U.S. patent application number 10/976352 was filed with the patent office on 2005-09-29 for psma formulations and uses thereof.
This patent application is currently assigned to PSMA Development Company, LLC. Invention is credited to Maddon, Paul J., Olson, William C., Schulke, Norbert.
Application Number | 20050215472 10/976352 |
Document ID | / |
Family ID | 46303153 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050215472 |
Kind Code |
A1 |
Schulke, Norbert ; et
al. |
September 29, 2005 |
PSMA formulations and uses thereof
Abstract
The invention includes stable multimeric, particularly dimeric,
forms of PSMA protein, compositions and kits containing dimeric
PSMA protein as well as methods of producing, purifying and using
these compositions. Such methods include methods for eliciting or
enhancing an immune response to cells expressing PSMA, including
methods of producing antibodies to dimeric PSMA, as well as methods
of treating cancer, such as prostate cancer.
Inventors: |
Schulke, Norbert; (New City,
NY) ; Maddon, Paul J.; (Scarsdale, NY) ;
Olson, William C.; (Ossining, NY) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Assignee: |
PSMA Development Company,
LLC
Tarrytown
NY
|
Family ID: |
46303153 |
Appl. No.: |
10/976352 |
Filed: |
October 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10976352 |
Oct 27, 2004 |
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10695667 |
Oct 27, 2003 |
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10695667 |
Oct 27, 2003 |
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10395894 |
Mar 21, 2003 |
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10395894 |
Mar 21, 2003 |
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PCT/US02/33944 |
Oct 23, 2002 |
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60335215 |
Oct 23, 2001 |
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60362747 |
Mar 7, 2002 |
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60412618 |
Sep 20, 2002 |
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Current U.S.
Class: |
424/185.1 ;
514/19.5; 514/19.8 |
Current CPC
Class: |
G01N 33/57434 20130101;
A61K 51/1072 20130101; C07K 2317/21 20130101; A61K 51/1093
20130101; A61K 39/001195 20180801; A61K 2039/505 20130101; A61K
47/6869 20170801; C07K 16/40 20130101; A61K 51/1096 20130101; C07K
16/3069 20130101; C07K 14/4748 20130101; C07K 2317/622 20130101;
A61K 39/39558 20130101; A61K 47/6825 20170801; A61P 35/00 20180101;
A61P 37/04 20180101; A61K 38/1709 20130101; C07K 2317/565
20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
1. A composition comprising isolated PSMA protein, wherein at least
5% of the isolated PSMA protein is an isolated PSMA protein
multimer.
2. The composition of claim 1, wherein the isolated PSMA protein
multimer is an isolated PSMA protein dimer.
3-12. (canceled)
13. The composition of claim 1, wherein the composition further
comprises at least 0.25 molar equivalents of metal ion to PSMA
protein.
14-16. (canceled)
17. The composition of claim 1, wherein the composition is in a
liquid or lyophilized form.
18. The composition of claim 1, wherein the composition further
comprises an adjuvant.
19. The composition of claim 18, wherein the adjuvant is alum;
monophosphoryl lipid A; a saponin; QS-7; QS-17; QS-18; QS-21; a
saponin fraction; a saponin-based adjuvant; SaponImmune.TM.;
PolysaccImmune.TM.; SynthImmune.TM.; an immunostimulatory
oligonucleotide; incomplete Freund's adjuvant; complete Freund's
adjuvant; montanide; MONTANIDE ISA51; MONTANIDE ISA720; vitamin E,
a water-in-oil emulsions prepared from a biodegradable oil; Quil A;
a micellular mixture of Quil A and cholesterol known as
immunostimulating complexes (ISCOMS); a MPL and mycobacterial cell
wall skeleton combination; ENHANZYN.TM.; RC-529; RC-552; CRL-1005,
L-121, alpha-galactosylceramide; a composition of biodegradable
particles composed of poly-lactide-co-glycolide (PLG); a
composition of aluminum or iron oxide beads or a combination
thereof.
20-21. (canceled)
22. The composition of claim 1, wherein the composition further
comprises a cancer therapeutic agent
23-24. (canceled)
25. The composition of claim 1, wherein the composition further
comprises a cytokine.
26. (canceled)
27. The composition of claim 1, wherein the composition is free of
chelating agents.
28. The composition of claim 1, wherein the composition further
comprises at least one buffer.
29. (canceled)
30. The composition of claim 1, wherein the composition further
comprises a free amino acid, wherein the free amino acid is
naturally occurring or non-naturally occurring.
31-32. (canceled)
33. The composition of claim 1, wherein the composition further
comprises a surfactant.
34. (canceled)
35. The composition of claim 1, wherein the composition further
comprises a cryoprotectant, an antioxidant, a preservative or a
combination thereof.
36-46. (canceled)
47. A composition comprising isolated multimeric PSMA protein,
wherein the composition comprises less than 35% of a monomeric PSMA
protein.
48. The composition of claim 47, wherein the isolated multimeric
PSMA protein is an isolated dimeric PSMA protein.
49-51. (canceled)
52. A composition comprising PSMA protein in a solution that
promotes or preserves multimeric association of PSMA protein.
53. The composition of claim 52, wherein the solution that promotes
or preserves multimeric association of PSMA protein is a solution
that promotes or preserves dimeric association of PSMA protein.
54. The composition of claim 52, wherein the solution that promotes
or preserves dimeric association of PSMA protein has a pH that
ranges from 4 to 8.
55-57. (canceled)
58. The composition of claim 52, wherein the solution that promotes
or preserves dimeric association of PSMA protein comprises a
salt.
59-63. (canceled)
64. The composition of claim 58, wherein the composition further
comprises an adjuvant.
65. (canceled)
66. The composition of claim 52, wherein the solution that promotes
or preserves dimeric association of PSMA protein comprises metal
ions.
67. The composition of claim 66, wherein the metal ions are zinc
ions, calcium ions, magnesium ions, cobalt ions, manganese ions or
a combination thereof.
68. The composition of claim 67, wherein the metal ions are zinc
ions and calcium ions.
69. (canceled)
70. The composition of claim 68, wherein the zinc ions are present
at a concentration that is lower than the concentration of the
calcium ions.
71-74. (canceled)
75. The composition of claim 67, wherein the metal ions are
magnesium ions and calcium ions.
76. The composition of claim 52, wherein the solution that promotes
or preserves dimeric association of PSMA protein is free of
chelating agents.
77-99. (canceled)
100. A composition comprising isolated PSMA protein in a solution
that promotes or preserves dimeric association of PSMA protein
wherein the solution comprises: (a) 5 to 20 mM of sodium phosphate,
sodium acetate or a combination thereof, (b) 100 to 300 mM sodium
chloride or sodium sulfate, and (c) 0.1 to 2 mM of at least one
metal ion.
101. The composition of claim 100, wherein the solution has a pH in
the range of 4 to 8.
102-103. (canceled)
104. The composition of claim 101, wherein the composition further
comprises an adjuvant.
105. The composition of claim 104, wherein the adjuvant is alum or
a saponin-based adjuvant.
106. (canceled)
107. The composition of claim 100, wherein the composition further
comprises a cancer therapeutic agent.
108-110. (canceled)
111. A composition comprising isolated PSMA protein in a solution
that promotes or preserves dimeric association of PSMA protein
wherein the solution comprises: (a) 1.47 mM potassium phosphate,
monobasic, (b) 8.1 mM sodium phosphate, dibasic, (c) 2.68 mM
potassium chloride, (d) 0.14 M sodium chloride, (e) 0.9 mM calcium
chloride, and (f) 0.49 mM magnesium chloride; and wherein the
solution has a pH of 7.0.
112. The composition of claim 111, wherein the isolated PSMA
protein is at a concentration of between 0.2 mg/mL and 10
mg/mL.
113-115. (canceled)
116. The composition of claim 111, wherein the composition further
comprises an adjuvant.
117. The composition of claim 116, wherein the adjuvant is a
saponin-based adjuvant.
118-121. (canceled)
122. A method of promoting or preserving dimeric association of
PSMA protein in a solution comprising: obtaining a solution of PSMA
protein, and adjusting the pH to be in the range of 4 to 8.
123. The method of claim 122, wherein the pH is adjusted to be in
the range of 5 to 7.
124-125. (canceled)
126. A method of processing a PSMA protein comprising: contacting
the PSMA protein in a solution with a first agent that promotes or
preserves dimeric association of PSMA protein in an amount
effective to promote or preserve PSMA protein dimer formation.
127. The method of claim 126, wherein the amount effective to
promote or preserve PSMA protein dimer formation is enough to
promote or maintain at least 5%, 25%, 50%, 75% or 95% of the PSMA
protein in PSMA dimer form.
128. The method of claim 126, wherein the first agent that promotes
or preserves dimeric association of PSMA protein is a salt, metal
ion or a pH adjusting agent.
129-132. (canceled)
133. The method of claim 126, further comprising combining the PSMA
protein solution with an adjuvant or diluent.
134. The method of claim 133, wherein the adjuvant or diluent is
combined with the PSMA protein in an amount to dilute the salt
concentration to 100 mM to 300 mM.
135-143. (canceled)
144. The method of claim 128, wherein the pH of the solution is
adjusted to be in the range of 4 to 8.
145-147. (canceled)
148. The method of claim 128, wherein the method further comprises
contacting the PSMA protein with a second agent that promotes or
preserves dimeric association of PSMA protein, and wherein the
second agent is different than the first agent.
149. The method of claim 148, wherein the second agent that
promotes or preserves dimeric association of PSMA protein is a
metal ion, salt or pH adjusting agent.
150-152. (canceled)
153. The method of claim 148, wherein the pH of the solution is
adjusted to be in the range of 4 to 8.
154-156. (canceled)
157. A method of purifying a sample containing PSMA protein
comprising: subjecting the sample containing PSMA to chromatography
in the presence of an agent that preserves or promotes the dimeric
association of PSMA.
158. The method of claim 157, wherein the agent that promotes or
preserves the dimeric association of PSMA is a metal ion, a salt or
a solution with a pH in the range of 4 to 8 or a combination
thereof.
159. The method of claim 158, wherein the metal ion is a zinc ion,
calcium ion, magnesium ion, cobalt ion, manganese ion or a
combination thereof.
160. The method of claim 159, wherein the metal ion is a
combination of calcium ion and magnesium ion.
161-168. (canceled)
169. A method of purifying a sample containing PSMA protein
comprising: applying the sample to a first column, washing the
first column with a first wash solution containing salt and metal
ions, and collecting the PSMA protein that elutes from the first
column.
170. The method of claim 169, wherein the metal ions are zinc ions,
calcium ions, magnesium ions, cobalt ions, manganese ions or a
combination thereof.
171. The method of claim 170, wherein the metal ions are calcium
and magnesium ions.
172-173. (canceled)
174. The method of claim 169, wherein the cationic component of the
salt is sodium, potassium, ammonium, magnesium, calcium, zinc or a
combination thereof, and wherein the anionic component of the salt
is chloride, sulfate acetate or a combination thereof.
175. The method of claim 174, wherein the salt is ammonium sulfate
at a saturation of no more than 35% in the wash solution.
176. The method of claim 169, further comprising dialyzing or
diafiltering the eluted PSMA protein with a first salt solution at
a pH in the range of 6 to 7.5 to yield a dialyzed or diafiltrated
solution containing PSMA protein.
177. The method of claim 176, wherein the first salt solution has a
salt concentration of at least 5 mM.
178. (canceled)
179. The method of claim 169, further comprising: loading the
eluted PSMA protein, dialyzed or diafiltrated solution containing
PSMA protein onto a second column, washing the second column with a
second salt solution, and collecting the PSMA eluted by the second
salt solution.
180. The method of claim 179, wherein the second salt solution has
a salt concentration of 100 mM to 2 M.
181. (canceled)
182. The method of claim 179, wherein the second salt solution has
a pH in the range of 6 to 7.5.
183. The method of claim 179, further comprising dialyzing or
diafiltrating the PSMA eluted by the second salt solution with a
metal ion solution, applying the dialyzed or diafiltrated PSMA
eluted by the second salt solution onto a third column, washing the
third column with a second wash solution containing salt and metal
ions and collecting the PSMA eluted.
184. The method of claim 183, wherein the pH is maintained in the
range of 6 to 7.5 through all of the purification steps.
185. The method of claim 183, further comprising separating the
different forms of PSMA protein, wherein the different forms of
PSMA protein are monomeric, dimeric or other multimeric forms of
PSMA.
186. (canceled)
187. A method of identifying an agent which promotes or preserves
dimeric association of PSMA protein comprising: determining the
amount of a form of PSMA protein in a sample prior to exposure to a
candidate agent, exposing the sample to the candidate agent,
determining the amount of the form of PSMA protein in the sample
after the exposure, and comparing the amount of the form of PSMA
protein in the sample prior to and after the exposure.
188. (canceled)
189. A method of treating a subject to elicit or enhance an immune
response to cells in the subject expressing PSMA, comprising
administering to the subject an effective amount of the composition
of any one of claims 1, 47, 52, 100 and 111.
190. The method of claim 189, wherein the expressed PSMA is
expressed on the cell surface.
191. The method of claim 189, wherein the method further comprises
administering one or more booster doses of a composition comprising
PSMA protein.
192. The method of claim 191, wherein the composition comprising
PSMA protein is a composition of PSMA protein dimer.
193. The method of claim 191, wherein the booster dose composition
further comprises an adjuvant.
194-196. (canceled)
197. The method of claim 189, wherein the subject has cancer or has
been treated for cancer.
198. (canceled)
199. The method of claim 197, wherein the subject has prostate
cancer.
200. A method of eliciting an immune response, comprising
administering to a subject an effective amount of the composition
of any one of claims 1, 47, 52, 100 and 111.
201. The method of claim 200, wherein the method further comprises
administering one or more booster doses of a composition comprising
PSMA protein.
202. The method of claim 201, wherein the composition comprising
PSMA protein is a composition PSMA protein dimer.
203. The method of claim 201, wherein the booster dose composition
further comprises an adjuvant.
204-206. (canceled)
207. A kit which comprises the composition of any one of claims 1,
47, 52, 100 and 111 and instructions for use.
208. A kit which comprises the composition of any one of claims 1,
47, 52, 100 and 111, an adjuvant and instructions for mixing.
209-210. (canceled)
211. A kit which comprises the composition of any one of claims 1,
47, 52, 100 and 111, a diluent and instructions for mixing.
212-213. (canceled)
214. A pharmaceutical composition comprising the composition of any
one of the compositions of claims 1, 47, 52, 100 and 111, and a
pharmaceutically acceptable carrier.
215. A method of treating prostate cancer in a subject, comprising:
administering to the subject a therapeutically effective amount of
a composition comprising isolated PSMA protein in a solution that
promotes or preserves dimeric association of the PSMA protein,
wherein the composition is effective in treating prostate
cancer.
216. The method of claim 215, wherein the composition that is
administered further comprises an adjuvant.
217. The method of claim 216, wherein the adjuvant is a
saponin-based adjuvant.
218. The method of claim 215, wherein the method further comprises
administering to the subject a conventional prostate cancer
therapy.
219. (canceled)
220. A method of inhibiting metastasis in a subject with prostate
cancer, comprising: administering to the subject a therapeutically
effective amount of a composition comprising isolated PSMA protein
in a solution that promotes or preserves dimeric association of the
PSMA protein, wherein the composition is effective in inhibiting
metastasis.
221. The method of claim 220, wherein the composition that is
administered further comprises an adjuvant.
222. The method of claim 221, wherein the adjuvant is a
saponin-based adjuvant.
223. The method of claim 220, wherein the method further comprises
administering to the subject a conventional prostate cancer
therapy.
224. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of United States
nonprovisional application Ser. No. 10/695,667, filed on Oct. 27,
2003, which is a continuation-in-part of United States
nonprovisional application Ser. No. 10/395,894, filed on Mar. 21,
2003, which is a continuation-in-part of International application
PCT/US02/33944 designating the United States, filed on Oct. 23,
2002, which claims the benefit under 35 U.S.C. .sctn. 119 of U.S.
provisional application 60/335,215, filed Oct. 23, 2001, U.S.
provisional application 60/362,747, filed Mar. 7, 2002, and U.S.
provisional application 60/412,618, filed Sep. 20, 2002, each of
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of cancer
associated polypeptides and formulations of and kits including
these polypeptides. In particular, the invention relates, in part,
to formulations of multimeric forms of PSMA proteins, particularly
dimeric PSMA, and methods of their processing, purification,
production and use.
BACKGROUND OF THE INVENTION
[0003] Prostate cancer is the most prevalent type of cancer and the
second leading cause of death from cancer in American men, with an
estimated 179,000 cases and 37,000 deaths in 1999, (Landis, S. H.
et al. CA Cancer J. Clin. 48:6-29 (1998)). The number of men
diagnosed with prostate cancer is steadily increasing as a result
of the increasing population of older men as well as a greater
awareness of the disease leading to its earlier diagnosis (Parker
et al., 1997, CA Cancer J. Clin. 47:5-280). The life time risk for
men developing prostate cancer is about 1 in 5 for Caucasians, 1 in
6 for African Americans. High risk groups are represented by those
with a positive family history of prostate cancer or African
Americans.
[0004] Over a lifetime, more than 2/3 of the men diagnosed with
prostate cancer die of the disease (Wingo et al., 1996, CA Cancer
J. Clin. 46:113-25). Moreover, many patients who do not succumb to
prostate cancer require continuous treatment to ameliorate symptoms
such as pain, bleeding and urinary obstruction. Thus, prostate
cancer also represents a major cause of suffering and increased
health care expenditures.
[0005] Where prostate cancer is localized and the patient's life
expectancy is 10 years or more, radical prostatectomy offers the
best chance for eradication of the disease. Historically, the
drawback of this procedure is that most cancers had spread beyond
the bounds of the operation by the time they were detected.
Patients with bulky, high-grade tumors are less likely to be
successfully treated by radical prostatectomy.
[0006] Radiation therapy has also been widely used as an
alternative to radical prostatectomy. Patients generally treated by
radiation therapy are those who are older and less healthy and
those with higher-grade, more clinically advanced tumors.
Particularly preferred procedures are external-beam therapy which
involves three dimensional, confocal radiation therapy where the
field of radiation is designed to conform to the volume of tissue
treated; interstitial-radiation therapy where seeds of radioactive
compounds are implanted using ultrasound guidance; and a
combination of external-beam therapy and interstitial-radiation
therapy.
[0007] For treatment of patients with locally advanced disease,
hormonal therapy before or following radical prostatectomy or
radiation therapy has been utilized. Hormonal therapy is the main
form of treating men with disseminated prostate cancer. Orchiectomy
reduces serum testosterone concentrations, while estrogen treatment
is similarly beneficial. Diethylstilbestrol from estrogen is
another useful hormonal therapy which has a disadvantage of causing
cardiovascular toxicity. When gonadotropin-releasing hormone
agonists are administered testosterone concentrations are
ultimately reduced. Flutamide and other nonsteroidal, anti-androgen
agents block binding of testosterone to its intracellular
receptors. As a result, it blocks the effect of testosterone,
increasing serum testosterone concentrations and allows patients to
remain potent--a significant problem after radical prostatectomy
and radiation treatments.
[0008] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. Its toxicity makes such therapy unsuitable for
elderly patients. In addition, prostate cancer is relatively
resistant to cytotoxic agents.
[0009] Relapsed or more advanced disease is also treated with
anti-androgen therapy. Unfortunately, almost all tumors become
hormone-resistant and progress rapidly in the absence of any
effective therapy.
[0010] Accordingly, there is a need for effective therapeutics for
prostate cancer which are not overwhelmingly toxic to normal
tissues of a patient, and which are effective in selectively
eliminating prostate cancer cells.
SUMMARY OF THE INVENTION
[0011] The present invention relates, in part, to multimeric,
particularly dimeric, forms of PSMA protein, compositions and kits
containing dimeric PSMA protein as well as methods of producing,
purifying, processing and using these compositions.
[0012] In one aspect compositions comprising multimeric forms of
PSMA protein are provided. In some embodiments, these compositions
contain isolated PSMA protein, at least 5% of which is in the form
of PSMA protein multimer. In other embodiments at least 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or more of the isolated PSMA protein is in the form
of a PSMA protein multimer. In other embodiments the PSMA protein
multimer is a PSMA protein dimer, wherein the PSMA protein dimer is
formed by the covalent or non-covalent association of two PSMA
proteins. In some embodiments the PSMA protein dimer is engineered
to form a stable PSMA dimer through covalent bonds. In some
embodiments the covalent bonds are disulfide bonds. Preferably, the
PSMA protein dimer is associated in the same way as that of native
PSMA dimer or is associated in such a way as to form at least one
antigenic epitope that can be used to generate antibodies that
recognize the native PSMA dimer. These antibodies, preferably,
recognize the native PSMA dimer and not PSMA monomer or recognize
the native PSMA dimer with greater specificity. In some embodiments
of the invention the percent dimer can be calculated in terms of
the number of PSMA protein molecules in the dimeric form versus the
total number of PSMA protein (monomer, dimer or other multimer). In
other embodiments the percent dimer can be calculated in terms of
the number of PSMA dimers relative to the number of PSMA monomers,
PSMA dimers and PSMA multimers.
[0013] In some embodiments the PSMA protein multimers comprise the
full-length PSMA protein (SEQ ID NO: 1) or a fragment thereof. In
other embodiments the PSMA protein multimer comprises the
extracellular portion of PSMA (amino acids 44-750 of SEQ ID NO: 1)
or a fragment thereof. In still other embodiments the PSMA protein
multimer comprises the amino acids 58-750 of SEQ ID NO: 1 or a
fragment thereof. In yet other embodiments the PSMA protein
multimer comprises the amino acids 610-750 of SEQ ID NO: 1 or a
fragment thereof. The fragments are capable of forming a PSMA
multimer that can be used to generate antibodies that recognize
PSMA, preferably native PSMA dimer. Typically, the PSMA multimers
are homomultimers, meaning that the two or more PSMA molecules are
the same. In other embodiments, the PSMA multimers are
heteromultimers, whereby at least two of the PSMA proteins are not
the same. In still other embodiments the PSMA proteins can be
functionally equivalent proteins, whereby the PSMA protein is
conservatively substituted.
[0014] In another aspect of the invention compositions comprising
isolated multimeric PSMA protein, wherein the composition comprises
less than 35% of a monomeric PSMA protein are provided. In still
other embodiments the composition comprises less than 20% of the
monomeric PSMA protein. In yet other embodiments the composition
comprises less than 15% of the monomeric PSMA protein. In still
other embodiments the composition comprises less than 5% of the
monomeric PSMA protein. In some preferred embodiments the isolated
multimeric PSMA protein is an isolated dimeric PSMA protein.
[0015] In some aspects of the invention, agents and compositions
thereof that preserve or promote multimeric association of PSMA,
particularly dimeric association, are provided. In some embodiments
these agents include metal ions, salts, or pH adjusting agents.
These agents that preserve or promote multimeric PSMA associations
can do so individually or do so in combination. Therefore, in
another aspect of the invention, a composition comprising PSMA
protein multimers in conjunction with metal ion are provided. In
some embodiments these compositions comprise at least 0.25 molar
equivalents of metal ion to PSMA protein (total PSMA protein
regardless of its form). In other embodiments at least 0.3, 0.4,
0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.3, 1.5, 1.7, 2, 3, 4, 5, or more
molar equivalents of metal ion to PSMA protein are present in the
composition. In other embodiments the metal ion is in molar excess
to PSMA protein. In some preferred embodiments the compositions
provided are free of chelating agents.
[0016] In yet another aspect of the invention compositions
comprising PSMA protein in a solution that promotes or preserves
multimeric association of PSMA protein are provided. In some
embodiments the solution that promotes or preserves multimeric
association of PSMA protein is a solution that promotes or
preserves dimeric association of PSMA protein. In other embodiments
the solution that promotes or preserves dimeric association of PSMA
protein has a pH that ranges from 4 to 8. In still other
embodiments the solution that promotes or preserves dimeric
association of PSMA protein has a pH that ranges from 5 to 7. Other
embodiments include compositions wherein the solution that promotes
or preserves dimeric association of PSMA protein has a pH that
ranges from 5.5 to 7. In still other embodiments the solution that
promotes or preserves dimeric association of PSMA protein has a pH
of 6.
[0017] In still another aspect of the invention compositions
comprising PSMA protein in a solution that promotes or preserves
multimeric association of PSMA protein, wherein the solution
comprises a salt, are provided. In some embodiments, the cationic
component of the salt is sodium, potassium, ammonium, magnesium,
calcium, zinc or a combination thereof, and the anionic component
of the salt is chloride, sulfate, acetate or a combination thereof.
In preferred embodiments the salt is sodium chloride, sodium
sulfate, sodium acetate or ammonium sulfate. In some embodiments
the salt is present at a concentration in the range of 50 mM to 2
M. In other embodiments the salt is present at a concentration in
the range of 100 mM to 300 mM. In still other embodiments the salt
is present at a concentration of 150 mM.
[0018] In yet another aspect of the invention a composition
comprising PSMA protein in a solution that promotes or preserves
dimeric association of PSMA protein, wherein the solution comprises
metal ions, are provided. In some embodiments the metal ions are
zinc ions, calcium ions, magnesium ions, cobalt ions, manganese
ions or a combination thereof. In still other embodiments the metal
ions are zinc ions and calcium ions. In yet other embodiments the
zinc ions and calcium ions are present at a concentration in the
range of 0.1 mM to 5 mM. In still other embodiments the zinc ions
are present at a concentration that is lower than the concentration
of the calcium ions. In some embodiments the zinc ions are present
at a concentration of 0.1 mM and the calcium ions are present at a
concentration of 1 mM. In other embodiments the metal ions are
magnesium ions. In some of these embodiments the magnesium ions are
present at a concentration in the range of 0.1 mM to 5 mM. In other
embodiments the magnesium ions are present at a concentration of
0.5 mM. In another embodiment the metal ions are magnesium and
calcium ions. In a preferred embodiment the compositions are free
of chelating agents.
[0019] In still a further aspect of the invention a composition
comprising isolated PSMA protein in a solution that promotes or
preserves dimeric association of PSMA protein wherein the solution
comprises (a) 5 to 20 mM of sodium phosphate, sodium acetate or a
combination thereof, (b) 100 to 300 mM sodium chloride or sodium
sulfate, and (c) 0.1 to 2 mM of at least one metal ion is provided.
In one embodiment the solution has a pH in the range of 4 to 8. In
another embodiment the solution has a pH in a range of 5 to 7. In
still another embodiment the solution has a pH in a range of 6 to
6.5. The metal ion in some embodiments is a zinc ion, calcium ion,
magnesium ion, cobalt ion, manganese ion or a combination
thereof.
[0020] In still another aspect of the invention a composition
comprising isolated PSMA protein in a solution that promotes or
preserves dimeric association of PSMA protein wherein the solution
comprises (a) 1.47 mM potassium phosphate, monobasic, (b) 8.1 mM
sodium phosphate, dibasic, (c) 2.68 mM potassium chloride, (d) 0.14
M sodium chloride, (e) 0.9 mM calcium chloride, and (f) 0.49 mM
magnesium chloride; and wherein the solution has a pH of 7.0 is
provided. In one embodiment the isolated PSMA protein is at a
concentration of between 0.2 mg/mL and 10 mg/mL. In still a further
embodiment the isolated PSMA protein is at a concentration of
between 2 mg/mL and 5 mg/mL. In other embodiments the isolated PSMA
protein is at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.5, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14,
15, 17, 20, 22, 25, 30, 35, 40, 45, 50 mg/mL or more. In another
embodiment the isolated PSMA protein is at a concentration of 0.2
mg/mL. In still a further embodiment the isolated PSMA protein is
at a concentration of 2 mg/mL. In some embodiments the compositions
provided further comprise an adjuvant. In one embodiment the
adjuvant is a saponin-based adjuvant. In another embodiment the
saponin-based adjuvant is QS-21. The adjuvant in some embodiments
is in an amount of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, 175, 200,
225, 250, 300 .mu.g or more. In one embodiment the QS-21 is in an
amount of between 50 .mu.g and 150 .mu.g. In another embodiment the
QS-21 is in an amount of 50 .mu.g. In another embodiment the QS-21
is in an amount of 100 .mu.g. When such compositions are
administered to a subject, in some embodiments, the amount of the
adjuvant is the amount of the adjuvant per dose to the subject.
[0021] In another aspect of the invention a composition comprising
PSMA protein which also comprises an agent that promotes or
preserves multimeric association, particularly dimeric association
of PSMA protein, is provided, wherein the composition is stable
when stored at -80.degree. C. In other aspects of the invention the
composition is stable when stored at -20.degree. C. In still other
aspects the composition is stable when stored at 4.degree. C. In
yet another aspect of the invention the composition is stable when
stored at room temperature.
[0022] Another aspect of the invention provides a method of
promoting or preserving dimeric association of PSMA protein in a
solution by obtaining a solution of PSMA protein, and adjusting the
pH to be in the range of 4 to 8. In some embodiments the pH is
adjusted to be in the range of 5 to 7. In other embodiments the pH
is adjusted to be in the range of 5.5 to 7. In yet other
embodiments the pH is adjusted to be 6.
[0023] In another aspect of the invention a method of processing a
PSMA protein by contacting the PSMA protein in a solution with a
first agent that promotes or preserves dimeric association of PSMA
protein in an amount effective to promote or preserve PSMA protein
dimer formation is provided. In some embodiments the amount
effective to promote or preserve PSMA protein dimer formation is
enough to promote or maintain at least 5% of the PSMA protein in
the solution in dimer form. In other embodiments at least 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or more of the PSMA protein in the solution is in
dimer form. The percentage of the dimer form of PSMA is calculated
in terms of the total amount of the various forms of PSMA protein.
In other words the percentage is calculated according to the number
of PSMA dimers relative to the number of PSMA monomers, dimers and
other multimers. In some embodiments the first agent that promotes
or preserves dimeric association of PSMA protein is a salt, metal
ion or a pH adjusting agent. The cationic components of the salt
can include sodium, potassium, ammonium, magnesium, calcium, zinc
or a combination thereof, while the anionic component of the salt
can include chloride, sulfate, acetate or a combination thereof. In
some embodiments the salt is sodium chloride, sodium sulfate,
sodium acetate or ammonium sulfate. In other embodiments the salt
is present at a concentration in the range of 50 mM to 2 M. In
still other embodiments the salt is present at a concentration in
the range of 100 mM to 300 mM. In yet other embodiments the salt is
present at a concentration of 150 mM. In some embodiments of the
invention the method further includes combining the PSMA protein
solution with an adjuvant or diluent. The adjuvant or diluent can
be combined with the PSMA protein in an amount to dilute the salt
concentration to 100 mM to 300 mM. In some embodiment the salt
concentration is diluted to 150 mM. In certain embodiments this is
done prior to administering the solution to a subject. In other
embodiments the first agent is a metal ion and the metal ion is a
zinc ion, calcium ion, magnesium ion, cobalt ion, manganese ion or
a combination thereof. In some embodiments the metal ion is a
combination of zinc ion and calcium ion. In still other embodiments
the zinc ion and calcium ion are present at a concentration in the
range of 0.1 mM to 5 mM. In yet other embodiments the zinc ion is
present at a concentration that is lower than the concentration of
the calcium ion. In still further embodiments the zinc ion is
present at a concentration of 0.1 mM, and the calcium ion is
present at a concentration of 1 mM. In other embodiments the metal
ion is a magnesium ion. In some of these embodiments the magnesium
ion is present at a concentration in the range of 0.1 mM to 5 mM.
In still other of these embodiments the magnesium ion is present at
a concentration of 0.5 mM. In the embodiments where the first agent
is a solution of a certain pH, the pH of the solution can be
adjusted to be in the range of 4 to 8. In some embodiments the pH
of the solution is adjusted to be in the range of 5 to 7. In still
other embodiments the pH of the solution is adjusted to be in the
range of 5.5 to 7. In yet other embodiments the pH of the solution
is adjusted to be 6.
[0024] In some embodiments the method further comprises contacting
the PSMA protein with a second agent that promotes or preserves
dimeric association of PSMA protein, wherein the second agent is
different than the first agent. A second agent that is different
than the first agent includes agents that are of a different type
or different class. The second agent, therefore, can be a metal
ion, salt or pH adjusting agent. In some embodiments where the
first agent is a metal ion the second agent can be a salt, pH
adjusting agent or a solution with a certain pH. In other
embodiments the first agent is a salt, and the second agent is a
metal ion, pH adjusting agent or a solution with a certain pH. In
still another embodiment the first agent is a pH adjusting agent or
a solution with a certain pH and the second agent is a metal ion or
a salt. In yet other embodiments the first agent can be a salt,
metal ion, pH adjusting agent or a solution with a certain pH and
the second agent can be of the same class but a different type
within the same class of agents. For instance if the first agent is
a salt such as sodium chloride, the second agent can also be a salt
but a different type, e.g., ammonium sulfate.
[0025] In another aspect of the invention a method of purifying a
sample containing PSMA protein by subjecting the sample containing
PSMA to chromatography in the presence of an agent that preserves
or promotes the dimeric association of PSMA is provided. In some
embodiments the agent that promotes or preserves the dimeric
association of PSMA is a metal ion, a salt or a solution with a pH
in the range of 4 to 8 or a combination thereof. In a preferred
embodiment the metal ion is a combination of calcium ion and
magnesium ion. In one such embodiment the calcium ion and magnesium
ion are each present at a concentration in the range of 0.1 mM to 5
mM. In a further embodiment the calcium ion and magnesium ion are
present at a concentration of 1 mM and 0.5 mM, respectively. In
other embodiments wherein the agent that promotes or preserves the
dimeric association of PSMA is a salt, the salt is present at a
concentration in the range of 50 mM to 2 M. In some of these
embodiments the salt is present at a concentration of 2 M. In still
other embodiments where the agent that promotes or preserves the
dimeric association of PSMA is a solution with a pH in the range of
4 to 8, the pH of the solution is in the range of 5 to 7. In still
other embodiments the pH of the solution is in the range of 6 to
7.5.
[0026] In other aspects of the invention a method of purifying a
sample containing PSMA protein by applying the sample to a first
column, washing the first column with a first wash solution
containing salt and metal ions, and collecting the PSMA protein
that elutes from the first column is provided. In some embodiments
the salt is ammonium sulfate at a saturation of no more than 35% in
the wash solution.
[0027] In embodiments of the invention the method further comprises
dialyzing or diafiltering the eluted PSMA protein with a first salt
solution at a pH in the range of 6 to 7.5 to yield a dialyzed or
diafiltrated solution containing PSMA protein. In some of these
embodiments the first salt solution has a salt concentration of at
least 5 mM. In still other of these embodiments the first salt
solution is a 10 mM sodium phosphate solution with a pH of 7.
[0028] In still other embodiments of the invention the method
further comprises loading the eluted PSMA protein, dialyzed or
diafiltrated solution containing PSMA protein onto a second column,
washing the second column with a second salt solution, and
collecting the PSMA eluted by the second salt solution. In some
embodiments the second salt solution has a salt concentration of
100 mM to 2 M. In certain of these embodiments the second salt
solution is 2 M sodium chloride in 10 mM sodium phosphate. In still
other embodiments the second salt solution has a pH in the range of
6 to 7.5.
[0029] In yet another embodiment of the invention the method
further comprises dialyzing or diafiltrating the PSMA eluted by the
second salt solution with a metal ion solution, applying the
dialyzed or diafiltrated PSMA eluted by the second salt solution
onto a third column, washing the third column with a second wash
solution containing salt and metal ions and collecting the PSMA
eluted. In some of these embodiments the pH is maintained in the
range of 6 to 7.5 through all of the purification steps.
[0030] In other embodiments the method further comprises separating
the different forms of PSMA protein, wherein the different forms of
PSMA protein are monomeric, dimeric or other multimeric forms of
PSMA. In some of these embodiments the different forms of PSMA
protein are separated by size exclusion chromatography.
[0031] In yet another aspect of the invention a method of
identifying an agent which promotes or preserves dimeric
association of PSMA protein by determining the amount of a form of
PSMA protein in a sample prior to exposure to a candidate agent,
exposing the sample to the candidate agent, determining the amount
of the form of PSMA protein in the sample after the exposure, and
comparing the amount of the form of PSMA protein in the sample
prior to and after the exposure is provided. In some embodiments
the form of PSMA protein is monomer or dimer. In other embodiments
the form of PSMA can be another multimer form with three or more
associated PSMA proteins.
[0032] In another aspect of the invention a method of treating a
subject to elicit or enhance an immune response to cells in the
subject expressing PSMA, comprising administering to the subject an
effective amount of any of the compositions given herein is
provided. In some embodiments the expressed PSMA is expressed on
the cell surface. In other embodiments the method further comprises
administering one or more booster doses of a composition comprising
PSMA protein. In some of these embodiments the composition
comprising PSMA protein is a composition of PSMA protein dimer. In
still other embodiments the booster dose composition further
comprises an adjuvant. In yet other embodiments the booster dose
composition can be any of the compositions provided herein. In
still other embodiments the composition is administered by
intravenous, intramuscular, subcutaneous, parenteral, spinal,
intradermal or epidermal administration. In this aspect of the
invention the subject has cancer or is at risk of having cancer. In
some embodiments the subject has also been treated for cancer. In
some embodiments the cancer is a primary tumor or is metastatic
cancer. In a preferred embodiment the subject has prostate cancer.
In some embodiments the subject is a non-castrate patient who,
preferably, has received primary therapy, such as prostatectomy or
radiation therapy. In one embodiment the non-castrate patient has a
serum testosterone level that is greater than or equal to 180
ng/mL. In other embodiments the subject is a castrate patient who,
preferably, has completed a course of hormonal therapy. In one
embodiment the castrate patient has a serum testosterone level of
less than 50 ng/mL. In still other embodiments the subject is a
patient who has received a conventional cancer therapy for prostate
cancer.
[0033] In another aspect of the invention methods of treating a
subject with cancer, such as prostate cancer, are provided. Such
methods comprise administering to a subject at least one of the
compositions provided herein. In one embodiment the method
comprises administering to the subject a therapeutically effective
amount of a composition comprising isolated PSMA protein in a
solution that promotes or preserves dimeric association of the PSMA
protein, wherein the composition is effective in treating prostate
cancer. In another embodiment the method further includes the
administration of an adjuvant, which preferably, is contained in
the composition comprising the isolated PSMA protein. In some
embodiments the adjuvant is a saponin-based adjuvant. In other
embodiments the methods further include administering to the
subject a conventional cancer therapy. Conventional cancer therapy
includes, but is not limited to, surgery, radiation, cryosurgery,
thermotherapy, hormone therapy or chemotherapy.
[0034] In still another aspect of the invention a method of
inhibiting metastasis in a subject with cancer is also provided.
One example of such a method includes administering to the subject
a therapeutically effective amount of a composition comprising
isolated PSMA protein in a solution that promotes or preserves
dimeric association of the PSMA protein, wherein the composition is
effective in inhibiting metastasis. In one embodiment the method
also includes the administration of an adjuvant, which preferably,
is a component of the composition of the isolated PSMA protein. In
another embodiment the method further comprises administering to
the subject a conventional prostate cancer therapy.
[0035] In another aspect of the invention a method of eliciting an
immune response by administering to a subject an effective amount
of any of the compositions provided is given. In some embodiments
the method further comprises administering one or more booster
doses of a composition comprising PSMA protein. In certain of these
embodiments the composition comprising PSMA protein is a
composition comprising PSMA protein dimer. In still other
embodiments the booster dose composition is any of the compositions
given herein. In yet another embodiment the booster dose
compositions can also include an adjuvant.
[0036] In other aspects of the invention kits which contain any of
the compositions provided and instructions for use are provided. In
some aspects the kit contains a multimeric composition provided
herein, an adjuvant and instructions for mixing. In other aspects
the kit includes one of the compositions provided herein, a diluent
and instructions for mixing. In some embodiments the composition is
provided in a vial or ampoule with a septum or a syringe. In other
embodiments the composition is in lyophilized form.
[0037] The compositions provided herein can further comprise a
therapeutic agent (e.g., a cytokine, an anti-cancer agent, an
adjuvant, etc.). In some embodiments the adjuvant is alum;
monophosphoryl lipid A; a saponin; a saponin fraction; a
saponin-based adjuvant, such as SaponImmune.TM.; a chemically
modified saponin; QS-7; QS-17; QS-18; QS-21; a polysaccharide-based
adjuvant, such as PolysaccImmune.TM.; a synthetic adjuvant, such as
SynthImmune.TM.; an immunostimulatory oligonucleotide; incomplete
Freund's adjuvant; complete Freund's adjuvant; vitamin E; a
water-in-oil emulsions prepared from a biodegradable oil;
montanide, such as MONTANIDE ISA51 and MONTANIDE ISA720; Quil A;
micellular mixtures of Quil A and cholesterol known as
immunostimulating complexes (ISCOMS); a MPL and mycobacterial cell
wall skeleton combination; ENHANZYN.TM.; RC-529; RC-552; CRL-1005;
L-121; alpha-galactosylceramide; a composition of biodegradable
particles composed of poly-lactide-co-glycolide (PLG) or other
similar polymers; a composition of aluminum or iron oxide beads or
a combination thereof. Other specific examples of adjuvants include
QS-21 fractions, such as crude QA-21, a QA-21H form, QA-21-V1;
QA-21-V2; a combination of QA-21-V1 and QA-21-V2, and chemically
modified forms or combinations thereof. In some embodiments the
preferred adjuvant is QS-21.
[0038] As used herein a "saponin-based adjuvant" is any adjuvant
that is based on or includes a saponin or portion thereof.
Therefore, saponin-based adjuvants include saponins, saponin
fractions and modified saponins (e.g., chemically modified
saponins).
[0039] In another embodiment the compositions provided herein can
also include a cancer therapeutic agent. Cancer therapeutic agents
include any agent used to treat cancer in a subject. In some
embodiments the cancer therapeutic agent is a chemotherapeutic
agent, such as, for example, docetaxel. Cancer therapeutic agents
also include anti-inflammatory agents and immunomodulatory agents.
The anti-inflammatory agent in one embodiment is prednisone. In
another embodiment the immunomodulatory agent is a cytokine.
[0040] In other embodiments the compositions provided can also
include at least one buffer. Buffers include PBS (phosphate
buffered saline), citric acid, sodium citrate, sodium acetate,
acetic acid, sodium phosphate, phosphoric acid, sodium ascorbate,
tartartic acid, maleic acid, glycine, sodium lactate, lactic acid,
ascorbic acid, imidazole, sodium bicarbonate, carbonic acid, sodium
succinate, succinic acid, histidine, sodium benzoate, benzoic acid
and combinations thereof.
[0041] In some embodiments the compositions provided further
include a free amino acid. These free amino acids can be naturally
or non-naturally occurring. In some embodiments the free amino
acids are non-acidic free amino acids. Examples of non-acidic free
amino acids include glycine, proline, isoleucine, leucine, alanine,
arginine and combinations thereof.
[0042] Compositions of PSMA protein multimers including a
surfactant are also provided. Such surfactants include Tween20,
Tween80, Triton X-100, dodecylmaltoside, cholic acid, CHAPS and
combinations thereof.
[0043] Also provided are compositions of PSMA protein multimers
that comprise a cryoprotectant, an antioxidant, a preservative or a
combination thereof. Examples of cryoprotectants include a sugar, a
polyol, an amino acid, a polymer, an inorganic salt, an organic
salt, trimethylamine N-oxide, sarcosine, betaine,
gamma-aminobutyric acid, octapine, alanopine, strombine,
dimethylsulfoxide and ethanol. When the cryoprotectant is a sugar
the sugar can be sucrose, lactose, glucose, trehalose or maltose.
In other embodiments when the cryoprotectant is a polyol the polyol
can be inositol, ethylene glycol, glycerol, sorbitol, xylitol,
mannitol or 2-methyl-2,4-pentane-diol. When the cryoprotectant is
an amino acid the amino acid can be Na glutamate, proline,
alpha-alanine, beta-alanine, glycine, lysine-HCl or
4-hydroxyproline. When the cryoprotectant is a polymer the polymer
can be polyethylene glycol, dextran or polyvinylpyrrolidone. When
the cryoprotectant is an inorganic salt the cryoprotectant can be
sodium sulfate, ammonium sulfate, potassium phosphate, magnesium
sulfate or sodium fluoride. Finally, when the cryoprotectant is an
organic salt the organic salt can be sodium acetate, sodium
polyethylene, sodium caprylate, proprionate, lactate or succinate.
Examples of antioxidants that are part of these composition in some
embodiments include ascorbic acid, an ascorbic acid derivative,
butylated hydroxy anisole, butylated hydroxy toluene, alkylgallate,
dithiothreitol (DTT), sodium meta-bisulfite, sodium bisulfite,
sodium dithionite, sodium thioglycollic acid, sodium formaldehyde
sulfoxylate, tocopherol, a tocopherol derivative, monothioglycerol
and sodium sulfite. Ascorbic acid derivatives, in some embodiments,
include ascorbylpalmitate, ascorbylstearate, sodium ascorbate and
calcium ascorbate, while tocopherol derivatives include d-alpha
tocopherol, d-alpha tocopherol acetate, dl-alpha tocopherol
acetate, d-alpha tocopherol succinate, beta tocopherol, delta
tocopherol, gamma tocopherol and d-alpha tocopherol polyoxyethylene
glycol 1000 succinate. Examples of preservatives present in the
compositions in some embodiments include benzalkonium chloride,
chlorobutanol, parabens, thimerosal, benzyl alcohol and phenol.
[0044] The composition in some embodiments are physiologically
acceptable compositions.
[0045] The compositions provided are, in some embodiments, in
liquid or lyophilized form.
[0046] In some other embodiments the compositions provided are
sterile.
[0047] In other aspects of the invention pharmaceutical
compositions are provided which contain any of the compositions
provided herein and a pharmaceutically acceptable carrier.
[0048] The present invention also relates, in part, to antibodies
or antigen-binding fragments thereof which specifically bind the
extracellular domain of prostate specific membrane antigen (PSMA),
compositions containing one or a combination of such antibodies or
antigen-binding fragments thereof, hybridoma cell lines that
produce the antibodies, and methods of using the antibodies or
antigen-binding fragments thereof for cancer diagnosis and
treatment.
[0049] According to one aspect of the invention, isolated
antibodies or an antigen-binding fragments thereof are provided.
The antibodies or fragments thereof specifically bind to an
extracellular domain of prostate specific membrane antigen (PSMA),
and competitively inhibit the specific binding of a second antibody
to its target epitope on PSMA. In a second aspect of the invention,
isolated antibodies or antigen-binding fragments thereof are
provided which specifically bind to an epitope on prostate specific
membrane antigen (PSMA) defined by a second antibody. In each of
the forgoing aspects of the invention, the second antibody is
selected from the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9,
PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3,
PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix
4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix
4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix
4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix
4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix
4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, Abgenix 4.152.1, and
antibodies comprising (a) a heavy chain encoded by a nucleic acid
molecule comprising the coding region or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 8-13.
[0050] In certain embodiments, the antibody or antigen-binding
fragment thereof is selected from the group consisting of PSMA 3.7,
PSMA 3.8, PSMA 3.9, PSMA 3.11 PSMA 5.4, PSMA 7.3, PSMA 10.3, PSMA
1.8.3, PSMA A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3,
Abgenix 4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1,
Abgenix 4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3,
Abgenix 4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1,
Abgenix 4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3,
Abgenix 4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix
4.152.1. In other embodiments, the antibody or antigen-binding
fragment thereof is selected from the group consisting of
antibodies comprising (a) a heavy chain encoded by a nucleic acid
molecule comprising the coding region or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as SEQ ID NOs: 2-7, and (b) a light chain encoded by a
nucleic acid molecule comprising the coding region or regions of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as SEQ ID NOs: 8-13, and
antigen-binding fragments thereof.
[0051] In further embodiments, the antibody or antigen-binding
fragments thereof is encoded by a nucleic acid molecule comprising
a nucleotide sequence that is at least about 90% identical to the
nucleotide sequence encoding the foregoing antibodies, preferably
at least about 95% identical, more preferably at least about 97%
identical, still more preferably at least about 98% identical, and
most preferably is at least about 99% identical.
[0052] In some embodiments of the foregoing aspects,
antigen-binding fragments of the isolated antibodies are provided.
The antigen-binding fragments include (a) a heavy chain variable
region encoded by a nucleic acid molecule comprising the coding
regions or regions of a nucleotide sequence selected from the group
consisting of nucleotide sequences set forth as: SEQ ID NOs: 14,
18, 22, 26 and 30, and (b) a light chain variable region encoded by
a nucleic acid molecule comprising the coding region or region of a
nucleotide sequence selected from the group consisting of
nucleotide sequences set forth as: SEQ ID NOs: 16, 20, 24, 28 and
32. In other embodiments, the antigen-binding fragment includes (a)
a heavy chain variable region comprising an amino acid sequence
selected from the group consisting of amino acid sequences set
forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and (b) a light chain
variable region comprising an amino acid sequence selected from the
group consisting of nucleotide sequences set forth as: SEQ ID NOs:
17, 21, 25, 29 and 33.
[0053] In a further embodiments of the invention, isolated
antigen-binding fragments of antibodies, which include a CDR of the
foregoing antigen-binding fragments are provided. Preferably the
CDR is CDR3.
[0054] According to another aspect of the invention, expression
vectors including an isolated nucleic acid molecule encoding the
foregoing isolated antibodies or antigen-binding fragments are
provided. Host cells transformed or transfected by these expression
vectors also are provided.
[0055] In certain embodiments, the antibody or antigen-binding
fragment thereof is selected for its ability to bind live cells,
such as a tumor cell or a prostate cell, preferably LNCaP cells. In
other embodiments, the antibody or antigen-binding fragment thereof
mediates cytolysis of cells expressing PSMA. Preferably cytolysis
of cells expressing PSMA is mediated by effector cells or is
complement mediated in the presence of effector cells.
[0056] In other embodiments, the antibody or antigen-binding
fragment thereof inhibits the growth of cells expressing PSMA.
Preferably the antibody or antigen-binding fragment thereof does
not require cell lysis to bind to the extracellular domain of
PSMA.
[0057] In further embodiments, the antibody or antigen-binding
fragment thereof is selected from the group consisting of IgG1,
IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or has
immunoglobulin constant and/or variable domain of IgG1, IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgAsec, IgD or IgE. In other embodiments,
the antibody is a bispecific or multispecific antibody.
[0058] In still other embodiments, the antibody is a recombinant
antibody, a polyclonal antibody, a monoclonal antibody, a humanized
antibody or a chimeric antibody, or a mixture of these. In
particularly preferred embodiments, the antibody is a human
antibody, e.g., a monoclonal antibody, polyclonal antibody or a
mixture of monoclonal and polyclonal antibodies. In still other
embodiments, the antibody is a bispecific or multispecific
antibody.
[0059] Preferred antigen-binding fragments include a Fab fragment,
a F(ab').sub.2 fragment, and a Fv fragment CDR3.
[0060] In further embodiments, the isolated antibody or
antigen-binding fragment is a monoclonal antibody produced by a
hybridoma cell line selected from the group consisting of PSMA 3.7
(PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269),
PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA
10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904),
PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3
(PTA-4428), Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556),
Abgenix 4.177.3 (PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix
4.22.3 (PTA-4358), Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2
(PTA-4360), Abgenix 4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362),
Abgenix 4.209.3 (PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix
4.288.1 (PTA-4367), Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1
(PTA-4363), Abgenix 4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389),
Abgenix 4.292.3 (PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix
4.78.1 (PTA-4652), and Abgenix 4.152.1(PTA-4653).
[0061] In certain other embodiments, the antibody or
antigen-binding fragment thereof binds to a conformational epitope
and/or is internalized into a cell along with the prostate specific
membrane antigen. In other embodiments, the isolated antibody or
antigen-binding fragment thereof is bound to a label, preferably
one selected from the group consisting of a fluorescent label, an
enzyme label, a radioactive label, a nuclear magnetic resonance
active label, a luminescent label, and a chromophore label.
