U.S. patent application number 16/198276 was filed with the patent office on 2019-03-21 for rs7 antibodies.
The applicant listed for this patent is Immunomedics, Inc.. Invention is credited to David M. Goldenberg, Serengulam V. Govindan, Hans J. Hansen, Zhengxing Qu.
Application Number | 20190083621 16/198276 |
Document ID | / |
Family ID | 27788969 |
Filed Date | 2019-03-21 |
View All Diagrams
United States Patent
Application |
20190083621 |
Kind Code |
A1 |
Govindan; Serengulam V. ; et
al. |
March 21, 2019 |
RS7 Antibodies
Abstract
This invention relates to monovalent and multivalent,
monospecific binding proteins and to multivalent, multispecific
binding proteins. One embodiment of these binding proteins has one
or more binding sites where each binding site binds with a target
antigen or an epitope on a target antigen. Another embodiment of
these binding proteins has two or more binding sites where each
binding site has affinity towards different epitopes on a target
antigen or has affinity towards either a target antigen or a
hapten. The present invention further relates to recombinant
vectors useful for the expression of these functional binding
proteins in a host. More specifically, the present invention
relates to the tumor-associated antigen binding protein designated
RS7, and other EGP-1 binding-proteins. The invention further
relates to humanized, human and chimeric RS7 antigen binding
proteins, and the use of such binding proteins in diagnosis and
therapy.
Inventors: |
Govindan; Serengulam V.;
(Summit, NJ) ; Qu; Zhengxing; (Warren, NJ)
; Hansen; Hans J.; (Picayune, MS) ; Goldenberg;
David M.; (Mendham, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunomedics, Inc. |
Morris Plains |
NJ |
US |
|
|
Family ID: |
27788969 |
Appl. No.: |
16/198276 |
Filed: |
November 21, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15980057 |
May 15, 2018 |
10179171 |
|
|
16198276 |
|
|
|
|
15816635 |
Nov 17, 2017 |
9999668 |
|
|
15980057 |
|
|
|
|
15613928 |
Jun 5, 2017 |
9849176 |
|
|
15816635 |
|
|
|
|
14259469 |
Apr 23, 2014 |
9833511 |
|
|
15613928 |
|
|
|
|
14040024 |
Sep 27, 2013 |
8758752 |
|
|
14259469 |
|
|
|
|
13293608 |
Nov 10, 2011 |
8574575 |
|
|
14040024 |
|
|
|
|
12389503 |
Feb 20, 2009 |
8084583 |
|
|
13293608 |
|
|
|
|
11745896 |
May 8, 2007 |
7517964 |
|
|
12389503 |
|
|
|
|
10377121 |
Mar 3, 2003 |
7238785 |
|
|
11745896 |
|
|
|
|
60360229 |
Mar 1, 2002 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/1051 20130101;
A61P 37/02 20180101; C07K 16/30 20130101; A61K 2039/505 20130101;
A61P 31/00 20180101; A61P 35/00 20180101; C07K 2317/24 20130101;
C07K 16/3015 20130101; A61K 45/06 20130101; C07K 2317/21 20130101;
A61K 51/1045 20130101; A61K 39/39558 20130101 |
International
Class: |
A61K 45/06 20060101
A61K045/06; C07K 16/30 20060101 C07K016/30; A61K 51/10 20060101
A61K051/10; A61K 39/395 20060101 A61K039/395 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Number CA072324 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A pharmaceutical composition comprising: a) a chimeric,
humanized or human anti-TROP-2 antibody, wherein the anti-TROP-2
antibody comprises the light chain complementarity determining
region (CDR) sequences CDR1 (KASQDVSIAVA, SEQ ID NO:28), CDR2
(SASYRYT, SEQ ID NO:29), and CDR3 (QQHYITPLT, SEQ ID NO:30) and the
heavy chain CDR sequences CDR1 (NYGMN, SEQ ID NO:31), CDR2
(WINTYTGEPTYTDDFKG, SEQ ID NO:32), and CDR3 (GGFGSSYWYFDV, SEQ ID
NO:33); and b) at least one excipient selected from the group
consisting of water, a buffer, a salt, a detergent, and a
stabilizing agent.
2. The composition of claim 1, wherein the buffer is a Good's
biological buffer.
3. The composition of claim 2, wherein the buffer is selected from
the group consisting of N-(2-acetamido)-2-aminoethanesulfonic acid
(ACES); N-(2-acetamido)iminodiacetic acid (ADA);
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);
2-(N-morpholino)ethanesulfonic acid (MES);
3-(N-morpholino)propanesulfonic acid (MOPS);
3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); and
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES).
4. The composition of claim 1, wherein the buffer is MES.
5. The composition of claim 1, wherein the salt is sodium
chloride.
6. The composition of claim 1, wherein the detergent is polysorbate
80.
7. The composition of claim 1, wherein the stabilizing agent is
trehalose.
8. The composition of claim 1, wherein the composition is
lyophilized.
9. The composition of claim 1, wherein the antibody is
unconjugated.
10. The composition of claim 1, wherein the antibody is conjugated
to at least one therapeutic agent selected from the group
consisting of an antigen-binding antibody fragment, a drug, a
toxin, a radionuclide, a cytotoxic agent, an pro-apoptotic agent,
an immunomodulator, a photoactive agent, an anti-angiogenic agent,
and an siRNA.
11. The composition of claim 10, wherein the radionuclide is
selected from the group consisting of .sup.131I, .sup.123I,
.sup.124I, .sup.86Y, .sup.62Cu, .sup.64Cu, .sup.67Ga, .sup.68Ga,
.sup.99mTc, .sup.94mTc, .sup.90Y, .sup.111In, .sup.125I,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.177Lu, .sup.67Cu,
.sup.212Bi, .sup.213Bi, .sup.211At, .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.125mTe, .sup.165Tm, .sup.167Tm, .sup.168Tm, .sup.111Ag,
.sup.197Pt, .sup.109Pd, .sup.32P, .sup.33P, .sup.47Sc, .sup.153Sm,
.sup.105Rh, .sup.142Pr, .sup.143Pr, .sup.161Tb, .sup.166Ho,
.sup.199Au, .sup.57Co, .sup.58Co, .sup.51Cr, .sup.59Fe, .sup.18F,
.sup.75Se, .sup.201Tl, .sup.225Ac, .sup.76Br, .sup.86Y, .sup.169Yb,
.sup.166Dy, .sup.212Pb, and .sup.223Ra.
12. The composition of claim 10, wherein the immunomodulator is
selected from the group consisting of a cytokine, a stem cell
growth factor, a lymphotoxin, a tumor necrosis factor (TNF), an
hematopoietic factor, interleukin-1 (IL-1), IL-2, IL-3, IL-6,
IL-10, IL-12, IL-18, IL-21, a colony stimulating factor,
granulocyte-colony stimulating factor (G-CSF), granulocyte
macrophage-colony stimulating factor (GM-CSF), interferon-.alpha.,
interferon-.beta., interferon-.gamma., stem cell growth factor S1,
erythropoietin and thrombopoietin.
13. The composition of claim 10, wherein the therapeutic agent is
selected from the group consisting of a nitrogen mustard,
ethylenimine derivative, alkyl sulfonate, nitrosourea, triazene,
folic acid analog, anthracycline, taxane, COX-2 inhibitor, tyrosine
kinase inhibitor, pyrimidine analog, purine analog, antibiotic,
enzyme, epipodophyllotoxin, platinum coordination complex, vinca
alkaloid, substituted urea, methyl hydrazine derivative,
adrenocorticol suppressant, endostatin, taxol, camptothecin,
doxorubicin and doxorubicin analog.
14. The composition of claim 1, wherein the antibody comprises the
framework regions (FRs) of the light and heavy chain regions of one
or more human antibodies.
15. The composition of claim 14, wherein the FRs of the light and
heavy chain variable regions of said antibody comprise at least one
amino acid residue selected from the group consisting of amino acid
residues 38, 46, 68 and 91 of SEQ ID NO:4 and amino acid residues
20, 85, 60 and 100 of SEQ ID NO:2.
16. The composition of claim 14, wherein the FRs of the light and
heavy chain variable regions of said antibody comprise all of the
amino acid residues 38, 46, 68 and 91 of SEQ ID NO:4 and amino acid
residues 20, 85, 60 and 100 of SEQ ID NO:2.
17. The composition of claim 1, wherein the antibody comprises the
light chain variable region amino acid sequence SEQ ID NO:12 and
the heavy chain variable region amino acid sequence SEQ ID NO:14.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/980,057, filed May 15, 2018, which was a divisional of
U.S. patent application Ser. No. 15/816,635 (now issued U.S. Pat.
No. 9,999,668), filed Nov. 17, 2017, which was a divisional of U.S.
patent application Ser. No. 15/613,928 (now issued U.S. Pat. No.
9,849,176), filed Jun. 5, 2017, which was a divisional of U.S.
patent application Ser. No. 14/259,469 (now issued U.S. Pat. No.
9,833,511), filed Apr. 23, 2014, which was a continuation of U.S.
patent application Ser. No. 14/040,024 (now issued U.S. Pat. No.
8,758,752), filed Sep. 27, 2013, which was a divisional of U.S.
patent application Ser. No. 13/293,608 (now issued U.S. Pat. No.
8,574,575), filed Nov. 10, 2011, which was a divisional of U.S.
patent application Ser. No. 12/389,503 (now issued U.S. Pat. No.
8,084,583), filed Feb. 20, 2009, which was a continuation of U.S.
patent application Ser. No. 11/745,896 (now issued U.S. Pat. No.
7,517,964), filed May 8, 2007, which was a divisional of U.S.
patent application Ser. No. 10/377,121 (now issued U.S. Pat. No.
7,238,785), filed Mar. 3, 2003, which claimed priority to U.S.
Provisional Application No. 60/360,229, filed Mar. 1, 2002, which
is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to monovalent and multivalent,
monospecific binding proteins and to multivalent, multispecific
binding proteins. One embodiment of these binding proteins has one
or more binding sites where each binding site binds with a target
antigen or an epitope on a target antigen. Another embodiment of
these binding proteins has two or more binding sites where each
binding site has affinity towards different epitopes on a target
antigen or has affinity towards either a target antigen or a
hapten. The present invention further relates to recombinant
vectors useful for the expression of these functional binding
proteins in a host. More specifically, the present invention
relates to the tumor-associated antigen binding protein designated
RS7. The invention further relates to humanized RS7 antigen binding
proteins, and the use of such binding proteins in diagnosis and
therapy.
BACKGROUND OF THE INVENTION
[0004] Man-made binding proteins, in particular monoclonal
antibodies and engineered antibodies or antibody fragments, have
been tested widely and shown to be of value in detection and
treatment of various human disorders, including cancers, autoimmune
diseases, infectious diseases, inflammatory diseases, and
cardiovascular diseases (Filpula and McGuire, Exp. Opin. Ther.
Patents (1999) 9: 231-245). For example, antibodies labeled with
radioactive isotopes have been tested to visualize tumors after
injection to a patient using detectors available in the art. The
clinical utility of an antibody or an antibody-derived agent is
primarily dependent on its ability to bind to a specific targeted
antigen. Selectivity is valuable for delivering a diagnostic or
therapeutic agent, such as isotopes, drugs, toxins, cytokines,
hormones, growth factors, enzymes, conjugates, radionuclides, or
metals, to a target location during the detection and treatment
phases of a human disorder, particularly if the diagnostic or
therapeutic agent is toxic to normal tissue in the body.
[0005] The potential limitations of antibody systems are discussed
in Goldenberg, The American Journal of Medicine (1993) 94: 298-299.
The important parameters in the detection and treatment techniques
are the amount of the injected dose specifically localized at the
site(s) where target cells are present and the uptake ratio, i.e.
the ratio of the concentration of specifically bound antibody to
that of the radioactivity present in surrounding normal tissues.
When an antibody is injected into the blood stream, it passes
through a number of compartments as it is metabolized and excreted.
The antibody must be able to locate and bind to the target cell
antigen while passing through the rest of the body. Factors that
control antigen targeting include location, size, antigen density,
antigen accessibility, cellular composition of pathologic tissue,
and the pharmacokinetics of the targeting antibodies. Other factors
that specifically affect tumor targeting by antibodies include
expression of the target antigens, both in tumor and other tissues,
and bone marrow toxicity resulting from the slow blood-clearance of
the radiolabeled antibodies. The amount of targeting antibodies
accreted by the targeted tumor cells is influenced by the
vascularization and barriers to antibody penetration of tumors, as
well as intratumoral pressure. Non-specific uptake by non-target
organs such as the liver, kidneys or hone-marrow is another
potential limitation of the technique, especially for
radioimmunotherapy, where irradiation of the bone marrow often
causes the dose-limiting toxicity.
[0006] One suggested approach, referred to as direct targeting, is
a technique designed to target specific antigens with antibodies
carrying diagnostic or therapeutic radioisotopes. In the context of
tumors, the direct targeting approach utilizes a radiolabeled
anti-tumor monospecific antibody that recognizes the target tumor
through its antigens. The technique involves injecting the labeled
monospecific antibody into the patient and allowing the antibody to
localize at the target tumor to obtain diagnostic or therapeutic
benefits. The unbound antibody clears the body. This approach can
be used to diagnose or treat additional mammalian disorders.
[0007] Another suggested solution, referred to as the "Affinity
Enhancement System" (AES), is a technique especially designed to
overcome deficiencies of tumor targeting by antibodies carrying
diagnostic or therapeutic radioisotopes (U.S. Pat. No. 5,256,395
(1993), Barbet et al., Cancer Biotherapy & Radiopharmaceuticals
(1999) 14: 153-166). The AES utilizes a radiolabeled hapten and an
anti-tumor/anti-hapten bispecific binding protein that recognizes
both the target tumor and the radioactive hapten. Haptens with
higher valency and binding proteins with higher specificity may
also be utilized for this procedure. The technique involves
injecting the binding protein into the patient and allowing it to
localize at the target tumor. After a sufficient amount of time for
the unbound binding protein to clear from the blood stream, the
radiolabeled hapten is administered. The hapten binds to the
antibody-antigen complex located at the site of the target cell to
obtain diagnostic or therapeutic benefits. The unbound hapten
clears the body. Barbet mentions the possibility that a bivalent
hapten may crosslink with a bispecific antibody, when the latter is
bound to the tumor surface. As a result, the radiolabeled complex
is more stable and stays at the tumor for a longer period of time.
This system can be used to diagnose or treat mammalian
disorders.
[0008] There remains a need in the art for production of
multivalent, monospecific binding proteins that are useful in a
direct targeting system and for production of multivalent,
multispecific binding proteins that are useful in an affinity
enhancement system. Specifically, there remains a need for a
binding protein that exhibits enhanced uptake at targeted antigens,
decreased concentration in the blood, and optimal protection of
normal tissues and cells from toxic pharmaceuticals.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is an object of the present invention to
provide a monospecific monoclonal antibody and fragments thereof
that recognizes a tumor-associated antigen, defined as epithelial
glycoprotein-1 (EGP-1) by the murine MAb RS7-3G11 raised against
human non-small-cell lung carcinoma. The RS7 antigen has been
designated as EGP-1 (epithelial glycoprotein-1) following the
proposal of the 3.sup.rd International IASLC Workshop on Lung Tumor
and Differentiation Antigens. At least one epitope associated with
EGP-1 is alternatively referred to as TROP2 in the literature. In a
preferred embodiment, the antibody or antibody fragment of the
present invention binds the same epitope as the murine RS7 antibody
disclosed by Stein (infra) and other earlier studies.
Alternatively, the antibody or fragment may bind an epitope
distinct from the epitope that the murine RS7 antibody disclosed by
Stein binds. In a preferred embodiment, the anti-EGP-1, or
anti-TROP2 antibody or fragment thereof is a chimeric, humanized,
or fully human RS7 antibody or fragment thereof.
[0010] For example, contemplated in the present invention is a
humanized antibody or fragment thereof, wherein the complementarity
determining regions (CDRs) of the light chain variable region of
the humanized RS7 MAb comprises CDR1 comprising an amino acid
sequence of KASQDVSIAVA (SEQ ID NO:28); CDR2 comprising an amino
acid sequence of SASYRYT (SEQ ID NO:29); and CDR3 comprising an
amino acid sequence of QQHYITPLT (SEQ ID NO:30). Another embodiment
of the present invention is a humanized antibody or fragment
thereof, wherein the CDRs of the heavy chain variable region of the
humanized RS7 MAb comprises CDR1 comprising an amino acid sequence
of NYGMN (SEQ ID NO:31); CDR2 comprising an amino acid sequence of
WINTYTGEPTYTDDFKG (SEQ ID NO:32) and CDR3 comprising an amino acid
sequence of GGFGSSYWYFDV (SEQ ID NO:33). Also preferred, the
humanized antibody or fragment thereof of comprises the CDRs of a
murine RS7 MAb and the framework region (FR) of the light and heavy
chain variable regions of a human antibody, wherein the CDRs of the
light chain variable region of the humanized RS7 MAb comprises CDR1
comprising an amino acid sequence of KASQDVSIAVA (SEQ ID NO:28);
CDR2 comprising an amino acid sequence of SASYRYT (SEQ ID NO:29);
and CDR3 comprising an amino acid sequence of QQHYITPLT (SEQ ID
NO:30); and the CDRs of the heavy chain variable region of the
humanized RS7 MAb comprises CDR1 comprising an amino acid sequence
of NYGMN (SEQ ID NO:31); CDR2 comprising an amino acid sequence of
WINTYTGEPTYTDDFKG (SEQ ID NO:32) and CDR3 comprising an amino acid
sequence of GGFGSSYWYFDV (SEQ ID NO:33). Still preferred, the
humanized antibody or fragment thereof further comprises the FRs of
the light and heavy chain constant regions of a human antibody.
[0011] In a preferred embodiment, the humanized RS7 antibody or
fragment comprises a FR of a light and/or heavy chain that
comprises at least one amino acid substituted by an amino acid
residue found at a corresponding location in the RS7 murine
antibody. For example, at least one of the substituted amino acids
is preferably at a location selected from the group consisting of
residue 38, 46, 68 and 91 of the murine heavy chain variable region
of SEQ ID NO:4, and/or at least one of the substituted amino acids
is preferably at a location selected from the group consisting of
residue 20, 85 and 100 of the murine light chain variable region of
SEQ ID NO:2.
[0012] Also described in the present invention is an antibody
fission protein or fragment thereof that comprises at least two
anti-EGP-1 MAb or fragments thereof, wherein the MAb or fragments
thereof are selected from the anti-EGP-1 MAb or fragments thereof
of the present invention. In a related vein, the antibody fusion
protein or fragment thereof comprises at least one first anti-EGP-1
MAb or fragment thereof of any of the anti-EGP-1 antibodies of the
present invention and at least one second MAb or fragment thereof,
other than the anti-EGP antibodies or fragment thereof in the
present invention. For example, the second antibody or fragment
thereof may be a carcinoma-associated antibody or fragment thereof.
