CZ20004159A3 - The combination suitable for use in the treatment of gastrin-dependent tumor and its use - Google Patents

Abstract

A combination suitable for use in the treatment of a gastrin-dependent tumor, comprising i) an immunogen directed against the growth of said tumor and ii) one or more chemotherapeutic agents for the treatment thereof. Use of said combination for the manufacture of a preparation for use in the treatment of said gastrin-dependent tumor.

Description

Technical area

The invention relates to a combination suitable for use in the treatment of gastrin-dependent tumors and its use for the manufacture of a pharmaceutical preparation. Treatment with the combination according to the invention results in neutralization of growth-stimulating peptide hormones in combination with chemotherapy.

State of the art

Gastrin is a peptide hormone that exists in two mature forms, tetratriacontagastrin (G34) and heptadekagastrin (G17), and is synthesized and secreted by specialized cells found in the antrum of the stomach. In the gastrin-producing cells, these gastrin hormones are post-secreted from a common precursor molecule called preprogastrin, which contains a signal peptide. The pre signal peptide is removed in the endoplasmic reticulum of the cells, resulting in the formation of the progastrin peptide, which is further processed by the cells to form the mature gastrins G34 and G17 before being secreted into the bloodstream (Dickinson, 1991) (full references are given in the References section before the claims). Both the mature forms G34 and G17 are amidated at their carboxy-termini (-NH2). Many forms of G17 have been identified in humans, resulting from different processing of the precursor molecule, and each of them has different biological activities (Dickinson, 1995, and Ciccotosto et al.,

1995). In post-translational processing of gastrin, it is the mature carboxy-terminally amidated form that binds to a specific cellular receptor, the so-called CCK-B/gastrin receptor, via the carboxy-terminus of the peptide (Kopin et al.,

1992) .

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Gastrin hormones are secreted into the circulating blood and bind to specific cells in the stomach, namely enterochromaffin-like (ECL) cells and parietal cells, which indirectly or directly affect gastric acid production. Historically, both gastrin hormones have been associated with the stimulation of gastric acid secretion (Edkins, J.S., 1905). In recent years, evidence has accumulated that gastrin also acts as a trophic factor in the gastrointestinal tract (Johnson, L., 1997) and that it promotes the growth of gastrointestinal tumors (Watson et al., 1989, Dickinson, C.J., 1995), as well as non-gastrointestinal tumors, including small cell lung cancer (Rehfeld et al., 1989).

Several types of tumors, including colorectal adenocarcinoma and adenocarcinomas of the stomach, pancreas, and hepatocellular carcinoma, contain CCK-B/gastrin receptors on their plasma membranes, and tumor cells respond to gastrin with significant proliferation (Rehfeld, J.F., 1972, Upp et al., 1989, and Watson et al., 1993). Elevated plasma concentrations of total gastrin are found in patients with colorectal carcinoma, and high levels of the hormonal precursor progastrin have been detected in many colorectal carcinomas using antisera to gastrin (Ciccotosto et al., 1995). More recently, many of these tumor cells have also been shown to secrete gastrin, thereby causing autonomous proliferation stimulation (Van-Solinge et al., 1993, Nemeth et al., 1993, and Seva et al., 1994).

The peptide hormones G17 and G34 bind to CCK-B/gastrin receptors on the cell membranes of normal cells. However, G17, but not G34, has been shown to stimulate gastrin-dependent tumor cells. In particular, serum G17 has the potential to endocrine-stimulate the growth of colorectal tumors via CCK-B/gastrin receptors on tumor cells (Watson et al., 1993). In particular, G17 has been suggested to be involved in the stimulation of the growth of colorectal adenocarcinomas because of its increased affinity for CCK-B/gastrin receptors on tumor cells, compared with other types of gastrin (Rehfeld, 1972, and 1993). CCK-B/gastrin receptors were found to be expressed in a high-affinity form on 56.7% of human primary colorectal tumors (Upp et al., 1989).

Many studies have shown that, in addition to being able to respond to exogenous gastrin, human colorectal and gastric tumor cells are able to 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), thereby autocrinely stimulating growth. Gastrin production in tumor cells differs from that in endocrine G cells. Specifically, these tumor cells contain a higher ratio of the precursor progastrin together with lower concentrations of the mature peptide. This abnormal ratio is thought to be due to sustained unregulated gastrin release together with reduced peptidoglycine-1-amidating monooxygenase activity (Ciccotosto et al., 1995; Kelly, 1985). Thus, unregulated gastrin release leads to abnormal production and secretion of various molecular forms of the hormone. In particular, colon tumor cells do not process gastrin efficiently, resulting in less conversion of gastrin to mature peptides and thus the production of mostly incomplete or aberrant gastrin (Dickinson, 1993, and Rehfeld et al., 1993). In addition, elevated gastrin concentrations in colorectal tumors are partly due to aberrant expression of the gastrin gene in colorectal tumor cells (Hoosein et al., 1990, Baldwin et al., 1992, and Finley et al., 1993). Gastrin-like peptides have been identified in these cells (Hoosein et al., 1988, Watson et al., 1991) and have been confirmed to be precursors of gastrin (Van Solinge et al., 1993, and Nemeth et al., 1993).

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The presence of amidated G17 (G17-NH2) in some colorectal tumors (Ciccotosto et al., 1995; Van Solinge et al., 1993) suggests that some tumors retain an intact processing pathway, as gastrin amidation occurs only in secretory granules (Varro et al., 1994). Endogenously produced gastrin also acts as an autocrine growth factor, as inhibition of basal growth of a colorectal cell line was observed with an anti-gastrin antibody (Hoosein et al., 1988). This was confirmed in a second study, in which the presence of gastrin mRNA was detected in some cell lines by northern blotting and a radioimmunoassay showed gastrin-like immunoreactivity in the cell culture supernatant (Hoosein et al., 1990). Gastrin peptides also have a paracrine role (Watson et al., 1991b), which was confirmed in experiments (Finley et al., 1993) showing gastrin immunoreactivity primarily in subpopulations of malignant colorectal mucosal cells.