[0062] In still other embodiments, the isolated antibody or
antigen-binding fragment thereof is bound to at least one
therapeutic moiety, such as a drug, preferably a cytotoxic drug, a
replication-selective virus, a toxin or a fragment thereof, or an
enzyme or a fragment thereof. Preferred cytotoxic drug include:
calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan,
chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum,
etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine,
etoposide, doxorubicin, paclitaxel, docetaxel, dolastatin 10,
auristatin E and auristatin PHE. In other embodiments, the
therapeutic moiety is an immunostimulatory or immunomodulating
agent, preferably one selected from the group consisting of: a
cytokine, chemokine and adjuvant.
[0063] In some embodiments, the antibodies or antigen-binding
fragments of the invention specifically bind cell-surface PSMA
and/or rsPSMA with a binding affinity of about 1.times.10.sup.-9 M
or less. Preferably, the binding affinity is about
1.times.10.sup.-10 M or less, more preferably the binding affinity
is about 1.times.10.sup.-11 M or less. In other embodiments the
binding affinity is less than about 5.times.10.sup.-10 M.
[0064] In additional embodiments, the antibodies or antigen-binding
fragments of the invention mediate specific cell killing of
PSMA-expressing cells with an IC.sub.50 of less than about
1.times.10.sup.-10 M. Preferably the IC.sub.50 is less than about
1.times.10.sup.-11 M. More preferably the IC.sub.50 is less than
about 1.times.10.sup.-12 M. In other embodiments the IC.sub.50 is
less than about 1.5.times.10.sup.-11 M.
[0065] In yet other embodiments, the isolated antibody or
antigen-binding fragment thereof is bound to a radioisotope. The
radioisotope can emit .alpha. radiations, .beta. radiations, or
.gamma. radiations. Preferably the radioisotope is selected from
the group consisting of .sup.225Ac, .sup.211At, .sup.212Bi,
.sup.213Bi, .sup.186Rh, .sup.188Rh, .sup.177Lu, .sup.90Y,
.sup.131I, .sup.67Cu, .sup.125I, .sup.123I, .sup.77Br, .sup.153Sm,
.sup.166Ho, .sup.64Cu, .sup.212Pb, .sup.224Ra and .sup.223Ra.
[0066] According to another aspect of the invention, hybridoma cell
lines are provided that produce an antibody selected from the group
consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4,
PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3, PSMA
A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix 4.7.1, Abgenix
4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix 4.22.3, Abgenix
4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix 4.49.1, Abgenix
4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix 4.333.1, Abgenix
4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix 4.292.3, Abgenix
4.304.1, Abgenix 4.78.1 and Abgenix 4.152.1. In some embodiments,
the hybridoma cell line is selected from the group consisting of
PSMA 3.7 (PTA-3257), PSMA 3.8, PSMA 3.9 (PTA-3258), PSMA 3.11
(PTA-3269), PSMA 5.4 (PTA-3268), PSMA 7.1 (PTA-3292), PSMA 7.3
(PTA-3293), PSMA 10.3 (PTA-3247), PSMA 1.8.3 (PTA-3906), PSMA
A3.1.3 (PTA-3904), PSMA A3.3.1 (PTA-3905), Abgenix 4.248.2
(PTA-4427), Abgenix 4.360.3 (PTA-4428), Abgenix 4.7.1 (PTA-4429),
Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3 (PTA-4557), Abgenix
4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358), Abgenix 4.28.3
(PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix 4.48.3 (PTA-4361),
Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3 (PTA-4365), Abgenix
4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367), Abgenix 4.333.1
(PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix 4.153.1 (PTA-4388),
Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3 (PTA-4390), Abgenix
4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652), and Abgenix
4.152.1(PTA-4653).
[0067] According to a further aspect of the invention, compositions
are provided that include the foregoing antibodies or
antigen-binding fragments thereof and a pharmaceutically acceptable
carrier, excipient, or stabilizer. Other compositions include a
combination of two or more of the foregoing antibodies or
antigen-binding fragments thereof and a pharmaceutically acceptable
carrier, excipient, or stabilizer. In some embodiments, the
compositions also include an antitumor agent, an immunostimulatory
agent, an immunomodulator, or a combination thereof. Preferred
antitumor agents include a cytotoxic agent, an agent that acts on
tumor neovasculature, or a combination thereof. Preferred
immunomodulators include .alpha.-interferon, .gamma.-interferon,
tumor necrosis factor-.alpha. or a combination thereof. Preferred
immunostimulatory agents include interleukin-2, immunostimulatory
oligonucleotides, or a combination thereof.
[0068] According to another aspect of the invention antibodies or
antigen-binding fragments thereof that mediate antibody-dependent
cellular cytotoxicity (ADCC) are provided. In some embodiments
these antibodies or antigen-binding fragments thereof mediate ADCC
of human prostate cancer cells. In other embodiments the antibodies
are human antibodies. In still other embodiments the antibodies are
capable of causing at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
50%, 75% or more cell lysis in vitro with an effector to target
ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1 or more. In
other embodiments these antibodies mediate more ADCC than control
antibodies.
[0069] According to another aspect of the invention, kits for
detecting prostate cancer for diagnosis, prognosis or monitoring
are provided. The kits include the foregoing isolated labeled
antibody or antigen-binding fragment thereof, and one or more
compounds for detecting the label. Preferably the label is selected
from the group consisting of a fluorescent label, an enzyme label,
a radioactive label, a nuclear magnetic resonance active label, a
luminescent label, and a chromophore label.
[0070] The invention in another aspect provides one or more of the
foregoing isolated antibodies or antigen-binding fragments thereof
packaged in lyophilized form, or packaged in an aqueous medium.
[0071] In another aspect of the invention, methods for detecting
the presence of PSMA, or a cell expressing PSMA, in a sample are
provided. The methods include contacting the sample with any of the
foregoing antibodies or antigen-binding fragments thereof which
specifically bind to an extracellular domain of PSMA, for a time
sufficient to allow the formation of a complex between the antibody
or antigen-binding fragment thereof and PSMA, and detecting the
PSMA-antibody complex or PSMA-antigen-binding fragment complex. The
presence of a complex in the sample is indicative of the presence
in the sample of PSMA or a cell expressing PSMA.
[0072] In another aspect, the invention provides other methods for
diagnosing a PSMA-mediated disease in a subject. The methods
include administering to a subject suspected of having or
previously diagnosed with PSMA-mediated disease an amount of any of
the foregoing antibodies or antigen-binding fragments thereof which
specifically bind to an extracellular domain of prostate specific
membrane antigen. The method also includes allowing the formation
of a complex between the antibody or antigen-binding fragment
thereof and PSMA, and detecting the formation of the PSMA-antibody
complex or PSMA-antigen-binding fragment antibody complex to the
target epitope. The presence of a complex in the subject suspected
of having or previously diagnosed with prostate cancer is
indicative of the presence of a PSMA-mediated disease.
[0073] In certain embodiments of the methods, the PSMA-mediated
disease is prostate cancer. In other embodiments, the PSMA-mediated
disease is a non-prostate cancer, such as those selected from the
group consisting of bladder cancer including transitional cell
carcinoma; pancreatic cancer including pancreatic duct carcinoma;
lung cancer including non-small cell lung carcinoma; kidney cancer
including conventional renal cell carcinoma; sarcoma including soft
tissue sarcoma; breast cancer including breast carcinoma; brain
cancer including glioblastoma multiforme; neuroendocrine carcinoma;
colon cancer including colonic carcinoma; testicular cancer
including testicular embryonal carcinoma; and melanoma including
malignant melanoma.
[0074] In preferred embodiments of the foregoing methods, the
antibody or antigen-binding fragment thereof is labeled. In other
embodiments of the foregoing methods, a second antibody is
administered to detect the first antibody or antigen-binding
fragment thereof.
[0075] In a further aspect of the invention, methods for assessing
the prognosis of a subject with a PSMA-mediated disease are
provided. The methods include administering to a subject suspected
of having or previously diagnosed with PSMA-mediated disease an
effective amount of an antibody or antigen-binding fragment thereof
according to claim A1 or B1, allowing the formation of a complex
between the antibody or antigen-binding fragment thereof and PSMA,
and detecting the formation of the complex to the target epitope.
The amount of the complex in the subject suspected of having or
previously diagnosed with PSMA-mediated disease is indicative of
the prognosis.
[0076] In another aspect of the invention, methods for assessing
the effectiveness of a treatment of a subject with a PSMA-mediated
disease are provided. The methods include administering to a
subject suspected treated for a PSMA-mediated disease an effective
amount of the foregoing antibodies or antigen-binding fragments
thereof, allowing the formation of a complex between the antibody
or antigen-binding fragment thereof and PSMA, and detecting the
formation of the complex to the target epitope. The amount of the
complex in the subject suspected of having or previously diagnosed
with PSMA-mediated disease is indicative of the effectiveness of
the treatment.
[0077] In certain embodiments of these two aspects of the
invention, the PSMA-mediated disease is prostate cancer. In other
embodiments, the PSMA-mediated disease is a non-prostate cancer. In
those embodiments, the non-prostate cancer preferably is selected
from the group consisting of bladder cancer including transitional
cell carcinoma; pancreatic cancer including pancreatic duct
carcinoma; lung cancer including non-small cell lung carcinoma;
kidney cancer including conventional renal cell carcinoma; sarcoma
including soft tissue sarcoma; breast cancer including breast
carcinoma; brain cancer including glioblastoma multiforme;
neuroendocrine carcinoma; colon cancer including colonic carcinoma;
testicular cancer including testicular embryonal carcinoma; and
melanoma including malignant melanoma. In still other embodiments,
the antibody or antigen-binding fragment thereof is labeled. In
further embodiments, a second antibody is administered to detect
the first antibody or antigen-binding fragment thereof.
[0078] According to yet another aspect of the invention, methods
for inhibiting the growth of a cell expressing PSMA are provided.
The methods include contacting a cell expressing PSMA with an
amount of at least one of the foregoing antibodies or
antigen-binding fragments thereof which specifically binds to an
extracellular domain of PSMA effective to inhibit the growth of the
cell expressing PSMA.
[0079] According to another aspect of the invention, methods for
inducing cytolysis of a cell expressing PSMA are provided. The
methods include contacting a cell expressing PSMA with an amount of
at least one of the foregoing antibodies or antigen-binding
fragments thereof which specifically binds to an extracellular
domain of PSMA effective to induce cytolysis of the cell expressing
PSMA. In certain embodiments, the cytolysis occurs in the presence
of an effector cell. In other embodiments, the cytolysis is
complement mediated.
[0080] According to still another aspect of the invention, methods
for treating or preventing a PSMA-mediated disease are provided.
The methods include administering to a subject having a
PSMA-mediated disease an effective amount of at least one of the
forgoing antibodies or antigen-binding fragments thereof to treat
or prevent the PSMA-mediated disease. In some embodiments, the
PSMA-mediated disease is a cancer, such as prostate cancer or a
non-prostate cancer (including the nonprostate cancers described
elsewhere herein).
[0081] In yet a further aspect of the invention, methods for
treating or preventing a PSMA-mediated disease are provided. The
methods include administering to a subject having a PSMA-mediated
disease or at risk of having a PSMA-mediated disease an amount of
at least one of the foregoing antibodies or antigen-binding
fragments thereof effective to treat or prevent the PSMA-mediated
disease.
[0082] In some embodiments, the PSMA-mediated disease is a cancer,
such as prostate cancer or a non-prostate cancer (including the
nonprostate cancers described elsewhere herein).
[0083] In other embodiments, the method also includes administering
another therapeutic agent to treat or prevent the PSMA-mediated
disease at any time before, during or after the administration of
the antibody or antigen-binding fragment thereof. In some of these
embodiments, the therapeutic agent is a vaccine, and preferably the
vaccine immunizes the subject against PSMA.
[0084] In still other embodiments, the antibody or antigen-binding
fragment thereof is bound to at least one therapeutic moiety,
preferably a cytotoxic drug, a drug which acts on the tumor
neovasculature and combinations thereof. Preferred cytotoxic drugs
are selected from the group consisting of: calicheamicin,
esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil,
ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin,
5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin,
paclitaxel, docetaxel, dolastatin 10, auristatin E and auristatin
PHE.
[0085] In other embodiments, the antibody or antigen-binding
fragment thereof is bound to a radioisotope and the radiations
emitted by the radioisotope is selected from the group consisting
of .alpha., .beta. and .gamma. radiations. Preferably, the
radioisotope is selected from the group consisting of .sup.225Ac
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.186Rh, .sup.188Rh,
.sup.177Lu, .sup.90Y, .sup.131I, .sup.67Cu, .sup.125I, .sup.123I,
.sup.77Br, .sup.153Sm, .sup.166Ho, .sup.64Cu, .sup.212Pb,
.sup.224Ra and .sup.223Ra.
[0086] The present invention provides methods for modulating at
least one enzymatic activity of PSMA. As used in preferred
embodiments of the methods, "modulating" an enzymatic activity of
PSMA means enhancing or inhibiting the enzymatic activity. Thus in
certain aspects of the invention, methods for inhibiting an
enzymatic activity of PSMA are provided, and in other aspects of
the invention, methods for enhancing an enzymatic activity of PSMA
are provided. The terms "enhancing" and "inhibiting" in this
context indicate that the enzymatic activity of PSMA is enhanced or
inhibited in the presence of an antibody that specifically binds
PSMA, or antigen-binding fragment thereof, relative to the level of
activity in the absence of such an antibody or antigen-binding
fragment thereof. Enzymatic activities of PSMA include folate
hydrolase activity, N-acetylated .alpha.-linked acidic dipeptidase
(NAALADase) activity, dipeptidyl dipeptidase IV activity and
.gamma.-glutamyl hydrolase activity.
[0087] Thus the invention in another aspect provides methods for
modulating folate hydrolase activity. In certain embodiments of
these methods, the activity is inhibited and in other embodiments,
the activity is enhanced. The methods include contacting a folate
hydrolase polypeptide with an amount of the foregoing isolated
antibody or antigen-binding fragment thereof, under conditions
wherein the isolated antibody or antigen-binding fragment thereof
modulates the folate hydrolase activity. The folate hydrolase
polypeptide can be isolated, contained in a sample such as a cell,
a cell homogenate, a tissue, or a tissue homogenate, or contained
in an organism. The organism preferably is an animal, particularly
preferably a mammal.
[0088] In another aspect of the invention, methods for modulating
N-acetylated .alpha.-linked acidic dipeptidase (NAALADase) activity
are provided. In certain embodiments of these methods, the activity
is inhibited and in other embodiments, the activity is enhanced.
The methods include contacting a NAALADase polypeptide with an
amount of the foregoing isolated antibody or antigen-binding
fragment thereof under conditions wherein the isolated antibody or
antigen-binding fragment thereof modulates NAALADase activity. The
NAALADase polypeptide can be isolated, contained in a sample such
as a cell, a cell homogenate, a tissue, or a tissue homogenate, or
contained in an organism. The organism preferably is an animal,
particularly preferably a mammal.
[0089] In yet another aspect of the invention, methods for
modulating dipeptidyl dipeptidase IV activity are provided. In
certain embodiments of these methods, the activity is inhibited and
in other embodiments, the activity is enhanced. The methods include
contacting a dipeptidyl dipeptidase IV polypeptide with an amount
of the foregoing isolated antibody or antigen-binding fragment
thereof under conditions wherein the isolated antibody or
antigen-binding fragment thereof modulates dipeptidyl dipeptidase
IV activity. The dipeptidyl dipeptidase IV polypeptide can be
isolated, contained in a sample such as a cell, a cell homogenate,
a tissue, or a tissue homogenate, or contained in an organism. The
organism preferably is an animal, particularly preferably a
mammal.
[0090] In yet another aspect of the invention, methods for
modulating .gamma.-glutamyl hydrolase activity are provided. In
certain embodiments of these methods, the activity is inhibited and
in other embodiments, the activity is enhanced. The methods include
contacting a .gamma.-glutamyl hydrolase polypeptide with an amount
of the foregoing isolated antibody or antigen-binding fragment
thereof under conditions wherein the isolated antibody or
antigen-binding fragment thereof modulates .gamma.-glutamyl
hydrolase activity. The .gamma.-glutamyl hydrolase polypeptide can
be isolated, contained in a sample such as a cell, a cell
homogenate, a tissue, or a tissue homogenate, or contained in an
organism. The organism preferably is an animal, particularly
preferably a mammal.
[0091] Methods of specific delivery of at least one therapeutic
agent to PSMA-expressing cells are provided according to another
aspect of the invention. The methods include administering an
effective amount of at least one of the foregoing antibodies or
antigen-binding fragments thereof conjugated to the at least one
therapeutic agent. In some embodiments, the therapeutic agent is a
nucleic acid molecule, an antitumor drug, a toxin or a fragment
thereof, an enzyme or a fragment thereof, a replication-selective
virus, or an immunostimulatory or immunomodulating agent. Preferred
antitumor drugs include cytotoxic drugs, drugs which act on the
tumor neovasculature and combinations thereof. Preferred cytotoxic
drugs include calicheamicin, esperamicin, methotrexate,
doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin
C, cis-platinum, etoposide, bleomycin, 5-fluorouracil,
estramustine, vincristine, etoposide, doxorubicin, paclitaxel,
docetaxel, dolastatin 10, auristatin E and auristatin PHE.
Preferred immunostimulatory or immunomodulating agent included
cytokines, chemokines and adjuvants.
[0092] In still another aspect of the invention, isolated
antibodies that selectively bind a PSMA protein multimer are
provided. In preferred embodiments, the PSMA protein multimer is a
dimer, and preferably at least one of the PSMA proteins forming the
multimer is a recombinant, soluble PSMA (rsPSMA) polypeptide.
Preferably the rsPSMA polypeptide consists essentially of amino
acids 44-750 of SEQ ID NO:1.
[0093] In a further aspect of the invention, isolated antibodies
are provided that selectively bind a PSMA protein multimer and
modulate one or more enzymatic activities of the PSMA protein
multimer. As used in preferred embodiments of this aspect of the
invention, "modulating" an enzymatic activity of a PSMA multimer
means enhancing or inhibiting the enzymatic activity. Thus in
certain aspects of the invention, antibodies that inhibit an
enzymatic activity of PSMA multimers are provided, and in other
aspects of the invention, antibodies that inhibit an enzymatic
activity of PSMA multimers are provided. The terms "enhancing" and
"inhibiting" in this context indicate that the enzymatic activity
of a PSMA multimer is enhanced or inhibited in the presence of an
antibody that specifically binds the PSMA multimers, or
antigen-binding fragment thereof, relative to the level of activity
in the absence of such an antibody or antigen-binding fragment
thereof. In some embodiments, the enzymatic activity is selected
from the group consisting of folate hydrolase activity, NAALADase
activity, dipeptidyl dipeptidase IV activity and .gamma.-glutamyl
hydrolase activity. In other embodiments, the enzymatic activity is
in the extracellular domain of the PSMA molecule. In still other
embodiments, the antibody or antigen-binding fragment thereof
specifically binds to an extracellular domain of PSMA.
[0094] In a further aspect, an isolated antibody or antigen-binding
fragment thereof is provided that selectively binds a PSMA protein
multimer. In this aspect, the isolated antibody is raised by
immunizing an animal with a preparation comprising a PSMA protein
multimer. Preferred preparations used in raising the antibody
include those having at least about 10%, 20%, 30%, 40%, 50%, 75%,
90%, or 95% PSMA protein multimer. Preferably the PSMA protein
multimer is a dimer.
[0095] In yet another aspect of the invention, compositions are
provided that include one or more of the foregoing isolated
antibodies, and an immunostimulatory molecule, such as an adjuvant
and/or and a cytokine. Preferably the immunostimulatory molecule is
IL-2 or an immunostimulatory oligonucleotide. In certain
embodiments, the foregoing compositions also include a
pharmaceutically-acceptable carrier.
[0096] The invention also includes methods for inducing an immune
response, including administering to a subject in need of such
treatment an effective amount of the foregoing isolated antibodies
or compositions.
[0097] The invention provides, in another aspect, isolated
antibodies or antigen-binding fragments thereof that selectively
bind a PSMA protein multimer and modulate at least one enzymatic
activity of PSMA. As used in preferred embodiments of this aspect
of the invention, "modulating" an enzymatic activity of a PSMA
means enhancing or inhibiting the enzymatic activity. Thus in
certain aspects of the invention, antibodies that inhibit an
enzymatic activity of PSMA are provided, and in other aspects of
the invention, antibodies that inhibit an enzymatic activity of
PSMA are provided. The terms "enhancing" and "inhibiting" in this
context indicate that the enzymatic activity of PSMA is enhanced or
inhibited in the presence of an antibody that specifically binds
PSMA, or antigen-binding fragment thereof, relative to the level of
activity in the absence of such an antibody or antigen-binding
fragment thereof. The enzyme, in certain embodiments, is selected
from the group consisting of hydrolases and peptidases. Preferred
hydrolases include folate hydrolase and .gamma.-glutamyl hydrolase.
In a particularly preferred embodiment of PSMA inhibition, the
hydrolase is folate hydrolase and the antibody is mAb 5.4 or mAb
3.9. Preferred peptidases include NAALADase and dipeptidyl
dipeptidase IV. In some embodiments, the enzyme is active in cancer
cells and has lesser activity in normal cells than in cancer cells
or, preferably, no activity in normal cells. In preferred
embodiments, the cancer cells in which the enzyme is active are
prostate cancer cells. Compositions including the foregoing
isolated antibodies or antigen-binding fragments thereof, and a
pharmaceutically acceptable carrier, also are provided by the
invention.
[0098] In another aspect of the invention, compositions are
provided that include an isolated PSMA protein multimer. Preferably
the PSMA protein multimer is a dimer. In certain embodiments, the
compositions include at least about 10%, 20%, 30%, 40%, 50%, 75%,
90%, or 95% PSMA protein multimer. In other embodiments, the PSMA
protein multimer comprises noncovalently associated PSMA proteins.
The PSMA proteins preferably are noncovalently associated under
nondenaturing conditions.
[0099] In certain embodiments of the foregoing compositions, at
least one of the PSMA proteins forming the multimer is a
recombinant, soluble PSMA (rsPSMA) polypeptide. In other
embodiments, the PSMA protein multimer is reactive with a
conformation-specific antibody that specifically recognizes PSMA.
Preferably, the PSMA protein multimer comprises PSMA proteins in a
native conformation and/or the PSMA multimer is enzymatically
active. In preferred embodiments, the enzymatic activity is folate
hydrolase activity, NAALADase activity, dipeptidyl dipeptidase IV
activity and/or .gamma.-glutamyl hydrolase activity.
[0100] In still other embodiments, the foregoing compositions also
include an adjuvant and/or a cytokine or other immunostimulatory
molecule. Preferred cytokines include IL-2, IL-12, IL-18 and
GM-CSF. In further embodiments, the foregoing compositions also
include a pharmaceutically acceptable carrier.
[0101] According to yet another aspect of the invention, methods
for inducing an immune response are provided. The methods include
administering to a subject in need of such treatment an effective
amount of one or more of the foregoing compositions.
[0102] In a further aspect, the invention includes isolated
recombinant soluble PSMA (rsPSMA) protein multimers and isolated
rsPSMA protein dimers. In some embodiments, the dimer includes
noncovalently associated rsPSMA proteins, and preferably the rsPSMA
proteins are noncovalently associated under nondenaturing
conditions. In other embodiments, the isolated rsPSMA dimer is
reactive with a conformation-specific antibody that specifically
recognizes PSMA.
[0103] In a certain preferred embodiment, the isolated rsPSMA dimer
is enzymatically active, with the enzymatic activity selected from
the group consisting of folate hydrolase activity, NAALADase
activity, dipeptidyl dipeptidase IV activity and .gamma.-glutamyl
hydrolase activity.
[0104] In still another aspect of the invention, methods of
screening for a candidate agent that modulates at least one
enzymatic activity of a PSMA enzyme are provided. As used in
preferred embodiments of the methods, "modulating" an enzymatic
activity of PSMA means enhancing or inhibiting the enzymatic
activity. Thus in certain aspects of the invention, methods for
screening for a candidate agent that inhibits an enzymatic activity
of PSMA are provided, and in other aspects of the invention,
methods for screening for a candidate agent that enhances an
enzymatic activity of PSMA are provided. The terms "enhancing" and
"inhibiting" in this context indicate that the enzymatic activity
of PSMA is enhanced or inhibited in the presence of a candidate
agent relative to the level of activity in the absence of such an
agent. The methods include mixing the candidate agent with an
isolated PSMA protein multimer to form a reaction mixture, followed
by adding a substrate for the PSMA enzyme to the reaction mixture,
and determining the amount of a product formed from the substrate
by the PSMA enzyme. A change in the amount of product formed in
comparison to a control is indicative of an agent capable of
modulating at least one enzymatic activity of the PSMA enzyme. A
decrease in the amount of product formed in comparison to a control
is indicative of an agent capable of inhibiting at least one
enzymatic activity of the PSMA enzyme. An increase in the amount of
product formed in comparison to a control is indicative of an agent
capable of enhancing at least one enzymatic activity of the PSMA
enzyme. In some embodiments the PSMA enzyme is selected from the
group consisting of NAALADase, folate hydrolase, dipeptidyl
dipeptidase IV and .gamma.-glutamyl hydrolase. In other embodiments
the PSMA multimer comprises recombinant, soluble PSMA. In yet other
embodiments the candidate agent is selected from the group
consisting of an antibody, a small organic compound, or a
peptide.
[0105] In another aspect of the invention, candidate agents that
modulate at least one enzymatic activity of PSMA are provided. The
candidate agents are identified according to the foregoing methods.
Thus in certain aspects of the invention, candidate agents that
inhibit an enzymatic activity of PSMA are provided, and in other
aspects of the invention, candidate agents that enhance an
enzymatic activity of PSMA are provided. In certain embodiments,
the agent is selected from a combinatorial antibody library, a
combinatorial protein library, or a small organic molecule
library.
[0106] The invention also provides methods for identifying
compounds that promote dissociation of PSMA dimers. The methods
include contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound, measuring the amount of PSMA monomer and/or dimer;
and comparing the amount of PSMA monomer and/or dimer measured in
the presence of the compound with that observed in the absence of
the compound. An increase in the amount of PSMA monomer measured in
the presence of the compound indicates that the compound is capable
of promoting dissociation of the PSMA dimer. A decrease in the
amount of PSMA dimer measured in the presence of the compound
indicates that the compound is capable of promoting dissociation of
the PSMA dimer. When the amounts of PSMA monomer and PSMA dimer are
measured, the methods can include calculating a ratio of PSMA
monomer to PSMA dimer and comparing the ratio obtained in the
presence of the compound with that obtained in the absence of the
compound. In such methods, an increase in the ratio measured in the
presence of the compound indicates that the compound is capable of
promoting dissociation of the PSMA dimer.
[0107] The use of the foregoing compositions, molecules and agents
in the preparation of medicaments also is provided. In preferred
embodiments, the medicaments are useful in the treatment of
conditions related to hyperproliferative diseases including cancer,
and diseases of inappropriate NAALADase activity, folate hydrolase
activity, dipeptidyl dipeptidase IV activity and/or
.gamma.-glutamyl hydrolase activity.
[0108] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention.
[0109] These and other aspects of the invention will be described
in further detail in connection with the detailed description of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0110] FIG. 1 depicts PSMA reactivity of mAbs as determined by flow
cytometry. Anti-PSMA mAbs (3.7, 3.9, 3.11, 3.12, 5.4, and 10.3)
incubated with either parental 3T3 cells (denoted by black lines)
or 3T3 cells engineered to express cell-surface PSMA (3T3-PSMA;
gray lines).
[0111] FIG. 2 shows a digitized image of immunoprecipitation of
PSMA by mAbs. Lysates from 3T3-PSMA cells or parental 3T3 cells
were incubated with each mAb and then precipitated using Protein
A/G agarose beads. After washing, proteins were resolved on a
polyacrylamide gel, blotted onto nitrocellulose membranes and
visualized using the MAB544 anti-PSMA mAb.
[0112] FIG. 3 shows the recognition of non-denatured PSMA by
several PSMA antibodies that recognize PSMA conformation.
[0113] FIG. 4 is a digitized image of a Western blot that shows the
recognition of denatured PSMA by two PSMA antibodies and shows that
antibodies that recognize PSMA conformation do not recognize
denatured PSMA.
[0114] FIG. 5 is a digitized image of a polyacrylamide gel that
shows an analysis of purified recombinant, soluble PSMA (rsPSMA)
and of full-length PSMA from 3T3 cells (3T3 PSMA) or LNCaP cells
(LNCaP PSMA) by reduced and non-reduced SDS-PAGE.
[0115] FIG. 6 provides the results for the determination of the
dimeric structure of PSMA. FIG. 6A is a digitized image of a
polyacrylamide gel that depicts a Blue Native PAGE analysis of
purified recombinant, soluble PSMA (Purified rsPSMA) and of
full-length PSMA extracted from 3T3 cells (3T3 PSMA) or LNCaP cells
(LNCaP PSMA). FIG. 6B shows the results of the analytical size
exclusion chromatography (SEC) of purified rsPSMA in neutral PBS
buffer. The arrows indicate the retention times of protein
standards. The retention time of 260 kDa for rsPSMA is consistent
with that of a homodimer.
[0116] FIG. 7 illustrates that the dimeric but not monomeric rsPSMA
(also referred to as PSMA.sub.ECTO) is enzymatically active.
Dimeric and monomeric PSMA were tested for folate hydrolase
activity (FIG. 7A) and NAALADase activity (FIG. 7B). The background
activity observed for PSMA monomer is consistent with residual
amount (approximately 4%) of dimer present in the preparation.
[0117] FIG. 8 shows the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase through measuring the rate of cleavage of glutamate from
methotrexate di-gamma glutamate by folate hydrolase present in
0.0002 .mu.g rsPSMA #7.
[0118] FIG. 9 shows the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase through measuring the rate of cleavage of glutamate from
methotrexate di-gamma glutamate by folate hydrolase present in
0.0002 .mu.g rsPSMA #8.
[0119] FIG. 10 shows the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase through measuring the rate of cleavage of glutamate from
methotrexate di-gamma glutamate by folate hydrolase present in
lysates of C4-2 cells.
[0120] FIG. 11 shows the impact of four antibodies (human mAbs 006,
026 and 4.40.2 as well as murine mAb 5.4) on PSMA folate hydrolase
activity.
[0121] FIG. 12 illustrates the rapid and efficient internalization
of .sup.111In labeled mAb 026 incubated with C4-2 cells.
[0122] FIG. 13 depicts the cloning protocol for IgG1 antibody
cloning into pcDNA.
[0123] FIG. 14 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-006.
[0124] FIG. 15 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-026.
[0125] FIG. 16 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-051.
[0126] FIG. 17 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PG1-XG1-069.
[0127] FIG. 18 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody AB-PGL-XG1-077.
[0128] FIG. 19 provides the plasmid map of a nucleic acid molecule
encoding the heavy chain of antibody PSMA 10.3.
[0129] FIG. 20 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-006.
[0130] FIG. 21 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-026.
[0131] FIG. 22 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-051.
[0132] FIG. 23 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-069.
[0133] FIG. 24 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody AB-PG1-XG1-077.
[0134] FIG. 25 provides the plasmid map of a nucleic acid molecule
encoding the light chain of antibody PSMA 10.3.
[0135] FIG. 26 depicts the cytotoxicity of .sup.225Ac-3.9 on LNCaP
target cells.
[0136] FIG. 27 illustrates the reactivity of anti-PSMA monoclonal
antibodies XG-006, XG-051, 4.40.1, 4.49.1, 4.292.1 and 4.304.1
incubated with either parent 3T3 cells (black histogram) or 3T3
cells engineered to express cell-surface human PSMA (red histogram)
and analyzed by flow cytometry.
[0137] FIG. 28 illustrates the binding of the anti-PSMA Abs. FIG.
28A shows that anti-PSMA mAbs bind to 3T3-PSMA cells and not 3T3
cells. One representative experiment from at least ten
determinations is shown. FIG. 28B illustrates that binding to
cell-surface PSMA using serial dilutions of anti-PSMA
mAb-containing culture supernatants occurred. One representative
experiment from five is shown. FIG. 28C shows binding to
cell-surface PSMA using serial dilutions of purified anti-PSMA
mAbs, XG-006 and 10.3 One representative experiment is shown.
[0138] FIG. 29 illustrates the immunotoxin cytotoxicity of murine
anti-PSMA antibodies on C4-2 prostate cancer cells. SJ25C-1 as a
control antibody is a murine anti-CD19 IgG. The LD 50s (M) for 5.4,
3.9, and mJ591 antibodies were 2.27.times.10.sup.-11,
2.29.times.10.sup.-11 and 8.82.times.10.sup.-11, respectively.
[0139] FIG. 30 illustrates the immunotoxin cytotoxicity of murine
anti-PSMA antibodies on PSMA-3T3 cells. SJ25C-1 as a control
antibody is a murine anti-CD19 IgG. The LD 50s (M) for 5.4, 3.9,
and mJ591 antibodies were 1.64.times.10.sup.-11,
1.96.times.10.sup.-11 and 8.90.times.10.sup.-11, respectively.
[0140] FIG. 31 provides the cytotoxicity of direct conjugated human
4.304 anti-PSMA antibodies with saporin on PSMA-3T3. The LD50 was
1.48.times.10.sup.-11 M for direct conjugated 4.304 anti-PSMA
antibodies with saporin.
[0141] FIG. 32 illustrates the results of the competition assay of
unmodified 4.304, 4.40, mJ591 anti-PSMA antibodies used to compete
with In-111 radiolabeled 4.40 and 4.304 anti-PSMA antibodies.
[0142] FIG. 33 illustrates the results of the competition assay of
unmodified 4.304, mJ591 anti-PSMA antibodies used to compete with
In-111 radiolabeled mJ591 anti-PSMA antibodies.
[0143] FIG. 34 shows an analysis of antibody PRGX1-XG-006 in
association phase and dissociation phase at different
concentrations of rsPSMA from 100 nM to 6.25 nM.
[0144] FIG. 35 shows the results of the comparison of the fully
human anti-PSMA antibodies 4.40.1, 4.49.1, 051 and 006 and the
murine anti-PSMA antibody 3.9 performed using Biacore analysis.
[0145] FIG. 36 provides results from the Scatchard analysis using
In-111 labeled anti-PSMA antibody 3.9 of the PSMA-3T3, LNCaP and
C4-2 cell lines.
[0146] FIG. 37 shows in vitro cytotoxicity of Ac-225 labeled human
anti-PSMA antibody 4.40 on prostate cancer cells.
[0147] FIG. 38 shows the specific killing of PSMA expressing cells
(C4-2) vs. PSMA non-expressing cells (PC-3) treated with .sup.225Ac
labeled mAb 026.
[0148] FIG. 39 shows the in vitro cytotoxicity of .sup.225Ac
labeled mAb 026 on human prostate cancer cell lines (C4-2 and
LNCaP).
[0149] FIG. 40 shows the in vitro cytotoxicity of .sup.225Ac
labeled mAb 026 on human prostate cancer cell line, C4-2, evaluated
by .sup.3H thymidine incorporation.
[0150] FIG. 41 shows the results of in vivo radioimmunotherapy with
Lu-177 labeled human anti-PSMA antibodies.
[0151] FIG. 42 provides the radio-HPLC profile and cell-based
immunoreactivity of .sup.177Lu labeled antibodies (006, 026, mJ591
and HuIgG (control)).
[0152] FIG. 43 shows the specific binding of .sup.177Lu labeled
antibodies (006, 026, mJ591 and IgG (control)) to PSMA positive
tumors in vivo.
[0153] FIG. 44 shows the preferential retention of radiolabeled
antibodies (006, 026, mJ591 and HuIgG) in PSMA+ tumors vs. PSMA-
tumors as assessed by the percent activity in the tumors.
[0154] FIG. 45 provides data for normal organ (blood, liver,
spleen, lungs, bone, heart and muscle) uptake (injected dose per
gram of tissue, % ID/g) for the antibodies (006, 026, mJ591 and
HuIgG).
[0155] FIG. 46 illustrates the therapeutic efficacy of .sup.177Lu
labeled mAb 026 in PSMA-3T3 and 3T3 tumor-bearing mice.
[0156] FIG. 47 shows the preferential binding of mAb 006 to rsPSMA
dimer.
[0157] FIG. 48 shows the preferential binding of mAb 026 to rsPSMA
dimer.
[0158] FIG. 49 shows the binding of mAb 4.40 to rsPSMA dimer and
monomer.
[0159] FIG. 50 shows the binding of mAb mJ591 to rsPSMA dimer and
monomer.
[0160] FIG. 51 is a series of graphs that show flow cytometry data
for the binding of anti-PSMA antisera to PSMA-3T3 cells. Antisera
from mice immunized with a rsPSMA dimer preparation (ABIM151,
ABIM152, ABIM153, ABIM154 and ABIM155) exhibited strong binding to
PSMA-expressing cells. Antisera from mice immunized with a rsPSMA
monomer preparation (ABIM156, ABIM157, ABIM158, ABIM159 and
ABIM160) exhibited little or no binding to PSMA-expressing
cells.
[0161] FIG. 52 provides the results showing antibody dependent
cell-mediated cytotoxicity (ADCC) of human prostate cancer cells
mediated by mAbs 006 and 026.
[0162] FIG. 53 shows the results of PSMA monomer-dimer equilibrium
analysis. Purified dimeric (FIG. 53A) and monomeric (FIG. 53B)
rsPSMA were subjected to various buffer conditions and analyzed for
size by analytical size exclusion chromatography (SEC). The
percentages of monomer (M) and dimer (D) are indicated. The monomer
and dimer were initially contained in PBS+ buffer at a
concentration of 0.2 mg/ml. The buffer conditions were adjusted as
indicated, and the proteins were incubated at ambient temperature
for the indicated time periods before SEC analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0163] The present invention provides, in part, multimeric,
particularly dimeric, forms of PSMA protein, compositions and kits
containing dimeric PSMA protein as well as methods of producing,
purifying, processing and using these compositions. Such methods
include methods for eliciting or enhancing an immune response to
PSMA and/or cells expressing PSMA. Such methods include methods of
producing antibodies to dimeric PSMA as well as methods of treating
cancer, such as prostate cancer.
[0164] Prostate specific membrane antigen (PSMA) is a 100 kD Type
II membrane glycoprotein expressed in prostate tissues and was
originally identified by reactivity with a monoclonal antibody
designated 7E11-C5 (Horoszewicz et al., 1987, Anticancer Res.
7:927-935; U.S. Pat. No. 5,162,504). PSMA was obtained in purified
form (Wright et al., 1990, Antibody Immunoconjugates and Radio
Pharmaceuticals 3: Abstract 193) and characterized as a type II
transmembrane protein having sequence identity with the transferrin
receptor (Israeli et al., 1994, Cancer Res. 54:1807-1811) and with
NAALADase activity (Carter et al., 1996, Proc. Natl. Acad. Sci.
U.S.A. 93:749-753). More importantly, PSMA is expressed in
increased amounts in prostate cancer, and elevated levels of PSMA
are also detectable in the sera of these patients (Horoszewicz et
al., 1987; Rochon et al., 1994, Prostate 25:219-223; Murphy et al.,
1995, Prostate 26:164-168; and Murphy et al., 1995, Anticancer Res.
15:1473-1479). PSMA expression increases with disease progression,
becoming highest in metastatic, hormone-refractory disease for
which there is no present therapy. Provocative recent data
indicates that PSMA is also abundantly expressed on the
neovasculature of a variety of other important tumors, including
bladder, pancreas, sarcoma, melanoma, lung, and kidney tumor cells,
but not on normal vasculature.
[0165] It has been discovered that PSMA in its native form is a
homodimer. When ordinary isolation techniques are followed,
however, the native form of PSMA is not typically maintained.
Compositions of isolated PSMA protein that include isolated
multimeric PSMA, particularly dimeric PSMA, therefore, are
provided. These compositions include isolated PSMA protein, wherein
at least about 5% of the isolated PSMA protein is in multimeric
form. Other compositions are provided where at least about 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95% or more of the isolated PSMA protein is in
multimeric form. In a preferred embodiment, the PSMA protein
multimer composition contains substantially pure PSMA protein
multimer, with substantially no PSMA protein monomer. It is
understood that the list of specific percentages includes by
inference all of the unnamed percentages between the recited
percentages. It has further been discovered that certain agents
preserve or promote the multimeric, particularly the dimeric,
association of isolated PSMA. Compositions of isolated PSMA protein
that include these agents as well as methods of purifying and
processing isolated PSMA protein compositions are, therefore, also
provided.
[0166] As used herein "PSMA protein" includes the full-length PSMA
protein (provided as SEQ ID NO: 1) or a portion thereof. These
proteins are capable of forming multimers or aggregates of PSMA
protein. As used herein, a "multimer or aggregate of PSMA protein"
refers to the association of two or more PSMA proteins. Preferably,
the PSMA proteins described herein are those that are capable of
forming a dimer like that of native PSMA by non-covalent
interactions or engineered to form a stable native-like dimer
through covalent bonds, such as disulfide bonds. "A dimer like that
of native PSMA" includes two PSMA proteins that are associated in
the same way as the protein as found in nature or in such a way as
to allow for the generation of antibodies that recognize at least
one antigenic epitope of the native dimer (i.e., associate in a way
such as to form an antigenic region as found in the native PSMA
dimer or one capable of generating cross-reacting antibodies). The
antibodies generated to the dimers provided herein are, therefore,
capable of recognizing the native dimer. Preferably, the antibodies
generated recognize native PSMA dimer but not PSMA monomer or have
greater specificity for the native PSMA dimer than the monomer. In
some embodiments, the PSMA proteins provided herein are larger
aggregates of PSMA (i.e., three or more PSMA protein that are
associated). These aggregates are likewise capable of generating
antibodies that recognize PSMA. In some embodiments, these
antibodies do not recognize PSMA monomer but do recognize native
PSMA dimer. In other embodiments, these antibodies have greater
specificity for the native PSMA dimer rather than PSMA monomer.
[0167] PSMA multimers are typically homomultimers (i.e., the
associated PSMA proteins are the same). However, in some
embodiments the PSMA multimers can be heteromultimers, particularly
heterodimers. As used herein a "PSMA heteromultimer" is a multimer
of PSMA proteins that is composed of at least two different PSMA
proteins. Examples include two PSMA fragments, where one is
slightly longer than the other or when one has a conservative amino
acid substitution and the other does not. The heteromultimers
provided herein, like homomultimers, are capable of generating
antibodies that recognize native PSMA dimer. In preferred
embodiments the antibodies raised against the PSMA heteromultimers
recognize native PSMA dimer but not PSMA monomer. In still other
preferred embodiments these antibodies have greater specificity for
native PSMA dimer rather than PSMA monomer.
[0168] PSMA protein capable of forming multimers, particularly
dimers, include the full-length protein (SEQ ID NO: 1). In some
embodiments the PSMA protein capable of forming a multimer is the
extracellular portion of PSMA (amino acids 44-750 of SEQ ID NO: 1).
In other embodiments the PSMA protein capable of forming a multimer
is PSM' (amino acids 58-750 of SEQ ID NO: 1), an alternatively
spliced form of PSMA. In yet other embodiments fragments of the
full-length protein, the extracellular portion or PSM' are capable
of forming multimers. For example, these fragments include
truncated PSMA proteins that begin at amino acid 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, etc. of SEQ ID NO: 1 and end at amino acid 750 of SEQ ID NO: 1.
Other such truncated proteins begin at amino acid 44 of SEQ ID NO:
1 and end at amino acid 749, 748, 747, 746, 745, 744, 743, 742,
741, 740, etc. of SEQ ID NO: 1. Still other truncated proteins
include those that begin at amino acid 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, etc. of
SEQ ID NO: 1 and end at amino acid 749, 748, 747, 746, 745, 744,
743, 742, 741, 740, etc. of SEQ ID NO: 1. In some embodiments the
truncated PSMA protein includes the amino acids 601-750 of SEQ ID
NO: 1 or a functional portion thereof capable of forming dimers. As
provided herein, the PSMA proteins are intended to encompass any
fragment of the PSMA protein that is capable of forming a multimer
as provided herein. Therefore, any portion of SEQ ID NO: 1 is
included in this definition as well as its functional variant.
Functional variants are described further herein below.
[0169] In some embodiments the isolated PSMA protein is not
full-length PSM' (amino acids 58-750 of SEQ ID NO: 1) or the
full-length extracellular portion of PSMA (amino acids 44-750 of
SEQ ID NO: 1). In other instances, the isolated PSMA protein is not
full-length PSMA (SEQ ID NO: 1). The fragment can have a size of at
least about 25, 50, 100, 125, 150, 175, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, or 749 amino acids and every integer
length therebetween. In some embodiments, these fragments include
amino acids 63-68, 132-137 or 482-487 of SEQ ID NO:1. In some other
preferred instances, the PSMA protein is not membrane-bound.
[0170] Compositions of PSMA protein with agents and/or solutions
that preserve or promote the multimeric association, particularly
the dimeric association, of PSMA also are provided. In some
instances the agents are in a solution along with the PSMA protein
but are not necessarily so. An agent or solution that "preserves or
promotes the dimeric association of PSMA" is one that either
maintains the dimeric association (dimerization) of PSMA over time
or facilitates the dimeric association of monomeric forms of the
PSMA protein. For example, any solution that increases the amount
of PSMA dimers, maintains the amount of PSMA dimers or retards the
disassociation of PSMA dimers is encompassed by the above
definition. Although the dimeric state is specifically recited,
these terms are also intended to encompass other multimeric states
of PSMA, and therefore, compositions, kits and methods of
production and use of other multimers of PSMA.
[0171] Preferably, the "preservation or promotion of dimeric PSMA"
refers to the maintenance of the dimeric state of PSMA protein for
at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%
or more of the PSMA dimers initially present in a composition.
Preferred compositions comprising the dimeric form of PSMA have
less than about 35% of the monomeric form of PSMA, preferably less
than about 20%, more preferably less than about 15% of the
monomeric form. In one embodiment the composition has less than
about 5% of the monomeric PSMA protein. The preservation or
promotion of dimeric PSMA also refers to the conversion of about
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the
initially monomeric PSMA to dimeric form.
[0172] The promotion of dimerization or maintenance of the dimeric
state can occur at any of a number of experimental or storage
temperatures. In some instances the promotion or preservation of
dimeric PSMA occurs at a temperature of about 45.degree. C. or
lower. In other instances the promotion or preservation of dimeric
PSMA is at a temperature of about 37.degree. C. The promotion or
preservation can also be at a temperature range of about 20.degree.
C. to about 30.degree. C. or about or below room temperature. In
other instances the promotion or preservation is at a range of
about 4.degree. C. to about 20.degree. C. In still other instances
the promotion or preservation is at about -20.degree. C. to about
4.degree. C. or about -80.degree. C. to about -20.degree. C. The
promotion or preservation of the dimeric state of PSMA can also
occur in a composition of PSMA protein that is in solution or in a
freeze-dried form, e.g., lyophilized form. The dimeric state can
also be promoted or preserved over any period of time. In some
instances the period of time is at least about 1, 2, 3, 4, 5, 6,
10, 15, 20, 24, 48, 72 or more hours. In other instances the period
of time is at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 or more
days. In still other instances the period of time is at least about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30 or more weeks. In
yet other instances, the period of time is at least about 1, 2, 3,
6, 9, 12 or more months or as long as 2 years or more. The
formulations provided herein are stable during long-term storage,
i.e., the formulations preserve or promote the dimeric PSMA
state.
[0173] It was surprisingly discovered that pH alone can influence
the dimeric state of PSMA. As described below in the Examples
section, the pH at which a PSMA solution is incubated can influence
the multimeric form of PSMA as well as its recovery. Incubation at
various pHs for 4 days at a temperature of about 45.degree. C.
influenced the dimerization or aggregation of PSMA protein as well
as the recovery of PSMA protein by analytical TSK gel filtration
chromatography. The benefits of pH on the preservation of dimeric
rsPSMA (2 mg/ml in PBS+) are retained when the protein solution is
diluted 10-fold in a variety of buffer solutions, each containing 2
mM glycine, 2 mM citric acid, 2 mM Hepes, 2 mM MES and 2 mM Tris
Base.
[0174] The dimeric structure of PSMA according to the invention is
preserved at a pH in the range of about 4 to about 8. Therefore, a
solution that preserves or promotes the dimerization of PSMA is one
with a pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8. Recovery
of dimeric PSMA from a column was better at a pH in the range of
about 5 to about 7, and these pH values are preferred. In some
instances, a pH of about 6 is preferred. Thus, the invention
provides formulations of PSMA in solution, wherein the pH is in the
range from about 4 to about 8, preferably in the range from about 5
to about 7, more preferably in the range from about 5.5 to about 7,
and most preferably in the range from about 6 to about 7.
[0175] An "agent that preserves or promotes the dimeric association
of PSMA" is meant to encompass an agent that promotes or maintains
the dimerization of PSMA. Such agents have been found to include pH
adjusting agents (as discussed above), metal ions and salts. It has
been discovered that these agents, individually or in combination,
are able to preserve or promote dimeric association of PSMA. In
some embodiments it is the combination of the metal ion, salt or pH
adjusting agent that can promote or preserve dimeric association of
PSMA, while the individual metal ion, salt or pH adjusting agent
cannot. As provided in the Examples, the use of chelating agents,
such as EDTA, converted dimeric PSMA into the monomer. This result
indicated that the presence of metal ions can positively affect the
stability of the dimer. Additionally, PSMA shares modest sequence
and structural homology with human transferrin receptor (TfR),
which contains additional metal-binding sites within its helical
domains (Lawrence, C. M., et al. (1999) Science 286, 779-782).
Therefore, metal ions are considered to be agents which promote or
preserve the dimeric state of PSMA protein. Such metals ions
include, but are not limited to, zinc ions (e.g., Zn.sup.2+),
calcium ions (e.g., Ca.sup.2+), magnesium ions (e.g., Mg.sup.2+),
cobalt ions (e.g., Co.sup.2+), manganese ions (e.g., Mn.sup.2+) or
combinations thereof.
[0176] In some instances these metal ions can be added to a
composition of PSMA protein in the form of a salt. Such salts
include zinc chloride, calcium chloride, magnesium chloride, cobalt
chloride or manganese chloride. It has been further determined that
compositions of PSMA protein, wherein the dimeric state is promoted
or preserved, include at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5 or more molar equivalents of metal ion
to PSMA protein (total PSMA protein, i.e., total amount of PSMA
protein molecules). In some instances, the molar equivalent of
metal ions should be in molar excess to PSMA protein. In some
specific solutions of PSMA protein (2 mg/ml in PBS+; diluted
10-fold), as provided in the Examples, it has further been found,
that the metal ions are preferably present at a concentration in
the range of about 0.1 mM to about 5 mM. The metal ions in some
instances are present at a concentration in the range of about 0.1
mM to about 1 mM. In other embodiments the metal ions are present
at a concentration in the range of about 0.1 mM to about 0.5 mM. In
solutions where there is a combination of one or more types of
metal ions, the one or more metal ions can be at the same
concentration or at a different concentration. For example, one
such solution can contain a concentration of calcium ions of about
0.5 mM and a concentration of zinc ions of a concentration that is
greater than 0.1 mM but less than 0.5 mM. Because of the importance
of metal ions in the dimerization of PSMA in some compositions, in
some instances, it is preferred that the compositions do not
contain a chelating agent.
[0177] It has also been found that salts preserve or promote PSMA
dimerization. As shown below in the Examples, a dimer preparation
that contained approximately 5% monomer initially was converted to
100% dimer upon incubation for 72 hours at ambient temperature in
PBS+(phosphate-buffered saline containing 1 mM Ca.sup.2+ and 0.5 mM
Mg.sup.2+, pH 7.2) supplemented with 2 M sodium chloride. For a
preparation that initially comprised >95% monomer, high salt
similarly drove the equilibrium to mostly (81%) dimer within 72
hours.
