
regeneration of surface glycoproteins. Burdick et al., 287 AM. J.
PHYSIOL. CELL PHYSIOL. C539-547 (2004); Mannori et al., 55 CANCER
RES. 4425-4431 (1995); and McCarty et al., 96 BLOOD 1789-1797
(2000). CHO cells, stably transfected with cDNA encoding
full-length E-selectin (CHO-E) or with the phosphatidylinositol
glycan-linked extracellular domain of P-selectin (CHO--P), were
kindly donated by Affymax (Palo Alto, Calif.), and processed as
previously described. Hanley et al., 65 CANCER RES. 5812-5817
(2005); Napier et al., 282 J. BIOL. CHEM. 3433-3441 (2007). Cell
lines were routinely checked and confirmed to be negative for
mycoplasma infection. Jadhav et al., 167J. IMMUNOL. 5986-5993
(2001).
[0239] Colon Carcinoma Cell Lysis and Immunoprecipitation Assays.
Whole cell lysate was prepared by membrane disruption using 2%
NP-40 followed by differential centrifugation. Aigner et al., 89
BLOOD 3385-3395 (1997); and Hofmann et al., 51 CANCER RES.
5292-5297 (1991). PCLP was immunoprecipitated from colon carcinoma
cell lysate with an anti-PCLP mAb, 3D3, using recombinant Protein G
agarose beads (Invitrogen, Carlsbad, Calif.). Hanley et al., 20
FASEB J. 337-339 (2006); Napier et al., 282 J. BIOL. CHEM.
3433-3441 (2007); and Thomas et al., 283 J BIOL CHEM 15647-15655
(2008). In view of immunoblot assays showing that LS174T cells do
not express CD66d, CEA (CD66e) and CD66c were immunoprecipitated
from LS174T colon carcinoma cell lysate with an anti-CD66de mAb,
Col-1, and an anti-CD66c mAb, B6.2, respectively, using recombinant
Protein G agarose beads (Invitrogen, Carlsbad, Calif.).
[0240] Purification and Mass Spectrometry Analysis of 180 kDa,
HECA-452-Reactive LSI 74T Protein. The putative selectin ligands
corresponding to the HECA-452-reactive 180-kDa protein was isolated
by performing affinity chromatography on the whole cell lysate of
CD44-knockdown LS174T cells using KappaLock.TM.-agarose supports
(Invitrogen, Carlsbad, Calif.) crosslinked with
bis(sulfosuccinimidyl)suberate (BS.sup.3) (Pierce Biotechnology,
Rockford, Ill.) to HECA-452 mAb. Thomas et al., 283 J BIOL CHEM
15647-15655 (2008). Eluted proteins were then separated by SDS-PAGE
and stained in gel with ProQ Emerald 300 glycoprotein stain
(Invitrogen), which only binds to carbohydrate groups at
glycosylation sites, thereby leaving the polypeptide core intact.
Thomas et al., 283 J BIOL CHEM 15647-15655 (2008). The stained band
at 180-kDa was then excised and trypsin-digested gel fragments were
submitted for analysis by nano-flow HPLC interfaced to electrospray
ionization tandem mass spectrometry (HPLC-MS/MS) using a
ThermoFinnigan LTQ mass spectrometer. Id. The MS data were searched
against all taxonomies in the NCBI non-redundant protein database
with a 95% significance threshold (p<0.05) using Mascot (Matrix
Science) and with a p<0.01 confidence using the BioWorks 3.3
software featuring the SEQUEST algorithm (ThermoFinnigan).
[0241] SDS-PAGE and Western Blotting. Whole cell lysate or
immunopurified PCLP or CD66e was diluted with reducing sample
buffer, and separated using 4-20% SDS-PAGE gels (Bio-Rad
Laboratories, Hercules, Calif.). Napier et al., 282 J. BIOL. CHEM.
3433-3441 (2007). Resolved proteins were transferred to Sequi-blot
or Immun-blot polyvinylidene difluoride (PVDF) and blocked with
StartingBlock (Pierce Biotechnology, Rockford, Ill.) for 15 min.
Immunoblots were stained with HECA-452, MECA-79, anti-PCLP (3D3),
anti-CD66de (Col-1), or anti-CD66c (B6.2) mAbs, and rinsed with TB
S/0.1% Tween 20. Subsequently, blots were incubated with
appropriate AP- or HRP-conjugated secondary antibodies. Western
Blue AP substrate (Promega, Madison, Wis.) and SuperSignal West
Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford,
Ill.) were used to develop the AP- and HRP-conjugated
antibody-stained immunoblots, respectively.
[0242] Blot Rolling Assay. Blots of immunopurified PCLP or CD66e
from wild-type or CD44-knockdown LS174T whole cell lysate were
stained with anti-PCLP (3D3), anti-CD66de (Col-1), anti-CD66c, or
HECA-452 mAbs, and rendered translucent by immersion in 90%
D-PBS/10% glycerol. Fuhlbrigge et al., 168 J. IMMUNOL. 5645-5651
(2002). The blots were placed under a parallel-plate flow chamber,
and human peripheral blood lymphocytes or CHO transfectants,
re-suspended at 5.times.10.sup.6 cells/ml in 90% D-PBS/10%
glycerol, were perfused at the shear stress of 0.5 dyn/cm.sup.2.
Hanley et al., 20 FASEB J. 337-339 (2006); Napier et al., 282 J.
BIOL. CHEM. 3433-3441 (2007); and Thomas et al., 283 J BIOL CHEM
15647-15655 (2008). Molecular weight markers were used as guides to
aid placement of the flow chamber over stained bands of interest.
The number of interacting cells per lane was averaged over
10.times. fields of view (0.55 mm.sup.2 each) for 5 mM within each
stained region. Non-specific adhesion was assessed by perfusing 5
mM EDTA in the flow medium.
[0243] Preparation of PCLP-Coated and CD66e-Coated Microspheres.
