CZ399899A3 - Immunogenic preparations to receptor for CCK-B/gastrin and methods of treating tumors - Google Patents

Abstract

The present invention relates to immunogenic, immunogenic treatment of gastrin-dependent tumors. Immunogens are peptides from the receptor molecule CCK-B / gastrin conjugated to spacer molecule α immunogenic carrier. Immunogens are capable of inducing antibody production under in vivo conditions and bind the antibodies for CCK-B / gastrin receptors on tumor cells, thereby they prevent peptide hormone from stimulating growth in binding receptors and thus inhibit the growth of tumor cells. Immunogens they are also formed by anti-CCKB / gastrin receptor antibodies for passive immunization. The solution also applies diagnostic methods for gastrindependent detection tumors under in vivo or biopsy conditions tissues using antibodies that are the subject solution.

Description

Immunogenic preparations for CCK-B / gastrin tumor treatment receptors and methods

BACKGROUND OF THE INVENTION

The gastrin hormone binds with high affinity to gastrin / cholecystokinin (CCK) -B receptors through its 5 carboxy-terminal amino acids. The CCK-B / gastrin receptor is a cytoplasmic membrane protein that is coupled via a G-protein to an intracellular signaling pathway that controls the expression of various genes.

Gastrin is a peptide hormone that occurs in two forms, tetratriacontagastrin (G34) and heptadecagastrin (G17), and is synthesized and secreted by specialized cells, G cells, which are located in the stomach anthra. The hormone is secreted into the circulating blood and binds to specific gastric cells, especially enterochromaffin cell-like cells (ECL) and parietal cells that indirectly or directly affect gastric acid secretion. Gastrin hormones have historically been associated with stimulation of gastric acid secretion (Edkins, J.S. 1905). (A complete citation of the references herein is given in the Reference section before the Claims section). In recent years, evidence has accumulated that gastrin may also function as a trophic factor for the gastrointestinal tract (Johnson, L. 1997) and may initiate the growth of gastrointestinal tumors (Watson et al.

C.J. 1995) as well as tumors

1989, Dickinson, not based on the gastrointestinal tract, including small cell lung cancer (Rehfeld et al. 1989). Through the carboxy terminus, a specific "mature" carboxy-amidated form of gastrin, which results from its post-translational processing, binds to the gastrin / CCK-B receptor (Kopin et al. 1992).

Several types of tumors, such as colorectal cancer, gastric cancer, pancreatic cancer, and hepatocellular adenocarcinoma, have been shown to have CCK-B / gastrin receptors on their

plasma membranes, and also that they respond to the action of gastrin by massive cell proliferation (Rehfeld, J.F. 1972, Upp et al. 1989 and Watson et al. 1993). In addition, it has recently been discovered that many of these tumors secrete gastrin, thereby affecting the autonomous proliferative metabolic pathway (Van Solinge et al. 1993, Nemeth et al. 1993 and Seva et al. 1994).

The CCK-B / gastrin receptor is a family of G protein-coupled receptors with seven transmembrane domains with equal affinity for CCK and gastrin (Soli et al. 1984). This receptor was named CCK type B receptor because it was found most in brain tissue (Wank et al. 1992). This receptor was subsequently found to be identical to the peripheral CCK / gastrin receptor in the parietal and ECL cells of the stomach (Nakata et al. 1992). This receptor has been well characterized in a variety of normal (Fourmy et al. 1984, Grider et al. 1990) and tumor tissues (Singh et al. 1990, Watson et al. 1993) and has also been extensively studied using rat pancreatic adenocarcinoma cell lines. AR42J (Scemama et al. 1987). The gastrin receptor cDNA expressed on AR42J cell lines has been cloned and sequenced and is over 90% homologous to the CCK-B / gastrin receptor DNA sequence in rat and human brain tissue, and over 84% homologous to the CCKB cDNA DNA sequence / gastrin receptor parietal cells of the dog (Wank, SA 1995), indicating high sequence homology even between species.

Peptide hormones G17 and G34 bind to the CCKB / gastrin receptor located on the cell membrane of normal cells. However, it has been discovered that G17, but not G34, stimulates the growth of gastrin-dependent tumor cells. Serum G17 has a particular potential to stimulate the growth of colorectal tumors, which is mediated by CCK-B / gastrin receptors (Watson et al. 1993) on tumor cells. In particular, gastrin17 appears to be involved in stimulating the growth of colorectal adenocarcinomas due to the possible increased affinity of this hormone

compared to other gastrin hormones for the CCK-B / gastrin receptor on tumor cells (Rehfeld 1972 and 1993). It has been discovered that CCK-B / gastrin receptors are expressed in high affinity form in 56.7% of human primary colorectal tumors (Upp et al. 1989). It is believed that there may also be a potential autocrine loop caused by the endogenous production of gastrin precursor peptides produced by these tumors (Van-Sollinge et al. 1993 and Nemth et al. 1993). The resulting G17 ligand / receptor complex stimulates cell growth through secondary messengers regulating cellular functions (Ullrich et al. 1990). Binding of G17 to the CCK-B / gastrin receptor leads to activation of phosphatidyl inositol breakdown, activation of protein kinase C resulting in increased intracellular calcium ion concentration, as well as induction of c-fos and c-jun genes by mitogen activated protein kinase, which it plays a role in the regulation of cell proliferation (Tadisco et al. 1995). In addition, gastrin binding to the CCK-B / gastrin receptor has been shown to be associated with increased phosphorylation of tirosin kinase ppl25FADK (focal adhesion kinase), which may also play a role in mitogenic signal transduction (Tanaguchi et al. 1994).

A variety of high-affinity CCK-B / gastrin receptor antagonists have been evaluated therapeutically in both in vitro and in vivo studies on a large number of experimental gastrointestinal tumors. For example, it has been shown that proglumide, a glutamic acid derivative (Seva et al. 190; Harrison et al. 1990 and Watson et al. 1991a), Benzotript, an N-acyl tryptophan derivative, L-365260, an Aspercilin derivative (Bock et al. 1989 and Cl-998, a molecule mimicking the C-terminal pentapeptide sequence of CCK (Hughes et al. 1990), effectively neutralizes the effects of exogenous gastrin on gastrointestinal tumor growth in both in vitro and in vivo studies (Watson et al., Romani et al. 1994) ). However, these antagonists have severe toxic side effects and lack specificity because they block the action of all potential receptor ligands, such as

for example, G34 and CCK on normal cells. Recently, highly potent and selective CCK-B / gastrin receptor antagonists YM022 (Yuki et al., 1997) and YF476 (Takinami et al. 1997) have also been described.

Proglumide and Benzotript have been extensively studied in preclinical studies. The main problem with these compounds is the loss of potency and also relatively high concentrations are required for their G17 antagonistic activity (Watson et al. 1992a, Watson et al. 1992b). Nevertheless, proglumide and benzotript inhibited basal and gastrin-stimulated proliferation of a variety of cell lines (Seva et al. 1990, Watson et al. 1991a). In addition, proglumide increased the survival of treated gastrin-sensitive gastrin-sensitive colon tumor-bearing mice xenografted to 39 days compared to 25 days in control animals.