[0178] Salts that preserve or promote PSMA dimerization can include
those with a cationic component selected from the group consisting
of sodium, potassium, ammonium, magnesium, calcium, zinc and
combinations thereof, and those with an anionic component selected
from the group consisting of chloride, sulfate, acetate and
combinations thereof. In preferred embodiments the salt is sodium
chloride, sodium sulfate, sodium acetate or ammonium sulfate. The
salt can be present in a PSMA-containing composition at any
concentration that preserves or promotes the dimerization of PSMA.
In some instances the salt is present at a concentration in the
range of about 50 mM to about 2 M. The salt preferably is present
at a concentration of about 100 mM to 300 mM. The salt more
preferably is present at a concentration of about 150 mM.
[0179] In some cases where a high salt concentration is used to
promote or preserve PSMA dimerization, the salt concentration can
be diluted to within a physiologically acceptable range suitable
for parenteral use prior to administration. As an example, the salt
concentration can be diluted with an adjuvant or a diluent.
Diluents and adjuvants are both well known in the art. An adjuvant
is a substance which potentiates the immune response. Specific
examples of adjuvants include monophosphoryl lipid A (MPL,
SmithKline Beecham); saponins, including QS-7, QS-17, QS-18, QS-21
(Antigenics, New York, N.Y.; U.S. Pat. Nos. 6,524,584 and
6,645,495); saponin-based adjuvants, such as SaponImmune.TM.
(GPI-0100) Series (Galenica Pharmaceuticals, Birmingham, Ala.; U.S.
Pat. Nos. 5,977,081 and 6,080,725) and chemically modified saponins
(Galenica Pharmaceuticals, U.S. Pat. No. 6,262,029);
polysaccharide-based adjuvants, such as PolysaccImmune.TM.
(GPI-0200) Series (Galenica Pharmaceuticals); synthetic adjuvants,
such as SynthImmune.TM. (GPI-0300) Series (Galenica
Pharmaceuticals); biodegradable particles composed of
poly-lactide-co-glycolide (PLG) or other similar polymers;
immunostimulatory oligonucleotides (e.g., CpG oligonucleotides
described by Kreig et al., Nature 374:546-9, 1995); incomplete
Freund's adjuvant; complete Freund's adjuvant; vitamin E and
various water-in-oil emulsions prepared from biodegradable oils
such as squalene and/or tocopherol; montanide, such as MONTANIDE
ISA51 and MONTANIDE ISA720, which are water-in-oil emulsions
provided by Seppic (Paris, France); Quil A; micellular mixtures of
Quil A and cholesterol known as immunostimulating complexes
(ISCOMS); MPL and cell wall skeleton from mycobacterium
combinations such as ENHANZYN.TM. (Corixa, Seattle, Wash.); RC-529
(Corixa); RC-552 (Corixa); CRL-1005; L-121;
alpha-galactosylceramide (Fujii et al., J. Exp. Med., 2003, Jul.
21; 198(2): 267-79); aluminum or iron oxide beads and combinations
thereof. Other specific examples of adjuvants include QS-21
fractions, such as crude QA-21; a QA-21H form; QA-21-V1; QA-21-V2;
a combination of QA-21-V1 and QA-21-V2; and chemically modified
forms or combinations thereof. Preferred adjuvants include alum and
QS-21. Other diluents include water suitable for injection, saline,
PBS, solubilizing agents and emulsifiers such as ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl
formamide, oils (in particular, cottonseed, groundnut, corn, germ,
olive, castor and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan and
mixtures thereof.
[0180] Therefore, in some aspects of the invention a preferred
composition comprising isolated PSMA protein is a solution that
promotes or preserves multimeric, particularly dimeric, association
of PSMA protein comprising 5 to 20 mM sodium phosphate, sodium
acetate or a combination thereof; 100 to 300 mM sodium chloride or
sodium sulfate; and 0.1 to 2 mM of at least one metal ion. The
metal ions can be chosen from zinc ions, calcium ions, magnesium
ions, cobalt ions, manganese ions or a combination thereof. The pH
of such a solution can also be adjusted to be in a range of about 4
to 8, preferable 5 to 7 and most preferable 6 to 6.5. Such a
solution can also, optionally, include an adjuvant such as alum or
a saponin-based adjuvant, such as QS-21.
[0181] Agents that preserve or promote PSMA dimerization can be
used in compositions of PSMA protein or methods of processing such
compositions. Furthermore, a method for identifying such agents is
provided herein. Such a method includes the following steps:
determining the amount of a form of PSMA in a sample prior to
exposure to a candidate agent; exposing the sample to the candidate
agent; determining the amount of the form of PSMA in the sample
after the exposure; and comparing the amount of the form of PSMA in
the sample prior to and after the exposure to the candidate agent.
The form of PSMA can be a monomer or multimer, preferably the
dimer. An agent which preserves and/or promotes dimer formation of
PSMA protein is suitable for use in the compositions comprising
PSMA protein dimers.
[0182] As described below the effect of buffering agent on the
ability of PSMA to dimerize or maintain its dimerization was also
tested. It was found that many can be used in a solution of PSMA
without negatively impacting the dimeric state of PSMA. The sole
exception for solutions of PSMA protein with 150 mM NaCl at a pH of
6 was citrate buffer. Interestingly, citrate buffer is known to
function as a chelating agent. Therefore the formulations of PSMA
described herein can include any buffer so long as the buffer is
not one with a chelating effect that outweighs the preservation or
promoting effect of the other properties of the formulation.
Preferably optimal buffers include those with buffering capacity at
a pH in the range of about 4 to about 8. More preferably, buffers
are those with buffering capacity at a pH in the range of about 5
to about 7. Most preferably the buffers are those that have
buffering capacity at a pH in the range of about 5.5 to about 7.
Buffers in general are well known to those of ordinary skill in the
art. Buffer systems include citrate buffers, acetate buffers,
borate buffers, and phosphate buffers. Specific examples of buffers
include citric acid, sodium citrate, sodium acetate, acetic acid,
sodium phosphate and phosphoric acid, sodium ascorbate, tartartic
acid, maleic acid, glycine, sodium lactate, lactic acid, ascorbic
acid, imidazole, sodium bicarbonate and carbonic acid, sodium
succinate and succinic acid, histidine, and sodium benzoate and
benzoic acid. Buffers also include PBS and Hepes.
[0183] The effect of free amino acids on the dimeric state of
rsPSMA (2 mg/ml in PBS+) dialyzed into 20 mM sodium acetate and 150
mM NaCl at a pH of about 6 was also tested. In general it was found
that free amino acids did not have a strong negative effect on
dimer association of PSMA and/or column recovery, with the
exception of histidine, glutamic acid and aspartic acid used
individually at the specific experimental conditions. Therefore,
the formulations provided herein can also include a free amino acid
or combination of free amino acids, provided that the free amino
acid does not have a negative effect that outweighs the dimeric
association promoting or preserving nature of the specific
formulation. Such free amino acids can be naturally occurring,
modified or non-naturally occurring free amino acids (i.e.,
compounds that do not occur in nature but that can be incorporated
into a polypeptide chain; see, for example,
http://www.cco.caltech.edu/.about.dadgrp/Unnatstruct.gi- f, which
displays structures of non-natural amino acids that have been
successfully incorporated into functional ion channels). Modified
or non-naturally occurring free amino acids also include but are
not limited to 2-aminoadipic acid; 3-aminoadipic acid;
beta-alanine, beta-aminopropionic acid; 2-aminobutyric acid;
4-aminobutyric acid, piperidinic acid; 6-aminocaproic acid;
2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine;
2,2'-diaminopimelic acid; 2,3-diaminopropionic acid;
N-ethylglycine; N-ethylasparagine; hydroxylysine;
allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline;
isodesmosine; allo-isoleucine; N-methylglycine, sarcosine;
N-methylisoleucine; 6-N-methyllysine; N-methylvaline; norvaline;
norleucine and ornithine. In particular, free amino acids that do
not have a negative effect on dimeric association of PSMA and/or
column recovery include those that are non-acidic. Examples of
these non-acidic free amino acids include glycine, proline,
isoleucine, leucine, alanine and arginine.
[0184] In addition to free amino acids, surfactants and other
excipients were also found not to have a negative impact on the
dimeric state of PSMA. Therefore, surfactants as well as other
excipients can be included in the compositions provided herein.
Examples of surfactants include those known in the art and
described herein. For example, surfactants include Triton X-100,
dodecylmaltoside, cholic acid and CHAPS.
[0185] Examples of excipients include binders, coatings,
compression/encapsulation aids, disintegrants, creams and lotions,
lubricants, materials for chewable tablets, parenterals,
plasticizers, powder lubricants, soft gelatin capsules, spheres for
coating, spheronization agents, suspending/gelling agents,
sweeteners and wet granulation agents. Specific examples of such
excipients include acetyltriethyl citrate (ATEC); acetyltri-n-butyl
citrate (ATBC); aspartame; aspartame and lactose; alginates;
calcium carbonate; carbopol; carrageenan; cellulose acetate
phthalate-based coatings; cellulose-based coatings; cellulose and
lactose combinations; colorants for film coating systems;
croscarmellose sodium; crospovidone; dextrose; dibutyl sebacate;
ethylcellulose-based coatings; fructose; gellan gum; glyceryl
behenate; honey; lactose; anhydrous; lactose; monohydrate; lactose
and aspartame; lactose and cellulose; lactose and microcrystalline
cellulose; L-HPC (Low-substituted HydroxyPryopl Cellulose);
magnesium stearate; maltodextrin; maltose DC; mannitol DC;
methylcellulose-based coatings; microcrystalline cellulose;
methacrylate-based coatings; microcrystalline cellulose and
carrageenan; microcrystalline cellulose and guar gum;
microcrystalline cellulose and lactose; microcrystalline cellulose
and sodium carboxymethylcellulose; molasses DC; polyvinyl acetate
phathalate (PVAP); povidone; shellac; sodium starch glycolate;
sorbitol, crystalline; sorbitol, special solution; starch DC;
sucrose DC; sugar spheres; triacetin; triethylcitrate and xanthan
gum. Other excipients include antioxidants and cryoprotectants.
[0186] Antioxidants are substances capable of inhibiting oxidation
by removing free radicals from solution. Antioxidants are well
known to those of ordinary skill in the art and include materials
such as ascorbic acid, ascorbic acid derivatives (e.g.,
ascorbylpalmitate, ascorbylstearate, sodium ascorbate, calcium
ascorbate, etc.), butylated hydroxy anisole, butylated hydroxy
toluene, alkylgallate, dithiothreitol (DTT), sodium meta-bisulfite,
sodium bisulfite, sodium dithionite, sodium thioglycollic acid,
sodium formaldehyde sulfoxylate, tocopherol and derivatives thereof
(e.g., d-alpha tocopherol, d-alpha tocopherol acetate, dl-alpha
tocopherol acetate, d-alpha tocopherol succinate, beta tocopherol,
delta tocopherol, gamma tocopherol, and d-alpha tocopherol
polyoxyethylene glycol 1000 succinate) monothioglycerol, and sodium
sulfite. Such materials are typically added in ranges from about
0.01 to about 2%.
[0187] For a lyophilized product or a product stored in the cold,
one or more cryoprotectants can be added. Typical cryoprotectants
for proteins include but are not limited to: sugars such as
sucrose, lactose, glucose, trehalose, maltose, and the like;
polyols such as inositol, ethylene glycol, glycerol, sorbitol,
xylitol, mannitol, 2-methyl-2,4-pentane-diol and the like; amino
acids such as Na glutamate, proline, alpha-alanine, beta-alanine,
glycine, lysine-HCl, 4-hydroxyproline; polymers such as
polyethylene glycol, dextran, polyvinylpyrrolidone and the like;
inorganics salts such as sodium sulfate, ammonium sulfate,
potassium phosphate, magnesium sulfate, and sodium fluoride and the
like; organics salts such as sodium acetate, sodium polyethylene,
sodium caprylate, proprionate, lactate, succinate and the like; as
well as agents such as trimethylamine N-oxide, sarcosine, betaine,
gamma-aminobutyric acid, octapine, alanopine, strombine,
dimethylsulfoxide, and ethanol.
[0188] The invention also involves methods for preparing or
processing compositions of PSMA protein. Aqueous solutions of PSMA
protein are included in these methods. Some of these methods
include the step of adjusting the pH so that it is in the range of
about 4 to about 8. In some methods the pH is adjusted to be in the
range of about about 5 to 7, and more preferably the pH is adjusted
to be in the range of about 5.5 to 7. Most preferably the pH is
adjusted to be about 6. The compositions can also contain any one
or combination of an isotonicity agent, a buffering agent, a
surfactant, an antioxidant, a cryoprotectant or other excipients.
Preferably the compositions do not include a chelating agent.
[0189] According to another aspect of the invention, a composition
of PSMA protein is processed by contacting the composition of PSMA
protein with an agent that promotes or preserves the dimeric
association of PSMA such as pH adjusting agents, metal ions and/or
salts as provided above. Compositions that include these agents can
also include agents selected from an isotonicity agent, a buffering
agent, a surfactant, an antioxidant, a cryoprotectant and other
excipients, but preferably, not a chelating agent. Such methods can
also include further steps of contacting the composition of PSMA
protein with other dimer promoting or preserving agents and/or pH
adjusting steps when the PSMA protein is in a solution.
[0190] Additionally, in another aspect of the invention, a method
of purifying PSMA protein is also provided. The methods of
purifying PSMA include the use of any of the agents and/or
solutions described herein that preserve or promote the multimeric,
particularly dimeric, association of PSMA in conjunction with any
of the separation techniques that are known to those in the art.
Such separation techniques include chromatography (e.g., TSK gel
filtration chromatography) and are described in more detail in the
Examples below. For instance, a separation technique encompassed
within this aspect of the invention can include the steps of
loading a sample onto a column, eluting or washing the sample from
the column and collecting the eluted fractions. Such steps can be
repeated any of a number of times to produce the desired PSMA
protein composition. These steps can, optionally, also include
steps whereby the sample containing PSMA protein is dialyzed.
Preferably, the sample containing PSMA protein is dialyzed into a
solution that preserves or promotes the multimeric association of
PSMA. In one embodiment, the solutions used in these methods
contain a metal ion or a salt. In other embodiments, the solution
is at a pH that preserves or promotes PSMA multimerization. The
metal ion and salts, including concentration ranges, as well as pH
ranges that can be used in these purification methods have been
provided above. In some preferred embodiments, the pH of the
solution can be at about 7 or 7.5. In other preferred embodiments,
the metals are calcium ions, magnesium ions or combinations
thereof. The calcium and magnesium ions are present, for instance,
at a concentration of about 1 mM and of about 0.5 mM, respectively.
In other preferred embodiments the salt is present at a
concentration of about 2 M.
[0191] The amount of dimeric PSMA in the compositions provided
herein is effective to elicit or enhance an immune response to
cells expressing PSMA. The compositions can, therefore, be used to
immunize an animal for the purpose of raising antibodies to dimeric
PSMA. The compositions provided herein can also be used to treat a
subject suffering from a cancer, wherein the cancer cells or
proximate neovasculature express PSMA. Such cancers can include
prostate, bladder, pancreas, lung, colon, kidney, melanomas and
sarcomas. In a preferred embodiment the cancer cell is a prostate
cancer cell. The cancer cells can be cells of a primary tumor or
can be those of a metastatic tumor.
[0192] The subject can be a non-castrate patient who has, in some
embodiments, received primary therapy, such as prostatectomy and/or
radiation therapy. As used herein, "non-castrate patient" refers to
a patient in some embodiments with a serum testosterone level that
is greater than or equal to about 180 ng/mL. The subject can also
be a castrate patient who has, in some embodiments, completed a
course of hormonal therapy. As used herein, the term "castrate
patient" refers to a patient in some embodiments with a serum
testosterone level of less than about 50 ng/mL. The compositions
provided herein can also be administered to a patient who has
received conventional cancer therapy. Another aspect of the
invention provides an isolated antibody or an antigen-binding
fragment thereof which specifically binds to an extracellular
domain of PSMA wherein the antibody or the antigen-binding fragment
thereof competitively inhibits the specific binding of a second
antibody to its target epitope on PSMA, and wherein the second
antibody is selected from the group consisting of PSMA 3.7, PSMA
3.8, PSMA 3.9, PSMA 3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3,
PSMA 1.8.3, PSMA A3.1.3, PSMA A3.3.1, 4.248.2, 4.360.3, 4.7.1,
4.4.1, 4.177.3, 4.16.1, 4.22.3, 4.28.3, 4.40.2, 4.48.3, 4.49.1,
4.209.3, 4.219.3, 4.288.1, 4.333.1, 4.54.1, 4.153.1, 4.232.3,
4.292.3, 4.304.1, 4.78.1, and 4.152.1.
[0193] Another aspect of the invention provides an isolated
antibody or an antigen-binding fragment thereof that specifically
binds to an epitope on PSMA defined by an antibody selected from
the group consisting of PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA 3.11,
PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA A3.1.3,
PSMA A3.3.1, 4.248.2, 4.360.3, 4.7.1, 4.4.1, 4.177.3, 4.16.1,
4.22.3, 4.28.3, 4.40.2, 4.48.3, 4.49.1, 4.209.3, 4.219.3, 4.288.1,
4.333.1, 4.54.1, 4.153.1, 4.232.3, 4.292.3, 4.304.1, 4.78.1, and
4.152.1.
[0194] In particular embodiments, these antibodies are produced by
hybridomas referred to herein as PSMA 3.7, PSMA 3.8, PSMA 3.9, PSMA
3.11, PSMA 5.4, PSMA 7.1, PSMA 7.3, PSMA 10.3, PSMA 1.8.3, PSMA
A3.1.3, PSMA A3.3.1, Abgenix 4.248.2, Abgenix 4.360.3, Abgenix
4.7.1, Abgenix 4.4.1, Abgenix 4.177.3, Abgenix 4.16.1, Abgenix
4.22.3, Abgenix 4.28.3, Abgenix 4.40.2, Abgenix 4.48.3, Abgenix
4.49.1, Abgenix 4.209.3, Abgenix 4.219.3, Abgenix 4.288.1, Abgenix
4.333.1, Abgenix 4.54.1, Abgenix 4.153.1, Abgenix 4.232.3, Abgenix
4.292.3, Abgenix 4.304.1, Abgenix 4.78.1, and Abgenix 4.152.1,
respectively. These hybridomas were deposited with ATCC as an
International Depository Authority and given the following Patent
Deposit Designations (Table 1):
1TABLE 1 Patent Antibody Hybridoma/Plasmid Deposit Designation Date
of Deposit PSMA 3.7 PSMA 3.7 PTA-3257 Apr. 5, 2001 PSMA 3.9 PSMA
3.9 PTA-3258 Apr. 5, 2001 PSMA 3.11 PSMA 3.11 PTA-3269 Apr. 10,
2001 PSMA 5.4 PSMA 5.4 PTA-3268 Apr. 10, 2001 PSMA 7.1 PSMA 7.1
PTA-3292 Apr. 18, 2001 PSMA 7.3 PSMA 7.3 PTA-3293 Apr. 18, 2001
PSMA 10.3 PSMA 10.3 PTA-3347 May 1, 2001 PSMA 10.3 HC in PTA-4413
May 29, 2002 pcDNA (SEQ ID NO: 7) PSMA 10.3 Kappa in PTA-4414 May
29, 2002 pcDNA (SEQ ID NO: 13) PSMA 1.8.3 PSMA 1.8.3 PTA-3906 Dec.
5, 2001 PSMA A3.1.3 PSMA A3.1.3 PTA-3904 Dec. 5, 2001 PSMA A3.3.1
PSMA A3.3.1 PTA-3905 Dec. 5, 2001 Abgenix 4.248.2 Abgenix 4.248.2
PTA-4427 Jun. 4, 2002 Abgenix 4.360.3 Abgenix 4.360.3 PTA-4428 Jun.
4, 2002 Abgenix 4.7.1 Abgenix 4.7.1 PTA-4429 Jun. 4, 2002 Abgenix
4.4.1 Abgenix 4.4.1 PTA-4556 July 18, 2002 Abgenix 4.177.3 Abgenix
4.177.3 PTA-4557 July 18, 2002 Abgenix 4.16.1 Abgenix 4.16.1
PTA-4357 May 16, 2002 Abgenix 4.22.3 Abgenix 4.22.3 PTA-4358 May
16, 2002 Abgenix 4.28.3 Abgenix 4.28.3 PTA-4359 May 16, 2002
Abgenix 4.40.2 Abgenix 4.40.2 PTA-4360 May 16, 2002 Abgenix 4.48.3
Abgenix 4.48.3 PTA-4361 May 16, 2002 Abgenix 4.49.1 Abgenix 4.49.1
PTA-4362 May 16, 2002 Abgenix 4.209.3 Abgenix 4.209.3 PTA-4365 May
16, 2002 Abgenix 4.219.3 Abgenix 4.219.3 PTA-4366 May 16, 2002
Abgenix 4.288.1 Abgenix 4.288.1 PTA-4367 May 16, 2002 Abgenix
4.333.1 Abgenix 4.333.1 PTA-4368 May 16, 2002 Abgenix 4.54.1
Abgenix 4.54.1 PTA-4363 May 16, 2002 Abgenix 4.153.1 Abgenix
4.153.1 PTA-4388 May 23, 2002 Abgenix 4.232.3 Abgenix 4.232.3
PTA-4389 May 23, 2002 Abgenix 4.292.3 Abgenix 4.292.3 PTA-4390 May
23, 2002 Abgenix 4.304.1 Abgenix 4.304.1 PTA-4391 May 23, 2002
AB-PG1-XG1-006 AB-PG1-XG1-006 Heavy PTA-4403 May 29, 2002 Chain
(SEQ ID NO: 2) AB-PG1-XG1-006 Light PTA-4404 Chain (SEQ ID NO: 8)
AB-PG1-XG1-026 AB-PG1-XG1-026 Heavy PTA-4405 May 29, 2002 Chain
(SEQ ID NO: 3) AB-PG1-XG1-026 Light PTA-4406 Chain (SEQ ID NO: 9)
AB-PG1-XG1-051 AB-PG1-XG1-051 Heavy PTA-4407 May 29, 2002 Chain
(SEQ ID NO: 4) AB-PG1-XG1-051 Light PTA-4408 Chain (SEQ ID NO: 10)
AB-PG1-XG1-069 AB-PG1-XG1-069 Heavy PTA-4409 May 29, 2002 Chain
(SEQ ID NO: 5) AB-PG1-XG1-069 Light PTA-4410 Chain (SEQ ID NO: 11)
AB-PG1-XG1-077 AB-PG1-XG1-077 Heavy PTA-4411 May 29, 2002 Chain
(SEQ ID NO: 6) AB-PG1-XG1-077 Light PTA-4412 Chain (SEQ ID NO:
12)
[0195] In another aspect of the invention, antibodies having
particular sequences are provided. Specifically, the antibodies are
selected from the group consisting of antibodies comprising: a
heavy chain encoded by a nucleic acid molecule comprising the heavy
chain coding region or regions of a nucleotide sequence selected
from the group consisting of nucleotide sequences set forth as SEQ
ID NOs: 2-7, and a light chain encoded by a nucleic acid molecule
comprising the light chain coding region or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as SEQ ID NOs: 8-13. Also provided are antigen-binding
fragments of the foregoing antibodies.
[0196] The plasmids encoding the heavy and light chains of
antibodies PSMA 10.3, AB-PG1-XG1-006, AB-PG1-XG1-026,
AB-PG1-XG1-051, AB-PG1-XG1-069, AB-PG1-XG1-077 were also deposited
with ATCC and are shown in Table 1 above. As used herein, the names
of the deposited hybridomas or plasmids may be used interchangeably
with the names of the antibodies. It would be clear to one of skill
in the art when the name is intended to refer to the antibody or
when it refers to the plasmids or hybridomas that encode or produce
the antibodies, respectively. Additionally, the antibody names may
be an abbreviated form of the name shown in Table 1. For instance
antibody AB-PG1-XG1-006 may be referred to as AB-PG1-XG1-006,
PG1-XG1-006, XG1-006, 006, etc. In another example, the antibody
name PSMA 4.232.3 may be referred to as PSMA 4.232.1, 4.232.3,
4.232.1, 4.232, etc. It is intended that all of the variations in
the name of the antibody refer to the same antibody and not a
different one.
[0197] Antibodies are also provided that are encoded by particular
sets of heavy and light chain sequences. In one embodiment an
antibody (AB-PG1-XG1-006) encoded by a nucleic acid molecule which
comprises the coding region or regions of the nucleic acid
sequences set forth as: SEQ ID NOs: 2 and 8 is provided. In another
embodiment the antibody (AB-PG1-XG1-026) is encoded by the nucleic
acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 3 and 9. In still
another embodiment the antibody (AB-PG1-XG1-051) is encoded by the
nucleic acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 4 and 10. In yet
another embodiment the antibody (AB-PG1-XG1-069) is encoded by the
nucleic acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 5 and 11. In another
embodiment the antibody (AB-PG1-XG1-077) is encoded by the nucleic
acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 6 and 12. In yet
another embodiment the antibody (PSMA 10.3) is encoded by the
nucleic acid molecules comprising the coding region or regions of
nucleotide sequences set forth as: SEQ ID NOs: 7 and 13.
[0198] In particularly preferred embodiments, the antibodies
include a heavy chain variable region encoded by a nucleic acid
molecule comprising the coding regions or regions of a nucleotide
sequence selected from the group consisting of nucleotide sequences
set forth as: SEQ ID NOs: 14, 18, 22, 26 and 30, and a light chain
variable region encoded by a nucleic acid molecule comprising the
coding region or region of a nucleotide sequence selected from the
group consisting of nucleotide sequences set forth as: SEQ ID NOs:
16, 20, 24, 28 and 32. As used herein, a "coding region" refers to
a region of a nucleotide sequence that encodes a polypeptide
sequence; the coding region can include a region coding for a
portion of a protein that is later cleaved off, such as a signal
peptide.
[0199] Those of skill in the art will appreciate that the invention
includes nucleic acids and polypeptides that include nucleotide and
amino acid sequences presented herein. In some instances, the
nucleotide and amino acid sequences may include sequences that
encode or that are signal peptides. The invention embraces each of
these sequences with, or without, the portion of the sequence that
encodes or is a signal peptide.
[0200] Antibodies also are provided that include particular sets of
heavy and light chain variable sequences. In one embodiment an
antibody (AB-PG1-XG1-006) includes an immunoglobulin variable
sequence encoded by nucleic acid molecules which included the
coding region or regions of the nucleic acid sequences set forth as
SEQ ID NOs: 14 and 16 is provided. Likewise the antibody may
include an immunoglobulin variable sequence which comprises the
amino acid sequences set forth as SEQ ID NOs: 15 and 17. In another
embodiment the antibody (AB-PG1-XG1-026) includes an immunoglobulin
variable sequence encoded by nucleic acid molecules comprising the
coding region or regions of nucleotide sequences set forth as: SEQ
ID NOs: 18 and 20 or includes an immunoglobulin variable sequence
which comprises the amino acid sequences set forth as SEQ ID NOs:
19 and 21. In still another embodiment the antibody
(AB-PG1-XG1-051) includes an immunoglobulin variable sequence
encoded by the nucleic acid molecules comprising the coding region
or regions of nucleotide sequences set forth as: SEQ ID NOs: 22 and
24 or includes an immunoglobulin variable sequence which comprises
the amino acid sequences set forth as SEQ ID NOs: 23 and 25. In yet
another embodiment the antibody (AB-PG1-XG1-069) includes an
immunoglobulin variable sequence encoded by the nucleic acid
molecules comprising the coding region or regions of nucleotide
sequences set forth as: SEQ ID NOs: 26 and 28 or includes an
immunoglobulin variable sequence which comprises the amino acid
sequences set forth as SEQ ID NOs: 27 and 29. In another embodiment
the antibody (AB-PG1-XG1-077) includes an immunoglobulin variable
sequence encoded by the nucleic acid molecules comprising the
coding region or regions of nucleotide sequences set forth as: SEQ
ID NOs: 30 and 32 or includes an immunoglobulin variable sequence
which comprises the amino acid sequences set forth as SEQ ID NOs:
31 and 33.
[0201] In certain embodiments, the antibody is encoded by a nucleic
acid molecule that is highly homologous to the foregoing nucleic
acid molecules. Preferably the homologous nucleic acid molecule
comprises a nucleotide sequence that is at least about 90%
identical to the nucleotide sequence provided herein. More
preferably, the nucleotide sequence is at least about 95%
identical, at least about 97% identical, at least about 98%
identical, or at least about 99% identical to the nucleotide
sequence provided herein. The homology can be calculated using
various, publicly available software tools well known to one of
ordinary skill in the art. Exemplary tools include the BLAST system
available from the website of the National Center for Biotechnology
Information (NCBI) at the National Institutes of Health.
[0202] One method of identifying highly homologous nucleotide
sequences is via nucleic acid hybridization. Thus the invention
also includes antibodies having the PSMA-binding properties and
other functional properties described herein, which are encoded by
nucleic acid molecules that hybridize under high stringency
conditions to the foregoing nucleic acid molecules. Identification
of related sequences can also be achieved using polymerase chain
reaction (PCR) and other amplification techniques suitable for
cloning related nucleic acid sequences. Preferably, PCR primers are
selected to amplify portions of a nucleic acid sequence of
interest, such as a CDR.
[0203] The term "high stringency conditions" as used herein refers
to parameters with which the art is familiar. Nucleic acid
hybridization parameters may be found in references that compile
such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. One example of high-stringency
conditions is hybridization at 65.degree. C. in hybridization
buffer (3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone,
0.02% Bovine Serum Albumin, 2.5 mM NaH.sub.2PO.sub.4 (pH7), 0.5%
SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium
citrate, pH7; SDS is sodium dodecyl sulphate; and EDTA is
ethylenediaminetetracetic acid. After hybridization, a membrane
upon which the nucleic acid is transferred is washed, for example,
in 2.times.SSC at room temperature and then at
0.1-0.5.times.SSC/0.1.times.SDS at temperatures up to 68.degree.
C.
[0204] In other preferred embodiments, the antibodies include a
heavy chain variable region comprising an amino acid sequence
selected from the group consisting of amino acid sequences set
forth as: SEQ ID NOs: 15, 19, 23, 27 and 31, and a light chain
variable region comprising an amino acid sequence selected from the
group consisting of nucleotide sequences set forth as: SEQ ID NOs:
17, 21, 25, 29 and 33. Antigen-binding fragments of the foregoing
also are provided, as described elsewhere herein.
[0205] As used herein, the term "antibody" refers to a glycoprotein
comprising at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. Each heavy chain is comprised
of a heavy chain variable region (abbreviated herein as HCVR or
V.sub.H) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, C.sub.H1, C.sub.H2
and C.sub.H3. Each light chain is comprised of a light chain
variable region (abbreviated herein as LCVR or V.sub.L) and a light
chain constant region. The light chain constant region is comprised
of one domain, CL. The V.sub.H and V.sub.L regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies may mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system.
[0206] The term "antigen-binding fragment" of an antibody as used
herein, refers to one or more portions of an antibody that retain
the ability to specifically bind to an antigen (e.g., PSMA). It has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
fragment" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the V.sub.H and CH1
domains; (iv) a Fv fragment consisting of the V.sub.L and V.sub.H
domains of a single arm of an antibody, (v) a dAb fragment (Ward et
al., (1989) Nature 341:544-546) which consists of a V.sub.H domain;
and (vi) an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, V and
V.sub.H, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the V.sub.L and V.sub.H
regions pair to form monovalent molecules (known as single chain Fv
(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and
Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such
single chain antibodies are also intended to be encompassed within
the term "antigen-binding portion" of an antibody. These antibody
fragments are obtained using conventional procedures, such as
proteolytic fragmentation procedures, as described in J. Goding,
Monoclonal Antibodies: Principles and Practice, pp 98-118 (N.Y.
Academic Press 1983), which is hereby incorporated by reference as
well as by other techniques known to those with skill in the art.
The fragments are screened for utility in the same manner as are
intact antibodies.
[0207] An "isolated antibody", as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to PSMA is substantially free of
antibodies that specifically bind antigens other than PSMA). An
isolated antibody that specifically binds to an epitope, isoform or
variant of PSMA may, however, have cross-reactivity to other
related antigens, e.g., from other species (e.g., PSMA species
homologs). Moreover, an isolated antibody may be substantially free
of other cellular material and/or chemicals. As used herein,
"specific binding" refers to antibody binding to a predetermined
antigen. Typically, the antibody binds with an affinity that is at
least two-fold greater than its affinity for binding to a
non-specific antigen (e.g., BSA, casein) other than the
predetermined antigen or a closely-related antigen.
[0208] The isolated antibodies of the invention encompass various
antibody isotypes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,
IgAsec, IgD, IgE. As used herein, "isotype" refers to the antibody
class (e.g. IgM or IgG1) that is encoded by heavy chain constant
region genes. The antibodies can be full length or can include only
an antigen-binding fragment such as the antibody constant and/or
variable domain of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec,
IgD or IgE or could consist of a Fab fragment, a F(ab').sub.2
fragment, and a Fv fragment.
[0209] The antibodies of the present invention can be polyclonal,
monoclonal, or a mixture of polyclonal and monoclonal antibodies.
The antibodies can be produced by a variety of techniques well
known in the art. Procedures for raising polyclonal antibodies are
well known. For example anti-PSMA polyclonal antibodies are raised
by administering PSMA protein subcutaneously to New Zealand white
rabbits which have first been bled to obtain pre-immune serum. The
PSMA can be injected at a total volume of 100 .mu.l per site at six
different sites, typically with one or more adjustments. The
rabbits are then bled two weeks after the first injection and
periodically boosted with the same antigen three times every six
weeks. A sample of serum is collected 10 days after each boost.
Polyclonal antibodies are recovered from the serum, preferably by
affinity chromatography using PSMA to capture the antibody. This
and other procedures for raising polyclonal antibodies are
disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory
Manual (1988), which is hereby incorporated by reference.
[0210] Monoclonal antibody production may be effected by techniques
which are also well known in the art. The term "monoclonal
antibody," as used herein, refers to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
displays a single binding specificity and affinity for a particular
epitope. The process of monoclonal antibody production involves
obtaining immune somatic cells with the potential for producing
antibody, in particular B lymphocytes, which have been previously
immunized with the antigen of interest either in vivo or in vitro
and that are suitable for fusion with a B-cell myeloma line.
[0211] Mammalian lymphocytes typically are immunized by in vivo
immunization of the animal (e.g., a mouse) with the desired protein
or polypeptide, e.g., with PSMA in the present invention. Such
immunizations are repeated as necessary at intervals of up to
several weeks to obtain a sufficient titer of antibodies. Once
immunized, animals can be used as a source of antibody-producing
lymphocytes. Following the last antigen boost, the animals are
sacrificed and spleen cells removed. Mouse lymphocytes give a
higher percentage of stable fusions with the mouse myeloma lines
described herein. Of these, the BALB/c mouse is preferred. However,
other mouse strains, rabbit, hamster, sheep and frog may also be
used as hosts for preparing antibody-producing cells. See; Goding
(in Monoclonal Antibodies: Principles and Practice, 2d ed., pp.
60-61, Orlando, Fla., Academic Press, 1986). In particular, mouse
strains that have human immunoglobulin genes inserted in the genome
(and which cannot produce mouse immunoglobulins) are preferred.
Examples include the HuMAb mouse strains produced by
Medarex/GenPharm International, and the XenoMouse strains produced
by Abgenix. Such mice produce fully human immunoglobulin molecules
in response to immunization.
[0212] Those antibody-producing cells that are in the dividing
plasmablast stage fuse preferentially. Somatic cells may be
obtained from the lymph nodes, spleens and peripheral blood of
antigen-primed animals, and the lymphatic cells of choice depend to
a large extent on their empirical usefulness in the particular
fusion system. The antibody-secreting lymphocytes are then fused
with (mouse) B cell myeloma cells or transformed cells, which are
capable of replicating indefinitely in cell culture, thereby
producing an immortal, immunoglobulin-secreting cell line. The
resulting fused cells, or hybridomas, are cultured, and the
resulting colonies screened for the production of the desired
monoclonal antibodies. Colonies producing such antibodies are
cloned, and grown either in vivo or in vitro to produce large
quantities of antibody. A description of the theoretical basis and
practical methodology of fusing such cells is set forth in Kohler
and Milstein, Nature 256:495 (1975), which is hereby incorporated
by reference.
[0213] Alternatively, human somatic cells capable of producing
antibody, specifically B lymphocytes, are suitable for fusion with
myeloma cell lines. While B lymphocytes from biopsied spleens,
tonsils or lymph nodes of an individual may be used, the more
easily accessible peripheral blood B lymphocytes are preferred. The
lymphocytes may be derived from patients with diagnosed prostate
carcinomas or another PSMA-expressing cancer. In addition, human B
cells may be directly immortalized by the Epstein-Barr virus (Cole
et al., 1995, Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96). Although somatic cell hybridization
procedures are preferred, in principle, other techniques for
producing monoclonal antibodies can be employed such as viral or
oncogenic transformation of B lymphocytes.
[0214] Myeloma cell lines suited for use in hybridoma-producing
fusion procedures preferably are non-antibody-producing, have high
fusion efficiency, and enzyme deficiencies that render them
incapable of growing in certain selective media which support the
growth of the desired hybridomas. Examples of such myeloma cell
lines that may be used for the production of fused cell lines
include P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4.1, Sp2/0-Ag14, FO,
NSO/U, MPC-11, MPC11-X45-GTG 1.7, S194/5XX0 Bul, all derived from
mice; R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 derived from rats
and U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, all derived from
humans (Goding, in Monoclonal Antibodies: Principles and Practice,
2d ed., pp. 65-66, Orlando, Fla., Academic Press, 1986; Campbell,
in Monoclonal Antibody Technology, Laboratory Techniques in
Biochemistry and Molecular Biology Vol. 13, Burden and Von
Knippenberg, eds. pp. 75-83, Amsterdam, Elseview, 1984).
[0215] Fusion with mammalian myeloma cells or other fusion partners
capable of replicating indefinitely in cell culture is effected by
standard and well-known techniques, for example, by using
polyethylene glycol ("PEG") or other fusing agents (See Milstein
and Kohler, Eur. J. Immunol. 6:511 (1976), which is hereby
incorporated by reference).
[0216] In other embodiments, the antibodies can be recombinant
antibodies. The term "recombinant antibody", as used herein, is
intended to include antibodies that are prepared, expressed,
created or isolated by recombinant means, such as antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
another species' immunoglobulin genes, antibodies expressed using a
recombinant expression vector transfected into a host cell,
antibodies isolated from a recombinant, combinatorial antibody
library, or antibodies prepared, expressed, created or isolated by
any other means that involves splicing of immunoglobulin gene
sequences to other DNA sequences.
[0217] In yet other embodiments, the antibodies can be chimeric or
humanized antibodies. As used herein, the term "chimeric antibody"
refers to an antibody, that combines the murine variable or
hypervariable regions with the human constant region or constant
and variable framework regions. As used herein, the term "humanized
antibody" refers to an antibody that retains only the
antigen-binding CDRs from the parent antibody in association with
human framework regions (see, Waldmann, 1991, Science 252:1657).
Such chimeric or humanized antibodies retaining binding specificity
of the murine antibody are expected to have reduced immunogenicity
when administered in vivo for diagnostic, prophylactic or
therapeutic applications according to the invention.
[0218] According to an alternative embodiment, the monoclonal
antibodies of the present invention can be modified to be in the
form of a bispecific antibody, or a multispecific antibody. The
term "bispecific antibody" is intended to include any agent, e.g.,
a protein, peptide, or protein or peptide complex, which has two
different binding specificities which bind to, or interact with (a)
a cell surface antigen and (b) an Fc receptor on the surface of an
effector cell. The term "multispecific antibody" is intended to
include any agent, e.g., a protein, peptide, or protein or peptide
complex, which has more than two different binding specificities
which bind to, or interact with (a) a cell surface antigen, (b) an
Fc receptor on the surface of an effector cell, and (c) at least
one other component. Accordingly, the invention includes, but is
not limited to, bispecific, trispecific, tetraspecific, and other
multispecific antibodies which are directed to cell surface
antigens, such as PSMA, and to Fc receptors on effector cells. The
term "bispecific antibodies" further includes diabodies. Diabodies
are bivalent, bispecific antibodies in which the V.sub.H and
V.sub.L domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen-binding sites (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poijak, R. J., et al. (1994)
Structure 2:1121-1123).
[0219] A bispecific antibody can be formed of an antigen-binding
region specific for the extracellular domain of PSMA and an
antigen-binding region specific for an effector cell which has
tumoricidal or tumor inhibitory activity. The two antigen-binding
regions of the bispecific antibody are either chemically linked or
can be expressed by a cell genetically engineered to produce the
bispecific antibody. (See generally, Fanger et al., 1995 Drug News
& Perspec. 8(3): 133-137). Suitable effector cells having
tumoricidal activity include but are not limited to cytotoxic
T-cells (primarily CD8.sup.+ cells), natural killer cells, etc. An
effective amount of a bispecific antibody according to the
invention is administered to a prostrate cancer patient and the
bispecific antibody kills and/or inhibits proliferation of the
malignant cells after localization at sites of primary or
metastatic tumors bearing PSMA.
[0220] In certain embodiments, the antibodies are human antibodies.
The term "human antibody", as used herein, is intended to include
antibodies having variable and constant regions derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse
have been grafted onto human framework sequences (referred to
herein as "humanized antibodies"). Human antibodies directed
against PSMA are generated using transgenic mice carrying parts of
the human immune system rather than the mouse system.
[0221] Fully human monoclonal antibodies also can be prepared by
immunizing mice transgenic for large portions of human
immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat.
Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and
references cited therein, the contents of which are incorporated
herein by reference. These animals have been genetically modified
such that there is a functional deletion in the production of
endogenous (e.g., murine) antibodies. The animals are further
modified to contain all or a portion of the human germ-line
immunoglobulin gene locus such that immunization of these animals
results in the production of fully human antibodies to the antigen
of interest. Following immunization of these mice (e.g., XenoMouse
(Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies
are prepared according to standard hybridoma technology. These
monoclonal antibodies have human immunoglobulin amino acid
sequences and therefore will not provoke human anti-mouse antibody
(HAMA) responses when administered to humans.
[0222] Preferably, the mice are 6-16 weeks of age upon the first
immunization. For example, a purified or enriched preparation of
PSMA antigen (e.g., recombinant PSMA, PSMA-expressing cells,
dimeric PSMA) is used to immunize the mice intraperitoneally (IP),
although other routes of immunization known to one of ordinary
skill in the art are also possible. PSMA antigen is injected in
combination with an adjuvant, such as complete Freund's adjuvant,
and preferably the initial injection is followed by booster
immunizations with antigen in an adjuvant, such as incomplete
Freund's adjuvant. The immune response is monitored over the course
of the immunization protocol with plasma samples obtained by, for
example, retroorbital bleeds. The plasma is screened by ELISA (as
described below), and mice with sufficient titers of anti-PSMA
human immunoglobulin are used for fusions. Mice are boosted
intravenously with antigen 3 days before sacrifice and removal of
the spleen.
[0223] In particular embodiments, the antibodies are produced by
hybridomas referred to herein as PSMA 3.7 (PTA-3257), PSMA 3.8,
PSMA 3.9 (PTA-3258), PSMA 3.11 (PTA-3269), PSMA 5.4 (PTA-3268),
PSMA 7.1 (PTA-3292), PSMA 7.3 (PTA-3293), PSMA 10.3 (PTA-3247),
PSMA 1.8.3 (PTA-3906), PSMA A3.1.3 (PTA-3904), PSMA A3.3.1
(PTA-3905), Abgenix 4.248.2 (PTA-4427), Abgenix 4.360.3 (PTA-4428),
Abgenix 4.7.1 (PTA-4429), Abgenix 4.4.1 (PTA-4556), Abgenix 4.177.3
(PTA-4557), Abgenix 4.16.1 (PTA-4357), Abgenix 4.22.3 (PTA-4358),
Abgenix 4.28.3 (PTA-4359), Abgenix 4.40.2 (PTA-4360), Abgenix
4.48.3 (PTA-4361), Abgenix 4.49.1 (PTA-4362), Abgenix 4.209.3
(PTA-4365), Abgenix 4.219.3 (PTA-4366), Abgenix 4.288.1 (PTA-4367),
Abgenix 4.333.1 (PTA-4368), Abgenix 4.54.1 (PTA-4363), Abgenix
4.153.1 (PTA-4388), Abgenix 4.232.3 (PTA-4389), Abgenix 4.292.3
(PTA-4390), Abgenix 4.304.1 (PTA-4391), Abgenix 4.78.1 (PTA-4652),
and Abgenix 4.152.1 (PTA-4653). These hybridomas were deposited
pursuant to, and in satisfaction of, the requirements of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure with the
American Type Culture Collection ("ATCC") as an International
Depository Authority and given the Patent Deposit Designations
shown above and in Table 1.
[0224] The present invention further provides nucleic acid
molecules encoding anti-PSMA antibodies and vectors comprising the
nucleic acid molecules as described herein. The vectors provided
can be used to transform or transfect host cells for producing
anti-PSMA antibodies with the specificity of antibodies described
herein. In a preferred embodiment the antibodies produced will have
the specificity of the antibodies AB-PG1-XG1-006, AB-PG1-XG1-026,
AB-PG1-XG1-051, AB-PG1, XG1-069, AB-PG1-XG1-077 and PSMA 10.3. In
one embodiment the vectors can comprise an isolated nucleic acid
molecule encoding the heavy chain of the antibodies listed above
encoded by a nucleic acid molecules comprising the coding region or
regions of the nucleic acid sequences set forth as SEQ ID NO: 2-7.
In another embodiment, the vectors can comprise the nucleic acid
sequences encoding the light chain of the antibodies set forth as
SEQ ID NOs: 8-13. In a further embodiment the vectors of the
invention may comprise a heavy chain and a light chain sequence. In
a further embodiment, plasmids are given which produce the
antibodies or antigen binding fragments described herein. Plasmids
of the invention include plasmids selected from the group
consisting of: AB-PG1-XG1-006 Heavy Chain (SEQ ID NO: 2),
AB-PG1-XG1-006 Light Chain (SEQ ID NO: 8), AB-PG1-XG1-026 Heavy
Chain (SEQ ID NO: 3), AB-PG1-XG1-026 Light Chain (SEQ ID NO: 9),
AB-PG1-XG1-051 Heavy Chain (SEQ ID NO: 4), AB-PG1-XG1-051 Light
Chain (SEQ ID NO: 10), AB-PG1-XG1-069 Heavy Chain (SEQ ID NO: 5),
AB-PG1-XG1-069 Light Chain (SEQ ID NO: 11), AB-PG1-XG1-077 Heavy
Chain (SEQ ID NO: 6), AB-PG1-XG1-077 Light Chain (SEQ ID NO: 12),
PSMA 10.3 Heavy Chain (SEQ ID NO: 7), and PSMA 10.3 Kappa (SEQ ID
NO: 13).
[0225] The isolated antibody or antigen-binding fragment thereof
preferably is selected for its ability to bind live cells
expressing PSMA. In order to demonstrate binding of monoclonal
antibodies to live cells expressing the PSMA, flow cytometry can be
used. For example, cell lines expressing PSMA (grown under standard
growth conditions) or prostate cancer cells that express PSMA are
mixed with various concentrations of monoclonal antibodies in PBS
containing 0.1% Tween 80 and 20% mouse serum, and incubated at
37.degree. C. for 1 hour. After washing, the cells are reacted with
fluorescein-labeled anti-human IgG secondary antibody (if human
anti-PSMA antibodies were used) under the same conditions as the
primary antibody staining. The samples can be analyzed by a
fluorescence activated cell sorter (FACS) instrument using light
and side scatter properties to gate on single cells. An alternative
assay using fluorescence microscopy may be used (in addition to or
instead of) the flow cytometry assay. Cells can be stained exactly
as described above and examined by fluorescence microscopy. This
method allows visualization of individual cells, but may have
diminished sensitivity depending on the density of the antigen.
[0226] Binding of the antibody or antigen-binding fragment thereof
to live cells expressing PSMA can inhibit the growth of the cells
or mediate cytolysis of the cells. Cytolysis can be complement
mediated or can be mediated by effector cells. In a preferred
embodiment, the cytolysis is carried out in a living organism,
preferably a mammal, and the live cell is a tumor cell. Examples of
tumors which can be targeted by the antibodies of the invention
include, any tumor that expresses PSMA, such as, prostate, bladder,
pancreas, lung, colon, kidney, melanomas and sarcomas. In a
preferred embodiment the tumor cell is a prostate cancer cell.
[0227] The testing of antibody cytolytic activity in vitro by
chromium release assay can provide an initial screening prior to
testing in vivo models. This testing can be carried out using
standard chromium release assays. Briefly, polymorphonuclear cells
(PMN), or other effector cells, from healthy donors can be purified
by Ficoll Hypaque density centrifugation, followed by lysis of
contaminating erythrocytes. Washed PMNs can be suspended in RPMI
supplemented with 10% heat-inactivated fetal calf serum and mixed
with .sup.51Cr labeled cells expressing PSMA, at various ratios of
effector cells to tumor cells (effector cells:tumor cells).
Purified anti-PSMA IgGs can then be added at various
concentrations. Irrelevant IgG can be used as negative control.
Assays can be carried out for 0-120 minutes at 37.degree. C.
Samples can be assayed for cytolysis by measuring .sup.51Cr release
into the culture supernatant. Anti-PSMA monoclonal antibodies can
also be tested in combinations with each other to determine whether
cytolysis is enhanced with multiple monoclonal antibodies.
[0228] Antibodies which bind to PSMA also can be tested in an in
vivo model (e.g., in mice) to determine their efficacy in mediating
cytolysis and killing of cells expressing PSMA, e.g., tumor cells.
These antibodies can be selected, for example, based on the
following criteria, which are not intended to be exclusive:
[0229] 1) binding to live cells expressing PSMA;
[0230] 2) high affinity of binding to PSMA;
[0231] 3) binding to a unique epitope on PSMA (to eliminate the
possibility that antibodies with complimentary activities when used
in combination would compete for binding to the same epitope);
[0232] 4) opsonization of cells expressing PSMA;
[0233] 5) mediation of growth inhibition, phagocytosis and/or
killing of cells expressing PSMA in the presence of effector
cells;
[0234] 6) modulation (inhibition or enhancement) of NAALADase,
folate hydrolase, dipeptidyl peptidase IV and/or .gamma.-glutamyl
hydrolase activities;
[0235] 7) growth inhibition, cell cycle arrest and/or cytotoxicity
in the absence of effector cells;
[0236] 8) internalization of PSMA;
[0237] 9) binding to a conformational epitope on PSMA;
[0238] 10) minimal cross-reactivity with cells or tissues that do
not express PSMA; and
[0239] 11) preferential binding to dimeric forms of PSMA rather
than monomeric forms of PSMA.
[0240] Preferred antibodies of the invention meet one or more, and
preferably all, of these criteria. In a particular embodiment, the
antibodies are used in combination, e.g., as a pharmaceutical
composition comprising two or more different anti-PSMA antibodies
or binding fragments thereof. For example, anti-PSMA antibodies
having different, but complementary activities can be combined in a
single therapy to achieve a desired therapeutic or diagnostic
effect. An illustration of this would be a composition containing
an anti-PSMA antibody that mediates highly effective killing of
target cells in the presence of effector cells, combined with
another anti-PSMA antibody that inhibits the growth of cells
expressing PSMA.
[0241] In a preferred aspect of the invention, the antibody or
antigen-binding fragment thereof binds to a conformational epitope
within the extracellular domain of the PSMA molecule. To determine
if the selected human anti-PSMA antibodies bind to conformational
epitopes, each antibody can be tested in assays using native
protein (e.g., non-denaturing immunoprecipitation, flow cytometric
analysis of cell surface binding) and denatured protein (e.g.,
Western blot, immunoprecipitation of denatured proteins). A
comparison of the results will indicate whether the antibodies bind
conformational epitopes. Antibodies that bind to native protein but
not denatured protein are those antibodies that bind conformational
epitopes, and are preferred antibodies.
