U.S. patent application number 15/235482 was filed with the patent office on 2016-12-01 for antibodies reactive with an epitope located in the n-terminal region of muc5ac comprising cysteine-rich subdomain 2 (cys2).
The applicant listed for this patent is Immunomedics, Inc.. Invention is credited to Chien-Hsing Chang, David M. Goldenberg, Donglin Liu.
Application Number | 20160347857 15/235482 |
Document ID | / |
Family ID | 54354760 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160347857 |
Kind Code |
A1 |
Liu; Donglin ; et
al. |
December 1, 2016 |
ANTIBODIES REACTIVE WITH AN EPITOPE LOCATED IN THE N-TERMINAL
REGION OF MUC5AC COMPRISING CYSTEINE-RICH SUBDOMAIN 2 (CYS2)
Abstract
The present invention concerns compositions and methods of use
of antibodies or antibody fragments that bind to an epitope located
within the second cysteine-rich domain (Cys2, amino acid residues
1575-1725) of MUC5AC. The antibodies bind with high specificity and
selectivity to pancreatic cancer and are of use for therapy,
detection and/or diagnosis of pancreatic cancer. In preferred
embodiments, therapeutic antibody may be conjugated to at least one
therapeutic agent, such as .sup.90Y. Both in vivo and in vitro
detection of pancreatic cancer may be performed with the subject
methods and compositions. Specific dosages of radiolabeled antibody
and/or gemcitabine, of use in human pancreatic cancer patients, are
disclosed herein.
Inventors: |
Liu; Donglin; (Kendall Park,
NJ) ; Chang; Chien-Hsing; (Downingtown, PA) ;
Goldenberg; David M.; (Mendham, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunomedics, Inc. |
Morris Plains |
NJ |
US |
|
|
Family ID: |
54354760 |
Appl. No.: |
15/235482 |
Filed: |
August 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14753249 |
Jun 29, 2015 |
9452228 |
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15235482 |
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14632480 |
Feb 26, 2015 |
9238084 |
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14753249 |
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14242138 |
Apr 1, 2014 |
9005613 |
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14632480 |
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61807176 |
Apr 1, 2013 |
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61818708 |
May 2, 2013 |
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61896909 |
Oct 29, 2013 |
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62018989 |
Jun 30, 2014 |
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62091932 |
Dec 15, 2014 |
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62148428 |
Apr 16, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/28 20130101;
A61K 45/06 20130101; A61K 51/08 20130101; C07K 2317/77 20130101;
C07K 2317/34 20130101; A61B 5/055 20130101; A61K 31/7068 20130101;
B82Y 5/00 20130101; C07K 2317/54 20130101; A61K 31/519 20130101;
A61K 38/29 20130101; A61K 39/39558 20130101; A61K 51/1093 20130101;
C07K 2317/24 20130101; A61K 51/109 20130101; A61K 51/1096 20130101;
A61K 51/1045 20130101; A61K 38/212 20130101; A61K 51/0495 20130101;
C07K 2317/31 20130101; G01N 2333/4725 20130101; A61K 38/20
20130101; C07K 2317/55 20130101; A61K 2300/00 20130101; A61K 38/193
20130101; C07K 2317/33 20130101; A61K 31/5377 20130101; A61K
31/7068 20130101; C07K 16/303 20130101; A61K 2300/00 20130101; A61K
38/21 20130101; C07K 2317/567 20130101; A61K 38/215 20130101; A61K
51/088 20130101; A61K 2039/507 20130101; A61K 51/1057 20130101;
A61K 51/0406 20130101; B82Y 15/00 20130101; G01N 33/57438 20130101;
A61K 38/19 20130101; A61K 31/404 20130101; A61K 39/39558 20130101;
A61K 2039/505 20130101; A61K 33/24 20130101; A61K 38/217 20130101;
C07K 2317/30 20130101; C07K 2317/565 20130101; C07K 16/44 20130101;
A61K 51/0491 20130101; C07K 16/3092 20130101; A61K 41/0038
20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 45/06 20060101 A61K045/06; C07K 16/44 20060101
C07K016/44; G01N 33/574 20060101 G01N033/574; A61K 47/48 20060101
A61K047/48; A61K 51/08 20060101 A61K051/08; A61K 41/00 20060101
A61K041/00; A61K 51/10 20060101 A61K051/10; A61K 31/7068 20060101
A61K031/7068 |
Claims
1. A method of treating a cancer that expresses MUC5ac comprising
administering to a human subject with a cancer that expresses
MUC5ac a humanized anti-MUC5ac monoclonal antibody or
antigen-binding fragment thereof that binds to an epitope located
within the second cysteine-rich domain (Cys2, amino acid residues
1575-1725) of MUC5ac, wherein the anti-MUC5ac antibody is
conjugated to at least one therapeutic agent.
2. The method of claim 1, wherein the cancer is selected from the
group consisting of gastric, colorectal, lung, biliary and
pancreatic adenocarcinoma.
3. The method of claim 1, wherein the cancer is pancreatic
adenocarcinoma.
4. The method of claim 1, wherein the therapeutic agent is selected
from the group consisting of a radionuclide, an immunomodulator, a
hormone, a hormone antagonist, an enzyme, an anti-sense
oligonucleotide, siRNA, an enzyme inhibitor, a photoactive
therapeutic agent, a cytotoxic agent, a drug, a toxin, an
angiogenesis inhibitor and a pro-apoptotic agent.
5. The method of claim 4, wherein the radionuclide is selected from
the group consisting of .sup.14C, .sup.13N, .sup.15O, .sup.32P,
.sup.33P, .sup.47Sc, .sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe,
.sup.62Cu, .sup.67Cu, .sup.67Ga, .sup.67Ga, .sup.75Br, .sup.75Se,
.sup.75Se, .sup.76Br, .sup.77As, .sup.77Br, .sup.80mBr, .sup.89Sr,
.sup.90Y, .sup.95Ru, .sup.97Ru, .sup.99Mo, .sup.99mTc, .sup.103mRh,
.sup.103Ru, .sup.105Rh, .sup.105Ru, .sup.107Hg, .sup.109Pd,
.sup.109Pt, .sup.111Ag, .sup.111In, .sup.113mIn, .sup.119Sb,
.sup.121mTe, .sup.122mTe, .sup.125I, .sup.125mTe, .sup.126I,
.sup.131I, .sup.133I, .sup.142Pr, .sup.143Pr, .sup.149Pm,
.sup.152Dy, .sup.153Sm, .sup.161Ho, .sup.161Tb, .sup.165Tm,
.sup.166Dy, .sup.166Ho, .sup.167Tm, .sup.168Tm, .sup.169Er,
.sup.169Yb, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.189mOs,
.sup.189Re, .sup.192Ir, .sup.194Ir, .sup.197Pt, .sup.198Au,
.sup.199Au, .sup.199Au, .sup.201Tl, .sup.203Hg, .sup.211At,
.sup.211Bi, .sup.211Pb, .sup.212Bi, .sup.212Pb, .sup.213Bi,
.sup.215Po, .sup.217At, .sup.219Rn, .sup.221Fr, .sup.223Ra,
.sup.224Ac, .sup.225Ac, .sup.255Fm and Th.sup.227.
6. The method of claim 5, wherein the radionuclide is .sup.90Y.
7. The method of claim 4, wherein the drug is selected from the
group consisting of 5-fluorouracil, afatinib, aplidin, azaribine,
anastrozole, anthracyclines, axitinib, AVL-101, AVL-291,
bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1,
busulfan, calicheamycin, camptothecin, carboplatin,
10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,
cisplatinum, Cox-2 inhibitors, irinotecan (CPT-11), SN-38,
carboplatin, cladribine, camptothecans, crizotinib,
cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,
docetaxel, dactinomycin, daunorubicin, doxorubicin,
2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX, cyano-morpholino
doxorubicin, doxorubicin glucuronide, epirubicin glucuronide,
erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat,
estrogen receptor binding agents, etoposide (VP16), etoposide
glucuronide, etoposide phosphate, exemestane, fingolimod,
floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine,
flutamide, farnesyl-protein transferase inhibitors, flavopiridol,
fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,
gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,
ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide,
leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,
nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel,
PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib,
streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an
aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vatalanib,
vinorelbine, vinblastine, vincristine, vinca alkaloids and
ZD1839.
8. The method of claim 4, wherein the toxin is elected from the
group consisting of ricin, abrin, alpha toxin, saporin,
ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, ranpirnase, gelonin, diphtheria toxin,
Pseudomonas exotoxin, and Pseudomonas endotoxin.
9. The method of claim 4, wherein the immunomodulator is selected
from the group consisting of a cytokine, a stem cell growth factor,
a lymphotoxin, a hematopoietic factor, a colony stimulating factor
(CSF), an interferon (IFN), erythropoietin, thrombopoietin tumor
necrosis factor (TNF), granulocyte-colony stimulating factor
(G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF),
interferon-.alpha., interferon-.beta., interferon-.gamma.,
interferon-.lamda., human growth hormone, N-methionyl human growth
hormone, bovine growth hormone, parathyroid hormone, thyroxine,
insulin, proinsulin, relaxin, prorelaxin, follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), luteinizing
hormone (LH), hepatic growth factor, prostaglandin, fibroblast
growth factor, prolactin, placental lactogen, OB protein, tumor
necrosis factor-.alpha., tumor necrosis factor-.beta.,
mullerian-inhibiting substance, mouse gonadotropin-associated
peptide, inhibin, activin, vascular endothelial growth factor,
integrin, thrombopoietin (TPO), NGF-.beta., platelet-growth factor,
TGF-.alpha., TGF-.beta., insulin-like growth factor-I, insulin-like
growth factor-II, erythropoietin (EPO), macrophage-CSF (M-CSF),
IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin,
endostatin, and lymphotoxin.
10. The method of claim 4, wherein the therapeutic agent is a
tyrosine kinase inhibitor selected from the group consisting of
canertinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib,
leflunomide, nilotinib, pazopanib, semaxinib, sorafenib, sunitinib,
sutent, vatalanib, PCI-32765 (ibrutinib), PCI-45292, GDC-0834,
LFM-A13 and RN486.
11. The method of claim 1, further comprising administering at
least one other therapeutic agent to the human subject, wherein the
at least one other therapeutic agent is selected from the group
consisting of a second antibody, a second antigen-binding antibody
fragment, an immunoconjugate, an immunomodulator, a hormone, a
hormone antagonist, an enzyme, an anti-sense oligonucleotide,
siRNA, an enzyme inhibitor, a photoactive therapeutic agent, a
cytotoxic agent, a drug, an angiogenesis inhibitor and a
pro-apoptotic agent.
12. The method of claim 11, wherein the second antibody, second
antigen-binding antibody fragment, or immunoconjugate binds to an
antigen selected from the group consisting of CA19.9, DUPAN2,
SPAN1, Nd2, B72.3, CC49, Le.sup.a, Le(y), CEACAM5, CEACAM6, CSAp,
MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC16, MUC17, HLA-DR, CD40, CD74,
CD138, HER2/neu, EGFR, EGP-1, EGP-2, VEGF, PlGF, insulin-like
growth factor, tenascin, platelet-derived growth factor, IL-6,
bcl-2, K-ras, p53 and cMET.
13. The method of claim 11, wherein the second antibody, second
antigen-binding antibody fragment, or immunoconjugate is selected
from the group consisting of hR1 (anti-IGF-1R), hPAM4
(anti-MUC5ac), hIMMU-31 (anti-AFP), hLL1 (anti-CD74), hMu-9
(anti-CSAp), hL243 (anti-HLA-DR), hL243 IgG4P (anti-HLA-DR), hMN-14
(anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1 or
anti-TROP-2), hMN-3 (anti-CEACAM6), Ab124 (anti-CXCR4) and Ab125
(anti-CXCR4).
14. A method of treating a cancer that expresses MUC5ac comprising:
a. administering to a human subject with a cancer that expresses
MUC5ac a bispecific antibody comprising (i) a humanized anti-MUC5ac
monoclonal antibody or antigen-binding fragment thereof that binds
to an epitope located within the second cysteine-rich domain (Cys2,
amino acid residues 1575-1725) of MUC5ac, and (ii) an anti-hapten
antibody or antigen-binding fragment thereof; and b. administering
to the individual a targetable construct comprising at least one
copy of the hapten, wherein the targetable construct is conjugated
to at least one therapeutic agent.
15. The method of claim 14, wherein the hapten is HSG or
In-DTPA.
16. The method of claim 14, wherein the cancer is selected from the
group consisting of gastric, colorectal, lung, biliary and
pancreatic adenocarcinoma.
17. The method of claim 14, wherein the cancer is pancreatic
adenocarcinoma.
18. The method of claim 14, wherein the therapeutic agent is
selected from the group consisting of a radionuclide, an
immunomodulator, a hormone, a hormone antagonist, an enzyme, an
anti-sense oligonucleotide, siRNA, an enzyme inhibitor, a
photoactive therapeutic agent, a cytotoxic agent, a drug, a toxin,
an angiogenesis inhibitor and a pro-apoptotic agent.
19. The method of claim 18, wherein the radionuclide is selected
from the group consisting of .sup.14C, .sup.13N, .sup.15O,
.sup.32P, .sup.33P, .sup.47Sc, .sup.51Cr, .sup.57Co, .sup.58Co,
.sup.59Fe, .sup.62Cu, .sup.67Cu, .sup.67Ga, .sup.67Ga, .sup.75Br,
.sup.75Se, .sup.75Se, .sup.76Br, .sup.77As, .sup.77Br, .sup.80mBr,
.sup.89Sr, .sup.90Y, .sup.95Ru, .sup.97Ru, .sup.99Mo, .sup.99mTc,
.sup.103mRh, .sup.103Ru, .sup.105Rh, .sup.105Ru, .sup.107Hg,
.sup.109Pd, .sup.109Pt, .sup.111Ag, .sup.111In, .sup.113mIn,
.sup.119Sb, .sup.121mTe, .sup.122mTe, .sup.125I, .sup.125mTe,
.sup.126I, .sup.131I, .sup.133I, .sup.142Pr, .sup.143Pr,
.sup.149Pm, .sup.152Dy, .sup.153Sm, .sup.161Ho, .sup.161Tb,
.sup.165Tm, .sup.166Dy, .sup.166Ho, .sup.167Tm, .sup.168Tm,
.sup.169Er, .sup.169Yb, .sup.177Lu, .sup.186Re, .sup.188Re,
.sup.189mOs, .sup.189Re, .sup.192Ir, .sup.194Ir, .sup.197Pt,
.sup.198Au, .sup.199Au, .sup.199Au, .sup.201Tl, .sup.203Hg,
.sup.211At, .sup.211Bi, .sup.211Pb, .sup.212Bi, .sup.212Pb,
.sup.213Bi, .sup.215Po, .sup.217At, .sup.219Rn, .sup.221Fr,
.sup.223Ra, .sup.224Ac, .sup.225Ac, .sup.255Fm and Th.sup.227.
20. The method of claim 18, wherein the radionuclide is
.sup.90Y.
21. The method of claim 18, wherein the drug is selected from the
group consisting of 5-fluorouracil, afatinib, aplidin, azaribine,
anastrozole, anthracyclines, axitinib, AVL-101, AVL-291,
bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1,
busulfan, calicheamycin, camptothecin, carboplatin,
10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil,
cisplatinum, Cox-2 inhibitors, irinotecan (CPT-11), SN-38,
carboplatin, cladribine, camptothecans, crizotinib,
cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,
docetaxel, dactinomycin, daunorubicin, doxorubicin,
2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX, cyano-morpholino
doxorubicin, doxorubicin glucuronide, epirubicin glucuronide,
erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat,
estrogen receptor binding agents, etoposide (VP16), etoposide
glucuronide, etoposide phosphate, exemestane, fingolimod,
floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine,
flutamide, farnesyl-protein transferase inhibitors, flavopiridol,
fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,
gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,
ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide,
leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,
nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel,
PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib,
streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an
aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vatalanib,
vinorelbine, vinblastine, vincristine, vinca alkaloids and
ZD1839.
22. The method of claim 18, wherein the toxin is elected from the
group consisting of ricin, abrin, alpha toxin, saporin,
ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas
exotoxin, and Pseudomonas endotoxin.
23. The method of claim 18, wherein the immunomodulator is selected
from the group consisting of a cytokine, a stem cell growth factor,
a lymphotoxin, a hematopoietic factor, a colony stimulating factor
(CSF), an interferon (IFN), erythropoietin, thrombopoietin tumor
necrosis factor (TNF), granulocyte-colony stimulating factor
(G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF),
interferon-.alpha., interferon-.beta., interferon-.gamma.,
interferon-.lamda., human growth hormone, N-methionyl human growth
hormone, bovine growth hormone, parathyroid hormone, thyroxine,
insulin, proinsulin, relaxin, prorelaxin, follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), luteinizing
hormone (LH), hepatic growth factor, prostaglandin, fibroblast
growth factor, prolactin, placental lactogen, OB protein, tumor
necrosis factor-.alpha., tumor necrosis factor-.beta.,
mullerian-inhibiting substance, mouse gonadotropin-associated
peptide, inhibin, activin, vascular endothelial growth factor,
integrin, thrombopoietin (TPO), NGF-.beta., platelet-growth factor,
TGF-.alpha., TGF-.beta., insulin-like growth factor-I, insulin-like
growth factor-II, erythropoietin (EPO), macrophage-CSF (M-CSF),
IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin,
endostatin, and lymphotoxin.
24. The method of claim 18, wherein the therapeutic agent is a
tyrosine kinase inhibitor selected from the group consisting of
canertinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib,
leflunomide, nilotinib, pazopanib, semaxinib, sorafenib, sunitinib,
sutent, vatalanib, PCI-32765 (ibrutinib), PCI-45292, GDC-0834,
LFM-A13 and RN486.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/753,249, filed Jun. 29, 2015, which was a
continuation-in-part of U.S. patent application Ser. No. 14/632,480
(now U.S. Pat. No. 9,238,084) filed Feb. 26, 2015, which was a
divisional of U.S. patent application Ser. No. 14/242,138 (now U.S.
Pat. No. 9,005,613), filed Apr. 1, 2014, which claimed the benefit
under 35 U.S.C. 119(e) of provisional U.S. Patent Application Ser.
No. 61/807,176, filed Apr. 1, 2013, 61/818,708, filed May 2, 2013,
and 61/896,909, filed Oct. 29, 2013. This application claims the
benefit under 35 U.S.C. 119(e) of provisional U.S. Patent
Application Ser. No. 62/018,989, filed Jun. 30, 2014, 62/091,932,
filed Dec. 15, 2014, and 62/148,428, filed Apr. 16, 2015, the
entire text of each priority application incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 16, 2015, is named IMM352US1_SL.txt and is 56,774 bytes in
size.
FIELD OF THE INVENTION
[0003] This invention relates to anti-pancreatic cancer antibodies
and antigen-binding fragments thereof that bind to MUC5AC mucin in
pancreatic cancer. Preferably, the antibodies or fragments thereof
bind to an epitope located within the second to fourth
cysteine-rich subdomains of MUC5AC (amino acid residues 1575-2052,
Cys2-Cys4). More preferably, the antibodies bind to an epitope
located in amino acid residues 1575-1725 and 1903-2052 (Cys2 and
Cys4). Even more preferably, the antibodies bind to an epitope
located in amino acid residues 1575-1725 (Cys2+). Most preferably,
the antibodies bind to an epitope located in the Cys2 subdomain of
MUC5AC. In preferred embodiments, the anti-pancreatic cancer
antibody binds to the same epitope as, or competes for binding to
MUC5AC with a PAM4 antibody comprising the light chain variable
region complementarity-determining region (CDR) sequences CDR1
(SASSSVSSSYLY, SEQ ID NO: 1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3
(HQWNRYPYT, SEQ ID NO:3); and the heavy chain CDR sequences CDR1
(SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and
CDR3 (GFGGSYGFAY, SEQ ID NO:6). The subject antibodies or antibody
fragments bind with high selectivity to pancreatic cancer cells to
allow detection and/or diagnosis of pancreatic adenocarcinoma at
the earliest stages of the disease. Most preferably, antibody-based
assays are capable of detecting about 85% or more of pancreatic
adenocarcinomas, with a false positive rate of about 5% or less for
healthy controls. In particular embodiments, the methods and
compositions can be used to detect and/or diagnose pancreatic
adenocarcinoma by screening serum samples from subjects and
preferably can detect 60% or more of stage I pancreatic cancers and
80% or more of stage II cancers by serum sample analysis. In still
other embodiments, reactivity with the anti-pancreatic cancer
antibody can be used to detect occult pancreatic cancer or
neoplastic precursor lesions against a background of pancreatitis
or benign pancreatic hyperplasia.
[0004] The anti-pancreatic cancer antibody is of use for diagnosis,
detection and/or treatment of pancreatic cancer. For therapy, the
antibody or fragment thereof may be administered as a naked
antibody, as an antibody complex, as an antibody fusion protein, or
conjugated to at least one therapeutic agent, such as a
radionuclide, an immunomodulator, a hormone, a hormone antagonist,
an enzyme, an oligonucleotide such as an anti-sense oligonucleotide
or a siRNA, an enzyme inhibitor, a photoactive therapeutic agent, a
cytotoxic agent such as a drug or toxin, an angiogenesis inhibitor
and a pro-apoptotic agent. Most preferably, the antibody is a
.sup.90Y-hPAM4 antibody and the radiolabeled antibody may be
administered in fractionated dosages for treating pancreatic
cancer.
BACKGROUND
[0005] The number of patients who succumb to pancreatic ductal
adenocarcinoma (PDAC) each year continues to rise, unlike other
leading cancers where surveillance and/or screening technologies
have led to a decrease in cancer-related mortality rates (Cardin
& Berlin, 2013, J Natl Cancer Inst 105:1675-6; Ma et al., 2013,
J Natl Cancer Inst 105:1694-1700; Siegel et al. 2012, Cancer
statistics, 62:10-29). Unfortunately, the mortality rate for PDAC
is nearly equal to the incidence. The overall survival rate for all
stages of pancreatic cancer diagnosed between 2001 and 2007 is only
20% after one year, and about 6% after 5 years (Siegel et al. 2012,
Cancer statistics, 62:10-29). With the alarming increase in PDAC
incidence, it is projected that by the year 2030, pancreatic cancer
will become the second leading cause of cancer deaths in the United
States (Rahib et al., 2014, Cancer Res 74:2913-21). The major
reason for this poor prognosis is the inability to detect the
disease at an early stage, when curative measures may have a
greater opportunity to provide successful outcomes.
[0006] Biomarkers, whether they are biological, chemical, or
physical in nature, have proven of significant value in providing
information leading to the earlier detection and diagnosis of
cancer, such as breast (Goldhirsch et al., 2003, Ann Oncol
14:1212-4), colon (Mandel et al., 1993, N Engl J Med 328:1365-71),
and prostate (Jacobsen et al., 1995, JAMA 274:1445-9), resulting in
improved patient outcomes. Unfortunately, this has not been the
case for PDAC. Despite considerable attention directed towards
discovery of biomarkers for PDAC (Lennon et al., 2014, Cancer
Research 74:1-9), to date no FDA-approved means for early
detection/diagnosis exists. A need exists for more effective
compositions and methods for early detection and/or diagnosis of
prostate cancer, preferably at the earliest stages of the
disease.
[0007] In addition to more effective and earlier means of
detection, a need also exists for better therapeutic treatments for
pancreatic cancer. The outlook for patients with advanced
pancreatic adenocarcinoma remains poor (Hidalgo, 2010, N Engl J Med
362:1605-17). In the frontline, median survival was 6.2-6.7 months
with gemcitabine alone (Burris et al., 1997, J Clin Oncol
15:2403-13) or with erlotinib (Moore et al., 2007, J Clin Oncol
25:1960-6), 8.5 months combined with albumin-bound paclitaxel (Von
Hoff et al., 2013, N Engl J Med 369:1691-1703), and 11.1 months for
those able to tolerate combination chemotherapy (FOLFIRINOX)
(Conroy et al., 2011, N Engl J Med 364:1817-25). Beyond 1st line,
the survival advantage with chemotherapy remains limited (Rahma et
al., 2013, Ann Oncol 24:1972-9; Oettle et al., 2014, J Clin Oncol
32:2423-9) and after two prior treatments (one usually
gemcitabine-based, the other fluoropyrimidine-based), there are no
accepted treatments (Seufferlein et al., 2012, Ann Oncol 23(suppl
7):vii33-40; Almhanna & Kim, 2008, Oncology (Williston Park)
22:1176-83).
[0008] There is an unmet need for more effective therapies in
pancreatic cancer patients who have received and shown resistance
to or relapsed from two or more prior therapies.
SUMMARY
[0009] In various embodiments, the present invention concerns
antibodies, antigen-binding antibody fragments and fusion proteins
that bind to the MUC5AC pancreatic cancer mucin. Preferably, the
antibodies or fragments thereof bind to an epitope located within
the second to fourth cysteine-rich subdomains of MUC5AC (amino acid
residues 1575-2052, Cys2-Cys4). More preferably, the antibodies
bind to an epitope located in amino acid residues 1575-1725 and
1903-2052 (Cys2 and Cys 4). Even more preferably, the antibodies
bind to an epitope located in amino acid residues 1575-1725
(Cys2+). Most preferably, the antibodies bind to an epitope located
in Cys2.
[0010] In preferred embodiments, the subject antibodies or
fragments thereof bind specifically to pancreatic cancer cells,
with little or no binding to normal or non-neoplastic pancreatic
cells. The antibodies are capable of binding to the earliest stages
of pancreatic cancer, with detection rates of about 50-60% for
PanIN-1A, 70-80% for Pan1B and 80-90% for PanIN-2. More preferably,
the antibodies bind to 80 to 90% or more of human invasive
pancreatic adenocarcinoma, intraductal papillary mucinous
neoplasia, PanIN-1A, PanIN-1B and PanIN-2 lesions. Most preferably,
the antibodies can distinguish between early stage pancreatic
cancer and non-malignant conditions such as pancreatitis.
[0011] Such antibodies are of particular use for early detection of
cancer and differential diagnosis between early stage pancreatic
cancer and benign pancreatic conditions. In preferred embodiments,
such antibodies are of use for in vivo or ex vivo analysis of
samples from individuals suspected of having early stage pancreatic
or certain other cancers. More preferably, the antibodies are of
use for detection and diagnosis of early stage pancreatic cancer by
analysis of serum samples.
[0012] In alternative embodiments, the antibodies, antibody
fragments or fusion proteins are capable of binding to synthetic
peptide sequences, for example to phage display peptides, such as
WTWNITKAYPLP (SEQ ID NO:7) and ACPEWWGTTC (SEQ ID NO:8). Such
synthetic peptides may be linear or cyclic and may or may not
compete with antibody binding to the endogenous pancreatic cancer
antigen. Amino acids in certain positions of the synthetic peptide
sequences may be less critical for antibody binding than others.
For example, in SEQ ID NO:7 the residues K, A and L at positions 7,
8 and 11 of the peptide sequence may be varied while still
retaining antibody binding. Similarly, in SEQ ID NO:8 the threonine
residues at positions 8 and 9 of the sequence may be varied,
although substitution of the threonine at position 9 may
significantly affect antibody binding to the peptide.
[0013] Binding of the antibodies to a target pancreatic cancer
antigen may be inhibited by treatment of the target antigen with
reagents such as dithiothreitol (DTT) and/or periodate. Thus,
binding of the antibodies to a pancreatic cancer antigen may be
dependent upon the presence of disulfide bonds and/or the
glycosylation state of the target antigen. In more preferred
embodiments, the epitope recognized by the subject antibodies is
not cross-reactive with other reported mucin-specific antibodies,
such as the MA5 antibody, the CLH2-2 antibody and/or the 45M1
antibody (see, e.g., Major et al., J Histochem Cytochem. 35:139-48,
1987; Dion et al., Hybridoma 10:595-610, 1991).
[0014] The subject antibodies or fragments may be naked antibodies
or fragments or preferably are conjugated to at least one
therapeutic and/or diagnostic agent for delivery of the agent to
target tissues. In alternative embodiments, the subject antibodies
or fragments may be part of a bispecific antibody with a first
binding site for an epitope of MUC5AC as discussed above and a
second binding site for a hapten conjugated to a targetable
construct. The targetable construct may in turn be attached to at
least one therapeutic and/or diagnostic agent, of use in
pretargeting techniques.
[0015] In preferred embodiments, the subject antibody, antibody
fragment or fusion protein is a humanized PAM4 antibody or
fragment, comprising the light chain variable region CDR sequences
CDR1 (SASSSVSSSYLY, SEQ ID NO: 1); CDR2 (STSNLAS, SEQ ID NO:2); and
CDR3 (HQWNRYPYT, SEQ ID NO:3); and the heavy chain variable region
CDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG,
SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6) and human antibody
framework region (FR) and constant region sequences. More
preferably, the FRs of the light and heavy chain variable regions
of the humanized PAM4 antibody or fragment thereof comprise at
least one amino acid substituted from amino acid residues 5, 27,
30, 38, 48, 66, 67 and 69 of the murine PAM4 heavy chain variable
region (SEQ ID NO: 12) and/or at least one amino acid selected from
amino acid residues 21, 47, 59, 60, 85, 87 and 100 of the murine
PAM4 light chain variable region (SEQ ID NO: 10). Most preferably,
the antibody or fragment thereof comprises the hPAM4 V.sub.H amino
acid sequence of SEQ ID NO: 19 and the hPAM4 VK amino acid sequence
of SEQ ID NO: 16.
[0016] In alternative embodiments, the anti-pancreatic cancer
antibody may be a chimeric, humanized or human antibody that binds
to the same antigenic determinant (epitope) as, or competes for
binding to MUC5AC with, a chimeric PAM4 (cPAM4) antibody. As
discussed below, the cPAM4 antibody is one that comprises the light
chain variable region CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:
1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3);
and the heavy chain variable region CDR sequences CDR1 (SYVLH, SEQ
ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3
(GFGGSYGFAY, SEQ ID NO:6). Antibodies that bind to the same
antigenic determinant may be identified by a variety of techniques
known in the art, such as by competitive binding studies using the
cPAM4 antibody as the competing antibody and human pancreatic mucin
or MUC5AC as the target antigen. Antibodies that block (compete
for) binding to human pancreatic mucin by a cPAM4 antibody are
referred to as cross-blocking antibodies. Preferably, such
cross-blocking antibodies are ones that bind to an epitope located
within the second to fourth cysteine-rich subdomains of MUC5AC, or
that compete for binding to amino acid residues 1575-2052 with a
PAM4 antibody. More preferably, the antibodies bind to an epitope
located in Cys2.
[0017] Other embodiments concern cancer cell-targeting therapeutic
immunoconjugates comprising an antibody or fragment thereof or
fusion protein bound to at least one therapeutic agent. Preferably,
the therapeutic agent is selected from the group consisting of a
radionuclide, an immunomodulator, a hormone, a hormone antagonist,
an enzyme, an oligonucleotide such as an anti-sense oligonucleotide
or a siRNA, an enzyme inhibitor, a photoactive therapeutic agent, a
cytotoxic agent such as a drug or toxin, an angiogenesis inhibitor
and a pro-apoptotic agent. In embodiments where more than one
therapeutic agent is used, the therapeutic agents may comprise
multiple copies of the same therapeutic agent or else combinations
of different therapeutic agents. More preferably, the therapeutic
agent is a radionuclide, such as .sup.90Y. The labeled antibody may
be administered alone, or in combination with one or more other
therapeutic agents, such as low-dose gemcitabine.
[0018] In certain embodiments, the therapeutic agent is a cytotoxic
agent, such as a drug or a toxin. Also preferred, the drug is
selected from the group consisting of nitrogen mustards,
ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
gemcitabine, triazenes, folic acid analogs, anthracyclines,
taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,
antibiotics, enzyme inhibitors, epipodophyllotoxins, platinum
coordination complexes, vinca alkaloids, substituted ureas, methyl
hydrazine derivatives, adrenocortical suppressants, hormone
antagonists, endostatin, taxols, camptothecins, SN-38, doxorubicins
and their analogs, antimetabolites, alkylating agents,
antimitotics, anti-angiogenic agents, tyrosine kinase inhibitors,
Bruton tyrosine kinase inhibitors, mTOR inhibitors, heat shock
protein (HSP90) inhibitors, proteosome inhibitors, HDAC inhibitors,
pro-apoptotic agents, methotrexate, CPT-11, SN-38, 2-PDOX,
pro-2-PDOX, and a combination thereof.
[0019] In another preferred embodiment, the therapeutic agent is a
toxin selected from the group consisting of ricin, abrin, alpha
toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria
toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin and
combinations thereof. Or an immunomodulator selected from the group
consisting of a cytokine, a stem cell growth factor, a lymphotoxin,
a hematopoietic factor, a colony stimulating factor (CSF), an
interferon (IFN), a stem cell growth factor, erythropoietin,
thrombopoietin and a combinations thereof.
[0020] Alternatively, the therapeutic agent is an enzyme selected
from the group consisting of malate dehydrogenase, staphylococcal
nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. Such enzymes may be used, for example, in
combination with prodrugs that are administered in relatively
non-toxic form and converted at the target site by the enzyme into
a cytotoxic agent. In other alternatives, a drug may be converted
into less toxic form by endogenous enzymes in the subject but may
be reconverted into a cytotoxic form by the therapeutic enzyme.
[0021] Other therapeutic agents include radionuclides such as
.sup.14C, .sup.13N, .sup.15O, .sup.32P, .sup.33P, .sup.47Sc,
.sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.62Cu, .sup.67Cu,
.sup.67Ga, .sup.67Ga, .sup.75Br, .sup.75Se, .sup.75Se, .sup.76Br,
.sup.77As, .sup.77Br, .sup.80mBr, .sup.89Sr, .sup.90Y, .sup.95Ru,
.sup.97Ru, .sup.99Mo, .sup.99mTc, .sup.103mRh, .sup.103Ru,
.sup.105Rh, .sup.105Ru, .sup.107Hg, .sup.109Pd, .sup.109Pt,
.sup.111Ag, .sup.111In, .sup.113mIn, .sup.119Sb, .sup.121mTe,
.sup.122mTe, .sup.125I, .sup.125mTe, .sup.126I, .sup.131I,
.sup.133I, .sup.142Pr, .sup.143Pr, .sup.149Pm, .sup.152Dy,
.sup.153Sm, .sup.161Ho, .sup.161Tb, .sup.165Tm, .sup.166Dy,
.sup.166Ho, .sup.167Tm, .sup.168Tm, .sup.169Er, .sup.169Yb,
.sup.177Lu, .sup.186Re, .sup.188Re, .sup.189mOs, .sup.189Re,
.sup.192Ir, .sup.194Ir, .sup.197Pt, .sup.198Au, .sup.199Au,
.sup.199Au, .sup.201Tl, .sup.203Hg, .sup.211At, .sup.211Bi,
.sup.211Pb, .sup.212Bi, .sup.212Pb, .sup.213Bi, .sup.215Po,
.sup.217At, .sup.219Rn, .sup.221Fr, .sup.223Ra, .sup.224Ac,
.sup.225Ac, .sup.255Fm or .sup.227Th.
[0022] A variety of tyrosine kinase inhibitors are known in the art
and any such known therapeutic agent may be utilized. Exemplary
tyrosine kinase inhibitors include, but are not limited to
canertinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib,
leflunomide, nilotinib, pazopanib, semaxinib, sorafenib, sunitinib,
sutent and vatalanib. A specific class oftyrosine kinase inhibitor
is the Bruton tyrosine kinase inhibitor. Bruton tyrosine kinase
(Btk) has a well-defined role in B-cell development. Bruton kinase
inhibitors include, but are not limited to, PCI-32765 (ibrutinib),
PCI-45292, GDC-0834, LFM-A13 and RN486.
[0023] The subject antibody or fragment may be conjugated to at
least one diagnostic (or detection) agent. Preferably, the
diagnostic agent is selected from the group consisting of a
radionuclide, a contrast agent, a fluorescent agent, a
chemiluminescent agent, a bioluminescent agent, a paramagnetic ion,
an enzyme and a photoactive diagnostic agent. Still more preferred,
the diagnostic agent is a radionuclide with an energy between 20
and 4,000 keV or is a radionuclide selected from the group
consisting of .sup.110In, .sup.111In, .sup.177Lu, .sup.18F,
.sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y, .sup.90Y, .sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc,
.sup.120I, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.32P, .sup.11C, .sup.13N, .sup.15O, .sup.186Re,
.sup.188Re, .sup.51Mn, .sup.52mMn, .sup.55Co, .sup.72As, .sup.75Br,
.sup.76Br, .sup.82mRb, .sup.83Sr, or other gamma-, beta-, or
positron-emitters. In a particularly preferred embodiment, the
diagnostic radionuclide .sup.18F is used for labeling and PET
imaging, as described in the Examples below. The .sup.18F may be
attached to an antibody, antibody fragment or peptide by
complexation to a metal, such as aluminum, and binding of the
.sup.18F-metal complex to a chelating moiety that is conjugated to
a targeting protein, peptide or other molecule.
[0024] Also preferred, the diagnostic agent is a paramagnetic ion,
such as chromium (III), manganese (II), iron (III), iron (II),
cobalt (II), nickel (II), copper (II), neodymium (III), samarium
(III), ytterbium (III), gadolinium (III), vanadium (II), terbium
(III), dysprosium (III), holmium (III) and erbium (III), or a
radiopaque material, such as barium, diatrizoate, ethiodized oil,
gallium citrate, iocarmic acid, iocetamic acid, iodamide,
iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol,
iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid,
iosulamide meglumine, iosemetic acid, iotasul, iotetric acid,
iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,
ipodate, meglumine, metrizamide, metrizoate, propyliodone, and
thallous chloride.
[0025] In still other embodiments, the diagnostic agent is a
fluorescent labeling compound selected from the group consisting of
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine, a
chemiluminescent labeling compound selected from the group
consisting of luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester, or a
bioluminescent compound selected from the group consisting of
luciferin, luciferase and aequorin. In another embodiment, the
diagnostic immunoconjugates are used in intraoperative, endoscopic,
or intravascular tumor diagnosis.
[0026] Also contemplated are multivalent, multispecific antibodies
or fragments thereof comprising at least one binding site that
binds to an epitope of MUC5AC as discussed above and one or more
hapten binding sites having affinity towards hapten molecules.
Preferably, the antibody or fragment thereof is a chimeric,
humanized or fully human antibody or fragment thereof. The hapten
molecule may be conjugated to a targetable construct for delivery
of one or more therapeutic and/or diagnostic agents. In certain
preferred embodiments, the multivalent antibodies or fragments
thereof may be prepared by the DOCK-AND-LOCK.TM. (DNL.TM.)
technique, as described below. An exemplary DNL.TM. construct
incorporating hPAM4 antibody fragments is designated TF10, as
described below.
[0027] Also contemplated is a bispecific antibody or fragment
thereof comprising at least one binding site with an affinity
toward an epitope of MUC5AC as discussed above and at least one
binding site with an affinity toward a targetable construct which
is capable of carrying at least one diagnostic and/or therapeutic
agent. Targetable constructs suitable for use are disclosed, for
example, in U.S. Pat. Nos. 6,576,746; 6,962,702; 7,052,872;
7,138,103; 7,172,751; 7,405,320; 7,597,876; 7,563,433; 7,993,626;
8,147,799; 8,153,100; 8,153,101; 8,202,509; 8,343,460; 8,444,956,
8,496,912; 8,545,809; 8,617,518; and 8,632,752, the Examples
section of each of which is incorporated herein by reference.
[0028] Other embodiments concern fusion proteins comprising at
least two anti-pancreatic cancer antibodies and fragments thereof
as described herein. Alternatively, the fusion protein or fragment
thereof may comprise at least one first antibody or fragment
thereof that binds to an epitope of MUC5AC as discussed above and
at least one second MAb or fragment thereof. Preferably, the second
MAb binds to a tumor-associated antigen, for example selected from
the group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49,
CEA (CEACAM5), CEACAM6, Le.sup.a, the Lewis antigen Le(y), CSAp,
insulin-like growth factor (IGF), epithelial glycoprotein-1
(EGP-1), epithelial glycoprotein-2 (EGP-2), CD-80, placental growth
factor (PlGF), carbonic anhydrase IX, tenascin, IL-6, HLA-DR, CD40,
CD74 (e.g., milatuzumab), CD138 (syndecan-1), CTLA-4, PD-1, PD-L1,
TIM-3, LAG-3, matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-7,
MMP-9, MMP-14, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC16, MUC17,
TAG-72, EGFR, platelet-derived growth factor (PDGF), angiogenesis
factors (e.g., VEGF and PlGF), products of oncogenes (e.g., bcl-2,
Kras, p53), cMET, HER2/neu, and antigens associated with gastric
cancer and colorectal cancer. The second MAb may also bind to a
different epitope of MUC5AC than the first MAb. The antibody fusion
protein or fragments thereof may further comprise at least one
diagnostic and/or therapeutic agent.
[0029] Also described herein are DNA sequences comprising a nucleic
acid encoding an anti-pancreatic cancer antibody, fusion protein,
multispecific antibody, bispecific antibody or fragment thereof as
described herein. Other embodiments concern expression vectors
and/or host cells comprising the antibody-encoding DNA sequences.
In certain preferred embodiments, the host cell may be an Sp2/0
cell line transformed with a mutant Bcl-2 gene, for example with a
triple mutant Bcl-2 gene (T69E, S70E, S87E), that has been adapted
to cell transformation and growth in serum free medium. (See, e.g.,
U.S. Pat. Nos. 7,531,327; 7,537,930; and 7,608,425, the Examples
section of each of which is incorporated herein by reference.)
[0030] Another embodiment concerns methods of delivering a
diagnostic or therapeutic agent, or a combination thereof, to a
target comprising (i) providing a composition that comprises an
anti-pancreatic cancer antibody or fragment that binds to an
epitope located within the second to fourth cysteine-rich
subdomains of MUC5AC (amino acid residues 1575-2052), more
preferably to an epitope located in amino acid residues 1575-1725
and 1903-2052 (Cys2 and Cys 4), even more preferably to an epitope
located in amino acid residues 1575-1725 (Cys2+), most preferably,
to an epitope located in Cys2, conjugated to at least one
diagnostic and/or therapeutic agent and (ii) administering to a
subject in need thereof the diagnostic or therapeutic conjugate of
any one of the antibodies, antibody fragments or fusion proteins
claimed herein.
[0031] Also contemplated is a method of delivering a diagnostic
agent, a therapeutic agent, or a combination thereof to a target,
comprising: (a) administering to a subject any one of the
multivalent, multispecific or bispecific antibodies or fragments
thereof that have an affinity toward an epitope of MUC5AC as
discussed above and comprising one or more hapten binding sites;
(b) waiting a sufficient amount of time for antibody that does not
bind to MUC5AC to clear the subject's blood stream; and (c)
administering to said subject a carrier molecule comprising a
diagnostic agent, a therapeutic agent, or a combination thereof,
that binds to a binding site of the antibody. Preferably, the
carrier molecule binds to more than one binding site of the
antibody.
[0032] Described herein is a method for diagnosing or treating
cancer, comprising: (a) administering to a subject any one of the
multivalent, multispecific antibodies or fragments thereof claimed
herein that have an affinity toward an epitope of MUC5AC as
discussed above and comprising one or more hapten binding sites;
(b) waiting a sufficient amount of time for an amount of the
non-bound antibody to clear the subject's blood stream; and (c)
administering to said subject a carrier molecule comprising a
diagnostic agent, a therapeutic agent, or a combination thereof,
that binds to a binding site of the antibody. In a preferred
embodiment the cancer is pancreatic cancer. Also preferred, the
method can be used for intraoperative identification of diseased
tissues, endoscopic identification of diseased tissues, or
intravascular identification of diseased tissues.
[0033] Another embodiment is a method of treating a malignancy in a
subject comprising administering to said subject a therapeutically
effective amount of an antibody or fragment thereof that binds to
an epitope of MUC5AC as discussed above, optionally conjugated to
at least one therapeutic agent, such as .sup.90Y. The antibody or
fragment thereof may alternatively be a naked antibody or fragment
thereof. In more preferred embodiments, the antibody or fragment is
administered either before, simultaneously with, or after
administration of another therapeutic agent as described above.
[0034] Contemplated herein is a method of diagnosing a malignancy
in a subject, particularly a pancreatic cancer, comprising (a)
administering to said subject a diagnostic conjugate comprising an
antibody or fragment thereof that binds to an epitope of MUC5AC as
discussed above, wherein said MAb or fragment thereof is conjugated
to at least one diagnostic agent, and (b) detecting the presence of
labeled antibody bound to pancreatic cancer cells or other
malignant cells, wherein binding of the antibody is diagnostic for
the presence of pancreatic cancer or another malignancy. In
preferred embodiments, the antibody or fragment binds to pancreatic
cancer and not to normal pancreatic tissue, pancreatitis or other
non-malignant conditions. In less preferred embodiments, the
antibody or fragment binds at a significantly higher level to
cancer cells than to non-malignant cells, allowing differential
diagnosis of cancer from non-malignant conditions. In a most
preferred embodiment, the diagnostic agent may be an F-18 labeled
molecule that is detected by PET imaging.
[0035] In more preferred embodiments, the use of anti-pancreatic
cancer antibodies that bind to an epitope located within the second
to fourth cysteine-rich subdomains of MUC5AC (amino acid residues
1575-2052), more preferably to an epitope located in amino acid
residues 1575-1725 and 1903-2052 (Cys2 and Cys 4), even more
preferably to an epitope located in amino acid residues 1575-1725
(Cys2+), most preferably, to an epitope located in Cys2, allows the
detection and/or diagnosis of pancreatic cancer with high
specificity and sensitivity at the earliest stages of malignant
disease. Preferably, the diagnostic antibody or fragment is capable
of labeling at least 70%, more preferably at least 80%, more
preferably at least 90%, more preferably at least 95%, most
preferably about 100% of well differentiated, moderately
differentiated and poorly differentiated pancreatic cancer and 90%
or more of invasive pancreatic adenocarcinomas. The anti-pancreatic
cancer antibody of use is preferably capable of detecting 85% or
more of PanIN-1A, PanIN-1B, PanIN-2, IPMN and MCN precursor
lesions. Most preferably, immunoassays using the anti-pancreatic
cancer antibody are capable of detecting 89% or more of total
PanIN, 86% or more of IPMN, and 92% or more of MCN.
[0036] An alternative embodiment is a method of detecting the
presence of PAM4-binding MUC5AC and/or diagnosing pancreatic cancer
in an individual by in vitro analysis of blood, plasma or serum
samples. Preferably, the sample is subjected to an organic phase
extraction, using an organic solvent such as butanol, before it is
processed for immunodetection using an anti-pancreatic cancer
antibody, such as a PAM4 antibody. Following organic phase
extraction, the extracted aqueous phase is analyzed for the
presence of the epitope of MUC5AC to which PAM4 binds in the
sample, using any of a variety of immunoassay techniques known in
the art, such as ELISA, sandwich immunoassay, solid phase RIA, and
similar techniques. Surprisingly, the organic phase extraction
results in the removal of an inhibitor of PAM4 binding to MUC5AC,
allowing detection of MUC5AC in fresh serum samples. More
surprisingly, using the in vitro analysis techniques described
herein, serum samples may be analyzed to detect and/or diagnose
pancreatic cancer in a subject at the earliest stages of pancreatic
adenocarcinoma. These unexpected results provide the first
serum-based assay technique that is diagnostic for the presence of
early stage pancreatic cancer.
[0037] Another embodiment is a method of treating a cancer cell in
a subject comprising administering to said subject a composition
comprising a naked antibody or fragment thereof or a naked antibody
fusion protein or fragment thereof that binds to an epitope of
MUC5AC as discussed above. Preferably, the method further comprises
administering a second naked antibody or fragment thereof selected
from the group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3,
CC49, anti-CEA, anti-CEACAM6, anti-EGP-1, anti-EGP-2,
anti-Le.sup.a, antibodies defined by the Lewis antigen Le(y), and
antibodies against CSAp, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC16,
MUC17, TAG-72, EGFR, CD40, HLA-DR, CD74, CD138, angiogenesis
factors (e.g., VEGF and placenta-like growth factor (PlGF),
insulin-like growth factor (IGF), tenascin, platelet-derived growth
factor, IL-6, products of oncogenes, cMET, and HER2/neu.
[0038] Still other embodiments concern a method of diagnosing a
malignancy in a subject comprising (i) performing an in vitro
diagnosis assay on a specimen from said subject with a composition
comprising an antibody or fragment thereof that binds to an epitope
of MUC5AC as discussed above; and (ii) detecting the presence of
antibody or fragment bound to malignant cells in the specimen.
Preferably, the malignancy is a cancer. More preferably, the cancer
is pancreatic cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The following drawings are provided to illustrate preferred
embodiments of the invention. However, the claimed subject matter
is in no way limited by the illustrative embodiments disclosed in
the drawings.
[0040] FIG. 1. Co-localization of PAM4 antigen with MUC5AC by
cytoflurometric staining. Mucin-expressing cell lines (Capan-1,
BxPC-3, HT-29, and MCF-7) were stained with DAPI, 2-11M1
(anti-MUC5AC), hPAM4, and examined by immunofluorescence microcopy.
BxPC-3 and HT-29 cells were also stained with anti-MUC1. PAM4
antigen was shown to co-localize with MUC5AC, not MUC1.
[0041] FIG. 2. Co-knockdown of PAM4 antigen and MUC5AC by
MUC5AC-specific siRNA. CFPAC-1 cells were treated with a
MUC5AC-specific siRNA, followed by immunostaining with DAPI, hPAM4,
and 2-11M1 (anti-MUC5AC), or with DAPI, hPAM4 and anti-MUC1.
Untreated Cells or cells treated with only the transfection agent
(Mock) served as controls. Cells treated with MUC5AC-specific siRNA
lost the binding to anti-MUC5AC and hPAM4 concurrently, with little
effect on the binding to anti-MUC1.
[0042] FIG. 3A. Immunoreactivity of fractions eluted from
SEPHAROSE.RTM. CL-2B. Capan-1 cell culture supernatant was
separated on a SEPHAROSE.RTM. CL2B column with the eluted fractions
analyzed by hPAM4 and anti-MUC1.
[0043] FIG. 3B. Immunoreactivity of fractions eluted from
SEPHAROSE.RTM. CL-2B. The void-volume (Vo) fractions of Capan-1
reacted positively with three anti-MUC5AC antibodies (45M1, 1-13M1
and H-160), but not with 2Q445, which recognizes the unglycosylated
tandem repeat region of MUC5AC.
[0044] FIG. 3C. Immunoreactivity of fractions eluted from
SEPHAROSE.RTM. CL-2B. The Capan-1 void-volume peak, following
capture by 2-11M1, could be detected directly by HRP-hPAM4, or
indirectly by biotin-45M1 plus SA-HRP.
[0045] FIG. 4A. Agarose gel electrophoresis. The Capan-1
void-volume peak displayed the characteristic banding pattern of
MUC5AC as revealed by Western blot analysis with hPAM4, 45M1, and
MAN-5ACI. In the left panel, samples in the lanes marked as 1, 1/2,
1/4, and 1/8 were tested undiluted, 2-, 4- and 8-fold diluted,
respectively. In the far right panel, the monomeric and dimeric
MUC5AC were indicated as M and D, respectively.
[0046] FIG. 4B. Agarose gel electrophoresis. The serum from a
pancreatic cancer patient (PS) tested positive for hPAM4-reactive
substance was differentially detected by hPAM4 and three
anti-MUC5AC antibodies (2-11M1, 45M1, and H-160). The Capan-1
void-volume peak (Vo) and normal serum sample (NS) were included as
controls.
[0047] FIG. 5A. Mapping the PAM4-reactive epitope on human MUC5AC.
Schematic diagram of different MUC5AC recombinant fragments (a-h)
generated in PANC-1 cells for mapping PAM4 epitope; Numbers are AA
positions in the MUC5AC protein sequence (UniProtKB/Swiss-Prot:
P98088).
[0048] FIG. 5B. Mapping the PAM4-reactive epitope on human MUC5AC.
Western blot of a-fragment (AA3992-5030), b-fragment (AA1-1218) and
c-fragment (AA1218-2199), which spans the five N-terminal
cysteine-rich subdomains (Cys1-2-3-4-5), with hPAM4 and 45M1
antibodies. Lane m indicates samples from untransfected cells.
[0049] FIG. 5C. Mapping the PAM4-reactive epitope on human MUC5AC.
Western blot of c-fragment (AA1218-2199), d-fragment (AA1218-1517)
and e-fragment (AA1575-2052), with hPAM4, H160 and 45M1 antibodies.
Lane m indicates samples from untransfected cells.
[0050] FIG. 5D. Mapping the PAM4-reactive epitope on human MUC5AC.
Western blot of e-fragment (AA1575-2052), f-fragment (AA1725-2052),
g-fragment (AA1575-1725 and 1903-2052) and h-fragment (AA1575-1853)
with hPAM4 and 45M1 antibodies. Lane m indicates samples from
untransfected cells.
[0051] FIG. 5E. Mapping the PAM4-reactive epitope on human MUC5AC.
Western blot of GFP-fused e*-fragment (AA1575-2052), f*-fragment
(AA1725-2052), g*-fragment (AA1575-1725 and 1903-2052) and
h*-fragment (AA1575-1853) with hPAM4 and anti-GFP antibodies. Lane
m indicates samples from untransfected cells.
[0052] FIG. 6A. SDS-PAGE and Western blot analyses of recombinant
MUC5AC fragments expressed in E. coli. Four gels were run under
similar conditions of SDS-PAGE. Gel was stained with coomassie
blue. Samples, either reduced (R) or non-reduced (NR), were loaded
at 500 ng/well; lane M, markers; lanes 1 & 3, Cys2-3-4
(AA1575-2052); lanes 2 &4, Cys2+(AA1575-1725).
[0053] FIG. 6B. SDS-PAGE and Western blot analyses of recombinant
MUC5AC fragments expressed in E. coli. Four gels were run under
similar conditions of SDS-PAGE. Gel was transferred onto
nitrocellulose membrane and stained with anti-Myc. Samples, either
reduced (R) or non-reduced (NR), were loaded at 500 ng/well; lane
M, markers; lanes 1 & 3, Cys2-3-4 (AA1575-2052); lanes 2
&4, Cys2+(AA1575-1725).
[0054] FIG. 6C. SDS-PAGE and Western blot analyses of recombinant
MUC5AC fragments expressed in E. coli. Four gels were run under
similar conditions of SDS-PAGE. Gel was transferred onto
nitrocellulose membrane and stained with hPAM4. Samples, either
reduced (R) or non-reduced (NR), were loaded at 500 ng/well; lane
M, markers; lanes 1 & 3, Cys2-3-4 (AA1575-2052); lanes 2
&4, Cys2+(AA1575-1725).
[0055] FIG. 6D. SDS-PAGE and Western blot analyses of recombinant
MUC5AC fragments expressed in E. coli. Four gels were run under
similar conditions of SDS-PAGE. Gel was transferred onto
nitrocellulose membrane and stained with 1-13M1. Samples, either
reduced (R) or non-reduced (NR), were loaded at 500 ng/well; lane
M, markers; lanes 1 & 3, Cys2-3-4 (AA1575-2052); lanes 2
&4, Cys2+(AA1575-1725).
[0056] FIG. 7. Overall survival. Kaplan-Meier curves and time-point
analyses for all 29 patients in Arm A (.sup.90Y-clivatuzumab
tetraxetan combined with low-dose gemcitabine) and all 29 patients
in Arm B (.sup.90Y-clivatuzumab tetraxetan alone).
[0057] FIG. 8A. Variable region cDNA sequences (SEQ ID NO:9) and
the deduced amino acid sequences (SEQ ID NO: 10) of the murine PAM4
Vk. Amino acid sequences encoded by the corresponding DNA sequences
are given as one-letter codes below the nucleotide sequence.
Numbering of the nucleotide sequence is on the right side. The
amino acid residues in the CDR regions are shown in bold and
underlined. Kabat's Ig molecule numbering is used for amino acid
residues as shown by the numbering above the amino acid residues.
The amino acid residues numbered by a letter are the insertion
residues defined by Kabat numbering scheme. The insertion residues
have the same preceding digits as that of the previous residue.
[0058] FIG. 8B. Variable region cDNA sequence (SEQ ID NO: 11) and
the deduced amino acid sequence (SEQ ID NO: 12) of the murine PAM4
VH. Amino acid sequences encoded by the corresponding DNA sequences
are given as one-letter codes below the nucleotide sequence.
Numbering of the nucleotide sequence is on the right side. The
amino acid residues in the CDR regions are shown in bold and
underlined. Kabat's Ig molecule numbering is used for amino acid
residues as shown by the numbering above the amino acid residues.
The amino acid residues numbered by a letter are the insertion
residues defined by Kabat numbering scheme. The insertion residues
have the same preceding digits as that of the previous residue.
[0059] FIG. 9A. Amino acid sequence (SEQ ID NO: 13) of the chimeric
PAM4 (cPAM4) Vk. The sequences are given as one letter codes. The
amino acid residues in the CDR regions are shown in bold and
underlined. Kabat's Ig molecule number scheme is used to number the
residues.
[0060] FIG. 9B. Amino acid sequence (SEQ ID NO: 14) of the cPAM4
VH. The sequences are given as one letter codes. The amino acid
residues in the CDR regions are shown in bold and underlined.
Kabat's Ig molecule number scheme is used to number the
residues.
[0061] FIG. 10A. Alignment of the VK amino acid sequences of the
human antibody Walker (SEQ ID NO:15) with PAM4 (SEQ ID NO:10) and
hPAM4 (SEQ ID NO:16). Dots indicate the residues of PAM4 that are
identical to the corresponding residues of the human or humanized
antibodies. Boxed regions represent the CDR regions. Both N- and
C-terminal residues (underlined) of hPAM4 are fixed by the staging
vectors used. Kabat's Ig molecule number scheme is used to number
the residues.
[0062] FIG. 10B. Alignment of the VH amino acid sequences of the
human antibody Wil2 (FR1-3) (SEQ ID NO:17) and NEWM (FR4) (SEQ ID
NO:18) with PAM4 (SEQ ID NO:12) and hPAM4 (SEQ ID NO: 19). Dots
indicate the residues of PAM4 that are identical to the
corresponding residues of the human or humanized antibodies. Boxed
regions represent the CDR regions. Both N- and C-terminal residues
(underlined) of hPAM4 are fixed by the staging vectors used.
