COMBINATION THERAPY FOR THE TREATMENT OF TUMORS

Abstract: The present invention relates to a combination therapy method for treating gastrin-dependent tumors. The method comprises the immunization of a patient with an anti-gastrin (17) immunogenic composition in combination with the administration of chemotherapeutic agents such as 5-fluorouracil and leucovorin.

Description: COMBINATION THERAPY FOR THE TREATMENT OF TUMORS FIELD OF INVENTION The invention is related to a tumor therapy for inhibiting growth by neutralizing immunologically the growth stimulating peptide hormones in combination with a chemotherapy applying a 5-fluorouracil derivative and leucovorin. BACKGROUND OF INVENTION Gastrin is a peptide hormone which occurs in two mature forms, tetratriacontagastrin (G34) and heptadecagastrin (G17), and is synthesized and secreted by specialized cells, G cells, that are located in the stomach antrum. In gastrin-producing cells, these gastrin hormones are posttranslationally processed from a common precursor molecule termed "preprogastrin" containing a signal peptide. The signal peptide "pre" is removed in the endoplasmic reticulum of the cell, resulting in the "progastrin" peptide, which is in turn further processed in the cell to yield the mature gastrins G34 and G17, before secretion into the bloodstream (Dickinson 1991). (The full citations for the references cited herein are provided in the Reference Section preceding the Claims). Both mature forms of G34 and G17 are amidated at their carboxy-terminal end (-NH 2 ). In humans, multiple forms of G17 have been found resulting from differential processing of the precursor molecule, each of which may have different biological activities (Dickinson 1995 and Ciccotosto et al. 1995). In the posttranslational processing of gastrin, it is the "mature" carboxy-amidated form that binds to a specific cell receptor, the so-called CCK-B/gastrin receptor, via the carboxy terminus of the peptide (Kopin et al. 1992). The gastrin hormones are secreted into the circulating blood and bind to specific cells in the stomach, namely, enterochromaffin-like (ECL) cells and parietal cells, that indirectly or directly affect stomach acid output. Historically, both gastrin hormones have been associated with the stimulation of gastric acid secretion (Edkins, J.S. 1905). In recent years, evidence has accumulated showing that gastrin also acts as a trophic factor within the gastrointestinal tract (Johnson, L. 1997) and that it promotes the growth of gastrointestinal cancers (Watson et al. 1989, Dickinson, C.J. 1995), as well as nongastrointestinal cancers, including small cell carcinoma of the lung (Rehfeld et al. 1989). Several types of tumors, including colorectal, stomach, pancreatic and hepatocellular adenocarcinomas possess CCK-B/gastrin receptors in their plasma membranes and the tumor cells respond to gastrin with powerful cellular proliferation (Rehfeld, J.F. 1972, Upp et al. 1989 and Watson et al. 1993). Elevated plasma levels of total gastrin occur in patients with colorectal cancers, and, in particular, increased amounts of the hormone precursor progastrin have been detected in many colorectal tumors using gastrin antisera (Ciccotosto et al.1995). More recently,it has been discovered that many of these cancer cells also secrete gastrin and thus effect an autonomous proliferative pathway(Van-Solinge et al.1993,Nemeth et al.1993 and Seva et al. 1994). The peptide hormones G17 and G34 bind to the CCK-B/gastrin receptors on the cell membranes of normal cells. However, it has been found that G17,but not G34, stimulates the growth of gastrin-dependent cancer cells. Serum-associated G17,in particular, has the potential to stimulate the growth of colorectal tumors in an endocrine manner mediated by CCK-B/gastrin receptors in tumor cells(Watson et al. 1993).G17 is particularly implicated in stimulating the growth of colorectal adenocarcinomas due to a possible increased affinity for the CCK-B/gastrin receptors on the tumor cells, as compared to other gastrin hormone species (Rehfeld 1972 and 1993). The CCK-B/gastπn receptors were found to be expressed in a high affinity form on 56.7% of human primary colorectal tumors (Upp et al. 1989). Numerous studies have shown that, in addition to being able to respond to exogenous endocrine gastrin, human gastric and colorectal tumors produce gastrin and its precursors (Ciccotosto et al., 1995; Finley et al., 1993; Kochman et al., 1992; Nemeth et al., 1993; Van Solinge et al.,1993),thus effecting an autocrine growth stimulatory pathway. Gastrin production in tumor cells differs from that of endocrine G cells. Specifically,those tumor cells contain a high proportion of the precursor progastrin along with a lower concentration of mature peptides.This abnormal ratio is postulated to be due to constitutive unregulated release of gastrin combined with a limited activity of peptidylglycine -amidating monooxygenase (Ciccotosto et al., 1995; Kelly, 1985). Thus, the unregulated release of gastrin leads to the abnormal production and secretion of different molecular forms of the hormone. Specifically, colon carcinoma cells do not efficiently process progastrin resulting in less conversion of precursor gastrin to the mature peptides and,thus,produce mostly incomplete or aberrant gastrins, (Dickinson 1993 and Rehfeld et al. 1993). In addition, the increased gastrin level in colorectal tumors is, in part, attributed to the aberrant expression of the gastrin gene in the colorectal tumor cells(Hoosein et al l 990, Baldwin et al. 1992 and Finley et al.1993).Gastrin-like peptides have been identified in such cells(Hoosein et al.1988,Watson et al. 1991 and Finley et al.1993),and were confirmed to be precursor gastrin species (Van-Solinge et al. 1993 and Nemeth et al. 1993). The presence of amidated-G17 (G17-NH ) in some colorectal cancers(Ciccotosto et al., 1995; Van Solinge et al., 1993) demonstrates that some tumors retain an intact processing -2- pathwav as gastrin amidation only occurs in secretory granules (Varro et al , 1994) Endogenously produced gastπn also acts as an autocπne growth factor, since the basal growth of a colorectal cell line was shown to be inhibited by an anti-gastπn antibody (Hoosein et al , 1988) This was confirmed in a second study in which Northern blot analysis revealed gastπn mRNA in the same cell lines and radioimmunoassay revealed gastπn-like lmmunoreactivity in cell culture supernatant (Hoosein et al , 1990) Gastπn peptides also possess paracπne roles (Watson et