After binding of G17 to its receptor, a G17/receptor complex is formed, which stimulates cell growth through second messengers regulating cell functions (Ullrich et al., 1990). Binding of G17 to CCK-B/gastrin receptors leads to activation of the phosphatidylinositol pathway, activation of protein kinase C with a subsequent increase in intracellular calcium ion concentration, and induction of c-fos and c-jun proto-oncogenes through mitogen-activated protein kinase, which is thought to be involved in the regulation of cell proliferation (Tadisco et al.,

1995). Furthermore, gastrin binding to CCK-B/gastrin receptors is associated with a subsequent increase in phosphorylation by the tyrosine kinase, ppl25FADK (focal adhesion kinase), which may also have a role in the transduction of mitogenic signals (Tanaguchi et al., 1994).

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Colorectal cancer remains a difficult disease to treat, with only small improvements in survival over the past few years. Surgery is an effective treatment for primary disease, but it is ineffective for the treatment of residual disease that is often present. Postoperative radiotherapy is usually recommended for patients with rectal cancer to reduce the risk of disease recurrence. Chemotherapy with 5-fluorouracil (5-FU) is the most commonly effective therapy after surgery for patients with more advanced colorectal cancer. However, 5-FU therapy has been shown to have little benefit for patients, as 5-FU is highly toxic and expensive, and it does not appear to significantly prolong survival, either alone or in combination with other cytotoxic drugs. In most cases, occult or inoperable colorectal cancer does not respond well to chemotherapy and radiation, and new treatments are needed to complement current treatments.

Recently, several studies have shown that adjuvant combination chemotherapy with 5-FU and leucovorin enhances the efficacy of 5-FU in patients with advanced colorectal cancer. Leucovorin is a derivative of folic acid, also known as folinic acid, citrovorum factor, or 5-formyl-5,6,7,8tetrahydrofolic acid. Studies have shown that in patients with Dukes stage C, combination therapy with 5-FU/leucovorin can reduce mortality by 10-15% (Moertel, 1994). In the same group of patients, combined intravenous and intraperitoneal 5-FU/leucovorin therapy resulted in a nonsignificant trend toward improved disease-free and overall survival (Scheithauer et al., 1995). In advanced disease, the same combination of drugs can improve survival (Taylor, 1993), which was 13.5 months (median) in the combination group compared with 7.5 months in the 5-FU group (Petrioli et al., 1995). However, this combination chemotherapy is not without significant morbidity and causes serious adverse effects including stomatitis, diarrhea and myelosuppression (Mahhod et al., 1991; Erlichman et al., 1988; Pietnelli et al., 1989), raising questions about quality of life, especially in patients with advanced disease.

Many high-affinity CCK-B/gastrin receptor antagonists have been tested for therapeutic efficacy both in vitro and in vivo in a variety of experimental gastrointestinal tumors. For example, proglumide, a glutamic acid derivative (Seva et al., 190; Harrison et al., 1990; and Watson et al., 1991a); benzotript, an N-acyl derivative of tryptophan; L365260, an aspercillin derivative (Bock et al., 1989) and CI-988, a molecule that mimics the C-terminal pentapeptide sequence of CCK (Hughes et al., 1990), effectively neutralize the effects of exogenous gastrin on gastrointestinal tumor growth both in vitro and in vivo (Watson et al., and Romani et al., 1994). However, these antagonists have significant toxic side effects and lack specificity because they block the action of all potential receptor ligands, such as G34 and CCK, on normal cells. Highly potent and selective molecules antagonizing CCK-B/gastrin receptors, such as YM022 (Yuki et al., 1997) and YF476 (Takinami et al., 1997), have also recently been described.

Proglumide and benzotript have been extensively tested in preclinical studies. The main problem with these compounds is their lack of efficacy, which requires relatively high concentrations to remove G17 (Watson et al., 1992a; Watson et al., 1992b). Nevertheless, proglumide and benzotript inhibit basal and gastrin-stimulated proliferation of many cell lines (Seva et al., 1990; Watson et al.,

1991a). In addition, proglumide increases the survival of xenotransplanted mice carrying gastrin-sensitive cells

Ί « · · · · 9 9 ···· • · · · · 9 9 9 9

9 9 9 · ·«···« • · · · · · · · · ·· · 9· 999 ··· ·· ·* murine intestinal tumors MC26 for 39 days compared to 25 days for control animals.

Because of the low specificity of this class of gastrin antagonists for the gastrin/CCK-B receptor, it is believed that growth inhibition is also induced by actions independent of gastrin receptors. Furthermore, the cellular receptors that recognize and bind gastrin do not bind all inhibitors tested (Seva et al., 1994). Therefore, unless gastrin binding to receptors in the autocrine growth cascade is completely inhibited, gastrin antagonists may be unable to block this mechanism of growth stimulation.

Therefore, there is a need for new therapeutic approaches, both for single-agent therapy and for combination therapeutic strategies with chemotherapy. Combination therapy may increase the therapeutic index and/or reduce the required doses of chemotherapy, thereby reducing adverse side effects.

A therapeutic method of selective immunological neutralization of the biological activity of the gastrin hormone will be an effective means for controlling and/or preventing pathological changes resulting from excessive production of the gastrin hormone, which are associated with colorectal carcinomas.

Related U.S. Patent Nos. 5,023,077; 5,468,494; 5,607,676; 5,609,870 and 5,622,702 describe immunogens and immunogenic compositions useful for controlling G17 and G34 levels in patients by generating anti-gastrin antibodies and also describe the use of such compositions for treating gastrin-induced gastric and duodenal ulcers and tumors.

The present invention relates to the use of the anti-G17 immunogen and immunogenic compositions described in Patent Nos. 5,023,077;

5468494; 5607676; 5609870 and 5622702 in combination therapy with ···· ·· ·* ·· ·· ··· chemotherapeutic agents for the treatment of gastrin-dependent colorectal carcinomas.