[0242] In another preferred aspect of the invention, the antibody
or antigen-binding fragment thereof binds to a dimer-specific
epitope on PSMA. Generally, antibodies or antigen-binding fragments
thereof which bind to a dimer-specific epitope preferentially bind
the PSMA dimer rather than the PSMA monomer. To determine if the
selected human anti-PSMA antibodies bind preferentially (i.e.,
selectively and/or specifically) to a PSMA dimer, each antibody can
be tested in assays (e.g., immunoprecipitation followed by Western
blotting) using native dimeric PSMA protein and dissociated
monomeric PSMA protein. A comparison of the results will indicate
whether the antibodies bind preferentially to the dimer or to the
monomer. Antibodies that bind to the PSMA dimer but not to the
monomeric PSMA protein are preferred antibodies.
[0243] Preferred antibodies include antibodies that competitively
inhibit the specific binding of a second antibody to its target
epitope on PSMA. To determine competitive inhibition, a variety of
assays known to one of ordinary skill in the art can be employed.
For example, the cross-competition assays set forth in Examples 4
and 21 can be used to determine if an antibody competitively
inhibits binding to PSMA by another antibody. These examples
provide cell-based methods employing flow cytometry or solid phase
binding analysis. Other assays that evaluate the ability of
antibodies to cross-compete for PSMA molecules that are not
expressed on the surface of cells, in solid phase or in solution
phase, also can be used. These assays preferably use the PSMA
multimers described herein.
[0244] Certain preferred antibodies competitively inhibit the
specific binding of a second antibody to its target epitope on PSMA
by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
or 99%. Inhibition can be assessed at various molar ratios or mass
ratios; for example competitive binding experiments can be
conducted with a 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold or
more molar excess of the first antibody over the second
antibody.
[0245] Other preferred antibodies include antibodies that
specifically (i.e., selectively) bind to an epitope on PSMA defined
by a second antibody. To determine the epitope, one can use
standard epitope mapping methods known in the art. For example,
fragments (peptides) of PSMA antigen (preferably synthetic
peptides) that bind the second antibody can be used to determine
whether a candidate antibody binds the same epitope. For linear
epitopes, overlapping peptides of a defined length (e.g., 8 or more
amino acids) are synthesized. The peptides preferably are offset by
1 amino acid, such that a series of peptides covering every 8 amino
acid fragment of the PSMA protein sequence are prepared. Fewer
peptides can be prepared by using larger offsets, e.g., 2 or 3
amino acids. In addition, longer peptides (e.g., 9-, 10- or 1-mers)
can be synthesized. Binding of peptides to antibodies can be
determined using standard methodologies including surface plasmon
resonance (BIACORE; see Example 22) and ELISA assays. For
examination of conformational epitopes, larger PSMA fragments can
be used. Other methods that use mass spectrometry to define
conformational epitopes have been described and can be used (see,
e.g., Baerga-Ortiz et al., Protein Science 11:1300-1308, 2002 and
references cited therein). Still other methods for epitope
determination are provided in standard laboratory reference works,
such as Unit 6.8 ("Phage Display Selection and Analysis of B-cell
Epitopes") and Unit 9.8 ("Identification of Antigenic Determinants
Using Synthetic Peptide Combinatorial Libraries") of Current
Protocols in Immunology, Coligan et al., eds., John Wiley &
Sons. Epitopes can be confirmed by introducing point mutations or
deletions into a known epitope, and then testing binding with one
or more antibodies to determine which mutations reduce binding of
the antibodies.
[0246] In one embodiment of the invention the antibody or
antigen-binding fragment thereof binds to and is internalized with
PSMA expressed on cells. The mechanism by which the antibody or
antigen-binding fragment thereof is internalized with the prostate
specific membrane antigen is not critical to the practice of the
present invention. For example, the antibody or antigen-binding
fragment thereof can induce internalization of PSMA. Alternatively,
internalization of the antibody or antigen-binding fragment thereof
can be the result of routine internalization of PSMA. The antibody
or antigen-binding fragment thereof can be used in an unmodified
form, alone or in combination with other compositions.
Alternatively, the antibody or antigen-binding fragment thereof can
be bound to a substance effective to kill the cells upon binding of
the antibody or antigen-binding fragment thereof to prostate
specific membrane antigen and upon internalization of the
biological agent with the prostate specific membrane antigen.
[0247] The human PSMA antibodies of the present invention
specifically bind cell-surface PSMA and/or rsPSMA with
sub-nanomolar affinity. The human PSMA antibodies of the present
invention have binding affinities of about 1.times.10.sup.-9 M or
less, preferably about 1.times.10.sup.-10 M or less, more
preferably 1.times.10.sup.-11 M or less. In a particular embodiment
the binding affinity is less than about 5.times.10.sup.-10 M.
[0248] An antibody can be linked to a detectable marker, an
antitumor agent or an immunomodulator. Antitumor agents can include
cytotoxic agents and agents that act on tumor neovasculature.
Detectable markers include, for example, radioactive or fluorescent
markers. Cytotoxic agents include cytotoxic radionuclides, chemical
toxins and protein toxins.
[0249] The cytotoxic radionuclide or radiotherapeutic isotope
preferably is an alpha-emitting isotope such as .sup.225Ac,
.sup.211At, .sup.212Bi, .sup.213Bi, .sup.212Pb, .sup.224Ra or
.sup.223Ra. Alternatively, the cytotoxic radionuclide may a
beta-emitting isotope such as .sup.186Rh, .sup.188Rh, .sup.177Lu,
.sup.90Y, .sup.131I, .sup.67Cu, .sup.64Cu, .sup.153Sm or
.sup.166Ho. Further, the cytotoxic radionuclide may emit Auger and
low energy electrons and include the isotopes .sup.125I, .sup.123I
or .sup.77Br.
[0250] Suitable chemical toxins or chemotherapeutic agents include
members of the enediyne family of molecules, such as calicheamicin
and esperamicin. Chemical toxins can also be taken from the group
consisting of methotrexate, doxorubicin, melphalan, chlorambucil,
ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin
and 5-fluorouracil. Other antineoplastic agents that may be
conjugated to the anti-PSMA antibodies of the present invention
include dolastatins (U.S. Pat. Nos. 6,034,065 and 6,239,104) and
derivatives thereof. Of particular interest is dolastatin 10
(dolavaline-valine-dolaisoleuine-dolaproine-dolaphenine) and the
derivatives auristatin PHE (dolavaline-valine-dolaisoleuine-dolap-
roine-phenylalanine-methyl ester) (Pettit, G. R. et al., Anticancer
Drug Des. 13(4):243-277, 1998; Woyke, T. et al., Antimicrob. Agents
Chemother. 45(12):3580-3584, 2001), and aurastatin E and the like.
Toxins that are less preferred in the compositions and methods of
the invention include poisonous lectins, plant toxins such as
ricin, abrin, modeccin, botulina and diphtheria toxins. Of course,
combinations of the various toxins could also be coupled to one
antibody molecule thereby accommodating variable cytotoxicity.
Other chemotherapeutic agents are known to those skilled in the
art.
[0251] Toxin-conjugated forms of the PSMA antibodies of the present
invention mediate specific cell killing of PSMA-expressing cells at
picomolar concentrations. The toxin-conjugated PSMA antibodies of
the present invention exhibit IC.sub.50s at concentrations of less
than about 1.times.10.sup.-10 M, preferably less than about
1.times.10.sup.-11 M, more preferably less than about
1.times.10.sup.-12 M. In a particular embodiment an IC.sub.50 is
achieved at a concentration of less than about 1.5.times.10.sup.-11
M.
[0252] Agents that act on the tumor vasculature can include
tubulin-binding agents such as combrestatin A4 (Griggs et al.,
Lancet Oncol. 2:82, 2001), angiostatin and endostatin (reviewed in
Rosen, Oncologist 5:20, 2000, incorporated by reference herein) and
interferon inducible protein 10 (U.S. Pat. No. 5,994,292). A number
of antiangiogenic agents currently in clinical trials are also
contemplated. Agents currently in clinical trials include: 2ME2,
Angiostatin, Angiozyme, Anti-VEGF RhuMAb, Apra (CT-2584), Avicine,
Benefin, BMS275291, Carboxyamidotriazole, CC4047, CC5013, CC7085,
CDC801, CGP-41251 (PKC 412), CM101, Combretastatin A-4 Prodrug, EMD
121974, Endostatin, Flavopiridol, Genistein (GCP), Green Tea
Extract, IM-862, ImmTher, Interferon alpha, Interleukin-12, Iressa
(ZD1839), Marimastat, Metastat (Col-3), Neovastat, Octreotide,
Paclitaxel, Penicillamine, Photofrin, Photopoint, PI-88,
Prinomastat (AG-3340), PTK787 (ZK22584), R0317453, Solimastat,
Squalamine, SU 101, SU 5416, SU-6668, Suradista (FCE 26644),
Suramin (Metaret), Tetrathiomolybdate, Thalidomide, TNP-470 and
Vitaxin. additional antiangiogenic agents are described by Kerbel,
J. Clin. Oncol. 19(18s):45s-51s, 2001, which is incorporated by
reference herein. Immunomodulators suitable for conjugation to
anti-PSMA antibodies include .alpha.-interferon,
.gamma.-interferon, and tumor necrosis factor alpha
(TNF.alpha.).
[0253] The coupling of one or more toxin molecules to the anti-PSMA
antibody is envisioned to include many chemical mechanisms, for
instance covalent binding, affinity binding, intercalation,
coordinate binding, and complexation. The toxic compounds used to
prepare the anti-PSMA immunotoxins are attached to the antibodies
or PSMA-binding fragments thereof by standard protocols known in
the art.
[0254] The covalent binding can be achieved either by direct
condensation of existing side chains or by the incorporation of
external bridging molecules. Many bivalent or polyvalent agents are
useful in coupling protein molecules to other proteins, peptides or
amine functions, etc. For example, the literature is replete with
coupling agents such as carbodiimides, diisocyanates,
glutaraldehyde, diazobenzenes, and hexamethylene diamines. This
list is not intended to be exhaustive of the various coupling
agents known in the art but, rather, is exemplary of the more
common coupling agents.
[0255] In preferred embodiments, it is contemplated that one may
wish to first derivatize the antibody, and then attach the toxin
component to the derivatized product. Suitable cross-linking agents
for use in this manner include, for example, SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate)- , and SMPT,
4-succinimidyl-oxycarbonyl-methyl-(2-pyridyldithio)toluene.
[0256] In addition, protein toxins can be fused to the anti-PSMA
antibody or PSMA binding fragment by genetic methods to form a
hybrid immunotoxin fusion protein. To make a fusion immunotoxin
protein in accordance with the invention, a nucleic acid molecule
is generated that encodes an anti-PSMA antibody, a fragment of an
anti-PSMA antibody, a single chain anti-PSMA antibody, or a subunit
of an anti-PSMA antibody linked to a protein toxin. Such fusion
proteins contain at least a targeting agent (e.g., anti-PSMA
antibody subunit) and a toxin of the invention, operatively
attached. The fusion proteins may also include additional peptide
sequences, such as peptide spacers which operatively attach the
targeting agent and toxin compound, as long as such additional
sequences do not appreciably affect the targeting or toxin
activities of the fusion protein. The two proteins can be attached
by a peptide linker or spacer, such as a glycine-serine spacer
peptide, or a peptide hinge, as is well known in the art. Thus, for
example, the C-terminus of an anti-PSMA antibody or fragment
thereof can be fused to the N-terminus of the protein toxin
molecule to form an immunotoxin that retains the binding properties
of the anti-PSMA antibody. Other fusion arrangements will be known
to one of ordinary skill in the art.
[0257] To express the fusion immunotoxin, the nucleic acid encoding
the fusion protein is inserted into an expression vector in
accordance with standard methods, for stable expression of the
fusion protein, preferably in mammalian cells, such as CHO cells.
The fusion protein can be isolated and purified from the cells or
culture supernatant using standard methodology, such as a PSMA
affinity column.
[0258] Radionuclides typically are coupled to an antibody by
chelation. For example, in the case of metallic radionuclides, a
bifunctional chelator is commonly used to link the isotope to the
antibody or other protein of interest. Typically, the chelator is
first attached to the antibody, and the chelator-antibody conjugate
is contacted with the metallic radioisotope. A number of
bifunctional chelators have been developed for this purpose,
including the diethylenetriamine pentaacetic acid (DTPA) series of
amino acids described in U.S. Pat. Nos. 5,124,471, 5,286,850 and
5,434,287, which are incorporated herein by reference. As another
example, hydroxamic acid-based bifunctional chelating agents are
described in U.S. Pat. No. 5,756,825, the contents of which are
incorporated herein. Another example is the chelating agent termed
p-SCN-Bz-HEHA
(1,4,7,10,13,16-hexaazacyclo-octadecane-N,N',N",N'",N"",N'"-
"-hexaacetic acid) (Deal et al., J. Med. Chem. 42:2988, 1999),
which is an effective chelator of radiometals such as .sup.225Ac.
Yet another example is DOTA (1,4,7,10-tetraazacyclododecane
N,N',N",N'"-tetraacetic acid), which is a bifunctional chelating
agent (see McDevitt et al., Science 294:1537-1540, 2001) that can
be used in a two-step method for labeling followed by
conjugation.
[0259] In another aspect, the invention provides compositions
comprising a multimeric (e.g., dimeric) PSMA protein, an isolated
antibody, an antibody derivatized or linked to other functional
moieties, or an antigen-binding fragment thereof or a combination
of one or more of the aforementioned multimeric PSMA proteins,
antibodies or antigen-binding fragments thereof. The compositions
include a physiologically or pharmaceutically acceptable carrier,
excipient, or stabilizer mixed with the isolated multimeric PSMA
protein, antibody or antigen-binding fragment thereof. In a
preferred embodiment, the compositions include a combination of
multiple (e.g., two or more) isolated multimeric PSMA proteins,
antibodies or antigen-binding portions thereof of the invention.
Preferably, each of the antibodies or antigen-binding portions
thereof of the composition binds to a distinct conformational
epitope of PSMA. In one embodiment, anti-PSMA antibodies having
complementary activities are used in combination, e.g., as a
pharmaceutical composition, comprising two or more anti-PSMA
antibodies. For example, an antibody that mediates highly effective
cytolysis of target cells in the presence of effector cells can be
combined with another antibody that inhibits the growth of cells
expressing PSMA. As used herein, "target cell" shall mean any
undesirable cell in a subject (e.g., a human or animal) that can be
targeted by a composition of the invention. In preferred
embodiments, the target cell is a cell expressing or overexpressing
PSMA. Cells expressing PSMA typically include tumor cells, such as
prostate, bladder, pancreas, lung, kidney, colon tumor cells,
melanomas, and sarcomas.
[0260] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include a
composition of the present invention with at least one anti-tumor
agent, immunomodulator, immunostimulatory agent, or other
conventional therapy. For instance, the agent may be bound or
conjugated to or formed as a recombinant fusion molecule with the
PSMA antibodies of the present invention for directed targeting of
the agent to PSMA-expressing cells.
[0261] In some embodiments the various agents can be administered
concomitantly. In other embodiments the agents are administered
separately (prior to or subsequent to each other). The compositions
provided herein can be given to any patient in need thereof. As one
example, the compositions provided herein can be given to a
conventional cancer treatment-experienced patient. For instance a
composition of dimeric PSMA can be administered to such a patient
at some time subsequent to a conventional cancer therapy.
Conventional cancer therapy, such as for prostate cancer, includes
one or more of the following: surgery, radiation, cryosurgery,
thermotherapy, hormone treatment, chemotherapy, etc. In one
embodiment the therapy received prior to administration of a
composition of dimeric PSMA is at least prostatectomy and/or
radiation. In another embodiment the therapy received prior to
administration of a composition of dimeric PSMA is at least
castration and hormonal therapy. In yet another embodiment the
therapy received prior to administration of a composition of
dimeric PSMA is at least chemotherapy. In one embodiment for
prostate cancer the chemotherapy is preferably the administration
of the chemotherapeutic agent, docetaxel, alone or in combination
with an anti-inflammatory compound. The anti-inflammatory compound
in one embodiment is prednisone. Therefore, in some embodiments
compositions and methods are provided for treating patients with a
composition containing dimeric PSMA that is administered
concomitantly with, subsequent to, or prior to conventional cancer
therapy. In one such embodiment the methods provided include the
administration of docetaxel (75 mg/m.sup.2 q3 weeks) plus the
anti-inflammatory agent, prednisone (5 mg po bid), concomitantly
with, subsequent to, or prior to the administration of dimeric PSMA
compositions as provided herein.
[0262] In one embodiment patients amenable to treatment using
dimeric PSMA include those who have not received conventional
cancer treatment. In another embodiment patients amenable to
treatment using dimeric PSMA include those who have evidence of
cancer despite having received one or more conventional cancer
therapies. Patients therefore can include patients with
biochemically progressive prostate cancer such as non-castrate
patients (serum testosterone greater than or equal to 180 ng/mL).
In some embodiments these patients have received definitive primary
therapy such as prostatectomy or radiation. Patients can also
include castrate patients (serum testosterone less than 50 ng/mL),
who in some embodiments have completed a course of hormonal
therapy. Patients can also include patients having radiographic
evidence of disease progression. In one embodiment such a treatment
regimen is indicated in hormone-refractory prostate cancer
patients.
[0263] The PSMA antibodies of the present invention may be used as
a targeting moiety for delivery of replication-selective virus to
PSMA-expressing cells for tumor therapy. Replication-competent
virus such as the p53 pathway targeting adenovirus mutant dl1520,
ONYX-015, kill tumor cells selectively (Biederer, C. et al., J.
Mol. Med. 80(3):163-175, 2002).
[0264] The compositions of the present invention may include or be
diluted into a pharmaceutically-acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" or "physiologically
acceptable carrier" means one or more compatible solid or liquid
fillers, diluents or encapsulating substances which are suitable
for administration to a human or other mammal such as a primate,
dog, cat, horse, cow, sheep, or goat. Such carriers include any and
all salts, solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. The term "carrier"
denotes an organic or inorganic ingredient, natural or synthetic,
with which the active ingredient is combined to facilitate the
application. The carriers are capable of being commingled with the
preparations of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy or stability.
Preferably, the carrier is suitable for oral, intranasal,
intravenous, intramuscular, subcutaneous, parenteral, spinal,
intradermal or epidermal administration (e.g., by injection or
infusion). Suitable carriers can be found in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
Depending on the route of administration, the active compound,
i.e., antibody or PSMA multimer may be coated in a material to
protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0265] When administered, the pharmaceutical preparations of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptable compositions. The term
"pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredients. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy. Such preparations may
routinely contain salts, buffering agents, preservatives,
compatible carriers, and optionally other therapeutic agents, such
as supplementary immune potentiating agents including adjuvants,
chemokines and cytokines. When used in medicine, the salts should
be pharmaceutically acceptable, but non-pharmaceutically acceptable
salts may conveniently be used to prepare
pharmaceutically-acceptable salts thereof and are not excluded from
the scope of the invention.
[0266] A salt retains the desired biological activity of the parent
compound and does not impart any undesired toxicological effects
(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19).
Examples of such salts include acid addition salts and base
addition salts. Acid addition salts include those derived from
nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,
sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as
well as from nontoxic organic acids such as aliphatic mono- and
dicarboxylic acids, phenyl substituted alkanoic acids, hydroxy
alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic
acids and the like. Base addition salts include those derived from
alkaline earth metals, such as sodium, potassium, magnesium,
calcium and the like, as well as from nontoxic organic amines, such
as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0267] The pharmaceutical preparations of the invention also may
include isotonicity agents. This term is used in the art
interchangeably with iso-osmotic agent, and is known as a compound
which is added to the pharmaceutical preparation to increase the
osmotic pressure to that of 0.9% sodium chloride solution, which is
iso-osmotic with human extracellular fluids, such as plasma.
Preferred isotonicity agents are sodium chloride, mannitol,
sorbitol, lactose, dextrose and glycerol.
[0268] Optionally, the pharmaceutical preparations of the invention
may further comprise a preservative, such as benzalkonium chloride.
Suitable preservatives also include but are not limited to:
chlorobutanol (0.3-0.9% W/V), parabens (0.01-5.0%), thimerosal
(0.004-0.2%), benzyl alcohol (0.5-5%), phenol (0.1-1.0%), and the
like.
[0269] The formulations provided herein also include those that are
sterile. Sterilization processes or techniques as used herein
include aseptic techniques such as one or more filtration (0.45 or
0.22 micron filters) steps.
[0270] An anti-PSMA antibody composition may be combined, if
desired, with a pharmaceutically-acceptable carrier.
[0271] The pharmaceutical compositions may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; and phosphoric acid in a salt.
[0272] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. All methods include the
step of bringing the active agent into association with a carrier
which constitutes one or more accessory ingredients. In general,
the compositions are prepared by uniformly and intimately bringing
the active compound into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary,
shaping the product.
[0273] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous or non-aqueous preparation
of PSMA multimers and/or anti-PSMA antibodies, which is preferably
isotonic with the blood of the recipient. This preparation may be
formulated according to known methods using suitable dispersing or
wetting agents and suspending agents. The sterile injectable
preparation also may be a sterile injectable solution or suspension
in a non-toxic parenterally-acceptable diluent or solvent, for
example, as a solution in 1,3-butane diol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or di-glycerides. In addition,
fatty acids such as oleic acid may be used in the preparation of
injectables. Carrier formulations suitable for oral, subcutaneous,
intravenous, intramuscular, etc. administration can be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa.
[0274] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0275] The therapeutics of the invention can be administered by any
conventional route, including injection or by gradual infusion over
time. The administration may, for example, be oral, subcutaneous,
intravenous, intraperitoneal, intramuscular, intracavity,
intratumor, or transdermal. In some embodiments subcutaneous or
intramuscular administration is preferred. When antibodies are used
therapeutically, preferred routes of administration include
intravenous and by pulmonary aerosol. Techniques for preparing
aerosol delivery systems containing antibodies are well known to
those of skill in the art. Generally, such systems should utilize
components which will not significantly impair the biological
properties of the antibodies, such as the paratope binding capacity
(see, for example, Sciarra and Cutie, "Aerosols," in Remington's
Pharmaceutical Sciences, 18th edition, 1990, pp. 1694-1712;
incorporated by reference). Those of skill in the art can readily
determine the various parameters and conditions for producing
antibody aerosols without resorting to undue experimentation.
[0276] The pharmaceutical preparations of the invention, when used
in alone or in cocktails, are administered in therapeutically
effective amounts. Effective amounts are well known to those of
ordinary skill in the art and are described in the literature. A
therapeutically effective amount will be determined by the
parameters discussed below; but, in any event, is that amount which
establishes a level of the drug(s) effective for treating a
subject, such as a human subject, having one of the conditions
described herein. An effective amount means that amount alone or
with multiple doses, necessary to delay the onset of, inhibit
completely or lessen the progression of or halt altogether the
onset or progression of the condition being treated. When
administered to a subject, effective amounts will depend, of
course, on the particular condition being treated; the severity of
the condition; individual patient parameters including age,
physical condition, size and weight; concurrent treatment;
frequency of treatment; and the mode of administration. These
factors are well known to those of ordinary skill in the art and
can be addressed with no more than routine experimentation. It is
preferred generally that a maximum dose be used, that is, the
highest safe dose according to sound medical judgment.
[0277] An "effective amount" is that amount of an anti-PSMA
antibody or PSMA multimer that alone, or together with further
doses, produces the desired response, e.g. treats a malignancy in a
subject. The term is also meant to encompass the amount of an
anti-PSMA antibody and/or PSMA multimer that in combination with a
chemotherapeutic agent produces the desired response. This may
involve only slowing the progression of the disease temporarily,
although more preferably, it involves halting the progression of
the disease permanently. This can be monitored by routine methods.
The desired response to treatment of the disease or condition also
can be delaying the onset or even preventing the onset of the
disease or condition.
[0278] Such amounts will depend, of course, on the particular
condition being treated, the severity of the condition, the
individual patient parameters including age, physical condition,
size and weight, the duration of the treatment, the nature of
concurrent therapy (if any), the specific route of administration
and like factors within the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary
skill in the art and can be addressed with no more than routine
experimentation. It is generally preferred that a maximum dose of
the individual components or combinations thereof be used, that is,
the highest safe dose according to sound medical judgment. It will
be understood by those of ordinary skill in the art, however, that
a patient may insist upon a lower dose or tolerable dose for
medical reasons, psychological reasons or for virtually any other
reasons.
[0279] The pharmaceutical compositions used in the foregoing
methods preferably are sterile and contain an effective amount of
anti-PSMA antibodies or PSMA multimers for producing the desired
response in a unit of weight or volume suitable for administration
to a patient. The response can, for example, be measured by
determining the physiological effects of the anti-PSMA antibody or
PSMA multimer, such as regression of a tumor or decrease of disease
symptoms. Other assays will be known to one of ordinary skill in
the art and can be employed for measuring the level of the
response.
[0280] The doses of anti-PSMA antibodies or PSMA multimers
administered to a subject can be chosen in accordance with
different parameters, in particular in accordance with the mode of
administration used and the state of the subject. Other factors
include the desired period of treatment. In the event that a
response in a subject is insufficient at the initial doses applied,
higher doses (or effectively higher doses by a different, more
localized delivery route) may be employed to the extent that
patient tolerance permits.
[0281] A variety of administration routes are available. The
particular mode selected will depend of course, upon the particular
drug selected, the severity of the disease state being treated and
the dosage required for therapeutic efficacy. The methods of this
invention, generally speaking, may be practiced using any mode of
administration that is medically acceptable, meaning any mode that
produces effective levels of the active compounds without causing
clinically unacceptable adverse effects. Such modes of
administration include oral, rectal, sublingual, topical, nasal,
transdermal or parenteral routes. The term "parenteral" includes
subcutaneous, intravenous, intramuscular, or infusion.
[0282] In general, doses can range from about 10 .mu.g/kg to about
100,000 .mu.g/kg. In one embodiment, the dose is about 50 mg. In
another embodiment, the dose is about 250 mg. In still another
embodiment, the dose is about 500 mg, 1000 mg or greater. Based
upon the composition, the dose can be delivered once, continuously,
such as by continuous pump, or at periodic intervals. The periodic
interval may be weekly, bi-weekly, or monthly. The dosing can occur
over the period of one month, two months, three months or more to
elicit an appropriate humoral and/or cellular immune response.
Desired time intervals of multiple doses of a particular
composition can be determined without undue experimentation by one
skilled in the art. Other protocols for the administration of
anti-PSMA antibody or PSMA multimers will be known to one of
ordinary skill in the art, in which the dose amount, schedule of
administration, sites of administration, mode of administration and
the like vary from the foregoing.
[0283] Dosage may be adjusted appropriately to achieve desired drug
levels, locally or systemically. Generally, daily oral doses of
active compounds will be from about 0.1 mg/kg per day to 30 mg/kg
per day. It is expected that IV doses in the range of 0.01-1.00
mg/kg will be effective. In the event that the response in a
subject is insufficient at such doses, even higher doses (or
effective higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits. Continuous IV dosing over, for example, 24 hours or
multiple doses per day also are contemplated to achieve appropriate
systemic levels of compounds.
[0284] In general, doses of radionuclide delivered by the anti-PSMA
antibodies of the invention can range from about 0.01 mCi/Kg to
about 10 mCi/kg. Preferably the dose of radionuclide ranges from
about 0.1 mCi/Kg to about 1.0 mCi/kg. The optimal dose of a given
isotope can be determined empirically by simple routine titration
experiments well known to one of ordinary skill in the art.
[0285] Administration of anti-PSMA antibody or PSMA multimer
compositions to mammals other than humans, e.g. for testing
purposes or veterinary therapeutic purposes, is carried out under
substantially the same conditions as described above.
[0286] The compositions (antibodies to PSMA and
derivatives/conjugates thereof and PSMA multimers) of the present
invention have in vitro and in vivo diagnostic and therapeutic
utilities. For example, these molecules can be administered to
cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g.,
in vivo, to treat, prevent or diagnose a variety of disorders. As
used herein, the term "subject" is intended to include humans and
non-human animals. Preferred subjects include a human patient
having a disorder characterized by expression, typically aberrant
expression (e.g., overexpression) of PSMA. Other preferred subjects
include subjects that are treatable with the compositions of the
invention. This includes those who have or are at risk of having a
cancer or who would otherwise would benefit from an enhanced or
elicited immune response to cells expressing PSMA. In preferred
embodiments these cells express PSMA on their surface.
[0287] One aspect of the present invention relates to a method of
detecting cancerous cells or portions thereof in a biological
sample (e.g., histological or cytological specimens, biopsies and
the like), and, in particular, to distinguish malignant tumors from
normal tissues and non-malignant tumors. This method involves
providing an antibody or an antigen-binding binding fragment
thereof, probe, or ligand, which binds to an extracellular domain
of PSMA of such cells, e.g., an anti-PSMA antibody. The anti-PSMA
antibody is bound to a label that permits the detection of the
cells or portions thereof (e.g., PSMA or fragments thereof
liberated from such cancerous cells) upon binding of the anti-PSMA
antibody to the cells or portions thereof. The biological sample is
contacted with the labeled anti-PSMA antibody under conditions
effective to permit binding of the anti-PSMA antibody to the
extracellular domain of PSMA of any of the cells or portions
thereof in the biological sample. The presence of any cells or
portions thereof in the biological sample is detected by detection
of the label. In one preferred form, the contact between the
anti-PSMA antibody and the biological sample is carried out in a
living mammal and involves administering the anti-PSMA antibody to
the mammal under conditions that permit binding of the anti-PSMA
antibody to PSMA of any of the cells or portions thereof in the
biological sample. Again, such administration can be carried out by
any suitable method known to one of ordinary skill in the art.
[0288] In addition, the anti-PSMA antibodies of the present
invention can be used in immunofluorescence techniques to examine
human tissue, cell and bodily fluid specimens. In a typical
protocol, slides containing cryostat sections of frozen, unfixed
tissue biopsy samples or cytological smears are air dried, formalin
or acetone fixed, and incubated with the monoclonal antibody
preparation in a humidified chamber at room temperature. The slides
are then washed and further incubated with a preparation of a
secondary antibody directed against the monoclonal antibody,
usually some type of anti-mouse immunoglobulin if the monoclonal
antibodies used are derived from the fusion of a mouse spleen
lymphocyte and a mouse myeloma cell line. This secondary antibody
is tagged with a compound, for instance rhodamine or fluorescein
isothiocyanate, that fluoresces at a particular wavelength. The
staining pattern and intensities within the sample are then
determined by fluorescent light microscopy and optionally
photographically recorded.
[0289] As yet another alternative, computer enhanced fluorescence
image analysis or flow cytometry can be used to examine tissue
specimens or exfoliated cells, i.e., single cell preparations from
aspiration biopsies of tumors using the anti-PSMA antibodies of
this invention. The anti-PSMA antibodies of the invention are
particularly useful in quantitation of live tumor cells, i.e.,
single cell preparations from aspiration biopsies of prostate
tumors by computer enhanced fluorescence image analyzer or with a
flow cytometer. The antibodies of the invention are particularly
useful in such assays to differentiate benign from malignant
prostate tumors since the PSMA protein to which the anti-PSMA
antibodies bind is expressed in increased amounts by malignant
tumors as compared to benign prostate tumors. The percent PSMA
positive cell population, alone or in conjunction with
determination of other attributes of the cells (e.g., DNA ploidy of
these cells), may, additionally, provide very useful prognostic
information by providing an early indicator of disease
progression.
[0290] In yet another alternative embodiment, the antibodies of the
present invention can be used in combination with other known
antibodies to provide additional information regarding the
malignant phenotype of a cancer.
[0291] The method of the present invention can be used to screen
patients for diseases associated with the presence of cancerous
cells or portions thereof. Alternatively, it can be used to
identify the recurrence of such diseases, particularly when the
disease is localized in a particular biological material of the
patient. For example, recurrence of prostatic disease in the
prostatic fossa may be encountered following radical prostatectomy.
Using the method of the present invention, this recurrence can be
detected by administering a short range radiolabeled antibody to
the mammal and then detecting the label rectally, such as with a
transrectal detector probe.
[0292] Alternatively, the contacting step can be carried out in a
sample of serum or urine or other body fluids, including but not
limited to seminal fluid, prostatic fluid, ejaculate, and the like,
such as to detect the presence of PSMA in the body fluid. When the
contacting is carried out in a serum or urine sample, it is
preferred that the biological agent recognize substantially no
antigens circulating in the blood other than PSMA. Since intact
cells do not excrete or secrete PSMA into the extracellular
environment, detecting PSMA in serum, urine, or other body fluids
generally indicates that cells are being lysed or shed. Thus, the
biological agents and methods of the present invention can be used
to determine the effectiveness of a cancer treatment protocol by
monitoring the level of PSMA in serum, urine or other body
fluids.
[0293] In a particularly preferred embodiment of the method of
detecting cancerous cells in accordance with the present invention,
the anti-PSMA antibodies or an antigen-binding fragment thereof,
binds to and is internalized with the prostate specific membrane
antigen of such cells. Again, the biological agent is bound to a
label effective to permit detection of the cells or portions
thereof upon binding of the biological agent to and internalization
of the biological agent with the prostate specific membrane
antigen.
[0294] Biological agents suitable for detecting cancerous cells
include anti-PSMA antibodies, such as monoclonal or polyclonal
antibodies. In addition, antibody fragments, half-antibodies,
hybrid derivatives, probes, and other molecular constructs may be
utilized. These biological agents, such as antibodies,
antigen-binding fragments thereof, probes, or ligands, bind to
extracellular domains of prostate specific membrane antigens or
portions thereof in cancerous cells. As a result, the biological
agents bind not only to cells which are fixed or cells whose
intracellular antigenic domains are otherwise exposed to the
extracellular environment. Consequently, binding of the biological
agents is concentrated in areas where there are prostate cells,
irrespective of whether these cells are fixed or unfixed, viable or
necrotic. Additionally or alternatively, these biological agents
bind to and are internalized with prostate specific membrane
antigens or portions thereof in normal, benign hyperplastic, and to
a greater degree in cancerous cells.
[0295] The PSMA multimers and antibodies or antigen-binding
fragments thereof can also be utilized in in vivo therapy of
cancer. The PSMA multimers and antibodies or antigen-binding
fragments thereof can be used with a compound which kills and/or
inhibits proliferation of malignant cells or tissues. For instance,
the antibodies can be covalently attached, either directly or via
linker, to such a compound following administration and
localization of the conjugates. When the antibody is used by
itself, it may mediate tumor destruction by complement fixation or
antibody-dependent cellular cytotoxicity. Alternatively, the PSMA
multimer or antibody may be administered in combination with a
chemotherapeutic drug to result in synergistic therapeutic effects
(Baslya and Mendelsohn, 1994 Breast Cancer Res. and Treatment
29:127-138). A variety of different types of substances can be
directly conjugated for therapeutic uses, including radioactive
metal and non-metal isotopes, chemotherapeutic drugs, toxins, etc.
as described above and known in the art (see, e.g., Vitetta and
Uhr, 1985, Annu. Rev. Immunol. 3:197).
[0296] The antibodies or antigen-binding fragments thereof of the
invention can also be administered together with complement.
Accordingly, within the scope of the invention are compositions
comprising antibodies or antigen-binding fragments thereof and
serum or complement. These compositions are advantageous in that
the complement is located in close proximity to the human
antibodies or antigen-binding fragments thereof. Alternatively, the
antibodies or antigen-binding fragments thereof of the invention
and the complement or serum can be administered separately.
[0297] The PSMA multimers or antibodies can be administered with
one or more immunostimulatory agents to induce or enhance an immune
response, such as IL-2 and immunostimulatory oligonucleotides
(e.g., those containing CpG motifs). Preferred immunostimulatory
agents stimulate specific arms of the immune system, such as
natural killer (NK) cells that mediate antibody-dependent cell
cytotoxicity (ADCC).
[0298] As provided elsewhere herein, the compositions provided can
be administered with one or more adjuvants to induce or enhance an
immune response. An adjuvant is a substance which potentiates the
immune response. Adjuvants of many kinds are well known in the art.
Specific examples of adjuvants include monophosphoryl lipid A (MPL,
SmithKline Beecham); saponins including QS-21 (Antigenics);
immunostimulatory oligonucleotides (e.g., CpG oligonucleotides
described by Kreig et al., Nature 374:546-9, 1995);incomplete
Freund's adjuvant; complete Freund's adjuvant; montanide; vitamin E
and various water-in-oil emulsions prepared from biodegradable oils
such as squalene and/or tocopherol, Quil A, Ribi Detox, CRL-1005,
L-121, and combinations thereof.
[0299] Other agents which stimulate the immune response of the
subject to PSMA multimer antigens can also be administered to the
subject. For example, cytokines are also useful in vaccination
protocols as a result of their lymphocyte regulatory properties.
Many cytokines useful for such purposes will be known to one of
ordinary skill in the art, including interleukin-2 (IL-2); IL-4;
IL-5; IL-12, which has been shown to enhance the protective effects
of vaccines (see, e.g., Science 268: 1432-1434, 1995); GM-CSF;
IL-15; IL-18; combinations thereof, and the like. Thus cytokines
can be administered in conjunction with antibodies, antigens,
chemokines and/or adjuvants to increase an immune response.
[0300] Chemokines useful in increasing immune responses include but
are not limited to SLC, ELC, MIP3.alpha., MIP3.beta., IP-10, MIG,
and combinations thereof.
[0301] The PSMA multimers or antibodies or antigen-binding
fragments thereof of the present invention can be used in
conjunction with other therapeutic treatment modalities. Current
standard or conventional treatments for cancer, such as prostate
cancer, include surgery, radiation, cryosurgery, thermotherapy,
hormone treatment and chemotherapy. Subjects receiving one or more
of the standard treatments may be referred to as
treatment-experienced subjects. Hormone therapy includes treatment
with one or more of the following modalities: a leutinizing
hormone-releasing hormone agonist such as leuprolide, goserelin or
buserelin; an antiandrogen, such as flutaminde or bicalutamide; a
drug that prevents adrenal glands from making androgens, such as
ketoconazole or aminoglutethimide; estrogens; and orchiectomy
(castration). Chemotherapy may use any
chemotherapeutic/antineoplastic agent known in the art. In some
preferred embodiments the chemotherapeutic agent is a taxane, such
as paclitaxel (Taxol.RTM.) or docetaxel (Taxotere.RTM.).
Chemotherapy may be used in combination with an anti-inflammatory
compound such as a corticosteroid. Corticosteroids include
cortisone, hydrocortisone, prednisone, prednisolone, triamcinolone,
methylprednisolone, dexamethasone, betamethasone and the like. A
preferred anti-inflammatory compound is prednisone. Other
therapeutic modalities that may be used in combination with PSMA
multimers include the use of other vaccines and
immunotherapies.
[0302] Also encompassed by the present invention is a method which
involves using the PSMA multimers or antibodies or antigen-binding
fragments thereof for prophylaxis. For example, these materials can
be used to prevent or delay development or progression of
cancer.
[0303] Use of the cancer therapy of the present invention has a
number of benefits. Since the anti-PSMA antibodies or
antigen-binding fragments thereof according to the present
invention preferentially target prostate cancer cells, other tissue
is spared. As a result, treatment with such biological agents is
safer, particularly for elderly patients. Treatment according to
the present invention is expected to be particularly effective,
because it directs high levels of anti-PSMA antibodies or
antigen-binding fragments thereof to the bone marrow and lymph
nodes where prostate cancer metastases predominate. Moreover, tumor
sites for prostate cancer tend to be small in size and, therefore,
easily destroyed by cytotoxic agents. Treatment in accordance with
the present invention can be effectively monitored with clinical
parameters such as serum prostate specific antigen and/or
pathological features of a patient's cancer, including stage,
Gleason score, extracapsular, seminal, vesicle or perineural
invasion, positive margins, involved lymph nodes, etc.
Alternatively, these parameters can be used to indicate when such
treatment should be employed.
[0304] Because the antibodies or antigen-binding fragments thereof
of the present invention bind to living cells, therapeutic methods
using these biological agents are much more effective than those
which target lysed cells. For the same reasons, diagnostic and
imaging methods which determine the location of living normal,
benign hyperplastic, or cancerous cells are much improved by
employing the antibodies or antigen-binding fragments thereof of
the present invention. In addition, the ability to differentiate
between living and dead cells can be advantageous, especially to
monitor the effectiveness of a particular treatment regimen.
[0305] Also within the scope of the invention are kits comprising
the compositions of the invention and instructions for use. The
kits can further contain at least one additional reagent, such as
complement, or one or more additional antibodies of the invention
(e.g., an antibody having a complementary activity which binds to
an epitope in PSMA antigen distinct from the first antibody). Other
kits can include the PSMA multimers described herein below.
[0306] The kits provided herein include any of the compositions
described and instructions for the use of these compositions. The
instructions can include instructions for mixing a particular
amount of an agent or solution that preserves or promotes the
multimerization of PSMA with a particular amount of a PSMA
composition. The instructions can also include instructions for
mixing a particular amount of a diluent with a particular amount of
a PSMA dimeric composition, whereby a final formulation for
injection or infusion is prepared. Therefore, kits are also
provided, which include the compositions of the invention and,
optionally, an adjuvant (e.g., alum) or diluent and instructions
for mixing. Kits are also provided wherein the compositions of the
inventions are provided in a vial or ampoule with a septum or a
syringe. Other kits where the composition is in lyophilized form
are also provided. The instructions, therefore, would take a
variety of forms depending on the presence or absence of diluent or
other agents (e.g., therapeutic agents). The instructions can
include instructions for treating a patient with an effective
amount of dimeric PSMA. It also will be understood that the
containers containing the pharmaceutical preparation, whether the
container is a bottle, a vial with a septum, an ampoule with a
septum, an infusion bag, and the like, can contain indicia such as
conventional markings which change color when the pharmaceutical
preparation has been autoclaved or otherwise sterilized.
[0307] Kits containing the antibodies or antigen-binding fragments
thereof of the invention can be prepared for in vitro diagnosis,
prognosis and/or monitoring cancer by the immunohistological,
immunocytological and immunoserological methods described above.
The components of the kits can be packaged either in aqueous medium
or in lyophilized form. When the antibodies or antigen-binding
fragments thereof are used in the kits in the form of conjugates in
which a label moiety is attached, such as an enzyme or a
radioactive metal ion, the components of such conjugates can be
supplied either in fully conjugated form, in the form of
intermediates or as separate moieties to be conjugated by the user
or the kit.
[0308] A kit may comprise a carrier being compartmentalized to
receive in close confinement therein one or more container means or
series of container means such as test tubes, vials, flasks,
bottles, syringes, or the like. A first of said container means or
series of container means may contain one or more anti-PSMA
antibodies or antigen-binding fragments thereof or PSMA. A second
container means or series of container means may contain a label or
linker-label intermediate capable of binding to the primary
anti-PSMA antibodies (or fragment thereof).
[0309] It should be understood that the pharmaceutical preparations
of the invention will typically be held in bottles, vials,
ampoules, infusion bags, and the like, any one of which may be
sparged to eliminate oxygen or purged with nitrogen. In some
embodiments, the bottles vials and ampoules are opaque, such as
when amber in color. Such sparging and purging protocols are well
known to those of ordinary skill in the art and should contribute
to maintaining the stability of the pharmaceutical preparations.
The pharmaceutical preparations also, in certain embodiments, are
expected to be contained within syringes.
[0310] Kits for use in in vivo tumor localization and therapy
method containing the anti-PSMA antibodies or antigen-binding
fragments thereof conjugated to other compounds or substances can
be prepared. The components of the kits can be packaged either in
aqueous medium or in lyophilized form. When the antibodies or
antigen-binding fragments thereof are used in the kits in the form
of conjugates in which a label or a therapeutic moiety is attached,
such as a radioactive metal ion or a therapeutic drug moiety, the
components of such conjugates can be supplied either in fully
conjugated form, in the form of intermediates or as separate
moieties to be conjugated by the user of the kit.
[0311] In one aspect of the invention, a method for modulating at
least one enzymatic activity of PSMA, the activity selected from
the group consisting of N-acetylated .alpha.-linked acidic
dipeptidase (NAALADase), folate hydrolase, dipeptidyl dipeptidase
IV and .gamma.-glutamyl hydrolase activity or combination thereof
in vitro or in vivo. The modulation may be enhancement or
inhibition of at least one enzymatic activity of PSMA.
[0312] In a preferred embodiment, the invention provides methods
for inhibiting at least one enzymatic activity of PSMA, the
activity selected from the group consisting of N-acetylated
.alpha.-linked acidic dipeptidase (NAALADase), folate hydrolase,
dipeptidyl dipeptidase IV and .gamma.-glutamyl hydrolase activity
or combination thereof in vitro or in vivo. The method comprises
contacting a NAALADase, a folate hydrolase, a dipeptidyl
dipeptidase IV and/or a .gamma.-glutamyl hydrolase with an amount
of an isolated antibody or antigen-binding fragment thereof of the
invention under conditions wherein the isolated monoclonal antibody
or antigen-binding fragment thereof inhibits NAALADase, folate
hydrolase, dipeptidyl dipeptidase IV or .gamma.-glutamyl hydrolase
activity.
[0313] Tissue levels of NAALADase can be determined by detergent
solubilizing homogenizing tissues, pelleting the insoluble material
by centrifugation and measuring the NAALADase activity in the
remaining supernatant. Likewise, the NAALADase activity in bodily
fluids can also be measured by first pelleting the cellular
material by centrifugation and performing a typical enzyme assay
for NAALADase activity on the supernatant. NAALADase enzyme assays
have been described by Frieden, 1959, J. Biol, Chem., 234:2891. In
this assay, the reaction product of the NAALADase enzyme is
glutamic acid. This is derived from the enzyme catalyzed cleavage
of N-acetylaspartylglutamate to yield N-acetylaspartic acid and
glutamic acid. Glutamic acid, in a NAD(P).sup.+ requiring step,
yields 2-oxoglutarate plus NAD(P)H in a reaction catalyzed by
glutamate dehydrogenase. Progress of the reaction can easily and
conveniently be measured by the change in absorbance at 340 nm due
to the conversion of NAD(P).sup.+ to NAD(P)H.
[0314] Folate hydrolase activity of PSMA can be measured by
performing enzyme assays as described by Heston and others (e.g.,
Clin. Cancer Res. 2(9):1445-51, 1996; Urology 49(3A
Suppl):104-12,1997). Folate hydrolases such as PSMA remove the
gamma-linked glutamates from polyglutamated folates. Folate
hydrolase activity can be measured using substrates such as
methotrexate tri-gamma glutamate (MTXGlu3), methotrexate di-gamma
glutamate (MTXGlu2) and pteroylpentaglutamate (PteGlu5), for
example using capillary electrophoresis (see Clin. Cancer Res.
2(9):1445-51, 1996). Timed incubations of PSMA with polyglutamated
substrates is followed by separation and detection of hydrolysis
products.
[0315] The invention also includes isolated antibodies and binding
fragments thereof that selectively bind PSMA multimers. As used
herein, particularly with respect to the binding of PSMA multimers
by the anti-PSMA antibodies and binding fragments, "selectively
binds" means that an antibody preferentially binds to a PSMA
protein multimer (e.g., with greater avidity, greater binding
affinity) rather than to a PSMA protein monomer. In preferred
embodiments, the antibodies of the invention bind to a PSMA protein
multimer with an avidity and/or binding affinity that is 1.1-fold,
1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,
1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 7-fold,
10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 70-fold, 100-fold,
200-fold, 300-fold, 500-fold, 1000-fold or more than that exhibited
by the antibody for a PSMA protein monomer. Preferably, the
antibody selectively binds a PSMA protein multimer, and not a PSMA
protein monomer, i.e., substantially exclusively binds to a PSMA
protein multimer. Most preferably, the antibody selectively binds a
PSMA protein dimer.
[0316] The isolated antibody or binding fragment that selectively
binds a PSMA protein multimer can, in some embodiments, modulate
enzymatic activity of the PSMA protein multimer. In one such
embodiment, the antibody inhibits at least one enzymatic activity
such as NAALADase activity, folate hydrolase activity, dipeptidyl
dipeptidase IV activity, .gamma.-glutamyl hydrolase activity, or
combinations thereof. In another embodiment, the antibody enhances
at least one enzymatic activity such as NAALADase activity, folate
hydrolase activity, dipeptidyl dipeptidase IV activity,
.gamma.-glutamyl hydrolase activity, or combinations thereof.
[0317] As described elsewhere herein, a PSMA protein multime is a
protein complex of at least two PSMA proteins or fragments thereof.
The PSMA protein multimers can be composed of various combinations
of full-length PSMA proteins (e.g., SEQ ID NO: 1), recombinant
soluble PSMA (rsPSMA, e.g., amino acids 44-750 of SEQ ID NO:1) and
fragments of the foregoing that form multimers (i.e., that retain
the protein domain required for forming dimers and/or higher order
multimers of PSMA). In preferred embodiments, at least one of the
PSMA proteins forming the multimer is a recombinant, soluble PSMA
(rsPSMA) polypeptide. Preferred PSMA protein multimers are dimers,
particularly those formed from recombinant soluble PSMA protein. A
particularly preferred embodiment is a rsPSMA homodimer.
[0318] The PSMA protein multimers referred to herein are believed
to assume a native conformation and preferably have such a
conformation. The PSMA proteins in certain embodiments are
noncovalently bound together to form the PSMA protein multimer. For
example, it has been discovered that PSMA protein noncovalently
associates to form dimers under non-denaturing conditions, as
described in the Examples below.
[0319] The PSMA protein multimers can, and preferably do, retain
the activities of PSMA. The PSMA activity may be an enzymatic
activity, such as folate hydrolase activity, NAALADase activity,
dipeptidyl peptidase IV activity and .gamma.-glutamyl hydrolase
activity. Methods for testing the PSMA activity of multimers are
well known in the art (reviewed by O'Keefe et al. in: Prostate
Cancer: Biology Genetics, and the New Therapeutics, L. W. K. Chung,
W. B. Isaacs and J. W. Simons (eds.) Humana Press, Totowa, N.J.,
2000, pp. 307-326), some of which are described in the Examples
herein below.
[0320] As used herein with respect to polypeptides, proteins or
fragments thereof, "isolated" means separated from its native
environment and present in sufficient quantity to permit its
identification or use. Isolated, when referring to a protein or
polypeptide, means, for example: (i) selectively produced by
expression cloning or (ii) purified as by chromatography or
electrophoresis. Isolated proteins or polypeptides may be, but need
not be, substantially pure. The term "substantially pure" means
that the proteins or polypeptides are essentially free of other
substances with which they may be found in nature or in vivo
systems to an extent practical and appropriate for their intended
use. Substantially pure polypeptides may be produced by techniques
well known in the art. Because an isolated protein may be admixed
with a pharmaceutically acceptable carrier in a pharmaceutical
preparation, the protein may comprise only a small percentage by
weight of the preparation. The protein is nonetheless isolated in
that it has been separated from the substances with which it may be
associated in living systems, i.e. isolated from other
proteins.
[0321] Fragments of a PSMA protein preferably are those fragments
which retain a distinct functional capability of the PSMA protein.
Functional capabilities which can be retained in a fragment include
binding of other PSMA molecules to form dimers and higher order
multimers, interaction with antibodies, interaction with other
polypeptides or fragments thereof, and enzymatic activity. Other
PSMA protein fragments, e.g., other recombinant soluble fragments
of SEQ ID NO:1, can be selected according to their functional
properties. For example, one of ordinary skill in the art can
prepare PSMA fragments recombinantly and test those fragments
according to the methods exemplified below.