al , 1991b)whιch was confirmed(Fιnley et al ,1993)ιn expeπments showing gastπn lmmunoreactivity more predominant in subpopulations of malignant colorectal mucosal cells When G17 binds to its receptor a G17/receptor complex is formed which stimulates cell growth by way of secondary messengers for regulating cell function (Ullπch et al 1990) The binding of G17 to the CCK- B/gastπn receptor leads to activation of phosphatidv nositol breakdown, the protein kinase C activation with a resultant increase in intracellular calcium ion concentration, and the induction oϊc-fos and c-jun protooncogenes via the mitogen-activated protein kinase, which has been implicated in the regulation of cell proliferation (Tadisco et al 1995) Additionally, gastπn binding to the CCK-B/gastπn receptor has been associated with the subsequent increase in phosphorylation by a tyrosine kinase, the ppl25FADK(focal adhesion kιnase),whιch may also have a role in the transmission of rruto- genic signals (Tanaguchi et al 1994) Colorectal cancer remains a formidable disease to treat, as only minor improvements in survival have been obtained in recent years Surgery is an effective treatment of the pπmary disease, but it is ineffectual against residual occult disease, which is frequently present Radiation therapy post-surgery is generally recommended for patients with rectal cancers to reduce the πsks of recurrence of the disease Chemotherapy with 5-fluorouracιl(5-FU) has been the most traditional effective therapy following surgery in patients with more advanc- ed colorectal cancers However,5-FU therapy has been shown to be only of marginal benefit to the patient, since 5-FU is highly toxic and the therapy is costly and does not appear, alone or in combination with other cytotoxic drugs, to significantly prolong survival In most instances, occult or inoperable colorectal tumors do not respond well to chemotherapy or radiation, and new treatments are needed to supplement present procedures Recently, several studies have shown that adjuvant combination chemotherapy with 5-FU and Leucovoπn improves the efficacy of 5-FU in patients with advanced colorectal cancer Leucovoπn is a fo c acid deπvative, also known as folinic acid. Citrovorum factor, or 5-formyl-5,6,7,8,-tetrahydrofohc acid The studies show that in Dukes ' stage C patients, 5- FU/Leucovoπn combination therapv mav reduce mortality bv 10 to 15% (Moertel 1994) In the same patient group, combined intravenous and lntrapeπtoneal therapy with 5-FU/leucovoπn resulted in a non-significant trend to disease-free survival and overall survival advantage (Scheithauer et al , 1995) In advanced disease the same drug combination may give πse to a survival advantage (Taylor, 1993), which has been shown to be 13 5 months of median survival in the combination group compared to 7 5 months in 5-FU-treated patients (Petπoli et al , 1995) However, this combination chemotherapy is not without significant morbidity and causes deleteπous side effects including stomatitis, diaπhea and myelosuppression (Mahood et al , 1991, Erlichman et al , 1988, Pietnel et al , 1989), making quality of life an issue, especially in patients with advanced disease A number of high affinity CCK-B/gastπn receptor antagonists have been evaluated therapeutically both m vitro and in vivo in a number of expeπmental gastrointestinal cancers For example, proglumide, a glutamic acid derivative (Seva et al 190 Harrison et al 1990 and Watson et al 1991a), Benzotπpt, an N-acvl derivative of tryptophan, L-365,260, a deπvative of Aspercillin (Bock et al 1989), and CI-988, a molecule that mimics the C-terminal pentapeptide sequence of CCK (Hughes et al 1990), have been shown to effectively neutralize the effects of exogenous gastπn on gastrointestinal tumor growth both in vitro and in vivo (Watson et al and Romani et al 1994) However, these antagonists have severe toxic side effects and lack specificity, as they block the action of all potential ligands of the receptor such as G34 and CCK in normal cells Recently, highly potent and selective CCK-B/gastπn receptor antagonists such as YM022 (Yuki et al , 1997) and YF476 (Takinami et al , 1997) have been also descπbed Proglumide and Benzotπpt have been widely assessed in prechnical studies The main problem with these compounds is their lack of potency, with relatively high concentrations required to displace G 7 (Watson et al , 1992a, Watson et al , 1992b) Despite this, proglumide and Benzotπpt inhibited the basal and gastnn-stimulated proliferation of a number of cell lines (Seva et al , 1990, Watson et al , 1991a) In addition, proglumide increased the survival of xenograft mice beaπng the gastπn-sensitive mouse colon tumor MC26 to 39 days in the treated animals from 25 days in the control animals Due to the low specificity of this class of gastπn antagonizing agents for the gastπn CCK-B receptor, the inhibition of growth is also thought to be induced by a gastπn- receptor-independent action Moreover, the cellular receptors which recognize and bind the gastπn do not bind all the inhibitors tested (Seva et al 1994) Thus, if complete inhibition of gastrin binding to the receptor does not occur in the autocπne growth cascade, the gastπn antagonists may be unable to block this mechanism of tumor growth promotion Thus, novel therapeutic approaches are needed both as modalities in their own πght and for combination strategies with chemotherapy Combined treatments offer the possibilities of enhancing the therapeutic index and/or reducing the dose of chemotherapy required, thereby limiting the disadvantageous side effects A therapeutic method of selectively immunologically neutralizing the biological activity of the gastπn hormone would provide an effective means of controlling or preventing the pathologic changes resulting from excessive gastπn hormone production associated with colorectal cancers Coassigned U S Patents Nos 5,023,077, 5,468,494, 5,607,676, 5,609,870 and 5,622.702 disclose immunogens and immunogenic compositions useful for controlling G17 and G34 levels in a patient bv generating anti-gastπn antibodies and also disclose the use of such compositions for the treatment of gastπc and duodenal ulcers and gastπn-induced cancers The present invention concerns the use of the antι-G17 immunogens and immunogenic compositions disclosed in Patent Nos 5,023,077, 5,468,494, 5,607,676, 5,609,870 and 5,662,702 in a combination therapy with chemotherapeutic agents for treating gastrin-dependent colorectal cancers The method of cancer therapy descπbed