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(12) Patent Application Publication (10) Pub. N0.: US 2001/0014334 A1 SEID et al. (54) METHODS AND MATERIALS FOR THE
(43) Pub. Date:
Aug. 16, 2001
(21) Appl. N0.:
Jun. 17, 1998
TREATMENT OF PROSTATIC CARCINOMA
(76) Inventors: CHRISTOPHER ALLEN SEID, THE WOODLANDS, TX (Us); GURPREET SINGH, HOUSTON, TX (Us)
Patent Application Publication Aug. 16, 2001 Sheet 8 0f 9 Figure B 18.00
US 2001/0014334 A1
Patent Application Publication Aug. 16, 2001 Sheet 9 0f 9
US 2001/0014334 A1
Aug. 16, 2001
US 2001/0014334 A1
METHODS AND MATERIALS FOR THE TREATMENT OF PROSTATIC CARCINOMA BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to methods for the
treatment of neoplastic diseases and, more speci?cally, to an
immunotherapeutic agent comprising a prostate-associated antigen in conjunction With a chitosan-based adjuvant and its use in the treatment of prostatic carcinoma.
 2. Related Technology  Prostatic carcinoma is one of the most common malignancies of men and a leading cause of death in this
population. WorldWide it is an important public health concern as over a quarter of a million neW cases Were
diagnosed in 1985 alone. Within the United States, it Was
predicted that for 1994, 38,000 individuals Would succumb to prostate cancer, While in the countries of the European Union it Was predicted the disease Would account for more
than 35,000 deaths. If detected at an early stage, prostate cancer is potentially curable. Its relatively sloW groWth and
androgen dependence distinguish it from most other carci nomas. The disease eXhibits an improved prognosis When detected in an early stage, although a majority of cases are
diagnosed at later stages, Where metastasis of the primary
localiZed Within the tissues surrounding the prostate capsule. Side effects such as hair loss and lethargy after irradiation have been Well documented.
 Another form of treatment for prostatic carcinoma involves hormone therapy. Speci?cally and as prostate can cer has been shoWn to be under the trophic in?uence of
androgen hormones, androgen deprivation may often pro duce a regression of the disease and improvement of symp toms. The goal of androgen deprivation is to achieve cas tration levels of testosterone and dihydrotestosterone. This
goal is usually attained by one of four methods: (1) surgical castration (orchiectomy); (2) administration of eXogenous estrogens such as diethylstilbestrol (DES); (3) use of analogs of luteiniZing hormone-releasing hormone (LHRH) that inhibit the release of pituitary gonadotropins; or (4) the use of anti-androgens, such as ?utamide, that block the action of
androgens at target tissues. Hormonal therapy is usually used When there is evidence that the cancer has spread beyond the prostate. It is important to note that hormonal therapy is not considered curative. Speci?cally, the cancer
eventually becomes resistant to hormone deprivation and continues to groW. Further, it has been noted in most patients
receiving anti-androgen therapy, such therapy Will result in the disease becoming hormone independent.  In vieW of the limitations of the current therapeutic approaches to the treatment of prostatic carcinoma, there remains a need for a more suitable treatment that could be
tumor has already occurred. Five year survival rates for patients With prostate cancer range from 88% for those With localiZed disease to 29% for those With metastatic disease.
used to alleviate prostate cancer (either localiZed or metas
 Present treatments for prostatic carcinoma include simply monitoring the disease, to radical prostatectomy, radiation therapy, or hormonal therapy. Simple monitoring
SUMMARY OF THE INVENTION
tasiZed) that is neither invasive nor produces the unWanted side effects of the currently available treatments.
of the disease is advocated as a reasonable approach for
 The present invention is directed to compositions
some patients With prostate cancer. Although untreated prostate cancer continues to groW, it may do so quite sloWly. Speci?cally, the groWth of the cancer may be sloW enough
chitosan-metal chelate adjuvant. The compositions may be used in methods for producing antibodies against prostate
that it causes no problems in a particular individuals life time, even if left untreated. While no one can predict exactly hoW long it Will take for a speci?c cancer to spread or hoW
long a particular individual’s lifespan Would be, unless an individual is eXpected to live at least 10 years, simply monitoring With no immediate treatment may be appropriate When the cancer is small and of loW grade. If the prostatic cancer is of a higher grade and thus more aggressive then the cancer may be a signi?cant threat to life or health Within 10 years and therefore a more aggressive approach to manage ment Would be Warranted.
 Surgery performed for treatment of localiZed pros
comprising one or more prostate-associated antigens and a
associated antigens and/or may be used as agents immuno therapeutic agent. In another aspect, the invention is directed to methods for producing compositions comprising one or more prostate-associated antigens and a chitosan-metal che late adjuvant as Well methods for inhibiting the groWth or alleviating prostate cancer or metastatic carcinoma of pro static origin. The invention is also directed to immunothera peutic agents comprising one or more prostate-associated antigens in combination With a chitosan-metal chelate adju vant.
 As yet another aspect, the present invention is directed to antibody substances (that speci?cally binds a
tate cancer is referred to as a radical prostatectomy. Through an incision in the loWer abdomen or beloW the scrotum, the
prostate-associated antigen) produced by the process of
entire prostate and seminal vesicles are removed. When the
antigens in combination With the chitosan-metal chelate adjuvant. The invention is further directed to methods for the
carcinoma is con?ned Within the tissues removed during surgery, a radical prostatectomy completely alleviates local iZed prostate cancer. Such surgery is of little therapeutic value once the cancer has metastasiZed to the areas sur
rounding the prostate as Well as distant areas of the body.
Further, such surgery may leave the individual permanently
administering the one or more speci?c prostate-associated
production of such antibody substances. 
The present invention is also directed to composi
tions comprising one or more prostate-associated antigens
and a chitosan/sodium hydroXide/oil/surfactant adjuvant. The compositions may be used in methods for producing
antibodies against prostate-associated antigens and/or may
 As an alternative to surgery, radiation therapy may be indicated. Speci?cally, radiation is recommended for men in Whom the disease has spread outside of the prostate
be used as immunotherapeutic agents. In another aspect, the invention is directed to methods for producing compositions
capsule (and thus making surgery more dif?cult) but is still
chitosan/sodium hydroXide/oil/surfactant adjuvant as Well
comprising one or more prostate-associated antigens and a
Aug. 16, 2001
US 2001/0014334 A1
methods for inhibiting the growth or alleviating prostate cancer or metastatic carcinoma of prostatic in origin. The invention is also directed to immunotherapeutic agents com prising one or more prostate-associated antigens and a
chitosan-Zinc chelate or native PSA, on the groWth of Du145 prostatic carcinoma cells in the presence and absence of
 As yet another aspect, the present invention pro vides antibody substances (that speci?cally binds a prostate associated antigen) produced by the process of administer
either partially puri?ed anti-recombinant PSA antibodies or partially puri?ed anti-native PSA antibodies in terms of
ing the speci?c prostate-associated antigen in combination With the chitosan/sodium hydroxide/oil/surfactant based adjuvant. The invention is further directed to methods for the
production of such antibody substances. 
FIG. 8 demonstrates the groWth of the tumors in
animals receiving adjuvant only versus animals receiving tumor diameter over time.
FIG. 9 illustrates the vector map for expression
vector pZ068, Which Was used in Example 1 for the expres
sion of prostate-speci?c antigen.