Another preferred embodiment is a fusion protein or fragment
thereof that comprises two different epitope-binding anti-EGP-1
antibodies or fragments thereof.
[0013] It is one object of this invention to provide a
multispecific antibody and fragments thereof that recognize more
than one epitope on the RS7 antigen or that has affinity for the
RS7 antigen and for a hapten molecule. The latter binding protein
is useful for pretargeting a target antigen. Accordingly, a method
of delivering a diagnostic agent, a therapeutic agent, or a
combination thereof to a target, comprising: (i) administering to a
subject a multivalent, multispecific MAb, or fragment thereof (ii)
waiting a sufficient amount of time for an amount of the
non-binding protein to clear the subject's blood stream; and (iii)
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 said antibody, is also
described.
[0014] It is a further object of this invention to provide a method
of delivering a diagnostic or therapeutic agent to a targeted
disease that expresses EGP-1 antigen. For example, 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-EGP-1 antibody or fragment thereof bound to at
least one therapeutic and/or diagnostic agent and (ii)
administering to a subject in need thereof said composition, is
described. Preferably, the diagnostic or therapeutic agent is
selected from the group consisting of an isotope, drug, toxin,
immuno, modulator, hormone, enzyme, growth factor, radionuclide,
metal, contrast agent, and detecting agent.
[0015] In another embodiment of the present invention, the method
for delivering a diagnostic agent, a therapeutic agent, or a
combination thereof to a target comprises (i) administering to a
subject a multivalent, multispecific antibody or fragment
comprising one or more antigen-binding sites having affinity toward
an EGP-1 target antigen and one or more hapten binding sites having
an affinity toward a hapten molecule, (ii) waiting a sufficient
amount of time for an amount of the non-binding antibody or
fragment to clear a subject's blood stream, and (iii) administering
to said subject a hapten comprising a diagnostic agent, a
therapeutic agent, or a combination thereof.
[0016] Another object of the present invention to provide a cancer
cell targeting diagnostic or therapeutic conjugate that comprises
an anti-EGP-1 MAb or fragment thereof or an antibody fusion protein
or fragment thereof of any one of antibodies of the present
invention and wherein the anti-EGP-1 antibody or fragment thereof
is bound to at least one diagnostic or therapeutic agent. A
suitable therapeutic agent is a drug that possesses the
pharmaceutical property selected from the group consisting of an
antimitotic, alkylating, antimetabolite, antiangiogenic, apoptotic,
alkaloid antibiotic, and combinations thereof. Also preferred is a
therapeutic agent selected from the group consisting of a nitrogen
mustard, ethylenimine derivative, alkyl sulfonate, nitrosourea,
triazene, folic acid analog, anthracycline, taxane, COX-2
inhibitor, tyrosine kinase inhibitor, pyrimidine analog, purine
analog, antibiotic, enzyme, epipodophyllotoxin, platinum
coordination complex, vinca alkaloid, substituted urea, methyl
hydrazine derivative, adrenocortical suppressant, antagonist,
endostatin taxol, camptothecins, doxorubicin, doxorubicin analog,
and a combination thereof. Preferably, the diagnostic agent is
selected from the group consisting of a photoactive radionuclide,
preferably between 25 and 4000 keV, and a contrast agent.
[0017] In a preferred embodiment, a DNA sequence comprising a
nucleic acid encoding a MAb or fragment that contains a anti-EGP-1
MAb or fragment thereof of the present invention; an antibody
fusion protein or fragment thereof containing at least two of said
MAbs or fragments thereof; an antibody fusion protein or fragment
thereof containing at least one first anti-EGP-1 MAb or fragment
thereof containing the MAb or fragment thereof of the anti-EGP-1
antibodies and fragments of the present invention and at least one
second MAb or fragment thereof, other than the anti-EGP-1 MAb or
fragment thereof described herein; or an antibody fusion protein or
fragment thereof comprising at least one first MAb or fragment
thereof comprising said MAb or fragment thereof of any of the
antibodies described herein and at least one second MAb or fragment
thereof, other than the MAb or fragment thereof of any one of the
antibodies described herein, wherein the second MAb is reactive
with an antigen selected from the group consisting of EGP-2, WC
1-4, A33, CSAp, CEA, Le(y), Tn, Tag-72, PSMA, PSA, EGFR, HER2/neu,
AFP, HCG, HCG-beta, ferritin, PAP, PLAP, EGP-2, histone,
cytokeratin, Tenascin, CanAg, kidney cancer G 250, VGFR1, VGFR2,
P4-antigen, oncogene products, or a combination thereof. The second
MAb may instead be reactive with vascular endothelial antigens
associated with tumors, such as VEGF (vascular endothelial growth
factor) and P1GF (placenta growth factor). Selection of the second
antibody is dependent on tumor cell type. For example, anti-PSMA or
anti-PSA antibodies may be used for treating or diagnosing prostate
cancer, anti-CEA or anti-MUC1, MUC2, MUC3 and MUC4 antibodies for
breast, ovarian, lung, and colon cancer, EGFR for colon and head
and neck cancers, anti-CSAp antibodies for colon and ovarian
cancer, and anti-HER/neu for breast, ovarian and other cancers.
These are merely given as examples, and are not intended to be
limiting. Expression vectors and host cells containing this DNA
sequence are also preferred embodiments of the present
invention.
[0018] Also provided herein are methods for diagnosing and treating
a malignancy. For example, a method for diagnosing or treating
cancer, comprises (i) administering to a subject in need thereof a
multivalent, multispecific antibody or fragment comprising one or
more antigen-binding sites having affinity toward an EGP-1 target
antigen and one or more hapten binding sites having an affinity
toward a hapten molecule; (ii) waiting a sufficient amount of time
for an amount of the non-binding protein to clear the subject's
blood stream; and (iii) administering to said subject a hapten
comprising a diagnostic agent, a therapeutic agent, or a
combination thereof, that binds to a binding site of said
antibody.
[0019] Likewise, the methods for diagnosing and treating a
malignancy may comprise administering a therapeutically effective
amount of an anti-EGP-1 fusion protein or fragment thereof or a
therapeutic conjugate comprising a EGP-1 MAb or fragment thereof,
wherein the EGP-1 MAb or fragment thereof or antibody fusion
protein or fragment thereof is bound to at least one therapeutic
agent in a pharmaceutically suitable excipient. In a related vein,
naked anti-EGP-1 antibodies and fragments thereof, including naked
anti-EGP-1 fusion proteins and fragments thereof, can also be used
for treating a malignancy. Naked anti-EGP-1 antibodies may be used
for in vitro diagnosis of a malignancy, for example with
immunoassays or immunohistochemistry, but not for in vivo
diagnosis, unless this involves a pretargeting technology, such as
AES. Labeled EGP-1 antibodies, however, may be used for in vivo
diagnosis and treatment of a malignancy. For example, described
herein is a method of treating a cancer cell in a subject
comprising (i) administering to a subject a therapeutically
effective amount of a composition containing an anti-EGP-1 MAb or
fragment thereof or an antibody fusion protein or fragment thereof
(ii) formulating the EGP-1 MAb or fragment thereof or antibody
fission protein or fragment thereof in a pharmaceutically suitable
excipient. Similarly, combinations of naked MAbs and fragments
thereof with conjugated MAbs or fragments thereof or fusion
proteins or fragments thereof for diagnosis and treatment are also
contemplated in the instant invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows a comparison of mRS7, cAb-V.kappa.#23 (cRS7),
and cAb-V.kappa.#1 in competitive binding assays. Varying
concentrations of competing Abs were used to compete with the
binding of a constant amount of biotinylated mRS7 antibody. Results
indicate that the V.kappa.#1 light chain does not bind the RS7
antigen.
[0021] FIG. 2A shows the DNA (SEQ ID NO:1) and amino acid (SEQ ID
NO:2) sequences encoding RS7 V.kappa. cloned by 5' RACE. The
putative CDR regions are underlined and indicated. Nucleotide
residues are numbered sequentially. Kabat's Ig molecule numbering
is used for amino acid residues.
[0022] FIG. 2B shows the DNA (SEQ ID NO:3) and amino acid (SEQ ID
NO:4) sequences encoding RS7 VH cloned by RT-PCR. The putative CDR
regions are underlined and indicated. Nucleotide residues are
numbered sequentially. Kabat's Ig molecule numbering is used for
amino acid residues. The numbering for the residues with a letter
(on top) is the number of preceding residues plus the letter, e.g.,
the number for T following N52 is 52A; the numbers for N, N and L
following 182 are 82A, 82B and 82C, respectively.
[0023] FIG. 3A shows the amino acid sequence alignment of human
SA-1A'cl (SEQ ID NO:5), murine RS7 (SEQ ID NO:2), and hRS7 (SEQ ID
NO:7) V.kappa. chains. Dots indicate the residues in RS7 are
identical to the corresponding residues in SA-1A'cl. Dashes
represent gaps introduced to aid the alignment. Boxed represent the
CDR regions. Both N- and C-terminal residues (underlined) of hRS7
are fixed by the staging vector used. Therefore, the corresponding
terminal residues of RS7 are not compared with that of the human
sequence. Kabat's numbering scheme is used.
[0024] FIG. 3B shows the amino acid sequence alignment of human
RF-TS3 (SEQ ID NO:8), murine RS7 (SEQ ID NO:4, SEQ ID NO:9), and
hRS7 (SEQ ID NO:10, SEQ ID NO:27) 0V.sub.H chains. Dots indicate
the residues in RS7 are identical to the corresponding residues in
RF-TS3. Dashes represent gaps introduced to aid the alignment.
Boxed represent the CDR regions. Both N- and C-terminal residues
(underlined) of hRS7 are fixed by the staging vector used.
Therefore, the corresponding terminal residues of RS7 are not
compared with that of the human VH sequence.
[0025] FIG. 4A shows the DNA (SEQ ID NO:11) and amino acid (SEQ ID
NO:12) sequences for humanized RS7 V.kappa.. The bold and
underlined sections of the amino acid sequences indicate the CDRs
as defined by the Kabat numbering scheme.
[0026] FIG. 4B shows the DNA (SEQ ID NO:13) and amino acid (SEQ ID
NO:14) sequences for humanized RS7 V.sub.H. The bold and underlined
sections of the amino acid sequences indicate the CDRs as defined
by the Kabat numbering scheme.
[0027] FIG. 5A shows the light chain cDNA (SEQ ID NO:15) and amino
acid (SEQ ID NO:16) sequences for humanized RS7 V.kappa.. The
underlined sections of the amino acid sequences indicate the leader
peptide sequence for secretion. "*" indicates the stop codon.
[0028] FIG. 5B shows the heavy chain cDNA (SEQ ID NO:17) and amino
acid (SEQ ID NO:18) sequences for humanized RS7 V.sub.H. The
underlined sections of the amino acid sequences indicate the leader
peptide sequence for secretion. "*" indicates the stop codon.
[0029] FIG. 6 shows a comparison of mRS7, cRS7, and hRS7 in
competitive binding assays. Varying concentrations of competing Abs
were used to compete with the binding of a constant amount of
Biotinylated RS7 to the Ag coated in 96-well ELISA plates. hRS7
showed comparable blocking activity as that of RS7 and cRS7.
[0030] FIG. 7 indicates the structure of the residualizing moieties
IMP-R4, IMP-R5 and IMP-R8.
[0031] FIG. 8 is a bar graph of dosimetry due to radioiodinated
hRS7 in the MDA-MB-468 tumor model.
[0032] FIG. 9A shows tumor growth control, as a plot of tumor
volume (cm.sup.3) in Y-axis versus days post-treatment in X-axis,
in individual NIH Swiss nude mice (female) subcutaneously carrying
MDA-MB-468 human breast carcinoma xenografts, and which were
untreated. Each line corresponds to tumor growth in a single
mouse.
[0033] FIG. 9B shows tumor growth control, as a plot of tumor
volume (cm.sup.3) in Y-axis versus days post-treatment in X-axis,
in individual NIH Swiss nude mice (female) subcutaneously carrying
MDA-MB-468 human breast carcinoma xenografts, and which were
treated with 0.175 mCi of hRS7 antibody radioiodinated with
.sup.131I-IMP-R4 which is a residualizing form of .sup.131I,
.sup.131I-IMP-R4-hR4S7. Each line corresponds to tumor growth in a
single mouse.
[0034] FIG. 9C shows tumor growth control, as a plot of tumor
volume (cm.sup.3) in Y-axis versus days post-treatment in X-axis,
in individual NIH Swiss nude mice (female) subcutaneously carrying
MDA-MB-468 human breast carcinoma xenografts, and which were
treated with 0.2 mCi of conventionally .sup.131I-radioiodinated
hRS7, .sup.131I-hRS7. Each line corresponds to tumor growth in a
single mouse.
[0035] FIG. 9D is a composite of the tumor growth controls in the
different groups, and represents mean tumor volumes, as a function
of time in days, in animals that were treated with 0.175 mCi of
.sup.131I-IMP-R4-hRS7 (solid square) or were treated with 0.2 mCi
of .sup.131I-hRS7 (open triangle) or were untreated (solid
diamond). Error bar represents standard deviation.
[0036] FIG. 9E is a different representation of tumor growth
control vs. time plots, showing mean relative tumor volumes as a
function of time in various groups with mean tumor volume at the
start of therapy taken as 100. Otherwise, the legend is the same as
for FIG. 9D. Error bar represents standard deviation.
[0037] FIG. 10A depicts determination of myelotoxicity of treatment
with radioiodinated hRS7 in MDA-MB-468 human tumor
xenograft-bearing Swiss nude mice (female). FIG. 10A shows the data
for treatment with 0.175 mCi of hRS7 radioiodinated with a
residualizing form of .sup.131I (i.e. .sup.131I-IMP-R4-hRS7). Mean
white blood cell counts (solid diamond), mean lymphocyte counts
(solid square), mean monocyte counts (open triangle), and mean
neutrophil counts (`X`), expressed as percentage of respective mean
values in untreated control animals, are shown as a function of
time in weeks.
[0038] FIG. 10B depicts determination of myelotoxicity of treatment
with radioiodinated hRS7 in MDA-MB-468 human tumor
xenograft-bearing Swiss nude mice (female). FIG. 10B shows data for
treatment with 0.2 mCi of hRS7 conventionally radioiodinated with
.sup.131I (i.e. .sup.131I-hRS7). Mean white blood cell counts
(solid diamond), mean lymphocyte counts (solid square), mean
monocyte counts (open triangle), and mean neutrophil counts (`X`),
expressed as percentage of respective mean values in untreated
control animals, are shown as a function of time in weeks.
[0039] FIG. 11 is a graph demonstrating relative mean tumor volumes
(MTV).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0040] Unless otherwise specified, "a" or "an" means "one or
more."
[0041] An RS7 antibody (previously designated RS7-3G11) is a murine
IgG.sub.1 raised against a crude membrane preparation of a human
primary squamous cell carcinoma from the lung. See Stein et al.,
Cancer Res. 50: 1330 (1990), which is fully incorporated by
reference. The RS7 antibody recognizes a tumor-associated antigen,
which was defined by the murine MAb RS7-3G11 raised against human
non-small-cell lung carcinoma. Stein et al. discloses that the RS7
antibody recognizes a 46-48 kDa glycoprotein, characterized as
cluster 13. Stein et al., Int. J. Cancer Supp. 8:98-102 (1994). See
also, Basu et al., Int. J. Cancer 52:472-479 (1995). The antigen
has been designated as EGP-1 (epithelial glycoprotein-1) following
the proposal of the 3.sup.rd International IASLC Workshop on Lung
Tumor and Differentiation Antigens. See, for example DeLeij et al.,
Int. J. Cancer Supp., 8:60-63 (1994). Accordingly, as described
herein, the RS7 and EGP-1 antigens are synonymous. The EGY-1
antigen is also referred to as TROP2 in the literature, but there
may be multiple epitopes of both EGP-1 and TROP2.
[0042] Flow cytometry and immunohistochemical staining studies have
shown that the RS7 MAb detects antigen on a variety of tumor types,
with limited binding to normal human tissue. (Stein et al., (1990),
supra). The RS7 antibody is reactive with an EGP-1 glycoprotein,
which can be rapidly internalized. EGP-1 is expressed primarily by
carcinomas such as carcinomas of the lung, stomach, urinary
bladder, breast, ovary, uterus, and prostate. Localization and
therapy studies using radiolabeled murine RS7 MAb in animal models
have demonstrated tumor targeting and therapeutic efficacy (Stein
et al., (1990), supra. Stein et al., (1991), supra).
[0043] A more recent study has demonstrated strong RS7 staining in
tumors from the lung, breast, bladder, ovary, uterus, stomach, and
prostate. See Stein et al., Int. J. Cancer 55: 938 (1993), which is
fully incorporated by reference. Moreover, the lung cancer cases in
this study comprised both squamous cell carcinomas and
adenocarcinomas. Id. Both cell types stained strongly, indicating
that the RS7 antibody does not distinguish between histologic
classes of non-small-cell carcinoma of the lung.
[0044] As discussed supra, the RS7 MAb is rapidly internalized into
target cells (Stein et al. (1993), supra). The internalization rate
constant for RS7 MAb is intermediate between the internalization
rate constants of two other rapidly internalizing MAbs, which have
been demonstrated to be useful for immunotoxin production. Id. It
is well documented that the internalization of immunotoxin
conjugates is an absolute requirement for anti-tumor activity.
(Pastan et al., Cell 47:641 (1986)). Internalization of drug
immunoconjugates also has been described as a major factor in
anti-tumor efficacy. (Yang et al., Proc. Nat'l Acad. Sci. USA 85:
1189 (1988)). Therefore, the RS7 antigen may be an important target
for those types of immunotherapy that require internalization of
the therapeutic agent.
[0045] Thus, studies with the RS7 MAb indicate that the antibody
exhibits several important properties, which make it a candidate
for clinical diagnostic and therapeutic applications. Since the RS7
antigen provides a useful target for diagnosis and therapy, it is
desirable to obtain a MAb that recognizes an epitope of the RS7
antigen. Moreover, the availability of chimeric, humanized and
human RS7 antibodies is essential for the development of a
double-determinant enzyme-linked immunosorbent assay (ELISA), which
is desirable for detecting the RS7 antigen in clinical samples, and
essential for in vivo applications in humans.
[0046] To this end, the present invention describes chimeric,
humanized and human antibodies and fragments thereof that bind the
RS7 antigen and can be used for diagnostic and therapeutic methods.
Humanized antibodies and antibody fragments are described in
Provisional U.S. Application titled "Anti-CD20 Antibodies And
Fusion Proteins Thereof And Methods Of Use", U.S. Provisional
Application No. 60/356,132, filed Feb. 14, 2002, (expired), and
U.S. Provisional Application No. 60/416,232, filed Oct. 7, 2002,
(expired), both now U.S. application Ser. No. 10/366,709, filed
Feb. 4, 2003 (PGP No. US 2003-0219433-A1, now issued U.S. Pat. No.