herein has several advantages over present colorectal cancer treatment methods The antι-G17 immunization, in combination with chemotherapeutic agents such as 5-FU and Leucovoπn, increases the therapeutic effects in controlling or inhibiting colorectal tumor growth over chemotherapy alone SUMMARY OF THE INVENTION The present invention provides a combination therapy for treating tumors compnsing immunologically neutralizing peptide hormones and factors which promote tumor cell division in combination with chemotherapy In particular, the present invention provides a method for treating gastπn-dependent cancers, such as colorectal adenocarcinomas The method compπses a combination therapy compπsing antι-G17 immunization of the patient in need of the therapy, in conjunction with the administration of one or more chemotherapeutic agents The antι-G17 immunization for treating gastπn-dependent tumors is surpπsingly effective in generating antι-G17 antibodies, despite the known myelo-suppressive effects of the chemotherapeutic agents used -3- The antι-G17 immunization compπses the active or passive immunization of a patient with an antι-G17 immunogen against the hormone G17 in order to control the patient's G17 levels Λ.s the result of induction of antι-G17 antibodies in a patient, the G17 hormone is neutralized in vivo and its physiological effects are inhibited, thereby inhibiting G17-dependent tumor cell growth Furthermore, the use of antι-G17 immunization in combination with standard chemotherapy increases the efficacy of colorectal cancer treatment, since in the combination, lower amounts of chemotherapeutic agents may be required to treat a patient, thereby loweπng their toxic effects on normal tissues In addition, the patient's quality of life may be improved and his survival time prolonged In a preferred embodiment, the method comprises the active immunization of a mammal possessing a gastπn-dependent tumor with an antι-G17 immunogen, in combination with the administration of one or more chemotherapeutic agents such as 5-fluorouracιl leucovoπn, levamisole cisplatin, tumor necrosis factor and proglumide The antι-G17 immunogen may be administered to a patient at the onset of therapy and at subsequent intervals as required by the patient The antι-G17 antibodies produced by the patient following immunization bind and neutralize G17 in its mature, amιdated-G17 as well as its precursor forms, e g , G17-Gly, in vivo and prevent the binding of G17 to its receptors, thereby preventing gastπn-dependent tumor cell growth The antι-G17 antibody titers produced by an immunized patient may be monitored at predetermined intervals using standard techniques In addition, the chemotherapeutic agents may be administered as directed by standard regimes or lower doses may be administered as required by the patient In another embodiment, the invention further provides a method of treating a gastrin-dependent tumor comprising the passive immunization of a patient possessing a gastπn- dependent tumor with antι-G17 antibodies in combination with one or more chemotherapeutic agents, such as 5-fluorouracιl leucovoπn, levamisole, cisplatin, tumor necrosis factor and proglumide In a prefeπed embodiment of this aspect of the invention, the antibodies may be chimeπc humamzed, or human monoclonal antibodies which may be produced by methods well known in the art The antibodies may be administered together with the chemotherapeutic agents at the onset of therapy and at subsequent intervals after the initial therapy, as required bv the patient BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts a graph showing a time scale of serum antibody titers after immunization of rats immunized with 500 μg/ml of rat antι-G17 (l-9)-DT immunogen FIG. 2 depicts a graph showing the effects of 30 mg/kg dose of 5-FU/leucovorm treatment on the antι-G17(l-9) antibody titers obtained in rats immunized with the immunogen of the invention FIG. 3 depicts a Scatchard plot showing the effects of treatment cycles of 30 mg/kg of 5- FU/leucovoπn on the mean white blood cell counts in BDIX rats FIG. 4 depicts a bar graph showing the median tumor weight of untreated, antι-G17(l-9) DT- treated and DT-treated rats FIG. 5 depicts a bar graph showing the median tumor weights of rats treated with 30mg/kg of 5-FU/leucovoπn,30 mg/kg of 5-FU/leucovoπn and DT ιmmunogen,30 mg/kg of 5-FU/leucov- oπn and antι-G17(l-9)-DT,25 mg/kg of 5-FU/leucovoπn and DT ιmmunogen,25 mg/kg of 5- FU/leucovoπn and antι-G17(l-9)DT, 20 mg/kg of 5-FU/leucovoπn and DT immunogen, 20 mg/kg of 5-FU/leucovoπn and antι-G17(l-9)DT, 12 5 mg/kg of 5-FU/leucovoπn and DT immunogen, and 12 5 mg/kg of 5-FU/leucovoπn and antι-G17(l-9)DT DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods of treating tumors, in particular those associated with gastπn-dependent colorectal cancer, with a combination therapy compπsing immunizing a patient with an antι-G17 immunogen and treating the patient with chemotherapeutic agents, such as 5-FU and leucovoπn The antι-G17 ιmmunιzatιon 5-FU-leucovoπn combination therapy, surpπsιngly,has been found to be more effective than previous therapies in treat- ing colorectal cancer The chemotherapeutic agents useful in the combination therapy do not significantly inhibit antι-G17 antibody production in an immunized patient and lower doses of chemotherapeutic agents can be used for treating the tumor growth In addition, the antι-G17 antibody titers produced by immunization are effective to neutralize all forms of G17 hormone In a preferred embodiment, the method compπses actively immunizing a patient afflicted with a gastπn-dependent colorectal cancer applying an antι-G17 immunogenic composition in conjunction with admimsteπng to the patient chemotherapeutic agents Subsequent booster antι-G17 immunizations may be administered as required by the patιent,as determined by analysis of the patient's serum antι-G17 antibody titers post-ιmmunιzatιon,usιng standard techniques and standard radiological assessments of the tumors Antι-G17 immunization may also be provided to a patient pπor to tumor surgery The antι-G17 immunogens compπse a natural or synthetic peptide fragment of the N-terminal amino acids of G17 as the lmmunomimic portion of the immunogen This peptide fragment is conjugated to an immunogemc earner such as Diphtheπa toxoid (DT) In a preferred embodiment of this aspect of the invention, the antι-G17 immunogen compπses the amino- terminal amino acids of G17 from positions 1 through 9, having the amino acid sequence