Kabat's Ig molecule number scheme is used to number the
residues.
[0063] FIG. 11A. DNA (SEQ ID NO:20) and amino acid (SEQ ID NO:16)
sequences of the humanized PAM4 (hPAM4) Vk. Numbering of the
nucleotide sequence is on the right side. Amino acid sequences
encoded by the corresponding DNA sequences are given as one-letter
codes. The amino acid residues in the CDR regions are shown in bold
and underlined. Kabat's Ig molecule numbering scheme is used for
amino acid residues.
[0064] FIG. 11B. DNA (SEQ ID NO:21) and amino acid (SEQ ID NO: 19)
sequences of the hPAM4 VH. Numbering of the nucleotide sequence is
on the right side. Amino acid sequences encoded by the
corresponding DNA sequences are given as one-letter codes. The
amino acid residues in the CDR regions are shown in bold and
underlined. Kabat's Ig molecule numbering scheme is used for amino
acid residues.
[0065] FIG. 12. Binding activity of humanized PAM4 antibody, hPAM4,
as compared to the chimeric PAM4, cPAM4. hPAM4 is shown by diamonds
and cPAM4 is shown by closed circles. Results indicate comparable
binding activity of the hPAM4 antibody and cPAM4 when competing
with .sup.125I-cPAM4 binding to CaPan1 antigens.
[0066] FIG. 13. PET/CT fusion images for a patient with inoperable
metastatic pancreatic cancer treated with fractionated
.sup.90Y-hPAM4 plus gemcitabine, before therapy (left side) and
post-therapy (right side). The circle indicates the location of the
primary lesion, which shows a significant decrease in PET/CT
intensity following therapy.
[0067] FIG. 14. 3D PET images for a patient with inoperable
metastatic pancreatic cancer treated with fractionated
.sup.90Y-hPAM4 plus gemcitabine, before therapy (left side) and
post-therapy (right side). Arrows point to the locations of the
primary lesion (on right) and metastases (on left), each of which
shows a significant decrease in PET image intensity after therapy
with radiolabeled hPAM4 plus gemcitabine.
[0068] FIG. 15A. In vivo imaging of tumors using an
.sup.111In-labeled diHSG peptide (IMP 288) with or without
pretargeting TF10 bispecific anti-pancreatic cancer MUC5AC
antibody. FIG. 15A illustrates mice showing the location of tumors
(arrow).
[0069] FIG. 15B In vivo imaging of tumors using an
.sup.111In-labeled diHSG peptide (IMP 288) with or without
pretargeting TF10 bispecific anti-pancreatic cancer MUC5AC
antibody. FIG. 15B shows the detected tumors with
.sup.111In-labeled IMP 288 in the presence (above) or absence
(below) of TF10 bispecific antibody.
[0070] FIG. 16. Exemplary binding curves for TF10, PAM4-IgG,
PAM4-F(ab').sub.2 and a monovalent bsPAM4 chemical conjugate
(PAM4-Fab'.times.anti-DTPA-Fab'). Binding to target mucin antigen
was measured by ELISA assay.
[0071] FIG. 17A. Immunoscintigraphy of CaPan1 human pancreatic
cancer xenografts (.about.0.25 g). An image of mice that were
injected with bispecific TF10 (80 Gig, 5.07.times.10.sup.-10 mol)
followed 16 h later by administration of .sup.111In-IMP-288 (30
.mu.Ci, 5.07.times.10.sup.-11 mol). The image was taken 3 h later.
The intensity of the image background was increased to match the
intensity of the image obtained when .sup.111In-IMP-288 was
administered alone (30 .mu.Ci, 5.07.times.10.sup.-11 mol).
[0072] FIG. 17B. Immunoscintigraphy of CaPan1 human pancreatic
cancer xenografts (.about.0.25 g). No targeting was observed in
mice given .sup.111In-IMP-288 alone.
[0073] FIG. 17C. Immunoscintigraphy of CaPan1 human pancreatic
cancer xenografts (.about.0.25 g). An image of mice that were given
.sup.111In-DOTA-PAM4-IgG (20 .mu.Ci, 50 .mu.g) with imaging done 24
h later. Although tumors are visible, considerable background
activity is still present at this time point.
[0074] FIG. 18A. Extended biodistribution of
.sup.111In-DOTA-PAM4-IgG (20 .mu.Ci, 50 .mu.g) and TF10-pretargeted
.sup.111In-IMP-288 (80 .mu.g, 5.07.times.10.sup.-10 mol TF10
followed 16 h later with 30 .mu.Ci, 5.07.times.10.sup.-11 mol
.sup.111In-IMP-288) in nude mice bearing CaPan1 human pancreatic
cancer xenografts (mean tumor weight+/-SD, 0.28+/-0.21 and
0.10+/-0.06 g for the pretargeting and IgG groups of animals,
respectively). FIG. 18A shows percent of initial dose per gram of
tissue in tumor with PAM4 IgG (open circles), blood with PAM4 IgG
(open squares), tumor with pretargeted peptide (closed circles) and
blood with pretargeted peptide (closed squares).
[0075] FIG. 18B. Extended biodistribution of
.sup.111In-DOTA-PAM4-IgG (20 .mu.Ci, 50 .mu.g) and TF10-pretargeted
.sup.111In-IMP-288 (80 .mu.g, 5.07.times.10.sup.-10 mol TF10
followed 16 h later with 30 .mu.Ci, 5.07.times.10.sup.-11 mol
.sup.111In-IMP-288) in nude mice bearing CaPan1 human pancreatic
cancer xenografts (mean tumor weight+/-SD, 0.28+/-0.21 and
0.10+/-0.06 g for the pretargeting and IgG groups of animals,
respectively). FIG. 18B shows percent of initial dose per gram of
tissue in liver with PAM4 IgG (open triangles), kidney with PAM4
IgG (open diamonds), liver with pretargeted peptide (closed
triangles) and kidney with pretargeted peptide (closed
diamonds).
[0076] FIG. 18C. Extended biodistribution of
.sup.111In-DOTA-PAM4-IgG (20 .mu.Ci, 50 .mu.g) and TF10-pretargeted
.sup.111In-IMP-288 (80 .mu.g, 5.07.times.10.sup.-10 mol TF10
followed 16 h later with 30 .mu.Ci, 5.07.times.10.sup.-11 mol
.sup.111In-IMP-288) in nude mice bearing CaPan1 human pancreatic
cancer xenografts (mean tumor weight+/-SD, 0.28+/-0.21 and
0.10+/-0.06 g for the pretargeting and IgG groups of animals,
respectively). FIG. 18C shows microcuries per gram of tissue in
tumor with PAM4 IgG (open circles), blood with PAM4 IgG (open
squares), tumor with pretargeted peptide (closed circles) and blood
with pretargeted peptide (closed squares).
[0077] FIG. 18D. Extended biodistribution of
.sup.111In-DOTA-PAM4-IgG (20 .mu.Ci, 50 .mu.g) and TF10-pretargeted
.sup.111In-IMP-288 (80 .mu.g, 5.07.sup.-10 mol TF10 followed 16 h
later with 30 .mu.Ci, 5.07.times.10.sup.-11 mol .sup.111In-IMP-288)
in nude mice bearing CaPan1 human pancreatic cancer xenografts
(mean tumor weight+/-SD, 0.28+/-0.21 and 0.10+/-0.06 g for the
pretargeting and IgG groups of animals, respectively). FIG. 18D
shows microcuries per gram of tissue in liver with PAM4 IgG (open
triangles), kidney with PAM4 IgG (open diamonds), liver with
pretargeted peptide (closed triangles) and kidney with pretargeted
peptide (closed diamonds).
[0078] FIG. 19. Therapeutic activity of a single treatment of
established (.about.0.4 cm.sup.3) CaPan1 tumors with 0.15 mCi of
.sup.90Y-hPAM4 IgG, or 0.25 or 0.50 mCi of TF10-pretargeted
.sup.90Y-IMP-288.
[0079] FIG. 20. Effect of gemcitabine potentiation of PT-RAIT
therapy.
[0080] FIG. 21. Effect of combination of cetuximab with gemcitabine
and PT-RAIT.
[0081] FIG. 22. Differential diagnosis of pancreatic cancer using
PAM4-based immunoassay. The horizontal line shows the cutoff level
selected for a positive result, based on ROC analysis.
[0082] FIG. 23. Frequency distribution of PAM4 antigen in patient
sera from healthy volunteers and individuals with varying stages of
pancreatic cancer.
[0083] FIG. 24. ROC curve for PAM4 serum immunoassay, showing
sensitivity for detection of 81.6% and specificity of 84.6%.
[0084] FIG. 25. Accuracy of the PAM4-immunoassay was determined to
be within 10% of the nominal concentrations examined at or above
the cutoff value of 2.40 units/mL. A linear trend was calculated
with an equation of y=0.965x+0.174, and goodness of fit
r.sup.2=0.999.
[0085] FIG. 26. Frequency distribution of PAM4-reactive antigen in
patient sera by stage of disease. Cutoff value=2.4 units/mL
(horizontal line). The median values (units/mL) are shown for each
study group.
[0086] FIG. 27. Receiver Operator Characteristics (ROC) curve for
the performance of the PAM4-based immunoassay; pancreatic
adenocarcinoma vs. healthy adults. Values for the area under the
curves (AUC) and 95% confidence limits are provided.
[0087] FIG. 28A. Circulating PAM4 antigen levels correlated with
progression/regression of tumor volume (CT) following treatment
with .sup.90Y-PAM4-IgG plus gemcitabine. Patient 076-001 was
responsive to therapy and serum PAM4 antigen decreased. Serum PAM4
levels correlated with tumor volume.
[0088] FIG. 28B. Circulating PAM4 antigen levels correlated with
progression/regression of tumor volume (CT) following treatment
with .sup.90Y-PAM4-IgG plus gemcitabine. Patient 1810002 showed an
initial response to therapy, followed by recurrence of the tumor.
Serum PAM4 levels correlated with tumor volume.
[0089] FIG. 29. Reactivity of PAM4 with mucin standards in the
presence or absence of palmitic acid.
[0090] FIG. 30A. Sensitivity and specificity for PAM4 detection of
PDAC vs. chronic pancreatitis (CP).
[0091] FIG. 30B. Sensitivity and specificity for PAM4 detection of
PDAC vs. all benign tissue samples.
[0092] FIG. 31. Comparative labeling of PDAC vs. non-neoplastic
prostate tissue with PAM4 vs. antibodies against MUC1, MUC4,
CEACAM6 and CA19-9.
[0093] FIG. 32. Reactivity of several anti-mucin MAbs with a high
molecular weight mucin containing fraction (CPM1) isolated from the
Capan-1, human pancreatic adenocarcinoma. MAbs are identified by
clone name with reactive species of mucin indicated by horizontal
bars beneath MAb clone names. In addition to PAM4, substantial
reactions were observed for anti-MUC1, MUC5AC, and CEACAM6
antibodies. All MAbs were employed at a constant 10 .mu.g/mL.
[0094] FIG. 33. Reaction of several anti-mucin MAbs with
PAM4-captured antigen. Mucin antigens were captured on hPAM4 coated
plates, and then probed with several murine anti-mucin MAbs for
reaction signal. Both anti-MUC5AC MAbs (2-11M1 and 45M1) bound to
the hPAM4-captured mucin, whereas the anti-MUC1 MAbs (MA5 and KC4)
did not bind. The homologous hPAM4/mPAM4, capture/probe immunoassay
gave no signal, suggesting the density of PAM4 epitopes within the
mucin may be low, possibly only a single site. A rabbit polyclonal
anti-CPM1, IgG, was used as a positive control for reaction with
hPAM4-captured antigen.
[0095] FIG. 34A. Inhibition of hPAM4/antigen binding reaction by
murine anti-mucin MAbs. Anti-mucin mMAbs (purified IgG) were added
to CPM1-coated plates as potential inhibitors prior to addition of
hPAM4. mPAM4 provided almost complete inhibition of the reaction
between hPAM4 and antigen with the 45M1 anti-MUC5AC providing
limited inhibitory affect (IC.sub.max=25.5%). Neither 2-11M1,
anti-MUC5AC nor MA5 and KC4, anti-MUC1 MAbs were able to inhibit
the specific hPAM4/antigen reaction.
[0096] FIG. 34B. Inhibition of hPAM4/antigen binding reaction by
murine anti-mucin MAbs. A similar inhibition study was performed
with several anti-MUC5AC MAbs obtained as ascites fluids. MAbs
21M1, 62M1, and 463M1, anti-MUC5AC provided substantial inhibitory
affect similar to that observed with mPAM4, IgG, self-inhibition.
The ascites form of 45M1 yielded an inhibitory affect similar to
that of the purified IgG. Ascites containing anti-alpha fetoprotein
was employed as a negative control.
[0097] FIG. 35. Representation of the domains of the MUC5AC
glycoprotein with reactive epitopes indicated for several
anti-MUC5AC MAbs. Data derived by transfection with plasmid vectors
containing the cDNA of the 3'-end of MUC5AC, along with derivative
cDNA vectors obtained by restriction enzyme digestion, have
identified the location of specific epitopes for anti-MUC5AC MAbs
employed in the current studies. Specific blocking studies suggest
the PAM4-epitope resides within the cysteine-rich C-terminus
domain.
DETAILED DESCRIPTION
Definitions
[0098] Unless otherwise specified, "a" or "an" means one or
more.
[0099] As used herein, "about" means plus or minus 10%. For
example, "about 100" would include any number between 90 and
110.
[0100] As described herein, the term "PAM4 antibody" includes
murine, chimeric, humanized and human PAM4 antibodies. In preferred
embodiments, the PAM4 antibody or antigen-binding fragment thereof
comprises the CDR sequences of SEQ ID NO: 1 to SEQ ID NO:6.
[0101] As used herein, an "anti-pancreatic cancer antibody" is an
antibody that exhibits the same diagnostic, therapeutic and binding
characteristics as the PAM4 antibody. In preferred embodiments, the
"anti-pancreatic cancer antibody" binds to an epitope located
within the second to fourth cysteine-rich subdomains of MUC5AC
(amino acid residues 1575-2052), more preferably to an epitope
located in amino acid residues 1575-1725 and 1903-2052 (Cys2 and
Cys 4), even more preferably to an epitope located in amino acid
residues 1575-1725 (Cys2+), most preferably, to an epitope located
in Cys2.
[0102] A "non-endocrine pancreatic cancer" generally refers to
cancers arising from the exocrine pancreatic glands. The term
excludes pancreatic insulinomas and includes pancreatic carcinoma,
pancreatic adenocarcinoma, adenosquamous carcinoma, squamous cell
carcinoma and giant cell carcinoma and precursor lesions such as
pancreatic intra-epithelial neoplasia (PanIN), mucinous cyst
neoplasms (MCN) and intrapancreatic mucinous neoplasms (IPMN),
which are neoplastic but not yet malignant. The terms "pancreatic
cancer" and "non-endocrine pancreatic cancer" are used
interchangeably herein.
[0103] An antibody, as described herein, refers to a full-length
(i.e., naturally occurring or formed by normal immunoglobulin gene
fragment recombinatorial processes) immunoglobulin molecule (e.g.,
an IgG antibody) or an immunologically active (i.e., specifically
binding) portion of an immunoglobulin molecule, like an antibody
fragment.
[0104] An antibody fragment is a portion of an antibody such as
F(ab').sub.2, Fab', Fab, Fv, sFv and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the full-length antibody. The term "antibody
fragment" also includes isolated fragments consisting of the
variable regions of antibodies, such as the "Fv" fragments
consisting of the variable regions of the heavy and light chains
and recombinant single chain polypeptide molecules in which light
and heavy variable regions are connected by a peptide linker ("scFv
proteins"). Another form of antibody fragment is a single domain
antibody (nanobody).
[0105] A naked antibody is an antibody or fragment thereof that is
not conjugated to a therapeutic or diagnostic agent. Generally, the
Fc portion of the antibody molecule provides effector functions,
such as complement-mediated cytotoxicity (CDC) and ADCC
(antibody-dependent cellular cytotoxicity), which set mechanisms
into action that may result in cell lysis. However, the Fc portion
may not be required for therapeutic function, with other
mechanisms, such as signaling-induced apoptosis, coming into play.
Naked antibodies include both polyclonal and monoclonal antibodies,
as well as fusion proteins and certain recombinant antibodies, such
as chimeric, humanized or human antibodies.
[0106] A chimeric antibody is a recombinant protein that contains
the variable domains including the complementarity determining
regions (CDRs) of an antibody derived from one species, preferably
a rodent antibody, while the constant domains of the antibody
molecule are derived from those of a human antibody. For veterinary
applications, the constant domains of the chimeric antibody may be
derived from that of other species, such as a cat or dog.
[0107] A humanized antibody is a recombinant protein in which the
CDRs from an antibody from one species; e.g., a rodent antibody,
are transferred from the heavy and light variable chains of the
rodent antibody into human heavy and light variable domains (e.g.,
framework region sequences). The constant domains of the antibody
molecule are derived from those of a human antibody. In certain
embodiments, a limited number of framework region amino acid
residues from the parent (rodent) antibody may be substituted into
the human antibody framework region sequences.
[0108] A human antibody is, e.g., an antibody obtained from
transgenic mice that have been "engineered" to produce specific
human antibodies in response to antigenic challenge. In this
technique, elements of the human heavy and light chain loci are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous murine
heavy chain and light chain loci. The transgenic mice can
synthesize human antibodies specific for particular antigens, and
the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described by Green et al., Nature Genet. 7:13 (1994),
Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.
Immun. 6:579 (1994). A fully human antibody also can be constructed
by genetic or chromosomal transfection methods, as well as phage
display technology, all of which are known in the art. See for
example, McCafferty et al., Nature 348:552-553 (1990) for the
production of human antibodies and fragments thereof in vitro, from
immunoglobulin variable domain gene repertoires from unimmunized
donors. In this technique, antibody variable domain genes are
cloned in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, 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. In this way, the phage mimics some of
the properties of the B cell. Phage display can be performed in a
variety of formats, for review, see e.g. Johnson and Chiswell,
Current Opinion in Structural Biology 3:5564-571 (1993). Human
antibodies may also be generated by in vitro activated B cells. See
U.S. Pat. Nos. 5,567,610 and 5,229,275, the Examples sections of
which are incorporated herein by reference.
[0109] A therapeutic agent is a compound, molecule or atom which is
administered separately, concurrently or sequentially with an
antibody moiety or conjugated to an antibody moiety, i.e., antibody
or antibody fragment, or a subfragment, and is useful in the
treatment of a disease. Examples of therapeutic agents include
antibodies, antibody fragments, cytotoxic agents, drugs, toxins,
nucleases, hormones, immunomodulators, pro-apoptotic agents,
anti-angiogenic agents, boron compounds, photoactive agents or dyes
and radioisotopes. Therapeutic agents of use are described in more
detail below.
[0110] A diagnostic agent is a molecule, atom or other detectable
moiety which may be administered conjugated to an antibody moiety
or targetable construct and is useful in detecting or diagnosing a
disease by locating cells containing the target antigen. Useful
diagnostic agents include, but are not limited to, radioisotopes,
dyes, contrast agents, fluorescent compounds or molecules and
enhancing agents (e.g., paramagnetic ions) for magnetic resonance
imaging (MRI) or positron emission tomography (PET) scanning.
Preferably, the diagnostic agents are selected from the group
consisting of radioisotopes, enhancing agents for use in magnetic
resonance or PET imaging, and fluorescent compounds. In order to
load an antibody component with radioactive metals, paramagnetic
ions or other diagnostic cations, it may be necessary to react it
with a reagent having a long tail to which are attached a
multiplicity of chelating groups for binding the ions. Such a tail
can be a polymer such as a polylysine, polysaccharide, or other
derivatized or derivatizable chain having pendant groups to which
can be bound chelating groups such as, e.g.,
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA,
porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,
polyoximes, and like groups known to be useful for this purpose.
Chelates are coupled to the antibodies using standard chemistries.
The chelate is normally linked to the antibody by a group which
enables formation of a bond to the molecule with minimal loss of
immunoreactivity and minimal aggregation and/or internal
cross-linking. Other, more unusual, methods and reagents for
conjugating chelates to antibodies are disclosed in U.S. Pat. No.
4,824,659, the Examples section of which is incorporated herein by
reference. Particularly useful metal-chelate combinations include
2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with
diagnostic isotopes in the general energy range of 60 to 4,000 keV,
such as .sup.125I, .sup.131I, .sup.123I, .sup.124I, .sup.62Cu,
.sup.64Cu, .sup.18F, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.99mTc,
.sup.94mTc, .sup.11C, .sup.13N, .sup.15O, .sup.76Br, for
radioimaging. The same chelates, when complexed with
non-radioactive metals, such as manganese, iron and gadolinium are
useful for MRI, when used along with the antibodies of the
invention. Macrocyclic chelates such as NOTA
(1,4,7-triazacyclononane-N,N',N''-triacetic acid), DOTA
(1,4,7,10-tetraazacyclododecanetetraacetic acid), and TETA
(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of
use with a variety of metals and radiometals, most particularly
with radionuclides of gallium, yttrium and copper, respectively.
Such metal-chelate complexes can be made very stable by tailoring
the ring size to the metal of interest. Other ring-type chelates
such as macrocyclic polyethers, which are of interest for stably
binding nuclides, such as .sup.223Ra for radioimmunotherapy (RAIT)
are encompassed by the invention. More recently, techniques of
general utility for labeling virtually any molecule with an
.sup.18F atom of use in PET imaging have been described in U.S.
Pat. Nos. 7,563,433; 7,597,876 and 7,993,626, the Examples section
of each incorporated herein by reference.
[0111] An immunoconjugate is an antibody, antibody fragment or
antibody fusion protein conjugated to at least one therapeutic
and/or diagnostic agent. The diagnostic agent and/or therapeutic
agent are as defined above.
[0112] An expression vector is a DNA molecule comprising a gene
that is expressed in a host cell. Typically, gene expression is
placed under the control of certain regulatory elements, including
constitutive or inducible promoters, tissue-specific regulatory
elements and enhancers. Such a gene is said to be "operably linked
to" the regulatory elements.
[0113] A recombinant host may be any prokaryotic or eukaryotic cell
that contains either a cloning vector or expression vector. This
term also includes those prokaryotic or eukaryotic cells, as well
as transgenic animals, that have been genetically engineered to
contain the cloned gene(s) in the chromosome or genome of the host
cell. Suitable mammalian host cells include myeloma cells, such as
SP2/0 cells, and NS0 cells, as well as Chinese Hamster Ovary (CHO)
cells, hybridoma cell lines and other mammalian host cell useful
for expressing antibodies. Also particularly useful to express mAbs
and other fusion proteins are Sp2/0 cells transfected with an
apoptosis inhibitor, such as a Bcl-EEE gene, and adapted to grow
and be further transfected in serum free conditions, as described
in U.S. Pat. Nos. 7,531,327; 7,537,930; and 7,608,425, the Examples
section of each of which is incorporated herein by reference.
[0114] Anti-Pancreatic Cancer Antibodies
[0115] Various embodiments of the invention concern antibodies that
react with very high selectivity with pancreatic cancer as opposed
to normal or benign pancreatic tissues. The anti-pancreatic cancer
antibodies and fragments thereof are preferably raised against a
crude mucin preparation from a tumor of the human pancreas,
although partially purified or even purified MUC5AC may be
utilized. A non-limiting example of such antibodies is the PAM4
antibody.
[0116] The murine PAM4 (mPAM4) antibody was developed by employing
pancreatic cancer mucin derived from the xenografted RIP-1 human
pancreatic carcinoma as immunogen. (Gold et al., Int. J. Cancer,
57:204-210, 1994.) As discussed below, antibody cross-reactivity
and immunohistochemical staining studies indicate that the PAM4
antibody recognizes a unique and novel epitope on MUC5AC.
Immunohistochemical staining studies have shown that the PAM4 MAb
binds to an antigen expressed by breast, pancreas and other cancer
cells, with limited binding to normal human tissue. However, the
highest expression is usually by pancreatic cancer cells. Thus, the
PAM4 antibodies are relatively specific to pancreatic cancer and
preferentially bind pancreatic cancer cells. The PAM4 antibody is
reactive with a target epitope which can be internalized. This
epitope is expressed primarily by antigens associated with
pancreatic cancer and not with focal pancreatitis or normal
pancreatic tissue. Binding of PAM4 antibody to the PAM4 epitope is
inhibited by treatment of the antigen with DTT or periodate.
Localization and therapy studies using a radiolabeled PAM4 MAb in
animal models have demonstrated tumor targeting and therapeutic
efficacy.
[0117] The PAM4 antibodies bind to an epitope located within the
second to fourth cysteine-rich subdomains of MUC5AC (amino acid
residues 1575-2052), more preferably to an epitope located in amino
acid residues 1575-1725 and 1903-2052 (Cys2 and Cys 4), even more
preferably to an epitope located in amino acid residues 1575-1725
(Cys2+), most preferably, to an epitope located in Cys2. The PAM4
epitope is expressed by many organs and tumor types, but is
preferentially expressed in pancreatic cancer cells. Studies with a
PAM4 MAb, as in the Examples below, indicate that the antibody
exhibits several important properties, which make it a good
candidate for clinical diagnostic and therapeutic applications. The
epitope provides a useful target for diagnosis and therapy of
pancreatic and other cancers. The PAM4 antibody apparently
recognizes an epitope of MUC5AC that is distinct from the epitopes
recognized by non-PAM4 anti-pancreatic cancer antibodies (e.g.,
CA19.9, DUPAN2, SPAN1, Nd2, CEACAM5, B72.3, anti-Le.sup.a, and
other anti-Lewis antigens).
[0118] Surprisingly, the Examples below indicate that the MUC5AC
epitope to which PAM4 binds is present in detectable concentrations
in serum of patients with very early stage pancreatic cancer. Also
surprisingly, it appears that an endogenous inhibitor of PAM4
antibody binding to MUC5AC is present in fresh human serum. The
inhibitor is removed by long-term frozen storage of serum samples,
or by organic phase extraction of fresh serum. These unexpected
results provide the basis of a relatively non-invasive, early
detection test for pancreatic cancer, using blood, serum or plasma
samples. In alternative embodiments, the PAM4 antibody may be used
alone, or else in conjunction with one or more other antibodies,
such as CA19.9 antibody, to detect pancreatic cancer markers in
serum.
[0119] At the tissue level, the reactivity of PAM4 is highly
restricted to PDAC, with the biomarker expressed (or becomes
accessible) at the earliest stages of neoplastic development (Gold
et al., 1994, Int J Cancer 57:204-10; Gold et al., 2007, Clin
Cancer Res 13:7380-7), including pancreatic intraepithelial
neoplasia (PanIN), and intraductal papillary mucinous neoplasm
(IPMN). Notably, the PAM4-biomarker is absent from normal pancreas
and benign, non-neoplastic lesions. In over 50 surgical specimens
of chronic pancreatitis, the PAM4-biomarker was identified only
within associated PanIN lesions and not by the inflamed parenchyma,
including ducts, acinar cells, and acinar-ductal metaplasia (Shi et
al., 2014, Arch Pathol Lab Med 138:220-8).
[0120] Preclinical studies have demonstrated the potential
applications of PAM4 for radioimmunoimaging and radioimmunotherapy
of pancreatic carcinoma (Gold et al., 2002, Crit Rev Oncol Hematol
39:147-54; Gold et al., 2004, Int J Cancer 109: 618-26). In
patients, .sup.90Y-labeled, humanized PAM4 (.sup.90Y-clivatuzumab
tetraxetan, hereafter referred to as .sup.90Y-hPAM4) was well
tolerated with manageable hematologic toxicity under maximal
tolerated .sup.90Y dosing, and produced objective responses in both
chemotherapy-naive and -refractory patients with advanced PDAC
(Gulec et al., 2011, Clin Cancer Res 17:4091-100). Further,
.sup.90Y-hPAM4 in combination with low-dose gemcitabine showed
enhanced therapeutic efficacy in patients with metastatic
pancreatic cancer (Ocean et al., 2012, Cancer 118:5497-506). In a
recently completed phase Ib study (Picozzi et al., 2014, J Clin
Oncol 132:4026) involving 58 patients with metastatic PDAC who had
at least 2 prior therapies, multiple cycles of fractionated
.sup.90Y-hPAM4 in combination with low radiosensitizing doses of
gemcitabine significantly (P=0.004) improved the Kaplan-Meier
median overall survival of this difficult-to-treat population to
7.9 months, compared to those receiving only .sup.90Y-hPAM4 (3.4
months). These promising results led to the ongoing phase III
registration trial of .sup.90Y-hPAM4 in combination with
gemcitabine (NCT01956812).
[0121] In addition, PAM4 or hPAM4-based ELISA has been devised and
evaluated for detection of PDAC, showing that nearly two-thirds of
patients having confirmed stage-1 disease had elevated PAM4 antigen
in their serum (Gold et al., 2010, Cancer Epidemiol Biomarkers Prev
19:2786-94). However, the current assay, which employs hPAM4 as the
capture antibody and a polyclonal rabbit anti-mucin antiserum (IgG
fraction) as a probe, is not optimal, because the polyclonal probe
is available in only limited quantities and, more importantly, is
not itself specific for the PAM4 antigen. Another concern for
further development of the assay has been the unknown nature of the
antigen marker to which PAM4 is reactive. Given the clinical merit
and ongoing evaluation of hPAM4 as a potential diagnostic and
therapeutic agent for PDAC, there is an urgent need to identify the
PAM4 epitope. Towards this end, we recently proposed (Gold et al.,
2013, Mol Cancer 12:143) that PAM4 was reactive with the human
MUC5AC, a polymeric gel-forming mucin with the monomeric form
consisting of more than 5,000 amino acid residues organized into
three major regions (Thornton et al., 2008, Annu Rev Physiol
70:459-86): a signal peptide and four von Willebrand factor
(vWF)-like cysteine-rich domains (D1, D2, D' and D3) in the
N-terminal region, a MUC11p15-type domain preceding the heavily
O-glycosylated mucin domain in the central region, and a cluster of
vWF-like cysteine-rich domains (D4, B, C, and CK) in the C-terminal
region. In addition, 9 cysteine-rich subdomains (designated Cys1,
Cys2, Cys3, Cys4, Cys5, Cys6, Cys7, Cys8, and Cys9) are
interspersed within the mucin domain. Herein we present further
evidence to support MUC5AC as the PAM4-reactive mucin and,
importantly, have mapped the PAM4 epitope to Cys2.
[0122] For therapeutic use, antibodies suitable for use in
combination or conjunction with PAM4 antibodies include, for
example, the antibodies CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49,
anti-CEA, anti-CEACAM6, anti-Le.sup.a, anti-HLA-DR, anti-CD40,
anti-CD74, anti-CD138, and antibodies defined by the Lewis antigen
Le(y), or antibodies against colon-specific antigen-p (CSAp),
CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, matrix metalloproteinase-1
(MMP-1), MMP-2, MMP-7, MMP-9, MMP-14, MUC1, MUC2, MUC3, MUC4,
MUC5AC, MUC16, MUC17, EGP-1, EGP-2, HER2/neu, EGFR, angiogenesis
factors (e.g., VEGF and PlGF), insulin-like growth factor (IGF),
tenascin, platelet-derived growth factor, and IL-6, as well as
products of oncogenes (bcl-2, Kras, p53), cMET, and antibodies
against tumor necrosis substances, such as described in patents by
Epstein et al. (U.S. Pat. Nos. 6,071,491, 6,017,514, 5,019,368 and
5,882,626). Such antibodies would be useful for complementing PAM4
antibody immunodetection and immunotherapy methods. These and other
therapeutic agents could act synergistically with anti-pancreatic
cancer antibodies, such as PAM4 antibody, when administered before,
together with or after administration of PAM4 antibody.
[0123] In therapeutic applications, antibodies that are agonistic
or antagonistic to immunomodulators involved in effector cell
function against tumor cells could also be useful in combination
with PAM4 antibodies alone or in combination with other
tumor-associated antibodies, one example being antibodies against
CD40. Todryk et al., J. Immunol Methods, 248:139-147 (2001); Turner
et al., J. Immunol, 166:89-94 (2001). Also of use are antibodies
against markers or products of oncogenes (e.g., bcl-2, Kras, p53,
cMET), or antibodies against angiogenesis factors, such as VEGFR
and placenta-like growth factor (PlGF).
[0124] The availability of another PAM4-like antibody that binds to
a different epitope of MUC5AC is important for the development of a
double-determinant enzyme-linked immunosorbent assay (ELISA), of
use for MUC5AC in clinical samples. ELISA experiments are described
in the Examples below.
[0125] The murine, chimeric, humanized and fully human PAM4
antibodies and fragments thereof described herein are exemplary of
anti-pancreatic cancer antibodies of use for diagnostic and/or
therapeutic methods. The Examples below disclose a preferred
embodiment of the construction and use of a humanized PAM4
antibody. Because non-human monoclonal antibodies can be recognized
by the human host as a foreign protein, and repeated injections can
lead to harmful hypersensitivity reactions, humanization of a
murine antibody sequences can reduce the adverse immune response
that patients may experience. For murine-based monoclonal
antibodies, this is often referred to as a Human Anti-Mouse
Antibody (HAMA) response. Preferably some human residues in the
framework regions of the humanized anti-pancreatic cancer antibody
or fragments thereof are replaced by their murine counterparts. It
is also preferred that a combination of framework sequences from
two different human antibodies is used for V.sub.H. The constant
domains of the antibody molecule are derived from those of a human
antibody.
[0126] Antibody Preparation
[0127] Monoclonal antibodies for specific antigens may be obtained
by methods known to those skilled in the art. See, for example,
Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al.
(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) (hereinafter "Coligan"). Briefly,
anti-pancreatic cancer MAbs can be obtained by injecting mice with
a composition comprising a mixture of pancreatic cancer mucins
comprising MUC5AC, or a purified MUC5AC, or a peptide or protein
corresponding to an epitope located within the second to fourth
cysteine-rich domains of MUC5AC (amino acid residues 1575-2052),
more preferably to an epitope located in amino acid residues
1575-1725 and 1903-2052 (Cys2 and Cys 4), even more preferably to
an epitope located in amino acid residues 1575-1725 (Cys2+), most
preferably, to an epitope located in Cys2, verifying the presence
of antibody production by removing a serum sample, removing the
spleen to obtain B-lymphocytes, fusing the B-lymphocytes with
myeloma cells to produce hybridomas, cloning the hybridomas,
selecting positive clones which produce antibodies to MUC5AC,
culturing the clones that produce antibodies to an epitope of
MUC5AC as discussed above, and isolating anti-pancreatic cancer
antibodies from the hybridoma cultures.
[0128] After the initial raising of antibodies to the immunogen,
the antibodies can be sequenced and subsequently prepared by
recombinant techniques to produce chimeric or humanized antibodies.
Chimerization of murine antibodies and antibody fragments are well
known to those skilled in the art. The use of antibody components
derived from chimerized monoclonal antibodies reduces potential
problems associated with the immunogenicity of murine constant
regions.
[0129] General techniques for cloning murine immunoglobulin
variable domains are described, for example, by the publication of
Orlandi et al., Proc Nat'l Acad. Sci. USA 86: 3833 (1989),
incorporated herein by reference. In general, the V.sub.K (variable
light chain) and V.sub.H (variable heavy chain) sequences for
murine antibodies can be obtained by a variety of molecular cloning
procedures, such as RT-PCR, 5'-RACE, and cDNA library screening.
Specifically, the V.sub.H and V.sub.K sequences of the murine PAM4
MAb were cloned by PCR amplification from the hybridoma cells by
RT-PCR, and their sequences determined by DNA sequencing. To
confirm their authenticity, the cloned V.sub.K and V.sub.H genes
can be expressed in cell culture as a chimeric Ab as described by
Orlandi et al., (Proc Natl. Acad. Sci., USA, 86: 3833, 1989).
[0130] In a preferred embodiment, a chimerized PAM4 antibody or
antibody fragment comprises the complementarity-determining regions
(CDRs) and framework regions (FR) of a murine PAM4 MAb and the
light and heavy chain constant regions of a human antibody, wherein
the CDRs of the light chain variable region of the chimerized PAM4
comprises CDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID
NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and the CDRs of the heavy
chain variable region of the chimerized PAM4 MAb comprises CDR1
(SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and
CDR3 (GFGGSYGFAY, SEQ ID NO:6). The use of antibody components
derived from chimerized monoclonal antibodies reduces potential
problems associated with the immunogenicity of murine constant
regions.
[0131] Humanization of murine antibodies and antibody fragments is
also well known to those skilled in the art. Techniques for
producing humanized MAbs are disclosed, for example, by Carter et
al., Proc Nat'l Acad. Sci. USA 89: 4285 (1992), Singer et al., J.
Immun. 150: 2844 (1992), Mountain et al. Biotechnol Genet Eng Rev.
10:1 (1992), and Coligan at pages 10.19.1-10.19.11, each of which
is incorporated herein by reference. For example, humanized
monoclonal antibodies may be produced by transferring murine
complementary determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting selected human residues in the framework
regions with their the murine FR counterparts. The use of human
framework region sequences, in addition to human constant region
sequences, further reduces the chance of inducing HAMA
reactions.
[0132] Humanized antibodies can be designed and constructed as
described by Leung et al. (Mol Immunol. 32: 1413 (1995)). Example 3
describes the humanization process utilized for construction of the
hPAM4 MAb.
[0133] The nucleotide sequences of the primers used to prepare the
hPAM4 antibodies are discussed in Example 3, below. In a preferred
embodiment, a humanized PAM4 antibody or antibody fragment
comprises the light and heavy chain CDR sequences (SEQ ID NO: 1 to
SEQ ID NO:6) disclosed above. Also preferred, the FRs of the light
and heavy chain variable regions of the humanized antibody comprise
at least one amino acid substituted from said corresponding FRs of
the murine PAM4 MAb.
[0134] A fully human antibody, e.g., human PAM4 can be obtained
from a transgenic non-human animal. See, e.g., Mendez et al.,
Nature Genetics, 15: 146-156 (1997); U.S. Pat. No. 5,633,425. For
example, a human antibody can be recovered from a transgenic mouse
possessing human immunoglobulin loci. The mouse humoral immune
system is humanized by inactivating the endogenous immunoglobulin
genes and introducing human immunoglobulin loci. The human
immunoglobulin loci are exceedingly complex and comprise a large
number of discrete segments which together occupy almost 0.2% of
the human genome. To ensure that transgenic mice are capable of
producing adequate repertoires of antibodies, large portions of
human heavy- and light-chain loci must be introduced into the mouse
genome. This is accomplished in a stepwise process beginning with
the formation of yeast artificial chromosomes (YACs) containing
either human heavy- or light-chain immunoglobulin loci in germline
configuration. Since each insert is approximately 1 Mb in size, YAC
construction requires homologous recombination of overlapping
fragments of the immunoglobulin loci. The two YACs, one containing
the heavy-chain loci and one containing the light-chain loci, are
introduced separately into mice via fusion of YAC-containing yeast
spheroblasts with mouse embryonic stem cells. Embryonic stem cell
clones are then microinjected into mouse blastocysts. Resulting
chimeric males are screened for their ability to transmit the YAC
through their germline and are bred with mice deficient in murine
antibody production. Breeding the two transgenic strains, one
containing the human heavy-chain loci and the other containing the
human light-chain loci, creates progeny which produce human
antibodies in response to immunization. However, these techniques
are not limiting and other methods known in the art for producing
human antibodies, such as the use of phage display, may also be
utilized to produce human anti-pancreatic cancer antibodies.
[0135] Antibodies can be produced by cell culture techniques using
methods known in the art. In one example transfectoma cultures are
adapted to serum-free medium. For production of humanized antibody,
cells may be grown as a 500 ml culture in roller bottles using
HSFM. Cultures are centrifuged and the supernatant filtered through
a 0.2 m membrane. The filtered medium is passed through a protein-A
column (1.times.3 cm) at a flow rate of 1 ml/min. The resin is then
washed with about 10 column volumes of PBS and protein A-bound
antibody is eluted from the column with 0.1 M glycine buffer (pH
3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in
tubes containing 10 .mu.l of 3 M Tris (pH 8.6), and protein
concentrations determined from the absorbance at 280/260 nm. Peak
fractions are pooled, dialyzed against PBS, and the antibody
concentrated, for example, with a Centricon 30 filter (Amicon,
Beverly, Mass.). The antibody concentration is determined by ELISA
and its concentration adjusted to about 1 mg/ml using PBS. Sodium
azide, 0.01% (w/v), is conveniently added to the sample as
preservative.
[0136] Antibodies can be isolated and purified from hybridoma
cultures by a variety of well-established techniques. Such
isolation techniques include affinity chromatography with Protein-A
SEPHAROSE.RTM., size-exclusion chromatography, and ion-exchange
chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and
pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10,
pages 79-104 (The Humana Press, Inc. 1992).
[0137] Anti-pancreatic cancer MAbs can be characterized by a
variety of techniques that are well-known to those of skill in the
art. For example, the ability of an antibody to bind to an epitope
of MUC5AC as discussed above can be verified using an indirect
enzyme immunoassay, flow cytometry analysis, ELISA or Western blot
analysis.
[0138] Antibody Fragments
[0139] Antibody fragments are antigen binding portions of an
antibody, such as F(ab').sub.2, Fab', F(ab).sub.2, Fab, Fv, sFv,
scFv and the like. Antibody fragments which recognize specific
epitopes can be generated by known techniques. F(ab').sub.2
fragments, for example, can be produced by pepsin digestion of the
antibody molecule. These and other methods are described, for
example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and
references contained therein. Also, see Nisonoff et al., Arch
Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119
(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422
(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4. Alternatively, Fab' expression libraries can be
constructed (Huse et al., 1989, Science, 246:1274-1281) to allow
rapid and easy identification of monoclonal Fab' fragments with the
desired specificity.
[0140] A single chain Fv molecule (scFv) comprises a V.sub.L domain
and a V.sub.H domain. The V.sub.L and V.sub.H domains associate to
form a target binding site. These two domains are further
covalently linked by a peptide linker (L). A scFv molecule is
denoted as either V.sub.L-L-V.sub.H if the V.sub.L domain is the
N-terminal part of the scFv molecule, or as V.sub.H-L-V.sub.L if
the V.sub.H domain is the N-terminal part of the scFv molecule.
Methods for making scFv molecules and designing suitable peptide
linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.
4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol
9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain
Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).
[0141] Other antibody fragments, for example single domain antibody
fragments, are known in the art and may be used in the claimed
constructs. Single domain antibodies (VHH) may be obtained, for
example, from camels, alpacas or llamas by standard immunization
techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001;
Yau et al., J Immunol Methods 281:161-75, 2003; Maass et al., J
Immunol Methods 324:13-25, 2007). The VHH may have potent
antigen-binding capacity and can interact with novel epitopes that
are inaccessible to conventional V.sub.H-V.sub.L pairs.
(Muyldermans et al., 2001). Alpaca serum IgG contains about 50%
camelid heavy chain only IgG antibodies (HCAbs) (Maass et al.,
2007). Alpacas may be immunized with known antigens, such as
TNF-.alpha., and VHHs can be isolated that bind to and neutralize
the target antigen (Maass et al., 2007). PCR primers that amplify
virtually all alpaca VHH coding sequences have been identified and
may be used to construct alpaca VHH phage display libraries, which
can be used for antibody fragment isolation by standard biopanning
techniques well known in the art (Maass et al., 2007). Commercially
available single domain antibodies, also known as nanobodies, may
be purchased for example from Ablynx (Ghent, Belgium).
[0142] An antibody fragment can also be prepared by proteolytic
hydrolysis of a full-length antibody or by expression in E. coli or
another host of the DNA coding for the fragment. An antibody
fragment can be obtained by pepsin or papain digestion of
full-length antibodies by conventional methods. For example, an
antibody fragment can be produced by enzymatic cleavage of
antibodies with pepsin to provide an approximate 100 Kd fragment
denoted F(ab').sub.2. This fragment can be further cleaved using a
thiol reducing agent, and optionally a blocking group for the
sulfhydryl groups resulting from cleavage of disulfide linkages, to
produce an approximate 50 Kd Fab' monovalent fragment.
Alternatively, an enzymatic cleavage using papain produces two
monovalent Fab fragments and an Fc fragment directly.
[0143] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0144] Antibody Fusion Proteins and Multivalent Antibodies
[0145] Fusion proteins comprising the anti-pancreatic cancer
antibodies of interest can be prepared by a variety of conventional
procedures, ranging from glutaraldehyde linkage to more specific
linkages between functional groups. The antibodies and/or antibody
fragments that comprise the fusion proteins described herein are
preferably covalently bound to one another, directly or through a
linker moiety, through one or more functional groups on the
antibody or fragment, e.g., amine, carboxyl, phenyl, thiol, or
hydroxyl groups. Various conventional linkers in addition to
glutaraldehyde can be used, e.g., diisocyanates,
diiosothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides,
maleimidehydroxy succinimide esters, and the like.
[0146] A simple method for producing fusion proteins is to mix the
antibodies or fragments in the presence of glutaraldehyde. The
initial Schiff base linkages can be stabilized, e.g., by
borohydride reduction to secondary amines. A diiosothiocyanate or
carbodiimide can be used in place of glutaraldehyde as a
non-site-specific linker. In one embodiment, an antibody fusion
protein comprises an anti-pancreatic cancer MAb, or fragment
thereof, wherein the MAb binds to an epitope located within the
second to fourth cysteine-rich domains of MUC5AC (amino acid
residues 1575-2052), more preferably to an epitope located in amino
acid residues 1575-1725 and 1903-2052 (Cys2 and Cys 4), even more
preferably to an epitope located in amino acid residues 1575-1725
(Cys2+), most preferably, to an epitope located in Cys2. This
fusion protein and fragments thereof preferentially bind pancreatic
cancer cells. This monovalent, monospecific MAb is useful for
direct targeting of an antigen, where the MAb is attached to a
therapeutic agent, a diagnostic agent, or a combination thereof,
and the protein is administered directly to a patient.
[0147] The fusion proteins may instead comprise at least two
anti-pancreatic cancer MAbs that bind to distinct epitopes of
MUC5AC. For example, the MAbs can produce antigen specific
diabodies, triabodies and tetrabodies, which are multivalent but
monospecific to the MUCSAC. The non-covalent association of two or
more scFv molecules can form functional diabodies, triabodies and
tetrabodies. Monospecific diabodies are homodimers of the same
scFv, where each scFv comprises the V.sub.H domain from the
selected antibody connected by a short linker to the V.sub.L domain
of the same antibody. A diabody is a bivalent dimer formed by the
non-covalent association of two scFvs, yielding two Fv binding
sites. A triabody results from the formation of a trivalent trimer
of three scFvs, yielding three binding sites, and a tetrabody is a
tetravalent tetramer of four scFvs, resulting in four binding
sites. Several monospecific diabodies have been made using an
expression vector that contains a recombinant gene construct
comprising V.sub.H1-linker-V.sub.L1. See Holliger et al., Proc
Natl. Acad. Sci USA 90: 6444-6448 (1993); Atwell et al., Molecular
Immunology 33: 1301-1302 (1996); Holliger et al., Nature
Biotechnology 15: 632-631 (1997); Helfrich et al., Int J Cancer 76:
232-239 (1998); Kipriyanov et al., Int J Cancer 77: 763-772 (1998);
Holiger et al., Cancer Research 59: 2909-2916 (1999)). Methods of
constructing scFvs are disclosed in U.S. Pat. No. 4,946,778 (1990)
and U.S. Pat. No. 5,132,405 (1992), the Examples section of each of
which is incorporated herein by reference. Methods of producing
multivalent, monospecific antibodies based on scFv are disclosed in
U.S. Pat. No. 5,837,242 (1998), U.S. Pat. No. 5,844,094 (1998) and
WO-98/44001 (1998), the Examples section of each of which is
incorporated herein by reference. The multivalent, monospecific
antibody fusion protein binds to two or more of the same type of
epitopes that can be situated on the same antigen or on separate
antigens. The increased valency allows for additional interaction,
increased affinity, and longer residence times. These antibody
fusion proteins can be utilized in direct targeting systems, where
the antibody fusion protein is conjugated to a therapeutic agent, a
diagnostic agent, or a combination thereof, and administered
directly to a patient in need thereof.
[0148] A preferred embodiment is a multivalent, multispecific
antibody or fragment thereof comprising one or more antigen binding
sites having an affinity toward a PAM4 target epitope and one or
more additional binding sites for other epitopes associated with
pancreatic cancer. This fusion protein is multispecific because it
binds at least two different epitopes, which can reside on the same
or different antigens. For example, the fusion protein may comprise
more than one antigen binding site, the first with an affinity
toward an epitope of MUC5AC as discussed above and the second with
an affinity toward another target antigen such as TAG-72 or CEA.
Another example is a bispecific antibody fusion protein which may
comprise a CA19.9 MAb (or fragment thereof) and a PAM4 MAb (or
fragment thereof). Such a fusion protein will have an affinity
toward CA19.9 as well as MUC5AC. The antibody fusion proteins and
fragments thereof can be utilized in direct targeting systems,
where the antibody fusion protein is conjugated to a therapeutic
agent, a diagnostic agent, or a combination thereof, and
administered directly to a patient in need thereof.
[0149] Another preferred embodiment is a multivalent, multispecific
antibody comprising at least one binding site having affinity
toward a PAM4 target epitope and at least one hapten binding site
having affinity towards hapten molecules. For example, a bispecific
fusion protein may comprise the 679 MAb (or fragment thereof) and
the PAM4 MAb (or fragment thereof). The monoclonal 679 antibody
binds with high affinity to molecules containing the tri-peptide
moiety histamine succinyl glycyl (HSG). Such a bispecific PAM4
antibody fusion protein can be prepared, for example, by obtaining
a F(ab').sub.2 fragment from 679, as described above. The
interchain disulfide bridges of the 679 F(ab').sub.2 fragment are
gently reduced with DTT, taking care to avoid light-heavy chain
linkage, to form Fab'-SH fragments. The SH group(s) is (are)
activated with an excess of bis-maleimide linker
(1,1'-(methylenedi-4,1-phenylene)b-is-maleimide). The PAM4 MAb is
converted to Fab'-SH and then reacted with the activated 679
Fab'-SH fragment to obtain a bispecific antibody fusion protein.
Bispecific antibody fusion proteins such as this one can be
utilized in affinity enhancing systems, where the target antigen is
pretargeted with the fusion protein and is subsequently targeted
with a diagnostic or therapeutic agent attached to a carrier moiety
(targetable construct) containing one or more HSG haptens. In
alternative preferred embodiments, a DNL.TM.-based hPAM4-679
construct, such as TF10, may be prepared and used as described in
the Examples below.
[0150] Bispecific antibodies can be made by a variety of
conventional methods, e.g., disulfide cleavage and reformation of
mixtures of whole IgG or, preferably F(ab').sub.2 fragments,
fusions of more than one hybridoma to form polyomas that produce
antibodies having more than one specificity, and by genetic
engineering. Bispecific antibody fusion proteins have been prepared
by oxidative cleavage of Fab' fragments resulting from reductive
cleavage of different antibodies. This is advantageously carried
out by mixing two different F(ab').sub.2 fragments produced by
pepsin digestion of two different antibodies, reductive cleavage to
form a mixture of Fab' fragments, followed by oxidative reformation
of the disulfide linkages to produce a mixture of F(ab').sub.2
fragments including bispecific antibody fusion proteins containing
a Fab' portion specific to each of the original epitopes. General
techniques for the preparation of antibody fusion proteins may be
found, for example, in Nisonoff et al., Arch Biochem Biophys. 93:
470 (1961), Hammerling et al., J Exp Med. 128: 1461 (1968), and
U.S. Pat. No. 4,331,647.
[0151] More selective linkage can be achieved by using a
heterobifunctional linker such as maleimidehydroxysuccinimide
ester. Reaction of the ester with an antibody or fragment will
derivatize amine groups on the antibody or fragment, and the
derivative can then be reacted with, e.g., an antibody Fab fragment
having free sulfhydryl groups (or, a larger fragment or intact
antibody with sulfhydryl groups appended thereto by, e.g., Traut's
Reagent). Such a linker is less likely to crosslink groups in the
same antibody and improves the selectivity of the linkage.
[0152] It is advantageous to link the antibodies or fragments at
sites remote from the antigen-binding sites. This can be
accomplished by, e.g., linkage to cleaved interchain sulfhydryl
groups, as noted above. Another method involves reacting an
antibody having an oxidized carbohydrate portion with another
antibody that has at lease one free amine function. This results in
an initial Schiff base linkage, which is preferably stabilized by
reduction to a secondary amine, e.g., by borohydride reduction, to
form the final composite. Such site-specific linkages are
disclosed, for small molecules, in U.S. Pat. No. 4,671,958, and for
larger addends in U.S. Pat. No. 4,699,784, the Examples section of
each of which is incorporated herein by reference.
[0153] ScFvs with linkers greater than 12 amino acid residues in
length (for example, 15- or 18-residue linkers) allow interactions
between the V.sub.H and V.sub.L domains on the same chain and
generally form a mixture of monomers, dimers (termed diabodies) and
small amounts of higher mass multimers, (Kortt et al., Eur J
Biochem. (1994) 221: 151-157). ScFvs with linkers of 5 or less
amino acid residues, however, prohibit intramolecular pairing of
the V.sub.H and V.sub.L domains on the same chain, forcing pairing
with V.sub.H and V.sub.L domains on a different chain. Linkers
between 3- and 12-residues form predominantly dimers (Atwell et
al., Protein Engineering (1999) 12: 597-604). With linkers between
0 and 2 residues, trimeric (termed triabodies), tetrameric (termed
tetrabodies) or higher oligomeric structures of scFvs are formed;
however, the exact patterns of oligomerization appear to depend on
the composition as well as the orientation of the V-domains, in
addition to the linker length.
[0154] More recently, a novel technique for construction of
mixtures of antibodies, antibody fragments and/or other effector
moieties in virtually any combination has been described in U.S.
Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787; and
7,666,400, the Examples section of each of which is incorporated
herein by reference. The technique, known generally as
DOCK-AND-LOCK.TM. (DNL.TM.) involves the production of fusion
proteins that comprise at their N- or C-terminal ends one of two
complementary peptide sequences, called dimerization and docking
domain (DDD) and anchoring domain (AD) sequences. In preferred
embodiments, the DDD sequences are derived from the regulatory
subunits of cAMP-dependent protein kinase and the AD sequence is
derived from the sequence of A-kinase anchoring protein (AKAP). The
DDD sequences form dimers that bind to the AD sequence, which
allows formation of trimers, tetramers, hexamers or any of a
variety of other complexes. By attaching effector moieties, such as
antibodies or antibody fragments, to the DDD and AD sequences,
complexes may be formed of any selected combination of antibodies
or antibody fragments. The DNL.TM. complexes may be covalently
stabilized by formation of disulfide bonds or other linkages.
[0155] Pretargeting
[0156] Bispecific or multispecific antibodies may be utilized in
pre-targeting techniques. Pre-targeting is a multistep process
originally developed to resolve the slow blood clearance of
directly targeting antibodies, which contributes to undesirable
toxicity to normal tissues such as bone marrow. With pre-targeting,
a radionuclide or other therapeutic agent is attached to a small
delivery molecule (targetable construct or targetable conjugate)
that is cleared within minutes from the blood. A pre-targeting
bispecific or multispecific antibody, which has binding sites for
the targetable construct as well as a target antigen, is
administered first, free antibody is allowed to clear from
circulation and then the targetable construct is administered.
[0157] Pre-targeting methods are well known in the art, for
example, as disclosed in Goodwin et al., U.S. Pat. No. 4,863,713;
Goodwin et al., J. Nucl. Med. 29:226, 1988; Hnatowich et al., J.
Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl. Med. 29:728, 1988;
Klibanov et al., J. Nucl. Med. 29:1951, 1988; Sinitsyn et al., J.
Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl. Med. 31:1791,
1990; Schechter et al., Int. J. Cancer 48:167, 1991; Paganelli et
al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl. Med.
Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,
Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991;
U.S. Pat. No. 6,077,499; U.S. Pat. No. 6,090,381; U.S. Pat. No.
6,472,511; and U.S. Pat. No. 6,962,702.
[0158] A pre-targeting method of treating or diagnosing a disease
or disorder in a subject may be provided by: (1) administering to
the subject a bispecific antibody or antigen binding antibody
fragment; (2) optionally administering to the subject a clearing
composition, and allowing the composition to clear the antibody
from circulation; and (3) administering to the subject the
targetable construct, containing one or more chelated or chemically
bound therapeutic or diagnostic agents. The technique may also be
utilized for antibody dependent enzyme prodrug therapy (ADEPT) by
administering an enzyme conjugated to a targetable construct,
followed by a prodrug that is converted into active form by the
enzyme.
[0159] Known Antibodies
[0160] In various embodiments, the claimed methods and compositions
may utilize any of a variety of antibodies known in the art, for
example for combination antibody therapy. Antibodies of use may be
commercially obtained from a number of known sources. For example,
a variety of antibody secreting hybridoma lines are available from
the American Type Culture Collection (ATCC, Manassas, Va.). A large
number of antibodies against various disease targets, including but
not limited to tumor-associated antigens, have been deposited at
the ATCC and/or have published variable region sequences and are
available for use in the claimed methods and compositions. See,
e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403;
7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803; 7,041,802;
7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598; 6,998,468;
6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854;
6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129;
6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533; 6,919,433;
6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625; 6,887,468;
6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568;
6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282;
6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206; 6,793,924;
6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681; 6,764,679;
6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966; 6,709,653;
6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737;
6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852; 6,635,482;
6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279; 6,596,852;
6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618; 6,545,130;
6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404;
6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408; 6,479,247;
6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356; 6,455,044;
6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402;
6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276;
6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654; 6,372,215;
6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244; 6,346,246;
6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868;
6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499;
5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554; 5,776,456;
5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953; 5,525,338,
the Examples section of each of which is incorporated herein by
reference. These are exemplary only and a wide variety of other
antibodies and their hybridomas are known in the art. The skilled
artisan will realize that antibody sequences or antibody-secreting
hybridomas against almost any disease-associated antigen may be
obtained by a simple search of the ATCC, NCBI and/or USPTO
databases for antibodies against a selected disease-associated
target of interest. The antigen binding domains of the cloned
antibodies may be amplified, excised, ligated into an expression
vector, transfected into an adapted host cell and used for protein
production, using standard techniques well known in the art (see,
e.g., U.S. Pat. Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880,
the Examples section of each of which is incorporated herein by
reference).
