U.S. patent application number 10/829388 was filed with the patent office on 2005-01-06 for polyvalent protein complex.
Invention is credited to Chang, Chien Hsing, McBride, William John, Rossi, Edmund A..
Application Number | 20050003403 10/829388 |
Document ID | / |
Family ID | 33313482 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050003403 |
Kind Code |
A1 |
Rossi, Edmund A. ; et
al. |
January 6, 2005 |
Polyvalent protein complex
Abstract
The invention provides for a polyvalent protein complex (PPC)
comprising two polypeptide chains generally arranged laterally to
one another. Each polypeptide chain typically comprises 3 or 4
"v-regions", which comprise amino acid sequences capable of forming
an antigen binding site when matched with a corresponding v-region
on the opposite polypeptide chain. Up to about 6 "v-regions" can be
used on each polypeptide chain. The v-regions of each polypeptide
chain are connected linearly to one another and may be connected by
interspersed linking regions. When arranged in the form of the PPC,
the v-regions on each polypeptide chain form individual antigen
binding sites.
Inventors: |
Rossi, Edmund A.; (Nutley,
NJ) ; Chang, Chien Hsing; (Downingtown, PA) ;
McBride, William John; (Boonton, NJ) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
1666 K STREET,NW
SUITE 300
WASHINGTON
DC
20006
US
|
Family ID: |
33313482 |
Appl. No.: |
10/829388 |
Filed: |
April 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60464532 |
Apr 22, 2003 |
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60525391 |
Nov 24, 2003 |
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Current U.S.
Class: |
435/6.16 ;
435/183; 435/320.1; 435/325; 435/69.7; 530/351; 530/391.1; 530/399;
536/23.2 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 25/14 20180101; A61P 31/00 20180101; A61P 29/00 20180101; C07K
2319/00 20130101; A61P 43/00 20180101; A61P 1/04 20180101; A61P
17/00 20180101; A61P 37/02 20180101; A61K 2039/505 20130101; A61P
33/10 20180101; A61P 5/14 20180101; C07K 16/468 20130101; A61P
21/00 20180101; A61P 37/06 20180101; C07K 16/44 20130101; A61P 7/04
20180101; A61P 33/06 20180101; A61P 5/40 20180101; A61P 11/00
20180101; A61P 37/08 20180101; A61P 7/06 20180101; A61P 13/12
20180101; A61P 35/00 20180101; A61P 9/10 20180101; A61P 31/04
20180101; A61P 1/16 20180101; A61P 3/10 20180101; C07K 2317/24
20130101; A61P 31/10 20180101; A61P 25/28 20180101; A61P 31/12
20180101; A61P 35/02 20180101; A61P 21/04 20180101; A61P 9/00
20180101; C07K 16/3007 20130101; C07K 2317/626 20130101; A61P 19/00
20180101; A61P 25/00 20180101; C07K 2318/20 20130101 |
Class at
Publication: |
435/006 ;
435/069.7; 435/320.1; 435/325; 530/351; 435/183; 530/399;
536/023.2; 530/391.1 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/04; A61K 039/395; C07K 014/52; C07K 014/475 |
Claims
1. A polyvalent protein complex (PPC) comprising a first and a
second polypeptide chain, wherein said first polypeptide chain
comprises a polypeptide sequence represented, by the formula
a.sub.1-1.sub.1-a.sub.2-- 1.sub.2-a.sub.3, wherein a.sub.1,
a.sub.2, and a.sub.3 are immunoglobulin variable domains and
1.sub.1 and 1.sub.2 are peptide linkers, and a.sub.1 is N-terminal
of a.sub.2, which in turn is N-terminal of a.sub.3, wherein said
second polypeptide chain comprises a polypeptide sequence
represented by the formula b.sub.1-1.sub.3-b.sub.2-1.sub.4-b.sub.3,
wherein b.sub.1, b.sub.2, and b.sub.3 are immunoglobulin variable
domains and 1.sub.3 and 1.sub.4 are peptide linkers, and b.sub.3 is
N-terminal of b.sub.2, which in turn is N-terminal of b.sub.1,
wherein said first and second polypeptide chain together form a
complex comprising at least three antigen binding sites, wherein
each of said antigen binding sites comprises a variable domain from
said first polypeptide chain and a variable domain from said second
polypeptide chain, and wherein each binding site comprises an
immunoglobulin heavy chain variable domain and an immunoglobulin
light chain variable domain.
2. The complex according to claim 1 wherein each polypeptide chain
further comprises 1-3 additional immunoglobulin variable domains,
wherein each domain is linked via a peptide linker, wherein said
first and second polypeptide chain together form a complex
comprising 4-6 antigen binding sites, and wherein each of said
antigen binding sites comprises a variable domain from said first
polypeptide chain and a variable domain from said second
polypeptide chain.
3. The complex according to claim 1, wherein at least one
polypeptide chain further comprises an amino acid sequence selected
from the group consisting of a toxin, a cytokine, a lymphokine, a
enzyme, a growth factor, and an affinity purification tag.
4. The complex according to claim 1, wherein at least two of said
antigen binding sites have the same binding specificity.
5. The complex according to claim 1, wherein each of said antigen
binding sites has a different binding specificity.
6. The complex according to claim 4, wherein said antigen binding
sites have the same binding specificity.
7. The complex according to claim 2 wherein said antigen binding
sites have at least two different binding specificities.
8. The complex according to claim 7 wherein at least 3 of said
antigen binding sites have different binding specificities.
9. The polyvalent protein complex of claim 7 wherein at least 4 of
said antigen binding sites have different binding
specificities.
10. The complex according to claim 7 comprising at least 5 antigen
binding sites wherein at least 5 of said binding sites have
different binding specificities.
11. The complex according to claim 7 comprising 6 antigen binding
sites each having a different binding specificity.
12. The complex according to claim 7 comprising at least 5 antigen
binding sites wherein at least 5 of said binding sites have
different binding specificities.
13. The complex according to claim 1, wherein two of said antigen
binding sites are specific for epitopes of tumor associated
antigens, and wherein said third antigen binding sites is reactive
with a targetable construct.
14. The polyvalent protein complex of claim 13, wherein two antigen
binding sites are specific for epitopes of tumor associated
antigens, and wherein the third antigen binding sites is reactive
with a targetable construct, and wherein the epitope on the
targetable construct is a hapten.
15. A complex comprising at least one complex according to claim 1
bound to a targetable construct, wherein said complex is bound to a
first hapten on said construct and wherein said construct further
comprises a second hapten capable of binding simultaneously to a
second polyvalent protein complex.
16. The polyvalent protein complex of claim 14, wherein the tumor
associated antigen, or antigens are selected from the group
consisting of antigens associated with carcinomas, melanomas,
sarcomas, gliomas, leukemias and lymphomas.
17. The polyvalent protein complex of claim 14, wherein the tumor
associated antigen is selected from the group consisting of
.alpha.-fetoprotein, A3, CA125, carcinoembryonic antigen (CEA),
CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD45, CD74, CD80,
colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2, folate
receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin, Ia,
IL-2, IL-6, insulin-like growth factor, KS-1, Le(y), MAGE, MUC1,
MUC2, MUC3, MUC4, NCA66, necrosis antigens, PAM-4, placental growth
factor, prostatic acid phosphatase PSA, PSMA, S100, T101, TAC,
TAG-72, tenascin and VEGF.
18. The polyvalent protein complex of claim 16, comprising at least
two tumor antigen binding sites, wherein both tumor antigen binding
sites are specific for CEA and wherein the third binding site is
specific for the hapten, histamine-succinyl-glycine (HSG).
19. The polyvalent protein complex of claim 16, wherein the
polyvalent protein is BS14HP, or hBS14.
20. A complex comprising a polyvalent protein complex according to
claim 19, bound to IMP 241, or IMP 245
21. A pretargeting method of treating or diagnosing or treating and
diagnosing a neoplastic condition comprising (a) administering to
said subject the polyvalent protein complex of claim 1, wherein two
antigen binding sites are directed to a tumor associated antigen,
and one antigen binding sites is directed to a targetable construct
comprising a bivalent hapten; (b) optionally, administering to said
subject a clearing composition, and allowing said composition to
clear the polyvalent complex from circulation; and (c)
administering to said subject said targetable construct comprising
a bivalent hapten, wherein said targetable construct further
comprises one or more chelated or chemically bound therapeutic or
diagnostic agents.
22. The method of claim 21, wherein the diagnostic agent is a
radionuclide selected from the group consisting of .sup.18F,
.sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y, .sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc, .sup.111In,
.sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.154-158Gd,
.sup.177Lu, .sup.32P, .sup.188Re, and .sup.90Y or a combination
thereof.
23. The method of claim 22, wherein said radioactive labels are
imaged using computed tomography (CT), single photon emission
computed tomography (SPECT), or positron emission tomography
(PET).
24. The method of claim 22, wherein the application is for
intraoperative diagnosis to identify occult neoplastic tumors.
25. The method of claim 21, wherein said targetable construct
comprises one or more image enhancing agents for use in magnetic
resonance imaging (MRI).
26. The method of claim 25, wherein said image enhancing agent is a
metal selected from the group consisting of chromium (III),
manganese (II), iron (III), iron (II), cobalt (II), nickel (II),
copper (III), neodymium (III), samarium (III), ytterbium (III),
gadolinium (III), vanadium (II), terbium (III), dysprosium (III),
holmium (III) and erbium (III).
27. The method of claim 21, wherein said targetable construct
comprises one or more image enhancing agents for use in ultrasound
imaging.
28. The method of claim 21, wherein said targetable construct is a
liposome with a bivalent HSG-peptide covalently attached to the
outside surface of the liposome lipid membrane.
29. The method of claim 28, wherein said liposome is gas
filled.
30. The method of claim 21, wherein said targetable construct
comprises one or more radioactive isotopes useful for killing
neoplastic cells.
31. The method of claim 30, wherein said radioactive isotope is
selected from the group consisting of .sup.32P, .sup.33P,
.sup.47Sc, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.90Y, .sup.111Ag,
.sub.111In, .sup.125I, .sup.131I, .sup.142Pr, .sup.153Sm,
.sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.177Lu, .sup.186Re,
.sup.188Re, .sup.189Re, .sup.212Pb, .sup.212Bi, .sup.213Bi,
.sup.211At, .sup.223Ra and .sup.225Ac or a combination thereof.
32. The method of claim 30, wherein the pretargeted therapy is
administered prior to, with or after one or more therapeutic
agents.
33. The method of claim 32, wherein said therapeutic agent is a
cytokine or a chemotherapeutic agent, or a colony-stimulating
growth factor.
34. The method of claim 33, wherein said therapeutic agent is a
chemotherapeutic agent selected from the group consisting of
taxanes, nitrogen mustards, ethylenimine derivatives, alkyl
sulfonates, nitrosoureas, triazenes; folic acid analogs, pyrimidine
analogs, purine analogs, vinca alkaloids, antibiotics, enzymes,
platinum coordination complexes, substituted urea, methyl hydrazine
derivatives, adrenocortical suppressants, and antagonists.
35. The method of claim 33, wherein said therapeutic agent is a
chemotherapeutic agent selected from the group consisting of
steroids, progestins, estrogens, antiestrogens, and androgens.
36. The method of claim 33, wherein said therapeutic agent is a
chemotherapeutic agent selected from the group consisting of
azaribine, bleomycin, bryostatin-1, busulfan, carmustine,
chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, ethinyl estradiol, etoposide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, L-asparaginase, leucovorin, lomustine,
mechlorethamine, medroprogesterone acetate, megestrol acetate,
melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin,
mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine,
semustine streptozocin, tamoxifen, taxanes, taxol, testosterone
propionate, thalidomide, thioguanine, thiotepa, uracil mustard,
vinblastine, and vincristine.
37. The method of claim 33, wherein said therapeutic agent is a
cytokine selected from the group consisting of interleukin-1
(IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, interferon-alpha,
interferon-beta, and interferon-gamma.
38. The method of claim 33, wherein said therapeutic agent is a
colony-stimulating growth factor selected from the group consisting
of granulocyte-colony stimulating factor (G-CSF), granulocyte
macrophage-colony stimulating factor (GM-CSF), erthropoietin and
thrombopoietin.
39. A method of treating a neoplastic disorder in a subject,
comprising administering to said subject a "naked" polyvalent
protein complex according to claim 1, wherein at least one of said
antigen binding sites binds to an antigen selected from the group
consisting of alpha fetoprotein, A3, CA125, carcinoembryonic
antigen (CEA), CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD45,
CD74, CD80, colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2,
folate receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin,
Ia, IL-2, IL-6, insulin-like growth factor, KS-1, Le(y), MAGE,
MUC1, MUC2, MUC3, MUC4, NCA66, necrosis antigens, PAM-4, placental
growth factor, prostatic acid phosphatase PSA, PSMA, S100, T101,
TAC, TAG-72, tenascin and VEGF.
40. The method of claim 39, wherein the neoplastic disorder is
selected from the group consisting of carcinomas, sarcomas,
gliomas, lymphomas, leukemias, and melanomas.
41. A method for treating a B-cell malignancy, or B-cell immune or
autoimmune disorder in a subject, comprising administering to said
subject one or more dosages of a therapeutic composition comprising
a polyvalent protein complex of claim 1 and a pharmaceutically
acceptable carrier.
42. A method for treating a B-cell malignancy, or B-cell immune or
autoimmune disorder in a subject, comprising administering to said
subject one or more dosages of a therapeutic composition comprising
a polyvalent protein complex of claim 2 and a pharmaceutically
acceptable carrier, wherein each antigen binding site binds a
distinct epitope of CD19, CD20 or CD22.
43. The method of claim 42, wherein said polyvalent protein complex
is parenterally administered in a dosage of 20 to 1500 milligrams
protein per dose.
44. The method of claim 42, wherein said polyvalent protein complex
is parenterally administered in a dosage of 20 to 500 milligrams
protein per dose.
45. The method of claim 42, wherein said polyvalent protein complex
is parenterally administered in a dosage of 20 to 100 milligrams
protein per dose.
46. The method of claim 42, wherein said subject receives the
polyvalent protein complex as repeated parenteral dosages of 20 to
100 milligrams protein per dose.
47. The method of claim 42, wherein said subject receives the
polyvalent protein complex as repeated parenteral dosages of 20 to
1500 milligrams protein per dose.
48. The method of claim 42, wherein a sub-fraction of the
polyvalent protein complex is labeled with a radioactive
isotope.
49. The method of claim 48, wherein said radioactive isotope is
selected from the group consisting of .sup.32P, .sup.33P,
.sup.47Sc, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.90Y, .sup.111Ag,
.sub.111In, .sup.125I, .sup.131I, .sup.142Pr, .sup.153Sm,
.sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.177Lu, .sup.186Re,
.sup.188Re, .sup.189Re, .sup.212Pb, .sup.212Bi, .sup.213Bi,
.sup.211At, .sup.223Ra and .sup.225Ac or a combination thereof.
50. A method for detecting or diagnosing a B-cell malignancy, or
B-cell immune or autoimmune disorder in a subject, comprising
administering to said subject a diagnostic composition comprising a
polyvalent protein complex of claim 2 and a pharmaceutically
acceptable carrier, wherein each antigen binding site binds a
distinct epitope of CD19, CD20 or CD22, and wherein said complex is
radiolabeled with a radionuclide selected from the group consisting
of .sup.18F, .sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga,
.sup.68Ga, .sup.86Y, .sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc,
.sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.177Lu, .sup.32P, 188Re, and .sup.90Y or a
combination thereof.
51. The method of claim 50, wherein said radioactive labels are
imaged using computed tomography (CT), single photon emission
computed tomography (SPECT), or positron emission tomography
(PET).
52. The method of claim 50, wherein the application is for
intraoperative diagnosis to identify occult neoplastic tumors.
53. A method for detecting or diagnosing a B-cell malignancy, or
B-cell immune or autoimmune disorder in a subject, comprising
administering to said subject a diagnostic composition comprising a
polyvalent protein complex of claim 2 and a pharmaceutically
acceptable carrier, wherein each antigen binding site binds a
distinct epitope of CD19, CD20 or CD22, and wherein said complex is
labeled with one or more image enhancing agents for use in magnetic
resonance imaging (MRI).
54. The method of claim 53, wherein said image enhancing agent is a
paramagnetic ion selected from the group consisting of chromium
(III), manganese (II), iron (II), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) and erbium (III).
55. A method of diagnosing a non-neoplastic disease or disorder,
comprising administering to a subject suffering from said disease
or disorder a complex according to claim 1, wherein a detectable
label is attached to said complex, and wherein one or more of said
antigen binding sites is specific for a marker substance of the
disease or disorder.
56. The method of claim 55, wherein said disease or disorder is
caused by a fungus.
57. The method of claim 56, wherein said fungus is selected from
the group consisting of Microsporum, Trichophyton, Epidermophyton,
Sporothrix schenckii, Cryptococcus neoformans, Coccidioides
immitis, Histoplasma capsulatum, Blastomyces dermatitidis, and
Candida albicans.
58. The method of claim 55 wherein said disease or disorder is
caused by a virus.
59. The method of claim 58, wherein said virus is selected from the
group consisting of human immunodeficiency virus (HIV), herpes
virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B
virus, Sendai virus, feline leukemia virus, Reo virus, polio virus,
human serum parvo-like virus, simian virus 40, respiratory
syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,
Dengue virus, rubella virus, measles virus, adenovirus, human
T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus,
mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic
choriomeningitis virus, wart virus and blue tongue virus.
60. The method of claim 55 wherein said disease or disorder is
caused by a bacterium.
61. The method of claim 60, wherein said bacterium is selected from
the group consisting of Anthrax bacillus, Streptococcus agalactiae,
Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli,
Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus,
Hemophilis influenzae B, Treponema pallidum, Lyme disease
spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella
abortus, and Mycobacterium tuberculosis
62. The method of claim 55 wherein said disease or disorder is
caused by a Mycoplasma.
63. The method of claim 55 wherein said disease or disorder is
caused by a parasite.
64. The method of claim 55 wherein said disease or disorder is
malaria.
65. The method of claim 55, wherein said disease or disorder is an
autoimmune disease.
66. The method of claim 65, wherein said autoimmune disease is
selected from the group consisting of acute idiopathic
thrombocytopenic purpura, chronic idiopathic thrombocytopenic
purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis,
systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcalnephritis, erythema
nodosurn, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme, IgA nephropathy, polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome,
thromboangitisubiterans, Sjogren's syndrome, primary biliary
cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,
chronic active hepatitis, polymyositis/dermatomyositis,
polychondritis, pamphigus vulgaris, Wegener's granulomatosis,
membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant cell arteritis/polymyalgia, pernicious anemia,
rapidly progressive glomerulonephritis, psoriasis, and fibrosing
alveolitis.
67. The method of claim 55, wherein said the disease or disorder is
myocardial infarction, ischemic heart disease, or atherosclerotic
plaques.
68. The method of claim 55, wherein said disease or disorder is
graft rejection.
69. The method of claim 55, wherein said disease or disorder is
Alzheimer's disease.
70. The method of claim 55, wherein said disease or disorder is
caused by atopic tissue.
71. The method of claim 55, wherein said disease or disorder is
inflammation caused by accretion of activated granulocytes,
monocytes, lymphoid cells or macrophages at the site of
inflammation, and wherein the inflammation is caused by an
infectious agent.
72. The method of claim 55, wherein said detectable label is a
radionuclide selected from the group consisting of .sup.18F,
.sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y, .sup.89 Zr .sup.94mTc, .sup.94Tc, .sup.99mTc, .sup.111In,
.sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.154-158Gd,
.sup.177Lu, .sup.32P, .sup.188Re, and .sup.90Y or a combination
thereof.
73. The method of claim 72, wherein said radioactive labels are
imaged using computed tomography (CT), single photon emission
computed tomography (SPECT), or positron emission tomography
(PET).
74. The method of claim 73, wherein the application is for
intraoperative diagnosis of said disease or disorder.
75. The method of claim 55, wherein at least one of said antigen
binding sites is specific for a targetable construct, and wherein
said construct comprises one or more image enhancing agents for use
in magnetic resonance imaging (MRI).
76. The method of claim 75, wherein said image enhancing agent is a
paramagnetic ion selected from the group consisting of chromium
(III), manganese (II), iron (III), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) and erbium (III).
77. The method of claim 55, wherein at least one of said antigen
binding sites is specific for a targetable construct, and wherein
said targetable construct comprises one or more image enhancing
agents for use in ultrasound imaging.
78. The method of claim 55, wherein at least one of said antigen
binding sites is specific for a targetable construct and wherein
said targetable construct comprises a liposome with a bivalent
HSG-peptide covalently attached to the outside surface of the
liposome lipid membrane.
79. The method of claim 74, wherein said liposome is gas
filled.
80. A pretargeting method of treating or diagnosing a
non-neoplastic disease or disorder in a subject comprising (a)
administering to said subject the polyvalent protein complex of
claim 1, wherein two antigen binding sites are directed to a marker
substance, or marker substances specific for the disorder, and one
antigen binding sites is directed to a targetable construct
comprising a bivalent hapten; (b) optionally administering to said
subject a clearing composition, and allowing said composition to
clear the polyvalent complex from circulation; and (c)
administering to said subject said targetable construct comprising
a bivalent hapten, wherein the targetable construct further
comprises one or more chelated or chemically bound therapeutic or
diagnostic agents.
81. The method of claim 80, wherein said disease or disorder is
caused by a fungus.
82. The method of claim 81, wherein the species of fungus is
selected from the group consisting of Microsporum, Trichophyton,
Epidermophyton, Sporothrix schenckii, Cryptococcus neoformans,
Coccidioides immitis, Histoplasma capsulatum, Blastomyces
dermatitidis, or Candida albicans.
83. The method of claim 80 wherein said disease or disorder is
caused by a virus.
84. The method of claim 83, wherein the species of virus is
selected from the group consisting of human immunodeficiency virus
(HIV), herpes virus, cytomegalovirus, rabies virus, influenza
virus, hepatitis B virus, Sendai virus, feline leukemia virus, Reo
virus, polio virus, human serum parvo-like virus, simian virus 40,
respiratory syncytial virus, mouse mammary tumor virus,
Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,
adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,
murine leukemia virus, mumps virus, vesicular stomatitis virus,
Sindbis virus, lymphocytic choriomeningifis virus, wart virus and
blue tongue virus.
85. The method of claim 80 wherein said disease or disorder is
caused by a bacterium.
86. The method of claim 85, wherein the bacterium is selected from
the group consisting of Anthrax bacillus, Streptococcus agalactiae,
Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli,
Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus,
Hemophilis influenzae B, Treponema pallidum, Lyme disease
spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella
abortus, and Mycobacterium tuberculosis.
87. The method of claim 80 wherein said disease or disorder is
caused by a Mycoplasma.
88. The method of claim 80 wherein said disease or disorder is
caused by a parasite.
89. The method of claim 80 wherein the disease or disorder is
malaria.
90. The method of claim 80, wherein said disease or disorder is an
autoimmune disease.
91. The method of claim 90, wherein the autoimmune disease is
selected from the group consisting of acute idiopathic
thrombocytopenic purpura, chronic idiopathic thrombocytopenic
purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis,
systemic lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcalnephritis, erythema
nodosurn, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme, IgA nephropathy, polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome,
thromboangitisubiterans, Sjogren's syndrome, primary biliary
cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,
chronic active hepatitis, polymyositis/dermatomyositis,
polychondritis, pamphigus vulgaris, Wegener's granulomatosis,
membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant cell arteritis/polymyalgia, pernicious anemia,
rapidly progressive glomerulonephritis, psoriasis, and fibrosing
alveolitis.
92. The method of claim 80, wherein the disease or disorder is
selected from the group consisting of myocardial infarction,
ischemic heart disease, and atherosclerotic plaques.
93. The method of claim 80, wherein the disease or disorder is
graft rejection.
94. The method of claim 80, wherein the disease or disorder is
Alzheimer's disease.
95. The method of claim 80, wherein the disease or disorder is
caused by atopic tissue.
96. The method of claim 80, wherein the disease or disorder is
inflammation caused by accretion of activated granulocytes,
monocytes, lymphoid cells or macrophages at the site of
inflammation, and wherein the inflammation is caused by an
infectious agent.
97. The method of claim 80, wherein said targetable construct is
labeled with a radionuclide selected from the group consisting of
.sup.18F, .sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga,
.sup.68Ga, .sup.86Y, .sup.89Zr, .sup.94mTc, .sup.94Tc, .sup.99mTc,
.sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.177LU, .sup.32P, .sup.188Re, and .sup.90Y or a
combination thereof.
98. The method of claim 97, wherein said radioactive labels are
imaged using computed tomography (CT), single photon emission
computed tomography (SPECT), or positron emission tomography
(PET).
99. The method of claim 97, wherein the application is for
intraoperative diagnosis of the disorder.
100. The method of claim 80, wherein said targetable construct
comprises one or more image enhancing agents for use in magnetic
resonance imaging (MRI).
101. The method of claim 100, wherein image enhancing agent is a
paramagnetic ion selected from the group consisting of chromium
(III), manganese (II), iron (III), iron (II), cobalt (II), nickel
(II), copper (II), neodymium (III), samarium (III), ytterbium
(III), gadolinium (III), vanadium (II), terbium (III), dysprosium
(III), holmium (III) and erbium (III).
102. The method of claim 80, wherein said targetable construct
comprises one or more image enhancing agents for use in ultrasound
imaging.
103. The method of claim 102, wherein said targetable construct is
a liposome with a bivalent HSG-peptide covalently attached to the
outside surface of the liposome lipid membrane.
104. The method of claim 103, wherein said liposome is gas
filled.
105. A method of antibody dependent enzyme prodrug therapy (ADEPT)
comprising; (a) administering to a patient with a neoplastic
disorder the polyvalent protein complex of claim 3, wherein said
complex comprises a covalently attached enzyme capable of
activating a prodrug, (b) optionally administering to said subject
a clearing composition, and allowing said composition to clear the
polyvalent complex from circulation, and (c) administering said
prodrug to the patient.
106. An assay method comprising detecting a target molecule using
one or more polyvalent protein complexes of claim 1.
107. An immunostaining method comprising staining a cell using one
or more polyvalent protein complexes of claim 1.
108. An isolated nucleic acid molecule encoding a first or second
polypeptide according to claim 1.
109. A nucleic acid expression cassette comprising the isolated
nucleic acid of claim 108.
110. An episome comprising: (a) a first promoter operationally
connected to a first nucleic acid encoding a first polypeptide
comprising a polypeptide chain represented by the formula
a.sub.1-1.sub.1-a.sub.2-1.su- b.2-a.sub.3, wherein a.sub.1,
a.sub.2, and a.sub.3 are immunoglobulin variable domains and
1.sub.1 and 1.sub.2 are peptide linkers, (b) a second promoter
operationally connected to a second nucleic acid encoding a
polypeptide comprising a second polypeptide chain represented by
the formula b.sub.1-1.sub.3-b.sub.2-1.sub.4-b.sub.3, wherein
b.sub.1, b.sub.2, and b.sub.3 are immunoglobulin variable domains
and 1.sub.3 and 1.sub.4 are peptide linkers, wherein said first and
second polypeptide chain together form a complex comprising at
least three antigen binding sites, wherein each of said antigen
binding sites comprises a variable domain from said first
polypeptide chain and a variable domain from said second
polypeptide chain, wherein said first nucleic acid and said second
nucleic acid are coexpressed when the episome is transformed into a
host cell.
111. The episome of claim 110 which is a plasmid or a cosmid.
112. A host cell comprising an episome according to claim 110.
113. The host cell of claim 112, wherein said host cell is selected
from the group consisting of E. coli, yeast, a plant cell and a
mammalian cell.
114. A method of preparing a polyvalent protein complex, comprising
culturing a host cell according to claim 112.
115. The host cell of claim 112, wherein said cell is a murine
myeloma cell line.
116. The episome of claim 111, wherein the plasmid is pdHL2.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/464,532, filed Apr. 22, 2003, and 60/525,391,
filed Nov. 24, 2003, the contents of which are hereby incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to polyvalent protein
complexes, including trivalent bispecific proteins, useful for the
treatment and diagnosis of diseases, and to methods of producing
such proteins.
BACKGROUND OF THE INVENTION
[0003] Throughout this specification, various patents, published
applications and scientific references are cited to describe the
state and content of the art. Those disclosures, in their
entireties, are hereby incorporated into the present specification
by reference.
[0004] The present invention is directed to a novel protein
structures, termed a "polyvalent protein complex" or PPC, that
comprise three or four antigen binding sites (ABS). These PPC
comprise novel properties, such as trivalence and tetravalence,
when compared to immunoglobulins and can substitute for
immunoglobulins or other engineered antibodies in applications such
as diagnosis, detection, and therapy of normal (ectopic) or
diseased tissues. These diseased tissues include cancers,
infections, autoimmune diseases, cardiovascular diseases, and
neurological diseases. Normal tissues can be detected and/or
ablated, such as when they are ectopic (misplaced, such as
parathyroid, thymus, endometrium) or if they need to be ablated as
a therapy measure (e.g., bone marrow ablation in cancer
therapies).
[0005] Discrete V.sub.H and V.sub.L domains of antibodies produced
by recombinant DNA technology may pair with each other to form a
heterodimer (recombinant Fv fragment) with binding capability (U.S.
Pat. No. 4,642,334). However, such non-covalently associated
molecules are not sufficiently stable under physiological
conditions to have any practical use. Cognate V.sub.H and V.sub.L
domains can be joined with a peptide linker of appropriate
composition and length (usually consisting of more than 12 amino
acid residues) to form a single-chain Fv (scFv) with binding
activity. Methods of manufacturing scFvs are disclosed in U.S. Pat.
No. 4,946,778 and U.S. Pat. No. 5,132,405. Reduction of the peptide
linker length to less than 12 amino acid residues prevents pairing
of V.sub.H and V.sub.L domains on the same chain and forces pairing
of V.sub.H and V.sub.L domains with complementary domains on other
chains, resulting in the formation of functional multimers.
Polypeptide chains of V.sub.H and V.sub.L domains joined with
linkers between 3 and 12 amino acid residues form predominantly
dimers (termed diabodies). With linkers between 0 and 2 amino acid
residues, trimers (termed triabody) and tetramers (termed
tetrabody) are in favor, but the exact patterns of oligomerization
appear to depend on the composition as well as the orientation of
V-domains (V.sub.H-linker-V.sub.L or V.sub.L-linker-V.sub.H), in
addition to the linker length. Monospecific diabodies, triabodies,
and tetrabodies with multiple valencies have been obtained using
peptide linkers consisting of 5 amino acid residues or less.
Bispecific diabodies, which are heterodimers of two different
polypeptides, each polypeptide consisting of the V.sub.H domain
from one antibody connected by a short peptide linker to the
V.sub.L domain of another antibody, have also been made using a
dicistronic expression vector that contains in one cistron a
recombinant gene construct comprising V.sub.H1-linker-V.sub.L2 and
in the other cistron a second recombinant gene construct comprising
V.sub.H2-linker-V.sub.L1. (Holliger et al., Proc. Natl. Acad. Sci.
USA (1993) 90: 6444-6448; Atwell et al., Molecular Immunology
(1996) 33: 1301-1302; Holligeret al., Nature Biotechnology (1997)
15: 632-631; Helfrich et al., Int. J. Cancer (1998) 76: 232-239;
Kipriyanov et al., Int. J. Cancer (1998) 77: 763-772; Holiger et
al., Cancer Research (1999) 59: 2909-2916]. More recently, a
tetravalent tandem diabody (termed tandab) with dual specificity
has also been reported (Cochlovius et al., Cancer Research (2000)
60: 4336-4341]. The bispecific tandab is a dimer of two homologous
polypeptides, each containing four variable domains of two
different antibodies (V.sub.H1, V.sub.L1, V.sub.H2, V.sub.L2)
linked in an orientation to facilitate the formation of two
potential binding sites for each of the two different specificities
upon self-association.
[0006] Methods of manufacturing monospecific diabodies,
monospecific triabodies, monospecific tetrabodies and bispecific
diabodies by varying the length of the peptide linker as described
above are disclosed in U.S. Pat. No. 5,844,094, U.S. Pat. No.
5,837,242, and WO 98/44001.
[0007] Alternative methods of manufacturing multispecific and
multivalent antigen-binding proteins from V.sub.H and V.sub.L
domains are disclosed in U.S. Pat. No. 5,989,830 and U.S. Pat. No.
6,239,259. Such multivalent and multispecific antigen-binding
proteins are obtained by expressing a dicistronic vector which
encodes two polypeptide chains, with one polypeptide chain
consisting of two or more V.sub.H domains (from the same or
different antibodies) connected in series by a peptide linker and
the other polypeptide chain consisting of complementary V.sub.L
domains connected in series by a peptide linker.
[0008] Increasing the valency of a binding protein is of interest
as it enhances the functional affinity of that protein due to the
avidity effect. The increased affinity enables the resulting
protein to bind more strongly to target cells. Furthermore, the
multivalency may, via crosslinking, induce growth inhibition of
target cells (Ghetie, et al, Blood, 97: 1392-8, 2001) or facilitate
internalization (Yarden, Proc. Natl. Acad. Sci., USA, 94: 9637,
1990), either property is desirable for an anti-tumor agent. The
present invention addresses the continuous need to develop
multivalent, multispecific agents for use in therapeutic and
diagnostic applications.