Immunoprecipitated PCLP or CD66e from wild-type or CD44-knockdown
LS174T whole cell lysate was diluted to desired concentrations with
binding buffer (0.2 M carbonate/bicarbonate buffer, pH 9.2), and
incubated with 10 .mu.m polystyrene microspheres
(2.5.times.10.sup.7 microspheres/ml; Polysciences Inc., Warrington,
Pa.) overnight at 4.degree. C. with constant rotation. Hanley et
al., 20 FASEB J. 337-339 (2006); Napier et al., 282 J. BIOL. CHEM.
3433-3441 (2007); and Thomas et al., 283 J BIOL CHEM 15647-15655
(2008). Microspheres were washed 2.times. with D-PBS, and
subsequently blocked with D-PBS/1% BSA for 30 min at RT.
Microspheres were resuspended (2.times.10.sup.6 microspheres/ml) in
D-PBS/0.1% BSA for use in flow cytometric and flow chamber assays.
Site densities of PCLP-coated microspheres were determined by flow
cytometry. Hanley et al., 20 FASEB J. 337-339 (2006); Napier et
al., 282 J. BIOL. CHEM. 3433-3441 (2007); and Thomas et al., 283 J
BIOL CHEM 15647-15655 (2008).
[0244] Enzymatic Treatments. To remove terminal sialic acid
residues, wild-type LS174T PCLP-coated or CD66e-coated microspheres
were incubated with 0.1 U/ml Vibrio cholerae sialidase (Roche
Molecular Biochemicals) for 90 mM at 37.degree. C. Hanley et al.,
20 FASEB J. 337-339 (2006); Napier et al., 282 J. BIOL. CHEM.
3433-3441 (2007). In select experiments, PCLP-coated microsphere
suspensions (5.times.10.sup.6 microspheres/ml) were incubated for 2
h at 37.degree. C. with 120 mg/ml of O-sialoglycoprotein
endopeptidase (OSGE; Accurate Chemical & Scientific, Westbury,
N.Y.) to specifically cleave glycoproteins with O-glycosylation on
serine and threonine residues. Hanley et al., 117 J CELL SCI.
2503-2511 (2004). To cleave N-glycans from CD66e, LS174T whole cell
lysate was pretreated with 8 U/ml N-glycosidase F (EMD Biosciences,
San Diego, Calif.) for 48 h at 37.degree. C., as described
previously before immunoprecipitation. Hanley, et al., 20(2) FASEB
J. 20(2), 337-339 (2006); and Napier, et al., 282(6) J. BIOL. CHEM.
3433-3441 (2007). Site densities of PCLP or CD66e adsorbed onto
microspheres following enzymatic treatments were determined by flow
cytometry before use in flow-based adhesion assays.
[0245] Inhibitor Treatments. Prior to metabolic inhibitor studies,
LS174T cell suspensions (10.sup.7 cells/ml) were pre-treated with
0.1 U/ml Vibrio cholerae sialidase for 60 min at 37.degree. C. to
remove terminal sialic acid residues, and ensure de novo synthesis
of newly generated HECA-452 reactive carbohydrate structures.
Hanley et al., 20 FASEB J. 337-339 (2006); and Napier et al., 282J.
BIOL. CHEM. 3433-3441 (2007). Complete removal of sialylated
structures was confirmed via flow cytometry using the mAb HECA-452
that recognizes sialic acid-bearing epitopes. Subsequently, LS174T
cells were cultured for 48 h at 37.degree. C. in medium containing
either 2 mM benzyl-2-acetamido-2-deoxy-.alpha.-D-galactopyranoside
(benzyl-Ga1NAc) to inhibit O-linked glycosylation, or 1 mM
deoxymannojirimycin (DMJ) to disrupt N-linked processing. Hanley et
al., 20 FASEB J. 337-339 (2006); and Napier et al., 282 J. BIOL.
CHEM. 3433-3441 (2007). D-PBS diluting was used for control
untreated cells.
[0246] Flow Cytometry. PCLP, CD66e, CD66c, and HECA-452 site
densities on microspheres were quantified by single-color
immunofluorescence and flow cytometry (FACSCalibur, BD Biosciences)
using FITC-conjugated anti-PCLP (53D11), PE-conjugated antiCD66
(B1.1), anti-CD66c (B6.2), or HECA-452 mAbs. Similarly, PCLP
expression by colon carcinoma cells was monitored by using the
FITC-conjugated anti-PCLP antibody 53D11. CEACAM expression on
colon carcinoma cells was studied by using primary anti-CD66 mAbs
(CD66a, GM8G5; CD66b, 80H3; CD66c, 9A6; CD66de, Col-1; CD66f, BAP3;
CEACAM7, BAC2) with appropriate PE-conjugated secondary and isotype
control antibodies. Background levels were determined by incubating
cell or microsphere suspensions with properly matched
FITC-conjugated isotype control antibodies. Hanley et al., 20 FASEB
J. 337-339 (2006); and Napier et al., 282 J. BIOL. CHEM. 3433-3441
(2007).
[0247] Flow-Based Adhesion Assays. To simulate the physiological
shear environment of the vasculature, PCLP-coated or CD66e-coated
microspheres suspended in D-PBS/0.1% BSA were perfused over
immobilized IgG- or E-, L- or P-selectin-coated dishes at
prescribed wall shear stresses using a parallel-plate flow chamber
(250 .mu.m channel depth, 5.0 mm channel width). Hanley et al., 20
FASEB J. 337-339 (2006); and Napier et al., 282 J. BIOL. CHEM.
3433-3441 (2007). The extent of adhesion was quantified by
perfusing cells/microspheres at 1.times.10.sup.6/ml and enumerating
the total number of tethering events in a single 10.times. field of
view during a 2 min period. Average rolling velocities were
computed as the distance traveled by the centroid of the
translating cell/microsphere divided by the time interval at the
given wall shear stress. Burdick et al., 287 AM. J. PHYSIOL. CELL
PHYSIOL. C539-547 (2004); Burdick et al., 284 AM. J. PHYSIOL. CELL
PHYSIOL. C977-987 (2003); and McCarty et al., 96 BLOOD 1789-1797
(2000). A minimum of 30 cells was tracked for each condition. In
select experiments, PCLP-coated microspheres, wildtype and
CD44-knockdown LS174T cells, or CD66e-coated microspheres were
perfused over substrates with 5 mM EDTA in the flow medium.