In another of its aspects, the present invention is
DETAILED DESCRIPTION OF THE INVENTION
further directed to a composition comprising one or more
prostate-associated antigens and a phosphate buffer/chito san-based adjuvant. The composition may be used in meth ods for producing antibodies against prostate-associated antigens and/or may be used as immunotherapeutic agents.
system. It is a Walnut-siZed organ, made up largely of
In another aspect, the invention is directed to methods for producing compositions comprising one or more prostate
and the urethra. Its main function is to produce ?uid for semen. Speci?cally, the prostate secretes a thin, milky,
associated antigens in combination With a phosphate buffer/ chitosan-based adjuvant as Well methods for inhibiting the groWth or alleviating prostate cancer or metastatic carci noma of prostatic in origin. The invention is also directed to an immunotherapeutic agent comprising one or more pros
tate-associated antigens and a phosphate buffer/chitosan
based adjuvant.  As yet another aspect, the present invention is directed to antibody substances (that speci?cally binds a
The prostate is a gland of the male reproductive
muscular and glandular tissues, located betWeen the bladder
alkaline ?uid containing citric acid, calcium, acid phosphate, a clotting factor, and a pro?brinolysin. During emission, the capsule of the prostate gland contracts simultaneously With the contractions of the vas deferens so that the thin, milky ?uid of the prostate gland adds to the bulk of the semen.
 Over 95% of the prostatic carcinomas are adeno carcinomas that arise in the prostatic acini. Adenocarcinoma may begin anyWhere in the prostate but has a predilection for the periphery. The tumors are frequently multifocal. Vari
prostate-associated antigen) produced by the process of
ability in cellular siZe, nuclear and nucleolar shape, glandu
administering the one or more prostate-associated antigens
lar differentiation, and the content of acid phosphatase and
in combination With the phosphate buffer/chitosan-based
mucin may occur Within a single specimen, but the most poorly differentiated area of tumor (i.e., its area With the
adjuvant. The invention is further directed to methods for the
production of such antibody substances.
highest histologic grade) appears to determine its biologic
 Other objectives and advantages of the invention
behavior. The remaining prostatic cancers are divided among squamous cell and transitional cell carcinomas that
may be apparent to those skilled in the art from a revieW of
the folloWing detailed description, including any draWings, as Well as the approved claims.
BRIEF DESCRIPTION OF THE DRAWINGS
arise in the prostatic ducts, carcinoma of the prostatic utricle, carcinosarcomas that arise in the mesenchymal elements of the gland, and occasional metastatic tumors (usually carci noma of the lung, melanoma, or lymphoma).
FIG. 1 illustrates the antibody titers obtained from
 Although given the enormous antigenic diversity
rats immunized With recombinant PSA/chitosan-Zinc chelate
of the many forms of cancer, the identi?cation of all tumor
as Well as the titer of the antisera obtained With native PSA.
antigens that possess protective potential is a formidable
 FIGS. 2 and 3 demonstrate the effect of rat anti recombinant PSA antibodies on prostatic carcinoma cell
task, a number of human clinical trials are currently under Way to assess the ef?cacy of treating human tumors by immunotherapeutic means.
lines LnCap and Du145, respectively, to incorporate 3H-thy
 This type of therapy (cancer immunotherapy) is
FIG. 4 sets forth the titers in the serum of monkeys
to the recombinant PSA/chitosan-Zinc chelate as Well as
native PSA subsequent to immuniZation
based upon the recent and expanding identi?cation of spe
ci?c therapeutic targets: tumor antigens With various degrees of association With their normal counterparts. These targets
can be grouped into four general categories: (1) the “cancer/
 FIG. 5 sets forth the prostate organ Weight of the monkeys immuniZed With either recombinant PSA/chitosan
testis” antigens, such as the MAGE gene family, Whose
Zinc chelate or native PSA/chitosan-oil emulsion
Whose genes have been mapped to the X chromosome [van
 FIG. 6 shoWs the effect of monkey antisera, from animals immuniZed With either recombinant PSA/chitosan Zinc chelate or native PSA, on the groWth of LnCap prostatic carcinoma cells in the presence and absence of guinea pig
complement.  FIG. 7 illustrates the effect of monkey antisera, from animals immuniZed With either recombinant PSA/
expression is tumor speci?c except for spermatogonia and
der Bruggen et al., Science, 25411643 (1991); Gaugler et al.,
J. Exp. Med, 1791921 (1994)]; (2) virally derived antigens, including herpesvirus [Ressing et al., J. ImmunoL, 15415934 (1995)] and Epstein-Barr virus [Chen et al., Hum. ImmunoL, 18:75 (1987)]; (3) differentiation antigens, including pros tate speci?c antigen [Murphy et al., Anticancer Res., 1511473 (1995)] and prostate speci?c membrane antigen [Murphy et al., Prostate, 281266 (1996)]; and (4) antigens
Aug. 16, 2001
US 2001/0014334 A1
existing in a modi?ed or mutated form in tumor as compared
prostatic acid phosphatase (PAP) Was used to aid in the
to normal cells [Robbins et al., J. Exp. Med, 183:1185
diagnosis of prostate cancer as Well as to monitor the
ef?cacy of therapy for prostate cancer. PAP has noW been replaced by PSA for use in the diagnosis of prostate cancer because of PSA’s greater sensitivity. PSA is useful for
Whereas vaccination aims to prevent tumor forma
tion, the goal of immunotherapy is to augment a patient’s immune responses to an established tumor. Because speci?c tumor antigens have not yet been identi?ed or isolated from most human cancers, most forms of immune intervention
monitoring therapy, particularly surgical prostectomey, because complete removal of the prostate gland should
nonspeci?c immunopotentiators. HoWever, several recent
result in undetectable levels of PSA. Measurable PSA after radical prostectomy indicates residual prostate tissue or metastasis and increasing PSA concentrations indicate recur
advances in basic biology have revived interest in active
attempt to enhance a patient’s overall level of immunity With
speci?c immunotherapy. It has become possible to identify, isolate, and characteriZe tumor antigens that are recogniZed by T cells in the context of MHC (major histocompability complex) class I or class II determinants. Such T cell-de?ned tumor antigens are likely to be better candidates for use in
immunotherapeutic methods than epitopes identi?ed With
tumor-speci?c antibodies, given the greater over-all impor tance of cells over antibodies in antitumor immunity. Some
of the T cell-de?ned antigens are expressed in different tumors of related tissue and cellular origins; this eliminates the need for autologous tumor tissue for immunotherapeutic
development.  Types of cancer immunotherapy include (1) Whole
cell vaccines, (2) viral oncolysates, (3) partially puri?ed tumor antigen vaccines, and (4) highly puri?ed or synthetic tumor antigen vaccines. Whole tumor cells display not one
but all of the potential antigens expressed by the cancer cells, as such they can function as antigen presenting cells. In addition they can be engineered to express tumor antigens or to produce cytotoxic cytokines. Viral oncolysates, used as cancer vaccines, are virus augmented tumor cell lysates. Tumor cells are infected With an appropriate virus utiliZing
 A primary concern involving the development of an immunotherapeutic agent for the treatment of prostate cancer is Whether an active immune response can be gen
erated to a self-polypeptide such as PSA. Controlled immu
niZation for the purpose of stimulating antibody production by B cells is dependent upon a myriad of factors inherent to both the antigen itself and the immuniZed individual. In general, the farther removed in evolutionary terms the antigen, or its source, is from the invaded host, the more
effective the immune response elicited by the antigen. Anti gens derived from closely related species are less competent in eliciting antibody production due to the fact that the host immune system is unable to clearly distinguish the foreign antigen from endogenous, or self antigens. In addition, the dosage of the antigen, the purity of the antigen, and the frequency With Which the antigen is administered are also factors Which signi?cantly contribute to the resulting anti body titer and speci?city of the resulting antibodies. Still other factors include the form, or complexity, of the antigen, and hoW the antigen is administered. Finally, both the genetic makeup and overall physiological state of the immu niZed animal contribute to the extent to Which an immune
modern cell culture techniques. Partially puri?ed tumor
response is mounted. Of these factors, the form or complex
antigen vaccines are prepared from material shed into a
ity of the antigen is directly affected by immuniZation With
culture medium from multiple cell, Which express tumor antigens. In excluding much of the cellular material the vaccine is said to be partially puri?ed. While the foregoing approaches to cancer immunotherapy have been evaluated
for a number of different cancer types, none have resulted in success in clinical trials.