7,151,164); hMN-14 antibodies, such as those disclosed in U.S. Pat.
No. 5,874,540, which is a Class III anti-carcinoembryonic antigen
antibody (anti-CEA antibody); Mu-9 antibodies, such as those
described in U.S. application Ser. No. 10/116,116, filed Apr. 5,
2002, titled "Chimeric, Human And Humanized Anti-CSAP Monoclonal
Antibodies;" AFP antibodies, such as those described in U.S.
Provisional Application No. 60/399,707, filed Aug. 1, 2002, titled
"Alpha-Fetoprotein IMMU31 Antibodies And Fusion Proteins And
Methods Of Use Thereof," (expired), now U.S. application Ser. No.
10/631,722, filed Aug. 1, 2003 (PGP No. US 2004-0235065 A1, now
issued U.S. Pat. No. 7,300,655); PAM4 antibodies, such as those
described in Provisional U.S. Application No. 60/388,313, filed
Jun. 14, 2002 (expired), titled "Monoclonal Antibody cPAM4, now
U.S. application Ser. No. 10/461,878, filed Jun. 16, 2003 (PGP No.
2004/0057902, now issued U.S. Pat. No. 7,238,786)"; RS7 antibodies,
such as those described in U.S. Provisional Application No.
60/360,229, filed Mar. 1, 2002 (expired), from which this
application claims priority; and CD22 antibodies, such as those
disclosed in U.S. Pat. Nos. 5,789,554 and 6,187,287 and U.S.
application Ser. No. 09/741,843 (PGP No. US-2002-0102254-A1) and
Ser. No. 09/988,013 (PGP No. US 2003-0103979-A1), all of which are
incorporated herein by reference in their entirety. A chimeric
antibody as disclosed herein 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 is 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. A humanized antibody is a
recombinant protein in which the CDRs from an antibody of one
species, e.g., a rodent antibody, are transferred from the heavy
and variable chains of the rodent antibody into human heavy and
light variable domains.
[0047] In a preferred embodiment, the RS7 antibody is humanized.
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 RS7
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. Another
embodiment of the present invention is an anti-EGF-1 antibody or
fragment thereof that is a subhuman primate anti-EGP-1 antibody,
murine monoclonal anti-EGP-1 antibody (restricted to veterinary
applications), chimeric anti-EGP-1 antibody, human anti-EGP-1
antibody, and humanized anti-EGP-1 antibody. Preferably, the
chimeric, human and humanized anti-EGP-1 antibody comprises
constant and hinge regions of a human IgG1. Also preferred, some
human residues in the framework regions of the humanized RS7
antibody or fragments thereof are replaced by their murine
counterparts. It is also preferred that a combination of framework
sequences from 2 different human antibodies are used for V.sub.H.
The constant domains of the antibody molecule are derived from
those of a human antibody.
[0048] Another preferred embodiment of the present invention is a
human RS7 antibody. A human antibody is 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 locus are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous heavy
chain and light chain loci. The transgenic mice can synthesize
human antibodies specific for human 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 their review, see e.g. Johnson and
Chiswell, Current Opinion in Structural Biology 3:5564-571
(1993).
[0049] The antibody and fragments thereof of the present invention
is preferably raised against a crude membrane preparation from a
human primary squamous cell carcinoma of the lung. Also preferred,
the RS7 antibody and fragments thereof is raised against a membrane
preparation of viable cells from a human ovarian carcinoma cell
line. Still preferred, the RS7 antigen is provided by viable Colo
316 cells. In a related vein, the RS7 antibody can be obtained
using a substantially pure preparation of the RS7 antigen. A
substantially pure protein is a protein that is essentially free
from contaminating cellular components, which are associated with
the protein in nature. As described herein, the term "RS7 antibody"
also includes chimeric, human and humanized RS7 antibodies.
Preparation of Chimeric, Humanized and Human RS7 Antibodies
[0050] Monoclonal antibodies to 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, RS7
antigen MAbs, such as RS7, can be obtained by injecting mice with a
composition comprising the RS7 antigen, 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 RS7 antigen, culturing
the clones that produce antibodies to RS7 antigen, and isolating
RS7 antibodies from the hybridoma cultures.
[0051] After the initial raising of antibodies to the immunogen,
the antibodies can be sequenced and subsequently prepared by
recombinant techniques. Humanization and chimerization of murine
antibodies and antibody fragments are well known to those skilled
in the art. For example, humanized monoclonal antibodies are
produced by transferring mouse complementary determining regions
from heavy and light variable chains of the mouse immunoglobulin
into a human variable domain, and then, substituting human residues
in the framework regions of the murine counterparts. The use of
antibody components derived from humanized monoclonal antibodies
obviates potential problems associated with the immunogenicity of
murine constant regions.
[0052] A human antibody of the present invention, i.e., human EGP-1
MAbs or other human antibodies, such as anti-EGP-2, MUC1-4, CEA,
CC49, CSAp, PSMA, PSA, EGFR, A33 and HER2/neu MAbs for combination
therapy with humanized, chimeric or human RS7 antibodies, 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,
which are incorporated in their entirety by reference. A human
antibody of the present invention that can be used for combination
therapy may also be reactive with an antigen selected from the
group consisting of Le(y), Tn, Tag-72, AFP, HCG, HCG-beta,
ferritin, PAP, EGP-2, histone, cytokeratin, Tenascin, CanAg, kidney
cancer G 250, VGFR1, VGFR2, or a combination thereof. 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.
[0053] 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), which
is incorporated by reference in its entirety. Techniques for
producing humanized MAbs are described, 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 hereby incorporated by reference.
[0054] In general, the V.kappa. (variable light chain) and V.sub.H
(variable heavy chain) sequences for RS7 antibodies can be obtained
by a variety of molecular cloning procedures, such as RT-PCR,
5'-RACE, and cDNA library screening. Specifically, the VH and
V.kappa. genes of the MAb RS7 were cloned by PCR amplification from
the hybridoma cells by RT-PCR and 5' RACE, respectively, and their
sequences determined by DNA sequencing. To confirm their
authenticity, the cloned V.sub.L 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)) which is
incorporated by reference. Based on the V gene sequences, a
humanized RS7 antibody can then be designed and constructed as
described by Leung et at. (Mol. Immunol., 32: 1413 (1995)), which
is incorporated by reference. cDNA can be prepared from any known
hybridoma line or transfected cell line producing a murine or
chimeric RS7 antibody by general molecular cloning techniques
(Sambrook et al., Molecular Cloning, A laboratory manual, 2.sup.nd
Ed (1989)). In a preferred embodiment, the RS7 hybridoma line is
used. The V.kappa. sequence for the mAb may be amplified using the
primers VK1BACK and VK1FOR (Orlandi et al., 1989) or the extended
primer set described by Leung et at. (BioTechniques, 15: 286
(1993)), which is incorporated by reference, while V.sub.H
sequences can be amplified using the primer pair VH1BACK/VH1FOR
(Orlandi et al., 1989 above), or the primers annealing to the
constant region of murine IgG described by Leung et al. (Hybridoma,
13:469 (1994)), which is incorporated by reference. The PCR
reaction mixtures containing 10 .mu.l of the first strand cDNA
product, 10 .mu.l of 10.times.PCR buffer [500 mM KCl, 100 mM
Tris-HCl (pH 8.3), 15 mM MgCl.sub.2, and 0.01% (w/v) gelatin]
(Perkin Elmer Cetus, Norwalk, Conn.), 250 .mu.M of each dNTP, 200
nM of the primers, and 5 units of Taq DNA polymerase (Perkin Elmer
Cetus) can be subjected to 30 cycles of PCR. Each PCR cycle
preferably consists of denaturation at 94.degree. C. for 1 min,
annealing at 50.degree. C. for 1.5 min, and polymerization at
72.degree. C. for 1.5 min. Amplified V.kappa. and V.sub.H fragments
can be purified on 2% agarose (BioRad, Richmond, Calif.).
Similarly, the humanized V genes can be constructed by a
combination of long oligonucleotide template syntheses and PCR
amplification as described by Leung et al. (Mol. Immunol., 32: 1413
(1995)).
[0055] PCR products for V.kappa. can be subcloned into a staging
vector, such as a pBR327-based staging vector, VKpBR, that contains
an Ig promoter, a signal peptide sequence and convenient
restriction sites to facilitate in-frame ligation of the V.kappa.
PCR products. PCR products for V.sub.H can be subcloned into a
similar staging vector, such as the pBluescript-based VHpBS.
Individual clones containing the respective PCR products may be
sequenced by, for example, the method of Sanger et al. (Proc. Natl.
Acad. Sci., USA, 74: 5463 (1977)), which is incorporated by
reference.
[0056] The DNA sequences described herein are to be taken as
including all alleles, mutants and variants thereof, whether
occurring naturally or induced.
[0057] The expression cassettes containing the V.kappa. and VH,
together with the promoter and signal peptide sequences can be
excised from VKpBR and VHpBS, respectively, by double restriction
digestion as HindIII-BamHI fragments. The V.kappa. and VH
expression cassettes can then be ligated into appropriate
expression vectors, such as pKh and pG1g, respectively (Leung et
al., Hybridoma, 13:469 (1994)). The expression vectors can be
co-transfected into an appropriate cell, e.g., myeloma Sp2/0-Ag14
(ATCC, VA), colonies selected for hygromycin resistance, and
supernatant fluids monitored for production of a chimeric or
humanized RS7 MAb by, for example, an ELISA assay, as described
below. Alternately, the V.kappa. and VH expression cassettes can be
assembled in the modified staging vectors, VKpBR2 and VHpBS2,
excised as XbaI/BamHI and XhoI/BamHI fragments, respectively, and
subcloned into a single expression vector, such as pdHL2, as
described by Gilles et al. (J. Immunol. Methods 125:191 (1989) and
also shown in Losman et al., Cancer, 80:2660 (1997)) for the
expression in Sp2/0-Ag14 cells. Another vector that is useful in
the present invention is the GS vector, as described in Barnes et
al., Cytotechnology 32:109-123 (2000), which is preferably
expressed in the NS0 cell line and CHO cells. Other appropriate
mammalian expression systems are described in Werner et al.,
Arzneim.-Forsch./Drug Res. 48(11), Nr. 8, 870-880 (1998).
[0058] Co-transfection and assay for antibody secreting clones by
ELISA, can be carried out as follows. About 10 .mu.g of VKpKh
(light chain expression vector) and 20 .mu.g of VHpG1g (heavy chain
expression vector) can be used for the transfection of
5.times.10.sup.6 SP2/0 myeloma cells by electroporation (BioRad,
Richmond, Calif.) according to Co et al., J. Immunol., 148: 1149
(1992) which is incorporated by reference. Following transfection,
cells may be grown in 96-well microtiter plates in complete HSFM
medium (Life Technologies, Inc., Grand Island, N.Y.) at 37.degree.
C., 5% CO.sub.2. The selection process can be initiated after two
days by the addition of hygromycin selection medium (Calbiochem,
San Diego, Calif.) at a final concentration of 500 units/ml of
hygromycin. Colonies typically emerge 2-3 weeks
post-electroporation. The cultures can then be expanded for further
analysis.
[0059] Suitable host cells include microbial or mammalian host
cells. A preferred host is the human cell line, PER.C6, which was
developed for production of MAbs, and other fusion proteins.
Accordingly, a preferred embodiment of the present invention is a
host cell comprising a DNA sequence encoding and anti-EGP-1 MAb,
conjugate, fusion protein or fragments thereof. PER.C6 cells (WO
97/00326) were generated by transfection of primary human embryonic
retina cells, using a plasmid that contained the Adserotype 5 (Ad5)
E1A- and E1B-coding sequences (Ad5 nucleotides 459-3510) under the
control of the human phosphoglycerate kinase (PGK) promoter. E1A
and E1B are adenovirus early gene activation protein 1A and 1B,
respectively. The methods and compositions are particularly useful
for generating stable expression of human recombinant proteins of
interest that are modified post-translationally, e.g. by
glycosylation. Several features make PER.C6 particularly useful as
a host for recombinant protein production, such as PER.C6 is a
fully characterized human cell line and it was developed in
compliance with good laboratory practices. Moreover, PER.C6 can be
grown as a suspension culture in defined serum-free medium devoid
of any human- or animal-derived proteins and its growth is
compatible with roller bottles, shaker flasks, spinner flasks and
bioreactors with doubling times of about 35 hrs. Finally, the
presence of E1A causes an up regulation of expression of genes that
are under the control of the CMV enhancer/promoter and the presence
of E13 prevents p53-dependent apoptosis possibly enhanced through
over expression of the recombinant transgene. In one embodiment,
the cell is capable of producing 2 to 200-fold more recombinant
protein and/or proteinaceous substance than conventional mammalian
cell lines.
[0060] Transfectoma clones that are positive for the secretion of
chimeric or humanized heavy chain can be identified by ELISA assay.
Briefly, supernatant samples (.about.100 .mu.l) from transfectoma
cultures are added in triplicate to ELISA microtiter plates
precoated with goat anti-human (GAH)-IgG, F(ab').sub.2
fragment-specific antibody (Jackson ImmunoResearch, West Grove,
Pa.). Plates are incubated for 1 hr at room temperature. Unbound
proteins are removed by washing three times with wash buffer (PBS
containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)
conjugated GAH-IgG, Fc fragment-specific antibodies (Jackson
ImmunoResearch) are added to the wells, (100 .mu.l of antibody
stock diluted.times.10.sup.4, supplemented with the unconjugated
antibody to a final concentration of 1.0 .mu.g/ml). Following an
incubation of 1 h, the plates are washed, typically three times. A
reaction solution, [100 .mu.l, containing 167 .mu.g of
orthophenylene-diamine (OPD) (Sigma, St. Louis, Mo.), 0.025%
hydrogen peroxide in PBS], is added to the wells. Color is allowed
to develop in the dark for 30 minutes. The reaction is stopped by
the addition of 50 .mu.l of 4 N HCl solution into each well before
measuring absorbance at 490 nm in an automated ELISA reader
(Bio-Tek instruments, Winooski, Vt.). Bound chimeric antibodies are
than determined relative to an irrelevant chimeric antibody
standard (obtainable from Scotgen, Ltd., Edinburg, Scotland).
[0061] Antibodies can be isolated from cell culture media as
follows. Transfectoma cultures are adapted to serum-free medium.
For production of chimeric antibody, cells are grown as a 500 ml
culture in roller bottles using HSFM. Cultures are centrifuged and
the supernatant filtered through a 0.2.mu. 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 the Centricon 30 (Amicon, Beverly, Mass.). The antibody
concentration is determined by ELISA, as before, 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.
[0062] The nucleotide sequences of the primers used to prepare the
RS7 antibodies are listed in Example 2, below. In a preferred
embodiment, a humanized RS7 antibody or antibody fragment comprises
the complementarity-determining regions (CDRs) of a murine RS7 MAb
and the framework (FR) regions of the light and heavy chain
variable regions of a human antibody and the light and heavy chain
constant regions of a human antibody, wherein the CDRs of the light
chain variable region of the humanized RS7 comprises CDR1
comprising an amino acid sequence of KASQDVSIAVA (SEQ ID NO:28);
CDR2 comprising an amino acid sequence of SASYRYT (SEQ ID NO:29);
and CDR3 comprising an amino acid sequence of QQHYITPLT (SEQ ID
NO:30); and the CDRs of the heavy chain variable region of the
humanized RS7 MAb comprises CDR1 comprising an amino acid sequence
of NYGMN (SEQ ID NO:31); CDR2 comprising an amino acid sequence of
WINTYTGEPTYTDDFKG (SEQ ID NO:32) and CDR3 comprising an amino acid
sequence of GGFGSSYWYFDV (SEQ ID NO:33). 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 RS7 MAb.
[0063] RS7 MAbs 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, 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).
[0064] RS7 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 RS7 MAb to bind to the RS7 antigen can be verified
using an indirect immunofluorescence assay, flow cytometry
analysis, or Western analysis.
Production of RS7 Antibody Fragments
[0065] The present invention contemplates the use of fragments of
RS7 and hRS7 antibodies. Antibody fragments, which recognize
specific epitopes, can be generated by known techniques. The
antibody fragments are antigen binding portions of an antibody,
such as F(ab')2, Fab', Fab, Fv, sFv and the like. Other antibody
fragments include, but are not limited to: the F(ab)'.sub.2
fragments which can be produced by pepsin digestion of the antibody
molecule and the Fab' fragments, which can be generated by reducing
disulfide bridges of the F(ab)'2 fragments. These methods are
described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and
4,331,647 and references contained therein, which patents are
incorporated herein in their entireties by reference. 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. The present invention
encompasses antibodies and antibody fragments.
[0066] A single chain Fv molecule (scFv) comprises a VL domain and
a VH domain. The VL and VH 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 VL-L-VH if
the VL domain is the N-terminal part of the scFv molecule, or as
VH-L-VL if the VH 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. Nos. 4,704,692,
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). These
references are incorporated herein by reference.
[0067] An antibody fragment can be prepared by proteolytic
hydrolysis of the 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 a 5S 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 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using papain produces two
monovalent Fab fragments and an Fc fragment directly. These methods
are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945
and 4,331,647 and references contained therein, which patents are
incorporated herein in their entireties by reference. 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.
[0068] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). A CDR is a
segment of the variable region of an antibody that is complementary
in structure to the epitope to which the antibody binds and is more
variable than the rest of the variable region. Accordingly, a CDR
is sometimes referred to as hypervariable region. A variable region
comprises three CDRs. CDR peptides can be obtained by constructing
genes encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology 2: 106 (1991); Courtenay-Luck, "Genetic
Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies:
Production, Engineering and Clinical Application, Ritter et al.
(eds.), pages 166-179 (Cambridge University Press 1995); and Ward
et al., "Genetic Manipulation and Expression of Antibodies," in
Monoclonal Antibodies: Principles and Applications, Birch et al.,
(eds.), pages 137-185 (Wiley-Liss, Inc. 1995).
[0069] 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.
Production of Chimeric, Humanized and Human RS7 Antibody Fusion
Proteins
[0070] Antibody fusion proteins and fragments thereof 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 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., disiocyanates, diiosothiocyanates,
bis(hydroxysuccinimide) esters, carbodiimides,
maleimidehydroxysuccinimide esters, and the like.
[0071] A simple method to produce chimeric, humanized and human RS7
antibody fusion proteins is to mix the antibodies or fragments in
the presence of glutaraldehyde to form an antibody fusion protein.
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. Antibody fusion proteins are expected to
have a greater binding specificity than MAbs, since the fusion
proteins comprise moieties that bind to at least two epitopes of
the RS7 antigen. Thus, antibody fusion proteins arc the preferred
form of RS7 antigen binding protein for therapy.