[0322] Modifications to a PSMA polypeptide are typically made to
the nucleic acid which encodes the PSMA polypeptide, and can
include deletions, point mutations, truncations, amino acid
substitutions and additions of amino acids or non-amino acid
moieties. Alternatively, modifications can be made directly to the
polypeptide, such as by cleavage, addition of a linker molecule,
addition of a detectable moiety, such as biotin, addition of a
fatty acid, and the like. Modifications also embrace fusion
proteins comprising all or part of the PSMA amino acid
sequence.
[0323] In general, modified PSMA polypeptides include polypeptides
which are modified specifically to alter a feature of the
polypeptide unrelated to its physiological activity. For example,
cysteine residues can be added or substituted or deleted to promote
or prevent unwanted disulfide linkages, respectively. Similarly,
certain amino acids can be changed to enhance expression of a PSMA
polypeptide by eliminating proteolysis by proteases in an
expression system (e.g., dibasic amino acid residues in yeast
expression systems in which KEX2 protease activity is present).
[0324] Modifications conveniently are prepared by altering a
nucleic acid molecule that encodes the PSMA polypeptide. Mutations
of a nucleic acid which encode a PSMA polypeptide preferably
preserve the amino acid reading frame of the coding sequence, and
preferably do not create regions in the nucleic acid which are
likely to hybridize to form secondary structures, such a hairpins
or loops, which can be deleterious to expression of the modified
polypeptide.
[0325] Modifications can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the PSMA polypeptide. Modified PSMA
polypeptides then can be expressed and tested for one or more
activities (e.g., antibody binding, enzymatic activity, multimeric
stability) to determine which mutation provides a modified
polypeptide with the desired properties. Further mutations can be
made to modified PSMA polypeptides (or to non-modified PSMA
polypeptides) which are silent as to the amino acid sequence of the
polypeptide, but which provide preferred codons for translation in
a particular host. The preferred codons for translation of a
nucleic acid in, e.g., E. coli, are well known to those of ordinary
skill in the art. Still other mutations can be made to the
noncoding sequences of a PSMA coding sequence or cDNA clone to
enhance expression of the polypeptide. The activity of modified
PSMA polypeptides can be tested by cloning the gene encoding the
modified PSMA polypeptide into a bacterial or mammalian expression
vector, introducing the vector into an appropriate host cell,
expressing the modified PSMA polypeptide, and testing for a
functional capability of the PSMA polypeptides as disclosed herein.
The foregoing procedures are well known to one of ordinary skill in
the art.
[0326] The skilled artisan will also realize that conservative
amino acid substitutions may be made in PSMA polypeptides to
provide functionally equivalent PSMA polypeptides, i.e., modified
PSMA polypeptides that retain the functional capabilities of PSMA
polypeptides. These functionally equivalent PSMA polypeptides
include those PSMA polypeptides or proteins that are capable of
associating to form multimers, particularly dimers. As used herein,
a "conservative amino acid substitution" refers to an amino acid
substitution which does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Modified PSMA polypeptides can be prepared according to
methods for altering polypeptide sequence known to one of ordinary
skill in the art such as are found in references which compile such
methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,
et al., eds., Second Edition, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular
Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc.,
New York. Exemplary functionally equivalent PSMA polypeptides
include conservative amino acid substitutions of SEQ ID NO:1, or
fragments thereof, such as the recombinant soluble PSMA polypeptide
(amino acids 44-750 of SEQ ID NO:1). Conservative substitutions of
amino acids include substitutions made amongst amino acids within
the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d)
A, G; (e) S, T; (f) Q, N; and (g) E, D.
[0327] Conservative amino-acid substitutions in PSMA polypeptides
typically are made by alteration of a nucleic acid encoding a PSMA
polypeptide. Conservatively substituted PSMA polypeptides include
those with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more
substitutions. Such substitutions can be made by a variety of
methods known to one of ordinary skill in the art. For example,
amino acid substitutions may be made by PCR-directed mutation,
site-directed mutagenesis, or by chemical synthesis of a gene
encoding a PSMA polypeptide. Where amino acid substitutions are
made to a small fragment of a PSMA polypeptide, the substitutions
can be made by directly synthesizing the peptide. The activity of
functionally equivalent fragments of PSMA polypeptides can be
tested by cloning the gene encoding the altered PSMA polypeptide
into a bacterial or mammalian expression vector, introducing the
vector into an appropriate host cell, expressing the altered PSMA
polypeptide, and testing for a functional capability of the PSMA
polypeptides as disclosed herein.
[0328] The PSMA protein multimers as described herein have a number
of uses, some of which are described elsewhere herein. The
multimers are useful for testing of compounds that modulate PSMA
enzymatic activity or PSMA multimerization. The multimers can be
used to isolate antibodies that selectively bind PSMA, including
those selective for conformational epitopes, those selective for
binding PSMA multimers and not PSMA monomers, and those that
selectively modulate an enzymatic activity of PSMA. The multimers,
particularly dimeric PSMA, also can be used to induce or increase
immune responses to PSMA, as vaccine compositions.
[0329] Agents that selectively modulate an enzymatic activity of
PSMA include agents that inhibit or enhance at least one enzymatic
activity of PSMA, such as NAALADase activity, folate hydrolase
activity, dipeptidyl dipeptidase IV activity, .gamma.-glutamyl
hydrolase activity, or combinations thereof.
[0330] Thus methods of screening for candidate agents that modulate
at least one enzymatic activity of a PSMA enzyme are provided in
accordance with the invention. The methods can include mixing the
candidate agent with an isolated PSMA protein multimer to form a
reaction mixture, thereby contacting the PSMA enzyme with the
candidate agent. The methods also include adding a substrate for
the PSMA enzyme to the reaction mixture, and determining the amount
of a product formed from the substrate by the PSMA enzyme. Such
methods are adaptable to automated, high-throughput screening of
compounds. A decrease in the amount of product formed in comparison
to a control is indicative of an agent capable of inhibiting at
least one enzymatic activity of the PSMA enzyme. An increase in the
amount of product formed in comparison to a control is indicative
of an agent capable of enhancing at least one enzymatic activity of
the PSMA enzyme. The PSMA enzyme can be NAALADase, folate
hydrolase, dipeptidyl dipeptidase IV and/or .gamma.-glutamyl
hydrolase. The PSMA enzyme preferably is a PSMA multimer that
includes recombinant soluble PSMA, most preferably a noncovalently
associated dimer of PSMA in a native conformation.
[0331] The reaction mixture comprises a candidate agent. The
candidate agent is preferably an antibody, a small organic
compound, or a peptide, and accordingly can be selected from
combinatorial antibody libraries, combinatorial protein libraries,
or small organic molecule libraries. Typically, a plurality of
reaction mixtures are run in parallel with different agent
concentrations to obtain a different response to the various
concentrations. Typically, one of these concentrations serves as a
negative control, i.e., at zero concentration of agent or at a
concentration of agent below the limits of assay detection.
[0332] Candidate agents encompass numerous chemical classes,
although typically they are organic compounds, proteins or
antibodies (and fragments thereof that bind antigen). In some
preferred embodiments, the candidate agents are small organic
compounds, i.e., those having a molecular weight of more than 50
yet less than about 2500, preferably less than about 1000 and, more
preferably, less than about 500. Candidate agents comprise
functional chemical groups necessary for structural interactions
with polypeptides and/or nucleic acids, and typically include at
least an amine, carbonyl, hydroxyl, or carboxyl group, preferably
at least two of the functional chemical groups and more preferably
at least three of the functional chemical groups. The candidate
agents can comprise cyclic carbon or heterocyclic structure and/or
aromatic or polyaromatic structures substituted with one or more of
the above-identified functional groups. Candidate agents also can
be biomolecules such as peptides, saccharides, fatty acids,
sterols, isoprenoids, purines, pyrimidines, derivatives or
structural analogs of the above, or combinations thereof and the
like.
[0333] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides, synthetic organic
combinatorial libraries, phage display libraries of random or
non-random peptides, combinatorial libraries of proteins or
antibodies, and the like. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant, and animal
extracts are available or readily produced. Additionally, natural
and synthetically produced libraries and compounds can be readily
be modified through conventional chemical, physical, and
biochemical means. Further, known agents may be subjected to
directed or random chemical modifications such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs of the agents.
[0334] A variety of other reagents also can be included in the
mixture. These include reagents such as salts, buffers, neutral
proteins (e.g., albumin), detergents, etc. which may be used to
facilitate optimal protein-protein and/or protein-agent binding.
Such a reagent may also reduce non-specific or background
interactions of the reaction components. Other reagents that
improve the efficiency of the assay such as protease inhibitors,
nuclease inhibitors, antimicrobial agents, and the like may also be
used.
[0335] The mixture of the foregoing reaction materials is incubated
under conditions whereby, the candidate agent interacts with the
PSMA enzyme. The order of addition of components, incubation
temperature, time of incubation, and other parameters of the assay
may be readily determined. Such experimentation merely involves
optimization of the assay parameters, not the fundamental
composition of the assay. Incubation temperatures typically are
between 4.degree. C. and 40.degree. C. Incubation times preferably
are minimized to facilitate rapid, high throughput screening, and
typically are between 0.1 and 10 hours.
[0336] After incubation, the presence or absence of PSMA enzyme
activity is detected by any convenient method available to the
user. For example, the reaction mixture can contain a substrate for
the PSMA enzyme. Preferably the substrate and/or the product formed
by the action of the PSMA enzyme are detectable. The substrate
usually comprises, or is coupled to, a detectable label. A wide
variety of labels can be used, such as those that provide direct
detection (e.g., radioactivity, luminescence, optical, or electron
density, etc) or indirect detection (e.g., epitope tag such as the
FLAG epitope, enzyme tag such as horseradish peroxidase, etc.). The
label may be bound to the substrate, or incorporated into the
structure of the substrate.
[0337] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the substrate
or subsequent to separation from the substrate. Labels may be
directly detected through optical or electron density, radioactive
emissions, nonradiative energy transfers, etc. or indirectly
detected with antibody conjugates, strepavidin-biotin conjugates,
etc. Methods for detecting a variety of labels are well known in
the art.
[0338] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
[0339] Materials and Methods
[0340] DNA Constructs. All secreted PSMA constructs were derived
from the original human PSMA clone p55A provided by Dr. W. D. W.
Heston (Israeli et al., Cancer Res. 53: 227-230, 1993). The
constructs were subcloned into expression vector PPI4 (Trkola et
al., Nature 384: 184-187, 1996) for high-level expression and
secretion in mammalian cells. Recombinant soluble PSMA (rsPSMA)
corresponds to the entire extracellular domain of PSMA (amino acids
44-750 of SEQ ID NO: 1 (GenBank Protein Accession number
AAA60209)).
[0341] pcDNA Plasmid Constructs: Nucleic acid molecules encoding
the anti-PSMA antibodies 10.3, 006, 026, 051, 069 and 077 were
cloned into plasmid pcDNA. The cloning protocol is given in FIG.
13. Primers (SEQ ID NOs: 33-36, sense and anti-sense) used for the
variable region amplifications are also shown. The plasmids
constructed for anti-PSMA antibodies 006, 026, 051, 069, 077 and
10.3 contain nucleotide sequences encoding the heavy chain of the
antibodies (SEQ ID NOs: 2-7; PTA-4403, PTA-4405, PTA-4407,
PTA-4409, PTA-4411, PTA-4413, respectively) or contain nucleotide
sequences encoding light chain of the antibodies (SEQ ID NOs: 8-13;
PTA-4404, PTA-4406, PTA-4408, PTA-4410, PTA-4412 and PTA-4414,
respectively). Plasmid maps are given in FIGS. 14-25.
[0342] Western Blots. Cells were lysed in PBS containing 1 mM EDTA,
1% NP-40, 1% Triton X-100, and 5 mg/ml aprotinin and cell debris
was removed by centrifugation at 3000g for 30 min at 4.degree. C.
Lysates were separated on a 5-20% gradient gel before transfer to
nitrocellulose membranes. The resulting blots were blocked in PBS
containing 5% milk, 0.02% SDS and 0.1% Triton X-100 before
incubation with MAB544 primary antibody (Maine Biotechnologies) at
a concentration of 2 mg/ml. After three washes, blots were
incubated with a goat anti-mouse HRP-conjugated secondary antibody
at a concentration of 0.2 mg/ml. Blots are visualized using the
Renaissance chemiluminescence system (Perkin-Elmer Life Sciences,
Boston, Mass.).
[0343] ELISA. Cells were lysed in PBS containing 1 mM EDTA, 1%
NP-40, 1% Triton X-100, and 5 mg/ml aprotinin. The resulting cell
membranes were plated onto 96-well plates and dried in a sterile
hood overnight. The plates were then blocked with PBS containing
casein and Tween-20 before addition of mouse sera or hybridoma
supernatants, using purified MAB544 (Maine Biotechnologies) or 7E11
(Cytogen) as a standard. After washing in PBS, an alkaline
phosphatase conjugated secondary antibody (subclass specific) was
incubated and subsequently washed in PBS. The pNPP substrate was
then added for colorimetric detection at a wavelength of 405 nm.
Flow Cytometry. Wild-type 3T3 or PSMA-expressing 3T3 cells
(10.sup.6 cells per condition) were washed in PBS containing 0.1%
NaN.sub.3. Antibodies or sera were then added (1:100 dilution in
PBS) and incubated on ice for 30 minutes. After washing in PBS+0.1%
NaN.sub.3, the cells were incubated with anti-mouse IgG+IgM
(Calbiotech) for 30 minutes on ice. Cells were washed again in
PBS+0.1% NaN.sub.3 and analyzed by flow cytometry.
Example 1
Generation of a Panel of Monoclonal Antibodies (mAbs) to
Conformational Epitopes on PSMA
[0344] A panel of anti-PSMA mAbs that represent promising
candidates for therapy was created. Briefly, the mAbs were
generated as follows: BALB/c mice were immunized subcutaneously
with recombinant PSMA at approximately three-week intervals. After
a total of 4 injections, mice were sacrificed and their splenocytes
fused with a myeloma cell line using standard techniques in order
to create hybridomas. Individual hybridoma supernatants were
screened by ELISA for reactivity with PSMA derived from either
LNCaP human prostate tumor cells or from 3T3 cells engineered to
express full-length human PSMA (3T3-PSMA cells). Positive clones
were secondarily screened by flow cytometry for specific reactivity
with intact 3T3-PSMA and LNCaP cells so as to select antibodies
that recognize native, cell-surface PSMA and thus have the greatest
therapeutic potential.
[0345] Mice having the ability to produce human antibodies
(XenoMouse, Abgenix; Mendez et al., Nature Genetics 15:146, 1997)
were immunized subcutaneously once or twice weekly with
5.times.10.sup.6 LNCaP cells adjuvanted with alum or Titermax Gold
(Sigma Chemical Co., St. Louis, Mo.). Animals were boosted twice
with 10 .mu.g of recombinant PSMA protein immunoaffinity captured
onto protein G magnetic microbeads (Miltenyi Biotec, Auburn,
Calif.). PSMA mAb 3.11 was used for capture. Splenocytes were fused
with NSO myeloma cells and the hybridomas that resulted were
screened as above by flow cytometry to detect clones producing
antibodies reactive with the extracellular portion of PSMA. One
clone, 10.3 (PTA-3347), produced such antibodies.
[0346] These methods have yielded a high proportion of mAbs that
react exclusively with conformation-specific epitopes on
cell-surface PSMA. As shown in FIG. 1, several (mAbs 3.7, 3.9,
3.11, 5.4, and 10.3) but not all (mAb 3.12) mAbs specifically bind
viable PSMA-expressing cells. Using recombinant soluble PSMA
proteins expressed in Chinese hamster ovary (CHO) cell lines, it
further was demonstrated that the mAbs bind epitopes in the
extracellular region of PSMA. The mAbs were also tested for their
ability to immunoprecipitate native PSMA from 3T3-PSMA cell
lysates. The mAbs positive in flow cytometry (FIG. 1) were also
effective in immunoprecipitation (FIG. 2), whereas mAb 3.12 was
unreactive. FIG. 3 shows the recognition of non-denatured
full-length PSMA and recombinant soluble PSMA by several PSMA
antibodies that recognize PSMA conformation. This further confirms
that these methods yield a preponderance of mAbs that efficiently
recognize native PSMA.
[0347] The mAbs were tested for reactivity with denatured PSMA by
Western blot analysis (FIG. 4). Lysates from the indicated cells
and samples (controls: 3T3 cells, PSMA-negative human prostate cell
lines PC-3 and DU145, mock supernatant; PSMA-positive samples:
PSMA-expressing 3T3 cells, PSMA-positive human prostate cell line
LNCaP, rsPSMA-positive supernatant) were resolved by SDS-PAGE,
electroblotted, and probed with anti-PSMA mAbs 3.1 and 3.12 (ATCC
Patent Deposit Designations PTA-3639 and PTA-3640, respectively).
Four mAbs tested in parallel (3.7, 3.8, 3.9, 3.11) showed no
reactivity to either full-length or secreted rsPSMA proteins. 7E11
mAb immunoprecipitated full-length but not secreted rsPSMA.
[0348] The mAbs reactive in flow cytometry and immunoprecipitation
(mAbs 3.7, 3.9, 3.11, 5.4, and 10.3) were all unreactive in Western
blot analysis, indicating that the mAbs do not recognize linear
epitopes. Taken together, the data strongly suggest that these 5
mAbs recognize conformation-specific epitopes located in the
extracellular domain of PSMA. Since mAbs to conformational epitopes
typically possess the greatest affinity and specificity for
antigen, they represent preferred candidates for therapy.
[0349] The reactivities of certain anti-PSMA antibodies are
described in Table 2:
2TABLE 2 Anti-PSMA Antibody Properties Reactivity Flow West- mAb
ELISA Cytometry IP ern Epitope 3.1 + + + + Linear, Extracellular,
exposed on native PSMA 3.7 + + + - Conformational, extracellular
3.8 + + + - Conformational, extracellular 3.9 + + + -
Conformational, extracellular 3.11 + + + - Conformational,
extracellular 3.12 + - - + Linear, Extracellular, not exposed on
native PSMA 5.4 + + + - Conformational, extracellular 7.1 + - - +
Linear, Extracellular, not exposed on native PSMA 7.3 + + + -
Conformational, extracellular 10.3 + + + - Conformational,
extracellular 1.8.3 + + - Extracellular A3.1.3 + + - Extracellular
A3.3.1 + + - Extracellular
[0350] The mAbs were determined by ELISA to be primarily of the
mouse IgG2a, mouse IgG2b and human IgG1 isotypes, which mediate
potent effector functions. Although a number of anti-PSMA mAbs have
been described over the years and evaluated for therapeutic
potential (see, e.g., Liu, H. et al. Cancer Res. 57: 3629-3634,
1997; Chang, S. S. et al. Cancer Res. 59: 3192-3198, 1999; Murphy,
G. P. et al. J. Urology 160: 2396-2401, 1998), none inhibit the
enzymatic activity of PSMA and few recognize conformational
determinants on PSMA.
Example 2
Production of Anti-PSMA mAbs
[0351] To accurately and quantitatively assess the therapeutic
potential of these mAbs, the mAbs are produced in a quantity and
quality suitable for extensive in vitro and in vivo
characterization. Briefly, the mAb-secreting hybridomas are
cultured in roller bottles in DMEM/F12 medium supplemented with 10%
FBS that has been depleted of bovine IgG (Life Technologies).
During the production phase of the culture, cells are maintained at
.about.5.times.10.sup.6 cells/mL via twice-weekly exchanges of
media. Collected media are clarified by filtration through a 0.22
micron filter and stored at -95.degree. C. prior to purification.
Given an average antibody expression levels of .about.25 mg/L,
approximately 3L of roller bottle supernatants are required for
each antibody to allow for losses in purification.
[0352] Culture supernatants from a given hybridoma are pooled and
loaded onto a Protein A Sepharose affinity column. Mouse IgG2a,
mouse IgG2b and human IgG1 antibodies are loaded directly, but
supernatants containing mouse IgG1 antibodies are adjusted to pH
8.5 and 1M NaCl prior to loading in order to promote binding. After
washing the column, the mAb is eluted with low pH buffer into
fractions using 1M Tris, pH 8.0. Elution peak fractions are pooled,
dialyzed against PBS buffer, concentrated to 5 mg/mL and stored in
sterile aliquots at -95.degree. C. All purification procedures are
carried out using endotoxin-free buffers and sanitized
chromatography columns. Purified mAbs are tested for purity by
reducing and nonreducing SDS-PAGE, for PSMA binding affinity by
ELISA, and for endotoxin levels by the limulus amebocyte lysate
assay. These procedures routinely yield "animal-grade" antibody at
>95% purity and <0.5 endotoxin units per milligram of
protein.
Example 3
Evaluation of the Therapeutic Potential of the Unlabeled mAbs In
Vitro
[0353] Purified mAbs are tested in a battery of assays for
therapeutically relevant properties, including affinity,
specificity, enzyme inhibitory activity and effector functions. The
ideal product candidate binds and inhibits PSMA activity at
subnanomolar concentrations and mediates potent cell-killing
through Fc-related effector functions.
[0354] First, the mAbs' affinity for cell-surface and secreted
forms of PSMA is measured by flow cytometry and ELISA,
respectively. In the flow cytometry assay, varying amounts of mAbs
are incubated with 5.times.10.sup.5 3T3-PSMA cells in FACS buffer
(PBS containing 1% FBS and 0.1% NaN.sub.3) for 2 hr to allow for
saturation binding. Cells are washed and incubated with a
phycoerythrin-coupled goat antibody to mouse IgG (ICN/Cappel) for
detection of bound mAb by flow cytometry. Specific binding is
calculated by subtracting the fluorescence intensity observed with
parental 3T3 cells.
[0355] For ELISA, CHO cell-derived recombinant soluble PSMA protein
(rsPSMA, Progenics, Tarrytown, N.Y.) is diluted to 1 .mu.g/ml in 50
mM carbonate buffer, pH 9.4, and coated overnight at 4.degree. C.
onto 96-well Immulon II microtiter plates at 100 .mu.l/well. The
plates are then blocked for 2 hr with PBS buffer containing 5% BSA.
mAbs are added in a range of concentrations in ELISA buffer (PBS
buffer containing 2% BSA, 1% FBS and 0.5% Tween 20) for 2 hours at
room temperature. The plates are washed, and horseradish peroxidase
conjugated goat antibody to mouse IgG is added for 1 hr at room
temperature. The plates are washed again and
3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB) substrate
(Pierce, Rockford, Ill.) is added for colorimetric readout at 450
nm using an ELISA plate reader (Molecular Devices, Sunnyvale,
Calif.).
Example 4
mAb Cross-Competition Binding Assay
[0356] To identify whether a given group of mAbs recognize distinct
or overlapping epitopes on PSMA, cross-competition binding assays
are performed (Liu, H. et al. Cancer Res 57: 3629-3634, 1997). In
this flow cytometry assay, a biotinylated test mAb is incubated
with 3T3-PSMA cells in the presence or absence of varying
concentrations of unlabeled competitor mAbs as described above.
Following washing, phycoerythrin-conjugated streptavidin is added
to determine the amount of bound biotinylated mAb. The percent
inhibition is defined relative to that observed in the presence of
an isotype-matched mAb of irrelevant specificity (0% inhibition)
and to that observed using excess unlabeled test mAb (100%
inhibition).
Example 5
Effects of mAbs on PSMA Enzymatic Activity
[0357] PSMA has been shown to possess both folate hydrolase
(pteroyl-glutamyl carboxypeptidase) and N-acetylated .alpha.-linked
acidic dipeptidase (NAALADase) enzymatic activities, which may
influence the proliferation and malignancies of the tumor cell
(Heston, W. D. W. Prostate: Basic and Clinical Aspects (R. K. Naz,
ed.). CRC Press, New York: 219-243, 1997). A first set of mAbs
described above (mAb 3.9, mAb 5.4 and mAb 7.3) and mAb J591 (ATCC
#HB-12126) were tested for folate hydrolase modulating activity
using previously described assays for measuring PSMA enzymatic
activity (Pinto, J. T. et al. Clinical Cancer Res 2: 1445-1451,
1996).
[0358] Briefly, folate hydrolase activity was measured as follows.
Fifty .mu.M methotrexate di-gamma glutamate and 10 .mu.g/ml rsPSMA
(premixed with anti-PSMA or irrelevant mAb) was incubated in pH 4.5
acetate buffer in a volume of 100 .mu.l for 2 hr at 37.degree. C.
Reactions were terminated by boiling for 5 minutes prior to
separation of free, mono- and di-gamma glutamate forms of
methotrexate by capillary electrophoresis on a Spectra Phoresis
1000 (Thermo Separation, San Jose, Calif.). The various
methotrexate derivatives were quantified based on their retention
times and absorbance at 300 nm.
[0359] The data show that mAb 5.4 potently blocks the enzymatic
activity of purified rsPSMA protein and in lysates of C4-2 cells.
C4-2 is an androgen independent derivative of the LNaCP cell line
(human prostate cancer line) which expresses endogenous PSMA. More
details regarding the C4-2 cell line may be found in O'Keefe D. S.
et al. Prostate 45: 149-157, 2000). FIGS. 8 and 9 provide the
results for two production lots of rsPSMA (rsPSMA #7 and rsPSMA
#8). The results for the C4-2 cell lysates are shown in FIG. 10.
The figures illustrate the effect of four antibodies (mAb 3.9, mAb
5.4, mAb 7.3 and mAb J591) on the enzymatic activity of folate
hydrolase by way of the rate of cleavage of glutamate from
methotrexate di-gamma glutamate (MTXGlu2) by folate hydrolase
present in the two production lots of rsPSMA and in the C4-2 cell
lysates. In addition to the inhibitory effects of mAb 5.4, mAb 3.9
was also found to inhibit folate hydrolase activity.
[0360] Another set of mAbs (mAb 4.40.2, mAb 006, mAb 026 and mAb
5.4) was also tested for folate hydrolase modulating activity. The
data confirm that mAb 5.4 potently blocks folate hydrolase activity
of PSMA (FIG. 11). The concentration of mAb 5.4 which inhibited
PSMA enzymatic activity by 50% (IC50, also referred to as EC50 or
"effective concentration") was determined to be
4.902.times.10.sup.-4 mg/mL. The data further show that mAb 006 and
mAb 026 also block PSMA folate hydrolase activity, while mAb 4.40.2
did not (FIG. 11). The IC50 values for mAb 006 and mAb 026 were
9.338.times.10.sup.-3 mg/mL and 1.385.times.10.sup.-3 mg/mL,
respectively.
[0361] For NAALADase activity assays, rsPSMA protein is incubated
with varying amounts of anti-PSMA or control mAbs in 50 mM Tris pH
7.4, 1 mM CoCl.sub.2 for 10 minutes at 37.degree. C. before adding
50 .mu.l of 0.6 .mu.M N-acetylaspartyl-[.sup.3H]glutamate. After 15
minutes, the reaction is stopped by adding 1 ml of 100 mM
NaPO.sub.4. Cleaved glutamate is separated from the substrate by
ion exchange chromatography and detected by scintillation counting.
Each measurement is performed in triplicate.
Example 6
Reactivity with Normal and Malignant Human Tissues by
Immunohistochemistry
[0362] Anti-PSMA mAbs are tested by immunohistochemistry for
reactivity with both normal and malignant human tissues using an
avidin-biotin peroxidase method (Silver, D. A. et al. Clin Cancer
Res 3: 81-85,1997). Frozen or paraffin-embedded tissues can be
used. Paraffin-embedded tissue sections are deparaffinized and
endogenous peroxidase activity is blocked by incubation with 1%
H.sub.2O.sub.2 for 15 minutes. Sections are blocked in a 1:10
dilution of horse serum in 2% PBS-BSA (Sigma Chemical, St Louis,
Mo.) for 30 minutes before overnight incubation with 2 .mu.g/ml
anti-PSMA mAb in 2% PBS-BSA. After washing, sections are incubated
with biotinylated secondary antibody, washed, and incubated with
avidin:biotin peroxidase complexes (Vector Laboratories,
Burlingame, Calif.) diluted 1:25 in PBS for 30 minutes. After
washing, sections are visualized by immersion in PBS containing
0.05% diaminobenzidine tetrachloride, 0.01% H.sub.2O.sub.2, and
0.5% Triton X-100. Negative control sections are incubated with
isotype-matched mAbs of irrelevant specificity. As a positive
control, 7E11 (Cytogen, Princeton, N.J.), a well-characterized
anti-PSMA mAb, is used.
Example 7
Antibody-Dependent Cellular Cytotoxicity (ADCC)
[0363] In the ADCC assay, mAbs are serially diluted and combined
with .sup.51Cr-labeled 3T3-PSMA cells or human prostate PC-3 cells
that have been engineered to express human PSMA (PC-3-PSMA cells).
NK effector cells are purified from lymph nodes or spleens using
anti-NK microbeads (Miltenyi Biotec). Sera, NK effector cells, and
.sup.51Cr-loaded target cells are co-incubated at effector:target
cell ratios of 10:1, 20:1, and 40:1, with each condition performed
in triplicate. Cells are incubated 4-5 hours at 37.degree. C.
before supernatants are collected for measurement of 51 Cr release
by gamma counting. The percent specific lysis is determined
relative to that observed in the presence of isotype-matched
non-specific mAb (0% lysis) to that obtained using 10% sodium
dodecyl sulfate (100% lysis).
Example 8
Complement-Mediated Lysis (CML)
[0364] For CML, .sup.51Cr-loaded 3T3-PSMA or PC-3-PSMA cells serve
as target cells. Serial dilutions of mAbs are co-incubated with
rabbit complement and target cells for 4-5 hours at 37.degree. C.,
with each condition being performed in triplicate. Supernatants are
then collected and counted with a gamma counter. Specific lysis is
computed as previously done with the ADCC assay data.
Example 9
Anti-Proliferative Effects
[0365] To test anti-proliferative effects of these antibodies,
anti-PSMA mAbs are serially diluted and incubated with LNCaP,
PC-3-PSMA and parental PC-3 cells in log-phase growth. At 4 hr, 24
hr, and 72 hr intervals, cells are removed and analyzed for density
and viability by trypan blue staining and WST-1 assay (Roche
Biochemicals).
Example 10
Optimization of Chelation and Radiolabeling Procedures
[0366] The most promising mAbs identified using the procedures
described in the foregoing examples will be optimized for
biochemical and biological stability and activity after labeling
prior to evaluation in animals. Success in in vitro experiments is
defined as identification of a radiolabeled mAb that specifically
kills PSMA-expressing tumor cells at >10-fold lower
concentrations than unlabeled or similarly labeled isotype control
mAb.
[0367] Because the preferred .alpha.- and .beta.-emitting isotopes
are all radiometals, each of the mAbs is first conjugated with an
appropriate metal chelating agent. Based on the favorable in vivo
stability data and its proven use in human clinical trials, the
bifunctional chelating agent C-functionalized trans
cyclohexyldiethylenetriaminepentaacetic acid (p-SCN-CHX-A"-DTPA) is
the preferred agent for attaching either .sup.90Y or .sup.213Bi to
the antibody (Brechbiel, M. W. et al. J. Chem. Soc. Chem. Commun.
1169-1170, 1991). A form of this chelate has previously been tested
in more than 70 doses in humans in ongoing trials at Memorial-Sloan
Kettering Cancer Center (McDevitt, M. R. et al. J. Nucl. Med.
40:1722-1727, 1999). For 225Ac, our initial studies will examine a
novel bifunctional chelating agent termed p-SCN-Bz-HEHA
(1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N",N'",N"",N'""-hexaacetic
acid) (Deal, K. A. et al. J. Med. Chem. 42:2988-2992, 1999). The
objective is to optimize the antibody conjugation and chelation
ratios to maximize labeling yield and activity while maintaining
suitable stability for in vivo utilization. Additional chelating
agents also are used as they become available from the N.I.H. and
other sources.
[0368] Initially, the antibody is rendered metal-free by incubation
with a large molar excess of EDTA at pH=5. The EDTA and any metals
scavenged from the antibody preparation are removed via continuous
buffer exchange/dialysis so as to replace the pH=5 buffer with the
conjugation buffer (Nikula, T. K. et al. Nucl. Med. Biol.
22:387-390, 1995). Conditions that yield optimal chelator to
antibody ratio but still remain immunoreactive are identified by
systematically varying the chelator:antibody ratio, reaction time,
temperature, and/or buffer systems about initial conditions that
employ a 40-fold molar excess of chelator to antibody in HEPES
buffer, pH 8.5. The number of chelates bound per antibody is
determined using an established spectrophotometric method (Pippin,
C. G. et al. Bioconjugate Chemistry 3: 342-345, 1992).
[0369] For .sup.90Y and .sup.225Ac constructs, labeling efficiency
is measured directly. For .sup.213Bi, initial antibody constructs
are tested for chelation efficiency using .sup.111In, which has
similar chelation chemistry as .sup.213Bi but possesses the
advantages of a longer half life (t.sub.1/2=3 days), ready
availability, and traceable .gamma.-emission. Once optimized using
.sup.111In, labeling efficiency is determined for .sup.213Bi.
[0370] Radiolabeled mAb is purified over a BioRad 10DG desalting
column using 1% HSA as the mobile phase and evaluated by instant
thin layer liquid chromatography (ITLC) and/or high performance
liquid chromatography (HPLC) to determine the percent incorporation
of radionuclide (Zamora, P. O. et al. Biotechniques 16: 306-311,
1994). ITLC and HPLC provide a means of establishing purity and
identifying the percent of low molecular weight radiochemical
impurities (i.e., metal chelates, colloids, and free metal).
Duplicate ITLC strips for each mobile phase are developed, dried,
and cut at the R.sub.f of 0.5 mark and counted in a gamma counter.
The HPLC system is equipped with both an online UV absorption
detector and radioactivity detector. The HPLC elution profile
directly correlates radioactivity with protein and low molecular
weight species as a function of the elution time. A TSK
SW3000.sub.XL column (TosoHaas, Montgomeryville, Pa.) is used and
calibrated using a range of protein molecular weight standards.
Example 11
Affinity and Immunoreactivity of Radiolabeled mAbs
[0371] Once radiolabeled constructs are obtained, purified, and
assessed for biochemical and radiochemical purity, biological
activity is determined. Binding activity of the radioconstruct is
performed by Scatchard analysis of binding data obtained using
whole LNCaP and 3T3-PSMA cells and/or membrane fractions as
previously described (Scheinberg, D. A. et al. Leukemia 3: 440-445
(1991).
[0372] The immunoreactivity of the synthetic constructs is
evaluated in order to correlate the chelate:antibody molar ratio
with the biological activity. Briefly, 2 ng of labeled mAb is
incubated with a 25-fold excess of PSMA as expressed on 3T3-PSMA
cells. After a 30 min incubation at 0.degree. C., the cells are
collected by centrifugation and the supernatant containing unbound
mAb is added to fresh 3T3-PSMA cells for an additional 30 min at
0.degree. C. Both sets of cells are centrifuged and washed twice
with cold PBS. The cell pellets, supernatant and wash fractions are
counted for radioactivity. Immunoreactivity is defined as the
amount of radioactivity in the cell pellets divided by the total
radioactivity in the cell pellets, supernatant and wash
fractions.
Example 12
mAb Internalization
[0373] The activity of radiolabeled mAbs can be significantly
modulated by their internalization rates. Based upon previous
results by other groups (Smith-Jones P. M. et al. Cancer Res 60:
5237-5243, 2000), significant internalization of PSMA after binding
with one or more of the mAb constructs was expected.
Internalization of the cell surface antibody-antigen complex was
measured using .sup.111In radiolabeled antibody (mAb 026)
constructs (Caron, P. C. et al. Cancer Res 52: 6761-6767, 1992).
Briefly, 5.times.10.sup.5 C4-2 cells were incubated at 37.degree.
C. in 5% CO.sub.2 with .sup.111In radiolabeled antibody. At
different times, cells were washed with PBS and cell-surface bound
radiolabeled constructs were stripped with 1 ml of 50 mM
glycine/150 mM NaCl, pH=2.8. Total cell-associated radioactivity
and acid-resistant (internalized) radioactivity were determined by
.gamma.-counting. Percent internalization and total binding were
calculated. .sup.111In labeled mAb 026 was found to be rapidly and
efficiently internalized. FIG. 12 shows the percent internalization
and total binding of .sup.111In labeled mAb 026 as a function of
incubation time. Cells (such as parental 3T3 cells) that do not
express PSMA can be used as a control to determine non-specific
binding.
Example 13
In Vitro Cytotoxicity Studies
[0374] Assessment of in vitro cytotoxicity of .alpha.-labeled mAbs
was undertaken once the immunoreactivity of the
radioimmunoconjugate was established. Approximately 50,000 target
cells (either LNCaP or 3T3-PSMA cells) were treated in 96 well
plates and analyzed 24-96 hours later. Quantification of cell death
due to .sup.225Ac-labeled constructs (or .sup.213Bi) was
accomplished by determining the uptake of .sup.3H-thymidine by
surviving cells (Nikula, T. K. et al. J. Nucl. Med. 40: 166-176,
1999). Specificity was determined by use of control cells
(PSMA-negative human prostate cell lines PC-3 and DU-145, as well
as control 3T3 cells), blocking with excess unlabeled antibody, and
control radioconjugates.
[0375] The cytotoxic effects of antibody conjugate concentration,
specific activity, and time of exposure were then assessed.
Cytotoxicity was expressed relative to that seen with 1M HCl (100%
cell death) and media (background cell death). LD.sub.50 values
were calculated by plotting cell viability as a function of the
number of .sup.225Ac atoms bound on the cells (McDevitt, M. R. et
al. (1998) Eur. J. Nucl. Med. 25: 1341-1351 (1998).
[0376] Multicellular spheroids of LNCaP-FGC cells had been
established and were used to investigate the potential of
radioimmunotherapy (RIT) to eradicate minimal disease in vitro.
These three-dimensional spheroids mimic tissue structures more
accurately than monolayer cultures and thus provide a more relevant
model of solid tumors (O'Connor, K. C. Pharm. Res. 16: 486-493,
1999). LNCaP-FGC is a fast growing clone of the original LNCaP cell
line, and the cells were grown using a liquid overlay technique to
a size of 200-600 .mu.m (Ballangrud, A. M. et al. Clin. Cancer Res.
5: 3171s-3176s, 1999). In larger spheroids, the inner mass of cells
becomes necrotic, while the outer rim consists of proliferating
tumor cells. Antibody penetration was measured by confocal
microscopy, and prior results suggested that an anti-PSMA antibody
should penetrate to a depth of 40-50 .mu.m (Ballangrud, A. M. et
al. 7th Conference on Radioimmunodetection and Radioimmunotherapy
of Cancer, Princeton N.J., 1998). The in vitro cytotoxicity of
.sup.225Ac-3.9 on LNCaP target cells is shown in FIG. 26. The
percentage of viable PSMA.sup.+ LNCaP cells was plotted as a
function of activity of the radioconjugate. Addition of a 100-fold
excess of unlabeled antibody was used as a control for
specificity.
Example 14
Evaluation of the In Vivo Efficacy of Unlabeled and Radiolabeled
mAbs in Mouse Xenograft Models of Human Prostate Cancer
[0377] Antibodies that are successful in the foregoing assays
demonstrate significant specificity and functional properties that
suggest they will be useful for therapeutic use. The most promising
of these radiolabeled and "naked" mAb constructs are evaluated in
the best available mouse models of prostate cancer. The studies
employ an established xenograft model in which the LNCaP human
prostate tumor cell line is injected into immunocompromised nude
mice and allowed to form solid tumors (Ellis, W. J. et al. Clin
Cancer Res 2: 1039-1048 (1996), which then are treated with both
radiolabeled and unlabeled anti-PSMA mAb constructs. Follow-on
studies also utilize a mouse xenograft model, CWR22, which
reproduces many of the key biological features of human prostate
cancer.
[0378] Lncap Tumor Cell Xenograft Model
[0379] A construct showing high affinity and high specificity is
taken into the LNCaP tumor cell xenograft in vivo model for
biodistribution and pharmacokinetic analysis. .sup.111In-labeled
anti-PSMA antibody is used for these studies due to its favorable
chelation chemistry, radioactive half-life and traceable gamma
emission. Timepoints are evaluated as appropriate for the
half-lives of .sup.213Bi, .sup.225Ac, .sup.177Lu and .sup.90Y,
which are the nuclides of therapeutic interest. Labeled
radioconstructs (1-5 .mu.g) are injected i.v. into nude mice
(normal and tumor bearing) and the mice are sacrificed at 5 min, 15
min, 30 min, 60 min, 2 hrs, 4 hrs, 18 hrs, and 24 hrs
post-injection. Blood and major organs are taken from animals,
weighed, and the percent radioactivity injected per gram of tissue
is determined (Nikula, T. K. et al. J. Nucl. Med. 40: 166-176,
1999). Specificity is addressed by pre-injection with excess
unlabeled construct. Macroscopic tumor volume and animal survival
rates is recorded throughout the experiments.
[0380] A dose-ranging study is also conducted to determine the
toxicity of the constructs when administered via i.v. or i.p.
injection to normal and tumor-bearing mice. These animals are
routinely examined for toxic side effects during the course of the
studies by blood chemistry and physical examination. Animals are
sacrificed during and at the conclusion of the study in order to
collect blood and body tissues for further evaluation. Previous
data has demonstrated an approximate maximum tolerated dose of 250
.mu.Ci/mouse, so total doses are kept below that level.
[0381] Once i.v. biodistribution and toxicity is documented,
radiotherapy of tumors is assessed. Groups of five mice are
injected with <1 .mu.g radiolabeled anti-PSMA mAb construct both
pre- and post-tumor challenge to assess anti-tumor activity.
Antigen negative (RAJI or RAMOS) xenografted tumors are also used
as a control. Other controls include (1) treatment with unlabeled
anti-PSMA mAb only and (2) excess unlabeled anti-PSMA mAb
pretreatment before .sup.213Bi, .sup.225Ac, .sup.177Lu and/or
.sup.90Y-labeled anti-PSMA to block specific targeting.
[0382] Groups of tumor bearing mice are injected with unlabeled
anti-PSMA mAbs (at equimolar concentrations) and several dose
levels of radiolabeled anti-PSMA or a similarly labeled isotype
control antibody. The effect on tumor growth is assessed over time.
Statistical differences between therapy groups is determined using
an analysis of variance (ANOVA) method and animal survival is
illustrated using Kaplan-Meier plots. The efficacy of .sup.213Bi,
.sup.225Ac, .sup.177Lu and/or .sup.90Y-labeled anti-PSMA constructs
is correlated to the data obtained in vitro. Success in these
experiments is defined as the ability to significantly (p<0.05)
increase life-span and/or decrease tumor volume as compared to a
radiolabeled isotype control mAb.
[0383] Furthermore, the tumor models are used to test whether
predosing with unlabeled antibody prior to injection of
radiolabeled antibody improves delivery of the radiolabeled
antibody to the tumor. The tumor-bearing mice are injected with
<1 .mu.g radiolabeled anti-PSMA antibody with or without a prior
single injection of 5-100 .mu.g of unlabeled antibody. After
several days, animals are sacrificed for evaluation of the
distribution of radioactivity in the tumor, normal tissue, and
blood. If predosing with unlabeled antibody improves delivery and
targeting of radiolabeled antibody to the tumors, this approach is
applied and optimized in toxicity and therapeutic studies.
[0384] In addition to overall survival, the role of timing of the
injection after tumor transplantation (Day 1 vs 3 vs 7), the role
of dosage (dose-response curves using 3-4 dose levels), the role of
schedule (single vs multiple divided daily injections) and the
specificity of the treatment (pre-treatment with unlabeled
anti-PSMA to block targeting) is examined.
[0385] These in vivo studies are designed to address the maximum
tolerated dose of radiolabeled antibody, the activity of the
antibody, the optimal dosing schedule (single or multiple
injections), and the effect on tumor size. Successful completion of
this work enables determination of the feasibility of PSMA-targeted
alpha particle radioimmunotherapy (RIT) of prostate cancer and
identifies the optimal .sup.213Bi and/or .sup.225Ac-labeled
constructs to enter into clinical development.
[0386] CWR22 Mouse Xenograft Model
[0387] The most promising anti-PSMA mAbs in unlabeled,
toxin-labeled and/or radiolabeled form are tested in the CWR22
human prostate cancer xenograft mouse model, (Wainstein, M. A. et
al. Cancer Res 54:6049-6052 (1994); Nagabhushan, M. et al. Cancer
Res 56:3042-3046 (1996); Pretlow, T. G. et al. J Natl Cancer Inst
85:394-398 (1993)). This model has many features of the human
condition including a dependence on androgens, a correlation
between measured levels of PSA in serum and tumor size, and
high-level expression of PSMA. Following androgen withdrawal, PSA
levels decrease to nearly undetectable levels and tumor volume
decreases. Later, the tumor regrows as an androgen-independent
neoplasm, manifest initially by a rise in PSA and later, measurable
tumor growth. After androgen withdrawal, tumors regrow at variable
time periods.
[0388] Four to six week old nude athymic BALB/c male mice are
obtained from the National Cancer Institute-Frederick Cancer Center
and maintained in pressurized ventilated caging. While
immunodeficient in many respects, these mice mediate wild-type
levels of ADCC and CML. The CWR22 tumor line is propagated in the
animals by the injection of minced tumor tissue from an established
tumor into the subcutaneous tissue of the flanks of athymic nude
mice together with reconstituted basement membrane (Matrigel,
Collaborative Research, Bedford, Mass.). To maintain serum androgen
levels, the mice are administered 12.5-mg sustained-release
testosterone pellets (Innovative Research of America, Sarasota,
Fla.) subcutaneously before receiving tumors. Three to four weeks
after inoculation, tumors of approximately 1.5.times.1.0.times.1.0
cm are measured. Androgens are withdrawn by surgical castration
under pentobarbital anesthesia and removal of the sustained-release
testosterone pellets. Tumor size is determined by caliper
measurements of height, width and depth. PSA values are performed
on the serum of the mice after tail bleeding using a Tandem-R PSA
immuno-radiometric assay (Hybritech, San Diego, Calif.).
[0389] Groups of five mice are injected with anti-PSMA mAb or a
similar isotype control mAb at dosages from 5-100 .mu.g to assess
anti-tumor activity. The effect of scheduling single doses vs.
multiple divided daily injections is also examined. Macroscopic
tumor volume and animal survival rates are recorded throughout the
experiments. Statistical differences between therapy groups are
determined using an analysis of variance (ANOVA) method and animal
survival are illustrated using Kaplan-Meier plots, with success
defined as a difference of p<0.05. Similarly, the efficacy of
"naked" mAbs is compared to that seen with .sup.90Y, .sup.177Lu,
.sup.213Bi and/or .sup.225Ac-labeled anti-PSMA constructs.
[0390] These in vivo studies are designed to address the maximum
tolerated dose of mAb, the activity of the antibody, the optimal
dosage and dosing schedule (single or multiple divided injections),
and the effect of treatment on tumor size. Successful completion of
this work will enable determination of the feasibility of
PSMA-targeted immunotherapy of prostate cancer and identification
of the optimal constructs to enter into clinical development.
Example 15
Investigation of Native PSMA Protein Conformation
[0391] Extraction of PSMA from the Cell Surface of LNCaP and 3T3
Cells
[0392] LNCaP or 3T3 cells were grown to confluency in a T150 cell
culture flask, detached using cell dissociation solution
(Mediatech, Herndon, Va.) and transferred to a 15 ml conical tube.
The cells were washed twice with PBS and resuspended with 2 ml of
M-Per.TM. Mammalian Protein Extraction Reagent (Pierce, Rockford,
Ill.). Following incubation for 10 min at 4.degree. C., cell debris
and insoluble aggregates were removed by centrifugation at 15,000
rpm for 30 min at 4.degree. C. The supernatant was transferred to a
cryogenic vial and stored at -80.degree. C. until further use.
[0393] Production of Recombinant, Soluble PSMA (rsPSMA)
[0394] The extracellular domain of PSMA (amino acids 44-750 of the
full-length protein, SEQ ID NO:1) was obtained as a secreted
protein from a DXB11 Chinese hamster ovary (CHO) cell line, stably
transfected with an rsPSMA expression vector. The cells were grown
in a Celligen Plus 2.2L Packed Bed Bioreactor (New Brunswick
Scientific, Edison, N.J.) in protein-free media. The Bioreactor was
operated in perfusion mode, and supernatant was collected
aseptically into collection bags maintained at 4.degree. C. The
protease inhibitor aprotinin was added to the harvest supernatant,
which was concentrated 25-fold prior to storage at -90.degree. C.
In some instances for purification, the concentrate was thawed and
purified using subsequent steps of Concanavalin A lectin affinity
chromatography and Butyl-Sepharose hydrophobic interaction
chromatography or according to the steps shown below.
[0395] The purified rsPSMA protein is dimeric, and possesses folate
hydrolase enzymatic activity when tested according to published
procedures (Pinto et al., Clinical Cancer Research 2:1445, 1996)
and reacts with each of a panel of conformation-specific monoclonal
antibodies, indicating that rsPSMA adopts a native
conformation.
[0396] Purification of Recombinant, Soluble PSMA (rsPSMA)
[0397] Cell culture supernatants were concentrated 25-fold by
tangential flow ultrafiltration and adjusted to 35% saturation with
ammonium sulfate. Under these conditions, rsPSMA remains in the
supernatant. Precipitated proteins were removed by centrifugation
(20,000.times.g for 30 min, SS-34, Sorvall) and the clarified
supernatant was applied to a Butyl-Sepharose resin (BioRad,
Hercules, Calif.) followed by a wash with 35% ammonium sulfate in
neutral phosphate-buffered saline containing 1 mM Ca.sup.2+ and 0.5
mM Mg.sup.2+ (PBS+). rsPSMA eluted in the flow-through and wash
fractions of the column. The fractions containing the rsPSMA
protein were pooled, dialyzed into 10 mM sodium phosphate, pH 7.0,
and loaded onto a Ceramic Hydroxyapatite column (BioRad, Hercules,
Calif.). rsPSMA was eluted from the resin using 2M sodium chloride
in 10 mM sodium phosphate, pH 7.0. The fractions containing the
protein were pooled, dialyzed into 20 mM Tris, pH 7.5 containing 1
mM Ca.sup.2+ and 0.5 mM Mg.sup.2+, and applied to a Q650-Sepharose
column (TosoHaas, Montgomeryville, Pa.). rsPSMA was eluted from the
resin with 150 mM NaCl in 20 mM Tris, pH 7.5 containing 1 mM
Ca.sup.2+ and 0.5 mM Mg.sup.2+. Monomeric and dimeric forms of
rsPSMA present after this step were separated using preparative
size exclusion chromatography on a Superdex 200 resin (Amersham
Biosciences, Piscataway, N.J.) and PBS+ (containing 1 mM Ca.sup.2+
and 0.5 mM Mg.sup.2+) as the running buffer. Purified rsPSMA was
stored at -80.degree. C. in PBS+. Unless otherwise indicated, PSMA
monomers represent spontaneously dissociated protein recovered over
SEC rather than forcibly denatured material.
[0398] Polyacrylamide Gel Electrophoresis (PAGE) and Western
Blotting of the Different PSMA Proteins
[0399] For each individual PAGE analysis, 15 .mu.l of each cell
lysate and 5 .mu.l of the purified rsPSMA were used.
[0400] SDS-PAGE was performed using standard procedures. Samples
were prepared by boiling for 5 minutes in the presence of Laemmli
sample buffer (with or without the reducing agent dithiothreitol
[DTT]). Samples were then applied on a 4-15% Tris-Glycine gel
(BioRad, Hercules, Calif.). After electrophoresis for 1 h at 200V,
the proteins were transferred onto nitrocellulose (BioRad) and
analyzed by Western blotting.
[0401] The oligomeric nature of the different PSMA proteins was
analyzed using Blue Native PAGE (BN-PAGE). Each sample was diluted
with an equal volume of 2.times.BN-PAGE sample buffer (0.1M
MOPS/0.1M Tris/40% glycerol/0.1% Coomassie G-250) prior to loading
onto the gel. BN-PAGE was performed using 4-12% BisTris gels
(Invitrogen, Carlsbad, Calif.) and 50 mM MOPS/50 mM Tris, pH 7.7 as
running buffer. Coomassie Blue was omitted from the cathode buffer
to avoid interference with protein binding during the transfer of
the proteins onto nitrocellulose. Following electrophoresis for 2.5
hrs at 125V, the proteins were transferred onto a nitrocellulose
membrane (BioRad) and analyzed by Western blotting.