[0009] Another area of the present invention is in the field of
bio-assays. Virtually every area of biomedical sciences is in need
of a system to assay chemical and biochemical reactions and
determine the presence and quantity of particular analytes. This
need ranges from the basic science research lab, where biochemical
pathways are being mapped out and their functions correlated to
disease processes, to clinical diagnostics, where patients are
routinely monitored for levels of clinically relevant analytes.
Other areas include pharmaceutical research, military applications,
veterinary, food, and environmental applications. In all of these
cases, the presence and quantity of a specific analyte or group of
analytes, needs to be determined.
[0010] For analysis in the fields of chemistry, biochemistry,
biotechnology, molecular biology and numerous others, it is often
useful to detect the presence of one or more molecular structures
and measure binding between structures. The molecular structures of
interest typically include, but are not limited to, cells,
antibodies, antigens, metabolites, proteins, drugs, small
molecules, proteins, enzymes, nucleic acids, and other ligands and
analytes. In medicine, for example, it is very useful to determine
the existence of a cellular constituents such as receptors or
cytokines, or antibodies and antigens which serve as markers for
various disease processes, which exists naturally in physiological
fluids or which has been introduced into the system. Additionally,
DNA and RNA analysis is very useful in diagnostics, genetic testing
and research, agriculture, and pharmaceutical development. Because
of the rapidly advancing state of molecular cell biology and
understanding of normal and diseased systems, there exists an
increasing need for methods of detection, which do not require
labels such as fluorophores or radioisotopes, are quantitative and
qualitative, specific to the molecule of interest, highly sensitive
and relatively simple to implement.
[0011] Numerous methodologies have been developed over the years to
meet the demands of these fields, such as Enzyme-Linked
Immunosorbent Assays (ELISA), Radio-Immunoassays (RIA), numerous
fluorescence assays, mass spectroscopy, colorimetric assays, gel
electrophoresis, as well as a host of more specialized assays. Most
of these assay techniques require specialized preparations,
especially attaching a label or greatly purifying and amplifying
the sample to be tested. To detect a binding event between a ligand
and an antiligand, a detectable signal is required which relates to
the existence or extension of binding. Usually the signal is
provided by a label that is conjugated to either the ligand or
antiligand of interest. Physical or chemical effects which produce
detectable signals, and for which suitable labels exist, include
radioactivity, fluorescence, chemiluminescence, phosphorescence and
enzymatic activity to name a few. The label can then be detected by
spectrophotometric, radiometric, or optical tracking methods.
SUMMARY OF THE INVENTION
[0012] This invention provides a polyvalent protein complex (PPC),
a dimer, comprising at least three antigen binding sites (ABS) in a
linear array. The invention also provides a fusion PPC, which is a
PPC chemically bonded to a second molecule such as a conjugate. It
is understood that "fusion PPC" is a subset of all PPC and that
references to PPC in this disclosure is also meant to refer to
"fusion PPC."
[0013] The invention also provides a nucleic acid that encodes at
least one polypeptide of a PPC. A host cell that comprise the
polypeptide is also an embodiment of the invention.
[0014] In addition, the invention also provides a method for
reducing a symptom of a disorder, such as a cancer, an infection, a
cardiological disorder or an autoimmune disorder by administering a
PPC or fusion PPC to a patient.
[0015] Specifically, there is provided a polyvalent protein complex
(PPC) containing a first and a second polypeptide chain, where the
first polypeptide chain contains a polypeptide sequence
represented, by the formula
a.sub.1-1.sub.1-a.sub.2-1.sub.2-a.sub.3, where a.sub.1, a.sub.2,
and a.sub.3 are immunoglobulin variable domains and 1.sub.1 and
1.sub.2 are peptide linkers, and a.sub.1 is N-terminal of a.sub.2,
which in turn is N-terminal of a.sub.3, where the second
polypeptide chain contains a polypeptide sequence represented by
the formula b.sub.1-1.sub.3-b.sub.2-1- .sub.4-b.sub.3, where
b.sub.1, b.sub.2, and b.sub.3 are immunoglobulin variable domains
and 1.sub.3 and 1.sub.4 are peptide linkers, and b.sub.3 is
N-terminal of b.sub.2, which in turn is N-terminal of b.sub.1,
where the first and second polypeptide chain together form a
complex containing at least three antigen binding sites, where each
of the antigen binding sites contains a variable domain from the
first polypeptide chain and a variable domain from the second
polypeptide chain, and where each binding site contains an
immunoglobulin heavy chain variable domain and an immunoglobulin
light chain variable domain.
[0016] Each polypeptide chain may further contain 1-3 additional
immunoglobulin variable domains, where each domain is linked via a
peptide linker, where the first and second polypeptide chain
together form a complex containing 4-6 antigen binding sites, and
where each of the antigen binding sites contains a variable domain
from the first polypeptide chain and a variable domain from the
second polypeptide chain. At least one of the polypeptide chains
may further contain an amino acid sequence selected from the group
consisting of a toxin, a cytokine, a lymphokine, a enzyme, a growth
factor, and an affinity purification tag.
[0017] The complex may contain any of the possible combinations of
binding affinities, for example, at least two of the antigen
binding sites may have the same binding specificity, each of the
antigen binding sites may have a different or the same binding
specificity, the antigen binding sites may have at least two
different binding specificities, at least 3 of the antigen binding
sites may have different binding specificities, at least 4 of the
antigen binding sites may have different binding specificities, the
complex may contain at least 5 antigen binding sites where at least
5 of the binding sites have different binding specificities, or the
complex may contain 6 antigen binding sites each having a different
binding specificity. In another example two of the antigen binding
sites are specific for epitopes of tumor associated antigens, and
the third antigen binding sites is reactive with a targetable
construct. In another example, two antigen binding sites are
specific for epitopes of tumor associated antigens, and the third
antigen binding sites is reactive with a targetable construct,
where the epitope on the targetable construct is a hapten. In still
another complex, the complex is bound to a first hapten on the
construct and the construct further contains a second hapten
capable of binding simultaneously to a second polyvalent protein
complex.
[0018] In each of these examples, the complex may bind tumor
associated antigen, or antigens are selected from the group
consisting of antigens associated with carcinomas, melanomas,
sarcomas, gliomas, leukemias and lymphomas, such as
.alpha.-fetoprotein, A3, CA125, carcinoembryonic antigen (CEA),
CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD45, CD74, CD80,
colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2, folate
receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin, Ia,
IL-2, IL-6, insulin-like growth factor, KS-1, Le(y), MAGE, MUC1,
MUC2, MUC3, MUC4, NCA66, necrosis antigens, PAM-4, placental growth
factor, prostatic acid phosphatase PSA, PSMA, S100, T101, TAC,
TAG-72, tenascin and/or VEGF.
[0019] In another example, the complex contains at least two tumor
antigen binding sites, where both tumor antigen binding sites are
specific for CEA and where the third binding site is specific for
the hapten, histamine-succinyl-glycine (HSG).
[0020] The polyvalent protein may be BS14HP, or hBS14, which may be
bound to IMP 241, or IMP 245
[0021] In another embodiment, any of the complexes described above
may be used in a pretargeting method of treating or diagnosing or
treating and diagnosing a neoplastic condition by (a) administering
to the subject a complex as above, where two antigen binding sites
are directed to a tumor associated antigen, and one antigen binding
sites is directed to a targetable construct containing a bivalent
hapten; (b) optionally, administering to the subject a clearing
composition, and allowing the composition to clear the polyvalent
complex from circulation; and (c) administering to the subject the
targetable construct containing a bivalent hapten, where the
targetable construct further contains one or more chelated or
chemically bound therapeutic or diagnostic agents.
[0022] The diagnostic agent may be a radionuclide selected from the
group consisting of .sup.18F, .sup.52Fe, .sup.62Cu, .sup.64Cu,
.sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.86Y, .sup.89Zr .sup.94mTc,
.sup.94Tc, .sup.99mTc, .sup.111In, .sup.123I, .sup.124I, .sup.125I,
.sup.131I, .sup.154-158Gd, .sup.177Lu, .sup.32P, .sup.188Re, and
.sup.90Y or a combination thereof, which may be detected, for
example, by computed tomography (CT), single photon emission
computed tomography (SPECT), or positron emission tomography (PET).
The application may be for intraoperative diagnosis to identify
occult neoplastic tumors. The targetable construct may contain one
or more image enhancing agents for use in magnetic resonance
imaging (MRI), such as a metal selected from the group consisting
of chromium (III), manganese (II), iron (III), iron (II), cobalt
(II), nickel (II), copper (II), neodymium (III), samarium (III),
ytterbium (III), gadolinium (III), vanadium (II), terbium (III),
dysprosium (III), holmium (III) and erbium (III). The targetable
construct may contains one or more image enhancing agents for use
in ultrasound imaging.
[0023] The targetable construct may be a liposome with a bivalent
HSG-peptide covalently attached to the outside surface of the
liposome lipid membrane. The liposome may be gas filled.
[0024] The targetable construct may contain one or more radioactive
isotopes useful for killing neoplastic cells, such as .sup.32P,
.sup.33P, .sup.47Sc, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.90Y,
.sup.111Ag, .sub.111In, .sup.125I, .sup.131I, .sup.142Pr,
.sup.153Sm, .sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.177Lu,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.212Pb, .sup.212Bi,
.sup.213Bi, .sup.211At, .sup.223Ra and .sup.225Ac or a combination
thereof.
[0025] The pretargeted therapy may be administered prior to, with
or after one or more therapeutic agents. The therapeutic agent may
be a cytokine or a chemotherapeutic agent, or a colony-stimulating
growth factor. The therapeutic agent may be a chemotherapeutic
agent selected from the group consisting of taxanes, nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes; folic acid analogs, pyrimidine analogs, purine analogs,
vinca alkaloids, antibiotics, enzymes, platinum coordination
complexes, substituted urea, methyl hydrazine derivatives,
adrenocortical suppressants, and antagonists, or may be selected
from the group consisting of steroids, progestins, estrogens,
antiestrogens, and androgens. The therapeutic agent may be a
chemotherapeutic agent selected from the group consisting of
azaribine, bleomycin, bryostatin-1, busulfan, carmustine,
chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, ethinyl estradiol, etoposide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, L-asparaginase, leucovorin, lomustine,
mechlorethamine, medroprogesterone acetate, megestrol acetate,
melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin,
mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine,
semustine streptozocin, tamoxifen, taxanes, taxol, testosterone
propionate, thalidomide, thioguanine, thiotepa, uracil mustard,
vinblastine, and vincristine. The therapeutic agent may be a
cytokine selected from the group consisting of interleukin-1
(IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, interferon-alpha,
interferon-beta, and interferon-gamma, or may be a
colony-stimulating growth factor selected from the group consisting
of granulocyte-colony stimulating factor (G-CSF), granulocyte
macrophage-colony stimulating factor (GM-CSF), erthropoietin and
thrombopoietin.
[0026] Also provided is a method of treating a neoplastic disorder
in a subject, by administering to the subject a "naked" polyvalent
protein complex as described above, where at least one of the
antigen binding sites binds to an antigen selected from the group
consisting of alpha fetoprotein, A3, CA125, carcinoembryonic
antigen (CEA), CD19, CD20, CD21, CD22, CD23, CD30, CD33, CD45,
CD74, CD80, colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2,
folate receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin,
Ia, IL-2, IL-6, insulin-like growth factor, KS-1, Le(y), MAGE,
MUC1, MUC2, MUC3, MUC4, NCA66, necrosis antigens, PAM-4, placental
growth factor, prostatic acid phosphatase PSA, PSMA, S100, T101,
TAC, TAG-72, tenascin and VEGF.
[0027] The neoplastic disorder may be selected from the group
consisting of carcinomas, sarcomas, gliomas, lymphomas, leukemias,
and melanomas.
[0028] Also provided is a method for treating a B-cell malignancy,
or B-cell immune or autoimmune disorder in a subject, containing
administering to the subject one or more dosages of a therapeutic
composition containing a polyvalent protein complex as described
above and a pharmaceutically acceptable carrier.
[0029] Also provided is a method for treating a B-cell malignancy,
or B-cell immune or autoimmune disorder in a subject, by
administering to the subject one or more dosages of a therapeutic
composition containing a polyvalent protein complex and a
pharmaceutically acceptable carrier, where each antigen binding
site binds a distinct epitope of CD19, CD20 or CD22. The complex
may be parenterally administered in a dosage of 20 to 1500
milligrams protein per dose, or 20 to 500 milligrams protein per
dose, or 20 to 100 milligrams protein per dose. The subject may
receive repeated parenteral dosages of 20 to 100 milligrams protein
per dose, or repeated parenteral dosages of 20 to 1500 milligrams
protein per dose. In these methods, a sub-fraction of the
polyvalent protein complex is labeled with a radioactive isotope,
such as .sup.32P, .sup.33P, .sup.47Sc, .sup.64Cu, .sup.67Cu,
.sup.67Ga, .sup.90Y, .sup.111Ag, .sub.111In, .sup.125I, .sup.131I,
.sup.142Pr, .sup.153Sm, .sup.161Tb, .sup.166Ho, .sup.177Lu,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.212Pb, .sup.212Bi,
.sup.213Bi, .sup.211At, .sup.223Ra and .sup.225Ac or a combination
thereof.
[0030] Also provided is a method for detecting or diagnosing a
B-cell malignancy, or B-cell immune or autoimmune disorder in a
subject, by administering to the subject a diagnostic composition
containing a polyvalent protein complex as above and a
pharmaceutically acceptable carrier, where each antigen binding
site binds a distinct epitope of CD19, CD20 or CD22, and where the
complex is radiolabeled with a radionuclide selected from the group
consisting of .sup.18F, .sup.52Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu,
.sup.67Ga, .sup.68Ga, .sup.86Y, .sup.89Zr .sup.94mTc, .sup.94Tc,
.sup.99mTc, .sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.154-158Gd, .sup.177Lu, .sup.32P, .sup.188Re, and .sup.90Y or a
combination thereof. Detection may be as described above. The
application may be for intraoperative diagnosis to identify occult
neoplastic tumors.
[0031] Also provided is a method for detecting or diagnosing a
B-cell malignancy, or B-cell immune or autoimmune disorder in a
subject, containing administering to the subject a diagnostic
composition containing a polyvalent protein complex as above and a
pharmaceutically acceptable carrier, where each antigen binding
site binds a distinct epitope of CD19, CD20 or CD22, and where the
complex is labeled with one or more image enhancing agents for use
in magnetic resonance imaging (MRI). The image enhancing agent may
be as described above
[0032] Also provided is a method of diagnosing a non-neoplastic
disease or disorder, by administering to a subject suffering from
the disease or disorder a complex as above, where a detectable
label is attached to the complex, and where one or more of the
antigen binding sites is specific for a marker substance of the
disease or disorder. The disease or disorder may be caused by a
fungus, such as Microsporum, Trichophyton, Epidermophyton,
Sporothrix schenckii, Cryptococcus neoformans, Coccidioides
immitis, Histoplasma capsulatum, Blastomyces dermatitidis, and
Candida albican, or a virus, such as human immunodeficiency virus
(HIV), herpes virus, cytomegalovirus, rabies virus, influenza
virus, hepatitis B virus, Sendai virus, feline leukemia virus, Reo
virus, polio virus, human serum parvo-like virus, simian virus 40,
respiratory syncytial virus, mouse mammary tumor virus,
Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,
adenovirus, human T-cell leukemia-viruses, Epstein-Barr virus,
murine leukemia virus, mumps virus, vesicular stomatitis virus,
Sindbis virus, lymphocytic choriomeningitis virus, wart virus and
blue tongue virus. The disease or disorder may be caused by a
bacterium, such as Anthrax bacillus, Streptococcus agalactiae,
Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli,
Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus,
Hemophilis influenzae B, Treponema pallidum, Lyme disease
spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella
abortus, and Mycobacterium tuberculosis, or a Mycoplasma. The
disease or disorder may be caused by a parasite, such as malaria.
The disease or disorder may be an autoimmune disease, such as acute
idiopathic thrombocytopenic purpura, chronic idiopathic
thrombocytopenic purpura, dermatomyositis, Sydenham's chorea,
myasthenia gravis, systemic lupus erythematosus, lupus nephritis,
rheumatic fever, polyglandular syndromes, bullous pemphigoid,
diabetes mellitus, Henoch-Schonlein purpura,
post-streptococcalnephritis, erythema nodosum, Takayasu's
arteritis, Addison's disease, rheumatoid arthritis, multiple
sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme,
IgA nephropathy, polyarterifis nodosa, ankylosing spondylitis,
Goodpasture's syndrome, thromboangitisubiterans, Sjogren's
syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,
thyrotoxicosis, scleroderma, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, parnphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, giant cell
arteritis/polymyalgia, pernicious anemia, rapidly progressive
glomerulonephritis, psoriasis, and fibrosing alveolitis. The
disease or disorder may be myocardial infarction, ischemic heart
disease, or atherosclerotic plaques, or graft rejection, or
Alzheimer's disease, or caused by atopic tissue. The disease or
disorder may be inflammation caused by accretion of activated
granulocytes, monocytes, lymphoid cells or macrophages at the site
of inflammation, and where the inflammation is caused by an
infectious agent.
[0033] Also provided is a pretargeting method of treating or
diagnosing a non-neoplastic disease or disorder in a subject by (a)
administering to the subject the polyvalent protein complex of
claim 1, where two antigen binding sites are directed to a marker
substance, or marker substances specific for the disorder, and one
antigen binding sites is directed to a targetable construct
containing a bivalent hapten; (b) optionally administering to the
subject a clearing composition, and allowing the composition to
clear the polyvalent complex from circulation; and (c)
administering to the subject the targetable construct containing a
bivalent hapten, where the targetable construct further contains
one or more chelated or chemically bound therapeutic or diagnostic
agents. The disease or disorder may be as described above.
[0034] Also provided is a method of antibody dependent enzyme
prodrug therapy (ADEPT) by; a) administering to a patient with a
neoplastic disorder the polyvalent protein complex as above, where
the complex contains a covalently attached enzyme capable of
activating a prodrug, (b) optionally administering to the subject a
clearing composition, and allowing the composition to clear the
polyvalent complex from circulation, and (c) administering the
prodrug to the patient.
[0035] Also provided are assay and immunostaining methods using one
or more polyvalent protein complexes as described above.
[0036] Further provided is an isolated nucleic acid molecule
encoding a first or second polypeptide as described above, and a
nucleic acid expression cassette containing such an isolated
nucleic acid. Also provided is an episome containing: (a) a first
promoter operationally connected to a first nucleic acid encoding a
first polypeptide containing a polypeptide chain represented by the
formula a.sub.1-1.sub.1-a.sub.2-1.- sub.2-a.sub.3, where a.sub.1,
a.sub.2, and a.sub.3 are immunoglobulin variable domains and
1.sub.1 and 1.sub.2 are peptide linkers, (b) a second promoter
operationally connected to a second nucleic acid encoding a
polypeptide containing a second polypeptide chain represented by
the formula b.sub.1-1.sub.3-b.sub.2-14-b.sub.3, where b.sub.1,
b.sub.2, and b.sub.3 are immunoglobulin variable domains and
1.sub.3 and 1.sub.4 are peptide linkers, where the first and second
polypeptide chain together form a complex containing at least three
antigen binding sites, where each of the antigen binding sites
contains a variable domain from the first polypeptide chain and a
variable domain from the second polypeptide chain, where the first
nucleic acid and the second nucleic acid are coexpressed when the
episome is transformed into a host cell. The episome may be a
plasmid or a cosmid. Also provided is a host cell containing a
nucleic acid, a expression cassette and/or an episome as described
above. The host cell may be, for example, E. coli, yeast, a plant
cell and a mammalian cell.
[0037] Also provided are methods of preparing a polyvalent protein
complex, containing culturing a host cell as described above. The
host cell may be, for example, a murine myeloma cell line. The
episome may contain the plasmid is pdHL2.
[0038] Other aspects and advantages of the present invention are
described further in the following detailed description of
preferred embodiments of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 Panel A shows an expression cassette in
BS14HP-GAP+vector, which codes for a two species of mRNA
synthesized from the constitutive GAP promoters.
[0040] Panel B shows a drawings of the two mature heterologous
polypeptides,
h679V.sub.H-GGGGS-hMN-14V.sub.K-LEGGGS-hMN-14V.sub.H-6His (Left)
and hMN-14V.sub.K-GGGQFM-hMN-14V.sub.H-GGGGS-h679V.sub.K-6His
(Right), following cleavage of the .alpha. factor signal peptides
by Kex2 protease.
[0041] Panel C shows a drawing of a trivalent protein structure
formed by the heterodimerization of polypeptides 1 and 2 possessing
two binding sites for CEA and one for HSG.
[0042] Panel D shows the amino acid sequence and cDNA sequence of
EAEAEFM-h679VH-GGGGS-hMN-14VK-LEGGGS-hMN-14VH-6His.
[0043] Panel E shows the amino acid sequence and cDNA sequence of
EAEAEF-hMN-14VK-GGGQFM-hMN-14VH-GGGGS-h679VK-6His.
[0044] FIG. 2 depicts the construction of the modified Pichia
expression vector pGAPZ.alpha.+ used for the co-expression of two
heterologous polypeptides from the same host cell.
[0045] FIG. 3 shows one of many BIAcore sensorgrams used to
evaluate expression of BS14HP in the culture media of Pichia
pastoris clones. Following growth to stationary phase, culture
media was diluted ten-fold in BIAcore eluent buffer and injected
over an HSG-coupled sensorchip. A subsequent injection of WI2 IgG
(anti-id to hMN-14) confirmed the bispecificity of the samples.
[0046] FIG. 4 shows a Coomassie blue-stained SDS-PAGE gel of
BS14HP. Purified protein samples were subjected to reducing
SDS-PAGE on 4-20% polyacrylamide Tris-Glycine gels. 1, 4 and 10
.mu.g were loaded in indicated lanes. Arrows indicate the positions
of molecular weight standards.
[0047] FIG. 5 shows the size exclusion HPLC profile of purified
BS14HP.
[0048] FIG. 6(a) shows a graphical representation of the results of
a competitive ELISA experiment. HRP-conjugated hMN14 IgG (1 nM) was
mixed with either BS14HP, BS1.5H (a bispecific diabody, monovalent
for CEA and monovalent for HSG, derived from the same variable
domains as BS14HP) or hMN14 F(ab').sub.2 at concentrations ranging
from 1-250 nM, prior to incubation in CEA-coated (0.5 .mu.g/well)
wells. The % inhibition is plotted vs. nM concentration of sample.
The 50% inhibitory concentration (IC.sub.50) is given for each and
(b) shows the results of SE-HPLC analysis of BS14HP
immunoreactivity with CEA.
[0049] FIG. 7 shows a graphical representation of the tumor
residence and blood clearance of .sup.125I, labeled BS14HP in
GW39tumor bearing nude mice. The % injected dose/gram (% ID/g) is
plotted versus time (hours).
[0050] FIG. 8 shows the biodistribution (A) and tumor/non-tumor
ratios after 3 hours (B) of .sup.111In-IMP-241 in GW-39 tumor
bearing mice pretargeted with three bispecific constructs and (C)
and tumor/non-tumor ratios after 24 hours. Standard deviations are
shown as error bars (A and C) or as .+-. in parentheses (B).
[0051] FIG. 9 panel A shows the molecular structure of IMP 281,
panel B shows the molecular structure if IMP 284, panel C shows the
figure of IMP 288.
[0052] FIG. 10 depicts constructs of SV3 construct and ORF1 and
ORF2 polypeptide.
[0053] FIG. 11 is a schematic representation of hBS14-pDHL2
expression vector.
[0054] FIG. 12 depicts the results of MTX amplification of hBS14
SP2/0 clone 1H6.
[0055] FIG. 13 depicts the results of SE-HPLC analysis of purified
hBS14.
[0056] FIG. 14 depicts the results of SDS-PAGE analysis of purified
hBS14.
[0057] FIG. 15 depicts the results of IEF analysis of purified
hBS14.
[0058] FIG. 16 depicts the results of BIAcore analysis of
hBS14.
[0059] FIG. 17 depicts the results of BIAcore analysis of HSG
binding of hBS14 produced in either SP2/0 or YB2/0 cells.
[0060] FIG. 18 shows the structure of the peptide IMP 291.
[0061] FIG. 19 shows the structure of the peptide IMP 245.
[0062] FIG. 20 shows the tumor uptake of .sup.125I-hBS14 and
.sup.99mTc-IMP-245 in mice when the hBS14 was given 4 hrs (top
panel) or 24 hrs (bottom panel) to clear prior to administration of
peptide (Groups I and II respectively).
[0063] FIG. 21 the top panel shows the tumor uptake of
.sup.125I-hBS14 and .sup.99mTc-IMP-245 in mice given 48 hrs to
clear the hBS114 prior the administration of the peptide (Group
III). The bottom panel shows peptide uptake in imaged mice at 24 hr
post-injection.
[0064] FIG. 22 is a table showing percent ID/g and tumor/non-tumor
ratios of .sup.99mTc-IMP-245 peptide at 1 h post injection.
[0065] FIG. 23 shows imaging data in mice. The first pair of images
shows the location of the tumors in the mice. The second pair of
images shows the image at 1 hr post-peptide administration. The
third pair of images show imaging data at 3 hrs post-peptide
administration. The final pair of images shows the image at 24 hrs
post-peptide administration.
DETAILED DESCRIPTION OF THE INVENTION
[0066] Definitions:
[0067] As used herein, the term "engineered antibody" encompasses
all biochemically or recombinately produced functional derivatives
of antibodies. A protein is a functional derivative of an antibody
if it has at least one antigen binding site (ABS) or a
complementarity-determining region (CDR) that when combined with
other CDR regions (on the same polypeptide chain or on a different
polypeptide chain) can form an ABS. The definition of engineered
antibody would include, at least, recombinant antibodies, tagged
antibodies, labeled antibodies, Fv fragments, Fab fragments,
recombinant (as opposed to natural) multimeric antibodies, single
chain antibodies, diabodies, triabodies, tetravalent multimers
(dimer of diabodies), pentavalent multimers (dimer of diabody and
triabody), hexavalent multimers (dimer of triabodies) and other
higher multimeric forms of antibodies.
[0068] As used herein, the term "single-chain antibody (scFv),"
refers to engineered antibody constructs prepared by isolating the
binding domains (both heavy and light chain) of a binding antibody,
and supplying a linking moiety which permits preservation of the
binding function. This forms, in essence, a radically abbreviated
antibody, having only the variable domain necessary for binding the
antigen. Determination and construction of single chain antibodies
are described in many prior publications including U.S. Pat. No.
4,946,778; Bird et al., Science 242:423 (1988) and Huston et al.,
Proc. Nat'l Acad. Sci. USA85:5879 (1988).
[0069] The term "humanized" means that at least a portion of the
framework regions of an immunoglobulin or engineered antibody
construct (including the PPC of this invention that comprise an
immunoglobulin or engineered antibody) is derived from human
immunoglobulin sequences. It should be clear that any method to
humanize antibodies or antibody constructs, as for example by
variable domain resurfacing as described by Roguska et al., (1994)
Proc. Natl. Acad. Sci. USA 91: 969-973 would be applicable to the
PPC of this invention. Alternatively, CDR grafting (also called CDR
shuffling) or reshaping as reviewed by Hurle and Gross ((1994)
Curr. Opin. Biotech. 5:428-433), can be used. Manipulation of the
complementarity-determining regions (CDR) is a way of achieving
humanized antibodies. The use of antibody components derived from
humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions. See,
for example, U.S. Pat. Nos. 5,874,540 and 6,254,868. 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). Techniques for producing
humanized MAbs are described, for example, by Jones et al., Nature
321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),
Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc.
Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech.
12: 437 (1992), Singer et al., J. Immun. 150: 2844 (1993), Winter
& Milstein, Nature 349:293 (1991).
[0070] The terms "recombinant nucleic acid" or "recombinantly
produced nucleic acid" refer to nucleic acids such as DNA or RNA
which has been isolated from its native or endogenous source and
modified either chemically or enzymatically by adding, deleting or
altering naturally-occurring flanking or internal nucleotides.
Flanking nucleotides are those nucleotides which are either
upstream or downstream from the described sequence or sub-sequence
of nucleotides, while internal nucleotides are those nucleotides
which occur within the described sequence or subsequence.
[0071] The term "recombinant means" refers to techniques where
proteins are isolated, the cDNA sequence coding the protein
identified and inserted into an expression vector. The vector is
then introduced into a cell and the cell expresses the protein.
Recombinant means also encompasses the ligation of coding or
promoter DNA from different sources into one vector for expression
of a PPC, constitutive expression of a protein, or inducible
expression of a protein.
[0072] The term "promoter" refers to a DNA sequence which directs
the transcription of a structural gene to produce mRNA. Typically,
a promoter is located in the 5' region of a gene, proximal to the
start codon 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.
[0073] The term "enhancer" refers to a promoter element. An
enhancer can increase the efficiency with which a particular gene
is transcribed into mRNA irrespective of the distance or
orientation of the enhancer relative to the start site of
transcription.
[0074] "Complementary DNA (cDNA)" refers to 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 complement.
[0075] "Expression" refers to the process by which a polypeptide is
produced from a structural gene. The process involves transcription
of the gene into mRNA and the translation of such mRNA into
polypeptide(s).
[0076] "Cloning vector" refers to a DNA molecule, such as a
plasmid, cosmid, phagemid, or bacteriophage, which has the
capability of replicating autonomously in a host cell and which is
used to transform cells for gene manipulation. Cloning vectors
typically contain one or a small number of restriction endonuclease
recognition sites at which foreign DNA sequences may be inserted in
a determinable fashion without loss of an essential biological
function of the vector, as well as a marker gene which 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.
[0077] "Expression vector" refers to a DNA molecule comprising a
cloned structural gene encoding a foreign protein which provides
the expression of the foreign protein in a recombinant host.
Typically, the expression of the cloned gene is placed under the
control of (i.e., operably linked to) certain regulatory sequences
such as promoter and enhancer sequences. Promoter sequences may be
either constitutive or inducible.
[0078] "Recombinant Host" or "Host cell" refers to a prokaryotic or
eukaryotic cell which contains either a cloning vector or
expression vector. This term is also meant to include 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 host cell is not limited to a
unicellular organism such as E. coli and yeast. Cells from
multicellular organisms such as mammals, insects, and plants are
also contemplated as host cells in the context of this invention.
For examples of suitable hosts, see Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989). A mammalian host cell
may be of any mammalian origin and include, at least cells of
human, bovine, canine, murine, rattus, equine, porcine, feline and
non-human primate origin.
[0079] A "tumor-associated antigen" is a protein normally not
expressed, or expressed at very low levels, by a normal cell.
However, in a neoplastic or preneoplastic cell (a cell predisposed
to becoming a cancer cell), the tumor-associated antigen is
expressed at a level that is higher than that of a normal cell. The
preferred tumor-associated antigens are the ones that are expressed
at very high levels in neoplastic and preneoplastic cells but at
very low levels or not expressed in normal cells. "Antigens" and
"tumor-associated antigens" are well known and include at least
.alpha.-fetoprotein, A3, A33 (GI cancers, particularly colon
cancer), CA125, carcinoembryonic antigen (CEA), CD19, CD20, CD21,
CD22, CD23, CD30, CD33, CD45, CD52 (associated with chronic
lymphocytic leukemia and other lymphomas), CD74, CD66, CD80,
colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2, folate
receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin, Ia,
IL-2, IL-6 (prostate cancer), insulin-like growth factor, KS-1,
Le(y), MAGE, MUC1, MUC2, MUC3, MUC4, necrosis antigens, PAM-4,
placental growth factor, prostatic acid phosphatase (PAP), prostate
specific antigen (PSA), PSMA, S100, T101, TAC, TAG-72, tenascin,
and VEGF. Furthermore, the ABS of the invention includes, at least,
an ABS that binds to an epitope of the above listed antigens.
Tumor-associated antigens may be either produced by the tumor cells
themselves or by adjacent structures, such as the tumor's vascular
endothelium. B-cell, T-cell and other such "lineage" antigens which
are present in both normal and malignant cell types may still be
useful targets because of a differential expression by or
sensitivity of the malignant cells to antibodies against these
lineage antigens (e.g., CD19, CD20, CD21, CD22 in normal and
malignant B cells). Many other illustrations of tumor-associated
antigens are known to those of skill in the art. See, e.g., Urban
et al., Ann. Rev. Immunol. 10:617 (1992). The list above is
illustrative only and cites the cancers most closely associated to
the tumor-associated antigen. In most cases, each tumor-associated
antigen may have up to 2, 3, 4, 5, 6 or more epitopes.
[0080] Known tumors that are associated with tumor-associated
antigens include, at least, carcinomas, melanomas, sarcomas,
gliomas, myelomas, leukemias and lymphomas.