[0161] Particular antibodies that may be of use for therapy of
cancer within the scope of the claimed methods and compositions
include, but are not limited to, LL1 (anti-CD74), LL2 or RFB4
(anti-CD22), veltuzumab (hA20, anti-CD20), rituxumab (anti-CD20),
obinutuzumab (GA101, anti-CD20), lambrolizumab (anti-PD-1
receptor), nivolumab (anti-PD-1 receptor), ipilimumab
(anti-CTLA-4), RS7 (anti-epithelial glycoprotein-1 (EGP-1, also
known as TROP-2)), KC4 (anti-mucin), MN-14 (anti-carcinoembryonic
antigen (anti-CEA, also known as CD66e or CEACAM5), MN-15 or MN-3
(anti-CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 31 (an
anti-alpha-fetoprotein), R1 (anti-IGF-1R), A19 (anti-CD19), TAG-72
(e.g., CC49), Tn, J591 or HuJ591 (anti-PSMA (prostate-specific
membrane antigen)), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B
(anti-PSMA), G250 (an anti-carbonic anhydrase IX MAb), L243
(anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),
cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan
(anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20); PAM4
(aka clivatuzumab, anti-MUC5AC) and trastuzumab (anti-ErbB2). Such
antibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;
5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;
6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;
7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;
7,612,180; 7,642,239; and U.S. Patent Application Publ. No.
20050271671; 20060193865; 20060210475; 20070087001; the Examples
section of each incorporated herein by reference.) Specific known
antibodies of use include hPAM4 (U.S. Pat. No. 7,282,567), hA20
(U.S. Pat. No. 7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU-31
(U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No. 7,312,318), hLL2
(U.S. Pat. No. 7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243
(U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hMN-15
(U.S. Pat. No. 7,541,440), hR1 (U.S. patent application Ser. No.
12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3 (U.S. Pat. No.
7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser. No.
11/983,372, deposited as ATCC PTA-4405 and PTA-4406), D2/B (WO
2009/130575), BWA-3 (anti-histone H4), LG2-1 (anti-histone H3) and
LG2-2 (anti-histone H2B) (U.S. patent application Ser. No.
14/180,646, filed Feb. 14, 2014) the text of each recited patent or
application is incorporated herein by reference with respect to the
Figures and Examples sections.
[0162] Other useful antigens that may be targeted using the
described conjugates include carbonic anhydrase IX, B7, CCL19,
CCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8,
CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5
MAbs), CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38,
CD40, CD40L, CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD59, CD64,
CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138,
CD147, CD154, CEACAM5, CEACAM6, CTLA-4, CXCR4, alpha-fetoprotein
(AFP), VEGF (e.g., AVASTIN.RTM., fibronectin splice variant), ED-B
fibronectin (e.g., L19), EGP-1 (TROP-2), EGP-2 (e.g., 17-1A), EGF
receptor (ErbB1) (e.g., ERBITUX.RTM.), ErbB2, ErbB3, Factor H,
FHL-1, Flt-3, folate receptor, Ga 733, GRO-.beta., HMGB-1, hypoxia
inducible factor (HIF), HM1.24, HER-2/neu, histone H2B, histone H3,
histone H4, insulin-like growth factor (ILGF), IFN-.gamma.,
IFN-.alpha., IFN-.beta., IFN-.lamda., IL-2R, IL-4R, IL-6R, IL-13R,
IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17,
IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the
HLA-DR antigen to which L243 binds, CD66 antigens, i.e., CD66a-d or
a combination thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B,
macrophage migration-inhibitory factor (MIF), MUC1, MUC2, MUC3,
MUC4, MUC5AC, placental growth factor (PlGF), PSA
(prostate-specific antigen), PSMA, PD-1, PD-L1, TIM-3, LAG-3,
matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-7, MMP-9, MMP-14,
NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1, Le(y), mesothelin, S100,
tenascin, TAC, Tn antigen, Thomas-Friedenreich antigens, tumor
necrosis antigens, tumor angiogenesis antigens, TNF-.alpha., TRAIL
receptor (R1 and R2), TROP-2, VEGFR, RANTES, T101, as well as
cancer stem cell antigens, complement factors C3, C3a, C3b, C5a,
C5, and an oncogene product.
[0163] A comprehensive analysis of suitable antigen (Cluster
Designation, or CD) targets on hematopoietic malignant cells, as
shown by flow cytometry and which can be a guide to selecting
suitable antibodies for drug-conjugated immunotherapy, is Craig and
Foon, Blood prepublished online Jan. 15, 2008; DOL
10.1182/blood-2007-11-120535.
[0164] The CD66 antigens consist of five different glycoproteins
with similar structures, CD66a-e, encoded by the carcinoembryonic
antigen (CEA) gene family members, BCG, CGM6, NCA, CGM1 and CEA,
respectively. These CD66 antigens (e.g., CEACAM6) are expressed
mainly in granulocytes, normal epithelial cells of the digestive
tract and tumor cells of various tissues. Also included as suitable
targets for cancers are cancer testis antigens, such as NY-ESO-1
(Theurillat et al., Int. J. Cancer 2007; 120(11):2411-7), as well
as CD79a in myeloid leukemia (Kozlov et al., Cancer Genet.
Cytogenet. 2005; 163(1):62-7) and also B-cell diseases, and CD79b
for non-Hodgkin's lymphoma (Poison et al., Blood 110(2):616-623). A
number of the aforementioned antigens are disclosed in U.S.
Provisional Application Ser. No. 60/426,379, entitled "Use of
Multi-specific, Non-covalent Complexes for Targeted Delivery of
Therapeutics," filed Nov. 15, 2002. Cancer stem cells, which are
ascribed to be more therapy-resistant precursor malignant cell
populations (Hill and Perris, J. Natl. Cancer Inst. 2007;
99:1435-40), have antigens that can be targeted in certain cancer
types, such as CD133 in prostate cancer (Maitland et al., Ernst
Schering Found. Sympos. Proc. 2006; 5:155-79), non-small-cell lung
cancer (Donnenberg et al., J. Control Release 2007; 122(3):385-91),
and glioblastoma (Beier et al., Cancer Res. 2007; 67(9):4010-5),
and CD44 in colorectal cancer (Dalerba er al., Proc. Natl. Acad.
Sci. USA 2007; 104(24)10158-63), pancreatic cancer (Li et al.,
Cancer Res. 2007; 67(3):1030-7), and in head and neck squamous cell
carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;
104(3)973-8). Another useful target for breast cancer therapy is
the LIV-1 antigen described by Taylor et al. (Biochem. J. 2003;
375:51-9).
[0165] For multiple myeloma therapy, suitable targeting antibodies
have been described against, for example, CD38 and CD138
(Stevenson, Mol Med 2006; 12(11-12):345-346; Tassone et al., Blood
2004; 104(12):3688-96), CD74 (Stein et al., ibid.), CS1 (Tai et
al., Blood 2008; 112(4):1329-37, and CD40 (Tai et al., 2005; Cancer
Res. 65(13):5898-5906).
[0166] Macrophage migration inhibitory factor (MIF) is an important
regulator of innate and adaptive immunity and apoptosis. It has
been reported that CD74 is the endogenous receptor for MIF (Leng et
al., 2003, J Exp Med 197:1467-76). The therapeutic effect of
antagonistic anti-CD74 antibodies on MIF-mediated intracellular
pathways may be of use for treatment of a broad range of disease
states, such as cancers of the bladder, prostate, breast, lung,
colon and chronic lymphocytic leukemia (e.g., Meyer-Siegler et al.,
2004, BMC Cancer 12:34; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54); autoimmune diseases such as rheumatoid arthritis and
systemic lupus erythematosus (Morand & Leech, 2005, Front
Biosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma
52:1446-54); kidney diseases such as renal allograft rejection
(Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and numerous
inflammatory diseases (Meyer-Siegler et al., 2009, Mediators
Inflamm epub Mar. 22, 2009; Takahashi et al., 2009, Respir Res
10:33; Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of
therapeutic use for treatment of MIF-mediated diseases.
[0167] Anti-TNF-.alpha. antibodies are known in the art and may be
of use to treat immune diseases, such as autoimmune disease, immune
dysfunction (e.g., graft-versus-host disease, organ transplant
rejection) or diabetes. Known antibodies against TNF-.alpha.
include the human antibody CDP571 (Ofei et al., 2011, Diabetes
45:881-85); murine antibodies MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI,
M302B and M303 (Thermo Scientific, Rockford, Ill.); infliximab
(Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,
Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and
many other known anti-TNF-.alpha. antibodies may be used in the
claimed methods and compositions. Other antibodies of use for
therapy of immune dysregulatory or autoimmune disease include, but
are not limited to, anti-B-cell antibodies such as veltuzumab,
epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6
receptor); basiliximab (anti-CD25); daclizumab (anti-CD25);
efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3 receptor);
anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-.alpha.4
integrin) and omalizumab (anti-IgE).
[0168] Checkpoint inhibitor antibodies have been used primarily in
cancer therapy. Immune checkpoints refer to inhibitory pathways in
the immune system that are responsible for maintaining
self-tolerance and modulating the degree of immune system response
to minimize peripheral tissue damage. However, tumor cells can also
activate immune system checkpoints to decrease the effectiveness of
immune response against tumor tissues. Exemplary checkpoint
inhibitor antibodies against cytotoxic T-lymphocyte antigen 4
(CTLA-4, also known as CD152), programmed cell death protein 1
(PD-1, also known as CD279) and programmed cell death 1 ligand 1
(PD-L1, also known as CD274), may be used in combination with one
or more other agents to enhance the effectiveness of immune
response against disease cells, tissues or pathogens. Exemplary
anti-PD1 antibodies include lambrolizumab (MK-3475, MERCK),
nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), and
pidilizumab (CT-011, CURETECH LTD.). Anti-PD1 antibodies are
commercially available, for example from ABCAM.RTM. (AB137132),
BIOLEGEND.RTM. (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE
(J105, J116, MIH4). Exemplary anti-PD-L1 antibodies include
MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and
BMS-936559 (BRISTOL-MYERS SQUIBB). Anti-PD-L1 antibodies are also
commercially available, for example from AFFYMETRIX EBIOSCIENCE
(MIH1). Exemplary anti-CTLA4 antibodies include ipilimumab
(Bristol-Myers Squibb) and tremelimumab (PFIZER). Anti-PD1
antibodies are commercially available, for example from ABCAM.RTM.
(AB134090), SINO BIOLOGICAL INC. (11159-H03H, 11159-H08H), and
THERMO SCIENTIFIC PIERCE (PA5-29572, PA5-23967, PA5-26465,
MA1-12205, MA1-35914). Ipilimumab has recently received FDA
approval for treatment of metastatic melanoma (Wada et al., 2013, J
Transl Med 11:89). More recently, other checkpoint inhibitory
receptors have been identified, including TIM-3 and LAG-3 (Stagg,
2013, Ther Adv Med Oncol 5:169-81). Antibodies against TIM-3 and
LAG-3 may also be used in combination with the anti-MUC5AC
antibodies disclosed herein.
[0169] Antibodies against matrix metalloproteinases, for example
matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-7, MMP-9 and MMP-14,
are also of use in combination anti-cancer therapies. (See, e.g.,
Agarwal A, et al., Mol Cancer Ther 2008; 7:2746-57; Freije J M, et
al. Adv Exp Med Biol 2003; 532:91-107; Coticchia C M, et al.
Gynecol Oncol 2011; 123:295-300; Boiire D, et al., Cell 2005;
120:303-13; Belotti D, et al., Cancer Res 2003; 63:5224-9;
Barbolina M V, et al., J Biol Chem 2007; 282:4924-31; Kaimal R, et
al., Cancer Res 2013; 73:2457-67; Denzel S, et al, Int J Exp Pathol
2012; 93:341-53.)
[0170] Other antibodies of use may include anti-histone antibodies
and/or antigen-binding fragments thereof, such as the BWA-3
(anti-H4), LG2-1 (anti-H3) and LG2-2 (anti-H2B) antibodies.
Exemplary anti-histone antibodies are disclosed, for example, in
U.S. patent application Ser. No. 14/180,646, filed Feb. 14, 2014
(the Examples section of which is incorporated herein by
reference).
[0171] In another preferred embodiment, antibodies are used that
internalize rapidly and are then re-expressed, processed and
presented on cell surfaces, enabling continual uptake and accretion
of circulating conjugate by the cell. An example of a
most-preferred antibody/antigen pair is LL1, an anti-CD74 MAb
(invariant chain, class II-specific chaperone, Ii) (see, e.g., U.S.
Pat. Nos. 6,653,104; 7,312,318; the Examples section of each
incorporated herein by reference). The CD74 antigen is highly
expressed on B-cell lymphomas (including multiple myeloma) and
leukemias, certain T-cell lymphomas, melanomas, colonic, lung, and
renal cancers, glioblastomas, and certain other cancers (Ong et
al., Immunology 98:296-302 (1999)). A review of the use of CD74
antibodies in cancer is contained in Stein et al., Clin Cancer Res.
2007 Sep. 15; 13(18 Pt 2):5556s-5563s, incorporated herein by
reference.
[0172] The diseases that are preferably treated with anti-CD74
antibodies include, but are not limited to, non-Hodgkin's lymphoma,
Hodgkin's disease, melanoma, lung, renal, colonic cancers,
glioblastome multiforme, histiocytomas, myeloid leukemias, and
multiple myeloma. Continual expression of the CD74 antigen for
short periods of time on the surface of target cells, followed by
internalization of the antigen, and re-expression of the antigen,
enables the targeting LL1 antibody to be internalized along with
any chemotherapeutic moiety it carries. This allows a high, and
therapeutic, concentration of LL1-chemotherapeutic drug conjugate
to be accumulated inside such cells. Internalized
LL1-chemotherapeutic drug conjugates are cycled through lysosomes
and endosomes, and the chemotherapeutic moiety is released in an
active form within the target cells.
[0173] Antibody Use for Treatment and Diagnosis
[0174] Certain embodiments concern methods of diagnosing or
treating a malignancy in a subject, comprising administering to the
subject an anti-pancreatic cancer MAb, fusion protein or fragment
thereof, wherein the MAb, fusion protein or fragment is bound to at
least one diagnostic and/or therapeutic agent. The antibody
preferably binds to an epitope located within the second to fourth
cysteine-rich domains of MUC5AC (amino acid residues 1575-2052),
more preferably to an epitope located in amino acid residues
1575-1725 and 1903-2052 (Cys2 and Cys 4), even more preferably to
an epitope located in amino acid residues 1575-1725 (Cys2+), most
preferably, to an epitope located in Cys2
[0175] Also preferred is a method for diagnosing or treating
cancer, comprising administering to a subject a multivalent,
multispecific antibody or fragment thereof comprising one or more
antigen binding sites toward an epitope of MUC5AC as discussed
above and one or more hapten binding sites, waiting a sufficient
amount of time for non-bound antibody to clear the subject's blood
stream; and then administering to the subject a carrier molecule
comprising a diagnostic agent, a therapeutic agent, or a
combination thereof, that binds to the hapten-binding site of the
localized antibody. In a more preferred embodiment, the cancer is a
non-endocrine pancreatic cancer.
[0176] The use of MAbs for in vitro diagnosis is well-known. See,
for example, Carlsson et al., Bio/Technology 7 (6): 567 (1989). For
example, MAbs can be used to detect the presence of a
tumor-associated antigen in tissue from biopsy samples. MAbs also
can be used to measure the amount of tumor-associated antigen in
clinical fluid samples, such as blood or serum, using techniques
such as radioimmunoassay, enzyme-linked immunosorbent assay, and
fluorescence immunoassay. In vitro and in vivo methods of diagnosis
are discussed in further detail below.
[0177] Conjugates of tumor-targeted MAbs and toxins can be used to
selectively kill cancer cells in vivo (Spalding, Bio/Technology
9(8): 701 (1991); Goldenberg, Scientific American Science &
Medicine 1(1): 64 (1994)). For example, therapeutic studies in
experimental animal models have demonstrated the anti-tumor
activity of antibodies carrying cytotoxic radionuclides.
(Goldenberg et al., Cancer Res. 41: 4354 (1981), Cheung et al., J.
Nat'l Cancer Inst. 77: 739 (1986), and Senekowitsch et al., J.
Nucl. Med. 30: 531 (1989)). In a preferred embodiment, the
conjugate comprises a .sup.90Y-labeled hPAM4 antibody. The
conjugate may optionally be administered in conjunction with one or
more other therapeutic agents. In a preferred embodiment,
.sup.90Y-labeled hPAM4 is administered together with gemcitabine or
5-fluorouracil to a patient with pancreatic cancer. In a further
preferred embodiment, .sup.90Y is conjugated to a DOTA chelate for
attachment to hPAM4. In a more preferred embodiment, the
.sup.90Y-DOTA-hPAM4 is combined with gemcitabine in fractionated
doses comprising a treatment cycle, such as with repeated, lower,
less-toxic doses of gemcitabine combined with lower, fractionated
doses of .sup.90Y-DOTA-hPAM4. Alternatively, a radiolabeled or
other conjugated PAM4 antibody may be administered in combination
with another immunoconjugate, such as an SN-38 conjugated antibody.
A particularly preferred combination is .sup.90Y-hPAM4 and
SN-38-hRS7 (anti-TROP2 antibody) (see, e.g., U.S. Pat. No.
8,586,050, the Examples section incorporated herein by
reference).
[0178] As tolerated, repeated cycles of a fractionated dose
schedule are indicated. By way of example, 4 weekly doses of 200
mg/m.sup.2 of gemcitabine are combined with three weekly doses of 8
mg/m.sup.2 of .sup.90Y-DOTA-hPAM4, with the latter commencing in
the second week of gemcitabine administration, constitutes a single
therapy cycle. Still other doses, higher or lower of each
component, may constitute a fractionated dose, which is determined
by conventional means of assessing hematopoietic toxicity (see,
e.g., U.S. Pat. Nos. 6,649,352; 7,112,409; 7,279,289; 7,465,551),
since myelosuppressive effects of both agents can be cumulative. A
skilled physician in such therapy interventions can adjust these
doses based on the patient's bone marrow status and general health
status based on many factors, including prior exposure to
myelosuppressive therapeutic agents. These principles can also
apply to combinations of radiolabeled hPAM4 with other therapeutic
agents, including radiosensitizing drugs such as 5-fluorouracil and
cisplatin.
[0179] Chimeric, humanized and human antibodies and fragments
thereof have been used for in vivo therapeutic and diagnostic
methods. Accordingly contemplated is a method of delivering a
diagnostic or therapeutic agent, or a combination thereof, to a
target comprising (i) providing a composition that comprises an
anti-pancreatic cancer antibody or fragment thereof, such as a
chimeric, humanized or human PAM4 antibody, conjugated to at least
one diagnostic and/or therapeutic agent and (ii) administering to a
subject the diagnostic or therapeutic antibody conjugate. In a
preferred embodiment, the anti-pancreatic cancer antibodies and
fragments thereof are humanized or fully human.
[0180] Another embodiment concerns a method for treating a
malignancy comprising administering a naked or conjugated
anti-pancreatic cancer antibody, antibody fragment or fusion
protein that binds to an epitope located within the second to
fourth cysteine-rich domains of MUC5AC (amino acid residues
1575-2052), more preferably to an epitope located in amino acid
residues 1575-1725 and 1903-2052 (Cys2 and Cys 4), even more
preferably to an epitope located in amino acid residues 1575-1725
(Cys2+), most preferably, to an epitope located in Cys2, such as a
PAM4 antibody, either alone or in conjunction with one or more
other therapeutic agents. The other therapeutic agent may be added
before, simultaneously with or after the antibody. In a preferred
embodiment, the therapeutic agent is gemcitabine, and in a more
preferred embodiment, gemcitabine is given with the hPAM4
radioconjugate in a fractionated dose schedule at lower doses than
the conventional 800-1,000 mg/m.sup.2 doses of gemcitabine given
weekly for 6 weeks. For example, when combined with fractionated
therapeutic doses of .sup.90Y-PAM4, repeated fractionated doses
intended to function as a radiosensitizing agent of 200-380
mg/m.sup.2 gemcitabine are infused. The skilled artisan will
realize that the antibodies, fusion proteins and/or fragments
thereof described and claimed herein may be administered with any
known or described therapeutic agent, including but not limited to
heat shock protein 90 (Hsp90).
[0181] In another form of multimodal therapy, subjects receive
immunoconjugates in conjunction with standard cancer chemotherapy.
For example, "CVB" (1.5 g/m.sup.2 cyclophosphamide, 200-400
mg/m.sup.2 etoposide, and 150-200 mg/m.sup.2 carmustine) is a
regimen used to treat non-Hodgkin's lymphoma. Patti et al., Eur. J.
Haematol. 51: 18 (1993). Other suitable combination
chemotherapeutic regimens are well-known to those of skill in the
art. See, for example, Freedman et al., "Non-Hodgkin's Lymphomas,"
in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.),
pages 2028-2068 (Lea & Febiger 1993). As an illustration, first
generation chemotherapeutic regimens for treatment of
intermediate-grade non-Hodgkin's lymphoma (NHL) include C-MOPP
(cyclophosphamide, vincristine, procarbazine and prednisone) and
CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone).
A useful second generation chemotherapeutic regimen is m-BACOD
(methotrexate, bleomycin, doxorubicin, cyclophosphamide,
vincristine, dexamethasone and leucovorin), while a suitable third
generation regimen is MACOP-B (methotrexate, doxorubicin,
cyclophosphamide, vincristine, prednisone, bleomycin and
leucovorin). Additional useful drugs include phenyl butyrate,
bendamustine, and bryostatin-1.
[0182] The present invention contemplates the administration of
anti-pancreatic cancer antibodies and fragments thereof, including
fusion proteins and fragments thereof, alone, as a naked antibody
or antibody fragment, or administered as a multimodal therapy.
Preferably, the antibody is a humanized or fully human PAM4
antibody or fragment thereof. Multimodal therapies further include
immunotherapy with a naked anti-pancreatic cancer antibody
supplemented with administration of other antibodies in the form of
naked antibodies, fusion proteins, or as immunoconjugates. For
example, a humanized or fully human PAM4 antibody may be combined
with another naked antibody, or a humanized PAM4 or other antibody
conjugated to an isotope, one or more chemotherapeutic agents,
cytokines, toxins or a combination thereof. For example, the
present invention contemplates treatment of a naked or conjugated
PAM4 antibody or fragments thereof before, in combination with, or
after other pancreatic tumor associated antibodies such as CA19.9,
DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-Le.sup.a antibodies, and
antibodies to other Lewis antigens (e.g., Le(y)), as well as
antibodies against carcinoembryonic antigen (CEA or CEACAM5),
CEACAM6, colon-specific antigen-p (CSAp), MUC1, MUC2, MUC3, MUC4,
MUC5AC, MUC16, MUC17, HLA-DR, CD40, CD74, CD138, HER2/neu, EGFR,
EGP-1, EGP-2, angiogenesis factors (e.g., VEGF, PlGF), insulin-like
growth factor (IGF), tenascin, platelet-derived growth factor, and
IL-6, as well as products of oncogenes (e.g., bcl-2, Kras, p53),
cMET, and antibodies against tumor necrosis substances.
[0183] These solid tumor antibodies may be naked or conjugated to,
inter alia, drugs, toxins, isotopes, radionuclides or
immunomodulators. Many different antibody combinations may be
constructed and used in either naked or conjugated form.
Alternatively, different naked antibody combinations may be
employed for administration in combination with other therapeutic
agents, such as a cytotoxic drug or with radiation, given
consecutively, simultaneously, or sequentially.
[0184] Administration of the antibodies and their fragments can be
effected by intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, intrapleural, intrathecal, perfusion
through a regional catheter, or direct intralesional injection.
When administering the antibody by injection, the administration
may be by continuous infusion or by single or multiple boluses.
[0185] The immunoconjugate of the present invention can be
formulated for intravenous administration via, for example, bolus
injection or continuous infusion. Preferably, the antibody of the
present invention is infused over a period of less than about 4
hours, and more preferably, over a period of less than about 3
hours. For example, the first 25-50 mg could be infused within 30
minutes, preferably even 15 min, and the remainder infused over the
next 2-3 hrs. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0186] Additional pharmaceutical methods may be employed to control
the duration of action of the therapeutic conjugate. Control
release preparations can be prepared through the use of polymers to
complex or adsorb the immunoconjugate. For example, biocompatible
polymers include matrices of poly(ethylene-co-vinyl acetate) and
matrices of a polyanhydride copolymer of a stearic acid dimer and
sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The
rate of release of an immunoconjugate or antibody from such a
matrix depends upon the molecular weight of the immunoconjugate or
antibody, the amount of immunoconjugate or antibody within the
matrix, and the size of dispersed particles. Saltzman et al.,
Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid
dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE
FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger
1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th
Edition (Mack Publishing Company 1990), and revised editions
thereof.
[0187] More generally, the dosage of an administered
immunoconjugate for humans will vary depending upon such factors as
the patient's age, weight, height, sex, general medical condition
and previous medical history. It may be desirable to provide the
recipient with a dosage of immunoconjugate, antibody fusion protein
that is in the range of from about 1 mg/kg to 25 mg/kg as a single
intravenous infusion, although a lower or higher dosage also may be
administered as circumstances dictate. A dosage of 1-20 mg/kg for a
70 kg patient, for example, is 70-1,400 mg, or 41-824 mg/m.sup.2
for a 1.7-m patient. The dosage may be repeated as needed, for
example, once per week for 4-10 weeks, once per week for 8 weeks,
or once per week for 4 weeks. It may also be given less frequently,
such as every other week for several months, or monthly or
quarterly for many months, as needed in a maintenance therapy.
[0188] Alternatively, an antibody may be administered as one dosage
every 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,
the antibodies may be administered twice per week for 4-6 weeks. If
the dosage is lowered to approximately 200-300 mg/m.sup.2 (340 mg
per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient),
it may be administered once or even twice weekly for 4 to 10 weeks.
Alternatively, the dosage schedule may be decreased, namely every 2
or 3 weeks for 2-3 months. It has been determined, however, that
even higher doses, such as 20 mg/kg once weekly or once every 2-3
weeks can be administered by slow i.v. infusion, for repeated
dosing cycles. The dosing schedule can optionally be repeated at
other intervals and dosage may be given through various parenteral
routes, with appropriate adjustment of the dose and schedule.
[0189] Immunoconjugates
[0190] Anti-pancreatic cancer antibodies and fragments thereof may
be conjugated to at least one therapeutic and/or diagnostic agent
for therapy or diagnosis. For immunotherapy, the objective is to
deliver cytotoxic doses of radioactivity, toxin, antibody and/or
drug to target cells, while minimizing exposure to non-target
tissues. Preferably, anti-pancreatic cancer antibodies are used to
diagnose and/or treat pancreatic tumors.
[0191] Any of the antibodies, antibody fragments and fusion
proteins can be conjugated with one or more therapeutic or
diagnostic agents, using a variety of techniques known in the art.
One or more therapeutic or diagnostic agents may be attached to
each antibody, antibody fragment or fusion protein, for example by
conjugating an agent to a carbohydrate moiety in the Fc region of
the antibody. If the Fc region is absent (for example with certain
antibody fragments), it is possible to introduce a carbohydrate
moiety into the light chain variable region of either an antibody
or antibody fragment to which a therapeutic or diagnostic agent may
be attached. See, for example, Leung et al., J Immunol. 154: 5919
(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et
al., U.S. Pat. No. 6,254,868, the Examples section of each patent
incorporated herein by reference.
[0192] Methods for conjugating peptides to antibody components via
an antibody carbohydrate moiety are well-known to those of skill in
the art. See, for example, Shih et al., Int J Cancer 41: 832
(1988); Shih et al., Int J Cancer 46: 1101 (1990); and Shih et al.,
U.S. Pat. No. 5,057,313, the Examples section of which is
incorporated herein by reference. The general method involves
reacting an antibody component having an oxidized carbohydrate
portion with a carrier polymer that has at least one free amine
function and that is loaded with a plurality of therapeutic agents,
such as peptides or drugs. This reaction results in an initial
Schiff base (imine) linkage, which can be stabilized by reduction
to a secondary amine to form the final conjugate.
[0193] Antibody fusion proteins or multispecific antibodies
comprise two or more antibodies or fragments thereof, each of which
may be attached to at least one therapeutic agent and/or diagnostic
agent. Accordingly, one or more of the antibodies or fragments
thereof of the antibody fusion protein can have more than one
therapeutic and/or diagnostic agent attached. Further, the
therapeutic agents do not need to be the same but can be different
therapeutic agents, for example, one can attach a drug and a
radioisotope to the same fusion protein. For example, an IgG can be
radiolabeled with .sup.131I and attached to a drug. The .sup.131I
can be incorporated into the tyrosine of the IgG and the drug
attached to the epsilon amino group of the IgG lysines. Both
therapeutic and diagnostic agents also can be attached to reduced
SH groups and to the carbohydrate side chains of antibodies.
Alternatively, a bispecific antibody may comprise one antibody or
fragment thereof against a disease antigen and another against a
hapten attached to a targetable construct, for use in pretargeting
techniques as discussed above.
[0194] A therapeutic or diagnostic agent can be attached at the
hinge region of a reduced antibody component via disulfide bond
formation. As an alternative, such agents can be attached to the
antibody component using a heterobifunctional cross-linker, such as
N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int.
J. Cancer 56: 244 (1994). General techniques for such conjugation
are well-known in the art. See, for example, Wong, CHEMISTRY OF
PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis
et al., "Modification of Antibodies by Chemical Methods," in
MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al.
(eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production
and Characterization of Synthetic Peptide-Derived Antibodies," in
MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995).
[0195] Click Chemistry
[0196] An alternative method for attaching chelating moieties,
drugs or other functional groups to an antibody, fragment or fusion
protein involves use of click chemistry reactions. The click
chemistry approach was originally conceived as a method to rapidly
generate complex substances by joining small subunits together in a
modular fashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed
40:3004-31; Evans, 2007, Aust J Chem 60:384-95.) Various forms of
click chemistry reaction are known in the art, such as the Huisgen
1,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al.,
2002, J Organic Chem 67:3057-64), which is often referred to as the
"click reaction." Other alternatives include cycloaddition
reactions such as the Diels-Alder, nucleophilic substitution
reactions (especially to small strained rings like epoxy and
aziridine compounds), carbonyl chemistry formation of urea
compounds and reactions involving carbon-carbon double bonds, such
as alkynes in thiol-yne reactions.
[0197] The azide alkyne Huisgen cycloaddition reaction uses a
copper catalyst in the presence of a reducing agent to catalyze the
reaction of a terminal alkyne group attached to a first molecule.
In the presence of a second molecule comprising an azide moiety,
the azide reacts with the activated alkyne to form a
1,4-disubstituted 1,2,3-triazole. The copper catalyzed reaction
occurs at room temperature and is sufficiently specific that
purification of the reaction product is often not required.
(Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe et al.,
2002, J Org Chem 67:3057.) The azide and alkyne functional groups
are largely inert towards biomolecules in aqueous medium, allowing
the reaction to occur in complex solutions. The triazole formed is
chemically stable and is not subject to enzymatic cleavage, making
the click chemistry product highly stable in biological systems.
Although the copper catalyst is toxic to living cells, the
copper-based click chemistry reaction may be used in vitro for
immunoconjugate formation.
[0198] A copper-free click reaction has been proposed for covalent
modification of biomolecules. (See, e.g., Agard et al., 2004, J Am
Chem Soc 126:15046-47.) The copper-free reaction uses ring strain
in place of the copper catalyst to promote a [3+2] azide-alkyne
cycloaddition reaction (Id.) For example, cyclooctyne is a 8-carbon
ring structure comprising an internal alkyne bond. The closed ring
structure induces a substantial bond angle deformation of the
acetylene, which is highly reactive with azide groups to form a
triazole. Thus, cyclooctyne derivatives may be used for copper-free
click reactions (Id.)
[0199] Another type of copper-free click reaction was reported by
Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving
strain-promoted alkyne-nitrone cycloaddition. To address the slow
rate of the original cyclooctyne reaction, electron-withdrawing
groups are attached adjacent to the triple bond (Id.) Examples of
such substituted cyclooctynes include difluorinated cyclooctynes,
4-dibenzocyclooctynol and azacyclooctyne (Id.) An alternative
copper-free reaction involved strain-promoted alkyne-nitrone
cycloaddition to give N-alkylated isoxazolines (Id.) The reaction
was reported to have exceptionally fast reaction kinetics and was
used in a one-pot three-step protocol for site-specific
modification of peptides and proteins (Id.) Nitrones were prepared
by the condensation of appropriate aldehydes with
N-methylhydroxylamine and the cycloaddition reaction took place in
a mixture of acetonitrile and water (Id.) These and other known
click chemistry reactions may be used to attach chelating moieties
to antibodies or other targeting molecules in vitro.
[0200] Therapeutic Agents
[0201] A wide variety of therapeutic reagents can be administered
concurrently or sequentially, or advantageously conjugated to the
antibodies of the invention, for example, drugs, toxins,
oligonucleotides (e.g., siRNA), immunomodulators, hormones, hormone
antagonists, enzymes, enzyme inhibitors, radionuclides,
angiogenesis inhibitors, pro-apoptotic agents, etc. The therapeutic
agents recited here are those agents that are useful for either
conjugated to an antibody, fragment or fusion protein or for
administration separately with a naked antibody as described
above.
[0202] Therapeutic agents include, for example, chemotherapeutic
drugs such as vinca alkaloids, anthracyclines, gemcitabine,
epipodophyllotoxins, taxanes, antimetabolites, alkylating agents,
antibiotics, SN-38, COX-2 inhibitors, antimitotics, antiangiogenic
and apoptotic agents, particularly doxorubicin, methotrexate,
taxol, CPT-11, camptothecans, proteosome inhibitors, mTOR
inhibitors, HDAC inhibitors, tyrosine kinase inhibitors, and others
from these and other classes of anticancer agents.
[0203] Other useful cancer chemotherapeutic drugs include nitrogen
mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid
analogs, COX-2 inhibitors, antimetabolites, pyrimidine analogs,
purine analogs, platinum coordination complexes, mTOR inhibitors,
tyrosine kinase inhibitors, proteosome inhibitors, HDAC inhibitors,
camptothecins and hormones. Suitable chemotherapeutic agents are
described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack
Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE
PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan
Publishing Co. 1985), as well as revised editions of these
publications. Other suitable chemotherapeutic agents, such as
experimental drugs, are known to those of skill in the art.
[0204] Specific drugs of use may include 5-fluorouracil, afatinib,
aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101,
AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,
bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,
10-hydroxycamptothecin, carmustine, celebrex, chlorambucil,
cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38,
carboplatin, cladribine, camptothecans, crizotinib,
cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,
docetaxel, dactinomycin, daunorubicin, doxorubicin,
2-pyrrolinodoxorubicine (2-PDOX), pro-2PDOX, cyano-morpholino
doxorubicin, doxorubicin glucuronide, epirubicin glucuronide,
erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat,
estrogen receptor binding agents, etoposide (VP16), etoposide
glucuronide, etoposide phosphate, exemestane, fingolimod,
floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine,
flutamide, farnesyl-protein transferase inhibitors, flavopiridol,
fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib,
gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib,
ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide,
leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan,
mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,
mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,
nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel,
PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib,
streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an
aqueous form of DTIC), transplatinum, thalidomide, thioguanine,
thiotepa, teniposide, topotecan, uracil mustard, vatalanib,
vinorelbine, vinblastine, vincristine, vinca alkaloids and
ZD1839.
[0205] In a preferred embodiment, conjugates of camptothecins and
related compounds, such as SN-38, may be conjugated to hPAM4 or
other anti-pancreatic cancer antibodies, for example as disclosed
in U.S. Pat. No. 7,591,994; and U.S. patent application Ser. No.
11/388,032, filed Mar. 23, 2006, the Examples section of each of
which is incorporated herein by reference.
[0206] In another preferred embodiment, prodrug forms of 2-PDOX, as
disclosed in U.S. patent application Ser. No. 14/175,089 (the
Examples section of which is incorporated herein by reference) may
be used as an immunoconjugate with an anti-pancreatic cancer
antibody that binds to an epitope of MUC5AC as discussed above.
[0207] In another preferred embodiment, an hPAM4 antibody is given
with gemcitabine, which may be given before, after, or concurrently
with a naked or conjugated chimeric, humanized or human PAM4
antibody. Preferably, the conjugated hPAM4 antibody or antibody
fragment is conjugated to a radionuclide.
[0208] A toxin can be of animal, plant or microbial origin. A
toxin, such as Pseudomonas exotoxin, may also be complexed to or
form the therapeutic agent portion of an immunoconjugate of the
anti-pancreatic cancer and hPAM4 antibodies. Other toxins suitably
employed in the preparation of such conjugates or other fusion
proteins, include ricin, abrin, ribonuclease (RNase), DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtheria toxin, ranpirnase, Pseudomonas exotoxin, and Pseudomonas
endotoxin. See, for example, Pastan et al., Cell 47:641 (1986),
Goldenberg, C A--A Cancer Journal for Clinicians 44:43 (1994),
Sharkey and Goldenberg, C A--A Cancer Journal for Clinicians 56:226
(2006). Additional toxins suitable for use are known to those of
skill in the art and are disclosed in U.S. Pat. No. 6,077,499, the
Examples section of which is incorporated herein by reference.
[0209] An immunomodulator, such as a cytokine, may also be
conjugated to, or form the therapeutic agent portion of the
immunoconjugate, or may be administered with, but unconjugated to,
an antibody, antibody fragment or fusion protein. The fusion
protein may comprise one or more antibodies or fragments thereof
binding to different antigens. For example, the fusion protein may
bind to an epitope of MUC5AC as discussed above as well as to
immunomodulating cells or factors. Alternatively, subjects can
receive a naked antibody, antibody fragment or fusion protein and a
separately administered cytokine, which can be administered before,
concurrently or after administration of the naked antibodies. As
used herein, the term "immunomodulator" includes a cytokine, a
lymphokine, a monokine, a stem cell growth factor, a lymphotoxin, a
hematopoietic factor, a colony stimulating factor (CSF), an
interferon (IFN), parathyroid hormone, thyroxine, insulin,
proinsulin, relaxin, prorelaxin, follicle stimulating hormone
(FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH),
hepatic growth factor, prostaglandin, fibroblast growth factor,
prolactin, placental lactogen, OB protein, a transforming growth
factor (TGF), TGF-.alpha., TGF-.beta., insulin-like growth factor
(IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF),
TNF-.alpha., TNF-.beta., a mullerian-inhibiting substance, mouse
gonadotropin-associated peptide, inhibin, activin, vascular
endothelial growth factor, integrin, interleukin (IL),
granulocyte-colony stimulating factor (G-CSF), granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon-.alpha.,
interferon-.beta., interf.lamda.eron-.gamma., S1 factor, IL-1,
IL-1cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and
IL-25, LIF, kit-ligand, FLT-3, angiostatin, thrombospondin,
endostatin and lymphotoxin.
[0210] The therapeutic agent may comprise one or more radioactive
isotopes useful for treating diseased tissue. Particularly useful
therapeutic radionuclides include, but are not limited to
.sup.111In, .sup.177Lu, .sup.212Bi, .sup.213Bi, .sup.211At,
.sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.90Y, .sup.125I, .sup.131I,
.sup.32P, .sup.33P, .sup.47Sc, .sup.111Ag, .sup.67Ga, .sup.142Pr,
.sup.153Sm, .sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.186Re,
.sup.188Re, .sup.189Re, .sup.212Pb, .sup.223Ra, .sup.225Ac,
.sup.59Fe, .sup.75Se, .sup.77As, .sup.89Sr, .sup.99Mo, .sup.105Rh,
.sup.109Pd, .sup.143Pr, .sup.149Pm, .sup.169Er, .sup.194Ir,
.sup.198Au, .sup.199Au, .sup.211Pb and .sup.227Th. The therapeutic
radionuclide preferably has a decay energy in the range of 20 to
6,000 keV, preferably in the ranges 60 to 200 keV for an Auger
emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for
an alpha emitter. Maximum decay energies of useful
beta-particle-emitting nuclides are preferably 20-5,000 keV, more
preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also
preferred are radionuclides that substantially decay with
Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,
Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m and
Ir-192. Decay energies of useful beta-particle-emitting nuclides
are preferably <1,000 keV, more preferably <100 keV, and most
preferably <70 keV. Also preferred are radionuclides that
substantially decay with generation of alpha-particles. Such
radionuclides include, but are not limited to: Dy-152, At-211,
Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217,
Bi-213, Th-227 and Fm-255. Decay energies of useful
alpha-particle-emitting radionuclides are preferably 2,000-10,000
keV, more preferably 3,000-8,000 keV, and most preferably
4,000-7,000 keV.
[0211] For example, .sup.67Cu, considered one of the more promising
radioisotopes for radioimmunotherapy due to its 61.5-hour half-life
and abundant supply of beta particles and gamma rays, can be
conjugated to an antibody using the chelating agent,
p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA).
Alternatively, .sup.90Y, which emits an energetic beta particle,
can be coupled to an antibody, antibody fragment or fusion protein,
using diethylenetriaminepentaacetic acid (DTPA), or more preferably
using DOTA. Methods of conjugating .sup.90Y to antibodies or
targetable constructs are known in the art and any such known
methods may be used. (See, e.g., U.S. Pat. No. 7,259,249, the
Examples section of which is incorporated herein by reference. See
also Linden et al., Clin Cancer Res. 11:5215-22, 2005; Sharkey et
al., J Nucl Med. 46:620-33, 2005; Sharkey et al., J Nucl Med.
44:2000-18, 2003.)
[0212] Additional potential therapeutic radioisotopes include
.sup.11C, .sup.13N, .sup.15O, .sup.75Br, .sup.198Au, .sup.224Ac,
.sup.126I, .sup.133I, .sup.77Br, .sup.113mIn, .sup.95Ru, .sup.97Ru,
.sup.103Ru, .sup.105Ru, .sup.107Hg, .sup.203Hg, .sup.121mTe,
.sup.122mTe, .sup.125mTe, .sup.165Tm, .sup.167Tm, .sup.168Tm,
.sup.197Pt, .sup.109Pd, .sup.105Rh, .sup.142Pr, .sup.143Pr,
.sup.161Tb, .sup.166Ho, .sup.199Au, .sup.57Co, .sup.58Co,
.sup.51Cr, .sup.59Fe, .sup.75Se, .sup.201Tl, .sup.225Ac, .sup.76Br,
.sup.169Yb, and the like.
[0213] In another embodiment, a radiosensitizer can be used in
combination with a naked or conjugated antibody or antibody
fragment. For example, the radiosensitizer can be used in
combination with a radiolabeled antibody or antibody fragment. The
addition of the radiosensitizer can result in enhanced efficacy
when compared to treatment with the radiolabeled antibody or
antibody fragment alone. Radiosensitizers are described in D. M.
Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRC
Press (1995). Other typical radionsensitizers of interest for use
with this technology include gemcitabine, 5-fluorouracil, and
cisplatin, and have been used in combination with external
irradiation in the therapy of diverse cancers, including pancreatic
cancer. Therefore, we have studied the combination of gemcitabine
at what is believed to be radiosensitizing doses (once weekly 200
mg/m.sup.2 over 4 weeks) of gemcitabine combined with fractionated
doses of .sup.90Y-hPAM4, and have observed objective evidence of
pancreatic cancer reduction after a single cycle of this
combination that proved to be well-tolerated (no grade 3-4
toxicities by NCI-CTC v. 3 standard).
[0214] Antibodies or fragments thereof that have a boron
addend-loaded carrier for thermal neutron activation therapy will
normally be affected in similar ways. However, it will be
advantageous to wait until non-targeted immunoconjugate clears
before neutron irradiation is performed. Clearance can be
accelerated using an anti-idiotypic antibody that binds to the
anti-pancreatic cancer antibody. See U.S. Pat. No. 4,624,846 for a
description of this general principle. For example, boron addends
such as carboranes, can be attached to antibodies. Carboranes can
be prepared with carboxyl functions on pendant side chains, as is
well-known in the art. Attachment of carboranes to a carrier, such
as aminodextran, can be achieved by activation of the carboxyl
groups of the carboranes and condensation with amines on the
carrier. The intermediate conjugate is then conjugated to the
antibody. After administration of the antibody conjugate, a boron
addend is activated by thermal neutron irradiation and converted to
radioactive atoms which decay by alpha-emission to produce highly
toxic, short-range effects.
[0215] Interference RNA
[0216] Another type of therapeutic agent is RNAi or siRNA. RNA
interference (RNAi) is mediated by the RNA-induced silencing
complex (RISC) and is initiated by short double-stranded RNA
molecules that interact with the catalytic RISC component argonaute
(Rand et al., 2005, Cell 123:621-29). Types of RNAi molecules
include microRNA (miRNA) and small interfering RNA (siRNA). RNAi
species can bind with messenger RNA (mRNA) through complementary
base-pairing and inhibits gene expression by post-transcriptional
gene silencing. Upon binding to a complementary mRNA species, RNAi
induces cleavage of the mRNA molecule by the argonaute component of
RISC. Among other characteristics, miRNA and siRNA differ in the
degree of specificity for particular gene targets, with siRNA being
relatively specific for a particular target gene and miRNA
inhibiting translation of multiple mRNA species.
[0217] Therapeutic use of RNAi by inhibition of selected gene
expression has been attempted for a variety of disease states, such
as macular degeneration and respiratory syncytial virus infection
(Sah, 2006, Life Sci 79:1773-80). It has been suggested that siRNA
functions in host cell defenses against viral infection and siRNA
has been widely examined as an approach to antiviral therapy (see,
e.g., Zhang et al., 2004, Nature Med 11:56-62; Novina et al., 2002,
Nature Med 8:681-86; Palliser et al., 2006, Nature 439:89-94). The
use of siRNA for cancer therapy has also been attempted. Fujii et
al. (2006, Int J Oncol 29:541-48) transfected HPV positive cervical
cancer cells with siRNA against HPV E6 and E7 and suppressed tumor
growth. siRNA-mediated knockdown of metadherin expression in breast
cancer cells was reported to inhibit experimental lung metastasis
(Brown and Ruoslahti, 2004, Cancer Cell 5:365-74).
[0218] Attempts have been made to provide targeted delivery of
siRNA to reduce the potential for off-target toxicity. Song et al.
(2005, Nat Biotechnol 23:709-17) used protamine-conjugated Fab
fragments against HIV envelope protein to deliver siRNA to
circulating cells. Schiffelers et al. (2004, Nucl Acids Res
32:e149) conjugated RGD peptides to nanoparticles to deliver
anti-VEGFR2 siRNA to tumors and inhibited tumor angiogenesis and
growth rate in nude mice. Dickerson et al. (2010, Cancer 10:10)
used nanogels functionalized with anti-EphA2 receptor peptides to
chemosensitize ovarian cancer cells with siRNA against EGFR.
Dendrimer-conjugated magnetic nanoparticles have been applied to
the targeted delivery of antisense survivin oligodeoxynucleotides
(Pan et al., 2007, Cancer Res 67:8156-63).
[0219] The skilled artisan will realize that any siRNA or
interference RNA species may be attached to the subject antibodies.
siRNA and RNAi species against a wide variety of targets are known
in the art, and any such known oligonucleotide species may be
utilized in the claimed methods and compositions.
[0220] Known siRNA species of potential use include those specific
for IKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR
(U.S. Pat. No. 7,148,342); Bcl2 and EGFR (U.S. Pat. No. 7,541,453);
CDC20 (U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S.
Pat. No. 7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic
anhydrase II (U.S. Pat. No. 7,579,457); complement component 3
(U.S. Pat. No. 7,582,746); interleukin-1 receptor-associated kinase
4 (IRAK4) (U.S. Pat. No. 7,592,443); survivin (U.S. Pat. No.
7,608,7070); superoxide dismutase 1 (U.S. Pat. No. 7,632,938); MET
proto-oncogene (U.S. Pat. No. 7,632,939); amyloid beta precursor
protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R (U.S. Pat. No.
7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complement factor B
(U.S. Pat. No. 7,696,344); p53 (U.S. Pat. No. 7,781,575), and
apolipoprotein B (U.S. Pat. No. 7,795,421), the Examples section of
each of which is incorporated herein by reference.
[0221] Additional siRNA species are available from known commercial
sources, such as Sigma-Aldrich (St Louis, Mo.), Invitrogen
(Carlsbad, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.),
Ambion (Austin, Tex.), Dharmacon (Thermo Scientific, Lafayette,
Colo.), Promega (Madison, Wis.), Mirus Bio (Madison, Wis.) and
Qiagen (Valencia, Calif.), among many others. Other publicly
available sources of siRNA species include the siRNAdb database at
the Stockholm Bioinformatics Centre, the MIT/ICBP siRNA Database,
the RNAi Consortium shRNA Library at the Broad Institute, and the
Probe database at NCBI. For example, there are 30,852 siRNA species
in the NCBI Probe database. The skilled artisan will realize that
for any gene of interest, either an siRNA species has already been
designed, or one may readily be designed using publicly available
software tools. Any such siRNA species may be delivered using the
subject DNL.TM. complexes.
[0222] Exemplary siRNA species that have been reported are listed
in Table 1. Although siRNA is delivered as a double-stranded
molecule, for simplicity only the sense strand sequences are shown
in Table 1.
TABLE-US-00001 TABLE 1 Exemplary siRNA Sequences Target Sequence
SEQ ID NO VEGF R2 AATGCGGCGGTGGTGACAGTA SEQ ID NO: 22 VEGF R2
AAGCTCAGCACACAGAAAGAC SEQ ID NO: 23 CXCR4 UAAAAUCUUCCUGCCCACCdTdT
SEQ ID NO: 24 CXCR4 GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 25 PPARC1
AAGACCAGCCUCUUUGCCCAG SEQ ID NO: 26 Dynamin 2 GGACCAGGCAGAAAACGAG
SEQ ID NO: 27 Catenin CUAUCAGGAUGACGCGG SEQ ID NO: 28 E1A binding
protein UGACACAGGCAGGCUUGACUU SEQ ID NO: 29 Plasminogen
GGTGAAGAAGGGCGTCCAA SEQ ID NO: 30 activator K-ras
GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID NO: 31 CAAGAGACTCGCCAACAGCTCCAACT
TTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAG SEQ ID NO: 32
Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 33 Apolipoprotein
E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 34 Bcl-X UAUGGAGCUGCAGAGGAUGdTdT
SEQ ID NO: 35 Raf-1 TTTGAATATCTGTGCTGAGAACACA SEQ ID NO: 36
GTTCTCAGCACAGATATTCTTTTT Heat shock aatgagaaaagcaaaaggtgccctgtctc
SEQ ID NO: 37 transcription factor 2 IGFBP3 AAUCAUCAUCAAGAAAGGGCA
SEQ ID NO: 38 Thioredoxin AUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 39
CD44 GAACGAAUCCUGAAGACAUCU SEQ ID NO: 40 MMP14
AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO: 41 MAPKAPK2
UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 42 FGFR1 AAGTCGGACGCAACAGAGAAA
SEQ ID NO: 43 ERBB2 CUACCUUUCUACGGACGUGdTdT SEQ ID NO: 44 BCL2L1
CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 45 ABL1 TTAUUCCUUCUUCGGGAAGUC SEQ
ID NO: 46 CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO: 47 CD9
GAGCATCTTCGAGCAAGAA SEQ ID NO: 48 CD151 CATGTGGCACCGTTTGCCT SEQ ID
NO: 49 Caspase 8 AACTACCAGAAAGGTATACCT SEQ ID NO: 50 BRCA1
UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 51 p53 GCAUGAACCGGAGGCCCAUTT SEQ
ID NO: 52 CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO: 53
Diagnostic Agents
[0223] In the context of this application, the terms "diagnosis" or
"detection" can be used interchangeably. Whereas diagnosis usually
refers to defining a tissue's specific histological status,
detection recognizes and locates a tissue, lesion or organism
containing a particular antigen.
[0224] The subject antibodies and fragments can be detectably
labeled by linking the antibody to an enzyme. When the
antibody-enzyme conjugate is incubated in the presence of the
appropriate substrate, the enzyme moiety reacts with the substrate
to produce a chemical moiety which can be detected, for example, by
spectrophotometric, fluorometric or visual means. Examples of
enzymes that can be used to detectably label antibody include
malate dehydrogenase, staphylococcal nuclease, delta-V-steroid
isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
alpha-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0225] The immunoconjugate may comprise one or more radioactive
isotopes useful for detecting diseased tissue. Particularly useful
diagnostic radionuclides include, but are not limited to,
.sup.110In, .sup.111In, .sup.177Lu, .sup.18F, .sup.52Fe, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.86Y, .sup.90Y,
.sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc, .sup.120I, .sup.123I,
.sup.124I, .sup.125I, .sup.131I, .sup.154-158Gd, .sup.32P,
.sup.11C, .sup.13N, .sup.15O, .sup.186Re, .sup.188Re, .sup.51Mn,
.sup.52mMn, .sup.55Co, .sup.72As, .sup.75Br, .sup.76Br, .sup.82mRb,
.sup.83Sr, or other gamma-, beta-, or positron-emitters, preferably
with a decay energy in the range of 20 to 4,000 keV, more
preferably in the range of 25 to 4,000 keV, and even more
preferably in the range of 25 to 1,000 keV, and still more
preferably in the range of 70 to 700 keV. Total decay energies of
useful positron-emitting radionuclides are preferably <2,000
keV, more preferably under 1,000 keV, and most preferably <700
keV. Radionuclides useful as diagnostic agents utilizing gamma-ray
detection include, but are not limited to: .sup.51Cr, .sup.57Co,
.sup.58Co, .sup.59Fe, .sup.67Cu, .sup.67Ga, .sup.75Se, .sup.97Ru,
.sup.99mTc, .sup.111In, .sup.114mIn, .sup.123I, .sup.125I,
.sup.131I, .sup.169Yb, .sup.197Hg, and .sup.201Tl. Decay energies
of useful gamma-ray emitting radionuclides are preferably 20-2000
keV, more preferably 60-600 keV, and most preferably 100-300
keV.
[0226] Methods of diagnosing cancer in a subject may be
accomplished by administering a diagnostic immunoconjugate and
detecting the diagnostic label attached to an immunoconjugate that
is localized to a cancer or tumor. The antibodies, antibody
fragments and fusion proteins may be conjugated to the diagnostic
agent or may be administered in a pretargeting technique using
targetable constructs attached to a diagnostic agent. Radioactive
agents that can be used as diagnostic agents are discussed above. A
suitable non-radioactive diagnostic agent is a contrast agent
suitable for magnetic resonance imaging, X-rays, computed
tomography or ultrasound. Magnetic imaging agents include, for
example, non-radioactive metals, such as manganese, iron and
gadolinium, complexed with metal-chelate combinations that include
2-benzyl-DTPA and its monomethyl and cyclohexyl analogs. See U.S.
Ser. No. 09/921,290 (now abandoned) filed on Oct. 10, 2001, the
Examples section of which is incorporated herein by reference.
Other imaging agents such as PET scanning nucleotides, preferably
.sup.18F, may also be used.
[0227] Contrast agents, such as MRI contrast agents, including, for
example, gadolinium ions, lanthanum ions, dysprosium ions, iron
ions, manganese ions or other comparable labels, CT contrast
agents, and ultrasound contrast agents may be used as diagnostic
agents. Paramagnetic ions suitable for use include chromium (III),
manganese (II), iron (III), iron (II), cobalt (II), nickel (II),
copper (II), neodymium (III), samarium (III), ytterbium (III),
gadolinium (III), vanadium (II), terbium (III), dysprosium (III),
holmium (III) and erbium (III), with gadolinium being particularly
preferred.
[0228] Ions useful in other contexts, such as X-ray imaging,
include but are not limited to lanthanum (III), gold (III), lead
(II) and bismuth (III). Fluorescent labels include rhodamine,
fluorescein and renographin. Rhodamine and fluorescein are often
linked via an isothiocyanate intermediate.
[0229] Metals are also useful in diagnostic agents, including those
for magnetic resonance imaging techniques. These metals include,
but are not limited to: gadolinium, manganese, iron, chromium,
copper, cobalt, nickel, dysprosium, rhenium, europium, terbium,
holmium and neodymium. In order to load an antibody with
radioactive metals or paramagnetic ions, it may be necessary to
react it with a reagent having a long tail to which are attached a
multiplicity of chelating groups for binding the ions. Such a tail
can be a polymer such as a polylysine, polysaccharide, or other
derivatized or derivatizable chain having pendant groups to which
can be bound chelating groups such as, e.g.,
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines,
crown ethers, bis-thiosemicarbazones, polyoximes, and like groups
known to be useful for this purpose.
[0230] Chelates are coupled to an antibody, fusion protein, or
fragments thereof using standard chemistries. The chelate is
normally linked to the antibody by a group which enables formation
of a bond to the molecule with minimal loss of immunoreactivity and
minimal aggregation and/or internal cross-linking. Other, more
unusual, methods and reagents for conjugating chelates to
antibodies are disclosed in U.S. Pat. No. 4,824,659 to Hawthorne,
entitled "Antibody Conjugates", issued Apr. 25, 1989, the Examples
section of which is incorporated herein by reference. Particularly
useful metal-chelate combinations include 2-benzyl-DTPA and its
monomethyl and cyclohexyl analogs, used with diagnostic isotopes in
the general energy range of 20 to 2,000 keV. The same chelates,
when complexed with non-radioactive metals, such as manganese, iron
and gadolinium are useful for MRI. Macrocyclic chelates such as
NOTA, DOTA, and TETA are of use with a variety of metals and
radiometals, most particularly with radionuclides of gallium,
yttrium and copper, respectively. Such metal-chelate complexes can
be made very stable by tailoring the ring size to the metal of
interest. Other ring-type chelates such as macrocyclic polyethers,
which are of interest for stably binding nuclides, such as 223Ra
for RAIT are encompassed by the invention.
[0231] Radiopaque and contrast materials are used for enhancing
X-rays and computed tomography, and include iodine compounds,
barium compounds, gallium compounds, thallium compounds, etc.
Specific compounds include barium, diatrizoate, ethiodized oil,
gallium citrate, iocarmic acid, iocetamic acid, iodamide,
iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol,
iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid,
iosulamide meglumine, iosemetic acid, iotasul, iotetric acid,
iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,
ipodate, meglumine, metrizamide, metrizoate, propyliodone, and
thallous chloride.