Because of the low specificity of this class of gastrin antagonists acting on the gastrin / CCK-B receptor, inhibition of tumor growth cannot be effectively controlled by gastrin antagonists. In addition, cellular receptors that recognize and bind gastrin molecules do not bind all tested inhibitors (Seva et al. 1994). Therefore, unless complete inhibition of gastrin binding to its receptor occurs within an autocrine growth cascade, gastrin antagonists may not be able to block this mechanism of tumor growth.

SUMMARY OF THE INVENTION

The present invention relates to immunogenic preparations and immunological methods for the treatment of gastrin-dependent tumors. The method consists of active and passive immunization of a patient with an anti-CCK-B / gastrin receptor immunogen or antibodies against the CCK-B / gastrin receptor. The antibodies produced by the immunogens are specific for the CCK-B / gastrin receptor on tumor cells and block the proliferative effects of gastrin at the receptors. Antibodies prevent peptide hormones from binding

to CCK-B / gastrin receptors occurring on gastrindependent tumor cells. In this way, tumor growth is stopped.

Moreover, antibodies specific for the NH ₂ -terminal end of the receptor are surprisingly internalized after their binding and rapidly translocated into the cytoplasm and tumor cell nucleus. Internalization occurs as early as 10 seconds after the cells are exposed to the antibody. This rapid internalization of the antibody / receptor complex results in apoptosis of the affected tumor cells.

The immunogens of the present invention form natural or synthetic peptides derived from the human CCK-B / gastrin receptor as an immunogenic portion of the immunogen. The immunogens may also comprise a spacer peptide sequence attached to the end of the immunogenic peptide. The immunogen may also be conjugated to a protein carrier such as diphtheria or tetanus toxoid, bovine serum albumin and the like.

In one embodiment of the present invention, a method of immunizing against a CCK-B / gastrin receptor is provided by active immunization wherein the patient is immunized with the immunogen of the present invention. Immunogen stimulates the production of anti-CCK-B / gastrin receptor antibodies occurring on tumor cells.

Antibodies produced by CCKB / gastrin receptor immunogens bind to these tumor cell receptors and effectively prevent the binding of peptide hormones to receptors, thus inhibiting the autocrine growth-stimulating metabolic pathway of tumor cell division and hence tumor growth.

In another embodiment of the present invention, the method of treatment is formed by passive immunization, wherein the antibodies against the CCK6 receptor are:

Β Β Β

Β Β Β Β

B / gastrin is administered to patients in sufficient concentration to bind to CCK-B / gastrin receptors on tumor cells and block the binding of peptide hormones to the receptor. Preventing the binding of hormones to their receptors inhibits the proliferative metabolic pathway of tumor cells, thereby inhibiting the growth of hormone-dependent tumors. In a preferred embodiment of the present invention, the antibodies for human therapy may be chimeric, humanized, or human monoclonal antibodies, which may be generated by methods well known in the art. In addition, anti-CCK-B / gastrin receptor antibodies may be conjugated to cytotoxic molecules such as cholera toxin, or radionuclide-labeled radioactive molecules, such as ¹²⁵ I and ¹³¹ I, to enhance the tumoricidal effect.

The present invention also relates to diagnostic methods of gastrin-dependent tumors, which consist of immunochemical detection of gastrin-dependent (CCK-B / gastrin-containing) tumors from biopsy samples using the antibodies of the present invention. Specific anti-CCK-B / gastrin receptor antibodies of the present invention can be labeled using a detection system using compounds such as biotin, horseradish peroxidase and fluorescein to detect CCKB / gastrin receptors in tumor tissue using standard immunochemical procedures.

The invention also relates to a diagnostic method for detecting gastrin-dependent tumors, which is formed by in vivo detection of gastrin-dependent (containing CCK-B / gastrin receptors) tumors. The method comprises administering an effective dose of radionuclide-labeled anti-CCK-B / gastrin receptor antibodies to a patient with colorectal cancer by intravenous injection, and imaging or detecting tumor cells bound to the CCK-B / gastrin receptor to their cell membranes using standard scintigraphic procedures. In this • 999

An aspect of the present invention may be antibodies against the receptors for the following: < tb > ______________________________________ < tb > ______________________________________ < tb > CCK-B / gastrin labeled with a radionuclide such as ^{11 L} Indium, ⁹⁰ Ytrium and ¹³¹ L.

Description of the invention

Brief description of the illustrations

Giant. 1A and 1B illustrate a schematic view of the CCKB / gastrin receptor and its 7 transmembrane domains.

In FIG. 2 shows ELISA data with antibodies generated by immunizing rabbits with an immunogen against Peptide 1 from the CCK-B / gastrin receptor.

In FIG. 3 shows ELISA data with antibodies generated by immunizing rabbits with an immunogen against Peptide 4 from the CCK-B / gastrin receptor.

In FIG. 4 shows data obtained from an inhibitory ELISA assay that was used to assess the specificity of affinity purified antibodies raised against the GRP1-DT immunogen. Giant. 5 shows a bar graph depicting inhibition of ¹²⁵ human G17 binding to AR42J cells by peptide inhibitors.

In FIG. 6 is a bar graph showing the cell distribution of immuno-gold labeled AR4-2J tumor cells.

In FIG. 7 is a photograph of Western blot analysis of protein extracts from nuclear membranes of adenocarcinoma cells using antibodies against Peptide 1.

In FIG. 8 is a photograph of Western blot analysis of protein extracts from extranuclear and plasma membranes of adenocarcinoma cells using antibodies against Peptide 1.

In FIG. 9 is a graph illustrating C170HM2 tumor weight in control animals and animals treated with antibodies against the CCK-B receptor / gastrin.

In FIG. 10 is a graph illustrating the tumor cross-sectional area

C170HM2 in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

9 -. - 9 -. · 999 9 9 99 99 9 3 ······ 9 9 9 9

999 99 99

In FIG. 11 is a bar graph showing the average weight of C170HM2 tumors in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

In FIG. 12 is a bar graph showing the average cross-sectional area of C170HM2 tumors in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

In FIG. 13 is a bar graph showing the average number of C170HM2 tumors in control animals and animals treated with antibodies against the CCK-B receptor / gastrin.

In FIG. 14 is a bar graph showing the median weight of liver metastasis of C170HM2 tumors in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

In FIG. 15 is a bar graph showing the median cross-sectional area of C170HM2 tumors in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

In FIG. 16 is a bar graph showing the median number of C170HM2 tumors in control animals and animals treated with antibodies against the CCK-B receptor / gastrin.

In FIG. 17 is a bar graph showing the mean and median number of C170HM2 liver tumors in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

In FIG. 18 is a bar graph showing the mean and median weight of C170HM2 liver tumors in control animals and animals treated with antibodies against the CCK-B / gastrin receptor.

In FIG. 19 is a bar graph showing the mean and median cross-sectional area of liver metastases of C170HM2 tumors in control animals and animals treated with antibodies against the CCKB / gastrin receptor.

Giant. 20 is a graph showing the concentration of ¹²⁵ I-labeled radionuclide antibodies in C170HM2 liver tumor xenografts in controls (normal rabbit serum) and anti-GRP1 treated nude mice.

In FIG. 21 is a bar graph showing the average number of liver tumors of C170HM2 in liver xenografts of control nude mice and nude mice treated with anti-GRP1.

