BACKGROUND OF THE INVENTION In the United States, lung cancer remains the leading cause of cancer death in men, and has surpassed breast cancer as the leading cause of death in women. Small cell lung cancer (SCLC) accounts for approximately 20% of all lung cancer cases, and is the fifth leading cause of death from cancer (Wingo et al., 1995). Distant metastases are present in more than two-thirds of patients with SCLC at diagnosis, and in the absence of treatment, tumor progression is rapid, with a median survival of only 2 to 4 months. However, SCLC is responsive to chemotherapy, with over 80% of patients with limited stage disease (LD) and over 60% of patients with extensive stage disease (ED) achieving a major response to treatment. Despite these results, relapses are common, and most patients die within two years of their diagnosis. The median survival of patients with LD is 14-20 months, and those with ED is 8-12 months. Over the past decade, no additional therapy has been shown to improve overall survival, and standard therapy is observation alone for patients who have achieved a major response after 4 to 6 cycles of chemotherapy. Hence, new approaches to adjuvant therapy are needed.

Antibodies produced by B cells are the primary mechanism for the elimination of circulating pathogens from the bloodstream. They can cause rejection of allografts by both acute and chronic mechanisms. Antibodies induce destruction of cells by several mechanisms including opsonification and removal by the reticuloendothelial system, complement mediated lysis, and antibody-dependent cell mediated lysis.

Thus, antibodies appear ideally suited for eradication of circulating tumor cells and micrometastases in the adjuvant setting (Livingston, 1995).

A study using monoclonal antibodies (mAbs) against polysialic acid demonstrated that this molecule is distributed on SCLC and neuroblastoma cells (Zhang, 1997). For example, polysialic acid was detected on 6 of 6 SCLCs and 5 of 5 neuroblastomas but not on any of the other cancer cells tested. Moreover, polysialic acid (as detected by mAbs 735 and NP-4) had a more restricted distribution on normal tissues than most of the other antigens, being detectable in grey matter of the brain by 735 but not NP-4, and in bronchial epithelia and some alveolar pneumocytes by both mAbs. NP-4 also showed some reactivity with secretory epithelia of stomach, colon and pancreas. While the possibility exists that immunization with polysialic acid could induce an autoimmune response against various subpopulations of cells, expression of polysialylated proteins on normal tissues seems to be minimal and in privileged sites. In fact, individuals having high titer antibodies against these polysialic acid antigens and some patients with paraproteins against polysialic acid have been identified, with no evidence of autoimmunity (Pon et al., 1997).

Polysialic acid chains associated with certain cancers have been characterized and generally contain 14 or more sialic acid units linked by alpha-2, 8 linkages. These long polysialic acid chains are covalently attached to the extracellular regions of the neural cell adhesion molecule (N-CAM) by post-translational modification. The median number of sialic acid units in the polymers may be as high as 50. The embryonal form of N-CAM, modified with long sialic acid polymers, is found on the great majority of neuroblastomas and SCLC cells (Troy, 1992 ; Zhang et al., 1997).

Long polysialic acid chains are also found in developing neural tissues of the fetus.

Apart from mammalian cells, long polysialic acid chains are found to be associated

with the outer membrane of Escherichia coli Kl and Neisseria meni7lgiíidis Group B (Rougon et al., 1986). The difference between polysialic acid chains of bacteria and embryonal N-CAM relative to polysialic acid chains attached to N-CAM in mature tissue is the number of sialic acid units in the chain which may average 50 or more for the bacterial/embryonal form but fewer than 8 for the mature form. During post-natal maturation, the embryonal form is down-regulated (Friedlander et al., 1985), but can be found to be expressed on tumor cells (Roth et al., 1988 ; Komminoth et al., 1991) and certain types of normal cells (see, e. g., Husmann et al., 1989). For reasons that appear related to the secondary structure assumed by these longer polysialic acid chains, a series of monoclonal antibodies including mAbs 735 and NP-4 react strongly with embryonal neural tissue, neuroblastomas and SCLC tissues but only weakly or not at all with most normal tissues, which are known to express N-CAM with the shorter mature form polysialic acid (Hayrinen et al., 1995). Thus, the presence of antigenically distinct long polysialic acid chains on SCLC cells and neuroblastomas and the limited distribution of long polysialic acid chains in mature tissue suggests that it is an excellent target for immunotherapy.