pyroGlu-Glv-Pro-Trp-Leu-Glu-Glu-Glu-Glu, conjugated to Diphtheria toxoid Other suitable immunogemc protein earners, include bovine serum albumin, key mpet hemocyain, hemocy- amn and tetanus toxoid The immunogens of the invention may also compπse an extension or a spacer peptide sequence suitable for projecting the lmmunomimic peptide away from the protein earner and for enhancing its capacity to bind the lymphocyte receptors A suitable spacer peptide sequence is the amino acid sequence SSPPPPC (SEQ ID NO 2 in the Sequence Listing) However, other spacer peptides would be suitable as well In a prefeπed embodiment of this aspect of the invention, the prefeπed spacer sequence is attached to the carboxy-termmal end of the lmmunomimic peptide The immunogens of the invention are produced by standard techniques and are disclosed in U S Pat Nos 5,023,077, 5,468,494, 5,607,676, 5,609,870, 5,688,506 and 5,662,702, the disclosures of which are hereby incorporated by reference Following immumzation, the immunogens of the invention produce high affinity, neutralizing antibodies for inhibiting the effects of G17 in its mature and precursor forms on tumor growth in immunized animals The antι-G17 antibodies produced bind and neutralize mature and precursor G17, thereby preventing the binding of G17 to the receptors on tumor cells and ultimately inhibiting tumor cell growth The immunogens raise antibodies which neutralize both the carboxy-amidated and glycine-extended G17, and show no cross-reactivity with G34 or CCK The compositions in which the immunogens for active immunization are administered for the treatment of gastπn-dependent tumors in patients may be in a vaπety of forms These include, for example, solid, semi-solid and liquid dosage forms, such as powders, liquid solutions, suspensions, suppositoπes, and mjectable and infusible solutions The prefeπed form depends on the intended mode of administration and therapeutic applications The compositions compπse the present immunogens and suitable pharmaceutically acceptable components, and may include other medicinal agents, earners, adjuvants excipients. etc ,whιch can be mixed using standard procedures Preferably, the compositions are in the form of unit doses The amount of active compound admimstered for immunization or as a medicament at one time, or over a peπod of time, will depend on the subject being treated, the manner and form of administration, and the judgment of the treating physician An effective dosage ranging from 0 001 to 2 mg of the immunogemc composition is administered to the patient for the treatment of the gastrointestinal cancer The effective dosage of the immunogenic composition is capable of eliciting an immune response in a patient of effective levels of antibody titer to bind and neutralize mature and precursor G17 for 1-3 months after immunization Following the immunization and the chemotherapeutic agent treatment, with, for example, 5-FU/Leucovoπn, of a patient with colorectal cancer, the effectiveness of the therapy on tumor growth is assayed by standard clinical procedures, such as ultrasound and magnetic resonance imaging (MRI) to detect the presence and size of tumors, if any The anti- G17 antibody titers may also be monitored from a sample of blood taken from the patient Booster immunizations should be given as required to maintain an effective antibody titer Effective treatment of gastπn-dependent colorectal adenocarcinoma and other gastπn-dependent cancers such as stomach, liver, pancreatic and small cell carcinoma of the lungs according to this method should result in inhibition of tumor growth and a decrease in size of the tumor For passive immumzation, the antι-G17 antibodies are administered to a patient intravenously using a pharmaceutically acceptable earner, such as saline solution, for example, phosphate-buffered saline The chemotherapeutic agents are administered at doses recommended in standard regimes and may be administered at the onset of therapy simultaneously with antι-G17 immunogen, pπor to immunization or after immunization In some cases, it may be beneficial to administer the chemotherapeutic agent both before and after immunization Subsequent chemotherapeutic treatments may also be administered as required by the patient following evaluation by MRI and ultrasound imaging The following expenments were conducted to demonstrate the effects of the present combination therapy on colorectal cancers EXAMPLE 1 The following expenments were conducted to determine the potential clinical benefit offered by antι-G17(l-9)-DT The aims of this study were as follows (a) to determine the long term effect of specific rat antι-G17(l-9)-DT immunization on the histological appearance of the rat GI tract (b) to evaluate the effect of 5-FU/Leucovoπn combinations on antibody titers raised bv antι-G17(l-9)-DT, and (c) to determine the therapeutic effect of antι-G17(l-9)-DT and 5- FU/leucovoπn combinations on a rat colon model Cell line DHDK12 is a rat colomc epithelial tumor cell line (Martin, 1983) The cell line was maintained in RPMI 1640 growth medium (Gibco, Paisley, Scotland) contaimng 10% fetal calf serum (FCS, Sigma, Poole, UK) in humidified conditions at 37°C and 5% CO 2 Immunogen The antι-G17(l-9)-DT immunogen consists of amino acid residues 1-9 of G17 linked via the carboxy-terminus to the peptide spacer SSPPPPC (SEQ ID NO 1 in the Sequence Listing) which in turn is conjugated to DT The immunogen used in these studies was made specific for rat G17 by replacing the human G17 epitope with the amino terminal 9 amino acids of rat G17 linked through a peptide spacer to dφhthena toxoid (DT) Antiserum raised by rat anti- G17(l-9)-DT was denoted as anti-rat G17 (1-9) DT Experimental animals Male and female BDIX rats were provided by the Cancer Studies Unit, Umversity of Nottingham, UK and were 6-10 weeks old, weighing 340-420 g The rats were housed in pairs and maintained in a cycle of 12-hour light and 12-hour dark at 25°C with 50% humidity Prior to each expenment, the animals were grouped to equalize weight distπbution Group sizes ranged from 6-13 animals The UK Coordinating Committee for Cancer Research (UKCCCR) guidelines were adhered to throughout all animal expeπmentation Immunization procedure Rat antι-G17(l-9)-DT was dissolved in sterile saline (0 9%), pH 7 3 to lmg/ml The adjuvant, nor-muramyl dipeptide (Peninsula Labs , Belmont, CA, USA), was added to the conjugate solution to give a final conjugate concentration of between 200 and 500 μg ml The aqueous solution was