> · ♦ ·

• 9 9 9 9

9 9 9 9 9

9 9 9 9 9

999 99 99

In FIG. 22 is a bar graph showing the mean weight of C170HM2 liver tumors in liver xenografts of control nude mice and nude mice treated with anti GRP1.

In FIG. 23 shows Western blots of C170HM2 liver tumor xenograft proteins in control nude mice and nude mice treated with anti GRP1.

In FIG. 24 is a light microscopy histological section photo showing a cross-section of C170HM2 liver xenografts stained with hematoxylin-eosin in a control mouse.

In FIG. 25 is a light microscopy histology sectional view of a C170HM2 liver xenograft section stained with hematoxylin eosin in mice treated with rabbit anti-GRP1 antibodies.

Detailed description of the invention

The methods of the present invention are intended for the treatment of gastrin-dependent tumors in animals including humans and comprise administering to the patient an immunogen against the CCK-B receptor / gastrin, resulting in the production of antibodies in an immunized patient binding to the CCKB receptor / gastrin on tumor cells to prevent hormone-receptor binding, thereby inhibiting the proliferative effects of the hormone. In addition, from a clinical perspective, the receptor / anti-GRP1 complex is rapidly internalized, transferred to the cytoplasm and subsequently to the nucleus. This apparently leads to apoptosis of the affected tumor cells.

Immunogens consist of natural or synthetic peptides that form the human CCK-B / gastrin receptor and are capable of immunization. In particular, two synthetic peptides have been developed with this in mind. Developed according to the amino acid sequence of the CCK-B receptor / gastrin, these peptides are immunogenic and cross-react with the endogenous CCK receptor.

9 99 99 9999

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999 99 99

B / gastrin on tumor cells under in vivo and in vitro conditions. Peptide 1 consists of amino acids 5 to 21 of the CCK-B / gastrin receptor sequence: KLNRSVQGTGPGPGASL (Peptide 1. SEQ ID NO: 1 in the Sequence Protocol). Peptide 1 forms the amino terminal end of the receptor and is located on the extracellular surface of the cell membrane (see Figure 1).

In another embodiment of the present invention, the immunogen is comprised of Peptide 4, which consists of the following CCK-B / gastrin receptor amino acid sequence: GPGAHRALSGAPISF (Peptide 4. SEQ ID NO: 2 in the Sequence Protocol). Peptide 4 is part of the fourth extracellular domain of the receptor and is also located on the outside of the plasma membrane (see Figure 1).

peptide) is (Serine (Ser)

Immunogens may also be produced by extending a peptide projecting into space from a proteinaceous carrier with an immunizing peptide at its end. This increases the capacity of immunogens to bind to lymphocyte receptors. Such a suitable peptide (spacer peptide with the amino acid sequence SSPPPPC spacer. SEQ ID NO: 3 in the Sequence Protocol). However, other spacer peptides may also be suitable. Immunizing peptides with or without a spacer peptide are then conjugated to a carrier protein, such as a diphtheria toxoid, via a cysteine residue at the carboxyterminal end. Spacer peptides are not immunologically related to CCKB / gastrin receptor-derived peptides and should therefore increase, but not determine, the specific immunogenicity of the receptor-derived peptide.

The presence and density of the CCK-B / gastrin receptor on tumor cells of a patient can be determined by reacting the labeled antireceptor antibodies with a biopsy tumor sample. Antibodies to the receptor may be labeled with either a radionuclide, a color indicator, or fluorescence. Moreover, the responsiveness of the tumor cells to gastrin can be assessed under in vitro conditions from a biopsy of a patient's tumor using standard techniques. Tumor patients whose biopsy specimens are positive in the CCK-B / gastrin receptor antibody test are typical candidates for treatment using the methods of the present invention.

An effective dose in the range of 0.001 to 2 mg of the immunogenic preparation is administered to a patient to treat gastrointestinal tumors. An effective dose of the immunogenic preparation should be capable of eliciting an immune response in the patient by increasing effective levels of CCK-B / gastrin receptor antibody titer 1-3 months after immunization. Following immunization of the patient, the efficacy of the immunogens is monitored by standard clinical procedures, such as ultrasound and nuclear magnetic resonance (NMR) testing to detect the presence and size of tumors. The titer of anti-receptor antibody levels can also be monitored from a blood sample taken from a patient. Revaccination immunization should be performed to maintain an effective antibody titer. Effective treatment of gastrin-dependent tumors, such as gastric, liver, pancreatic and colorectal adenocarcinomas, should, according to the method of the present invention, result in tumor growth inhibition and tumor size reduction.

Antibodies generated by CCKB / gastrin receptor immunogens of the present invention may have antitrophic effects against gastrin-dependent tumors due to three possible mechanisms: (i) inhibition of gastrin binding to its receptor, (ii) degradation or disruption of the signal transduction pathway tumor cell proliferation; and (iii) induction of apoptosis (cell suicide) in cells where the receptor / antibody complexes are internalized and migrate to the core.

In another embodiment of the present invention, anti-CCK-B / gastrin antibodies are administered to cancer patients with CCKB / gastrin receptors. Antibodies specifically bind to CCKB / gastrin receptors on tumor cells. Binding of antibodies to receptors prevents the binding of gastrin to its ligand in cell membranes and therefore the growth signal for gastrin-dependent tumor cells is inhibited and tumor growth is arrested. The antibodies are preferably chimeric or humanized, or are fragments thereof that effectively bind to a target receptor and can be generated using standard techniques, such as described in U.S. Pat. These exogenously engineered antibodies may also be useful for destroying tumor cells that carry the CCK-B / gastrin receptor on their plasma membrane by inhibiting tumor cell growth or transporting toxic cells. substances into the tumor cell.

Anti-CCK-B / gastrin antibodies preferred for treatment are those antibodies that react with the extracellular domains 1 and 4 of the receptor protein depicted in FIG. 1 such as GRP-1 and GRP-4. Particularly preferred are antibodies that specifically recognize and bind to the amino acid sequences of the receptor proteins corresponding to Peptides 1 and 4. Inhibition of tumor growth in this method is also monitored by ultrasound examination and NMR and repeated immunizations are performed as needed by each patient

The efficacy of antibodies to inhibit tumor cell growth and destruction can be enhanced by cytotoxic molecules conjugated to anti-CCK-B / gastrin antibodies. The cytotoxic molecules may be toxins, for example cholera toxin, ricin, α-amanitin, or radioactive molecules labeled, for example, with a ¹²⁵ I or ¹³¹ I radioisotope, or chemotherapeutic agents, such as cytosine with arabinoside or 5-fluorouridine.

• · · · · · · · · ···

In addition to antibodies labeled with a radioisotope ¹²⁵ I or ¹³¹ I can be an antibody against the receptor for CCK-B / gastrin radionuclides and labeled as ^Iu Indium or ⁹⁰ Yttrium. In this aspect of the present invention, the antibodies are useful for detecting and diagnosing tumors with CCK-B / gastrin receptors under in vivo conditions by administering these antibodies to a patient and detecting bound antibodies to tumor cells bearing CCK-B / gastrin receptors. Upon reaching radiolabeled antibodies against CCK-B / gastrin tumor tissue, approximately 1-2 hours after injection, radioactive sites are imaged using standard and known scintigraphic procedures (Harrison's Principles of Internal Medicine, Isselbacher et al. Eds. 13. Edition, 1994).