[0081] As used herein, an "infectious agent" and "pathogen" denotes
both microbes and parasites. A "microbe" includes viruses,
bacteria, rickettsia, mycoplasma, protozoa, fungi and like
microorganisms. A "parasite" denotes infectious, generally
microscopic or very small multicellular invertebrates, or ova or
juvenile forms thereof, which are susceptible to antibody-induced
clearance or lytic or phagocytic destruction, such as malarial
parasites, spirochetes, and the like. Examples of infectious agents
include, for example, a fungus, virus, parasite, bacterium,
protozoan, or mycoplasm. The fungus may be from the species of
Microsporum, Trichophyton, Epidermophyton, Ssporothrix schenckii,
Cyrptococcus neoformans, Coccidioides immitis, Histoplasma
capsulatum, Blastomyces dermatitidis, or Candida albicans. The
virus may be from the species of human immunodeficiency virus
(HIV), herpes virus, cytomegalovirus, rabies virus, influenza
virus, hepatitis B virus, Sendai virus, feline leukemia virus, Reo
virus, polio virus, human serum parvo-like virus, simian virus 40,
respiratory syncytial virus, mouse mammary tumor virus,
Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,
adenovirus, human T-cell leukemia viruses, Epstein-Barr virus,
murine leukemia virus, mumps virus, vesicular stomatitis virus,
Sindbis virus, lymphocytic choriomeningitis virus, wart virus and
blue tongue virus. The bacterium may be, for example, Anthrax
bacillus, Streptococcus agalactiae, Legionella pneumophilia,
Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,
Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B,
Treponema pallidum, Lyme disease spirochetes, Pseudomonas
aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium
tuberculosis and Tetanus toxin. The parasite may be a helminth or a
malarial parasite. The protozoan may be Plasmodium falciparum,
Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli,
Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei,
Schistosoma mansoni, Schistosoma japanicum, Babesia bovis, Elmeria
tenella, Onchocerca volvulus, Leishmania tropica, Trichinella
spiralis, Onchocerca volvulus, Theileria parva, Taenia hydatigena,
Taenia ovis, Taenia saginata, Echinococcus granulosus or
Mesocestoides corti. The mycoplasma may be Mycoplasma arthritidis,
Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini,
Acholeplasma laidlawii, Mycoplasma salivarum, and Mycoplasma
pneumoniae. Other examples of infectious agents and pathogens that
may be treated by the product (PPC) and methods of this invention
are contained in the second and subsequent editions of Davis et al,
"Microbiology" (Harper & Row, New York, 1973 and later), and
are well known to the ordinary skilled art worker.
[0082] The term "treating" in its various grammatical forms in
relation to the present invention refers to preventing, curing,
reversing, attenuating, alleviating, minimizing, suppressing or
halting the deleterious effects of a disease state, disease
progression, disease causative agent (e.g., bacteria or viruses) or
other abnormal condition. Because some of the inventive methods
involve the physical removal of the etiological agent, the artisan
will recognize that they are equally effective in situations where
the inventive compound is administered prior to, or simultaneous
with, exposure to the etiological agent (prophylactic treatment)
and situations where the inventive compounds are administered after
(even well after) exposure to the etiological agent.
[0083] Unless otherwise noted, use of the term "antibody" or
"immunoglobulin" herein will be understood to include antibody
fragments and functional derivatives (i.e., engineered antibody)
thereof. Antibodies can be whole immunoglobulin of any class, e.g.,
IgG, IgM, IgA, IgD, IgE, or hybrid antibodies with dual or multiple
antigen or epitope specificities, or fragments, e.g., F(ab').sub.2,
F(ab).sub.2, Fab', Fab.sub.1 and the like, including hybrid
fragments. Functional derivatives include engineered
antibodies.
[0084] The terms "recombinant protein," "recombinantly produced
protein" or "recombinantly produced immunotoxin" refer to a peptide
or protein produced using non-native cells that do not have an
endogenous copy of DNA able to express the protein. The cells
produce the protein because they have been genetically altered by
the introduction of the appropriate nucleic acid sequence. The
recombinant protein will not be found in association with proteins
and other subcellular components normally associated with the cells
producing the protein.
[0085] The term "selective cytotoxic reagent" refers to a compound
that when added to a population of different cells, e.g., within an
organism, kills one type of cell in the population based on some
physical characteristic of the cell, i.e., a surface ligand or
marker to which the cytotoxic reagent binds and then becomes
internalized.
[0086] The term "surface marker" refers to various constituents,
such as a protein, carbohydrate, or glycoprotein, that are present
on the surface of a cell. Different types of cells express
different cell surface markers and therefore cells can be
identified by the presence of a cell surface marker. For example, B
cells express CD19, CD20 (See, Ansell et al., J. Clin. Oncology,
20:3885-3890 (2002) and Witzig et al., J. Clin. Oncology
20:2453-2463) and CD22. Thus, the binding of an antibody that
recognizes CD19, CD20 or CD22 identifies that cell as a B cell,
either normal or malignant. As another example, the B-cell may be a
multiple myeloma, in which case the B cells may express the
tumor-associated antigen MUC1 or CD74. B cell surface markers may
be used for ablation of B cells and B cell tumor-associated
antigens may be used to ablate B cell tumors such as the multiple
myeloma described above.
[0087] The term "CD22" refers to a lineage-restricted B-cell
antigen belonging to the Ig superfamily, is expressed on the
surface of many types of malignant B cells, including but not
limited to, acute lymphocytic leukemia (B-ALL), chronic
B-lymphocytic cells (B-CLL), B lymphoma cells such as Burkitt's,
AIDS-associated and follicular lymphomas, and hairy cell leukemias,
as well as on normal mature B lymphocytes. See, U.S. Pat. Nos.
6,183,744 and 6,306,393. CD22 is not expressed in early stages of
B-cell development, nor is it found on the surface of stem cells or
terminal stage plasma cells. Vaickus et al., Crit. Rev.
Oncol/Hematol. 11:267-297 (1991). Additionally, no shed antigen is
detected in normal human serum or serum from patients with CLL. Li
et al., Cell. Immunol. 118:85-99 (1989).
[0088] According to the specific case, the "therapeutically
effective amount" of an agent should be determined as being the
amount sufficient to improve the symptoms of the patient in need of
treatment or at least to partially arrest the disease and its
complications. Amounts effective for such use will depend on the
severity of the disease and the general state of the patient's
health. Single or multiple administrations may be required
depending on the dosage and frequency as required and tolerated by
the patient.
[0089] As used herein, the term "a method to detect" refers to any
assay (including immunoassays and colorimetric assays) known in the
art for the measurement of a detectable label. These assays
include, at least, assays utilizing biotin and avidin (including
streptavidin), ELISA's and immunoprecipitation, immunohistochemical
techniques and agglutination assays. A detailed description of
these assays is given in WO 96/13590 to Maertens & Stuyver. The
term "biological sample" relates to any possible sample taken from
an animal (including humans), such as blood (which also encompasses
serum and plasma samples), sputum, cerebrospinal fluid, urine,
lymph or any possible histological section, and other body fluid.
Detection may also include methods of imaging a lesion, such as
with immunoscintigraphy, computed tomography (CT), ultrasonography,
X-rays, and the like.
[0090] The terms "binding specificity," "specifically binds to" or
"specifically immunoreactive with," when referring to a protein or
ABS of the invention, refers to a binding reaction which is
determinative of the presence of the protein or carbohydrate in the
presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified PPC bind to a particular protein or carbohydrate and do
not bind in a significant amount to other proteins or carbohydrates
present in the sample. Specific binding to a PPC under such
conditions may require a PPC selected for its specificity towards a
particular protein or carbohydrate. For example, PPCs specific for
the CD22 antigen may be selected to provide PPC that are
specifically immunoreactive with CD22 protein and not with other
proteins. A variety of immunoassay formats may be used to select
PPC specifically immunoreactive with a particular protein or
carbohydrate. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein or carbohydrate. See Harlow & Lane, Antibodies,
A Laboratory Manual, Cold Spring Harbor Publication, New York
(1988) for a description of immunoassay formats and conditions that
can be used to determine specific immunoreactivity.
[0091] The terms "isolated" or "substantially purified," when
applied to a nucleic acid or protein, denotes that the nucleic acid
or protein is essentially free of other cellular components with
which it is associated in the natural state. It is preferably in a
homogeneous state, although it can be in either a dry or aqueous
solution. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein which is the predominant species present in a preparation
is substantially purified.
[0092] The terms "nucleic acid encoding" or "nucleic acid sequence
encoding" refer to a nucleic acid which directs the expression of a
specific protein or peptide. The nucleic acid sequences include
both the DNA strand sequence that is transcribed into RNA and the
RNA sequence that is translated into protein. The nucleic acid
sequences include both full length nucleic acid sequences as well
as shorter sequences derived from the full length sequences. It is
understood that a particular nucleic acid sequence includes the
degenerate codons of the native sequence or sequences which may be
introduced to provide codon preference in a specific host cell. The
nucleic acid includes both the sense and antisense strands as
either individual single strands or in the duplex form.
[0093] "Pharmaceutical composition" refers to formulations of
various preparations. Parenteral formulations are known and are
preferred for use in the invention. The formulations containing
therapeutically effective amounts of the immunotoxins are either
sterile liquid solutions, liquid suspensions or lyophilized
versions and optionally contain stabilizers or excipients.
Lyophilized compositions are reconstituted with suitable diluents,
e.g., water for injection, saline, 0.3% glycine and the like, at a
level of about from 0.01 mg/kg of host body weight to 10 mg/kg or
more.
[0094] The term "crosslinker" is well known in the art and include
at least ABH(21509), AEDP(22101), AMAS(22295), ANB-NOS(21451),
APDP(27720), APG(20108), ASBA(21512), BASED(21564), BMB(22331),
BMDB(22332), BMH(22330), BMOE(22323), BMPA(22296), BMPH(22297),
BMPS(22298), BM[PEO].sub.3(22336), BM[PEO].sub.4(22337),
BSOCOES(21600), BS3(21580), DFDNB(21525), DMA(20663), DMP(21666),
DMS(20700), DPDPB(21702), DSG(20593), DSP(22585), DSS(21555),
DST(20589), DTBP(20665), DTME(22335), DTSSP(21578), EDC(22980),
EGS(21565), EMCA(22306), EMCH(22106), EMCS(22308), GMBS(22309),
HBVS(22334), KMUA(22211), KMUH(22111), LC-SMCC(22362),
LC-SPDP(21651), MBS(22311), M2C2H(22303), MPBH(22305), MSA(22605),
NHS-ASA(27714), PDPH(22301), PMPI(28100), SADP(21533), SAED(33030),
SAND(21549), SANPAH(22600), SASD(27716), SATA(26102), SATP(26100),
SBAP(22339), SFAD(27719), SIA(22349), SIAB(22329), SMCC(22360),
SMPB(22416), SMPH(22363), SMPT(21558), SPDP(21857),
Sulfo-BSOCOES(21556), Sulfo-DST(20591), Sulfo-EGS(21566),
Sulfo-EMCS(22307), Sulfo-GMBS(22324), Sulfo-HSAB(21563),
Sulfo-KMUS(21111), Sulfo-LC-SPDP(21650), Sulfo-MBS(22312),
Sulfo-NHS-LC-ASA(27735), Sulfo-SADP(21553), Sulfo-SANPAH(22589),
Sulfo-SIAB(22327), Sulfo-SMCC(22322), Sulfo-SMPB(22317),
Sulfo-LC-SMPT(21568), Sulfo-SBED(33033), SVSB(22358), TFCS(22299),
THPP(22607), TMEA(33043), and TSAT(33063) (Pierce Chemical,
Rockford, Ill. catalog number in parenthesis). See, also U.S. Pat.
No. 4,680,338 and provisional patent application 60/436,359 filed
Dec. 24, 2002, for additional linker descriptions.
[0095] The term "chemotherapeutic agent" may be any
chemotherapeutic agent known in the art and includes, at least,
taxanes, nitrogen mustards, ethylenimine derivatives, alkyl
sulfonates, nitrosoureas, triazenes; folic acid analogs, pyrimidine
analogs, purine analogs, vinca alkaloids, antibiotics, enzymes,
platinum coordination complexes, substituted urea, methyl hydrazine
derivatives, adrenocortical suppressants, or antagonists.
Specifically, the chemotherapeutic agent may be from the group of
steroids, progestins, estrogens, antiestrogens, and androgens. More
specifically, the chemotherapeutic agent may be azaribine,
bleomycin, bryostatin-1, busulfan, carmustine, celebrex,
chlorambucil, cisplatin, CPT-11, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, dexamethasone,
diethylstilbestrol, doxorubicin, ethinyl estradiol, etoposide,
fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone
caproate, hydroxyurea, L-asparaginase, leucovorin, lomustine,
mechlorethamine, medroprogesterone acetate, megestrol acetate,
melphalan, mercaptopurine, methotrexate, methotrexate, mithramycin,
mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine,
semustine streptozocin, tamoxifen, taxanes, taxol, testosterone
propionate, thalidomide, thioguanine, thiotepa, uracil mustard,
vinblastine, and vincristine.
[0096] The term "cytotoxic agents" includes all known cytotoxic and
cytostatic agents. Examples of these agents are listed in Goodman
et al., "THE PHARMACOLOGICAL BASIS OF THERAPEUTICS," Sixth Edition,
A. G. Gilman et al, eds./Macmillan Publishing Co. New York, 1980,
as well as a more current edition (See also, U.S. Pat. Nos.
6,083,477 and 6,395,276) These agents include, at least the
following: antiapoptotic agents, antimetabolites, alkaloids,
antimitotic agents, enzyme inhibitors, COX-inhibitors,
chemotherapeutic agents; antibiotics, such as dactinomycin,
daunorubicin, doxorubicin, bleomycin, mithramycin and mitomycin;
enzymes, such as L-asparaginase; platinum coordination complexes,
such as cisplatin; substituted urea, such as hydroxyurea; methyl
hydrazine derivatives, such as procarbazine; adrenocortical
suppressants, such as mitotane; hormones and antagonists, such as
adrenocortisteroids (prednisone), progestins (hydroxyprogesterone
caproate, medroprogesterone acetate and megestrol acetate),
estrogens (diethylstilbestrol and ethinyl estradiol), antiestrogens
(tamoxifen), and androgens (testosterone propionate and
fluoxymesterone). Other examples of cytotoxic agents include ricin,
abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A,
pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas
exotoxin, Pseudomonas endotoxin and radionuclides. See, for
example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA--A
Cancer Journal for Clinicians 44:43 (1994). Other suitable toxins
are known to those of skill in the art.
[0097] Therapeutic agents are as defined in the specification but
include at least, an immunoe modulator, an enzyme, a hormone.
[0098] Radionuclide include any radioactive isotope useful for
medical diagnostic, therapeutic and imaging methods (i.e.,
detectable labels). Examples of radionuclides include .sup.225Ac,
.sup.111Ag, .sup.72As, .sup.77As, .sup.211At, .sup.198Au,
.sup.199Au, .sup.212Bi, .sup.213Bi, .sup.75Br, .sup.76Br, .sup.11C,
.sup.55Co, .sup.62Cu, .sup.67Cu, .sup.166Dy, .sup.169Er, .sup.18F,
.sup.52Fe, .sup.59Fe, .sup.67Ga, .sup.68Ga, .sup.154-158Gd,
.sup.166Ho, .sup.120I, .sup.121I, .sup.123I, .sup.124I, .sup.125I,
.sup.131I, .sup.110In, .sup.111In, .sup.194Ir, .sup.177Lu,
.sup.51Mn, .sup.99Mn, .sup.99Mo, .sup.13N, .sup.15O, .sup.32P,
.sup.33P, .sup.211Pb, .sup.212Pb, .sup.109Pd, .sup.149Pm,
.sup.142Pr, .sup.143Pr, .sup.223Ra, .sup.82mRb, .sup.186Re,
.sup.188Re, .sup.189Re, .sup.105Rh, .sup.47Sc, .sup.75Se,
.sup.153Sm, .sup.83Sr, .sup.89Sr, .sup.161Tb, .sup.94mTc,
.sup.99mTc, .sup.86Y, .sup.90Y and .sup.89Zr. Of these
radionuclides .sup.225Ac, .sup.111Ag, .sup.77As, .sup.211At,
.sup.198Au, .sup.199Au, .sup.212Bi, .sup.213Bi, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.166Dy, .sup.169Er, .sup.59Fe, .sup.67Ga,
.sup.166Ho, .sup.125I, .sup.131I, .sup.111In, .sup.194Ir,
.sup.177Lu, .sup.99Mo, .sup.32P, .sup.33P, .sup.211Pb, .sup.212Pb,
.sup.109Pd, .sup.149Pm, .sup.142Pr, .sup.143Pr, .sup.223Ra,
.sup.186Re, .sup.188Re, .sup.189Re, .sup.105Rh, .sup.47Sc,
.sup.75Se, .sup.153Sm, .sup.89Sr, .sup.161Tb and .sup.90Y are
particularly useful as therapeutic radionuclides and therapeutic
cations. Further, .sup.72As, .sup.75Br, .sup.76Br, .sup.11C,
.sup.55Co, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.18F, .sup.52Fe,
.sup.67Ga, .sup.68Ga, .sup.154-158Gd, .sup.120I, .sup.123I,
.sup.124I, .sup.125I, .sup.131I, .sup.110In, .sup.111In,
.sup.177Lu, .sup.51Mn, .sup.52Mn, .sup.13N, .sup.15O, .sup.32P,
.sup.223Ra, .sup.82Rb, .sup.186Re, .sup.188Re, .sup.83Sr,
.sup.94Tc, .sup.99Tc, .sup.86Y, .sup.90Y and .sup.89Zr are
particularly useful as diagnostic radionuclides and diagnostic
cations.
[0099] "Antimicrobial agents" are any agents that has a cytotoxic
or cytostatic effect on microbes. Antimicrobial agents may be
conventionally classified into four main groups, based upon their
affecting (1) bacterial cell-wall synthesis, (2) the cytoplasmic
membrane, (3) protein synthesis, and (4) nucleic acid synthesis,
and often each of these groups can be subdivided into several
classes. Reviews of antimicrobial chemotherapy can be found in the
chapter by M. P. E. Slack (In: Oxford Textbook of Medicine, Second
Ed., Vol. 1, edited by D. J. Weatherall, J. G. G. Lidingham, and D.
A. Warrell, pp. 5.35-5.53; Oxford University Press,
Oxford/Melbourne/New York, 1987) and in Section XII, Chemotherapy
of Microbial Diseases (In: Goodman and Gilman's THE PHARMACOLOGICAL
BASIS OF THERAPEUTICS, 6th Ed., Goodman et al., Eds., pp.
1080-1248; Macmillan Publishing Co., New York, 1980--and also the
2001 edition). As indicated in these texts, some antimicrobial
agents are selective in their toxicity, since they kill or inhibit
the microorganism at concentrations that are tolerated by the host
(i.e., the drug acts on microbial structures or biosynthetic
pathways that differ from those of the host's cells). Other agents
are only capable of temporarily inhibiting the growth of the
microbe, which may resume growth when the inhibitor is removed.
Often, the ability to kill or inhibit a microbe or parasite is a
function of the agent's concentration in the body and its
fluids.
[0100] Cytokines are known to those of skill in the art and
includes, at least, "immune modulators" such as IL-1, IL-2, IL-3,
IL-6, IL-1, IL-12, IL-18, IL-21, interferon-.alpha.,
interferon-.beta., and interferon-.gamma..
[0101] It is understood that the definitions provided above are not
mutually exclusive. For example, one molecule may be a cytotoxic
agent, a radionuclide and a detectable label.
[0102] Structure of the Polyvalent Protein Complex (PPC)
[0103] The invention provides for a polyvalent protein complex
(PPC) comprising two polypeptide chains generally arranged
laterally to one another. Each polypeptide chain typically
comprises 3 or 4 "v-regions", which comprise amino acid sequences
capable of forming an antigen binding site when matched with a
corresponding v-region on the opposite polypeptide chain. Up to
about 6 "v-regions" can be used on each polypeptide chain, however.
The v-regions of each polypeptide chain are connected linearly to
one another and may be connected by interspersed linking regions.
When arranged in the form of the PPC, the v-regions on each
polypeptide chain form individual antigen binding sites. Thus, for
example, a PPC with 4 antigen binding sites and three linking
regions can be depicted as follows:
[0104] [amino
terminus]-a.sub.1-1-a.sub.2-1.sub.2-a.sub.3-1.sub.3-a.sub.4--
[carboxyl terminus]
[0105] [carboxyl
terminus]-b.sub.1-1.sub.4-b.sub.2-1.sub.5-b.sub.3-1.sub.6-
-b.sub.4-[amino terminus]
[0106] As shown here, the first polypeptide comprises 4 v-regions,
a.sub.1, a.sub.2, a.sub.3 and a.sub.4, connected by three linker
regions, 1.sub.1, 1.sub.2 and 1.sub.3. The second polypeptide of
the PPC comprises 4 corresponding v-regions b.sub.1, b.sub.2,
b.sub.3 and b.sub.4 and three interspersed linker regions, 1.sub.4,
1.sub.5 and 1.sub.6. The individual polypeptide chains of the PPC
are bound to one another by the complementarity binding of the
corresponding v-regions on each chain. Thus, as depicted above,
a.sub.1 binds to b.sub.1, a.sub.2 binds to b.sub.2, a.sub.3 binds
to b.sub.3, etc. to form the PPC.
[0107] The PPC of the invention can comprise v-regions of various
amino acid sequences so long as the arrangement of corresponding
v-regions on the two polypeptide chains (i.e., a.sub.n to b.sub.n)
provides for an antigen binding site. The binding of the
corresponding v-regions forms the individual antigen binding sites
of the PPC. A preferred method for forming each antigen binding
site on the PPC is to arrange corresponding V.sub.H and V.sub.L
regions of known antigen binding regions from antibodies or
antibody fragments. However the practice of the invention is not
limited to incorporation of such known antigen binding regions. If
corresponding V.sub.H and V.sub.L regions are used, there are no
limitations on which of the two v-regions (i.e., a.sub.n or
b.sub.n) encode V.sub.H or V.sub.L. For example, where n=3, any
combination of V.sub.H and V.sub.L listed below are possible:
1 Combination a.sub.1 a.sub.2 a.sub.3 b.sub.1 b.sub.2 b.sub.3 1
V.sub.H V.sub.H V.sub.H V.sub.L V.sub.L V.sub.L 2 V.sub.H V.sub.H
V.sub.L V.sub.L V.sub.L V.sub.H 3 V.sub.H V.sub.L V.sub.H V.sub.L
V.sub.H V.sub.L 4 V.sub.L V.sub.H V.sub.H V.sub.H V.sub.L V.sub.L 5
V.sub.H V.sub.L V.sub.L V.sub.L V.sub.H V.sub.H 6 V.sub.L V.sub.H
V.sub.L V.sub.H V.sub.L V.sub.H 7 V.sub.L V.sub.L V.sub.H V.sub.H
V.sub.H V.sub.L 8 V.sub.L V.sub.L V.sub.L V.sub.H V.sub.H
V.sub.H
[0108] As a further example, in the case where there are four
V-regions, any of the following are possible.
2 Combination a.sub.1 a.sub.2 a.sub.3 a.sub.4 b.sub.1 b.sub.2
b.sub.3 b.sub.4 1 V.sub.H V.sub.H V.sub.H V.sub.H V.sub.L V.sub.L
V.sub.L V.sub.L 2 V.sub.H V.sub.H V.sub.L V.sub.H V.sub.L V.sub.L
V.sub.H V.sub.L 3 V.sub.H V.sub.L V.sub.H V.sub.H V.sub.L V.sub.H
V.sub.L V.sub.L 4 V.sub.L V.sub.H V.sub.H V.sub.H V.sub.H V.sub.L
V.sub.L V.sub.L 5 V.sub.H V.sub.L V.sub.L V.sub.H V.sub.L V.sub.H
V.sub.H V.sub.L 6 V.sub.L V.sub.H V.sub.L V.sub.H V.sub.H V.sub.L
V.sub.H V.sub.L 7 V.sub.L V.sub.L V.sub.H V.sub.H V.sub.H V.sub.H
V.sub.L V.sub.L 8 V.sub.L V.sub.L V.sub.L V.sub.H V.sub.H V.sub.H
V.sub.H V.sub.L 9 V.sub.H V.sub.H V.sub.H V.sub.L V.sub.L V.sub.L
V.sub.L V.sub.H 10 V.sub.H V.sub.H V.sub.L V.sub.L V.sub.L V.sub.L
V.sub.H V.sub.H 11 V.sub.H V.sub.L V.sub.H V.sub.L V.sub.L V.sub.H
V.sub.L V.sub.H 12 V.sub.L V.sub.H V.sub.H V.sub.L V.sub.H V.sub.L
V.sub.L V.sub.H 13 V.sub.H V.sub.L V.sub.L V.sub.L V.sub.L V.sub.H
V.sub.H V.sub.H 14 V.sub.L V.sub.H V.sub.L V.sub.L V.sub.H V.sub.L
V.sub.H V.sub.H 15 V.sub.L V.sub.L V.sub.H V.sub.L V.sub.H V.sub.H
V.sub.L V.sub.H 16 V.sub.L V.sub.L V.sub.L V.sub.L V.sub.H V.sub.H
V.sub.H V.sub.H
[0109] In one embodiment, one polypeptide of the PPC may be SEQ ID
NO:1 (FIG. 1D). In another embodiment, one polypeptide of the PPC
may be SEQ ID NO:2 (FIG. 1E). In a preferred embodiment, one
polypeptide of the PPC is SEQ ID NO:1 while the other polypeptide
is SEQ ID NO:2.
[0110] Because each of the v-regions of the polypeptides of a PPC
are independent, each of the antigen binding sites can
independently have the same or different affinity or specificity.
In separately preferred embodiments, the antigen binding sites of a
PPC bind different epitopes or the same epitope. In the practice of
this invention, in either such embodiment, it is likely and
acceptable that binding affinity for each individual antigen
binding site will differ.
[0111] As noted above, a preferred embodiment of the PPC of this
invention comprises known V.sub.H and V.sub.L sequences for the
v-regions. For example, if it is desired for a PPC to have an ABS
with the same specificity as a target antibody. The gene for the
target antibody may be cloned or the target antibody may be
subjected to protein sequencing. Then the V.sub.H and V.sub.L
sequence of the target antibody may be determined. A nucleic acid
construct may be made to coexpress both polypeptides of the PPC in
a host where at least one of the PPC's antigen binding sites would
comprise the corresponding V.sub.H and V.sub.L regions as the
target antibody. These antigen binding sites would be expected to
have similar, if not identical, antigen binding specificity and
affinity with the target antibody. In the practice of this
embodiment of the invention, the target antibody may be human,
nonhuman or an engineered antibody. Furthermore, the antibody may
be any antibody whose sequence is in the public domain.
[0112] Methods of producing a target antibody of any specificity
are known in the art. For example, a monoclonal antibody may be
made from an antigen. Recombinant antibody libraries expression
libraries, which express a repertoire of antibodies on different
host cells may be screened. Furthermore, antibodies may be purified
and their protein sequences determined using antigen affinity
columns.
[0113] In another embodiment of the invention, the V.sub.H and
V.sub.L regions of the PPC may be derived from a "humanized"
monoclonal antibody or from a human antibody. Alternatively, the
V.sub.H and/or V.sub.L regions may comprise a sequence derived from
human antibody fragments isolated from a combinatorial
immunoglobulin library. See, for example, Barbas et al., METHODS: A
companion to Methods in Enzymology 2: 119 (1991), and Winter et
al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and expression
vectors that are useful for producing a human immunoglobulin phage
library can be obtained, for example, from STRATAGENE Cloning
Systems (La Jolla, Calif.).
[0114] The human antibody V.sub.H or V.sub.L sequence may be
derived from a human monoclonal antibody produced in a mouse. Such
antibodies are 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).
[0115] The linker regions may comprise any amino acid sequence that
are of sufficient length to allow for arrangement of corresponding
v-regions on the individual polypeptide chains of the PPC into
antigen binding sites (i.e. a.sub.1/b.sub.1, b.sub.2/a.sub.2,
etc.), for example, due to steric constraints. However, the linker
sequences should not be so long as to allow two adjacent v-regions
on the polypeptide chains to fold back on one another (i.e.,
a.sub.1/a.sub.2, b.sub.1/b.sub.2, etc.). Typically, linkers longer
than 10 amino acids are more likely to demonstrate folding back
problems. In a preferred embodiment, the linkers comprise a
polypeptide of between 3 to 8 amino acids in length. While any
amino acid may be used in the linker, the preferred amino acids are
those that are flexible and hydrophilic (e.g., glycine and serine).
Examples of such linkers include, for example, the linkers of the
invention as shown in FIGS. 1D and 1E. In some embodiments where
steric hindrance is not a constraint, the linker regions may be
omitted.
[0116] Tagged PPC
[0117] PPCs of the present invention may also be modified in a way
to form chimeric molecules (referred to herein as "tagged PPC")
comprising a fusion of a PPC with a "epitope tag" which provides an
epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino or carboxyl terminus
of the target protein. Provision of the epitope tag enables the
target protein to be readily detected, as well as readily purified
by affinity purification. Various tag epitopes are well known in
the art. Examples include poly-histidine (poly-his) or
poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 (see, Field et al. (1988) Mol.
Cell. Biol. 8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto (see, Evans et al., (1985) Molecular
and Cellular Biology, 5:3610); and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody (see, Paborsky et al.,
(1990) Protein Engineering, 3:547). Other tag polypeptides include
the Flag-peptide (see, Elnhauer et al., J. Biochem. Biophys.
Methods, 2001 Oct. 30, 49(1-3), 455-65; Song et al., Int. J. Oncol.
2003 Jan., 22(1).sub.93-8; Werkmeister et al., Biochim. Biophys
Acta 1993 May 7, 1157(1): 50-4; Hopp et al. (1988) BioTechnology
6:1204); the KT3 epitope peptide (see, Martine et al. (1992)
Science, 255:192); tubulin epitope peptide (see, Skinner (1991) J.
Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag
(see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA
87:6393.). It is understood that tagged PPC is a subset of all PPC
and any reference to PPC in this disclosure also comprise tagged
PPC.
[0118] In one embodiment of the invention, the three or four ABS of
a PPC may be specific for an epitope of a tumor-associated antigen.
Each ABS of a PPC may be specific for a different tumor-associated
antigen. For example, one tumor-associated antigen may be CEA while
another tumor associate antigen may be a non-CEA antigen. In
another embodiment of the invention, the PPC has at least one ABS
specific for an epitope of a hapten. The hapten may be, for
example, histamine-succinyl-glycine (HSG).
[0119] In another embodiment of the invention, the PPC is linked,
via a chemical bond, to a second molecule. These linkages may be
made using a crosslinker. Alternatively, the linkage may be a
binding pair such as antigen-antibody, hormone-receptor,
drug-receptor, cell surface antigen-lectin, biotin-avidin,
substrate/enzyme, peptide-receptor, and complementary nucleic acid
strands, hapten-anti-hapten systems and the like. The avidin
described includes reduced affinity avidin and reduced
immunogenicity avidin as described by U.S. Pat. No. 5,698,405.
[0120] In one embodiment, the PPC may be linked to peptides (which
includes proteins) to form a fusion PPC. The linkage may be any
linkage that could be used to join two peptides since the PPC is
itself comprised of peptides. For example, one method would be to
synthesize the fusion PPC in a peptide synthesizer. In this case,
the bond would be a peptide bond (also referred to as an amide
bond). Another method would be to synthesize or clone a DNA to
encode both polypeptides of the fusion PPC. The DNA is placed into
an expression vector and transformed into a host cell permanently
or transiently. Yet another method would be to use a chemical
crosslinker to join two peptides.
[0121] The PPC molecule of the invention may further comprise a
"detectable label" such as a "diagnostic agent." Detectable labels
and diagnostic agents may include radiolabels, fluorescent labels,
luminescent (chemiluminescent and bioluminescent) labels,
positron-emission tomography (PET) labels and SPECT labels. The
choice of labels are well known but specific examples are provided
below. Methods of detecting labels are generally known and are also
described in U.S. Pat. Nos. 4,595,654, 4,735,210, 4,792,521,
5,364,612, 5,439,665, 5,632,968, 5,697,902, 5,753,206, 6,071,490,
6,120,768, 6,126,916, and 6,187,284. The discussion of various
labels in this segment of the disclosure is applicable to all
references to labels in this invention.
[0122] Radiolabels may be further classified as therapeutic cations
and diagnostic cations. Diagnostic cations may emit particles
and/or positrons having 25-10,000 keV. Therapeutic cations may emit
particles and/or positrons having 20 to 10,000 keV. Any
conventional method of radiolabeling which is suitable for labeling
proteins for in vivo use will be generally suitable for labeling
the PPC of the invention. Such methods are known to the ordinary
skilled artisan and are disclosed inter alia in, e.g., Childs et
al., J. Nucl. Med., 26:293 (1985); and in U.S. Pat. Nos.