The described method of antitumor therapy has several advantages over existing methods of treating colorectal carcinomas. AntiG17 immunization, in combination with chemotherapeutic agents such as 5-FU and leucovorin, enhances the therapeutic effects in controlling or inhibiting the growth of colorectal tumors, compared to chemotherapy alone.

The essence of the invention

The subject of the invention is a combination suitable for use in the treatment of gastrin-dependent tumors, the essence of which consists in the fact that it contains

i) an immunogen directed against the growth of said tumor and ii) one or more chemotherapeutic agents for treating it.

The invention further provides the combination as described above for use in the treatment of a gastrin-dependent tumor in a patient.

Finally, the invention also provides the use of the combination mentioned above for the manufacture of a preparation for use in the treatment of a gastrin-dependent tumor.

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The present invention provides a combination therapy for the treatment of tumors that comprises immunological neutralization of peptide hormones and cell division-promoting factors in combination with chemotherapy. More specifically, the present invention provides a method for the treatment of gastrin-dependent tumors, such as colorectal carcinomas. The method comprises a combination therapy comprising anti-G17 immunization of a patient in need of therapy, together with the administration of one or more chemotherapeutic agents. Anti-G17 immunization for the treatment of gastrin-dependent tumors is surprisingly effective in inducing the formation of anti-G17 antibodies, despite the known myelosuppressive effects of the chemotherapeutic agents used.

Anti-Gl7 immunization involves active or passive immunization of a patient with an anti-Gl7 immunogen against the hormone G17, with the aim of controlling the concentrations of G17 in the patient. As a result of the induction of anti-Gl7 antibodies in the patient, the hormone G17 is neutralized in vivo and its physiological effects are inhibited, which inhibits the growth of G17-dependent tumor cells.

Furthermore, anti-Gl7 immunization in combination with standard chemotherapy increases the efficacy of colorectal cancer treatment, ♦ · ♦ • · * • · 0 • · ·

because lower doses of chemotherapeutic agents can be used when used in combination, reducing their toxic effects on normal tissues. In addition, the patient's quality of life and survival time can be improved.

In a preferred embodiment, the method comprises actively immunizing a mammal with a gastrin-dependent tumor with an anti-G17 immunogen, in combination with the administration of one or more chemotherapeutic agents, such as 5-fluorouracil, leucovorin, levamisole, cis-platinum, tumor necrosis factor, and proglumide. The anti-G17 immunogen can be administered to the patient at the initiation of therapy and at designated intervals thereafter. The anti-G17 antibodies generated in the patient following immunization can bind to G17 and can neutralize G17 in its mature, amidated, as well as precursor forms, e.g., G17-Gly, in vivo, and can prevent G17 from binding to its receptors, thereby inhibiting the growth of gastrin-dependent tumor cells. The titers of anti-G17 antibodies generated in the immunized patient can be monitored at predetermined intervals using standard techniques. Furthermore, the chemotherapeutic agents can be administered at standard doses or at lower doses, according to the needs of the patient.

In another embodiment, the invention provides a method of treating gastrin-dependent tumors, which comprises passively immunizing a mammal with a gastrin-dependent tumor with anti-G17 antibodies, in combination with the administration of one or more chemotherapeutic agents, such as 5-fluorouracil, leucovorin, levamisole, cisplatin, tumor necrosis factor, and proglumide. In a preferred aspect of this embodiment of the invention, the antibodies may be chimeric, humanized, or human monoclonal antibodies, which may be prepared by methods well known in the art. The antibodies may be administered to the patient at the initiation of therapy and at designated intervals thereafter, according to the needs of the patient.

Description of images in the attached drawings

Fig. 1 is a graph showing the time course of serum antibody titers following immunization of rats with 500 pg/ml rat anti-G17 (1-9)-DT immunogen.

Fig. 2 is a graph showing the effect of 5-FU/leucovorin treatment at a dose of 30 mg/kg on anti-G17(1-9) antibody titers in rats immunized with an immunogen of the present invention.

Fig. 3 is a Scatchard plot showing the effect of treatment cycles of 30 mg/kg 5-FU/leucovorin on mean leukocyte counts in BDIX rats.

Fig. 4 is a bar graph showing the median tumor weight in untreated rats, rats treated with anti-G17(1-9)-DT, and DT-alone.

Fig. 5 is a bar graph showing median tumor weight in rats treated with 30 mg/kg 5-FU/leucovorin; 30 mg/kg 5-FU/leucovorin and DT immunogen; 30 mg/kg 5-FU/leucovorin and anti-G17(1-9)-DT; 25 mg/kg 5-FU/leucovorin and DT immunogen;

mg/kg 5-FU/leucovorin and anti-G17(1-9)-DT; 20 mg/kg 5-FU/leucovorin and DT immunogen; 20 mg/kg 5-FU/leucovorin and anti-G17(1-9)-DT; 12.5 mg/kg 5-FU/leucovorin and DT immunogen; and 12.5 mg/kg 5-FU/leucovorin and anti-G17(1-9)-DT.

The present invention provides a method for treating tumors, particularly gastrin-dependent colorectal carcinomas, by a combination therapy comprising immunizing a patient with an anti-G17 immunogen and treating the patient with chemotherapeutic agents such as 5-FU and leucovorin. Surprisingly, it has been found that the combination therapy of anti-G17 immunization/5-FU/leucovorin is more effective than previous methods of treating colorectal carcinomas. The chemotherapeutic agents used in the combination therapy do not significantly inhibit the production of anti-G17 antibodies in the immunized patients and lower doses of the chemotherapeutic agents can be used to treat the tumors. Furthermore, the titers of anti-G17 antibodies elicited by immunization are effective in neutralizing all forms of the G17 hormone.

In a preferred embodiment, the method comprises actively immunizing a patient with gastrin-dependent colorectal carcinoma by administering an anti-G17 immunogenic composition in conjunction with the administration of chemotherapeutic agents. Subsequent booster anti-G17 immunizations may be administered as needed, as determined by analyzing serum anti-G17 antibody titers after immunization in the patient, using standard techniques and standard radiological assessment of the tumors. Anti-G17 immunization may also be administered to the patient prior to surgical removal of the tumor.