[0072] In the present context, an antibody fusion protein comprises
at least two chimeric, humanized or human RS7 MAbs, or fragments
thereof, wherein at least two of the MAbs or fragments bind to
different epitopes of the RS7 antigen or against an RS7 epitope and
that of a totally different antigen. For example, a bispecific RS7
antibody fusion protein may comprise a CEA antibody or fragment
thereof and the RS7 MAb or fragment thereof. Such a bispecific RS7
antibody fusion protein can be prepared, for example, by obtaining
an F(ab').sub.2 fragment from CEA as described above. The
interchain disulfide bridges of the antibody F(ab')2 fragment are
gently reduced with cysteine, 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)bis-malemide). The RS7 MAb is
converted to Fab'-SH and then reacted with the activated CEA
Fab'-SH fragment to obtain a bispecific RS7 antibody fusion
protein.
[0073] A polyspecific RS7 antibody fusion protein can be obtained
by adding RS7 antigen binding moieties to a bispecific chimeric,
humanized or human RS7 antibody fusion protein. For example, a
bispecific antibody fusion protein can be reacted with
2-iminothiolane to introduce one or more sulfhydryl groups for use
in coupling the bispecific fusion protein to a third RS7 antigen
MAb or fragment, using the bis-maleimide activation procedure
described above. These techniques for producing antibody composites
are well known to those of skill in the art. See, for example, U.S.
Pat. No. 4,925,648, which is incorporated by reference in its
entirety.
[0074] 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')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')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., 1 Exp. Med. 128: 1461 (1968), and U.S. Pat. No.
4,331,647. Contemplated in the present invention is an antibody
fusion protein or fragment thereof comprising at least one first
anti-EGP-1 MAb or fragment thereof and at least one second MAb or
fragment thereof, other than the anti-EGP-1 MAbs or fragments
thereof of the present invention.
[0075] 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., and 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.
[0076] 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 sulfydryl
groups, as noted above. Another method involves reacting an
antibody having an oxidized carbohydrate portion with another
antibody, which has at lease one free amine function. This results
in an initial Schiff base (mime) 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--incorporated by
reference.
[0077] ScFvs with linkers greater than 12 amino acid residues in
length (for example, 15- or 18-residue linkers) allow interacting
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. For example, scFvs of the
anti-neuraminidase antibody NC 10 formed predominantly trimers
(V.sub.H to V.sub.L orientation) or tetramers (V.sub.L to V.sub.H
orientation) with 0-residue linkers (Dolezal et al., Protein
Engineering (2000) 13: 565-574). For scFvs constricted from NC10
with 1- and 2-residue linkers, the V.sub.H to V.sub.L orientation
formed predominantly diabodies (Atwell et al., Protein Engineering
(1999) 12: 597-604); in contrast, the V.sub.L, to V.sub.H
orientation formed a mixture of tetramers, trimers, dimers, and
higher mass multimers (Dolezal et al., Protein Engineering (2000)
13: 565-574). For scFvs constructed from the anti-CD 19 antibody
HD37 in the V.sub.H to V.sub.L, orientation, the 0-residue linker
formed exclusively trimers and the 1-residue linker formed
exclusively tetramers (Le Gall et al., FEBS Letters (1999) 453:
164-168).
[0078] The RS7 antibodies and fragments thereof of the present
invention can also be used to produce antigen-specific diabodies,
triabodies and tetrabodies, which are multivalent but monospecific.
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).
Methods of producing multivalent, monospecific binding proteins
based on scFv are disclosed in U.S. Pat. No. 5,837,242 (1998) and
U.S. Pat. No. 5,844,094 (1998) and WO-98/44001 (1998). A preferred
embodiment of the instant invention is a multivalent, multispecific
antibody or fragment thereof comprising one or more antigen binding
sites having affinity toward an EGP-1 target antigen and one or
more hapten binding sites having affinity towards hapten
molecules.
Determining Antibody Binding Affinity
[0079] Comparative binding affinities of the mRS7, cRS7 and hRS7
antibodies thus isolated may be determined by direct
radioimmunoassay. RS7 can be labeled with .sup.131I or .sup.125I
using the chloramines-T method (see, for example, Greenwood et al.,
Biochem. J., 89: 123 (1963) which is incorporated by reference).
The specific activity of the iodinated antibody is typically
adjusted to about 10 .mu.Ci/.mu.g. Unlabeled and labeled antibodies
are diluted to the appropriate concentrations using reaction medium
(HSFM supplemented with 1% horse serum and 100 .mu.g/ml
gentamicin). The appropriate concentrations of both labeled and
unlabeled antibodies are added together to the reaction tubes in a
total volume of 100 .mu.l. A culture of ME180 cells (a human
cervical carcinoma cell line) is sampled and the cell concentration
determined. The culture is centrifuged and the collected cells
washed once in reaction medium followed by resuspension in reaction
medium to a final concentration of about 10.sup.7 cells/ml. All
procedures are carried out in the cold at 4.degree. C. The cell
suspension, 100 .mu.l, is added to the reaction tubes. The reaction
is carried out at 4.degree. C. for 2 h with periodic gentle shaking
of the reaction tubes to resuspend the cells. Following the
reaction period, 5 ml of wash buffer (PBS containing 1% BSA) is
added to each tube. The suspension is centrifuged and the cell
pellet washed a second time with another 5 ml of wash buffer.
Following centrifugation, the amount of remaining radioactivity
remaining in the cell pellet is determined in a gamma counter
(Minaxi, Packard Instruments, Sterling, Va.).
Expression Vectors
[0080] 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. A promoter is a DNA sequence that
directs the transcription of a structural gene. A structural gene
is a DNA sequence that is transcribed into messenger RNA (mRNA)
which is then translated into a sequence of amino acids
characteristic of a specific polypeptide. Typically, a promoter is
located in the 5' region of a gene, proximal to the transcriptional
start site of a structural gene. If a promoter is an inducible
promoter, then the rate of transcription increases in response to
an inducing agent. In contrast, the rate of transcription is not
regulated by an inducing agent if the promoter is a constitutive
promoter. An enhancer is a DNA regulatory element that can increase
the efficiency of transcription, regardless of the distance or
orientation of the enhancer relative to the start site of
transcription.
[0081] An isolated DNA molecule is a fragment of DNA that is not
integrated in the genomic DNA of an organism. For example, a cloned
RS7 antigen gene is a DNA fragment that has been separated from the
genomic DNA of a mammalian cell. Another example of an isolated DNA
molecule is a chemically-synthesized DNA molecule that is not
integrated in the genomic DNA of an organism. Complementary DNA
(cDNA) is a single-stranded DNA molecule that is formed from an
mRNA template by the enzyme reverse transcriptase. Typically, a
primer complementary to portions of mRNA is employed for the
initiation of reverse transcription. Those skilled in the art also
use the term "cDNA" to refer to a double-stranded DNA molecule
consisting of such a single-stranded DNA molecule and its
complementary DNA strand.
[0082] A cloning vector is a DNA molecule, such as a plasmid,
cosmid, or bacteriophage that has the capability of replicating
autonomously in a host cell. Cloning vectors typically contain one
or a small number of restriction endonuclease recognition sites at
which foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function of the
vector, as well as a marker gene that is suitable for use in the
identification and selection of cells transformed with the cloning
vector. Marker genes typically include genes that provide
tetracycline resistance or ampicillin resistance. 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 that have been genetically
engineered to contain the cloned gene(s) in the chromosome or
genome of the host cell. The term expression refers to the
biosynthesis of a gene product. For example, in the case of a
structural gene, expression involves transcription of the
structural gene into mRNA and the translation of mRNA into one or
more polypeptides.
Humanized, Human and Chimeric RS7 Antibodies Use for Treatment and
Diagnosis
[0083] Contemplated in the present invention is a method of
diagnosing or treating a malignancy in a subject comprising
administering to the subject a therapeutically effective amount of
a therapeutic conjugate comprising an EGP-1 MAb or fragment thereof
or an antibody fusion protein or fragment thereof, wherein the
EGP-1MAb or fragment thereof or antibody fusion protein or fragment
thereof is bound to at least one therapeutic agent and then
formulated in a pharmaceutically suitable excipient. It is also
contemplated that an unconjugated (naked) EGP-1 MAb or fusion
construct with other antigen-binding moieties also can be sued as a
therapeutic for cancer cells expressing EGP-1. These unconjugated`
antibodies may be given advantageously in combination with other
therapeutic modalities, such as chemotherapy, radiotherapy, and/or
immunotherapy, either together or in various sequences and
schedules. Also preferred is a method for diagnosing or treating
cancer, comprising: administering a multivalent, multispecific
antibody or fragment thereof comprising one or more antigen binding
sites toward a EGP-1 antigen and one or more hapten binding sites
to a subject in need thereof, waiting a sufficient amount of time
for an amount of the non-binding protein 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 binding site of the
multivalent, multispecific antibody or fragment thereof. In a
preferred embodiment, the cancer is a lung, breast, head and neck,
ovarian, prostate, bladder or colon cancer.
[0084] Hybridoma technology for the production of monoclonal
antibodies (MAbs) has provided a method for the production of
molecular probes capable of locating or killing cancer cells. Tumor
imaging techniques using radiolabeled MAbs have been used to
delineate cancerous invasion in a number of malignancies. In
experimental animals and in humans, antibodies have been used for
the radioimmunodetection of carcinoembryonic antigen in diverse
tumors that express carcinoembryonic antigen, and also tumors such
as melanoma, colon carcinoma, and breast carcinoma with other
targeting antibodies. Goldenberg et al., Cancer Res. 40: 2984
(1980); Hwang et al., Cancer Res. 45: 4150 (1985); Zalcberg et al.,
J. Nat'l Cancer Inst. 71: 801 (1983); Colcher et al., Cancer Res.
43: 736 (1983); (Larson et al., J. Nucl. Med. 24: 123 (1983);
DeLand et al., Cancer Res. 40: 3046 (1980); Epenetos et al., Lancet
2: 999 (1982).
[0085] 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 using techniques such as radioimmunoassay,
enzyme-linked immunosorbent assay, and fluorescence
immunoassay.
[0086] 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)). Also, see Stein et al., Antibody Immunoconj.
Radiopharm. 4: 703 (1991), which is fully incorporated by
reference. Moreover, Phase-I therapeutic trials with some of these
MAbs have been initiated for treatment of lymphoma, melanoma, and
other malignancies. See, for example, DeNardo et al., Int. J Cancer
Suppl. 3: 96 (1988), and Goldenberg et al., J Clin. Oncol. 9: 548
(1991).
[0087] Humanized, chimeric and fully human antibodies and fragments
thereof are suitable for use in therapeutic methods and diagnostic
methods. Accordingly, contemplated in the present invention 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-EGP-1 antibody and (ii)
administering to a subject in need thereof the diagnostic or
therapeutic antibody conjugate. Preferably, the chimeric, humanized
and fully human RS7 antibodies and fragments thereof of the present
invention are used in methods for treating malignancies.
[0088] Also described herein is a cancer cell targeting diagnostic
or therapeutic conjugate comprising an antibody component
comprising an anti-EGP-1 mAb or fragment thereof or an antibody
fusion protein or fragment thereof that hinds to the cancer cell,
wherein the antibody component is bound to at least one diagnostic
or at least one therapeutic agent. Preferably, the diagnostic
conjugate comprises at least a photoactive diagnostic agent or an
MRI contrast agent. Still preferred, the diagnostic agent is a
radioactive label with an energy between 60 and 4,000 keV.
[0089] The compositions for treatment contain at least one naked or
conjugated humanized, chimeric or human RS7 antibody alone, or in
combination with other naked or conjugated humanized, chimeric,
human or other antibodies of the present invention, or other naked
or conjugated humanized, chimeric or human antibodies not disclosed
herein. The present invention also contemplates administration of a
conjugated or naked antibody with a therapeutic agent such as an
immunomodulator, or diagnostic agent that is not conjugated to the
anti-EGP-1 antibody. Naked or conjugated antibodies to the same or
different epitope or antigen may be also combined with one or more
of the antibodies of the present invention.
[0090] Accordingly, the present invention contemplates the
administration anti-EGP-1 antibodies and fragments thereof alone,
as a naked antibody or antibody fragment, or administered as a
multimodal therapy. Preferably, the antibody is a humanized,
chimeric or fully human RS7 antibody or fragment thereof.
Multimodal therapies of the present invention further include
immunotherapy with a naked anti-EGP-1 antibody supplemented with
administration of other antibodies in the form of naked antibodies,
fusion proteins, or as immunoconjugates. For example, a humanized,
chimeric or fully human RS7 antibody may be combined with another
naked humanized, chimeric RS7 or other antibody, or a humanized,
chimeric RS7 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 EGP-1 or RS7 antibody or fragments thereof
before, in combination with, or after other solid tumor/carcinoma
associated antibodies such as anti-EGP-2, CEA, CSAp, MUC1-4, EGFR,
HER2/neu, PSA, CC49 (anti-Tag 72 antibody) and PSMA antibodies.
These solid tumor antibodies may be naked or conjugated to, inter
alia, drugs, enzymes, hormones, toxins, isotopes, or
immunomodulators. A fusion protein of a humanized, chimeric or
fully human RS7 antibody and a toxin or may also be used in this
invention. Many different antibody combinations may be constructed,
either as naked antibodies or as partly naked and partly conjugated
with a therapeutic agent or immunomodulator. Alternatively,
different naked antibody combinations may be employed for
administration in combination with other therapeutic agents, such
as a cytotoxic drug or with radiation. Combinations of such
antibodies can also be made, advantageously, with antisense
oligonucleotides, as are known in the art. As such, the therapeutic
conjugates may comprise an oligonucleotide, especially an antisense
oligonucleotide that preferably are directed against oncogenes and
oncogene products of B-cell malignancies. For example, antisense
molecules inhibiting bcl-2 expression that are described in U.S.
Pat. No. 5,734,033 (Reed) which is incorporated by reference in its
entirety, may also be conjugated to, or form the therapeutic agent
portion of an antibody fusion protein or be administered with a
humanized RS7 antibody of the present invention.
[0091] The monospecific binding proteins described herein that are
linked to diagnostic or therapeutic agents directly target RS7
positive tumors. The monospecific molecules bind selectively to
targeted antigens and as the number of binding sites on the
molecule increases, the affinity for the target cell increases and
a longer residence time is observed at the desired location.
Moreover, non-antigen bound molecules are cleared from the body
quickly and exposure of normal tissues is minimized. A use of
multispecific binding proteins is pre-targeting RS7 positive tumors
for subsequent specific delivery of diagnostic or therapeutic
agents. The agents arc carried by histamine succinyl glycyl (HSG)
containing peptides. The murine monoclonal antibody designated 679
(an IgG1, K) binds with high affinity to molecules containing the
tri-peptide moiety, HSG (Morel et al., Molecular immunology, 27,
995-1000, 1990). 679 MAb can form a bispecific binding protein with
hRS7 that binds with HSG and the target antigen. Alternative
haptens may also be utilized. These binding proteins bind
selectively to targeted antigens allowing for increased affinity
and a longer residence time at the desired location. Moreover,
non-antigen bound diabodies are cleared from the body quickly and
exposure of normal tissues is minimized.
[0092] RS7 antibodies and fragments thereof can be used to treat
mammalian disorders such as cancer. The cancer includes, but is not
limited to, lung, breast, bladder, ovarian prostate and colon
cancers.
[0093] Delivering a diagnostic or a therapeutic agent to a target
for diagnosis or treatment in accordance with the invention
includes providing the anti-EGP-1 antibody or fragments thereof
with a diagnostic or therapeutic agent and administering to a
subject in need thereof with the binding protein. Diagnosis further
requires the step of detecting the bound proteins with known
techniques.
[0094] Administration of the antibodies and their fragments of the
present invention with diagnostic or therapeutic agents can be
effected in a mammal by intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, perfusion through a regional catheter, or direct
intralesional injection. When administering the binding protein by
injection, the administration may be by continuous infusion or by
single or multiple boluses. Doses in the range of 20 to 800
mg/m.sup.2 are feasible, with doses between 100 and 500 mg/m.sup.2
preferably, for therapy, and commensurately lower doses recommended
for diagnostic imaging, such as 0.5 mg to 100 mg/patient. Such
doses may be repeated at different frequencies, depending on the
clinical situation and patient tolerance.
[0095] The antibody with the diagnostic or therapeutic agent may be
provided as a kit for human or mammalian therapeutic and diagnostic
use in a pharmaceutically acceptable injection vehicle, preferably
phosphate-buffered saline (PBS) at physiological pH and
concentration. The preparation preferably will be sterile,
especially if it is intended for use in humans. Optional components
of such kits include stabilizers, buffers, labeling reagents,
radioisotopes, paramagnetic compounds, second antibody for enhanced
clearance, and conventional syringes, columns, vials and the
like.
Naked Antibody Therapy
[0096] A therapeutically effective amount of the naked chimeric,
humanized and fully human RS7 antibodies, or their fragments, can
be formulated in a pharmaceutically acceptable excipient. The
efficacy of the naked chimeric, humanized and fully human RS7
antibodies can also be enhanced by supplementing these naked
antibodies with one or more other naked antibodies, with one or
more immunoconjugates of chimeric, humanized and fully human RS7
antibodies conjugated to a therapeutic agent, such as a drug,
toxin, immunomodulator, hormone, growth factor, enzyme or
therapeutic radionuclides, or with one or more therapeutic agent,
including a drug, toxin, immunomodulator, hormone, growth factor,
enzyme, oligonucleotide, or therapeutic radionuclide, administered
concurrently or sequentially or according to a prescribed dosing
regimen, with the RS7 antibodies or fragments thereof.
[0097] In a preferred embodiment, the naked or conjugated RS7
antibodies of the present invention are combined with at least one
cancer drug. Such combination therapy can improve the effect of the
drug or lower drug dose that is needed. For example, the IC50 value
was determined for Dox-RS7 and 2P-Dox-RS7 on a lung cancer cell
line, Calu3, and two breast cancer cell lines, MDA468 and T47D,
respectively. Calu3 and T47D cells are positive for an EGP-1
antigen and negative for a CEA antigen, and MDA468 is positive for
both the EGP-1 and CEA antigens. Results indicate that the IC50
value for Dox-RS7 is 0.04 .mu.g/ml and for 2P-Dox-RS7 is 0.023
.mu.g/ml. Therefore, conjugating a naked, human, humanized or
chimeric anti-EGP-1 antibody or fragment of the present invention
to a particular drug, such as 2P-Dox may help overcome multidrug
resistance. This is also possible when the antibody is given in a
combination with a particular drug, as described.