The formulations in which the immunogens are administered to treat gastrindependent tumors in patients may take various forms. These include, for example, solid, semi-solid and liquid dosage forms such as powders, liquid solutions, suspensions, suppositories, and solutions for injection and infusion. The preferred form depends on the intended route of administration and therapeutic applications. The preparations are comprised of the present immunogens and suitable pharmaceutically acceptable components, and may include other drug substances, carriers, adjuvants, excipients, etc. Suitable adjuvants may include nor-muramyl dipeptide (nor-MDP, Peninsula Labs., CA) and oils such as Montanide ISA 703 (Seppic, Inc, Paris, France), which can be mixed using standard procedures. Preferred preparations are in unit dosage form. The amount of active compound administered for immunization as a medicament at a given time or over a period of time will depend on the subject being treated, the mode and form of administration, and the evaluation by the treating physician.

The anti-CCK-B / gastrin receptor antibodies of the present invention for passive immunization are preferably administered to a patient by injection using a pharmaceutically acceptable carrier, such as a salt solution, for example a phosphate buffer.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of GRP1-DT and GRP4-DT conjugates.

CCK-B / gastrin receptor peptides were prepared using standard peptide synthesis techniques. To generate immunogens capable of inducing a specific immune response, each of Peptides 1 and 4 was synthesized with a spacer peptide of sequence SSPPPPC (SEQ ID NO: 3 in the Sequence Protocol) at its carboxy-terminal end. These peptides were then conjugated to the amino acids present on the carrier, diphtheria toxoid (DT), through the terminal amino acid residue of the spacer peptide of cysteine using a heterobifunctional binder containing a succimidyl ester at one end and a maleimide at the other end. below.

Method A: As previously described in US Patent No. 5023077, the binding of the aforementioned Peptides 1 or 4 is performed as follows. The dry peptide was dissolved in 0.1 M sodium phosphate buffer, pH 8.0, with a thirty-fold excess of dithiotreitol (DTT). The solution was stirred under a nitrogen atmosphere saturated with water vapor for four hours. The reduced cysteine containing peptide was chromatographically separated from the other components on Sephadex G10 equilibrated with 0.2 M acetic acid. The peptide was lyophilized and stored under vacuum until use. The carrier was activated in reaction with a heterobifunctional binder, for example, epsilon-maleimido caproic acid N-hydroxysuccimide ester (EMCS) in an amount sufficient to activate about 25 free amino groups per 10 ⁵ molecular weight of the carrier. In the particular case of diphtheria toxoid, this was achieved by adding 6.18 mg of EMCS (75% purity) to each 20 mg of diphtheria toxoid.

Activation of diphtheria toxoid was achieved by dissolving each 20 mg aliquot of diphtheria toxoid in 1 ml of 0.2 M sodium phosphate buffer, pH 6.45. Aliquots containing 6.18 mg EMCS were dissolved in 0.2 ml dimethylformamide (DMF) In the dark, 50 µl (µl) was added dropwise to DT EMCS with stirring. The protein content of the concentrate was determined by either the Lowry method or the Bradford method. EMCS carrier content was determined by incubating the activated carrier with cysteine-HCl followed by reaction with 10 mM Ellman's reagent (5,5'-thiobis (2-nitrobenzoic acid). Difference in optical density between the cysteine-HCl-containing blank tube and the cysteine-containing sample tube. HCl and the carrier was converted to EMCS content using a 13.6x10 ³ molar extinction coefficient for 5-thio-2-nitrobenzoic acid at 412 nm.

The reduced cysteine (-SH) peptide content was also determined using an Ellman reagent. Approximately 1 mg of the peptide was dissolved in 1 mL of nitrogen saturated water, and a 0.1 mL aliquot of this solution was treated with Ellman's reagent. Using the molar extinction coefficient for 5-thio-2-nitrobenzoic acid (13.6x10 ³ ), the amount of free SH groups was calculated. An amount of peptide containing sufficient free SH groups to react with each of the 25 EMCS activated amino acids on the carrier was dissolved in 0.1 M sodium citrate buffer, pH 6.0 containing 0.1 mM EDTA, and this solution was added dropwise in the dark to the EMCS activated carrier. After the entire peptide solution was added to the support, the mixture was incubated in a vapor-saturated nitrogen atmosphere overnight.

The peptide conjugate bound to the carrier by EMCS was separated from the other components of the mixture by Sephadex G50 column equilibration with 0.2 M ammonium bicarbonate.

* 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

The conjugate eluted in void volume was lyophilized and stored dried at 20 ° C until use.

The resulting conjugate can be characterized as a peptide content using a variety of methods known to one skilled in the art. These methods include determination of weight gain, amino acid analysis, etc. For conjugates of Peptides 1 and 4 with spacer peptide and diphtheria toxin that have been generated by this method, they have been determined to have an effective peptide / carrier ratio of 5-35 moles peptide per 100 kD molecular weight carrier. and that they are suitable immunogens for immunizing test animals. Preferably, the peptide produced an effective immune response at a concentration ranging from 10 to 30 moles per 100 kD molecular weight DT.

Method B: In a preferred method, conjugates consisting of DT bound GRP1 and DT bound GRP4 peptide were prepared at room temperature as follows. Purified DT (400 mg) was dissolved in 20 ml of 0.5 M phosphate buffer, pH = 6.6, saturated with nitrogen to give a 20 mg / ml DT solution. The DT solution was placed in a 60 ml dark amber glass bottle (serving as reaction vessel and filter reservoir). EMCS binder (123.6 mg) was dissolved in 2.0 mL of dimethylformamide. The EMCS solution was added dropwise over 15 minutes to the DT solution with continuous stirring. The bottle was sealed and the mixture was stirred at room temperature for an additional 1 hour and 45 minutes to give activated DT (M-DT). M-DT was then purified by diafiltration using the Amicon Model TFC10 Thin-Chanel Ultrafiltration System to an I113G Operator Manual with XM50 diaflow ultrafiltration membrane. The M-DT solution was further washed twice with 420 ml phosphate buffer and concentrated to 20 ml each time. Next, the M-DT solution was washed once against 0.1 M sodium citrate buffer, pH - 6.0 containing 0.1 M EDTA, and the solution was again concentrated to 20 ml.

To prepare the GRP1-DT conjugate, 2.02 ml of M-DT solution (containing 22.3 mg of M-DT) was placed in a 10 ml vial of dark

99% amber glass, then 13 mg of GRP1 peptide was dissolved in citrate buffer to give 40 mg / ml peptide, and the solution was added dropwise to the M-DT solution with stirring.

The reaction was overnight in the dark. Each of the conjugates was removed from the reaction vessel and separately dialyzed through a dialysis membrane with a 12000-14000 molecular weight cut-off against a 5-fold 500 mL 0.1 M ammonium bicarbonate solution. Each conjugate was lyophilized. The conjugates were then lyophilized by amino acid analysis and their peptide-to-DT substitution ratios were determined as 21.8 peptides per 10 ⁵ molecular weight DT for GRP1-DT and 21.1 peptides per 10 ⁵ molecular weight DT for GRP4DT.