 Ideally, an adjuvant should potentiate long-lasting expression of functionally active antibodies, elicit cell mediated immunity (CMI), and enhance production of memory T- and B-lymphocytes With highly speci?c immu noreactivity against an invading antigen. More important is
to cancer antigens in patients that must be overcome in order
the ability of an adjuvant to augment the immune response With a minimum of toxic side effects. Therefore, efficacy of an adjuvant is described in terms of hoW it balances positive
to ensure effectiveness. In order to overcome immune tol
(potentiated immunity) and negative (toxicity) effects.
erance, vaccine design should be directed toWard enhancing the host’s immune response to puri?ed or synthetic antigens.
 Current understanding suggests that adjuvants act
 With respect to the use of highly puri?ed or syn thetic tumor antigen vaccines, there is an inherent tolerance
The present invention addresses this problem by providing a novel antigen-adjuvant composition (i.e., an immunothera
peutic agent) comprising a prostate-associated antigen in conjunction With an adjuvant system, Which results in the inhibition of groWth and incidence of prostate carcinoma.
 The tissue speci?city of prostate-related antigens such as PSA, PSMA, HKZ, PCTA-1, PTI-1, PSCA, PAP, PTEN/MMAC1, and the LH receptor make them potential target antigens for active speci?c immunotherapy. Prostate speci?c antigen (PSA) is a 240 amino acid glycoprotein produced by normal prostate tissue. It is a serine protease
and belongs to the glandular kallikrien gene family [Lund Wall et al, Biochem. Biophys Res. Comm, 161:1151-1159 (1988)]. PSA’s absolute tissue speci?city makes it a valu able as a tumor marker for prostate cancer. Previously,
to augment the immune response by a variety of different mechanisms. In one mechanism, the adjuvant directly stimu lates one of either CD4+helper T-cell subpopulations desig
nated TH1 or THZ [Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173 (1989)]. Helper T cells are required for B-cell antibody responses to most antigens. Alum, an alu minum salt adjuvant approved for clinical use in humans, has been reported to selectively activate THZ cells in mice
[Grun and Maurer, Cell. Immunol. 121:134-145 (1989)], While Freund’s complete adjuvant (FCA), an emulsion of mineral oil With killed mycobacteria [Freund, et al., Proc. Soc. Exp. Biol. Med. 37:509 (1937)], preferentially activates murine TH1 cells [Grun and Maurer, Cell. Immunol. 121:134-145 (1989)]. The nature of the cytokine production in activated T-cells may also be in?uenced in part by the choice of adjuvant.
Aug. 16, 2001
US 2001/0014334 A1
 Another mechanism by Which the immune
molecule, and not from the o-speci?c polysaccharide or core
response is augmented involves the direct stimulation of B
oligosaccharide regions of the molecule. LPS, Which aug ments both humoral [Johnson, et al., J. Exp. Med. 103:225
cells by certain antigens, for example, lipopolysaccharide (LPS) from Gram-negative bacteria. [Gery, et al., J. Immu n0l. 10811088 (1972)]. LPS has also been shoWn to stimulate
246 (1956)] and cell-mediated immunity [Ohta, et al., Immu nobiology 53:827 (1984)], possesses numerous biological
secretion of interferon-y (INF-y) [Tomai and Johnson, J.
activities, but is impractical for use in humans due to its
Biol. Resp. Med. 8:625-643 (1989)], Which both inhibits proliferation of TH2 cells and stimulates differentiation of TH1 cells [GajeWski, et al., J. Immunol. 143:15-22 (1989); GajeWski, et al., J. Immunol. 146:1750-1758 (1991)]. The mechanism by Which LPS potentiates the immune response is therefore through direct stimulation of B cells, and indi rect regulation of both TH1 and TH2 cell populations.
inherent toxicity [See Gupta, et al., Vaccine 11:291-306 (1993)]. Attention has therefore shifted to other polysaccha rides including, among others, chitosan.  Chitosan [[3-(1-4)-2-amino-2-deoxy-D-glucan] is a
 Still other modes of immunopotentiation have been reported for other adjuvants. Oil emulsions (i.e., Freund’s
derivative of chitin and has been Widely used in biomedical
applications, due in part to is biodegradability by lysoZyme and loW toxicity in humans. These same properties have resulted in increased interest in chitosan as an immunopo
tentiating agent. For example, Matuhashi, et al., in US. Pat. No. 4,372,883, disclosed conjugation of soluble polysaccha rides, including chitosan, to normally toxic antigens, con
[FIA]) and liposomes act through depot formation as does alum, thus alloWing for sloW release of antigen. SloW release of antigen permits extended exposure of the antigen to the
jugation thereby detoxifying the antigen and permitting its
immune system and also alloWs for initial immuniZation With a dosage of antigen that, if delivered at one time, Would
san, nor did Matuhashi compare the resulting serum anti
use as an immunogen. Matuhashi et al., hoWever, did not
address the use of insoluble polysaccharide forms of chito
ordinarily be counterproductive to antibody formation. It has been previously reported that While a large initial dose of antigen results in the production of a higher immediate titer of antibody, the increase in antibody titer and increase in
body titer With that obtained from immuniZation With other
antibody speci?city as a function of time is not as great as
therapeutics for treatment of bacterial and fungal infections,
knoWn adjuvants.  Likewise, Suzuki, et al., in US. Pat. No. 4,971,956, disclosed the use of Water soluble chitosan-oligomers as
observed With loWer and more frequent doses of antigen
as Well as for the treatment of tumors. SuZuki, et al,
[Siskind, G., Pharm. Rev. 25:319-324 (1973)]. Therefore, adjuvants Which control presentation of an antigen to the
discussed the dif?culty in modifying chitosan to produce an appropriate Water soluble form, disclosing that Water-in
immune system modulate antigen dosage in addition to altering the form, or complexity, of the antigen.
soluble forms are impractical for therapeutic application. In
 To date, only one adjuvant, alum [AlK(SO4)2H2O],
addition, SuZuki et al., does not disclose conjugation of an antigen to chitosan to effect enhanced immune response.
has proven sufficiently non-toxic to permit its use in humans.
 Mitsuhashi, et al., in US. Pat. No. 4,814,169,
Alum not only acts through TH2 cell activation, depot formation and sloW release of antigen folloWing immuniZa tion [Edelman, Rev. Infect. Dis. 2:370-383 (1980); Warren,
polysaccharides to generate antibodies against human pro
disclosed the use of human protein conjugated to soluble
through granuloma formation by attracting immunocompe
tein in non-human animals. Although Mitsuhashi et al. included chitosan in an exemplary list of polysaccharides, the patent merely predicted that chitosan may be useful for
tent cells [White, et al., J. Exp. Med. 102:73-82 (1955)] and activation of complement [Ramanathan, et al., Immunol. 37:881-888 (1979)]. HoWever, alum is not Without its nega tive side effects Which include erythema, subcutaneous
the nature of the expected immune response. Further, the ’169 patent set forth non-ionic polysaccharides as the pre ferred saccharides, While chitosan is highly cationic.
nodules, contact hypersensitivity, and granulomatous
 Nishimura et al. [Vaccine 2:93-99 (1984)] reported
the immunological properties of derivatives of chitin in terms of activation of peritoneal macrophages in vivo,
et al., Ann. Rev. Immunol. 4:369-388 (1986)], but also
Other adjuvants, Which are Widely employed out
side of human application, are also the focus of continuing research to develop acceptable alternatives for use in humans. Included in this group are bacterial products (i.e.,
LPS, cholera toxin, mycobacterial components and Whole killed Corynebacterium parvum, Corynebacterium granu losum, and Bordetella pertussis, liposomes, immunostimu
lating complexes (ISCOMs), and naturally occurring and derivatiZed polysaccharides from other than bacterial
antibody production, and did not provide details regarding
suppression of tumor groWth in mice, and protection against bacterial infection. Results obtained from their single-injec tion system shoWed that both chitin and chitosan Were
ineffective stimulators of host resistance against challenge With tumor cells or bacteria.