The poor immunogenicity of polysialic acid is borne out by studies of high molecular weight polysaccharides extracted from group B strains of N. meningitidis (MenB) of which sialic acid was the major constituent (Gotschlich et al., 1969). In a study where antibody responses to group B polysaccharides were tested in human volunteers, out of 51 men tested, only one showed a confirmed rise in antibody (Wyle et al., 1972). On the basis of rabbit studies using MenB polysaccharide mixed with various adjuvants, only one of four volunteers immunized with MenB polysaccharide mixed with influenza vaccine developed a significant increase in antibody titer (Wyle et al., 1972). MenB polysaccharides coupled to tetanus toxoid failed to elicit polysaccharide specific antibodies in rabbits and mice (Jennings and Lugowski, 1981) and those antibodies actually produced were specific for the area of coupling between the MenB polysaccharide and the tetanus toxoid, rather than for the polysaccharide itself. To increase immunogenicity, N. group B polysaccharides were modified by N-propionylation prior to immunization (Jennings et al., 1986).

However, not all of these antibodies raised against N-propionylated MenB polysaccharide were specific for native MenB polysaccharide (Hayrinen et al., 1995).

Many antibodies reacted with propionyl moiety. Accordingly, there remains a need for an immunogen and a method to induce an immune response against polysialic acid linked to N-CAM SUMMARY OF THE INVENTION This invention is directed to an immunogenic composition comprising an a- (2-8)-polysialic acid-carrier conjugate and a saponin, wherein the a- (2- 8)-polysialic acid-carrier conjugate comprises one or more a- (2- 8)-polysialic acid polymers covalently linked to an immunogenic carrier, and wherein the median number of sialic acid units in each of the polymers is at least about 10. The polysialic acid polymers can also be modified to increase immunogenicity. Modifications include, but are not limited to, N-acylation and N-propionylation. Polysialic acid polymers have at least about 10 sialic acid units in order to stimulate immunity against polysialic acid polymers of the type found attached to embryonal N-CAM and can comprise as many as 200 or more sialic acid units. Polysialic acid polymers of the invention which are obtained from bacteria can average about 50 to 100 subunits or more and have a molecular weight of about 10, 000 or more. Preferred immunogenic carriers include keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Preferred saponins are QS-21 and the semi-synthetic saponin GPI 0100.

Another aspect of the invention is directed to compositions useful as vaccines comprising amounts of the immunogenic compositions of the invention effective to induce antibodies specific for polysialic acid moieties of embryonal neural cell adhesion molecule (N-CAM) and a pharmaceutically-acceptable vehicle. The preferred adjuvant is a saponin and more preferably is QS-21 or GPI 0100.

The invention also provides a method for stimulating antibodies specific for polysialic acid of embryonal N-CAM by administering an amount of an immunogenic composition of the invention sufficient to stimulate antibodies specifically reactive

with embryonal N-CAM. Such antibodies are also reactive with SCLC or neuroblastoma cells having long chain polysialic acids.

Yet another aspect of the invention is directed to a method of treating SCLC or neuroblastoma in a subject or a patient by administering an amount of a composition comprising an a- (2-8)-polysialic acid-carrier conjugate and adjuvant in amount effective to ameliorate the cancer or neuroblastoma wherein the a- (2-8)-polysialic acid-carrier conjugate comprises one or more a- (2- 8)-polysialic acid polymers covalently linked to an immunogenic carrier. This method is useful for further treatment of patients who have previously undergone primary treatment for SCLC or neuroblastoma (e. g., tumor removal and/or one or more rounds of chemotherapy).