Peptide 1 and 4 conjugates with a spacer peptide and DT that have been produced by this method have an effective peptide / carrier ratio of 5-35 moles of peptide per 100 kD molecular weight carrier and all are considered suitable immunogens. A preferred range of ratio for generating an effective immune response is 10-25 moles of peptide per 100 kD molecular weight DT.

Preparation of immunogens

The present immunogens containing Peptide 1 or Peptide 4 with a spacer peptide conjugated to DT as described above were used to immunize rabbits. Immunogens were prepared as follows. The conjugate was dissolved in 0.15 M sodium phosphate buffer, pH 7.3 at a concentration of 3.79 mg / ml. The conjugate solution was added to the Montanide ISA adjuvant (703) Seppic, Inc.) at a 30:70 weight ratio of the conjugate solution to the Montanide ISA 703, then the mixture was homogenized using a Silverson homogenizer for 3 minutes at 8000 rpm at formation of an emulsion containing 1 mg / ml conjugate.

Immunization and sample preparation

Rabbits were injected intramuscularly with 0.1 ml of an immunogen consisting of either GRP1-DT or GRP4-DT conjugate.

· 9 · ·

9 9 · 99 9900 • · «99 9 ···

Each rabbit was injected with immunogen at 0 and 4 weeks. Each rabbit was bled 6 and 8 weeks after the start of the experiment. Serum was withdrawn from the blood samples and stored at -20 ° C until the samples were used in assays to determine the presence of antibodies against the CCK-B / gastrin receptor.

Enzyme-linked immunosorbent analysis (ELISA)

A solid phase ELISA was used to evaluate the reaction or cross-reaction of the antisera against Peptide 1 and Peptide 4 of each immunized rabbit. ELISA analysis was performed by coating polystyrene 96-well plates (Immulon II, Dynatech) with 25 µl / well of 10 µg / ml Peptide 1 bound to Bovine Serum Albumin (BSA) (GRP1-BSA) or Peptide 4 bound to BSA (GRP4- BSA) in 0.1 M glycine-HCl buffer, pH 9.5. Plates were incubated overnight at 4 ° C and subsequently washed with buffer.

Antisera obtained from the immunized rabbits were serially diluted in the range of 10 ^-1 to 10 ⁸ in 1% BSA-FTA hemagglutination buffer, pH 7.2. Twenty-five µl of test antiserum per well was incubated with each test peptide for 1 hour at room temperature. After incubation, the plates were washed with buffer to remove unbound antibody. Each well was reacted with 25 µl biotinylated goat anti-rabbit IgG (H + L) diluted 1: 1000 in 1% BSA-FTA dilution buffer. The reaction was run at room temperature for one hour. After washing the plates to remove unbound rabbit reagent, each well was incubated for one hour at room temperature with 25 µl avidin / alkaline phosphatase conjugate diluted 1: 1000 in 1% BSA-FTA buffer. Plates were washed to remove unbound avidin / alkaline phosphatase reagent and incubated with 25 µl p-nitrophenyl phosphate (PNPP) in 10% diethanolamine buffer containing 0.01% MgCl ₂ x 6 H ₂ O, pH 9.8. Plates were developed until the absorbance of the reaction mixture was between 0.8 to 1.5.

• 9 φφ • 9 9 Φ

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Κ To verify the specificity of the antiserum produced by rabbits, rabbits were also immunized with DT and for ELISA analysis, the plates were coated with DT as antigen to determine the reactivity of the antiserum against the carrier.

Giant. 2 shows the results of an ELISA analysis using Peptide 1 / GRP1 and FIG. 3 shows the results of an ELISA analysis using Peptide 4 / GRP4 as the antigen. As seen in FIG. 2, ELISA results show that rabbits immunized with Peptide 1-spacer-DT conjugate produce high antibody titers that specifically bind to Peptide 1 as seen from antibody binding to Peptide 1, even at high dilutions of antiserum (1: 100,000). Similarly, FIG. 3 that rabbits immunized with Peptide 1spacer-DT conjugate produce high titers of antibodies against Peptide 4.

As shown in Figures 2 and 3, rabbits immunized with each of the peptides produce antibodies that specifically bind to each of the peptides at low antiserum concentrations. The results show that antibodies against Peptides 1 and 4 have a large binding capacity to Peptides 1 and 4 for the CCKB / gastrin receptor. The results also show that immunization of rabbits with the present conjugates induces a significant immune response against Peptides 1 and 4. In addition, rabbits immunized with either Peptide 1 or Peptide 4 appear and behave normally and show no disease symptoms throughout the experiment.

Example 2

The following experiments were performed to verify the specificity of rabbits antibodies raised against the GRP1-DT peptide containing the Ser spacer peptide described in Example 1 using Method B.

A series of tests were performed to assess the specificity of rabbit antibodies induced by immunization with GRP1-DT and affinity purified immunoadsorption on a GRP1-Ser-Sepharose column.

4 4 4 ·· · 4 4

4 4 4444 4 ··· • 4 · 4 4 4 4 4 4

444 4 4 4 44 ··

444 44 44

Inhibitory ELISA analysis was used to assess the specificity of affinity purified antibodies to the GRP1-Ser peptide. The assay was performed as follows: A 96 well plate (Immulon, U-shaped bottom) was coated with GRP1-Ser-BSA conjugate by incubating overnight with 50 µl of a 2 µg conjugate solution in glycine buffer (0.1 M, pH = 9.5). / ml at 4 ° C. Affinity purified anti-GRP1 antibodies (at a final concentration of 10 ng / ml) were combined with various inhibitors (1:10 dilution series) and incubated for one hour at room temperature. Inhibitors included GRP1-Ser, GRP1 EPT, Ser, human gastrin 17 (1-9) -Ser spacer (hG17 (9) -Ser), GRP1EPT + Ser, and buffer (non-inhibiting). The incubation buffer consisted of PBS + 0.5% BSA + 0.05% Tween 20 + 0.02 NaN ₃ . In subsequent steps, the same buffer was used, but without BSA. The 96-well plate was washed to remove unbound GRP1-Ser-BSA and an antibody + inhibitor mixture (50 µl / well) was added. After one hour, the plates were washed and a goat anti-rabbit Ig (H + L) alkaline phosphatase conjugate (Zymed) was added (1: 2000 dilution). After 1 hour incubation, the plates were washed to remove unbound reagent and a 50 µl / well solution of pNPP substrate (Sigma, 1 mg / ml) in substrate buffer (PBS + 0.1 mg / ml MgCl 2 + 10% diethanolamine +) was added. 0.02% NaN _3. After 60 minutes incubation, absorbance was measured on MRX (Dynatech Laboratories), samples were measured in doublets, and the average was calculated for each concentration. all samples were then calculated and the percent inhibition relative to the added blank sample (anti-GRPl + buffer) for each inhibitor tested:% inhibition = (100) (1- ((a _n i _not inhibited by

Ainhibited (Aneinhibited), where A = absorbance. The results are shown in Figure 4.