[0232] The antibodies, antibody fragments and fusion proteins also
can be labeled with a fluorescent compound. The presence of a
fluorescent-labeled MAb is determined by exposing the antibody to
light of the proper wavelength and detecting the resultant
fluorescence. Fluorescent labeling compounds include Alexa 350,
Alexa 430, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665,
BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl amino,
Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansyl chloride, Fluorescein,
fluorescein isothiocyanate, fluorescamine, HEX, 6-JOE, NBD
(7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green
500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic
acid, isophthalic acid, cresyl fast violet, cresyl blue violet,
brilliant cresyl blue, para-aminobenzoic acid, erythrosine,
phthalocyanines, phthaldehyde, azomethines, cyanines, xanthines,
succinylfluoresceins, rare earth metal cryptates, europium
trisbipyridine diamine, a europium cryptate or chelate, diamine,
dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,
phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,
phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,
Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine
isothiol), Tetramethylrhodamine, and Texas Red.
Fluorescently-labeled antibodies are particularly useful for flow
cytometry analysis, but can also be used in endoscopic and
intravascular detection methods.
[0233] Alternatively, the antibodies, antibody fragments and fusion
proteins can be detectably labeled by coupling the antibody to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged MAb is determined by detecting the presence
of luminescence that arises during the course of a chemical
reaction. Examples of chemiluminescent labeling compounds include
luminol, isoluminol, an aromatic acridinium ester, an imidazole, an
acridinium salt and an oxalate ester.
[0234] Similarly, a bioluminescent compound can be used to label
the antibodies and fragments there. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Bioluminescent compounds that are
useful for labeling include luciferin, luciferase and aequorin.
[0235] Accordingly, a method of diagnosing a malignancy in a
subject is described, comprising performing an in vitro diagnosis
assay on a specimen (fluid, tissue or cells) from the subject with
a composition comprising an anti-pancreatic cancer MAb, fusion
protein or fragment thereof. Immunohistochemistry can be used to
detect the presence of PAM4 antigen in a cell or tissue by the
presence of bound antibody. Preferably, the malignancy that is
being diagnosed is a cancer. Most preferably, the cancer is
pancreatic cancer.
[0236] Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA
or a suitable peptide, to which a detectable label, such as a
fluorescent molecule, or cytotoxic agent, such as a heavy metal or
radionuclide, can be conjugated to a subject antibody. For example,
a therapeutically useful immunoconjugate can be obtained by
conjugating a photoactive agent or dye to an antibody fusion
protein. Fluorescent compositions, such as fluorochrome, and other
chromogens, or dyes, such as porphyrins sensitive to visible light,
have been used to detect and to treat lesions by directing the
suitable light to the lesion. In therapy, this has been termed
photoradiation, phototherapy, or photodynamic therapy (Jori et al.
(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria
Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)).
Moreover, monoclonal antibodies have been coupled with
photoactivated dyes for achieving phototherapy. Mew et al., J.
Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380 (1985);
Oseroff et al., Proc Natl. Acad. Sci. USA 83:8744 (1986); idem.,
Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin. Biol.
Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422
(1989); Pelegrin et al., Cancer 67:2529 (1991).
[0237] Fluorescent and radioactive agents conjugated to antibodies
or used in bispecific, pretargeting methods, are particularly
useful for endoscopic, intraoperative or intravascular detection of
the targeted antigens associated with diseased tissues or clusters
of cells, such as malignant tumors, as disclosed in Goldenberg U.S.
Pat. Nos. 5,716,595; 6,096,289 and 6,387,350, the Examples section
of each incorporated herein by reference, particularly with gamma-,
beta- and positron-emitters. Endoscopic applications may be used
when there is spread to a structure that allows an endoscope, such
as the colon. Radionuclides useful for positron emission tomography
include, but are not limited to: F-18, Mn-51, Mn-52m, Fe-52, Co-55,
Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86,
Zr-89, Tc-94m, In-110, I-120, and I-124. Total decay energies of
useful positron-emitting radionuclides are preferably <2,000
keV, more preferably under 1,000 keV, and most preferably <700
keV. Radionuclides useful as diagnostic agents utilizing gamma-ray
detection include, but are not limited to: Cr-51, Co-57, Co-58,
Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-111, In-114m, I-123,
I-125, I-131, Yb-169, Hg-197, and Tl-201. Decay energies of useful
gamma-ray emitting radionuclides are preferably 20-2000 keV, more
preferably 60-600 keV, and most preferably 100-300 keV.
[0238] In Vitro Diagnosis
[0239] The present invention contemplates the use of
anti-pancreatic cancer antibodies to screen biological samples in
vitro for the presence of the PAM4 antigen. In such immunoassays,
the antibody, antibody fragment or fusion protein may be utilized
in liquid phase or bound to a solid-phase carrier, as described
below. For purposes of in vitro diagnosis, any type of antibody
such as murine, chimeric, humanized or human may be utilized, since
there is no host immune response to consider.
[0240] One example of a screening method for determining whether a
biological sample contains MUC5AC is the radioimmunoassay (RIA).
For example, in one form of RIA, the substance under test is mixed
with PAM4 MAb in the presence of radiolabeled MUC5AC. In this
method, the concentration of the test substance will be inversely
proportional to the amount of labeled MUC5AC bound to the MAb and
directly related to the amount of free, labeled MUC5AC. Other
suitable screening methods will be readily apparent to those of
skill in the art.
[0241] Alternatively, in vitro assays can be performed in which an
anti-pancreatic cancer antibody, antibody fragment or fusion
protein is bound to a solid-phase carrier. For example, MAbs can be
attached to a polymer, such as aminodextran, in order to link the
MAb to an insoluble support such as a polymer-coated bead, a plate
or a tube.
[0242] Other suitable in vitro assays will be readily apparent to
those of skill in the art. The specific concentrations of
detectably labeled antibody and MUC5AC, the temperature and time of
incubation, as well as other assay conditions may be varied,
depending on various factors including the concentration of MUC5AC
in the sample, the nature of the sample, and the like. The binding
activity of a sample of anti-pancreatic cancer antibody may be
determined according to well-known methods. Those skilled in the
art will be able to determine operative and optimal assay
conditions for each determination by employing routine
experimentation.
[0243] The presence of the PAM4 antigen in a biological sample can
be determined using an enzyme-linked immunosorbent assay (ELISA)
(e.g., Gold et al. J Clin Oncol. 24:252-58, 2006). In the direct
competitive ELISA, a pure or semipure antigen preparation is bound
to a solid support that is insoluble in the fluid or cellular
extract being tested and a quantity of detectably labeled soluble
antibody is added to permit detection and/or quantitation of the
binary complex formed between solid-phase antigen and labeled
antibody.
[0244] In contrast, a "double-determinant" ELISA, also known as a
"two-site ELISA" or "sandwich assay," requires small amounts of
antigen and the assay does not require extensive purification of
the antigen. Thus, the double-determinant ELISA is preferred to the
direct competitive ELISA for the detection of an antigen in a
clinical sample. See, for example, the use of the
double-determinant ELISA for quantitation of the c-myc oncoprotein
in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);
Spandidos et al., AntiCancer Res. 9: 821 (1989).
[0245] In a double-determinant ELISA, a quantity of unlabeled MAb
or antibody fragment (the "capture antibody") is bound to a solid
support, the test sample is brought into contact with the capture
antibody, and a quantity of detectably labeled soluble antibody (or
antibody fragment) is added to permit detection and/or quantitation
of the ternary complex formed between the capture antibody,
antigen, and labeled antibody. In the present context, an antibody
fragment is a portion of an anti-pancreatic cancer MAb that binds
to an epitope of MUC5AC. Methods of performing a double-determinant
ELISA are well-known. See, for example, Field et al., supra,
Spandidos et al., supra, and Moore et al., "Twin-Site ELISAs for
fos and myc Oncoproteins Using the AMPAK System," in METHODS IN
MOLECULAR BIOLOGY, VOL. 10, pages 273-281 (The Humana Press, Inc.
1992).
[0246] In the double-determinant ELISA, the soluble antibody or
antibody fragment must bind to a MUC5AC epitope that is distinct
from the epitope recognized by the capture antibody. The
double-determinant ELISA can be performed to ascertain whether the
PAM4 antigen is present in a biopsy sample. Alternatively, the
assay can be performed to quantitate the amount of MUC5AC that is
present in a clinical sample of body fluid. The quantitative assay
can be performed by including dilutions of purified MUC5AC.
[0247] The anti-pancreatic cancer MAbs, fusion proteins, and
fragments thereof also are suited for the preparation of an assay
kit. Such a kit may comprise a carrier means that is
compartmentalized to receive in close confinement one or more
container means such as vials, tubes and the like, each of said
container means comprising the separate elements of the
immunoassay.
[0248] The subject antibodies, antibody fragments and fusion
proteins also can be used to detect the presence of the PAM4
antigen in tissue sections prepared from a histological specimen.
Such in situ detection can be used to determine the presence of
MUC5AC and to determine the distribution of MUC5AC in the examined
tissue. In situ detection can be accomplished by applying a
detectably-labeled antibody to frozen tissue sections. Studies
indicate that the PAM4 antigen is preserved in paraffin-embedded
sections. General techniques of in situ detection are well-known to
those of ordinary skill. See, for example, Ponder, "Cell Marking
Techniques and Their Application," in MAMMALIAN DEVELOPMENT: A
PRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987), and Coligan
at pages 5.8.1-5.8.8.
[0249] Antibodies, antibody fragments and fusion proteins can be
detectably labeled with any appropriate marker moiety, for example,
a radioisotope, an enzyme, a fluorescent label, a dye, a chromogen,
a chemiluminescent label, a bioluminescent labels or a paramagnetic
label.
[0250] The marker moiety can be a radioisotope that is detected by
such means as the use of a gamma counter or a scintillation counter
or by autoradiography. In a preferred embodiment, the diagnostic
conjugate is a gamma-, beta- or a positron-emitting isotope. A
marker moiety in the present description refers to a molecule that
will generate a signal under predetermined conditions. Examples of
marker moieties include radioisotopes, enzymes, fluorescent labels,
chemiluminescent labels, bioluminescent labels and paramagnetic
labels.
[0251] The binding of marker moieties to anti-pancreatic cancer
antibodies can be accomplished using standard techniques known to
the art. Typical methodology in this regard is described by Kennedy
et al., Clin Chim Acta 70: 1 (1976), Schurs et al., Clin. Chim.
Acta 81: 1 (1977), Shih et al., Int J Cancer 46: 1101 (1990).
[0252] The above-described in vitro and in situ detection methods
may be used to assist in the diagnosis or staging of a pathological
condition. For example, such methods can be used to detect tumors
that express the PAM4 antigen such as pancreatic cancer.
[0253] In Vivo Diagnosis/Detection
[0254] Various methods of in vivo diagnostic imaging with
radiolabeled MAbs are well-known. In the technique of
immunoscintigraphy, for example, antibodies are labeled with a
gamma-emitting radioisotope and introduced into a patient. A gamma
camera is used to detect the location and distribution of
gamma-emitting radioisotopes. See, for example, Srivastava (ed.),
RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum
Press 1988), Chase, "Medical Applications of Radioisotopes," in
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al.
(eds.), pp. 624-652 (Mack Publishing Co., 1990), and Brown,
"Clinical Use of Monoclonal Antibodies," in BIOTECHNOLOGY AND
PHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall
1993).
[0255] For diagnostic imaging, radioisotopes may be bound to
antibody either directly or indirectly by using an intermediary
functional group. Useful intermediary functional groups include
chelators such as ethylenediaminetetraacetic acid and
diethylenetriaminepentaacetic acid. For example, see Shih et al.,
supra, and U.S. Pat. No. 5,057,313.
[0256] The radiation dose delivered to the patient is maintained at
as low a level as possible through the choice of isotope for the
best combination of minimum half-life, minimum retention in the
body, and minimum quantity of isotope which will permit detection
and accurate measurement. Examples of radioisotopes that can be
bound to anti-pancreatic cancer antibody and are appropriate for
diagnostic imaging include .sup.99mTc, .sup.111In and .sup.18F.
[0257] The subject antibodies, antibody fragments and fusion
proteins also can be labeled with paramagnetic ions and a variety
of radiological contrast agents for purposes of in vivo diagnosis.
Contrast agents that are particularly useful for magnetic resonance
imaging comprise gadolinium, manganese, dysprosium, lanthanum, or
iron ions. Additional agents include chromium, copper, cobalt,
nickel, rhenium, europium, terbium, holmium, or neodymium.
Antibodies and fragments thereof can also be conjugated to
ultrasound contrast/enhancing agents. For example, one ultrasound
contrast agent is a liposome. Also preferred, the ultrasound
contrast agent is a liposome that is gas filled.
[0258] In a preferred embodiment, a bispecific antibody can be
conjugated to a contrast agent. For example, the bispecific
antibody may comprise more than one image-enhancing agent for use
in ultrasound imaging. In another preferred embodiment, the
contrast agent is a liposome. Preferably, the liposome comprises a
bivalent DTPA-peptide covalently attached to the outside surface of
the liposome.
[0259] Pharmaceutically Suitable Excipients
[0260] Additional pharmaceutical methods may be employed to control
the duration of action of an anti-pancreatic cancer antibody in a
therapeutic application. Control release preparations can be
prepared through the use of polymers to complex or adsorb the
antibody, antibody fragment or fusion protein. For example,
biocompatible polymers include matrices of poly(ethylene-co-vinyl
acetate) and matrices of a polyanhydride copolymer of a stearic
acid dimer and sebacic acid. Sherwood et al., Bio/Technology 10:
1446 (1992). The rate of release of an antibody, antibody fragment
or fusion protein from such a matrix depends upon the molecular
weight of the antibody, antibody fragment or fusion protein, the
amount of antibody within the matrix, and the size of dispersed
particles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et
al., supra. Other solid dosage forms are described in Ansel et al.,
PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition
(Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S
PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company
1990), and revised editions thereof.
[0261] The antibodies, fragments thereof or fusion proteins to be
delivered to a subject can comprise one or more pharmaceutically
suitable excipients, one or more additional ingredients, or some
combination of these. The antibody can be formulated according to
known methods to prepare pharmaceutically useful compositions,
whereby the immunoconjugate or naked antibody is combined in a
mixture with a pharmaceutically suitable excipient. Sterile
phosphate-buffered saline is one example of a pharmaceutically
suitable excipient. Other suitable excipients are well-known to
those in the art. See, for example, Ansel et al., PHARMACEUTICAL
DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea &
Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL
SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised
editions thereof.
[0262] The immunoconjugate or naked antibody can be formulated for
intravenous administration via, for example, bolus injection or
continuous infusion. Formulations for injection can be presented in
unit dosage form, e.g., in ampules or in multi-dose containers,
with an added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0263] The immunoconjugate, naked antibody, fragment thereof or
fusion protein may also be administered to a mammal subcutaneously
or by other parenteral routes. In a preferred embodiment, the
antibody or fragment thereof is administered in a dosage of 20 to
2000 milligrams protein per dose. Moreover, the administration may
be by continuous infusion or by single or multiple boluses. In
general, the dosage of an administered immunoconjugate, fusion
protein or naked antibody for humans will vary depending upon such
factors as the patient's age, weight, height, sex, general medical
condition and previous medical history. Typically, it is desirable
to provide the recipient with a dosage of immunoconjugate, antibody
fusion protein or naked antibody that is in the range of from about
1 mg/kg to 20 mg/kg as a single intravenous or infusion, although a
lower or higher dosage also may be administered as circumstances
dictate. This dosage may be repeated as needed, for example, once
per week for four to ten weeks, preferably once per week for eight
weeks, and more preferably, once per week for four weeks. It may
also be given less frequently, such as every other week for several
months, or more frequently, such as two- or three-time weekly. The
dosage may be given through various parenteral routes, with
appropriate adjustment of the dose and schedule.
[0264] Kits
[0265] Various embodiments may concern kits containing components
suitable for treating or diagnosing diseased tissue in a patient.
Exemplary kits may contain at least one antibody, antigen binding
fragment or fusion protein as described herein. If the composition
containing components for administration is not formulated for
delivery via the alimentary canal, such as by oral delivery, a
device capable of delivering the kit components through some other
route may be included. One type of device, for applications such as
parenteral delivery, is a syringe that is used to inject the
composition into the body of a subject. Inhalation devices may also
be used. In certain embodiments, an anti-pancreatic cancer antibody
or antigen binding fragment thereof may be provided in the form of
a prefilled syringe or autoinjection pen containing a sterile,
liquid formulation or lyophilized preparation of antibody (e.g.,
Kivitz et al., Clin. Ther. 2006, 28:1619-29).
[0266] The kit components may be packaged together or separated
into two or more containers. In some embodiments, the containers
may be vials that contain sterile, lyophilized formulations of a
composition that are suitable for reconstitution. A kit may also
contain one or more buffers suitable for reconstitution and/or
dilution of other reagents. Other containers that may be used
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers. Another component that can be included is
instructions for use of the kit.
EXAMPLES
[0267] The examples below are illustrative of embodiments of the
current invention and are not limiting to the scope of the claims.
The examples discuss studies employing an exemplary anti-pancreatic
cancer monoclonal antibody (e.g., PAM4). Clinical studies with the
PAM4 MAb have shown that a majority of pancreatic cancer lesions
were targeted in patients and there was no indication of uptake in
normal tissues. Dosimetry indicated that it was possible to deliver
10 to 20 cGy/mCi to tumors, with a tumor to red marrow dose ratio
of 3:1 to 10:1. The data show that PAM4 is useful for the treatment
of pancreatic cancer.
Example 1
Epitope of MUC5AC that Binds to hPAM4 Antibody
[0268] PAM4 is a murine monoclonal antibody showing high
specificity for pancreatic ductal adenocarcinoma (PDAC) compared
with normal tissues and other cancers. Humanized PAM4 labeled with
.sup.90Y in combination with low-dose gemcitabine has shown
promising therapeutic activity in patients with metastatic PDAC,
and is being evaluated in a phase III registration trial. Prior
efforts have suggested the mucin species recognized by PAM4 is
human MUC5AC, a secretory mucin expressed de novo in early
pancreatic intraepithelial neoplasia and retained throughout
disease progression. In the present study, we provide further
evidence validating MUC5AC as the PAM4 antigen, and locate the
PAM4-reactive epitope within the N-terminal cysteine-rich subdomain
2 (Cys2), thus differentiating PAM4 from anti-MUC5AC antibodies
known to-date. Specifically, we show (i) PAM4-antigen and MUC5AC
were co-localized in the immunocytochemical analysis of multiple
human cancer cell lines, including Capan-1, BxPC-3, HT-29, and
MCF-7; (ii) MUC5AC-specific siRNA prominently reduced the
expression of both MUC5AC and PAM4-antigen in CFPAC-1 cells; (iii)
ELISA performed on Capan-1 culture supernatants following
SEPHAROSE.RTM.-CL2B chromatography depicted the preferential
binding of PAM4 to the void-volume fractions, which were further
revealed by agarose gel electrophoresis and Western blot to display
the ladder pattern characteristic of oligomeric MUC5AC; and (iv) by
testing the reactivity of PAM4 with a panel of recombinant
fragments of MUC5AC, we demonstrated the N-terminal region
comprising Cys2 is essential for binding to PAM4.
[0269] Materials and Methods
[0270] Antibodies and Reagents--
[0271] Humanized PAM4 (hPAM4) was provided by Immunomedics, Inc.
Horseradish peroxidase (HRP)-hPAM4 conjugate was generated using
the SureLINK HRP Conjugation Kit (Kirkegaard & Perry
Laboratories). MAN-5ACI, a rabbit antiserum against MUC5AC
(Thornton et al., 1996, Biochem J 316:967-75) was a generous gift
from Dr. David J. Thornton (University of Manchester). Commercially
available antibodies acquired include the following: four mouse
mAbs against MUC5AC (45M1, 2-11M1, 2-12M1, and 1-13M1) from Thermo
Fisher Scientific, one mouse monoclonal (2Q445) and one rabbit
polyclonal (H-160) antibodies against MUC5AC from Santa Cruz
Biotechnology, one mouse mAb against human MUC1 (MAB6298, hereafter
referred to as .alpha.-MUC1) from R&D Systems, one rabbit
polyclonal antibody against full-length GFP (.alpha.-GFP) from
Clontech Laboratories, one rabbit polyclonal Myc-tag antibody
(.alpha.-Myc) from Cell Signaling Technology, one FITC-labeled goat
anti-human IgG (FITC-GAH) from Jackson ImmunoResearch Laboratories,
and one Cy3-labeled goat anti-mouse IgG (Cy3-GAM) from EMD
Millipore. The MUC5AC double-strand siRNA targeting sequence
5'-GGAGCCTGATCATCCAGCA-3' (SEQ ID NO:54) was synthesized by
GenScript. SEPHAROSE.RTM..RTM. CL-2B was purchased from
Sigma-Aldrich.
[0272] Cell Culture--
[0273] All cell lines were obtained from the American Type Culture
Collection (ATCC) and have been authenticated by Promega using
Short Tandem Repeat (STR) analysis. BxPC-3, HT-29, LS174T, MCF-7,
and Calu-3 were grown in RPMI 1640 medium (Life Technologies) with
10% fetal bovine serum (FBS, Thermo Scientific HyClone); Capan-1
was grown in RPMI 1640 medium with 20% FBS; CFPAC-1 was grown in
ATCC-formulated Iscove's Modified Dulbecco's Medium (IMDM) with 10%
FBS; SW1990 was grown in ATCC-formulated Leibovitz's L-15 Medium
with 10% FBS; and PANC-1 was grown in Dulbecco's Modified Eagle
Medium (Life technologies) plus 10% FBS. All cell lines were
incubated at 37.degree. C. in 5% CO.sub.2 except SW1990, which was
cultured in 100% air.
[0274] Immunocytochemistry--
[0275] Cells were plated on 8-chamber slides (Thermo Fisher
Scientific) at approximately 2.times.10.sup.4 cells/chamber and
incubated overnight at 37.degree. C. Following removal of the
medium, cells were fixed in 4% formalin (Sigma-Aldrich) for 15 min
at RT, and then treated with 0.1% Triton X-100 in PBS for another
15 min. After washing twice with PBS, cells were incubated with 10
.mu.g/ml of either hPAM4 or a murine mAb against MUC5AC or MUC1 in
PBS plus 1% BSA for 45 min at RT. Afterwards, cells were washed
twice and incubated with a mixture of FITC-GAH and Cy3-GAM in PBS
plus 1% BSA for 30 min at RT. After three washes, chambers were
dissembled. Slides were mounted with an antifade solution
(VectaShield, Vector Laboratories) containing the nuclear
counterstain, 4,6-diamidino-2-phenylindole (DAPI). Image
acquisition and analyses were performed using an Olympus
fluorescence microscope with a Kodak camera system.
[0276] RNA Interference--
[0277] CFPAC-1 cells grown to 90% confluence were used for
transfection. MUC5AC SiRNA or PBS alone (Mock) was 1:100 diluted
into Opti-MEM I Medium (Life Technologies) prior to the addition of
1/100 volume of LIPOFECTAMINE.RTM. RNAiMAX Reagent (Life
Technologies). After 20 min incubation at RT, the siRNA or Mock
mixture was dispersed onto 8-chamber slides (80 d/chamber).
Meanwhile, cells were trypsinized, washed, diluted in complete
growth medium, and then added at 8.times.10.sup.3 cells/400
.mu.l/chamber. The final RNA concentration was 15.6 nM in a total
volume of 480 .mu.l. After 48-h incubation, cells were stained with
hPAM4 and anti-MUC5AC mAbs and examined under fluorescence
microscope as described above.
[0278] Gel Chromatography of Cell Culture Supernatant--
[0279] Capan-1 cells were cultured for 3-4 days to reach over 90%
confluence. The spent media were collected, mixed with an equal
volume of 8 M guanidine hydrochloride (GdmCl) in 20 mM sodium
phosphate buffer (pH 7), and 10-fold concentrated using the Amicon
ultrafiltration membrane with 30 kDa normal molecular weight limit
(EMD Millipore). Gel chromatography was performed on a
SEPHAROSE.RTM. CL-2B column (78 cm.times.2.6 cm) using 4 M GdmCl as
the eluent and a flow rate of 40 ml/h. Fractions of 8 mL were
collected and each analyzed for reactivity with hPAM4 and
.alpha.-MUC-1 by ELISA as follows. Briefly, MaxiSorp 96-well plates
(Nunc, Roskilde, Denmark) were coated with CL-2B-eluted fractions
(100 .mu.l/well) at 37.degree. C. overnight, washed twice with PBS,
and blocked with Casein Blocking Buffers (Thermo Fisher Scientific)
for 1 h. HRP-hPAM4 or .alpha.-MUC1 was diluted in PBS and added at
100 l/well. After 1-h incubation at RT, plates with .alpha.-MUC1
were washed and incubated further with HRP-GAM for 1 h. Plates were
washed and bound HRP-hPAM4 or HRP-GAM was detected with
o-phenylenediamine dihydrochloride (0.4 mg/ml) in PBS plus 0.03%
hydrogen peroxide as a substrate. The optical density was read at
490 nm using the EnVision 2100 Multilabel Reader (PerkinElmer). The
fractions eluted in the void-volume peak were also pooled, dialyzed
against the PBS-AG buffer (35.2 mM Na.sub.2PO.sub.4.7H.sub.2O; 0.4
M NaCl; 6.5 mM NaH.sub.2PO.sub.4.H.sub.2O; 150 mM arginine; 150 mM
monosodium glutamate, pH 8.0), and concentrated with 30 kDa Amicon
Ultra centrifugal filters (EMD Millipore) for further analysis.
[0280] MUC5AC Sandwich ELISA--
[0281] MaxiSorp 96-well plates were coated with 100 .mu.l of 2-11M1
(20 .mu.g/ml) in PBS and incubated at 4.degree. C. overnight. After
blocking with casein buffer, a 5-fold concentrated void-volume peak
pooled from the CL-2B fractionation of Capan-1 supernatant
(hereafter referred to as the Capan-1 void-volume peak) was 2-fold
serially diluted and added to the plate at 100 .mu.l/well. After
overnight incubation at RT, plates were washed and detected by
HRP-PAM4, or by Biotin-45M1 plus HRP-streptavidin as a positive
control.
[0282] Agarose Gel Electrophoresis--
[0283] Agarose gel electrophoresis was performed as described
(Sheehan et al., 2000, Biochem J 347:37-44), with modifications.
Briefly, the Capan-1 void-volume peak was concentrated in PB S-AG
buffer and diluted with gel running buffer (40 mM Tris-acetate/1 mM
EDTA, 0.1% SDS, pH 8.0). In selective experiments, serum samples
from normal subjects or pancreatic cancer patients were mixed with
an equal volume of 8 M guanidine hydrochloride (GdmCl) and dialyzed
into gel running buffer. All samples were supplemented with 1 M
urea, 3% glycerol and 0.02% bromophenol blue before loading into
thin wells shaped with a 0.8 mm-thick comb in 0.7% agarose gel (5.7
cm.times.8.3 cm). Electrophoresis was performed at 30 V for 4 to 8
h in the Horizon 58 Electrophoresis Apparatus (LABRepCo).
[0284] Construction of Expression Vectors for MUC5AC Recombinant
Fragments--
[0285] The pSM-MUC5AC-CH-long expression vector (Lidell et al.,
2008, FEBS J 275:481-9; Lidell & Hansson, 2006, Biochem J 399:
121-9), which encodes a signal sequence, a Myc tag (EQKLISEEDL, SEQ
ID NO:55), the human MUC5AC (Swiss-Prot accession no. P98088)
C-terminal cysteine-rich part (AA3993-5030), and a histidine tag,
was kindly provided by Dr. Gunner Hansson of Gothenburg University
(Gothenburg, Sweden). Additional vectors were constructed from
pSM-MUC5AC-CH-long by replacing the DNA sequence of AA3993-5030
with that of AA1-1217, AA1218-2199, AA1218-1517, AA1575-2052,
AA1725-2052, AA1575-1723/1903-2052, AA1575-1853, and AA1575-1725,
to express D1-D2-D'-D3 (b-fragment), 11P15-Cys1-2-3-4-5
(c-fragment), 11P15-Cys1 (d-fragment), Cys2-3-4 (e-fragment),
Cys3-4 (f-fragment), Cys2/4 (g-fragment), Cys2-3 (h-fragment), and
Cys2+(i-fragment), respectively, as listed in Table 2. In addition,
four GFP-fused fragments were produced by replacing the Myc tag
with a full GFP sequence in the vectors encoding Cys2-3-4, Cys3-4,
Cys2/4, and Cys2-3, resulting in the e*-, f*-, g*- and h*-fragment,
respectively. Myc-tagged Cys2-3-4 and Cys2+ were also expressed in
E. coli, and purified from the inclusion body using HIS-Select
Nickel Affinity Gel (Sigma-Aldrich), and refolded.
TABLE-US-00002 TABLE 2 Recombinant MUC5AC fragments MUC5AC MW
Expression Fragment Tag Domains AA # (P98088) .sup.a(Da) PANC-1 E.
coli a Myc Cys9-D4-B-C-CK 3992-5030 116,140 + b Myc D1-D2-D'-D3
1-1217 136,727 + c Myc 11P15-Cys1-2-3-4-5 1218-2199 109,380 + d Myc
11P15-Cys1 1218-1517 35,704 + e Myc Cys2-3-4 1575-2052 56,444 + + f
Myc Cys3-4 1725-2052 39,686 + g Myc Cys2/4 1575-1725/1903-2052
37,171 + h Myc Cys2-3 1575-1853 35,452 + I Myc Cys2+ 1575-1725
20,504 + e* GFP Cys2-3-4 1575-2052 81,742 + f* GFP Cys3-4 1725-2052
64,983 g* GFP Cys2/4 1575-1725/1903-2052 62,468 + h* GFP Cys2-3
1575-1853 60,749 +
[0286] Transient Expression of Recombinant MUC5AC Fragments--
[0287] One day prior to transfection, PANC-1 cells were seeded in a
24-well plate at 2.times.10.sup.5/well and held at 37.degree. C.
overnight. Transfection was performed using Lipofectamine 2000
(Life Technologies) with and without the recombinant plasmid DNA of
interest. After 72 h, the spent media were collected and analyzed
by Western blot following gel electrophoresis.
[0288] Western Blot--
[0289] Samples were electrophoresed in the same gel or different
gels under the same conditions. After electrophoresis, samples were
transferred (100V, 1 h) onto a nitrocellulose membrane using the
Mini TRANS-BLOT.RTM. cell system (Bio-Rad Laboratories) and probed
with hPAM4, an anti-MUC5AC antibody, .alpha.-GFP, or .alpha.-Myc,
as indicated. The signals were developed with SUPERSIGNAL.TM. West
Dura Chemiluminescent Substrate (Thermo Fisher Scientific).
[0290] Results
[0291] Co-Localization of the hPAM4 Antigen and MUC5AC in Different
Cell Lines--
[0292] Several cell lines were subjected to cytofluorometry in
order to evaluate localization patterns (heterogeneous and/or
homogenous) of MUC1, MUC5AC, and/or MUC17, as detected by hPAM4 and
other mucin-specific mAbs. The cell lines examined included those
derived from human pancreatic (CaPan-1, BxPC3, CFPAC-1, and
AsPC-1), colorectal (HT-29 and LS174 T), breast (MCF-7), and lung
(A549) carcinomas. As shown in FIG. 1, in each of the cell lines
examined, hPAM4 exclusively co-localized with MUC5AC (as identified
by two anti-MUC5AC mAbs, 2-11M1 and 2-12M1), but not with MUC1 or
MUC17 (data not shown), suggesting that MUC5AC is the
hPAM4-reactive antigen.
[0293] Co-Knockdown of the hPAM4 Antigen and MUC5AC by
MUC5AC-Specific siRNA--
[0294] The disparate localization between PAM4 and anti-MUC1 or
anti-MUC17 indicates that PAM4 reacts with neither MUC1 nor MUC17.
On the other hand, the co-localization of PAM4 and the two
anti-MUC5AC mAbs (2-11M1 and 2-12M1) is consistent with PAM4 being
specific for MUC5AC (Gold et al., 2013, Mol Cancer 12:143). To
investigate if hPAM4 associates with MUC5AC, we employed the RNAi
method to specifically knockdown MUC5AC. As shown in the upper
panel of FIG. 2, hPAM4 and 2-11M1 are co-localized in untreated
CFPAC-1 cells, as well as the mock-treated (transfection agent
alone) cells. In contrast, treatment with MUC5AC-specific siRNA
resulted in substantially reduced immunostaining for both 2-11M1
and hPAM4. Moreover, as shown in the bottom panel of FIG. 2, siRNA
knockdown of MUC5AC did not alter the anti-MUC1 immunostaining,
providing further evidence that hPAM4 is not reactive with
MUC1.
[0295] Presence of the hPAM4 Antigen in the Culture Supernatant of
Mucin-Producing Carcinoma Cell Lines--
[0296] MUC5AC is a highly oligomeric secretory mucin that has been
isolated from cell culture and in vivo mucous secretions (Sheehan
et al., 2000, Biochem J 347:37-44; Hovenberg et al., 1996,
Glycoconj J 13:839-47). Our early studies showed that hPAM4 reacts
with mucin derived from the CaPan-1 xenografted human PDAC (Gold et
al., 1994, Int J Cancer 57:204-10). In the current study, we used
SEPHAROSE.RTM. CL-2B molecular sieve chromatography to separate the
mucin species secreted into the supernatant of CaPan-1. The eluted
fractions were then examined for immunoreactivity with hPAM4 and
.alpha.-MUC1. As shown in FIG. 3A, PAM4-reactive substance was
present predominantly in the void-volume peak, whereas only
subsequently eluted fractions were found reactive with
.alpha.-MUC1. When the Capan-1 void-volume peak was probed with
anti-MUC5AC antibodies, we found a positive response with 45M1,
1-13M1, and H-160, but not 2Q445, as shown in FIG. 3B. It is noted
that the void-volume peaks obtained from other cancer cell lines
known to secret MUC5AC, such as HT-29 (Sheehan et al., 2000,
Biochem J 347:37-44), LS 174T (Asker et al., 1998, Biochem J
335:381-7), SW1990 (Hoshi et al, 2011, Int J Oncol 38:619-27),
CFPAC-1 (Luka et al., 2011, J Biomed Biotechnol 2011:93475728), and
Calu-3 (Rose et al., 2000, J Aerosol Med 13:245-61), all tested
positive for reactivity with hPAM4 (data not shown).
[0297] Direct evidence that correlates the hPAM4-reactive substance
in the Capan-1 void-volume peak with MUC5AC is provided by a
sandwich ELISA formatted to quantify the MUC5AC captured by 2-11M1,
which reacts with the N-terminal domains of MUC5AC (Nollet et al.,
2004, Hybrid Hybridomics 23:93-930). As shown in FIG. 3C,
2-11M1-captured MUC5AC could be detected by hPAM4 in a
dose-dependent manner, demonstrating that hPAM4 binds to a
different region of MUC5AC from 2-11M1. The additional results
obtained with 45M1, which serves as a positive control, also
support the previous conclusions that the epitopes of 45M1 (Lidell
et al., 2008, FEBS J 275:481-9) and 2-11M1 on MUC5AC are
non-overlapping.
[0298] Electrophoretic Resolution of the hPAM4-Reactive Void-Volume
Fractions on Agarose Gel--
[0299] To further verify that the hPAM4-reactive substance is
MUC5AC, the Capan-1 void-volume peak was separated by
electrophoresis on 0.7% agarose gel, and subsequently probed with
hPAM4 (FIG. 4A, left panel), 45M1 (FIG. 4A, middle panel), or
MAN-5ACI (FIG. 4A, right panel) by Western blotting. Under
non-reducing conditions, a group of bands resembling the
ladder-like pattern reported for MUC5AC (Sheehan et al., 2000,
Biochem J 347:37-44, Sheehan et al., 2004, J Biol Chem
279:15698-705) was clearly discerned by all three antibodies in the
same gel. In contrast, under reducing conditions, two bands were
revealed by MAN-5ACI, but undetectable by either hPAM4 or 45M1,
which corroborate the previous findings that the predominant
fast-migrating band and the minor band trailing behind represent
the MUC5AC monomer and a reduction-resistant dimer, respectively
(Sheehan et al., 2000, Biochem J 347:37-44), and that neither 45M1
(Lidell et al., 2008, FEBS J 275:481-9) nor hPAM4 (Gold et al.,
1994, Int J Cancer 57:204-10) should react with a reduced
mucin.
[0300] Detection of hPAM4-Reactive Substance in Serum Samples of
Pancreatic Cancer Patients--
[0301] The visualization of MUC5AC by hPAM4 as a characteristic
ladder in Western blot following agarose gel electrophoresis
prompted us to examine whether such a pattern could be demonstrated
for patient serum found positive with the presently formulated
PAM4-based assay (Picozzi et al., 2014, J Clin Oncol 132:4026; Gold
et al., 2010, Cancer Epidemiol Biomarkers Prev 19:2786-94). As
shown in FIG. 4B, a broad band migrating faster than MUC5AC monomer
was detected by hPAM4 and several MUC5AC-specific antibodies, such
as 2-11M and H-160, but not by 45M1, suggesting the PAM4-reactive
antigen in patient serum could be derived from an immature MUC5AC
variant, or a breakdown product of mature MUC5AC.
[0302] Mapping of the hPAM4 Epitope on MUC5AC--
[0303] The disparity in the reactivity of hPAM4 and 2Q445 with the
Capan-1 void-volume peak, as noted in FIG. 3B, suggests that the
hPAM4 epitope is not in the tandem repeat region of MUC5AC
recognized by 2Q445 (Perez-Vilar et al., 2006, J Biol Chem
281:4844-55). Therefore, we excluded the tandem repeat region
(AA2199-3992) and decided to express in PANC-1 cells three large
recombinant fragments (designated as a, b, and c) that comprise the
remainder of MUC5AC (FIG. 5A). We found that hPAM4 did not react
with the C-terminal a-fragment (AA3992-5030) or the N-terminal
b-fragment (AA1-1217), suggesting its epitope was located outside
the N-terminal D1-D2-D'-D3 domains and the C-terminal region
encompassing Cys9-D4-B-C-CK domains. In contrast, the c-fragment
(AA1218-2199), which spans the five N-terminal cysteine-rich
subdomains (Cys1-2-3-4-5), reacted with hPAM4 as shown by Western
blot (FIG. 5B, left panel). Expectedly, the c-fragment was found to
react also with 1-13M1 (data not shown) and 45M1 (FIG. 5B, right
panel), which recognize cysteine-rich subdomains of class-2 (Cys2
and Cys4) and class-3 (Cys3, 5, 6, 7 and 9), respectively. We next
expressed two sub-fragments (d and e) within the c-fragment and
showed (FIG. 5C, left panel) that hPAM4 failed to react with the
d-fragment (AA1218-1517) comprising 11P15-Cys1, but strongly
stained the e-fragment (AA1575-2052) comprising Cys2-3-4. We then
expressed three overlapping sub-fragments (f, g, and h) of the
e-fragment and showed (FIG. 5D, left panel) hPAM4 stained the
g-fragment (AA1575-1725 joined to AA1903-2052, comprising Cys2 and
Cys4 with Cys3 deleted), but barely the f-fragment (AA1725-2052,
comprising Cys3-4) or the h-fragment (AA1575-1853, comprising
Cys2-3). The differential reactivity of hPAM4 observed for the e-,
f-, g-, and h-fragments was confirmed (FIG. 5E, left panel) with
the respective GFP-fused counterparts (the e*-, f*-, g*- and
h*-fragments); the expression of each was clearly shown by Western
blot with anti-GFP (FIG. 5E, right panel). Together, these results
indicate that (i) the hPAM4 epitope resides within the e-fragment,
which contains the Cys2-3-4 region; (ii) the presence of Cys2 or
Cys4, or both, is needed for recognition by hPAM4; (iii) Cys3 is
essential for the binding of 45M1, since it stained each of the c-
(FIG. 5B, right panel; FIG. 5C, rightmost panel), e-, f-, and
h-fragments (FIG. 5C, rightmost panel; FIG. 5D, right panel), all
of which contains Cys3; but not the g-fragment (FIG. 5D, right
panel), which lacks Cys3; and (iv) the validity of the d-fragment
was supported by its positive staining with H-160 (FIG. 5C, middle
panel), whose epitope was reported to reside in AA1214-1373 (33)
contained in the d-fragment.
[0304] The successful expression of Cys2-3-4 (AA1575-2052) and
Cys2+ (AA1575-1725) in E. coli, as evidenced by the coomassie blue
staining (FIG. 6A) and Western blot using anti-Myc (FIG. 6B), was
instrumental in further defining the location of the hPAM4 epitope
to the Cys2 subdomain. The unglycosylated Cys2-3-4 and Cys2+ were
isolated predominantly as monomeric species of 55.4 and 20.5 kDa,
respectively. As shown in FIG. 6C, hPAM4 reacts with non-reduced,
but not the reduced, Cys2-3-4 and Cys2+. Although 1-13M1 also
targets Cys2 or Cys4, its binding to both non-reduced and reduced
Cys2+ (FIG. 6D) differentiates it from hPAM4. Thus, we further
establish that the hPAM4 epitope, being reduction-sensitive, is
conformational, located within the Cys2 subdomain, and unlikely
involving carbohydrates. We speculate that the weakly positive
bands observed for hPAM4 in lanes 3 and 4 of FIG. 6C could result
from reformation of the disulfide bond to a varying degree in the
process of blotting, which would restore the hPAM4 epitope.
[0305] Discussion
[0306] In the past decade, concerted efforts in the search of
biomarkers for PDAC have produced compelling evidence that mucins
are aberrantly expressed in this devastating malignancy, and have
diverse biological functions in tumor development, progression,
metastasis, and drug resistance (Kaur et al., 2013, Nat Rev
Gastroenterol Hepatol 10:607-20). Moreover, a number of studies
(Kaur et al., 2013, Nat Rev Gastroenterol Hepatol 10:607-20; Lau et
al., 2004, Am J Clin Pathol 122:61-9; Remmers et al., 2013, Clin
Cancer Res 19:1981-93) have shown that both cell-tethered and
secreted mucins display different expression profiles in pancreatic
cancer when compared to normal pancreas. As a de novo mucin in
pancreatic cancer, MUC5AC could be detected as early as the
pre-malignant/dysplastic stages (Nagata et al., 2007, J
Hepatobiliary Pancreat Surg 14:243-54), and was identified in a
high percentage of PDAC (Remmers et al., 2013, Clin Cancer Res
19:1981-93; Yamazoe et al., 2011, Pancreas 40:896-904; Kanno et
al., 2006, Pancreas 33:391-6).
[0307] Our own endeavors for over 20 years have focused on the
exploration of mucin-reactive PAM4 as a potential diagnostic and
therapeutic agent for PDAC. Although we have recently proposed
MUC5AC to be the PAM4 antigen (Gold et al., 2013, Mol Cancer
12:143), the identification of the PAM4 epitope has lagged behind
its clinical development, mainly due to the challenges encountered
in characterizing MUC5AC, which is polymeric, heavily
O-glycosylated, and present in several variant forms (Thornton et
al., 2008, Annu Rev Physiol 70:459-86; Silverman et al., 2001,
Glycobiology 2001; 11:459-71; Guo et al., 2014, Am J Respir Cell
Mol Biol 50:223-32).
[0308] In the current Example, we provide additional evidence from
immunocytochemistry, RNA interference, and biochemical studies that
authenticates MUC5AC as the hPAM4 antigen; and more importantly,
have located the PAM epitope to the N-terminal region comprising
Cys2 through the recombinant expression of MUC5AC domains
(Backstrom et al., 2013, Mol Biotechnol 54:250-6). We should note
that DEGYTFCESPR (SEQ ID NO:56), one of the 6 MUC5AC peptides most
frequently detected in the pancreatic cystic lesions with malignant
potential, and not in the benign lesions, is located in the Cys2
and Cys4 subdomains, as reported in a very recent study of mucin
proteomics (Jabbar et al., 2014, J Natl Cancer Inst
106:djt439).
[0309] Based on their sequence similarity (Escande et al., 2001,
Biochem J 358:763-72), the 9 Cys subdomains of MUC5AC have been
characterized (Guo et al., 2014, Am J Respir Cell Mol Biol
50:223-32) as Class I (Cys1), Class II (Cys2, Cys4; 98% identical),
and Class III (Cys3, Cys5-9; 96% identical). Whereas each subdomain
contains about 110 amino acid residues, including 10 remarkably
conserved cysteine residues involved in intramolecular disulfide
bonds, there is only one potential O-glycosylation site and no
potential N-glycosylation site. These structural features appear to
match the characteristics of the hPAM4 epitope. Earlier work (Gold
et al., Int J Cancer 1994, 57:204-10) showed that the reactivity
between PAM4 and its mucin antigen was negatively affected by
heating, reduction of disulfide bonds, or certain protease
digestion, suggesting that the PAM4 epitope is a conformational
glycopeptide. While we have confirmed that reduced MUC5AC no longer
reacts with hPAM4, the results obtained from the unglycosylated
Cys2-3-4 and Cys2+ of this study also indicate that the hPAM
epitope is retained under denaturing conditions (or can be readily
restored following blotting or immobilization and washing), and
unlikely to involve carbohydrates. Because Cys2 and Cys4 are 98%
identical in amino acid sequence, including all of the 10 conserved
cysteine residues, we expect the hPAM4 epitope is present on Cys4
also.
[0310] It is worthy of note that among the various anti-MUC5AC
antibodies with mapped epitopes, which include the mouse mAbs of
the M1 series: 1-13M1 (Rose et al., 2000, J Aerosol Med 13:245-61),
2-11M1 (Rose et al., 2000), 9-13M1 (Rose et al., 2000), 19M1 (Rose
et al., 2000), 21M1 (Rose et al., 2000), 62M1 (Rose et al., 2000),
45M1 (Sheehan et al., 2000, Biochem J 347:37-44), and 2-12M1
(Sheehan et al., 2000); other murine mAbs such as CLH2 (Reis et
al., 1997, Int J Cancer 74:112-21), SOMU1 (Rose et al., 2000, J
Aerosol Med 13:245-61), 2Q445 (Sheehan et al., 2004, J Biol Chem
279:15698-705), and NPC-1C (U.S. Pat. No. 7,763,720); and two
rabbit polyclonal antibodies, H-160 (Perez-Vilar et al., 2006, J
Biol Chem 281:4844-55) and MAN-5ACI (Thornton et al., 1996, Biochem
J 316:967-75), 1-13M1 is the only mAb reported to react with Cys2/4
subdomains of MUC5AC. Our data, however, indicate that 1-13M1 binds
to a reduction-insensitive epitope, thus being different from that
of hPAM4.
[0311] Because the Cys2, Cys3 and Cys4 subdomains are flanked by
threonine/serine/proline (TSP)-rich sequences, which contain
numerous O-glycosylation sites, we further note that the
accessibility of hPAM4 to its epitope on Cys2 (or Cys4) could be
masked by the surrounding oligosaccharides either structurally or
in a conformation-dependent manner, or both. Accordingly, hPAM4
would prevail for underglycosylated MUC5AC, whose expression in
epithelial cancers in general, and PDAC in particular, including
the early-stage pancreatic cancer precursors, has not been as well
studied as that of underglycosylated MUC1 (Reis et al., 1998, Int J
Cancer 79:402-10).
[0312] In conclusion, we have located the hPAM4 antigen to the
N-terminal Cys2 of MUC5AC and characterized it as a
reduction-sensitive, carbohydrate-free epitope, whose access may be
restricted by the surrounding oligosaccharides in the flanking
TSP-domains. We believe the ultimate delineation of the hPAM4
epitope may lead to its exploration as a candidate for vaccine
development, while providing valuable insight for diagnosis and
treatment of MUC5AC-expressing cancers, such as biliary tract
cancer (Wongkham et al., 2003, Cancer Lett 195:93-9), colorectal
cancer (Bu et al., 2010, World J Gastroenterol 16:4089-94), and
gastric cancer (Wang et al., 2003, J Surg Oncol 83:453-60), in
addition to PDAC.
Example 2
Therapeutic Dosages of .sup.90Y-Labeled Anti-MUC5AC Antibody for
Human Pancreatic Cancer
[0313] For patients with metastatic pancreatic adenocarcinoma,
there are no approved or established treatments beyond 2.sup.nd
line. A study of fractionated radioimmunotherapy was undertaken,
administering .sup.90Y-clivatuzumab tetraxetan
(yttrium-90-radiolabeled hPAM4 anti-MUC5AC antibody) with or
without low radiosensitizing doses of gemcitabine.
[0314] Methods:
[0315] Fifty-eight patients with 3 median (range 2-7) prior
treatments were treated on Arm A (N=29, .sup.90Y-clivatuzumab
tetraxetan, weekly 6.5 mCi/m.sup.2 doses.times.3, plus gemcitabine,
weekly 200 mg/m.sup.2 doses.times.4 starting one week earlier) or
Arm B (N=29, .sup.90Y-clivatuzumab tetraxetan alone, weekly 6.5
mCi/m.sup.2 doses.times.3), repeating cycles after 4-week delays.
Safety and efficacy were evaluated.
[0316] Results:
[0317] Cytopenias (predominantly transient thrombocytopenia) were
the only significant toxicities. Fifty-three patients (27 Arm A, 26
Arm B, 91% overall) completed .gtoreq.1 full treatment cycles, with
23 (12 Arm A, 11 Arm B; 40%) receiving multiple cycles, including 7
(6 Arm A, 1 Arm B; 12%) given 3-9 cycles. Two patients in Arm A had
partial responses by RECIST criteria. Kaplan-Meier overall survival
(OS) appeared improved in Arm A v B (hazard ratio [HR] 0.55, 95%
CI: 0.29-0.86; P=0.017, log-rank) and the median OS for Arm A v Arm
B increased to 7.9 v 3.4 months with multiple cycles (HR 0.32,
P=0.004), including 3 patients in Arm A surviving >1 year.
[0318] Conclusions:
[0319] With these surprising results, clinical use of
.sup.90Y-clivatuzumab tetraxetan and low-dose gemcitabine is
feasible in metastatic pancreatic cancer patients beyond 2.sup.nd
line. A Phase III trial is now underway in this setting.
[0320] Introduction
[0321] The outlook for patients with advanced pancreatic
adenocarcinoma remains poor (Hidalgo, 2010, N Engl J Med
362:1605-17). In the frontline, median survival was 6.2-6.7 months
with gemcitabine alone (Burris et al., 1997, J Clin Oncol
15:2403-13) or with erlotinib (Moore et al., 2007, J Clin Oncol
25:1960-6), 8.5 months combined with albumin-bound paclitaxel (Von
Hoff et al., 2013, N Engl J Med 369:1691-1703), and 11.1 months for
those able to tolerate combination chemotherapy (FOLFIRINOX)
(Conroy et al., 2011, N Engl J Med 364:1817-25). Beyond 1st line,
the survival advantage with chemotherapy remains limited (Rahma et
al., 2013, Ann Oncol 24:1972-9; Oettle et al., 2014, J Clin Oncol
32:2423-9) and after two prior treatments (one usually
gemcitabine-based, the other fluoropyrimidine-based), there are no
accepted treatments (Seufferlein et al., 2012, Ann Oncol 23(suppl
7):vii33-40; Almhanna & Kim, 2008, Oncology (Williston Park)
22:1176-83).
[0322] We pursued radioimmunotherapy to target and directly
irradiate tumor sites without needing to physically overcome
transport barriers in pancreatic cancer (high interstitial
pressure, dense stromal reaction) or be incorporated into the tumor
cells to be effective (Koay et al., 2014, J Clin Invest
124:1525-36). PAM4, an anti-MUC5AC monoclonal antibody selectively
binding to pancreatic adenocarcinoma mucin, proved active when
radiolabeled in preclinical models of human pancreatic cancer
(Cardillo et al., 2001, Clin Cancer Res 7:3186-92; Gold et al.,
1997, Int J Cancer 71:660-7). However, dosage studies in animal
model systems are generally not predictive of effective therapeutic
dosages in humans (Reagan-Shaw et al., 2007, FASEB J 22:659-61),
requiring clinical studies in human subjects to establish safe and
effective therapeutic dosages.
[0323] After humanization and conjugation with DOTA
(1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid),
the chelate-hPAM4 conjugate (clivatuzumab tetraxetan) was labeled
with 90-yttrium (.sup.90Y), a beta-emitting radionuclide with a
radiation path-length of .about.5 mm suitable for bulky tumors.
.sup.90Y-clivatuzumab tetraxetan was initially administered as a
single dose (Gulec et al., 2011, Clin Cancer Res 17:4091-100), but
fractionated doses should be more effective (DeNardo et al., 2002,
Cancer 94:1332-48). Gemcitabine is a known radiosensitizer (Morgan
et al., 2008, Clin Cancer Res 14:6744-50), tolerated clinically at
low doses with external radiotherapy (Pauwels et al., 2005,
Oncologist 10:34-51), and preclinical studies showed enhanced
anti-tumor activity combining .sup.90Y-labeled PAM4 with
gemcitabine (Cardillo et al., 2002, Int J Cancer 97:386-92; Gold et
al., 2003, Clin Cancer Res 9:3929S-37S; Gold et al., 2004, Int J
Cancer 109:618-26).
[0324] In the frontline, fractionated doses of
.sup.90Y-clivatuzumab tetraxetan combined with 200 mg/m.sup.2 doses
of gemcitabine achieved 11.8 months median survival for those
patients given repeated treatment cycles; manageable
myelosuppression was the principal side-effect (Ocean et al., 2012,
Cancer 118:5497-505). There is an unmet medical need for further
therapy in pancreatic cancer patients who have received and shown
resistance to or relapsed from two or more prior therapies.
Radioimmunotherapy may be particularly attractive for patients
considering continued treatment, but unable or unwilling to
tolerate the side effects of further chemotherapy.
[0325] Methods
[0326] This Example reports the results of an open-label,
multicenter phase Ib study of .sup.90Y-clivatuzumab tetraxetan
administered with or without 200 mg/m.sup.2 gemcitabine in patients
with metastatic pancreatic adenocarcinoma after .gtoreq.2 prior
therapies. Primary study objectives included evaluating treatment
safety and tolerability in this setting. Additional objectives were
to obtain evidence of efficacy based on survival, CT imaging and
CA19-9 serum levels, assess the contribution of gemcitabine to this
treatment regimen, and evaluate any immunogenicity toward this
antibody-based regimen.
[0327] Population.
[0328] Adults .gtoreq.18 years old with metastatic pancreatic
adenocarcinoma had received .gtoreq.2 prior chemotherapy regimens
for their advanced disease and had measureable disease by CT
imaging, but no CNS metastases or bulky disease (no single mass
.gtoreq.10 cm). Other requirements included Karnofsky performance
status .gtoreq.70%, hemoglobin .gtoreq.9 g/dL, neutrophils
[ANC].gtoreq.1500/mm.sup.3, platelets .gtoreq.100,000/mm.sup.3,
creatinine and bilirubin .gtoreq.1.5.times.IULN (institutional
upper limit of normal), AST and ALT.gtoreq.2.0.times.IULN, with any
prior external radiation therapy <2000 cGy to lungs and kidneys,
<3000 cGy to liver, <30% of red marrow, and with
.ltoreq.Grade 2 nausea/vomiting, anorexia, or signs of intestinal
obstruction.
[0329] Treatment and Assessments.
[0330] Immunomedics, Inc., (Morris Plains, N.J.) provided
clivatuzumab tetraxetan to local commercial radiopharmacies for
.sup.90Y-radiolabeling. Based on the prior study (Ocean et al.,
2012, Cancer 118:5497-5506), a 6.5 mCi/m.sup.2 dose was selected
for this population, with .sup.90Y-clivatuzumab tetraxetan
administered in the hospital nuclear medicine department by slow
injection over 5-10 minutes. Commercially available gemcitabine
prepared by the hospital pharmacy was given intravenously over 30
minutes.
[0331] Patients were alternately assigned to treatment arms,
without consideration of prior history other than meeting
eligibility criteria. In Arm A, patients received 6.5 mCi/m.sup.2
90Y-clivatuzumab tetraxetan once-weekly for 3 weeks together with
200 mg/m.sup.2 gemcitabine once-weekly for 4 weeks starting 5 days
before beginning .sup.90Y-clivatuzumab tetraxetan and then 2 days
after each dose. In Arm B, patients received only 6.5 mCi/m.sup.2
90Y-clivatuzumab tetraxetan once-weekly for 3 weeks. For both arms,
treatment cycles were repeated after 4 weeks following the last
dose until unacceptable toxicity, progressive disease, or patient
withdrawal. The full .sup.90Y-clivatuzumab tetraxetan dose was
administered for ANC.gtoreq.1000/mm.sup.3 and platelets
.gtoreq.100,000/mm.sup.3; otherwise, a 75% dose for
ANC.gtoreq.750/mm.sup.3 and platelets .gtoreq.75,000/mm.sup.3, or a
50% dose for lower values with ANC.gtoreq.500/mm.sup.3 and
platelets .gtoreq.50,000/mm.sup.3, with the dose held if
ANC<500/mm.sup.3 or platelets <50,000/mm.sup.3 and treatment
delayed on a weekly basis until blood levels permitted dosing to
continue. Gemcitabine doses were held if .sup.90Y-clivatuzumab
tetraxetan doses were held, but were otherwise given without
reduction.
[0332] Adverse events were graded by NCI-CTCAE v4.0 (National
Cancer Institute (NCI). Common terminology criteria for adverse
events (CTCAE) version 4.0). Vital signs, physical examination,
blood counts, serum chemistries, and CA19-9 serum levels were
evaluated during treatment cycles, then 4, 8, and 12 weeks after
last cycle. Serum samples were evaluated by enzyme immunoassay for
any human anti-hPAM4 antibodies (HAHA). CT scans were interpreted
by local radiologists every 4 weeks after each cycle until
progression or 12 weeks post-treatment. Tumor lesion changes were
categorized as a complete response (CR), partial response (PR),
stable (SD), or progressive disease (PD) by RECIST v1.0 [27].
.sup.18F-FDG-PET or PET/CT imaging was optional. All patients were
followed for survival.
[0333] Statistical Analysis.
[0334] Overall survival (OS) from first dose to death or last
contact was analyzed by Kaplan-Meier methods. Other results were
summarized by descriptive statistics. To determine if this approach
is sufficiently active in this population, and if low-dose
gemcitabine adds to the therapeutic activity, a SWOG two-stage
design was used with a treatment target of .gtoreq.25% disease
control (PR or CR, or else SD for at least 8 weeks from treatment
initiation) versus .gtoreq.5% for an inactive therapy, resulting in
a sample size of .gtoreq.50 patients (25 per arm) for this
study.