Anti-G17 immunogens comprise natural or synthetic peptide fragments of the N-terminal amino acids of G17 as the immunogenic portion of the immunogen. This peptide fragment is conjugated to an immunogenic carrier, such as diphtheria toxoid (DT). In a preferred embodiment of this aspect of the invention, the anti-G17 immunogen comprises the amino-terminal amino acids of G17 from position 1 to position 9, which have the amino acid sequence pyroGlu-GlyPro-Trp-Leu-Glu-Glu-Glu-Glu, which is conjugated to diphtheria toxoid. Other suitable immunogenic protein carriers include bovine serum albumin, keyhole limpet hemocyanin, hemocyanin and tetanus toxoid.

The immunogens of the present invention may comprise an extension or spacer peptide sequence that extends the immunogenic peptide from the protein carrier and which

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increases its binding capacity to lymphocyte receptors. A suitable spacer peptide sequence is the amino acid sequence SSPPPPC (SEQQ ID NO: 2 in the Sequence Listing). However, other spacer peptides may be used. In a preferred embodiment of this aspect of the invention, the spacer peptide sequence is linked to the carboxyl terminus of the immunogenic peptide. The immunogens of the present invention are prepared by standard techniques and are described in U.S. Patent Nos. 5,023,077;

5,468,494; 5,607,676; 5,609,870; 5,688,506 and 5,622,702, the disclosures of which are incorporated herein by reference. Upon immunization, the immunogens of the present invention elicit the production of high-affinity, neutralizing antibodies to inhibit the action of G17 in mature and precursor forms on tumor growth in immunized animals. The anti-G17 antibodies produced bind to and neutralize mature and precursor G17, preventing G17 from binding to receptors on tumor cells and ultimately inhibiting tumor cell growth. The immunogens elicit antibodies that neutralize both carboxy-amidated and glycine-extended G17, and have no cross-reactivity with G34 or CCK.

Compositions in which immunogens for active immunization are administered to patients for the treatment of gastrin-dependent tumors may be in a variety of forms. Such forms include, for example, solid, semi-solid and liquid dosage forms such as powders, liquid solutions, suspensions, suppositories and injectable or infusible solutions. The preferred form depends on the selected route of administration and therapeutic application. The compositions contain immunogens of the present invention and suitable pharmaceutically acceptable ingredients and may contain other medicinal agents, carriers, adjuvants, etc., which may be admixed using standard methods. The compositions are preferably in unit dosage form. The dose of active compound administered for immunization or as a medicament in a single administration or over a period of time depends on the individual being treated, the route and form of administration and the discretion of the attending physician.

In the treatment of gastrointestinal tumors, a dose in the range of 0.001 to 2 mg of the immunogenic composition is administered to the patient. An effective dose of the immunogenic composition is capable of eliciting an immune response in the patient that consists of the formation of effective antibody titers to bind and neutralize mature and precursor G17 within 1-3 months after immunization. Following immunization and chemotherapy, for example 5-FU/leucovorin, in a patient with colorectal cancer, the effectiveness of the therapy on tumor growth is evaluated by standard clinical procedures, such as ultrasound and magnetic resonance imaging (MRI), to detect the presence and size of the tumor. Anti-G17 antibody titers can also be monitored from blood samples from the patient.

If necessary, a booster dose should be administered to maintain an effective antibody titer. Effective treatment of gastrin-dependent colorectal adenocarcinoma and other gastrin-dependent carcinomas, such as gastric, liver, pancreatic, and small cell lung carcinoma, using the method of the present invention should result in inhibition of tumor growth and reduction in tumor size.

For passive immunization, anti-G17 antibodies are administered to a patient intravenously using a pharmaceutically acceptable carrier such as saline, for example, phosphate-buffered saline.

Chemotherapeutic agents are administered at doses recommended in standard regimens and may be administered at the initiation of therapy concurrently with the anti-G17 immunogen, before immunization, or after immunization. In some cases, it may be advantageous to administer the chemotherapeutic agent both before and after immunization. Additional courses of chemotherapy may also be administered, based on the patient's needs as assessed by MRI and ultrasound.

The following experiments were performed to demonstrate the effects of the combination therapy of the present invention on colorectal carcinomas.

Examples of embodiments of the invention

Example 1

The following experiments were conducted to determine the potential clinical benefit of anti-G17(1-9)-DT. The objectives of the study were as follows:

(a) to determine the long-term effect of specific anti-G17(1-9)-DT immunization of rats on the histological findings in the Gl tract of rats;

(b) to evaluate the effect of the combination of 5-FU/leucovorin on the titers of antibodies to anti-G17(1-9)-DT; and (c) to determine the therapeutic effect of the combination of anti-G17(1-9)-DT and 5-FU/leucovorin in a rat model.

Cell line

DHDK12 is a rat colon epithelial tumor cell line (Martin, 1983). The cell line was cultured in RPMI 1640 culture medium (Gibco, Paisley, Scotland) containing 10% fetal calf serum (FCS, Sigma, Poole, UK) in a humidified environment at 37°C and 5% CO2.

Immunogen

The anti-G17(1-9)-DT immunogen consisted of amino acid residues 1-9 of G17 linked at its carboxyl terminus to the spacer peptide SSPPPPC (SEQ ID NO: 1 in the Sequence Listing), «· 9 · ·· • · · * · ···· ··· ♦ · · « * · • · ♦ · «····» • ·

Experimental animals

Male and female BDIX rats were obtained from the Cancer Studies Unit, University of Nottingham, UK, and were 6-10 weeks old, weighing 340-420 g. Rats were housed in pairs under a 12-hour light/12-hour dark cycle at 25°C and 50% humidity.

Before each experiment, animals were divided into groups with equal weight distribution. Group sizes were 6-13 animals. The UK Coordinating Committee for Cancer Research (UKCCCR) approved all animal experiments.