[0248] Preparation of CD66e siRNA Oligonucleotides. Short
interfering (si) RNA oligonucleotides targeting CD66e were
generated using the WI siRNA design program (Whitehead Institute,
Massachusetts Institute of Technology). Napier, et al., 282(6) J.
BIOL. CHEM. 3433-3441 (2007). The siRNA sequences were used to
construct 60-mer short hairpin (sh)RNA oligonucleotides, which were
then synthesized (Operon, Inc., Huntsville, Ala.), and ligated into
the pSUPER.puro.gfp expression vector (Oligoengine, Inc, Seattle,
Wash.) under the control of the H1 promoter. The following
oligonucleotide was used (underlined: sense and antisense
sequences; bold: restriction enzyme sites; italicized: Pol III
termination signal; bold/italicized: loon with linker):
TABLE-US-00002 (SEQ ID NO: 5) (5'-GATCCCCGGACCCTCACTCTATTCAA
TTGAATA GAGTGAGGGTCCTTTTTC-3').
The ligated product was transformed into competent DH5a E. coli
cells, amplified in the presence of ampicillin, and the plasmid was
purified using the EndoFree Maxi Kit (Qiagen, Valencia, Calif.).
Sequence insertion was verified by restriction digestion, and
confirmed by direct sequencing. An empty vector was used as a
negative control in all shRNA experiments.
[0249] Generation of Stable CD66e-knockdown and
CD66e/CD44-double-knockdown Colon Carcinoma Cell Lines. About
8.times.10.sup.6 wildtype or CD44-knockdown LS174T cells were
plated in 100-mm dishes and grown overnight reaching a .about.50%
confluency. The cells were then transfected with 32 .mu.g of
pSUPER.puro.gfp.CD66e using Lipofectamine 2000 for 24 h. Upon
reaching confluency, transfected cells were passed and
5.times.10.sup.6 cells seeded per Petri dish in growth medium in
triplicate. After 24 h, the medium was replaced by a fresh aliquot
containing 2.5 .mu.g/ml puromycin. Cells were then grown
continually without passaging for 15 days, replenishing the
puromycin-containing medium every 2-3 days. Single cell colonies
were isolated and cultured using standard techniques.
[0250] Statistical Analysis. Data are expressed as the mean.+-.SEM
for at least 3 independent experiments. Statistical significance of
differences between means was determined by ANOVA. If means were
shown to be significantly different (p<0.05), multiple
comparisons were performed by the Tukey test.
Example 1
Sialofucosylated PCLP Expressed by LS174T Colon Carcinoma Cells is
an E-/L-, but not P-, Selectin Ligand
[0251] Blot rolling assays revealed the presence of alternative
sialofucosylated glycoprotein(s) with an apparent molecular mass of
.about.170-180-kDa, which can mediate selectin binding in
CD44-knockdown LS174T colon carcinoma cells. Id. To identify and
characterize the putative selectin ligand(s), immunoaffinity
chromatography was performed in order to purify the .about.180-kDa
sialofucosylated glycoprotein from whole cell lysates of
CD44-knockdown LS174T cells using KappaLock.TM.-agarose supports
crosslinked with a HECA-452 mAb. See FIG. 1. Eluted proteins were
separated by SDS-PAGE and stained in gel using ProQ Emerald 300
glycoprotein stain, which fluorescently labels periodate-oxidized
glycans while leaving the polypeptide backbone intact.
Alternatively, the proteins were transferred to PVDF membranes and
immunoblotted using HECA-452 to confirm the retention of the
putative glycoprotein targets throughout purification. This
purification process retained the .about.180 kDa HECA-452-reactive
band in both stained gels and western blots. See FIG. 3A. This band
was then excised and submitted for HPLC-MS/MS analysis of
trypsin-digested fragments. Bioinformatics analysis of the MS data
revealed peptide fragment matches for PCLP (podocalyxin-like
isoform 1 precursor, accession number NP.sub.--001018121;
podocalyxin-like isoform 2 precursor, accession number
NP.sub.--005388).
[0252] A series of experiments were next performed to validate that
PCLP is indeed a selectin ligand in LS174T colon carcinoma cells.
As a first step, Western blots of HECA-452 immunoaffinity product
from CD44-knockdown LS174T cell lysate stained with either an
anti-PCLP mAb, 3D3, or HECA-452 mAb revealed that PCLP is an
.about.180-kDa protein recovered by affinity chromatography (FIG.
3A). Immunoblot analysis using an anti-PCLP mAb, 3D3, also
disclosed the presence of PCLP with an apparent molecular mass of
.about.180-kDa in whole cell lysates from both wild-type and
CD44-knockdown LS174T colon carcinoma cells (FIG. 3B). Using
immunopurified PCLP from wild-type (data not shown) and
CD44-knockdown LS174T cell lysates (FIG. 3C) blotted with HECA-452
mAb, it was demonstrated that PCLP expressed by these cells is
sialofucosylated.
[0253] Using a blot rolling assay, the ability of immunopurified
PCLP to support selectin-dependent adhesion in shear flow was
evaluated. To this end, E- and P-selectin-expressing CHO cells as
well as L-selectin-expressing human peripheral blood lymphocytes
were perfused over the SDS-PAGE resolved immunopurified PCLP
protein band from CD44-knockdown LS174T cells under physiologically
relevant levels of shear stress. This assay revealed that E- and
L-, but not P-, selectin-expressing cells tethered appreciably over
the .about.180 kDa region (Table 2, below), which corresponds to
sialofucosylated PCLP, thereby suggesting that PCLP possesses
E-/L-, but not P-, selectin ligand activity.