[0402] Western blotting was performed as follows: Subsequent to
transfer, the nitrocellulose membrane was blocked with 5% milk in
PBS/0.1% Triton X-100/0.02% SDS, which was also used for the
subsequent wash and antibody incubation steps. PSMA proteins were
detected using the anti-PSMA mAbs 3.1 or 3.9 (Progenics
Pharmaceuticals) as primary antibody and HRP-labeled anti-mouse IgG
as secondary antibody and 1 h incubation at room temperature. The
membranes were colorimetrically developed using chemiluminescence
(NEN Plus, Perkin Elmer Life Sciences, Boston, Mass.).
[0403] Analytical size exclusion chromatography (SEC) was performed
using a TSK G3000SW.sub.XL (TosoHaas, Montgomeryville, Pa.) column
equilibrated in PBS+. The column was calibrated using bovine serum
albumin (67 kDa), immunoglobulin G (150 kDa), ferritin (440 kDa)
and thyroglobulin (670 kDa) as standards.
[0404] Results
[0405] Both full-length PSMA and recombinant, soluble PSMA (rsPSMA)
migrated on reducing and non-reducing SDS-PAGE with a molecular
weight of .about.100 kDa (FIG. 5). Thus, like full-length PSMA,
rsPSMA is a monomer in the presence of denaturing agents, and no
disulfide or other covalent bonds are present to mediate
oligomerization. The result for full-length PSMA is in accordance
with prior observations (Israeli et al., U.S. Pat. No. 5,538,866;
Murphy et al., U.S. Pat. No. 6,158,508; Israeli, et al., Cancer
Research 54:1807, 1994; Troyer et al. Int. J. Cancer 62:552, 1995;
Troyer et al., The Prostate 30:233, 1997; Grauer et al., Cancer
Research 58:4787, 1998). In each of these reports, full-length PSMA
migrated as a major band of 100-120 kDa, with a minor (typically
<5% of the total PSMA protein) 180-200 kDa band observed in a
subset of reports (U.S. Pat. No. 6,158,508; Troyer et al., 1995;
Troyer et al., 1997). Troyer et al. (1995) describe the 180-200 kDa
species as being a noncovalently associated PSMA dimer that can be
disrupted with increasing concentrations of SDS detergent.
[0406] rsPSMA contains 94% (707 of 750) of the amino acids present
in full-length PSMA, and the two proteins were not clearly resolved
in this analysis, as expected.
[0407] SDS-PAGE allows the analysis of denatured proteins only. In
order to examine native proteins in their native state, other
techniques have to be employed, such as Blue Native PAGE (BN-PAGE).
BN-PAGE is used to determine the native molecular weight of
proteins and their noncovalent complexes (Schgger & v. Jagow,
Anal. Biochem. 199:223-231, 1991; Schgger et al., Anal. Biochem.
217:220-230, 1994). The dye Coomassie Blue G-250 binds to the
hydrophobic domains on the surface of most proteins, enhances
solubility, and introduces a charge shift on the native proteins
resulting in migration towards the anode at pH 7.5 irrespective of
the isoelectric point of the protein. Although the migration
velocity of proteins in BN-PAGE varies somewhat, the molecular mass
of proteins can be determined by their respective end points of
migration due to the decreasing pore size of the acrylamide
gradient present in the gels.
[0408] When analyzed by BN-PAGE, full-length PSMA (extracted from
LNCaP or 3T3 cells with nonionic detergents) as well as purified
rsPSMA migrate with a molecular weight of .about.190 kDa (FIG. 6A).
This surprising observation for full-length PSMA indicates that the
predominant form of cell-surface PSMA is a noncovalently associated
dimer. This unexpected result can be contrasted with that of
previous reports (U.S. Pat. No. 6,158,508; Troyer et al. 1995;
Troyer et al., 1997), where the PSMA dimer represents a minor
species in SDS-PAGE analyses. Presumably, the noncovalent PSMA
dimer is largely dissociated by boiling in the presence of the
denaturing detergent SDS.
[0409] Moreover, the result for the purified rsPSMA protein
indicates that the dimer is stabilized via interactions between
extracellular amino acids in addition to or exclusive of amino
acids in the transmembrane or intracellular segments, which are not
present in rsPSMA.
[0410] rsPSMA was subjected to analytical size exclusion
chromatography (SEC) as a second sizing method. When analyzed in
neutral PBS+ buffer, purified rsPSMA eluted as a single major peak
with an apparent molecular mass of 260 kDa (FIG. 6B), slightly
higher than expected. However, glycoproteins (such as rsPSMA) are
typically nonglobular in shape and run at higher apparent molecular
mass than standard SEC calibration proteins (Schulke, N., et al.
(2002) J. Virol. 76, 7760-7776). Therefore, an apparent molecular
mass of 260 kDa is consistent with the proposed homodimeric
structure of rsPSMA. In contrast, purified monomeric rsPSMA eluted
with an apparent molecular mass of 130 kDa. The studies demonstrate
that the extracellular domain of PSMA is sufficient for
dimerization, and the similarities between rsPSMA (amino acids
44-750) and PSM' (amino acids 58-750) suggest that the latter
protein is likely to dimerize as well.
Example 16
Homodimerization is Required for Enzymatic Activity
[0411] Enzyme Assays
[0412] Pteroyl .gamma.-glutamyl carboxypeptidase (folate hydrolase)
activity was determined by monitoring the cleavage of poly
.gamma.-glutamylated methotrexate as described (Pinto, J. T., et
al. (1996) Clin. Cancer Res. 2, 1445-1451) with the following
exceptions. Di-.gamma.-glutamylated methotrexate (MTXglu2) was used
as substrate and HPLC was used rather than capillary
electrophoresis. At the completion of the incubation (50 .mu.M
methotrexate di-gamma glutamate and 10 .mu.g/ml rsPSMA in pH 4.5
acetate buffer in a volume of 100 .mu.l for 2 hr at 37.degree. C.),
100 .mu.l of 0.5 M Na.sub.2HPO.sub.4 was added to stop the
reaction. Samples were loaded at a flow rate of 1.25 ml/min through
a 50.times.4.6 mm, 3 .mu.m PRISM reversed-phase column (Thermo
Hypersil-Keystone, Bellefonte, Pa.) with a PRISM 10.times.4-mm
guard column, eluted with 15% methanol in 85% 0.5M
K.sub.2HPO.sub.4, pH 7.0, and quantitated based on relative peak
area observed at a wavelength of 313 nm.
[0413] For NAALADase assays, rsPSMA was incubated with
N-acetyl-.alpha.-L-aspartyl-L-glutamate for 22 h at 37.degree. C.
in the presence of 20 mM sodium phosphate, 50 mM NaCl, 10 mM
ZnCl.sub.2, pH 7.1. Released L-glutamic acid was quantitated by
using a commercial kit (R-Biopharm, Marshall, MI);
2-(phosphonomethyl) pentanedioic acid and
Gly-Pro-7-amido-4-methylcoumarin were purchased from Sigma. Porcine
kidney dipeptidyl peptidase IV (DPP IV) was used according to the
manufacturer's instructions (Sigma).
[0414] Results
[0415] PSMA has been reported to possess folate hydrolase,
NAALADase, and DPP IV activities (Pinto, J. T., et al. (1996) Clin.
Cancer Res. 2, 1445-1451; Carter, R. E., et al. (1996) Proc. Natl.
Acad. Sci. USA 93, 749-753; Pangalos, M. N., et al. (1999) J. Biol.
Chem. 274, 8470-8483). The first two activities involve the
hydrolysis of a carboxyl-terminal peptide bond to liberate a
glutamic acid residue, whereas DPP IV cleaves downstream of an
amino-terminal Aaa-Pro dipeptide sequence. The folate hydrolase
activities of purified monomeric and dimeric forms of rsPSMA were
evaluated. Whereas the dimer demonstrated high-level folate
hydrolase activity, the monomer was essentially inactive (FIG. 7A).
In fact, the residual activity of the monomer could be attributed
to the residual amount (approximately 4%) of dimeric rsPSMA present
in the preparation. High-level folate hydrolase activity was also
observed for LNCaP cell lysates, consistent with prior observations
(Pinto, J. T., et al. (1996) Clin. Cancer Res. 2, 1445-1451).
Similarly, dimeric but not monomeric forms of rsPSMA possessed
high-level NAALADase activity (FIG. 7B), which was abrogated by
using 5 nM of the inhibitor 2-(phosphonomethyl)pentanedioic acid.
Neither monomer nor dimer demonstrated DPP IV activity under
conditions where porcine DPP IV efficiently hydrolyzed the
substrate Gly-Pro-7-amido-4-methylcoumarin. This is consistent with
the results reported by Barinka et al. (Barinka, C., et al. (2002)
J. Neurochem. 80, 477-487), who similarly failed to confirm the DPP
IV activity previously reported for PSMA (Pangalos, M. N., et al.
(1999) J. Biol. Chem. 274, 8470-8483).
Example 16
Dissociation of PSMA Multimers
[0416] PSMA is a putative zinc metalloprotease, and site-directed
mutagenesis of amino acids implicated in zinc binding results in a
profound loss of enzymatic activity (Speno et al., Molecular
Pharmacology, 55:179, 1999). These amino acids include His-377,
Asp-387, Glu-425, Asp-453 and His-553. Ethylenediaminetetraacetic
acid (EDTA) is a strong chelating agent for Zn.sup.2+ and other
divalent cations, and thus has the potential to remove Zn.sup.2+ or
other coordinate divalent cations from PSMA. We have determined
that EDTA treatment causes the PSMA homodimer to dissociate into
monomeric subunits. Similar results can be expected for other
agents that possess similar chelating properties, such as
ethyleneglycol-bis(beta-aminoethyl ether) (EGTA).
[0417] The purified rsPSMA protein was incubated with or without 10
mM EDTA for 16 hr at 4.degree. C. and then analyzed by BN-PAGE.
Under these conditions, the EDTA-treated protein was monomeric,
whereas rsPSMA remained dimeric in the absence of EDTA. Although
the dissociation of the PSMA dimer into monomer was essentially
complete, any residual dimeric protein can be removed if desired by
gel filtration, ultracentrifugation or other size-based separation
methods that are well-known to those skilled in the art.
Example 17
Methods for Identifying Promoters of PSMA Dissociation
[0418] Compounds are screened for the ability to promote
dissociation of PSMA dimers using a method that includes:
[0419] (a) contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound;
[0420] (b) measuring the amount of PSMA monomer; and
[0421] (c) comparing the amount of PSMA monomer measured in the
presence of the compound with that observed in the absence of the
compound.
[0422] An increase in the amount of PSMA monomer measured in the
presence of the compound indicates that the compound is capable of
promoting dissociation of the PSMA dimer.
[0423] In a further embodiment, compounds are screened for the
ability to promote dissociation of PSMA dimers using a method that
includes:
[0424] (a) contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound;
[0425] (b) measuring the amount of PSMA dimer; and
[0426] (c) comparing the amount of PSMA dimer measured in the
presence of the compound with that observed in the absence of the
compound.
[0427] A decrease in the amount of PSMA dimer measured in the
presence of the compound indicates that the compound is capable of
promoting dissociation of the PSMA dimer.
[0428] In a further embodiment, compounds are screened for the
ability to promote dissociation of PSMA dimers using a method that
includes:
[0429] (a) contacting a PSMA dimer with a compound under conditions
that do not promote dissociation of the PSMA dimer in the absence
of the compound;
[0430] (b) measuring the amounts of PSMA monomer and PSMA
dimer;
[0431] (c) calculating a ratio of PSMA monomer to PSMA dimer;
and
[0432] (d) comparing the ratio obtained in (c) with that obtained
in the absence of the compound.
[0433] An increase in the ratio measured in the presence of the
compound indicates that the compound is capable of promoting
dissociation of the PSMA dimer.
Example 18
Cell Surface PSMA Binding Studies
[0434] Flow Cytometry
[0435] Parent 3T3 cells or PSMA-expressing 3T3 cells
(2.times.10.sup.5 cells per condition) were washed in PBS and
incubated with PBS containing goat serum (10% v/v) for 20 minutes
on ice to block non-specific binding sites. Anti-PSMA monoclonal
antibodies (unpurified form in supernatants or purified mAbs) were
added in serial dilutions to cells in 100 .mu.l PBS and incubated
on ice for 30 minutes. Control anti-human IgG (Caltag, Burlingame,
Calif.) was used to establish background binding. After two washes
in PBS, the cells were incubated with anti-human IgG (BD
Pharmingen, San Diego, Calif.) for 30 minutes on ice. Cells were
washed twice in PBS, resuspended in 250 .mu.l PBS and analyzed by
flow cytometry using a FACScan machine (Becton Dickinson, Franklin
Lakes, N.J.) and CellQuest software. Viable cells were gated by
forward scatter and side scatter parameters, and binding was
quantified using histogram plots of mean fluorescence intensity
(MFI) levels.
[0436] Anti-PSMA mAbs XG-006 (PTA-4403 and PTA-4404, heavy and
light chain plasmids), XG-051 (PTA-4407 and PTA-4408), 4.40.1
(PTA-4360; 4.40, 4.40.1 and 4.40.2 are the same antibody that
represent different stages of subcloning the hybridoma), 4.49.1,
4.292.1 (PTA-4390) and 4.304.1 were found to avidly bind to cell
surface PSMA (FIG. 27).
[0437] Maximal Binding
[0438] Flow cytometry data (mean fluorescence intensity v. antibody
concentration) were transposed and plotted using Excel software
(Microsoft, Redmond, Wash.). Results from representative
experiments of at least three determinations are depicted in FIGS.
28A-28C. Binding was compared by calculation of 50% effective
concentration (EC50) using the Forecast function in Excel. The EC50
value represents the concentration of antibody required for
half-maximal binding.
[0439] Anti-PSMA mAbs 10.3 (PSMA 10.3) and XG-006 were found to
bind to 3T3-PSMA cells and not 3T3 cells (FIG. 28A). Antibody (26
nM) was added to cells, which were analyzed by flow cytometry.
Binding to cell-surface PSMA using serial dilutions of anti-PSMA
mAb-containing culture supernatants of XG-006, 4.304.1, XG-026
(PTA-4405 and PTA-4406) and 4.49.1 also was demonstrated (FIG.
28B). Binding to cell-surface PSMA using serial dilutions of
purified anti-PSMA mAbs XG-006 and 10.3 is represented by FIG.
28C.
Example 19
Cytotoxicity of Toxin-Labeled Antibody
[0440] PSMA-3T3, LNCaP, and/or C4-2 cells (and control cell lines
3T3 and PC3 that do not express PSMA) were plated at 2,500
cells/100 .mu.L/well in 96-well microplates (Falcon) and were
incubated overnight at 37.degree. C. in the presence of 5%
CO.sub.2. The media used for PSMA-3T3 (and 3T3) and LNCaP (and C4-2
and PC3) was DMEM or RMPI 1640, respectively, containing 2 mM
L-glutamine, 10% FBS, and 1% penicillin-streptomycin. 50 ng (in 50
.mu.L) of Mab-Zap or Hum-ZAP (Advanced Targeting Systems, San
Diego, Calif.) in medium was added in each well. Mab-Zap and
Hum-Zap are goat anti-mouse IgG antibody or goat anti-human IgG
antibody covalently linked to saporin, the most potent of the plant
ribosome-inactivating proteins (RIP) from the seeds of the plant
Saponaria officinalis. Saporin induces cell death by apoptosis
(Bergamaschi, G., Perfetti, V., Tonon, L., Novella, A., Lucotti,
C., Danova, M., Glennie, M.J., Merlini, G., Cazzola, M. Saporin, a
ribosome-inactivating protein used to prepare immunotoxins, induces
cell death via apoptosis. Br J Haematol 93, 789-94. (1996)). The
Mab-Zap did not bind to or internalize in cells in the absence of
an appropriate primary antibody.
[0441] Murine 3.9, 5.4, mJ591 (ATCC# HB-12126) and human 006, 4.40,
4.304 anti-PSMA antibodies (and control IgG antibodies) were added
into plates at different concentrations to bring the total volume
to 200 .mu.L in triplicate. The plates were kept cold on ice for at
least 30 min to maximize Map-Zap or Hum-Zap binding to PSMA
antibodies before internalization. The plates were incubated for 2
days and then the medium was changed and incubated for another 2
days. After 4 days incubation, the medium was withdrawn and fresh
medium containing 10% Alamar Blue (20 .mu.L, Bioscience, Camarillo,
Calif.) was added into each well and incubated for 2 hrs. A
CytoFlour plate reader was used to measure fluorescence in 96-well
plates at wavelengths of 530 nm excitation and 590 nm emission.
Internalization of toxin was mediated by anti-PSMA antibodies. The
cell kill is illustrated in FIG. 29 on C4-2 cells and in FIG. 30 on
PSMA-3T3 cells.
[0442] Human 4.304 anti-PSMA antibody was directly conjugated with
saporin (Wrenn et al., Brain Res. 740:175-184, 1996), and its
cytotoxicity was demonstrated using a similar protocol as described
above (see FIG. 31).
Example 20
Immunoreactivity
[0443] PSMA-3T3, LNCaP and C4-2 were used as PSMA expressing cell
lines and 3T3 was used as a control cell line not expressing PSMA.
The cells were blocked with 10% goat serum on ice to reduce
non-specific binding in this assay.
[0444] A small amount (1-5 ng) of labeled mAb was added into a cell
pellet of 10 million cells and incubated at 0.degree. C. (on ice)
with gentle mixing. After a 1 hour incubation, the cells were
collected by centrifugation and the supernatant containing unbound
mAb was transferred to a fresh cell pellet for an additional 1 hour
incubation at 0.degree. C. Both sets of cells were centrifuged and
washed twice with cold PBS. The cell pellets, supernatant and wash
fractions were counted for radioactivity. Immunoreactivity is
defined as the amount of radioactivity in the cell pellets divided
by the total radioactivity in the cell pellets, supernatant and
wash fractions. These data are shown below in Table 3.
3TABLE 3 Immunoreactivity of .sup.111In Radiolabeled Antibody on
PSMA Expressing Cells Radiolabeled mAb Immunoreactivity (%) Cell
line .sup.111In 4.304 92.6 (1.4) PSMA-3T3 (3T3) 92.6 PSMA-3T3 91.4
(1.7) PSMA-3T3 (3T3) 89.1 LNCaP 92.4 C4-2 Average= 91.6 .+-. 1.5
.sup.111In 4.40 87.7 (0.5) PSMA-3T3 (3T3) 86.8 PSMA-3T3 89.4 (1.5)
PSMA-3T3 (3T3) Average= 88.0 .+-. 1.3 .sup.111In mJ591 58.5
PSMA-3T3 54.9 (1.1) PSMA-3T3 (3T3) Average= 56.7 .+-. 2.5
.sup.111In 3.9 88 LNCaP 87 C4-2 89 (2) PSMA-3T3 (3T3) 95.3 (0.5)
PSMA-3T3 (3T3) 88.6 PSMA-3T3 84.8 C4-2 89.3 PSMA-3T3 Average= 88.6
.+-. 3.2
[0445] Antibodies 4.40, 4.304 and mJ591 were conjugated to the
bifunctional chelate CHX-A"-DTPA and antibody 3.9 was conjugated to
C-DOTA.
[0446] Immunoreactivity of .sup.225Ac radiolabeled antibody (026
and 4.40) was also assessed with a methodology similar to that
described above for the .sup.111In labeled antibodies. .sup.225Ac
was chelated with the bifunctional DOTA at 50.degree. C. for 30
minutes. The chelated .sup.225Ac was then conjugated to antibodies
026 and 4.40 at 35.degree. C. for 30 minutes. Unconjugated
.sup.225Ac was removed by a PD10 column (Amersham Biosciences,
Picataway, N.J.). The immunoreactivity of the radiolabeled
antibodies was then determined. The data are presented below in
Table 4. In addition to the assessment of the immunoreactivity of
these antibodies, the yield of the labeling procedure was also
assessed, and these data are also provided below in Table 4.
4TABLE 4 Yield and Immunoreactivity of .sup.225Ac Radiolabeled
Antibody Antibody Yield Immunoreactivity 026 9.3 +/- 0.8 (n = 2)
61.3 +/- 1.1 (n = 2) 4.40 14.3 +/- 0.6 (n = 2) 78.1 +/- 0.1 (n =
2)
Example 21
Competitive Binding Assay to Identify Binding Epitopes
[0447] To identify whether a given group of mAbs recognize distinct
or overlapping epitopes on PSMA, competition binding assays were
performed with .sup.111In radiolabeled antibodies. 2.times.10.sup.5
cells (100 .mu.L) of PSMA-3T3 were plated into 96-well microplates,
and antibodies 4.40, 4.304 and mJ591 (100 .mu.L) at different
concentrations (series dilution) were added. The cells were
incubated at 0.degree. C. for 30 min. 20 .mu.L of In-111
radiolabeled CHX-A"-DTPA antibody constructs were added into each
well. After a 2 hour incubation on ice for competition binding, the
cells were washed 5 times using cold PBS. The cells containing
bound .sup.111In antibodies were recovered from microplates into
test tubes and counted in a gamma counter.
[0448] Results detailed in FIG. 32 show that mJ591 blocked
.sup.111In 4.40 binding to PSMA-3T3 cells and did not block
.sup.111In 4.304. In addition, 4.40 and 4.304 did not block each
other. Unmodified antibodies 4.304 and mJ591 were also used to
compete with .sup.111In radiolabeled mJ591. Human 4.304 did not
compete with .sup.111In mJ591 for binding to PSMA-3T3 (FIG.
33).
Example 22
Binding Affinity Using Biacore 3000
[0449] To determine the kinetics and affinity of the antibodies,
the antibodies in crude supernatants, in purified form and in
bifunctional chelate modified forms were analyzed using a Biacore
3000 instrument (Biacore Inc., Piscataway, N.J.). Biacore 3000 is a
fully automated surface plasmon resonance (SPR)-based biosensor
system that is designed to provide real-time kinetic data from
assay formats that require no tags or labeling of compounds for
biomolecular interactions. It is ideal for screening crude
supernatants.
[0450] The streptavidin-coated sensor chips (SA chips, Biacore)
were used to capture biotinylated anti-human IgG antibody (Sigma,
St. Louis, Mo.). The entire sensor chip surface was conditioned
with five injections of conditioning solution (1 M NaCl, 50 mM
NaOH) and equilibrated with PBS buffer containing 0.005%
polysorbate 20. Two to three thousand resonance units (RU) of
biotinylated anti-human IgG antibody (Sigma) were immobilized onto
the SA chip followed by an injection of regeneration buffer
(glycine-HCl, pH 2.2). Antibodies in supernatants were diluted to 2
.mu.g/mL in PBS buffer and captured onto one anti-human IgG flow
cell, while isotype-matched control human antibody (Sigma) was
similarly captured on a second flow cell. rsPSMA at different
concentrations in PBS buffer was flowed over the cells at 30
.mu.L/min for 3 min in an "association phase" followed by a
"dissociation phase" for 10 min. SPR was monitored and displayed as
a function of time. For each antibody at one concentration, the
chip was regenerated and equilibrated. Examples of the analysis of
antibody PRGX1-XG-006 in association phase and dissociation phase
at different concentrations of rsPSMA from 100 nM to 6.25 nM are
shown in FIG. 34. Thermodynamic and kinetic rate constants of
binding were calculated using the Biacore Evaluation software. For
example, the affinity of XG-006 antibodies in a supernatant to
rsPSMA was determined to be 4.92.times.10.sup.-10 M with a K.sub.a
of 1.3.times.10.sup.5 M.sup.-1s.sup.-1 and a K.sub.d of
6.4.times.10.sup.-5 s.sup.-1. Selective data for several human PSMA
antibodies in crude supernatant, purified form, and modified with
bifunctional chelate is listed in Table 5 for comparison.
[0451] Binding activity of .sup.111In radiolabeled antibodies was
determined by Scatchard analysis of binding data obtained using
PSMA-expressing cells (LNCaP, C4-2, PSMA-3T3 and parental 3T3 as a
control). The experimental procedures and methods of data analysis
have been described previously (Scheinberg, D. A. et al. Leukemia
3: 440-445 (1991).
5TABLE 5 Kinetic Rate Constants of Antibodies in Crude Supernatant,
Purified, Bifunctional Chelate Modified Forms along with KD
Determined Using .sup.111In Radiolabeled Scatchard Analysis Ka
Antibodies (M.sup.-1, s.sup.-1) Kd (s.sup.-1) KD (M.sup.-1) Avg KD
006 Supernatant 1.30E+05 6.40E-05 4.92E-10 4.92E-10 Purified 006-1
2.94E+05 1.37E-04 4.66E-10 Purified 006-2 2.26E+05 1.27E-04
5.62E-10 5.14E-10 4.40 Supernatant 2.10E+05 1.25E-04 5.95E-10
5.95E-10 Purified 4.40-1 2.54E+05 1.52E-04 5.98E-10 Purified 4.40-2
2.43E+05 2.37E-04 9.75E-10 7.87E-10 CHX-4.40-1 2.57E+05 1.60E-04
6.23E-10 CHX-4.40-2 2.47E+05 1.55E-04 6.28E-10 6.25E-10
IN-111CHX-4.40-1 4.44E-09 IN-111CHX-4.40-2 4.95E-09 4.70E-09 4.304
Supernatant 1.40E+05 1.25E-04 8.93E-10 8.93E-10 Purified 4.304-1
8.31E+04 1.20E-04 1.44E-09 Purified 4.304-2 1.06E+05 6.33E-05
5.97E-10 1.02E-09 CHX-4.304-1 6.19E+04 1.21E-04 1.95E-09
CHX-4.304-2 6.79E+04 1.49E-04 2.19E-09 2.07E-09 IN-111CHX4.304-1-
9.63E-09 IN-111CHX-4.304-2 5.97E-09 7.80E-09 10.3 Supernatant
1.90E+05 3.63E-04 1.91E-09 1.91E-09 Purified 10.3-1 3.28E+05
6.32E-05 1.93E-10 Purified 10.3-2 2.96E+05 6.43E-05 2.17E-10
2.05E-10
[0452] A comparison of the fully human antibodies 4.40.1, 4.49.1,
051 and 006 and the murine antibody 3.9 was performed by Biacore.
For each antibody for comparison, response was normalized to 100
RU. The graph of time vs. response difference for these antibodies
is given in FIG. 35. The binding affinities for these antibodies
were determined to be 6.1, 6.7, 5.8, 4.8 and 13.7.times.10.sup.-10
M, respectively.
Example 23
Characterization of Cell Lines for In Vitro and In Vivo Studies
[0453] Results from a Scatchard analysis using .sup.111In labeled
anti-PSMA antibody 3.9 are represented in FIG. 36. Transfected
murine 3T3 cells express >1 million copies of PSMA per cell,
LNCAP cells (androgen dependent human prostate cancer cell line)
express 0.64 million copies, while C4-2 cells (androgen
independent) express 0.25 million copies per cell. The affinity of
3.9 for cell surface PSMA is 6.4 nM for PSMA-3T3, 4.0 nM for LNCAP
and 3.3 nM for C4-2 (4.6 nM is the average of these data).
[0454] A summary of the analyses of crude supernatants for the
human anti-PSMA antibodies is given in Table 6 below.
6TABLE 6 Characterization of Anti-PSMA Monoclonal Antibodies
Binding to 3T3- Ab Conc PSMA (FACS) Biacore studies (.mu.g/mL) AVG
Ka, Lysate PGNX Max AVG C4.2 Anti-PSMA KD, M - 1 M - 1s- 1 Kd, s -
1 Supernatant PGNX EIA FACS binding EC50 FACS Western
(.times.10.sup.-10) (.times.10.sup.5) (.times.10 - 5) PRGX1-XG1-
4.7 ND.sup.1 ND 148 2.4 ND Conf..sup.2 2.0 1.5 2.9 026 4.4.1 4.7
0.08 7 8 ND 5.2 Conf. 4.2 2.3 9.7 PRGX1-XG1- 1.8 0.39 114 183 3.4
9.5 Conf. 4.8 1.3 6.4 006 PRGX1-XG1- 3.5 0.48 83 202 2.0 9.9 Conf.
5.8 1.4 8.2 051 4.40.1 4.3 0.33 53 163 2.3 10.8 Conf. 6.1 2.1 12.5
4.49.1 2.6 0.36 362 162 0.9 16.2 Conf. 6.7 3.1 20.7 4.292.1 2.7
0.18 75 195 6.0 9.2 Conf. 6.8 1.2 8.5 4.304.1 4.1 0.39 92 184 9.1
8.4 Conf. 8.7 1.4 12.5 4.232.1 2.4 0.49 97 138 2.7 6.0 Linear.sup.3
9.4 1.5 13.8 4.153.1 5.9 0.29 279 182 5.3 14.8 Conf. 9.5 1.2 11.8
4.333.1 2.9 0.18 82 168 3.1 6.6 Conf. 11 0.7 8.5 PRGX1-XG1- 3.9
0.45 392 227 6.0 12.4 Conf. 16 0.6 10.4 077 10.3 8.5 1.06 ND ND ND
ND ND 19 1.9 36.4 pure 10.3 0.44 130 181 7.5 ND Conf. ND 4.7 4.22.1
2.8 0.08 7 ND ND 4.7 ND 20 1.7 33 4.248.1 3.5 0.37 7 ND ND 4.1
Conf. 27 1.0 28 4.54.1 10 0.14 267 162 3.9 13.6 ND 30 1.9 56 4.7.1
5 0.23 156 141 1.6 10.2 Conf. 32 1.7 56 4.78.1 5.3 0.00 205 118 1.0
7.9 Conf. 53 2.4 125 4.48.1 4.9 0.06 14 ND ND 7.7 ND 62 0.9 59
4.209.1 3.5 0.22 60 ND ND 6.7 ND 142 0.9 125 4.177.1 1.1 0.15 236
174 2.4 10.6 ND 155 0.6 93 4.152.1 3.4 0.38 81 85 4.0 7.5 ND 163
0.8 126 4.28.1 4.2 0.04 112 155 4.2 11.3 ND 167 1.2 192 4.16.1 5.3
0.00 8 ND ND 7.8 ND 177 1.8 313 4.360.1 1.5 0.02 112 130 2.2 7.9 ND
197 1.0 201 4.288.1 15.4 0.02 67 141 4.1 6.5 ND 198 1.3 257 4.219.2
0.5 0.34 69 ND ND 5.9 ND ND PRGX1-XG1- 6.5 ND ND 71 7.9 ND ND No
Binding 069 Murine 3.9 13.7 0.7 9.7 Control 6.34 2.24 14.2 .sup.1ND
= not determined .sup.2conf. = conformational epitope .sup.3linear
= linear epitope
Example 24
Cytotoxicity of Radiolabeled Antibody
[0455] The in vitro cytotoxicity of .sup.225Ac labeled anti-PSMA
antibody (4.40 and 026) was determined using methodology similar to
that used in Example 19. Prostate cancer cells (100 .mu.L of C4-2,
LNCaP, and PC3 cells at a concentration of 2.times.10.sup.4
cells/mL) were placed into separate wells of a 96 well microplate.
For tests with the 026 antibody, C4-2 and PC3 cells were placed
into separate wells of a 96 well microplate. After overnight
incubation, the cells were treated with .sup.225Ac labeled human
anti-PSMA antibody at different concentrations for over 4 days.
Cell cytotoxicity was quantified using Alamar Blue (Biosource
International, Camarillo, Calif.).
[0456] FIG. 37 shows a plot of cell survival vs. .sup.225Ac
activity concentration using .sup.225Ac labeled 4.40 antibody. The
EC50 for PSMA expressing cells (C4-2 and LNCaP) was <2 nCi/mL.
However, the EC50 was 420 nCi/mL for PC3 cells, which do not
express PSMA on the cell surface. Therefore, the .sup.225Ac labeled
human anti-PSMA 4.40 antibody shows >200-fold selectivity in
killing PSMA expressing prostate cancer cells (C4-2 and LNCaP) vs.
control cells (PC3).
[0457] FIG. 38 shows a plot of cell survival vs. .sup.225Ac
activity concentration using .sup.225Ac labeled 026 antibody. The
.sup.225Ac labeled human anti-PSMA 026 antibody shows >50-fold
selectivity in killing PSMA expressing prostate cancer cells (C4-2)
vs. control cells (PC3).
Example 25
Cytotoxicity of .sup.225Ac Labeled Antibody vs. Control
Antibody
[0458] The in vitro cytotoxicity of .sup.225Ac labeled anti-PSMA
antibody was determined using methodology similar to that used in
Example 19 and Example 24 above. Human prostate cancer cells (100
.mu.L of C4-2 and LNCaP cells at a concentration of
2.times.10.sup.4 cells/mL) were placed into separate wells of a 96
well microplate. After overnight incubation, the cells were treated
with .sup.225Ac labeled human anti-PSMA 026 antibody at different
concentrations for 4 days. Cell cytotoxicity was quantified using
Alamar Blue (Biosource International, Camarillo, Calif.). Human IgG
(HuIgG) was used as a control. The cytotoxicity of an anti-PSMA mAb
026 "2 hour wash" was also determined. A 2 hour wash means that the
cells were incubated with .sup.225Ac labeled antibody for 2 hours.
After 2 hours, the media was removed and fresh media was added for
the 4 day incubation.
[0459] FIG. 39 shows a plot of cell survival vs. the .sup.225Ac
activity concentration for both C4-2 and LNCaP cells using
radiolabeled mAb 026, mAb 026 2 hour wash and HuIgG. .sup.225Ac
labeled mAb 026 showed an IC50 of <1 nCi/mL. Therefore, the
.sup.225Ac labeled human anti-PSMA 026 antibody showed >50-fold
selectivity in killing the prostate cancer cells vs. the control
antibody.
Example 26
Cytotoxicity of .sup.225Ac Labeled Antibody vs. Control Antibody
Evaluated by .sup.3H Thymidine Incorporation
[0460] Human prostate cancer cells (C4-2) in a 96 microplate were
treated with .sup.225Ac labeled mAbs at different concentrations
for 4 days. Cell survival was assessed using .sup.3H thymidine
incorporation (Nikula, T. K, et al. J. Nucl. Med. 40: 166-176,
1999).
[0461] FIG. 40 shows a plot of cell survival vs. the .sup.225Ac
activity concentration for C4-2 cells using radiolabeled mAb 026
and control mAb (HuM195). The IC50 was 0.12 nCi/mL using 225Ac
labeled 026 vs. 13 nCi/ml with the control mAb (HuM195). The
radiolabeled 026 antibody, therefore, showed >100-fold
selectivity in killing the PSMA expressing C4-2 cells vs. the
control antibody.
Example 27
In Vivo Radioimmunotherapy with .sup.177Lu Labeled Antibodies
[0462] Athymic nude mice from the National Cancer Institute were
implanted subcutaneously with 2.times.10.sup.6 PSMA-3T3 cells.
After measurable tumors appeared at day 7 post implantation, the
mice were treated by injection with either a single 250 .mu.Ci dose
human anti-PSMA antibody 4.40 or 4.304 labeled with .sup.177Lu
(University of Missouri Research Reactor), or were injected with
buffer only as control. The tumor size of individual animals was
measured using an electronic caliper. FIG. 41 shows a plot of the
median tumor size in each group over time. Tumor growths were
substantially reduced in .sup.177Lu antibody treated groups
compared to the control group.
Example 28
In Vivo Biodistribution Study with .sup.177Lu Labeled
Antibodies
[0463] Athymic nude mice from the National Cancer Institute (male,
approximately 6 weeks old) were injected subcutaneously with
4.times.10.sup.6 PSMA-3T3 cells and 2.8.times.10.sup.6 3T3 cells in
0.2 mL in the right and left flank of each animal, respectively.
Anti-PSMA antibodies 006, 026, mJ591 and HuIgG (control) modified
with CHX-A"-DTPA were labeled with .sup.177Lu. FIG. 42 shows the
radio-HPLC profile of the radiolabeled antibodies as well as the
cell-based immunoreactivity performed as quality control. On day 6
after tumor implantation, .sup.177Lu labeled antibodies (10 .mu.Ci
and 1 .mu.g in 0.15 mL) were injected retro-orbitally. The animals
were randomized before antibody injection. Mice (30 per antibody, 5
per time point) were sacrificed at different times (days 0.17, 1,
2, 4, 7 and 12). Tumors and individual organs (PSMA+ tumor, PSMA-
tumor, blood, liver, kidneys, spleen, lungs, heart, bone, muscle,
carcass) were taken and weighed. Activity in each organ along with
standards prepared from injection solutions were counted using a
multi-channel gamma counter.
[0464] Results of this study show that .sup.177Lu labeled
antibodies specifically bound to tumors expressing PSMA in vivo in
the animal model. The percent injected dose per gram of tissue (%
ID/g) was calculated and plotted over time for the different
antibodies in the PSMA+ and PSMA- tumors (FIG. 43A). PSMA specific
tumor targeting (ratio of PSMA+/PSMA- tumor uptake) is provided in
FIG. 43B.
[0465] FIG. 44 shows the percent activity in the tumors with the
various radiolabeled antibodies (006, 026, mJ591 and HuIgG) over
time (% tumor retention vs. total body retention). The data again
illustrate the specificity by which the radiolabeled antibodies
target the PSMA expressing tumors. FIG. 44A shows the activity over
time in the PSMA+ tumors while FIG. 44B shows the percent activity
over time in the PSMA- tumors for the different antibodies. FIG. 45
shows the data for normal organ (blood, liver, kidneys, spleen,
lungs, bone, heart and muscle) uptake (% ID/g) plotted over
time.
Example 29
In Vivo Therapeutic Efficacy of .sup.177Lu Radiolabeled
Antibodies
[0466] Athymic nude mice from the National Cancer Institute (male,
approximately 6 weeks old) were injected subcutaneously with
4.times.10.sup.6 PSMA-3T3 cells and 2.8.times.10.sup.6 3T3 cells in
0.2 mL in the right and left flank of each animal, respectively.
.sup.177Lu labeled mAb 026 (0 .mu.Ci, n=5; 300 .mu.Ci and 10 .mu.g,
n=9; and 400 .mu.Ci and 13.5 .mu.g, n=5) were injected into the
mice on day 6 after tumor implantation. Animals were weighed and
tumors were measured over time. Tumor size (mm.sup.3) was
calculated using the formula: length.times.(width).sup.2/2. Mice
were sacrificed if tumor size reached 1000 mm.sup.3. Animal
survival was also assessed, and the Kaplan-Meier plot was
created.
[0467] The results of the study show that treatment decreased tumor
size and increased survival in the mice. FIG. 46A shows the tumor
size in the mice treated with the radiolabeled antibodies (177Lu
labeled mAb 026) at all three dose levels. The mice treated with
300 .mu.Ci and 400 .mu.Ci had consistently smaller tumors than the
mice in the control group (0 Cci). FIG. 46B shows that the mice
treated with 300 .mu.Ci and 400 .mu.Ci had increased survival
relative to the control mice. Median survival was increased by
2.4-fold in mice treated with 300 .mu.Ci and 3.5-fold in mice
treated with 400 .mu.Ci using time after treatment. Treatment with
400 .mu.Ci was found to be non-toxic. Additionally, at the end of
the experiment (48 days after tumor implantation), one animal from
each treated group remained PSMA-3T3 tumor free but had large 3T3
tumors.
Example 30
Binding of Antibodies to rsPSMA Dimer and Monomer
[0468] A Biacore 3000 instrument was used to monitor, in real time,
binding of rsPSMA dimer and monomer to anti-PSMA mAbs. Antibodies
were immobilized at approximately 10,000 resonance units to CM5
sensor chips according to the manufacturer's instructions for amine
coupling (Biacore, Inc., Piscataway, N.J.). A reference surface of
isotype-matched antibody of irrelevant specificity was used as a
background control. Binding experiments were performed at
25.degree. C. in PBS buffer with 0.005% [vol/vol] Surfactant P20.
Purified rsPSMA dimer (50 nM) or monomer (100 nM) was passed over
control and test flow cells at a flow rate of 5 .mu.L/min. The
sensor surface was regenerated with two pulses of 20 nM HCl.
[0469] FIGS. 47 and 48, respectively, show that anti-PSMA mAbs 006
and 026 bind preferentially to the rsPSMA dimer rather than the
rsPSMA monomer. Anti-PSMA antibodies 4.40 and mJ591, however, were
shown to bind both the rsPSMA dimer and monomer at significant
levels (FIGS. 49 and 50, respectively). This study illustrates that
anti-PSMA mAbs 006 and 026 are PSMA dimer-specific antibodies and
bind dimer-specific epitopes on PSMA. The results also indicate
that the native conformation of PSMA is a homodimer, and that the
monomer possesses a partially denatured conformation or exposes
epitopes located at the dimer surface and/or dimer interface that
are not accessible in the dimer.
Example 31
Immunization with rsPSMA Dimer Preparations
[0470] Immunization
[0471] BALB/c mice were immunized by subcutaneous injection at days
0, 7, 14, and 42 with either 5 .mu.g clinical rsPSMA lot #
4019-C001 (75% dimer/25% monomer) or 5 .mu.g rsPSMA batch #
TD045-003 run 1/peak 2 (100% monomer) on alum (250 .mu.g per dose,
Sigma) or adjuvanted with 50 .mu.g alhydrogel per dose. Serum was
drawn 10 days after the fourth immunization and analyzed by
enzyme-linked immunoassay (EIA) and flow cytometry.
[0472] EIA
[0473] rsPSMA lot # 4019-C001 or rsPSMA batch # TD045-003 run
1/peak 2 was passively adsorbed to 96-well microtiter plates.
Remaining binding sites on the plate were blocked with a
PBS/Casein/Tween 20 buffer. Serially diluted mouse serum or
controls were added and bound antibody was detected using a goat
anti-mouse IgG antibody conjugated to alkaline phosphatase. The EIA
was developed with the substrate pNPP which produces a color change
that is directly proportional to the amount of anti-PSMA antibody
bound. Absorbance was read at 405 nm with a correction of 620 nm.
Antibody titer was defined as the highest dilution of mouse serum
yielding a blank corrected absorbance of 0.1. Immune mouse serum
with a known anti-PSMA titer or normal mouse serum with no
anti-PSMA reactivity was used as controls.
[0474] Flow Cytometry Analysis
[0475] PSMA-3T3 cells were incubated with 200 .mu.L of immune serum
at a dilution of 1/50 in PBS with 0.1% sodium azide on ice for 30
minutes. Immune mouse serum with known anti-PSMA titer or normal
mouse serum with no anti-PSMA reactivity was used as controls. The
cells were washed twice with PBS with 0.1% sodium azide and
incubated for 30 minutes on ice with FITC-conjugated goat
anti-mouse IgG. Cells were washed once, resuspended in PBS with
0.1% sodium azide and subjected to flow cytometric analysis on
FACScaliber (Becton Dickinson).
[0476] Results
[0477] 5/5 mice immunized with rsPSMA lot # 4019-C001 showed an
anti-PSMA antibody response by EIA. Antibody titer was similar for
assay plates coated with rsPSMA lot # 4019-C001 (75% dimer/25%
monomer) and assay plates coated with rsPSMA batch # TD045-003 run
1/peak 2 (100% monomer). Median response for the group was
1/6400.
[0478] 4/5 mice immunized with rsPSMA batch # TD045-003 run 1/peak
2 showed an anti-PSMA antibody response by EIA. One mouse was
negative. Antibody titer was similar for assay plates coated with
rsPSMA lot # 4019-C001 (75% dimer/25% monomer) and assay plates
coated with rsPSMA batch # TD045-003 run 1/peak 2 (100% monomer).
Median response for the group was 1/6400.
[0479] The results of the EIA analysis are provided in Table 7.
[0480] The results of the flow cytometry analysis are provided in
FIG. 51.
7TABLE 7 Specificity of the Anti-PSMA Antibody Response in Mice
Vaccinated 4 Times with rsPSMA 5 .mu.g/dose and 50 .mu.g/dose
Alhydrogel EIA Titer EIA Titer vs. Median RFI Mouse vs. Lot Batch
TD045- vs. PSMA- ID # Immunogen 4019-C001 003 run1/peak 2 3T3 cells
ABIM151 4019-C001 1/3200 1/3200 84 Dimer ABIM152 4019-C001 1/3200
1/3200 41 Dimer ABIM153 4019-C001 1/25600 1/25600 76 Dimer ABIM154
4019-C001 1/12800 1/12800 63 Dimer ABIM155 4019-C001 1/6400 1/6400
74 Dimer ABIM156 Monomer 1/1600 1/1600 5 ABIM157 Monomer 1/6400
1/12800 8 ABIM158 Monomer 0 0 6 ABIM159 Monomer 1/6400 1/6400 6
ABIM160 Monomer 1/6400 1/6400 12
[0481] When tested by ELISA, sera from both monomer and dimer
immunized animals showed similar levels of anti-PSMA antibodies,
indicating that each protein was immunogenic when formulated on
alum. For dimer immunized animals, the median endpoint titers were
1/6,400 (range 1/3,200 to 1/12,800) regardless of whether rsPSMA
monomer or dimer was used as the coating antigen. Similarly,
monomer-immunized animals had median endpoint titers of 1/6,400 in
both assay formats, although the range varied depending on whether
the monomer (range <1/400 to 1/12,800) or dimer (range <1/400
to 1/6,400) was used for coating.
[0482] However, a difference between sera was observed with
cell-based flow cytometry (FIG. 51). Anti-PSMA antibody in the
serum of mice immunized with a dimer preparation of rsPSMA (lot #
4019-C001) showed strong binding to PSMA-3T3 cells. Anti-PSMA
antibody in the serum of mice immunized with a 100% monomer
preparation of rsPSMA (batch # TD045-003 run 1/peak 2) showed no
binding to PSMA-3T3 cells.
[0483] Each dimer immunized animal elicited high-titered antibodies
to PSMA-3T3 cells (median mean fluorescence intensity (MFI)=74,
range 41-84), but such antibodies were very weak to absent in
monomer-immunized animals (median MFI=6, range 5-12). The level of
binding observed for monomer immunized animals was comparable to
that for nave animals. Similar background levels of binding to
parental 3T3 cells were observed for all sera (median MFI=6 in all
cases).
[0484] An identical pattern of reactivity was observed with human
prostate cancer cell lines. Consistent, high-level reactivity with
PSMA-expressing C4-2 cells was observed for sera from dimer
immunized animals (median MFI=28.0, range 23.1-28.8) but not
monomer immunized (median MFI=12.8, range 11.2-14.5) or control
animals (median MFI=12.3, range 8.6-16.0). Background levels of
binding to PSMA-negative PC-3 cells were observed for all sera
(median MFI=7 in all cases).
[0485] Thus, while it is possible to elicit the production of
antibodies that recognize native PSMA using monomeric forms of the
PSMA protein or fragments thereof, these results speak to the
relative efficiency of eliciting an immune response to native PSMA
using dimeric forms of PSMA protein. Additionally, flow cytometry
but not ELISA was able to reveal the differences in the humoral
immune responses elicited by monomeric and dimeric forms of PSMA.
The inability of the ELISA to uncover such differences suggests
that rsPSMA adopts a partially denatured conformation upon
adsorption to plastic.
Example 32
mAbs 006 and 026 Mediate Efficient ADCC of Human Prostate Cancer
Cells
[0486] .sup.51Cr labeled C4-2 cells (1.times.10.sup.4/well, target
cells) were incubated in triplicates with 10 .mu.g/mL mAb at
4.degree. C. for 1 hour. Fresh human PBMCs (effector cells) were
added to washed target cells at effector to target (E/T) ratios of
40:1, 20:1, and 10:1 and incubated at 37.degree. C. overnight.
.sup.51Cr in harvested supernatants was measured using a
.gamma.-scintillation counter and % cell lysis was calculated. mAbs
006 and 026 demonstrated statistically significant antibody
dependent cell-mediated cytotoxicity (ADCC) of C4-2 cells compared
to isotype matched human IgG1 mAb control (FIG. 52). No effect was
observed when PSMA-negative human prostate tumor cells (PC-3) were
used.
Example 33
Monomer-Dimer Equilibrium
[0487] Purified dimeric and monomeric forms of rsPSMA were resolved
by preparative size exclusion chromatography (SEC) in PBS+ buffer
and collected in separate fractions. To assess whether dimer and
monomer exist in a reversible equilibrium, the buffer conditions
were perturbed, and the monomer-dimer ratio was analyzed by SEC. As
indicated in FIG. 53A, a dimer preparation that contained
approximately 5% monomer initially was converted to 100% dimer upon
incubation for 72 h at ambient temperature in PBS+ supplemented
with 2M sodium chloride (FIG. 53A). Conversely, the addition of 2
mM of the metal-chelating agent EDTA converted the dimer into
monomer with a half-life of approximately 2 days (FIG. 53A),
indicating that dimer stability is dependent upon the presence of
metal ions, such as Zn.sup.2+ in the active site of PSMA.
[0488] For a preparation that initially comprised >95% monomer,
high salt similarly drove the equilibrium to mostly (81%) dimer
within 72 h (FIG. 53B). EDTA had little influence on the oligomeric
state of the monomer. Thus, regardless of the initial oligomeric
state of the protein, high salt concentrations promoted
dimerization, whereas metal-chelating agents dissociated dimers
into monomers.
[0489] PSMA shares modest sequence and structural homology with
human transferrin receptor (TfR), which contains a vestigial
catalytic domain but lacks enzymatic activity. TfR is expressed as
a type II membrane protein that forms a disulfide-linked homodimer,
but the intermolecular disulfides are not required for dimerization
(Alvarez, E., et al. (1989) EMBO J. 8, 2231-2240.0). The
high-resolution crystal structure of the TfR ectodomain reveals
that the protein is organized into three distinct domains known as
the protease-like, apical, and helical domains, with the last
domain being principally responsible for dimerization (Lawrence, C.
M., et al. (1999) Science 286, 779-782). PSMA and TfR share 30%,
30%, and 24% sequence identity within these domains, respectively.
The helical dimerization domain of PSMA are amino acids 601-750 of
SEQ ID NO: 1.
Example 34
rsPSMA Formulation Studies
[0490] pH Stability of rsPSMA
[0491] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into a
broad-range base buffer solution (2 mM glycine, 2 mM citric acid, 2
mM Hepes, 2 mM MES, 2 mM Tris Base) that was adjusted to cover pH 4
to pH 8.5 in steps of 0.5 pH units. Following incubation for 4 days
at 45.degree. C., the individual samples were subjected to
analytical TSK gel filtration chromatography (run at pH 7.5) and
analyzed for protein recovery and the preservation of the dimeric
structure of rsPSMA. The findings are summarized in Table 8.
8TABLE 8 Recovery and Structure of rsPSMA at Various pHs Dimer
Monomer Aggregate Recovery from pH Content.sup.1 Content.sup.1
Content.sup.1 column.sup.2 4.0 +++++ - - + 4.5 - - - - 5.0 ++ - +++
++ 5.5 ++++ + - +++ 6.0 +++++ - - +++++ 6.5 ++++ + - ++++ 7.0 ++++
+ - ++++ 7.5 ++++ + - + 8.0 +++ + + + 8.5 - - - - .sup.1of
recovered protein .sup.2of total protein at t = 0 - <5% + 5%-25%
++ 25-50% +++ 50-75% ++++ 75-95% +++++ >95%
[0492] Base Buffer Evaluation
[0493] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
the following buffer solutions:
[0494] PBS+
[0495] 20 mM Hepes, pH 7.0
[0496] 20 mM sodium phosphate+150 mM NaCl, pH 6.5
[0497] 20 mM histidine+150 mM NaCl, pH 6.0
[0498] 20 mM sodium phosphate+150 mM NaCl, pH 6.0
[0499] 20 mM sodium acetate+150 mM NaCl, pH 6.0
[0500] 20 mM sodium citrate+150 mM NaCl, pH 6.0
[0501] Each sample was incubated for 3 or 4 days at 45.degree. C.
and subsequently analyzed by analytical TSK gel filtration
chromatography for protein recovery and the preservation of the
dimeric structure of rsPSMA. The findings are summarized in Table
9.