[0335] Results
[0336] Patients and Study Treatment.
[0337] Fifty-eight patients with a median of 3 (range, 2-7) prior
chemotherapy regimens were enrolled. All had received
gemcitabine-containing regimens (21% with nab-paclitaxel) while 97%
had received fluoropyrimidine-containing regimens (50% with
FOLFIRINOX). Twenty-nine patients were treated in both Arm A
(gemcitabine) and Arm B (no gemcitabine). Table 3 summarizes
patient demographics and baseline characteristics for each arm.
TABLE-US-00003 TABLE 3 Demographics and baseline characteristics
Arm A Arm B Patients, N 29 29 Sex, M/F 19/10 14/15 Age, median
(range) 62 (39-73) 66 (51-80) Karnofsky performance status, N
90-100 14 10 70-80 15 19 Hematology, median (range) Platelets
(.times.1000/.mu.L) 211 (100-577) 224 (113-570) Neutrophils
(.times.1000/.mu.L) 5.2 (1.9-15.1) 5.4 (3.0-13.4) Hemoglobin (g/dL)
11.3 (9.2-15.1) 11.6 (9.4-15.8) Years from diagnosis, median 1.8
(0.3-4.1) 1.5 (0.4-3.7) (range) Stage IV disease, N (%) 29 (100%)
29 (100%) Extent of disease, median (range) CA19-9 (U/mL) 863
(0.8-UL*) 2720 (6.5-UL*) Sum of index lesions (cm) 9.8 (1.7-19.0)
7.1 (1.2-22.2) Tumor location, N (%) Pancreas, including resection
bed 11 (38%) 15 (52%) Liver 14 (48%) 17 (59%) Other abdomen sites
13 (45%) 16 (55%) Lung 13 (45%) 12 (41%) Prior therapies
Pancreatectomy, N (%) 15 (52%) 9 (31%) External radiation, N (%) 10
(34%) 8 (28%) Chemotherapy regimens Median (range) 3 (2-7) 3 (2-6)
Most frequent, N (%) Gemcitabine-containing 29 (100%) 29 (100%)
Gemcitabine/nab-paclitaxel 6 (21%) 6 (21%)
Fluoropyrimidine-containing 28 (97%) 28 (97%)
FOLFIRINOX.sup..dagger. 17 (59%) 12 (41%) *UL = upper limit of
quantitation, nominally >200,000 U/mL. .sup..dagger.Irinotecan,
oxaliplatin, and 5-fluorouracil/leucovorin combination.
[0338] No drug-related interruptions, discontinuations or adverse
reactions occurred with treatment administrations. Five patients
rapidly deteriorated before finishing one cycle (stroke, entered
hospice, biliary obstruction, pulmonary embolism, severe
constipation). Patients terminated further treatment due to disease
progression/clinical deterioration or other treatment-unrelated
events, with 30/58 (52%) patients (15 per arm) completing only one
cycle, and 23 (40%) patients (12 Arm A, 11 Arm B) receiving
multiple cycles, including 16 (6 Arm A, 10 Arm B) given 2 cycles
and 7 (6 Arm A, 1 Arm B) given 3 to 9 cycles. For cycle 1, 8/58
(14%) patients had .gtoreq.1 doses of .sup.90Y-clivatuzumab
tetraxetan reduced to 75%; but, besides the 5 patients who rapidly
deteriorated, no doses were held. For repeated cycles, 16/23 (70%)
patients had .gtoreq.1 doses reduced to 75% (N=7) or 50% (N=9),
including 11 (48%) who also had .gtoreq.1 doses held.
[0339] Adverse Events.
[0340] Events considered at least possibly treatment-related were
thrombocytopenia, 50% of patients; fatigue, 26%; anemia, 22%;
nausea, 16%; leukopenia, neutropenia, 12% each; abdominal pain,
anorexia, vomiting, diarrhea, 9% each; bleeding, fever, chills, 7%
each; dyspnea, hyperbilirubinemia, headache, 5% each; others
<5%. These included Grade .gtoreq.3 events of thrombocytopenia,
19%; anemia, leukopenia, neutropenia, 7% each; others
.ltoreq.2%.
[0341] Comparison of events regardless of assumed treatment
relationship shows limited differences between treatment arms, and
AEs occurring more frequently (>10% difference) in arm A
(fatigue, neutropenia, leukopenia, nausea, diarrhea, dyspnea,
alkaline phosphatase, headache) or arm B (ascites, asthenia,
gastrointestinal pain or tenderness) were primarily limited to
Grade 1-2 events (Table 4).
TABLE-US-00004 TABLE 4 Patient Incidence of Most Frequent Adverse
Events Regardless of Assumed Relationship to Treatment.* Arm A Arm
B All Grade All Adverse event, % Grades .gtoreq.3 Grades Grade
.gtoreq.3 Laboratories Thrombocytopenia 62 21 52 17 Anemia 45 14 38
10 Neutropenia 28 10 3 3 Leukopenia 24 14 3 3 Alkaline phosphatase
24 3 10 0 Hyponatremia 21 7 14 7 Aspartate aminotransferase 17 0 7
7 Lymphopenia 14 10 17 10 Hyperbilirubinemia 14 3 21 3
Hyperglycemia 14 3 14 7 Hypoalbumemia 14 0 3 3 Clinical Events
Fatigue 76 3 38 7 Nausea 41 0 24 3 Anorexia 31 0 24 7
Abdominal/gastrointestinal 31 3 48 10 pain or tenderness
Constipation 28 0 28 3 Diarrhea 28 0 10 0 Dyspnea 28 7 14 0
Infection 24 7 17 7 Vomiting 21 3 28 3 Abdominal distension 17 0 7
0 Bleeding 17 3 14 7 Back Pain 17 7 14 0 Cough 17 0 7 0 Dehydration
14 3 17 3 Headache 14 0 0 0 Hypertension 14 7 3 0 Fever 14 0 14 0
Pleural effusion 10 7 17 7 Peripheral edema 10 0 14 0 Ascites 0 0
14 3 Asthenia 0 0 14 3 *Events occurring in >10% of the 29
patients in either treatment arm
[0342] Six patients (3 per arm) had serious events considered at
least possibly treatment-related. Two patients had cerebrovascular
accidents due to thromboembolic or watershed events, one after the
first dose and the other one month after cycle 1, and two other
patients developed consumptive coagulopathies, one after cycle 1
with thrombocytopenia, deep venous thrombosis and sub-acute
cerebral infarcts, the other after cycle 2 with thrombocytopenia,
acute renal failure and fatal gastrointestinal hemorrhage. One
patient developed fever after cycle 3 with negative cultures but
responded to antibiotics, while another patient with a history of
severe infections developed fatal Gram-negative bacteremia during
cycle 2; both had undergone recent biliary stent placements and
were not neutropenic at time of event.
[0343] Overall, 20/58 (34%) patients (10 per arm) developed Grade
.gtoreq.3 thrombocytopenia (4 given platelets), 14 (11 Arm A, 3 Arm
B; 24% overall) developed Grade .gtoreq.3 neutropenia (4 given
cytokine support), and 11 (7 Arm A, 4 Arm B; 19% overall) developed
Grade .gtoreq.3 anemia (8 transfused). In patients with follow-up
data, only 4 events (all thrombocytopenia) remained at Grade 4
levels >7 days, while all Grade 3 cytopenias recovered to Grade
2 levels within 12 weeks. Grade .gtoreq.3 cytopenias generally
increased with repeated cycles but Grade 4 occurrences generally
remaining limited (Table 5).
TABLE-US-00005 TABLE 5 Grade 3 and 4 Hematological Toxicity by
Treatment Cycle Thrombo- cytopenia Neutropenia Anemia Grade Grade
Grade Grade Grade Grade Cycle N 3 4 3 4 3 4 Cycle 58 4 (7%) 6 (10%)
10 (17%) 1 (2%) 5 (9%) 0 (0%) 1 Cycle 23 7 (30%) 2 (9%) 3 (13%) 0
(0%) 4 (17%) 0 (0%) 2 Cycle 7 3 (43%) 2 (29%) 1 (14%) 1 (14%) 3
(43%) 0 (0%) 3-9
[0344] Infections occurred in 11 (19%) patients, including 4
serious events [fatal septic entercolitis from pre-study
pancreatectomy (Sump syndrome); fatal septic bacteremia from
unidentified source; post-interventional Grade 3 acute cholangitis;
Grade 3 pneumonia responding to antibiotics]; and 10 Grade 1-2
events [upper respiratory infection (URI).times.4, urinary tract
infection (UTI).times.3, superficial fungal infection.times.2,
pneumonia, Lyme disease). The fatal bacteremia was considered
possibly-related although the patient had a history of severe
infections and recent biliary stent placement; other infections
were considered unrelated by the investigators. Furthermore, only 3
patients were neutropenic (700-900 cells/.mu.L) at time of
infection (pneumonia, UTI, URI).
[0345] Bleeding occurred in 9 (16%) patients, including 3 serious
events (fatal GI bleeding from consumptive coagulopathy, Grade 3
melena from concomitant medications, Grade 3 GI bleeding from
underlying disease), and 7 minor Grade 1 events (bruising.times.4,
epistaxis, hemorrhoids, conjunctival). Minor bruising was
considered at least possibly study drug related, but the other
bleeding events were all considered unrelated by the investigators,
and only 3 patients had Grade 3-4 thrombocytopenia at time of event
(consumptive coagulopathy, bruising.times.2).
[0346] Efficacy.
[0347] The median overall survival (OS) for all 58 patients was 2.7
months, with 2 patients (both Arm A) currently alive 22 and 23
months from treatment initiation. Kaplan-Meier survival curves
(FIG. 7) showed improvement in Arm A v B beginning at about 3
months, with the relative number of patients remaining alive in Arm
A v B then progressively increasing with time (hazard ratio [HR]
0.55, 95% CI: 0.29-0.86; P=0.017, log-rank test). Median OS for Arm
A v B (Table 6) was only 2.7 v 2.6 months overall, but increased to
7.9 v 3.4 months for those patients who received multiple cycles
(HR 0.32, P=0.004), including 3 patients in Arm A surviving >1
year (one for 1.5 years, the others still alive). OS generally
increased with better performance status and lower CA19-9 serum
levels at study entry, and to a lesser degree with fewer prior
therapies and smaller tumor burden estimated by summing lengths of
index lesions (Table 6).
TABLE-US-00006 TABLE 6 Dependence of overall survival (OS) on
treatment and patient factors N Median (range), months Treatment
Arm A, Overall 29 2.7 (0.4-22.8+) Single Cycle 17 1.9 (0.4-11.0)
Multiple Cycles 12 7.9 (3.9-22.8+) Arm B, Overall 29 2.6 (0.7-9.4)
Single Cycle 18 1.7 (0.7-4.1) Multiple Cycles 11 3.4 (1.7-9.4)
Patient Factors Karnofsky Performance Status 90-100 24 4.0
(0.4-22.8+) 70-80 34 2.0 (0.7-21.7+) Number of Prior Systemic
Treatments 2 19 2.9 (0.4-21.7+) 3 18 3.1 (0.9-22.8+) >3 21 2.4
(0.7-17.5) Sum of Index Lesions (cm) 1.2-8.1 29 2.9 (0.7-22.8+)
8.3-22.2 29 2.6 (0.4-21.7+) Serum CA19-9 (U/mL) .ltoreq.1257 29 3.9
(0.4-22.8+) >1257 28* 2.1 (0.7-8.4) f.n. +indicates value from
patient currently alive *Baseline CA19-9 unavailable in one
patient.
[0348] There was >25% disease control (PR+SD) in both treatment
arms at interim evaluation, thus meeting the SWOG two-stage
criteria to complete enrollment. By RECIST criteria, there were 2
PRs (both Arm A) and 22 SDs (10 Arm A, 12 Arm B) as best response,
with other patients having progressed by first CT evaluation 4
weeks after cycle 1. Median OS for those with PR, SD and PD was
11.5, 5.1 and 1.8 months, respectively. Eleven patients had
elevated CA19-9 levels at baseline that decreased with treatment,
either 20-50% and considered a minor response (N=8), or >50% and
considered an objective response (N=3). Median OS for patients with
CA19-9 responses was 3.9 v 2.5 months for the remaining
population.
[0349] Immunogenicity.
[0350] Five patients (1 arm A, 4 arm B) with baseline serum samples
that were HAHA-negative (<50 ng/mL) became HAHA-positive after
their first (N=3) or second (N=2) cycle, developing maximum titers
of 135-21,611 ng/mL. These were isolated laboratory findings
without event and of uncertain clinical significance.
[0351] Discussion
[0352] Despite having 3 median (2 to 7) prior chemotherapies, 58
patients with metastatic pancreatic cancer were enrolled within 8
months, demonstrating the feasibility of using
.sup.90Y-clivatuzumab tetraxetan in this setting. All had received
gemcitabine-containing regimens (21% with nab-paclitaxel), and 97%
had received fluoropyrimidine-containing regimens (50% with
FOLFIRINOX), underscoring the importance of developing treatments
beyond 2nd line for pancreatic cancer.
[0353] There were no infusion reactions and, as expected,
cytopenias (predominantly thrombocytopenia) were the only
significant toxicities. Even in this population, these were mostly
transient and reversible events, with infrequent hematologic
support required. Treatment-related myelosuppression may have
exacerbated two cases of consumptive coagulopathy, but otherwise,
the few infections or major bleeding events that occurred could be
attributed to complications of underlying disease. Most other AEs
were mild-moderate constitutional and gastrointestinal events also
expected in advanced pancreatic cancer, and comparison of events
between treatment arms showed no substantial differences. Thus this
combination approach appears to be an acceptable regimen in this
advanced population.
[0354] Although median survival was only 2.7 months overall, the
addition of low-dose gemcitabine to this regimen showed survival
progressively improving with time (48% v 35% alive at 3 months, 35%
v 10% at 6 months, 21% v 3% at 9 months, 10% v 0% at 1 year). For
patients receiving only one cycle, gemcitabine made little
difference, but with multiple cycles median OS increased (3.4 v 7.9
months), including 3 patients surviving >1 year. Patients
undergoing multiple cycles could reflect a healthier population,
but this would not explain survival results favoring combining
radioimmunotherapy with low-dose gemcitabine. Although more limited
than previously seen with .sup.90Y-clivatuzumab tetraxetan in less
heavily treated patients (Gulec et al., 2011, Clin Cancer Res
17:4091-100; Ocean et al., 2012, Cancer 118:5497-505), tumor
response assessed by CT imaging or CA19-9 levels still showed
treatment activity, and the improvement of survival with better
responses and patient risk factors also supported the consistency
of results in this advanced population. However, to additionally
examine the role of low-dose gemcitabine in the treatment regimen
before pursuing a large, randomized, controlled trial, the study
was powered to demonstrate prespecified criteria for treatment arm
activity, not survival.
[0355] In conclusion, this trial demonstrated the feasibility of
using .sup.90Y-clivatuzumab tetraxetan in metastatic pancreatic
cancer patients after .gtoreq.2 prior chemotherapy regimens
(3.sup.rd line and beyond). This is important, because the benefits
of current second-line therapies appear modest, at the cost of drug
toxicity (Rahma et al., 2013, Ann Oncol 24:1972-9; Seufferlein et
al., 2012, Ann Oncol 23(suppl 7):vii33-40; Almhanna & Kim,
2008, Oncology (Williston Park) 22:1176-83). .sup.90Y-clivatuzumab
tetraxetan combined with low-dose gemcitabine appears promising in
this difficult-to-treat population.
Example 3
Humanized PAM4 MAb
[0356] In preferred embodiments, the claimed methods and
compositions utilize the antibody hPAM4 which is a humanized IgG of
the murine PAM4 MAb raised against pancreatic cancer mucin.
Humanization of the murine PAM4 sequences was utilized to reduce
the human antimouse antibody (HAMA) response. To produce the
humanized PAM4, murine complementarity determining regions (CDR)
were transferred from heavy and light variable chains of the mouse
immunoglobulin into human framework region (FR) antibody sequences,
followed by the replacement of some human FR residues with their
murine counterparts. Humanized monoclonal antibodies are suitable
for use in in vitro and in vivo diagnostic and therapeutic
methods.
[0357] Comparison of the variable region framework sequences of the
murine PAM4 MAb (FIG. 8A and FIG. 8B) to known human antibodies in
the Kabat database showed that the FRs of PAM4 VK and V.sub.H
exhibited the highest degree of sequence homology to that of the
human antibodies Walker VK (FIG. 10A) and Wil2 V.sub.H (FIG. 10B),
respectively. Therefore, the Walker VK (FIG. 10A) and Wil2 V.sub.H
(FIG. 10B) FRs were selected as the human frameworks into which the
murine CDRs for PAM4 VK and V.sub.H were grafted, respectively. The
FR4 sequence of the human antibody, NEWM, however, was used to
replace the Wil2 FR4 sequence for the humanization of the PAM4
heavy chain (FIG. 10B). A few amino acid residues in PAM4 FRs that
flank the putative CDRs were maintained in hPAM4 based on the
consideration that these residues have more impact on Ag binding
than other FR residues. These residues were 21M, 47W, 59P, 60A,
85S, 87F, and 100G of VK (FIG. 10A) and 27Y, 30P, 38K, 48I, 66K,
67A, and 69L of V.sub.H (FIG. 10B). The DNA and amino acid
sequences of hPAM4 VK (SEQ ID NO:16) and V.sub.H (SEQ ID NO: 19)
are shown in FIGS. 11A and 11B, respectively.
[0358] A modified strategy as described by Leung et al. (Leung et
al., 1994)) was used to construct the designed VK (FIG. 11A) and
V.sub.H (FIG. 11B) genes for hPAM4 using a combination of long
oligonucleotide syntheses and PCR. For the construction of the
hPAM4 V.sub.H domain, two long oligonucleotides, hPAM4 V.sub.H A
(173-mer) and hPAM4 V.sub.H B (173-mer) were synthesized on an
automated DNA synthesizer (Applied Biosystems). hPAM4 V.sub.H A
represents nt 17 to 189 of the hPAM4 V.sub.H domain.
TABLE-US-00007 (SEQ ID NO: 57) 5'-AGTCTGGGGC TGAGGTGAAG AAGCCTGGGG
CCTCAGTGAA GGTCTCCTGC GAGGCTTCTG GATACACATT CCCTAGCTAT GTTTTGCACT
GGGTGAAGCA GGCCCCTGGA CAAGGGCTTG AGTGGATTGG ATATATTAAT CCTTACAATG
ATGGTACTCA GTACAATGAG AAG-3'
[0359] hPAM4 V.sub.HB represents the minus strand of the hPAM4
V.sub.H domain complementary to nt 169 to 341.
TABLE-US-00008 (SEQ ID NO: 58) 5'-AGGGTTCCCT GGCCCCAGTA AGCAAATCCG
TAGCTACCAC CGAAGCCTCT TGCACAGTAA TACACGGCCG TGTCGTCAGA TCTCAGCCTG
CTCAGCTCCA TGTAGGCTGT GTTGATGGAC GTGTCCCTGG TCAGTGTGGC CTTGCCTTTG
AACTTCTCAT TGTACTGAGT ACC-3'
[0360] The 3'-terminal sequences (21 nt residues) of hPAM4 V.sub.HA
and V.sub.HB are complementary to each other. Under defined PCR
condition, the 3'-ends of hPAM4 V.sub.HA and V.sub.HB anneal to
form a short double stranded DNA flanked by the rest of the long
oligonucleotides. Each annealed end serves as a primer for the
transcription of the single stranded DNA, resulting in a double
strand DNA composed of the nt 17 to 341 of hPAM4 V.sub.H. This DNA
was further amplified in the presence of two short
oligonucleotides, hPAM4 V.sub.HBACK and hPAM4 V.sub.HFOR to form
the full-length hPAM4 V.sub.H. The underlined portions are
restriction sites for subcloning as shown in FIG. 11B.
TABLE-US-00009 hPAM4 V.sub.HBACK (SEQ ID NO: 59) 5'-CAG GTG CAG CTG
CAG CAG TCT GGG GCT GAG GTG A-3' hPAM4 V.sub.HFOR (SEQ ID NO: 60)
5'-TGA GGA GAC GGT GAC CAG GGT TCC CTG GCC CCA-3'
[0361] A minimal amount of hPAM4 V.sub.HA and V.sub.HB (determined
empirically) was amplified in the presence of 10 .mu.L of
10.times.PCR Buffer (500 mM KCl, 100 mM Tris HCl buffer, pH 8.3, 15
mM MgCl.sub.2), 2 .mu.mol of hPAM4 V.sub.HBACK and hPAM4
V.sub.KFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer
Cetus, Norwalk, Conn.). This reaction mixture was subjected to
three cycles of polymerase chain reaction (PCR) consisting of
denaturation at 94.degree. C. for 1 minute, annealing at 45.degree.
C. for 1 minute, and polymerization at 72.degree. C. for 1.5
minutes. This procedure was followed by 27 cycles of PCR reaction
consisting of denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1 minute, and polymerization at 72.degree. C.
for 1 minute. Double-stranded PCR-amplified product for hPAM4
V.sub.H was gel-purified, restriction-digested with PstI and BstEII
restriction sites and cloned into the complementary PstI/BstEII
restriction sites of the heavy chain staging vector, V.sub.HpBS2,
in which the V.sub.H sequence was fully assembled with the DNA
sequence encoding the translation initiation codon and a secretion
signal peptide in-frame ligated at the 5'-end and an intron
sequence at the 3'-end. V.sub.HpBS2 is a modified staging vector of
V.sub.HpBS (Leung et al., Hybridoma, 13:469, 1994), into which a
XhoI restriction site was introduced at sixteen bases upstream of
the translation initiation codon to facilitate the next subcloning
step. The assembled V.sub.H gene was subcloned as a XhoI-BamHI
restriction fragment into the expression vector, pdHL2, which
contains the expression cassettes for both human IgG heavy and
light chains under the control of IgH enhancer and MT1 promoter, as
well as a mouse d/fr gene as a marker for selection and
amplification. Since the heavy chain region of pdHL2 lacks a BamHI
restriction site, this ligation requires use of a linker to provide
a bridge between the BamHI site of the variable chain and the
HindIII site present in the pdHL2 vector. The resulting expression
vectors were designated as hPAM4 V.sub.HpdHL2.
[0362] For constructing the full length DNA of the humanized VK
sequence (FIG. 11A), hPAM4 V.sub.KA (157-mer) and hPAM4 V.sub.KB
(156-mer) were synthesized as described above. hPAM4 V.sub.KA and
V.sub.KB were amplified by two short oligonucleotides hPAM4
V.sub.KBACK and hPAM4 V.sub.KFOR as described above. hPAM4 V.sub.KA
represents nt 16 to 172 of the hPAM4 VK domain.
TABLE-US-00010 (SEQ ID NO: 61) 5'-CAGTCTCCAT CCTCCCTGTC TGCATCTGTA
GGAGACAGAG TCACCATGAC CTGCAGTGCC AGCTCAAGTG TAAGTTCCAG CTACTTGTAC
TGGTACCAAC AGAAACCAGG GAAAGCCCCC AAACTCTGGA TTTATAGCAC ATCCAACCTG
GCTTCTG-3'
[0363] hPAM4 V.sub.KB represents the minus strand of the hPAM4 VK
domain complementary to nt 153 to 308.
TABLE-US-00011 (SEQ ID NO: 62) 5'-GTCCCCCCTC CGAACGTGTA CGGGTACCTA
TTCCACTGAT GGCAGAAATA AGAGGCAGAA TCTTCAGGTT GCAGACTGCT GATGGTGAGA
GTGAAGTCTG TCCCAGATCC ACTGCCACTG AAGCGAGCAG GGACTCCAGA AGCCAGGTTG
GATGTG-3'
[0364] The 3'-terminal sequences (20 nt residues) of hPAM4 V.sub.KA
and V.sub.KB are complementary to each other. Under defined PCR
condition, the 3'-ends of hPAM4 V.sub.KA and V.sub.KB anneal to
form a short double-stranded DNA flanked by the rest of the long
oligonucleotides. Each annealed end served as a primer for the
transcription of the single stranded DNA, resulting in a double
strand DNA composed of nt 16 to 308 of hPAM4 VK. This DNA was
further amplified in the presence of two short oligonucleotides,
hPAM4 V.sub.KBACK and hPAM4 V.sub.KFOR to form the full-length
hPAM4 VK. The underlined portions are restriction sites for
subcloning as described below.
TABLE-US-00012 hPAM4 V.sub.KBACK (SEQ ID NO: 63) 5'-GAC ATC CAG CTG
ACC CAG TCT CCA TCC TCC CTG-3' hPAM4 V.sub.KFOR (SEQ ID NO: 64)
5'-TTA GAT CTC CAG TCG TGT CCC CCC TCC GAA CGT-3'
[0365] Gel-purified PCR products for hPAM4 VK were
restriction-digested with PvuII and BglII and cloned into the
complementary PvuII/BclI sites of the light chain staging vector,
V.sub.KpBR2. V.sub.KpBR2 is a modified staging vector of V.sub.KpBR
(Leung et al., Hybridoma, 13:469, 1994), into which a XbaI
restriction site was introduced at sixteen bases upstream of the
translation initiation codon. The assembled VK genes were subcloned
as XbaI-BamHI restriction fragments into the expression vector
containing the V.sub.H sequence, hPAM4 V.sub.HpdHL2. The resulting
expression vectors were designated as hPAM4pdHL2.
[0366] Approximately 30 .mu.g of hPAM4pdHL2 was linearized by
digestion with SalI and transfected into Sp2/0-Ag14 cells by
electroporation at 450 V and 25 .mu.F. The transfected cells were
plated into 96-well plates and incubated in a CO.sub.2 cell culture
incubator for two days and then selected for MTX resistance.
Colonies surviving selection emerged in two to three weeks and were
screened for human antibody secretion by ELISA assay. Briefly,
supernatants (.about.100 ul) from the surviving colonies were added
into the wells of an ELISA microplate precoated with goat
anti-human IgG F(ab').sub.2 fragment-specific Ab. The plate was
incubated for one hour at room temperature. Unbound proteins were
removed by washing three times with wash buffer (PBS containing
0.05% Tween-20). Horseradish peroxidase-conjugated goat anti-human
IgG Fc fragment-specific Ab was added to the wells. Following
incubation for one hour, a substrate solution (100 .mu.L/well)
containing 4 mM o-phenylenediamine dihydrochloride (OPD) and 0.04%
H.sub.2O.sub.2 in PBS was added to the wells after washing. Color
was allowed to develop in the dark for 30 minutes and the reaction
was stopped by the addition of 50 .mu.L of 4 N H.sub.2SO.sub.4
solution. The bound human IgG was measured by reading the
absorbance at 490 nm on an ELISA reader. Positive cell clones were
expanded and hPAM4 was purified from cell culture supernatant by
affinity chromatography on a Protein A column.
[0367] The Ag-binding activity of hPAM4 was confirmed by ELISA
assay in a microtiter plate coated with pancreas cancer cell
extracts. An ELISA competitive binding assay using PAM4-antigen
coated plates was developed to assess the Ag-binding affinity of
hPAM4 in comparison with that of a chimeric PAM4 composed of murine
V and human C domains. Constant amounts of the HRP-conjugated cPAM4
mixed with varying concentrations of cPAM4 or hPAM4 were added to
the coated wells and incubated at room temperature for 1-2 h. The
amount of HRP-conjugated cPAM4 bound to the CaPan1 Ag was revealed
by reading the absorbance at 490 nm after the addition of a
substrate solution containing 4 mM o-phenylenediamine
dihydrochloride and 0.04% H.sub.2O.sub.2. As shown by the
competition assays in FIG. 12, hPAM4 and cPAM4 antibodies exhibited
similar binding activities.
Example 4
Immunohistochemistry Staining Studies
[0368] Immunohistochemistry on normal adult tissues showed that the
PAM4 reactive epitope was restricted to the gastrointestinal tract
where staining was weak, yet positive (Table 7). Normal pancreatic
tissue, including ducts, ductules, acini, and islet cells, were
negative for staining. A PAM4 based enzyme immunoassay with tissue
homogenates as antigens generally supported the immunohistology
data (Table 8). The PAM4 epitope was absent from normal pancreas
and other non-gastrointestinal tissues. In neoplastic tissues, PAM4
was reactive with twenty one out of twenty five (85%) pancreatic
cancers (Table 9 and Table 10) and ten out of twenty six colon
cancers, but only limited reactivity with tumors of the stomach,
lung, breast, ovary, prostate, liver or kidney (Table 10). PAM4
reactivity appeared to correlate with the stage of tumor
differentiation, with a greater percentage of staining observed in
well differentiated pancreatic cancers than in moderately
differentiated or poorly differentiated tumors. Generally, poorly
differentiated tumors represent less than 10% of all pancreatic
cancers.
[0369] These studies have shown the PAM4 reactivity and tissue
distribution (both normal and cancer) to be unlike that reported
for the CA19.9, DUPAN2, SPAN1, Nd2 and B72.3 antibodies and
antibodies against the Lewis antigens. Together with crossblocking
studies performed with certain of these MAbs, the data suggests
that the PAM4 MAb recognizes a unique and novel epitope. When
compared to the antigens recognized by the CA19.9, DUPAN2, and
anti-Le.sup.a antibodies, the PAM4 antigen appears to be more
restricted in its tissue distribution and is reactive with a higher
percentage of pancreatic tumors. Moreover, it gives a greater
overall intensity of reaction at equivalent concentrations and is
reactive with a higher percentage of cells within the pancreatic
tumors. Finally, PAM4 was found to be only weakly reactive with
three out of twelve chronic pancreatitis specimens, whereas CA19.9
and DUPAN2 were strongly reactive with all twelve specimens.
Although it is recognized that specificity is dependent in part
upon the type of assay employed and the range and number of tissues
examined, the ability of PAM4 to discriminate between normal and
neoplastic pancreatic tissue, its ability to react with a large
percentage of the cancer specimens, the high intensity of the
reactions, and the ability to distinguish between early stage
pancreatic cancer and benign conditions such as pancreatitis are
important characteristics of this exemplary anti-pancreatic cancer
antibody.
TABLE-US-00013 TABLE 7 Immunoperoxidase Staining of Normal Adult
Tissues with MAb PAM4 Staining Tissue Reaction Pancreas (22).sup.a
-- Ducts -- Acini -- Islets -- Submaxillary gland (2) -- Esophagus
(2) -- Stomach (3) + mucus secreting cells Duodenum (3) + goblet
cells Jejunum (3) + goblet cells Ileum (3) + goblet cells Colon (5)
+ goblet cells Liver (3) -- Gallbladder (2) -- Bronchus (3) -- Lung
(3) -- Heart (3) -- Spleen (3) -- Kidney (3) -- Bladder (3) --
Prostate (2) -- Testes (2) -- Uterus (2) -- Ovary (2) --
.sup.anumber of individual specimens examined in parentheses
TABLE-US-00014 TABLE 8 Monoclonal Antibody PAM4 Reactivity with
Normal Adult Tissue Homogenates by EIA Tissue .mu.g/g tissue.sup.a
Pancreas 6.4 Esophagus 8.1 Stomach 61.3 Duodenum 44.7 Jejunum 60.6
Colon 74.5 Liver 0.0 Gallbladder 5.6 Heart 3.7 Spleen 3.4 Kidney
6.6 Bladder 4.9 Thyroid 3.5 Adrenal 1.3 Ureter 2.6 Testes 3.9
CaPan1 Pancreatic Tumor 569 .sup.avalues are mean from two autopsy
specimens
TABLE-US-00015 TABLE 9 Immunohistochemical Reactivity of Several
Monoclonal Antibodies with Pancreatic Tumors Differentiation PAM4
CA19.9 Le.sup.a DUPAN2 1 W +++ - - +++ 2 M ++ +++ +++ + 3 M + - + +
4 M +++ +++ +++ + 5 M ++ + - - 6 M + ND ND ND 7 M* +++ +++ +++ +++
8 M + - - +++ 9 M ++ + ++ - 10 M* ++ ++ ++ +++ 11 M ++ +++ +++ + 12
M ++ + + +++ 13 M + +++ +++ + 14 M ++ + + ++ 15 M +++ + + ++ 16 M +
+ ++ - 17 M - + + - 18 M ++ ++ ++ ++ 19 M +++ + +++ ++ 20 M + - - -
21 M +++ +++ + ++ 22 P + + + +++ 23 P - - - - 24 P - - - - 25 P - -
+ - TOTAL 21/25 17/24 18/24 16/24 : Negative; +: 5-20% of tissue is
stained; ++: 21-50% of tissue is stained; +++: >50% of tissue is
stained; W, M, P: Well, moderate, or poor differentiation; :
Metastatic tissue; ND: Not Done
TABLE-US-00016 TABLE 10 Immunoperoxidase Staining of Neoplastic
Tissues with MAb PAM4 Cancer Tissue Positive/Total Pancreas 21/25
Colon 10/26 Stomach 1/5 Lung 1/15 Breast 0/30 Ovarian 0/10 Prostate
0/4 Liver 0/10 Kidney 0/4
Example 5
In Vivo Biodistribution and Tumor Targeting of Radiolabeled
PAM4
[0370] Initial biodistribution studies of PAM4 were carried out in
a series of four different xenografted human pancreatic tumors
covering the range of expected differentiation. Each of the four
tumor lines employed, AsPc1, BxPc3, Hs766T and CaPan1, exhibited
concentrations of .sup.131I-PAM4 within the tumors (range: 21%-48%
ID/g on day three) that were significantly (P<0.01-0.001) higher
than concomitantly administered nonspecific, isotype-matched Ag8
antibody (range: 3.6%-9.3% ID/g on day three). The biodistribution
data were used to estimate potential radiation doses to the tumor
of 12,230; 10,684; 6,835; and 15,843 cGy/mCi of injected dose to
AsPc1, BxPc3, Hs766T and CaPan1, respectively. With an actual
maximum tolerated dose (MTD) of 0.7 mCi, PAM4 could provide
substantial rad dose to each of the xenografted tumor models. In
each tumor line the blood levels of radiolabeled PAM4 were
significantly (P<0.01-0.001) lower than the nonspecific Ag8.
Potential radiation doses to the blood from PAM4 were 1.4-4.4 fold
lower than from Ag8. When radiation doses to the tumor from PAM4
were normalized to the blood doses from PAM4, the tumors received
doses that were 2.2; 3.3; 3.4; and 13.1-fold higher than blood,
respectively. Importantly, potential radiation doses to non-tumor
tissues were minimal.
[0371] The biodistribution of PAM4 was compared with an anti-CEA
antibody, MN-14, using the CaPan1 tumor model. The concentration of
PAM4 within the tumor was much greater than MN-14 at early
timepoints, yielding tumor:blood ratios at day three of 12.7.+-.2.3
for PAM4 compared to 2.7.+-.1.9 for MN-14. Although PAM4 uptake
within the tumor was significantly higher than for MN-14 at early
timepoints (day one--P<0.001; day three--P<0.01), dosimetry
analyses indicated only a 3.2-fold higher dose to the tumor from
PAM4 as compared to MN-14 over the fourteen day study period. This
was due to a rapid clearance of PAM4 from the tumor, such that at
later timepoints similar concentrations of the two antibodies were
present within the tumors. A rapid clearance of PAM4 from the tumor
was also noted in the BxPc3 and Hs766T but not AsPc1 tumor models.
These observations were unlike those reported for other anti-mucin
antibodies, as for example G9 and B72.3 in colorectal cancer, where
each exhibited longer retention times as compared to the MN-14
antibody. Results from studies on the metabolism of PAM4, indicate
that after initial binding to the tumor cell, antibody is rapidly
released, possibly being catabolized or being shed as an
antigen:antibody complex. The blood clearance is also very rapid.
These data suggest that .sup.131I may not be the appropriate choice
of isotope for therapeutic applications. A short-lived isotope,
such as .sup.90Y or .sup.188Re, which can be administered
frequently may be a more effective reagent.
[0372] PAM4 showed no evidence of targeting to normal tissues,
except in the CaPan1 tumor model, where a small but statistically
significant splenic uptake was observed (range 3.1-7.5% ID/g on
day-3). This type of splenic targeting has been observed in the
clinical application of the anti-mucin antibodies B72.3 and CC49.
Importantly, these studies also reported that splenic targeting did
not affect tumor uptake of antibody nor did it interfere with
interpretation of the nuclear scans. These studies suggested that
splenic targeting was not due to crossreactive antigens in the
spleen, nor to binding by Fc receptors, but rather to one or more
of the following possibilities: direct targeting of antigen trapped
in the spleen, or indirect uptake of antigen:antibody complexes
formed either in the blood or released from the tumor site. The
latter would require the presence of immune complexes in the blood.
However, these were not observed when specimens as early as five
minutes and as late as seven days were examined by gel filtration
(HPLC, GF-250 column); radiolabeled antibody eluted as native
material. The former explanation seems more likely in view of the
fact that the CaPan1 tumor produced large quantities of
PAM4-reactive antigen, 100- to 1000-fold higher than for the other
tumor cell lines examined. The lack of splenic targeting by PAM4 in
these other tumor lines suggests that this phenomenon was related
to excessive antigen production. Splenic targeting can be overcome
by increasing the protein dose to 10 .mu.g from the original 2
.mu.g dose. A greater amount of the splenic entrapped antigen
presumably was complexed with unlabeled PAM4 rather than
radiolabeled antibody. Increasing the protein dose had no adverse
effect upon targeting of PAM4 to the tumor or nontumor tissues. In
fact, an increase of the protein dose to 100 .mu.g more than
doubled the concentration of radiolabeled PAM4 within the CaPan1
tumor.
Example 6
Development of Orthotopic Pancreatic Tumor Model in Athymic Nude
Mice
[0373] In order to resemble the clinical presentation of pancreatic
cancer in an animal model more closely, we developed an orthotopic
model by injecting tumor cells directly into the head of the
pancreas. Orthotopic CaPan1 tumors grew progressively without overt
symptoms until the development of ascites and death at ten to
fourteen weeks. By three to four weeks post-implantation, animals
developed a palpable tumor of approximately 0.2 g. Within eight
weeks of growth, primary tumors of approximately 1.2 g along with
metastases to the liver and spleen were observed (1-3 metastatic
tumors/animal; each tumor <0.1 g). At ten to fourteen weeks
seeding of the diaphragm with development of ascites were evident.
Ascites formation and occasional jaundice were usually the first
overt indications of tumor growth. At this time tumors were quite
large, 1 to 2 g, and animals had at most only three to four weeks
until death occurred.
[0374] Radiolabeled .sup.131I-PAM4, administered to animals bearing
four week old orthotopic tumors (approximately 0.2 g) showed
specific targeting to the primary tumor with localization indices
of 7.9.+-.3.0 at day one increasing to 22.8.+-.15.3 at day
fourteen. No evidence of specific targeting to other tissues was
noted. In one case where tumor metastases to the liver and spleen
were observed, both metastases were targeted, and had high
concentrations of radiolabeled antibody. In addition, approximately
half of the animals developed a subcutaneous tumor at the incision
site. No significant differences were noted in the targeting of
orthotopic and subcutaneous tumors within the same animal, and no
significant differences were observed in the targeting of
orthotopic tumor whether or not the animal had an additional
subcutaneous tumor. The estimated radiation doses from PAM4 were
6,704 and 1,655 cGy/mCi to the primary tumor and blood,
respectively.
Example 7
Radioimmunotherapy of Pancreatic Cancer
[0375] The initial studies on the use of .sup.131I-PAM4 for therapy
were carried out with the CaPan1 tumor, which was grown as a
subcutaneous xenograft in athymic mice. Animals bearing a 0.25 g
tumor were administered 350 .mu.Ci, .sup.131I-PAM4 in an experiment
that also compared the therapeutic effects of a similar dose of
nonspecific Ag8. The MTD for administration of .sup.131I-PAM4 to
animals bearing 1 cm.sup.3 tumors is 700 .mu.Ci. By weeks five and
six, the PAM4 treated animals showed a dramatic regression of
tumor, and even at week twenty seven, five out of eight remained
tumor free. The untreated, as well as Ag8-treated animals, showed
rapid progression of tumor growth although a significant difference
was noted between these two control groups. At seven weeks, tumors
from the untreated group had grown 20.0.+-.14.6-fold from the
initial timepoint whereas the .sup.131I-Ag8-treated tumors had
grown only 4.9.+-.1.8-fold. At this time point, the PAM4 tumors had
regressed to 0.1.+-.0.1-fold of their original size, a significant
difference from both untreated (P<0.001) and nonspecific
Ag8-treated (P<0.01) animals.
[0376] These data show that CaPan1 tumors were sensitive to
treatment with .sup.131I-PAM4. The outcome, that is, regression or
progression of the tumor, was dependent upon several factors
including initial tumor size. Thus, groups of animals bearing
CaPan1 tumor burdens of 0.25 g, 0.5 g, 1.0 g, or 2.0 g were treated
with a single dose of the 350 .mu.Ci .sup.131I-PAM4. The majority
of animals having tumors of initial size 0.25 g and 0.5 g (nine of
ten animals in each group) showed tumor regression or growth
inhibition for at least sixteen weeks post treatment. In the 1.0 g
tumor group five out of seven showed no tumor growth for the
sixteen week period and in the 2.0 g tumor group six out of nine
showed no tumor growth for a period of six weeks before progression
occurred. Although a single 350 .mu.Ci dose was not as effective
against larger tumors, a single dose may not be the appropriate
regimen for large tumors.
[0377] Toxicity studies indicate the ability to give multiple
cycles of radioimmunotherapy, which may be more effective with a
larger tumor burden. Animals bearing CaPan1 tumors averaging 1.0 g,
were given either a single dose of 350 .mu.Ci .sup.131I-PAM4, two
doses given at times zero and four weeks or were left untreated.
The untreated group had a mean survival time of 3.7.+-.1.0 weeks
(survival defined as time for tumor to reach 5 cm.sup.3). Animals
died as early as three weeks, with no animal surviving past six
weeks. A single dose of 350 .mu.Ci .sup.131I-PAM4 produced a
significant increase in the survival time to 18.8.+-.4.2 weeks
(P<0.0001). The range of animal deaths extended from weeks
thirteen to twenty five. None of the animals were alive at the end
of the study period of twenty six weeks.
[0378] A significant increase in survival time was observed for the
two dose group as compared to the single dose group. Half of the
animals were alive at the twenty six week timepoint with tumor
sizes from 1.0-2.8 cm.sup.3, and a mean tumor growth rate of
1.6.+-.0.7 fold from initial tumor size. For those animals that
were non-survivors at twenty six weeks, the mean survival time
(17.7.+-.5.3 weeks) was similar to the single dose group.
[0379] Therapy studies with PAM4 were also conducted using the
orthotopic tumor model. Groups of animals bearing four week old
orthotopic tumors (estimated tumor weight of 0.25 g) were either
left untreated or treated with a single dose of either 350 .mu.Ci
.sup.131I-PAM4 or 350 .mu.Ci of .sup.131I-nonspecific Ag8. The
untreated animals had a 50% death rate by week ten with no
survivors at week fifteen. Animals administered nonspecific
.sup.131I-Ag8 at four weeks of tumor growth, showed a 50% death
rate at week seven with no survivors at week fourteen. Although
statistically (logrank analysis) there were no differences between
these two groups, it is possible that radiation toxicity had
occurred in the Ag8 treated animals. Radiolabeled PAM4 provided a
significant survival advantage (P<0.001) as compared to the
untreated or Ag8 treated animals, with 70% survival at sixteen
weeks, the end of the experiment. At this time the surviving
animals were sacrificed to determine tumor size. All animals had
tumor with an average weight of 1.2 g, as well as one or two small
(<0.1 g) metastases evident in four of the seven animals. At
sixteen weeks of growth, these tumors were more representative of
an eight-week-old tumor.
Example 8
Combined Modality GEMZAR.RTM. Chemotherapy and .sup.131I-PAM4
Experimental Radioimmunotherapy
[0380] Initial studies into the combined use of gemcitabine
(GEMZAR.RTM.) with .sup.131I-PAM4 radioimmunotherapy were performed
as a checkerboard array; a single dose of gemcitabine (0, 100, 200,
500 mg/kg) versus a single dose of .sup.131I-PAM4 ([MTD=700 .mu.Ci]
100%, 75%, 50%, 0% of the MTD). The combined MTD was found to be
500 mg/kg gemcitabine with 350 .mu.Ci .sup.131I-PAM4 (50% MTD).
Toxicity, as measured by loss of body weight, went to the maximum
considered as nontoxic; that is 20% loss in body weight. Although
the combined treatment protocol was significantly more effective
than gemcitabine alone, the treatment was no more effective than
radioimmunotherapy alone. The next studies were performed at a low
dose of gemcitabine and radioimmunotherapy to examine if a true
synergistic therapeutic effect would be observed. Athymic nude mice
bearing tumors of approximately 1 cm.sup.3 (approximately 5% of
body weight) were administered gemcitabine, 100 mg/kg on days zero,
three, six, nine, and twelve, with 100 .mu.Ci of .sup.131I-PAM4
given on day zero. A therapeutic effect was observed with
statistically significant (P<0.0001) regression (two of five
tumors less than 0.1 cm.sup.3) and/or growth inhibition of the
tumors compared to gemcitabine alone. Thus, at lower dosages of
therapeutic agent, there surprisingly appears to be a synergistic
effect of the combination of gemcitabine and radioimmunotherapy. Of
additional note, in terms of body weight, toxicity was not
observed. The combination treatment protocol can, if necessary, be
delivered in multiple cycles, with the second treatment cycle
beginning in week-four, as was done with the
radioimmunotherapy-alone studies described above.
Example 9
Effects of Reagent Treatment on Immunoreactivity of PAM4
Antigen
[0381] Treatment of pancreatic mucin with DTT (15 min at room
temp), completely abolished reactivity with PAM4 (DTT-EC.sub.50,
0.60.+-.0.0 .mu.M). The only cysteines (cystine bridges) within
MUC-1 are present within the transmembrane domain and should not be
accessible to DTT. The secreted form of MUC-1 does not contain the
transmembrane domain and therefore has no intramolecular cystine
bridges. Data from periodate oxidation treatment of pancreatic
cancer mucin with 0.05 M sodium periodate for 2 hrs at room
temperature yielded 40% loss of immunoreactivity with PAM4 antibody
(not shown). Further periodate studies have shown as high as a 60%
loss of immunoreactivity with PAM4 antibody (not shown). The
results of periodate and DTT studies suggest that the PAM4 epitope
is conformationally dependent upon some minimal form of
glycosylation, and may be affected by intermolecular disulfide bond
formation.
Example 10
Distribution and Cross-Reactivity of the PAM4 Antigen
[0382] The expression of the PAM4-epitope within PanINs is atypical
for MUC-1. It is similar to the expression reported for MUC5AC as
detected by the commercially available MAb-CLH2-2. However, an
attempted sandwich immunoassay with PAM4 capture and MAb-CLH2-2 as
probe gave negative results. Although this possibly suggests the
PAM4 and CLH2-2 epitopes may overlap and thus block each other, the
CLH2-2 was reported to be reactive with 42/66 (64%) gastric
carcinomas whereas the PAM4 MAb showed reactivity with only 6/40
(15%) of gastric carcinomas and, of these, only in focal
reactivity.
[0383] Use of the commercially available 45M1, an anti-MUC5AC MAb,
as a probe reagent in EIA (with PAM4 as capture) provided positive
results, indicating that the two epitopes may be present on the
same antigenic molecule. Blocking studies (either direction)
indicated that the epitopes bound by 45M1 and PAM4 are in fact two
distinct epitopes, as no blocking was observed. Labeling of tissue
microarrays consisting of cores from invasive pancreatic carcinoma
has demonstrated significant differences for expression of the 45M1
and PAM4 epitopes in individual patient specimens. Of 28 specimens,
concordance was observed in only 17 cases (61%). PAM4 was reactive
with 24/28 cases (86%) while 45M1 was reactive with 13/28 (46%)
cases (not shown).
[0384] The results of periodate studies are consistent with
glycosylation as a factor in MUC5AC immunoreactivity with the PAM4
antibody. Thus, results of studies with apomucins may not be
definitive for antigen determination.
[0385] Although based on EIA capture, the PAM4 antibody appears to
bind to the same antigenic protein as the 45M1 anti-MUC5AC MAb, it
is noted that MUC5AC is not specific to pancreas cancer and it is
found in a number of normal tissues (other than the gastric mucosa
with which PAM4 is reactive). For example, MUC5AC is found in
normal lung, colon and other tissues. PAM4 antibody does not bind
to normal lung tissues, except as indicated above in few samples
and to a limited or minimal amount.
[0386] With respect to the effects of DTT and periodate, it is
probable that the peptide core disulfide bridges are identical no
matter what tissue produces the protein. A specific amino acid
sequence should fold in a specific manner, independent of the
tissue source. However, glycosylation patterns may differ dependent
upon tissue source.
Example 11
Phage Display Peptide Binding of PAM4 Antibody
[0387] PAM4 antibody binding was examined with two different phage
display peptide libraries. The first was a linear peptide library
consisting of 12 amino acid sequences and the second was a cyclic
peptide consisting of 7 amino acid sequences cyclized by a
disulfide bridge. We panned the individual libraries alternately
against hPAM4 and hLL2 (negative selection with anti-CD22 antibody)
for a combined total of 4 rounds, and then screened the phage
displayed peptide for reactivity with both hPAM4 and mPAM4 with
little to no reactivity against hLL2. Phage binding in a
non-specific manner (i.e., binding to epratuzumab [hLL2]) were
discarded.
[0388] For the linear phage-displayed peptide, the sequence
WTWNITKAYPLP (SEQ ID NO:7) was identified 30 times (in 35 sequenced
phage), each of which were shown to have reactivity with PAM4
antibodies. A mutational analysis was conducted in which a library
based on this sequence and having 7.5% degeneracy at each position,
was constructed, panned and screened as before. Variability was
noted in the 19 obtained peptide sequences that were positive for
PAM4 binding with 7 being identical to the parental sequence, 5
having the sequence WTWNITKEYPQP (SEQ ID NO:65) and the rest being
uniquely present. Table 11 shows the results of this mutational
analysis. The upper row lists the sequences identified and the
lower row lists the frequency with which each of the amino acids
was identified in that position. The parent sequence is most
frequent (bold) with the next highest variation a substitution of E
for A at position 8 and a substitution of Q for L at position 11.
It does not appear that these substitutions had any great effect
upon immunoreactivity.
TABLE-US-00017 TABLE 11 Phage Display Amino Acid Sequence (SEQ ID
NO: 116) Variation with Linear Peptide Binding to PAM4 Antibody A K
E L R T Q T R I N T N F Y P M W T W D I R G C T R C P number of 19
19 19 18 19 17 14 10 18 17 11 19 occurrences 1 2 1 5 1 2 5 (out of
19 2 1 1 sequences 1 1 1 analyzed) 1 1 1 1
[0389] Results with the phage displayed cyclic library were
significantly different from the linear library (Table 12). The
sequence ACPEWWGTTC (SEQ ID NO:66) was present in 33 of 35 peptide
sequences examined. Analysis of the cyclic library presented the
following results (positions with an asterisk were invariant and
not subject to selective pressure in the library).
TABLE-US-00018 TABLE 12 Phage Display Amino Acid Sequence (SEQ ID
NO: 117) Variation with Linear Peptide Binding to PAM4 Antibody T M
T S P G G Q A C Y E W W S S P C number of * * 33 35 35 35 34 29 28
* occurrences 2 1 5 4 (out of 19 1 1 sequences 1 analyzed) 1
[0390] The two cysteines (at positions 2 and 10) formed a disulfide
bridge. Substitution of T at position 9 with any amino acid greatly
affected immunoreactivity. The sequence GTTGTTC (SEQ ID NO:67) is
present within the MUC5AC protein towards the amino terminus as
compared to the cyclic peptide sequence shown above, which shows
homology at the C-terminal end of the consensus peptide sequence.
However, the cyclic peptide only showed approximately 10% of the
immunoreactivity of the linear sequence with the PAM4 antibody.
Both linear and cyclic consensus sequences are associated with a
cysteine, which may or may not relate to the effect of DTT on
MUC5AC immunoreactivity.
[0391] The results reported herein indicate that the PAM4 epitope
is dependent upon a specific conformation which may be produced by
disulfide bridges, as well as a specific glycosylation pattern.
Example 12
Immunohistology of Pancreatic Cancer in a Pancreatitis Specimen
[0392] Several pathologic conditions predispose patients to the
development of pancreatic carcinoma, such as pancreatitis,
diabetes, smoking and others. Within this pre-selected group of
patients, screening measures are particularly important for the
early detection of pancreatic neoplasia. We examined 9 specimens of
chronic pancreatitis tissue from patients having primary diagnosis
of this disease. We employed an anti-CD74 MAb, LL1, as an indicator
of inflammatory infiltrate, and MAb-MA5 as a positive control for
pancreatic ductal and acinar cells. Whereas the two control MAbs
provided immunohistologic evidence consistent with pancreatitis, in
no instance did PAM4 react with inflamed pancreatic tissue.
However, in one case, a moderately differentiated pancreatic
adenocarcinoma was also present within the tissue specimen. PAM4
gave an intense stain of the neoplastic cells within this tumor. In
a second case, while the inflamed tissue was negative with PAM4, a
small PanIN precursor lesion was identified that was labeled with
PAM4. Labeling of the PanIN within this latter specimen is
consistent with early detection of pancreatic neoplasia in a
patient diagnosed with a non-malignant disease. These results show
that detection and/or diagnosis using the PAM4 antibody may be
performed with high sensitivity and selectivity for pancreatic
neoplasia against a background of benign pancreatic tissues.
Example 13
Therapy of a Patient with Inoperable and Metastatic Pancreatic
Carcinoma
[0393] Patient 118-001, CWG, is a 63-year-old man with Stage-IV
pancreatic adenocarcinoma with multiple liver metastases, diagnosed
in November of 2007. He agreed to undertake combined
radioimmunotherapy and gemcitabine chemotherapy as a first
treatment strategy, and was then given a first therapy cycle of 6.5
mCi/m.sup.2 of .sup.90Y-hPAM4, combined with 200 mg/m.sup.2
gemcitabine, whereby the gemcitabine was given once weekly on weeks
1-4 and .sup.90Y-hPAM4 was given once-weekly on weeks 2-4 (3
doses). Two months later, the same therapy cycle was repeated,
because no major toxicities were noted after the first cycle.
Already 4 weeks after the first therapy cycle, CT evidence of a
reduction in the diameters of the primary tumor and 2 of the 3
liver metastases surprisingly was noted, and this was consistent
with significant decreases in the SUV values of FDG-PET scans, with
3 of the 4 tumors returning to normal background SUV levels at this
time (FIG. 13 and FIG. 14). The patient's pre-therapy CA-19.9 level
of 1,297 dropped to a low level of 77, further supportive of the
therapy being effective. Table 13 shows the effects of combined
radioimmunotherapy with .sup.90Y-hPAM4 and gemcitabine chemotherapy
in this patient. It was surprising and unexpected that such low
doses of the radionuclide conjugated to the antibody combined with
such low, nontoxic, doses of gemcitabine showed such antitumor
activity even after only a single course of this therapy.
TABLE-US-00019 TABLE 13 Effects of Combined Radioimmunotherapy with
.sup.90Y-hPAM4 and Gemcitabine Chemotherapy in Metastatic
Pancreatic Carcinoma Baseline Longest 4 wk post-Tx Baseline
Diameter Longest PET 4 wk post-Tx Tumor Location (cm) Diameter (cm)
(SUV) PET (SUV) Pancreatic tail 4.5 4.3 9.2 4.2 (primary) L hepatic
met 1.9 1.9 4.1 Background R post hepatic 1.7 1.6 3.7 Background
met R central hepatic 1.9 1.2 3.2 Background met
Example 14
Therapy of a Patient with Inoperable Metastatic Pancreatic
Carcinoma
[0394] A 56-year-old male with extensive, inoperable adenocarcinoma
of the pancreas, with several liver metastases ranging from 1 to 4
cm in diameter, substantial weight loss (30 lbs of weight or more),
mild jaundice, lethargy and weakness, as well as abdominal pains
requiring medication, is given 4 weekly infusions of gemcitabine at
doses of 200 mg/m.sup.2 each. On the last three gemcitabine
infusions, .sup.90Y-DOTA-hPAM4 radiolabeled humanized antibody is
administered at a dose of 10 mCi/m.sup.2 of .sup.90Y and 20 mg
antibody protein, in a two-hour i.v. infusion. Two weeks later, the
patient is given a course of gemcitabine chemotherapy consisting of
3 weekly doses of 600 mg/m.sup.2 by i.v. infusion. The patient is
then evaluated 4 weeks later, and has a mild leukopenia (grade-2),
no other major blood or enzyme changes over baseline, but shows an
improvement in the blood CA19.9 titer from 5,700 to 1,200 and a
decrease in jaundice, with an overall subjective improvement. This
follows 3 weeks later with a repeat of the cycle of lower-dose
gemcitabine (weekly.times.4), with 3 doses of .sup.90Y-DOTA-hPAM4.
Four weeks later, the patient is reevaluated, and the CT and PET
scans confirm an approximately 40% reduction of total tumor mass
(primary cancer and metastases), with a further reduction of the
CA19.9 titer to 870. The patient regains appetite and activity, and
is able to return to more usual daily activities without the need
for pain medication. He gains 12 lbs after beginning this
experimental therapy. A repeat of the scans and blood values
indicates that this response is maintained 6 weeks later.
Example 15
Preparation of DNL.TM. Constructs for Pretargeting
[0395] DDD and AD Fusion Proteins
[0396] The DNL.TM. technique can be used to make dimers, trimers,
tetramers, hexamers, etc. comprising virtually any antibodies or
fragments thereof or other effector moieties. For certain preferred
embodiments, IgG antibodies or Fab antibody fragments may be
produced as fusion proteins containing either a dimerization and
docking domain (DDD) or anchoring domain (AD) sequence. Although in
preferred embodiments the DDD and AD moieties are produced as
fusion proteins, the skilled artisan will realize that other
methods of conjugation, such as chemical cross-linking or click
chemistry, may be utilized within the scope of the claimed methods
and compositions.