TABLE-US-00003 TABLE 2 E-, P- and L-selectin-dependent adhesion to
SDS-PAGE resolved immunopurified PCLP protein band under flow.
CHO-E CHO-P Lymphocyte Blot Condition (E-selectin) (P-selectin)
(L-selectin) Immunopurified +++++ -- *** PCLP E- or
P-selectin-expressing CHO cells or L-selectin-expressing peripheral
blood lymphocytes were perfused at 0.5 dyn/cm.sup.2 over SDS-PAGE
immunoblots of immunopurified PCLP from whole cell lysate of
CD44-knockdown LS174T colon carcinoma cells. The number of
interacting cells per mm.sup.2 was quantified over five fields of
view surrounding the 180-kDa region that marked the center of the
PCLP band. Each "+" represents 50 stationary cells/mm.sup.2, while
an "*" represents 50 transient tethered/rolling cells/mm.sup.2. A
"--" indicates no adhesion.
Example 2
PCLP Serves as an Alternative Glycosylation Acceptor on Colon
Carcinoma Cells
[0254] A cell-free flow-based adhesion assay was used to validate
that PCLP is an E-/L-, but not P-, selectin ligand, and to compare
the adhesion of microbeads coated with PCLP immunoprecipitated from
wild-type versus CD44-knockdown LS174T colon carcinoma cells to
purified selectin substrates under flow. Burdick et al., 287 AM. J.
PHYSIOL. CELL PHYSIOL. C539-547 (2004); Napier et al., 282 J. BIOL.
CHEM. 3433-3441 (2007); and Thomas et al., 283 J BIOL CHEM
15647-15655 (2008). This technique allows quantitative comparisons
of PCLP-mediated adhesion to selectin substrates at prescribed PCLP
and selectin site densities in shear flow. Burdick et al., 287
.mu.m. J. PHYSIOL. CELL PHYSIOL. C539-547 (2004); Napier et al.,
282 J. BIOL. CHEM. 3433-3441 (2007); and Thomas et al., 283 J BIOL
CHEM 15647-15655 (2008). By coating microbeads with equivalent
levels of PCLP from each cell type (FIG. 4A), it was found that
PCLP on CD44-knockdown LS174T cells exhibits higher FIECA-452
immunoreactivity than PCLP on wild-type LS174T cells (FIG. 4B),
thereby suggesting that PCLP serves as an alternative glycosylation
acceptor on colon carcinoma cells. Because sialofucosylated
structures are pivotal to selectin binding function (Burdick et
al., 284 AM. J. PHYSIOL. CELL PHYSIOL. C977-987 (2003); Simon et
al., 7 ANNU REV BIOMED ENG 151-185 (2005); and Varki et al., 100 J
CLIN INVEST S31-35 (1997)), it was hypothesized that this
difference in biochemical reactivity would directly impact the
biophysics of selectin-PCLP interactions. This hypothesis was
tested by perfusing PLCP-coated microbeads from each cell type over
selectin substrates at a wall shear stress of 1 dyn/cm.sup.2. As
expected from blot rolling assays, microbeads coated with PCLP from
either cell type were capable of tethering and rolling over E- and
L-, but not P-, selectin, albeit with varying efficiencies (FIG.
4C). The specificity of PCLP-selectin binding in these assays was
disclosed through the use of nonspecific IgG-bearing microbeads and
by preincubating the selectin-coated dishes with the respective
function-blocking anti-selectin mAb prior to the perfusion of
PCLP-coated microbeads. As an additional control, EDTA (5 mM) was
added to the perfusion medium in select experiments. In all three
control experiments, no microbeads tethered to the selectin
substrates in shear flow. It is noteworthy that the extent of
interaction of CD44-knockdown LS174T PCLP-decorated microbeads with
E-selectin was significantly less than that of microbeads coated
with PCLP from wild-type LS174T cells (FIG. 4C). This difference is
ascribed to the lower average rolling velocities of the former
microbeads (and thus lower number of microspheres entering the
field of observation (41)) relative to wild-type LS174T PCLP-coated
microspheres over a wide range of wall shear stresses (FIG. 4D). On
the other hand, no difference was noted in either the extent of
microbead binding (FIG. 4C) or the average rolling velocities (FIG.
4E) of microbead coated with PCLP from either cell type over
L-selectin.
Example 3
The Selectin Binding Determinants of PCLP are Displayed on
Sialofucosylated O-Linked Glycans
[0255] To characterize the structural linkage-bearing selectin
binding determinants on PCLP, PCLP-coated microbeads were treated
with highly selective enzymes that cleave specific carbohydrate
moieties from the PCLP glycoprotein. Treatment of wild-type LS174T
PCLP-coated microbeads with sialidase eliminated HECA-452
reactivity (FIG. 5B) without affecting the PCLP site density on the
bead surface (FIG. 5A). This intervention nearly abolished
microbead adhesion to L-selectin and reduced binding to E-selectin
by .about.80% (FIG. 5C). It is noteworthy that sialidase treatment
converted the remaining interactions between PCLP-bearing
microbeads and E-selectin from stable rolling to swift tethers.
[0256] To assess the potential contribution of N-linked glycans to
PLCP-selectin interactions, microbeads were generated using PCLP
immunopurified from LS174T cells cultured for 48 h in medium
containing deoxymannojirimycin (DMJ) (1 mM) to disrupt N-linked
processing (17, 44). This treatment did not alter the PCLP site
density on the bead surface (FIG. 5A) or the HECA-452-reactivity
(FIG. 4B), or the extent of bead tethering to E- and L-selectin
substrates under flow (FIGS. 5C, 5D).