4,331,647,4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,624,846,
5,334,708, 5670,132, 5,514,363, 5,976,492, 6,358,489, and
6,440,386. A wide range of labeling techniques are disclosed in
Feteanu, "LABELED ANTIBODIES IN BIOLOGY AND MEDICINE", pages
214-309 (McGraw-Hill Int. Book Co., New York et al, 1978). The
introduction of various metal radioiosotopes may be accomplished
according to the procedures of Wagner et al., J. Nucl. Med., 20,428
(1979); Sundberg et al, J. Med. Chem., 17, 1304 (1974); and Saha et
al. J. Nucl. Med., 6, 542 (1976). Some of these methods describe
the use of labeled antibodies. The methods may be used in the
present invention by the substitution of PPC of the invention for
the antibodies described in these methods.
[0123] The detectable label may be a fluorescent label, a
chemiluminescent label, or a biolumincent label. Examples of
fluorescent labels include fluorescein isothiocyanate, rhodamine,
phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde or
fluorescamine. Examples of chemiluminescent labels include luminol,
isoluminol, an aromatic acridinium ester, an imidazole, an
acridinium salt and an oxalate ester. Examples of biolumincent
labels include luciferin, luciferase or aequorn.
[0124] The detectable labels may include one or more image
enhancing agents. Image enhancing agents are useful for magnetic
resonance imaging (MRI). Magnetic resonance imaging (MRI) agents
are described, for example, in Pykett, Scientific American, 246,
78(1982); Runge et al., Am. J. Radiol., 141, 1209(1983). Examples
of compounds useful for MRI image enhancement include complexes of
paramagnetic ions, e.g., Gd(III), Eu(III), Dy(III), Pr(III),
Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III),
and V(IV) ions, or radicals, e.g., nitroxides, and these may be
further attached to a substrate via a suitable linker. The MRI
enhancing agent must be present in sufficient amounts to enable
detection by an external camera, using magnetic field strengths
which are reasonably attainable and compatible with patient safety
and instrumental design. The requirements for such agents are well
known in the art for those agents which have their effect upon
water molecules in the medium, and are disclosed, inter alia, in,
e.g., Pykett, Scientific American, 246:78 (1982); and Runge et al.,
Am. J. Radiol, 141:1209 (1987).
[0125] The detectable label may comprise one or more radiopaque or
contrast agents for X-ray or computed tomography. Radiopaque or
contrast agents may be barium, diatrizoate, ethiodized oil, gallium
citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide,
iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid,
ioprocemic acid, iosefamic acid, ioseric acid, iosulamide
meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid,
iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine,
metrizamide, metrizoate, propyliodone, or thallous chloride. See
U.S. Pat. Nos. 5,120,525, 5,128,119, 5,328,679.
[0126] The detectable label may comprise one or more ultrasound
contrast agents such as, for example, a liposome (including gas
filled liposome) or dextran.
[0127] In addition to the described detectable label, the PPC may
comprise a therapeutic agent such as a radionuclide. Other
applicable methods for labeling the PPC of this invention are
disclosed in U.S. Pat. Nos. 5,061,641, 5,101,827.
[0128] Additional examples for using radiolabeled antibodies and
engineered antibodies for detection or therapy may be found in U.S.
Pat. Nos. 4,624,846, 5,482,698, 5,525,338, 5,609,846, 5,716,595,
5,728,369, 5,736,119, 5,746,996, 5,772,981, 5,776,093, 5,776,094,
5,776,095, 5,843,397, 5,851,527, 5,958,408, 5,965,131, 6,010,680,
6,077,499, 6,096,289, 6,331,175, 6,361,774, 6,387,350, 6,399,068,
and 6,458,933. The PPC of the invention may be substituted for any
of the antibodies mentioned in these patents.
[0129] The invention also provides for PPC where at least one ABS
of the PPC is specific for an epitope on a cancer associated
antigen and at least one ABS is specific for an epitope on a
hapten.
[0130] Another embodiment of the invention is directed to a PPC
linked to a conjugate (See U.S. Pat. No. 4,824,659 for a
description of an antibody conjugate). The linkage may by a
crosslinker. The conjugate may be a radionuclide or a cytotoxic
agent, a drug, a chemotherapeutic agent, and a radionuclide.
[0131] In another embodiment of the invention, the PPC may have at
least one ABS with specificity for an antigen on the surface of
effector cells and at least one ABS specific for an antigen on a
target cell or a virus. Examples of the first antigen may be an
antigen of the surface of T-cells, natural killer cells,
granulocytes, monocytes, or macrophages. In this case, the binding
of the PPC to these two antigens may result in the killing or the
mitotic arrest of the target cell. The following articles make
reference to the utility of a polyvalent protein with these
characteristics: Takemura et al., Cancer Immunol. Immunother. 2002
March; 51(1): 33-44; Kipriyanov et al., J. Immunol. 2002 Jul. 1;
169(1): 137-44; Stockmeyer et al., J. Immunol 2000 Nov. 15;
165(10): 5954-61.
[0132] In another embodiment of the invention, the PPC may have at
least one ABS with specificity for an antigen on the surface of a
cells and at least one ABS specific for an antigenic substance.
Examples of the first antigen may be an antigen of the surface of
B-cells, monocytes, dendritic cells and macrophages. In this case,
the binding of the PPC to the cell surface antigen and the
antigenic substance results in the induction of an immune response
to the antigenic substance.
[0133] In another embodiment, the invention is directed to a PPC
wherein one of the polypeptides comprise an additional V-region.
The V-region may be linked to the other V-regions by an additional
linker. This additional V-region may comprise an amino acid
sequence of a toxin, a hapten or a detectable moiety. Many examples
of toxins, haptens, and detectable moiety are proteins and peptides
with known amino acid sequences. Many of these peptides are cited
as examples throughout this specification. These amino acid
sequences may be used in the V-regions described in this
paragraph.
[0134] Bispecific and Multispecific PPC
[0135] The invention provides for methods of using the PPC. In
general, any method that require the use of an antibody or
engineered antibody (see, e.g., Cao Y and Lam, L. Adv Drug Deliv
Rev 2003 Feb. 10;55(2): 171-97) may be performed using a PPC with a
similar binding affinity and specificity. These methods includes
any of the methods described in this disclosure and in the
references, patents and patent applications cited therein.
Descriptions of specific embodiments are described below.
[0136] Bispecific and multispecific PPC are effective for the
recruitment of effector functions and treatment of tumor cells.
Multispecificity refers to the ability of a engineered antibody,
like the PPCs of the invention, to have multiple ABS where each ABS
binds a different epitope. As discussed above, the fusion PPC of
the invention may have at least 3 to 10 or more ABS and each ABS
may have specificity to a different epitope. Further each different
epitope may be on the same or different antigen. Bispecific
antibodies have found particular use in recruiting the powerful
effector functions of cytotoxic T cells or natural killer (NK)
cells. Thus bispecific antibodies have been used to bridge the T
cell coreceptor (CD3) (Staerz et al., Nature 314: 628-631, 1985) or
FcRIII (CD16) (De Palazzo et al., Cell Immunol. 142: 338-347, 1992)
and the cell surface antigen of a target cell to mediate the
killing of target cells by cytotoxic T cells or NK cells. In mice,
such anti-CD3 bisAbs can inhibit the growth of solid tumors (Titus
et al., J. Immunol. 138: 4018-4022, 1987, Garrido et al., Cancer
Res. 50: 4227-4232, 1990) or even eradicate lymphoma (Brissinck et
al., J. Immunol 147: 4019-4026, 1991); in humans, they have been
used against malignant glioma (Brissinck et al., J. Immunol. 147:
4019-4026, 1991). Bispecific antibodies have also been used for ex
vivo purging of leukaemia cells from bone marrow (T. Kaneko et al.,
Blood 81: 1333-1341, 1993). Bispecific antibodies synthesized in
vitro have also been used to deliver enzymes, antigens, toxins,
radionuclides and cytotoxic drugs to tumor cells (see Bonardi et
al., Cancer Res. 53: 187-199 1992). Any of the above method, and
any of the methods in the cited references in this disclosure, may
be performed using the bispecific PPCs of the invention as a
substitute for the multispecific antibody (or functional
derivatives) specified in these method.
[0137] The multispecific PPC of the invention may be used for
imaging of tumors. Bispecific anti-tumor marker, anti-hapten
antibodies have been used to image tumors (J. M. Le Doussal et al.
Int. J. Cancer Supplement 7: 58-62, 1992; P. Peltier et al. J.
Nucl. Med. 34: 1267-1273 1993; C. Somasundaram et al. Cancer
Immunol. Immunother. 36: 337-345, 1993; A. Bruynck et al. Br. J.
Cancer 67: 436-440, 1993). The method comprises two steps. In the
first step, a bispecific antibody is injected and localize to the
tumor by binding to a tumor-associated antigen. In the second step,
a radioactively labeled hapten is then injected which
preferentially localizes to the tumor, by binding to the bispecific
antibody, enabling imaging of the tumor. Multispecific PPC of the
invention with at least one ABS specific for tumor cells and one
ABS specific for the hapten could be used to in place of the
bispecific antibody to achieve the same results.
[0138] As another example, the PPC of the invention may be used to
deliver cytotoxic drugs to tumor cells, using one binding site to
deliver the drug and the other to bind to the tumor, or using
systems analogous to that described for the delivery of doxorubicin
to tumors by P. A. Trail et al. (Science 261: 212-215, 1993). These
authors used an antibody directed to the Lewis Y antigen,
covalently linked to doxorubicin, which was internalized into
lysosomes and endosomes. The linkage was cleavable in these
environments leading to delivery of the drug to these cells.
Bivalent PPCs may be particularly useful to increase the avidity of
the antibody for the tumor cell. The specificity may be increased
by using a bispecific antibody directed against two (or more)
different tumor-associated antigen on the same tumor or two (or
more) epitopes on the same tumor-associated antigen.
[0139] The multispecific PPCs of the invention may be used to
deliver therapeutic agents across the blood brain barrier. In this
method, a multispecific PPC with one ABS directed against either
FHA, an adhesin of the bacterium Bordetella pertussis or against
the natural ligand for the leucocyte adhesion molecule CR3 (E. I
Tuomanen et al. Proc. Natl. Acad. Sci. USA 90: 7824-7828, 1993) and
the other ABS may then be directed against a target to provide the
therapeutic function.
[0140] Multivalent PPCs may be particularly useful for imaging
purposes for instance when localizing tumors by binding to two
different epitopes of a tumor-associated antigen with a
radiolabeled PPC. The presence of two ABS for one tumor-associated
antigen would give an avidity component which may increase the
signal to noise ratio of the detection method.
[0141] The multispecific PPCs of the invention may be used in
retargetting of antibodies to a site or antigen for which they have
no specificity under normal circumstances. The PPC would possess
two ABSs; one ABS is specific for a target site, the other ABS is
capable of binding to selected parts of an antibody molecule. In
this manner, antibodies with no specificity for the antigen target
are brought into proximity with the antigen via the PPC. This
principle is advantageous for re-targeting antibodies in the
circulation to sites within the body such as tumors and to block
inappropriate immune responses exemplified by autoimmune disease
and would allow recruitment of effector functions.
[0142] In this way, multispecific PPCs could be used to recruit
effector functions through binding to whole antibody chains. One
ABS of the PPC would be directed against antigen for therapy and
the second arm against whole antibodies for the recruitment of
effector functions.
[0143] In a preferred embodiment of the invention, the ABS in a PPC
may be specific for an epitope of a tumor-associated antigen. The
tumor-associated antigen may be associated with, for example,
carcinomas, melanomas, sarcomas, gliomas, leukemias or lymphomas. A
tumor-associated antigen may have more than one epitope. For
example, a tumor-associated antigen may have at least 1, 2, 3, 4
epitopes. Other target antigens present in more than once cell type
and useful in this invention are CD74, HLA-DR, Where the construct
contain more than one ABS, the ABS may be specific for epitopes on
the same tumor antigen or different tumor antigens. Thus, a PPC
with 3 or 4 ABSs may bind from 1 to 3 or 4 of tumor antigens.
[0144] In a preferred embodiment, the ABS of a PPC has the same
binding specificity as monoclonal antibody (Mab) Mu-9 and MAb 679.
This can be achieved, for example, by using the sequence of the
monoclonal antibodies to construct the V.sub.H and V.sub.L regions
of the PPC.
[0145] In addition, the PPC of the invention may comprise one or
more ABS which bind an epitope on a hapten. The hapten may be a
histamine-succinyl-glycine (HSG) or indium-DTPA. Naturally, the ABS
of the PPC may bind multiple epitopes of one hapten or different
epitopes of different haptens. The three or more ABS of a PPC can
bind any combination of tumor-associated antigens or haptens
without limitation. As an example, one ABS may bind CEA while
another ABS may bind a non-CEA tumor-associated antigen. For
example, where the number of ABS is equal to N, the number of ABS
that binds tumor-associated antigen may range from zero to N. The
remainer of the ABS may all bind hapten.
[0146] As the above examples illustrate, the multispecific PPC of
the invention may serve as a substitute for multi specific
engineered antibodies in any method. These methods includes any of
the methods described in this disclosure and in the references,
patents and patent applications cited therein. More specific
examples of the use of PPC are discussed below.
[0147] Methods for Treating, Diagnosing, and Detecting
Disorders
[0148] The invention also provides for methods for treating,
diagnosing, and detecting a symptom of a neoplastic disorder by
administering any of the PPC of this disclosure with an ABS
directed to a cancer associated antigen. The PPC may be
administered with one or more therapeutic agents, diagnostic
agents, or detecting agents and one or more cytokines. The
therapeutic agent may be a chemotherapeutic agent or a combination
of chemotherapy agents. The administration of the therapeutic agent
or cytokine may be before, during or after the administration of
the PPC.
[0149] When more than one therapeutic agents are used, the
therapeutic agents may be the same or different, and may be, for
example, therapeutic radionuclides, drugs, hormones, hormone
antagonists, receptor antagonists, enzymes or proenzymes activated
by another agent, autocrines or cytokines. Toxins also can be used
in the methods of the present invention. Other therapeutic agents
useful in the present invention include anti-DNA, anti-RNA,
radiolabeled oligonucleotides, such as anti-sense oligonucleotides,
anti-protein and anti-chromatin cytotoxic or antimicrobial agents.
Other therapeutic agents are known to those skilled in the art, and
the use of such other therapeutic agents in accordance with the
present invention is specifically contemplated.
[0150] In a preferred embodiment, the therapeutic agents comprise
different isotopes, which are effective over different distances as
a result of their individual energy emissions, are used as first
and second therapeutic agents. This process achieves more effective
treatment of tumors, and is useful in patients presenting with
multiple tumors of differing sizes, as in normal clinical
circumstances.
[0151] Few of the available isotopes are useful for treating the
very smallest tumor deposits and single cells, and a drug or toxin
may be a more useful therapeutic agent in these situations.
Accordingly, in preferred embodiments of the present invention,
isotopes are used in combination with non-isotopic species such as
drugs, toxins, and neutron capture agents. Many drugs and toxins
are known which have cytotoxic effects on cells, and can be used in
connection with the present invention. They are to be found in
compendia of drugs and toxins, such as the Merck Index, Goodman and
Gilman, and the like, and in the references cited above.
[0152] Drugs that interfere with intracellular protein synthesis
can also be used in the methods of the present invention; such
drugs are known to those skilled in the art and include puromycin,
cycloheximide, and ribonuclease.
[0153] The therapeutic agents may be linked to the PPC. Methods of
making linked proteins in which one recombinant protein comprises a
cytotoxic agent, therapeutic agent or chemotherapeutic agent also
are known to those of skill in the art. These methods can be
applied to the PPC of the invention. For example,
antibody-Pseudomonasexotoxin A PPCs have been described by
Chaudhary et al., Nature 339: 394 (1989), Brinkmann et al., Proc.
Nat'l Acad. Sci. USA 88: 8616 (1991), Batra et al., Proc. Nat'l
Acad. Sci. USA 89: 5867 (1992), Friedman et al., J. Immunol. 150:
3054 (1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et
al., J. Biol. Chem. 271: 10560 (1996), Kuan et al., Biochemistry
35: 2872 (1996), and Schmidt et al., Int. J. Can. 65: 538 (1996).
Antibody-toxin PPCs containing a diphtheria toxin moiety have been
described by Kreitman et al., Leukemia 7: 553 (1993), Nicholls et
al., J. Biol. Chem. 268: 5302 (1993), Thompson et al., J. Biol.
Chem. 270: 28037 (1995), and Vallera et al., Blood 88: 2342 (1996).
Deonarain et al., Tumor Targeting 1: 177 (1995), have described an
antibody-toxin PPC having an RNase moiety, while Linardou et al.,
Cell Biophys. 24-25: 243 (1994), produced an antibody-toxin PPC
comprising a DNase I component. As a further example, Dohlsten et
al., Proc. Nat'l Acad. Sci. USA 91: 8945 (1994), reported an
antibody-toxin PPC comprising Staphylococcal enterotoxin-A. These
methods are also applicable for making the PPCs comprising a toxin
of the invention. Other suitable cytotoxic agents are listed in the
definitions section of this disclosure.
[0154] It is to be understood that any combination of the above
described therapeutic agents may be used. For example, a PPC may be
conjugated to two or more radioisotopes, or drugs. When a mixture
of therapeutic agents is used, a plurality of therapeutic agents
are delivered to the tumor sites, thereby enhancing the benefits of
the method. The use of mixtures of nuclides has the further
advantage that a greater percentage of the injected biotinylated
chelates delivers a toxic payload to the tumor target.
[0155] The present invention also contemplates dyes used, for
example, in photodynamic therapy, and used in conjunction with
appropriate non-ionizing radiation. The use of light and porphyrins
in methods of the present invention is also contemplated and their
use in cancer therapy has been reviewed (van den Bergh, Chemistry
in Britain, 22: 430-437 (1986)).
[0156] The invention also provides for methods of reducing a
symptom of a neoplastic disorder in a subject. The subject can be
any animal including horses, mice, rats, pigs, bovines, chickens
etc. In a preferred embodiment, the animal is a human. In the
method, a PPC is administered to a patient displaying a symptom of
the neoplastic disorder to reduce the symptom. The neoplastic
disorder may be a carcinomas, sarcomas, gliomas, lymhomas,
leukemias, melanomas or the like. In a preferred embodiment, the
neoplastic disorder is a B-cell malignancy such as indolent forms
of B-cell lymphomas, aggressive forms of B-cell lymphomas
(including non-Hodgkin's lymphoma), chronic lymphatic leukemias, or
acute lymphatic leukemias.
[0157] Another embodiment of the invention is directed to a method
for treating B cell malignancies. The method involves administering
to a subject having a B cell malignancy one or more dosages of a
therapeutic composition which contains a pharmaceutically
acceptable carrier and at least one PPC of the invention. The
B-cell malignancies may be any B-cell malignancy including, at
least, carcinomas, sarcomas, gliomas, lymphomas, leukemias, and
melanomas. In the method, the PPC may be parenterally administered
in a dosage of 20 to 1500 milligrams protein per dose. In a
preferred embodiment, the PPC may be administered in a dosage of 20
to 500 milligrams protein per dose. In a most preferred embodiment,
the PPC may be parenterally administered in a dosage of 20 to 100
milligrams protein per dose. Any of these dosages may be repeatedly
administered to achieve an even higher dosage. As discussed above,
the PPC of the invention, including the PPC in the methods of the
invention, may be radiolabeled. In administering a PPC that is
radiolabeled, the dosage of the radiolabel may be between 15 to 40
mCi. In a preferred embodiment, the dosage is between 10 and 30
mCi. In a more preferred embodiment, the dosage may be between 20
and 30 mCi. In another more preferred embodiment, the dosage may be
between 10 and 20 mCi.
[0158] In another embodiment, where a method of the calls for the
administration of PPC, the PPC may be administered before, after or
concurrently with a chemotherapeutic agent, cytokine, or colony
stimulating factor. Specific examples of chemotherapeutic agents
and cytokines are enumerated in another section of the
specification.
[0159] Any of the methods of the invention, including methods for
treating autoimmune disorders and neoplastic disorders may be used
to treat disorders such as cardiovascular diseases and
inflammation. These disorders include clots, emboli, myocardial
infarction, ischemic heart disease, and atherosclerotic plaques.
PPCs that are suitable for treating these disorders include those
PPCs with an ABS specifc for CD74 (e.g., hLL1), NCA (or -CD66) and
NCA90. This would include ABS with the same specificity as hMN3.
The diagnostic imaging methods of the invention are particularly
adaptable for using the above stated PPC. In particular, the
detection methods, diagnostic methods, and the cell ablation
methods may be applied to cardiovascular disorders. For example,
the detection may be used to detect damaged heart and vascular
tissue. The cell ablation methods may be used for targeting
diseased heart tissue. Inflammation can be detected or treated with
anti-granulocyte (e.g., anti-CD66, anti-CD33, anti-CD45),
anti-lymphocyte (anti-B- or anti-T-cell antibodies), and/or
anti-monocyte antibodies (e.g., anti-Ia or anti-CD74 antibody).
[0160] In another embodiment of the invention, the treatment
methods of the invention can be used in combination with other
compounds or techniques for preventing, mitigating or reversing the
side effects of cytotoxic agents. Examples of such combinations
include, e.g., administration of IL-1 together with a second
antibody for rapid clearance, as described. e.g., U.S. Pat. No.
4,624,846, from 3 to 72 hours after administration of a targeted
primary PPC antibody fragment conjugate (with a radioisotope, drug
or toxin as the cytotoxic component of the immunoconjugate) or of a
non-conjugated drug or toxin, to enhance clearance of the
conjugate, drug or toxin from the circulation and to mitigate or
reverse myeloid and other hematopoietic toxicity caused by the
therapeutic agent. This method is also applicable to the PPC of the
invention.
[0161] In another aspect, cancer therapy often involves a
combination of more than one tumoricidal agent, e.g., a drug and a
radioisotope, or a radioisotope and a Boron-10 agent for
neutron-activated therapy, or a drug and a biological response
modifier, or a PPC conjugate and a biological response modifier.
The cytokine can be integrated into such a therapeutic regimen to
maximize the efficacy of each component thereof.
[0162] Similarly, certain antileukemic and antilymphoma antibodies
conjugated with radioisotopes that are .beta. or .alpha. emitters
can induce myeloid and other hematopoietic side effects when these
agents are not solely directed to the tumor cells, particularly
when the tumor cells are in the circulation and in the
blood-forming organs. Concomitant and/or subsequent administration
of the hematopoietic cytokine (growth factor, such as colony
stimulating factors (e.g., G-CSF and GM-CSF) is preferred to reduce
or ameliorate the hematopoietic side effects, while augmenting the
anticancer effects.
[0163] In addition to preventing, mitigating or reversing the
myelosuppressive or other hematopoietic side effects of the
therapy, cytokines such as, e.g., IL-1, can have anticancer effects
(Nakamura et al., Gann 77:1734-1739, 1986; Nakata et al., Cancer
Res. 48:584-588, 1988), as well as IL-12, and therefore are capable
of enhancing the therapeutic effect of the targeted agents when
used in combination with these other therapeutic modalities. Thus,
another aspect of the present invention is to maximize the
antiproliferative activity of the cytokine by conjugating it to the
targeting PPC to form a heteroconjugate. Since the cytokines are
polypeptides, conjugation to the PPC can be performed using any of
the conventional methods for linking polypeptides to antibodies.
These include, e.g., use of the heterobifunctional reagent
N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), according to
published procedures, e.g., that of Carlsson et al., Biochem. J.
173:723-737, 1978, use of glutaraldehyde, carbodiimide or like
condensing and/or linking reagents.
[0164] It is preferable to achieve a high ratio of the cytokine to
the PPC without affecting the latter's immunoreactivity and
targeting properties. Thus, it may be advantageous to use a carrier
for the cytokine and to link a plurality of cytokine molecules to
the carrier, which is then linked to the PPC. A particularly
effective method for achieving this result is to use the method of
Shih et al., PCT/US WO 87/005031, wherein an addend is conjugated
to a polymer such as an aminodextran, which is then
site-specifically linked to the oxidized carbohydrate portion of a
PPC. Depending upon the cytokine and PPC used, 20 to more than 100
cytokine molecules per PPC can be attached without affecting the
PPC appreciably, and in some circumstances 100 to 1,000 molecules
of cytokine per PPC molecule can be achieved.
[0165] Use of IL-1 or G-CSF as the cytokine is preferable if a
cytokine with antitumor activity is desired to potentiate the
targeting PPC's effects, especially if the latter is conjugated
with a toxic radioisotope or drug. If the targeting PPC circulates
and deposits in other normal organs, such as the bone marrow, then
the presence of the cytokine is important to prevent, mitigate or
reverse the hematologic side effects that would normally result.
Since some of the cytokines have lymphoid effector cell functions
for tumor cell killing (e.g., IL-2), the heteroconjugate of this
invention provides a multimodal therapy to the target, whether it
be a cancer, an infection, or another lesion that is unresponsive
to more traditional measures.
[0166] An appropriate dose of the cytokine can be administered
prior to, simultaneously with or subsequent to the administration
of the therapeutic agent. The object will be to maximize the
cytotoxic activity of the agent on the pathological lesion, such as
cancer cells or infectious organisms, while minimizing toxicity to
the myeloid and other hematopoietic cells. Careful monitoring of
the WBC and other blood elements, including but not limited to
erythrocyte (red blood cell/RBC) count, thrombocyte (platelet)
count, and including a differential WBC analysis to monitor the
myloid/lymphoid series, as well as the bone marrow hematological
picture during the course of therapy, with particular attention to
possible depletion of myeloid lymphoid forms, but also the status
of immature erythrocytes, myelocytes, lymphocytes and thrombocytes,
will permit optimization of the cytokine treatment. Depending upon
which hematologic element is adversely affected, the choice of
cytokine and administration schedule can be individualized in each
circumstance, including the combination of cytokines, such as IL-1
and IL-3; IL-1 and IL-2; IL-1 and GM-CSF; IL-1, erythropoietin, and
platelet growth factor and the like.
[0167] Correlation of the choice of cytokine, singly or in
combinations, and doses thereof, to hematotoxicity is important,
since each cytokine generally has its effect mostly on particular
hematopoietic elements. The following guidelines may be used for
choosing cytokines in the methods of the invention. For example, if
a cytotoxic agent has both severe myeloid and thrombocytic
toxicity, the combination of IL-1 and IL-3 in a 1:1 or 2:1 (or
higher) ratio will be advantageous. Thus, reduction in the WBC
count to a level below about 2,000 and platelets to a level below
about 20,000 can be reversed by administration of from about 1 ug
to about 500 ug, preferably 5-100 ug, more preferably about 10 ug
of rIL-1 in a single dose, together with or followed by
administration of from about 1 ug to about 200 ug, preferably 5-50
ug, more preferably about 5 ug of IL-3. The applications can be
repeated, with the reversal of the myeloid and platelet depressions
occurring within about 5-20 days, usually about 7 days. The
ordinary skilled clinician will appreciate that variations in the
timing and dosage of cytokine administration and cytokine
combinations and dosages are a function of the cytokine used, the
nature of the bone marrow and/or other hematopoietic element
depressed, and the nature of the patient (e.g., prior toxicity
affecting bone marrow status) and the cytotoxic agent and
protocol.
[0168] Examples of autoimmune diseases that could be treated by the
methods of the invention include acute idiopathic thrombocytopenic
purpura, chronic idiopathic thrombocytopenic purpura,
dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic
lupus erythematosus, lupus nephritis, rheumatic fever,
polyglandular syndromes, bullous pemphigoid, diabetes mellitus,
Henoch-Schonlein purpura, post-streptococcalnephritis, erythema
nodosum, Takayasu's arteritis, Addison's disease, rheumatoid
arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis,
erythema multiforme, IgA nephropathy, polyarteritis nodosa,
ankylosing spondylitis, Goodpasture's syndrome,
thromboangitisubiterans, Sjogren's syndrome, primary biliary
cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma,
chronic active hepatitis, polymyositis/dermatomyositis,
polychondritis, pamphigus vulgaris, Wegener's granulomatosis,
membranous nephropathy, amyotrophic lateral sclerosis, tabes
dorsalis, giant cell arteritis/polymyalgia, pemiciousanemia,
rapidly progressive glomerulonephritis, psoriasis, and fibrosing
alveolitis.
[0169] The method of treating autoimmune disease may comprise an
additional step of administering a secondary antibody or PPC with
an ABS specific for an epitope on T-cells, plasma cells, or
macrophages or inflammatory cytokines. This additional step may be
performed before, during or after the administration the PPC.
[0170] Another major application of the methods and PPCs of the
invention is to depress host immunity in certain autoimmune
diseases such as, for example, systemic lupus erythematosis, and in
patients receiving organ transplants. In these applications, the
PPC is associated with cytotoxic drugs. These cytotoxic drugs are
similar to those often used in cancer chemotherapy, with the
attendant myeloid and other hematopoietic side effects. In addition
to these drugs, specific PPCs targeted against these lymphoid cells
(particularly T-cell), (e.g., a PPC with an ABS derived from the
anti-Tac monoclonal antibody of Uchiyama et al., J. Immunol.
126:1393 and 1398 (1981), which specifically binds to the human
IL-2 receptor of activated T-cells) can be conjugated to cytotoxic
agents, such as drugs, toxins or radioisotopes, to effect a
relatively select killing of these cells involved in organ
rejection. For example, a T-cell specific PPC can be conjugated
with .alpha., .beta. or .gamma. emitting radioisotope, and this can
be administered to the patient prior to undertaking organ
transplantation and, if needed, also thereafter.
[0171] In order to effect a high T-cell killing dose without the
concomitant limiting side effects to the hematopoietic system, this
treatment can be combined with the use of cytokines, according to
the present invention. This method is preferred for the long-term
survival of many organ transplants, such as the kidney, heart,
liver, etc., where a critical period of organ rejection needs to be
overcome.
[0172] The dosage level of the cytokine will be a function of the
extent of compromise of the hematopoietic cells, correlated
generally with the white blood cell (WBC) level in the patient.
Periodic monitoring of the WBC and other blood cell counts and
adjustment of the rate and frequency of infusion or the dosage of
the cytokine administered to achieve a relatively constant level of
WBC's will ensure that the patient does not sustain undue marrow
toxicity from the therapy. Experience will permit anticipation of
WBC lowering and in fusion of the cytokine at a time and in an
amount sufficient to substantially prevent WBC-depression.
Importantly, this also insures that excessive side effects due to
the cytokine itself are not produced, but only such side effects as
are necessary to prevent compromise of the patient's myeloid and
other hematopoietic cell systems.
[0173] Correlation of cytokine dosage to WBC count suggests that,
in general, reduction of WBC count from a normal range of
8-12,000/mm.sup.3 to a level of about 2,000 can be reversed by
administration of from about 1 ug to about 500 ug, preferably 5-100
ug, more preferably about 10 ug of recombinant human IL-1 in a
single dose, the reversal of WBC count depression occurring within
about 2-12 days, usually about 4 days. The clinician will
appreciate that variations in the timing and dosage of cytokine
administration as a function of the type of cytokine used, the
extent and rate of compromise of the bone marrow and/or other
components of the myeloid and/or other hematopoietic elements and
the individual characteristics of the patient and the therapy
protocol will be possible and often desirable. These can easily be
made by the clinician using conventional monitoring techniques and
dosimetric principles.
[0174] The methods of the invention, including methods for treating
autoimmune disorders and neoplastic disorders may be used to treat
disorders such as cardiovascular diseases and inflammation. These
disorders include myocardial infarction, ischemic heart disease,
and atherosclerotic plaques. PPCs that are suitable for treating
these disorders include those PPCs with an ABS specific for CD74
(e.g., hLL1), NCA (or -CD66) and NCA90. This would include ABS with
the same specificity as hMN3. The diagnostic imaging methods of the
invention are particularly adaptable for using the above stated
PPCs. In particular, the detection methods, diagnostic methods, and
the cell ablation methods may be applied to cardiovascular
disorders. For example, the detection may be used to detect damaged
heart and vascular tissue. The cell ablation methods may be used
for targeting diseased heart tissue. Inflammation can be detected
or treated with anti-granulocyte (e.g., anti-CD66, anti-CD33,
anti-CD45), anti-lymphocyte (anti-B- or anti-T-cell antibodies),
and/or anti-monocyte antibodies (e.g., anti-Ia or anti-CD74
antibody).
[0175] Any of the methods of the invention, including methods for
treating autoimmune disorders and neoplastic disorders may be used
to treat disorders such as neurological diseases such as
Alzheimer's disease. PPC that are suitable for treating these
disorders include those PPC with an ABS specific for the amyloid
plaques of Alzheimer patients. The diagnostic imaging methods of
the invention are particularly adaptable for using the above stated
PPC. In particular, the detection methods, diagnostic methods, and
the cell ablation methods may be applied to neurological disorders.
For example, the detection may be used to detect damaged brain
tissue or brain tissue with amyloid. The cell ablation methods may
be used for targeting amyloid. Inflammation can be detected or
treated with anti-amyloid PPCs.
[0176] This invention also provides for methods of detecting and
diagnosing a diseased tissue or a disease. For example, any of the
methods of treatment presented may be performed with a PPC that has
a detectable label, such as, for example, a radiolabel. The PPC can
be detected after administration to the patient. Thus, any of the
treatment methods can be used as detecting methods by the
additional step of detecting the PPC after administration to the
patient. Furthermore, by using a PPC with a specificity to a known
pathogen, diseased cell, tumor associated antigen, disease
associated antigen (e.g., amyloid) and the like, the presence of a
disease may be diagnosed.