[0397] Bispecific antibodies may be formed by combining a Fab-DDD
fusion protein of a first antibody with a Fab-AD fusion protein of
a second antibody. Alternatively, constructs may be made that
combine IgG-AD fusion proteins with Fab-DDD fusion proteins. The
technique is not limiting and any protein or peptide of use may be
produced as an AD or DDD fusion protein for incorporation into a
DNL.TM. construct. Where chemical cross-linking is utilized, the AD
and DDD conjugates are not limited to proteins or peptides and may
comprise any molecule that may be cross-linked to an AD or DDD
sequence using any cross-linking technique known in the art. In
certain exemplary embodiments, a polyethylene glycol (PEG) or other
polymeric moiety may be incorporated into a DNL.TM. construct, as
described in further detail below.
[0398] For pretargeting applications, an antibody or fragment
containing a binding site for an antigen associated with a diseased
tissue, such as a tumor-associated antigen (TAA), may be combined
with a second antibody or fragment that binds a hapten on a
targetable construct, to which a therapeutic and/or diagnostic
agent is attached. The DNL.TM.-based bispecific antibody is
administered to a subject, circulating antibody is allowed to clear
from the blood and localize to target tissue, and the conjugated
targetable construct is added and binds to the localized antibody
for diagnosis or therapy.
[0399] Independent transgenic cell lines may be developed for each
Fab or IgG fusion protein. Once produced, the modules can be
purified if desired or maintained in the cell culture supernatant
fluid. Following production, any DDD-fusion protein module can be
combined with any AD-fusion protein module to generate a bispecific
DNL.TM. construct. For different types of constructs, different AD
or DDD sequences may be utilized. Exemplary DDD and AD sequences
are provided below.
TABLE-US-00020 DDD1: (SEQ ID NO: 68)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2: (SEQ ID NO: 69)
CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1: (SEQ ID NO: 70)
QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 71) CGQIEYLAKQIVDNAIQQAGC
[0400] The skilled artisan will realize that DDD1 and DDD2 comprise
the DDD sequence of the human RII.alpha. form of protein kinase A.
However, in alternative embodiments, the DDD and AD moieties may be
based on the DDD sequence of the human RI.alpha. form of protein
kinase A and a corresponding AKAP sequence, as exemplified in DDD3,
DDD3C and AD3 below.
TABLE-US-00021 DDD3 (SEQ ID NO: 72)
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK DDD3C (SEQ ID
NO: 73) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLE KEEAK
AD3 (SEQ ID NO: 74) CGFEELAWKIAKMIWSDVFQQGC
[0401] Expression Vectors
[0402] The plasmid vector pdHL2 has been used to produce a number
of antibodies and antibody-based constructs. See Gillies et al., J
Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila)
(1997), 80:2660-6. The di-cistronic mammalian expression vector
directs the synthesis of the heavy and light chains of IgG. The
vector sequences are mostly identical for many different IgG-pdHL2
constructs, with the only differences existing in the variable
domain (V.sub.H and V.sub.L) sequences. Using molecular biology
tools known to those skilled in the art, these IgG expression
vectors can be converted into Fab-DDD or Fab-AD expression vectors.
To generate Fab-DDD expression vectors, the coding sequences for
the hinge, CH2 and CH3 domains of the heavy chain are replaced with
a sequence encoding the first 4 residues of the hinge, a 14 residue
Gly-Ser linker and the first 44 residues of human RII.alpha.
(referred to as DDD1). To generate Fab-AD expression vectors, the
sequences for the hinge, CH2 and CH3 domains of IgG are replaced
with a sequence encoding the first 4 residues of the hinge, a 15
residue Gly-Ser linker and a 17 residue synthetic AD called AKAP-IS
(referred to as AD1), which was generated using bioinformatics and
peptide array technology and shown to bind RII.alpha. dimers with a
very high affinity (0.4 nM). See Alto, et al. Proc. Natl. Acad.
Sci., U.S.A (2003), 100:4445-50.
[0403] Two shuttle vectors were designed to facilitate the
conversion of IgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1
expression vectors, as described below.
Preparation of CH1
[0404] The CH1 domain was amplified by PCR using the pdHL2 plasmid
vector as a template. The left PCR primer consisted of the upstream
(5') end of the CH1 domain and a SacII restriction endonuclease
site, which is 5' of the CH1 coding sequence. The right primer
consisted of the sequence coding for the first 4 residues of the
hinge (PKSC (SEQ ID NO: 118)) followed by four glycines and a
serine, with the final two codons (GS) comprising a Bam HI
restriction site. The 410 bp PCR amplimer was cloned into the
PGEMT.RTM. PCR cloning vector (PROMEGA.RTM., Inc.) and clones were
screened for inserts in the T7 (5') orientation.
[0405] A duplex oligonucleotide, designated (G.sub.4S).sub.2DDD1
(`(G.sub.4S).sub.2` disclosed as SEQ ID NO: 119), was synthesized
by Sigma GENOSYS.RTM. (Haverhill, UK) to code for the amino acid
sequence of DDD1 preceded by 11 residues of the linker peptide,
with the first two codons comprising a BamHI restriction site. A
stop codon and an EagI restriction site are appended to the 3'end.
The encoded polypeptide sequence is shown below.
TABLE-US-00022 (SEQ ID NO: 75)
GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA
[0406] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44
bottom, which overlap by 30 base pairs on their 3' ends, were
synthesized and combined to comprise the central 154 base pairs of
the 174 bp DDD1 sequence. The oligonucleotides were annealed and
subjected to a primer extension reaction with Taq polymerase.
Following primer extension, the duplex was amplified by PCR. The
amplimer was cloned into PGEMT.RTM. and screened for inserts in the
T7 (5') orientation.
[0407] A duplex oligonucleotide was synthesized to code for the
amino acid sequence of AD1 preceded by 11 residues of the linker
peptide with the first two codons comprising a BamHI restriction
site. A stop codon and an EagI restriction site are appended to the
3'end. The encoded polypeptide sequence is shown below.
TABLE-US-00023 (SEQ ID NO: 76) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA
[0408] Two complimentary overlapping oligonucleotides encoding the
above peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom,
were synthesized and annealed. The duplex was amplified by PCR. The
amplimer was cloned into the PGEMT.RTM. vector and screened for
inserts in the T7 (5') orientation.
Ligating DDD1 with CH1
[0409] A 190 bp fragment encoding the DDD1 sequence was excised
from PGEMT.RTM. with BamHI and NotI restriction enzymes and then
ligated into the same sites in CH1-PGEMT.RTM. to generate the
shuttle vector CH1-DDD1-PGEMT.RTM..
Ligating AD1 with CH1
[0410] A 110 bp fragment containing the AD1 sequence was excised
from PGEMT.RTM. with BamHI and NotI and then ligated into the same
sites in CH1-PGEMT.RTM. to generate the shuttle vector
CH1-AD1-PGEMT.RTM..
[0411] Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors
[0412] With this modular design either CH1-DDD1 or CH1-AD1 can be
incorporated into any IgG construct in the pdHL2 vector. The entire
heavy chain constant domain is replaced with one of the above
constructs by removing the SacII/EagI restriction fragment
(CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment
of CH1-DDD1 or CH1-AD1, which is excised from the respective pGemT
shuttle vector.
[0413] Construction of h679-Fd-AD1-pdHL2
[0414] h679-Fd-AD1-pdHL2 is an expression vector for production of
h679 Fab with AD1 coupled to the carboxyl terminal end of the CH1
domain of the Fd via a flexible Gly/Ser peptide spacer composed of
14 amino acid residues. A pdHL2-based vector containing the
variable domains of h679 was converted to h679-Fd-AD1-pdHL2 by
replacement of the SacII/EagI fragment with the CH1-AD1 fragment,
which was excised from the CH1-AD1-SV3 shuttle vector with SacII
and EagI.
[0415] Construction of C-DDD1-Fd-hMN-14-pdHL2
[0416] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for
production of a stable dimer that comprises two copies of a fusion
protein C-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at
the carboxyl terminus of CH1 via a flexible peptide spacer. The
plasmid vector hMN-14(I)-pdHL2, which has been used to produce
hMN-14 IgG, was converted to C-DDD1-Fd-hMN-14-pdHL2 by digestion
with SacII and EagI restriction endonucleases to remove the CH1-CH3
domains and insertion of the CH1-DDD1 fragment, which was excised
from the CH1-DDD1-SV3 shuttle vector with SacII and EagI.
[0417] The same technique has been utilized to produce plasmids for
Fab expression of a wide variety of known antibodies, such as hLL1,
hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others.
Generally, the antibody variable region coding sequences were
present in a pdHL2 expression vector and the expression vector was
converted for production of an AD- or DDD-fusion protein as
described above. The AD- and DDD-fusion proteins comprising a Fab
fragment of any of such antibodies may be combined, in an
approximate ratio of two DDD-fusion proteins per one AD-fusion
protein, to generate a trimeric DNL.TM. construct comprising two
Fab fragments of a first antibody and one Fab fragment of a second
antibody.
C-DDD2-Fd-hMN-14-pdHL2
[0418] C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for
production of C-DDD2-Fab-hMN-14, which possesses a dimerization and
docking domain sequence of DDD2 appended to the carboxyl terminus
of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide
linker. The fusion protein secreted is composed of two identical
copies of hMN-14 Fab held together by non-covalent interaction of
the DDD2 domains.
[0419] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides, which comprise the
coding sequence for part of the linker peptide and residues 1-13 of
DDD2, were made synthetically. The oligonucleotides were annealed
and phosphorylated with T4 PNK, resulting in overhangs on the 5'
and 3' ends that are compatible for ligation with DNA digested with
the restriction endonucleases BamHI and PstI, respectively.
[0420] The duplex DNA was ligated with the shuttle vector
CH1-DDD1-PGEMT.RTM., which was prepared by digestion with BamHI and
PstI, to generate the shuttle vector CH1-DDD2-PGEMT.RTM.. A 507 bp
fragment was excised from CH1-DDD2-PGEMT.RTM. with SacII and EagI
and ligated with the IgG expression vector hMN-14(I)-pdHL2, which
was prepared by digestion with SacII and EagI. The final expression
construct was designated C-DDD2-Fd-hMN-14-pdHL2. Similar techniques
have been utilized to generated DDD2-fusion proteins of the Fab
fragments of a number of different humanized antibodies.
h679-Fd-AD2-pdHL2
[0421] h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14
as A. h679-Fd-AD2-pdHL2 is an expression vector for the production
of h679-Fab-AD2, which possesses an anchoring domain sequence of
AD2 appended to the carboxyl terminal end of the CH1 domain via a
14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteine
residue preceding and another one following the anchor domain
sequence of AD1.
[0422] The expression vector was engineered as follows. Two
overlapping, complimentary oligonucleotides (AD2 Top and AD2
Bottom), which comprise the coding sequence for AD2 and part of the
linker sequence, were made synthetically. The oligonucleotides were
annealed and phosphorylated with T4 PNK, resulting in overhangs on
the 5' and 3' ends that are compatible for ligation with DNA
digested with the restriction endonucleases BamHI and SpeI,
respectively.
[0423] The duplex DNA was ligated into the shuttle vector
CH1-AD1-PGEMT.RTM., which was prepared by digestion with BamHI and
SpeI, to generate the shuttle vector CH1-AD2-PGEMT.RTM.. A 429 base
pair fragment containing CH1 and AD2 coding sequences was excised
from the shuttle vector with SacII and EagI restriction enzymes and
ligated into h679-pdHL2 vector that prepared by digestion with
those same enzymes. The final expression vector is
h679-Fd-AD2-pdHL2.
Example 16
Production of AD- and DDD-Linked Fab and IgG Fusion Proteins from
Multiple Antibodies
[0424] Using the techniques described in the preceding Example, the
IgG and Fab fusion proteins shown in Table 14 were constructed and
incorporated into DNL.TM. constructs. The fusion proteins retained
the antigen-binding characteristics of the parent antibodies and
the DNL.TM. constructs exhibited the antigen-binding activities of
the incorporated antibodies or antibody fragments.
TABLE-US-00024 TABLE 14 Fusion proteins comprising IgG or Fab
Fusion Protein Binding Specificity C-AD1-Fab-h679 HSG
C-AD2-Fab-h679 HSG C-(AD).sub.2-Fab-h679 HSG C-AD2-Fab-h734
Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20
C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22
C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1
C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5
C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19
C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DR
C-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6
C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1
IGF-1R C-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5
Example 17
Sequence Variants for DNL.TM.
[0425] In certain preferred embodiments, the AD and DDD sequences
incorporated into the DNL.TM. construct comprise the amino acid
sequences of AD1, AD2, AD3, DDD1, DDD2, DDD3 or DDD3C as discussed
above. However, in alternative embodiments sequence variants of AD
and/or DDD moieties may be utilized in construction of the DNL.TM.
complexes. For example, there are only four variants of human PKA
DDD sequences, corresponding to the DDD moieties of PKA RI.alpha.,
RII.alpha., RI.beta. and RII.beta.. The RII.alpha. DDD sequence is
the basis of DDD1 and DDD2 disclosed above. The four human PKA DDD
sequences are shown below. The DDD sequence represents residues
1-44 of RII.alpha., 1-44 of RII.beta., 12-61 of RI.alpha. and 13-66
of RI.beta.. (Note that the sequence of DDD1 is modified slightly
from the human PKA RII.alpha. DDD moiety.)
TABLE-US-00025 PKA RI.alpha. (SEQ ID NO: 77)
SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RI.beta.
(SEQ ID NO: 78) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR
QILA PKA RII.alpha. (SEQ ID NO: 79)
SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RII.beta. (SEQ ID
NO: 80) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER
[0426] The structure-function relationships of the AD and DDD
domains have been the subject of investigation. (See, e.g.,
Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Carr et al.,
2001, J Biol Chem 276:17332-38; Alto et al., 2003, Proc Natl Acad
Sci USA 100:4445-50; Hundsrucker et al., 2006, Biochem J
396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Gold et
al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell
24:397-408, the entire text of each of which is incorporated herein
by reference.)
[0427] For example, Kinderman et al. (2006) examined the crystal
structure of the AD-DDD binding interaction and concluded that the
human DDD sequence contained a number of conserved amino acid
residues that were important in either dimer formation or AKAP
binding, underlined in SEQ ID NO:68 below. (See FIG. 1 of Kinderman
et al., 2006, incorporated herein by reference.) The skilled
artisan will realize that in designing sequence variants of the DDD
sequence, one would desirably avoid changing any of the underlined
residues, while conservative amino acid substitutions might be made
for residues that are less critical for dimerization and AKAP
binding.
TABLE-US-00026 (SEQ ID NO: 68)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0428] Alto et al. (2003) performed a bioinformatic analysis of the
AD sequence of various AKAP proteins to design an RII selective AD
sequence called AKAP-IS (SEQ ID NO:70), with a binding constant for
DDD of 0.4 nM. The AKAP-IS sequence was designed as a peptide
antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence
where substitutions tended to decrease binding to DDD are
underlined in SEQ ID NO:70. The skilled artisan will realize that
in designing sequence variants of the AD sequence, one would
desirably avoid changing any of the underlined residues, while
conservative amino acid substitutions might be made for residues
that are less critical for DDD binding.
TABLE-US-00027 AKAP-IS sequence (SEQ ID NO: 70)
QIEYLAKQIVDNAIQQA
[0429] Gold (2006) utilized crystallography and peptide screening
to develop a SuperAKAP-IS sequence (SEQ ID NO:81), exhibiting a
five order of magnitude higher selectivity for the RII isoform of
PKA compared with the RI isoform. Underlined residues indicate the
positions of amino acid substitutions, relative to the AKAP-IS
sequence, which increased binding to the DDD moiety of RII.alpha..
In this sequence, the N-terminal Q residue is numbered as residue
number 4 and the C-terminal A residue is residue number 20.
Residues where substitutions could be made to affect the affinity
for RII.alpha. were residues 8, 11, 15, 16, 18, 19 and 20 (Gold et
al., 2006). It is contemplated that in certain alternative
embodiments, the SuperAKAP-IS sequence may be substituted for the
AKAP-IS AD moiety sequence to prepare DNL.TM. constructs. Other
alternative sequences that might be substituted for the AKAP-IS AD
sequence are shown in SEQ ID NO:82-84. Substitutions relative to
the AKAP-IS sequence are underlined. It is anticipated that, as
with the AD2 sequence shown in SEQ ID NO:68, the AD moiety may also
include the additional N-terminal residues cysteine and glycine and
C-terminal residues glycine and cysteine.
TABLE-US-00028 SuperAKAP-IS (SEQ ID NO: 81) QIEYVAKQIVDYAIHQA
Alternative AKAP sequences (SEQ ID NO: 82) QIEYKAKQIVDHAIHQA (SEQ
ID NO: 83) QIEYHAKQIVDHAIHQA (SEQ ID NO: 84) QIEYVAKQIVDHAIHQA
[0430] FIG. 2 of Gold et al. disclosed additional DDD-binding
sequences from a variety of AKAP proteins, shown below.
TABLE-US-00029 RII-SPECIFIC AKAPS AKAP-KL (SEQ ID NO: 85)
PLEYQAGLLVQNAIQQAI AKAP79 (SEQ ID NO: 86) LLIETASSLVKNAIQLSI
AKAP-LBC (SEQ ID NO: 87) LIEEAASRIVDAVIEQVK RI-SPECIFIC AKAPS
AKAPCE (SEQ ID NO: 88) ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 89)
LEQVANQLADQIIKEAT PV38 (SEQ ID NO: 90) FEELAWKIAKMIWSDVF
DUAL-SPECIFICITY AKAPS AKAP7 (SEQ ID NO: 91) ELVRLSKRLVENAVLKAV
MAP2D (SEQ ID NO: 92) TAEEVSARIVQVVTAEAV DAKAP1 (SEQ ID NO: 93)
QIKQAAFQLISQVILEAT DAKAP2 (SEQ ID NO: 94) LAWKIAKMIVSDVMQQ
[0431] Stokka et al. (2006) also developed peptide competitors of
AKAP binding to PKA, shown in SEQ ID NO:95-97. The peptide
antagonists were designated as Ht31 (SEQ ID NO:95), RIAD (SEQ ID
NO:96) and PV-38 (SEQ ID NO:97). The Ht-31 peptide exhibited a
greater affinity for the RII isoform of PKA, while the RIAD and
PV-38 showed higher affinity for RI.
TABLE-US-00030 Ht31 (SEQ ID NO: 95) DLIEEAASRIVDAVIEQVKAAGAY RIAD
(SEQ ID NO: 96) LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 97)
FEELAWKIAKMIWSDVFQQC
[0432] Hundsrucker et al. (2006) developed still other peptide
competitors for AKAP binding to PKA, with a binding constant as low
as 0.4 nM to the DDD of the RII form of PKA. The sequences of
various AKAP antagonistic peptides are provided in Table 1 of
Hundsrucker et al., reproduced in Table 15 below. AKAPIS represents
a synthetic RII subunit-binding peptide. All other peptides are
derived from the RII-binding domains of the indicated AKAPs.
TABLE-US-00031 TABLE 15 AKAP Peptide sequences Peptide Sequence
AKAPIS QIEYLAKQIVDNAIQQA (SEQ ID NO: 70) AKAPIS-P QIEYLAKQIPDNAIQQA
(SEQ ID NO: 98) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 99)
Ht31-P KGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 100)
AKAP7.delta.-wt-pep PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 101)
AKAP7.delta.-L304T-pep PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 102)
AKAP7.delta.-L308D-pep PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 103)
AKAP7.delta.-P-pep PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 104)
AKAP7.delta.-PP-pep PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 105)
AKAP7.delta.-L314E-pep PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 106)
AKAP1-pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 107) AKAP2-pep
LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 108) AKAP5-pep
QYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 109) AKAP9-pep
LEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 110) AKAP10-pep
NTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 111) AKAP11-pep
VNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 112) AKAP12-pep
NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 113) AKAP14-pep
TQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 114) Rab32-pep
ETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 115)
[0433] Residues that were highly conserved among the AD domains of
different AKAP proteins are indicated below by underlining with
reference to the AKAP IS sequence (SEQ ID NO:70). The residues are
the same as observed by Alto et al. (2003), with the addition of
the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al.
(2006), incorporated herein by reference.) The sequences of peptide
antagonists with particularly high affinities for the RII DDD
sequence were those of AKAP-IS, AKAP7.delta.-wt-pep,
AKAP7.delta.-L304T-pep and AKAP7.delta.-L308D-pep.
TABLE-US-00032 AKAP-IS (SEQ ID NO: 70) QIEYLAKQIVDNAIQQA
[0434] Carr et al. (2001) examined the degree of sequence homology
between different AKAP-binding DDD sequences from human and
non-human proteins and identified residues in the DDD sequences
that appeared to be the most highly conserved among different DDD
moieties. These are indicated below by underlining with reference
to the human PKA RII.alpha. DDD sequence of SEQ ID NO:68. Residues
that were particularly conserved are further indicated by italics.
The residues overlap with, but are not identical to those suggested
by Kinderman et al. (2006) to be important for binding to AKAP
proteins. The skilled artisan will realize that in designing
sequence variants of DDD, it would be most preferred to avoid
changing the most conserved residues (italicized), and it would be
preferred to also avoid changing the conserved residues
(underlined), while conservative amino acid substitutions may be
considered for residues that are neither underlined nor
italicized.
TABLE-US-00033 (SEQ ID NO: 68)
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA
[0435] The skilled artisan will realize that these and other amino
acid substitutions in the antibody moiety or linker portions of the
DNL.TM. constructs may be utilized to enhance the therapeutic
and/or pharmacokinetic properties of the resulting DNL.TM.
constructs.
Example 18
Generation of TF2 DNL.TM. Pretargeting Construct
[0436] A trimeric DNL.TM. construct designated TF2 was obtained by
reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2
was generated with >90% yield as follows. Protein L-purified
C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60 mg) at a
1.4:1 molar ratio. The total protein concentration was 1.5 mg/ml in
PBS containing 1 mM EDTA. Subsequent steps involved TCEP reduction,
HIC chromatography, DMSO oxidation, and IMP 291 affinity
chromatography. Before the addition of TCEP, SE-HPLC did not show
any evidence of a.sub.2b formation. Addition of 5 mM TCEP rapidly
resulted in the formation of a.sub.2b complex consistent with a 157
kDa protein expected for the binary structure. TF2 was purified to
near homogeneity by IMP 291 affinity chromatography (not shown).
IMP 291 is a synthetic peptide containing the HSG hapten to which
the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res
11:7122s-29s). SE-HPLC analysis of the IMP 291 unbound fraction
demonstrated the removal of a.sub.4, a.sub.2 and free kappa chains
from the product (not shown).
[0437] Non-reducing SDS-PAGE analysis demonstrated that the
majority of TF2 exists as a large, covalent structure with a
relative mobility near that of IgG (not shown). The additional
bands suggest that disulfide formation is incomplete under the
experimental conditions (not shown). Reducing SDS-PAGE shows that
any additional bands apparent in the non-reducing gel are
product-related (not shown), as only bands representing the
constituent polypeptides of TF2 were evident (not shown). However,
the relative mobilities of each of the four polypeptides were too
close to be resolved. MALDI-TOF mass spectrometry (not shown)
revealed a single peak of 156,434 Da, which is within 99.5% of the
calculated mass (157,319 Da) of TF2.
[0438] The functionality of TF2 was determined by BIACORE.RTM.
assay. TF2, C-DDD1-hMN-14+h679-AD1 (used as a control sample of
noncovalent a.sub.2b complex), or C-DDD2-hMN-14+h679-AD2 (used as a
control sample of unreduced a.sub.2 and b components) were diluted
to 1 g/ml (total protein) and passed over a sensorchip immobilized
with HSG. The response for TF2 was approximately two-fold that of
the two control samples, indicating that only the h679-Fab-AD
component in the control samples would bind to and remain on the
sensorchip. Subsequent injections of WI2 IgG, an anti-idiotype
antibody for hMN-14, demonstrated that only TF2 had a
DDD-Fab-hMN-14 component that was tightly associated with
h679-Fab-AD as indicated by an additional signal response. The
additional increase of response units resulting from the binding of
WI2 to TF2 immobilized on the sensorchip corresponded to two fully
functional binding sites, each contributed by one subunit of
C-DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind
two Fab fragments of W12 (not shown).
Example 19
Production of TF10 Bispecific Antibody for Pretargeting
[0439] A similar protocol was used to generate a trimeric TF10
DNL.TM. construct, comprising two copies of a C-DDD2-Fab-hPAM4 and
one copy of C-AD2-Fab-679. The cancer-targeting antibody component
in TF10 was derived from hPAM4, a humanized anti-MUC5AC MAb that
has been studied in detail as a radiolabeled MAb (e.g., Gold et
al., Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding
component was derived from h679, a humanized
anti-histaminyl-succinyl-glycine (HSG) MAb. The TF10 bispecific
([hPAM4].sub.2.times.h679) antibody was produced using the method
disclosed for production of the (anti CEA).sub.2.times.anti HSG
bsAb TF2, as described above. The TF10 construct bears two
humanized PAM4 Fabs and one humanized 679 Fab.
[0440] The two fusion proteins (hPAM4-DDD and h679-AD2) were
expressed independently in stably transfected myeloma cells. The
tissue culture supernatant fluids were combined, resulting in a
two-fold molar excess of hPAM4-DDD. The reaction mixture was
incubated at room temperature for 24 hours under mild reducing
conditions using 1 mM reduced glutathione. Following reduction, the
DNL.TM. reaction was completed by mild oxidation using 2 mM
oxidized glutathione. TF10 was isolated by affinity chromatography
using IMP 291-affigel resin, which binds with high specificity to
the h679 Fab.
[0441] A full tissue histology and blood cell binding panel has
been examined for hPAM4 IgG and for an anti-CEA.times.anti-HSG
bsMAb that is entering clinical trials. hPAM4 binding was
restricted to very weak binding to the urinary bladder and stomach
in 1/3 specimens (no binding was seen in vivo), and no binding to
normal tissues was attributed to the anti-CEA.times.anti-HSG bsMAb.
Furthermore, in vitro studies against cell lines bearing the H1 and
H2 histamine receptors showed no antagonistic or agonistic activity
with the IMP 288 di-HSG peptide, and animal studies in 2 different
species showed no pharmacologic activity of the peptide related to
the histamine component at doses 20,000 times higher than that used
for imaging. Thus, the HSG-histamine derivative does not have
pharmacologic activity.
Example 20
Imaging Studies Using Pretargeting with TF10 Bispecific Antibody
and .sup.111In-Labeled Peptides
[0442] The following study demonstrates the feasibility of in vivo
imaging using the pretargeting technique with bispecific antibodies
incorporating hPAM4 and labeled peptides. The TF10 bispecific
antibody, comprising two copies of a C-DDD2-Fab-hPAM4 and one copy
of C-AD2-Fab-679, was prepared as described in the preceding
Example. Nude mice bearing 0.2 to 0.3 g human pancreatic cancer
xenografts were imaged, using pretargeting with TF10 and
.sup.111In-IMP-288 peptide. The results, shown in FIG. 15A and FIG.
15B, demonstrate how clearly delineated tumors can be detected in
animal models using a bsMAb pretargeting method, with
.sup.111In-labeled di-HSG peptide, IMP-288. The six animals in the
top of FIG. 15A and FIG. 15B received 2 different doses of TF10
(10:1 and 20:1 mole ratio to the moles of peptide given), and the
next day they were given an .sup.111In-labeled diHSG peptide (IMP
288). The 3 other animals on the bottom of FIG. 15A and FIG. 15B
received only the .sup.111In-IMP-288 (no pretargeting). The images
shown in FIG. 15B were taken 3 h after the injection of the labeled
peptide and show clear localization of 0.2-0.3 g tumors in the
pretargeted animals, with no localization in the animals given the
.sup.111In-peptide alone. Tumor uptake averaged 20-25% ID/g with
tumor/blood ratios exceeding 2000:1, tumor/liver ratios of 170:1,
and tumor/kidney ratios of 18/1.
Example 21
Production of Targeting Peptides for Use in Pretargeting and
.sup.18F Labeling
[0443] In a variety of embodiments, .sup.18F-labeled proteins or
peptides are prepared by a novel technique and used for diagnostic
and/or imaging studies, such as PET imaging. The novel technique
for .sup.18F labeling involves preparation of an .sup.18F-metal
complex, preferably an .sup.18F-aluminum complex, which is chelated
to a chelating moiety, such as DOTA, NOTA or NETA or derivatives
thereof. Chelating moieties may be attached to proteins, peptides
or any other molecule using conjugation techniques well known in
the art. In certain preferred embodiments, the .sup.18F--Al complex
is formed in solution first and then attached to a chelating moiety
that is already conjugated to a protein or peptide. However, in
alternative embodiments the aluminum may be first attached to the
chelating moiety and the .sup.18F added later.
[0444] Peptide Synthesis
[0445] Peptides were synthesized by solid phase peptide synthesis
using the Fmoc strategy. Groups were added to the side chains of
diamino amino acids by using Fmoc/Aloc protecting groups to allow
differential deprotection. The Aloc groups were removed by the
method of Dangles et. al. (J. Org. Chem. 1987, 52:4984-4993) except
that piperidine was added in a 1:1 ratio to the acetic acid used.
The unsymmetrical tetra-t-butyl DTPA was made as described in
McBride et al. (US Patent Application Pub. No. 2005/0002945, the
Examples section of which is incorporated herein by reference).
[0446] The tri-t-butyl DOTA, symmetrical tetra-t-butyl DTPA,
ITC-benzyl DTPA, p-SCN-Bn-NOTA and TACN were obtained from
MACROCYCLICS.RTM. (Dallas, Tex.). The DiBocTACN, NODA-GA(tBu).sub.3
and the NO2AtBu were purchased from CheMatech (Dijon, France). The
Aloc/Fmoc Lysine and Dap (diaminopropionic acid derivatives (also
Dpr)) were obtained from CREOSALUS.RTM. (Louisville, Ky.) or
BACHEM.RTM. (Torrance, Calif.). The Sieber Amide resin was obtained
from NOVABIOCHEM.RTM. (San Diego, Calif.). The remaining Fmoc amino
acids were obtained from CREOSALUS.RTM., BACHEM.RTM., PEPTECH.RTM.
(Burlington, Mass.), EMD BIOSCIENCES.RTM. (San Diego, Calif.), CHEM
IMPEX.RTM. (Wood Dale, Ill.) or NOVABIOCHEM.RTM.. The aluminum
chloride hexahydrate was purchased from SIGMA-ALDRICH.RTM.
(Milwaukee, Wis.). The remaining solvents and reagents were
purchased from FISHER SCIENTIFIC.RTM. (Pittsburgh, Pa.) or
SIGMA-ALDRICH.RTM. (Milwaukee, Wis.). .sup.18F was supplied by IBA
MOLECULAR.RTM. (Somerset, N.J.)
[0447] .sup.18F-Labeling of IMP 272
[0448] The first peptide that was prepared and .sup.18F-labeled was
IMP 272: DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH.sub.2 MH.sup.+
1512
[0449] IMP 272 was synthesized as described (U.S. Pat. No.
7,534,431, the Examples section of which is incorporated herein by
reference).
[0450] Acetate buffer solution--Acetic acid, 1.509 g was diluted in
.about.160 mL water and the pH was adjusted by the addition of 1 M
NaOH then diluted to 250 mL to make a 0.1 M solution at pH
4.03.
[0451] Aluminum acetate buffer solution--A solution of aluminum was
prepared by dissolving 0.1028 g of AlCl.sub.3 hexahydrate in 42.6
mL DI water. A 4 mL aliquot of the aluminum solution was mixed with
16 mL of a 0.1 M NaOAc solution at pH 4 to provide a 2 mM Al stock
solution.
[0452] IMP 272 acetate buffer solution--Peptide, 0.0011 g,
7.28.times.10.sup.-7 mol IMP 272 was dissolved in 364 .mu.L of the
0.1 M pH 4 acetate buffer solution to obtain a 2 mM stock solution
of the peptide.
[0453] F-18 Labeling of IMP 272--A 3 .mu.L aliquot of the aluminum
stock solution was placed in a REACTI-VIAL.TM. and mixed with 50
.mu.L .sup.18F (as received) and 3 .mu.L of the IMP 272 solution.
The solution was heated in a heating block at 110.degree. C. for 15
min and analyzed by reverse phase HPLC. HPLC analysis (not shown)
showed 93% free .sup.18F and 7% bound to the peptide. An additional
10 .mu.L of the IMP 272 solution was added to the reaction and it
was heated again and analyzed by reverse phase HPLC (not shown).
The HPLC trace showed 8% .sup.18F at the void volume and 92% of the
activity attached to the peptide. The remainder of the peptide
solution was incubated at room temperature with 150 .mu.L PBS for
.about.1 hr and then examined by reverse phase HPLC. The HPLC (not
shown) showed 58% .sup.18F unbound and 42% still attached to the
peptide. The data indicate that .sup.18F--Al-DTPA complex may be
unstable when mixed with phosphate.
[0454] The labeled peptide was purified by applying the labeled
peptide solution onto a 1 cc (30 mg) WATERS.RTM. HLB column (Part
#186001879) and washing with 300 .mu.L water to remove unbound
F-18. The peptide was eluted by washing the column with 2.times.100
.mu.L 1:1 EtOH/H.sub.2O. The purified peptide was incubated in
water at 25.degree. C. and analyzed by reverse phase HPLC (not
shown). The HPLC analysis showed that the .sup.18F-labeled IMP 272
was not stable in water. After 40 min incubation in water about 17%
of the .sup.18F was released from the peptide, while 83% was
retained (not shown).
[0455] The peptide (16 .mu.L 2 mM IMP 272, 48 .mu.g) was labeled
with .sup.18F and analyzed for antibody binding by size exclusion
HPLC. The size exclusion HPLC showed that the peptide bound
hMN-14.times.679 but did not bind to the irrelevant bispecific
antibody hMN-14.times.734 (not shown).
[0456] IMP 272 .sup.18F Labeling with Other Metals
[0457] A .about.3 .mu.L aliquot of the metal stock solution
(6.times.10.sup.-9 mol) was placed in a polypropylene cone vial and
mixed with 75 .mu.L .sup.18F (as received), incubated at room
temperature for .about.2 min and then mixed with 20 .mu.L of a 2 mM
(4.times.10.sup.-8 mol) IMP 272 solution in 0.1 M NaOAc pH 4
buffer. The solution was heated in a heating block at 100.degree.
C. for 15 min and analyzed by reverse phase HPLC. IMP 272 was
labeled with indium (24%), gallium (36%), zirconium (15%), lutetium
(37%) and yttrium (2%) (not shown). These results demonstrate that
the .sup.18F metal labeling technique is not limited to an aluminum
ligand, but can also utilize other metals as well. With different
metal ligands, different chelating moieties may be utilized to
optimize binding of an F-18-metal conjugate.
[0458] Production and Use of a Serum-Stable .sup.18F-Labeled
Peptide IMP 449
[0459] The peptide, IMP 448
D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH.sub.2 MH.sup.+ 1009 was made
on Sieber Amide resin by adding the following amino acids to the
resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc
was cleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH,
the Aloc was cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to
make the desired peptide. The peptide was then cleaved from the
resin and purified by HPLC to produce IMP 448, which was then
coupled to ITC-benzyl NOTA. The peptide, IMP 448, 0.0757 g
(7.5.times.10.sup.-5 mol) was mixed with 0.0509 g
(9.09.times.10.sup.-5 mol) ITC benzyl NOTA and dissolved in 1 mL
water. Potassium carbonate anhydrous (0.2171 g) was then slowly
added to the stirred peptide/NOTA solution. The reaction solution
was pH 10.6 after the addition of all the carbonate. The reaction
was allowed to stir at room temperature overnight. The reaction was
carefully quenched with 1 M HCl after 14 hr and purified by HPLC to
obtain 48 mg of IMP 449.
[0460] .sup.18F Labeling of IMP 449
[0461] The peptide IMP 449 (0.002 g, 1.37.times.10.sup.-6 mol) was
dissolved in 686 .mu.L (2 mM peptide solution) 0.1 M NaOAc pH 4.02.
Three microliters of a 2 mM solution of Al in a pH 4 acetate buffer
was mixed with 15 .mu.L, 1.3 mCi of .sup.18F. The solution was then
mixed with 20 .mu.L of the 2 mM IMP 449 solution and heated at
105.degree. C. for 15 min. Reverse Phase HPLC analysis showed 35%
(t.sub.R.about.10 min) of the activity was attached to the peptide
and 65% of the activity was eluted at the void volume of the column
(3.1 min, not shown) indicating that the majority of activity was
not associated with the peptide. The crude labeled mixture (5
.mu.L) was mixed with pooled human serum and incubated at
37.degree. C. An aliquot was removed after 15 min and analyzed by
HPLC. The HPLC showed 9.8% of the activity was still attached to
the peptide (down from 35%). Another aliquot was removed after 1 hr
and analyzed by HPLC. The HPLC showed 7.6% of the activity was
still attached to the peptide (down from 35%), which was
essentially the same as the 15 min trace (data not shown).
[0462] High Dose .sup.18F Labeling
[0463] Further studies with purified IMP 449 demonstrated that the
.sup.18F-labeled peptide was highly stable (91%, not shown) in
human serum at 37.degree. C. for at least one hour and was
partially stable (76%, not shown) in human serum at 37.degree. C.
for at least four hours. Additional studies were performed in which
the IMP 449 was prepared in the presence of ascorbic acid as a
stabilizing agent. In those studies (not shown), the
metal-.sup.18F-peptide complex showed no detectable decomposition
in serum after 4 hr at 37.degree. C. The mouse urine 30 min after
injection of .sup.18F-labeled peptide was found to contain .sup.18F
bound to the peptide (not shown). These results demonstrate that
the .sup.18F-labeled peptides disclosed herein exhibit sufficient
stability under approximated in vivo conditions to be used for
.sup.18F imaging studies.
[0464] For studies in the absence of ascorbic acid,
.sup.18F.about.21 mCi in .about.400 .mu.L of water was mixed with 9
.mu.L of 2 mM AlCl.sub.3 in 0.1 M pH 4 NaOAc. The peptide, IMP 449,
60 .mu.L (0.01 M, 6.times.10.sup.-7 mol in 0.5 NaOH pH 4.13) was
added and the solution was heated to 110.degree. C. for 15 min. The
crude labeled peptide was then purified by placing the reaction
solution in the barrel of a 1 cc WATERS.RTM. HLB column and eluting
with water to remove unbound .sup.18F followed by 1:1 EtOH/H.sub.2O
to elute the .sup.18F-labeled peptide. The crude reaction solution
was pulled through the column into a waste vial and the column was
washed with 3.times.1 mL fractions of water (18.97 mCi). The HLB
column was then placed on a new vial and eluted with 2.times.200
.mu.L 1:1 EtOH/H.sub.2O to collect the labeled peptide (1.83 mCi).
The column retained 0.1 mCi of activity after all of the elutions
were complete. An aliquot of the purified .sup.18F-labeled peptide
(20 .mu.L) was mixed with 200 .mu.L of pooled human serum and
heated at 37.degree. C. Aliquots were analyzed by reverse phase
HPLC. The results showed the relative stability of .sup.18F-labeled
purified IMP 449 at 37.degree. C. at time zero, one hour (91%
labeled peptide), two hours (77% labeled peptide) and four hours
(76% labeled peptide) of incubation in human serum (not shown). It
was also observed that .sup.18F-labeled IMP 449 was stable in TFA
solution, which is occasionally used during reverse phase HPLC
chromatography. There appears to be a general correlation between
stability in TFA and stability in human serum observed for the
exemplary .sup.18F-labeled molecules described herein. These
results demonstrate that .sup.18F-labeled peptide, produced
according to the methods disclosed herein, shows sufficient
stability in human serum to be successfully used for in vivo
labeling and imaging studies, for example using PET scanning to
detect labeled cells or tissues. Finally, since IMP 449 peptide
contains a thiourea linkage, which is sensitive to radiolysis,
several products are observed by RP-HPLC. However, when ascorbic
acid is added to the reaction mixture, the side products generated
were markedly reduced.
Example 22
In Vivo Studies with Pretargeting TF10 DNL.TM. Construct and
.sup.18F-Labeled Peptide
[0465] .sup.18F-labeled IMP 449 was prepared as follows. The
.sup.18F, 54.7 mCi in .about.0.5 mL was mixed with 3 .mu.L 2 mM Al
in 0.1 M NaOAc pH 4 buffer. After 3 min 10 .mu.L of 0.05 M IMP 449
in 0.5 M pH 4 NaOAc buffer was added and the reaction was heated in
a 96.degree. C. heating block for 15 min. The contents of the
reaction were removed with a syringe. The crude labeled peptide was
then purified by HPLC on a C.sub.18 column. The flow rate was 3
mL/min. Buffer A was 0.1% TFA in water and Buffer B was 90%
acetonitrile in water with 0.1% TFA. The gradient went from 100% A
to 75/25 A:B over 15 min. There was about 1 min difference in
retention time (t.sub.R) between the labeled peptide, which eluted
first and the unlabeled peptide. The HPLC eluent was collected in
0.5 min (mL) fractions. The labeled peptide had a t.sub.R between 6
to 9 min depending on the column used. The HPLC purified peptide
sample was further processed by diluting the fractions of interest
two fold in water and placing the solution in the barrel of a 1 cc
WATERS.RTM. HLB column. The cartridge was eluted with 3.times.1 mL
water to remove acetonitrile and TFA followed by 400 .mu.L 1:1
EtOH/H.sub.2O to elute the .sup.18F-labeled peptide. The purified
[Al.sup.18F] IMP 449 eluted as a single peak on an analytical HPLC
C.sub.18 column (not shown).
[0466] TACONIC.RTM. nude mice bearing the four slow-growing sc
CaPan1 xenografts were used for in vivo studies. Three of the mice
were injected with TF10 (162 .mu.g) followed with [Al.sup.18F] IMP
449 18 h later. TF10 is a humanized bispecific antibody of use for
tumor imaging studies, with divalent binding to the PAM-4 defined
tumor antigen and monovalent binding to HSG (see, e.g., Gold et
al., 2007, J. Clin. Oncol. 25(18S):4564). One mouse was injected
with peptide alone. All of the mice were necropsied at 1 h post
peptide injection. Tissues were counted immediately. Comparison of
mean distributions showed substantially higher levels of
.sup.18F-labeled peptide localized in the tumor than in any normal
tissues in the presence of tumor-targeting bispecific antibody
(data not shown).
[0467] Tissue uptake was similar in animals given the [Al.sup.18F]
IMP 449 alone or in a pretargeting setting (data not shown). Uptake
in the human pancreatic cancer xenograft, CaPan1, at 1 h was
increased 5-fold in the pretargeted animals as compared to the
peptide alone (4.6.+-.0.9% ID/g vs. 0.89% ID/g). Exceptional
tumor/nontumor ratios were achieved at this time (e.g., tumor/blood
and liver ratios were 23.4.+-.2.0 and 23.5.+-.2.8,
respectively).
[0468] The results demonstrate that .sup.18F labeled peptide used
in conjunction with a PAM4 containing antibody construct, such the
TF10 DNL.TM. construct, provide suitable targeting of the .sup.18F
label to perform in vivo imaging, such as PET imaging analysis.
Example 23
Further Imaging Studies with TF10
[0469] Summary
[0470] Preclinical and clinical studies have demonstrated the
application of radiolabeled mAb-PAM4 for nuclear imaging and
radioimmunotherapy of pancreatic carcinoma. We have examined herein
the ability of the TF10 construct to pretarget a radiolabeled
peptide for improved imaging and therapy. Biodistribution studies
and nuclear imaging of the radiolabeled TF10 and/or
TF10-pretargeted hapten-peptide (IMP-288) were conducted in nude
mice bearing CaPan1 human pancreatic cancer xenografts.
.sup.125I-TF10 cleared rapidly from the blood, with levels
decreasing to <1% injected dose per gram (ID/g) by 16 hours.
Tumor uptake was 3.47.+-.0.66% ID/g at this time point with no
accumulation in any normal tissue. To show the utility of the
pretargeting approach, .sup.111In-IMP-288 was administered 16 hours
after TF10. At 3 hours postadministration of radiolabeled peptide,
imaging showed intense uptake within the tumors and no evidence of
accretion in any normal tissue. No targeting was observed in
animals given only the .sup.111In-peptide. Tumor uptake of the
TF10-pretargeted .sup.111In-IMP-288 was 24.3.+-.1.7% ID/g, whereas
for .sup.111In-IMP-288 alone it was only 0.12.+-.0.002% ID/g at 16
hours. Tumor/blood ratios were significantly greater for the
pretargeting group (.about.1,000:1 at 3 hours) compared with
.sup.111In-PAM4-IgG (.about.5:1 at 24 hours; P<0.0003).
Radiation dose estimates suggested that TF10/.sup.90Y-peptide
pretargeting would provide a greater antitumor effect than
.sup.90Y-PAM4-IgG. Thus, the results support that TF10 pretargeting
may provide improved imaging for early detection, diagnosis, and
treatment of pancreatic cancer as compared with directly
radiolabeled PAM4-IgG. (Gold et al., Cancer Res 2008,
68(12):4819-26)
[0471] We have identified a unique biomarker present on mucin
expressed by >85% of invasive pancreatic adenocarcinomas,
including early stage I disease and the precursor lesions,
pancreatic intraepithelial neoplasia and intraductal papillary
mucinous neoplasia (Gold et al., Clin Cancer Res 2007, 13:7380-87).
The specific epitope, as detected by mAb-PAM4 (Gold et al., Int J
Cancer 1994, 57:204-10), is absent from normal and inflamed
pancreatic tissues, as well as most other malignant tissues. Thus,
detection of the epitope provides a high diagnostic likelihood for
the presence of pancreatic neoplasia. Early clinical studies using
.sup.131I- and .sup.99mTc-labeled, murine PAM4 IgG or Fab',
respectively, showed specific targeting in 8 of 10 patients with
invasive pancreatic adenocarcinoma (Mariani et al., Cancer Res
1995, 55:5911s-15s; Gold et al., Crit Rev Oncol Hematol 2001,
39:147-54). Of the two negative patients, one had a poorly
differentiated pancreatic carcinoma that did not express the
PAM4-epitope, whereas the other patient was later found to have
pancreatitis rather than a malignant lesion.
[0472] Accordingly, the high specificity of PAM4 for pancreatic
cancer is of use for the detection and diagnosis of early disease.
In addition to improved detection, .sup.90Y-PAM4 IgG was found to
be effective in treating large human pancreatic cancer xenografts
in nude mice (Cardillo et al., Clin Cancer Res 2001, 7:3186-92),
and when combined with gemcitabine, further improvements in
therapeutic response were observed (Gold et al., Clin Cancer Res
2004, 10:3552-61; Gold et al., Int J Cancer 2004, 109:618-26). A
Phase I therapy trial in patients who failed gemcitabine treatment
was recently completed, finding the maximum tolerated dose of
.sup.90Y-humanized PAM4 IgG to be 20 mCi/m.sup.2 (Gulec et al.,
Proc Amer Soc Clin Onc, 43rd Annual Meeting, J Clin Oncol 2007,
25(18S):636s). Although all patients showed disease progression at
or after week 8, initial shrinkage of tumor was observed in several
cases. Clinical studies are now underway to evaluate a fractionated
dosing regimen of .sup.90Y-hPAM4 IgG in combination with a
radiosensitizing dose of gemcitabine.
[0473] We report herein the development of a novel recombinant,
humanized bispecific monoclonal antibody (mAb), TF10, based on the
targeting specificity of PAM4 to pancreatic cancer. This construct
also binds to the unique synthetic hapten,
histamine-succinyl-glycine (HSG), which has been incorporated in a
number of small peptides that can be radiolabeled with a wide range
of radionuclides suitable for single-photon emission computed
tomography (SPECT) and positron emission tomography (PET) imaging,
as well as for therapeutic purposes (Karacay et al., Clin Cancer
Res 2005, 11:7879-85; Sharkey et al., Leukemia 2005, 19:1064-9;
Rossi et al., Proc Natl Acad Sci USA 2006, 103:6841-6; McBride et
al., J Nucl Med 2006, 47:1678-88). These studies illustrate the
potential of this new construct to target pancreatic adenocarcinoma
for imaging or therapeutic applications.
Methods and Materials
[0474] The TF2 and TF10 bispecific DNL.TM. constructs and the IMP
288 targeting peptide were prepared as described above. Sodium
iodide (.sup.125I) and indium chloride (.sup.111In) were obtained
from PERKIN-ELMER.RTM.. TF10 was routinely labeled with .sup.125I
by the iodogen method, with purification by use of size-exclusion
spin columns. Radiolabeling of DOTA-peptide and DOTA-PAM4-IgG with
.sup.111InCl was done as previously described (Rossi et al., Proc
Natl Acad Sci USA 2006, 103:6841-6; McBride et al., J Nucl Med
2006, 47:1678-88). Purity of the radiolabeled products was examined
by size-exclusion high-performance liquid chromatography with the
amount of free, unbound isotope determined by instant TLC.
[0475] For TF10 distribution studies, female athymic nude mice
.about.20 g (TACONIC.RTM. Farms), bearing s.c. CaPan1 human
pancreatic cancer xenografts, were injected with .sup.125I-TF10 (10
.mu.Ci; 40 .mu.g, 2.50.times.10.sup.-10 mol). At various time
points, groups of mice (n=5) were necropsied, with tumor and
nontumor tissues removed and counted in a gamma counter to
determine the percentage of injected dose per gram of tissue (%
ID/g), with these values used to calculate blood clearance rates
and tumor/nontumor ratios.
[0476] For pretargeting biodistribution studies, a bispecific
mAb/radiolabeled peptide molar ratio of 10:1 was used. For example,
a group of athymic nude mice bearing s.c. CaPan1 human pancreatic
cancer xenografts was administered TF10 (80 .mu.g,
5.07.times.10.sup.-10 mol), whereas a second group was left
untreated. At 16 h postinjection of TF10, .sup.111In-IMP-288
hapten-peptide (30 .mu.Ci, 5.07.times.10.sup.-11 mol) was
administered. Mice were necropsied at several time points, with
tumor and nontumor tissues removed and counted in a gamma counter
to determine the % ID/g. Tumor/nontumor ratios were calculated from
these data. In a separate study, groups of mice were given
.sup.111In-DOTA-PAM4-IgG (20 .mu.C, 50 .mu.g, 3.13.times.10.sup.-10
mol) for the purpose of comparing biodistribution, nuclear imaging,
and potential therapeutic activity. Radiation dose estimates were
calculated from the time-activity curves with the assumption of no
activity at zero time. Student's t test was used to assess
significant differences.
[0477] To perform nuclear immunoscintigraphy, at 3 h postinjection
of radiolabeled peptide or 24 h postinjection of radiolabeled
hPAM4-IgG, tumor-bearing mice were imaged with a dual-head Solus
gamma camera fitted with medium energy collimator for .sup.111In
(ADAC Laboratories). Mice were imaged for a total of 100,000 cpm or
10 min, whichever came first.
Results
[0478] In Vitro Characterization of the Bispecific mAb TF10.
[0479] The binding of TF10 to the target mucin antigen was analyzed
by ELISA (FIG. 16). The results showed nearly identical binding
curves for the divalent TF10, PAM4-IgG, and PAM4-F(ab').sub.2
(half-maximal binding was calculated as 1.42.+-.0.10, 1.31.+-.0.12,
and 1.83.+-.0.16 nmol/L, respectively; P>0.05 for all), whereas
the monovalent bsPAM4 chemical conjugate
(PAM4-Fab'.times.anti-DTPA-Fab') had a significantly lower avidity
(half-maximal binding, 30.61.+-.2.05 nmol/L; P=0.0379, compared
with TF10), suggesting that TF10 binds in a divalent manner. The
immunoreactive fraction of .sup.125I-TF10 bound to MUC5AC was 87%,
with 9% found as unbound TF10 and 3% as free iodide (not shown).
Ninety percent of the .sup.111In-IMP-288 bound to TF10 (not shown).
Of the total .sup.111In-IMP-288 bound to TF10, 92% eluted at higher
molecular weight when excess mucin (200 .mu.g) was added, with only
3% eluting with the non-mucin-reactive TF10 fraction. An additional
5% of the radiolabeled peptide eluted in the free peptide volume.
None of the radiolabeled peptide bound to the mucin antigen in the
absence of TF10 (not shown).
[0480] Biodistribution of .sup.125I-TF10 in CaPan1 Tumor-Bearing
Nude Mice.
[0481] TF10 showed a rapid clearance from the blood, starting with
21.03.+-.1.93% ID/g at 1 hour and decreasing to just 0.13.+-.0.02%
ID/g at 16 hours. The biological half-life was calculated to be
2.19 hours [95% confidence interval (95% CI), 2.11-2.27 hours].
Tissue uptake revealed enhanced activity in the liver, spleen, and
kidneys at 1 hour, which cleared just as quickly by 16 hours
[T.sub.1/2=2.09 hours (95% CI, 2.08-2.10), 2.84 hours (95% CI,
2.49-3.29), and 2.44 hours (95% CI, 2.28-2.63) for liver, spleen,
and kidney, respectively]. Activity in the stomach most likely
reflects the accretion and excretion of radioiodine, suggesting
that the radioiodinated TF10 was actively catabolized, presumably
in the liver and spleen, thereby explaining its rapid clearance
from the blood. Nevertheless, by 16 hours, the concentration of
radioiodine within the stomach was below 1% ID/g. A group of five
non-tumor-bearing nude mice given .sup.125I-TF10 and necropsied at
16 hours showed similar tissue distribution, suggesting that the
tumor had not affected the bispecific mAb distribution and
clearance from normal tissues (data not shown). Of course, it is
possible that differences occurred before the initial time point
examined. Tumor uptake of TF10 peaked at 6 hours postinjection
(7.16.+-.1.10% ID/g) and had decreased to half maximum binding
(3.47.+-.0.66% ID/g) at 16 hours. Tumor uptake again decreased
nearly 2-fold over the next 32 hours, but then was stable over the
following 24 hours.
[0482] Biodistribution of TF10-Pretargeted, .sup.111In-Labeled
Peptide.
[0483] Although maximum tumor uptake of TF10 occurred at 6 hours,
previous experience indicated that the radiolabeled peptide would
need to be given at a time point when blood levels of TF10 had
cleared to <1% ID/g (i.e., 16 hours). Higher levels of TF10 in
the blood would lead to unacceptably high binding of the
radiolabeled peptide within the blood (i.e., low tumor/blood
ratios), whereas administering the peptide at a later time would
mean the concentration of TF10 in the tumor would be decreased with
consequently reduced concentration of radiolabeled peptide within
the tumor. Thus, an initial pretargeting study was done using a
16-hour interval. With the amount of the .sup.111In-IMP-288 held
constant (30 .mu.Ci, 5.07.times.10.sup.-11 mol), increasing amounts
of TF10 were given so that the administered dose of TF10 and
IMP-288 expressed as mole ratio varied from 5:1 to 20:1 (Table
16).
TABLE-US-00034 TABLE 16 Biodistribution of .sup.111In-IMP-288 alone
(no TF10) or pretargeted with varying amounts of TF10 % ID/g at 3 h
(mean .+-. SD) 5:1 10:1 20:1 No TF10 Tumor 19.0 .+-. 3.49 24.3 .+-.
1.71 28.6 .+-. 0.73 0.12 .+-. 0.00 Liver 0.09 .+-. 0.01 0.21 .+-.
0.12 0.17 .+-. 0.01 0.07 .+-. 0.00 Spleen 0.12 .+-. 0.04 0.16 .+-.
0.07 0.26 .+-. 0.10 0.04 .+-. 0.01 Kidneys 1.59 .+-. 0.11 1.72 .+-.
0.24 1.53 .+-. 0.14 1.71 .+-. 0.22 Lungs 0.19 .+-. 0.06 0.26 .+-.
0.00 0.29 .+-. 0.04 0.03 .+-. 0.00 Blood 0.01 .+-. 0.00 0.01 .+-.
0.01 0.01 .+-. 0.00 0.00 .+-. 0.00 Stomach 0.03 .+-. 0.02 0.02 .+-.
0.02 0.01 .+-. 0.00 0.02 .+-. 0.01 Small intestine 0.12 .+-. 0.08
0.08 .+-. 0.03 0.04 .+-. 0.01 0.06 .+-. 0.02 Large intestine 0.23
.+-. 0.10 0.39 .+-. 0.08 0.25 .+-. 0.08 0.33 .+-. 0.02 Pancreas
0.02 .+-. 0.00 0.02 .+-. 0.01 0.02 .+-. 0.00 0.02 .+-. 0.00 Tumor
0.12 .+-. 0.03 0.32 .+-. 0.09 0.27 .+-. 0.01 0.35 .+-. 0.03 weight
(g)
[0484] At 3 hours the amount of .sup.111In-IMP-288 in the blood was
barely detectable (0.01%). Tumor uptake increased from
19.0.+-.3.49% ID/g to 28.55.+-.0.73% ID/g as the amount of
bispecific mAb administered was increased 4-fold (statistically
significant differences were observed for comparison of each
TF10/peptide ratio, one group to another; P<0.03 or better), but
without any appreciable increase in normal tissue uptake. Tumor
uptake in the animals given TF10 was >100-fold higher than when
.sup.111In-IMP-288 was given alone. Comparison of .sup.111In
activity in the normal tissues of the animals that either received
or did not receive prior administration of TF10 indicated similar
absolute values, which in most instances were not significantly
different. This suggests that the bispecific mAb had cleared
sufficiently from all normal tissues by 16 hours to avoid
appreciable peptide uptake in these tissues. Tumor/blood ratios
were >2,000:1, with other tissue ratios exceeding 100:1. Even
tumor/kidney ratios exceeded 10:1. The highest tumor uptake of
radioisotope with minimal targeting to nontumor tissues resulted
from the 20:1 ratio; however, either of the TF10/peptide ratios
could be used to achieve exceptional targeting to tumor, both in
terms of signal intensity and contrast ratios. The 10:1 ratio was
chosen for further study because the absolute difference in tumor
uptake of radiolabeled peptide was not substantially different
between the 10:1 (24.3.+-.1.71% ID/g) and 20:1 (28.6.+-.0.73% ID/g)
ratios.
[0485] Images of the animals given TF10-pretargeted
.sup.111In-IMP-288 at a bispecific mAb/peptide ratio of 10:1, or
the .sup.111In-IMP-288 peptide alone, are shown in FIG. 17A, FIG.