formulated with an oily vehicle (montamde ISA 703, AMS Seppic Inc , Pans, France) in a 1 2 ratio (v/v) by emulsification After placing in a glass synnge which was attached to a second synnge through a three-way stopcock, the mixture was forced back and forth through the synnges 40 times to form an emulsion An emulsion containing DT peptide and muramyl dipeptide was similarly formulated for control rats A 200μl volume of emulsion (50 μg/rat) was injected s c (nght hand flank of the expenmental ammals) The animals were immunized with either a single injection or repeatedly at 21 dav intervals as detailed below Cytotoxic treatment regime Rats received 12 5 and 25 mg/kg of 5-Fluorouracιl (5-FU, David Bull Labs , Warwick UK) and 12 5-25 mg/kg Leucovoπn (Lederle Labs , Gosport, Hants, UK) admimstered intravenously (IV) on days 1, 3 and 5 with the cycle being repeated every 4 weeks over the duration of the study penod (Asao, 1992) The cytotoxic combination was administered to the rats either pnor to or after antι-G17(l-9)-DT immunization (200 μg/ml) Initiation of tumor growth DHDK12 cells were suspended in steπle phosphate buffered line (PBS, Oxoid, Hants , UK) at a cell concentration of 2 5xl0 7 /ml Rats were anesthetized by a 1 ml intrapeπtoneal injection of Hypnorm (0 315 ng/ml fenatanyl citrate and 10 mg/ml fluamsone, Jannsen. Beπse, Belgium), Hypnovel (5 ng/ml midazolam, Roche, Basel, Switzerland), and stenle distilled water in a 1 1 5 ratio Following a subcutaneous (s c ) incision on the nght flank, a 200 μl volume of cell suspension was miected into the muscle layer of the abdominal wall and the surgical incision closed by wound clips Each expenmental group was composed of between 6 and 13 animals Determination of specific antibody levels of rat antι-G17(I-9)-DT-ιmmunιzedrats To obtain blood samples for analysis, rats were tail-bled at various time points throughout the expenment and at termination by cardiac puncture under terminal anesthesia Serum antι-rat-G17 antibody levels were determined by enzyme-linked lmmunosorbent assay (ELISA) A rat G17-bovιne serum albumin (BSA) conjugate was dissolved to 2 μg ml in 0 IM glycine buffer (pH 9 5) and 25 μl per well was plated into 96-well Immunulon U plates (Dynatech Labs , Sussex, UK) The wells were incubated overnight at 4 U C after which the unadsorbed conjugate was flicked out and the wells were washed with buffer (0 9% saline, 0 5% Tween-20 [Sigma] 0 02% NaN 3 [Sigma], pH 7 3) This buffer was used for all washing steps and reagent dilutions Sera were treated at 10-fold serial dilutions, starting at a dilution of 1 100 The positive control was rat anti-rat G17(l-9)-DT antiserum from previously immunized animals and the negative controls were normal rat serum, and serum from rats immunized with DT only All control sera were used at the same dilutions as the test sera The diluted sera were added to the wells in 25 μl a quots in the presence or absence of 25 μl/well rat G17-BSA at 100 μg/ml (as a soluble inhibitor) Baseline control wells received 25 μl assay buffer onlv The plates were incubated for 60 minutes at room temperature before washing with the assay buffer Goat anti- rat lmmunoglobuhn (H+L)-bιotιn (Zymed, San Francisco, CA, USA) was added to the wells at a 1 500 dilution, 50 μl/well and incubated for 60 minutes in the dark at room temperature After washing, avidin-alka ne phosphatase (Zvmed), 1 100 dilution was added (50 ul well ) and the plates were incubated for 60 mrns at room temperature After further washing, p- mtrophenylphosphate (pNPP) substrate (Sigma) was added to the wells at 50 μl/well and after a 5-mιnute developing time, the absorbance was read at 405 nm The difference in absorbance between untreated sera and sera co-incubated with rat G17-BSA was calculated as the specific absorbance Determination of white blood cell counts Hepannized blood from the rats was collected by tail bleeds during the experiment and by cardiac puncture at the termination of the experiment The numbers of white blood cells were analyzed by the Hematology Depaπment at the University Hospital, Nottingham with the use of a FACScan Histology At termination of the long-term antι-G17(l-9)-DT-ιmmunιzed rats, representative areas of the stomach, colon and rectum from the immunized rats and age-matched controls were dissected and formalin-fixed The sections were then embedded in paraffin and 4 μm sections were cut by use of a microtome These were stained by hematoxy n and eosin and evaluated by a histopathologist who had no knowledge of the treatment groups Crypt cell proliferation rate One hour pnor to animal termination, vincπstine (2 mg/kg, Sigma) was injected mtrapentoneally to induce metaphase aπest in the colonic epithelium pnor to the assessment of colonic crypt cell proliferation (CCPR) The number of cells in metaphase per crypt were counted The colon and rectum were removed from each rat, opened longitudinally and mucosa from each fixed in Carnoys' solution Crypts were gently squashed, longitudinally, under a dissecting microscope and the number of cells in metaphase enumerated (magnification x 25) Statistical analysis In vivo results were analyzed by a Mann Whitney non-parametric test by use of the SPSS statistical package for the IBM PC Long term anti-G 17 ( 1-9) -DT studies Five male rats were immunized with rat antι-G17(l-9)-DT immunogen as descnbed above, and their antibody titers were measured for a period of 34 weeks following a single immunization At this point, the rats were boosted with a second injection of rat antι-G17(l-9)-DT The results are shown in FIG 1 FIG 1 shows the time-scale up to 40 weeks after immumzation of antibodv titers from rats immunized with 500 μg/ml of rat antι-G17(l-9)-DT Each point represents an individual ammal Antibody titers were measured by an ELISA assay as descnbed above using a 1 100 dilution of sera Immunizations are indicated by the arrow Following the pπmary immunization, 4 of the 5 rats responded to the rat antι-G17(l-9)-DT immunogen Antibodies, following this single injection, were detectable by week 7 in 3 of 5 rats and in 4 of 5 rats by week 9 This initial surge of antibodies was followed by a second surge between 15-20 weeks, after which the antibody titers steadily declined and were approaching zero by week 34 At this point, FIG 1 also shows that after a second immunization with rat antι-G17(l-9)-DT, all rats had detectable antι-rat-G-17 antibody titers within 1-2 weeks post-immunization EXAMPLE 2 Histological analysis of the long term anti-G 17 (I-9)-DT-ιmmunιzed