 MarcinkieWicZ, et al., [Arch. Immunol. Ther. Exp. 39:127-132 (1991)] examined the immunoadjuvant activity of Water-insoluble chitosan and reported signi?cant
enhancement of T-dependent humoral response, but only moderate augmentation of T-independent humoral response.
 The immunopotentiating capacity of polysaccha
The enhanced humoral response Was detected With chitosan
rides has been a focus of investigation over the past feW years as these compounds are Widespread in nature, e.g., as
structural components in the cell Walls of bacteria, and exoskeletons of insects and crustacea. Lipopolysaccharide (LPS) isolated from certain Gram-negative bacteria is one
at doses of 100 mg/kg administered either intravenously or intraperitoneally. Subcutaneous and oral administration Were speci?cally reported as being ineffective. In addition, MarcinkieWicZ, et al., does not suggest conjugation of an antigen to insoluble chitosan, stating that chitosan “resulted
such polysaccharide even though the adjuvant properties of
in the same response irrespective of the site of administra
LPS are derived mainly from the lipid A region of the
tion—either together or separately from antigen.”
Aug. 16, 2001
US 2001/0014334 A1
 In vieW of the foregoing discussion and by Way of
 Example 13 describes the effect of partially puri
illustration of the invention, the examples describe an immu
?ed monkey anti-recombinant PSA or anti-native PSA anti bodies on the in vivo groWth of human prostatic carcinoma
notherapeutic agent for the treatment of prostatic carcinoma comprising a prostate-associated antigen and a chitosan based adjuvant system, and in a preferred embodiment, PSA and a chitosan-Zinc chelate-based adjuvant, PSA and a
chitosan/sodium hydroxide/squalene/poloxamer 401, or PSA and a phosphate buffer/chitosan-based adjuvant. The Examples set out beloW demonstrate that this immunothera
cells (passive immunization).  Example 14 describes the effect of early adminis tration of puri?ed monkey anti-recombinant PSA or anti native PSA antibodies on Du145 tumor incidence in athymic mice.
peutic agent Was successful in reducing and alleviating prostate and prostatic related tumors in subject animals. The illustrative Examples also describe methods for reducing
 Example 15 describes administration of an immu notherapeutic agent of the present invention to humans to
and/or alleviating prostatic carcinoma and prostatic-related
carcinoma of prostatic origin.
inhibit groWth or alleviate prostate cancer or metastatic
carcinoma (metatastic in origin) via the administration of any of the foregoing immunotherapeutic agents
 The invention is illustrated by the folloWing
CLONING AND EXPRESSION OF HUMAN
examples, Which are not intended to limit the scope of the invention as recited in the claims.
PROSTATE SPECIFIC ANTIGEN (PSA)
expression of human PSA.
 Polymerase Chain Reaction  The human PSA gene (With signal sequence) Was
 Example 2 provides methods for the puri?cation of
lot#6060116; Clontech, Palo Alto, Calif.) using the poly
the recombinant form of human PSA.
merase chain reaction [PCR; Mullis et al., The Polymerase
of the primers used (see beloW) Was determined based on a
 Example 1 provides methods for the cloning and
Example 3 describes the preparation of a chitosan
cloned from human prostate cDNA (Quickclone cDNA
Chain Reaction, Birkhauser, Boston (1994)]. The sequence
chelated metal based immunotherapeutic agent involving the use of prostate-related antigens (e.g., PSA, PSMA).
published sequence for human PSA [Riegman et al., Bio
 Example 4 describes the preparation of a chitosan iron chelated based immunotherapeutic agent involving the
Bank Accession No. M24543] and subsequently modi?ed as described beloW.
use of prostate-related antigens (e.g., PSA, PSMA).
 Both polymerase chain reaction primers Were syn thesiZed using an Applied Biosystems Model 391 DNA
Example 5 describes the preparation of an immu
notherapeutic agent Wherein the prostate-related antigen (e.g., PSA, PSMA) is incorporated and lyophiliZed in phos
chem. Biophys. Res. Comm. 159(1):95-102 (1989); Gen
SynthesiZer (Applied Biosystems, Foster City, Calif.) and standard protocols for solid phase DNA synthesis. Speci?
phate buffer and reconstituted in a chitosan solution.
cally, The 5‘ primer contained 21 bases of 5‘ untranslated region With the addition of SmaI and EcoRI restriction
enZyme sites for subcloning into sequencing and/or expres
Example 6 describes the preparation of an immu
notherapeutic agent Wherein the prostate-associated antigen (e.g., PSA, PSMA) is incorporated into a chitosan-oil emul sion.
Example 7 describes the characteriZation of the
immune response in rats to administration of either native human PSA/chitosan-Zinc chelate or recombinant PSA/chi tosan-Zinc chelate.
 Example 8 demonstrates the speci?city of the anti bodies to PSA found in the sera of the rats of Example 7 via
Western blot analysis.  Example 9 describes the ability of antibodies to PSA found in the sera of the rats of Example 7 to bind to
human prostatic carcinoma cells.  Example 10 describes the effect of rat anti-PSA antisera on the proliferation of human prostate cells (in
Example 11 describes the characteriZation of the
immune response in rats to administration of either native human PSA or recombinant PSA bound to a chitosan-Zinc
sion vectors [all restrictions enZymes Were obtained from
NeW England Biolabs, Beverly, Mass.]. The 3‘ primer con sisted of a 34 base pair oligonucleotide containing 20 bases of 3‘ untranslated region 50 bases doWnstream of the stop codon. Similarly, SmaI and EcoRI restriction enZyme sites Were also included in this primer for subcloning purposes. The sequence of both PCR primers are as folloWs:
 5‘ TAACCCGGGAATTCATrCCGCCG GAGAGCTGTGTC 3‘ (SEQ ID NO. 1) 3‘ Primer
 5‘ TAACCCGGGAATTCCTTGAGTCTTG GCCTGGTCA 3‘ (SEQ ID NO. 2).  Conditions for the polymerase chain reaction Were as folloWs. 2 pl of prostate cDNA (2 ng; Clontech, Palo Alto, Calif.) Were added to a 0.5 ml microfuge tube containing 10
pl 5XPCR reaction buffer (15 mM MgCl2, 500 mM KCl, 100 mM Tris-HCL, pH 8.3, 0.01% gelatin, 2 mM each
dNTP), 1 pl 5‘ primer (20 pmol), 1 pl 3‘ primer (20 pmol), 1 pl Taq DNA polymerase (Perkin-Elmer Cetus, Branch burg, N.J.), and 35 pl nuclease-free dioniZed Water. The
 Example 12 describes the effect of monkey anti
polymerase chain reaction Was performed using a Perkin
PSA antisera on the in vitro proliferation of human prostatic carcinoma cells.
Elmer Cetus, Branchburg, N.J.) With 40 cycles of ampli?