[0177] Methods of Administration
[0178] The preferred route for administration of the invention is
parental injection. In parenteral administration, the compositions
of this invention will be formulated in a unit dosage injectable
form such as a solution, suspension or emulsion, in association
with a pharmaceutically acceptable excipient. Such excipients are
inherently nontoxic and nontherapeutic. Examples of such excipients
are saline, Ringer's solution, dextrose solution and Hank's
solution. Nonaqueous excipients such as fixed oils and ethyl oleate
may also be used. A preferred excipient is 5% dextrose in saline.
The excipient may contain minor amounts of additives such as
substances that enhance isotonicity and chemical stability,
including buffers and preservatives. Other methods of
administration, such as oral administration are also
contemplated.
[0179] The PPC of the present invention may be administered in
solution. The pH of the solution should be in the range of pH 5 to
9.5, preferably pH 6.5 to 7.5. The PPC thereof should be in a
solution having a suitable pharmaceutically acceptable buffer such
as phosphate, tris (hydroxymethyl) aminomethane-HCl or citrate and
the like. Buffer concentrations should be in the range of 1 to 100
mM. The solution of the immunoglobulin may also contain a salt,
such as sodium chloride or potassium chloride in a concentration of
50 to 150 mM. An effective amount of a stabilizing agent such as
glycerol, albumin, a globulin, a detergent, a gelatin, a protamine
or a salt of protamine may also be included and may be added to a
solution containing the PPC or to the composition from which the
solution is prepared. Systemic administration of the PPC is
typically made every two to three days or once a week if a
humanized form of the antibody is used as a template for the PPC.
Alternatively, daily administration is useful. Usually
administration is by either intramuscular injection or
intravascular infusion.
[0180] Administration may also be intranasal or by other
nonparenteral routes. The PPC may also be administered via
microspheres, liposomes or other microparticulate delivery systems
placed in certain tissues including blood.
[0181] The PPC may be administered by aerosol to achieve localized
delivery to the lungs. This is accomplished by preparing an aqueous
aerosol, liposomal preparation or solid particles containing or
derivatives thereof. A nonaqueous (e.g., fluorocarbon propellent)
suspension could be used. Sonic nebulizers preferably are used in
preparing aerosols. Sonic nebulizers minimize exposing the PPC to
shear, which can result in degradation of the PPC.
[0182] In general, the dosage of administered PPC will vary
depending upon such factors as the patient's age, weight, height,
sex, general medical condition and previous medical history.
Preferably, a saturating dose of PPC is administered to a
patient.
[0183] Typically, it is desirable to provide the recipient with a
dosage that is in the range of from about 50 to 500 milligrams of
PPC, although a lower or higher dosage also may be administered as
circumstances dictate. Examples of dosages include 20 to 1500
milligrams protein per dose, 20 to 500 milligrams protein per dose,
20 to 100 milligrams protein per dose, 20 to 100 milligrams protein
per dose, 20 to 1500 milligrams protein per dose. In the
embodiments where the PPC of PPC comprise a radio nuclide, the
dosage may be measured by millicurries. In that case, the dosage
may be between 15 and 40 mCi, 10 and 30 mCi, 20 and 30 mCi, or 10
and 20 mCi.
[0184] 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, 19th Ed. (Mack Publishing Co. 1995), and
Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS
(Goodman et al., Eds. Macmillan Publishing Co., New York, 1980 and
2001 editions).
[0185] For purposes of therapy, one or more PPCs and a
pharmaceutically acceptable carrier are administered to a patient
in a therapeutically effective amount. A combination of one or more
PPCs and a pharmaceutically acceptable carrier 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 the present
context, an agent is physiologically significant if its presence
results in the inhibition of the growth of target cells.
[0186] PPC linked to radionuclide are particularly effective for
microbial therapy. After it has been determined that PPC are
localized at infectious sites in a subject, higher doses of the
labeled PPC, generally from 20 mCi to 150 mCi per dose for I-131, 5
mCi to 30 mCi per dose for Y-90, or 5 mCi to 20 mCi Re-186, each
based on a 70 kg patient weight, are injected. Injection may be
intravenous, intraarterial, intralymphatic, intrathecal, or
intracavitary (i.e., parenterally), and may be repeated. It may be
advantageous for some therapies to administer multiple, divided
doses of PPC or PPC composite, thus providing higher microbial
toxic doses without usually effecting a proportional increase in
radiation of normal tissues.
[0187] A variety of radionuclides are useful for therapy, and they
may be incorporated into the specific PPC by the labeling
techniques discussed above, as well as other conventional
techniques well known to the art. Preferred therapeutically
effective radionuclides are actinium-225, astatine-211,
bismuth-212, yttrium-90, rhenium-186, rhenium-188, copper-67,
phosphorus-32, lutetium-177, iodine-131, and iodine-125, although
other radionuclides as well as photosensitizing agents are also
suitable.
[0188] As discussed above, the PPC may be labeled and the use of a
labeled PPC in the methods of the invention is also contemplated.
The dosage of the radiolabel may be in the range of between 10 and
60 mCi per dose for yttrium-90. Preferably, between 10 and 50 mCi
per dose. Most preferably, between 15 and 40 mCi, or between 20 to
30 mCi per dose or between 10 to 30 mCi per dose of yttrium-90.
[0189] In a preferred embodiment of the invention, the PPC to be
administered to a patient suffering from a neoplastic disorder is
an PPC comprising at least one ABS specific for an epitope from the
appropriate tumor-associated antigen. That is, the PPC can bind one
of these tumor-associated antigens at more than one site (epitope).
Bispecific and polyspecific immunoglobulin derivative have may uses
which are enumerated in WO02082041A2.
[0190] In practicing the methods of the invention the PPC may
further comprise any of the additional components described above
which includes, at least, toxins, radionuclide, chemotherapeutic
agents or antimicrobial agents. In some embodiments of the
invention, a chemotherapeutic agent may be physically linked to the
PPC. In other embodiments, the chemotherapeutic agent may be
unlinked. Unlinked chemotherapeutic agents may be administered
before, during, or after the administration of the PPC.
[0191] In another embodiment of the invention, the PPC may be
administered before, during, or after administration of a cytokine
moiety. Other agents that can be advantageously administered
before, during of after the administration of PPC, for any of the
methods of the invention, include at least, granulocyte-colony
stimulating factor (G-CSF), granulocyte macrophage-colony
stimulating factor (GM-CSF), erythropoietin, thrombopoietin, and
the like. Other useful agents include a hematopoietic growth
factors conjugated to a bispecific antibody.
[0192] The PPC of the invention may be substituted for
immunoglobulin used for cancer therapy. It is well known that
radioisotopes, drugs, and toxins can be conjugated to antibodies or
antibody fragments which specifically bind to markers which are
produced by or associated with cancer cells, and that such antibody
conjugates can be used to target the radioisotopes, drugs or toxins
to tumor sites to enhance their therapeutic efficacy and minimize
side effects. Examples of these agents and methods are reviewed in
Wawrzynczak and Thorpe (in Introduction to the Cellular and
Molecular Biology of Cancer, L. M. Franks and N. M. Teich, eds,
Chapter 18, pp. 378410, Oxford University Press. Oxford, 1986), in
Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy
of Cancer (C. W. Vogel, ed., 3-300, Oxford University Press, N.Y.,
1987), in Dillman, R. O. (CRC Critical Reviews in
Oncology/Hematology 1:357, CRC Press, Inc., 1984), in Pastan et al.
(Cell 47:641, 1986). in Vitetta et al. (Science 238:1098-1104,
1987) and in Brady et al. (Int. J. Rad. Oncol. Biol. Phys.
13:1535-1544, 1987). Other examples of the use of immunoconjugates
for cancer and other forms of therapy have been disclosed, inter
alia, in U.S. Pat. Nos. 4,331,647, 4,348,376,4,361,544, 4,468,457,
4,444,744, 4,460,459, 4,460,561 4,624,846, 4,818,709, 4,046,722,
4,671,958, 4,046,784, 5,332,567, 5,443,953, 5,541,297, 5,601,825,
5,635,603, 5,637,288, 5,677,427, 5,686,578, 5,698,178, 5,789,554,
5,922,302, 6,187,287, and 6,319,500. These methods are also
applicable to the methods of the invention by the substitution of
the immunoconjugated engineered antibodies and antibodies of the
previous methods with the PPC of this invention.
[0193] The PPC of the invention, for use in any of the methods of
the invention, may be associated or administered with antimicrobial
agents.
[0194] The PPC of the invention, for use in any of the methods of
the invention, may be associated or administered with cytokines and
immune modulators (defined elsewhere in this disclosure). These
cytokines and immune modulators, includes, at least, interferonss
alpha, beta and gamma, and colony stimulating factors.
[0195] The invention also provides for methods for stimulating the
immune response in a patient using the PPC of the invention. In one
embodiment, the PPC of the invention may comprise an ABS of an
anti-idiotype antibody. The PPC may mimic an epitope of a
tumor-associated antigen to enhance the body's immune response.
[0196] The PPC of the invention may be used for many immunological
procedures currently employing antibodies. These procedures include
the use of anti-idiotypic antibodies and epitope conjugated
antibodies to boost the immune system. See, U.S. Pat. Nos.
5,798,100 6,090,381, 6,132,718. Anti-idiotypic antibodies are also
employed as vaccines against cancers and infectious diseases U.S.
Pat. Nos. 6,440,416 and 6,472,511. Further polyspecific PPC may
bind multidrug transporter proteins and overcome multidrug
resistant phenotype in cells and pathogens. The antibodies in these
methods may be replaced by the PPC of this invention.
[0197] The invention also provides for a method for treating a
symtom of an autoimmune disorder. In the method, an PPC of the
invention is administered to a patient with an autoimmune disorder.
The PPC may be admixed with a pharmaceutically acceptable carrier
before administration. The PPC of this method should contain at
least one ABS with binding specificity to a B-cell antigen epitope.
The B cell antigen may be CD22 and the epitope may be epitope A,
epitope B, epitope C, epitope D and epitope E of CD22. The B
cell-associated antigen may also be another cell antigen such as CD
19, CD20, HLA-DR and CD74.
[0198] The ABS may contain a sequence of subhuman primate, murine
monoclonal antibody, chimeric antibody, humanized antibody, or
human origin. For example, the ABS may be of humanized LL2
(anti-CD22) or A20 (anti-CD20) monoclonal antibody origin.
[0199] The administration may be parenteral with dosages from 20 to
2000 mg per dose. Administration may be repeated until a degree of
reduction in symptom is achieved.
[0200] The patients who may be treated by the methods of the
invention include any animal including humans. Preferably, the
animal is a mammal such as humans, primates, equines, canines and
felines.
[0201] The method and PPCs of the invention may be used for the
treatment of diseases that are resistant or refractory towards
systemic chemotherapy. These include various viral, fungal,
bacterial and protozoan infections, as well as particular parasitic
infections. Other viral infections include those caused by
influenza virus, herpes virus, e.g., Epstein-Barr virus and
cytomegalovirus, rabies virus (Rhabdoviridae) and papovavirus, all
of which are difficult to treat with systemic antibiotic/cytotoxic
agents. Use of PPC conjugates, provides a significantly higher
therapeutic index for antiviral drugs and toxins, thus enhancing
their efficacy and reducing systemic side effects. Targeted
radioimmunotherapy with conjugates of PPC with therapeutic
radioisotopes (including boron addends activatable with thermal
neutrons) offers a new approach to antiviral therapy
[0202] Protozoans that may be treated by the methods and PPCs
include, e.g., Plasmodia (especially P. falciparum, the malaria
parasite), Toxoplasma gondii (the toxoplasmosis infectious agent),
Leishmaniae (infectious agent in leishmaniasis), and Escherichia
histolytica. Detection and treatment of malaria in its various
stages is significantly enhanced using the PPC of the invention. As
noted above, MAbs that bind to sporozoite antigens are known.
However, since sporozoite antigens are not shared by blood stage
parasites, the use of MAbs against sporozoite antigens for
targeting is limited to a relatively short period of time in which
the sporozoites are free in the circulation, prior to and just
after injection of and development in the host's hepatocytes. Thus,
it is preferable to use a mixture of PPCs. Alternatively, a PPC
with ABSs that target more than one parasite stage of a protozoan
(such as P. falciparum) is also useful. The MAbs are conjugated to
a suitable radionuclide for imaging (e.g., Tc-99m) or for therapy
(e.g., astatine-211; rhenium-186), or with an antimalarial drug
(e.g., pyrimethamine) for more selective therapy.
[0203] Toxoplasmosis is also resistant to systemic chemotherapy. It
is not clear whether MAbs that bind specifically to T. gondii, or
natural, host antibodies, can play a role in the immune response to
toxoplasmosis but, as in the case of malarial parasites,
appropriately targeted PPC are effective vehicles for the delivery
of therapeutic agents.
[0204] Schistosomiasis, a widely prevalent helminth infection, is
initiated by free-swimming cercariae that are carried by some
freshwater snails. As in the case of malaria, there are different
stages of cercariae involved in the infectious process. PPCs that
bind to a plurality of stages of cercariae, optionally to a
plurality of epitopes on one or more thereof, and preferably in the
form of a polyspecific composite, can be conjugated to an imaging
or therapy agent for effective targeting and enhanced therapeutic
efficacy.
[0205] PPCs that bind to one or more forms of Trypanosoma cruzi,
the causative agent of Chagas' disease, can be made and used for
detection and treatment of this microbial infection. PPCs which
reacts with a cell-surface glycoprotein, as well as PPCs reactive
with other surface antigens on differentiation stages of the
trypanosome, are suitable for directing imaging and therapeutic
agents to sites of parasitic infiltration in the body.
[0206] Another very difficult infectious organism to treat by
available drugs is the leprosy bacillus (Mycobacterium leprae).
PPCs that specifically bind to a plurality of epitopes on the
surface of M. leprae can be made and can be used, alone or in
combination, to target imaging agents and/or antibiotic/cytotoxic
agents to the bacillus.
[0207] Helminthic parasitic infections, e.g., Strongyloidosis and
Trichinosis, themselves relatively refractory towards
chemotherapeutic agents, are suitable candidates for PPC-targeted
diagnosis and therapy according to the invention, using PPCs that
bind specifically to one or, preferably, to a plurality of epitopes
on the parasites.
[0208] Antibodies are available or can easily be raised that
specifically bind to most of the microbes and parasites responsible
for the majority of infections in humans. Many of these have been
used previously for in vitro diagnostic purposes and the present
invention shows their utility as components of antibody conjugates
to target diagnostic and therapeutic agents to sites of infection.
Microbial pathogens and invertebrate parasites of humans and
mammals are organisms with complex life cycles having a diversity
of antigens expressed at various stages thereof. Therefore,
targeted treatment can best be effected when PPC conjugates which
recognize antigen determinants on the different forms are made and
used in combination, either as mixtures or as polyspecific
conjugates, linked to the appropriate therapeutic modality. The
production of PPC is not difficult because the antibody may be
purified and its sequence determined. The same principle applies to
using the reagents comprising PPCs for detecting sites of infection
by attachment of imaging agents, e.g., radionuclides and/or MRI
enhancing agents.
[0209] The invention also provides a method of intraoperatively
identifying diseased tissues by administering an effective amount
of a PPC; and a targetable construct where the PPC comprises at
least one antigen binding site that specifically binds a targeted
tissue and at least one other antigen binding site that
specifically binds the targetable construct; and wherein said at
least one antigen binding site is capable of binding to a
complementary binding moiety on the target cells, tissues or
pathogen or on a molecule produced by or associated therewith.
[0210] The invention also provides a method for the endoscopic
identification of diseased tissues, in a subject, by administering
an effective amount of a PPC and administering a targetable
construct. The PPC comprises at least one antigen binding site that
specifically binds a targeted tissue and at least one antigen
binding site that specifically binds the targetable construct; and
wherein said at least one antigen binding site shows specific
binding to a complementary binding moiety on the target cells,
tissues or pathogen or on a molecule produced by or associated
therewith.
[0211] An alternative method of detection suitable for use in the
present invention is wireless capsule endoscopy, using an ingested
capsule camera/detector of the type that is commercially available
from, for example, Given Imaging (Norcross Ga.).
[0212] The invention also provides a method for the endoscopic
identification of diseased tissues, in a subject, by administering
an effective amount of a PPC; and administering a targetable
construct. In this embodiment, the PPC comprises at least one
antigen binding site that specifically binds a targeted tissue and
at least one antigen binding site that specifically binds the
targetable construct; and wherein said at least one antigen binding
site shows specific binding to a complementary binding moiety on
the target cells, tissues or pathogen or on a molecule produced by
or associated therewith.
[0213] The invention also provides a method for the intravascular
identification of diseased tissues, in a subject by administering
an effective amount of a PPC and a targetable construct. The PPC
comprise at least one antigen binding site that specifically binds
a targeted tissue and at least one antigen binding site that
specifically binds a targetable construct. The at least one antigen
binding site is capable of binding to a complementary binding
moiety on the target cells, tissues or pathogen or on a molecule
produced by or associated with the cell, tissues or pathogen. The
target tissue may be a tissue from normal thyroid, liver, heart,
ovary, thymus, parathyroid, endometrium, bone marrow, or
spleen.
[0214] The invention also provides for a kit for practicing the
methods of the invention. The kit may include a targetable
construct. The targetable construct may be labeled by any of the
labels described as suitable for targetable constructs above.
Further, the targetable construct may be unlabeled but the kit may
comprise labeling reagents to label the targetable construct. The
labeling reagents, if included, may contain the label and a
crosslinker. The kit may also contain an PPC of the invention
comprising at least one ABS specific for the targetable construct
and at least one ABS specific for a targetable tissue. The kit may
optionally contain a clearing composition for clearing
non-localized PPC.
[0215] Nucleic Acid Encoding PPC
[0216] Another embodiment of the invention is directed to a nucleic
acid molecule with at least one open reading frame (ORF) that
encodes at least one polypeptide of any of the PPC of the
invention. The open reading frame of the nucleic acids of the
invention may be linked, in an operational manner, to one or more
nucleic acid elements that promote the expression of the open
reading frame. These elements are known to those of skill in the
art and include, at least, promoters, enhancers, proximal
stimulatory elements and the like. In a preferred embodiment, a
nucleic acid molecule may comprise two open reading frame that
together express both chains of a PPC (see, e.g., FIG. 1A).
[0217] The nucleic acids of the invention may be present in many
forms such as, for example, an expression cassette or an episome
(plasmids, cosmids, and the like). Thus, an expression cassette or
an episome, such as a plasmid or cosmid) is also envisioned as an
embodiment of the invention. Another embodiment of the invention is
directed to a host cell comprising a nucleic acid of the invention.
The host cell may be an E. Coli, a yeast, a plant cell or a
mammalian cell. Mammalian cells may be, for example, a human cell
or a mouse cell.
[0218] The nucleic acids of the invention may be expressed. Where
the nucleic acid is an RNA, the expression may involve a first step
of reverse transcribing the RNA into DNA. The DNA sequence may then
be operably linked to regulatory sequences controlling
transcriptional expression in an expression vector and then,
introduced into either a prokaryotic or eukaryotic host cell. In
addition to transcriptional regulatory sequences, such as promoters
and enhancers, expression vectors include translational regulatory
sequences and a marker gene which is suitable for selection of
cells that carry the expression vector.
[0219] Suitable promoters for expression in a prokaryotic host can
be repressible, constitutive, or inducible. Suitable promoters are
well-known to those of skill in the art and include promoters
capable of recognizing the T4, T3, Sp6 and T7 polymerases, the PR
and PL promoters of bacteriophage lambda, the trp, recA, heat
shock, and lacZ promoters of E. coli, the .alpha.-amylase and the
sigma-specific promoters of B. subtilis, the promoters of the
bacteriophages of Bacillus, Streptomyces promoters, the int
promoter of bacteriophage lambda, the bla promoter of the
.beta.-lactamase gene of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene. Prokaryotic promoters are
reviewed by Glick, J. Ind. Microbiol. 1: 277 (1987); Watson et al.,
MOLECULAR BIOLOGY OF THE GENE, 4th Ed., Benjamin Cummins (1987);
Ausubel et al., supra, and Sambrook et al., supra.
[0220] The invention also provides for a host cell carrying the
nucleic acids of the invention. A preferred prokaryotic host is E.
coli. Preferred strains of E. coli include Y1088, Y1089, CSH18,
ER1451, and ER1647 (see, for example, Brown (Ed.), MOLECULAR
BIOLOGY LABFAX, Academic Press (1991)). An alternative preferred
host is Bacillus subtilus, including such strains as BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA CLONING: A PRACTICAL APPROACH, Glover (Ed.), IRL
Press (1985)). Other host include mammalian cells.
[0221] Methods for expressing nucleic acids are well-known to those
of skill in the art. See, for example, Ward et al., "Genetic
Manipulation and Expression of Antibodies," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss,
Inc. 1995). Moreover, expression systems for cloning antibodies in
prokaryotic cells are commercially available. For example, the
IMMUNO ZAP.TM. Cloning and Expression System (Stratagene Cloning
Systems; La Jolla, Calif.) provides vectors for the expression of
antibody light and heavy chains in E. coli.
[0222] The nucleic acids of the invention is preferably expressed
in eukaryotic cells, and especially mammalian, insect, and yeast
cells. Especially preferred eukaryotic hosts are mammalian cells.
Mammalian cells provide post-translational modifications to the
cloned polypeptide including proper folding and glycosylation. For
example, such mammalian host cells include COS-7 cells (ATCC CRL
1651), non-secreting myeloma cells (SP2/0-AG14; ATCC CRL 1581), rat
pituitary cells (GH.sub.1; ATCC CCL 82), and rat hepatoma cells
(H-4-II-E; ATCC CRL 1548).
[0223] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, and simian virus. In addition,
promoters from mammalian expression products, such as actin,
collagen, or myosin, can be employed. Alternatively, a prokaryotic
promoter (such as the bacteriophage T3 RNA polymerase promoter) can
be employed, wherein the prokaryotic promoter is regulated by a
eukaryotic promoter (for example, see Zhou et al., Mol. Cell. Biol.
10:4529 (1990); Kaufinan et al., Nucl. Acids Res. 19:4485 (1991)).
Transcriptional initiation regulatory signals may be selected which
allow for repression or activation, so that expression of the genes
can be modulated. In general, eukaryotic regulatory regions will
include a promoter region sufficient to direct the initiation of
RNA synthesis. Such eukaryotic promoters include the promoter of
the mouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen.
1:273 (1982)); the TK promoter of Herpes virus (McKnight, Cell
31:355 (1982)); the SV40 early promoter (Benoist et al., Nature
(London) 290:304 (1981)); the Rous sarcoma virus promoter (Gorman
et al., supra); the cytomegalovirus promoter (Foecking et al., Gene
45:101 (1980)); the yeast gal4 gene promoter (Johnston, et al.,
Proc. Natl. Acad. Sci. (USA) 79:6971 (1982); Silver et al., Proc.
Natl. Acad. Sci. (USA) 81:5951 (1984)); and the IgG promoter
(Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833 (1989)).
[0224] Strong regulatory sequences are the most preferred
regulatory sequences of the present invention. Examples of such
preferred regulatory sequences include the SV40 promoter-enhancer
(Gorman, "High Efficiency Gene Transfer into Mammalian cells," in
DNA CLONING: A PRACTICAL APPROACH, Volume II, Glover (Ed.), IRL
Press pp. 143-190 (1985)), the hCMV-MIE promoter-enhancer
(Bebbington et al., Bio/Technology 10:169 (1992)), and antibody
heavy chain promoter (Orlandi et al., Proc. Natl. Acad. Sci. USA
86:3833 (1989)). Also preferred are the kappa chain enhancer for
the expression of the light chain and the IgH enhance (Gillies,
"Design of Expression Vectors and Mammalian Cell Systems Suitable
for Engineered Antibodies," in Antibody Engineering: A Practical
Guide, C. Borrebaeck (Ed.), W. H. Freeman and Company, pp. 139-157
(1992); Orlandi et al., supra).
[0225] The PPC sequence and an operably linked promoter may be
introduced into eukaryotic cells as a non-replicating DNA molecule,
which may be either a linear molecule or, more preferably, a closed
covalent circular molecule. Since such molecules are incapable of
autonomous replication, the expression of the protein may occur
through the transient expression of the introduced sequence.
Preferably, permanent expression occurs through the integration of
the introduced sequence into the host chromosome.
[0226] Preferably, the introduced sequence will be incorporated
into a plasmid or viral vector that is capable of autonomous
replication in the recipient host. Several possible vector systems
are available for this purpose. One class of vectors utilize DNA
elements which provide autonomously replicating extra-chromosomal
plasmids, derived from animal viruses such as bovine papilloma
virus, polyoma virus, adenovirus, or SV40 virus. A second class of
vectors relies upon the integration of the desired genomic or cDNA
sequences into the host chromosome.
[0227] Additional elements may also be needed for optimal synthesis
of mRNA. These elements may include splice signals, as well as
transcription promoters, enhancers, and termination signals. The
cDNA expression vectors incorporating such elements include those
described by Okayama, Mol. Cell. Biol. 3:280 (1983), Sambrook et
al., supra, Ausubel et al., supra, Bebbington et al., supra,
Orlandi et al., supra, and Fouser et al., Bio/Technology 10:1121
(1992); Gillies, supra. Genomic DNA expression vectors which
include intron sequences are described by Orlandi et al., supra.
Also, see generally, Lemer et al. (Eds.), NEW TECHNIQUES IN
ANTIBODY GENERATION, Methods 2(2) (1991).
[0228] Methods Involving Targetable Constructs
[0229] The invention also provides for a method of treating or
diagnosing a disorder. In the method, an PPC of the invention which
has at least (A) one ABS specific for an epitope of a targeted
tissue and (B) one ABS specific for a targetable construct is
provided is administered to the patient. Following the
administration of the PPC, the targetable construct is administered
to the patient. The PPC and the targetable construct may be
administered to the patient at substantially the same time.
[0230] The targetable construct, for the purposes of this
disclosure may be of two formulas.
[0231] In the first structure, the targetable construct is a
compound of the formula:
X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH.sub.2;
[0232] where the compound includes a hard acid cation chelator at X
or Y, and a soft acid cation chelator at remaining X or Y; and
wherein the compound further comprises at least one diagnostic or
therapeutic cation, and/or one or more chelated or chemically bound
therapeutic agent, diagnostic agent, or enzyme (described elsewhere
in this disclosure). The diagnostic agent could be, for example,
Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III),
Co(III), Fe(III), Cu(II), Ni(II), Ti(III), V(IV) ions or a
radical.
[0233] In the second formula, the targetable construct is a
compound of the formula:
X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH.sub.2;
[0234] where the compound includes a hard acid cation chelator or a
soft acid cheator at X or Y, and nothing at the remaining X or Y;
and wherein the compound further comprises at least one diagnostic
or therapeutic cation, and/or one or more chelated or chemically
bound therapeutic agent, diagnostic agent, or enzyme (described
elsewhere in this disclosure). The diagnostic agent could be, for
example, Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II),
Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III), V(IV) ions or a
radical.
[0235] Any method of the invention that uses a targetable construct
may also use a combination of targetable constructs. In a preferred
embodiment, the targetable constructs are IMP241, IMP281 (FIG. 9A),
IMP284 (FIG. 9B), IMP288, or a combination thereof.
[0236] In this method, a clearing composition may be optionally
administered to the patient to clear non-localized PPC from
circulation. The clearing compound is administered after the
administration of the PPC but before the administration of the
targetable construct. These methods are described in detail in U.S.
Pat. No. 4,624,846, WO 92/19273, and Sharkey et al., Int. J. Cancer
51: 266 (1992).
[0237] The described method may be used for in vivo diagnosis. The
method of diagnostic imaging with radiolabeled monoclonal
antibodies is well-known and is applicable for the PPC of this
invention. 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., 990), Brown, "Clinical Use of Monoclonal
Antibodies," in BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al.
(eds.) (Chapman & Hall 1993), and Goldenberg, CA--A Cancer
Journal for Clinicians 44: 43 (1994). The methods of the invention
may be practiced, for example, by the substitution of the
monoclonal antibodies of the above referenced techniques with the
PPCs of the invention.
[0238] For diagnostic imaging, radioisotopes may be bound to a PPC
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 U.S. Pat. No.
5,057,313 and U.S. Pat. No. 5,128,119.
[0239] For purely diagnostic purposes (as opposed to therapeutic or
diagnostic/therapeutic purposes) radiation dose delivered to the
patient is maintained at as low a level as possible by choosing an
isotope with 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 the PPC and are appropriate for
diagnostic imaging include .gamma.-emitters and positron-emitters
such as .sup.99Tc, .sup.67Ga, .sup.111In, .sup.123I, .sup.124I,
.sup.125I, .sup.131I, .sup.51Cr, .sup.89Zr, .sup.18F and .sup.68Ga.
Other suitable radioisotopes are known to those of skill in the
art. Preferred .gamma.-emitters have a .gamma. radiation emission
peak in the range of 50-500 Kev, primarily because the state of the
art for radiation detectors currently favors such labels. Examples
of such .gamma.-emitters include .sup.99Tc, .sup.67Ga, .sup.123I,
.sup.125I and .sup.131I.
[0240] The PPCs also can be labeled with paramagnetic ions for
purposes of in vivo diagnosis. Elements that are particularly
useful for magnetic resonance imaging include Gd, Mn, Dy and Fe
ions. Other methods for enhancing in vivo diagnosis may be found,
for example, in U.S. Pat. Nos. 6,096,089, 5,965,131 and
5,958,048.
[0241] In an alternate approach, detection methods are improved by
taking advantage of the binding between avidin/streptavidin and
biotin. Avidin, found in egg whites, has a very high binding
affinity for biotin, which is a B-complex vitamin. Streptavidin,
isolated from Streptomyces avidinii, is similar to avidin, but has
lower non-specific tissue binding and therefore, streptavidin often
is used in place of avidin. A basic diagnostic method comprises
administering a PPC composite conjugated with avidin/streptavidin
(or biotin), injecting a clearing composition comprising biotin (or
avidin/streptavidin), and administering a conjugate of a diagnostic
agent and biotin (or avidin/streptavidin). Preferably, the biotin
(or avidin/streptavidin) component of the clearing composition is
coupled with a carbohydrate moiety (such as dextran) or a polyol
group (e.g., polyethylene glycol) to decrease immunogenicity and
permit repeated applications.
[0242] A modification of the basic method is performed by
parenterally injecting a mammal with a PPC which has been
conjugated with avidin/streptavidin (or biotin), injecting a
clearing composition comprising biotin (or avidin/streptavidin),
and parenterally injecting a polyspecific PPC according to the
present invention, which further comprises avidin/streptavidin (or
biotin). See WO 94/04702.
[0243] In a further variation of this method, improved detection
can be achieved by conjugating multiple avidin/streptavidin or
biotin moieties to a polymer which, in turn, is conjugated to a PPC
component. Adapted to the present invention, monospecific or
polyspecific PPCs can be produced which contain multiple
avidin/streptavidin or biotin moieties. Techniques for constructing
and using multiavidin/multistreptavidin and/or multibiotin polymer
conjugates to obtain amplification of targeting are disclosed by
Griffiths, PCT application number PCT/US94/04295.
[0244] In another variation, improved detection is achieved by
injecting a targeting PPC composite conjugated to biotin (or
avidin/streptavidin), injecting at least one dose of an
avidin/streptavidin (or biotin) clearing agent, and injecting a
diagnostic composition comprising a conjugate of biotin (or
avidin/streptavidin) and a naturally occurring metal atom chelating
protein which is chelated with a metal detection agent. Suitable
targeting proteins according to the present invention would be
ferritin, metallothioneins, ferredoxins, and the like. See,
PCT/US94/05149.
[0245] In another embodiment, the methods of the invention may be
used for therapy. In the therapeutic methods, a suitable
therapeutic agent is selected from the group consisting of
radioisotope, boron addend, immunomodulator, toxin, photoactive
agent or dye, cancer chemotherapeutic drug, antiviral drug,
antifungal drug, antibacterial drug, antiprotozoal drug and
chemosensitizing agent (See, U.S. Pat. Nos. 4,925,648, 4,932,412).
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
(Goodman et al., Eds. Macmillan Publishing Co., New York, 1980 and
2001 editions). Moreover a suitable therapeutic radioisotope is
selected from the group consisting of .alpha.-emitters,
.beta.-emitters, ..gamma..-emitters, Auger electron emitters,
neutron capturing agents that emit .alpha.-particles and
radioisotopes that decay by electron capture. Preferably, the
radioisotope is selected from the group consisting of .sup.225Ac,
.sup.198Au, .sup.32P, .sup.125I, .sup.131I, .sup.90Y, .sup.186Re,
.sup.188Re, .sup.67Cu, .sup.177Lu, .sup.213Bi, .sup.10B, and
.sup.211At.