RS7 Immunoconjugates
[0098] The present invention also contemplates the use of
humanized, chimeric and human RS7 antibodies and fragments thereof
for therapy. The objective of immunotherapy is to deliver cytotoxic
doses of radioactivity, toxin, cytokine, enzyme, or hormone, or
drug to target cells, while minimizing exposure to non-target
tissues. The RS7 antigen binding proteins of the present invention
can be used to treat a variety of tumors, such as of the lung,
breast, bladder, ovary, uterus, stomach, and prostate.
[0099] Any of the antibodies or antibody fusion proteins and
fragments thereof of the present invention can be conjugated with
one or more therapeutic or diagnostic agents. Generally, one
therapeutic or diagnostic agent is attached to each antibody or
antibody fragment but more than one therapeutic agent or diagnostic
agent can be attached to the same antibody or antibody fragment. If
the Fc region is absent (for example when the antibody used as the
antibody component of the immunoconjugate is an antibody fragment),
it is possible to introduce a carbohydrate moiety into the light
chain variable region of a full-length antibody or antibody
fragment. 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, all of which are incorporated in
their entirety by reference. The engineered carbohydrate moiety is
used to attach the therapeutic or diagnostic agent.
[0100] 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, all of which are incorporated in
their entirety 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 peptide. 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.
Also, a chelator such as DTPA (such as Mx-DTPA), DOTA, TETA, or
NOTA can be attached to the antibody.
[0101] The antibody fusion proteins of the present invention
comprise two or more antibodies or fragments thereof and each of
the antibodies or fragments that compose this fission protein can
contain a therapeutic agent or diagnostic agent. Additionally, one
or more of the antibodies or fragments of the antibody fusion
protein can have more than one therapeutic of 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.
Particularly, 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.
[0102] A wide variety of diagnostic and therapeutic reagents can be
advantageously conjugated to the antibodies of the invention. The
therapeutic agents recited here are those agents that also are
useful for administration separately with the naked antibody as
described above. Therapeutic agents include, for example,
chemotherapeutic drugs such as vinca alkaloids, anthracyclines,
epidophyllotoxinw, taxanes, antimetabolites, alkylating agents,
antibiotics, substituted urea, enzymes, Cox-2 inhibitors,
antimitotics, antiangiogenic and apoptotoic agents, particularly
doxorubicin, doxorubicin analogs, methotrexate, taxol, CPT-11,
camptothecans, and others from these and other classes of
anticancer agents, methyl hydrazine derivative, adrenocortical
suppressant, antagonist, endostatin, taxol, and the like. Other
useful cancer chemotherapeutic drugs for the preparation of
immunoconjugates and antibody fusion proteins include nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine
analogs, purine analogs, platinum coordination complexes, hormones,
tyrosine kinase inhibitors, such as those that inhibit a
EGF-receptor tyrosine kinase, a BCR ABL tyrosine kinase or a
VEGF-receptor tyrosine kinase, and the like. 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.
[0103] A toxin, such as Pseudomonas exotoxin, may also be complexed
to or form the therapeutic agent portion of an immunoconjugate of
the RS7 and hRS7 antibodies of the present invention. 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, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas
endotoxin. See, for example, Pastan et al., Cell 47:641 (1986), and
Goldenberg, CA--A Cancer Journal for Clinicians 44:43 (1994).
Additional toxins suitable for use in the present invention are
known to those of skill in the art and are disclosed in U.S. Pat.
No. 6,077,499, which is incorporated in its entirety by
reference.
[0104] An immunomodulator, such as a cytokine may also be
conjugated to, or form the therapeutic agent portion of the EGP-1,
RS7 and hRS7 immunoconjugate, or be administered unconjugated to
the chimeric, humanized or human RS7 antibodies or fragments
thereof of the present invention. As used herein, the teen
"immunomodulator" includes cytokines, stem cell growth factors,
lymphotoxins, such as tumor necrosis factor (TNF), and
hematopoietic factors, such as interleukins (e.g., interleukin-1
(IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony
stimulating factors (e.g., granulocyte-colony stimulating factor
(G-CSF) and granulocyte macrophage-colony stimulating factor
(GM-CSF)), interferons (e.g., interferons-.alpha., -.beta. and
-.gamma.), the stem cell growth factor designated "S1 factor,"
erythropoietin and thrombopoietin, or a combination thereof.
Examples of suitable immunomodulator moieties include IL-2, IL-6,
IL-10, IL-12, IL-18, IL-21, interferon-.gamma., TNF-.alpha., and
the like. Alternatively, subjects can receive naked EGP-1 or RS7
antibodies and a separately administered cytokine, which can be
administered before, concurrently or after administration of the
naked RS7 antibodies. The RS7 antibody may also be conjugated to
the immunomodulator. The immunomodulator may also be conjugated to
a hybrid antibody consisting of one or more antibodies binding to
different antigens.
[0105] 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 peptides 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). Alternatively, the therapeutic or
diagnostic agent can be conjugated via a carbohydrate moiety in the
Fc region of the antibody. The carbohydrate group can be used to
increase the loading of the same peptide that is bound to a thiol
group, or the carbohydrate moiety can be used to bind a different
peptide.
[0106] Furthermore, a radiolabeled antibody, immunoconjugate, or
fragments thereof may comprise a .gamma.-emitting radioisotope or a
positron-emitter useful for diagnostic imaging. Suitable
radioisotopes, particularly in the energy range of 25 to 4,000 keV,
include .sup.131I, .sup.123I, .sup.124I, .sup.86Y, .sup.62Cu,
.sup.64Cu, .sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.94mTc, .sup.18F,
.sup.11C, .sup.13N, .sup.15O, .sup.75Br, and the like. See for
example, U.S. Patent Application entitled "Labeling Targeting
Agents with Gallium-68"--Inventors G. L. Griffiths and W. J.
McBride, (U.S. Provisional Application No. 60/342,104 (expired),
now U.S. Pat. No. 7,011,816), which discloses positron emitters,
such as .sup.18F, .sup.68Ga, .sup.94mTc and the like, for imaging
purposes and which is incorporated in its entirety by reference.
Preferably, the energy range for diagnostic and therapeutic
radionuclides is 25-4,000_keV. Other useful radionuclides include
.sup.90Y, .sup.111In, .sup.125I, .sup.3H, .sup.35S, .sup.14C,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.177Lu, .sup.67Cu,
.sup.212Bi, .sup.213Bi, .sup.211At, .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.94mTc,
.sup.121mTe, .sup.122mTe, .sup.125mTe, .sup.165Tm, .sup.167Tm,
.sup.168Tm, .sup.111Ag, .sup.197Pt, .sup.109Pd, .sup.32P, .sup.33P,
.sup.47Sc, .sup.153Sm, .sup.177Lu, .sup.105Ru, .sup.142Pr,
.sup.143Pr, .sup.161Tb, .sup.166Ho, .sup.199Au, .sup.57Co,
.sup.58Co, .sup.51Cr, .sup.59Fe, .sup.18F, .sup.75Se, .sup.201Tl,
.sup.225Ac .sup.76Br, .sup.86Y, .sup.169Yb, .sup.166Dy, .sup.212Pb,
and .sup.223Ra.
[0107] 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 RS7 antigen binding protein using the chelating
agent, p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid
(TETA). Chase, supra. Alternatively, .sup.90Y, which emits an
energetic beta particle, can be coupled to an RS7 antigen binding
protein using diethylenetriaminepentaacetic acid (DTPA). Moreover,
a method for the direct radiolabeling of the RS7 MAb with .sup.131I
is described by Stein et al. (1991), supra, and the patent by
Govindan et al., WO 9911294A1 entitled "Stable Radioiodine
Conjugates and Methods for Their Synthesis," and is incorporated
herein by reference in their entirety.
[0108] The RS7 antibodies or fragments thereof of the present
invention that have a boron addend-loaded carrier for thermal
neutron activation therapy will normally be effected in similar
ways. However, it will be advantageous to wait until non-targeted
RS7 immunoconjugate clears before neutron irradiation is performed.
Clearance can be accelerated using an antibody that binds to the
RS7 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 RS7 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 RS7 antibody.
After administration of the RS7 antibody conjugate, a boron addend
is activated by thermal neutron irradiation and converted to
radioactive atoms that decay by .alpha.-emission to produce highly
toxic, short-range effects.
[0109] Furthermore, the present invention includes methods of
diagnosing cancer in a subject. Diagnosis may be accomplished by
administering a diagnostically effective amount of a diagnostic
conjugate, formulated in a pharmaceutically suitable excipient, and
detecting said label. For example, radioactive and non-radioactive
agents can be used as diagnostic agents. A suitable non-radioactive
diagnostic agent is a contrast agent suitable for magnetic
resonance imaging, 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, when used along with the antibodies of the
invention. See U.S. Ser. No. 09/921,290 filed on Oct. 10, 2001 (PGP
No. US 2002/0041847 A1), which is incorporated in its entirety by
reference.
[0110] Accordingly, a method of diagnosing a malignancy in a
subject is described, comprising (i) performing an in vitro
diagnosis assay on a specimen from the subject with a composition
comprising a naked anti-EGP-1 MAb or fragment thereof or a naked
antibody fusion protein or fragment thereof. For example, RT-PCR
and immunoassay in vitro diagnosis methods can be used to detect
the presence of minute amounts of EGP-1 in tissues, blood and other
body fluids as a useful diagnostic/detection method.
Immunohistochemistry can be used to detect the presence of EGP-1 in
a cell or tissue. Preferably, the malignancy that is being
diagnosed is a cancer. Most preferably, the cancer is selected from
the group of lung, prostate, ovarian, breast, colon and
bladder.
[0111] 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. 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 (Joni 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). However, these
earlier studies did not include use of endoscopic therapy
applications, especially with the use of antibody fragments or
subfragments. Thus, the present invention contemplates the
therapeutic use of immunoconjugates comprising photoactive agents
or dyes.
[0112] Contrast agents such as a MRI contrast agent, a paramagnetic
ion and an ultrasound enhancing agent are also contemplated in the
present invention. For example, gadolinium ions, lanthanum ions,
manganese ions or other comparable label, CT contrast agents, and
ultrasound contrast agents are suitable for use in the present
invention. In a preferred embodiment, the ultrasound enhancing
agent is a liposome that comprises a humanized RS7 IgG or fragment
thereof. Also preferred, the liposome is gas filled.
[0113] For purposes of therapy, the RS7 antibodies and fragments
thereof of the present invention are administered to a patient in a
therapeutically effective amount. An antibody is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient.
In Vitro Diagnosis
[0114] The present invention contemplates the use of RS7
antibodies, including RS7 and hRS7 antibodies and fragments
thereof, to screen biological samples in vitro for the presence of
the RS7 antigen. In such immunoassays, the RS7 antibody may be
utilized in liquid phase or bound to a solid-phase carrier, as
described below. Also, see Stein et al. (1993), supra, and Stein et
al., Cancer Res. 49: 32 (1989), which is fully incorporated by
reference.
[0115] One example of a screening method for determining whether a
biological sample contains the RS7 antigen is the radioimmunoassay
(RIA). For example, in one form of RIA, the substance under test is
mixed with RS7 antigen MAb in the presence of radiolabeled RS7
antigen. In this method, the concentration of the test substance
will be inversely proportional to the amount of labeled RS7 antigen
bound to the MAb and directly related to the amount of free labeled
RS7 antigen. Other suitable screening methods will be readily
apparent to those of skill in the art.
[0116] Alternatively, in vitro assays can be performed in which an
RS7 antigen binding 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.
[0117] Other suitable in vitro assays will be readily apparent to
those of skill in the art. The specific concentrations of
detectably labeled RS7 antigen binding protein and RS7 antigen, the
temperature and time of incubation, as well as other assay
conditions maybe varied, depending on various factors including the
concentration of the RS7 antigen in the sample, the nature of the
sample, and the like. The binding activity of a sample of RS7
antigen binding protein 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.
[0118] Other such steps as washing, stirring, shaking, filtering
and the like may be added to the assays as is customary or
necessary for the particular situation.
[0119] The presence of the RS7 antigen in a biological sample can
be determined using an enzyme-linked immunosorbent assay (ELISA).
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.
[0120] 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).
[0121] 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. An antibody fragment is a portion of
an antibody such as F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the
like. In the present context, an antibody fragment is a portion of
an RS7 MAb that binds to an epitope of the RS7 antigen. The term
"antibody fragment" also includes any synthetic or genetically
engineered protein that acts like an antibody by binding to a
specific antigen to form a complex. For example, antibody fragments
include isolated fragments consisting of the light chain variable
region, "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. An antibody fusion protein is a polyspecific
antibody composition comprising at least two substantially
monospecific antibodies or antibody fragments, wherein at least two
of the antibodies or antibody fragments bind to different epitopes
of the RS7 antigen. An RS7 fusion protein also includes a conjugate
of an antibody fusion protein with a diagnostic or therapeutic
agent. The term RS7 antibody includes humanized, chimeric, human
and murine antibodies, antibody fragments thereof, immunoconjugates
and fragments thereof and antibody fusion proteins and fragments
thereof.
[0122] 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). For
example, in one method for the detection of RS7 antigen using the
double-determinant ELISA, finely minced tissue from a biopsy sample
is lyophilized and resuspended in lysis buffer (100 mM NaCl, 50 mM
Tris-HCl, pH 7.4) containing 1% nonidet-p40 (NP40), 0.6 .mu.l/ml
aprotinin, 0.2 mM phenyl methyl sulphonyl fluoride, 0.1 .mu.g/ml
leupeptin and 1 mM EDTA at a concentration of 10-20 mg tissue (wet
weight) per 500 .mu.l solution. The suspension is incubated for 60
minutes on ice, and then sonicated for approximately six 10-second
intervals. Insoluble material is removed by centrifugation.
[0123] The soluble extract is added to microliter plate wells
containing an adsorbed RS7 antigen MAb as the capture antibody.
Captured RS7 antigen is then recognized by a second RS7 antigen
MAb, which has been coupled with alkaline phosphatase. The amount
of bound alkaline phosphatase, proportional to the amount of RS7
antigen in the extract, is detected colormetrically using a
chromogenic substrate, such as p-nitrophenylphosphate.
[0124] Alternatively, a double-determinant ELISA for the RS7
antigen can be performed using horse radish peroxidase. Other
variations of sample preparation and the double-determinant ELISA
can be devised by those of skill in the art with routine
experimentation.
[0125] In the double-determinant ELISA, the soluble antibody or
antibody fragment must bind to an RS7 epitope that is distinct from
the epitope recognized by the capture antibody. For example, the
soluble antibody can be the RS7 MAb, while the capture antibody can
be MR23. Alternatively, the soluble antibody can be MR23, while the
capture antibody can be the RS7 MAb.
[0126] The double-determinant ELISA can be performed to ascertain
whether the RS7 antigen is present in a biopsy sample.
Alternatively, the assay can be performed to quantitate the amount
of RS7 antigen that is present in a clinical sample of body fluid.
The quantitative assay can be performed by including dilutions of
purified RS7 antigen. A method for purifying the RS7 antigen is
illustrated below.
[0127] The RS7 MAbs and fragments thereof of the present invention
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.
[0128] For example, there may be a container means containing the
capture antibody immobilized on a solid phase support, and a
further container means containing detectably labeled antibodies in
solution. Further container means may contain standard solutions
comprising serial dilutions of RS7 antigen. The standard solutions
of RS7 antigen may be used to prepare a standard curve with the
concentration of RS7 antigen plotted on the abscissa and the
detection signal on the ordinate. The results obtained from a
sample containing RS7 antigen may be interpolated from such a plot
to give the concentration of RS7 antigen in the biological
sample.
[0129] RS7 antibodies and their fragments of the present invention
also can be used to detect the presence of the RS7 antigen in
tissue sections prepared from a histological specimen. Such in situ
detection can be used to determine the presence of the RS7 antigen
and to determine the distribution of the RS7 antigen in the
examined tissue. In situ detection can be accomplished by applying
a detectably-labeled RS7 antigen binding protein to frozen tissue
sections. Studies indicate that the RS7 antigen is not preserved in
paraffin-embedded sections. Stein et al. (1993), supra. 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. Also, see Stein et al. (1989), supra, and Stein et al.
(1993), supra.
[0130] RS7 antibodies and their fragments can be detectably labeled
with any appropriate detection agent, for example, a radioisotope,
an enzyme, a fluorescent label, a chemiluminescent label, a
bioluminescent label or a paramagnetic label. Methods of making and
detecting such detectably-labeled RS7 antigen binding proteins are
well-known to those of ordinary skill in the art, and are described
in more detail below.
[0131] 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 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. As used herein, a diagnostic or therapeutic agent is a
molecule or atom, which is conjugated to an antibody moiety to
produce a conjugate, which is useful for diagnosis and for therapy.
Examples of diagnostic or therapeutic agents include drugs, toxins,
chelators, dyes, chromagens, boron compounds, and marker moieties.
Isotopes that are particularly useful for the purpose of the
present invention are .sup.3H, .sup.131I, .sup.35S, .sup.14C, and
preferably .sup.125I. Examples of other radionuclides are, for
example, .sup.90Y, .sup.111In, .sup.99mTc, .sup.186Re, .sup.188Re,
.sup.177Lu, .sup.67Cu, .sup.212Bi, .sup.213Bi, and .sup.211At.
Additional radionuclides are also available as diagnostic and
therapeutic agents. Suitable diagnostic imaging isotopes are
usually in the range of 25 to 4,000 keV, while suitable therapeutic
radionuclides are usually in the range of 60 to 700 keV.
[0132] The RS7 antibodies and their fragments of the present
invention also can be labeled with a fluorescent compound. The
presence of a fluorescently-labeled MAb is determined by exposing
the RS7 antigen binding protein to light of the proper wavelength
and detecting the resultant fluorescence. Fluorescent labeling
compounds include fluorescein isothiocyanate, rhodamine,
phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine. Fluorescently-labeled RS7 antigen binding proteins
are particularly useful for flow cytometry analysis.
[0133] Alternatively, RS7 antibodies and their fragments can be
detectably labeled by coupling the RS7 antigen binding protein 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.
[0134] Similarly, a bioluminescent compound can be used to label
RS7 antibodies and fragments thereof the present invention.
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.
[0135] Alternatively, RS7 antibodies and fragments thereof can be
detectably labeled by linking the RS7 antibody to an enzyme. When
the RS7 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 RS7
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, .beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase.
[0136] RS7 antibodies, fusion proteins, and fragments thereof also
can be labeled with paramagnetic ions for purposes of in vivo
diagnosis. Contrast agents that are particularly useful for
magnetic resonance imaging comprise Gd, Mn, Dy or Fe ions. RS7
antibodies and fragments thereof can also be conjugated to
ultrasound contrast/enhancing agents. For example, the ultrasound
contrast agent is a liposome that comprises a humanized RS7 IgG or
fragment thereof. Also preferred, the ultrasound contrast agent is
a liposome that is gas filled.
[0137] In a related vein, 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 a 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. Still preferred, the liposome is gas filled.