Immunization

Rat anti-G17(1-9)-DT was dissolved in sterile saline (0.9%), pH 7.3, at a concentration of 1 mg/ml. The adjuvant, nor-muramyl peptide (Penisula Labs, Belmont, CA, USA) was added to the conjugate solution to achieve a final conjugate concentration between 200 and 500 ug/ml. The aqueous solution was prepared with an oil vehicle (montanide ISA 703; AMS Seppic lne., Paris, France) in a ratio of 1:2 (v/v) by emulsification. After being placed in a glass syringe, which was connected to a second syringe via a three-way stopcock, the mixture was extruded 40 times and drawn into the syringe, obtaining an emulsion. For control rats, an emulsion containing DT peptide and muramyl dipeptide was prepared in the same way. 200 μΐ emulsion (50 • ·· · 9

9999

9 4 9 9 · 4 4 4 « • · 0 4 0 • 949 00 «0

Hg/rat) was administered by s.c. injection (on the right flank of the experimental animals). The animals were immunized either by a single injection or repeatedly at 21-day intervals, as described below.

Cytotoxic treatment

Rats were given 12.5 and 25 mg/kg 5-fluorouracil (5-FU, David Bull Labs., Warwick, UK) and 12.5-25 mg/kg leucovorin (Lederle Labs., Gosport, Hants, UK) intravenously on days 1, 3 and 5 in a cycle that was repeated every 4 weeks during the test period (Asao, 1992). The cytotoxic combination was given to rats before or after anti-G17(1-9)-DT immunization (200 μg/kg).

Initiation of tumor growth

DHDK 12 cells were suspended in sterile phosphate-buffered saline (PBS, Oxoid, Hants, UK) at a cell concentration of 2.5 x 10 ⁷ /ml. Rats were anesthetized by intraperitoneal injection of 1 ml of Hypnorm (0.315 ng/ml fentanyl citrate and 10 mg/ml fluanisole; Jannsen, Berrse,

Belgium), Hypnovel (5 ng/ml midazolam; Roche, Basel, Switzerland) and sterile distilled water in a ratio of 1:1:5. After subcutaneous injection on the right flank, 200 μί of the cell suspension was injected into the abdominal wall muscle and the surgical incision was closed with clips. Each experimental group consisted of 6 to 13 animals.

Determination of specific antibody concentrations in rats immunized with rat anti-G17(1-9)-DT

To obtain blood samples for analysis, blood was collected from the rats' tails at various times during the experiment and at the end of the experiment, blood was collected by cardiac puncture under terminal anesthesia. Serum

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Anti-rat G17 antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Rat G17-bovine serum albumin (BSA) conjugate was dissolved at a concentration of 2 pg/ml in 0.1 M glycine buffer (pH 9.5) and was added at 25 µl per well to 96-well Immunolon U plates (Dynatech Labs., Sussex, UK). The wells were incubated overnight at 4°C, then unadsorbed conjugate was removed and the wells were washed with buffer (0.9% saline, 0.5% Tween20 (Sigma), 0.02% NaN3 (Sigma), pH 7.3). This buffer was used for all washes and reagent dilutions. Serum was processed at 10-fold serial dilutions, starting at 1:100.

The positive control was anti-rat-G17(1-9)-DT antiserum from pre-immunized animals, and the negative controls were normal rat serum and serum from animals immunized with DT alone. All control sera were used at the same dilutions as the test sera. The diluted sera were added to the wells in 25 µl aliquots in the presence or absence of 25 µl/well of rat G17-BSA at a concentration of 100 µg/ml (as a soluble inhibitor). Basic controls received 25 µl of assay buffer alone. The plates were incubated for 60 minutes at ambient temperature and then washed with assay buffer. Goat anti-rat immunoglobulin (H+L)-biotin (Zymed, San Francisco,

CA, USA) was added to the wells at a dilution of 1:500, 50 µl/well and incubated for 60 minutes in the dark at ambient temperature.

After washing, avidin-alkaline phosphatase (Zymed) was added at a dilution of 1:100 (50 µl/well) and the plates were incubated for 60 minutes at ambient temperature. After further washing, p-nitrophenyl phosphate (pNPP) substrate (Sigma) was added to the wells at 50 µl/well and after 5 minutes of development, the absorbance was read at 405 nm. The difference in absorbance between untreated serum and serum incubated with rat G17-BSA was calculated as the specific absorbance.

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Leukocyte count determination

Heparinized blood from rats was collected from the tail during the experiment and at the end of the experiment by cardiac puncture. Leukocyte counts were analyzed in the Hematology Department at the University Hospital, Nottingham, using a FACScan.

Histology

After sacrifice of the chronic anti-G17(1-9)-DT-immunized rats, representative areas of the stomach, colon, and rectum were removed from the immunized rats and age-matched controls and fixed in formalin. The sections were then embedded in paraffin and 4 μm sections were prepared using a microtome. These sections were stained with hematoxylin and eosin and examined by a histopathologist who had no knowledge of the treatment groups.

Crypt cell proliferation rate

One hour before sacrifice, vincristine (2 mg/kg, Sigma) was injected intraperitoneally to induce metaphase cell cycle arrest in the intestinal epithelium before colonic crypt cell proliferation (CCPR) was assessed. The number of metaphase cells per crypt was determined. The colon and rectum were removed from each rat, sectioned longitudinally, and their mucosa was fixed in Carnoy's solution. The crypts were carefully crushed under a dissecting microscope, and the number of metaphase cells (at 25x magnification) was determined.

Statistical analyses

The results of the in vivo tests were analyzed by the Mann-Whitney nonparametric test using the SPSS statistical package for IBM PC.

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99 9 9

Long-term anti-G17(1-9)-DT study Male rats were immunized with rat anti-G17(1-9)-DT immunogen as described above and their antibody titers were measured for 34 weeks after a single immunization. At this time, the animals were given a second booster injection of rat anti-G17(1-9)-DT. The results are shown in Fig. 1. Fig. 1 shows antibody titers from rats immunized with 500 μρ/μηΐ rat anti-G17(1-9)-DT as a function of time up to 40 weeks. Each point represents one animal. Antibody titers were measured by ELISA as described above using a 1:100 serum dilution. Immunizations are indicated by an arrow. After primary immunization, 4 out of 5 rats responded to rat anti-G17(1-9)-DT immunogen. Antibodies were detectable at week 7 in 3 of 5 rats after this single injection and at week 9 in 4 of 5 rats. The initial antibody surge was followed by a second surge between weeks 15-20, and then antibody titers gradually declined, reaching zero at week 34. At this time, Figure 1 also shows that after the second immunization with rat anti-Gl7(1-9)-DT, all rats had detectable antibody titers against rat G17 within 1-2 weeks after immunization.