SpeI adaptor Was made palindromic to regenerate the 8 base
cation. Each cycle consisted of a 45 second denaturation step at 94° C., a 1 minute annealing step at 58° C., and a 1 minute extension step at 72° C. After completion of 40 cycles, a ?nal 5 minute extension at 72° C. Was performed. The polymerase chain reaction resulted in the ampli?cation of an
pairs betWeen the MscI site and stop codon (noW consisting of a SpeI site). The phosphorylated oligonucleotides used to make the SpeI adaptor Were purchased from Genosys (Woodlands, Tex.). The sequence of the SpeI adaptor is as
approximately 900 base pair product, as determined by gel
electrophoresis, Which is the correct siZe for DNA encoding
 Subcloning of Human PSA into pBluescript SK Sequencing Vector
5‘ CCAACCCCACTAGTGGGGTTGG 3‘ (SEQ ID
 The 900 base pair polymerase chain reaction prod uct described above Was puri?ed from a 1% TAE agarose gel
using a GeneClean® kit (BIO101, Vista, Calif.) into 25 pl TE (10 mM Tris, 1 mM EDTA, pH=8.0). The DNA Was subsequently digested With the SmaI restriction enZyme,
extracted With phenolzchloroform (1:1 vol/vol.), and pre cipitated With 1/2 volume of 7.5M ammonium acetate and 2 volumes of 95% ethanol at —70° C., for 30 minutes. The DNA Was pelleted by centrifugation at 14,000 rpm in an
Eppendorf model 5415C microcentrifuge (Eppendorf, Ham burg, Germany), Washed With 70% ethanol, and air dried. The DNA Was resuspended in 10 pl TE (pH=8.0). SmaI digested human PSA cDNA (PCR product) Was next ligated into the SmaI site of pBluescript SK vector (Stratagene, LaJolla, Calif.). After ligation, the pBluescript vector con taining the human PSA cDNA insert Was transformed into E.
coli strain TOPP2 (Stratagene, LaJolla, Calif.) by standard
The complete human PSA sequence Was then
removed from the pBluescript sequencing vector by digest ing With SmaI and SpeI, and subsequently ligating the insert into the PvuII/SpeI restriction enZyme sites of the expression vector pZ068 [Which has the DHFR gene cassette, see FIG. 9 for vector map of expression vector pZ068] Lastly, a
poly-histidine tag (His6X tag) folloWed by a stop codon [sequence of His?x: 5‘ CACCACCATCACCACCAT 3‘ (SEQ ID NO. 7)] Were inserted doWnstream of the generated SpeI site. The resulting PSA insert consisted of the sequence: human PSA-His6 tag-stop codon. This modi?cation resulted in a C-terminal His6X tag fusion on the expressed protein, Which alloWs for the puri?cation of the recombinant PSA
using ImmobiliZed Metal Af?nity Chromatography (IMAC)
transformation protocols. An E. coli clone Was isolated
as described beloW.
containing the above described pBluescript/human PSA
 Transfection of CHO dhfr- Cells With PSA Expres
vector, Which Was subsequently used to generate further
DNA for subsequent experimentation. The human PSA insert Was veri?ed by restriction enZyme mapping using knoWn internal restriction enZyme sites of the published human PSA sequence and restriction enZyme sites of the vector that ?ank the human PSA cDNA insert (either on 5‘ or 3‘ end), as Well as performing agarose gel electrophoresis
to verify observed band siZe With expected band siZe. The identity of the human PSA cDNA insert Was also veri?ed by
partial sequencing using the M13-20 and Reverse primer set Stratagene, LaJolla, Calif.). The sequences of the sequenc ing primers is as folloWs:
Reverse primer 5‘ GGAAACAGCTATGACCATG‘3 (SEQ ID NO.
3) M13-20 primer
 The PSA expression vector (5-10 pg; see directly above) Was transfected into dihydrofolate reductase de? cient (dhfr') Chinese Hamster Ovary Cells (ATCC, Manas
sas, Va.) using the folloWing methodology. The pZ068 human PSA expression vector Was lineariZed With the SalI
restriction enZyme folloWed by phenolzcholoroform (1:1) extraction and precipitation With 1/2 volume 7.5M ammo nium acetate and 2 volumes of 95% ethanol at —70° C. for
30 minutes. DNA Was pelleted by centrifugation at 14,000 rpm in microcentrifuge for 10 minutes, Washed With 70% ethanol, and air dried. DNA Was resuspended in 10 pl TE (pH=8.0). A transfection mix Was prepared by mixing Solu
tion A (10 pl DNA+90 pl Opti MEM (Gibco BRL, Gaith
ersburg, Md.) With Solution B (10 pl Lipofectamine (Gibco BRL)+90 pl Opti MEM) and incubating the resulting mix ture at room temperature for 20 minutes. After incubation, the transfection mix Was added to a 35 mm tissue culture
dish (Costar, Cambridge, Ma.) containing 2 ml Opti MEM
5‘ GTAAAACGACGGCCAGT3‘ (SEQ ID NO. 4)
and CH0 dhfr‘cells at 60-70% con?uency. The transfection Was alloWed to proceed overnight (12-16 hr) at 37° C. and
 The sequence obtained from the human PSA insert (approximately 200 base pairs at each end of the insert) Was 100% identical to the knoWn human PSA nucleotide
the transfection mix Was removed and replaced With 2 ml
fetal bovine serum (dFBS) and 2 mM L-glutamine (Gibco
 Insertion of Human PSA Sequence into A Mam malian Expression Vector
BRL). Incubation Was continued at 37° C. and 5% CO2 for 72 hours. After 72 hours, the cells Were removed from the dish by trypsiniZation and transferred to a T75 tissue culture
 For insertion of human PSA encoding cDNA into the mammalian expression vector, the folloWing methodol ogy Was used. A SpeI adaptor Was ligated into the unique MscI restriction enZyme site Within the pBluescript/human PSA construct, 8 base pairs upstream of the stop codon. The
?ask (Costar) containing complete Earles MEM With 5% DFBS (for selection of transfected cells) and 2 mM L-glutamine. The function of the dialyZed serum (Which lacks purines) is to select for the cells that contain the plasmid DNA (the expression vector) due to the presence of
5% CO2. Immediately folloWing the overnight incubation, complete Earles MEM (Gibco BRL) containing 5% dialyZed
Aug. 16, 2001
US 2001/0014334 A1
a DHFR gene cassette present on the vector Which compli
ments the dhfr‘genotype of the cells (purine biosynthesis de?ciency). The dhfr'gene cassette is under control of the constitutive SV40 promoter and codes for dihydrofolate reductase enzyme, Which alloWs for the recovery of purine biosynthesis in CHO dhfr‘cells. In the presence of medium
that lacks purines (i.e., dialyZed serum-containing medium) only cells transfected With plasmid DNA (expression vector With dhfr‘cassette) Will survive.  After selection (1-2 Weeks), secretion of PSA Was veri?ed by Western blot analysis. Speci?cally, medium containing PSA Was run on a 4-20% polyacrylamide gradi
ent gel (Bio-Rad, Hercules, Calif.) and transferred to nitro cellulose. The resulting blot Was blocked in phosphate
buffered saline (PBS) containing 0.05% TWeen 20 (PBST) and 5% non-fat dry milk, folloWed by a 1 hour incubation (at room temperature) With primary antibody against human PSA (DAKO Corp., Carpinteria, Calif.). The blot Was Washed for 5 minutes (3 times) in PBST and subsequently incubated With mouse anti-rabbit secondary antibody at
1:5000 dilution (Zymed, San Francisco, Calif.) for 30 min utes. After three 5 minute Washes in PBST, the blot Was
incubated in PBST containing streptavidin-HRP conjugate at 1:5000 dilution for 20 minutes. The blot Was Washed three
times in PBS for 5 minutes and color Was developed using
the chromogenic substrate 3-amino-9-ethyl carbaZol and
(BioRad, Hercules, Calif.). The resin Was alloWed to settle into the column and an additional 50 ml of binding buffer
Was passed through the column. After equilibration of the column, the dialyZed media containing human PSA Was passed through the column. The column Was Washed With 20
bed volumes (100 ml) of binding buffer, folloWed by elution of PSA in 2 bed volumes (10 ml) of PBS containing 200 mM imidaZole. ImidaZole Was removed from the eluate by dialy sis in PBS (pH 8.0) and the protein Was concentrated to
0.5-2.0 mg/ml using a Centiprep concentrator (Amicon, Beverly, Mass.). The puri?ed concentrated PSA Was stored at —70° C. for use in the examples set forth beloW. In a
typical small scale run 5-7 mg of puri?ed recombinant protein could be obtained from 1 liter of medium. Media
used for protein production Was purchased from Hyclone
(PFX-CHO). EXAMPLE 3 PREPARATION OF CHITOSAN-CHELATED METAL BASED IMMUNOTHERAPEUTIC AGENT
 The chitosan-chelated metal based adjuvant Was prepared according to the folloWing method. While the Zinc is exempli?ed as the chelated-metal, those of skill in the art Will recogniZe that other metals, such as Zinc or copper may
be used in the practice of the present invention. Further,
 The heterogeneous population of cells Were then cloned by limiting dilution to identify and isolate individual clones that expressed and secreted high levels of recombi nant human PSA protein. Once identi?ed and isolated, the
While recombinant PSA is exempli?ed beloW as the prostate related antigen, those of skill in the art Will recogniZe that
clones Were scaled up for protein production as described
AF007544], human glandular kallikrein-2 [HK2; Schedlich
beloW, as Well as froZen for future use.