rats Specimens from the stomach, colon and rectum were evaluated histologically following hematoxyhn and eosin staining as described in EXAMPLE 1 These were compared to specimens from age and sex-matched control rats All areas of the GI tract evaluated were identical in both antι-G17(l-9)-DT-treated and age-matched control rats with respect to length of villae/crypts/mucosal height In the stomach, enterochromaffin-hke (ECL) cells were similar in number and appearance in the two subject ammal groups However, there was some evidence of granulation of the G cells in the antι-G17(l-9)-DT-treated rat stomach mucosa EXAMPLE 3 Crypt cell proliferation rate (CCPR) of colonic epithelium from long term antι-G17(I-9)-DT- immumzed rats The CCPR of colonic epithelium and anti-rat G17-antιbody titers were analyzed as descnbed above Table I shows the results obtained from 4 of 5 rats evaluated companng CCPR to anti-rat G17 antibody titers The mean CCPR for control rats was 18 93 (standard deviation 3 2) and for the antι-G17(l-9)-DT-ιmmunιzed rats 23 7 (standard deviation 7 9) There was no statistical difference in CCPR between the antι-G17(l-9)-DT-ιmmunιzed and age-matched control rats These results indicate that the rate of crypt cell division in a colonic epithelium is the same for control and antι-G17(l-9) DT-immunized rats Table I A comparison of anti-rat G17:DT antibody titers with the crypt cell proliferation of the colon Rat Specific absorbance relating Crypt cell proliferation rate to anti-rat G17:DT (mean metaphases/crypt after 2 antibodies (1:1000 dilution) hours ™ cristine treatment) Control 1 0 21 9 Control 2 0 19 4 Control 3 0 14 4 Control 4 0 20 0 Immunized rat 1 0 280 29 7 Immunized rat 2 0 340 30 3 Immunized rat 3 0 415 13 6 Immunized rat 4 0 420 21 1 EXAMPLE 4 Effect of pre- and post-cytotoxic treatment on antibody levels raised by rat antι-G17(l-9)-DT Rats were injected intravenously with a 1 1 ratio of 5-FU/Leucovonn at 30 mg/kg as described in Example 1 pnor to or after antι-G17(l-9)-DT immumzation Each group consisted of 6 male and 6 female rats per group and the mean antibody titers were measured by an ELISA technique using a 1 100 dilution of sera Antibody levels in each rat were measured from blood samples as descnbed in Example 1 FIG 2 shows the effect of pre- and post-cytotoxic treatment with 30 mg/kg of 5- FU/Leucovoπn cycles on antibody titers raised by antι-G17(l-9)-DT ιmmunιzatιon(500 μg/ml) In the Figure, the data is represented as follows D no cytotoxics, 7 immunizations, ♦ 2 immunizations pnor to 4 cytotoxic treatments, o 1 immunization pnor to 4 cytotoxic treatments, Δ 1 cytotoxic pnor to 4 immunizations (2 cytotoxic treatments dunng immunizations), Lu 2 cytotoxic treatments prior to 4 immunizations, 3 cytotoxic treatments pnor to 3 immunizations, and • 4 cytotoxic treatments pnor to 2 immunizations FIG 2 shows the mean of 6 female and 6 male rats per group The standard deviations were around 10% of the mean There was no significant effect on antibody titers by pre-treatment with the cytotoxic 5-FU/Leucovonn combination on either the antibody levels achieved or the time taken to achieve those levels when compared to untreated anti-Gl 7(1-9)- DT-immumzed rats The maximum number of treatment cycles evaluated was 4 cytotoxic treatment cycles followed by 2 immunizations Figure 3 shows the effects of treatment on mean white blood cells (WBC) counts, The effect of cytotoxic treatment with 30 mg/kg 5-FU/Leucovoπn in BDIX rats receiving 4 cytotoxic treatments pnor to 2 immunizations on the mean white blood cell (WBC) counts is shown in FIG 3 As shown in the Figure, there was a significant reduction in WBC counts in the representative rats evaluated, post cytotoxic treatment (p<0 005, Students' t-test) The counts were reduced by the number of cytotoxic treatment cycles, indicative of some myelosuppression However, there was no effect on the antibody response to antι-G17(l-9)-DT produced by the rats, as shown in FIG 2 EXAMPLE 5 Effect of combination therapy of 5-FU Leucovorin and anti-G 17 ( 1 -9)-DT on the in vivo growth 0/DHDKI2 tumors The effects of combined therapies with 5-FU/Leucovoπn (12 5-30 mg/kg) and rat anti- G17(l-9)-DT (200μg/ml) on the growth of the rat colon tumor DHDK12 cell line in the muscle layer of the abdominal wall of BDIX rats were tested by compaπson to tumors in control animals as descnbed in the previous Examples At the end of the therapies the rats were killed, their tumors excised and weighed using standard procedures Each group consisted of 10-12 rats/group of mixed sex The median tumor weights are shown with the mter-quartile ranges above the columns Statistical assessment was done by a Mann Whitley U non-parametnc test as described in Example 1 FIGs 4 and 5 show the effect of antι-G17(l-9)-DT immunization on the median final tumor weights from BDIX rats implanted with DHDK12 tumor cells in the muscle layer of the abdominal wall This route of implantation results in a well-vasculanzed tumor amenable to therapies administered into the circulation (Watson, 1996) Rat antι-G17(l-9)-DT had previously been shown to inhibit final DHDK12 tumor weight by 56 5% when administered at a dose of 500 μg/ml (Watson, 1996) In the present expenments, to detect any benefits of combination therapy with 5-FU/Leucovonn, the antι-G17(l-9)-DT dose was dropped to 200 μg/ml, which resulted in a significant inhibition of tumor growth of 25 7% as shown in FIG 4 FIG 4 shows data from tumors excised from untreated control rats, antι-G17(l-9)-DT immunized rats and DT-immunized rats After a 50 day time penod untreated rats had a median tumor weight of 4 43 g DT immunization resulted in a median tumor weight of 4 7 g, which was not significantly different from the tumor weights of untreated rats but which was significantly greater than the median tumor weight of antι-G17(l-9)-DT-ιmmumzed rats (3 49 g, p=0 034, Mann Whitney) FIG 5 shows that 5-FU/Leucovoπn alone, given at 30 mg/kg, significantly reduced tumor weight to a median of 1 01 g (p=0 0106 when compared to untreated control rats) When rats were treated with the same cytotoxic dose of 5-FU/Leucovonn together with DT immumzation, the median tumor weight was not significantly different (0 945 g) A combination of 5-FU/Leucovonn at 30 mg/kg and rat antι-G17(l-9)-DT immumzation resulted in a median tumor weight of 0 68 g which was not significantly different from the 5-FU/Leucovonn/DT-treated group (p=0 27) The combination of 25 mg/kg 5- FU/Leucovonn and DT immunization resulted in