Similarly, this method can be used to treat metastases of SCLC or neuroblastoma.

The preferred carrier used in this method is KLH and the preferred adjuvant is a saponin.

A still further aspect of this invention relates to a method of stimulating immunity against group B N. meningitidis or E. coli KI which comprises administering an amount of a composition comprising an a- (2-8)-polysialic acid- carrier conjugate and saponin in an amount effective to stimulate immunity against group B N. meningitidis or E. coli KI, wherein the a- (2-88)-polysialic acid-carrier conjugate comprises one or more a- (2-8)-polysialic acid polymers covalently linked to an immunogenic carrier, and wherein the median number or sialic acid units in each of the polymers is at least about 10.

The present invention further provides a method of passive immunization for treating SCLC or neuroblastoma in a subject by administering human or humanized antibodies specific for embryonal N-CAM to the subject in an amount effective to ameliorate the SCLC or neuroblastoma. Such antibodies can be administered prior to, concurrent with or after the subject has undergone a primary treatment for SCLC or neuroblastoma and is also useful for treating metastases of SCLC or neuroblastoma.

In yet another embodiment, the invention is directed to a method of passively immunizing a subject to treat or ameliorate infection caused by group B

N. meningitidis or E. coli KI using antibodies raised in response to compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION The compositions of the invention comprise polysialic acid conjugated to an immunogenic carrier and a saponin, the amounts of each being effective to stimulate or enhance antibody production in a human subject. These compositions can be formulated as vaccines by admixture with one or more pharmaceutically acceptable vehicles.

Polysialic acid polymers of the invention are substantially homopolymers of sialic acid of sufficient length to stimulate the production of antibodies which bind to long chain polysialic acid. Long chain polysialic acid consists of 10 or more sialic acid subunits. This is in contrast to polysialic acid present on normal cells which generally consist of 8 or fewer sialic acid units. It has been determined that the affinity of certain known monoclonal antibodies which bind to polysialic acid is significantly reduced when the polymer length is 8 sialic acid units or fewer. Such antibodies bind more efficiently when the polymer length is 10 or more. It has been suggested that binding of such antibodies requires that the polysialic acid polymer be in an extended conformation which is available only as the chain length increases beyond 8 sialic acid units. Preferred polysialic acid polymers contain 14 or more sialic acid units and can have up to about 200 sialic acid units. Polysialic acids obtained from bacteria typically can have a molecular weight of at least about 10, 000 and can contain at least about 50 sialic acid units. Polysialic acid polymers can be modified to increase immunogenicity (e. g., N-propionylated a- (2-8)-polysialic acid).

In accordance with the invention, the amount of polysialic acid in an immunogenic composition used for immunization is between about 0. 1, ug and about 1000 kit, and preferably is between about 1, ug and 100 Hg and more preferably is about 30 Hg. Hence, the amount of polysialic acid conjugate to administer depends on the total amount of sialic acid units present in the polysialic acid polymers conjugated to the carrier. The amount carrier can be determined by the conjugation

ratio of the polysialic acid to the carrier. For example, for conjugates having polysialic acid moieties linked to KLH, the molar ratio of polysialic acid to KLH ranges from about 50 to 1 to about 250 to 1. The molar ratio can be about 25 to 1 to about 1000 to 1. Where polysialic acid moieties are linked to small immunogenic carrier molecules, the molar ratio of polysialic acid to carrier can be less than 25.