et al., DNA, 6:429-437(1987); GenBank Accession No. M18157; see also, US. Pat. No. 5,516,639], prostate carci noma tumor antigen-1 [PCTA-1; Hadari et al., J. Biol. Chem, 270:3447-3453 (1995); GenBank Accession No. L78132], prostate carcinoma tumor inducer-1 [PI-1; Shen et
EXAMPLE 2 PURIFICATION OF RECOMBINANT PSA
Individual clones of CHO cells secreting recombi
nant human PSA Were groWn to con?uency in 150 ml
Minimal Essential Media (MEM) (Gibco-BRL, Grand Island, NY.) supplemented With 5% dialyZed fetal bovine serum (FBS) Hyclone, Logan, Utah) and 2 mM L-glutamine
other prostate-related antigens, such as prostate-speci?c membrane antigen [PSMA; GenBank Accession No.
al., Proc. Natl. Acad. Sci., U.S.A., 92:6778-6782 (1995); GenBank Accession No. L41498], prostate stem cell antigen [PSCA; Reiter et al.,Pr0c. NatLAcad. Sci., U.SA., 95:1735 1740 (1998); GenBank Accession No. AF043498], PTEN/ MMAC1 [Kong, et al., Nature Genet, 17:143-144 (1997); Sakurada et al., Jpn. J. Cancer Res., 88:1025-1028 (1997);
in Triple Flasks (Nunc, Naperville, 111.). Once con?uent,
GenBank Accession No. AB009903 or 2723418], prostate
cells Were Washed With PBS, and media Was replaced With
acid phosphatase [PAP; Sharief et al., Biochem Biophys.
PFX-CHO medium (Hyclone). After 72 hours, the medium
Was removed and centrifuged for 5 min. at 1500>
M24902], and the LH (luteiniZing hormone) receptor [Jia et al., M01. EndocrinoL, 5:759-768 (1991); GenBank Acces
remove particulate material, and dialyZed at 4° C. against 5 volumes of 50 mM NaH2PO4, 20 mM Tris, 0.1M NaCl, pH 8.0, With 3 buffer changes.
 After dialysis, media containing secreted PSA Was passed through an IMAC column, prepared as folloWs. First, 5 ml of chelating Sepharose fast How (Pharmacia, Piscat aWay, N] was Washed 5 times at room temperature With 50
sion No. S57793] may be used in the practice of the present invention
 To prepare chitosan/metal complex adjuvants con taining either Zinc, copper or nickel, a 2% chitosan solution
Was initially prepared by dissolving 2 g chitosan (SeaSan Mer N-2000, CTC Organics, Atlanta, Ga.) in 100 ml 2%
ml of sterile deioniZed H2O (centrifuging at 1500>
acetic acid, and the resulting solution Was steriliZed by
minutes betWeen each Wash). Next, the resin Was resus pended in 50 ml 100 mM CoCl2 and mixed for 10 min. at room temperature. The resin Was again centrifuged for 5 min at 1500>
autoclaving. As an alternative, the chitosan solution can also
H2O (centrifuging for 5 min at 1500>
IMAC binding buffer (50 mM NaH2PO4 20 mM Tris, 100 mM NaCl, pH=8.0) and loaded into a 15 ml Econo column
be prepared by dissolving 2 g in 100 ml 0.5M sodium acetate pH 4.5. Solutions of either Zinc acetate, nickel sulfate, or copper sulfate solution (although other salts of the metals may be used) Were prepared in deioniZed Water at a molarity betWeen 0.001 to 0.2M and ?lter steriliZed. The 2% chitosan solution Was diluted 1:1 using deioniZed Water and 4 ml of the resulting 1% chitosan solution Was added to 10 ml of the
Aug. 16, 2001
US 2001/0014334 A1
desired metal salt solution (i.e., either the Zinc, nickel, or
Paris, Ky.) in 400 ml of deioniZed (18 mOhm: DI) Water. The
copper solutions). The resulting suspension Was mixed on an end to end shaker for 2 to 4 hours at room temperature. The mixture Was then sonicated using a Branson Soni?er 250 for 3 to 5 minutes and the pH of the mixture adjusted to 12.0-12.5 With 10N NaOH during sonication. When the Zinc salt Was employed, a White complex precipitate Was formed. When the nickel salt Was used, the complex Was light green. The copper salt resulted in a blue precipitate. After sonica tion, the mixture Was centrifuged at 2000 rpm (1000>
pH of the solution is adjusted to 7.3 With 10N sodium
Washed tWice With PBS, pH 7.2, centrifuged after each Wash, the Wet Weight of the pellet determined, and the metal/chitosan complex pellet resuspended in 8M urea, pH 7.8 to 8.0. The metal/chitosan complexes Were either imme diately coupled to an antigen, or stored in either 8M urea or PBS at room temperature. The stored metal/chitosan com
plexes have shoWn to be stable for up to six months When
stored by this method.  The PSA (may or may not contain a poly-HIS tag), as produced in Example 1, Was associated With the metal/
chitosan complex by the folloWing method. The chitosan Zinc chelate Was equilibrated With phosphate buffered saline
(PBS, pH 8.0). Puri?ed recombinant human PSA expressed in CHO cells (see Example 1) Was added to the chitosan-Zinc chelate suspension and incubated at room temperature for 4
hydroxide (Sigma Chemical Co., St. Louis, M0). The total volume of the solution is adjusted to 500 ml by the addition of deioniZed Water.
 Adilute chitosan solution is made by ?rst preparing a 1% chitosan in 2% acetic acid solution: 1 gm of chitosan
(practical grade; Sigma Chemical Co., St. Louis, M0 in 100 ml of 2% glacial acetic acid (Mallinkrodt Chemical, Paris, Ky.). The resulting 1% chitosan in 2% acetic acid solution is then diluted further by adding 7.4 ml of the solution to 2.6 ml of deioniZed Water to obtain a Working chitosan solution.
 A desired amount of prostate-related antigen, such as PSA, PAP, PSMA, HK2, PCTA-1, PTI-1, PSCA, PTEN/ MMAC1, or LH receptor (the antigen may or may not contain a poly-HIS tag) is added to a 10 ml vial containing 5 ml of the 0.5M phosphate buffer. After adding 0.5 gm of
d-sorbitol (Sigma Chemical Co., St. Louis, M0), the solu tion is rapidly froZen in liquid nitrogen and lyophiliZed.  LyophiliZed sample is reconstituted With 5 ml of the Working chitosan solution, mixed by vortex to form a
cloudy solution containing White particles, and used for immuniZation as described beloW. The pH of the ?nal solution is betWeen 6 and 7.
hours. FolloWing incubation, the slurry Was centrifuged at
1000 rpm the supernatant Was collected and the amount of
unbound protein Was calculated using the Bradford protein PREPARATION OF AN IMMUNOTHERAPEUTIC AGENT INCORPORATED INTO A CHITOSAN-OIL EMULSION
assay (Pierce, Rockford 111.). During a typical preparation 1.67-0.97 pg of recombinant PSAWas found to be bound per
mg Wet Weight of the chitosan-metal chelate. The pellet of
immunotherapeutic agent (PSA-poly-HIS and chitosan-Zinc chelate) Was then resuspended in PBS at a concentration of about 500 pg PSA per ml. The resulting composition Was then used in the immuniZation studies set forth beloW. The ?nal concentration of the metal in the composition Was about 0.7 mM to about 143 mM. EXAMPLE 4 PREPARATION OF CHITOSAN-IRON CHELATE BASED IMMUNOTHERAPEUTIC AGENT
For preparation of a chitosan-iron chelate, 4 g of
ferric ammonium citrate is dissolved in 10 ml distilled Water With 100 pl 11.6N HCl. Four ml of the 1% chitosan solution
prepared as described above (see Example 3) is sonicated and 200 pl of the ferric ammonium citrate solution is added
during sonication. The resulting solution is centrifuged and the pellet containing chitosan-iron chelate is Washed once in deioniZed Water and recentrifuged. Recombinant prostate associated antigen (Which may or may not be modi?ed to
include six histidine residues) is coupled With the chitosan iron chelate as above (see Example 3). As With the chitosan Zine chelate composition, the resulting chitosan-iron chelate composition agent may then be used to immuniZe individu als for the reduction or alleviation of prostatic carcinoma.