Example 2: Histological analysis of rats long-term immunized with anti-G17(1-9)-DT

Samples from the stomach, colon, and rectum were examined histologically after staining with hematoxylin and eosin as described in Example 1. These samples were compared with samples from age- and sex-matched control rats. All regions of the GI tract evaluated were identical in anti-G17(1-9)-DT-treated rats and age-matched controls in terms of villus length/crypt/mucosa height. In the stomach, enterochromaffin cell-like (ECL) cells were similar in appearance and number in both

groups of animals. However, some signs of granulation were evident in the G cells of the gastric mucosa of rats treated with anti-G17(1-9)-DT.

Example 3: Crypt cell proliferation rate (CCPR) of intestinal epithelium of rats immunized with anti-Gl7(1-9)-DT for a long time

The CCPR of the colonic epithelium and the titers of antibodies to rat G17 were analyzed as described above. Table I shows the results obtained from 4 of the 5 rats evaluated comparing the CCPR and the titers of antibodies to rat G17. The mean CCPR for control rats was 18.93 (standard deviation 3.2) and for rats immunized with anti-G17(1-9)-DT was 23.7 (standard deviation 7.9). No statistically significant difference in CCPR was observed between rats immunized with anti-G17(1-9)-DT and age-matched control rats. These results indicate that the rate of cell division in the crypts of the colonic epithelium is the same for both control and anti-G17(1-9)-DT immunized rats.

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Table 1: Comparison of anti-G17:DT antibody titers with colonic crypt cell proliferation

Rat Specific absorbance for anti-rat G17:DT antibody (dilution 1:1000) Crypt cell proliferation rate (mean metaphases/crypt at 2 hours after vincristine administration) Check 1 0 21, 9 Check 2 0 19, 4 Check 3 0 14.4 Check 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 titers elicited by anti-G17(1-9)-DT

Rats were intravenously administered a 1:1 ratio of 5-FU/leucovorin at a dose of 30 mg/kg as described in Example 1, before or after immunization with anti-G17(1-9)-DT. Each group consisted of 6 males and 6 females, and the mean antibody titers were measured by ELISA using a 1:100 serum dilution. The antibody titer of each rat was measured from a blood sample as described in Example 1.

Fig. 2 shows the effect of pre- and post-cytotoxic treatment with 30 mg/kg 5-FU/leucovorin cycles on antibody titers elicited by anti-G17(1-9)-DT (500 ug/ml) immunization. In this figure, the data are presented as follows: no cytotoxics, 7 immunizations;

« · · • ·

99 immunizations before 4 cycles of cytostatic treatment; -Ο-, 1 immunization before 4 cycles of cytostatic treatment; -Δ-, 1 cytostatic treatment before 4 immunizations (2 cytotoxic treatments during immunizations); -ffi-, 2 cytostatic treatments before 4 immunizations;

cytostatic treatments before 3 immunizations; 4 cytostatic treatments before 2 immunizations.

Fig. 2 shows the mean of 6 females and 6 males per group. Standard deviations are approximately 10% of the mean. No significant effect on antibody titers due to pretreatment with the 5-FU/leucovorin combination was observed for antibody titers or time to rise compared to untreated rats immunized with anti-G17(1-9)-DT. The maximum number of treatment cycles evaluated was 4 cytotoxic therapy followed by 2 immunizations.

Fig. 3 shows the effect of treatment on the average leukocyte count in BDIX rats.

The effect of cytotoxic treatment with 30 mg/kg 5-FU/leucovorin on the mean white blood cell (WBC) count in BDIX rats that received cycles of cytotoxic treatment prior to 2 immunizations is shown in Figure 3. As shown in this figure, a significant decrease in WBC count was observed in representative rats evaluated after cytotoxic treatment (p<0.005, Students' t-test). The counts decreased with the number of cycles of cytotoxic treatment, indicating some myelosuppression. However, there was no effect on the antibody response to anti-G17(1-9)-DT in these rats, as shown in Figure 2.

Example 5: Effect of combination therapy of 5-FU/leucovorin and antiGl7(l-9)-DT on the growth of DHDK12 tumors in vivo • · · « · ·

The effect of combination therapy with 5-FU/leucovorin (12.5-30 mg/kg) and rat anti-G17(1-9)-DT (200 ug/ml) on the growth of the rat colon tumor cell line DHDK12 in the muscularis propria of the abdominal wall of BDIX rats was tested by comparing tumors in control animals as described in previous examples, at the end of treatment the animals were sacrificed and their tumors were excised and weighed using standard procedures. Each group consisted of 10/12 rats of mixed sex. Median tumor weights are shown with ranges of values of the quarter variable above the bars. Statistical evaluation was performed using the Mann-Whitley U non-parametric test as described in Example 1.

Fig. 4 and 5 show the effect of anti-G17(1-9)-DT immunization on the median final tumor weight from BDIX rats implanted with DHDK12 tumor cells into the abdominal wall muscle. This method of implantation results in well-vascularized tumors suitable for treatment administered into the circulation (Watson,

1996). Rat anti-G17(1-9)-DT has previously been shown to reduce the final weight of DHDK12 tumors by 56.5% when administered at a dose of 500 ug/ml (Watson, 1996). In these experiments, conducted to detect any benefit of combination therapy with 5FU/leucovorin, the dose of anti-G17(1-9)-DT was reduced to 200 ug/ml, resulting in a significant inhibition of tumor growth of 25.7%, as shown in Fig. 4. Fig. 4 shows data for tumors excised from untreated control rats, rats immunized with anti-G17(1-9)-DT, and rats immunized with DT. After 50 days, untreated rats had tumors with a median weight of 4.43 g. Rats immunized with DT had a median tumor weight of 4.7 g, which is not statistically significantly different from tumor weights of untreated rats, but which is significantly higher than the median tumor weight from rats immunized with anti-G17(1-9)-DT (3.49 g, p=0.034, Mann Whitney).