As used herein,"immunogenic carrier"means a molecule which can initiate T lymphocyte activation that accompanies an antibody response. Immunogenic carriers have determinants that stimulate helper T cells to secrete cytokines which activate and stimulate proliferation of antibody-producing B cells. Hence, to stimulate or enhance an antibody response against an antigen of interest, particularly with poor immunogens, the antigen can be covalently linked (i. e., conjugated) to such immunogenic carriers. An immunogenic carrier of the invention can be a protein or portion of a protein which, when conjugated to polysialic acid, stimulates or enhances antibody production against the polysialic moieties in the subject. Examples of immunogenic carrier proteins are KLH and BSA. Immunogenic carriers also include polypeptides that are promiscuous class II activators (see, e. g., Panina-Bordignon et al., 1989) to which polysialic acid can be conjugated. In some cases, the number of polysialic acid polymers that can be conjugated to a polypeptide will not be large, but if the polypeptide carrier molecule is a particularly good promoter of a class II immune response, a sufficient immune response to polysialic acid can be obtained. In a preferred embodiment, the carrier is KLH or a derivative thereof Conjugate linkages are made by well known methods.

In addition to immunogenic proteins and polypeptides which comprise a T cell epitope, carriers can also be constructs to which other immunogenic moieties (e. g., cytokines, polypeptides bearing T cell epitopes, etc.) can be linked. Branched constructs such as lipo-thioester and branched polylysine allow for multiple covalent linkages of such immunogenic moieties as well as conjugation of multiple polysialic acid polymers. Carriers further include proteins and polypeptides which have been modified by the covalent addition of immunogenically active moieties.

Polysialic acid-carrier conjugates are formulated with the adjuvant saponin to form the compositions of the invention. Saponins are immunological adjuvants that promote non-specific immune responses. The saponins in the compositions of the invention are present in an amount from about 1 to about 2000 ag. Saponins include semi-synthetic and synthetic saponins. In a preferred embodiment, the adjuvant is a saponin derived from the bark of a Ouillaja saponaria Molina tree. A particularly preferred adjuvant is QS-21. QS-21 is preferably present in an amount between about 50, ug and about 500//g or between about 50 µg and about about 200/, Ig and more preferably in an amount of about 100, ug. Other saponins from Ouillaja saponaria Molina may also be used, as well as saponins from other plant sources.

Another example of a saponin source is Saponaria o, gicinalis. The monosaccharide composition, molecular weight, adjuvant effect and toxicity for a series of saponins has been described (Kensil et al., 1991). A second particularly preferred adjuvant is the semi-synthetic saponin GPI 0100. GPI 0100 is preferably present in an amount between about 10 and about 2000, ug or between about 100/g and about 2000 Hg or between about 100/g and about 1000/-ig and more preferably in an amount of about 500/. g to about 1000 lit.

The ratio of polysialic acid and adjuvant used in compositions of the invention can vary. One of ordinary skill in the art can readily determine the effective amounts of conjugated polysialic acid to be used for immunization. Positive immune responses are indicated, for example, by an increased titer of antibodies specific for the embryonal form of N-CAM or by increased complement mediated lysis of cells having long chain polysialic acid on the cell surface (e. g. H345 cells) by immune sera relative to preimmune sera.

Vaccines of the invention comprise the compositions of the invention and a pharmaceutically acceptable vehicle. The vaccines can be administered intradermally, subcutaneously, intramuscularly or by any other suitable route for eliciting an immune response. For the purposes of the invention,"pharmaceutically acceptable vehicle" means any of the standard pharmaceutical vehicles. Examples of suitable vehicles are well known in the art and include, but are not limited to, any of the standard vehicles

such as phosphate buffered saline, phosphate buffered saline containing Polysorb, water, emulsions such as oil/water emulsion, and various types of wetting agents. In a preferred embodiment, vaccines of the invention are for administration to a human.

Alternatively, a vaccine can be administered to any animal to induce the formation of antibodies which bind to a polysialic acid moiety of embryonal N-CAM or for producing humanized antibodies.

The invention further provides a method of stimulating the production of antibodies specific for polysialic acid moieties on N-CAM (or on bacteria carrying such moieties) in a subject which comprises administering a sufficient amount of a composition of the invention to stimulate antibody production. Stimulating antibody production means inducing the production of antibodies which bind to long-chain polysialic acid polymers in a subject wherein such antibodies were previously undetectable or detectable at only a low level or titer. Stimulation of antibody production as used herein includes producing increased amounts of polysialic acid- specific antibodies or antibodies with increased affinity for polysialic acid, or both.