[0257] To determine the potential role of O-glycans in
PCLP-selectin binding, microbeads were prepared using PCLP
immunoprecipitated from colon carcinoma cells cultured for 48 h in
medium containing 2 mM benzyl-GalNAc to inhibit O-linked
glycosylation. Hanley et al., 20 FASEB J. 337-339 (2006); and
Napier et al., 282 J. BIOL. CHEM. 3433-3441 (2007). Site densities
of adsorbed PCLP were verified by flow cytometry to be similar to
those of untreated to controls (FIG. 5A). However, benzyl-GalNAc
treatment eliminated HECA-452 reactive epitopes from LS174T PCLP
(FIG. 5B), suggesting that the majority of sLe.sup.x displayed on
PCLP are O-linked glycans. Moreover, PCLP-coated microspheres from
benzyl-GalNAc-treated LS174T cells bound minimally to E- and
L-selectin in shear flow (FIG. 5D), suggesting that the
selectin-binding determinants on PCLP from LS174T colon carcinoma
cells are sialofucosylated structures displayed on O-linked
glycans. The presence of a high level of O-linked glycans on the
LS174T PCLP is further substantiated by the fact that enzymatic
treatment of PCLP-bearing microbeads with OSGE completely
eliminated PCLP detection on the bead surface by flow cytometry
(FIG. 5A).
[0258] Prior work suggested that PCLP on HEVs is a MECA-79-reactive
(sulfated glycan) L-selectin ligand. Sassetti et al., 187 J EXP MED
1965-1975 (1998). To determine whether PCLP on colon carcinoma
cells is MECA-79-reactive, immunopurified PCLP from wild-type
LS174T cells was resolved by SDS-PAGE and stained with MECA-79 via
Western blotting. Surprising, in contrast to PCLP expressed by HEVs
as reported previously (Id.), PCLP expressed by LS174T colon
carcinoma cells is not MECA-79-reactive (data not shown).
Example 4
Identification of CEA as an E- and L-Selectin Ligand Expressed by
Wildtype and CD44-Knockdown LS174T Colon Carcinoma Cells
[0259] The presence of a .about.170-180 kDa sialofucosylated
glycoprotein(s) in CD44-knockdown LS174T colon carcinoma cells that
was capable of mediating selectin binding under flow was
identified. Napier, et al., 282(6) J. BIOL. CHEM. 3433-3441 (2007).
To delineate the identity of the selectin ligand(s), the putative
target was purified from CD44-knockdown LS174T colon carcinoma cell
lysates by affinity chromatography using KappaLock.TM. agarose
beads coated with a HECA-452 mAb (FIG. 1), which detects
sialofucosylated epitopes. Eluted samples were separated by
SDS-PAGE and immunoblotted with HECA-452 to confirm the retention
of the putative target throughout purification. Replica gels were
incubated with ProQ Emerald 300 glycoprotein stain, which
fluorescently labels periodate-oxidized glycans while leaving the
polypeptide backbone intact. The ProQ Emerald 300-stained gel band
corresponding to the .about.180 kDa HECA-452-reactive protein(s)
was excised and digested in-gel with trypsin (FIG. 1). Extracted
peptides were then analyzed by nano-flow HPLC interfaced to
electrospray ionization MS/MS (FIG. 1). Bioinformatics analysis of
the MS data revealed peptide fragment matches for carcinoembryonic
antigen (CEA; CD66e) in two separate sample submissions.
[0260] A series of experiments were performed to confirm the
identity of the selectin ligand. Immunoblot analysis using an
anti-CD66de mAb, Col-1, revealed the presence of CEA with an
apparent molecular mass of .about.180 kDa in CD44-knockdown LS174T
cell lysate, and the lack of CD66d immuno-reactivity at .about.35
kDa (FIG. 6A, lane 1). CEA is enriched in
HECA-452-immunoprecipitated specimens relative to whole cell
lysates (FIG. 6A, lane 3). The presence of HECA-452 reactivity on
CEA was also disclosed by staining immunopurified CEA with a
HECA-452 mAb (FIG. 6A, lanes 5 and 6).
[0261] Although previous studies have reported that CEA and
CEA-family members such as CD66c (Kuijpers, et al., 118(2) J CELL
BIOL. 457-466 (1992)) bind E-selectin under static/no-flow
conditions, the capacity of CEA to interact with E-selectin under
physiologically relevant flow conditions as well as its potential
cross-reactivity with L- and P-selectin have yet to be examined. To
address these issues, E-selectin- and P-selectin-transfected CHO
cells as well as L-selectin-expressing human peripheral blood
lymphocytes were perfused over the SDS-PAGE resolved immunopurified
CEA protein band. The data reveals that E- and L-, but not P-,
selectin expressing cells bind avidly and extensively to
immunopurified CEA from CD44-knockdown colon carcinoma cells (FIG.
6B), suggesting that CEA possesses E- and L-, but not P-, selectin
ligand activity. The specificity of these adhesive interactions was
assessed by incubating CHO-E cell suspensions or lymphocytes with
an anti-E-selectin or an anti-L-selectin function-blocking mAb,
respectively (FIG. 6B). Moreover, CHO-E cells or lymphocytes
suspended in flow medium containing 5 mM EDTA failed to adhere to
any region of the blot (data not shown).
[0262] Using mAbs specific for the various members of the CEA
family of immunoglobulins (CD66a, b, c, e, f, and CEACAM7) along
with indirect single-color immunofluorescence flow cytometry, it
was deteimined that CEA (CD66e) and CD66c, but not CD66a, b, for
CEACAM7, are expressed on the surface of wildtype LS174T colon
carcinoma cells (FIG. 3A). Similarly, only CEA and CD66c are
present on the surface of CD44-knockdown LS174T cells at levels
equivalent to those on wildtype controls (data not shown). The
presence of CEA and CD66c in wildtype LS174T cell lysate was
confirmed by immunoblot analysis (FIGS. 7B, 7C). In accord with our
data using CD44-knockdown colon carcinoma cells, CEA is HECA-452
positive with a molecular mass of .about.180 kDa (FIG. 7B). In
contrast, CD66c is HECA-452 negative (FIG. 7C).