a median tumor weight of 0 96 g compared to a mean tumor weight of 0 68g in the antι-G17(l-9)-DT-ιmmunιzed in conjunction with 5- FU/Leucovoπn combination therapy group which was not significant (p=0 409) When the 5- FU/Leucovonn dose was reduced to 20 mg/kg, the 5-FU/Leucovoπn/DT immunogen combination resulted in a median tumor weight of 1 23 g The median tumor weight was significantly reduced to 0 71 g when 20 mg/kg of 5FU/leucovonn was combined with antι-G17(l- 9)-DT immunization (p=0 027, Mann Whitney) Finally, FIG 5 also shows that a 5-FU/Leucovonn dose of 12 5 mg/kg combined with antι-G17(l-9)-DT immunization (p=0 015, Mann Whitney) reduces the median tumor weight from 1 34 g to 0 41 g 5-FU/Leucovonn-antι-G17(l-9)-DT combinations were compared and no statistically significant difference existed between antι-G17(l-9)-DT given in combination with either 12 5 20 or 30 mg/kg of 5 -FU/Leucovonn Due to the limited benefit shown for combination chemotherapy with 5- FU/Leucovoπn in both an advanced cancer state and, in particular, with an adjuvant therapy treatment setting (Moertel, 1994, Scheithauer, 1995, Taylor, 1993, Petnoh 1995) new therapeutic modalities may need to be given either in conjunction with 5 -FU/Leucovonn to enhance the therapeutic index (and possibly reduce the chemotherapeutic dose to limit toxicity) or as a second line treatment if chemotherapy fails to be effective Thus new treatments must be amenable for such use Immunotherapeutic approaches in conjunction with chemotherapy were previously thought to be problematic due to the myelosuppression associated with chemotherapeutic agents, such as that seen with 5 -FU/Leucovonn (Mahood 1991) In the present study, however, myelosuppression of rats induced with 5 -FU/Leucovonn combinations of 30 mg/kg, administered according to Asao et al at the maximum tolerated dose did not affect the level of and time to achieve anti-rat G17:DT antibody titers following immunization with the anti- G17(l-9)-DT immunogen. In therapy studies using 5-FU/Leucovorin in combination with anti-G17(l-9)-DT, a potentiation of the 20 mg/kg and 12.5 mg/kg dosages was achieved. The 20 mg/kg dose was as effective as the maximum tolerated dose when combined with anti-G17(l-9)-DT, and the 12.5 mg/kg dose showed a trend to a greater therapeutic effect. The reason for the latter trend is not known but it may be due to the cytotoxic dose affecting the immune system to a lesser degree than higher dose levels, which may aid in the general inflammatory response against the tumor. 5- FU/Leucovorin given in continuous cycles would appear to exert an 'all or nothing' effect on tumor growth as lowering the dose to 1 mg/kg was found to exert no inhibition of tumor growth. The therapeutic effect may be titrated out more gradually by reducing the number of toxic cycles (Watson, personal communication). Therefore, in the combinations according to the present invention, lower than usual doses of 5-FU/Leucovorin can be administered, thus reducing the side effects of the drugs, while, at the same time, effective killing of tumor cells can be achieved using the present combination, since the immune system is only minimally affected. Thus, the growth inhibitory effect of anti-G17(l-9)-DT immunization is enhanced. These characteristics of the combination therapy are unexpected and surprising in view of the myelosuppressive effects of the chemotherapeutic agents by themselves. Furthermore, by the absence of deleterious effects on the host, anti-G17(l-9)-DT immunization is likely to be a long-term treatment as shown by the length of time that measurable antibody levels were present in rats receiving a single immunization. The first immunization was shown to be 80% effective, in terms of anti-gastrin antibody induction, and 100% effective after the second immunization with an immediate rise in antibody levels. Although potentiation of chemotherapy may be achieved by a single anti-G17(l-9)-DT injection, in most hosts the absence of side effects, characteristic of anti-G17(l-9)-DT immunization, and the host response rate following boosts, indicate a multi-injection regime may be desirable. Despite the length of time that anti-rat-G17 antibodies remained in the circulation there appeared to be no long term deleterious effects on the GI tract, as determined by a simple histological assessment. Additionally, the crypt cell proliferation index of mucosal cells in the colon revealed no significant effect on their growth. Example 6 Treatment of human colon cancer patients with a combination therapy of 5- FU/Leucovorin andanti-G17 (l-9)-DT. Anti-Gl 7(l-9)-DT immunization alone has previously been shown to be a valua- ble and safe therapeutic option in the treatment of gastrin-dependent cancer. The present combinations of anti-G17 immunogens with 5-FU/Leucovorin enhance the effectiveness of cancer treatment,in particular colon cancer treatment,and the possible reduction in the dosage of the chemotherapeutic agent required in the combination should reduce the deleterious cytotoxic side effects of any of the chemotherapeutic agents now in use. The present combinations of an immunogen with chemotherapeutic agents may also be useful as a second-line therapy in patients who do not respond to chemotherapy alone. Human colorectal tumor or colon cancer patients are treated with a combination of chemotherapy and immunotherapy. Specifically,for patients with gastrin responsive colorectal tumors or colon cancer can be treated with concomitant administration of 5-FU/Leucovorin and an anti-Gl 7 immunogen composition or anti-Gl 7 antibodies. In particular, the preferred immunotherapy provides an immunogenic composition comprising an aminoterminal G17 (1-9) peptide: DT conjugate in a pharmaceutically acceptable carrier which may include an adjuvant to further stimulate the immune response. The prefeπed immunotherapeutic regimen can start before, during or after the chemotherapy course depending on clinical considerations. For example, in a patient with a large tumor burden it may be advantageous to start with several cycles of chemotherapy to reduce the tumor bulk and then start with immunotherapy. Alternatively, in a patient with a small tumor burden or after curative surgery, immunotherapy can be started before or during chemotherapy. The active immumzation dose can range between 300 μg up to 1200 μg of the anti-Gl 7 immunogen, depending on the immune status of the patient (or the capacity of an immune response). The injection intervals can be on days 1, 7 