[0138] Those of skill in the art will know of other suitable labels
that can be employed in accordance with the present invention. The
binding of marker moieties to RS7 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'l J. Cancer 46: 1101 (1990), Stein et al. (1990), supra, and
Stein et al. (1993), supra. Also, see generally, Coligan.
[0139] 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 RS7 antigen including tumors of the lung, breast,
bladder, ovary, uterus, stomach, and prostate.
[0140] In Vivo Diagnosis
[0141] The present invention also contemplates the use of RS7
antibodies for in vivo diagnosis. The method of diagnostic imaging
with radiolabeled MAbs is 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).
[0142] For diagnostic imaging, radioisotopes may be bound to the
RS7 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.
[0143] 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 RS7 antibody and are appropriate for diagnostic imaging
include .sup.99mTc and .sup.111In.
Pharmaceutically Suitable Excipient
[0144] Additional pharmaceutical methods may be employed to control
the duration of action of an RS7 antibody in a therapeutic
application. Control release preparations can be prepared through
the use of polymers to complex or adsorb the RS7 antibody. 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 RS7
antibody from such a matrix depends upon the molecular weight of
the RS7 antibody, the amount of RS7 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 Remington's Pharmaceutical Sciences, 18th ed.
(1990).
[0145] The humanized, chimeric and human RS7 antibodies to be
delivered to a subject can consist of the antibody alone,
immunoconjugate, fusion protein, or can comprise one or more
pharmaceutically suitable excipients, one or more additional
ingredients, or some combination of these.
[0146] The immunoconjugate, naked antibody, fusion protein, and
fragments thereof of the present invention 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.
[0147] The immunoconjugate or naked antibody of the present
invention 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.
[0148] Additional pharmaceutical methods may be employed to control
the duration of action of the therapeutic or diagnostic conjugate
or naked antibody. Control release preparations can be prepared
through the use of polymers to complex or adsorb the
immunoconjugate or naked antibody. 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, 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.
[0149] The immunoconjugate, antibody fusion protein, naked
antibody, and fragments thereof may also be administered to a
mammal subcutaneously or even by other parenteral routes. In a
preferred embodiment, the anti-EGP-1 antibody or fragment thereof
is administered in a dosage of 10 to 2000 milligrams protein per
dose, and preferably is repeatedly administered. 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 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 4-10 weeks,
preferably once per week for 8 weeks, and more preferably, once per
week for 4 weeks. It may also be given less frequently, such as
every other week for several months. The dosage may be given
through various parenteral routes, with appropriate adjustment of
the dose and schedule.
[0150] The RS7 antibodies of the present invention can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby RS7 antibodies are combined in a
mixture with a pharmaceutically acceptable carrier. A composition
is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient patient. Sterile
phosphate-buffered saline is one example of a pharmaceutically
acceptable carrier. Other suitable carriers are well known to those
in the art. See, for example, Remington's Pharmaceutical Sciences,
18th Ed. (1990).
[0151] For purposes of therapy, the immunoconjugate, fusion
protein, or naked antibody is administered to a mammal in a
therapeutically effective amount. A suitable subject for the
present invention is usually a human, although a non-human animal
subject is also contemplated. An antibody preparation is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient mammal.
[0152] It will be apparent to those skilled in the art that various
modifications and variations can be made to the compositions 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.
[0153] The disclosure of all publications, patents and patent
applications cited above are expressly incorporated herein by
reference in their entireties to the same extent as if each were
incorporated by reference individually.
[0154] The examples below are illustrative of embodiments of the
current invention and should not be used, in any way, to limit the
scope of the claims.
Example 1. Construction of a Chimeric RS7 Antibody
Molecular Cloning of RS7 V.kappa. and VH Genes
[0155] Total cytoplasmic RNA and m NA was prepared from
RS7-producing hybridoma cells.
[0156] The genes encoding V.kappa. and VH sequences were cloned by
RT-PCR and 5'RACE and the sequences were determined by DNA
sequencing. Multiple independent clones were sequenced to eliminate
possible errors resulting from the PCR reaction. Sequence analyses
revealed presence of two V.kappa. (#1 and #23) and one VH (RS7VH)
transcripts. Combining each of the putative murine V.kappa. with
the VH, two chimeric Abs (cAbs), containing human constant region
domains were generated and expressed in Sp2/0 cells by
transfection. cAb-producing clones were identified by screening the
cell culture supernatants of the transfected cell clones by ELISA.
Positive clones were expanded and cAbs were purified from the cell
culture supernatants. The Ag-binding assay showed that only the cAb
composed of V.kappa.#23 and VH, cAb-V.kappa.#23, bound to
microwells coated with the crude membrane fraction of ME180, a
human cervical carcinoma cell (ATCC, Rockville, Md.) (FIG. 1). The
cAb with the combination of V.kappa.#1 and VH, cAb-V.kappa.#1, did
not show binding to the Ag-coated wells. Therefore, the
immunoreactive cAb (with V.kappa.#23) was designated as cRS7. The
cloned murine V.sub.H and the functional V.kappa. (#23) sequences
as the final PCR products were designated as RS7V.kappa. (FIG. 2A,
SEQ ID NO:1) and RS7VH (FIG. 2B, SEQ ID NO:3), respectively.
Binding Activity Assay for RS7 Abs
[0157] A competitive ELISA binding assay was used to evaluate the
binding affinity of engineered cRS7. Briefly, constant amount of
biotinylated murine RS7 is mixed with varying concentrations
(0.01-100 .mu.g/ml) of testing Abs (RS7 or cRS7), and added into
Ag-coated microwells, and incubated at room temperature for 1 h.
After washing, HRP conjugated streptavidin is added and incubated
for 1 h at room temperature. The amount of HRP-conjugated
streptavidin bound to the Ag-bound biotinylated RS7 was revealed by
reading OD.sub.490 after the addition of a substrate solution
containing 4 mM ortho-phenylenediamine dihydrochloride and 0.04%
H.sub.2O.sub.2. By this type of competitive Ag-binding assay, it
was revealed that cRS7 and murine RS7 competed equally well for the
binding of biotinylated murine RS7 to the antigen coated wells,
thus confirmed the authenticity of the V.kappa. and VH sequences
obtained (FIG. 1).
Example 2. Method of hRS7 Antibody Construction
[0158] Sequence Design of hRS7 V Genes
[0159] By searching the human V.kappa. and VH sequences in the
Kabat database, the FRs of RS7 V.kappa. (SEQ ID NO:2) and VH (SEQ
ID NO:4) were found to exhibit the highest degree of sequence
homology to human SA-1A'cl V.kappa. (SEQ ID NO:5) and RF-TS3 VH
(SEQ ID NO:8), respectively. One exception is the FR4 of RS7VH,
which showed the highest sequence homology with that of NEWM VH
(SEQ ID NO:6). Therefore human SA-1A'CL framework sequences were
used as the scaffold for grafting the CDRs of RS7V.kappa. (FIG. 3A,
SEQ ID NO:5; SEQ ID NO:2; SEQ ID NO:7), and a combination of RF-TS3
and NEWM framework sequences were used for RS7V.sub.H (FIG. 4, SEQ
ID NO:11; SEQ ID NO:12). There are a number of amino acid changes
in each chain outside of the CDR regions when compared to the
starting human antibody frameworks. Several amino acid residues in
murine FRs that flank the putative CDRs were maintained in the
reshaped hRS7 Fv based on the guideline previously established Qu,
Z., Losman, M. J., Eliassen, K. C., Hansen, H. J., Goldenberg, D.
M., and Leung, S. O. (1999). Humanization of Immu31, an
alphafetoprotein-specific antibody. Clin. Cancer Res. 5,
3095s-3100s. These residues are S20, D60, V85, and A100 of
RS7V.kappa. and K38, K46, A78, and F91 of RS7VH (FIG. 3A, SEQ ID
NO:5; SEQ ID NO:2; SEQ ID NO:7) and 3B, SEQ ID NO:8; SEQ ID NO:4;
SEQ ID NO:10).
Construction of hRS7 V Sequences
[0160] A modified strategy as described by Leung et al. (Leung, S.
O., Shevitz, J., Pellegrini, M. C., Dion, A. S., Shih, L. B.,
Goldenberg, D. M., and Hansen, H. J. (1994) Chimerization of LL2, a
rapidly internalizing antibody specific for B cell lymphoma.
Hybridoma, 13: 469-476) was used to construct the designed VL and
VH genes for hRS7 using a combination of long oligonucleotide
syntheses and PCR as illustrated in FIG. 4 (SEQ ID NO:11-12; SEQ ID
NO:13-14). For the construction of the hRS7 VH domain, two long
oligonucleotides, hRS7VHA (176-mer) and hRS7VHB (168-mer) were
synthesized on an automated DNA synthesizer (Applied
Biosystem).
TABLE-US-00001 hRS7VHA (SEQ ID NO: 19) represents nt 23 to 198 of
the hRS7VH domain (SEQ ID NO: 19) 5'-GGTCTGAGTT GAAGAAGCCT
GGGGCCTCAG TGAAGGTTTC CTGCAAGGCT TCTGGATACA CCTTCACAAA CTATGGAATG
AACTGGGTGA AGCAGGCCCC TGGACAAGGG CTTAAATGGA TGGGCTGGAT AAACACCTAC
ACTGGAGAGC CAACATATAC TGATGACTTC AAGGGA-3' hRS7VHB (SEQ ID NO: 20)
represents the minus strand of the hRS7VH domain complementary to
nt 174 to 340. (SEQ ID NO: 20) 5'-ACCCTTGGCC CCAGACATCG AAGTACCAGT
AGCTACTACC GAACCCCCCT CTTGCACAGA AATACACGGC AGTGTCGTCA GCCTTTAGGC
TGCTGATCTG GAGATATGCC GTGCTGACAG AGGTGTCCAA GGAGAAGGCA AACCGTCCCT
TGAAGTCATC AGTATATG-3'
[0161] The 3'-terminal sequences (23 nt residues) of hRS7VHA and B
are complementary to each other. Under defined PCR condition,
3'-ends of hRS7VHA and B 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 23 to 340
of hRS7VH. This DNA was further amplified in the presence of two
short oligonucleotides, hRS7VHBACK (SEQ ID NO:21) and hRS7VHFOR
(SEQ ID NO:22) to form the full-length hRS7VH.
TABLE-US-00002 hRS7VHBACK (SEQ ID NO: 21) 5'-GTGGTGCTGC AGCAATCTGG
GTCTGAGTTG AAGAAGCC-3' hRS7VHFOR (SEQ ID NO: 22) 5'-TGAGGAGACG
GTGACCAGGG ACCCTTGGCC CCAGACAT-3'
[0162] Minimum amount of hRS7VHA and B (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 hRS7VHBACK and hRS7VHFOR, and 2.5 units of Taq DNA
polymerase (Perkin Elmer Cetus, Norwalk, Conn.). This reaction
mixture was subjected to 3 cycle of PCR reaction 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, and 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 hRS7VH was gel-purified,
restriction-digested with PstI and BstEII and cloned into the
complementary PstJBstEII sites of the heavy chain staging vector,
VHpBS2.
[0163] For constructing the full length DNA of the humanized
V.kappa. sequence, hRS7VKA (156-mer) and hRS7VKB (155-mer) were
synthesized as described above. hRS7VKA and B were amplified by two
short oligonucleotides hRS7VKBACK and hRS7VKFOR as described
above.
TABLE-US-00003 HRS7VKA (SEQ ID NO: 23) represents nt 20 to 175 of
the hRS7V.kappa. domain. (SEQ ID NO: 23) 5'-CTCCATCCTC CCTGTCTGCA
TCTGTAGGAG ACAGAGTCAG CATCACCTGC AAGGCCAGTC AGGATGTGAG TATTGCTGTA
GCCTGGTATC AGCAGAAACC AGGGAAAGCC CCTAAGCTCC TGATCTACTC GGCATCCTAC
CGGTACACTG GAGTCC-3' hRS7VKB (SEQ ID NO: 24) represents the minus
strand of the hRS7V.kappa. domain complementary to nt 155 to 320.
(SEQ ID NO: 24) 5'-CCTTGGTCCC AGCACCGAAC GTGAGCGGAG TAATATAATG
TTGCTGACAG TAATAAACTG CAAAATCTTC AGGTTGCAGA CTGCTGATGG TGAGAGTGAA
ATCTGTCCCA GATCCACTGC CACTGAACCT ATCAGGGACT CCAGTGTACC GGTAG-3'
hRS7VKBACK (SEQ ID NO: 25) 5'-GACATTCAGC TGACCCAGTC TCCATCCTCC
CTGTCTG-3' hRS7VKFOR (SEQ ID NO: 26) 5'-ACGTTAGATC TCCACCTTGG
TCCCAGCACC G-3'
[0164] Gel-purified PCR products for hRS7V.kappa. were
restriction-digested with PvuII and BglIII and cloned into the
complementary PvuI/BcII sites of the light chain staging vector,
VKpBR2. The final expression vector hRS7pdHL2 was constructed by
sequentially subcloning the XbaI-BamHI and XhoI/BamHI fragments of
hRS7V.kappa. and VH, respectively, into pdHL2 as described above.
The full-length cDNA and the encoded amino acid sequences of the
light and heavy chains of hRS7 are disclosed in FIG. 5A (SEQ ID
NO:15; SEQ ID NO:16) and 5B (SEQ ID NO:17; SEQ ID NO:18),
respectively.
Transfection and Expression of hRS7 Antibodies
[0165] Approximately 30 .mu.g of the expression vectors for hRS7
were linearized by digestion with SalI and transfected into
Sp2/0-Ag14 cells by electroporation (450V and 25 .mu.F). The
transfected cells were plated into 96-well plates for 2 days and
then selected for drug-resistance by adding MTX into the medium at
a final concentration of 0.025 MTX-resistant colonies emerged in
the wells 2-3 weeks. Supernatants from colonies surviving selection
were screened for human Ab secretion by ELISA assay. Briefly, 100
.mu.l supernatants were added into the wells of a microliter plate
precoated with GAH-IgG, F(ab')2 fragment-specific Ab and incubated
for 1 h at room temperature. Unbound proteins were removed by
washing three times with wash buffer (PBS containing 0.05%
polysorbate 20). HRP-conjugated GAH-IgG, Fc fragment-specific Ab
was added to the wells. Following an incubation of 1 h, the plate
was washed. The bound HRP-conjugated Ab was revealed by reading
A490 nm after the addition of a substrate solution containing 4 mM
OPD and 0.04% H.sub.2O.sub.2. Positive cell clones were expanded
and hRS7 IgG were purified from cell culture supernatant by
affinity chromatography on a Protein A column.
Binding Activity of the Humanized RS7 Antibody
[0166] An ELISA competitive binding assay using ME180 cell membrane
extract coated plate was used to assess the immunoreactivity of
hRS7 as described (Stein et al., Int. J. Cancer 55:938-946 (1993)).
ME180 cell membrane fraction was prepared by sonication and
centrifugation. The crude membrane extract was coated in 96-well
flat bottomed PVC plate by centrifugation and fixed with 0.1%
glutaraldehyde. Constant amount of the biotinylated murine RS7
mixed with varying concentrations of mRS7, cRS7 or hRS7 was added
to the membrane coated wells and incubated at room temperature for
1-2 h. After washing, HRP-conjugated streptavidin was added and
incubated for 1 h at room temperature. The amount of HRP-conjugated
streptavidin bound to the membrane-bound biotinylated mRS7 was
revealed by reading A.sub.490 nm after the addition of a substrate
solution containing 4 mM orthophenylenediamine dihydrochloride and
0.04% H.sub.2O.sub.2. As shown by the competition assays in FIG. 6,
hRS7 IgG exhibited comparable binding activities with that of mRS7
and cRS7, confirming the binding affinity of RS7 was preserved in
humanization.
Example 3. Radioiodinations of Humanized RS7 Using Residualizing
Labels
[0167] The residualizing moiety (IMP-R4, IMP-R5 or IMP-R8) was
radioiodinated, and coupled to disulfide-reduced hRS7 along the
procedure described elsewhere (Govindan S V, et al., Bioconjugate
Chem. 1999; 10:231-240). See FIG. 7. In residualizing radioiodine
labelings using .sup.125I to prepare .sup.125I-IMP-Rx-hRS7 where
x=4, 5 or 8), overall yields and specific activities (in
parentheses) of 87.1% (3.38 mCi/mg), 34.3% (0.97 mCi/mg), and 76.6%
(2.93 mCi/mg) were obtained using IMP-R4, IMP-R5 and IMP-R8,
respectively. In large-scale .sup.131I labelings using
.sup.131I-IMP-R4 entity, the following results were obtained. Using
20.4 mCi of .sup.131I, 35.7 nmol of IMP-R4 and 3.22 mg of
DTT-reduced hRS7, a 60% overall yield (3.80 mCi/mg) was obtained. A
different run using 30.3 mCi of .sup.131I, IMP-R4 and reduced hRS7
produced 69.7% yield (3.88 mCi/mg). A third run using 13.97 mCi of
.sup.131I gave 71.8% incorporation (4.42 mCi/mg). A
.sup.131I-IMP-R4 labeling using 13.6 mCi of .sup.131I and a
non-specific humanized antibody, hLL2, resulted in 64.4% yield
(3.67 mCi/mg).
Example 4. Preclinical Experiments in Breast Cancer Animal
Model
[0168] For tumor targeting studies, tumors were propagated in 5-8
week old female nude mice by subcutaneous injection of
.about.0.2.3.times.10.sup.7 cultured MDA-MB-468 cells, and the
animals were used after one month when the tumor size reached
.about.01-to-0.2 cm.sup.3. The mice were injected i.v. with a
mixture of .about.10 .mu.Ci of .sup.125I-[IMP-Rx]-hRS7 where x=4, 5
or 8, and 20-25 .mu.Ci of .sup.131I-MAb (CT method). Thus, each
experiment was a paired-label experiment with .sup.125I/.sup.131I.
At the indicated times, biodistributions in various organs and
blood were determined, and expressed as % injected dose per gram.
Corrections for backscatter of .sup.131I into .sup.125I window were
made in determining .sup.125I biodistributions.
[0169] For therapy studies, tumor growth patterns under various
formats were studied to determine the optimal method for steady
growth of tumor. It was concluded that the method used for
targeting experiments was optimal after about 8-weeks of tumor
growth, and 30-50% of the animals could be used based on the tumor
growth profiles. For therapy studies, the tumor-bearing animals
were injected i.v. with .sup.131I-IMPR4-hRS7 was the agent
examined, and compared with directly radioiodinated material,
.sup.131I-hRS7. Baseline body weights were compared with weekly
measurements of body weights and tumor volumes. Animals were
sacrificed when tumors reached 3 cm.sup.3. All animal experiments
were carried out in accord with IACUC-approved protocols.