Example 5
The Selectin Binding Determinants of CD66e are Displayed on
Sialofucosylated a-Linked Glycans
[0263] To characterize the structural linkage-bearing selectin
binding determinants on CD66e, CD66ecoated microspheres were
treated with highly selective enz ymes that cleave specific
carbohydrate moieties from the CD66e glycoprotein. Treatment of
wildtype LS174T CD66ecoated microspheres with sialidase eliminated
HECA-452 reactivity (FIG. 9B) without affecting the CD66e site
density on the bead surface (FIG. 9A). This pharmacological
intervention nearly abolished microsphere adhesion to Lselectin and
reduced binding to E-selectin by -70% (FIG. 9C). It is noteworthy
that sialidase treatment converted the remaining interactions
between CD66e-bearing microspheres and E-selectin from stable
rolling to swift tethers.
[0264] To assess the potential contribution of N-linked glycans to
CD66e-selectin interactions, microspheres were coated with wildtype
LS174T CD66e immunoprecipitated from N-glycosidase F-treated
membrane lysate prior to their perfusion over E- and L-selectin
substrates. Alternatively, microspheres were generated using CD66e
immunopurified from LS174T cells cultured for 48 h in medium
containing deoxymannojirimycin (DMJ) (1 mM) to disrupt N-linked
processing. Hanley, et al., 20(2) FASEB J. 20(2), 337-339 (2006);
and Napier, et al., 282(6) J. BIOL. CHEM. 3433-3441 (2007). These
treatments did not alter the CD66e site density on the bead surface
(FIG. 9A) or the HECA-452-reactivity (FIG. 9B), or the extent of
bead tethering to E- and L-selectin substrates under flow (FIGS.
9C, 9D).
[0265] To determine the potential role of O-glycans in
CD66e-selectin binding, microspheres were prepared using CD66e
immunoprecipitated from colon carcinoma cells cultured for 48 h in
medium containing 2 mM benzyl-Ga1NAc to inhibit a-linked
glycosylation. Hanley, et al., 20(2) FASEB J. 20(2), 337-339
(2006); and Napier, et al., 282(6) J. BIOL. CHEM. 3433-3441 (2007).
Site densities of adsorbed CD66e were verified by flow cytometry to
be similar to those of untreated to controls (FIG. 9A). However,
benzyl-GalNAc treatment eliminated HECA-452 reactive epitopes from
LS174T CD66e (FIG. 9B), suggesting that the majority of sLex
displayed on CD66e are O-linked glycans. Moreover, CD66e-coated
microspheres from be nzyl-Ga I NAc-treated LS174 T cells bound
minimally to E- and L-selectin in shear flow (FIG. 9D), suggesting
that the selectin-binding determinants on CD66e from LS174T colon
carcinoma cells are sialofucosylated structures displayed on
O-linked glycans.
Example 6
CEA, but not CD66c, is a Sialofucosylated Selectin Ligand on LS174T
Colon Carcinoma Cells
[0266] A cell-free flow-based adhesion assay (Hanley, et al., 20(2)
FASEB J. 20(2), 337-339 (2006); Napier, et al., 282(6) J. BIOL.
CHEM. 3433-3441 (2007)) was used to compare the adhesion of
microspheres coated with CEA immunopurified from wildtype versus
CD44-knockdown LS174T colon carcinoma cells to selectin substrates
in shear flow. This technique allows quantitative comparisons of
CEA-mediated adhesion to selectin substrates at prescribed CEA and
selectin site densities under physiological flow conditions. By
coating microspheres with equivalent levels of CEA from wildtype
and CD44-knockdown LS174T cells (FIG. 8A), it was determined that
CEA from CD44-knockdown cells is much more densely decorated with
HECA-452-reactive epitopes relative to wildtype LS174T CEA (FIG.
8B). Taken together, these data suggest that CEA serves as an
alternative glycosylation acceptor on colon carcinoma cells.
[0267] It was hypothesized that the difference in HECA-452
immunoreactivity of CEA in wildtype and CD44-knockdown cells could
impact the biophysics of CEA-selectin interactions in shear flow.
To test this hypothesis, the CEA-coated microspheres were perfused
over purified selectin substrates under prescribed wall shear
stress levels. As expected from blot rolling assays, microspheres
coated with CEA from either cell type were capable of tethering and
rolling over E- and L-, but not P-, selectin substrates, albeit
with varying efficiencies (FIG. 8C-E). Most importantly, the extent
of tethering of CD44-knockdown LS174T CEA-coated microspheres to
E-selectin was lower than that of microspheres decorated with CEA
from wildtype LS174T cells (FIG. 8C). This difference is attributed
to the slower average rolling velocities of the former microspheres
(and thus lower number of beads entering the field of observation
(McCarty, et al., 96 BLOOD 1789-1797 (2000))) relative to wildtype
LS174T CEA-coated beads over E-selectin over a wide range of wall
shear stresses varying from 0.5 to 1.5 dyn/cm.sup.2 (FIG. 8D). In
contrast, no difference was detected in either the extent of
tethering (FIG. 8C) or the average rolling velocities (FIG. 8E) of
microspheres coated with CEA from either cell type over L-selectin.
The specificity of CEA-selectin interactions in these assays was
evaluated through the use of nonspecific IgG-coated microspheres
and by pre-incubating the selectin-functionalized dishes with the
respective function-blocking anti-selectin mAb prior to the
perfusion of CEA-coated microspheres. In both cases, no microsphere
tethered to selectin substrates during the entire length of the
flow experiment (FIG. 8C). As an additional control experiment,
CEA-bearing microspheres, perfused over selectin substrates in the
presence of 5 mM EDTA in the perfusion buffer, failed to tether to
either E- or L-selectin under flow (FIG. 8C).
[0268] It was next determined whether CD66c on LS174T cells serves
as a selectin ligand. CD66c was immunopurified from the whole cell
lysate of wildtype and CD44-knockdown LS174T colon carcinoma cells
using the anti-CD66c mAb B6.2. Microspheres coated with CD66c from
either cell type failed to tether to E-selectin substrates beyond
background levels under flow (data not shown). Cumulatively, these
data suggest that the HECA-452-negative CD66c (FIG. 7C) does not
possess E-selectin-ligand activity in LS174T colon carcinoma
cells.