[0246] Boron, when used as a therapeutic agent is useful in boron
neutron capture therapy (BNCT). BNCT is based on the nuclear
reaction which occurs when a stable isotope, B-10 (present in 19.8%
natural abundance), is irradiated with thermal neutrons to produce
an a particle and a Li-7 nucleus. These particles have a path
length of about one cell diameter, resulting in high linear energy
transfer. Just a few of the short-range 1.7 MeV .alpha. particles
produced in this nuclear reaction are sufficient to target the cell
nucleus and destroy it. Barth et al., Cancer, 70: 2995-3007 (1992).
Since the .sup.10B(n,.alpha.).sub.7 Li reaction will occur, and
thereby produce significant biological effect, only when there is a
sufficient number of thermal neutrons and a critical amount of B-10
localized around or within the malignant cell, the radiation
produced is localized. The neutron capture cross section of B-10
far exceeds that of nitrogen and hydrogen found in tissues, which
also can undergo capture reactions, (relative numbers: 1 for N-14,
5.3 for H-1, and 11560 for B-10), so that once a high concentration
differential of B-10 is achieved between normal and malignant
cells, only the latter will be affected upon neutron irradiation.
This is the scientific basis for boron neutron capture therapy.
This method is described in more detail in Barth et al., supra;
Barth et al. Cancer Res., 50: 1061-70 (1990); Perks et al., Brit.
J. Radiol., 61: 1115-26 (1988).
[0247] Therapeutic preparations contemplated herein comprise PPC
comprising an ABS specific for an epitope of a pathogen. This PPC
is conjugated to a therapeutically effective radioisotope and/or
antibiotic/cytotoxic drug, in a suitable vehicle for parenteral
administration. A therapeutic preparation may likewise comprise a
polyspecific anti-pathogen PPC composite conjugated to a
radioisotope and/or antibiotic/cytotoxic drug.
[0248] It is advantageous in certain cases to combine a drug with a
radionuclide, especially where the pathogen "hides" or is somewhat
inaccessible. The longer range action of radionuclides can reach
hidden pathogen so long as some antigen is accessible to the
conjugate. Also, radiation can cause lysis of an infected cell and
expose intracellular pathogen to the antimicrobial drug component
of the conjugate.
[0249] The anti-microbial polyspecific imaging PPCs and
monospecific or polyspecific therapeutic PPCs according to the
invention also can be conveniently provided in a therapeutic or
diagnostic kit for PPC targeting to a focus of infection.
Typically, such a kit will comprise a vial containing the PPC
conjugate of the present invention, either as a lyophilized
preparation or in an injection vehicle. If the conjugate is to be
used for scintigraphic imaging or for radioisotope therapy, it will
generally be provided as a cold conjugate together with reagents
and accessories for radiolabeling, in separate containers, while
MRI agents and therapeutic drug/toxin conjugates will generally be
supplied with a paramagnetic species or an antibiotic/cytotoxic
agent already conjugated to the PPC. The kit may further contain a
second, separately packaged, unlabeled PPC specific the therapeutic
agent, a carrier therefor, or a chelating agent for the
radionuclide or paramagnetic ion.
[0250] It is well known in the art that various methods of
radionuclide therapy can be used for the treatment of cancer and
other pathological conditions, as described. e.g., in Harbert,
"Nuclear Medicine Therapy", New York, Thieme Medical Publishers,
1087, pp. 1-340. A clinician experienced in these procedures will
readily be able to adapt the cytokine adjuvant therapy described
herein to such procedures to mitigate the hematopoietic side
effects thereof. Similarly, therapy with cytotoxic drugs, either
administered alone or as PPC conjugates for more precisely targeted
therapy. e.g., for treatment of cancer, infectious or autoimmune
diseases, and for organ rejection therapy, is governed by analogous
principles to radioisotope therapy with isotopes or radiolabeled
antibodies. Thus, the ordinary skilled clinician will be able to
adapt the description of cytokine use to mitigate marrow
suppression and other such hematopoietic side effects by
administration of the cytokine before, during and/or after drug
therapy.
[0251] Therapeutically useful immunoconjugates can be obtained by
conjugating photoactive agents or dyes to a PPC of the invention.
Fluorescent and other chromogens, or dyes, such as porphyrins
sensitive to visible light, have been used to detect and to treat
lesions by directing the suitable light to the lesion. In therapy,
this has been termed photoradiation, phototherapy, or photodynamic
therapy (Jori et al., (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND
OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.
Britain 22:430 (1986)). Moreover, monoclonal antibodies have been
coupled with photoactivated dyes for achieving phototherapy. Mew et
al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380
(1985); Oseroffet 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). The present
invention contemplates the therapeutic use of PPC comprising
photoactive agents or dyes. Anti-CD 19 and anti-CD20 antibodies are
known to those of skill in the art. See, for example, Ghetie et al,
Cancer Res. 48:2610 (1988); Hekrnan et al., Cancer Immunol.
Immunother. 32:364 (1991); Kaminski et al., N. Engl. J. Med.
329:459 (1993); Press et al., N. Engl. J. Med. 329:1219 (1993);
Maloney et al., Blood 84:2457 (1994); Press et al., Lancet 346:336
(1995); Longo, Curr. Opin. Oncol. 8:353 (1996).
[0252] The targetable construct may contain .sup.10B atoms. In this
case, the method may comprise an additional step of effecting BNCT
of a diseased tissue (including neoplastic tissue) where the
targetable construct is located. See U.S. Pat. Nos. 5,846,741,
6,228,362 for a discussion of BNCT.
[0253] In another embodiment of the invention, the targetable
construct of the method may comprise an enzyme. The enzyme may be
one that can increase the cytotoxicity of a drug. For example, the
enzyme may convert a drug from a nontoxic form to a toxic form.
Alternatively, the enzyme may convert a toxic drug to an even more
toxic drug. Examples of such enzyme-prodrug binding partners are
I-131-antibody-carboxypeptidase G2 and topoisomerase-inhibiting
prodrug CPT-11; .beta.-lactamase and cephalosporin-doxorubicin;
alkaline phosphatase and etoposide phosphate; carboxypeptidase G2
and glutamic acid derivative of benzoic acid mustard; and
.beta.-glucuronidase and the glucuronide of any drug which can form
a glucuronide, such as p-hydroxyaniline mustard. Other examples of
targeted enzymes for prodrug activation are discussed in
Bioconjuate Chem., Vol. 4, (1), 3-9 (1993).
[0254] In the methods where the PPC is labeled or the targetable
construct is labeled, the method may be used to detect a target
cell, target tissue, or a pathogen (i.e., infectious agent) in a
patient.
[0255] Methods for Producing PPC
[0256] The invention also provides for methods for producing the
PPC of the invention. In one embodiment, the methods comprise
providing a first polypeptide having an amino acid sequence
comprising 3 or 4 v-regions (i.e., a.sub.1, a.sub.2, a.sub.3, etc.)
linearly arranged in the polypeptide sequence, optionally
comprising amino acid linking sequences interspersed between the
v-regions, and providing a second polypeptide having an amino acid
sequence comprising 3 or 4 v-regions (i.e., b.sub.1, b.sub.2,
b.sub.3, etc.) linearly arranged in the polypeptide sequence,
optionally comprising amino acid linking sequences interspersed
between the v-regions; and contacting the first and second
polypeptides under appropriate conditions such that the individual
polypeptide chains arrange laterally to one another and bind to one
another by the complemetarity binding of corresponding v-regions
(i.e. a.sub.1 to b.sub.1, a.sub.2 to b.sub.2, a.sub.3 to b.sub.3,
etc.) to form the PPC.
[0257] The methods of producing the PPC's may be performed, for
example, by producing the polypeptides on a peptide synthesizer and
combining them in solution under appropriate conditions to allow
for the complementarity binding of the individual polypeptide
chains. Those of ordinary skill in the art are aware of several
such methods for combining the individual polypeptide chains. For
example polypeptide 1 and polypeptide 2 of a PPC may be synthesized
on a peptide synthesizer using following the manufacturer's
instruction (e.g., Applied Biosystems). Alternatively, peptides may
be ordered by mail from a commercial laboratory (e.g.,
Sigma-Genosys, The Woodlands, Tex.). The dried peptides may be
mixed and solubilized in water or water with 5% NH.sub.4OH to
produce the PPC.
[0258] In a preferred embodiment, the two PPC polypeptides may be
coexpressing in a host cell. For example, an expression plasmid
(referred to herein as the coexpression plasmid) that can
co-express two different genes inserted into two different cloning
sites may be chosen (e.g., BS 14HP-GAP+). Nucleic acid molecules
with open reading frames that encode polypeptide 1 and polypeptide
2 of PPC may be cloned into the two cloning sites. The nucleic acid
molecules may be cloned by traditional techniques or they may be
synthesized using an oligonucleotide synthesizer. The coexpression
plasmid may be transfected into a eukaryotic host such as a yeast
cell for expression. PPC may be produced by culturing the
eukaryotic host cell culture until a desired quantity of PPC is
produced.
[0259] In any of the production methods of the invention, the
produced PPC may be a tagged PPC. Tagged PPC may comprise an
additional peptide sequence, such as the FLAG sequence or the
polyHIS sequence. This sequence would allow any expressed PPC to be
purified with the proper affinity column.
[0260] Another example of a suitable expression system for
diabodies and triabodies (which includes the PPC of this invention)
is the pdHL2 vector, which has an amplifiable murine dhfr gene that
allows subsequent selection and amplification by methotrexate
treatment. Gillies et al., J. Immunol. Methods 125:191 (1989). The
pdHL2 vector provides independent expression of two genes that are
independently controlled by two metallothionine promoters and IgH
enhancers. One example of using an amplifiable selectable marker to
increase expression in a mammalian recombinant host cell line is
shown in Example 2.
[0261] Suitable host cells or cell lines for the expression of the
PPC of the invention are known to one of skill in the art and are
also listed in the definition of "host cell" above. One host cell
is a human cell--which would enable any expressed molecules to be
modified with human glycosylation patterns. It should be noted that
there is no indication that a human host cell is essential or
preferred for the methods of the invention.
[0262] As an illustration, SP2/0 cells can be transfected by
electroporation with linearized pdHL2 vector that contains coding
sequences for two antibody components. Selection can be initiated
48 hours after transfection by incubating cells with medium
containing 0.05-0.1 .mu.M methotrexate. Amplification of the two
antibody sequences is achieved by a stepwise increase in
methotrexate concentration up to 5 .mu.M.
[0263] To ensure that the PPC was formed correctly, or for
purification, the PPC may be purified by an antigen affinity column
which is loaded with an antigen that is recognized by an ABS of the
PPC. An antigen affinity purification column can purify only those
PPC with properly formed ABS because PPC without the proper ABS
should not bind to the affinity column matrix. The antigen affinity
purification may be performed multiple times. For example, if the
ABSs of a PPC recognize antigen 1, antigen 2 and antigen 3, the PPC
may be purified by three antigen affinity purification columns each
loaded with one of the three antigens. Other methods of
purification, such as, for example, precipitation of proteins, size
exclusion chromatography, co-precipitation and co renaturation are
known to those of skill in the art.
[0264] The following examples are provided to illustrate, but not
to limit, the claimed invention.
EXAMPLES
Example 1
BS14HP, a Bispecific Trivalent Heterodimer
[0265] Design.
[0266] BS14HP was designed for the constitutive expression of
foreign genes in Pichia pastoris using the GAP promoter system.
Transfection of P. pastoris cells with a linearized DNA plasmid
(BS14HP-GAP+) results in the stable and site-specific integration
of the two DNA segments (FIG. 1A) into the GAP locus of the host's
chromosome. These two DNA segments contain open reading frames, SEQ
ID NO:1 and SEQ ID NO:2, which codes for polypeptide 1 (SEQ ID
NO:9) and polypeptide 2 (SEQ ID NO:10) respectively. As each of the
two DNA segments also contains nucleotide sequences for the GAP
promoter, two mRNA species that encode the amino acid sequences of
polypeptide 1 and polypeptide 2 are synthesized in the same host
cell.
[0267] Polypeptide 1.
[0268]
.alpha.-Factor-h679V.sub.H-GGGGS-hMN-14V.sub.K-LEGGGS-hMN-14V.sub.H-
-6His (SEQ ID NO:1)
[0269] Polypeptide 2.
[0270]
.alpha.-Factor-hMN-14V.sub.K-GGGQFM-hMN-14V.sub.H-GGGGS-h679V.sub.K-
-6His (SEQ ID NO:2)
[0271] The ".alpha.-factor," as shown in the schematic of
polypeptide 1 and 2 above, represents is a signal peptide that is
removed during synthesis and protein transport, which resulting in
secretion of the protein (without the signal peptide) into the
media. The carboxyl terminal hexa-histadine (6His) sequence allows
for rapid and efficient purification of the secreted protein with
commercially available immobilized metal affinity chromatography
(IMAC) material. hMN-14 V.sub.H represents the amino acid sequence
of the variable region of the heavy chain of (V.sub.H region) a
humanized monoclonal antibody (Mab) that binds specifically to
carcinoembryonic antigen (CEA; Shevitz et al, J. Nucl. Med.,
suppl., 34, 217P, 1993). h679VK represents the humanized murine
monoclonal antibody designated 679 (an antibody of the IgG1, kappa
class) binds with high affinity to molecules containing the
tri-peptide moiety histamine-succinyl-glycyl (referred to herein as
"HSG"; Morel et al, Molecular immunology, 27, 995-1000, 1990). The
nucleotide sequence pertaining to the variable domains (V.sub.H and
V.sub.K) of 679 has been determined (Qu et al, unpublished
results). Humanized versions of the 679 variable domains (Rossi. et
al, unpublished results) were used in the design of this
construct.
[0272] The short peptide linkers, GGGGS, LEGGGS, GGGQFM, and GGGGS,
between the variable domains in the constructs are designed to
discourage intra-polypeptide domain pairing. It is anticipated that
the two different polypeptides (FIG. 1B) would associate with each
other noncovalently by pairing the cognate V.sub.H and V.sub.K
domains and thereby forming two functional binding sites for CEA
and one functional binding site for HSG as shown in FIG. 1C.
[0273] PGAPZ.alpha.+ Modified Vector
[0274] The novel construct pGAPZ.alpha.+, depicted in FIG. 2, was
engineered to make bispecific constructs through the synthesis of
two heterologous polypeptides from a single Pichia host cell. Two
overlapping oligonucleotides, which constitute the SS1 linker, were
synthesized, phosphorylated with T4 polynucleotide kinase and
annealed by heating to 95.degree. C. and then slowly cooling to
room temperature over 30 minutes.
3 SS1 Linker Top 5'- gatcccctgc agggagctca ctagta -3' (SEQ ID NO:3)
SS1 Linker bottom 5'- gatctactag tgagctccct gcaggg -3' (SEQ ID
NO:4)
[0275] The oligonucleotide duplex was ligated into the BamHI site
of the pGAPZ.alpha.A vector (Invitrogen) and transformants were
screened for the presence of the linker in the proper
orientation
[0276] Construction of the Pichia Expression Plasmid
BS14HP-GAP+
[0277] Cloning of BS14-Orf-1-pGAPz.alpha.+
[0278] Using the plasmid construct
hMN-14V.sub.H-L5-h679VK-GAP+(Rossi et al, unpublished results) as a
template, a PCR reaction was performed to generate the amplimer
XhoI-L6-hMN-14V.sub.H-SalI using the following oligonucleotide
primers:
4 L6-hMN14VH Xho Left 5'-catactcgagggcggaggtagcgaggtccaact-
ggtggagagc-3' (SEQ ID NO:5) hMN14V.sub.H SalI Right 5'-
cttagtcgacggagacggtgaccggggtc -3' (SEQ ID NO:6)
[0279] The PCR amplimer was cloned into pGemT vector (Promega) and
screened for clones inserted in the 5'-T7 orientation. This
construct, L6-hMN-14-pGemT(T7), was digested with NcoI and XhoI
restriction enzymes and ligated with a DNA fragment containing
h679V.sub.H-L5-hMN14V.sub.K that was excised from the
h679V.sub.H-L5-hMN-14VK-GAP+ plasmid construct (Rossi et al,
unpublished results) with NcoI and XhoI restriction enzymes to
generate the construct to generate the staging plasmid construct
BS14HPorf1-pGemT. This staging construct was first digested with
NcoI restriction endonuclease and the ends were made blunt by
filling with the Klenow fragment of the DNA polymerase. Following
the Klenow fragment treatment, the DNA molecule was digested with
SalI restriction endonuclease to generate a fragment named
BS14HP-orf1. The pGAPz.alpha.+ vector (FIG. 2) was first digested
with EcORI restriction endonuclease and the ends were made blunt by
filling with Klenow enzyme, and then it was digested with SalI. The
digested vector was ligated with the insert fragment to generate
BS14orf1-pGAPZ.alpha.+.
[0280] Cloning of BS14-orf2-PGAPZ.alpha.+AVRX
[0281] A PCR reaction was performed to generate the amplimer
EcoRI-L5-hMN-14V.sub.K-L5-MfeI using the plasmid construct
h679VH-L5-hMN-14VK-GAP+ (Rossi et al, unpublished results) as a
template and the following primers:
5 HMN-14VK EcoRI Left 5'-ctaggaattc gacatccagc tgacccagag-3' (SEQ
ID NO:7) hMN14V.sub.K-L5 MfeI Right 5'-cgtacaattg gccacctcca
cgtttgattt ccaccttgg-3' (SEQ ID NO:8)
[0282] The amplimer was digested with EcoRI and MfeI restriction
enzymes and ligated with the plasmid construct
hMN-14VH-L5-h679VK-AvrX (Rossi et al, unpublished results) that was
digested with EcoRI to generate the construct
BS14HP-orf2-pGAPZ.alpha.+AVRX.
[0283] Final Assembly of BS14HP-GAP+
[0284] The construct BS14HP-orf2-pGAPZ.alpha.+AVRX was digested
with NsiI and SpeI and separated by agarose gel electrophoresis. A
2260 bp DNA fragment containing the BS14-orf2 coding sequence was
isolated from the agarose gel. This isolated nucleic acid molecule
was digested with SbfI and SpeI restriction endonuclease and
ligated with the NsiI/SpeI BS14-orf2 fragment (discussed above) to
generate the final construct BS14HP-GAP+
[0285] Constitutive Expression of BS14HP in Pichia Pastoris
[0286] The BS14HP-GAP+ construct was prepared for transfection by
digestion with AvrII restriction endonuclease. This linearized
construct was used to transfect the X-33 strain of Pichia pastoris
by electroporation using standard methods. Stable transfectants
were isolated on YPD-agar plates containing 100g/ml of zeocin. Nine
zeocin-resistant colonies were re-streaked on YPD-zeocin plates and
the isolated clones were used to inoculate baffled shake flasks
containing modified YPD media (1% yeast extract, 2% tryptone, 2%
dextrose, 0.4 .mu.g/ml biotin, 1.34% yeast nitrogen base, 100 mM
K.sub.2HPO.sub.4, pH 6.0). The flask cultures were shook at 250 RPM
and 30.degree. C. for 48-72 hours to stationary phase where the
optical density at 600 nm was between 18 and 25. The media, which
should contain the excreted recombinant protein, was clarified by
centrifugation and assayed for active protein using a BIAcore
sensorchip.
[0287] The analysis step is as follows. Samples of the culture
media were diluted 1:5 in EB buffer (150 mM NaCl; 50 .mu.M EDTA;
0.005% surfactant P20; 10 mM HEPES, pH 7.4) and injected (50 .mu.l)
over a high-density, HSG-coupled sensorchip in a BIAcoreX system.
Following injection of the diluted media, EB containing 20 .mu.g/ml
of WI2 IgG, an anti idiotypic antibody to hMN-14, was injected
(100111) over the sensorchip to confirm bispecific binding. The
initial binding slopes were used to quantitate yields. Seven of the
nine zeocin resistant clones tested produced bispecific protein
with a yield of up to 3 mg per liter of culture media.
[0288] To purify a larger amount of recombinant protein, the
culture media from the highest expressing clone was buffer
exchanged by diafiltration into Ni Binding buffer (300 mM NaCl; 10
mM imidazole; 50 mM NaH.sub.2PO.sub.4, pH 8.0). Following buffer
exchange, the media protein was loaded onto Ni-NTA IMAC affinity
column. The column was washed extensively with buffer containing 20
mM imidazole and eluded with a buffer containing 250 mM imidazole
(250 mM imidazole; 50 mM NaCl; 25 mM Tris, pH 7.5). The eluates
were analyzed by BIAcore, as described above, and all of the
binding activity was retained (FIG. 3).
[0289] Biochemical Analysis of BS14HP
[0290] To assay the expressed and purified BS14HP, a protein sample
was analyzed by reducing SDS-PAGE and visualized by Coomassie
blue-stained SDS-PAGE gel. The results, as shown in FIG. 4, showed
that the samples are were highly purified and lack significant
protein contamination. The purified protein complex were resolved
in the SDS-PAGE gel as comprising two similar sized c(i.e., closely
spaced bands) polypeptide chain that migrate in the gel at a rate
that is near expected molecular weights of 40,614 and 40,061
Daltons.
[0291] To further analyze the expressed product, the expressed
protein was analyzed by MALDI TOF mass spectrometry and size
exclusion HPLC analysis. Both analysis of expressed BS14HP gave a
single peak consistent with an 81 kDa dimeric protein structure
(FIG. 5).
[0292] The binding stoichiometry can be extrapolated from BIAcore
sensorgrams such as the one shown in FIG. 3. The WI2:BS14HP molar
binding ratio can be derived by comparison of the response units
(RU) attained from BS14HP binding to the HSG sensorchip with the
further RU increase from WI2 binding to BS14HP with normalization
for the respective molecular weights. Several BS14HP preps were
analyzed with the measured ratio ranging from 0.8-0.9. This ratio
indicates the presence of two functional CEA binding sites per
molecule, as the theoretical maximum ratio for such a protein is
1.0. As a comparison, a variety of monovalent CEA-binding
bispecific (hMN-14.times.679) constructs all gave molar ratios
between 0.4 and 0.45.
[0293] The CEA binding of BS14HP was analyzed in a competitive
ELISA and compared to BS1.5H and hMN-14 F(ab').sub.2, which have
one and two CEA binding groups, respectively (FIG. 6).
HRP-conjugated hMN-14 IgG (1 nM) was mixed with either BS14HP,
BS1.5H or hMN-14 F(ab').sub.2 at concentrations ranging from 1 to
250 nM, prior to incubation in CEA-coated (0.5 .mu.g/well) wells.
The IC.sub.50 for BS14HP was 2.7 nM which is close to 2.0 nM for
hMN14 F(ab').sub.2. The monovalent CEA binder, BS1.5H, had an
IC.sub.50 of 10 nM. These results are consistent with the BIAcore
analysis in demonstrating that BS14HP binds CEA divalently.
Further, the binding avidity is comparable to that of the native
hMN-14 F(ab').sub.2.
[0294] Confirmation that BS14HP has the ability to bind CEA
bivalently was provided by SE-HPLC analysis of in vitro
immunoreactivity. When increasing amounts of CEA were mixed with
.sup.125I-hBS14, two distinct peak shifts were evident by HPLC
corresponding to complexes of 125I-hBS14 bound to either one or two
CEAs. (FIG. 6B).
[0295] In Vivo Analysis of BS14HP
[0296] The utility of BS14HP for tumor pretargeting was evaluated
in GW-39 tumor-bearing mice using a bivalent HSG peptide (IMP-241)
labeled with .sup.111In. IMP-241 has a structure of
DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-N- H2, where the N-terminal amino
group of Phe is linked to a DOTA and the epsilon amino group of
each lysine is derivatived with an HSG group. The tetrapeptide
backbone (Phe-Lys-(D-Tyr)-Lys) contains a d-amino acid (D-Tyr) and
the carboxyl group of the C-terminal lys is amidated.
[0297] The results from this analysis were compared with those of
chemically linked hMN-14.times.679 (Fab'.times.Fab') and BS1.5H
(bispecific diabody). Nude mice bearing GW-39 (CEA positive) tumors
were pre-targeted with BS14HP, BS1.5H or hMN-14.times.679.
Initially, the bio-distribution was followed with .sup.131I-labeled
bispecific agent. The tumor residence and blood clearance of
.sup.131I-BS14HP is depicted in FIG. 7. As determined in
preliminary experiments, the time interval between administration
of bispecific targeting agent and of .sup.111In-IMP241 peptide is
the amount of time required for the former to clear the blood to a
concentration of 1% ID/g or less. A pre-targeting clearance time of
24 hours was used for BS14HP and hMN-14.times.679. A 15-hour
clearance time was used for the smaller BS1.5H, which clears the
blood more rapidly. IMP-241 (Immunomedics, Inc), a peptide
containing two HSG groups and a DOTA moiety, was loaded with
.sup.111Indium and injected in pre-targeted mice. The
bio-distribution of the .sup.111In-IMP-241 was examined at 3 hours
after injection (FIG. 8A) and the tumor/non-tumor ratios are shown
in FIG. 8B.
[0298] The results indicate that approximately three-fold more
.sup.111In-IMP241 peptide was specifically bound to the tumor in
mice pretargeted with BS14HP, as compared to mice pretargeted with
either BS1.5H or hMN14.times.679. The radioactivity in all
non-tumor organs was low and comparable amongst the three
pretargeting agents.
[0299] These experiments were performed again using polypeptide
IMP281 (FIG. 9A), IMP284 (FIG. 9B), and IMP288 (FIG. 9C) with the
same results.
Example 2
hBS14, a Bispecific Trivalent Heterodimer Expressed in Myeloma
Cells
[0300] To demonstrate that similar PPCs could be made from other
types of host cell systems we developed a scheme for production of
a fusion protein named hBS14 in mammalian cell culture. The hBS14
PPC produced in mammalian cell culture was designed to be
structurally and functionally similar to BS14HP, which was produced
in the yeast P. pastoris (example 1). A DNA plasmid vector was
engineered for hBS14 expression and used to generate transgenic
cell lines in SP2/0-Ag14 mouse myeloma cells, NS0 mouse myeloma,
and YB2/0 rat myeloma cells. While these particular cell lines were
used, it is understood that the vectors of the invention may be
used in any mammalian cell lines, such as, for example, a human
cell line.
[0301] Generation of an hBS14 DNA Expression Vector
[0302] The nucleic acid encoding the hBS14 polypeptides were
recombinantly inserted into the mammalian expression vector pdHL2,
which permits the amplification of antibody production. The pdHL2
vector contains the genes for IgG constant regions (CH an CK) and
was originally designed to accept variable domain cassettes and
direct the synthesis of whole IgG. Since we are interested in
expressing novel single chain-based constructs devoid of constant
region sequences, it was necessary to create a new shuttle vector
to facilitate the assembly and transfer of the hBS14 genes into the
pdHL2 vector. See FIG. 10A.
[0303] Overlapping synthetic oligonucleotides (85mers) were
annealed to form duplex DNA possessing the features shown in FIG.
10A. This duplex was ligated into the HindIII and EcoRI restriction
endonuclease sites of the pGEM3z cloning vector (Promega) to
generate the SV3 shuttle vector. The variable domain genes were
amplified by PCR from BS14HP-GAP+(example 1) and assembled into
open reading frames (ORFs) in the SV3 shuttle vector via the NcoI
and SalI restriction endonuclease sites. SV3 constructs were
generated for both ORF 1 and ORF 2, which encode polypeptides 1 and
2 (See FIG. 10B).
[0304] Each ORF includes the IgG light chain leader peptide, which
directs secretion of the nascent polypeptides, preceding the
variable domain genes, which are in turn followed by the codons for
six histidines and two stop codons. The variable domains are
separated by linker peptides consisting of 5 or 6 amino acid
residues. ORF1 and ORF2 were sub-cloned into a single pdHL2
expression vector. ORF1 was excised from its shuttle vector with
Xba I and Bgl II restriction endonucleases and cloned into the XbaI
and BamHlI sites of pdHL2 to generate the intermediate construct
hBS14ORF1-pdHL2. ORF2 was then excised from its shuttle vector with
XhoI and EagI restriction enzymes and cloned into those same sites
of the intermediate hBS14ORF1-pdHL2 construct to generate the final
di-cistronic expression vector hBS14-pdHL2 (FIG. 11).
Stable Transfection and Amplification of hBS14 Genes in SP2/0
Myeloma Cells
[0305] SP2/0-Ag14 mouse myeloma cells have been used previously in
conjunction with the pdHL2 expression vector for high-level
expression of recombinant IgG. NS0 mouse myeloma and YB2/0 rat
myeloma cells have been used for high-level expression of
recombinant IgG with other expression vectors. The hBS14-pdHL2 DNA
vector was linearized by digestion with EcoRI restriction
endonuclease and successfully transfected into each of the three
cell lines (4.times.10.sup.6 cells) by electroporation (450 volts,
25 .mu.F). The pdHL2 vector contains the gene for dihydrofolate
reductase (DHFR) allowing clonal selection as well as gene
amplification with methotrexate (MTX).
[0306] Transfectants were cloned by plating in 96-well plates in
the presence of 0.05 .mu.M MTX and the primary screening for
hBS14-expressing clones was accomplished by ELISA. The ELISA
screening format was as follows: A conjugate consisting of an
HSG-containing peptide (IMP239) and bovine serum albumin was first
adsorbed to micro-plate wells and then conditioned media from the
putative clones were transferred to the micro-plate wells to allow
hBS14 binding to the HSG groups of the conjugate. Bound hBS14 was
detected with WI2, a rat anti-idiotype IgG to hMN-14, and
HRP-conjugated goat anti-rat IgG. Several positive clones were
identified and expanded. Expression of hBS14 was confirmed by
BIAcore using an HSG (IMP239) sensorchip. An increase in response
units (RU) following injection of culture media signified
expression of hBS14. A further increase in RU with subsequent
injection of WI2 demonstrated that the hBS14 was bispecific and
fully functional. With this method, standard concentration curves
were generated using purified 679-proteins allowing for accurate
real time measurements of productivity. The initial productivity of
the highest terminal culture hBS14 producer in SP2/0, YB2/0 and NS0
was 0.8 mg/L, 3.7 mg/L, and 4.4 mg/L, respectively.
[0307] Gene amplification and the resulting increase in
productivity were accomplished by stepwise increase in MTX
concentration in the culture media over several months. An example
of the increase in productivity is shown for SP2/0 clone 1H6 in
FIG. 12. The MTX concentration has been increased from 0.05 .mu.M
to 1 .mu.M and the productivity has increased to 8 mg/L, 16 mg/L
and 9.3 mg/L for representative clones of SP2/0, YB2/0 and NS0,
respectively without adverse effects. We expect these yields can be
further improved with further MTX treatment and selection.
Typically, MTX concentrations can be increased up to 5 .mu.M with
significant additional increase in productivity.
[0308] Production and Purification of hBS14
[0309] Nearly 100 mg of hBS14 has been purified to near
homogeneity. Starting material was generated in terminal roller
bottle cultures of each representative cell line grown with 1 .mu.M
MTX. The purification process was greatly facilitated by the
generation of an HSG-based affinity purification resin. Initial
attempts using affigel-IMP239, the same peptide that was used in
both ELISA and BIAcore experiments, were less successful because
the strong binding affinity made elution without protein
denaturation. None of the myriad elution buffers tested eluted the
hBS14 effectively. A new peptide (IMP291), which was designed to
have {fraction (1/10)} to {fraction (1/100)} lower affinity for
679, was synthesized and conjugated to Affigel (BIO-RAD) by
standard methods. The high binding capacity (>20 mg/ml)
Affigel-IMP291 proved to be ideal for affinity purification of
hBS14 by providing high yield, high purification and high retention
of activity.
[0310] Briefly, culture media from roller bottles was clarified by
cross-flow microfiltration (0.2 .mu.M) and then pH adjusted to 4.5
with citric acid. The hBS14 in the clarified and pH adjusted media
was partly purified about 25 fold by loading the media onto a
S-sepharose cation exchange column. The S-sepharose column was
eluted with 2.times.PBS (0.3 M NaCl; 80 mM NaH.sub.2PO.sub.4, pH
7.4) and the eluate was loaded onto an Affigel-IMP291 column. The
column was eluted with 50 ml of 1M imidazole; 150 mM sucrose; 0.02%
Tween-20; 50 mM Citrate, pH 4.5. The eluded product was dialyzed
into formulation buffer (150 mM Sucrose; 0.02% Tween-20; 10 mM
NaAc, pH 4.5). This procedure allows elution of nearly 100% of the
hBS14 bound to the affinity column.
[0311] Biochemical Analysis of hBS14
[0312] Basic biochemical analysis demonstrated that the
purification process resulted in highly purified hBS14. The native
quaternary structure of the hBS14 was designed to be a 79.4 kDa
heterodimer of polypeptide 1 (39.94 kDa) and polypeptide 2 (39.5
kDa). The size exclusion HPLC profile of purified hBS14 (Fig C)
shows a major sharp peak with a retention time of 9.23 minutes,
consistent with the profile of an 80 kDa protein. BS1.5H diabody
(54 kDa), hMN-14 triabody (78 kDa), and hMN-14 F(ab').sub.2 (100
kDa) were run in the same column as molecular weight and size
standards. These proteins had retention times of 9.60, 9.35, and
8.77 minutes, respectively. The HPLC profiles are very similar
among batches purified from each cell line (Fig C.). The peak at
.about.11.4 minutes is a non-protein buffer peak. The small peak at
8.30 minutes constitutes 3% of the total protein and is likely
dimerized/aggregated hBS14. SDS-PAGE analysis was used to evaluate
the purity and quality of the polypeptide constituents of hBS14.