 While the folloWing is exempli?ed by the use of squalene, those of ordinary skill in the art Will appreciate that any oil that is readily metaboliZed by the recipient animal may be used (e.g., corn, canola, peanut). Further, While the folloWing is exempli?ed by the use of Pluronic® L121 (poloxamer 401), those of ordinary skill in the art Will again appreciate that other surfactants such as sorbitan
trioleate, polyoxyethylene sorbitan monooleate, polyoxyeth ylene sorbitan monolaurate, and polyoxyethylene-20-tri oleate may be used, as Well as others.
A 2% chitosan solution in 0.5M sodium acetate is
prepared by dissolving 4.1 g of sodium acetate (Sigma Chemical Co., St. Louis, M0.) in 50 ml of deioniZed (18 mOhin: DI) Water With mixing. The pH of the solution is adjusted to 4.5 With approximately 7 ml of glacial acetic acid (Mallinkrodt Chemical, Paris, Ky.) and an additional 1.5 ml of glacial acetic acid is added to compensate for the effect of the addition of chitosan on the pH of the solution. The total volume of the solution is adjusted to 100 ml by the addition of deioniZed Water. 2 grams of chitosan (Sigma Chemical Co., St. Louis, M0.) is sloWly added to the sodium acetate solution With stirring and the mixture is stirred for 2-3 hours until the chitosan dissolves. The chitosan solution is then
steriliZed by autoclaving during a 25 minute cycle. The
PREPARATION OF IMMUNOTHERAPEUTIC AGENT INCORPORATED AND LYOPHILIZED IN PHOSPHATE BUFFER
net. The chitosan solution is then clari?ed by centrifugation in an IEC clinical centrifuge (International Equipment Co., Needham Hts., Mass.) at setting 7 for 5 minutes. The supernatant is decanted from the pellet. 87 to 90% (by Weight) of the chitosan added is retained in the supernatant.
solution is cooled to room temperature in a biosafety cabi
 A 0.5M phosphate buffer is prepared by diluting 15.6 ml of phosphoric acid (16M; Mallinkrodt Chemical,
Aug. 16, 2001
US 2001/0014334 A1
 A 50% sodium hydroxide solution is prepared by dissolving 50 gm of sodium hydroxide (Sigma Chemical
incubated for 1 hour at 37° C. The plates Were then Washed
Co., St. Louis, M0.) in 100 ml of deioniZed Water, With mixing. A squalene/surfactant solution is prepared by com
and substrate solution, ortho-phenylenediamine hydrochlo ride (8 mg/ 10 ml in citrate phosphate buffer, pH 5.5 plus 100
bining 1500 pL of squalene (2,6,10,15,19,23-Hexamethyl
pl of H2O2O) Was added. The plates Were then read in an
dilution; Zymed, San Francisco, Calif.) Was added and
2,6,10,14,18,22-tetracosahexane; Sigma Chemical Co., St.
ELISA reader (at an optical density of 450 nm). The highest
Louis, Mo.) With 600 pL of the surfactant Pluronic® L121 (BASF Corp., Parsippany, N] and is vortexed until homo
dilution at Which no further decrease in absorption Was observed Was estimated as the titer of that particular serum.
 The results (shoWn in FIG. 1) indicate that the
 A chitosan/squalene/surfactant/antigen emulsion is
dilution curve runs almost parallel for native as Well as
prepared by adding a desired amount of a prostate-related
antigen, such as PSA, PAP, PSMA, HK2, PCTA-1, PT-1,
that antibodies raised by recombinant PSA bound to a chitosan-Zinc chelate recogniZed the native PSA as Well as
PSCA, PTEN/MMAC1, or LH receptor (With or Without a
poly-HIS tag) to approximately 370 pL of 2% chitosan in 0.5M sodium acetate and vortexing. The actual amount of antigen used may range from 1 pg to several milligrams. 10 pL of the 50% sodium hydroxide are then added to the
antigen/chitosan and the sample is vortexed. 10 pL aliquots of the 50% sodium hydroxide are added until a stable cloudy
precipitate forms. Approximately 140 ML of the previously prepared squalene/surfactant solution are added to the above solution of antigen/chitosan. The resulting solution is vor texed until a cloudy emulsion formed. The ?nal concentra tion of chitosan ranges from about 0.1% to about 10%, While the ?nal concentration of the oil ranges from about 1% to about 20%. Further, the ?nal concentration of surfactant ranges from about 1% to about 20%. Immediately prior to administration for immuniZation purposes, the resulting
the recombinant PSA. Rat sera generated by immuniZation of rats With native PSA recogniZed the recombinant PSA in ELISA and the dilution curves Were also parallel (data not
shoWn). The puri?ed recombinant PSA containing C-termi nal His6X tag Was also recogniZed in a radioimmunoassay system using a PSA IRMA kit (Diagnostic Products Corpo ration, , Los Angeles, Calif.; data not shoWn). EXAMPLE 8 RAT ANTI-PSA SPECIFICITY
In order to characteriZe the speci?city and purity of
the antibodies obtained from immuniZed rats, the rat sera
(anti-recombinant PSA antisera) Was used in Western immu
noblot analysis, performed according to the method of
solution of chitosan/squalene/surfactant/ antigen is mixed by vortexing or syringe aspiration.
ToWbin et al., Proc. Natl. Acad. Sci. USA. 76:4350-4354
PSA Were run on 4-20% SDS-PAGE gel and blotted to a
IMMUNIZATION AND CHARACTERIZATION OF THE IMMUNE RESPONSE IN RATS
nitrocellulose membrane. The rat antiserum (containing anti-recombinant PSA antibodies) obtained in Example 7 Was then used (at a dilution of 1:5000) in the Western
 Sprague-DaWley male rats (Harlan, Houston,
(1979). Speci?cally, puri?ed human PSA from Whole human seminal plasma and CH0 expressed recombinant human
analysis to determine its speci?city (see Example 1, for
Tex.), Weighing approximately 250 g, Were administered,
actual methods for Western analysis).
via intramuscular injection, 25 pg of the recombinant human
PSA /chitosan-Zinc chelate (see Examples 1-3 above; 1.67
(containing the anti-recombinant PSA antibodies) recog
Immunoblot results indicate that the rat antisera
pg of recombinant PSA per mg Wet Weight of chitosan-Zinc
niZed a band of similar molecular Weight to the puri?ed
chelate) per animal. A booster injection of recombinant PSA/chitosan-Zinc chelate (25 pig/animal) Was administered
recombinant PSA as Well as the native PSA isolated from
5 Weeks subsequent to the initial injection. Animals Were bled by retro-orbital puncture and their serum Was collected
folloWing centrifugation. The resulting antiserum Was used in the experiments described beloW.