Example 7
CEA and CD44 Cooperate to Mediate Colon Carcinoma Cell Adhesion to
E- and L-Selectin at Elevated Shear Stresses
[0269] To assess the functional role of CEA in the adhesion of
colon carcinoma cells to selectins under flow, stable CEA-knockdown
and CEA/CD44-double knockdown LS174T cell lines were generated by
transfecting wildtype and CD44-knockdown cells, respectively, with
a CEA shRNA plasmid, isolating single cell clones and propagating
these clones in puromycin-containing media. As shown in FIG. 10A,
this procedure resulted in the generation of CEA-knockdown and
CEA/CD44-double knockdown LS174T cells with markedly reduced CEA
surface expression (>95% decrease in MFI) relative to wildtype
and CD44-knockdown LS174T cells transfected with a control plasmid,
as evidenced by flow cytometry using the anti-CD66de mAb Col-1.
Evidence for the specificity of this genetic intervention was
provided by the flow cytometric analysis of other LS174T cell
surface adhesion molecules such as CD29 (FIG. 10A).
[0270] In flow-based adhesion assays, CEA-knockdown LS174T colon
carcinoma cells tethered to E-, L-, and P-selectin substrates under
flow at levels comparable to those of wildtype controls (FIGS. 10B,
10C, 10D). However, CEA knockdown significantly increased the
average rolling velocity of colon carcinoma cells on L-selectin
(Table 3), but not on E-selectin (Table 4) or P-selectin (data not
shown), relative to wildtype cells. On the other hand,
CEA/CD44-double knockdown LS174T colon carcinoma cells displayed a
markedly reduced capacity to tether and roll on purified E-selectin
(.about.50% of control) and L-selectin (.about.70% of control), but
not P-selectin, substrates at a wall shear stress of 2.0
dyn/cm.sup.2, whereas no difference was observed at 1.0
dyn/cm.sup.2 (FIGS. 10B, 10C, 10D). These results were reproducible
using two distinct CEA/CD44-double knockdown cell lines (FIG. 10B).
Moreover, CEA/CD44-knockdown LS174T colon carcinoma cells rolled
with higher rolling velocities over E-selectin relative to
wildtype, CD44-knockdown and CEA-knockdown LS174T cells at 2
dyn/cm.sup.2, while no difference was evident at 1.0 dyn/cm.sup.2
(Table 3). Although CEA/CD44-double knockdown rolled faster than
wildtype controls on L-selectin, no significant difference was
detected between double, CEA- or CD44-knockdown cells. Taken
altogether, these data indicate that CEA serves as an auxiliary
L-selectin ligand, which is engaged in the stabilization of LS174T
cell rolling on L-selectin against fluid shear. Moreover, CEA and
CD44 cooperate to mediate colon carcinoma cell adhesion to E- and
L-selectin at elevated shear stresses.
TABLE-US-00004 TABLE 3 Average Rolling Velocity (.mu.m/s) 1.0
dyn/cm.sup.2 2.0 dyn/cm.sup.2 Wildtype LS174T 182 .+-. 9 320 .+-.
10 CD44-knockdown LS174T 236 .+-. 8* 440 .+-. 10* CEA-knockdown
LS174T 245 .+-. 7* 400 .+-. 20* CEA/CD44-knockdown LS174T 238 .+-.
8* 440 .+-. 20* Average rolling velocities (.mu.m/s) of wildtype,
CD44-knockdown, CEA-knockdown, CEA/CD44-double knockdown LS174T
cells (10.sup.6/ml) perfused over a surface coated with 1.5
.mu.g/ml L-selectin at the physiological shear stress levels of 1.0
or 2.0 dyn/cm.sup.2. Data represent the mean .+-. S.E. *p < 0.05
with respect to wildtype LS174T cells.
TABLE-US-00005 TABLE 4 Average Rolling Velocity (.mu.m/s) 1.0
dyn/cm.sup.2 2.0 dyn/cm.sup.2 Wildtype LS174T 5.3 .+-. 0.3 7.2 .+-.
0.4 CD44-knockdown LS174T 5.1 .+-. 0.3 6.9 .+-. 0.5 CEA-knockdown
LS174T 5.2 .+-. 0.4 7.1 .+-. 0.5 CEA/CD44-knockdown LS174T 5.2 .+-.
0.4 10 .+-. 1*.dagger..sctn. Average rolling velocities (.mu.m/s)
of wildtype, CD44-knockdown, CEA-knockdown, CEA/CD44-double
knockdown LS174T cells (10.sup.6/ml) perfused over a surface coated
with 0.75 .mu.g/ml E-selectin at the physiological shear stress
levels of 1.0 or 2.0 dyn/cm.sup.2. Data represent the mean .+-.
S.E. *p < 0.05 with respect to wildtype LS174T cells. .dagger.p
< 0.05 with respect to CD44-knockdown LS174T cells. .sctn.p <
0.05 with respect to CEA-knockdown LS174T cells.
Example 8
Cancer Cell-Sorting to Prescribed Antibody-Coated Microdomains
[0271] Utilizing a microfabricated surface to capture
antigen-specific cells and triggering their release at later time
point could be a valuable research tool for immunology and cancer
research. Zhu et al., 64 COLLOIDS SURF. B. BIONTERFACES 260268
(2008). By using a serial combination of .mu.CP and microfluidics,
multifunctional surfaces presenting discrete patches of different
proteins on an inert poly(ethylene glycol) (PEG)-functionalized
background (FIG. 11) were prepared. Ghosh et al., 24 LANGMUIR
8134-8142 (2008). This method permitted the entrapment of
CD44-expressing LS174T colon cancer cells onto discrete
microregions of the anti-CD44-antibody patterned glass substrate
from a static adhesion assay, with only weak nonspecific adhesion
to the nonactive area. Using this .mu.CP/microfluidic approach, a
glass slide was also patterned with anti-PSGL1 and anti-CEA
monoclonal antibodies (targeting PSGL1 on leukocytes and CEA on
colon carcinoma cells, respectively), in discrete microdomains
surrounded by inert PEG-functionalized microdomains. Driven by the
high affinity antibody-antigen binding, LS174T colon carcinoma
cells were effectively sorted from PMNs into prescribed patches
under both static and physiological flow conditions (FIG. 12).