17B and FIG. 17C. The majority of these tumors were <0.5 cm in
diameter, weighing .about.0.25 g. The images show highly intense
uptake in the tumor of the TF10-pretargeted animals (FIG. 17A). The
intensity of the image background for the TF10-pretargeted animals
was increased to match the intensity of the image taken of the
animals given the .sup.111In-IMP-288 alone (FIG. 17B). However,
when the images were optimized for the TF10-pretargeted mice, the
signal intensity and contrast were so high that no additional
activity was observed in the body. No tumor localization was seen
in the animals given the .sup.111In-IMP-288 alone, even when image
intensity was enhanced (FIG. 17C).
[0486] An additional experiment was done to assess the kinetics of
targeting .sup.111In-hPAM4 whole-IgG compared with that of the
TF10-pretargeted .sup.111In-IMP-288 peptide. Tumor uptake of the
.sup.111In-peptide was highest at the initial time point examined,
3 hours (15.99.+-.4.11% ID/g), whereas the blood concentration of
radiolabeled peptide was only 0.02.+-.0.01% ID/g, providing a mean
tumor/blood ratio of 946.3.+-.383.0. Over time, radiolabeled
peptide cleared from the tumor with a biological half-life of 76.04
hours. Among nontumor tissues, uptake was highest in the kidneys,
averaging 1.89.+-.0.42% ID/g at 3 hours with a steady decrease over
time (biological half-life, 33.6 hours). Liver uptake started at
0.15.+-.0.06% ID/g and remained essentially unchanged over time. In
contrast to the TF10-pretargeted .sup.111In-IMP-288, the
.sup.111In-hPAM4-IgG had a slower clearance from the blood, albeit
there was a substantial clearance within the first 24 hours,
decreasing from 30.1% ID/g at 3 hours to just 11.5.+-.1.7% ID/g at
24 hours. Variable elevated uptake in the spleen suggested that the
antibody was likely being removed from the blood by targeting of
secreted mucin that had become entrapped within the spleen. Tumor
uptake peaked at 48 hours with 80.4.+-.6.1% ID/g, and remained at
an elevated level over the duration of the monitoring period. The
high tumor uptake, paired with a more rapid than expected blood
clearance for an IgG, produced tumor/blood ratios of 5.2.+-.1.0
within 24 hours. FIG. 17C shows the images of the animals at 24
hours postadministration of .sup.111In-PAM4-IgG, illustrating that
tumors could be visualized at this early time, but there was still
considerable activity within the abdomen. Tumor/nontumor ratios
were mostly higher for TF10-pretargeted .sup.111In-labeled
hapten-peptide as compared with .sup.111In-hPAM4-IgG, except for
the kidneys, where tumor/kidney ratios with the .sup.111In-IMP-288
and .sup.111In-hPAM4-IgG were similar at later times. However,
tumor/kidney ratios for the TF10-pretargeted .sup.111In-IMP-288
were high enough (e.g., .about.7:1) at 3 hours to easily discern
tumor from normal tissue.
[0487] FIG. 18A to FIG. 18D illustrates the potential therapeutic
capability of the direct and pretargeted methods to deliver
radionuclide (.sup.90Y). Although the concentration (% ID/g) of
radioisotope within the tumor seems to be much greater when
delivered by PAM4-IgG than by pretargeted TF10 at their respective
maximum tolerated dose (0.15 mCi for .sup.90Y-hPAM4 and 0.9 mCi for
TF10-pretargeted .sup.90Y-IMP-288) (FIG. 18A), the radiation dose
to tumor would be similar (10,080 and 9,229 cGy for
.sup.90Y-PAM4-IgG and TF10-pretargeted .sup.90Y-IMP-288,
respectively) (FIG. 18C). The advantage for the pretargeting method
would be the exceptionally low activity in blood (9 cGy), almost
200-fold less than with the .sup.90Y-hPAM4 IgG (1,623 cGy) (FIG.
18C). It is also important to note that the radiation dose to
liver, as well as other nontumor organs, would be much lower with
the TF10-pretargeted .sup.90Y-IMP-288 (FIG. 18B, FIG. 18D).
[0488] The exception would be the kidneys, where the radiation dose
would be similar for both protocols at their respective maximum
dose (612 and 784 cGy for .sup.90Y-PAM4-IgG and
TF10-.sup.90Y-IMP-288, respectively) (FIG. 18B, FIG. 18D). The data
suggest that for .sup.90Y-PAM4-IgG, as with most other radiolabeled
whole-IgG mAbs, the dose-limiting toxicity would be hematologic;
however, for the TF10 pretargeting protocol, the dose-limiting
toxicity would be the kidneys.
Discussion
[0489] Current diagnostic modalities such as ultrasound,
computerized tomography (CT), and magnetic resonance imaging (MRI)
technologies, which provide anatomic images, along with PET imaging
of the metabolic environment, have routinely been found to provide
high sensitivity in the detection of pancreatic masses. However,
these data are, for the most part, based on detection of lesions
>2 cm in a population that is already presenting clinical
symptoms. At this time in the progression of the pancreatic
carcinoma, the prognosis is rather dismal. To improve patient
outcomes, detection of small, early pancreatic neoplasms in the
asymptomatic patient is necessary.
[0490] Imaging with a mAb-targeted approach, such as is described
herein with mAb-PAM4, may provide for the diagnosis of these small,
early cancers. Of prime importance is the specificity of the mAb.
We have presented considerable data, including immunohistochemical
studies of tissue specimens (Gold et al., Clin Cancer Res 2007;
13:7380-7; Gold et al., Int J Cancer 1994; 57:204-10) and
immunoassay of patient sera (Gold et al., J Clin Oncol 2006;
24:252-8), to show that mAb-PAM4 is highly reactive with a
biomarker, the presence of which provides high diagnostic
likelihood of pancreatic neoplasia. Furthermore, we determined that
PAM4, although not reactive with normal adult pancreatic tissues
nor active pancreatitis, is reactive with the earliest stages of
neoplastic progression within the pancreas (pancreatic
intraepithelial neoplasia 1 and intraductal papillary mucinous
neoplasia) and that the biomarker remains at high levels of
expression throughout the progression to invasive pancreatic
adenocarcinoma (Gold et al., Clin Cancer Res 2007; 13:7380-7).
Preclinical studies with athymic nude mice bearing human pancreatic
tumor xenografts have shown specific targeting of radiolabeled
murine, chimeric, and humanized versions of PAM4.
[0491] In the current studies, we have examined a next-generation,
recombinant, bispecific PAM4-based construct, TF10, which is
divalent for the PAM4 arm and monovalent for the anti-HSG hapten
arm. There are several important characteristics of this
pretargeting system's constructs, named DOCK-AND-LOCK.TM.,
including its general applicability and ease of synthesis. However,
for the present consideration, the major differences from the
previously reported chemical construct are the valency, which
provides improved binding to tumor antigen, and, importantly, its
pharmacokinetics. TF10 clearance from nontumor tissues is much more
rapid than was observed for the chemical conjugate. Time for blood
levels of the bispecific constructs to reach less than 1% ID/g was
40 hours postinjection for the chemical construct versus 16 hours
for TF10. A more rapid clearance of the pretargeting agent has
provided a vast improvement of the tumor/blood ratio, while
maintaining high signal strength at the tumor site (% ID/g).
[0492] In addition to providing a means for early detection and
diagnosis, the results support the use of the TF10 pretargeting
system for cancer therapy. Consideration of the effective radiation
dose to tumor and nontumor tissues favors the pretargeting method
over directly radiolabeled PAM4-IgG. The dose estimates suggest
that the two delivery systems have different dose-limiting
toxicities: myelotoxicity for the directly radiolabeled PAM4 versus
the kidney for the TF10 pretargeting system. This is of
significance for the future clinical development of radiolabeled
PAM4 as a therapeutic agent.
[0493] Gemcitabine, the frontline drug of choice for pancreatic
cancer, can provide significant radiosensitization of tumor cells.
In previous studies, we showed that combinations of gemcitabine and
directly radiolabeled PAM4-IgG provided synergistic antitumor
effects compared with either arm alone (Gold et al., Clin Cancer
Res 2004, 10:3552-61; Gold et al., Int J Cancer 2004, 109:618-26).
The dose-limiting factor with this combination was overlapping
hematologic toxicity. However, because the dose-limiting organ for
TF10 pretargeting seems to be the kidney rather than hematologic
tissues, combinations with gemcitabine should be less toxic, thus
allowing increased administration of radioisotope with consequently
greater antitumor efficacy.
[0494] The superior imaging achieved with TF10 pretargeting in
preclinical models, as compared with directly radiolabeled
DOTA-PAM4-IgG, provides a compelling rationale to proceed to
clinical trials with this imaging system. The specificity of the
tumor-targeting mAb for pancreatic neoplasms, coupled with the
bispecific antibody platform technology providing the ability to
conjugate various imaging compounds to the HSG-hapten-peptide for
SPECT (.sup.111In), PET (.sup.68Ga), ultrasound (Au), or other
contrast agents, or for that matter .sup.90Y or other radionuclides
for therapy, provides high potential to improve overall patient
outcomes (Goldenberg et al., J Nucl Med 2008, 49:158-63). In
particular, we believe that a TF10-based ImmunoPET procedure will
have major clinical value to screen individuals at high-risk for
development of pancreatic cancer (e.g., genetic predisposition,
chronic pancreatitis, smokers, etc.), as well as a means for
follow-up of patients with suspicious abdominal images from
conventional technologies and/or with indications due to the
presence of specific biomarker(s) or abnormal biochemical findings.
When used as part of an ongoing medical plan for following these
patients, early detection of pancreatic cancer may be achieved.
Finally, in combination with gemcitabine, TF10 pretargeting may
provide a better opportunity for control of tumor growth than
directly radiolabeled PAM4-IgG.
Example 24
Therapy of Pancreatic Cancer Xenografts with Gemcitabine and
.sup.90Y-Labeled Peptide Pretargeted Using TF10
Summary
[0495] .sup.90Y-hPAM4 IgG is currently being examined in Phase I/II
trials in combination with gemcitabine in patients with Stage
III/IV pancreatic cancer. We disclose a new approach for
pretargeting radionuclides that is able to deliver a similar amount
of radioactivity to pancreatic cancer xenografts, but with less
hematological toxicity, which would be more amenable for
combination with gemcitabine. Nude mice bearing .about.0.4 cm.sup.3
sc CaPan1 human pancreatic cancer were administered a recombinant
bsMAb, TF10, followed 1 day later with a .sup.90Y-labeled
hapten-peptide (IMP-288). Various doses and schedules of
gemcitabine were added to this treatment, and tumor progression
monitored up to 28 weeks. 0.7 mCi PT-RAIT alone produce only a
transient 60% loss in blood counts, and animals given 0.9 mCi of
PT-RAIT alone and 0.7 mCi PT-RAIT+6 mg gemcitabine (human
equivalent .about.1000 mg/m.sup.2) had no histological evidence of
renal toxicity after 9 months. A single dose of 0.25 or 0.5 mCi
PT-RAIT alone can completely ablate 20% and 80% of the tumors,
respectively. Monthly fractionated PT-RAIT (0.25 mCi/dose given at
the start of each gemcitabine cycle) added to a standard
gemcitabine regimen (6 mg wkly.times.3; 1 wk off; repeat 3 times)
significantly increased the median time for tumors to reach 3.0
cm.sup.3 over PT-RAIT alone. Other treatment plans examining
non-cytotoxic radiosensitizing dose regimens of gemcitabine added
to PT-RAIT also showed significant improvements in treatment
response over PT-RAIT alone. The results show that PT-RAIT is a
promising new approach for treating pancreatic cancer. Current data
indicate combining PT-RAIT with gemcitabine will enhance
therapeutic responses.
Methods
[0496] TF10 bispecific antibody was prepared as described above.
For pretargeting, TF10 was given to nude mice bearing the human
pancreatic adenocarcinoma cell line, CaPan1. After allowing
sufficient time for TF10 to clear from the blood (16 h), the
radiolabeled divalent HSG-peptide was administered. The small
molecular weight HSG-peptide (.about.1.4 kD) clears within minutes
from the blood, entering the extravascular space where it can bind
to anti-HSG arm of the pretargeted TF10 bsMAb. Within a few hours,
>80% of the radiolabeled HSG-peptide is excreted in the urine,
leaving the tumor localized peptide and a trace amount in the
normal tissues.
Results
[0497] FIG. 19 illustrates the therapeutic activity derived from a
single treatment of established (.about.0.4 cm.sup.3) CaPan1 tumors
with 0.15 mCi of .sup.90Y-hPAM4 IgG, or 0.25 or 0.50 mCi of
TF10-pretargeted .sup.90Y-IMP-288. Similar anti-tumor activity was
observed for the 0.5-mCi pretargeted dose vs. 0.15-mCi dose of the
directly radiolabeled IgG, but hematological toxicity was severe at
this level of the direct conjugate (not shown), while the
pretargeted dose was only moderately toxic (not shown). Indeed, the
MTD for pretargeting using 90Y-IMP-288 is at least 0.9 mCi in nude
mice.
[0498] FIG. 20 shows that the combination of gemcitabine and
PT-RAIT has a synergistic effect on anti-tumor therapy. Human
equivalent doses of 1000 mg/m.sup.2 (6 mg) of gemcitabine (GEM)
were given intraperitoneally to mice weekly for 3 weeks, then after
resting for 1 week, this regimen was repeated 2 twice. PT-RAIT
(0.25 mCi of TF10-pretargeted .sup.90Y-IMP-288) was given 1 day
after the first GEM dose in each of the 3 cycles of treatment. Gem
alone had no significant impact on tumor progression (survival
based on time to progress to 3.0 cm.sup.3). PT-RAIT alone improved
survival compared to untreated animals, but the combined GEM with
PT-RAIT regimen increased the median survival by nearly 10 weeks.
Because hematological toxicity is NOT dose-limiting for PT-RAIT,
but it is one of the limitations for gemcitabine therapy, these
studies suggest that PT-RAIT could be added to a standard GEM
therapy with the potential for enhanced responses. The significant
synergistic effect of gemcitabine plus PT-RAIT was surprising and
unexpected.
[0499] A further study examined the effect of the timing of
administration on the potentiation of anti-tumor effect of
gemcitabine plus PT-RAIT. A single 6-mg dose of GEM was given one
day before or 1 day after 0.25 mCi of TF10-pretargeted
.sup.90Y-IMP-288 (not shown). This study confirmed what is already
well known with GEM, i.e., radiosensitization is best given in
advance of the radiation. Percent survival of treated mice showed
little difference in survival time between PT-RAIT alone and
PT-RAIT with gemcitabine given 22 hours after the radiolabeled
peptide. However, administration of gemcitabine 19 hours prior to
PT-RAIT resulted in a substantial increase in survival (not
shown).
[0500] Single PT-RAIT (0.25 mCi) combined with cetuximab (1 mg
weekly ip; 7 weeks) or with cetuximab+GEM (6 mg weekly.times.3) in
animals bearing CaPan1 showed the GEM+cetuximab combination with
PT-RAIT providing a better initial response (FIG. 21), but the
response associated with just cetuximab alone added to PT-RAIT was
encouraging (FIG. 21), since it was as good or better than
PT-RAIT+GEM. Because the overall survival in this study was
excellent, with only 2 tumors in each group progressing to >2.0
cm3 after 24 weeks when the study was terminated, these results
indicate a potential role for cetuximab when added to PT-RAIT.
Example 25
Effect of Fractionated Pretargeted Radioimmunotherapy (PT-RAIT) for
Pancreatic Cancer Therapy
[0501] We evaluated fractionated therapy with .sup.90Y-DOTA-di-HSG
peptide (IMP-288) and TF10. Studies using TF10 and radiolabeled
IMP-288 were performed in nude mice bearing s.c. CaPan1 human
pancreatic cancer xenografts, 0.32-0.54 cm.sup.3. For therapy,
TF10-pretargeted .sup.90Y-IMP-288 was given [A] once (0.6 mCi on wk
0) or [B] fractionated (0.3 mCi on wks 0 and 1), [C](0.2 mCi on wks
0, 1 and 2) or [D] (0.2 mCi on wks 0, 1 and 4).
[0502] Tumor regression (>90%) was observed in the majority of
mice, 9/10, 10/10, 9/10 and 8/10 in groups [A], [B], [C] and [D],
respectively. In group [A], maximum tumor regression in 50% of the
mice was reached at 3.7 wks, compared to 6.1, 8.1 and 7.1 wks in
[B], [C] and [D], respectively. Some tumors showed regrowth. At
week 14, the best therapeutic response was observed in the
fractionated group (2.times.0.3 mCi), with 6/10 mice having no
tumors (NT) compared to 3/10 in the 3.times.0.2 mCi groups and 1/10
in the 1.times.0.6 mCi group. No major body weight loss was
observed. Fractionated PT-RAIT provides another alternative for
treating pancreatic cancer with minimum toxicity.
Example 26
.sup.90Y-hPAM4 Radioimmunotherapy (RAIT) Plus Radiosensitizing
Gemcitabine (GEM) Treatment in Advanced Pancreatic Cancer (PC)
[0503] .sup.90Y-hPAM4, a humanized antibody highly specific for PC,
showed transient activity in patients with advanced disease, and
GEM enhanced RAIT in preclinical studies. This study evaluated
repeated treatment cycles of .sup.90Y-hPAM4 plus GEM in patients
with untreated, unresectable PC. The .sup.90Y-dose was escalated by
cohort, with patients repeating 4-wk cycles (once weekly 200
mg/m.sup.2 GEM, .sup.90Y-hPAM4 once-weekly wks 2-4) until
progression or unacceptable toxicity. Response assessments used CT,
FDG-PET, and CA19.9 serum levels.
[0504] Of 8 patients (3F/5M, 56-72 y.o.) at the 1.sup.st 2 dose
levels (6.5 and 9.0 mCi/m.sup.2 90Y-hPAM4.times.3), hematologic
toxicity has been transient Grade 1-2. Two patients responded to
initial treatment with FDG SUV and CA19.9 decreases, and lesion
regression by CT. Both patients continue in good performance status
now after 9 and 11 mo. and after a total of 3 and 4 cycles,
respectively, without additional toxicity. A 3.sup.rd patient with
a stable response by PET and CT and decreases in CA19.9 levels
after initial treatment is now undergoing a 2nd cycle. Four other
patients had early disease progression and the remaining patient is
still being evaluated. Dose escalation is continuing after
fractionated RAIT with .sup.90Y-hPAM4 plus low-dose gemcitabine
demonstrated therapeutic activity at the initial .sup.90Y-dose
levels, with minimal hematologic toxicity, even after 4 therapy
cycles.
Example 27
Early Detection of Pancreatic Carcinoma Using Mab-PAM4 and in Vitro
Immunoassay
[0505] Immunohistochemistry studies were performed with PAM4
antibody. Results obtained with stained tissue sections showed no
reaction of PAM4 with normal pancreatic ducts, ductules and acinar
tissues (not shown). In contrast, use of the MA5 antibody applied
to the same tissue samples showed diffuse positive staining of
normal pancreatic ducts and acinar tissue (not shown). In tissue
sections with well differentiated or moderately differentiated
pancreatic adenocarcinoma, PAM4 staining was positive, with mostly
cytoplasmic staining but intensification of at the cell surface.
Normal pancreatic tissue in the same tissue sections was
unstained.
[0506] Table 17 shows the results of immunohistochemical analysis
with PAM4 MAb in pancreatic adenocarcinoma samples of various
stages of differentiation. Overall, there was an 87% detection rate
for all pancreatic cancer samples, with 100% detection of well
differentiated and almost 90% detection of moderately
differentiated pancreatic cancers.
TABLE-US-00035 TABLE 17 PAM4 Labeling Pattern Cancer n Focal
Diffuse Total Well Diff. 13 2 11 13 (100%) Moderately Diff. 24 6 15
21 (88%) Poorly Diff. 18 5 9 14 (78%) Total 55 13 35 48 (87%)
[0507] Table 18 shows that PAM4 immunohistochemical staining also
detected a very high percentage of precursor lesions of pancreatic
cancer, including PanIn-1A to PanIN-3, IPMN (intraductal papillary
mucinous neoplasms) and MCN (mucinous cystic neoplasms). Overall,
PAM4 staining detected 89% of all pancreatic precursor lesions.
These results demonstrate that PAM4 antibody-based immunodetection
is capable of detecting almost 90% of pancreatic cancers and
precursor lesions by in vitro analysis. PAM4 expression was
observed in the earliest phases of PanIN development. Intense
staining was observed in IPMN and MCN samples (not shown). The PAM4
epitope was present at high concentrations (intense diffuse stain)
in the great majority of pancreatic adenocarcinomas. PAM4 showed
diffuse, intense reactivity with the earliest stages of pancreatic
carcinoma precursor lesions, including PanIN-1, IPMN and MCN, yet
was non-reactive with normal pancreatic tissue. Taken together,
these results show that diagnosis and/or detection with the PAM4
antibody is capable of detecting, with very high specificity, the
earliest stages of pancreatic cancer development.
TABLE-US-00036 TABLE 18 PAM4 Labeling Pattern n Focal Diffuse Total
PanIn-1A 27 9 15 24 (89%) PanIn-1B 20 4 16 20 (100%) PanIn-2 11 6 4
10 (91%) PanIn-3 5 2 0 2 (40%) Total PanIn 63 21 35 56 (89%) IPMN
36 6 25 31 (86%) MCN 27 3 22 25 (92%)
[0508] An enzyme based immunoassay for PAM4 antigen in serum
samples was developed. FIG. 22 shows the results of differential
diagnosis using PAM4 immunoassay for pancreatic cancer versus
normal tissues and other types of cancer. The results showed a
sensitivity of detection of pancreatic cancer of 77.4%, with a
specificity of detection of 94.3%, comparing pancreatic carcinoma
(n=53) with all other specimens (n=233), including pancreatitis and
breast, ovarian and colorectal cancer and lymphoma. The data of
FIG. 22 are presented in tabular form in Table 19.
TABLE-US-00037 TABLE 19 PAM4-Reactive MUC5AC in Patient Sera #
Positive n Mean SD Median Range (%) Normal 43 0.1 0.3 0.0 0-2.0 0
(0) Pancreatitis 87 3.0 11.5 0.0 0-63.6 4 (5) Pancreatic CA 53 171
317 31.7 0-1000 41 (77) Colorectal CA 36 3.3 7.7 0.0 0-37.8 5 (14)
Breast CA 30 3.7 10.1 0.0 0-53.5 2 (7) Ovarian CA 15 1.8 4.3 0.0
0-16.5 1 (7) Lymphoma 19 12.3 44.2 0.0 0-194 1 (5)
[0509] An ROC curve (not shown) was constructed with the data from
Table 19. Examining a total of 283 patients, including 53 with
pancreatic carcinoma, and comparing the presence of circulating
MUC5AC in patients with pancreatic cancer to all other samples, the
ROC curve provided an AUC of 0.88.+-.0.03 (95% ci, 0.84-0.92) with
a P value <0.0001, a highly significant difference for
discrimination of pancreatic carcinoma from non-pancreatic
carcinoma samples. Comparing pancreatic CA with other tumors and
normal tissue, the PAM4 based serum assay showed a sensitivity of
77% and a specificity of 95%.
[0510] A comparison was made of MUC5AC concentration in serum
samples from normal patients, "early" (stage 1) pancreatic
carcinoma and all pancreatic carcinoma samples. The specimens
included 13 sera from healthy volunteers, 12 sera from stage-1, 13
sera from stage-2 and 25 sera from stage-3/4 (advanced) pancreatic
carcinoma. A cutoff value of 8.8 units/ml (horizontal line) was
used, as determined by ROC curve statistical analysis. The
frequency distribution of PAM4 antigen concentration is shown in
FIG. 23, which shows that 92% of "early" stage-1 pancreatic
carcinomas were above the cutoff line for diagnosis of pancreatic
cancer. An ROC curve for the PAM4 based assay is shown in FIG. 24,
which demonstrates a sensitivity of 81.6% and specificity of 84.6%
for the PAM4 assay in detection of pancreatic cancer.
[0511] These results confirm that an enzyme immunoassay based on
PAM4 antibody binding can detect and quantitate PAM4-reactive
antigen in the serum of pancreatic carcinoma patients. The
immunoassay demonstrates high specificity and sensitivity for
pancreatic carcinoma. The majority of patients with stage 1 disease
were detectable using the PAM4 immunoassay.
[0512] In conclusion, an immunohistology procedure employing PAM4
antibody identified approximately 90% of invasive pancreatic
carcinoma and its precursor lesions, PanIN, IPMN and MCN. A PAM4
based enzyme immunoassay to quantitate MUC5AC in human patient sera
showed high sensitivity and specificity for detection of early
pancreatic carcinoma. Due to the high specificity of PAM4 for
pancreatic carcinoma, the mucin biomarker can also serve as a
target for in vivo targeting of imaging and therapeutic agents.
ImmunoPET imaging for detection of "early" pancreatic carcinoma is
of use for the early diagnosis of pancreatic cancer, when it can be
more effectively treated. Use of radioimmunotherapy with a
humanized PAM4 antibody construct, preferably in combination with a
radiosensitizing agent, is of use for the treatment of pancreatic
cancer.
Example 28
Further Studies of In Vitro Detection of PAM4 Antigen in Human
Serum
[0513] In certain embodiments, it is preferred to detect the
presence of MUC5AC and/or to diagnose the presence of pancreatic
cancer in a subject by in vitro analysis of samples that can be
obtained by non-invasive techniques, such as blood, plasma or serum
samples. Such ex vivo analysis may be preferred, for example, in
screening procedures where there is no a priori reason to believe
that an individual has a pancreatic tumor in a specific location.
The objective of the present study was to develop a reliable,
accurate, serum-based assay for detection of pancreatic cancer at
the earliest stages of the disease.
Summary
[0514] A PAM4-based immunoassay was used to quantitate antigen in
the serum of healthy volunteers (N=19), patients with known
diagnosis of pancreatic adenocarcinoma (N=68), and patients with a
primary diagnosis of chronic pancreatitis (N=29). Sensitivity for
the detection of pancreatic adenocarcinoma was 82%, with a
false-positive rate of 5% for the healthy controls. Patients with
advanced disease had significantly higher antigen levels than those
with early-stage disease (P<0.01), with a diagnostic sensitivity
of 91%, 86%, and 62% for stage 3/4 advanced disease, stage-2, and
stage-1, respectively. We also evaluated chronic pancreatitis sera,
finding 38% positive for antigen. However, this observation was
discordant with immunohistochemical findings that suggest the
PAM4-antigen is not produced by inflamed pancreatic tissue.
Furthermore, several of the serum-positive pancreatitis patients,
for whom tissue specimens were available for pathological
interpretation, had evidence of neoplastic precursor lesions.
Immunohistochemistry of additional pancreatitis specimens showed
90% to be PAM4-negative with the remainder only weakly positive.
This suggested that positive levels of PAM4-antigen within the
serum are not derived from inflamed pancreatic tissues, but may be
an early indicator of pancreatic cancer.
[0515] These results show that the PAM4-serum assay may be used to
detect early-stage pancreatic adenocarcinoma, and that positive
serum levels of PAM4-antigen are not derived from inflamed
pancreatic tissues, but rather may provide evidence of subclinical
pancreatic neoplasia.
Materials and Methods
[0516] Human Specimens
[0517] Sera (N=68) were obtained from patients with a confirmed
diagnosis of pancreatic adenocarcinoma being treated at the Johns
Hopkins Medical Center, Baltimore, Md., and stored frozen <5
yrs. Each of these patients underwent surgical resection of the
pancreas, providing an opportunity for accurate diagnosis and
staging. For stage-1 disease, no neoplastic cells were observed
outside of the pancreas. However, patients with pancreatic
adenocarcinoma are likely to have undetected micrometastatic
disease at presentation, including those patients reported with
stage-1 disease. For this reason, we evaluated follow-up survival
data. All patients described as having stage-1 disease survived at
least 1 year (time to last recorded follow-up visit), with a median
survival time of 2.70 years (25.sup.th percentile=1.32 years) in
comparison to the latest SEER data (2002-2006), which reports a
1.42-year median survival for patients having stage-1 disease
treated by surgical resection.
[0518] A total of 29 sera from patients with a diagnosis of chronic
pancreatitis were obtained from the Johns Hopkins Medical Center
and Zeptometrix Corp. (Franklin, Mass.). Healthy volunteers (N=19)
provided blood for control specimens at the Center for Molecular
Medicine and Immunology. All specimens were de-identified, with the
only clinical data provided to the investigators being the
diagnosis, stage of disease, follow-up survival time, and size of
the primary tumor.
[0519] Reagents
[0520] A human pancreatic mucin preparation was isolated from
CaPan1, a human pancreatic cancer grown as
http://jco.ascopubs.org/cgi/content/full/24/2/252-SEC1#SEC1xenografts
in athymic nude mice. Briefly, 1 g of tissue was homogenized in 10
mL of 0.1 M ammonium bicarbonate containing 0.5 M sodium chloride.
The sample was then centrifuged to obtain a supernatant that was
fractionated on a SEPHAROSE.RTM.-4B-CL column with the void volume
material chromatographed on hydroxyapatite. The unadsorbed fraction
was dialyzed extensively against deionized water and then
lyophilized. A 1 mg/mL solution was prepared in 0.01 M sodium
phosphate buffer (pH, 7.2) containing 0.15 M sodium chloride
(phosphate-buffered saline [PBS]), and used as the stock solution
for the immunoassay standards. A polyclonal, anti-mucin antiserum
was prepared by immunization of rabbits, as described previously
(Gold et al., Cancer Res 43:235-38, 1983). An IgG fraction was
purified and assessed for purity by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
molecular-sieve high-performance liquid chromatography. Murine MA5
antibody reactive with the MUC1 protein core was obtained from
Immunomedics, Inc. (Morris Plains, N.J.). A non-binding
isotype-matched control antibody, Ag8, was purified from the
P3X63-Ag8 murine myeloma.
[0521] Sample Preparation
[0522] All assays were performed in a blinded fashion. To prepare
the specimens for immunoassay, 300 .mu.L of serum were placed in a
2.0 mL microcentrifuge tube and extracted with an equal volume of
1-butanol. The tubes were vortexed vigorously for 2 min at which
time 300 .mu.L of chloroform were added and the tubes again
vortexed for 2 min; this latter step was included in the procedure
in order to invert the aqueous and organic layers. The tubes were
then centrifuged in a microfuge at a setting of 12,000 rpm for 5
min. The top aqueous layer was removed to a clean tube and the
sample diluted 1:2 in 2.0% (w/v) casein-sodium salt in 0.1 M sodium
phosphate buffer, pH 7.2, containing 0.15 M sodium chloride (PBS)
for immunoassay.
[0523] Enzyme Immunoassay
[0524] The immunoassay was performed in a 96-well polyvinyl plate
that had been coated with 100 .mu.L of humanized-PAM4 IgG at 20
.mu.g/mL in PBS with incubation at 4.degree. C. overnight. The
wells were then blocked by addition of 200 .mu.L of a 2.0% (w/v)
solution of casein in PBS and incubated for 1.5 h at 37.degree. C.
The blocking solution was removed from the wells and the plate
washed 5-times with 250 .mu.L of PBS containing 0.1% (v/v)
Tween-20. The standards, or unknown specimens, 100 .mu.L in
triplicate, were added to the appropriate wells and incubated at
37.degree. C. for 1.5 h. The plate was then washed 5-times with
PBS-Tween-20 as above.
[0525] The polyclonal, rabbit anti-mucin antibody, diluted to 5
.mu.g/mL in 1.0% (w/v) casein in PBS containing 50 .mu.g/mL
non-specific, human IgG, was added to each well and incubated for 1
h at 37.degree. C. The polyclonal antibody was then washed from the
wells as above, and peroxidase-labeled donkey anti-rabbit IgG
(Jackson ImmunoResearch Laboratories, West Grove, Pa.), at a 1:2000
dilution in 1.0% (w/v) casein in PBS, also containing 50 .mu.g/mL
human IgG, was added to the wells and incubated at 37.degree. C.
for 1 h. After washing the plate as above, 100 .mu.L of a
3,3',5,5'-tetramethylbenzidine substrate solution were added to the
wells and incubated at room temperature for 30 min. The reaction
was stopped by the addition of 50 .mu.L 4.0 N sulfuric acid, and
the optical density read at a wavelength of 450 nm using a
SPECTRA-MAX.RTM. 250 spectrophotometer (Molecular Devices,
Sunnyvale, Calif.). Because of the considerable microheterogeneity
of the PAM4 antigen, we chose to report our results in arbitrary
units/mL, based on an initial reference standard purified from
xenografted CaPan-1 human pancreatic tumor.
[0526] Immunohistochemistry
[0527] Paraffin-embedded specimens obtained from the Cooperative
Human Tissue Network were cut to 4 micron sections on superfrost
plus adhesive slides (Thermo Scientific, Waltham, Mass.). Tissue
sections were then heated to 95.degree. C. for 20 min in a pH 9.0
Tris buffer, Target Retrieval Solution (Dako, Carpinteria, Calif.),
allowed to cool to room temperature, and then quenched with 3%
H.sub.2O.sub.2 for 15 min at room temperature. Primary antibodies
were then used at 10 .mu.g/mL with an ABC VECTASTAIN.RTM. kit
(Vector Laboratories, Burlingame, Calif.) for labeling the tissues.
The slides were scored independently by two pathologists using a
paradigm consistent with that reported for earlier studies on
biomarkers in pancreatic adenocarcinoma (Gold et al., 2007, Clin
Cancer Res 13:7380-87): 0-negative, <1% of the tissue was
labeled; 1-a weak, focal labeling of between 1%-25% of the tissue;
2-a strong, focal labeling of between 1%-25% of the tissue; 3-a
weak, diffuse labeling >25% of the tissue; 4-a strong, diffuse
labeling >25% of the tissue. Only the appropriate tissue
components (e.g., adenocarcinoma cells, normal ducts, etc.) were
considered for assessment.
[0528] Statistical Analyses
[0529] Standard curves were generated from the immunoassay data,
with regression analyses performed to interpolate concentrations of
the unknown samples (Prism 4.0 software, GraphPad, La Jolla,
Calif.). Receiver operating characteristic (ROC) curves were
generated by use of the Med-Calc statistical software package
(version 7.5) (Med-Calc, Mariakerke Belgium). Student's t-test was
used to compare variables in any two groups. The Cochran-Armitage
test was used to detect a trend between detection rates and stage
of disease.
Results
[0530] Accuracy and Precision of the Immunoassay
[0531] A set of control standards with nominal concentrations of
15.60, 6.20, 2.50, and 1.00 units/mL was evaluated on several
nonconsecutive days (N=7) for determination of accuracy and
precision. Curve fitting for the standards generally gave resultant
goodness of fit values for r.sup.2>0.990. Accuracy was
calculated to be within 8% of the nominal value for the first three
concentrations, but fell to approximately 22% for the 1.00 units/mL
standard. Linear regression of nominal vs. measured units/mL in
this series of controls gave a trend-line with a slope of 0.965 and
y intercept of 0.174 (r.sup.2=0.999), where a slope of 1.00 with a
y intercept of 0.00 would constitute 100% accuracy (FIG. 25). An
average absolute difference between nominal and recovered mass
equal to 0.190.+-.0.173 units/mL for the two lowest concentration
standards suggested a minimum absolute error of approximately 0.2
units/mL for the EIA. Values for the coefficient of variation (CV)
were 6.40%, 4.85%, 12.0%, and 66.4%, respectively, for the 4
control standards. Taken together, the data suggest that the
PAM4-immunoassay provides levels of accuracy and reproducibility
that are within the guidelines suggested for an immunoassay
measurement of an analyte; accuracy and precision were within 15%
for concentrations above the cutoff value (2.40 units/mL), and
within 20% at the cutoff value. To further test this, we examined 3
sera, two of which were from healthy controls, on 3 separate days.
The two healthy controls gave average results of 0.27.+-.0.06 and
0.30.+-.0.27 units/mL, each of which was close to the minimum
absolute error for the EIA with consequent high CV of 21.65% and
88.19%, respectively. The other patient serum gave an average of
19.45.+-.2.51 units/mL with a CV of 12.9%.
[0532] Quantitation of Antigen in Patient Sera
[0533] In a preliminary study reported in the Example above, the
PAM4 serum-based immunoassay had an apparent sensitivity of 77% and
a specificity of 94% for pancreatic carcinoma. It should be noted
that the overwhelming majority of cancer specimens of pancreatic
and non-pancreatic origin had been obtained from patients enrolled
in IRB-approved clinical trials conducted by the Garden State
Cancer Center and stored frozen at -80.degree. C. for more than 10
yrs. However, the specimens of pancreatitis had been stored frozen
for a considerably shorter time. We evaluated a new group of 24
sera from patients diagnosed with pancreatic adenocarcinoma. Only
two of the sera had levels of PAM4-reactive antigen considered to
be positive. Therefore, we considered and evaluated reasons why the
immunoassay had not performed as expected, including the quality of
the immunoassay reagents, the possibility that the antigen was
being degraded and/or removed from the serum, its presence in the
form of immune complexes, or being bound by a blocking substance.
We discovered that there is a substance in fresh human serum and/or
specimens stored frozen for short periods of time (<5 yrs) that
apparently binds to the PAM4-reactive epitope and blocks its
binding to PAM4 antibody, thus preventing detection by immunoassay.
Percent recovery of antigen from fresh normal human serum (N=2)
spiked with PAM4-antigen at concentrations from 5-20 units/mL was
on the order of 33% or less.
[0534] In a series of reports, Slomiany and co-workers disclosed
that gastric mucin had covalenty bound and/or associated lipids and
fatty-acids (Slomiany et al., 1984, Arch Biochem Biophys
229:560-67; Slomiany et al., 1986, Biochem Biophys Res Commun
141:387-93; Zalesna et al., 1989, Biochem Int 18:775-84), and that
these lipids and fatty acids had specific effects upon the
physicochemical properties of the mucin. Furthermore, it was
reported that fatty-acid synthetase levels and activity are
significantly elevated in pancreatic adenocarcinoma, as is also the
case for other forms of cancer and other pathologic conditions
(Walter et al., 2009, Cancer Epidemiol Biomarkers Prev 19:2380-85).
Because the blocking substance might be lipid in nature, we
performed organic extraction of sera from the group of 24
pancreatic adenocarcinoma patients that had been stored frozen for
<5 years. As was noted above, without prior extraction, only 2
of the 24 specimens (8.3%) had levels of PAM4-antigen that were
considered positive, whereas after organic extraction, 22 of the 24
specimens (92%) had positive levels of the PAM4-antigen.
[0535] We were also able to re-evaluate, from the study reported in
the Example above, 10 pancreatic adenocarcinoma patient sera that
had been stored frozen for >15 years to confirm the prior
results. With or without extraction, all 10 specimens had levels of
antigen that were considered to be positive. Regression analysis to
compare paired results from extracted and non-extracted sera gave a
trendline with slope of 1.10 (r.sup.2=0.94), demonstrating that
with or without extraction of these long-term frozen sera, the
results were similar. It is considered that long-term storage of
the specimens resulted in degradation of the inhibiting substance
or decreased binding to and unmasking of the epitope. All further
testing of sera was performed with organic extraction of specimens
prior to immunoassay.
[0536] Specimens evaluated for PAM4-reactive antigen included 68
patients with confirmed pancreatic adenocarcinoma divided by stage:
21 from stage-1; 14 from stage-2; and 33 from stages-3 and -4
(advanced). In addition, 19 sera collected from healthy adult
volunteers and 29 patients diagnosed with chronic pancreatitis were
included as control groups. The maximum concentration shown in the
dot-plot (FIG. 26) was 80 units/mL, because there were insufficient
volumes of sera to perform additional dilution studies. Although a
cutoff value of 10.2 units/mL was reported in the Example above,
because of the use of an organic extraction procedure, as well as
differences in the EIA protocol (reagent concentrations, inclusion
of human IgG in buffers), we chose to treat the current data set
independently of prior results. A positive cutoff value of 2.4
units/mL was calculated by ROC curve statistics (FIG. 27) for the
comparison of all pancreatic adenocarcinoma specimens versus
healthy adults. The overall sensitivity for detection of pancreatic
adenocarcinoma was 82%, with an area under the curve of
0.92.+-.0.03 (95% CI=0.84-0.97). At this level of sensitivity, a
false-positive rate of 5% was observed for the healthy control
group, the single positive case having 3.65 units/mL of circulating
antigen, just above the cutoff value. Insufficient volumes of sera
precluded CA19-9 immunoassays for comparison to the
PAM4-immunoassay results.
[0537] As shown in Table 20, sensitivity for detection of early,
stage-1 pancreatic adenocarcinoma was relatively high, with 13 of
21 (62%) specimens above the cutoff value. As expected, this
detection rate was lower than that observed for the stage-2 (86%)
and advanced stage-3 and -4 (91%) patient groups. A statistically
significant trend (P<0.01) was noted for detection rate vs.
stage of disease. We considered that this was most likely due to
tumor size or burden. The average tumor sizes for stage-1, stage-2,
and stage-3/4 groups were 2.14.+-.1.02 cm.sup.3, 3.36.+-.1.18
cm.sup.3, and 3.45.+-.1.06 cm.sup.3, respectively. While there was
no statistically significant difference in tumor size between the
stage-2 and -3/4 groups (P>0.41), a statistically significant
difference was observed for each of these two groups when compared
to stage-1 tumor size (P<0.004 or better). However, it should be
noted that individual tumor size did not correlate with antigen
concentration in the serum (r.sup.2=0.0065).
[0538] Specimens reported as Stage-1 could be divided into stage-1A
(N=13) and stage-1B (N=8) subgroups based on tumor size, with
detection rates of 54% and 75%, respectively; however, caution is
emphasized since the number of patients in each subgroup is small.
The average tumor size for stage-1A was 1.41.+-.0.58 cm.sup.3
(range: 0.4 cm.sup.3-2.0 cm.sup.3) and for stage-1B was
3.15.+-.0.44 cm.sup.3 (range: 2.5 cm.sup.3-4 cm.sup.3); P<0.001
for comparison of the two groups. While, on the whole, tumor sizes
were smaller in stage-1A disease than in stage-1B, there was no
apparent statistical correlation between individual tumor size and
concentration of the PAM4-antigen in the blood (r.sup.2=0.03).
Furthermore, it is important to note that of the 13 stage-1A
specimens, 4 of the 7 positive cases had PAM4-antigen levels
considerably higher than the cutoff value, with a range of
17.65-32.65 units/mL.
TABLE-US-00038 TABLE 20 PAM4-reactive antigen in the sera of
patients Median T-test N (units/mL) True-Positive (P value).sup.a
Total PC 68 9.85 81% <0.001 Stage-1 21 4.53 62% <0.002
Stage-1A 13 3.96 54% <0.02 Stage-1B 8 6.05 75% <0.02 Stage-2
14 10.39 86% <0.005 Stage-3/4 33 13.37 91% <0.001 Chronic
Pancreatitis 29 1.28 (38% FP) Healthy Volunteers 19 1.18 (5% FP)
.sup.aAll comparisons are to healthy volunteers
[0539] We also evaluated a set of 29 patient sera with the primary
diagnosis of chronic pancreatitis. At the 2.4 units/mL cutoff
established by ROC evaluation of normal and pancreatic
adenocarcinoma patients, 11 pancreatitis patients (38%) were
positive. ROC curve analysis of pancreatitis sera compared directly
to the pancreatic adenocarcinoma specimens gave an area under the
curve of 0.77.+-.0.05 (95% CI=0.68-0.85). The median value for the
pancreatitis group was 1.28 units/mL, comparable to the healthy
volunteer group (1.18 units/mL), but considerably lower (3.5-fold)
than the stage-1 pancreatic adenocarcinoma group (4.53 units/mL).
It should be noted that our earlier results for pancreatitis
specimens suggested a considerably lower false-positive rate, only
5%. However, those pancreatitis specimens were stored frozen for
less than 5 years, and were not organic phase extracted prior to
analysis.
[0540] Biopsy and/or surgical specimens were available from 14 of
the chronic pancreatitis specimens, 6 of which were from patients
who were considered positive for circulating MUC5AC. In 3 of these
6 positive cases, precursor lesions were identified within the
tissue sections. It was then considered whether the positive serum
test was due to pancreatitis or the presence of neoplastic
precursor lesions. We performed immunohistochemistry on an
additional 30 biopsy specimens from patients diagnosed with
pancreatitis. Of the 30 specimens, one frank invasive pancreatic
adenocarcinoma and one large PanIN-2-3 lesion were identified (in
separate specimens) by use of PAM4 staining, while surrounding
acinar-ductal metaplasia (ADM) and normal tissues were negative
(data not shown). Of the remaining 28 specimens, 19 had sufficient
parenchyma to be evaluated, 16 of which had evidence of ADM. PAM4
was negative in all but two of these cases, and in each of these
gave only a very focal, weak labeling of ADM within the specimens
(data not shown).
[0541] Validation Studies--
[0542] We have begun putting together a panel of well-annotated
serum specimens from patients with known diagnoses. A first set of
patient sera (N.about.450) including healthy individuals and
patients having invasive pancreatic cancers (carcinoma,
neuroendocrine, and other forms), benign disease of the pancreas
(adenomatous lesions, pancreatitis, etc.) and non-pancreatic
cancers and benign disease (biliary, duodenal, ampullary
carcinomas, cholecystitis, gastritis, etc.) is being evaluated
(blind study) to both confirm and extend the prior results on PAM4
specificity in a much larger group of patients. Table 21 presents
an interim analysis based upon studies completed to date; overall,
the data are remarkably similar to our earlier data. Employing a
cutoff value determined by ROC analysis of PC vs Healthy Adults,
the overall sensitivity for detection of pancreatic carcinoma was
80% at a specificity of 96%. Only 2 of 16 neuroendocrine tumors
were positive, just over the cutoff value.
[0543] To date, 14 of 53 (26%) patients with primary diagnosis of
pancreatitis have been identified as PAM4 positive, lower than that
reported in our recent publication. We are now attempting to
correlate clinical data with results in this pancreatitis group, as
well as provide for clinical and laboratory follow-up of these
patients. Only 2 of 11 patients with benign adenomatous lesions
(both cystadenoma) were considered positive. One other cystadenoma
had PAM4-antigen levels greater than 200 units/mL. The pathology
report describes the biopsy as "very suspicious for cancer".
TABLE-US-00039 TABLE 21 PAM4-reactive antigen in the sera of
patients Positive ROC-AUC.sup.b N Median.sup.a Mean .+-. SD.sup.a
(%) (95% CI) P value.sup.c Pancreatic 145 7.84 35.61 .+-. 64.58 80
(comparisons Carcinoma are to PC) Neuroendocrine 16 0.00 1.73 .+-.
4.91 12.5 Healthy 27 0.40 0.54 .+-. 0.53 3.7 0.90 .+-. 0.02
<0.0001 (0.85-0.94) Pancreatitis 53 0.37 1.56 .+-. 3.35 26 0.85
.+-. 0.28 <0.0001 (0.79-0.90) Pancreatic 11 0.00 0.64 .+-. 0.78
18 0.90 .+-. 0.03 <0.0001 Adenoma (0.84-0.94) .sup.aValues for
Median and Mean .+-. SD are in Units/mL .sup.bReceiver Operating
Characteristic Curves (ROC); Area Under the Curve (AUC) with values
for 95% Confidence Intervals presented.
Discussion
[0544] Studies reported in the Example above that employed both
immunohistology of tissue specimens and EIA of circulating antigen
demonstrated that the PAM4-reactive epitope is a biomarker for
invasive pancreatic adenocarcinoma and is expressed at the earliest
stages of pancreatic neoplasia (i.e., PanIN-1). It was not
detectable within normal pancreatic tissues (ducts, acinar and
islet cells), nor the majority of non-pancreatic cancers examined
(breast, lung, gastric, and others). Thus, an elevation of the
PAM4-epitope concentration in the serum provided a high positive
likelihood ratio of 16.8 for pancreatic adenocarcinoma. Missing
from the prior study was clinical information regarding the stage
of disease. Consequently, we could not evaluate the value of the
immunoassay for detection of potentially curable early disease
until now.
[0545] We report herein that PAM4-based EIA using serum samples can
detect patients having early-stage pancreatic adenocarcinoma, and
can provide accurate discrimination from disease-free individuals.
The assay's sensitivity for detection of early pancreatic
adenocarcinoma was 62% for patients with stage-1 and 86% for
patients with stage-2 disease and serum levels generally increased
with advancing stage of disease. A high percentage of patients with
stage-1 and -2 disease are clinically asymptomatic. We conclude
that detection of tumor growth at these early stages using a PAM4
serum assay could provide improved prospects for survival.
[0546] The cancer patients in this study all underwent surgical
resection, providing an opportunity to accurately stage each
patient. However, many patients with pancreatic cancer are
suspected of having micrometastatic disease at presentation, even
if they do not have histologically-apparent regional lymph node
involvement. This highlights a general problem in the study of
early detection, particularly with a low-incidence disease such as
pancreatic adenocarcinoma. The accrual of specimens that are
well-defined is problematic. Further complicating the issue is that
many of these pancreatic cancers occur in the presence of chronic
pancreatitis, cholecystitis, and neoplastic precursor lesions,
amongst other conditions.
[0547] Of 29 sera with a primary diagnosis of chronic pancreatitis,
38% were identified as positive for PAM4-antigen. However, several
of these serum-positive patients, for whom tissue specimens for
pathological interpretation were available, had evidence of
neoplastic precursor lesions. Furthermore, a discrepancy was
observed in the comparison of tissue reactivity by immunohistology
and serum levels of antigen by immunoassay. By
immunohistochemistry, only 10% of the evaluable specimens showed
evidence of PAM4 staining within the ADM, although this was at
considerably lower intensity than observed for the overwhelming
majority of pancreatic adenocarcinoma specimens (Gold et al., 2007,
Clin Cancer Res 13:7380-87). Therefore, the results suggest that
positive levels of PAM4-antigen within the serum may not be derived
from inflamed pancreatic tissues, but rather could provide evidence
of subclinical pancreatic neoplasia, such as PanIN lesions, and
that, at the very least, positive results provide the rationale for
clinical follow-up of these patients.
[0548] Findings from genetically-engineered animal models of
pancreatic adenocarcinoma suggest that human pancreatic neoplasia
may arise before the PanIN-1 lesion (Leach, 2004, Cancer Cell
5:7-11). ADM was the earliest change observed in the mutant KRAS
targeted model described by Zhu et al. (2007, Am J Pathol
171:263-73). On the other hand, Shi et al. (2009, Mol Cancer Res
7:230-36) reported that although KRAS gene mutations can occur
within ADM, they occur predominantly within ADM that are associated
with PanIN lesions. The authors suggest this may occur by
retrograde extension of the PanIN to the surrounding ADM. As yet,
there is no conclusive evidence that ADM progress to PanIN. The
fact that PAM4 is reactive with ADM in two patients with
pancreatitis is of interest.
[0549] At the present time, screening the general population for
pancreatic cancer is not considered medically or economically
worthwhile, because the disease is simply too infrequent. However,
there is considerable interest in screening patients predicted to
have an increased risk of developing pancreatic adenocarcinoma.
Several studies have demonstrated that screening individuals with
strong family histories of pancreatic cancer can identify precursor
neoplasms of the pancreas that are amenable to surgical resection
(Canto et al., 2006, Clin Gastroenterol Hepatol 4:766-81; Canto,
2005, Clin Gastroenterol Hepatol 3:S46-58; Brentnall et al., 1999,
Ann Intern Med 131:247-55). For example, relatives of pancreatic
cancer patients have a significantly higher risk of developing
pancreatic cancer than the general population (Shi et al., 2009,
Arch Pathol Lab Med 133:365-74). A small percentage of patients
with familial pancreatic cancer harbor mutations of PALB2 (partner
and localizer of BRCA2), a susceptibility gene for pancreatic
cancer (Tischkowitz et al., 2009, Gastroenterology 137:1183-86).
Similarly, patients with long-standing chronic pancreatitis are at
increased risk of developing pancreatic cancer, and the risk is
over 30%, among patients with early-onset (teenage) hereditary
pancreatitis (Lowenfels et al., 1993, New Eng J Med 328:1433-37;
Lowenfels et al., 1997, J Natl Cancer Inst 89:442-46). A 20- to
34-fold higher risk has been observed in individuals with familial
atypical multiple mole (FAMMM) syndrome (Rutter et al., 2004,
Cancer 101:2809-16). Also, several studies have shown a
significantly increased risk of developing pancreatic cancer in
diabetic individuals who meet certain criteria (Pannala et al.,
2009, Lancet Oncol 10:88-95). Longitudinal surveillance of these
patients by use of the PAM4-immunoassay may provide for early
detection of neoplasia. A second potential use of the immunoassay
could be as a means to detect recurrence of disease post-therapy,
and in particular, following surgical resection for those patients
where the tumor is supposedly confined to the pancreas.
[0550] The relatively high specificity of the PAM4 antibody
provides a means to target both imaging and therapeutic agents with
high tumor uptake and high tumor/nontumor ratios. We have
demonstrated PAM4's potential as both a directly-radiolabeled or
bispecific, pretargeting reagent for nuclear imaging and
radioimmunotherapy of pancreatic cancer. Also, initial results of a
clinical phase 1b trial to evaluate a fractionated dosing of
.sup.90Y-PAM4 whole IgG (clivatuzumab tetraxetan), in combination
with a radiosensitizing regimen of gemcitabine, were reported
recently (Pennington et al., 2009, J Clin Oncol 27:15s, abstract
4620). Of 22 patients with stage-3/4 disease (mostly stage-4), 68%
showed evidence of disease control, with 23% of patients having
partial responses based on RECIST criteria. Thus, positive results
by the PAM4-based immunoassay provides a rationale to pursue
PAM4-targeted imaging and therapy, thus providing a personalized
therapy.
[0551] The PAM4-based immunoassay can identify the majority of
pancreatic adenocarcinoma patients of all stages. Although a direct
comparison with CA19.9 was not possible in the current study, a
prior comparison of the two biomarkers in a limited set of
pancreatic adenocarcinoma sera (N=41) demonstrated a statistically
significant difference (P<0.01) with PAM4-antigen levels
positive in 71% of patient specimens and CA19.9-antigen levels
positive in 59% of specimens. In general, it is thought that CA19.9
lacks the sensitivity and specificity to provide for early
detection and/or diagnosis of pancreatic adenocarcinoma. However,
the assay does have its use for management with continued elevation
in CA19.9 serum levels post treatment indicative of a poor
prognosis. Similarly, we recently reported in abstract form
(Pennington et al., 2009, J Clin Oncol 27:15s, abstract 4620), the
use of circulating PAM4-antigen levels for prediction of anti-tumor
response.
[0552] These results show that the conditions under which specimens
are stored (e.g., the length of time they are kept frozen) can have
significant effects upon accessibility of the epitope under study.
For the PAM4-based immunoassay, a fatty acid or lipid substance may
be able to bind the specific epitope and interfere with the
immunoassay. However, it is also possible this material was a
low-molecular weight peptide or other substance soluble in organic
solvents. The ability to remove this substance by organic
extraction of the serum makes the PAM4-immunoassay reproducible. In
addition, the question is raised as to the biological significance
of the circulating inhibitor:MUC5AC interaction. However, when
using the PAM4 antibody as an in vivo targeting agent (e.g.,
radioimmunotherapy), the presence of circulating PAM4-antigen is
not a factor, since targeting of radiolabeled-PAM4 to sites of
tumor growth has been observed in the majority of patients
evaluated to date. Thus, it appears that the PAM4-antigen within
tumor is free of the blocking substance.
Example 29
Phase IB/II Study of .sup.90Y-Labeled hPAM4 Antibody and
Gemcitabine in Advanced Pancreatic Cancer
[0553] A phase IB/II study of .sup.90Y-labeled hPAM4 antibody
(clivatuzumab tetraxetan) in advanced pancreatic cancer patients
was performed. A total of 100 patients with previously untreated
Stage III or IV pancreatic cancer were enrolled into this
open-label trial to receive gemcitabine once-weekly.times.4 with
.sup.90Y-clivatuzumab tetraxetan on weeks 2, 3 and 4 (therapy
cycle). The therapy cycle could be repeated until disease
progression or until the patient displayed unacceptable toxicity.
Ten patients withdrew early, while 90 patients, of whom 82 had the
Stage IV (metastatic) disease, received 1-4 therapy cycles. Tumor
responses were assessed by CT, FDG/PET and serum CA19.9 after each
cycle (initially every 4 wks).
[0554] In Part I of this study, 38 patients were treated with
.sup.90Y-clivatuzumab tetraxetan at 6.5, 9, 12 or 15
mCi/m.sup.2.times.3, and a low, fixed gemcitabine dose of 200
mg/m.sup.2.times.4 for radiosensitization. Thirteen patients were
retreated with the same cycle 1-3 times. The overall disease
control rate, which included complete response (CR), partial
response (PR) and stable disease (SD), by CT-based RECIST criteria,
was 58%, including 6 patients (16%) with PR and 16 patients (42%)
with SD as best response.
[0555] The median overall survival (OS) for the 38 treated patients
was 7.7 months, which compares favorably with other regimens for
advanced pancreatic cancer. At the higher therapy doses (12 and 15
mCi/m.sup.2 of .sup.90Y-clivatuzumab tetraxetan.times.3), a median
OS of 8.0 months was noted. For the 13 patients who received
repeated cycles of the combination therapy, median OS improved to
11.8 months. Extended survival of up to 14.8 months post therapy
onset has been observed, with 8 patients achieving a survival >6
months (3 patients >1 yr). Anecdotal reports indicate
performance status and pain level improved with therapy.
[0556] Fifty-two patients who were treated in Part II of this study
received 3 weekly .sup.90Y doses of 12 mCi/m.sup.2 and gemcitabine
doses of 200, 600 or 1000 mg/m.sup.2.times.4, with 14 patients
receiving repeated therapy cycles at the same gemcitabine dose but
.sup.90Y doses of 6.5, 9 or 12 mCi/m.sup.2. Results were available
from 47 of the 52 patients. The disease control rate for the 200
mg/m.sup.2 group was 72%, with 19% PR and 53% SD. For the 600 and
1000 mg/m.sup.2 groups, the disease control rates were 63% (0% PR)
and 68% (18% PR), respectively. Higher gemcitabine doses appeared
to offer no advantage in treatment response over the lowest dose of
200 mg/m.sup.2. At the time of reporting, survival data were not
available for this group of patients. Treatments were well
tolerated with no infusion reactions to radiolabeled clivatuzumab
and few non-hematologic side effects. Hematologic suppression was
transient after cycles 1 and 2.