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Fig. 5 shows that 5-FU/leucovorin alone, administered at a dose of 30 mg/kg, significantly reduced the median tumor weight to 1.01 g (p=0.0106 compared to untreated control rats). When rats were treated with the same cytotoxic dose of 5-FU/leucovorin together with DT immunization, the median tumor weight was not significantly different (0.945 g).

The combination of 5-FU/leucovorin (30 mg/kg) and anti-G17(1-9)-DT immunization in rats resulted in a median tumor weight of 0.68 g, which was not statistically significantly different from the 5FU/leucovorin/DT group (p=0.27). The combination of 5-FU/leucovorin (25 mg/kg) and DT immunization resulted in a median tumor weight of 0.96 g, compared to a median tumor weight of 0.68 in the anti-G17(1-9)-DT immunization in combination with 5-FU/leucovorin, which was not statistically significantly different (p=0.409). When the dose of 5-FU/leucovorin was reduced to 20 mg/kg, the combination of 5-FU/leucovorin/DT resulted in a median tumor weight of 1.23 g. The median tumor weight was significantly reduced to 0.71 g when combining 20 mg/kg of 5-FU/leucovorin with anti-G17(1-9)-DT immunization (p=0.027, Mann Whitney).

Fig. 5 finally shows that a dose of 12.5 mg/kg 5FU/leucovorin in combination with anti-G17(1-9)-DT immunization (p=0.015, Mann Whitney) reduces the median tumor weight from 1.34 to 0.41 g. The 5-FU/leucovorin/DT combinations were compared and no statistically significant differences were found between anti-G17(1-9)-DT administered in combination with 12.5, 20 or 30 mg/kg 5FU/leucovorin.

Given the limited benefit of combination chemotherapy with 5FU/leucovorin both in advanced stages and in the adjuvant setting (Moertel, 1994; Scheithauer, 1995; Taylor, 1993;

Petrioli, 1995), new therapeutic modalities should be administered either together with 5-FU/leucovorin to increase the therapeutic

index (and perhaps a reduction in the dose of chemotherapeutic agents to limit toxicity), or as a second-line treatment after chemotherapy failure. Such new treatment modalities must be suitable for such use. Immunotherapy in combination with chemotherapy was previously considered problematic because of the myelosuppression associated with the administration of chemotherapeutic agents such as 5-FU/leucovorin (Mahood, 1991). However, in the present studies, myelosuppression in rats induced by the combination of 5FU/leucovorin (30 mg/kg), administered according to Asao et al. at the maximum tolerated doses, did not affect the titers and times of antibody development against rat G17:DT following immunization with the anti-G17(1-9)-DT immunogen.

In studies of 5-FU/leucovorin therapy in combination with anti-G17(1-9)-DT, potentiation was achieved at doses of 20 mg/kg and 12.5 mg/kg. The 20 mg/kg dose was as effective as the maximum tolerated dose when combined with anti-G17(1-9)-DT, and the 12.5 mg/kg dose showed a trend towards greater therapeutic effect.

The reason for this trend is not known, but it may be that this dose of cytostatics affects the immune system less than higher doses, which may contribute to the overall inflammatory response to the tumor. 5-FU/leucovorin given in continuous cycles has an all-or-nothing effect on tumor growth, since reducing the dose to 1 mg/kg causes no growth inhibition. The therapeutic effect can be titrated more gradually by reducing the number of toxic cycles (Watson, personal communication). Therefore, in the combination therapy according to the present invention, lower than usual doses of 5-FU/leucovorin can be administered, which will reduce the side effects of the drug, while achieving effective tumor cell killing, because the immune system is minimally disturbed during such treatment. Thus, the growth inhibitory effect of anti-G17(l9)-DT immunization is enhanced. These characteristics of the combination therapy are unexpected in view of the myelosuppressive effects of chemotherapeutic agents.

Furthermore, due to the absence of adverse effects for the patient, anti-G17(1-9)-DT immunization is likely to be a long-term treatment, as evidenced by the duration of antibody persistence in rats after a single immunization. The first immunization was 80% effective in inducing anti-gastrin antibodies, and the second immunization was 100% effective with immediate rise in antibody titers. Although chemotherapy can be potentiated with a single injection of anti-G17(1-9)-DT in the majority of patients in the absence of adverse effects, the nature of the anti-G17(1-9)-DT immunization and the rapidity of response after a booster dose suggest that a multiple-dose regimen is appropriate. Despite the long duration of anti-rat G17 antibodies in circulation, they do not appear to have deleterious effects on the GI tract as assessed by simple histological examination. Furthermore, the proliferation index of mucosal crypt cells in the colon did not show a significant effect on their growth.

Example 6: Treatment of human patients with colon cancer with a combination of 5-FU/leucovorin and anti-G17(1-9)-DT

It has been previously reported that anti-G17(1-9)-DT immunization alone is a valuable and safe therapeutic option for the treatment of gastrin-dependent tumors. The combination of anti-G17 immunogen and 5-FU/leucovorin according to the present invention increases the efficacy of antitumor treatment, especially in the treatment of colon cancer, and probably reduces the doses of chemotherapeutic agents in combination, which reduces the side effects of cytostatic treatment of any of the chemotherapeutic agents currently used. The combination of the immunogen with chemotherapeutic agents according to the present invention can also be used as a second-line therapy in patients who do not respond to chemotherapy alone.

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Colorectal tumors in humans or patients with colon tumors are treated with a combination of chemotherapy and immunotherapy.

More specifically, patients with gastrin-responsive colorectal cancers or colon cancers may be treated with the concomitant administration of 5-FU/leucovorin and an anti-G17 immunogenic agent or anti-G17 antibodies.