Using a microfluidic platform, Toner and colleagues were able to
capture CTCs onto anti-epithelial cell adhesion molecule
(anti-EpCAM)-coated microposts from peripheral blood of cancer
patients. Nagrath et al., 450 NATURE 1235-1239 (2007). Novel
adhesion molecules that are selectively expressed on the surface of
different metastatic tumor cell types (e.g. pancreatic, colon
carcinoma, breast carcinoma and neuroblastoma cells) are used in
the design of bionsensor devices for detecting CTCs in the blood of
cancer patients.
Example 9
Iodide-Labelling of Antibodies
[0272] The antibodies of the present invention may be labeled with
a radioisotope like iodine. Iodide-125 (supplied as NaI) is
oxidized to form iodine-125, which attacks tyrosyl and histidyl
side chains. The iodinated antibodies are easily detected and
quantitated using gamma counters or film.
[0273] IODO-GEN (Thomson Fisher Scientific, Inc. (Rockford, Ill.))
is dissolved at 0.5 .mu.g/ml in chloroform. The IODO-GEN solution
is dispensed into appropriate tubes (e.g., two 6-mm soda glass or
1.5-ml conical tubes) at 100 .mu.l and 20 .mu.l per tube. The
chloroform is allowed to evaporate overnight in a fume hood.
Immediately before the iodination, a gel filtration column is
prepared to separate the labeled antibody from the iodotyrosine
generated during the addition of the IODO-GEN stop buffer. A gel
matrix with an exclusion limit of 20,000-50,000 for globular
proteins is used with medium-sized beads (.about.100 .mu.m in
diameter). The column is prepared with 1 ml of bead volume
according to the manufacturer's instructions (swelling, etc.). A
convenient column for one-time use is be prepared either in a 1-ml
or 2-ml syringe barrel with a glass fiber filter cut to fit the
bottom of the barrel, or in a disposable pipet with a portion of
the cotton plug or some glass wool pushed to the bottom of the
pipet.
[0274] To keep the nonspecific binding of the iodinated proteins to
a minimum, the column is pre-run with at least 10 column volumes of
1% BSA in PBS with 0.02% sodium azide. The column is then washed
with 10 column volumes of PBS with 0.02% sodium azide to remove the
BSA. Alternatively, the beads can be swelled in buffer containing
BSA, and washed with PBS with 0.02% sodium azide before use.
[0275] The column is allowed to run until the buffer level drops to
just below the top of the bed resin. The flow of the column is
stopped either by using a valve at the bottom of the column or by
plugging the end with modeling clay. Immediately before the
iodination, 50 .mu.l of IODO-GEN stop buffer is added to a 1.5-ml
conical tube. This is the "stop tube" that will be used to
terminate the oxidation and capture all of the unincorporated
iodine.
[0276] Behind appropriate shielding in a fume hood, 50 .mu.l of
antibody (0.2-1 mg/ml in 0.5 M sodium phosphate, pH 7.5) is added
to an IODO-GEN-coated tube at room temperature. Other buffers can
be used with the antibody, but no reducing agents are included. 500
.mu.Ci of Na125I is added to the IODO-GEN tube with the antibody.
The pipetting tip is disposed in a container for .sup.125I solid
waste. The tube is incubated for 2 minutes, although longer
incubation times can be used, but may increase the chances of
oxidative damage.
[0277] Using a Pasteur pipet, the contents of the tube are
transferred to the 50 .mu.l of IODO-GEN stop buffer prepared above
and gently mixed. The pipetting tip is disposed in a container for
.sup.125I solid waste. The reaction mixture is carefully applied in
the stop solution to the column prepared above. The pipetting tip
is disposed in a container for .sup.125I solid waste.
[0278] The flow of the column is released and the eluate is
collected in a 1.5-ml conical tube. After the iodinated proteins
has run into the beads, 0.3 ml of PBS with 0.02% sodium azide is
carefully added to the column. The eluate continues to be collected
in the first tube. When the buffer reaches the top of the beads,
the collection tube is changed to a second 1.5-ml conical tube, and
then a second 0.3 ml of PBS with 0.02% sodium azide is added to the
column. The eluate continues to be collected in steps.
[0279] The tubes are monitored using a minimonitor to identify the
peaks of .sup.125I-labeled antibody and unincorporated label. The
fractions containing the iodinated antibody are pooled. The
antibody should come off in approximately the second to fourth
fraction, well ahead of the blue xylenecyanol, which should run
with the unincorporated label. It is often convenient to dispose of
the unincorporated label by leaving it on the column and putting
the entire column into the .sup.125I solid waste.
[0280] The labeled antibody may be stored in the column buffer of
1% PBS in BSA with 0.02% sodium azide at 4.degree. C. The antibody
is stable, but the radioactive iodine is not, so the antibody is
used within 6 weeks of preparation. Up to 90% of the input iodine
can be incorporated. This labeling procedure yields specific
activities between 1 .mu.Ci/.mu.g and 45 .mu.Ci/.mu.g. These levels
can be adjusted by varying the input iodine and protein
concentrations.
[0281] See COLD SPRING HARE. PROTOC. (2006). See also Fraker, P. J.
and Speck Jr., J. C., 80 BIOCHEM. BIOPHYS. RES. COMMUN. 849-857
(1978) ("Protein and cell membrane iodinations with a sparingly
soluble chloroamide,
1,3,4,6-tetra-chloro-3a,6a-diphenylglycoluril").
* * * * *