[0557] These results showed that repeated cycles of fractionated
doses of clivatuzumab tetraxetan, labeled with yttrium-90
(.sup.90Y) and given in combination with gemcitabine, demonstrated
therapeutic activity in patients with advanced, inoperable,
pancreatic cancer. Therapy with repeated cycles of clivatuzumab
tetraxetan plus low-dose gemcitabine improved overall survival over
single-cycle therapy in patients with locally advanced or
metastatic pancreatic cancer.
Example 30
Detection of Early-Stage Pancreatic Ductal Adenocarcinoma (PDAC):
Sensitivity, Specificity, and Discriminatory Properties of
Serum-Based PAM4-Immunoassay
[0558] As disclosed in Example 28, a serum-based enzyme immunoassay
employing the PAM4 antibody was able to correctly identify 81% of
patients with known PDAC and this assay had promising sensitivity
for detecting early-stage disease. These findings have been
extended in a much larger patient population that included over 600
sera from both malignant and benign diseases of the pancreas and
surrounding tissues. In a blinded analysis, sera from patients with
confirmed PDAC (N=298), other cancers (N=99), benign disease of the
pancreas (N=126), and healthy adults (N=79) were evaluated by
enzyme immunoassay for concentration of PAM4-antigen levels.
[0559] Overall sensitivity for detection of PDAC was 76%, with 64%
of stage-1 patients testing positive and a higher sensitivity (85%)
for advanced disease. For the most part, sera from patients with
neuroendocrine tumors of the pancreas or cancers of other origin
(squamous, GIST, etc.) did not have elevated levels of the
PAM4-antigen. Approximately half of the patients with ampullary
(48%) and extrahepatic biliary (50%) adenocarcinomas had positive
levels of circulating PAM4-antigen. Of 126 patients diagnosed with
benign conditions of the pancreas, only 24 (19%) were positive and,
in particular, 18 of 80 (23%) patients with chronic pancreatitis
(CP) were positive. ROC curve analysis demonstrated a statistically
significant difference between the PDAC and CP groups
(P<0.0001), with an area under the curve of 0.84.+-.0.02 (95%
CI: 0.79-0.89). The positive- and negative-likelihood ratios for
differentiating PDAC from benign conditions of the pancreas were
4.00 and 0.30, respectively.
[0560] In conclusion, the PAM4-immunoassay detected nearly
two-thirds of stage-1 PDAC patients, and did so with high
discriminatory power with respect to benign pancreatic disease. The
results provide a rationale for longitudinal surveillance of
patients considered at high-risk for PDAC (e.g., familial
pancreatic cancer, new-onset diabetes, etc.) with the PAM4
assay.
Example 31
PAM4-Based Assay Differentiates Pancreatic Ductal Adenocarcinoma
(PDAC) from Chronic Pancreatitis and Benign Nonmucinous Pancreatic
Cysts
[0561] We examined the expression of PAM4-reactive MUC5AC in
chronic pancreatitis and benign non-mucinous cystic lesions of the
pancreas. A tissue microarray of PDAC (N=14), as well as surgical
specimens from chronic pancreatitis (N=32) and benign non-mucinous
cystic lesions of the pancreas (N=19), were assessed by
immunohistochemistry for expression of the PAM4-reactive MUC5AC, as
well as MUC1 (mAb-MA5), MUC4 (mAb-8G7), and CEACAM6
(mAb-MN-15).
[0562] PAM4-reactive MUC5AC, MUC1, MUC4 and CEACAM6 were expressed
in 79% (11/14), 100% (14/14), 86% (12/14) and 100% (14/14) of
invasive pancreatic adenocarcinoma. PAM4 only weakly labeled 6%
(1/19) of benign non-mucinous cystic lesions, 1 of 15 serous
cystadenomas (SCAs) and 0 of 4 cysts with squamous epithelial
lining (2 lymphoepithelial cysts, and 2 retention cysts with
squamous metaplasia). However, the expression of MUC1, MUC4 and
CEACAM6 was detected in 53% (8/15), 0% (0/15) and 13% (2/15) of
SCAs, and in 4, 3 and 3 of the 4 cysts with squamous epithelial
lining, respectively. PAM4 labeled 19% (6/32) of chronic
pancreatitis specimens; however, this PAM4 reactivity was
restricted to the PanIN precursor lesions associated with chronic
pancreatitis. Inflamed tissue was negative. The expression of MUC1,
MUC4 and CEACAM6 was detected in 90% (27/30), 78% (25/32), and 97%
(31/32) of chronic pancreatitis. In all of the positively-labeled
specimens, the reactivity was present in non-neoplastic inflamed
pancreatic tissue in addition to PanIN.
[0563] In conclusion, the expression of PAM4 was detected in only
6% of benign non-mucinous cystic lesions and in the precursor
lesions associated with chronic pancreatitis. These results suggest
that PAM4, in contrast to MUC1, MUC4, and CEACAM6, may be useful to
differentiate benign non-mucinous cystic lesions of the pancreas
and chronic pancreatitis from PDAC.
Example 32
Combination of the PAM4 and CA19-9 Biomarkers for Improved
Detection of Pancreatic Adenocarcinoma
[0564] Pancreatic ductal adenocarcinoma (PDAC) is almost
universally lethal, due mainly to the inability to detect
early-stage disease. Thus, identification of biomarkers that can
identify patients with early-stage PDAC may improve overall
survival. In a blinded study, PAM4 and CA19-9 immunoassays were
performed on sera from 480 patients, including those with confirmed
PDAC (N=234), other cancers (N=84), benign diseases of the pancreas
(N=89), and healthy adults (N=50).
[0565] Overall sensitivity for PDAC was similar, 74% and 77% for
PAM4 and CA19-9, respectively. Sensitivity for detection of early,
stage-1 disease (N=26), although somewhat higher for the
PAM4-antigen, was also statistically similar, 65% and 58% for PAM4
and CA19-9, respectively (P=0.5775). However, specificity was
significantly lower for CA19-9, particularly with respect to
chronic pancreatitis (CP): 68% vs. 86% for the PAM4 assay
(P=0.014). Furthermore, CA19-9 results showed considerably higher
detection rates for non-PDAC neoplasia, including patients with
other cancers that metastasized to the pancreas. Thus, positive
likelihood ratios (+LR) were lower for CA19-9 (+LR=2.41) than for
the PAM4 assay (+LR=5.29).
[0566] PAM4 and CA19-9 antigen levels in PDAC were independent of
each other (r.sup.2=0.003, P=0.410); however, the positive and
negative interpretations were concordant in 68% of the cases. Thus,
a combined biomarker analysis improved the overall PDAC detection
rate (84%), without a significant decrease in specificity (83%).
Comparison of the ROC curves for PDAC vs. CP and PDAC vs. benign
disease demonstrated a statistically significant improvement for
the combined immunoassay, as compared to either assay alone
(P<0.0001 in both comparisons), to detect and discriminate PDAC
from benign disease.
[0567] While the PAM4-immunoassay provided high sensitivity and
specificity for detection and diagnosis of PDAC, inclusion of the
CA19-9 biomarker significantly enhanced positive identification of
PDAC patients, from 74% to 84%.
Example 33
Use of PAM4-Immunoassay as a Correlate of Tumor Response
[0568] We investigated whether specific trends in PAM4-reactive
MUC5AC concentrations (within the individual patient) can be used
as an indicator of tumor response after therapy. Several patients
from a .sup.90Y-hPAM4 phase-1b/II clinical trial now in progress
were evaluated. When patients were evaluated 4 weeks after
treatment had ended (a treatment cycle is 4 weeks), a decrease in
serum antigen levels of >40% was suggestive of a response. All
of the patients who had progressive disease had levels of PAM4
antigen that continued to rise. Trends are presented for two
patients in FIG. 28A and FIG. 28B. In both cases, trends in the
level of circulating MUC5AC were concordant with the trend in tumor
volume as determined by CT. These results suggest that serum PAM4
levels are of use to monitor responsiveness to anti-cancer
treatments for pancreatic cancer.
Example 34
Identification of Target Antigen for PAM4 Antibody
[0569] We performed a set of blocking and capture/probe paired
enzyme immunoassays to evaluate the relationships between the PAM4
antibody and antibodies reactive with MUC1 (MA5, KC4, HMFG1, SM3,
H23), MUC2 (G9), MUC4 (8G7) and MUC5AC (45M1). A mucin standard
derived from the CaPan1 human tumor xenograft was shown to contain
the reactive mucin species for all of these antibodies except those
reactive with G9 (MUC2). Of all MAbs examined, only 1 (45M1)
reported to be reactive with MUC5AC provided a positive reaction in
sandwich EIA when PAM4 was used as the capture reagent. The 45M1
antibody is reactive with a much lower percentage of pancreatic
carcinomas than PAM4 (by IHC on TMA) and so cannot be used as a
single probe for the serum-based PAM4-immunoassay.
[0570] As described above, we performed a peptide-phage-display
study by consecutive biopanning with the murine and humanized
versions of PAM4-IgG. A consensus sequence (12mer--WTWNITKAYPLP
(SEQ ID NO: 7)) was generated which when input into a BLAST protein
search with query coverage set at 100%, identified MUC5AC and MUC16
with 7 of 12 and 5 of 12 identical amino acids within the 12mer
sequence, respectively.
[0571] Studies were performed using mass spectrometry to identify
PAM4-immunoprecipitated antigens from credentialed cyst fluids
(these fluids were previously analyzed by mass spectrometry to
identify specific MUCs present in the mixtures). By PAGE analyses
of the PAM4-immunoprecipitated materials from 3 individual cyst
fluid specimens, only two identical bands were present in each
specimen (not shown). Both of these bands contained MUC5AC as the
major mucin species.
[0572] We have investigated the nature of the substance within
human blood that binds to the PAM4-epitope, which necessitates
organic extraction prior to immunoassay. As discussed above,
Slomiany and co-workers have observed that gastric mucin had
covalently bound and/or associated lipids and fatty-acids. Further,
fatty-acid synthetase levels and activity are significantly
elevated in pancreatic adenocarcinoma, as is also the case for
other forms of cancer and other pathologic conditions. Speculating
that the blocking substance might be lipid in nature, we performed
an EIA (FIG. 29) in the presence and absence of 100 .mu.M palmitic
acid and observed a statistically significant 69% reduction in
reactivity at an OD450 equivalent of 1.0 (P<0.0001). It is noted
that the normal adult serum level of palmitic acid is in the range
of 1,480 to 3,730 .mu.M, considerably higher than the concentration
that was used in this EIA experiment.
Example 35
PAM4 Differentiates Between Pancreatic Ductal Adenocarcinoma (PDAC)
and Chronic Pancreatitis (CP)
[0573] Current practice guidelines suggest that patients who
present with signs and/or symptoms suspicious of pancreatic cancer
undergo a pancreatic protocol CT imaging study for detection of
tumor mass within the pancreas. Follow-up imaging by endoscopic
technologies (e.g., EUS, ERCP) can provide high sensitivity for
detection of disease, and when combined with fine-needle
aspiration/biopsy, can provide good diagnostic accuracy. However,
the majority of these procedures have been performed on patients
with advanced disease; that is, tumors greater than 2 cm. Detection
of early pancreatic cancer is still problematic, especially when
occurring in a background of pancreatitis. Thus, the current
reality is that only 7% of all cases detected are early disease.
With no effective treatment for advanced PC, the prognosis for
these patients is dismal.
[0574] Biomarkers that can reliably distinguish between cancer and
benign conditions, and/or provide means to prioritize patients for
follow-up evaluation, would be of significant clinical value,
especially if the biomarker is capable of detecting early disease.
We have developed monoclonal antibody PAM4 that demonstrates a high
degree of specificity for pancreatic ductal adenocarcinoma
(PDAC).
[0575] MAbs having defined reactivity with several mucin species,
including MUC1, MUC2, MUC3, MUC4, MUC5AC, etc., were evaluated for
signal response in a heterologous PAM4-capture sandwich EIA. The
only MAbs able to provide signal response (45M1, 2-11M1) are known
to react with specific domains of the MUC5AC mucin. Further, three
additional anti-MUC5AC MAbs (21M1, 62M1, and 463M1) were each able
to inhibit the interaction between PAM4 and its mucin antigen.
These data suggest MUC5AC as an antigen to which PAM4 is reactive.
PAM4, unlike other anti-MUC5AC MAbs (45M1, 2-11M1, CLH2, and
others), demonstrates greater specificity for PDAC than cancers
originating from other organs, and may serve as a useful biomarker
for PDAC, as well as a target for antibody-directed imaging and
therapy.
TABLE-US-00040 TABLE 22 PAM4-Antigen In the Serum of Patients with
Known Disease Number of Percent of Median Positive Positive
Pancreatic Cancer N (units/mL) Cases Cases Ductal Adenocarcinoma
298 10.40 225 76 Neuroendocrine 20 0.08 2 10 Other Morphology 7
0.51 1 14 Non-PC, Mets to the Pancreas 11 0.00 2 18 Ampullary
Adenocarcinoma 21 1.52 10 48 Biliary Adenocarcinoma 26 4.41 13 50
Cholangiocarcinoma 7 1.07 2 29 Duodenal Adenocarcinoma 7 2.80 4 57
All Biliary and Periampullary 61 1.78 29 48 Colon Carcinoma 32 0.15
5 16 Chronic Pancreatitis (CP) 80 0.41 18 23 Benign Cystadenoma 15
0.18 1 7 Benign - Other 25 0.20 5 20 All Benign Disease 120 0.26 24
20 Healthy Volunteers 79 0.27 3 4 All groups are statistically
different from the pancreatic adenocarcinoma group with P values
equal to or better than 0.0001; Mann-Whitney nonparametric test.
Gold DV, Gaedcke J, Ghadimi BM, et al. Cancer. 2013 Feb 1; 119(3):
522-8.
[0576] The PDAC group consisted of 40% early and 60% advanced stage
patients. Detection rates were 64% and 85%, respectively. The
sensitivity and specificity of the PAM4 assay was determined for
PDAC vs. CP (FIG. 30A) and for PDAC vs. all benign tissue samples
(FIG. 30B). The calculated values of AUC were 0.84 and 0.85,
respectively.
[0577] Approximately 20% of patients with chronic pancreatitis (CP)
are positive by use of the serum-based immunoassay. This issue is
critical to the interpretation of the results with PAM4-positive CP
patients being either false positives, or perhaps, the discovery of
occult neoplasia. Thus, we undertook an extensive
immunohistochemical evaluation of PAM4-reactivity in CP tissue
specimens.
[0578] FIG. 31 shows comparative labeling of PDAC vs.
non-neoplastic prostate tissue by PAM4 antibody vs. antibodies
against MUC1, MUC4, CEACAM6 and CA19-9. Each of the antibodies
reacted with PDAC. PAM4 showed no reactivity with normal tissue.
The same antibodies were compared in a sample showing a PanIN-2
lesion arising within a background of CP, with partial loss of
acinar cells, some fibrosis and PanIN-associated acinar-ductal
metaplasia (ADM) (not shown). No labeling was observed with PAM4 in
any of the tissues within CP, including isolated ADM (not shown).
Each of the other antibodies showed some binding to non-neoplastic
tissue (not shown). Table 23 and Table 24 show comparative results
of labeling with PAM4 vs. antibodies against MUC1, MUC4, CEACAM6
and CA19-9.
TABLE-US-00041 TABLE 23 Expression of Biomarkers in Pancreatic
Ductal Adenocarcinoma PAM4 MUC1 MUC4 CEACAM6 CA19-9 Number 43 43 43
42 43 Focal Labelinga 8 (24%)b 1 (2%) 4 (15%) 3 (8%) 2 (5%) Diffuse
Labeling 26 (76%) 42 (98%) 22 (85%) 35 (92%) 37 (95%) Total Labeled
34 (79%) 43 (100%) 26 (60%) 38 (90%) 39 (91%) Adjacent Normal 0
(0%) 14 (100%) 6 (43%) 14 (100%) 14 (100%) (N = 14) a-Focal
labeling, 5% to 25% of the appropriate tissue components labeled
with the indicated MAb; Diffuse, >25% of the appropriate tissue
components labeled with the indicated MAb; Total, focal + diffuse.
bvalue provided in parenthesis is the percentage of total N PDAC
specimens evaluated
TABLE-US-00042 TABLE 24 Expression of Biomarkers in Chronic
Pancreatitis N PAM4 MUC1 MUC4 CEACAM6 CA19-9 Chronic 32
Pancreatitis PanIN1 5 2 2 1 5 5 PanIN2 5 4 4 3 5 5 Ducts 32a 0 22
25 31 29 Acinar cells 32a 0 27 8 30 29 Isolated 32a 0 24 0 0 26
ADM
[0579] We conclude that PAM4 is not reactive with the
non-neoplastic tissues from chronic pancreatitis (CP) patients, but
rather with PDAC and its neoplastic precursor lesions, such as
PanINs, which are known to develop within the inflamed parenchyma.
Together with results from a prior study, we have evaluated a total
of 51 specimens of CP, finding that in no instance was PAM4
reactive with the inflamed parenchyma. On the other hand, each of
the other biomarkers investigated, MUC1, MUC4, CEACAM6, and CA19-9,
were unable to differentiate PDAC and benign, non-neoplastic
tissues. These latter biomarkers were expressed to varying extents
in CP-associated PanIN lesions, but also in non-neoplastic ducts
and isolated ADM. A PAM4-based EIA to quantitate antigen in patient
sera shows high sensitivity and specificity for detection of PDAC.
Approximately 2/3 of patients with stage-1 disease are positive for
circulating PAM4-antigen. We speculate that CP patients (and
perhaps others having disease with high risk for development of
PDAC), who are found to have positive levels of PAM4-reactive
antigen in the circulation, may have occult PDAC and/or significant
mass of precursor lesions producing the PAM4-biomarker.
Example 36
Mapping the PAM4 Epitope on MUC5AC
[0580] Summary
[0581] Indirect and sandwich enzyme immunoassays (EIA) were
performed to compare and contrast the reactivity of PAM4 with
several anti-mucin antibodies having known reactivity to specific
mucin species (e.g., MUC1, MUC4, MUC5AC, etc.). Studies designed to
block reactivity of PAM4 with its specific antigen also were
performed. We demonstrated that MAbs 2-11M1 and 45M1, each reactive
with MUC5AC, are able to provide signal in a heterologous sandwich
immunoassay where PAM4 is the capture antibody. Further, we
identified MAbs 21M1, 62M1, and 463M1, each reactive with MUC5AC,
as inhibiting the reaction of PAM4 with its specific epitope. MAbs
directed to MUC1, MUC3, MUC4, MUC16 and CEACAM6 were not reactive
with PAM4-captured antigen, nor are they able to block the reaction
of PAM4 with its antigen. We concluded that MUC5AC is the mucin
species to which PAM4 antibody is reactive.
Background
[0582] Mucin glycoproteins are high molecular weight, heavily
glycosylated, proteins that include at least 19 species categorized
on the basis of their unique protein cores. They can be found as
either transmembrane components of the cell or as secreted
products. Abnormal expression of mucins is a well-known occurrence
in many forms of cancer (see Hollingsworth & Swanson, 2004, Nat
Rev Cancer 4:45-60; Kufe, 2009, Nat Rev Cancer 9:874-85; Rachagani
et al., 2009, Biofactors 35:509-27), including pancreatic ductal
adenocarcinoma (PDAC) (Ringel & Lohr, 2003, Mo lancer 2:9-13;
Andrianifahanana et al., 2001, Clin Cancer Res 7:4033-40; Torres et
al., 2012, Curr Pharm Des 18:2472-81). Neo-expression and/or
upregulation/downregulation of specific mucin species, with and
without the generation of newly transcribed and translated splice
variants (Schmid, 2003, Oncol Rep 10:1981-85), have been
well-documented in the literature. Alteration of carbohydrate
moieties through the addition of new terminal sugars (e.g.,
neuraminic acids), underglycosylation, and other abnormal
biochemical pathways also have been observed (Brockhausen, 2006,
EMBO Rep 7:599-604; Yue et al., 2009, Mol Cell Proteomics
8:1697-707; Haab et al., 2010, Ann Surg 251:937-45). These
modifications may lead to changes in conformational structure
and/or appearance or disappearance of specific epitopes.
Additionally, changes may be observed for the intracellular
distribution of the mucin species under consideration, such as
MUC1, which in normal tissues is a transmembrane glycoprotein, but
with neoplastic transformation is found in the cytoplasm as well
(Jass et al., 1995, J Pathol 176:143-49; Cao et al., 1997, Virchows
Arch 431:159-66). These events may prove to be of biological and
clinical significance in the process of neoplastic development and
progression, as well as provide new biomarkers/targets for early
detection and targeted therapy of cancer.
[0583] Our laboratory initially reported the use of a polyclonal
antiserum to identify a pancreatic ductal mucin, which at the level
of sensitivity provided by indirect immunohistochemistry (IHC), was
shown to contain an epitope relatively specific to the pancreas
(Gold et al., 1983, Cancer Res 43:235-38), and ultimately resulted
in the development of monoclonal antibody (MAb), PAM4 (Gold et al.,
1994, Int J Cancer 57:204-10), also known as clivatuzumab in its
humanized form. PAM4 demonstrates high specificity for PDAC with
little to no reactivity towards normal and benign, non-neoplastic,
pancreatic tissues, although it does show limited reactivity
(approximately 10% of all specimens examined) with adenocarcinomas
originating in certain other organs (e.g., stomach, colon, lung)
(Gold et al., 1994, Int J Cancer 57:204-10; Gold et al., 2007, Clin
Cancer Res 13:7380-87; Gold et al., 2010, Cancer Epidemiol
Biomarkers Prev 19:2786-94). PAM4 identifies a biomarker that, if
present, provides a high diagnostic likelihood of the presence of
pancreatic neoplasia (Gold et al., 2010, Cancer Epidemiol
Biomarkers Prev 19:2786-94; Gold et al., 2006, J Clin Oncol
24:252-58; Gold et al., 2013, Cancer 119:522-28). Thus, clinical
applications for detection of early-stage disease (Gold et al.,
2010, Cancer Epidemiol Biomarkers Prev 19:2786-94; Gold et al.,
2013, Cancer 119:522-28), and antibody-targeted imaging and
therapy, are being pursued (Gulec et al., 2011, Clin Cancer Res
17:4091-4100; Ocean et al., 2012, Cancer 118:5497-5506). In
addition to PDAC, the PAM4-biomarker is expressed in the precursor
lesions, pancreatic intraepithelial neoplasia (PanIN, including the
earliest developing lesion, PanIN-1A), and intraductal papillary
mucinous neoplasia (IPMN), suggesting that there may be oncogenic
significance to its expression (Gold et al., 2007, Clin Cancer Res
13:7380-87). In the current study, we investigated the identity of
the mucin species to which this clinically-relevant antibody is
reactive, in order to understand what role this mucin may play in
the development and progression of pancreatic cancers.
Methods
[0584] Antigen and Antibodies--
[0585] A mucin containing fraction, designated CPM1, was isolated,
as described previously (Gold et al., 2006, J Clin Oncol
24:252-58), from the Capan-1 human PDAC xenograft in athymic nude
mice. Briefly, this consisted of homogenization of the dissected
tumor in 0.1M ammonium bicarbonate containing 0.5M sodium chloride.
Following high-speed centrifugation (20,000 g.times.45 min), the
soluble material was chromatographed on a SEPHAROSE.RTM. 4B-CL
column, and then eluted with the identical ammonium
bicarbonate-sodium chloride solution. The void volume material was
collected, dialyzed against 0.01M sodium phosphate, pH 7.2, and
then passed through hydroxyapatite to remove nucleic acids and
proteins. The non-binding, mucin-containing fraction was again
dialyzed extensively to remove salts and used as a source of
antigen.
[0586] Antibodies used in the current study are listed in Table 25
with clone and source information. For sandwich and blocking
studies, PAM4 was available in both murine (mPAM4) and humanized
(hPAM4; clivatuzumab) versions provided by Immunomedics, Inc.
(Morris Plains, N.J.). All other MAbs were murine IgG. Mouse
ascites fluids containing MAbs 21M1, 45M1, 62M1 and 463M1 were
kindly provided by Dr. J. Bara, INSERM, Paris, France. PAM4
antibodies and ascites fluid containing an anti-alpha-fetoprotein
antibody, employed as a negative control for the blocking studies
(reactive with Hep-G2, hepatoceullar carcinoma cells) were provided
by Immunomedics, Inc. (Morris Plains, N.J.). A rabbit polyclonal
anti-CPM1 (Gold et al., 1994, Int J Cancer 57:204-210; Gold et al.,
2010, Cancer Epidemiol Biomarkers Prev 19:2786-94) IgG served as
the positive control with detection by a horseradish peroxidase
(HRP)-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, West
Grove, Pa.).
TABLE-US-00043 TABLE 25 Monoclonal antibodies used Antigen Clone
name Source MUC1 MA5 Immunomedics MUC1 KC4 Immunomedics MUC1 CM1
Gene Tex MUC2 994/152 Abcam MUC3 M3.1 Abcam MUC3 M3A LifeSpan Bio
MUC4 8G7 Santa Cruz Biotech MUC5AC 2-11M1 Santa Cruz Biotech MUC5AC
45M1 Santa Cruz Biotech MUC5AC CLH2 Santa Cruz Biotech MUC16 X306
Novus Bio MUC16 X325 Abcam CEACAM5 MN14 Immunomedics CEACAM6 MN15
Immunomedics CA 19-9 CA 19-9 Santa Cruz Biotech Immunomedics, Inc.
- Morris Plains, NJ; GeneTex - Irvine, CA; Abcam - Cambridge, MA;
LifeSpan Biosciences, Inc. - Seattle, WA; Santa Cruz Biotechnology,
Inc. - Santa Cruz, CA; Novus Biologicals - Littleton, CO.
[0587] Enzyme Immunoassay--
[0588] Procedures have been described for both indirect and
sandwich enzyme immunoassays (Gold et al., 1994, Int J Cancer
57:204-210; Gold et al., 2010, Cancer Epidemiol Biomarkers Prev
19:2786-94). For indirect immunoassays, primary MAbs were used at a
concentration of 10 .mu.g/mL to provide high sensitivity for signal
detection. For sandwich immunoassays, the capture MAb was coated
onto the wells at a concentration of 10 .mu.g/mL, followed by the
addition of the CPM1 antigen at various concentrations up to 10
.mu.g/mL. The MAb probe was then added at a high concentration of
10 .mu.g/mL for detection of response to captured antigen.
Secondary HRP-labeled anti-species-specific IgG (Jackson
ImmunoResearch, West Grove, Pa.) was evaluated initially to
determine optimum concentrations for use in the assay (usually
1:1000 or 1:2000). MAb inhibition studies were performed by adding
the inhibiting MAb to wells coated with CPM1 antigen, starting at a
high concentration of 100 g/mL of pure MAb or 1:10 dilution of
ascites fluid, and titrating to lower amounts. After incubating
with the inhibiting antibody at 37.degree. C. for 1 h, the plates
were washed, and hPAM4 added to the wells at a concentration of
0.25 .mu.g/mL. hPAM4 binding was then detected with a secondary
probe, HRP-labeled anti-human IgG conjugate.
[0589] SDS-PAGE and Western-Blotting--
[0590] SDS-PAGE was performed under non-reducing conditions using
4-20% Tris-Glycine gels at 125V for about 2 h. Resolved proteins
were transferred onto a nitrocellulose membrane using the Mini
TRANS-BLOT.RTM. cell system (Bio-Rad Laboratories, Hercules,
Calif.) at 100 V for 1 h. To examine the identity of recombinant
proteins, triplicate samples were run in the same gel and membrane
with transferred samples were cut into three pieces for probing
with HRP-anti-Myc, HRP-hPAM4, and 45M1 plus HRP-GAM, respectively.
The signals were developed with SUPERSIGNAL.TM. West Dura
Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham,
Mass.).
Results
[0591] Several MAbs were evaluated by indirect EIA for reactivity
with plates coated with CPM1 (FIG. 32), a high molecular weight
mucin fraction isolated from the Capan-1 human pancreatic cancer
xenograft. Murine PAM4 and MAbs reactive specifically with MUC1 and
MUC5AC mucins provided elevated reactivity in this indirect
immunoassay, with minor reactivity also observed for MAbs directed
to MUC3 and CEACAM6. Essentially no reaction was seen with MAbs to
MUC2, MUC4, MUC16, and CEACAM5 glycoproteins, or the CA19-9
carbohydrate epitope.
[0592] It should be noted that a negative EIA reaction does not
necessarily indicate absence of the mucin-antigen, because the
specific epitope structure may be present, but inaccessible (i.e.,
cryptic). This is likely the case for MAb-CLH2 anti-MUC5AC
generated against a peptide derived from the mucin's tandem repeat
(Reis et al., 1997, Int J Cancer 74:112-21), since the other two
anti-MUC5AC MAbs were highly reactive. Similarly, CM1 anti-MUC1 was
considerably less reactive than MA5 and KC4 anti-MUC1 antibodies.
Capan-1 cells produce well-differentiated tumors with highly
glycosylated mucins. Thus, it is likely that both CLH2 and CM1,
reactive with the tandem repeat domains of their respective mucins,
would not be reactive with CPM1, since the tandem repeat epitopes
are inaccessible.
[0593] We then evaluated whether the anti-mucin MAbs were reactive
with PAM4-captured mucin. Humanized PAM4 (hPAM4)-coated plates were
used to capture the specific mucin-antigen from the CPM1 fraction,
which was then probed with various anti-mucin MAbs. Murine MAbs
(mMAbs) specifically reactive with MUC1, MUC3, MUC4, MUC16 and
CEACAM6 did not provide a signal in these heterologous sandwich
immunoassays (not shown). On the other hand, both anti-MUC5AC mMAbs
tested, 45M1 and 2-11M1, gave positive reactions with the
hPAM4-captured antigen (FIG. 33), with 45M1 showing significantly
greater reaction than 2-11M1 (Kd=14.32.+-.1.08 .mu.g/mL and
24.4.+-.7.83 .mu.g/mL, respectively, for MAbs 45M1 and 2-11M1;
P<0.001). However, neither of these individual anti-MUC5AC MAbs
provided as strong signal intensity as the rabbit anti-CPM1
polyclonal IgG fraction. Importantly, mPAM4 did not bind to the
hPAM4-captured antigen, nor did hPAM4 bind to mPAM4-captured
antigen, suggesting that the PAM4 epitope is present at low
density, possibly only a single site within the mucin-antigen.
[0594] Follow-up studies were designed to inhibit the binding of
hPAM4 to CPM1-coated plates (FIG. 34A-B). Although 2-11M1
anti-MUC5AC was unable to inhibit hPAM4-CPM1 binding, 45M1
anti-MUC5AC was able to provide a limited inhibitory effect, with
IC.sub.max=25.5% inhibition (FIG. 34A). mPAM4, included as a
positive control, provided IC.sub.max=92.4% self-inhibition at a
concentration 0.1 .mu.g/mL, while the MA5 and KC4 anti-MUC1
antibodies provided no inhibition, even at the highest
concentration evaluated (10 .mu.g/mL) (FIG. 34A). hPAM4 was unable
to completely block mPAM4 binding to the CPM1 antigen
(IC.sub.max=52.8%) (not shown), a not unexpected finding since the
humanized version of PAM4 is known to have a lower affinity than
the murine parent. Ascites fluids containing mMAbs with known
mapping to MUC5AC were serially diluted as inhibitory reagents,
with results shown in FIG. 34B. mMAbs 21M1, 62M1, and 463M1 each
provided inhibition similar to the results shown for mPAM4
self-blocking, with 45M1 ascites providing limited inhibition,
similar to what was observed with the commercially available
45M1-IgG (FIG. 34B). Ascites fluid containing a murine
anti-alpha-fetoprotein (AFP), included here as a negative control,
provided no inhibition of the hPAM4 binding to CPM1 (FIG. 34B).
Unfortunately, insufficient volumes of ascites precluded
determination of MAb concentrations, so that relative blocking
efficiency could not be calculated.
Discussion
[0595] The current Example suggests that PAM4 is reactive with the
MUC5AC mucin glycoprotein. FIG. 35 presents a map of the MUC5AC
mucin domains with reactive epitopes indicated for several of the
anti-MUC5AC MAbs employed in our studies (Nollet et al., 2002, Int
J Cancer 99:336-43; Nollet et al., 2004, Hybrid Hybridomics
23:93-99; Lidell et al., 2008, FEBS J 275:481-89). CLH2 is reactive
with the peptide core of the tandem repeat domain (Reis et al.,
1997, Int J Cancer 74:112-21), and is likely a cryptic epitope
within the Capan-1 tumor-derived MUC5AC. 2-11M1 is reactive with
the N-terminus of the mucin (Nollet et al., 2004, Hybrid Hyridomics
23:93-99), and 45M1 at the furthest N-terminal region of the
cysteine-rich, C-terminus (Lidell et al., 2008, FEBS J 275:481-89).
Both of these MAbs were reactive with PAM4-captured mucin, whereas
MAbs to MUCs 1, 3, 4, and 16 were not. We observed that 45M1
provides a significantly greater signal response than 2-11M1,
suggesting a greater density of 45M1-epitopes than 2-11M1-epitopes
within CPM1. However, this may simply be due to a loss of 2-11M1
epitopes through proteolytic digestion of the relatively
non-glycosylated N-terminus, and/or molecular shear of this very
large glycoprotein during purification. In any case, the 2-11M1
antibody provided no inhibition of the hPAM4-CPM1 interaction,
suggesting the epitope is located distant to the PAM4-epitope.
[0596] On the other hand, 45M1 did inhibit the hPAM4-CPM1
interaction, albeit only partially, suggesting that the
PAM4-epitope is within the C-terminal region of the mucin or
conformationally altered by interaction of this antibody with the
mucin molecule. MAbs 21M1, 62M1, and 463M1 have also been mapped to
the C-terminal region of the MUC5AC mucin (Nollet et al., 2002 Int
J Cancer 99:336-43; et al., 2004, Hybrid Hybridomics 23:93-99;
Lidell et al., 2008, FEBS J 275:481-89), and each provided
significant inhibition of the PAM4-mucin reaction. Taken together,
our data provide direct evidence that PAM4 is reactive with the
identical mucin (MUC5AC), and that the PAM4 epitope is either
directly-blocked, or conformationally modified, by interaction of
these MAbs with the MUC5AC antigen.
[0597] We had initially reported that PAM4 was reactive with the
MUC1 mucin species (Gold et al., 2007, Clin Cancer Res 13:7380-87;
Gold et al., 2006, J Clin Oncol 24:252-58). This was based upon
MUC1-gene transfection studies, whereby PAM4 was observed to react
with the gene-transfected, MUC1.sup.+ cell line, but not the
MUC1.sup.- parental cell line or vector control cell lines.
However, other evidence acquired since then has questioned this
interpretation, suggesting that MUC1 transfection may have
upregulated other mucins as well. Prior results from our laboratory
lend support to the current findings. The PAM4 epitope was found to
be highly sensitive to mild reduction with dithiothreitol (0.02M,
15 min, 20.degree. C.) or heat (100.degree. C., 2 min), suggesting
the epitope is peptide in nature, and highly dependent upon a
specific conformation of the protein core kept intact by disulfide
bridges (Gold et al., 1994, Int J Cancer 57:204-10). This is
unlikely to be MUC1 with all of the cysteines located within the
transmembrane domain of the mucin, but is consistent with the loss
of reactivity shown by several anti-MUC5AC MAbs upon reduction of
the mucin antigen. Further, employing immunohistochemical methods,
we reported that frequency of expression and morphologic
distribution of the PAM4-epitope within PDAC and its precursor
lesions shared greater similarity to those described for MUC5AC
than for MUC1 (Gold et al., 2007, Clin Cancer Res 13:7380-87).
[0598] In conclusion, antibodies that bind to the PAM4 epitope of
MUC5AC are of use for detection and differential diagnosis of
pancreatic cancer. Immunoconjugates of such antibodies are of use
for pancreatic cancer therapy.
Example 37
DOTA Conjugates of PAM4
[0599] The hPAM4 antibody was prepared as described in Example The
genes of CDR-grafted V.sub.H and V.kappa. chains of hPAM4 were
inserted into the pdHL2 plasmid vector, a DHFR-based amplifiable
expression system. The plasmid was transfected into the murine
myeloma cell line, Sp2/0-Ag14 (ATCC, Manassas, Va.) to generate the
cell clones producing hPAM4. The complete mature amino acid
sequence is shown below.
TABLE-US-00044 hPAM4 Heavy Chain (SEQ ID NO: 120)
QVQLQQSGAEVKKPGASVKVSCEASGYTFPSYVLHWVKQAPGQGLEWIGY
INPYNDGTQYNEKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGF
GGSYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hPAM4 light chain
(SEQ ID NO: 121) DIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLYWYQQKPGKAPKLWIY
STSNLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFG
GGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC
[0600] The DNA and amino acid sequences of hPAM4 V.kappa. and
V.sub.H are shown in FIG. 4A and FIG. 4B, respectively, with the
CDRs identified in bold and underlined.
[0601] The current cell clone name is hPAM4-2E3 and is produced in
Sp2/0 host cells, DHFR expression system. The antibody is a
humanized IgG.sub.1.kappa. glycoprotein. A glycosylation site on
the heavy chain (Asn299) has a composition per mole of hPAM4-DOTA:
0.5 Fuc, 6.3 GlcNAc, 6.3 Man, 0.3 Gal and 0.15 Neu5Gc;
glycosylation species: G0F 70%, G1F 23%, G2F 2%, G1FS1 4%, G2FS1
1%. There are 16 S--S bonds (32 SH), identified and located exactly
as theoretical prediction based on the above sequence.
[0602] An hPAM4-DOTA product was prepared from purified hPAM4 IgG
that was coupled with the 12-membered macrocyclic chelating agent
1,4,7,10-tetraazacyclododecane-N, N',N'',N'''-tetraacetic acid
(DOTA).
[0603] DOTA was conjugated via one of the carboxyl moieties to
reactive sites on the hPAM4 antibody to generate a stable
conjugate. The coupling is assumed to be via stable amide bond to
the antibody's lysine side-chain amino group.
[0604] The chemical conjugation was performed by first reacting
DOTA with N-hydroxysulfo-succinimide (sulfo-NHS) in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to generate
activated DOTA, then incubating activated DOTA with purified hPAM4
antibody. Conditions were optimized to yield a substitution ratio
of 4-7 DOTA moieties per antibody molecule, as determined by mass
spectrometry assays.
[0605] It will be apparent to those skilled in the art that various
modifications and variations can be made to the products,
compositions, methods and processes of this invention. Thus, it is
intended that the present invention cover such modifications and
variations, provided they come within the scope of the appended
claims and their equivalents.
Sequence CWU 1
1
121112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu Tyr
1 5 10 27PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Ser Thr Ser Asn Leu Ala Ser 1 5 39PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3His
Gln Trp Asn Arg Tyr Pro Tyr Thr 1 5 45PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Ser
Tyr Val Leu His 1 5 517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Tyr Ile Asn Pro Tyr Asn Asp
Gly Thr Gln Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly 610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Gly
Phe Gly Gly Ser Tyr Gly Phe Ala Tyr 1 5 10 712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Trp
Thr Trp Asn Ile Thr Lys Ala Tyr Pro Leu Pro 1 5 10 810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Ala
Cys Pro Glu Trp Trp Gly Thr Thr Cys 1 5 10 9324DNAMus
sp.CDS(1)..(324) 9gat att gtg atg acc cag tct cca gca atc atg tct
gca tct cct ggg 48Asp Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser
Ala Ser Pro Gly 1 5 10 15 gag aag gtc acc atg acc tgc agt gcc agc
tca agt gta agt tcc agc 96Glu Lys Val Thr Met Thr Cys Ser Ala Ser
Ser Ser Val Ser Ser Ser 20 25 30 tac ttg tac tgg tac cag cag aag
cca gga tcc tcc ccc aaa ctc tgg 144Tyr Leu Tyr Trp Tyr Gln Gln Lys
Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 att tat agc aca tcc aac
ctg gct tct gga gtc cct gct cgc ttc agt 192Ile Tyr Ser Thr Ser Asn
Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 ggc agt ggg tct
ggg acc tct tac tct ctc aca atc agc agc atg gag 240Gly Ser Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 gct gaa
gat gct gcc tct tat ttc tgc cat cag tgg aat agg tac ccg 288Ala Glu
Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Asn Arg Tyr Pro 85 90 95
tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa 324Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 10108PRTMus sp. 10Asp Ile
Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 20
25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu
Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala
Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Ser Tyr Phe Cys
His Gln Trp Asn Arg Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 11357DNAMus sp.CDS(1)..(357) 11gag gtt
cag ctg cag gag tct gga cct gag ctg gta aag cct ggg gct 48Glu Val
Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
tca gtg aag atg tcc tgc aag gct tct gga tac aca ttc cct agc tat
96Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Tyr
20 25 30 gtt ttg cac tgg gtg aag cag aag cct ggg cag ggc ctt gag
tgg att 144Val Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 gga tat att aat cct tac aat gat ggt act cag tac
aat gag aag ttc 192Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln Tyr
Asn Glu Lys Phe 50 55 60 aaa ggc aag gcc aca ctg act tca gac aaa
tcg tcc agc aca gcc tac 240Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys
Ser Ser Ser Thr Ala Tyr 65 70 75 80 atg gag ctc agc cgc ctg acc tct
gag gac tct gcg gtc tat tac tgt 288Met Glu Leu Ser Arg Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 gca aga ggc ttc ggt ggt
agc tac gga ttt gct tac tgg ggc caa ggg 336Ala Arg Gly Phe Gly Gly
Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 act ctg atc act
gtc tct gca 357Thr Leu Ile Thr Val Ser Ala 115 12119PRTMus sp.
12Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser
Tyr 20 25 30 Val Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Gly
Gly Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Ile
Thr Val Ser Ala 115 13109PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 13Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu
Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50
55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met
Glu 65 70 75 80 Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Asn
Arg Tyr Pro 85 90 95 Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 100 105 14119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 14Gln Val Gln Leu Gln Glu
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Tyr 20 25 30 Val Leu
His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln Tyr Asn Glu Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe Gly Gly Ser Tyr Gly Phe Ala
Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Ile Thr Val Ser Ser 115
15107PRTHomo sapiens 15Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Thr Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Leu Ile 85 90
95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
16109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Ser Ala
Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro
Glu Asp Ser Ala Ser Tyr Phe Cys His Gln Trp Asn Arg Tyr Pro 85 90
95 Tyr Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg 100 105
17108PRTHomo sapiens 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Pro Arg Leu Leu Ala Asp Val Leu Leu 100 105
1811PRTHomo sapiens 18Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1
5 10 19119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser
Gly Tyr Thr Phe Pro Ser Tyr 20 25 30 Val Leu His Trp Val Lys Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro
Tyr Asn Asp Gly Thr Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Phe Gly Gly Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 20327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
20gac atc cag ctg acc cag tct cca tcc tcc ctg tct gca tct gta gga
48Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 gac aga gtc acc atg acc tgc agt gcc agc tca agt gta agt tcc
agc 96Asp Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser
Ser 20 25 30 tac ttg tac tgg tac caa cag aaa cca ggg aaa gcc ccc
aaa ctc tgg 144Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Trp 35 40 45 att tat agc aca tcc aac ctg gct tct gga gtc
cct gct cgc ttc agt 192Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val
Pro Ala Arg Phe Ser 50 55 60 ggc agt gga tct ggg aca gac ttc act
ctc acc atc agc agt ctg caa 240Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 cct gaa gat tct gcc tct tat
ttc tgc cat cag tgg aat agg tac ccg 288Pro Glu Asp Ser Ala Ser Tyr
Phe Cys His Gln Trp Asn Arg Tyr Pro 85 90 95 tac acg ttc gga ggg
ggg aca cga ctg gag atc aaa cga 327Tyr Thr Phe Gly Gly Gly Thr Arg
Leu Glu Ile Lys Arg 100 105 21357DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 21cag gtg cag ctg
cag cag tct ggg gct gag gtg aag aag cct ggg gcc 48Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg
aag gtc tcc tgc gag gct tct gga tac aca ttc cct agc tat 96Ser Val
Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe Pro Ser Tyr 20 25 30
gtt ttg cac tgg gtg aag cag gcc cct gga caa ggg ctt gag tgg att
144Val Leu His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 gga tat att aat cct tac aat gat ggt act cag tac aat gag
aag ttc 192Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Gln Tyr Asn Glu
Lys Phe 50 55 60 aaa ggc aag gcc aca ctg acc agg gac acg tcc atc
aac aca gcc tac 240Lys Gly Lys Ala Thr Leu Thr Arg Asp Thr Ser Ile
Asn Thr Ala Tyr 65 70 75 80 atg gag ctg agc agg ctg aga tct gac gac
acg gcc gtg tat tac tgt 288Met Glu Leu Ser Arg Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 gca aga ggc ttc ggt ggt agc tac
gga ttt gct tac tgg ggc cag gga 336Ala Arg Gly Phe Gly Gly Ser Tyr
Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 acc ctg gtc acc gtc tcc
tca 357Thr Leu Val Thr Val Ser Ser 115 2221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22aatgcggcgg tggtgacagt a 212321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23aagctcagca cacagaaaga c 212421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24uaaaaucuuc cugcccacct t 212521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25ggaagcuguu ggcugaaaat t 212621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26aagaccagcc ucuuugccca g 212719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27ggaccaggca gaaaacgag 192817RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 28cuaucaggau gacgcgg 172921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29ugacacaggc aggcuugacu u 213019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30ggtgaagaag ggcgtccaa 193160DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31gatccgttgg agctgttggc gtagttcaag agactcgcca
acagctccaa cttttggaaa 603220DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 32aggtggtgtt
aacagcagag 203321DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 33aaggtggagc aagcggtgga g
213421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 34aaggagttga aggccgacaa a
213521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 35uauggagcug cagaggaugt t
213649DNAArtificial SequenceDescription of Artificial Sequence
Synthetic
oligonucleotide 36tttgaatatc tgtgctgaga acacagttct cagcacagat
attcttttt 493729DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 37aatgagaaaa gcaaaaggtg
ccctgtctc 293821RNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 38aaucaucauc aagaaagggc a
213921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 39augacuguca ggauguugct t
214021RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 40gaacgaaucc ugaagacauc u
214129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 41aagcctggct acagcaatat gcctgtctc
294221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 42ugaccaucac cgaguuuaut t
214321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43aagtcggacg caacagagaa a
214421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44cuaccuuucu acggacgugt t
214521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 45ctgcctaagg cggatttgaa t
214621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 46ttauuccuuc uucgggaagu c
214721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 47aaccttctgg aacccgccca c
214819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 48gagcatcttc gagcaagaa
194919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49catgtggcac cgtttgcct
195021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 50aactaccaga aaggtatacc t
215121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 51ucacaguguc cuuuauguat t
215221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 52gcaugaaccg gaggcccaut t
215319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 53ccggacagtt ccatgtata
195419DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 54ggagcctgat catccagca
195510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10
5611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Asp Glu Gly Tyr Thr Phe Cys Glu Ser Pro Arg 1 5
10 57173DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 57agtctggggc tgaggtgaag aagcctgggg
cctcagtgaa ggtctcctgc gaggcttctg 60gatacacatt ccctagctat gttttgcact
gggtgaagca ggcccctgga caagggcttg 120agtggattgg atatattaat
ccttacaatg atggtactca gtacaatgag aag 17358173DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
58agggttccct ggccccagta agcaaatccg tagctaccac cgaagcctct tgcacagtaa
60tacacggccg tgtcgtcaga tctcagcctg ctcagctcca tgtaggctgt gttgatggac
120gtgtccctgg tcagtgtggc cttgcctttg aacttctcat tgtactgagt acc
1735934DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 59caggtgcagc tgcagcagtc tggggctgag gtga
346033DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 60tgaggagacg gtgaccaggg ttccctggcc cca
3361157DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 61cagtctccat cctccctgtc tgcatctgta
ggagacagag tcaccatgac ctgcagtgcc 60agctcaagtg taagttccag ctacttgtac
tggtaccaac agaaaccagg gaaagccccc 120aaactctgga tttatagcac
atccaacctg gcttctg 15762156DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 62gtcccccctc
cgaacgtgta cgggtaccta ttccactgat ggcagaaata agaggcagaa 60tcttcaggtt
gcagactgct gatggtgaga gtgaagtctg tcccagatcc actgccactg
120aagcgagcag ggactccaga agccaggttg gatgtg 1566333DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 63gacatccagc tgacccagtc tccatcctcc ctg
336433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 64ttagatctcc agtcgtgtcc cccctccgaa cgt
336512PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 65Trp Thr Trp Asn Ile Thr Lys Glu Tyr Pro Gln Pro
1 5 10 6610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Ala Cys Pro Glu Trp Trp Gly Thr Thr Cys 1 5 10
677PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 67Gly Thr Thr Gly Thr Thr Cys 1 5
6844PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 68Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu
Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val Leu Arg Gln Gln Pro
Pro Asp Leu Val Glu Phe Ala 20 25 30 Val Glu Tyr Phe Thr Arg Leu
Arg Glu Ala Arg Ala 35 40 6945PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 69Cys Gly His Ile Gln Ile
Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly 1 5 10 15 Tyr Thr Val Glu
Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe 20 25 30 Ala Val
Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala 35 40 45
7017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 70Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn
Ala Ile Gln Gln 1 5 10 15 Ala 7121PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 71Cys Gly Gln Ile Glu Tyr
Leu Ala Lys Gln Ile Val Asp Asn Ala Ile 1 5 10 15 Gln Gln Ala Gly
Cys 20 7250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 72Ser Leu Arg Glu Cys Glu Leu Tyr Val Gln Lys
His Asn Ile Gln Ala 1 5 10 15 Leu Leu Lys Asp Ser Ile Val Gln Leu
Cys Thr Ala Arg Pro Glu Arg 20 25 30 Pro Met Ala Phe Leu Arg Glu
Tyr Phe Glu Arg Leu Glu Lys Glu Glu 35 40 45 Ala Lys 50
7355PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 73Met Ser Cys Gly Gly Ser Leu Arg Glu Cys Glu
Leu Tyr Val Gln Lys 1 5 10 15 His Asn Ile Gln Ala Leu Leu Lys Asp
Ser Ile Val Gln Leu Cys Thr 20 25 30 Ala Arg Pro Glu Arg Pro Met
Ala Phe Leu Arg Glu Tyr Phe Glu Arg 35 40 45 Leu Glu Lys Glu Glu
Ala Lys 50 55 7423PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 74Cys Gly Phe Glu Glu Leu Ala Trp Lys
Ile Ala Lys Met Ile Trp Ser 1 5 10 15 Asp Val Phe Gln Gln Gly Cys
20 7555PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 75Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser His Ile Gln Ile 1 5 10 15 Pro Pro Gly Leu Thr Glu Leu Leu Gln
Gly Tyr Thr Val Glu Val Leu 20 25 30 Arg Gln Gln Pro Pro Asp Leu
Val Glu Phe Ala Val Glu Tyr Phe Thr 35 40 45 Arg Leu Arg Glu Ala
Arg Ala 50 55 7629PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 76Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Ile Glu Tyr 1 5 10 15 Leu Ala Lys Gln Ile Val Asp
Asn Ala Ile Gln Gln Ala 20 25 7751PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 77Ser Leu Arg Glu Cys
Glu Leu Tyr Val Gln Lys His Asn Ile Gln Ala 1 5 10 15 Leu Leu Lys
Asp Val Ser Ile Val Gln Leu Cys Thr Ala Arg Pro Glu 20 25 30 Arg
Pro Met Ala Phe Leu Arg Glu Tyr Phe Glu Lys Leu Glu Lys Glu 35 40
45 Glu Ala Lys 50 7854PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 78Ser Leu Lys Gly Cys Glu
Leu Tyr Val Gln Leu His Gly Ile Gln Gln 1 5 10 15 Val Leu Lys Asp
Cys Ile Val His Leu Cys Ile Ser Lys Pro Glu Arg 20 25 30 Pro Met
Lys Phe Leu Arg Glu His Phe Glu Lys Leu Glu Lys Glu Glu 35 40 45
Asn Arg Gln Ile Leu Ala 50 7944PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 79Ser His Ile Gln Ile Pro
Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr 1 5 10 15 Thr Val Glu Val
Gly Gln Gln Pro Pro Asp Leu Val Asp Phe Ala Val 20 25 30 Glu Tyr
Phe Thr Arg Leu Arg Glu Ala Arg Arg Gln 35 40 8044PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
80Ser Ile Glu Ile Pro Ala Gly Leu Thr Glu Leu Leu Gln Gly Phe Thr 1
5 10 15 Val Glu Val Leu Arg His Gln Pro Ala Asp Leu Leu Glu Phe Ala
Leu 20 25 30 Gln His Phe Thr Arg Leu Gln Gln Glu Asn Glu Arg 35 40
8117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Gln Ile Glu Tyr Val Ala Lys Gln Ile Val Asp Tyr
Ala Ile His Gln 1 5 10 15 Ala 8217PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 82Gln Ile Glu Tyr Lys Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
8317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 83Gln Ile Glu Tyr His Ala Lys Gln Ile Val Asp His
Ala Ile His Gln 1 5 10 15 Ala 8417PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 84Gln Ile Glu Tyr Val Ala
Lys Gln Ile Val Asp His Ala Ile His Gln 1 5 10 15 Ala
8518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 85Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn
Ala Ile Gln Gln 1 5 10 15 Ala Ile 8618PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 86Leu
Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Gln Leu 1 5 10
15 Ser Ile 8718PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 87Leu Ile Glu Glu Ala Ala Ser Arg Ile
Val Asp Ala Val Ile Glu Gln 1 5 10 15 Val Lys 8818PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Ala
Leu Tyr Gln Phe Ala Asp Arg Phe Ser Glu Leu Val Ile Ser Glu 1 5 10
15 Ala Leu 8917PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 89Leu Glu Gln Val Ala Asn Gln Leu Ala
Asp Gln Ile Ile Lys Glu Ala 1 5 10 15 Thr 9017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 90Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe 9118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 91Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn
Ala Val Leu Lys 1 5 10 15 Ala Val 9218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 92Thr
Ala Glu Glu Val Ser Ala Arg Ile Val Gln Val Val Thr Ala Glu 1 5 10
15 Ala Val 9318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 93Gln Ile Lys Gln Ala Ala Phe Gln Leu
Ile Ser Gln Val Ile Leu Glu 1 5 10 15 Ala Thr 9416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Leu
Ala Trp Lys Ile Ala Lys Met Ile Val Ser Asp Val Met Gln Gln 1 5 10
15 9524PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 95Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp
Ala Val Ile Glu 1 5 10 15 Gln Val Lys Ala Ala Gly Ala Tyr 20
9618PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Leu Glu Gln Tyr Ala Asn Gln Leu Ala Asp Gln Ile
Ile Lys Glu Ala 1 5 10 15 Thr Glu 9720PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 97Phe
Glu Glu Leu Ala Trp Lys Ile Ala Lys Met Ile Trp Ser Asp Val 1 5 10
15 Phe Gln Gln Cys 20 9817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Gln Ile Glu Tyr Leu Ala Lys
Gln Ile Pro Asp Asn Ala Ile Gln Gln 1 5 10 15 Ala 9925PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 99Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala 1 5 10
15 Val Ile Glu Gln Val Lys Ala Ala Gly 20 25 10025PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Lys
Gly Ala Asp Leu Ile Glu Glu Ala Ala Ser Arg Ile Pro Asp Ala 1 5 10
15 Pro Ile Glu Gln Val Lys Ala Ala Gly 20 25 10125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 101Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 10225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 102Pro
Glu Asp Ala Glu Leu Val Arg Thr Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 10325PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Asp Val Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 10425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 104Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Val Leu Lys Ala Val Gln Gln Tyr 20 25 10525PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 105Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Pro Glu Asn 1 5 10
15 Ala Pro Leu Lys Ala Val Gln Gln Tyr 20 25 10625PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Pro
Glu Asp Ala Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn 1 5 10
15 Ala Val Glu Lys Ala Val Gln Gln Tyr 20 25 10725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 107Glu
Glu Gly Leu Asp Arg Asn Glu Glu Ile Lys Arg Ala Ala Phe Gln 1 5 10
15 Ile Ile Ser Gln Val Ile Ser Glu Ala 20 25 10825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 108Leu
Val Asp Asp Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn 1
5 10 15 Ala Ile Gln Gln Ala Ile Ala Glu Gln 20 25
10925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 109Gln Tyr Glu Thr Leu Leu Ile Glu Thr Ala Ser
Ser Leu Val Lys Asn 1 5 10 15 Ala Ile Gln Leu Ser Ile Glu Gln Leu
20 25 11025PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 110Leu Glu Lys Gln Tyr Gln Glu Gln Leu Glu Glu
Glu Val Ala Lys Val 1 5 10 15 Ile Val Ser Met Ser Ile Ala Phe Ala
20 25 11125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Asn Thr Asp Glu Ala Gln Glu Glu Leu Ala Trp
Lys Ile Ala Lys Met 1 5 10 15 Ile Val Ser Asp Ile Met Gln Gln Ala
20 25 11225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 112Val Asn Leu Asp Lys Lys Ala Val Leu Ala Glu
Lys Ile Val Ala Glu 1 5 10 15 Ala Ile Glu Lys Ala Glu Arg Glu Leu
20 25 11325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 113Asn Gly Ile Leu Glu Leu Glu Thr Lys Ser Ser
Lys Leu Val Gln Asn 1 5 10 15 Ile Ile Gln Thr Ala Val Asp Gln Phe
20 25 11425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Thr Gln Asp Lys Asn Tyr Glu Asp Glu Leu Thr
Gln Val Ala Leu Ala 1 5 10 15 Leu Val Glu Asp Val Ile Asn Tyr Ala
20 25 11525PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Glu Thr Ser Ala Lys Asp Asn Ile Asn Ile Glu
Glu Ala Ala Arg Phe 1 5 10 15 Leu Val Glu Lys Ile Leu Val Asn His
20 25 11612PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Trp Thr Trp Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa
Pro 1 5 10 11710PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 117Ala Cys Xaa Glu Trp Trp Xaa Xaa Xaa
Cys 1 5 10 1184PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 118Pro Lys Ser Cys1 11910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 119Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 120449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
120Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe Pro
Ser Tyr 20 25 30 Val Leu His Trp Val Lys Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr
Gln Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Arg
Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Phe
Gly Gly Ser Tyr Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130
135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250
255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys 121215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
121Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser
Ser Ser 20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly
Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ser Ala Ser
Tyr Phe Cys His Gln Trp Asn Arg Tyr Pro 85 90 95 Tyr Thr Phe Gly
Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130
135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu
Cys 210 215
* * * * *
References