The Coomassie blue-stained reducing SDS-PAGE gel shown in FIG. 14
demonstrates the high degree of purity achieved from this two-step
purification process. Only trace amounts of contaminating protein
were detected even when a lane was overloaded with 4 .mu.g of
protein. This SDS-PAGE analysis indicates that the minor HPLC peak
(8.30 min) is indeed hBS14 aggregate and not contaminating protein.
The molecular weights (MW) given for polypeptides 1 and 2 were
calculated from the deduced amino acid sequences of the
polypeptides. The Mrs of the two bands are consistent with the
calculated MW of the hBS14 polypeptides. As predicted, the two
bands appear to be of equal intensity as they should be in
equimolar concentration based on the molecular design. There is no
evidence of appreciable protein degradation. Isoelectric focusing
(IEF) of the purified hBS14 shows a major band near the isoelectric
point (pI) of hBS14 (pI=7.73) as calculated from the deduced amino
acid sequence (FIG. 15). There are trace bands at lower pI that are
likely product related and may be the result of negligible
deamidation of some basic amino acid residues. Taken together, this
combination of standard biochemical analyses suggests that the
transgenic myeloma cells correctly synthesize and secrete hBS14 as
designed and that we have developed a robust purification process
capable of generating highly purified material. The biochemical
properties of hBS14 were indistinguishable among batches prepared
from the different cell lines.
[0313] Functional characterization was provided by BIAcore
experiments to demonstrate bispecific binding properties (FIG. 16).
hBS14 bound tightly to HSG that was immobilized on a sensorchip.
The HSG-bound proteins were able to capture subsequently added CEA
or WI2, demonstrating that they can simultaneously bind both
antigens. If the WI2 binding is allowed to approach saturation, the
stoichiometry of the binding can be determined. The additional
increase in RU resulting from WI2 binding was compared to the
initial RU increase of the hBS14 upon binding to the
HSG-sensorchip. As each increase in RU level is directly
proportional to the mass bound, the WI2:bsAb molar binding ratio
can be calculated using the formula
(RU.sub.WI2/RU.sub.hBS14).times.(MW.sub.hBS14/MW.sub.WI2). hBS14
was designed to be bivalent for CEA (and monovalent for HSG) and as
such should bind WI2 (also bivalent) with a 1:1 molar ratio.
Indeed, the experimentally determined molar binding ratio of WI2 to
hBS14 was found to be between 0.7 and 0.8, approaching the
theoretical maximum of 1.0. When equal concentrations of hBS14 were
bound to an HSG-sensorchip, BIAcore sensorgrams are
indistinguishable between lots derived from either SP2/0 or YB2/0
cells (FIG. 17).
[0314] The data demonstrate that the primary amino acid sequence is
solely responsible for the structure and function of the PPC,
independent of the host cell from which it is produced. The PPC
hBS14 was not only equivalent among batches produced in three
different mammalian cell lines, it was also very similar with
respect to structure and function to BS14HP, which has similar
primary amino acid sequences but is produced in yeast.
[0315] Biopolymer Sequences:
[0316] The nucleic acid and amino acid sequence of the biopolymers
used in Example 2 are as follows:
6 hBS14 polypeptide 1 deduced amino acid sequence:
MEVQLVESGGDLVKPGGSLKLSCAASGFTFSIYTMSWLRQTPGKGLEWVATLSGDGDDIYYPDSVKGR
(SEQ ID NO:11) FTISRDNAKNSLYLQMNSLRAEDTALYYCARVRLGDWDFDVWGQGT-
TVSVSSGGGGSDIQLTQSPSSL SASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKL-
LIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQ PEDIATYYCQQYSLYRSFGQGTKV-
EIKRLEGGGSEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWM
SWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFG
FPWFAYWGQGTPVTVSVDHHHHHH or
MetGluValGlnLeuValGluSerGlyGlyAspLeuValLysProGlyGlySerLeuLysLeuSerCy
(SEQ ID NO:11) sAlaAlaSerGlyPheThrPheSerIleTyrThrMetSerTrpLeuA-
rgGlnThrProGlyLysGlyL euGluTrpValAlaThrLeuSerGlyAspGlyAspA-
spIleTyrTyrProAspSerValLysGlyArg PheThrIleSerArgAspAsnAlaL-
ysAsnSerLeuTyrLeuGlnMetAsnSerLeuArgAlaGluAs
pThrAlaLeuTyrTyrCysAlaArgValArgLeuGlyAspTrpAspPheAspValTrpGlyGlnGlyT
hrThrValSerValSerSerGlyGlyGlyGlySerAspIleGlnLeuThrGlnSerProSer-
SerLeu SerAlaSerValGlyAspArgValThrIleThrCysLysAlaSerGlnAsp-
ValGlyThrSerValAl aTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeu-
IleTyrTrpThrSerThrArgHisThrG lyValProSerArgPheSerGlySerGly-
SerGlyThrAspPheThrPheThrIleSerSerLeuGln
ProGluAspIleAlaThrTyrTyrCysGlnGlnTyrSerLeuTyrArgSerPheGlyGlnGlyThrLy
sValGluIleLysArgLeuGluGlyGlyGlySerGluValGlnLeuValGluSerGlyGlyG-
lyValV alGlnProGlyArgSerLeuArgLeuSerCysSerAlaSerGlyPheAspP-
heThrThrTyrTrpMet SerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpI-
leGlyGluIleHisProAspSerSerTh rIleAsnTyrAlaProSerLeuLysAspA-
rgPheThrIleSerArgAspAsnAlaLysAsnThrLeuP
heLeuGlnMetAspSerLeuArgProGluAspThrGlyValTyrPheCysAlaSerLeuTyrPheGly
PheProTrpPheAlaTyrTrpGlyGlnGlyThrProValThrValSerValAspHisHisHi-
sHisHi sHis hBS14 polypeptide 2 deduced amino acid sequence:
MDIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKA-
PKLLIYWTSTRHTGVPSRFSGSGS (SEQ ID NO:12)
GTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKRGGGQFMEVQLVESGGGVVQPGRSLRLSC
SASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMD-
SLRPED TGVYFCASLYFGFPWFAYWGQGTPVTVSGGGGSDIVMTQSPSSLAVSPGER-
VTLTCKSSQSLFNSRTR KNYLGWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGT-
DFTLTINSLQAEDVAVYYCTQVYYLCTF GAGTKLELKRLDHHHHHH. or
MetAspIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerVa-
lGlyAspArgValThrIleTh (SEQ ID NO:12)
rCysLysAlaSerGlnAspValGlyThrSerValAlaTrpTyrGlnGlnLysProGlyLysAlaProL
ysLeuLeuIleTyrTrpThrSerThrArgHisThrGlyValProSerArgPheSerGlySer-
GlySer GlyThrAspPheThrPheThrIleSerSerLeuGlnProGluAspIleAla-
ThrTyrTyrCysGlnGl nTyrSerLeuTyrArgSerPheGlyGlnGlyThrLysVal-
GluIleLysArgGlyGlyGlyGlnPheM etGluValGlnLeuValGluSerGlyGly-
GlyValValGlnProGlyArgSerLeuArgLeuSerCys
SerAlaSerGlyPheAspPheThrThrTyrTrpMetSerTrpValArgGlnAlaProGlyLysGlyLe
uGluTrpIleGlyGluIleHisProAspSerSerThrIleAsnTyrAlaProSerLeuLysA-
spArgP heThrIleSerArgAspAsnAlaLysAsnThrLeuPheLeuGlnMetAspS-
erLeuArgProGluAsp ThrGlyValTyrPheCysAlaSerLeuTyrPheGlyPheP-
roTrpPheAlaTyrTrpGlyGlnGlyTh rProValThrValSerGlyGlyGlyGlyS-
erAspIleValMetThrGlnSerProSerSerLeuAlaV
alSerProGlyGluArgValThrLeuThrCysLysSerSerGlnSerLeuPheAsnSerArgThrArg
LysAsnTyrLeuGlyTrpTyrGlnGlnLysProGlyGlnSerProLysLeuLeuIleTyrTr-
pAlaSe rThrArgGluSerGlyValProAspArgPheSerGlySerGlySerGlyTh-
rAspPheThrLeuThrI leAsnSerLeuGlnAlaGluAspValAlaValTyrTyrCy-
sThrGlnValTyrTyrLeuCysThrPhe GlyAlaGlyThrLysLeuGluLeuLysAr-
gLeuAspHisHisHisHisHisHis. hBS14 Open reading frame 1 (including
coding sequence for the leader peptide) nucleic acid sequence:
atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtgtccactc-
catggaagtgca (SEQ ID NO:13) gctggtggagtcagggggagacttagtga-
agcctggagggtccctgaaactctcctgtgcagcctctg
gattcactttcagtatttacaccatgtcttggcttcgccagactccgggaaaggggctggagtgggtc
gcaaccctgagtggtgatggtgatgacatctactatccagacagtgtgaagggtcgattcac-
catctc cagagacaatgccaagaacagcctatatctgcagatgaacagtctaagggc-
tgaggacacggccttgt attactgtgcaagggtgcgacttggggactgggacttcga-
tgtctggggccaagggaccacggtctcc gtctcctcaggaggtggcggatccgacat-
ccagctgacccagagcccaagcagcctgagcgccagcgt
gggtgacagagtgaccatcacctgtaaggccagtcaggatgtgggtacttctgtagcttggtaccagc
agaagccaggtaaggctccaaagctgctgatctactggacatccacccggcacactggtgtg-
ccaagc agattcagcggtagcggtagcggtaccgacttcaccttcaccatcagcagc-
ctccagccagaggacat cgccacctactactgccagcaatatagcctctatcggtcg-
ttcggccaagggaccaaggtggaaatca aacgtctcgagggcggaggtagcgaggtc-
caactggtggagagcggtggaggtgttgtgcaacctggc
cggtccctgcgcctgtcctgctccgcatctggcttcgatttcaccacatattggatgagttgggtgag
acaggcacctggaaaaggtcttgagtggattggagaaattcatccagatagcagtacgatta-
actatg cgccgtctctaaaggatagatttacaatatcgcgagacaacgccaagaaca-
cattgttcctgcaaatg gacagcctgagacccgaagacaccggggtctatttttgtg-
caagcctttacttcggcttcccctggtt tgcttattggggccaagggaccccggtca-
ccgtctcagtcgaccatcatcatcatcatcattga. HBS14 Open reading frame 2
(including coding sequence for the leader peptide) nucleic acid
sequence: atgggatggagctgtatcatcctcttcttggtagcaacagcta-
caggtgtccactccatggacatcca (SEQ ID NO:14)
gctgacccagagcccaagcagcctgagcgccagcgtgggtgacagagtgaccatcacctgtaaggcca
gtcaggatgtgggtacttctgtagcctggtaccagcagaagccaggtaaggctccaaagctg-
ctgatc tactggacatccacccggcacactggtgtgccaagcagattcagcggtagc-
ggtagcggtaccgactt caccttcaccatcagcagcctccagccagaggacatcgcc-
acctactactgccagcaatatagcctct atcggtcgttcggccaagggaccaaggtg-
gaaatcaaacgtggaggtggccaattcatggaggtccaa
ctggtggagagcggtggaggtgttgtgcaacctggccggtccctgcgcctgtcctgctccgcatctgg
cttcgatttcaccacatattggatgagttgggtgagacaggcacctggaaaaggtcttgagt-
ggattg gagaaattcatccagatagcagtacgattaactatgcgccgtcgctaaaag-
atagatttacaatatcg cgagacaacgccaagaacacattgttcctgcaaatggaca-
gcctgagacccgaagacaccggggtcta tttttgtgcaagcctttacttcggcttcc-
cctggtttgcttattggggccaagggaccccggtcaccg
tctccggaggcggtggatccgacattgtgatgacacaatctccatcctccctggctgtgtcacccggg
gagagggtcactctgacctgcaaatccagtcagagtctgttcaacagtagaacccgaaagaa-
ctactt gggttggtaccagcagaaaccagggcagtctcctaaacttctgatctactg-
ggcatctactcgggaat ctggggtccctgatcgcttctcaggcagtggatccggaac-
agatttcactctcaccatcaacagtctg caggctgaagacgtggcagtttattactg-
cactcaagtttattatctgtgcacgttcggtgctgggac
caagctggagctgaaacggctcgaccatcatcatcatcatcattga. Nucleic acid
sequence of hBS14-pDHL2 plasmid construct:
ttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaaccc
(SEQ ID NO:15) gacaggactataaagataccaggcgtttccccctggaagctccctc-
gtgcgctctcctgttccgaccc tgccgcttaccggatacctgtccgcctttctccct-
tcgggaagcgtggcgctttctcatagctcacgc tgtaggtatctcagttcggtgtag-
gtcgttcgctccaagctgggctgtgtgcacgaaccccccgttca
gcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagag-
ttcttg aagtggtggcctaactacggctacactagaaggacagtatttggtatctgc-
gctctgctgaagccagt taccttcggaaaaagagttggtagctcttgatccggcaaa-
caaaccaccgctggtagcggtggttttt ttgtttgcaagcagcagattacgcgcaga-
aaaaaaggatctcaagaagatcctttgatcttttctacg
gggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggat
cttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagt-
aaactt ggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct-
gtctatttcgttcatcc atagttgcctgactccccgtcgtgtagataactacgatac-
gggagggcttaccatctggccccagtgc tgcaatgataccgcgagacccacgctcac-
cggctccagatttatcagcaataaaccagccagccggaa
gggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaa
gctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctgcaggcat-
cgtggt gtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatc-
aaggcgagttacatgat cccccatgttgtgcaaaaaagcggttagctccttcggtcc-
tccgatcgttgtcagaagtaagttggcc gcagtgttatcactcatggttatggcagc-
actgcataattctcttactgtcatgccatccgtaagatg
cttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgct
cttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctcatc-
attgga aaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatcc-
agttcgatgtaacccac tcgtgcacccaactgatcttcagcatcttttactttcacc-
agcgtttctgggtgagcaaaaacaggaa ggcaaaatgccgcaaaaaagggaataagg-
gcgacacggaaatgttgaatactcatactcttccttttt
caatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaa
aaataaacaaataggggttccgcgcacatttccccgaaaaggccacctgacgtctaagaaac-
cattat tatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtct-
tcaagaattccgatcca gacatgataagatacattgatgagtttggacaaaccacaa-
ctagaatgcagtgaaaaaaatgctttat ttgtgaaatttgtgatgctattgctttat-
ttgtaaccattataagctgcaataaacaagttaacaaca
acaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaac
ctctacaaatgtggtatggctgattatgatctaaagccagcaaaagtcccatggtcttataa-
aaatgc atagctttaggaggggagcagagaacttgaaagcatcttcctgttagtctt-
tcttctcgtagacttca aacttatacttgatgcctttttcctcctggacctcagaga-
ggacgcctgggtattctgggagaagttt atatttccccaaatcaatttctgggaaaa-
acgtgtcactttcaaattcctgcatgatccttgtcacaa
agagtctgaggtggcctggttgattcatggcttcctggtaaacagaactgcctccgactatccaaacc
atgtctactttacttgccaattccggttgttcaataagtcttaaggcatcatccaaactttt-
ggcaag aaaatgagctcctcgtggtggttctttgagttctctactgagaactatatt-
aattctgtcctttaaag gtcgattcttctcaggaatggagaaccaggttttcctacc-
cataatcaccagattctgtttaccttcc actgaagaggttgtggtcattctttggaa-
gtacttgaactcgttcctgagcggaggccagggtcggtc
tccgttcttgccaatccccatattttgggacacggcgacgatgcagttcaatggtcgaaccatgaggg
caccaagctagctttttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaa-
tagctc agaggccgaggcggcctcggcctctgcataaataaaaaaaattagtcagcc-
atggggcggagaatggg cggaactgggcggagttaggggcgggatgggcggagttag-
gggcgggactatggttgctgactaattg agatgcatgctttgcatacttctgcctgc-
tggggagcctggggactttccacacctggttgctgacta
attgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctaactgac
acacattccacagtcgactagaatatggatagtgggtgtttatgactctggataagcctgaa-
caattg atgattaatgcccctgagctctgttcttagtaacatgtgaacatttacttg-
tgtcagtgtagtagatt tcacatgacatcttataataaacctgtaaatgaaagtaat-
ttgcattactagcccagcccagcccata ctaagagttatattatgtctgtctcacag-
cctgctgctgaccaatattgaaaagaatagaccttcgac
tggcaggaagcaggtcatgtggcaaggctatttggggaagggaaaataaaaccactaggtaaacttgt
agctgtggtttgaagaagtggttttgaaacactctgtccagccccaccaaaccgaaagtcca-
ggctga gcaaaacaccacctgggtaatttgcatttctaaaataagttgaggattcag-
ccgaaactggagaggtc ctcttttaacttattgagttcaaccttttaattttagctt-
gagtagttctagtttccccaaacttaag tttatcgacttctaaaatgtatttagaat-
ttcgaccaattctcatgtttgacagcttatcatcgctgc
actccgcccgaaaagtgcgctcggctctgccaaggacgcggggcgcgtgactatgcgtgggctggagc
aaccgcctgctgggtgcaaaccctttgcgcccggactcgtccaacgactataaagagggcag-
gctgtc ctctaagcgtcaccacgacttcaacgtcctgagtaccttctcctcacttac-
tccgtagctccagcttc accagatccctcgactctagacacaggccgccaccatggg-
atggagctgtatcatcctcttcttggta gcaacagctacaggtgtccactccatgga-
agtgcagctggtggagtcagggggagacttagtgaagcc
tggagggtccctgaaactctcctgtgcagcctctggattcactttcagtatttacaccatgtcttggc
ttcgccagactccgggaaaggggctggagtgggtcgcaaccctgagtggtgatggtgatgac-
atctac tatccagacagtgtgaagggtcgattcaccatctccagagacaatgccaag-
aacagcctatatctgca gatgaacagtctaagggctgaggacacggccttgtattac-
tgtgcaagggtgcgacttggggactggg acttcgatgtctggggccaagggaccacg-
gtctccgtctcctcaggaggtggcggatccgacatccag
ctgacccagagcccaagcagcctgagcgccagcgtgggtgacagagtgaccatcacctgtaaggccag
tcaggatgtgggtacttctgtagcttggtaccagcagaagccaggtaaggctccaaagctgc-
tgatct actggacatccacccggcacactggtgtgccaagcagattcagcggtagcg-
gtagcggtaccgacttc accttcaccatcagcagcctccagccagaggacatcgcca-
cctactactgccagcaatatagcctcta tcggtcgttcggccaagggaccaaggtgg-
aaatcaaacgtctcgagggcggaggtagcgaggtccaac
tggtggagagcggtggaggtgttgtgcaacctggccggtccctgcgcctgtcctgctccgcatctggc
ttcgatttcaccacatattggatgagttgggtgagacaggcacctggaaaaggtcttgagtg-
gattgg agaaattcatccagatagcagtacgattaactatgcgccgtctctaaagga-
tagatttacaatatcgc gagacaacgccaagaacacattgttcctgcaaatggacag-
cctgagacccgaagacaccggggtctat ttttgtgcaagcctttacttcggcttccc-
ctggtttgcttattggggccaagggaccccggtcaccgt
ctcagtcgaccatcatcatcatcatcattgataagatcccgcaattctaaactctgagggggtcggat
gacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagc-
cctcag aatggctgcaaagagctccaacaaaacaatttagaactttattaaggaata-
gggggaagctaggaaga aactcaaaacatcaagattttaaatacgcttcttggtctc-
cttgctataattatctgggataagcatg ctgttttctgtctgtccctaacatgccct-
gtgattatccgcaaacaacacacccaagggcagaacttt
gttacttaaacaccatcctgtttgcttctttcctcaggaactgtggctgcaccatctgtcttcatctt
cccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataact-
tctatc ccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggta-
actcccaggagagtgtc acagagcaggacagcaaggacagcacctacagcctcagca-
gcaccctgacgctgagcaaagcagacta cgagaaacacaaagtctacgcctgcgaag-
tcacccatcagggcctgagctcgcccgtcacaaagagct
tcaacaggggagagtgttagagggagaagtgcccccacctgctcctcagttccagcctgaccccctcc
catcctttggcctctgaccctttttccacaggggacctacccctattgcggtcctccagctc-
atcttt cacctcacccccctcctcctccttggctttaattatgctaatgttggagga-
gaatgaataaataaagt gaatctttgcacctgtggtttctctctttcctcatttaat-
aattattatctgttgttttaccaactac tcaatttctcttataagggactaaatatg-
tagtcatcctaaggcgcataaccatttataaaaatcatc
cttcattctattttaccctatcatcctctgcaagacagtcctccctcaaacccacaagccttctgtcc
tcacagtcccctgggccatggtaggagagacttgcttccttgttttcccctcctcagcaagc-
cctcat agtcctttttaagggtgacaggtcttacagtcatatatcctttgattcaat-
tccctgagaatcaacca aagcaaatttttcaaaagaagaaacctgctataaagagaa-
tcattcattgcaacatgatataaaataa caacacaataaaagcaattaaataaacaa-
acaatagggaaatgtttaagttcatcatggtacttagac
ttaatggaatgtcatgccttatttacatttttaaacaggtactgagggactcctgtctgccaagggcc
gtattgagtactttccacaacctaatttaatccacactatactgtgagattaaaaacattca-
ttaaaa tgttgcaaaggttctataaagctgagagacaaatatattctataactcagc-
aattcccacttctaggg gttcgactggcaggaagcaggtcatgtggcaaggctattt-
ggggaagggaaaataaaaccactaggta aacttgtagctgtggtttgaagaagtggt-
tttgaaacactctgtccagccccaccaaaccgaaagtcc
aggctgagcaaaacaccacctgggtaatttgcatttctaaaataagttgaggattcagccgaaactgg
agaggtcctcttttaacttattgagttcaaccttttaattttagcttgagtagttctagttt-
ccccaa acttaagtttatcgacttctaaaatgtatttagaatttcgaccaattctca-
tgtttgacagcttatca tcgctgcactccgcccgaaaagtgcgctcggctctgccaa-
ggacgcggggcgcgtgactatgcgtggg ctggagcaaccgcctgctgggtgcaaacc-
ctttgcgcccggactcgtccaacgactataaagagggca
ggctgtcctctaagcgtcaccacgacttcaacgtcctgagtaccttctcctcacttactccgtagctc
cagcttcaccagatccctcgagtctagacacaggccgccaccatgggatggagctgtatcat-
cctctt cttggtagcaacagctacaggtgtccactccatggacatccagctgaccca-
gagcccaagcagcctga gcgccagcgtgggtgacagagtgaccatcacctgtaaggc-
cagtcaggatgtgggtacttctgtagct tggtaccagcagaagccaggtaaggctcc-
aaagctgctgatctactggacatccacccggcacactgg
tgtgccaagcagattcagcggtagcggtagcggtaccgacttcaccttcaccatcagcagcctccagc
cagaggacatcgccacctactactgccagcaatatagcctctatcggtcgttcggccaaggg-
accaag gtggaaatcaaacgtggaggtggccaattcatggaggtccaactggtggag-
agcggtggaggtgttgt gcaacctggccggtccctgcgcctgtcctgctccgcatct-
ggcttcgatttcaccacatattggatga gttgggtgagacaggcacctggaaaaggt-
cttgagtggattggagaaattcatccagatagcagtacg
attaactatgcgccgtctctaaaggatagatttacaatatcgcgagacaacgccaagaacacattgtt
cctgcaaatggacagcctgagacccgaagacaccggggtctatttttgtgcaagcctttact-
tcggct tcccctggtttgcttattggggccaagggaccccggtcaccgtctccggag-
gcggtggatccgacatt gtgatgacacaatctccatcctccctggctgtgtcacccg-
gggagagggtcactctgacctgcaaatc cagtcagagtctgttcaacagtagaaccc-
gaaagaactacttgggttggtaccagcagaaaccagggc
agtctcctaaacttctgatctactgggcatctactcgggaatctggggtccctgatcgcttctcaggc
agtggatccggaacagatttcactctcaccatcaacagtctgcaggctgaagacgtggcagt-
ttatta ctgcactcaagtttattatctgtgcacgttcggtgctgggaccaagctgga-
gctgaaacggctcgacc atcatcatcatcatcattgataagatctcggccggcaagc-
ccccgctccccgggctctcgcggtcgca cgaggatgcttggcacgtaccccgtctac-
atacttcccaggcacccagcatggaaataaagcacccac
cactgccctgggcccctgcgagactgtgatggttctttccacgggtcaggccgagtctgaggcctgag
tggcatgagggaggcagagcgggtcccactgtccccacactggcccaggctgtgcaggtgtg-
cctggg ccgcctagggtggggctcagccaggggctgccctcggcagggtgggggatt-
tgccagcgtggccctcc ctccagcagcagctgcctcgcgcgtttcggtgatgacggt-
gaaaacctctgacacatgcagctcccgg agacggtcacagcttgtctgtaagcggat-
gccgggagcagacaagcccgtcagggcgcgtcagcgggt
gttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaac
tatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacag-
atgcgt aaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactc-
gctgcgctcggtcgttc ggctgcggcgagcggtatcagctcactcaaaggcggtaat-
acggttatccacagaatcaggggataac gcaggaaagaacatgtgagcaaaaggcca-
gcaaaaggccaggaaccgtaaaaaggccgcgttgctggc gttt
Example 3
Affinity Purification of hBS14
[0317] hBS14 was purified to homogeneity using a novel affinity
resin that was prepared and used as described below.
[0318] Activation and Coupling of IMP291-affigel
[0319] IMP291 peptide (see structure in FIG. 18) was coupled to
Affigel 102 (BIO-RAD Laboratories, Hercules Calif.) using
chloroacetic anhydride (CAA). CCA (1.5 g, 8.8 mmol) was dissolved
in acetonitrile and added to 30 ml of Affigel 102 slurry. The pH
was adjusted to 9.0 with triethylamine and reacted for 1 hour at
room temperature to allow coupling of CAA to amine groups on the
Affigel. The CAA-Affigel was washed and exchanged into 0.2M
NaBorate, pH 8.0. A total of 166 mg of IMP291 was dissolved in 10
ml of 0.2M NaBorate, pH 8.0 and then added to the slurry, which was
then rocked overnight at room temperature to allow coupling of the
peptide to the CAA-Affigel via thioether bond formation. The resin
was quenched by adding cysteine in 0.2M NaBorate, pH 8.0 to a final
concentration of 20 mM and incubated for 1 hour at room
temperature.
[0320] Qualification of the Affinity Resin
[0321] The IMP291-affigel resin was qualified as follows: A column
was packed with 0.5 ml of the resin and equilibrated with PBS. A
total of 23.5 mg of BS1.5HP in 10 ml of PBS was passed over the
column. A total of 14 mg of BS1.5HP was detected in the unbound
fraction indicating 9.5 mg had bound. A total of 9.2 mg was
recovered in 7 ml of elution with 1M Imidazole, 150 mM sucrose, 10
mM NaAc, pH 4.5. The binding capacity of the resin was determined
to be 330 pmol/ml. For hBS14, this is equivalent to a capacity of
26.7 mg/ml.
[0322] Single-Step Purification of hBS14 with IMP291-Affigel
[0323] A total of 22 liters of hBS14 YB2/0 clone #8 roller bottle
culture containing 144 mg of hBS14 (as estimated by BIAcore) was
centrifuged and brought to 2 mM EDTA, 0.02% Triton-X-100; and 10 mM
Na.sub.2HPO.sub.4 The supernatant fluid was sterile-filtered
through a 0.2 .mu.M Millipak-200 filter unit into an autoclaved
10-L bottle closed system. The filtered media was loaded over a 10
ml IMP291-affigel column (2.5 cm diameter) at flow rates ranging
from 2 to 4 ml/min. The column was washed to baseline with PBS and
then eluted with 107 ml of elution buffer (1 M imidazole, 150 mM
sucrose, 50 mM citrate, pH 4.5). A total of 93 mg of hBS14 was
eluted. Size exclusion HPLC, SDS-PAGE, IEF, and MALDI-TOF mass
spectrometry all indicated a highly purified homogeneous product
from the single step IMP291-affigel affinity chromatography.
BLAcore and in vivo analysis demonstrated that the product was
fully active.
Example 4
Use of hBS14 for Pre-Targeting of Human Colorectal Tumor Xenografts
in Nude Mice
[0324] This example demonstrates the ability of the trivalent,
bispecific hBS14 molecule (hMN-14.times.hMN-14.times.679) to
pre-target IMP-245, a .sup.99mTc-labeled peptide, to a human
colonic tumor (GW-39) xenograft. The structure of IMP 245 is shown
in FIG. 19. IMP-245 was prepared and labeled using standard
techniques known in the art. See, for example, published
application US20030198595, which is hereby incorporated by
reference in its entirety.
[0325] The experiment used 3 groups of 15 mice, each of which was
necropsied, and 1 group of 5 mice that was imaged. Three groups of
mice were administered 6 .mu.Ci .sup.125I-hBS14 (40 .mu.g,
5.0.times.10.sup.-10 moles). In the last group (imaging group), 3
mice received unlabeled hBS14. The amount of .sup.99mTc-IMP-245
administered to all the mice was .about.40 .mu.Ci (92 ng,
5.0.times.10.sup.-11 moles) for a bispecific:peptide ratio of 10:1.
Mice were necropsied at 1, 4, and 24 hours post-peptide
administration, and were divided into the following groups:
[0326] Group I: .sup.125I-hBS14 with 4-hr clearance followed by
.sup.99mTc-IMP-245 [15 mice; sac 5/time-point at 1-, 4-, and 24-hrs
post-DCS injection]
[0327] Group II: .sup.125I-hBS14 with 24-hr clearance followed by
.sup.99mTc-IMP-245 [15 mice; sac 5/time-point at 1-, 4-, and 24-hrs
post-DCS injection]
[0328] Group III: .sup.125I-hBS14 with 48-hr clearance followed by
.sup.99mTc-IMP-245 [15 mice; sac 5/time-point at 1-, 4-, and 24-hrs
post-DCS injection]
[0329] Group IV: hBS14 with 48-hr clearance (3 mice) followed by
.sup.99mTc-IMP-245 (all 5 mice) [5 mice; image mice at 1-, 3-, 6-,
and 24-hrs post-DCS injection]
[0330] Due to differences in tumor growth rates, only 20 mice were
initially available for administration of hBS14. Fifteen mice were
used to fill out Group II while 5 mice were used for Group I. The
remaining mice (including the imaged mice) were injected one week
later. Not all the mice implanted with GW-39 tumors developed
usable tumors and, therefore, Group I only had 10 mice and were
sacrificed at 1 hr post-peptide injection and 24 hrs
post-injection.
[0331] The graph in FIG. 20 (top panel) shows the tumor uptake of
the .sup.125I-hBS14 and .sup.99mTc-IMP-245 in mice when the hBS14
was given 4 hrs to clear prior to administration of peptide (Group
I). At 1 hr post-peptide administration there was 13.5.+-.5.94%
ID/g hBS14 in the tumor versus 2.9.+-.0.46% ID/g of IMP-245
(4.7-fold less peptide than hBS14). After 24 hrs this ratio
reversed with 2.4-fold more IMP-245 in the tumor versus the hBS14
(9.08.+-.4.94% ID/g vs. 3.79.+-.4.15% ID/g, respectively). Blood
levels for the hBS14 and peptide were high at 1 hr post-injection
(16.85.+-.2.95% ID/g and 36.87+6.42% ID/g for hBS114 and IMP-245,
respectively).
[0332] Data for the mice in Group II are shown in FIG. 20 (bottom
panel) Approximately 2-fold more IMP-245 than hBS14 was observed in
the tumors. The greatest amount of variation occurred in the 4 hr
post-IMP245 administration group (15.9.+-.16.3% ID/g IMP-245 in the
tumor). These differences do not appear to be due to hBS14 uptake
since one mouse had 4.6% ID/g hBS14 in its tumor and only 3.6% ID/g
IMP-245 while another mouse in this group also had 4.4% ID/g hBS14
but 18.1% ID/g IMP-245. There appears, however, to be a correlation
between tumor size. These data suggest that larger tumors have
better targeting in mice.
[0333] The graph in FIG. 21 (top panel) shows the tumor uptake of
.sup.125I-hBS14 and .sup.99mTc-IMP-245 in mice given 48 hrs to
clear the hBS14 prior the administration of the peptide (Group
III). Like the Group II mice (24 hr hBS14 clearance), consistent
targeting of the hBS14 to the tumor at all three time-points was
observed. At 1 hr post-peptide injection (49 hrs post-hBS14
administration) there was 3.50.+-.0.86% ID/g hBS14 in the tumor.
This level was maintained throughout two later time-points (52 hrs
and 72 hrs post-hBS14 administration) at 3.62.+-.1.59% ID/g and
6.97.+-.3.10% ID/g, respectively. These data suggest that the
bivalent hMN-14 portion of the hBS14 molecule increased its ability
to stay on the tumor without being shed or otherwise lost. This
stabilized binding of hBS14 to the tumor also resulted in a
relatively constant .sup.99mTc-IMP-245 signal at the tumor. At 1 hr
post-peptide injection there was 21.03.+-.2.47% ID/g at the tumor.