Preferably, immunization with a composition of the invention is performed using multiple injections administered over a time course which is selected to maximize the titer and/or function of induced anti-polysialic acid antibodies.

However, any suitable immunization regimen can be used. In a preferred embodiment, a composition of the invention for human administration comprises KLH, or a derivative thereof, conjugated to polysialic acid and mixed with the adjuvant saponin. The saponin is preferably QS-21.

Antibody titers in sera of immunized subjects can be determined by ELISA or by any other assay to measure antibodies as well known to one of skill in the art. One way to examine the function of such antibodies is to measure their ability to bind to polysialic acid on the surface of cells. For example, dilutions of antisera from immunized subjects can be added to cultured H345 cells, which express long chain polysialic acid polymers, and antibodies in the antisera detected with secondary antibodies which bind specifically to constant domains of the test antibodies. The secondary antibodies can be labeled, for example with an enzyme or a fluorescent tag.

A measure of the degree to which detectable label is associated with the H345 cells is a measure of the ability of induced antibodies to bind to polysialic acid on the surface of a cell. Another way to test antisera from immunized subjects is to add serial dilutions of antisera, together with human complement, to cultured H345 cells and observe killing of the H345 cells. Other ways to test for binding and function of anti- polysialic acid antibodies raised by immunization with compositions of the invention will be evident to those of ordinary skill in the art.

Another aspect of this invention provides a method of treating SCLC or neuroblastoma in a subject by administering to the subject an amount of a composition of the invention effective to ameliorate, reduce or otherwise treat the SCLC or neuroblastoma. In accordance with the invention, adjuvants other than saponin can be used in this method. For example, adjuvants which can be substituted for, or utilized in addition to, saponin include, but are not limited to, alum, oil-in- water emulsions, complete or incomplete Freund's Adjuvant, non-ionic block copolymers (e. g., TiterMax, L121, CRL1005, etc.), adjuvant compositions which comprise bacteria derived substances such as monophospholipid A or cell wall skeleton (CWS), DNA having immunostimulatory CpG motifs and immunostimulatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2). Other common adjuvants well known to those of ordinary skill in the art can also be used.

Ameliorating SCLC or neuroblastoma means elimination or reduction of remnants of operable tumors which remain following surgical excision or other means of obliterating the tumor (e. g., a primary treatment). Moreover, the method is also useful for treating (e. g., eliminating or reducing) metastases and circulating cells that may give rise to metastases. For example, by the time SCLC is detected and treated, distant metastases are present in more than two-thirds of patients. While immunotherapy can be used as an adjuvant to chemotherapy, the present invention contemplates using immunotherapy as a substitute for chemotherapy, particularly for cancer patients who are not responsive to chemotherapy. The present method may also aid in preventing relapses in patients who have undergone successful primary

treatments. Hence, the present methods for treating SCLC or neuroblastoma are useful in a subject who has developed and been treated for SCLC or neuroblastoma and no longer displays any detectable signs of the cancer (i. e., a subject in remission).

Many ways are available to assess a patient's or subject's response to the immunotherapeutic methods of the invention. For example, methods of detecting cancerous cells or observing amelioration of the cancer are well known. An example of such a method is the detection of parathyroid hormone-related protein (PTHrP), which is expressed in a variety of human cancers including lung cancer, the COOH terminal of which is stable and measurable in urine (Nishigaki et al., 1999).

Immunolabeling techniques are also available for detection of tumor cells in bone marrow of SCLC patients (Pelosi et al., 1999). Alternatively, reverse transcriptase polymerase chain reaction (RT-PCR) is increasingly used to detect small numbers of circulating tumor cells. Such cancer detection and surveillance techniques are applicable for both active and passive immunization methods.