In FIG. 4 shows the percent inhibition of antibody binding as a function of concentration inhibitor. As shown in the figure, the peptide

GRP1-Ser completely inhibited antibody binding to GRP1-Ser-BSA. The GRP1 EPT peptide, which does not contain the Ser spacer peptide sequence, as well as the equimolar mixture of GRP1 EPT plus Ser spacer, achieved about 60% inhibition. The failure of these peptides to fully inhibit suggests that part of the antibodies were specific for the epitope (s) formed by both the GRP1 elements and the Ser spacer sequences. Zero inhibition was achieved either by the Ser spacer sequence alone or by an unrelated peptide bearing the Ser spacer (hG17 (9) -Ser, consisting of nine amino-terminal residues of hG17 followed by the Ser spacer peptide). These ELISA results indicate that affinity purified antibodies are specific for the GRP1-Ser peptide and that 60% of the binding capacity was directed against the gastrin-receptor epitope component of the peptide.

Example 3

AR42J tumor cells (Porton Down, UK) are derived from rat pancreatic adenocarcinoma and are known to have well characterized CCK-B / gastrin receptors. These cells were assayed by radioligand inhibition to confirm CCK-B / gastrin receptor expression for hG17 receptor specificity. AR42J cells were cultured at 37 ° C with 7% CO ₂ in complete RPMI 1640 (Sigma) supplemented with 10% FCS (Gemini Bioproducts), 2 mM glutamine (JRH Biosciens), 1 mM sodium pyruvate (JRH B.) and 50 µg / ml. gentamicin (Gemini Bioproducts). Cells were harvested with PBS containing 0.25% PBS (without EDTA) using 175 cm T flasks (Falcon Plastics)

EDTA, then washed twice by centrifugation (400 g for 10 minutes). The cells were treated at 0-4 ° C at all times. A single-cell suspension was prepared in buffer and the cell concentration was adjusted to 10 ⁶ cells per ml. Aliquots of 1 ml cell suspension were added to 12x75 mm culture tubes, then the cells were centrifuged and the supernatants discarded. Cells were resuspended in PBS (0.1 • 4

444 4

4 44 44 444 ·

4,444,444

444 4 4

444444 4444

444 44 44 444 44 44 ml / tube) containing human G17 (hG17), gonadotropin relasing hormone (GnRH), or containing no peptide. Peptide concentrations were 1 ng / ml, 100 ng / ml and 10 µg / ml. An aliquot of 0.1 mL of ¹²⁵ L hG17 (NEN) containing approximately 26300 CPM (specific activity, 2200 Ci / mmol) was added to each tube. The tubes were vortexed and then incubated for 15 minutes. The cells were further washed twice with PBS and then counted in a γ counter (Wallac). Samples were measured in duplicate. Background was subtracted and then the calculated percent inhibition of binding ^{of 125} I-hGl7 each inhibitor using the formula:% inhibition - (100) (1- ((~ CPMneinhibovaný CPMI inhibited by _n) / CPM i _not inhibited by _n) ·

The results of radioligand binding inhibition assays are shown in Figure 5, where the results are presented as means (± SE) of each value. As can be seen from the figure, hG17 inhibited the binding of ¹²⁵ I-hG17 to AR42J. The degree of inhibition increased with the amount of inhibitor added to 32% inhibition at a concentration of 1 µg hG17 per tube, the highest concentration tested. On the other hand, no inhibition was achieved by GnRH even at the two highest concentrations (6% inhibition achieved by 100 µg GnRH was considered non-specific), indicating that hG17 inhibition was specific for gastrin. These results confirmed the expression of gastrin receptor on the cell surface of AR42J tumor cells.

Example 4

Binding of GRP1-Ser specific antibodies to AR42J cells was assessed by immunofluorescence. AR42J cells were cultured under the conditions described in the previous Examples, collected with a 175 cm ² T-flask cell scraper and washed twice with buffer (PBS with 0.02% NaN ₃ by centrifugation (400 g for 7 minutes). at 0-4 ° C. Single-cell 9 99 99 9 9 9999 9999

9 9 9 9 9 9 9

99 9 9999

The suspension was prepared in buffer and the cell concentration was adjusted to 10 ⁶ cells per ml. The cell suspension was added with 1.5 ml microcentrifuge tubes (1 ml / tube). The cells were pelleted by centrifugation and the supernatants aspirated. Cells were resuspended in buffer (0.1 ml / tube) containing peptide inhibitors (1.0 mg / ml). Inhibitors included GRP1Ser, GnRH, hG17 (9) -Ser, and buffer (not containing inhibitor).

Antibodies including rabbit antiGRP1-Ser (100 µg / ml), affinity purified rabbit anti-DT (negative control, 100 µg / ml), mouse anti-AR42J antiserum (positive control, 1: 100 dilution, heat inactivated) were added to appropriate tubes. ), or normal mouse serum and mixed. Cells were incubated for 1 hour with occasional agitation. The cells were then washed three times with buffer and 0.1 ml of fluorescein-labeled goat anti-rabbit IgG antibody (Incorporated antibodies diluted 1:50) was added to each tube. Cells reacted with mouse serum were developed with fluorescein labeled anti-mouse IgG (Zymed). Cells were resuspended by vortexing and then incubated for 1 hour. Cells were washed again three times, then resuspended in glycerol: PBS (1: 1, v: v), 50 µl / tube.

Wet preparations were prepared from the contents of each tube and cells were examined using a Laborlux 12 fluorescence microscope (Leitz). Fluorescence was scored on a scale of 0 to 4, with 0 indicating background fluorescence (achieved in normal mouse serum) and 4 indicating maximum fluorescence achieved in mouse anti-AR42J positive control antiserum).

The results of the immunofluorescence tests are shown in Table 1.

As can be seen from the Table, there was strong fluorescence of AR42J cells reacted with anti-GRP1-ser antibodies in the absence of peptide inhibitors showing antibody binding to cells. Rabbit anti-DT antibodies did not produce fluorescent radiation, demonstrating that the staining observed in <RTIgt; <9 </RTI> binding. Anti-GRP1-Ser antibodies were not due to non-specific rabbit immunoglobulin on the cell surface. In addition, the binding has been shown to be specific for the GRP1-Ser peptide. Addition of GRP1-Ser fully inhibited binding, while unrelated peptides including hG17 (9) -ser and GnRH were unable to inhibit. Since the GRP1 epitope is made up of 5-21 gastrin receptor residues, it has been concluded that anti-GRP1-Ser antibodies are specific for the gastrin receptor expressed by AR42J cells.

Table 1

Antibody Inhibitor Preparation GRP1-Ser HG17 (9) -Ser GnRH Puf r Rabbit anti-GRP1-Ser 0 3+ 2 + 3 + Rabbit anti-DT 0.5 + 0.5 + 0,5+ 0.5 + Mouse anti-AR42J 4 + Normal mouse serum 0

Example 5

AR42J cells, passages # 16-18 were cultured in RPMI-1640 medium containing 10% FCS and 2 mM glutamine. All cells were maintained at 37 ° C in air with 5% CO ₂ at 100% humidity, cultured to 80% accumulation in T75 flasks (Falcon, London, UK) and passaged after reaction with 0.02% EDTA to release adherent cells into suspension. Cells were incubated for 10 and 30 seconds, 30 minutes and 1 hour with an anti-CCK-B / gastrin receptor (aGR) antibody produced by rabbits immunized with CCK-B / gastrin receptor Peptide of the present invention as described in Example 1. This was purified by affinity chromatography on a column prepared with Peptide 1.