 EnZyme Linked Immunosorbent Assay (ELISA) plates (Microtest III Flexible Assay Plates, Falcon, Oxnard, Calif.) Were coated With either recombinant PSA (produced as described above) or native PSA (PSA puri?ed from
human seminal plasma. EXAMPLE 9 BINDING OF RAT ANTI-PSA ANTIBODIES TO HUMAN PROSTATE CANCER CELLS
 In order to assess the binding capabilities of the PSA antibodies contained in the rat antisera (see Example 7), the antisera Was used in immuno?uorescence studies to
human semen, Chemicon Inc., Temecula, Calif.), 50 ng/50
determine the pattern of localiZation on human prostate
pal/Well in a sodium carbonate buffer, pH=9.6. The nonspe ci?c sites Were blocked With 100 pal/Well of 2% non-fat dry milk in a sodium carbonate buffer, pH 9.6. The plates Were Washed 4 times With Phosphate buffered saline containing
tumor cell lines, Du145 and LnCap, (ATCC HTB81 and ATCC CRL1640; American Type Culture Collection,
0.05% TWeen 20, Sigma Chemical Co., St. Louis, Mo.; PBST). The antisera, 50 pal/Well, Was added to the Wells in a serially-doubling dilution and incubated at 37° C. for at least 3 hr. The plates Were Washed 4 times With PBST and
rabbit- anti-rat biotin conjugate (1:1000 dilution; Zymed, San Francisco, Calif.) Was added and further incubated for 3 hours at 37° C. The plates Were Washed 4 times With PBST
and strepavidin-horse radish peroxidase conjugate (1:1000
Manassas, Va.). LnCap and DU145 cells Were groWn on chamber slides (Nunc) for 48 hr 37° C. in a CO2 incubator. The cells Were Washed 3 times With Cold HBSS (Hank’s
Balanced Salt Solution) and Were subsequently ?xed by treatment With cold (4° C.) 90% ethanol for 5 minutes at room temperature. The ?xed cells Were then Washed (tWice) HBSS and immediately incubated With rat anti-recombinant PSA or rat anti-native PSA antisera at a dilution of 1:500. The control cells Were incubated With pre-immune sera at 1:500 dilution. The ?xed cells Were then incubated for 2
Aug. 16, 2001
US 2001/0014334 A1
hours at 37° C. in a humidi?ed chamber. Following incu bation with the antisera, the cells were washed 4 times with
HBSS. Rabbit anti-rat IgG FITC conjugate (Zymed, San Francisco, Calif.) was then added to the cells and further incubated for 1 hour at 37° C. Following incubation, the cells were washed with HBSS 3 times and mounted in
Aquamount (Zymed, San Francisco, Calif). The slides were then observed under ?uorescence microscope on the epif
injection after 6 months, after the titers had started to decline. Two control monkeys received the chitosan-Zinc chelate only. Another set of monkeys were immuniZed with native human PSA/chitosan-oil emulsion (adjuvant of
EXample 6) isolated from human semen, each receiving 250 pg per animal, once a month for three months, followed by ?nal injection at the 6 months. 
Animals were bled every 2 weeks and the serum
titers were measured via ELISA (as described in Example 7)
 Results indicate that LnCap cells showed a very intense staining of the cell membrane by the anti-PSA antibodies and that staining could also be seen in the
from Diagnostic Product Corp., Los Angeles, Calif.)
cytoplasm. With respect to Du145 cells, the staining
throughout the study period. The monkeys were euthaniZed
against recombinant as well as native PSA. Total serum
testosterone was also determined (using a kit purchased
appeared to weaker and very diffuse as compared to the
after the study and prostate, seminal vesicles and testis were
staining of the LnCap cells, although the actual staining was
obtained, weighed, and inspected for any gross abnormali
distinct and more intense as compared to the controls.
 FIG. 4, shows the antibody titers in the serum of the monkeys against recombinant PSA as well as against
EFFECT OF RAT ANTI-PSA ANTISERA ON THE PROLIFERATION OF HUMAN PROSTATE CELLS IN VITRO
similar, demonstrating that the recombinant human PSA was able to elicit an immune response not only against the
In order to assess the effect of PSA antibodies
native PSA. The titers to natural and recombinant PSA were
administered antigen (i.e. recombinant PSA) but the anti bodies also recogniZed native PSA as well. The immuno
therapeutic agent comprising the recombinant PSA /chito
contained in the rat antisera (see EXample 7) on the prolif eration of human prostatic carcinoma cells, cell lines LnCap and Du145, were plated (104 per ml) in 96-well tissue
san-Zinc chelate was found to be immunogenic in rhesus, even though human and rhesus monkey PSAs are approxi
culture plates and allowed to grow and attach for 24 hours at 37° C. in a CO2 incubator. Rat anti-recombinant PSA
Biochim Biophys. Acta, 1174:207-210 (1993)].
antisera, at various dilutions (?nal dilutions: 1: 10,1:20,1:40) was added to the cells, followed by the addition of 5%
Colo.).  Pre-immune sera served as the control. The cells were allowed to incubate with the antibodies (contained in
the antisera) for at least 72 hours. Following incubation with
antibodies, 3H-thymidine (Amersham, Arlington Heights,
mately 89% homologous at the protein level [Gauthier et al. 
No measurable effects were seen with the testoster
one levels of the monkeys, with respect to the immuniZation with PSA. There was a slight reduction in the weight of
prostate (see FIG. 5) of the monkeys immuniZed with the recombinant PSA/chitosan-Zinc chelate immunotherapy. The autopsy of these animals did not reveal any gross abnormalities in the prostate or other reproductive tissue such as seminal vesicles, testis or epidydmis.
Ill.) was added and cells were further incubated for 24 hours.
The cells were harvested and the incorporation of 3H-thy midine in cells was determined via scintillation counting.  FIGS. 2 and 3 show the results obtained and indicate that the antisera (containing anti-recombinant PSA antibodies) alone were cytotoxic in a dose-dependent man ner, as measured by the decreased ability of LnCap cells to incorporate thymidine. This effect was more pronounced when complement was present. However, in the case of Du145 cells, the ability of the cells to uptake thymidine was decreased by about 40% at 1:10 dilution when antisera containing antibodies alone was added, whereas in presence
of complement thymidine uptake was reduced by 95%. At higher dilutions (1:20 and 1:40) less cytotoxic effects were observed.
EXAMPLE 12 EFFECT OF MONKEY ANTI-PSA ANTISERA ON IN VITRO PROLIFERATION OF HUMAN PROSTATE CANCER CELLS
 Human prostate cell lines, LnCap and Du145, were plated (104 per ml) in 96 well tissue culture plates and allowed to attach and grow for 24 hours at 37° C. in a CO2
incubator. Monkey antisera (see EXample 14), at various dilutions (?nal dilutions: 1:10,1:20,1:40), was added to the
cells followed by addition of 5% guinea pig complement (Colorado Serum Co., Denver, Colo.). Pre-immune sera served as the control. The cells were allowed to incubate
with the antisera containing the anti-recombinant PSA anti bodies for at least 72 hours. Following the incubation with EXAMPLE 11
IMMUNIZATION AND CHARACTERIZATION OF THE IMMUNE RESPONSE IN RHESUS MONKEYS
 Two male rhesus monkeys were immuniZed with recombinant human PSA/chitosan-Zinc chelate prepared as described above. Each animals received 250 pg equivalent
the antisera, 3H-thymidine (Amersham, Arlington Heights, Ill.) was added and the cells were further incubated for 24 hours. The cells were harvested and the incorporation of
3H-thymidine in cells determined by scintillation counting.  Results (FIG. 6) indicate that the monkey antisera alone was effective in inducing cellular cytotoXicity, as
noted by the decreased incorporation of thymidine by the
of recombinant PSA as PSA/chitosan-Zinc chelate once
LnCap cells. The cytotoxic effect was enhanced in the presence of complement. Results with respect to Du145
every month for ?rst three months followed by another
cells (see FIG. 7) indicated that the monkey antisera alone