Compositions of the invention can also be used to stimulate immunity against group B N. meningitidis or E coli KI. As in methods described above, immunity can be shown by an increase in the titer and/or affinity of circulating antibodies capable of binding to the polysialic acid moieties on the embryonal form of N-CAM or to the polysialic acid moieties on the aforementioned bacteria. If necessary, increased titers of circulating antibodies can be maintained by a regular course of immunizations.

However, following an initial course of vaccination, it is usually sufficient to administer booster vaccinations when needed to maintain the immune system in a state where it can respond to subsequent infection with a vigorous secondary immune response. The timing of administering booster vaccines can be determined by one of ordinary skill in the art.

A further aspect of the invention provides methods of passive immunization using antibodies obtained against the compositions of the invention. Such antibodies can be obtained, for example, by immunizing a subject with a composition of the invention and recovering sera from the subject. If desired the anti-polysialic acid antibodies can be further purified from the sera. It is well within the abilities of one

of average skill in the art to purify such antibodies from antisera of subjects that have been vaccinated with compositions of the invention, as well as to collect and immortalize B cells secreting anti-polysialic acid antibodies from such subjects.

Antibodies useful for treatment of humans may be obtained from other sources as well. Methods for generating human and humanized antibodies are well known. For example, the binding site amino acid residues of a non-human antibody specific for polysialic acid which was obtained by immunizing an animal with a composition of the invention can be substituted into a human antibody to produce a chimeric antibody which is not foreign to the human immune system. Alternatively, transgenic animals which comprise human Ig genes can be vaccinated with compositions of the invention to obtain polyclonal antisera specific for polysialic acid, or for the production of hybridomas which secrete human monoclonal antibodies which are specific for polysialic acid. For a review of such methods, see, e. g., Vaughan et al. (1998).

Antibodies obtained by such methods can be used for treatment of SCLC and neuroblastoma immediately upon diagnosis and prior to the usual primary treatments.

Such antibodies can also be administered to treat or prevent the formation of metastatic tumors concurrent with or following primary treatments.

The antibodies can also be administered in methods of passive immunization against group B N. meningitidis or E. coli KI. Such methods of treatment are especially useful for treating patients who may have acquired such an infection but whose immune systems have not had sufficient time to respond to and to eliminate the infection. Passive immunization can, for example, reduce the severity and duration of the infection. Methods and routes of administration are well known to those of ordinary skill in the art. Examples and reviews of such methods are readily available (see, e. g., Schirrmacher et al., 1996 ; Saltzman, 1993 ; Masuho, 1992 ; Hammarstrom and Smith, 1990).

Throughout this application, various publications, patents, and patent applications have been referred to. The teachings and disclosures of these publications, patents, and patent applications in their entireties are hereby

incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles of invention herein disclosed may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.

Examples of the invention which follow are set forth to further illustrate the invention and should not be construed to limit the invention in any way.

EXAMPLE 1 Antigen Preparation Polysialic acid preparation-Poly-2, 8-N-acetylneuraminic acid (colominic acid) is supplied as a powder (Sigma Chemical Co., St. Louis, MO ; Nacalai Tesque, Inc., Kyoto, Japan) which contains a single major species of about 30, 000 molecular weight (a homopolymer from E. coli of over 50 sialic acid units) and multiple species of lower molecular weight. Prior to conjugation to KLH, long polysialic acid chains were purified from lower molecular weight chains by gel filtration chromatography.

The polysialic acid was purified using a Sephacryl S-200 column (flow rate 1 ml/min ; column dimensions 90 cm x 2. 5 cm) and eluted with sterile PBS by Fast Performance Liquid Chromatography (FPLC). The long chain fraction was used directly for conjugation to KLH.