and 14, or days 1, 14 and 21, or days 1, 14, then 28 and 56. All the schedules can result in similar antibody titers. The acceler- ated schedules of immunization provide the possibility of earlier onset of immune response. The prefeπed method of the anti-gastrin therapy provides that a booster is administered every 6 months after the initial immunization period, regardless of which protocol is used. Yet another preferred method for the effective neutralization of G17, Gly G17 and G17 NH 2 provides passive immunization with anti-Gl 7 antibodies, preferably in purified form.More specifically, the inoculation of 10-1000 μg anti-G17(l-9) antibodies is administered before, during and/or after the chemotherapy cycles for the control of gastrin activities. The passive immumzation can be admimstered daily, weekly or biweekly. Other protocols can be followed depending on the effectiveness of the treatment. A further combination of treatment provides for an initial passive immunization before and/or during the first cycle of chemotherapy followed by active immunization as described above. Many chemotherapy regimens are in use. These art recognized regimens, al- though not described herein, are not excluded from the combination treatment according to this invention. One preferred chemotherapy regimen provides for 5-FU i.v. bolus of 425 mg/m 2 with i.v. infusion of Leucovorin (folic acid, FA, 20 mg/m 2 ) for 1-5 days per period up to 4 weeks. Another prefeπed regimen provides for 200 mg/m 2 FA over a period of 2h, followed by 5-FU i.v. boles of 400 mg/m 2 + 5-FU of 600 mg/m 2 over 22 hours 1 or 2 days in a 2-week period. Yet another preferred regimen provides for continuous infusion of 5-FU at 250- 300 mg/m 2 day continuous i.v. for 4-6 weeks, followed by 2 weeks rest. REFERENCES ASAO T, TAKAYUKI A, SHIBATA HR, BATIST G and BRODT P Eradication of Hepatic Metastases of Carcinoma H-59 combination chemoimmunotherapy with Liposomal Muramyl Tπpeptide, 5-Fluorouracιl, and Leucovonn Cancer Research 52: 6254-6257, 1992 BALDWIN G Binding of the progastπn fragments to the 78kDa gastnn-binding protein FEBS Lett 1995, 359: 97-100 ERLICHMAN C, FINE S, WONG A, ELHAKEIM T A randomized tπal of fluorouracil and fo nic acid in patients with metastatic CRC JChn Oncol 1988, 6: 496-475 MAHOOD DJ, DOSE AM, LOPNIZ CC Inhibition of Fluorouracil stomatitis by oral cryotherapy J Clin Oncol 1991, 9: 449-452 MAKISHTMA R, LARKTN D, MICHAELI D, GAGINELLA TS Active immunization against gastnn-17 with an N-terminal denved immunogen inhibits gastπn and duodenal lesions in rats Gastroenterol 1995, 106: A824 MARTIN F, CAIGNARD A, JEANNTN JF, LECLERC A, MARTIN M Selection of trypsin of 2 sublines of rat colon cancer cells forming progressive or regressive tumors Int J Cancer 1983, 32: 623-627 MOERTEL GG Chemotherapy CRC NEJM 1994, 330: 1136- 1142 PETRIOLI R, LORENZI M, AQUINO A, MARSILI S, FREDIANI B, PALAZZUOLI V, MARZOCCA G Treatment of advanced colorectal cancer with high-dose intensity fohnic acid and 5 -Fluorouracil plus supportive care Eur J Cancer 1995, 31A: 2105-2108 PDΞTNELLI N, DOUGLAS HO, HARRAVA L The modulation of Fluorouracil with leucovonn in metastatic CRC a prospective randomized phase III tnal J Clin Oncol 1989, 7: 1419-1426 SCHEITHAUER W, KORNEK G, ROSEN H, SEBESTA C, MARCELL A, KWASNY W, KARALL M, DEPISCH D Combined intrapentoneal plus intravenous chemotherapy after curative resection for colonic adenocarcinoma Eur J Cancer 1995, 31A: 1981-1986 SEVA C, DICKINSON CJ, SAWADA M, YAMADA T Charactenzation of the glycine- extended gastπn (G-gly) receptor on AR4-2J cells Gastroenterol 1995, 108: A742 TAYLOR, I Chemotherapy, radiotherapy and immunology of colorectal neoplasia Current opinion in Gastroenterology 1993 , 9: 28-33 WATSON SA and STEELE RJC Gastnn receptors in gastrointestinal tumors WG Landes Company, Austin, USA, 1993 WATSON SA, MICHAELI D, GRIMES S, MORRIS T, ROBINSON G, VARRO A, JUSTIN TA, HARDCASTLE JD. Gastrimmune raises antibodies that neutralize amidated and glycine- extended gastrin- 17 and inhibit the growth of colon cancer. Cancer Res 1996; 56: 880-885. SEQUENCE LISTING <110> APHTON CORPORATION <120> Combination Therapy for the Treatment of Tumors <130> 1102865-0034 <140> US <141> 1999-05-14 <150> US 60/085,687 <151> 1998-05-15 <160> 2 <170> Patentln Ver. 2.0 <210> 1 <211> 9 <212> PRT <213> human or synthetic peptide <220> <221> MOD_RES <222> (1) <223> pyroglutamic acid <400> 1 Glu Gly Pro Trp Leu Glu Glu Glu Glu 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic peptide <400> 2 Ser Ser Pro Pro Pro Pro Cys 1 5

Claims: What we claim is 1 A method for treating a tumor in a patient, compnsing immunologically neutralizing a tumor- growth factor and administering to the patient an effecϋve amount of one or more chemotherapeutic agents 2 The method of claim 1 , wherein the tumor is a gastπn-dependent tumor 3 The method of claim 1 , wherem the tumor-growth factor is gastrin 4 A method for treating a gastrin-dependent tumor with a combination therapy, compnsing administering to a mammal m need of said treatment a therapeutically effective amount of an anti- gastrin 17 immunogen, m combination with one ore more chemotherapeutic agents 5 The method of claim 4, wherem the antigastnn-Gl 7 immunogen is conjugated to Diphtheπa toxoid 6 The method of claim 4, wherem the antigastnn-Gl 7 immunogen further compnses a spacer peptide 7 The method of claim 4, wherem the antigastnn-Gl 7 immunogen compnses a peptide consisting of ammo acid sequence pGlu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu (SEQ ID NO 1 in the Sequence Listing) 8 The method of claim 4, wherein the chemotherapeutic agents are 5 -fluorouracil and leucovonn 9 The method of claim 4, wherem the antigastnn-Gl 7 immunogen is administered pnor to administering 5 -fluorouracil and leucovoπn chemotherapy 10 The method of claim 4 or 9, wherem the chemotherapeutic agents are admimstered in several cycles during the therapy 11 The method of claim 1 or 4, further compnsing administenng one or more booster immunizations

Inventors: GEVAS PHILIP C, GRIMES STEPHEN, KARR STEPHEN L, MICHAELI DOV, WATSON SUSAN A, GEVAS, PHILIP, C., GRIMES, STEPHEN, KARR, STEPHEN, L., MICHAELI, DOV, WATSON, SUSAN, A.
Applicants: APHTON CORP, GEVAS PHILIP C, GRIMES STEPHEN, KARR STEPHEN L, MICHAELI DOV, WATSON SUSAN A, APHTON CORPORATION, GEVAS, PHILIP, C., GRIMES, STEPHEN, KARR, STEPHEN, L., MICHAELI, DOV, WATSON, SUSAN, A.
Application year: 1999
Main classification: A61K39
IPC codes: A61K31, A61K38, A61K39, A61K45, A61P35, A61P43