··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ♦ ·· ··

Cells were fixed in 1% glutaraldehyde for 1 hour and prepared for immunoelectron microscopy (ImunoEM) studies using standard techniques. The cell suspensions were centrifuged twice at 2000 rpm for 2 minutes and then the cell pellet was resuspended in phosphate buffer (PBS). The cell pellet was infiltrated with LR white plastic resin. Ultrathin sections of 70-90 nm thickness were placed on Pioloform coated nickel grates. The grates were placed in normal goat serum (Dako, High Wycombe, UK) in 0.1% bovine serum albumin (BSA) solution (Sigma, Poole, Dorset) and incubated at room temperature for 30 minutes. The grates were washed in PBS, then incubated with secondary biotin-conjugated anti-rabbit antibody (labeled gold) diluted 1:50 in 1% BSA for 1 hour at room temperature. Control experiments were performed without secondary antibody. After a final wash with PBS, the grates were stained in saturated aqueous uranyl acetate for 3 minutes and in Reynolds lead citrate for 3 minutes. Gold particles on the cell membrane, in the cytoplasm, on the nuclear membrane and in the nucleus were counted. Twenty-five cells / grid were counted by an independent observer. For control, AR42J cells were exposed to antibodies for less than one second and liver cells without CCK-B / gastrin receptor were used. Normal IgG-exposed AR42J cells were used as controls to determine non-specific binding of antibodies to CCKB / gastrin receptor. The results of these experiments are shown in Table 2 and Figure 6.

····

Table 2

Distribution of immuno-labeled CCKB / gastrin receptor particles in AR42J cells

Cellular membrane Cell matrix Nuclear membrane Nuclear matrix Number of particles of gold 14.2 (± 0.97) 43.3 (± 2.32) 9.3 (± 0.81 51.4 (± 3.32 Percent distribution in the cell 12% 36.6% 7, 9% 43, 5%

(percentage ± SEM for 25 cells, repeat count = 5)

As shown in Table 2 and FIG. 6, immunoglobulin-labeled particles were coupled to CCKB / gastrin receptors on the plasma membrane, cytoplasm, nuclear membrane, and nuclear matrix of adenocarcinoma cells, further demonstrating that the antibody / receptor complex is internalized by the cells.

As can be seen in Table 2, immunoEM studies using antisera directed against the amino-terminal end of CCKB / gastrin receptors show that after one hour incubation, the immuno-labeled antibodies against CCKB / gastrin receptors are rapidly internalized because 12% of the antibody-receptor complex is localized. on the cell membrane, 36.6% is in the cytoplasm, 7.9% is on the nuclear membrane and quite surprisingly 43.5% is in the cell nucleus. Regions of intense CCK-B / gastrin receptor immunoreactivity in the nucleus are found on chromatin, which may indicate specific binding sites for DNA regulation.

An electron microscopic study with anti-immunoglobulin conjugated with gold beads shows that in tumor cells an extremely rapid turnover of the antireceptor / receptor complex occurs, even as early as 10 seconds after exposure to the antibodies, the complexes are detectable in the cell nucleus as shown in FIG. 6.

· »» * * * * * * * * * * * * * * * * * * · · * * •

Example 6

Adenocarcinoma cell lines, in particular AR42J, HCT116, C170HM2, LoVo, ST16 and MGLVA1, were cultured under in vitro conditions and harvested as described in Examples 3. Cells from 30 x T-75 flasks were suspended in 5 ml homogenization buffer ( 1 mM NaH Carbonate, 2 mM magnesium chloride, 1 nM phenylmethylsulfonyl fluoride, 40 mM sodium chloride, 10 µl leupeptin, 1 µM pepstatin, 5 nM EDTA (Sigma)). Homogenization was performed with 5 cycles of five second homogenization in a homogenizer. To obtain extracellular membranes, tissue residue pellets were obtained by centrifugation at 500 g for 7 minutes at 4 ° C. The pellet was discarded and the supernatant was centrifuged at 500g at 4 ° C to remove residual residues. The supernatant was again centrifuged at 48,000 g for 1 hour at 4 ° C. The pellet containing the extracellular membrane preparation was suspended in Tris / NP-40 solution (0.1M TRIZMA, 0.5% NONIDET P40 (Sigma Chemical)).

To prepare the nuclear membrane pellets of tissue residues were prepared after homogenization in a second homogenization buffer (25 mM Tris-HCl, pH 7.4, 0.1% TRITON 100, 0.32 M sucrose, 3 mM MgCl 2, 2 mM EGTA, 0, 1 mM spermine tetrahydrochloride, 2 mM PMSF, 10 mM bezomidine hydrochloride, 3 mM EGTA aminoacetonitrile hydrochloride (Sigma)) after centrifugation at 400 g for 10 minutes at 4 ° C. The pellet was resuspended in 55% (0.2 M) sucrose dissolved in water for HPLC. This mixture was centrifuged at 60,000 g for 1 hour at 4 ° C. The pellet was washed with 0.4% NONIDET P40 in TRITON 100 free homogenization buffer. The pellet was centrifuged at 700 g for 15 minutes at 4 ° C and resuspended in TRITON 100 free homogenization buffer.

Protein content was determined by the Lowry method (using a kit from Pierce). Samples containing 10-15 µg protein were loaded onto 8-16% Tris / glycine gradient polyacrylamide

electrophoresis (PAGE) (Novex R and D systems) in Tris / glycine buffer and electrophoresis was performed for 90 minutes at a constant voltage of 125 volts, 36 mA. The gel was fixed in 10% glacial acetic acid for 1 hour and the samples were blotted onto a nitrocellulose membrane. Membranes were incubated in 1% BSA for 1 hour, followed by incubation with GRP1 antiserum (with and without preabsorption) for 1 hour. Antibody binding was detected by the avidin: biotin peroxidase complex method using diaminobezidene as substrate. The results of a Western blot analysis using rabbit antiserum against Peptide 1 (rabbit anti GRP1 antiserum) are shown in Figs. 7 and FIG. 8.

As shown in FIG. 7, the molecular weight markers of the proteins are in the range of 116, 66, 45 and 29 kDa. The blot shows a pronounced antiPeptide 1 immunoreactive band located about 43 kDa in all adenocarcinoma cells studied; HCT 116, C170HM2, LoVo, ST16 and MGLVA1, except for one cell line (AP5LV). This protein corresponds to a truncated form of the CCK-B receptor / gastrin. Some cell lines (HCT 116 and C170HM2) show at least 3 bands in the molecular weight range between 60 and 100 kDa. These results indicate that anti-CCKB / gastrin antibodies can recognize and bind to various isoforms of the CCK-B / gastrin receptor on tumor cells.