9TABLE 9 Recovery and Structure of rsPSMA with Various Buffers Base
Dimer Monomer Aggregate Recovery from Buffer Content.sup.1
Content.sup.1 Content.sup.1 column.sup.2 PBS+ +++ - + ++ Phosphate
+++++ - - +++++ Acetate +++++ - - +++++ Citrate N/A N/A N/A -
Histidine +++ + ++ + .sup.1of recovered protein .sup.2of total
protein at t = 0 - <5% + 5%-25% ++ 25-50% +++ 50-75% ++++ 75-95%
+++++ >95%
[0502] Excipients
[0503] Dimeric rsPSMA (2 mg/ml in PBS+) was dialyzed over night
into 20 mM sodium acetate, pH 6.0 and 150 mM NaCl. To evaluate the
effect of the individual amino acids, the protein was diluted
8-fold into 20 mM sodium acetate, pH 6.0 and 150 mM NaCl containing
50 mM of either glycine, histidine, proline, isoleucine, leucine,
alanine, lysine, arginine, threonine, glutamic acid, or aspartic
acid as excipients. Following incubation for 5 days at 45.degree.
C., each sample was analyzed by analytical TSK gel filtration
chromatography for protein recovery and the preservation of the
dimeric structure of the protein. The findings are summarized in
Table 10.
10TABLE 10 Recovery and Structure of rsPSMA with Various Amino
Acids Dimer Monomer Aggregate Recovery from Amino Acid
Content.sup.1 Content.sup.1 Content.sup.1 column.sup.2 Glycine ++++
+ - ++++ Histidine N/A N/A N/A + Proline ++++ + - ++++ Isoleucine
++++ + - ++++ Leucine ++++ + - ++++ Alanine ++++ + - ++++ Arginine
++++ + - ++++ Threonine ++ N/A +++ ++++ Glutamic Acid - - +++++
++++ Aspartic Acid - - +++++ +++ .sup.1of recovered protein
.sup.2of total protein at t = 0 - <5% + 5%-25% ++ 25-50% +++
50-75% ++++ 75-95% +++++ >95%
[0504] Surfactants
[0505] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
PBS+containing 0.5% (w/v) of either Triton X-100, dodecylmaltoside,
cholic acid, or CHAPS and incubated for 4 days at 4.degree. C. Each
sample was subsequently analyzed by analytical TSK gel filtration
chromatography for protein recovery and the preservation of the
dimeric structure of the protein. The findings are summarized in
Table 11.
11TABLE 11 Recovery and Structure of rsPSMA with Various
Surfactants Dimer Monomer Aggregate Recovery from Surfactant
Content.sup.1 Content.sup.1 Content.sup.1 column.sup.2 Triton X-100
++++ + + ++++ Dodecylmaltoside ++++ - + +++++ Cholic Acid ++++ - +
+++++ CHAPS ++++ + - +++++ .sup.1of recovered protein .sup.2of
total protein at t = 0 - <5% + 5%-25% ++ 25-50% +++ 50-75% ++++
75-95% +++++ >95%
[0506] Other Excipients
[0507] Dimeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
PBS+containing either 1.4 M (35% saturation) ammonium sulfate, 5 mM
EDTA, 1 mM DTT, or 10% glycerol and incubated for 4 days at
4.degree. C. Each sample was subsequently analyzed by analytical
TSK gel filtration chromatography for protein recovery and the
preservation of the dimeric structure of the protein. The findings
are summarized in Table 12.
12TABLE 12 Recovery and Structure of rsPSMA with Various Excipients
Dimer Monomer Aggregate Recovery Excipient Content.sup.1
Content.sup.1 Content.sup.1 from column.sup.2 Ammonium Sulfate ++++
- + +++++ EDTA ++ ++++ - +++++ DTT ++++ + - +++++ Glycerol ++++ + -
+++++ .sup.1of recovered protein .sup.2of total protein at t = 0 -
<5% + 5%-25% ++ 25-50% +++ 50-75% ++++ 75-95% +++++ >95%
[0508] Conversion of Monomers into Dimers
[0509] To evaluate the potential of reversing monomeric rsPSMA into
dimers, monomeric rsPSMA (2 mg/ml in PBS+) was diluted 10-fold into
PBS+containing either 1.4 M (35% saturation) ammonium sulfate, 2 M
NaCl, 1 mM DTT, 5 mM EDTA, or 10% glycerol and incubated for up to
4 days at 4.degree. C. Each sample was subsequently analyzed by
analytical TSK gel filtration chromatography for protein recovery
and the formation of the dimeric structure of the protein. The
findings are summarized in Table 13.
13TABLE 13 Conversion of rsPSMA Monomers Dimer Monomer Aggregate
Recovery from Excipient Content.sup.1 Content.sup.1 Content.sup.1
column.sup.2 Ammonium Sulfate ++ +++ + +++++ NaCl +++ ++ + +++++
DTT + ++++ - +++++ EDTA - +++++ - +++++ Glycerol + ++++ - +++++
.sup.1of recovered protein .sup.2of total protein at t = 0 - <5%
+ 5%-25% ++ 25-50% +++ 50-75% ++++ 75-95% +++++ >95%
[0510] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose and variations can be made by those skilled
in the art without departing from the spirit and scope of the
invention which is defined by the following claims.
[0511] The contents of all references, patents and published patent
applications cited throughout this application are incorporated
herein by reference.
Sequence CWU 1
1
33 1 750 PRT Homo sapiens 1 Met Trp Asn Leu Leu His Glu Thr Asp Ser
Ala Val Ala Thr Ala Arg 1 5 10 15 Arg Pro Arg Trp Leu Cys Ala Gly
Ala Leu Val Leu Ala Gly Gly Phe 20 25 30 Phe Leu Leu Gly Phe Leu
Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45 Ala Thr Asn Ile
Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55 60 Leu Lys
Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile 65 70 75 80
Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile 85
90 95 Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala
His 100 105 110 Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro
Asn Tyr Ile 115 120 125 Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe
Asn Thr Ser Leu Phe 130 135 140 Glu Pro Pro Pro Pro Gly Tyr Glu Asn
Val Ser Asp Ile Val Pro Pro 145 150 155 160 Phe Ser Ala Phe Ser Pro
Gln Gly Met Pro Glu Gly Asp Leu Val Tyr 165 170 175 Val Asn Tyr Ala
Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 180 185 190 Lys Ile
Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val 195 200 205
Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly 210
215 220 Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val
Lys 225 230 235 240 Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly
Val Gln Arg Gly 245 250 255 Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp
Pro Leu Thr Pro Gly Tyr 260 265 270 Pro Ala Asn Glu Tyr Ala Tyr Arg
Arg Gly Ile Ala Glu Ala Val Gly 275 280 285 Leu Pro Ser Ile Pro Val
His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys 290 295 300 Leu Leu Glu Lys
Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg 305 310 315 320 Gly
Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn 325 330
335 Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val
340 345 350 Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val
Glu Pro 355 360 365 Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser
Trp Val Phe Gly 370 375 380 Gly Ile Asp Pro Gln Ser Gly Ala Ala Val
Val His Glu Ile Val Arg 385 390 395 400 Ser Phe Gly Thr Leu Lys Lys
Glu Gly Trp Arg Pro Arg Arg Thr Ile 405 410 415 Leu Phe Ala Ser Trp
Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr 420 425 430 Glu Trp Ala
Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala 435 440 445 Tyr
Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 450 455
460 Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu
465 470 475 480 Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu
Tyr Glu Ser 485 490 495 Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser
Gly Met Pro Arg Ile 500 505 510 Ser Lys Leu Gly Ser Gly Asn Asp Phe
Glu Val Phe Phe Gln Arg Leu 515 520 525 Gly Ile Ala Ser Gly Arg Ala
Arg Tyr Thr Lys Asn Trp Glu Thr Asn 530 535 540 Lys Phe Ser Gly Tyr
Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu 545 550 555 560 Leu Val
Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val 565 570 575
Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val 580
585 590 Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr
Ala 595 600 605 Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu
Met Lys Thr 610 615 620 Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala
Val Lys Asn Phe Thr 625 630 635 640 Glu Ile Ala Ser Lys Phe Ser Glu
Arg Leu Gln Asp Phe Asp Lys Ser 645 650 655 Asn Pro Ile Val Leu Arg
Met Met Asn Asp Gln Leu Met Phe Leu Glu 660 665 670 Arg Ala Phe Ile
Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg 675 680 685 His Val
Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser 690 695 700
Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp 705
710 715 720 Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val
Ala Ala 725 730 735 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu
Val Ala 740 745 750 2 7570 DNA Artificial Sequence Plasmid 2
gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg
atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag
ggagacccaa gctggctaga 900 ggtaccaagc ttggatctca ccatggagtt
gggactgcgc tggggcttcc tcgttgctct 960 tttaagaggt gtccagtgtc
aggtgcaatt ggtggagtct gggggaggcg tggtccagcc 1020 tgggaggtcc
ctgagactct cctgtgcagc gtctggattc gccttcagta gatatggcat 1080
gcactgggtc cgccaggctc caggcaaggg gctggagtgg gtggcagtta tatggtatga
1140 tggaagtaat aaatactatg cagactccgt gaagggccga ttcaccatct
ccagagacaa 1200 ttccaagaac acgcagtatc tgcaaatgaa cagcctgaga
gccgaggaca cggctgtgta 1260 ttactgtgcg agaggcggtg acttcctcta
ctactactat tacggtatgg acgtctgggg 1320 ccaagggacc acggtcaccg
tctcctcagc ctccaccaag ggcccatcgg tcttccccct 1380 ggcaccctct
agcaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga 1440
ctacttcccc gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca
1500 caccttcccg gctgtcctac agtcctcagg actctactcc ctcagcagcg
tggtgaccgt 1560 gccctccagc agcttgggca cccagaccta catctgcaac
gtgaatcaca agcccagcaa 1620 caccaaggtg gacaagagag ttggtgagag
gccagcacag ggagggaggg tgtctgctgg 1680 aagccaggct cagcgctcct
gcctggacgc atcccggcta tgcagtccca gtccagggca 1740 gcaaggcagg
ccccgtctgc ctcttcaccc ggaggcctct gcccgcccca ctcatgctca 1800
gggagagggt cttctggctt tttccccagg ctctgggcag gcacaggcta ggtgccccta
1860 acccaggccc tgcacacaaa ggggcaggtg ctgggctcag acctgccaag
agccatatcc 1920 gggaggaccc tgcccctgac ctaagcccac cccaaaggcc
aaactctcca ctccctcagc 1980 tcggacacct tctctcctcc cagattccag
taactcccaa tcttctctct gcagagccca 2040 aatcttgtga caaaactcac
acatgcccac cgtgcccagg taagccagcc caggcctcgc 2100 cctccagctc
aaggcgggac aggtgcccta gagtagcctg catccaggga caggccccag 2160
ccgggtgctg acacgtccac ctccatctct tcctcagcac ctgaactcct ggggggaccg
2220 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 2280 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
aggtcaagtt caactggtac 2340 gtggacggcg tggaggtgca taatgccaag
acaaagccgc gggaggagca gtacaacagc 2400 acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 2460 tacaagtgca
aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 2520
gccaaaggtg ggacccgtgg ggtgcgaggg ccacatggac agaggccggc tcggcccacc
2580 ctctgccctg agagtgaccg ctgtaccaac ctctgtccct acagggcagc
cccgagaacc 2640 acaggtgtac accctgcccc catcccggga ggagatgacc
aagaaccagg tcagcctgac 2700 ctgcctggtc aaaggcttct atcccagcga
catcgccgtg gagtgggaga gcaatgggca 2760 gccggagaac aactacaaga
ccacgcctcc cgtgctggac tccgacggct ccttcttcct 2820 ctatagcaag
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc 2880
cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg
2940 taaatgagaa ttcctcgagt ctagagggcc cgtttaaacc cgctgatcag
cctcgactgt 3000 gccttctagt tgccagccat ctgttgtttg cccctccccc
gtgccttcct tgaccctgga 3060 aggtgccact cccactgtcc tttcctaata
aaatgaggaa attgcatcgc attgtctgag 3120 taggtgtcat tctattctgg
ggggtggggt ggggcaggac agcaaggggg aggattggga 3180 agacaatagc
aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 3240
cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
3300 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
ccgctccttt 3360 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
ccccgtcaag ctctaaatcg 3420 gggcatccct ttagggttcc gatttagtgc
tttacggcac ctcgacccca aaaaacttga 3480 ttagggtgat ggttcacgta
gtgggccatc gccctgatag acggtttttc gccctttgac 3540 gttggagtcc
acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc 3600
tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa
3660 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt
gtgtcagtta 3720 gggtgtggaa agtccccagg ctccccaggc aggcagaagt
atgcaaagca tgcatctcaa 3780 ttagtcagca accaggtgtg gaaagtcccc
aggctcccca gcaggcagaa gtatgcaaag 3840 catgcatctc aattagtcag
caaccatagt cccgccccta actccgccca tcccgcccct 3900 aactccgccc
agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 3960
agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
4020 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
gatctgatca 4080 gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc
gagaagtttc tgatcgaaaa 4140 gttcgacagc gtctccgacc tgatgcagct
ctcggagggc gaagaatctc gtgctttcag 4200 cttcgatgta ggagggcgtg
gatatgtcct gcgggtaaat agctgcgccg atggtttcta 4260 caaagatcgt
tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct 4320
tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
4380 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
tcgcggaggc 4440 catggatgcg atcgctgcgg ccgatcttag ccagacgagc
gggttcggcc cattcggacc 4500 gcaaggaatc ggtcaataca ctacatggcg
tgatttcata tgcgcgattg ctgatcccca 4560 tgtgtatcac tggcaaactg
tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 4620 cgatgagctg
atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 4680
tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
4740 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
ggaggccgtg 4800 gttggcttgt atggagcagc agacgcgcta cttcgagcgg
aggcatccgg agcttgcagg 4860 atcgccgcgg ctccgggcgt atatgctccg
cattggtctt gaccaactct atcagagctt 4920 ggttgacggc aatttcgatg
atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 4980 atccggagcc
gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac 5040
cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
5100 ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct
atgaaaggtt 5160 gggcttcgga atcgttttcc gggacgccgg ctggatgatc
ctccagcgcg gggatctcat 5220 gctggagttc ttcgcccacc ccaacttgtt
tattgcagct tataatggtt acaaataaag 5280 caatagcatc acaaatttca
caaataaagc atttttttca ctgcattcta gttgtggttt 5340 gtccaaactc
atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 5400
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
5460 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
tgagctaact 5520 cacattaatt gcgttgcgct cactgcccgc tttccagtcg
ggaaacctgt cgtgccagct 5580 gcattaatga atcggccaac gcgcggggag
aggcggtttg cgtattgggc gctcttccgc 5640 ttcctcgctc actgactcgc
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 5700 ctcaaaggcg
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 5760
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
5820 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
ggtggcgaaa 5880 cccgacagga ctataaagat accaggcgtt tccccctgga
agctccctcg tgcgctctcc 5940 tgttccgacc ctgccgctta ccggatacct
gtccgccttt ctcccttcgg gaagcgtggc 6000 gctttctcaa tgctcacgct
gtaggtatct cagttcggtg taggtcgttc gctccaagct 6060 gggctgtgtg
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 6120
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
6180 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
ggcctaacta 6240 cggctacact agaaggacag tatttggtat ctgcgctctg
ctgaagccag ttaccttcgg 6300 aaaaagagtt ggtagctctt gatccggcaa
acaaaccacc gctggtagcg gtggtttttt 6360 tgtttgcaag cagcagatta
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 6420 ttctacgggg
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 6480
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
6540 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
gtgaggcacc 6600 tatctcagcg atctgtctat ttcgttcatc catagttgcc
tgactccccg tcgtgtagat 6660 aactacgata cgggagggct taccatctgg
ccccagtgct gcaatgatac cgcgagaccc 6720 acgctcaccg gctccagatt
tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 6780 aagtggtcct
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 6840
agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
6900 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
gatcaaggcg 6960 agttacatga tcccccatgt tgtgcaaaaa agcggttagc
tccttcggtc ctccgatcgt 7020 tgtcagaagt aagttggccg cagtgttatc
actcatggtt atggcagcac tgcataattc 7080 tcttactgtc atgccatccg
taagatgctt ttctgtgact ggtgagtact caaccaagtc 7140 attctgagaa
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 7200
taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
7260 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
ctcgtgcacc 7320 caactgatct tcagcatctt ttactttcac cagcgtttct
gggtgagcaa aaacaggaag 7380 gcaaaatgcc gcaaaaaagg gaataagggc
gacacggaaa tgttgaatac tcatactctt 7440 cctttttcaa tattattgaa
gcatttatca gggttattgt ctcatgagcg gatacatatt 7500 tgaatgtatt
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 7560
acctgacgtc 7570 3 7597 DNA Artificial Sequence Plasmid 3 gacggatcgg
gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
gctggctaga 900 ggtaccaagc ttggatctca ccatggggtc aaccgccatc
ctcaccatgg agttggggct 960 gcgctgggtt ctcctcgttg ctcttttaag
aggtgtccag tgtcaggtgc agctggtgga 1020 gtctggggga ggcgtggtcc
agcctgggag gtccctgaga ctctcctgtg cagcgtctgg 1080 attcaccttc
agtaactatg tcatgcactg ggtccgccag gctccaggca aggggctgga 1140
gtgggtggca attatatggt atgatggaag taataaatac tatgcagact ccgtgaaggg
1200 ccgattcacc atctccagag acaattccaa gaacacgctg tatctgcaaa
tgaacagcct 1260 gagagccgag gacacggctg tgtattactg tgcgggtgga
tataactgga actacgagta 1320 ccactactac ggtatggacg tctggggcca
agggaccacg gtcaccgtct cctcagcctc 1380 caccaagggc ccatcggtct
tccccctggc accctctagc aagagcacct ctgggggcac 1440 agcggccctg
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa 1500
ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
1560 ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat 1620 ctgcaacgtg aatcacaagc ccagcaacac caaggtggac
aagagagttg gtgagaggcc 1680 agcacaggga gggagggtgt ctgctggaag
ccaggctcag cgctcctgcc tggacgcatc 1740 ccggctatgc agtcccagtc
cagggcagca aggcaggccc cgtctgcctc ttcacccgga 1800 ggcctctgcc
cgccccactc atgctcaggg agagggtctt ctggcttttt ccccaggctc 1860
tgggcaggca caggctaggt gcccctaacc caggccctgc acacaaaggg gcaggtgctg
1920 ggctcagacc tgccaagagc catatccggg aggaccctgc ccctgaccta
agcccacccc 1980 aaaggccaaa ctctccactc cctcagctcg gacaccttct
ctcctcccag attccagtaa 2040 ctcccaatct tctctctgca gagcccaaat
cttgtgacaa aactcacaca tgcccaccgt 2100 gcccaggtaa gccagcccag
gcctcgccct ccagctcaag gcgggacagg tgccctagag 2160 tagcctgcat
ccagggacag gccccagccg ggtgctgaca cgtccacctc catctcttcc 2220
tcagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac
2280 accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt
gagccacgaa 2340 gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg
aggtgcataa tgccaagaca 2400 aagccgcggg aggagcagta caacagcacg
taccgtgtgg tcagcgtcct caccgtcctg 2460 caccaggact ggctgaatgg
caaggagtac aagtgcaagg tctccaacaa agccctccca 2520 gcccccatcg
agaaaaccat ctccaaagcc aaaggtggga cccgtggggt gcgagggcca 2580
catggacaga ggccggctcg gcccaccctc tgccctgaga gtgaccgctg taccaacctc
2640 tgtccctaca gggcagcccc gagaaccaca ggtgtacacc ctgcccccat
cccgggagga 2700 gatgaccaag aaccaggtca
gcctgacctg cctggtcaaa ggcttctatc ccagcgacat 2760 cgccgtggag
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt 2820
gctggactcc gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg
2880 gcagcagggg aacgtcttct catgctccgt gatgcatgag gctctgcaca
accactacac 2940 gcagaagagc ctctccctgt ctccgggtaa atgagaattc
ctcgagtcta gagggcccgt 3000 ttaaacccgc tgatcagcct cgactgtgcc
ttctagttgc cagccatctg ttgtttgccc 3060 ctcccccgtg ccttccttga
ccctggaagg tgccactccc actgtccttt cctaataaaa 3120 tgaggaaatt
gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg 3180
gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
3240 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
cccacgcgcc 3300 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
cgcagcgtga ccgctacact 3360 tgccagcgcc ctagcgcccg ctcctttcgc
tttcttccct tcctttctcg ccacgttcgc 3420 cggctttccc cgtcaagctc
taaatcgggg catcccttta gggttccgat ttagtgcttt 3480 acggcacctc
gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 3540
ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
3600 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
tataagggat 3660 tttggggatt tcggcctatt ggttaaaaaa tgagctgatt
taacaaaaat ttaacgcgaa 3720 ttaattctgt ggaatgtgtg tcagttaggg
tgtggaaagt ccccaggctc cccaggcagg 3780 cagaagtatg caaagcatgc
atctcaatta gtcagcaacc aggtgtggaa agtccccagg 3840 ctccccagca
ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 3900
gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
3960 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
ctgagctatt 4020 ccagaagtag tgaggaggct tttttggagg cctaggcttt
tgcaaaaagc tcccgggagc 4080 ttgtatatcc attttcggat ctgatcagca
cgtgatgaaa aagcctgaac tcaccgcgac 4140 gtctgtcgag aagtttctga
tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc 4200 ggagggcgaa
gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg 4260
ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
4320 ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
gcctgaccta 4380 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
ctgcctgaaa ccgaactgcc 4440 cgctgttctg cagccggtcg cggaggccat
ggatgcgatc gctgcggccg atcttagcca 4500 gacgagcggg ttcggcccat
tcggaccgca aggaatcggt caatacacta catggcgtga 4560 tttcatatgc
gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac 4620
cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
4680 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
cggacaatgg 4740 ccgcataaca gcggtcattg actggagcga ggcgatgttc
ggggattccc aatacgaggt 4800 cgccaacatc ttcttctgga ggccgtggtt
ggcttgtatg gagcagcaga cgcgctactt 4860 cgagcggagg catccggagc
ttgcaggatc gccgcggctc cgggcgtata tgctccgcat 4920 tggtcttgac
caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc 4980
gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
5040 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
ccgatagtgg 5100 aaaccgacgc cccagcactc gtccgagggc aaaggaatag
cacgtgctac gagatttcga 5160 ttccaccgcc gccttctatg aaaggttggg
cttcggaatc gttttccggg acgccggctg 5220 gatgatcctc cagcgcgggg
atctcatgct ggagttcttc gcccacccca acttgtttat 5280 tgcagcttat
aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 5340
tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
5400 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
ttcctgtgtg 5460 aaattgttat ccgctcacaa ttccacacaa catacgagcc
ggaagcataa agtgtaaagc 5520 ctggggtgcc taatgagtga gctaactcac
attaattgcg ttgcgctcac tgcccgcttt 5580 ccagtcggga aacctgtcgt
gccagctgca ttaatgaatc ggccaacgcg cggggagagg 5640 cggtttgcgt
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 5700
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
5760 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
ggaaccgtaa 5820 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
cctgacgagc atcacaaaaa 5880 tcgacgctca agtcagaggt ggcgaaaccc
gacaggacta taaagatacc aggcgtttcc 5940 ccctggaagc tccctcgtgc
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 6000 cgcctttctc
ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag 6060
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
6120 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
acgacttatc 6180 gccactggca gcagccactg gtaacaggat tagcagagcg
aggtatgtag gcggtgctac 6240 agagttcttg aagtggtggc ctaactacgg
ctacactaga aggacagtat ttggtatctg 6300 cgctctgctg aagccagtta
ccttcggaaa aagagttggt agctcttgat ccggcaaaca 6360 aaccaccgct
ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 6420
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
6480 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
agatcctttt 6540 aaattaaaaa tgaagtttta aatcaatcta aagtatatat
gagtaaactt ggtctgacag 6600 ttaccaatgc ttaatcagtg aggcacctat
ctcagcgatc tgtctatttc gttcatccat 6660 agttgcctga ctccccgtcg
tgtagataac tacgatacgg gagggcttac catctggccc 6720 cagtgctgca
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 6780
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
6840 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
gtttgcgcaa 6900 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
tcgtttggta tggcttcatt 6960 cagctccggt tcccaacgat caaggcgagt
tacatgatcc cccatgttgt gcaaaaaagc 7020 ggttagctcc ttcggtcctc
cgatcgttgt cagaagtaag ttggccgcag tgttatcact 7080 catggttatg
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 7140
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
7200 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
taaaagtgct 7260 catcattgga aaacgttctt cggggcgaaa actctcaagg
atcttaccgc tgttgagatc 7320 cagttcgatg taacccactc gtgcacccaa
ctgatcttca gcatctttta ctttcaccag 7380 cgtttctggg tgagcaaaaa
caggaaggca aaatgccgca aaaaagggaa taagggcgac 7440 acggaaatgt
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 7500
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
7560 tccgcgcaca tttccccgaa aagtgccacc tgacgtc 7597 4 7579 DNA
Artificial Sequence Plasmid 4 gacggatcgg gagatctccc gatcccctat
ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat
ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac
tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900
ggtaccaagc ttggatctca ccatggagtt gggacttagc tgggttttcc tcgttgctct
960 tttaagaggt gtccagtgtc aggtccagct ggtggagtct gggggaggcg
tggtccagcc 1020 tgggaggtcc ctgagactct cctgtgcagc gtctggattc
accttcagta gctatggcat 1080 gcactgggtc cgccaggctc caggcaaggg
gctggactgg gtggcaatta tttggcatga 1140 tggaagtaat aaatactatg
cagactccgt gaagggccga ttcaccatct ccagagacaa 1200 ttccaagaag
acgctgtacc tgcaaatgaa cagtttgaga gccgaggaca cggctgtgta 1260
ttactgtgcg agagcttggg cctatgacta cggtgactat gaatactact tcggtatgga
1320 cgtctggggc caagggacca cggtcaccgt ctcctcagcc tccaccaagg
gcccatcggt 1380 cttccccctg gcaccctcta gcaagagcac ctctgggggc
acagcggccc tgggctgcct 1440 ggtcaaggac tacttccccg aaccggtgac
ggtgtcgtgg aactcaggcg ccctgaccag 1500 cggcgtgcac accttcccgg
ctgtcctaca gtcctcagga ctctactccc tcagcagcgt 1560 ggtgaccgtg
ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa 1620
gcccagcaac accaaggtgg acaagagagt tggtgagagg ccagcacagg gagggagggt
1680 gtctgctgga agccaggctc agcgctcctg cctggacgca tcccggctat
gcagtcccag 1740 tccagggcag caaggcaggc cccgtctgcc tcttcacccg
gaggcctctg cccgccccac 1800 tcatgctcag ggagagggtc ttctggcttt
ttccccaggc tctgggcagg cacaggctag 1860 gtgcccctaa cccaggccct
gcacacaaag gggcaggtgc tgggctcaga cctgccaaga 1920 gccatatccg
ggaggaccct gcccctgacc taagcccacc ccaaaggcca aactctccac 1980
tccctcagct cggacacctt ctctcctccc agattccagt aactcccaat cttctctctg
2040 cagagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccaggt
aagccagccc 2100 aggcctcgcc ctccagctca aggcgggaca ggtgccctag
agtagcctgc atccagggac 2160 aggccccagc cgggtgctga cacgtccacc
tccatctctt cctcagcacc tgaactcctg 2220 gggggaccgt cagtcttcct
cttcccccca aaacccaagg acaccctcat gatctcccgg 2280 acccctgagg
tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 2340
aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
2400 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 2460 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
cagcccccat cgagaaaacc 2520 atctccaaag ccaaaggtgg gacccgtggg
gtgcgagggc cacatggaca gaggccggct 2580 cggcccaccc tctgccctga
gagtgaccgc tgtaccaacc tctgtcccta cagggcagcc 2640 ccgagaacca
caggtgtaca ccctgccccc atcccgggag gagatgacca agaaccaggt 2700
cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag
2760 caatgggcag ccggagaaca actacaagac cacgcctccc gtgctggact
ccgacggctc 2820 cttcttcctc tatagcaagc tcaccgtgga caagagcagg
tggcagcagg ggaacgtctt 2880 ctcatgctcc gtgatgcatg aggctctgca
caaccactac acgcagaaga gcctctccct 2940 gtctccgggt aaatgagaat
tcctcgagtc tagagggccc gtttaaaccc gctgatcagc 3000 ctcgactgtg
ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3060
gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
3120 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
gcaaggggga 3180 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
ggctctatgg cttctgaggc 3240 ggaaagaacc agctggggct ctagggggta
tccccacgcg ccctgtagcg gcgcattaag 3300 cgcggcgggt gtggtggtta
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 3360 cgctcctttc
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 3420
tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
3480 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
cggtttttcg 3540 ccctttgacg ttggagtcca cgttctttaa tagtggactc
ttgttccaaa ctggaacaac 3600 actcaaccct atctcggtct attcttttga
tttataaggg attttgggga tttcggccta 3660 ttggttaaaa aatgagctga
tttaacaaaa atttaacgcg aattaattct gtggaatgtg 3720 tgtcagttag
ggtgtggaaa gtccccaggc tccccaggca ggcagaagta tgcaaagcat 3780
gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
3840 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
ctccgcccat 3900 cccgccccta actccgccca gttccgccca ttctccgccc
catggctgac taattttttt 3960 tatttatgca gaggccgagg ccgcctctgc
ctctgagcta ttccagaagt agtgaggagg 4020 cttttttgga ggcctaggct
tttgcaaaaa gctcccggga gcttgtatat ccattttcgg 4080 atctgatcag
cacgtgatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct 4140
gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
4200 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
gctgcgccga 4260 tggtttctac aaagatcgtt atgtttatcg gcactttgca
tcggccgcgc tcccgattcc 4320 ggaagtgctt gacattgggg aattcagcga
gagcctgacc tattgcatct cccgccgtgc 4380 acagggtgtc acgttgcaag
acctgcctga aaccgaactg cccgctgttc tgcagccggt 4440 cgcggaggcc
atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc 4500
attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
4560 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
cgtccgtcgc 4620 gcaggctctc gatgagctga tgctttgggc cgaggactgc
cccgaagtcc ggcacctcgt 4680 gcacgcggat ttcggctcca acaatgtcct
gacggacaat ggccgcataa cagcggtcat 4740 tgactggagc gaggcgatgt
tcggggattc ccaatacgag gtcgccaaca tcttcttctg 4800 gaggccgtgg
ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga 4860
gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
4920 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
gatgcgacgc 4980 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa
atcgcccgca gaagcgcggc 5040 cgtctggacc gatggctgtg tagaagtact
cgccgatagt ggaaaccgac gccccagcac 5100 tcgtccgagg gcaaaggaat
agcacgtgct acgagatttc gattccaccg ccgccttcta 5160 tgaaaggttg
ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg 5220
ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
5280 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
tgcattctag 5340 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
tgtataccgt cgacctctag 5400 ctagagcttg gcgtaatcat ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac 5460 aattccacac aacatacgag
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 5520 gagctaactc
acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5580
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
5640 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
ggcgagcggt 5700 atcagctcac tcaaaggcgg taatacggtt atccacagaa
tcaggggata acgcaggaaa 5760 gaacatgtga gcaaaaggcc agcaaaaggc
caggaaccgt aaaaaggccg cgttgctggc 5820 gtttttccat aggctccgcc
cccctgacga gcatcacaaa aatcgacgct caagtcagag 5880 gtggcgaaac
ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 5940
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
6000 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
aggtcgttcg 6060 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg 6120 taactatcgt cttgagtcca acccggtaag
acacgactta tcgccactgg cagcagccac 6180 tggtaacagg attagcagag
cgaggtatgt aggcggtgct acagagttct tgaagtggtg 6240 gcctaactac
ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 6300
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
6360 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
aagaagatcc 6420 tttgatcttt tctacggggt ctgacgctca gtggaacgaa
aactcacgtt aagggatttt 6480 ggtcatgaga ttatcaaaaa ggatcttcac
ctagatcctt ttaaattaaa aatgaagttt 6540 taaatcaatc taaagtatat
atgagtaaac ttggtctgac agttaccaat gcttaatcag 6600 tgaggcacct
atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 6660
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
6720 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
ccggaagggc 6780 cgagcgcaga agtggtcctg caactttatc cgcctccatc
cagtctatta attgttgccg 6840 ggaagctaga gtaagtagtt cgccagttaa
tagtttgcgc aacgttgttg ccattgctac 6900 aggcatcgtg gtgtcacgct
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 6960 atcaaggcga
gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 7020
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
7080 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
gtgagtactc 7140 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaat 7200 acgggataat accgcgccac atagcagaac
tttaaaagtg ctcatcattg gaaaacgttc 7260 ttcggggcga aaactctcaa
ggatcttacc gctgttgaga tccagttcga tgtaacccac 7320 tcgtgcaccc
aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 7380
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
7440 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
tcatgagcgg 7500 atacatattt gaatgtattt agaaaaataa acaaataggg
gttccgcgca catttccccg 7560 aaaagtgcca cctgacgtc 7579 5 7558 DNA
Artificial Sequence Plasmid 5 gacggatcgg gagatctccc gatcccctat
ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat
ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac
tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900
ggtaccaagc ttggatccca ccatggggtc aaccgtcatc ctcgccctcc tcctggctgt
960 tctccaagga gtctgtgccg aggtgcagct ggtgcagtct ggagcagagg
tgaaaaagcc 1020 cggggagtct ctgaagatct cctgtaaggg ttctggatac
agctttacca gttactggat 1080 cggctgggtg cgccagatgc ccgggaaagg
cctggagtgg atggggatca tctatcctgg 1140 tgactctgat accagataca
gcccgtcctt ccaaggccag gtcaccatct cagccgacaa 1200 gtccatcagc
accgcctacc tgcagtggag cagcctgaag gcctcggaca ccgccatgta 1260
ttactgtgcg agacggatgg cagcagctgg cccctttgac tactggggcc agggaaccct
1320 ggtcaccgtc tcctcagcct ccaccaaggg cccatcggtc ttccccctgg
caccctctag 1380 caagagcacc tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga 1440 accggtgacg gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc 1500 tgtcctacag tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag 1560 cttgggcacc
cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga 1620
caagagagtt ggtgagaggc cagcacaggg agggagggtg tctgctggaa gccaggctca
1680 gcgctcctgc ctggacgcat cccggctatg cagtcccagt ccagggcagc
aaggcaggcc 1740 ccgtctgcct cttcacccgg aggcctctgc ccgccccact
catgctcagg gagagggtct 1800 tctggctttt tccccaggct ctgggcaggc
acaggctagg tgcccctaac ccaggccctg 1860 cacacaaagg ggcaggtgct
gggctcagac ctgccaagag ccatatccgg gaggaccctg 1920 cccctgacct
aagcccaccc caaaggccaa actctccact ccctcagctc ggacaccttc 1980
tctcctccca gattccagta actcccaatc ttctctctgc agagcccaaa tcttgtgaca
2040 aaactcacac atgcccaccg tgcccaggta agccagccca ggcctcgccc
tccagctcaa 2100 ggcgggacag gtgccctaga gtagcctgca tccagggaca
ggccccagcc gggtgctgac 2160 acgtccacct ccatctcttc ctcagcacct
gaactcctgg ggggaccgtc agtcttcctc 2220 ttccccccaa aacccaagga
caccctcatg atctcccgga cccctgaggt cacatgcgtg 2280 gtggtggacg
tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 2340
gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg
2400 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta
caagtgcaag 2460
gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaaggtggg
2520 acccgtgggg tgcgagggcc acatggacag aggccggctc ggcccaccct
ctgccctgag 2580 agtgaccgct gtaccaacct ctgtccctac agggcagccc
cgagaaccac aggtgtacac 2640 cctgccccca tcccgggagg agatgaccaa
gaaccaggtc agcctgacct gcctggtcaa 2700 aggcttctat cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc cggagaacaa 2760 ctacaagacc
acgcctcccg tgctggactc cgacggctcc ttcttcctct atagcaagct 2820
caccgtggac aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
2880 ggctctgcac aaccactaca cgcagaagag cctctccctg tctccgggta
aatgagaatt 2940 cctcgagtct agagggcccg tttaaacccg ctgatcagcc
tcgactgtgc cttctagttg 3000 ccagccatct gttgtttgcc cctcccccgt
gccttccttg accctggaag gtgccactcc 3060 cactgtcctt tcctaataaa
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 3120 tattctgggg
ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 3180
gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
3240 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
tggtggttac 3300 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
gctcctttcg ctttcttccc 3360 ttcctttctc gccacgttcg ccggctttcc
ccgtcaagct ctaaatcggg gcatcccttt 3420 agggttccga tttagtgctt
tacggcacct cgaccccaaa aaacttgatt agggtgatgg 3480 ttcacgtagt
gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3540
gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
3600 ttcttttgat ttataaggga ttttggggat ttcggcctat tggttaaaaa
atgagctgat 3660 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
gtcagttagg gtgtggaaag 3720 tccccaggct ccccaggcag gcagaagtat
gcaaagcatg catctcaatt agtcagcaac 3780 caggtgtgga aagtccccag
gctccccagc aggcagaagt atgcaaagca tgcatctcaa 3840 ttagtcagca
accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 3900
ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
3960 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
gcctaggctt 4020 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
tctgatcagc acgtgatgaa 4080 aaagcctgaa ctcaccgcga cgtctgtcga
gaagtttctg atcgaaaagt tcgacagcgt 4140 ctccgacctg atgcagctct
cggagggcga agaatctcgt gctttcagct tcgatgtagg 4200 agggcgtgga
tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta 4260
tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
4320 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca
cgttgcaaga 4380 cctgcctgaa accgaactgc ccgctgttct gcagccggtc
gcggaggcca tggatgcgat 4440 cgctgcggcc gatcttagcc agacgagcgg
gttcggccca ttcggaccgc aaggaatcgg 4500 tcaatacact acatggcgtg
atttcatatg cgcgattgct gatccccatg tgtatcactg 4560 gcaaactgtg
atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat 4620
gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
4680 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg
aggcgatgtt 4740 cggggattcc caatacgagg tcgccaacat cttcttctgg
aggccgtggt tggcttgtat 4800 ggagcagcag acgcgctact tcgagcggag
gcatccggag cttgcaggat cgccgcggct 4860 ccgggcgtat atgctccgca
ttggtcttga ccaactctat cagagcttgg ttgacggcaa 4920 tttcgatgat
gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg 4980
gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
5040 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg
caaaggaata 5100 gcacgtgcta cgagatttcg attccaccgc cgccttctat
gaaaggttgg gcttcggaat 5160 cgttttccgg gacgccggct ggatgatcct
ccagcgcggg gatctcatgc tggagttctt 5220 cgcccacccc aacttgttta
ttgcagctta taatggttac aaataaagca atagcatcac 5280 aaatttcaca
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 5340
caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
5400 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
acatacgagc 5460 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
agctaactca cattaattgc 5520 gttgcgctca ctgcccgctt tccagtcggg
aaacctgtcg tgccagctgc attaatgaat 5580 cggccaacgc gcggggagag
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 5640 tgactcgctg
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 5700
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
5760 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
ggctccgccc 5820 ccctgacgag catcacaaaa atcgacgctc aagtcagagg
tggcgaaacc cgacaggact 5880 ataaagatac caggcgtttc cccctggaag
ctccctcgtg cgctctcctg ttccgaccct 5940 gccgcttacc ggatacctgt
ccgcctttct cccttcggga agcgtggcgc tttctcaatg 6000 ctcacgctgt
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 6060
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
6120 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
ttagcagagc 6180 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
cctaactacg gctacactag 6240 aaggacagta tttggtatct gcgctctgct
gaagccagtt accttcggaa aaagagttgg 6300 tagctcttga tccggcaaac
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 6360 gcagattacg
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 6420
tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
6480 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
aaagtatata 6540 tgagtaaact tggtctgaca gttaccaatg cttaatcagt
gaggcaccta tctcagcgat 6600 ctgtctattt cgttcatcca tagttgcctg
actccccgtc gtgtagataa ctacgatacg 6660 ggagggctta ccatctggcc
ccagtgctgc aatgataccg cgagacccac gctcaccggc 6720 tccagattta
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 6780
aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
6840 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
tgtcacgctc 6900 gtcgtttggt atggcttcat tcagctccgg ttcccaacga
tcaaggcgag ttacatgatc 6960 ccccatgttg tgcaaaaaag cggttagctc
cttcggtcct ccgatcgttg tcagaagtaa 7020 gttggccgca gtgttatcac
tcatggttat ggcagcactg cataattctc ttactgtcat 7080 gccatccgta
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 7140
gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
7200 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
aactctcaag 7260 gatcttaccg ctgttgagat ccagttcgat gtaacccact
cgtgcaccca actgatcttc 7320 agcatctttt actttcacca gcgtttctgg
gtgagcaaaa acaggaaggc aaaatgccgc 7380 aaaaaaggga ataagggcga
cacggaaatg ttgaatactc atactcttcc tttttcaata 7440 ttattgaagc
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 7500
gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
7558 6 7576 DNA Artificial Sequence Plasmid 6 gacggatcgg gagatctccc
gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt
aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc
attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa
atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata
atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca
atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900 ggtaccaagc ttggatctca ccatggagtt tgggctgtgc tggattttcc
tcgttgctct 960 tttaagaggt gtccagtgtc aggtgcagct ggtggagtct
gggggaggcg tggtccagcc 1020 tgggaggtcc ctgagactct cctgtgcagc
ctctggattc accttcatta gctatggcat 1080 gcactgggtc cgccaggctc
caggcaaggg gctggagtgg gtggcagtta tatcatatga 1140 tggaagtaat
aaatactatg cagactccgt gaagggccga ttcaccatct ccagagacaa 1200
ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga gctgaggaca cggctgtgta
1260 ttactgtgcg agagtattag tgggagcttt atattattat aactactacg
ggatggacgt 1320 ctggggccaa gggaccacgg tcaccgtctc ctcagcctcc
accaagggcc catcggtctt 1380 ccccctggca ccctctagca agagcacctc
tgggggcaca gcggccctgg gctgcctggt 1440 caaggactac ttccccgaac
cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg 1500 cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt 1560
gaccgtgccc tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc
1620 cagcaacacc aaggtggaca agagagttgg tgagaggcca gcacagggag
ggagggtgtc 1680 tgctggaagc caggctcagc gctcctgcct ggacgcatcc
cggctatgca gtcccagtcc 1740 agggcagcaa ggcaggcccc gtctgcctct
tcacccggag gcctctgccc gccccactca 1800 tgctcaggga gagggtcttc
tggctttttc cccaggctct gggcaggcac aggctaggtg 1860 cccctaaccc
aggccctgca cacaaagggg caggtgctgg gctcagacct gccaagagcc 1920
atatccggga ggaccctgcc cctgacctaa gcccacccca aaggccaaac tctccactcc
1980 ctcagctcgg acaccttctc tcctcccaga ttccagtaac tcccaatctt
ctctctgcag 2040 agcccaaatc ttgtgacaaa actcacacat gcccaccgtg
cccaggtaag ccagcccagg 2100 cctcgccctc cagctcaagg cgggacaggt
gccctagagt agcctgcatc cagggacagg 2160 ccccagccgg gtgctgacac
gtccacctcc atctcttcct cagcacctga actcctgggg 2220 ggaccgtcag
tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 2280
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
2340 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 2400 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 2460 aaggagtaca agtgcaaggt ctccaacaaa
gccctcccag cccccatcga gaaaaccatc 2520 tccaaagcca aaggtgggac
ccgtggggtg cgagggccac atggacagag gccggctcgg 2580 cccaccctct
gccctgagag tgaccgctgt accaacctct gtccctacag ggcagccccg 2640
agaaccacag gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag
2700 cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt
gggagagcaa 2760 tgggcagccg gagaacaact acaagaccac gcctcccgtg
ctggactccg acggctcctt 2820 cttcctctat agcaagctca ccgtggacaa
gagcaggtgg cagcagggga acgtcttctc 2880 atgctccgtg atgcatgagg
ctctgcacaa ccactacacg cagaagagcc tctccctgtc 2940 tccgggtaaa
tgagaattcc tcgagtctag agggcccgtt taaacccgct gatcagcctc 3000
gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
3060 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
catcgcattg 3120 tctgagtagg tgtcattcta ttctgggggg tggggtgggg
caggacagca agggggagga 3180 ttgggaagac aatagcaggc atgctgggga
tgcggtgggc tctatggctt ctgaggcgga 3240 aagaaccagc tggggctcta
gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc 3300 ggcgggtgtg
gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 3360
tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
3420 aaatcggggc atccctttag ggttccgatt tagtgcttta cggcacctcg
accccaaaaa 3480 acttgattag ggtgatggtt cacgtagtgg gccatcgccc
tgatagacgg tttttcgccc 3540 tttgacgttg gagtccacgt tctttaatag
tggactcttg ttccaaactg gaacaacact 3600 caaccctatc tcggtctatt
cttttgattt ataagggatt ttggggattt cggcctattg 3660 gttaaaaaat
gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt 3720
cagttagggt gtggaaagtc cccaggctcc ccaggcaggc agaagtatgc aaagcatgca
3780 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
gcagaagtat 3840 gcaaagcatg catctcaatt agtcagcaac catagtcccg
cccctaactc cgcccatccc 3900 gcccctaact ccgcccagtt ccgcccattc
tccgccccat ggctgactaa ttttttttat 3960 ttatgcagag gccgaggccg
cctctgcctc tgagctattc cagaagtagt gaggaggctt 4020 ttttggaggc
ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc 4080
tgatcagcac gtgatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
4140 cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
aatctcgtgc 4200 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
gtaaatagct gcgccgatgg 4260 tttctacaaa gatcgttatg tttatcggca
ctttgcatcg gccgcgctcc cgattccgga 4320 agtgcttgac attggggaat
tcagcgagag cctgacctat tgcatctccc gccgtgcaca 4380 gggtgtcacg
ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 4440
ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
4500 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
cgattgctga 4560 tccccatgtg tatcactggc aaactgtgat ggacgacacc
gtcagtgcgt ccgtcgcgca 4620 ggctctcgat gagctgatgc tttgggccga
ggactgcccc gaagtccggc acctcgtgca 4680 cgcggatttc ggctccaaca
atgtcctgac ggacaatggc cgcataacag cggtcattga 4740 ctggagcgag
gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 4800
gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
4860 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
aactctatca 4920 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
cagggtcgat gcgacgcaat 4980 cgtccgatcc ggagccggga ctgtcgggcg
tacacaaatc gcccgcagaa gcgcggccgt 5040 ctggaccgat ggctgtgtag
aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 5100 tccgagggca
aaggaatagc acgtgctacg agatttcgat tccaccgccg ccttctatga 5160
aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
5220 tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
atggttacaa 5280 ataaagcaat agcatcacaa atttcacaaa taaagcattt
ttttcactgc attctagttg 5340 tggtttgtcc aaactcatca atgtatctta
tcatgtctgt ataccgtcga cctctagcta 5400 gagcttggcg taatcatggt
catagctgtt tcctgtgtga aattgttatc cgctcacaat 5460 tccacacaac
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 5520
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
5580 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
ttgggcgctc 5640 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
cggctgcggc gagcggtatc 5700 agctcactca aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa 5760 catgtgagca aaaggccagc
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 5820 tttccatagg
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 5880
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
5940 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
cttcgggaag 6000 cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
tcggtgtagg tcgttcgctc 6060 caagctgggc tgtgtgcacg aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa 6120 ctatcgtctt gagtccaacc
cggtaagaca cgacttatcg ccactggcag cagccactgg 6180 taacaggatt
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 6240
taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
6300 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
gtagcggtgg 6360 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
ggatctcaag aagatccttt 6420 gatcttttct acggggtctg acgctcagtg
gaacgaaaac tcacgttaag ggattttggt 6480 catgagatta tcaaaaagga
tcttcaccta gatcctttta aattaaaaat gaagttttaa 6540 atcaatctaa
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 6600
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
6660 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
tgataccgcg 6720 agacccacgc tcaccggctc cagatttatc agcaataaac
cagccagccg gaagggccga 6780 gcgcagaagt ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga 6840 agctagagta agtagttcgc
cagttaatag tttgcgcaac gttgttgcca ttgctacagg 6900 catcgtggtg
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 6960
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
7020 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
cagcactgca 7080 taattctctt actgtcatgc catccgtaag atgcttttct
gtgactggtg agtactcaac 7140 caagtcattc tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg 7200 ggataatacc gcgccacata
gcagaacttt aaaagtgctc atcattggaa aacgttcttc 7260 ggggcgaaaa
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 7320
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
7380 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
gaatactcat 7440 actcttcctt tttcaatatt attgaagcat ttatcagggt
tattgtctca tgagcggata 7500 catatttgaa tgtatttaga aaaataaaca
aataggggtt ccgcgcacat ttccccgaaa 7560 agtgccacct gacgtc 7576 7 7561
DNA Artificial Sequence Plasmid 7 gacggatcgg gagatctccc gatcccctat
ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat
ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac
tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900
ggtaccggat ctcaccatgg agttggggct gagctgggtt ttcctcgttg ctcttttaag
960 aggtgtccag tgtcaggagc agctggtgga gtctggggga ggcgtggtcc
agcctgggag 1020 gtccctgaga ctctcctgtg cagcgtctgg attcaccttc
agtacctatg gcatgcactg 1080 ggtccgccag gctccaggca aggggctgga
gtgggtggca gttacatggc atgatggaag 1140 taataaatac tatgcagact
ccgtgaaggg ccgattcacc atctccagag acaactccaa 1200 gaacacgctg
tatctgcaaa tgaacagcct gagagccgag gacacggctg tgtattactg 1260
tgcgagagga ggagtgggag caacttacta ctactactac ggtatggacg tctggggcca
1320 agggaccacg gtcaccgtct cctcagcctc caccaagggc ccatcggtct
tccccctggc 1380 accctctagc aagagcacct ctgggggcac agcggccctg
ggctgcctgg tcaaggacta 1440 cttccccgaa ccggtgacgg tgtcgtggaa
ctcaggcgcc ctgaccagcg gcgtgcacac 1500 cttcccggct gtcctacagt
cctcaggact ctactccctc agcagcgtgg tgaccgtgcc 1560 ctccagcagc
ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac 1620
caaggtggac aagagagttg gtgagaggcc agcacaggga gggagggtgt ctgctggaag
1680 ccaggctcag cgctcctgcc tggacgcatc ccggctatgc agtcccagtc
cagggcagca 1740 aggcaggccc cgtctgcctc ttcacccgga ggcctctgcc
cgccccactc atgctcaggg 1800 agagggtctt ctggcttttt ccccaggctc
tgggcaggca caggctaggt gcccctaacc 1860 caggccctgc acacaaaggg
gcaggtgctg ggctcagacc tgccaagagc catatccggg 1920 aggaccctgc
ccctgaccta agcccacccc aaaggccaaa ctctccactc cctcagctcg 1980
gacaccttct ctcctcccag attccagtaa ctcccaatct tctctctgca gagcccaaat
2040 cttgtgacaa aactcacaca tgcccaccgt gcccaggtaa gccagcccag
gcctcgccct 2100 ccagctcaag gcgggacagg tgccctagag tagcctgcat
ccagggacag gccccagccg 2160 ggtgctgaca cgtccacctc catctcttcc
tcagcacctg aactcctggg gggaccgtca 2220 gtcttcctct tccccccaaa
acccaaggac accctcatga
tctcccggac ccctgaggtc 2280 acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa ctggtacgtg 2340 gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta caacagcacg 2400 taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 2460
aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
2520 aaaggtggga cccgtggggt gcgagggcca catggacaga ggccggctcg
gcccaccctc 2580 tgccctgaga gtgaccgctg taccaacctc tgtccctaca
gggcagcccc gagaaccaca 2640 ggtgtacacc ctgcccccat cccgggagga
gatgaccaag aaccaggtca gcctgacctg 2700 cctggtcaaa ggcttctatc
ccagcgacat cgccgtggag tgggagagca atgggcagcc 2760 ggagaacaac
tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta 2820
tagcaagctc accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt
2880 gatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt
ctccgggtaa 2940 atgactcgag tctagagggc ccgtttaaac ccgctgatca
gcctcgactg tgccttctag 3000 ttgccagcca tctgttgttt gcccctcccc
cgtgccttcc ttgaccctgg aaggtgccac 3060 tcccactgtc ctttcctaat
aaaatgagga aattgcatcg cattgtctga gtaggtgtca 3120 ttctattctg
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag 3180
caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
3240 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
gtgtggtggt 3300 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
cccgctcctt tcgctttctt 3360 cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc ggggcatccc 3420 tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg attagggtga 3480 tggttcacgt
agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 3540
cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
3600 ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa
aaaatgagct 3660 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
tgtgtcagtt agggtgtgga 3720 aagtccccag gctccccagg caggcagaag
tatgcaaagc atgcatctca attagtcagc 3780 aaccaggtgt ggaaagtccc
caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3840 caattagtca
gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc 3900
cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
3960 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
gaggcctagg 4020 cttttgcaaa aagctcccgg gagcttgtat atccattttc
ggatctgatc agcacgtgat 4080 gaaaaagcct gaactcaccg cgacgtctgt
cgagaagttt ctgatcgaaa agttcgacag 4140 cgtctccgac ctgatgcagc
tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 4200 aggagggcgt
ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 4260
ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg
4320 ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
tcacgttgca 4380 agacctgcct gaaaccgaac tgcccgctgt tctgcagccg
gtcgcggagg ccatggatgc 4440 gatcgctgcg gccgatctta gccagacgag
cgggttcggc ccattcggac cgcaaggaat 4500 cggtcaatac actacatggc
gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 4560 ctggcaaact
gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 4620
gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc
4680 caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
gcgaggcgat 4740 gttcggggat tcccaatacg aggtcgccaa catcttcttc
tggaggccgt ggttggcttg 4800 tatggagcag cagacgcgct acttcgagcg
gaggcatccg gagcttgcag gatcgccgcg 4860 gctccgggcg tatatgctcc
gcattggtct tgaccaactc tatcagagct tggttgacgg 4920 caatttcgat
gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 4980
cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg
5040 tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
gggcaaagga 5100 atagcacgtg ctacgagatt tcgattccac cgccgccttc
tatgaaaggt tgggcttcgg 5160 aatcgttttc cgggacgccg gctggatgat
cctccagcgc ggggatctca tgctggagtt 5220 cttcgcccac cccaacttgt
ttattgcagc ttataatggt tacaaataaa gcaatagcat 5280 cacaaatttc
acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 5340
catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
5400 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
acaacatacg 5460 agccggaagc ataaagtgta aagcctgggg tgcctaatga
gtgagctaac tcacattaat 5520 tgcgttgcgc tcactgcccg ctttccagtc
gggaaacctg tcgtgccagc tgcattaatg 5580 aatcggccaa cgcgcgggga
gaggcggttt gcgtattggg cgctcttccg cttcctcgct 5640 cactgactcg
ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 5700
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
5760 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
ataggctccg 5820 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
aggtggcgaa acccgacagg 5880 actataaaga taccaggcgt ttccccctgg
aagctccctc gtgcgctctc ctgttccgac 5940 cctgccgctt accggatacc
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6000 atgctcacgc
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6060
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
6120 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
ggattagcag 6180 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
tggcctaact acggctacac 6240 tagaaggaca gtatttggta tctgcgctct
gctgaagcca gttaccttcg gaaaaagagt 6300 tggtagctct tgatccggca
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6360 gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6420
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
6480 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
tctaaagtat 6540 atatgagtaa acttggtctg acagttacca atgcttaatc
agtgaggcac ctatctcagc 6600 gatctgtcta tttcgttcat ccatagttgc
ctgactcccc gtcgtgtaga taactacgat 6660 acgggagggc ttaccatctg
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 6720 ggctccagat
ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 6780
tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
6840 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
tggtgtcacg 6900 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
cgatcaaggc gagttacatg 6960 atcccccatg ttgtgcaaaa aagcggttag
ctccttcggt cctccgatcg ttgtcagaag 7020 taagttggcc gcagtgttat
cactcatggt tatggcagca ctgcataatt ctcttactgt 7080 catgccatcc
gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7140
atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
7200 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
gaaaactctc 7260 aaggatctta ccgctgttga gatccagttc gatgtaaccc
actcgtgcac ccaactgatc 7320 ttcagcatct tttactttca ccagcgtttc
tgggtgagca aaaacaggaa ggcaaaatgc 7380 cgcaaaaaag ggaataaggg
cgacacggaa atgttgaata ctcatactct tcctttttca 7440 atattattga
agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 7500
ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
7560 c 7561 8 6082 DNA Artificial Sequence Plasmid 8 gacggatcgg
gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
gctggctaga 900 aagcttggat ctcaccatga gggtccctgc tcagctcctg
ggactcctgc tgctctggct 960 cccagatacc agatgtgaca tccagatgac
ccagtctcca tcctccctgt ctgcatctgt 1020 aggagacaga gtcaccatca
cttgccgggc gagtcagggc attagcaatt atttagcctg 1080 gtatcagcag
aaaacaggga aagttcctaa gttcctgatc tatgaagcat ccactttgca 1140
atcaggggtc ccatctcggt tcagtggcgg tggatctggg acagatttca ctctcaccat
1200 cagcagcctg cagcctgaag atgttgcaac ttattactgt caaaattata
acagtgcccc 1260 attcactttc ggccctggga ccaaagtgga tatcaaacga
actgtggctg caccctctgt 1320 cttcatcttc ccgccatctg atgagcagtt
gaaatctgga actgctagcg ttgtgtgcct 1380 gctgaataac ttctatccca
gagaggccaa agtacagtgg aaggtggata acgccctcca 1440 atcgggtaac
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 1500
cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga
1560 agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg
gagagtgtta 1620 ggaattcgcg gccgctcgag tctagagggc ccgtttaaac
ccgctgatca gcctcgactg 1680 tgccttctag ttgccagcca tctgttgttt
gcccctcccc cgtgccttcc ttgaccctgg 1740 aaggtgccac tcccactgtc
ctttcctaat aaaatgagga aattgcatcg cattgtctga 1800 gtaggtgtca
ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1860
aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
1920 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
agcgcggcgg 1980 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
cgccctagcg cccgctcctt 2040 tcgctttctt cccttccttt ctcgccacgt
tcgccggctt tccccgtcaa gctctaaatc 2100 ggggcatccc tttagggttc
cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2160 attagggtga
tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2220
cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
2280 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc
tattggttaa 2340 aaaatgagct gatttaacaa aaatttaacg cgaattaatt
ctgtggaatg tgtgtcagtt 2400 agggtgtgga aagtccccag gctccccagg
caggcagaag tatgcaaagc atgcatctca 2460 attagtcagc aaccaggtgt
ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2520 gcatgcatct
caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 2580
taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
2640 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
ggcttttttg 2700 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat
atccattttc ggatctgatc 2760 aagagacagg atgaggatcg tttcgcatga
ttgaacaaga tggattgcac gcaggttctc 2820 cggccgcttg ggtggagagg
ctattcggct atgactgggc acaacagaca atcggctgct 2880 ctgatgccgc
cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940
acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
3000 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
agggactggc 3060 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
tcaccttgct cctgccgaga 3120 aagtatccat catggctgat gcaatgcggc
ggctgcatac gcttgatccg gctacctgcc 3180 cattcgacca ccaagcgaaa
catcgcatcg agcgagcacg tactcggatg gaagccggtc 3240 ttgtcgatca
ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3300
ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
3360 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
tgtggccggc 3420 tgggtgtggc ggaccgctat caggacatag cgttggctac
ccgtgatatt gctgaagagc 3480 ttggcggcga atgggctgac cgcttcctcg
tgctttacgg tatcgccgct cccgattcgc 3540 agcgcatcgc cttctatcgc
cttcttgacg agttcttctg agcgggactc tggggttcga 3600 aatgaccgac
caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 3660
ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
3720 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
cttataatgg 3780 ttacaaataa agcaatagca tcacaaattt cacaaataaa
gcattttttt cactgcattc 3840 tagttgtggt ttgtccaaac tcatcaatgt
atcttatcat gtctgtatac cgtcgacctc 3900 tagctagagc ttggcgtaat
catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960 cacaattcca
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 4020
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
4080 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg 4140 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc 4200 ggtatcagct cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg 4260 aaagaacatg tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4320 ggcgtttttc
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 4380
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
4440 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
ttctcccttc 4500 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt 4560 tcgctccaag ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc 4620 cggtaactat cgtcttgagt
ccaacccggt aagacacgac ttatcgccac tggcagcagc 4680 cactggtaac
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 4740
gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
4800 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
ccgctggtag 4860 cggtggtttt tttgtttgca agcagcagat tacgcgcaga
aaaaaaggat ctcaagaaga 4920 tcctttgatc ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac gttaagggat 4980 tttggtcatg agattatcaa
aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040 ttttaaatca
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 5100
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
5160 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
ctgcaatgat 5220 accgcgagac ccacgctcac cggctccaga tttatcagca
ataaaccagc cagccggaag 5280 ggccgagcgc agaagtggtc ctgcaacttt
atccgcctcc atccagtcta ttaattgttg 5340 ccgggaagct agagtaagta
gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5400 tacaggcatc
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 5460
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
5520 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc 5580 actgcataat tctcttactg tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta 5640 ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc 5700 aatacgggat aataccgcgc
cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760 ttcttcgggg
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 5820
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
5880 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
aatgttgaat 5940 actcatactc ttcctttttc aatattattg aagcatttat
cagggttatt gtctcatgag 6000 cggatacata tttgaatgta tttagaaaaa
taaacaaata ggggttccgc gcacatttcc 6060 ccgaaaagtg ccacctgacg tc 6082
9 6082 DNA Artificial Sequence Plasmid 9 gacggatcgg gagatctccc
gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt
aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc
attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa
atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata
atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca
atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900 aagcttggat ctcaccatga gggtccccgc tcagctcctg gggctcctgc
tgctctgttt 960 cccaggtgcc agatgtgaca tccagatgac ccagtctcca
tcctcactgt ctgcatctgt 1020 aggagacaga gtcaccatca cttgtcgggc
gagtcagggc attaccaatt atttagcctg 1080 gtttcagcag aaaccaggga
aagcccctaa gtcccttatc tatgctgcat ccagtttgca 1140 aagtggggtc
ccatcaaagt tcagcggcag tggatctggg acagatttca gtctcaccat 1200
cagcagcctg cagcctgaag attttgcaac ttattactgc caacagtata atagttaccc
1260 gatcaccttc ggccaaggga cacgactgga gattaaacga actgtggctg
caccatctgt 1320 cttcatcttc ccgccatctg atgagcagtt gaaatctgga
actgctagcg ttgtgtgcct 1380 gctgaataac ttctatccca gagaggccaa
agtacagtgg aaggtggata acgccctcca 1440 atcgggtaac tcccaggaga
gtgtcacaga gcaggacagc aaggacagca cctacagcct 1500 cagcagcacc
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 1560
agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta
1620 ggaattcgcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca
gcctcgactg 1680 tgccttctag ttgccagcca tctgttgttt gcccctcccc
cgtgccttcc ttgaccctgg 1740 aaggtgccac tcccactgtc ctttcctaat
aaaatgagga aattgcatcg cattgtctga 1800 gtaggtgtca ttctattctg
gggggtgggg tggggcagga cagcaagggg gaggattggg 1860 aagacaatag
caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 1920
ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
1980 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
cccgctcctt 2040 tcgctttctt cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa gctctaaatc 2100 ggggcatccc tttagggttc cgatttagtg
ctttacggca cctcgacccc aaaaaacttg 2160 attagggtga tggttcacgt
agtgggccat cgccctgata gacggttttt cgccctttga 2220 cgttggagtc
cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2280
ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa
2340 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
tgtgtcagtt 2400 agggtgtgga aagtccccag gctccccagg caggcagaag
tatgcaaagc atgcatctca 2460 attagtcagc aaccaggtgt ggaaagtccc
caggctcccc agcaggcaga agtatgcaaa 2520 gcatgcatct caattagtca
gcaaccatag tcccgcccct aactccgccc atcccgcccc 2580 taactccgcc
cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 2640
cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
2700 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
ggatctgatc 2760 aagagacagg atgaggatcg tttcgcatga ttgaacaaga
tggattgcac gcaggttctc 2820 cggccgcttg ggtggagagg ctattcggct
atgactgggc acaacagaca atcggctgct 2880 ctgatgccgc cgtgttccgg
ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 2940 acctgtccgg
tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca 3000
cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
3060 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
cctgccgaga 3120 aagtatccat catggctgat gcaatgcggc ggctgcatac
gcttgatccg gctacctgcc 3180 cattcgacca ccaagcgaaa catcgcatcg
agcgagcacg tactcggatg gaagccggtc 3240 ttgtcgatca ggatgatctg
gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3300 ccaggctcaa
ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3360
gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
3420 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
gctgaagagc 3480 ttggcggcga atgggctgac
cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3540 agcgcatcgc
cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 3600
aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
3660 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
tcctccagcg 3720 cggggatctc atgctggagt tcttcgccca ccccaacttg
tttattgcag cttataatgg 3780 ttacaaataa agcaatagca tcacaaattt
cacaaataaa gcattttttt cactgcattc 3840 tagttgtggt ttgtccaaac
tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 3900 tagctagagc
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
4020 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
cgggaaacct 4080 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg 4140 gcgctcttcc gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc 4200 ggtatcagct cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg 4260 aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4320
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
4380 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
gaagctccct 4440 cgtgcgctct cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc 4500 gggaagcgtg gcgctttctc aatgctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt 4560 tcgctccaag ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 4620 cggtaactat
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 4680
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
4740 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
tgctgaagcc 4800 agttaccttc ggaaaaagag ttggtagctc ttgatccggc
aaacaaacca ccgctggtag 4860 cggtggtttt tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga 4920 tcctttgatc ttttctacgg
ggtctgacgc tcagtggaac gaaaactcac gttaagggat 4980 tttggtcatg
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
5100 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
cctgactccc 5160 cgtcgtgtag ataactacga tacgggaggg cttaccatct
ggccccagtg ctgcaatgat 5220 accgcgagac ccacgctcac cggctccaga
tttatcagca ataaaccagc cagccggaag 5280 ggccgagcgc agaagtggtc
ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5340 ccgggaagct
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5400
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
5460 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
gctccttcgg 5520 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
tcactcatgg ttatggcagc 5580 actgcataat tctcttactg tcatgccatc
cgtaagatgc ttttctgtga ctggtgagta 5640 ctcaaccaag tcattctgag
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 5700 aatacgggat
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
5820 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc 5880 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat 5940 actcatactc ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag 6000 cggatacata tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6060 ccgaaaagtg
ccacctgacg tc 6082 10 6082 DNA Artificial Sequence Plasmid 10
gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg
atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag
ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccctgc
tcagctcctg gggctcctgc tgctctgttt 960 cccaggtgcc agatgtgaca
tccagatgac ccagtctcca tcctcactgt ctgcatctgt 1020 aggagacaga
gtcaccatca cttgtcgggc gagtcagggc attagccatt atttagcctg 1080
gtttcagcag aaaccaggga aagcccctaa gtccctgatc tatgctgcat ccagtttgca
1140 aagtggggtc ccatcaaagt tcagcggcag tggatctggg acagatttca
ctctcaccat 1200 cagcagccta cagcctgaag attttgcaac ttattactgc
caacagtata atagtttccc 1260 gctcactttc ggcggaggga ccaaggtgga
gatcaaacga actgtggctg caccatctgt 1320 cttcatcttc ccgccatctg
atgagcagtt gaaatctgga actgctagcg ttgtgtgcct 1380 gctgaataac
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 1440
atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct
1500 cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct
acgcctgcga 1560 agtcacccat cagggcctga gctcgcccgt cacaaagagc
ttcaacaggg gagagtgtta 1620 ggaattcgcg gccgctcgag tctagagggc
ccgtttaaac ccgctgatca gcctcgactg 1680 tgccttctag ttgccagcca
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1740 aaggtgccac
tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1800
gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
1860 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
gcggaaagaa 1920 ccagctgggg ctctaggggg tatccccacg cgccctgtag
cggcgcatta agcgcggcgg 1980 gtgtggtggt tacgcgcagc gtgaccgcta
cacttgccag cgccctagcg cccgctcctt 2040 tcgctttctt cccttccttt
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2100 ggggcatccc
tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2160
attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
2220 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
acactcaacc 2280 ctatctcggt ctattctttt gatttataag ggattttggg
gatttcggcc tattggttaa 2340 aaaatgagct gatttaacaa aaatttaacg
cgaattaatt ctgtggaatg tgtgtcagtt 2400 agggtgtgga aagtccccag
gctccccagg caggcagaag tatgcaaagc atgcatctca 2460 attagtcagc
aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 2520
gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
2580 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
tttatttatg 2640 cagaggccga ggccgcctct gcctctgagc tattccagaa
gtagtgagga ggcttttttg 2700 gaggcctagg cttttgcaaa aagctcccgg
gagcttgtat atccattttc ggatctgatc 2760 aagagacagg atgaggatcg
tttcgcatga ttgaacaaga tggattgcac gcaggttctc 2820 cggccgcttg
ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 2880
ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
2940 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
tggctggcca 3000 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
tgaagcggga agggactggc 3060 tgctattggg cgaagtgccg gggcaggatc
tcctgtcatc tcaccttgct cctgccgaga 3120 aagtatccat catggctgat
gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 3180 cattcgacca
ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 3240
ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
3300 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
ggcgatgcct 3360 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
attcatcgac tgtggccggc 3420 tgggtgtggc ggaccgctat caggacatag
cgttggctac ccgtgatatt gctgaagagc 3480 ttggcggcga atgggctgac
cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3540 agcgcatcgc
cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga 3600
aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
3660 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
tcctccagcg 3720 cggggatctc atgctggagt tcttcgccca ccccaacttg
tttattgcag cttataatgg 3780 ttacaaataa agcaatagca tcacaaattt
cacaaataaa gcattttttt cactgcattc 3840 tagttgtggt ttgtccaaac
tcatcaatgt atcttatcat gtctgtatac cgtcgacctc 3900 tagctagagc
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 3960
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
4020 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
cgggaaacct 4080 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
agaggcggtt tgcgtattgg 4140 gcgctcttcc gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc 4200 ggtatcagct cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg 4260 aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 4320
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
4380 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
gaagctccct 4440 cgtgcgctct cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc 4500 gggaagcgtg gcgctttctc aatgctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt 4560 tcgctccaag ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 4620 cggtaactat
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 4680
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
4740 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
tgctgaagcc 4800 agttaccttc ggaaaaagag ttggtagctc ttgatccggc
aaacaaacca ccgctggtag 4860 cggtggtttt tttgtttgca agcagcagat
tacgcgcaga aaaaaaggat ctcaagaaga 4920 tcctttgatc ttttctacgg
ggtctgacgc tcagtggaac gaaaactcac gttaagggat 4980 tttggtcatg
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 5040
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
5100 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
cctgactccc 5160 cgtcgtgtag ataactacga tacgggaggg cttaccatct
ggccccagtg ctgcaatgat 5220 accgcgagac ccacgctcac cggctccaga
tttatcagca ataaaccagc cagccggaag 5280 ggccgagcgc agaagtggtc
ctgcaacttt atccgcctcc atccagtcta ttaattgttg 5340 ccgggaagct
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 5400
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
5460 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
gctccttcgg 5520 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
tcactcatgg ttatggcagc 5580 actgcataat tctcttactg tcatgccatc
cgtaagatgc ttttctgtga ctggtgagta 5640 ctcaaccaag tcattctgag
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 5700 aatacgggat
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 5760
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
5820 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
ctgggtgagc 5880 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
gcgacacgga aatgttgaat 5940 actcatactc ttcctttttc aatattattg
aagcatttat cagggttatt gtctcatgag 6000 cggatacata tttgaatgta
tttagaaaaa taaacaaata ggggttccgc gcacatttcc 6060 ccgaaaagtg
ccacctgacg tc 6082 11 6085 DNA Artificial Sequence Plasmid 11
gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg
atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa
tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta
catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag
ggagacccaa gctggctaga 900 aagcttggat ctcaccatga gggtccccgc
tcagcttctc ttccttctgc tactctggct 960 cccagatacc actggaggaa
tagtgatgac gcagtctcca gccaccctgt ctgtgtctcc 1020 aggggaaaga
gccaccctct cctgcaggac cagtcagagt attggctgga acttagcctg 1080
gtaccaacag aaacctggcc aggctcccag gctcctcatc tatggtgcat cttccaggac
1140 cactggtatc ccagccaggt tcagtggcag tgggtctggg acagagttca
ctctcaccat 1200 cagcagcctg cagtctgaag attctgcagt ttattactgt
cagcattatg ataactggcc 1260 catgtgcagt tttggccagg ggaccgagct
ggagatcaaa cgaactgtgg ctgcaccatc 1320 tgtcttcatc ttcccgccat
ctgatgagca gttgaaatct ggaactgcta gcgttgtgtg 1380 cctgctgaat
aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct 1440
ccaatcgggt aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag
1500 cctcagcagc accctgacgc tgagcaaagc agactacgag aaacacaaag
tctacgcctg 1560 cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag
agcttcaaca ggggagagtg 1620 ttaggaattc gcggccgctc gagtctagag
ggcccgttta aacccgctga tcagcctcga 1680 ctgtgccttc tagttgccag
ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 1740 tggaaggtgc
cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 1800
tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
1860 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
gaggcggaaa 1920 gaaccagctg gggctctagg gggtatcccc acgcgccctg
tagcggcgca ttaagcgcgg 1980 cgggtgtggt ggttacgcgc agcgtgaccg
ctacacttgc cagcgcccta gcgcccgctc 2040 ctttcgcttt cttcccttcc
tttctcgcca cgttcgccgg ctttccccgt caagctctaa 2100 atcggggcat
ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 2160
ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
2220 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
acaacactca 2280 accctatctc ggtctattct tttgatttat aagggatttt
ggggatttcg gcctattggt 2340 taaaaaatga gctgatttaa caaaaattta
acgcgaatta attctgtgga atgtgtgtca 2400 gttagggtgt ggaaagtccc
caggctcccc aggcaggcag aagtatgcaa agcatgcatc 2460 tcaattagtc
agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 2520
aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
2580 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
ttttttattt 2640 atgcagaggc cgaggccgcc tctgcctctg agctattcca
gaagtagtga ggaggctttt 2700 ttggaggcct aggcttttgc aaaaagctcc
cgggagcttg tatatccatt ttcggatctg 2760 atcaagagac aggatgagga
tcgtttcgca tgattgaaca agatggattg cacgcaggtt 2820 ctccggccgc
ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct 2880
gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
2940 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
tcgtggctgg 3000 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
cactgaagcg ggaagggact 3060 ggctgctatt gggcgaagtg ccggggcagg
atctcctgtc atctcacctt gctcctgccg 3120 agaaagtatc catcatggct
gatgcaatgc ggcggctgca tacgcttgat ccggctacct 3180 gcccattcga
ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg 3240
gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
3300 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
catggcgatg 3360 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
tggattcatc gactgtggcc 3420 ggctgggtgt ggcggaccgc tatcaggaca
tagcgttggc tacccgtgat attgctgaag 3480 agcttggcgg cgaatgggct
gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt 3540 cgcagcgcat
cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt 3600
cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
3660 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
tgatcctcca 3720 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
ttgtttattg cagcttataa 3780 tggttacaaa taaagcaata gcatcacaaa
tttcacaaat aaagcatttt tttcactgca 3840 ttctagttgt ggtttgtcca
aactcatcaa tgtatcttat catgtctgta taccgtcgac 3900 ctctagctag
agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 3960
gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
4020 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
agtcgggaaa 4080 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
gggagaggcg gtttgcgtat 4140 tgggcgctct tccgcttcct cgctcactga
ctcgctgcgc tcggtcgttc ggctgcggcg 4200 agcggtatca gctcactcaa
aggcggtaat acggttatcc acagaatcag gggataacgc 4260 aggaaagaac
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4320
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
4380 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
ctggaagctc 4440 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
tacctgtccg cctttctccc 4500 ttcgggaagc gtggcgcttt ctcaatgctc
acgctgtagg tatctcagtt cggtgtaggt 4560 cgttcgctcc aagctgggct
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4620 atccggtaac
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4680
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
4740 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
ctctgctgaa 4800 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
ggcaaacaaa ccaccgctgg 4860 tagcggtggt ttttttgttt gcaagcagca
gattacgcgc agaaaaaaag gatctcaaga 4920 agatcctttg atcttttcta
cggggtctga cgctcagtgg aacgaaaact cacgttaagg 4980 gattttggtc
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 5040
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
5100 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact 5160 ccccgtcgtg tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat 5220 gataccgcga gacccacgct caccggctcc
agatttatca gcaataaacc agccagccgg 5280 aagggccgag cgcagaagtg
gtcctgcaac tttatccgcc tccatccagt ctattaattg 5340 ttgccgggaa
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5400
tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
5460 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
ttagctcctt 5520 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
ttatcactca tggttatggc 5580 agcactgcat aattctctta ctgtcatgcc
atccgtaaga tgcttttctg tgactggtga 5640 gtactcaacc aagtcattct
gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5700 gtcaatacgg
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5760
acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
5820 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
tttctgggtg 5880 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
agggcgacac ggaaatgttg 5940 aatactcata ctcttccttt ttcaatatta
ttgaagcatt tatcagggtt attgtctcat 6000 gagcggatac atatttgaat
gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6060 tccccgaaaa
gtgccacctg acgtc 6085 12 6097 DNA Artificial Sequence Plasmid 12
gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
60 ccgcatagtt aagccagtat
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat
ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggac
tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag
gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg
gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga 900
aagcttggat ctcaccatga gggtccctgc tcagctcctg gggctgctaa tgctctggat
960 acctggatcc agtgcagata ttgtgatgac ccagactcca ctctctctgt
ccgtcacccc 1020 tggacagccg gcctccatct cctgcaagtc tagtcagagc
ctcctgcata gtgatggaaa 1080 gacctttttg tattggtatc tgcagaagcc
aggccagcct ccacagctcc tgatctatga 1140 ggtttccaac cggttctctg
gagtgccaga taggttcagt ggcagcgggt cagggacaga 1200 tttcacactg
aaaatcagcc gggtggaggc tgaggatgtt gggctttatt actgcatgca 1260
aagtatacag cttccgctca ctttcggcgg agggaccaag gtggagatca aacgaactgt
1320 ggctgcacca tctgtcttca tcttcccgcc atctgatgag cagttgaaat
ctggaactgc 1380 tagcgttgtg tgcctgctga ataacttcta tcccagagag
gccaaagtac agtggaaggt 1440 ggataacgcc ctccaatcgg gtaactccca
ggagagtgtc acagagcagg acagcaagga 1500 cagcacctac agcctcagca
gcaccctgac gctgagcaaa gcagactacg agaaacacaa 1560 agtctacgcc
tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa 1620
caggggagag tgttaggaat tcgcggccgc tcgagtctag agggcccgtt taaacccgct
1680 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
tcccccgtgc 1740 cttccttgac cctggaaggt gccactccca ctgtcctttc
ctaataaaat gaggaaattg 1800 catcgcattg tctgagtagg tgtcattcta
ttctgggggg tggggtgggg caggacagca 1860 agggggagga ttgggaagac
aatagcaggc atgctgggga tgcggtgggc tctatggctt 1920 ctgaggcgga
aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg 1980
cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
2040 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
ggctttcccc 2100 gtcaagctct aaatcggggc atccctttag ggttccgatt
tagtgcttta cggcacctcg 2160 accccaaaaa acttgattag ggtgatggtt
cacgtagtgg gccatcgccc tgatagacgg 2220 tttttcgccc tttgacgttg
gagtccacgt tctttaatag tggactcttg ttccaaactg 2280 gaacaacact
caaccctatc tcggtctatt cttttgattt ataagggatt ttggggattt 2340
cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
2400 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccaggcaggc
agaagtatgc 2460 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
gtccccaggc tccccagcag 2520 gcagaagtat gcaaagcatg catctcaatt
agtcagcaac catagtcccg cccctaactc 2580 cgcccatccc gcccctaact
ccgcccagtt ccgcccattc tccgccccat ggctgactaa 2640 ttttttttat
ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 2700
gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
2760 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
caagatggat 2820 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
cggctatgac tgggcacaac 2880 agacaatcgg ctgctctgat gccgccgtgt
tccggctgtc agcgcagggg cgcccggttc 2940 tttttgtcaa gaccgacctg
tccggtgccc tgaatgaact gcaggacgag gcagcgcggc 3000 tatcgtggct
ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 3060
cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
3120 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
catacgcttg 3180 atccggctac ctgcccattc gaccaccaag cgaaacatcg
catcgagcga gcacgtactc 3240 ggatggaagc cggtcttgtc gatcaggatg
atctggacga agagcatcag gggctcgcgc 3300 cagccgaact gttcgccagg
ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga 3360 cccatggcga
tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 3420
tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
3480 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
tacggtatcg 3540 ccgctcccga ttcgcagcgc atcgccttct atcgccttct
tgacgagttc ttctgagcgg 3600 gactctgggg ttcgaaatga ccgaccaagc
gacgcccaac ctgccatcac gagatttcga 3660 ttccaccgcc gccttctatg
aaaggttggg cttcggaatc gttttccggg acgccggctg 3720 gatgatcctc
cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 3780
tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
3840 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
atcatgtctg 3900 tataccgtcg acctctagct agagcttggc gtaatcatgg
tcatagctgt ttcctgtgtg 3960 aaattgttat ccgctcacaa ttccacacaa
catacgagcc ggaagcataa agtgtaaagc 4020 ctggggtgcc taatgagtga
gctaactcac attaattgcg ttgcgctcac tgcccgcttt 4080 ccagtcggga
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 4140
cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
4200 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
ccacagaatc 4260 aggggataac gcaggaaaga acatgtgagc aaaaggccag
caaaaggcca ggaaccgtaa 4320 aaaggccgcg ttgctggcgt ttttccatag
gctccgcccc cctgacgagc atcacaaaaa 4380 tcgacgctca agtcagaggt
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4440 ccctggaagc
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 4500
cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
4560 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
ttcagcccga 4620 ccgctgcgcc ttatccggta actatcgtct tgagtccaac
ccggtaagac acgacttatc 4680 gccactggca gcagccactg gtaacaggat
tagcagagcg aggtatgtag gcggtgctac 4740 agagttcttg aagtggtggc
ctaactacgg ctacactaga aggacagtat ttggtatctg 4800 cgctctgctg
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 4860
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
4920 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
ggaacgaaaa 4980 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
atcttcacct agatcctttt 5040 aaattaaaaa tgaagtttta aatcaatcta
aagtatatat gagtaaactt ggtctgacag 5100 ttaccaatgc ttaatcagtg
aggcacctat ctcagcgatc tgtctatttc gttcatccat 5160 agttgcctga
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 5220
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
5280 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
cctccatcca 5340 gtctattaat tgttgccggg aagctagagt aagtagttcg
ccagttaata gtttgcgcaa 5400 cgttgttgcc attgctacag gcatcgtggt
gtcacgctcg tcgtttggta tggcttcatt 5460 cagctccggt tcccaacgat
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 5520 ggttagctcc
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 5580
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
5640 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
gaccgagttg 5700 ctcttgcccg gcgtcaatac gggataatac cgcgccacat
agcagaactt taaaagtgct 5760 catcattgga aaacgttctt cggggcgaaa
actctcaagg atcttaccgc tgttgagatc 5820 cagttcgatg taacccactc
gtgcacccaa ctgatcttca gcatctttta ctttcaccag 5880 cgtttctggg
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 5940
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
6000 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
aaataggggt 6060 tccgcgcaca tttccccgaa aagtgccacc tgacgtc 6097 13
6094 DNA Artificial Sequence Plasmid 13 gacggatcgg gagatctccc
gatcccctat ggtcgactct cagtacaatc tgctctgatg 60 ccgcatagtt
aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc
attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa
atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata
atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca
atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt
acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
900 aagcttggat ctcaccatgg tgttgcagac ccaggtcttc atttctctgt
tactctggat 960 ctctggtgcc tacggggaca tcgtgatgac ccagtctcca
gactccctgg ctgtgtctct 1020 gggcgagagg gccaccatca actgcaagtc
caaccagagt gtcttacaca gctccaacaa 1080 taagaactat ttagcttggt
accagcagaa accaggacag cctcctaaat tgctcattta 1140 ttgggcattc
ctccgggaat ccggggtccc tgaccgcttc agtggcagcg ggtctgggac 1200
agatttcact ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca
1260 ccaatattat tctactttat atactttcgg cggagggacc aaggtagaga
tcaaacgaac 1320 ygtggctgca ccatctgtct tcatcttccc gccatctgat
gagcagttga aatctggaac 1380 tgctagcgtt gtgtgcctgc tgaataactt
ctatcccaga gaggccaaag tacagtggaa 1440 ggtggataac gccctccaat
cgggtaactc ccaggagagt gtcacagagc aggacagcaa 1500 ggacagcacc
tacagcctca gcagcaccct gacgctgagc aaagcagact acgagaaaca 1560
caaagtctac gcctgcgaag tcacccatca gggcctgagc tcgcccgtca caaagagctt
1620 caacagggga gagtgttagg cggccgctcg agtctagagg gcccgtttaa
acccgctgat 1680 cagcctcgac tgtgccttct agttgccagc catctgttgt
ttgcccctcc cccgtgcctt 1740 ccttgaccct ggaaggtgcc actcccactg
tcctttccta ataaaatgag gaaattgcat 1800 cgcattgtct gagtaggtgt
cattctattc tggggggtgg ggtggggcag gacagcaagg 1860 gggaggattg
ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 1920
aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
1980 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
agcgccctag 2040 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
gttcgccggc tttccccgtc 2100 aagctctaaa tcggggcatc cctttagggt
tccgatttag tgctttacgg cacctcgacc 2160 ccaaaaaact tgattagggt
gatggttcac gtagtgggcc atcgccctga tagacggttt 2220 ttcgcccttt
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 2280
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg
2340 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
ttctgtggaa 2400 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
ggcaggcaga agtatgcaaa 2460 gcatgcatct caattagtca gcaaccaggt
gtggaaagtc cccaggctcc ccagcaggca 2520 gaagtatgca aagcatgcat
ctcaattagt cagcaaccat agtcccgccc ctaactccgc 2580 ccatcccgcc
cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 2640
tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
2700 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
atatccattt 2760 tcggatctga tcaagagaca ggatgaggat cgtttcgcat
gattgaacaa gatggattgc 2820 acgcaggttc tccggccgct tgggtggaga
ggctattcgg ctatgactgg gcacaacaga 2880 caatcggctg ctctgatgcc
gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 2940 ttgtcaagac
cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat 3000
cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
3060 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
tctcaccttg 3120 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
gcggctgcat acgcttgatc 3180 cggctacctg cccattcgac caccaagcga
aacatcgcat cgagcgagca cgtactcgga 3240 tggaagccgg tcttgtcgat
caggatgatc tggacgaaga gcatcagggg ctcgcgccag 3300 ccgaactgtt
cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 3360
atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
3420 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
acccgtgata 3480 ttgctgaaga gcttggcggc gaatgggctg accgcttcct
cgtgctttac ggtatcgccg 3540 ctcccgattc gcagcgcatc gccttctatc
gccttcttga cgagttcttc tgagcgggac 3600 tctggggttc gaaatgaccg
accaagcgac gcccaacctg ccatcacgag atttcgattc 3660 caccgccgcc
ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat 3720
gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
3780 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
aagcattttt 3840 ttcactgcat tctagttgtg gtttgtccaa actcatcaat
gtatcttatc atgtctgtat 3900 accgtcgacc tctagctaga gcttggcgta
atcatggtca tagctgtttc ctgtgtgaaa 3960 ttgttatccg ctcacaattc
cacacaacat acgagccgga agcataaagt gtaaagcctg 4020 gggtgcctaa
tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 4080
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
4140 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
cggtcgttcg 4200 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca cagaatcagg 4260 ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa aaggccagga accgtaaaaa 4320 ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc acaaaaatcg 4380 acgctcaagt
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4440
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
4500 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
atctcagttc 4560 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
ccccccgttc agcccgaccg 4620 ctgcgcctta tccggtaact atcgtcttga
gtccaacccg gtaagacacg acttatcgcc 4680 actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg gtgctacaga 4740 gttcttgaag
tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4800
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
4860 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 4920 atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagtgga acgaaaactc 4980 acgttaaggg attttggtca tgagattatc
aaaaaggatc ttcacctaga tccttttaaa 5040 ttaaaaatga agttttaaat
caatctaaag tatatatgag taaacttggt ctgacagtta 5100 ccaatgctta
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5160
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
5220 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
caataaacca 5280 gccagccgga agggccgagc gcagaagtgg tcctgcaact
ttatccgcct ccatccagtc 5340 tattaattgt tgccgggaag ctagagtaag
tagttcgcca gttaatagtt tgcgcaacgt 5400 tgttgccatt gctacaggca
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5460 ctccggttcc
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5520
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
5580 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
gcttttctgt 5640 gactggtgag tactcaacca agtcattctg agaatagtgt
atgcggcgac cgagttgctc 5700 ttgcccggcg tcaatacggg ataataccgc
gccacatagc agaactttaa aagtgctcat 5760 cattggaaaa cgttcttcgg
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 5820 ttcgatgtaa
cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 5880
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
5940 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
atcagggtta 6000 ttgtctcatg agcggataca tatttgaatg tatttagaaa
aataaacaaa taggggttcc 6060 gcgcacattt ccccgaaaag tgccacctga cgtc
6094 14 481 DNA Artificial Sequence Includes BamHI/Bg1II cloning
junction, signal peptide, V region, portion of C region and
3'XbaI/NheI (heavy) or NheI (light) cloning junction 14 ggatctcacc
atggagttgg gactgcgctg gggcttcctc gttgctcttt taagaggtgt 60
ccagtgtcag gtgcaattgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct
120 gagactctcc tgtgcagcgt ctggattcgc cttcagtaga tatggcatgc
actgggtccg 180 ccaggctcca ggcaaggggc tggagtgggt ggcagttata
tggtatgatg gaagtaataa 240 atactatgca gactccgtga agggccgatt
caccatctcc agagacaatt ccaagaacac 300 gcagtatctg caaatgaaca
gcctgagagc cgaggacacg gctgtgtatt actgtgcgag 360 aggcggtgac
ttcctctact actactatta cggtatggac gtctggggcc aagggaccac 420
ggtcaccgtc tcctcagcct ccaccaaggg cccatcggtc ttccccctgg caccctctag
480 c 481 15 142 PRT Homo sapiens 15 Met Glu Leu Gly Leu Arg Trp
Gly Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe 35 40 45 Ser
Arg Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala
65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn 85 90 95 Thr Gln Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Gly Gly Asp Phe Leu
Tyr Tyr Tyr Tyr Tyr Gly 115 120 125 Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 130 135 140 16 463 DNA Artificial Sequence
Includes BamHI/Bg1II cloning junction, signal peptide, V region,
portion of C region and 3'XbaI/NheI (heavy) or NheI (light) cloning
junction 16 ggatctcacc atgagggtcc ctgctcagct cctgggactc ctgctgctct
ggctcccaga 60 taccagatgt gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga 120 cagagtcacc atcacttgcc gggcgagtca
gggcattagc aattatttag cctggtatca 180 gcagaaaaca gggaaagttc
ctaagttcct gatctatgaa gcatccactt tgcaatcagg 240 ggtcccatct
cggttcagtg gcggtggatc tgggacagat ttcactctca ccatcagcag 300
cctgcagcct gaagatgttg caacttatta ctgtcaaaat tataacagtg ccccattcac
360 tttcggccct gggaccaaag tggatatcaa acgaactgtg gctgcaccct
ctgtcttcat 420 cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 463
17 127 PRT Homo sapiens 17 Met Arg Val Pro Ala Gln Leu Leu Gly Leu
Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Arg Cys Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly 35 40 45 Ile Ser Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Thr Gly Lys Val Pro 50 55 60 Lys Phe
Leu Ile Tyr Glu Ala Ser Thr Leu Gln Ser Gly Val Pro Ser 65 70 75 80
Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85
90
95 Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn Tyr Asn
100 105 110 Ser Ala Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 115 120 125 18 508 DNA Artificial Sequence Includes BamHI/Bg1II
cloning junction, signal peptide, V region, portion of C region and
3'XbaI/NheI (heavy) or NheI (light) cloning junction 18 ggatctcacc
atggggtcaa ccgccatcct caccatggag ttggggctgc gctgggttct 60
cctcgttgct cttttaagag gtgtccagtg tcaggtgcag ctggtggagt ctgggggagg
120 cgtggtccag cctgggaggt ccctgagact ctcctgtgca gcgtctggat
tcaccttcag 180 taactatgtc atgcactggg tccgccaggc tccaggcaag
gggctggagt gggtggcaat 240 tatatggtat gatggaagta ataaatacta
tgcagactcc gtgaagggcc gattcaccat 300 ctccagagac aattccaaga
acacgctgta tctgcaaatg aacagcctga gagccgagga 360 cacggctgtg
tattactgtg cgggtggata taactggaac tacgagtacc actactacgg 420
tatggacgtc tggggccaag ggaccacggt caccgtctcc tcagcctcca ccaagggccc
480 atcggtcttc cccctggcac cctctagc 508 19 143 PRT Homo sapiens 19
Met Glu Leu Gly Leu Arg Trp Val Leu Leu Val Ala Leu Leu Arg Gly 1 5
10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45 Ser Asn Tyr Val Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Ile Ile Trp Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Gly Gly Tyr Asn Trp Asn Tyr Glu Tyr His Tyr Tyr 115 120 125 Gly
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140
20 463 DNA Artificial Sequence Includes BamHI/Bg1II cloning
junction, signal peptide, V region, portion of C region and
3'XbaI/NheI (heavy) or NheI (light) cloning junction 20 ggatctcacc
atgagggtcc ccgctcagct cctggggctc ctgctgctct gtttcccagg 60
tgccagatgt gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
120 cagagtcacc atcacttgtc gggcgagtca gggcattacc aattatttag
cctggtttca 180 gcagaaacca gggaaagccc ctaagtccct tatctatgct
gcatccagtt tgcaaagtgg 240 ggtcccatca aagttcagcg gcagtggatc
tgggacagat ttcagtctca ccatcagcag 300 cctgcagcct gaagattttg
caacttatta ctgccaacag tataatagtt acccgatcac 360 cttcggccaa
gggacacgac tggagattaa acgaactgtg gctgcaccat ctgtcttcat 420
cttcccgcca tctgatgagc agttgaaatc tggaactgct agc 463 21 127 PRT Homo
sapiens 21 Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys
Phe Pro 1 5 10 15 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly 35 40 45 Ile Thr Asn Tyr Leu Ala Trp Phe
Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Ser Leu Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser 65 70 75 80 Lys Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser 85 90 95 Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110
Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120
125 22 490 DNA Artificial Sequence Includes BamHI/Bg1II cloning
junction, signal peptide, V region, portion of C region and
3'XbaI/NheI (heavy) or NheI (light) cloning junction 22 ggatctcacc
atggagttgg gacttagctg ggttttcctc gttgctcttt taagaggtgt 60
ccagtgtcag gtccagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct
120 gagactctcc tgtgcagcgt ctggattcac cttcagtagc tatggcatgc
actgggtccg 180 ccaggctcca ggcaaggggc tggactgggt ggcaattatt
tggcatgatg gaagtaataa 240 atactatgca gactccgtga agggccgatt
caccatctcc agagacaatt ccaagaagac 300 gctgtacctg caaatgaaca
gtttgagagc cgaggacacg gctgtgtatt actgtgcgag 360 agcttgggcc
tatgactacg gtgactatga atactacttc ggtatggacg tctggggcca 420
agggaccacg gtcaccgtct cctcagcctc caccaagggc ccatcggtct tccccctggc
480 accctctagc 490 23 145 PRT Homo sapiens 23 Met Glu Leu Gly Leu
Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40
45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60 Asp Trp Val Ala Ile Ile Trp His Asp Gly Ser Asn Lys Tyr
Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Lys 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Ala Trp Ala
Tyr Asp Tyr Gly Asp Tyr Glu Tyr 115 120 125 Tyr Phe Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser 130 135 140 Ser 145 24 463
DNA Artificial Sequence Includes BamHI/Bg1II cloning junction,
signal peptide, V region, portion of C region and 3'XbaI/NheI
(heavy) or NheI (light) cloning junction 24 ggatctcacc atgagggtcc
ctgctcagct cctggggctc ctgctgctct gtttcccagg 60 tgccagatgt
gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga 120
cagagtcacc atcacttgtc gggcgagtca gggcattagc cattatttag cctggtttca
180 gcagaaacca gggaaagccc ctaagtccct gatctatgct gcatccagtt
tgcaaagtgg 240 ggtcccatca aagttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag 300 cctacagcct gaagattttg caacttatta
ctgccaacag tataatagtt tcccgctcac 360 tttcggcgga gggaccaagg
tggagatcaa acgaactgtg gctgcaccat ctgtcttcat 420 cttcccgcca
tctgatgagc agttgaaatc tggaactgct agc 463 25 127 PRT Homo sapiens 25
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys Phe Pro 1 5
10 15 Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly 35 40 45 Ile Ser His Tyr Leu Ala Trp Phe Gln Gln Lys
Pro Gly Lys Ala Pro 50 55 60 Lys Ser Leu Ile Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser 65 70 75 80 Lys Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn 100 105 110 Ser Phe Pro
Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 26 469
DNA Artificial Sequence Includes BamHI/Bg1II cloning junction,
signal peptide, V region, portion of C region and 3'XbaI/NheI
(heavy) or NheI (light) cloning junction 26 ggatcccacc atggggtcaa
ccgtcatcct cgccctcctc ctggctgttc tccaaggagt 60 ctgtgccgag
gtgcagctgg tgcagtctgg agcagaggtg aaaaagcccg gggagtctct 120
gaagatctcc tgtaagggtt ctggatacag ctttaccagt tactggatcg gctgggtgcg
180 ccagatgccc gggaaaggcc tggagtggat ggggatcatc tatcctggtg
actctgatac 240 cagatacagc ccgtccttcc aaggccaggt caccatctca
gccgacaagt ccatcagcac 300 cgcctacctg cagtggagca gcctgaaggc
ctcggacacc gccatgtatt actgtgcgag 360 acggatggca gcagctggcc
cctttgacta ctggggccag ggaaccctgg tcaccgtctc 420 ctcagcctcc
accaagggcc catcggtctt ccccctggca ccctctagc 469 27 138 PRT Homo
sapiens 27 Met Gly Ser Thr Val Ile Leu Ala Leu Leu Leu Ala Val Leu
Gln Gly 1 5 10 15 Val Cys Ala Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys 20 25 30 Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Ser Phe 35 40 45 Thr Ser Tyr Trp Ile Gly Trp Val
Arg Gln Met Pro Gly Lys Gly Leu 50 55 60 Glu Trp Met Gly Ile Ile
Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser 65 70 75 80 Pro Ser Phe Gln
Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser 85 90 95 Thr Ala
Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met 100 105 110
Tyr Tyr Cys Ala Arg Arg Met Ala Ala Ala Gly Pro Phe Asp Tyr Trp 115
120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 28 466 DNA
Artificial Sequence Includes BamHI/Bg1II cloning junction, signal
peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or
NheI (light) cloning junction 28 ggatctcacc atgagggtcc ccgctcagct
tctcttcctt ctgctactct ggctcccaga 60 taccactgga ggaatagtga
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga 120 aagagccacc
ctctcctgca ggaccagtca gagtattggc tggaacttag cctggtacca 180
acagaaacct ggccaggctc ccaggctcct catctatggt gcatcttcca ggaccactgg
240 tatcccagcc aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag 300 cctgcagtct gaagattctg cagtttatta ctgtcagcat
tatgataact ggcccatgtg 360 cagttttggc caggggaccg agctggagat
caaacgaact gtggctgcac catctgtctt 420 catcttcccg ccatctgatg
agcagttgaa atctggaact gctagc 466 29 128 PRT Homo sapiens 29 Met Arg
Val Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15
Asp Thr Thr Gly Gly Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser 20
25 30 Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln
Ser 35 40 45 Ile Gly Trp Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Thr
Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu Gln Ser Glu Asp Ser
Ala Val Tyr Tyr Cys Gln His Tyr Asp 100 105 110 Asn Trp Pro Met Cys
Ser Phe Gly Gln Gly Thr Glu Leu Glu Ile Lys 115 120 125 30 487 DNA
Artificial Sequence Includes BamHI/Bg1II cloning junction, signal
peptide, V region, portion of C region and 3'XbaI/NheI (heavy) or
NheI (light) cloning junction 30 ggatctcacc atggagtttg ggctgtgctg
gattttcctc gttgctcttt taagaggtgt 60 ccagtgtcag gtgcagctgg
tggagtctgg gggaggcgtg gtccagcctg ggaggtccct 120 gagactctcc
tgtgcagcct ctggattcac cttcattagc tatggcatgc actgggtccg 180
ccaggctcca ggcaaggggc tggagtgggt ggcagttata tcatatgatg gaagtaataa
240 atactatgca gactccgtga agggccgatt caccatctcc agagacaatt
ccaagaacac 300 gctgtatctg caaatgaaca gcctgagagc tgaggacacg
gctgtgtatt actgtgcgag 360 agtattagtg ggagctttat attattataa
ctactacggg atggacgtct ggggccaagg 420 gaccacggtc accgtctcct
cagcctccac caagggccca tcggtcttcc ccctggcacc 480 ctctagc 487 31 144
PRT Homo sapiens 31 Met Glu Phe Gly Leu Cys Trp Ile Phe Leu Val Ala
Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ile Ser Tyr Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala 65 70 75 80 Asp Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100
105 110 Tyr Tyr Cys Ala Arg Val Leu Val Gly Ala Leu Tyr Tyr Tyr Asn
Tyr 115 120 125 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 130 135 140 32 478 DNA Artificial Sequence Includes
BamHI/Bg1II cloning junction, signal peptide, V region, portion of
C region and 3'XbaI/NheI (heavy) or NheI (light) cloning junction
32 ggatctcacc atgagggtcc ctgctcagct cctggggctg ctaatgctct
ggatacctgg 60 atccagtgca gatattgtga tgacccagac tccactctct
ctgtccgtca cccctggaca 120 gccggcctcc atctcctgca agtctagtca
gagcctcctg catagtgatg gaaagacctt 180 tttgtattgg tatctgcaga
agccaggcca gcctccacag ctcctgatct atgaggtttc 240 caaccggttc
tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac 300
actgaaaatc agccgggtgg aggctgagga tgttgggctt tattactgca tgcaaagtat
360 acagcttccg ctcactttcg gcggagggac caaggtggag atcaaacgaa
ctgtggctgc 420 accatctgtc ttcatcttcc cgccatctga tgagcagttg
aaatctggaa ctgctagc 478 33 132 PRT Homo sapiens 33 Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15 Gly Ser
Ser Ala Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser 20 25 30
Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser 35
40 45 Leu Leu His Ser Asp Gly Lys Thr Phe Leu Tyr Trp Tyr Leu Gln
Lys 50 55 60 Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Glu Val Ser
Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala
Glu Asp Val Gly Leu Tyr Tyr 100 105 110 Cys Met Gln Ser Ile Gln Leu
Pro Leu Thr Phe Gly Gly Gly Thr Lys 115 120 125 Val Glu Ile Lys
130
* * * * *
References