In Vivo Animal Biodistributions
[0170] These experiments were carried out using dual-labeled hRS7
preparations (.sup.125I-IMP-Rx-hRS7 where x=4, 5 or 8, with each
agent mixed with direct label .sup.131I-hRS7) in the tumors grown
in NIH Swiss nude mice. Tables IA, 1B and 1C describe detailed
biodistributions showing the superior performance using the
residualizing labels. For instance, % injected dose per gram of
tumor on day-7 were 41.6.+-.3.0%, 32.2.+-.11.6% and 24.7.+-.8.5%
for .sup.125I-IMP-R4-hRS7, .sup.125I-IMP-R5-hRS7 and
.sup.125I-IMP-R8-hRS7, respectively, while that for directly
labeled .sup.131I-hRS7 at the same time-point in each of the
dual-labeled experiments were 5.9.+-.0.9%, 6.2.+-.2.1% and
6.7.+-.2.3%. Tumor-to-nontumor ratios for the same time-point were
1.7-to-7.6-fold higher with .sup.125I-IMP-R4-hRS7, 1.7-to-6.0-fold
higher with .sup.125I-IMP-R5-57, and 2.0-to-4.8-fold higher with
.sup.125I-MP-R8-S7 compared to the ratios with .sup.131I-hRS7 (data
not shown).
[0171] Table-1. Biodistributions of Humanized RS7, Dual-Labeled
with .sup.125I-IMP-R (R4 or R5 or R8) and .sup.131I-hRS7 CT Method
in NIH Swiss Nude Mice Bearing MDA-MB-468 Tumor Xenografts
TABLE-US-00004 TABLE 1A .sup.125I-IMP-R4-hRS7 versus .sup.131I-hRS7
(CT) % ID/g .+-. SD.sup.1, n = 5 Tissue Label 24 h 72 h 168 h, n =
4 336 h MDA-MB-468 .sup.125I-IMP-R4 32.8 .+-. 6.3 46.8 .+-. 11.0
41.6 .+-. 3.0 25.1 .+-. 3.8 .sup.131I (CT) 8.6 .+-. 1.5 8.6 .+-.
2.3 5.9 .+-. 0.9 4.4 .+-. 0.8 Tumor wt. (0.19 .+-. 0.06) (0.19 .+-.
0.08) (0.13 .+-. 0.07) (0.18 .+-. 0.04) Liver .sup.125I-IMP-R4 5.7
.+-. 0.7 4.7 .+-. 1.5 2.8 .+-. 0.4 1.3 .+-. 0.2 .sup.131I (CT) 4.1
.+-. 0.3 2.01 .+-. 0.1 1.5 .+-. 0.2 0.7 .+-. 0.1 Spleen
.sup.125I-IMP-R4 3.6 .+-. 0.6 3.3 .+-. 0.6 2.6 .+-. 0.8 1.9 .+-.
0.2 .sup.131I (CT) 2.6 .+-. 0.5 1.7 .+-. 0.4 1.1 .+-. 0.4 0.6 .+-.
0.1 Kidney .sup.125I-IMP-R4 7.8 .+-. 0.7 6.8 .+-. 0.4 5.6 .+-. 0.8
3.0 .+-. 0.5 .sup.131I (CT) 3.5 .+-. 0.3 2.1 .+-. 0.3 1.4 .+-. 0.3
0.7 .+-. 0.1 Lungs .sup.125I-IMP-R4 4.5 .+-. 1.0 3.2 .+-. 0.6 2.2
.+-. 0.7 0.8 .+-. 0.2 .sup.131I (CT) 3.1 .+-. 0.8 2.2 .+-. 0.4 1.6
.+-. 0.6 0.6 .+-. 0.2 Blood .sup.125I-IMP-R4 15.1 .+-. 1.4 9.5 .+-.
0.7 6.0 .+-. 1.5 1.9 .+-. 0.6 .sup.131I (CT) 10.8 .+-. 1.0 7.3 .+-.
0.6 5.3 .+-. 1.2 2.2 .+-. 0.6 Stomach .sup.125I-IMP-R4 1.3 .+-. 0.2
0.6 .+-. 0.1 0.4 .+-. 0.1 0.2 .+-. 0.1 .sup.131I (CT) 1.6 .+-. 0.5
0.7 .+-. 0.1 0.4 .+-. 0.1 0.2 .+-. 0.1 Sm. Int. .sup.125I-IMP-R4
1.5 .+-. 0.2 0.9 .+-. 0.1 0.6 .+-. 0.2 0.2+ .+-. 0.1 .sup.131I (CT)
1.0 .+-. 0.1 0.6 .+-. 0.1 0.4 .+-. 0.1 0.2 .+-. 0.04 Lg. Int.
.sup.125I-1MP-R4 1.3 .+-. 0.3 1.0 .+-. 0.1 0.8 .+-. 0.1 0.3 .+-.
0.1 .sup.131I (CT) 0.8 .+-. 0.2 0.5 .+-. 0.1 0.5 .+-. 0.1 0.2 .+-.
0.03 Muscle .sup.125I-IMP-R4 1.2 .+-. 0.2 0.7 .+-. 0.1 0.5 .+-. 0.1
0.3 .+-. 0.2 .sup.131I (CT) 0.9 .+-. 0.1 0.5 .+-. 0.05 0.3 .+-. 0.1
0.2 .+-. 0.1 Bone .sup.125I-IMP-R4 2.3 .+-. 0.3 2.1 .+-. 0.3 2.4
.+-. 0.6 2.3 .+-. 1.2 .sup.131I (CT) 1.4 .+-. 0.1 0.8 .+-. 0.1 0.5
.+-. 0.1 0.3 .+-. 0.1
TABLE-US-00005 TABLE 1B .sup.125I-IMP-R5-hRS7 versus .sup.131I-hRS7
(CT method) % ID/g .+-. SD.sup.1, n = 5 Tissue Label 24 h 72 h 168
h 336 h, n = 4 MDA-MB-468 .sup.125I-IMP-R5 29.1 .+-. 4.6 39.6 .+-.
2.7 32.2 .+-. 11.6 17.8 .+-. 7.0 .sup.131I (CT) 9.2 .+-. 1.0 9.1
.+-. _0.6 6.2 .+-. 2.1 4.9 .+-. 2.0 Tumor wt. (0.14 .+-. 0.02)
(0.20 .+-. 0.05) (0.11 .+-. 0.03) (0.13 .+-. 0.06) Liver
.sup.125I-IMP-R5 4.8 .+-. 1.4 2.5 .+-. 0.1 1.8 .+-. 0.3 0.8 .+-.
0.3 .sup.131I (CT) 5.1 .+-. 1.5 2.4 .+-. 0.2 1.7 .+-. 0.2 0.8 .+-.
0.3 Spleen .sup.125I-IMP-R5 4.1 .+-. 1.0 2.0 .+-. 0.4 1.9 .+-. 0.4
0.8 .+-. 0.4 .sup.131I (CT) 3.8 .+-. 1.2 1.7 .+-. 0.5 1.3 .+-. 0.3
0.7 .+-. 0.4 Kidney .sup.125I-IMP-R5 10.0 .+-. 1.4 6.3 .+-. 0.5 5.0
.+-. 0.5 1.1 .+-. 0.3 .sup.131I (CT) 3.7 .+-. 0.5 1.9 .+-. 0.3 1.7
.+-. 0.3 0.8 .+-. 0.2 Lungs .sup.125I-IMP-R5 5.4 .+-. 1.8 3.2 .+-.
0.8 2.3 .+-. 0.2 0.9 .+-. 0.4 .sup.131I (CT) 3.9 .+-. 1.2 2.5 .+-.
0.7 2.0 .+-. 0.3 0.9 .+-. 0.5 Blood .sup.125I-IMP-R5 16.5 .+-. 4.0
8.8 .+-. 0.6 6.5 .+-. 1.0 2.7 .+-. 1.4 .sup.131I (CT) 12.2 .+-. 3.0
7.8 .+-. 0.5 6.3 .+-. 0.8 3.1 .+-. 1.4 Stomach .sup.125I-IMP-R5 0.9
.+-. 0.2 0.5 .+-. 0.1 0.4 .+-. 0.1 0.2 .+-. 0.1 .sup.131I (CT) 1.1
.+-. 0.1 0.6 .+-. 0.1 0.5 .+-. 0.1 0.2 .+-. 0.1 Sm. Int.
.sup.125I-IMP-R5 1.5 .+-. 0.3 0.8 .+-. 0.04 0.6 .+-. 0.1 0.2 .+-.
0.1 .sup.131I (CT) 1.1 .+-. 0.2 0.6 .+-. 0.02 0.5 .+-. 0.1 0.3 .+-.
0.1 Lg. Int. .sup.125I-IMP-R5 1.4 .+-. 0.2 0.9 .+-. 0.1 0.6 .+-.
0.1 0.2 .+-. 0.04 .sup.131I (CT) 0.7 .+-. 0.1 1.4 .+-. 0.03 0.4
.+-. 0.1 0.2 .+-. 0.04 Muscle .sup.125I-IMP-R5 1.3 .+-. 0.3 0.7
.+-. 0.2 0.5 .+-. 0.1 0.2 .+-. 0.1 .sup.131I (CT) 0.9 .+-. 0.2 0.6
.+-. 0.2 0.4 .+-. 0.1 0.2 .+-. 0.1 Bone .sup.125I-IMP-R5 2.2 .+-.
0.6 1.3 .+-. 0.2 1.2 .+-. 0.5 1.0 .+-. 0.6 .sup.131I (CT) 1.9 .+-.
0.7 0.9 .+-. 0.1 0.6 .+-. 0.2 0.3 .+-. 0.2
TABLE-US-00006 TABLE 1C .sup.125I-IMP-R8-hRS7 versus .sup.131I-hRS7
(CT method) % ID/g .+-. SD.sup.1, n = 5 Tissue Label 24 h 72 h 168
h 336 h MDA-MB-468 .sup.125I-IMP-R8 29.1 .+-. 5.4 29.6 .+-. 3.9
24.7 .+-. 8.5 11.0 .+-. 6.4 .sup.131I (CT) 8.8 .+-. 1.6 8.8 .+-.
1.0 6.7 .+-. 2.3 2.4 .+-. 1.3 Tumor wt. (0.17 .+-. 0.04) (0.12 .+-.
0.05) (0.10 .+-. 0.04) (0.15 .+-. 0.05) Liver .sup.125I-IMP-R8 4.6
.+-. 0.7 3.3 .+-. 0.4 1.8 .+-. 0.2 0.7 .+-. 0.2 .sup.131I (CT) 4.1
.+-. 0.6 3.3 .+-. 0.4 1.8 .+-. 0.2 0.8 .+-. 0.2 Spleen
.sup.125I-IMP-R8 2.6 .+-. 0.7 2.3 .+-. 0.2 1.9 .+-. 0.2 1.0 .+-.
0.1 .sup.131I (CT) 2.4 .+-. 0.8 2.2 .+-. 0.3 2.0 .+-. 0.3 0.7 .+-.
0.1 Kidney .sup.125I-IMP-R8 7.2 .+-. 0.8 4.6 .+-. 0.8 2.6 .+-. 1.0
1.8 .+-. 0.1 .sup.131I (CT) 2.5 .+-. 0.3 3.0 .+-. 0.7 1.8 .+-. 0.5
0.8 .+-. 0.3 Lungs .sup.125I-IMP-R8 3.0 .+-. 0.7 4.7 .+-. 0.5 2.3
.+-. 0.6 1.0 .+-. 0.4 .sup.131I (CT) 2.4 .+-. 0.4 4.4 .+-. 0.5 2.1
.+-. 0.5 1.0 .+-. 0.4 Blood .sup.125I-IMP-R8 10.8 .+-. 1.2 9.6 .+-.
0.9 6.3 .+-. 1.4 2.2 .+-. 0.6 .sup.131I (CT) 9.2 .+-. 1.6 9.5 .+-.
0.8 6.4 .+-. 1.4 2.6 .+-. 0.6 Stomach .sup.125I-IMP-R8 0.9 .+-. 0.2
0.7 .+-. 0.2 0.3 .+-. 0.1 0.2 .+-. 0.1 .sup.131I (CT) 1.1 .+-. 0.2
0.9 .+-. 0.3 0.4 .+-. 0.1 0.3 .+-. 0.1 Sm. Int. .sup.125I-IMP-R8
1.0 .+-. 0.1 0.8 .+-. 0.2 0.5 .+-. 0.1 0.2 .+-. 0.1 .sup.131I (CT)
0.8 .+-. 0.1 0.8 .+-. 0.1 0.5 .+-. 0.1 0.2 .+-. 0.1 Lg. Int.
.sup.125I-IMP-R8 1.0 .+-. 0.1 0.9 .+-. 0.1 0.5 .+-. 0.1 0.3 .+-.
0.1 .sup.131I (CT) 0.6 .+-. 0.1 0.6 .+-. 0.1 0.4 .+-. 0.1 0.2 .+-.
0.1 Muscle .sup.125I-IMP-R8 0.8 .+-. 0.1 0.6 .+-. 0.1 0.4 .+-. 0.1
0.2 .+-. 0.1 .sup.131I (CT) 0.6 .+-. 0.04 0.6 .+-. 0.1 0.4 .+-. 0.1
0.2 .+-. 0.1 Bone .sup.125I-IMP-R8 1.4 .+-. 0.2 1.2 .+-. 0.3 1.4
.+-. 0.2 0.8 .+-. 0.2 .sup.131I (CT) 1.1 .+-. 0.2 0.9 .+-. 0.2 0.7
.+-. 0.1 0.3 .+-. 0.1
[0172] Dosimetry calculations, based on biodistributions using
.sup.125I in place of .sup.131I, were performed using the method of
Siegel, J A and Stabin, M G (Journal of Nuclear Medicine, 1994;
35:152-156). Table-2 compares sets of residualizing and
conventional radioiodine labels, and FIG. 8 describes the data
graphically. All of the residualizing agents are seen to perform
optimally in terms of dose delivered to tumor and tumor-to-nontumor
ratios; .sup.131I-IMP-R4-hRS7 was chosen for therapy experiments in
view of the advantageous radiochemical yields and specific
activities obtainable for the same agent.
TABLE-US-00007 TABLE 2 Calculated radiation doses due to variously
radioiodinated hRS7 in the MDA-MB-468 tumor model cGy normalized to
1500 cGy to Blood Group I Group II Group III Organ Model IMP-R4 CT
IMP-R5 CT IMP-R8 CT Tumor (Trap 0 6995 1613 5187 1506 4000 1206
point 0) Liver Exp 674 456 398 449 497 505 Spleen Exp 535 315 336
313 384 356 Kidney Exp 1063 402 867 361 761 394 Lungs Exp 450 392
450 422 506 473 Blood(org) Exp 1500 1500 1500 1500 1500 1500
Stomach Exp 104 144 84 118 101 128 Sm Int Exp 148 124 131 119 130
121 Lg Int Exp 163 108 136 86 140 97 Muscle Exp 112 99 105 100 97
93 Bone Exp 486 151 244 149 245 151 mCi for 1500 cGy to 0.231 0.285
0.213 0.239 0.248 0.255 blood
Therapy of MDA-MB-468 Human Breast Carcinoma Xenografts in Nude
Mice
[0173] Maximum-tolerated-dose (MTD): From dosimetry data (Table-2,
group-1), the mCi amounts of .sup.131I-IMP-R4-hRS7 and
.sup.131I-hRS7, producing a radiation dose of 1500 cGy to blood
(estimated MTD) were calculated to be 0.231 mCi and 0.285 mCi,
respectively. Experimental determination of MTD was carried out
using increasing doses of each agent in Swiss nude mice. For
.sup.131I-IMP-R4-hRS7, groups of animals were administered 200,
225, 250, 275, 300 and 325 .mu.Ci; 1 out of five animals in the 250
.mu.Ci dose group died by week 4, while 3 out of 4 animals in the
300 .mu.Ci dose group died between weeks 2 and 4. Although the
survival of animals in the 275 and 325 .mu.Ci dose groups at five
weeks was unexpected, we concluded that the MTD was between 231
.mu.Ci (calculated from dosimetry data) and 250 .mu.Ci of
administered dose. For the .sup.131I-hRS7 (`CT`-based
radioiodination), groups of animals were injected with 250, 280,
310, 340, 370 and 400 between weeks 2 and 3, six out of six animals
of 340 .mu.Ci dose group, three out of six animals of 370 .mu.Ci
dose group, and four out of four animals of 400 .mu.Ci dose group
died. Based on these, the MTD was projected to be in the 280-310
.mu.Ci range.
Therapy Study-1
[0174] For this first therapy experiment, comparing the efficacy of
.sup.131I-IMPR-4-hRS7 with that of .sup.131I-hRS7 (CT method), each
agent used at .about.70% of its maximum-tolerated dose. A single
dose of 175 .mu.Ci of the residualizing agent is seen to be
significantly more effective than 200 .mu.Ci of conventional
radioiodine agent. In this experiment, which also included
untreated controls, 10 or 11 animals were used per group, and all
the three groups were randomized such that the distribution of
starting tumor sizes were very similar. Mean tumor volumes for the
three groups before therapy (day -2) were 0.312.+-.0.181,
0.308.+-.0.203, and 0.303.+-.0.212.
[0175] In this experiment, interim data to day 49 are depicted in
FIG. 9 below. The top panel in FIG. 9 shows tumor volumes
(cm.sup.3) for individual animals in each group, and the bottom
panel indicates mean tumor volumes in two formats. There were three
deaths in the untreated group. Tumor growth control is
significantly better for the residualizing label group compared to
the conventional label and the untreated groups, as determined by
the student-t test on the area under the curves (AUC) for mean
tumor volumes (MTV) up to day-49. On day 49, significance (p
values) for differences in AUCs of MTVs due to therapy with
.sup.131I-IMP-R4-hRS7, with the respective p values for tumor
volume differences before therapy (day -2) given in parentheses,
are as follows. Versus untreated: 0.05 (0.78); versus
.sup.131I-hRS7 (CT): 0.03 (0.98); for -hRS7 (CT) versus untreated:
0.14 (0.81). There is continuing divergence in mean tumor volumes
between the conventional and the residualizing radioiodine groups
on day 49, with the latter group leading to continued decrease. At
8-weeks post-therapy, complete remissions were observed in 5 of 11
mice treated with .sup.131I-IMP-R4-hRS7, and the MTV was 20% of the
starting value. MTV in the untreated and .sup.131I-hRS7-treated
mice at 8 weeks were 280% and 163% of the respective starting
values, respectively, with 1 complete remission of 11 mice in the
.sup.131I-hRS7 group.
[0176] The treatments were well tolerated. The mean body weights of
IMP-R4 group on day -2 was 21.93.+-.2.03 and that on day 49 was
23.68.+-.1.81; for `CT` group, the mean body weights were
21.77.+-.2.21 and 23.90.+-.2.64 on days -2 and 49, respectively.