Conjugation of polysialic acid to KLH-Conjugation of polysialic acid to KLH (Intracel Corp., Rockville, MD) was accomplished by the periodate method which involves the reaction of sodium metaperiodate with the vicinal hydroxyl groups in the polysialic acid chain to form a dialdehyde at the end of each chain. The dialdehyde is reacted with free amino groups of KLH in aqueous solution to yield diamine linkages which in turn are stabilized by reaction with sodium cyanoborohydride.

Preparation of polysialic acid dialdehyde-All glassware was rinsed with distilled water and autoclaved prior to use. 10 mg of purified polysialic acid was dissolved in 1 ml sodium phosphate buffer. 2. 5 mg of sodium metaperiodate (freshly prepared) was added and the solution was stirred at room temperature (25°C) for 90 minutes. At the end of the incubation period, 2. 9 mg (46. 6 mole) of ethylene glycol (2. 6 lil ; 1. 11 mg/, may be added to neutralize unreacted sodium metaperiodate and the solution stirred for 15 min. at 25°C. Whether neutralized or not, excess sodium metaperiodate was removed by passage over a Sephadex G-10 column equilibrated with PBS buffer. Fractions were collected at one minute intervals and those fractions shown to be positive by reaction with 0. 2% resorcinol were combined.

Conjugation of polysialic acid dialdehyde to KLH-The polysialic acid dialdehyde was transferred to a sterile glass bottle containing 5 mg of sterile, pyrogen-free KLH (Intracel Corp., Rockville, MD) dissolved in PBS (5 mg/ml). The flask was rinsed two times with 2. 5 ml of PBS, and these washes were added to the KLH/polysialic acid dialdehyde mixture and allowed to incubate at 37°C for 60 minutes with gentle stirring. A 20 mg/ml solution of sodium cyanoborohydride (NaBH3CN) in PBS was prepared and sterile filtered. 1 ml of the NaBH3CN solution was added to the KLH/polysialic acid dialdehyde mixture and incubated at 37°C for 48 hr.

Purification of polysialic acid-KLH glycoconjugate-The contents of the polysialic acid-KLH reaction vial were loaded on a Sephacryl S-200 column (flow rate 1 ml/min ; column dimensions 90 cm x 2. 5 cm) and eluted with sterile saline. The fractions positive for both KLH and polysialic acid were combined and sterile filtered with a 0. 22 um low protein-binding sterile, pyrogen-free filter and stored at-20°C.

Protein and sialic acid content were determined by BioRad protein assay and resorcinol reaction, respectively. QS-21 was added to yield 100, ug/ml and the mixture was filtered through a 0. 22, um filter. Aliquots of 1 ml were added to sterile 2 ml vials (Nunc) and lyophilized, capped and stored at-30°C.

EXAMPLE 2 Immunization CB6/F1 mice were immunized subcutaneously with KLH-conjugated polysialic acid and 10, ug of QS-21 adjuvant. The amount of polysialic acid administered, as determined by reaction with resorcinol, was 3 plu. The initial vaccination was followed by two identical booster vaccinations at one week intervals.

Blood was collected and the presence of antibodies capable of binding to sialic acid was determined in two ways. First, antibody titers were determined by ELISA for both IgG and IgM. Table 1 shows the titers of IgM and IgG in the serum of five mice immunized according to the above protocol and indicates the highest serum dilutions which yielded a signal in an ELISA assay using microtiter dishes coated with a polysialic acid-BSA conjugate. All 5 mice displayed significant increases in antibody titer for both IgM and combined IgG classes. Table 1 Ab ELISA Titers Induced by Polysialic acid-KLH conjugate with QS-21 Mouse Number Preimmune Serum Immune Serum IgM IgG IgM IgG 1 0 0 200 6, 400 2 0 0 1, 600 6, 400 3 0 0 1, 600 25, 600 4 0 0 200 12, 800 5 0 0 1, 600 12, 800 The capacity of the serum antibodies to bind to surface antigen of H345 tumor cells (ATCC catalog no. HTB-180) was also tested. H345 cells were incubated with serum preparations from the five immunized mice and with preimmune serum. IgG and IgM antibodies were determined using fluorescein isothiocyanate (FITC)- conjugated secondary reagents specific for the selected antibody classes. Antibody bound to the H345 cells was determined by measuring fluorescence with a fluorescence-activated cell sorter (FACS). As shown in Table 2, the results of the FACS analysis demonstrate that the sera of all five mice produced antibodies capable of binding to polysialic acid on tumor cells. Furthermore, the antisera promoted complement-induced tumor cell lysis ? vitro.