After 4 hrs there was 14.53.+-.4.90% ID/g and 15.47.+-.9.31% ID/g
at 24 hrs post-injection. The differences between any of these
three time-points are not significany but, since .sup.99mTc has
such a short half-life (6.02 hrs) the relative amount of actual
signal in the tumors at 24 hrs was 13-fold less.
[0334] The table shown in FIG. 22 summarizes the % ID/g of the
.sup.99mTc-IMP-245 and the tumor to non-tumor ratios (T:NT) in the
various tissues at 1 hr post-peptide administration for all three
groups of mice (4, 24, and 48 hr hBS14 clearance). One hour
post-peptide injection was used since early time-points for imaging
are clinically desirable.
[0335] The data from the imaged mice are shown in FIG. 23. The
first pair of images shows the location of the tumors in the mice.
hBS14 (5.times.10.sup.-10 moles) was administered, followed after
48 hours by .sup.99mTc-IMP-245 (40 .mu.Ci; 5.times.10.sup.-11
moles). Animals 1 & 2 were given peptide only, while animals 3,
4, & 5 were administered hBS14 followed by peptide. The animals
had the following tumor sizes:
[0336] Animal 1: 1.68 cm.sup.3 tumor
[0337] Animal 2: 0.62 cm.sup.3 tumor
[0338] Animal 3: 1.22 cm.sup.3 tumor
[0339] Animal 4: 0.62 cm.sup.3 tumor
[0340] Animal 5: 0.56 cm.sup.3 tumor
[0341] The second pair of images shows the image at 1 hr
post-peptide administration. After only 1 hour the tumors in the
mice pre-targeted with the hBS14 were clearly visible. The majority
of the signal was located in the bladder at this early time-point,
as expected, and the kidneys also were evident in the images.
External radioactivity was found on the foot of Animal 4 (circled)
and was removed by washing the foot.
[0342] The third pair of images show imaging data at 3 hrs
post-peptide administration. At the 3 hr time-point, Animal 3 was
removed. This mouse had very high tumor uptake and adjusting the
image for this mouse decreased the sensitivity in the remaining
four mice. The outline of the tumors was visible in the mice that
received only peptide, but this was due to the blood pool and not
direct targeting as can be seen with Animals 4 & 5. The mice
were still under the effects of the anesthesia from the first
time-point and were unable to void their bladders, resulting in the
high signal observed in the bladded.
[0343] The final pair of images shows the image at 24 hrs
post-peptide administration. Little signal remained in the mice at
this time-point, which therefore were imaged for 20 minutes rather
than the 10 minutes used at earlier time-points. The only signal
detected was located in the tumors of the mice pre-targeted with
hBS14 prior to the administration of the .sup.99mTc-IMP-245.
[0344] All three pre-targeted mice and both mice that received
peptide alone were necropsied after the 24-hr imaging and the
results are shown in FIG. 21 (bottom panel). Tumor and kidney
uptake was the highest in the pre-targeted mice (19.01.+-.2.80%
ID/g and 3.81.+-.0.80% ID/g, respectively). There was very little
peptide in the tumor of the control mice (0.30.+-.0.08% ID/g), but
the same amount in the kidney as the pre-targeted mice
(3.71.+-.0.43% ID/g).
Sequence CWU 1
1
20 1 370 PRT Artificial Sequence Chimeric sequence from multiple
species 1 Glu Ala Glu Ala Glu Phe Met Glu Val Gln Leu Val Glu Ser
Gly Gly 1 5 10 15 Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser
Cys Ala Ala Ser 20 25 30 Gly Phe Thr Phe Ser Ile Tyr Thr Met Ser
Trp Leu Arg Gln Thr Pro 35 40 45 Gly Lys Gly Leu Glu Trp Val Ala
Thr Leu Ser Gly Asp Gly Asp Asp 50 55 60 Ile Tyr Tyr Pro Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70 75 80 Asn Ala Lys Asn
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 85 90 95 Asp Thr
Ala Leu Tyr Tyr Cys Ala Arg Val Arg Leu Gly Asp Trp Asp 100 105 110
Phe Asp Val Trp Gly Gln Gly Thr Thr Val Ser Val Ser Ser Gly Gly 115
120 125 Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
Ala 130 135 140 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Gln Asp Val 145 150 155 160 Gly Thr Ser Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys 165 170 175 Leu Leu Ile Tyr Trp Thr Ser Thr
Arg His Thr Gly Val Pro Ser Arg 180 185 190 Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 195 200 205 Leu Gln Pro Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu 210 215 220 Tyr Arg
Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Leu Glu 225 230 235
240 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
245 250 255 Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly
Phe Asp 260 265 270 Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly 275 280 285 Leu Glu Trp Ile Gly Glu Ile His Pro Asp
Ser Ser Thr Ile Asn Tyr 290 295 300 Ala Pro Ser Leu Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys 305 310 315 320 Asn Thr Leu Phe Leu
Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly 325 330 335 Val Tyr Phe
Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr 340 345 350 Trp
Gly Gln Gly Thr Pro Val Thr Val Ser Val Asp His His His His 355 360
365 His His 370 2 363 PRT Artificial Sequence Chimeric sequence
from multiple species 2 Glu Ala Glu Ala Glu Phe Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser 1 5 10 15 Leu Ser Ala Ser Val Gly Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser 20 25 30 Gln Asp Val Gly Thr Ser Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu
Ile Tyr Trp Thr Ser Thr Arg His Thr Gly Val 50 55 60 Pro Ser Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr 65 70 75 80 Ile
Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 85 90
95 Tyr Ser Leu Tyr Arg Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg Gly Gly Gly Gln Phe Met Glu Val Gln Leu Val Glu Ser
Gly Gly 115 120 125 Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser
Cys Ser Ala Ser 130 135 140 Gly Phe Asp Phe Thr Thr Tyr Trp Met Ser
Trp Val Arg Gln Ala Pro 145 150 155 160 Gly Lys Gly Leu Glu Trp Ile
Gly Glu Ile His Pro Asp Ser Ser Thr 165 170 175 Ile Asn Tyr Ala Pro
Ser Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp 180 185 190 Asn Ala Lys
Asn Thr Leu Phe Leu Gln Met Asp Ser Leu Arg Pro Glu 195 200 205 Asp
Thr Gly Val Tyr Phe Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp 210 215
220 Phe Ala Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Gly Gly Gly
225 230 235 240 Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ala Val Ser 245 250 255 Pro Gly Glu Arg Val Thr Leu Thr Cys Lys Ser
Ser Gln Ser Leu Phe 260 265 270 Asn Ser Arg Thr Arg Lys Asn Tyr Leu
Gly Trp Tyr Gln Gln Lys Pro 275 280 285 Gly Gln Ser Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg Glu Ser 290 295 300 Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 305 310 315 320 Leu Thr
Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 325 330 335
Thr Gln Val Tyr Tyr Leu Cys Thr Phe Gly Ala Gly Thr Lys Leu Glu 340
345 350 Leu Lys Arg Leu Asp His His His His His His 355 360 3 26
DNA Artificial Sequence Synthesized Oligonucleotide 3 gatcccctgc
agggagctca ctagta 26 4 26 DNA Artificial Sequence Synthesized
oligonucleotide 4 gatcccctgc agggagctca ctagta 26 5 43 DNA
Artificial Sequence Synthesized oligonucleotide 5 catactcgag
ggcggaggta gcgaggtcca actggtggag agc 43 6 29 DNA Artificial
Sequence Synthesized oligonucleotide 6 cttagtcgac ggagacggtg
accggggtc 29 7 30 DNA Artificial Sequence Synthesized
oligonucleotide 7 ctaggaattc gacatccagc tgacccagag 30 8 39 DNA
Artificial Sequence Synthesized oligonucleotide 8 cgtacaattg
gccacctcca cgtttgattt ccaccttgg 39 9 1110 DNA Artificial Sequence
Chimeric sequence from multiple organisms 9 gaggctgaag ctgaattcat
ggaagtgcag ctggtggagt cagggggaga cttagtgaag 60 cctggagggt
ccctgaaact ctcctgtgca gcctctggat tcactttcag tatttacacc 120
atgtcttggc ttcgccagac tccgggaaag gggctggagt gggtcgcaac cctgagtggt
180 gatggtgatg acatctacta tccagacagt gtgaagggtc gattcaccat
ctccagagac 240 aatgccaaga acagcctata tctgcagatg aacagtctaa
gggctgagga cacggccttg 300 tattactgtg caagggtgcg acttggggac
tgggacttcg atgtctgggg ccaagggacc 360 acggtctccg tctcctcagg
aggtggcgga tccgacatcc agctgaccca gagcccaagc 420 agcctgagcg
ccagcgtggg tgacagagtg accatcacct gtaaggccag tcaggatgtg 480
ggtacttctg tagcttggta ccagcagaag ccaggtaagg ctccaaagct gctgatctac
540 tggacatcca cccggcacac tggtgtgcca agcagattca gcggtagcgg
tagcggtacc 600 gacttcacct tcaccatcag cagcctccag ccagaggaca
tcgccaccta ctactgccag 660 caatatagcc tctatcggtc gttcggccaa
gggaccaagg tggaaatcaa acgtctcgag 720 ggcggaggta gcgaggtcca
actggtggag agcggtggag gtgttgtgca acctggccgg 780 tccctgcgcc
tgtcctgctc cgcatctggc ttcgatttca ccacatattg gatgagttgg 840
gtgagacagg cacctggaaa aggtcttgag tggattggag aaattcatcc agatagcagt
900 acgattaact atgcgccgtc tctaaaggat agatttacaa tatcgcgaga
caacgccaag 960 aacacattgt tcctgcaaat ggacagcctg agacccgaag
acaccggggt ctatttttgt 1020 gcaagccttt acttcggctt cccctggttt
gcttattggg gccaagggac cccggtcacc 1080 gtctccgtcg accatcatca
tcatcatcat 1110 10 1089 DNA Artificial Sequence Chimeric sequence
from multiple organisms 10 gaggctgaag ctgaattcga catccagctg
acccagagcc caagcagcct gagcgccagc 60 gtgggtgaca gagtgaccat
cacctgtaag gccagtcagg atgtgggtac ttctgtagct 120 tggtaccagc
agaagccagg taaggctcca aagctgctga tctactggac atccacccgg 180
cacactggtg tgccaagcag attcagcggt agcggtagcg gtaccgactt caccttcacc
240 atcagcagcc tccagccaga ggacatcgcc acctactact gccagcaata
tagcctctat 300 cggtcgttcg gccaagggac caaggtggaa atcaaacgtg
gaggtggcca attcatggag 360 gtccaactgg tggagagcgg tggaggtgtt
gtgcaacctg gccggtccct gcgcctgtcc 420 tgctccgcat ctggcttcga
tttcaccaca tattggatga gttgggtgag acaggcacct 480 ggaaaaggtc
ttgagtggat tggagaaatt catccagata gcagtacgat taactatgcg 540
ccgtctctaa aggatagatt tacaatatcg cgagacaacg ccaagaacac attgttcctg
600 caaatggaca gcctgagacc cgaagacacc ggggtctatt tttgtgcaag
cctttacttc 660 ggcttcccct ggtttgctta ttggggccaa gggaccccgg
tcaccgtctc cggaggcggt 720 ggatccgaca ttgtgatgac acaatctcca
tcctccctgg ctgtgtcacc cggggagagg 780 gtcactctga cctgcaaatc
cagtcagagt ctgttcaaca gtagaacccg aaagaactac 840 ttgggttggt
accagcagaa accagggcag tctcctaaac ttctgatcta ctgggcatct 900
actcgggaat ctggggtccc tgatcgcttc tcaggcagtg gatccggaac agatttcact
960 ctcaccatca acagtctgca ggctgaagac gtggcagttt attactgcac
tcaagtttat 1020 tatctgtgca cgttcggtgc tgggaccaag ctggagctga
aacggctcga ccatcatcat 1080 catcatcat 1089 11 364 PRT Artificial
Sequence Chimeric sequence from multiple species 11 Met Glu Val Gln
Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile 20 25 30
Tyr Thr Met Ser Trp Leu Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp 35
40 45 Val Ala Thr Leu Ser Gly Asp Gly Asp Asp Ile Tyr Tyr Pro Asp
Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Arg Val Arg Leu Gly Asp Trp
Asp Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Ser Val Ser
Ser Gly Gly Gly Gly Ser Asp Ile Gln 115 120 125 Leu Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr
Cys Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala Trp 145 150 155 160
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Thr 165
170 175 Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser 180 185 190 Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
Glu Asp Ile 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr
Arg Ser Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Arg Leu
Glu Gly Gly Gly Ser Glu Val 225 230 235 240 Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg Ser Leu 245 250 255 Arg Leu Ser Cys
Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr Trp Met 260 265 270 Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu 275 280 285
Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu Lys Asp 290
295 300 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe Leu
Gln 305 310 315 320 Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr
Phe Cys Ala Ser 325 330 335 Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr
Trp Gly Gln Gly Thr Pro 340 345 350 Val Thr Val Ser Val Asp His His
His His His His 355 360 12 358 PRT Artificial Sequence Chimeric
sequence from multiple species 12 Met Asp Ile Gln Leu Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Gln Asp Val Gly Thr 20 25 30 Ser Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr
Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln 65
70 75 80 Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu
Tyr Arg 85 90 95 Ser Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Gly Gly Gly Gln 100 105 110 Phe Met Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro 115 120 125 Gly Arg Ser Leu Arg Leu Ser Cys
Ser Ala Ser Gly Phe Asp Phe Thr 130 135 140 Thr Tyr Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 145 150 155 160 Trp Ile Gly
Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro 165 170 175 Ser
Leu Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 180 185
190 Leu Phe Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr
195 200 205 Phe Cys Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr
Trp Gly 210 215 220 Gln Gly Thr Pro Val Thr Val Ser Gly Gly Gly Gly
Ser Asp Ile Val 225 230 235 240 Met Thr Gln Ser Pro Ser Ser Leu Ala
Val Ser Pro Gly Glu Arg Val 245 250 255 Thr Leu Thr Cys Lys Ser Ser
Gln Ser Leu Phe Asn Ser Arg Thr Arg 260 265 270 Lys Asn Tyr Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys 275 280 285 Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 290 295 300 Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser 305 310
315 320 Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln Val Tyr
Tyr 325 330 335 Leu Cys Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
Arg Leu Asp 340 345 350 His His His His His His 355 13 1152 DNA
Artificial Sequence Chimeric sequence from multiple organisms 13
atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt ccactccatg
60 gaagtgcagc tggtggagtc agggggagac ttagtgaagc ctggagggtc
cctgaaactc 120 tcctgtgcag cctctggatt cactttcagt atttacacca
tgtcttggct tcgccagact 180 ccgggaaagg ggctggagtg ggtcgcaacc
ctgagtggtg atggtgatga catctactat 240 ccagacagtg tgaagggtcg
attcaccatc tccagagaca atgccaagaa cagcctatat 300 ctgcagatga
acagtctaag ggctgaggac acggccttgt attactgtgc aagggtgcga 360
cttggggact gggacttcga tgtctggggc caagggacca cggtctccgt ctcctcagga
420 ggtggcggat ccgacatcca gctgacccag agcccaagca gcctgagcgc
cagcgtgggt 480 gacagagtga ccatcacctg taaggccagt caggatgtgg
gtacttctgt agcttggtac 540 cagcagaagc caggtaaggc tccaaagctg
ctgatctact ggacatccac ccggcacact 600 ggtgtgccaa gcagattcag
cggtagcggt agcggtaccg acttcacctt caccatcagc 660 agcctccagc
cagaggacat cgccacctac tactgccagc aatatagcct ctatcggtcg 720
ttcggccaag ggaccaaggt ggaaatcaaa cgtctcgagg gcggaggtag cgaggtccaa
780 ctggtggaga gcggtggagg tgttgtgcaa cctggccggt ccctgcgcct
gtcctgctcc 840 gcatctggct tcgatttcac cacatattgg atgagttggg
tgagacaggc acctggaaaa 900 ggtcttgagt ggattggaga aattcatcca
gatagcagta cgattaacta tgcgccgtct 960 ctaaaggata gatttacaat
atcgcgagac aacgccaaga acacattgtt cctgcaaatg 1020 gacagcctga
gacccgaaga caccggggtc tatttttgtg caagccttta cttcggcttc 1080
ccctggtttg cttattgggg ccaagggacc ccggtcaccg tctcagtcga ccatcatcat
1140 catcatcatt ga 1152 14 1134 DNA Artificial Sequence Chimeric
sequence from multiple organisms 14 atgggatgga gctgtatcat
cctcttcttg gtagcaacag ctacaggtgt ccactccatg 60 gacatccagc
tgacccagag cccaagcagc ctgagcgcca gcgtgggtga cagagtgacc 120
atcacctgta aggccagtca ggatgtgggt acttctgtag cctggtacca gcagaagcca
180 ggtaaggctc caaagctgct gatctactgg acatccaccc ggcacactgg
tgtgccaagc 240 agattcagcg gtagcggtag cggtaccgac ttcaccttca
ccatcagcag cctccagcca 300 gaggacatcg ccacctacta ctgccagcaa
tatagcctct atcggtcgtt cggccaaggg 360 accaaggtgg aaatcaaacg
tggaggtggc caattcatgg aggtccaact ggtggagagc 420 ggtggaggtg
ttgtgcaacc tggccggtcc ctgcgcctgt cctgctccgc atctggcttc 480
gatttcacca catattggat gagttgggtg agacaggcac ctggaaaagg tcttgagtgg
540 attggagaaa ttcatccaga tagcagtacg attaactatg cgccgtcgct
aaaagataga 600 tttacaatat cgcgagacaa cgccaagaac acattgttcc
tgcaaatgga cagcctgaga 660 cccgaagaca ccggggtcta tttttgtgca
agcctttact tcggcttccc ctggtttgct 720 tattggggcc aagggacccc
ggtcaccgtc tccggaggcg gtggatccga cattgtgatg 780 acacaatctc
catcctccct ggctgtgtca cccggggaga gggtcactct gacctgcaaa 840
tccagtcaga gtctgttcaa cagtagaacc cgaaagaact acttgggttg gtaccagcag
900 aaaccagggc agtctcctaa acttctgatc tactgggcat ctactcggga
atctggggtc 960 cctgatcgct tctcaggcag tggatccgga acagatttca
ctctcaccat caacagtctg 1020 caggctgaag acgtggcagt ttattactgc
actcaagttt attatctgtg cacgttcggt 1080 gctgggacca agctggagct
gaaacggctc gaccatcatc atcatcatca ttga 1134 15 9116 DNA Artificial
Sequence Chimeric sequence from multiple organisms 15 ttccataggc
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 60
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
120 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
ttcgggaagc 180 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
cggtgtaggt cgttcgctcc 240 aagctgggct gtgtgcacga
accccccgtt cagcccgacc gctgcgcctt atccggtaac 300 tatcgtcttg
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 360
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
420 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
gccagttacc 480 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg tagcggtggt 540 ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga agatcctttg 600 atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg gattttggtc 660 atgagattat
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 720
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
780 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
ccccgtcgtg 840 tagataacta cgatacggga gggcttacca tctggcccca
gtgctgcaat gataccgcga 900 gacccacgct caccggctcc agatttatca
gcaataaacc agccagccgg aagggccgag 960 cgcagaagtg gtcctgcaac
tttatccgcc tccatccagt ctattaattg ttgccgggaa 1020 gctagagtaa
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc 1080
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
1140 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
cggtcctccg 1200 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
tggttatggc agcactgcat 1260 aattctctta ctgtcatgcc atccgtaaga
tgcttttctg tgactggtga gtactcaacc 1320 aagtcattct gagaatagtg
tatgcggcga ccgagttgct cttgcccggc gtcaacacgg 1380 gataataccg
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 1440
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
1500 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
agcaaaaaca 1560 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
ggaaatgttg aatactcata 1620 ctcttccttt ttcaatatta ttgaagcatt
tatcagggtt attgtctcat gagcggatac 1680 atatttgaat gtatttagaa
aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 1740 ggccacctga
cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 1800
tcacgaggcc ctttcgtctt caagaattcc gatccagaca tgataagata cattgatgag
1860 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
aatttgtgat 1920 gctattgctt tatttgtaac cattataagc tgcaataaac
aagttaacaa caacaattgc 1980 attcatttta tgtttcaggt tcagggggag
gtgtgggagg ttttttaaag caagtaaaac 2040 ctctacaaat gtggtatggc
tgattatgat ctaaagccag caaaagtccc atggtcttat 2100 aaaaatgcat
agctttagga ggggagcaga gaacttgaaa gcatcttcct gttagtcttt 2160
cttctcgtag acttcaaact tatacttgat gcctttttcc tcctggacct cagagaggac
2220 gcctgggtat tctgggagaa gtttatattt ccccaaatca atttctggga
aaaacgtgtc 2280 actttcaaat tcctgcatga tccttgtcac aaagagtctg
aggtggcctg gttgattcat 2340 ggcttcctgg taaacagaac tgcctccgac
tatccaaacc atgtctactt tacttgccaa 2400 ttccggttgt tcaataagtc
ttaaggcatc atccaaactt ttggcaagaa aatgagctcc 2460 tcgtggtggt
tctttgagtt ctctactgag aactatatta attctgtcct ttaaaggtcg 2520
attcttctca ggaatggaga accaggtttt cctacccata atcaccagat tctgtttacc
2580 ttccactgaa gaggttgtgg tcattctttg gaagtacttg aactcgttcc
tgagcggagg 2640 ccagggtcgg tctccgttct tgccaatccc catattttgg
gacacggcga cgatgcagtt 2700 caatggtcga accatgaggg caccaagcta
gctttttgca aaagcctagg cctccaaaaa 2760 agcctcctca ctacttctgg
aatagctcag aggccgaggc ggcctcggcc tctgcataaa 2820 taaaaaaaat
tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg 2880
ggatgggcgg agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca
2940 tacttctgcc tgctggggag cctggggact ttccacacct ggttgctgac
taattgagat 3000 gcatgctttg catacttctg cctgctgggg agcctgggga
ctttccacac cctaactgac 3060 acacattcca cagtcgacta gaatatggat
agtgggtgtt tatgactctg gataagcctg 3120 aacaattgat gattaatgcc
cctgagctct gttcttagta acatgtgaac atttacttgt 3180 gtcagtgtag
tagatttcac atgacatctt ataataaacc tgtaaatgaa agtaatttgc 3240
attactagcc cagcccagcc catactaaga gttatattat gtctgtctca cagcctgctg
3300 ctgaccaata ttgaaaagaa tagaccttcg actggcagga agcaggtcat
gtggcaaggc 3360 tatttgggga agggaaaata aaaccactag gtaaacttgt
agctgtggtt tgaagaagtg 3420 gttttgaaac actctgtcca gccccaccaa
accgaaagtc caggctgagc aaaacaccac 3480 ctgggtaatt tgcatttcta
aaataagttg aggattcagc cgaaactgga gaggtcctct 3540 tttaacttat
tgagttcaac cttttaattt tagcttgagt agttctagtt tccccaaact 3600
taagtttatc gacttctaaa atgtatttag aatttcgacc aattctcatg tttgacagct
3660 tatcatcgct gcactccgcc cgaaaagtgc gctcggctct gccaaggacg
cggggcgcgt 3720 gactatgcgt gggctggagc aaccgcctgc tgggtgcaaa
ccctttgcgc ccggactcgt 3780 ccaacgacta taaagagggc aggctgtcct
ctaagcgtca ccacgacttc aacgtcctga 3840 gtaccttctc ctcacttact
ccgtagctcc agcttcacca gatccctcga ctctagacac 3900 aggccgccac
catgggatgg agctgtatca tcctcttctt ggtagcaaca gctacaggtg 3960
tccactccat ggaagtgcag ctggtggagt cagggggaga cttagtgaag cctggagggt
4020 ccctgaaact ctcctgtgca gcctctggat tcactttcag tatttacacc
atgtcttggc 4080 ttcgccagac tccgggaaag gggctggagt gggtcgcaac
cctgagtggt gatggtgatg 4140 acatctacta tccagacagt gtgaagggtc
gattcaccat ctccagagac aatgccaaga 4200 acagcctata tctgcagatg
aacagtctaa gggctgagga cacggccttg tattactgtg 4260 caagggtgcg
acttggggac tgggacttcg atgtctgggg ccaagggacc acggtctccg 4320
tctcctcagg aggtggcgga tccgacatcc agctgaccca gagcccaagc agcctgagcg
4380 ccagcgtggg tgacagagtg accatcacct gtaaggccag tcaggatgtg
ggtacttctg 4440 tagcttggta ccagcagaag ccaggtaagg ctccaaagct
gctgatctac tggacatcca 4500 cccggcacac tggtgtgcca agcagattca
gcggtagcgg tagcggtacc gacttcacct 4560 tcaccatcag cagcctccag
ccagaggaca tcgccaccta ctactgccag caatatagcc 4620 tctatcggtc
gttcggccaa gggaccaagg tggaaatcaa acgtctcgag ggcggaggta 4680
gcgaggtcca actggtggag agcggtggag gtgttgtgca acctggccgg tccctgcgcc
4740 tgtcctgctc cgcatctggc ttcgatttca ccacatattg gatgagttgg
gtgagacagg 4800 cacctggaaa aggtcttgag tggattggag aaattcatcc
agatagcagt acgattaact 4860 atgcgccgtc tctaaaggat agatttacaa
tatcgcgaga caacgccaag aacacattgt 4920 tcctgcaaat ggacagcctg
agacccgaag acaccggggt ctatttttgt gcaagccttt 4980 acttcggctt
cccctggttt gcttattggg gccaagggac cccggtcacc gtctcagtcg 5040
accatcatca tcatcatcat tgataagatc ccgcaattct aaactctgag ggggtcggat
5100 gacgtggcca ttctttgcct aaagcattga gtttactgca aggtcagaaa
agcatgcaaa 5160 gccctcagaa tggctgcaaa gagctccaac aaaacaattt
agaactttat taaggaatag 5220 ggggaagcta ggaagaaact caaaacatca
agattttaaa tacgcttctt ggtctccttg 5280 ctataattat ctgggataag
catgctgttt tctgtctgtc cctaacatgc cctgtgatta 5340 tccgcaaaca
acacacccaa gggcagaact ttgttactta aacaccatcc tgtttgcttc 5400
tttcctcagg aactgtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt
5460 tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc
agagaggcca 5520 aagtacagtg gaaggtggat aacgccctcc aatcgggtaa
ctcccaggag agtgtcacag 5580 agcaggacag caaggacagc acctacagcc
tcagcagcac cctgacgctg agcaaagcag 5640 actacgagaa acacaaagtc
tacgcctgcg aagtcaccca tcagggcctg agctcgcccg 5700 tcacaaagag
cttcaacagg ggagagtgtt agagggagaa gtgcccccac ctgctcctca 5760
gttccagcct gaccccctcc catcctttgg cctctgaccc tttttccaca ggggacctac
5820 ccctattgcg gtcctccagc tcatctttca cctcaccccc ctcctcctcc
ttggctttaa 5880 ttatgctaat gttggaggag aatgaataaa taaagtgaat
ctttgcacct gtggtttctc 5940 tctttcctca tttaataatt attatctgtt
gttttaccaa ctactcaatt tctcttataa 6000 gggactaaat atgtagtcat
cctaaggcgc ataaccattt ataaaaatca tccttcattc 6060 tattttaccc
tatcatcctc tgcaagacag tcctccctca aacccacaag ccttctgtcc 6120
tcacagtccc ctgggccatg gtaggagaga cttgcttcct tgttttcccc tcctcagcaa
6180 gccctcatag tcctttttaa gggtgacagg tcttacagtc atatatcctt
tgattcaatt 6240 ccctgagaat caaccaaagc aaatttttca aaagaagaaa
cctgctataa agagaatcat 6300 tcattgcaac atgatataaa ataacaacac
aataaaagca attaaataaa caaacaatag 6360 ggaaatgttt aagttcatca
tggtacttag acttaatgga atgtcatgcc ttatttacat 6420 ttttaaacag
gtactgaggg actcctgtct gccaagggcc gtattgagta ctttccacaa 6480
cctaatttaa tccacactat actgtgagat taaaaacatt cattaaaatg ttgcaaaggt
6540 tctataaagc tgagagacaa atatattcta taactcagca attcccactt
ctaggggttc 6600 gactggcagg aagcaggtca tgtggcaagg ctatttgggg
aagggaaaat aaaaccacta 6660 ggtaaacttg tagctgtggt ttgaagaagt
ggttttgaaa cactctgtcc agccccacca 6720 aaccgaaagt ccaggctgag
caaaacacca cctgggtaat ttgcatttct aaaataagtt 6780 gaggattcag
ccgaaactgg agaggtcctc ttttaactta ttgagttcaa ccttttaatt 6840
ttagcttgag tagttctagt ttccccaaac ttaagtttat cgacttctaa aatgtattta
6900 gaatttcgac caattctcat gtttgacagc ttatcatcgc tgcactccgc
ccgaaaagtg 6960 cgctcggctc tgccaaggac gcggggcgcg tgactatgcg
tgggctggag caaccgcctg 7020 ctgggtgcaa accctttgcg cccggactcg
tccaacgact ataaagaggg caggctgtcc 7080 tctaagcgtc accacgactt
caacgtcctg agtaccttct cctcacttac tccgtagctc 7140 cagcttcacc
agatccctcg agtctagaca caggccgcca ccatgggatg gagctgtatc 7200
atcctcttct tggtagcaac agctacaggt gtccactcca tggacatcca gctgacccag
7260 agcccaagca gcctgagcgc cagcgtgggt gacagagtga ccatcacctg
taaggccagt 7320 caggatgtgg gtacttctgt agcttggtac cagcagaagc
caggtaaggc tccaaagctg 7380 ctgatctact ggacatccac ccggcacact
ggtgtgccaa gcagattcag cggtagcggt 7440 agcggtaccg acttcacctt
caccatcagc agcctccagc cagaggacat cgccacctac 7500 tactgccagc
aatatagcct ctatcggtcg ttcggccaag ggaccaaggt ggaaatcaaa 7560
cgtggaggtg gccaattcat ggaggtccaa ctggtggaga gcggtggagg tgttgtgcaa
7620 cctggccggt ccctgcgcct gtcctgctcc gcatctggct tcgatttcac
cacatattgg 7680 atgagttggg tgagacaggc acctggaaaa ggtcttgagt
ggattggaga aattcatcca 7740 gatagcagta cgattaacta tgcgccgtct
ctaaaggata gatttacaat atcgcgagac 7800 aacgccaaga acacattgtt
cctgcaaatg gacagcctga gacccgaaga caccggggtc 7860 tatttttgtg
caagccttta cttcggcttc ccctggtttg cttattgggg ccaagggacc 7920
ccggtcaccg tctccggagg cggtggatcc gacattgtga tgacacaatc tccatcctcc
7980 ctggctgtgt cacccgggga gagggtcact ctgacctgca aatccagtca
gagtctgttc 8040 aacagtagaa cccgaaagaa ctacttgggt tggtaccagc
agaaaccagg gcagtctcct 8100 aaacttctga tctactgggc atctactcgg
gaatctgggg tccctgatcg cttctcaggc 8160 agtggatccg gaacagattt
cactctcacc atcaacagtc tgcaggctga agacgtggca 8220 gtttattact
gcactcaagt ttattatctg tgcacgttcg gtgctgggac caagctggag 8280
ctgaaacggc tcgaccatca tcatcatcat cattgataag atctcggccg gcaagccccc
8340 gctccccggg ctctcgcggt cgcacgagga tgcttggcac gtaccccgtc
tacatacttc 8400 ccaggcaccc agcatggaaa taaagcaccc accactgccc
tgggcccctg cgagactgtg 8460 atggttcttt ccacgggtca ggccgagtct
gaggcctgag tggcatgagg gaggcagagc 8520 gggtcccact gtccccacac
tggcccaggc tgtgcaggtg tgcctgggcc gcctagggtg 8580 gggctcagcc
aggggctgcc ctcggcaggg tgggggattt gccagcgtgg ccctccctcc 8640
agcagcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
8700 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
ccgtcagggc 8760 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
cccagtcacg tagcgatagc 8820 ggagtgtata ctggcttaac tatgcggcat
cagagcagat tgtactgaga gtgcaccata 8880 tgcggtgtga aataccgcac
agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg 8940 cttcctcgct
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 9000
actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
9060 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgttt
9116 16 5 PRT Artificial Sequence Description of Artificial
Sequence Linker peptide 16 Gly Gly Gly Gly Ser 1 5 17 6 PRT
Artificial Sequence Description of Artificial Sequence Linker
peptide 17 Leu Glu Gly Gly Gly Ser 1 5 18 6 PRT Artificial Sequence
Description of Artificial Sequence Linker peptide 18 Gly Gly Gly
Gln Phe Met 1 5 19 5 PRT Artificial Sequence Description of
Artificial Sequence Linker peptide 19 Gly Gly Gly Gly Ser 1 5 20 6
PRT Artificial Sequence Description of Artificial Sequence 6-His
tag 20 His His His His His His 1 5
* * * * *