Myelotoxicities of the treated groups, as determined by blood cell
counts, are shown in FIG. 10A and FIG. 10B. Briefly: With
.sup.131I-IMP-R4-hRS7, nadirs of 34%, 7% and 61% of the control
levels for WBC, lymphocite and neutrophil counts, respectively,
were reached one week after the administration of the agent. By
week-5, these recovered to 74%, 58% and 92% of the control levels,
respectively, and remained at 45%, 36% and 51% of the control
levels on day-49; and for .sup.131I-hRS7 (CT): nadirs of 41%, 13%
and 67% of the control levels for WBC, lymphocite and neutrophil
counts, respectively, were reached one week after the
administration of the agent. By week-5, these recovered to 85%, 67%
and 103% of the control levels, respectively, and remained at 42%,
32% and 49% of the control levels on day-49.
Therapy Study-2
Specificity of RAIT Using .sup.131I-IMP-R4-hRS7 in the MDA-MB-468
Tumor Model
[0177] The efficacy of .sup.131I-IMP-R4-hRS7 was compared with that
of non-specific control humanized antibody, hLL2 (anti-CD-22 MAb),
labeled with .sup.131I-IMP-R4. In this experiment, 175 .mu.Ci of
each agent was administered. This represents .about.70% of the
maximum-tolerated dose of .sup.131I-IMP-R4-hRS7. In this
experiment, which included untreated controls, 7-to-8 animals were
used per group, and the groups were randomized with regard to the
starting tumor volume distributions as in therapy experiment-1.
FIG. 11, showing the relative mean tumor volumes (MTV) for the
three groups (MTV before therapy: 100), is indicative of the growth
control specificity.
Example 5. Treatment of a Breast Cancer Patient with Y-90 Humanized
RS7 mAb and with Naked Humanized RS7 mAb
[0178] A 56-year-old woman with a history of recurrent
adencarcinoma of the breast presents with cervical lymph node and
left lung metastases. She relapses twice after chemotherapy and
hormonal therapies. She is then given two therapeutic injections,
two weeks apart, of Y-90-conjugated humanized RS7 mAb i.v., at a
dose each of 20 mCi Y-90 in a protein dose of antibody of 100 mg.
Four weeks after therapy, her white blood cell and platelet counts
have decreased by approximately 50%, but recuperate by 9 weeks
post-therapy. At the restaging 12 weeks post-therapy, a ca. 30%
decrease in pulmonary and nodal metastases has been measured by
computed tomography. Thereafter, she receives 4 weekly infusions,
over 3 hours each, of naked humanized RS7, which is tolerated well,
except for some transient rigors and chills, and without any
adverse effects on her blood counts or blood chemistries. The naked
antibody dose for each infusion was 400 mg/m.sup.2. Approximately 8
weeks later, restaging by computed tomography indicates an
additional decrease in measurable lesions by about 20 percent. At
the followup examination 3 months later, her disease appears to be
stable (i.e., no evidence of additional, or progressive growth).
Sequence CWU 1
1
331324DNAMus sp.CDS(1)..(324) 1gac att cag ctg acc cag tct cac aaa
ttc atg tcc aca tca gta gga 48Asp Ile Gln Leu Thr Gln Ser His Lys
Phe Met Ser Thr Ser Val Gly1 5 10 15gac agg gtc agc atc acc tgc aag
gcc agt cag gat gtg agt att gct 96Asp Arg Val Ser Ile Thr Cys Lys
Ala Ser Gln Asp Val Ser Ile Ala 20 25 30gta gcc tgg tat caa cag aaa
cca gga caa tct cct aaa cta ctg att 144Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45tac tcg gca tcc tac cgg
tac act gga gtc cct gat cgc ttc act ggc 192Tyr Ser Ala Ser Tyr Arg
Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60agt gga tct ggg acg
gat ttc act ttc acc atc agc agt gtg cag gct 240Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80gaa gac ctg
gca gtt tat tac tgt cag caa cat tat att act ccg ctc 288Glu Asp Leu
Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95acg ttc
ggt gct ggg acc aag ctg gag ctg aaa cgg 324Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys Arg 100 1052108PRTMus sp. 2Asp Ile Gln Leu Thr
Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100
1053360DNAMus sp.CDS(1)..(360) 3gtg aag ctg cag gag tca gga cct gag
ctg aag aag cct gga gag aca 48Val Lys Leu Gln Glu Ser Gly Pro Glu
Leu Lys Lys Pro Gly Glu Thr1 5 10 15gtc aag atc tcc tgc aag gct tct
gga tat acc ttc aca aac tat gga 96Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr Gly 20 25 30atg aac tgg gtg aag cag gct
cca gga aag ggt tta aag tgg atg ggc 144Met Asn Trp Val Lys Gln Ala
Pro Gly Lys Gly Leu Lys Trp Met Gly 35 40 45tgg ata aac acc tac act
gga gag cca aca tat act gat gac ttc aag 192Trp Ile Asn Thr Tyr Thr
Gly Glu Pro Thr Tyr Thr Asp Asp Phe Lys 50 55 60gga cgg ttt gcc ttc
tct ttg gaa acc tct gcc acc act gcc tat ttg 240Gly Arg Phe Ala Phe
Ser Leu Glu Thr Ser Ala Thr Thr Ala Tyr Leu65 70 75 80cag atc aac
aac ctc aaa agt gag gac atg gct aca tat ttc tgt gca 288Gln Ile Asn
Asn Leu Lys Ser Glu Asp Met Ala Thr Tyr Phe Cys Ala 85 90 95aga ggg
ggg ttc ggt agt agc tac tgg tac ttc gat gtc tgg ggc caa 336Arg Gly
Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln 100 105
110ggg acc acg gtc acc gtc tcc tca 360Gly Thr Thr Val Thr Val Ser
Ser 115 1204120PRTMus sp. 4Val Lys Leu Gln Glu Ser Gly Pro Glu Leu
Lys Lys Pro Gly Glu Thr1 5 10 15Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr Gly 20 25 30Met Asn Trp Val Lys Gln Ala Pro
Gly Lys Gly Leu Lys Trp Met Gly 35 40 45Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Thr Asp Asp Phe Lys 50 55 60Gly Arg Phe Ala Phe Ser
Leu Glu Thr Ser Ala Thr Thr Ala Tyr Leu65 70 75 80Gln Ile Asn Asn
Leu Lys Ser Glu Asp Met Ala Thr Tyr Phe Cys Ala 85 90 95Arg Gly Gly
Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser 115 1205106PRTHomo sapiens 5Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser
Thr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100
105611PRTHomo sapiens 6Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser1
5 107108PRTHomo sapiens 7Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Ile Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Arg Tyr
Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95Thr Phe Gly
Ala Gly Thr Lys Val Glu Ile Lys Arg 100 1058108PRTHomo sapiens 8Val
Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala Ser1 5 10
15Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Ala
20 25 30Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
Gly 35 40 45Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly
Phe Thr 50 55 60Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr
Ala Tyr Leu65 70 75 80Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Arg Glu Asp Ser Asn Gly Tyr Lys Ile Phe
Asp Tyr 100 105911PRTMus sp. 9Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser1 5 1010109PRTHomo sapiens 10Gln Val Gln Leu Gln Gln Ser Gly
Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys
Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr
Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60Lys Gly Arg Phe
Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80Leu Gln
Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala
Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Val 100 10511324DNAHomo
sapiensCDS(1)..(324) 11gac 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 Gly1 5 10 15gac aga gtc agc atc acc tgc aag gcc agt
cag gat gtg agt att gct 96Asp Arg Val Ser Ile Thr Cys Lys Ala Ser
Gln Asp Val Ser Ile Ala 20 25 30gta gcc tgg tat cag cag aaa cca ggg
aaa gcc cct aag ctc ctg atc 144Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45tac tcg gca tcc tac cgg tac act
gga gtc cct gat agg ttc agt ggc 192Tyr Ser Ala Ser Tyr Arg Tyr Thr
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60agt gga tct ggg aca gat ttc
act ctc acc atc agc agt ctg caa cct 240Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80gaa gat ttt gca gtt
tat tac tgt cag caa cat tat att act ccg ctc 288Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu 85 90 95acg ttc ggt gct
ggg acc aag gtg gag atc aaa cgt 324Thr Phe Gly Ala Gly Thr Lys Val
Glu Ile Lys Arg 100 10512108PRTHomo sapiens 12Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Ser
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu
85 90 95Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg 100
10513363DNAHomo sapiensCDS(1)..(363) 13cag gtc caa ctg cag caa tct
ggg tct gag ttg aag aag cct ggg gcc 48Gln Val Gln Leu Gln Gln Ser
Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15tca gtg aag gtt tcc tgc
aag gct tct gga tac acc ttc aca aac tat 96Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30gga atg aac tgg gtg
aag cag gcc cct gga caa ggg ctt aaa tgg atg 144Gly Met Asn Trp Val
Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45ggc tgg ata aac
acc tac act gga gag cca aca tat act gat gac ttc 192Gly Trp Ile Asn
Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55 60aag gga cgg
ttt gcc ttc tcc ttg gac acc tct gtc agc acg gca tat 240Lys Gly Arg
Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65 70 75 80ctc
cag atc agc agc cta aag gct gac gac act gcc gtg tat ttc tgt 288Leu
Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90
95gca aga ggg ggg ttc ggt agt agc tac tgg tac ttc gat gtc tgg ggc
336Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly
100 105 110caa ggg tcc ctg gtc acc gtc tcc tca 363Gln Gly Ser Leu
Val Thr Val Ser Ser 115 12014121PRTHomo sapiens 14Gln Val Gln Leu
Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Met
Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35 40 45Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe 50 55
60Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr65
70 75 80Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe
Cys 85 90 95Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val
Trp Gly 100 105 110Gln Gly Ser Leu Val Thr Val Ser Ser 115
12015702DNAHomo sapiensCDS(1)..(702) 15atg gga tgg agc tgt atc atc
ctc ttc ttg gta gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile
Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15gtc cac tcc gac atc cag
ctg acc cag tct cca tcc tcc ctg tct gca 96Val His Ser Asp Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30tct gta gga gac aga
gtc agc atc acc tgc aag gcc agt cag gat gtg 144Ser Val Gly Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val 35 40 45agt att gct gta
gcc tgg tat cag cag aaa cca ggg aaa gcc cct aag 192Ser Ile Ala Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 50 55 60ctc ctg atc
tac tcg gca tcc tac cgg tac act gga gtc cct gat agg 240Leu Leu Ile
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg65 70 75 80ttc
agt ggc agt gga tct ggg aca gat ttc act ctc acc atc agc agt 288Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 85 90
95ctg caa cct gaa gat ttt gca gtt tat tac tgt cag caa cat tat att
336Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile
100 105 110act ccg ctc acg ttc ggt gct ggg acc aag gtg gag atc aaa
cgt act 384Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys
Arg Thr 115 120 125gtg gct gca cca tct gtc ttc atc ttc ccg cca tct
gat gag cag ttg 432Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu 130 135 140aaa tct gga act gcc tct gtt gtg tgc ctg
ctg aat aac ttc tat ccc 480Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro145 150 155 160aga gag gcc aaa gta cag tgg
aag gtg gat aac gcc ctc caa tcg ggt 528Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175aac tcc cag gag agt
gtc aca gag cag gac agc aag gac agc acc tac 576Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190agc ctc agc
agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac 624Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200 205aaa
gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc 672Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 210 215
220aca aag agc ttc aac agg gga gag tgt tag 702Thr Lys Ser Phe Asn
Arg Gly Glu Cys225 23016233PRTHomo sapiens 16Met Gly Trp Ser Cys
Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asp
Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30Ser Val Gly
Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val 35 40 45Ser Ile
Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 50 55 60Leu
Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg65 70 75
80Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
85 90 95Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr
Ile 100 105 110Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Val Glu Ile
Lys Arg Thr 115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200
205Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
210 215 220Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230171410DNAHomo
sapiensCDS(1)..(1410) 17atg gga tgg agc tgt atc atc ctc ttc ttg gta
gca aca gct aca ggt 48Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15gtc cac tcc gtc caa ctg cag caa tct ggg
tct gag ttg aag aag cct 96Val His Ser Val Gln Leu Gln Gln Ser Gly
Ser Glu Leu Lys Lys Pro 20 25 30ggg gcc tca gtg aag gtt tcc tgc aag
gct tct gga tac acc ttc aca 144Gly Ala Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr 35 40 45aac tat gga atg aac tgg gtg aag
cag gcc cct gga caa ggg ctt aaa 192Asn Tyr Gly Met Asn Trp Val Lys
Gln Ala Pro Gly Gln Gly Leu Lys 50 55 60tgg atg ggc tgg ata aac acc
tac act gga gag cca aca tat act gat 240Trp Met Gly Trp Ile Asn Thr
Tyr Thr Gly Glu Pro Thr Tyr Thr Asp65 70 75 80gac ttc aag gga cgg
ttt gcc ttc tcc ttg gac acc tct gtc agc acg 288Asp Phe Lys Gly Arg
Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr 85 90
95gca tat ctc cag atc agc agc cta aag gct gac gac act gcc gtg tat
336Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr
100 105 110ttc tgt gca aga ggg ggg ttc ggt agt agc tac tgg tac ttc
gat gtc 384Phe Cys Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe
Asp Val 115 120 125tgg ggc caa ggg tcc ctg gtc acc gtc tcc tca gcc
tcc acc aag ggc 432Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly 130 135 140cca tcg gtc ttc ccc ctg gca ccc tcc tcc
aag agc acc tct ggg ggc 480Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly145 150 155 160aca gcg gcc ctg ggc tgc ctg
gtc aag gac tac ttc ccc gaa ccg gtg 528Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175acg gtg tcg tgg aac
tca ggc gcc ctg acc agc ggc gtg cac acc ttc 576Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190ccg gct gtc
cta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg 624Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205acc
gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg 672Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215
220aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc aaa
720Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys225 230 235 240tct tgt gac aaa act cac aca tgc cca ccg tgc cca
gca cct gaa ctc 768Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 245 250 255ctg ggg gga ccg tca gtc ttc ctc ttc ccc
cca aaa ccc aag gac acc 816Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 260 265 270ctc atg atc tcc cgg acc cct gag
gtc aca tgc gtg gtg gtg gac gtg 864Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 275 280 285agc cac gaa gac cct gag
gtc aag ttc aac tgg tac gtg gac ggc gtg 912Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300gag gtg cat aat
gcc aag aca aag ccg cgg gag gag cag tac aac agc 960Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320acg
tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 1008Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330
335aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc
1056Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
340 345 350ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga
gaa cca 1104Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro 355 360 365cag gtg tac acc ctg ccc cca tcc cgg gag gag atg
acc aag aac cag 1152Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 370 375 380gtc agc ctg acc tgc ctg gtc aaa ggc ttc
tat ccc agc gac atc gcc 1200Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala385 390 395 400gtg gag tgg gag agc aat ggg
cag ccg gag aac aac tac aag acc acg 1248Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415cct ccc gtg ctg gac
tcc gac ggc tcc ttc ttc ctc tat agc aag ctc 1296Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430acc gtg gac
aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc 1344Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445gtg
atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc 1392Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455
460ctg tct ccg ggt aaa tga 1410Leu Ser Pro Gly Lys46518469PRTHomo
sapiens 18Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala
Thr Gly1 5 10 15Val His Ser Val Gln Leu Gln Gln Ser Gly Ser Glu Leu
Lys Lys Pro 20 25 30Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr 35 40 45Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro
Gly Gln Gly Leu Lys 50 55 60Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Pro Thr Tyr Thr Asp65 70 75 80Asp Phe Lys Gly Arg Phe Ala Phe
Ser Leu Asp Thr Ser Val Ser Thr 85 90 95Ala Tyr Leu Gln Ile Ser Ser
Leu Lys Ala Asp Asp Thr Ala Val Tyr 100 105 110Phe Cys Ala Arg Gly
Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val 115 120 125Trp Gly Gln
Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly145 150
155 160Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val 165 170 175Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe 180 185 190Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val 195 200 205Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val 210 215 220Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys225 230 235 240Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265
270Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
275 280 285Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val 290 295 300Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser305 310 315 320Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu 325 330 335Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390
395 400Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 405 410 415Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu 420 425 430Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser 435 440 445Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 450 455 460Leu Ser Pro Gly
Lys46519176DNAArtificialDescription of Artificial Sequence
Synthetic oligonucleotide 19ggtctgagtt gaagaagcct ggggcctcag
tgaaggtttc ctgcaaggct tctggataca 60ccttcacaaa ctatggaatg aactgggtga
agcaggcccc tggacaaggg cttaaatgga 120tgggctggat aaacacctac
actggagagc caacatatac tgatgacttc aaggga
17620168DNAArtificialDescription of Artificial Sequence Synthetic
Oligonucelotide 20acccttggcc ccagacatcg aagtaccagt agctactacc
gaacccccct cttgcacaga 60aatacacggc agtgtcgtca gcctttaggc tgctgatctg
gagatatgcc gtgctgacag 120aggtgtccaa ggagaaggca aaccgtccct
tgaagtcatc agtatatg 1682138DNAArtificialDescription of Artificial
Sequence Synthetic oligonucleotide 21gtggtgctgc agcaatctgg
gtctgagttg aagaagcc 382238DNAArtificialDescription of Artificial
Sequence Synthetic Oligonucleotide 22tgaggagacg gtgaccaggg
acccttggcc ccagacat 3823156DNAArtificialDescription of Artificial
Sequence Synthetic oligonucleotide 23ctccatcctc cctgtctgca
tctgtaggag acagagtcag catcacctgc aaggccagtc 60aggatgtgag tattgctgta
gcctggtatc agcagaaacc agggaaagcc cctaagctcc 120tgatctactc
ggcatcctac cggtacactg gagtcc 15624155DNAArtificialDescription of
Artificial Sequence Synthetic oligonucleotide 24ccttggtccc
agcaccgaac gtgagcggag taatataatg ttgctgacag taataaactg 60caaaatcttc
aggttgcaga ctgctgatgg tgagagtgaa atctgtccca gatccactgc
120cactgaacct atcagggact ccagtgtacc ggtag
1552537DNAArtificialDescription of Artificial Sequence Synthetic
oligonucleotide 25gacattcagc tgacccagtc tccatcctcc ctgtctg
372631DNAArtificialDescription of Artificial Sequence Synthetic
oligonucleotide 26acgttagatc tccaccttgg tcccagcacc g 312711PRTHomo
sapiens 27Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser1 5
102811PRTMus sp. 28Lys Ala Ser Gln Asp Val Ser Ile Ala Val Ala1 5
10297PRTMus sp. 29Ser Ala Ser Tyr Arg Tyr Thr1 5309PRTMus sp. 30Gln
Gln His Tyr Ile Thr Pro Leu Thr1 5315PRTMus sp. 31Asn Tyr Gly Met
Asn1 53217PRTMus sp. 32Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr
Thr Asp Asp Phe Lys1 5 10 15Gly3312PRTMus sp. 33Gly Gly Phe Gly Ser
Ser Tyr Trp Tyr Phe Asp Val1 5 10
* * * * *