Table 2 <BR> <BR> Anisera Reactivity with Tumor Cells by FACS Analysis I Mouse Number % Positive IgM % Positive IgG 1 36. 44 81. 63 2 90. 02 93. 37 3 91. 72 89. 41 4 93. 83 88. 78 5 92. 37 94. 96 Preimmune Serum 8. 25 10. 28 EXAMPLE 3 Immunization Patients having completed initial therapy for SCLC and having achieved either a complete or partial response to therapy without subsequent disease progression were immunized subcutaneously with 30 ug of polysialic acid in polysialic acid-KLH conjugate and 100, ug of QS-21 adjuvant or 30, ug of N-proprionylated polysialic acid in N-proprionylated polysialic acid-KLH conjugate and 100 Hg of QS-21 adjuvant.

Four vaccinations were administered at one week intervals. A fifth vaccination was then administered following a four week interval (week 8) and a sixth vaccination was administered after a further eight week interval (week 16). Peripheral blood (20-30 ml) was drawn immediately before each vaccination and 2 weeks after the fourth, fifth and sixth vaccinations (weeks 6, 10 and 18).

Antibody responses were measured as described above in Example 2. As measured by ELISA assay using microtiter dishes coated with a polysialic acid-BSA conjugate and blocked with human serum albumin, four patients showed significant IgM responses to the N-propionylated polysialic acid-KLH conjugate administered with QS-21 (Table 3).

Table 3 Ab ELISA Titers against Polysialic acid-BSA conjugate Induced by Polysialic acid-KLH conjugate with QS-21 or N-propionylated Polysialic acid - KLH conjugate with QS-21 Median ELISA titer Vaccine Patient"" Preimmune Immune Preimmune Immune Serum Serum Serum Serum 1 20 40 40 40 2 10 10 20 20 PSA-KLH 3 80 80 0 0 + QS-21 4 0 20 0 0 50000 1 10 320 0 0 2 10 320 0 0 N-propionylated 40 1280 40 20 PSA-KLH + QS-21 4 10 20 0 0 5 10 0 0 0 6 0 80 0 40 H69 SCLC tumor cells (Carney et al., 1985 ; ATCC catalog no. HTB-180) were used to test the serum of immunized subjects for the presence of antibodies capable of binding to SCLC tumor cells (Table 4). Because preimmune sera usually shows binding to a variety of antigens present on the surface of the cells, a serum dilution was chosen for each subject which resulted in approximately 10% of H69 cells testing positive in FACS analysis with preimmune serum. This same dilution was applied to immune serum which was later obtained from that subject. The increases in % positive cells observed for immune sera as compared to preimmune sera should primarily reflect an increase in antibodies specific for polysialic acid displayed on H69 cells. In three subjects, the response to N-propionylated polysialic acid-KLH conjugate plus QS-21 is detectable above the background level of serum antibodies which bind to other H69 antigens.

Table 4 Antisera Reactivity with Tumor Cells by FACS Analysis Median Percent Positive Cells Vaccine Patinent"" Preimmune Immune Preimmune Immune Serum Serum Serum Serum 1 10 18 10 16 2 10 8 11 12 PSA-KLH 3 10 16 11 14 + QS-21 4 10 6 11 15 5 10 11 11 9 1 11 48 11 14 2 11 27 11 8 N-propionylated 3 10 15 10 24 PSA-KLH + QS-21 4 10 8 10 16 5 11 10 11 5 6 10 12 10 7

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