Specifically, a preferred immunotherapy provides an immunogenic composition comprising an amino-terminal G17(1-9):DT conjugate in a pharmaceutically acceptable carrier, which may contain an adjuvant to further stimulate an immune response.

A preferred immunotherapy regimen may be initiated before, during, or after chemotherapy, depending on the clinical circumstances. For example, for a patient with a bulky tumor, it may be advantageous to begin treatment with several cycles of chemotherapy to reduce the tumor volume and then initiate immunotherapy.

Alternatively, in a patient with a small tumor or after curative surgery, immunotherapy may be initiated before or during chemotherapy.

Active immunization doses may range between 300 ug and 1200 ug of anti-G17 immunogen, depending on the patient's immune status (or immune response capacity). Injections may be given on days 1, 7 and 14 or 1, 14 and 21 or 1, 14 and 28 and 56. All administration protocols result in similar antibody titers. Accelerated immunization protocols may allow for earlier onset of immune response.

• ·

In a preferred method of anti-gastrin therapy, booster doses are administered every 6 months after the initial immunization, regardless of the protocol used.

Yet another preferred method for effective neutralization of G17, GlyG17 and G17-HN2 is passive immunization with anti-G17 antibodies, preferably in purified form. More specifically, 10-1000 μg of anti-G17(1-9) antibodies are administered before, during and/or after cycles of chemotherapy, in order to control gastrin activity. Passive immunization can be administered daily, once a week or once every two weeks. Other protocols can be used, depending on the effectiveness of the treatment.

Another therapeutic combination is initial passive immunization before and/or during the first cycle of chemotherapy, followed by active immunization as described above.

Many chemotherapy regimens are used. These known regimens, although not listed herein, are not excluded from the combination treatment of the present invention. One preferred chemotherapy regimen is a bolus i.v. dose of 425 mg/m 5-FU with i.v. infusion of leucovorin (folic acid, FA, 20 2 mg/m ) on days 1-5, repeated every 4 weeks.

Another preferred regimen is 200 mgm ² FA over 2 hours followed by 5-FU iv bolus dose of 400 mg/m ² + 5-FU 600 mg/m ² over 22 hours on days 1 and 2, repeated 2 weeks later.

Yet another preferred regimen is continuous i.v. infusion of 5-FU (250-300 mg/m) for 4-6 weeks, followed by a 2-week break.

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99

Links:

ASAO T, TAKAYUKI A, SHIBATA HR, BATIST G and BRODT P. Eradication of Hepatic Metastases of Carcinoma H-59 combination chemoimmunotherapy with Liposomal Muramyl Tripeptide, 5-Fluorouracil, and Leucovorin. Cancer Research 52: 6254-6257, 1992 BALD WIN G. Binding of the progastrin fragments to the 78kDa gastrin-binding protein. FEBS Ze// 1995; 359: 97-100.

ERLICHMAN C, FINE S, WONG A, ELHAKEIM T. A randomized trial of fluorouracil and folinic acid in patients with metastatic CRC. J Clin 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.

MAKISHEMLA R, LARKIN D, MICHAELI D, GAGINELLA TS. Active immunization against gastrin-17 with an N-terminal derived immunogen inhibits gastrin and duodenal lesions in rats. Gastroenterol 1995; 106: A824.

MARTIN F, CAIGNARD A, JEANNIN 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. CRC Chemotherapy. NEJM1994; 330: 1136-1142.

PETRIOLIR, LORENZIM, AQUINO A, MARSELI S, FREDIANIB, PALAZZUOLI V, MARZOCCA G. Treatment of advanced colorectal cancer with high-dose intensity folinic acid and 5-Fluorouracil plus supportive care. Eur J Cancer 1995; 31A: 2105-2108.

PIETNELLIN, DOUGLAS HO, HARRAVA L . The modulation of Fluorouracil with leucovorin in metastatic CRC: a prospective randomized phase III trial. JClin Oncol 1989; 7: 1419-1426.

SCHEITHAUER W, KORNEK G, ROSEN H, SEBESTA C, MARCELL A, KWASNY W, KARALL M, DEPISCH D. Combined intraperitoneal plus intravenous chemotherapy after curative resection for colonic adenocarcinoma. Eur J Cancer 1995; 31A: 1981-1986.

SEVA C, DICKINSON CJ, SAWADA M, YAMADA T. Characterization of the glycineextended gastrin (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. Gastrin receptors in gastrointestinal tumors. WG Landes

Company, Austin, USA, 1993.

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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.

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Claims (10)

PATENT CLAIMS A combination suitable for use in the treatment of a gastrin-dependent tumor comprising (i) an immunogen directed against the growth of the tumor; and (ii) one or more chemotherapeutic agents for its treatment. The combination of claim 1, wherein the immunogen comprises an immunogen comprising an anti-gastrin G17 peptide. The combination according to claim 2 or 3, wherein the anti-gastrin G17 peptide is conjugated to a diphtheria toxoid carrier. 4. The combination of claim 2, wherein the immunogen comprising the anti-gastrin G17 peptide comprises the anti-gastrin G17 peptide, a protein carrier, and a spacer peptide suitable for detaching the anti-gastrin G17 peptide from the protein carrier and increasing its capacity for lymphocyte receptor binding. The combination of claim 2, 3 or 4, wherein the anti-gastrin G17 peptide has the sequence of SEQ ID NO: 1. The combination of any one of claims 2 to 5, wherein the chemotherapeutic agent or agents is selected from the group consisting of 5-fluorouracil, leucovorin, levamisole, cisplatin, tumor necrosis factor and proglumide. ······· · · ··· · · A combination according to any preceding claim, further comprising a pharmaceutically acceptable carrier. The combination of any preceding claim for use in treating a gastrin-dependent tumor in a patient. The combination of claim 6 comprising as chemotherapeutic agents 5-fluorouracil and leucovorin for use in the treatment of a gastrin-dependent colorectal tumor in a patient. Use of a combination according to any one of claims 1 to 7 for the manufacture of a composition for use in the treatment of a gastrin-dependent tumor.