Figure 8 shows Western blot from extracellular (ENM and plasma membranes of C170HM2 and HCT116 adenocarcinoma cells. As shown in Figure 8, adenocarcinoma cell lines tested for ENM receptors for CCK-B / gastrin demonstrate the existence of two strongly staining bands: one around 43 kDa and others around 66 kDa When only the plasma membrane fraction was stained, only one band of about 66 kDa was detected, and Western blot studies therefore confirm immunoEM results suggesting that the CCK-B / gastrin receptor is present Adenocarcinoma tumor cells, although immunoEM studies do not differentiate CCK-B / gastrin receptor isoforms, the results show that the present immunogens induce the production of antibodies • · ♦ • • • «• • •« · CCK-B / gastrin receptor, which can recognize and bind to various i receptor forms useful in the treatment of these tumors.

Example 7

C170HM2 adenocarcinoma cells were administered intraperitoneally to nude mice and tumors were allowed to grow into the liver. Control mice were infused with phosphate buffer solution (PBS) and experimental mice were infused with one of the antibodies against the CCK-B receptor / gastrin. In Group 1, each mouse was infused 0.5 mg of rabbit anti-CCK-B / gastrin receptor antibodies raised against Peptide 1 (rabbit anti-Peptide 1, Rabbit @ GRP1). In Group 2, each mouse received 0.5 mg of rabbit anti-CCK-B / gastrin receptor antibodies raised against Peptide 4 (rabbit anti-Peptide 4, Rabbit @ GRP4). Mice were studied for 40 days after antibody infusion, then sacrificed and tumors removed for study. The weight, size and cross-section of the tumors were evaluated by standard techniques. The results are shown in Figures 9 and 10.

As seen in Figures 9 and 10, implantation of the C170HM2 colorectal carcinoma cell line into mice without therapy results in the rapid growth of a large tumor mass as determined by tumor weight or size and cross-section. However, infusion of rabbit antibodies against Peptide 1 or Peptide 4 into animals results in a noticeable reduction in the number of animals with a detectable tumor, as well as the weights and sizes of the tumors in the animals with these tumors compared to the control animals. The same effect is evident when calculating the average tumor weight, average tumor size, or average number of tumor foci. These data are shown in Figures 11, 12 and 13.

Another view of population distribution is obtained by calculating medians of tumor number, weight and size. The results are • · 9

shown in Figures 14, 15 and 16. As can be seen from these figures, the rabbit immunogen against Peptide 1 was clearly more effective in inhibiting tumor growth compared to the rabbit immunogen against Peptide 4. However, both rabbit antibodies against Peptides 1 and 4 showed control treatment with potent tumor inhibitory activity.

Example 8

In nude mice, greater tumor burden was generated using the C170HM2 cell line using the method described in Example 7, but with a higher initial cell inoculation. The C170HM2 cell line is a model of invading xenograft. Control and experimental mice were treated as described in Example 7.

Forty days after antibody infusion, mice were sacrificed and their liver tumors removed and examined. Figures 17, 18 and 19 show the results of these experiments. Figure 17 shows the mean and median number of liver tumors of both control and CCK-B / gastrin receptor treated mice. The results show that rabbit antibodies against CCK-B / gastrin receptor (Rabbit® GRP) effectively inhibit the growth of metastatic tumors in the liver. Using rabbit anti-Peptide 1 antibody in mice, there was a significant reduction in mean liver tumor count (Student's T test), p = 0.0084, and median liver tumor count, p = 0.0016 (Mann Whitney) compared to controls. Mice treated with anti-Peptide 4 antibodies also showed a decrease in the average number of liver tumors, but there was no difference in the average number of liver tumors in these animals compared to controls.

· · E e e e e e e e e e e e e e e e e e e e e e

In FIG. 18, it is shown that antibodies against Peptide 1 and Peptide 4 also reduced mean and median weight of liver metastasis compared to control animals.

The data in FIG. 19 shows that mice treated with antibodies against CCK-B / gastrin receptors also significantly decreased mean and median sections of liver tumors compared to control animals.

The results show that antibodies against CCKB / gastrin receptors effectively control the spread and growth of gastrin-dependent colon tumors to the liver, which are a major site of metastatic spread of this tumor.

Example 9

These studies were performed to confirm the immunoreactivity of GRP1 on C170HM2 cells. The aim of the study was to evaluate the localization of antiserum against GRP1 in the tumor and to determine its therapeutic effect on the growth of C170HM2 cells in the liver of nude mice. C170HM2 cells were administered intraperitoneally to nude mice as described in Examples 7 above. GRP1 antisera was created on rabbits. The antiserum was labeled with a ¹²⁵ I radioisotope and administered to nude mice bearing C170HM2 xenograft using a tail vein injection. Control mice received normal ¹²⁵ I-labeled rabbit serum. Mice were sacrificed at increasing time after injection of a single dose of ¹²⁵ I-labeled antibodies. Radioactivity was measured as units per minute per gram of tissue (CPM / g) and liver / liver tumor.

Figure 20 is a graph showing radiolabelled rabbit anti-GRP1 antibodies bound to liver tumors versus controls. As shown in the figure, more rabbit GRP1 antibodies are bound to liver tumor tissue compared to controls. Figure 20 also shows the hepatic • 9 · 9 ratio

9 9 ·

9

9 9 9 · 99 tumor / liver on the y-axis with increasing time on the x-axis for normal radioisotope-labeled rabbit antibodies as compared to GRP1 antiserum. In normal rabbit serum a ratio of 1 at day 1 was reached, and this ratio remained constant until day 5. This indicates that the concentration of radioisotope in liver tumor and normal liver tissue was the same. The ratio for GRP1 antiserum increased exponentially, reaching 2 on the fifth day, indicating that radioisotope-labeled GRP1 antiserum is taken up specifically in C170HM2 liver tumors

Example 10

Therapeutic effect of GRP 1 antiserum on C170HM2 xenografts

C170HM2 tumor xenotransplantation was initiated by intraperitoneal cell administration. Three different cell inocula were used to generate three levels of tumor burden. GRP1 antiserum was passively administered daily from day 0 by injection into the hepatic vein. The therapy was terminated on day 40.

Effect of GRP1 antiserum on tumor attachment

The initial endpoint was the number of liver tumors in the liver, as shown in FIG. 21. Control animals treated with normal rabbit antisera are grouped according to the degree of cell inoculation. As can be seen from FIG. 21, the mean number of liver tumors in the liver in the control group was between 1 and 3. In the group of animals treated with GRP1 antiserum, the average number of liver tumors in the liver was less than 1 in all three cell inoculation groups, which was significant in all three experiments. inoculum, n = 18, p = 0.003, 2 inocula, n = 12, p = 0.0001 and 3 inocula, n = 20, p = 0.0068, Mann-Whitey analysis).

Effect of GRP1 antiserum on tumor weight in established tumors

Giant. 22 shows in the left panel the average tumor weight in control animals treated with normal rabbit serum for all three cell inocula. The figure also shows the average

BB Β Β BB BB BB BB BBBB B BBBB • • • ♦ ♦ Β

Β ΒΒΒΒ ΒΒΒΒΒΒ

ΒΒΒΒ Β Β Β ΒΒΒΒ

The tumor weight of nude mice after treatment with GRP1 antiserum decreased by 60% in all 3 cell inoculations. This reduction was significant in all three experiments (1 inoculum, p = 0.0016, 2 inoculum, p = 0.0084 and 3 inoculum, p = 0.0001, MannWhitey analysis).

Immunoreactivity of GRP1 in C170HM2 xenograft as determined by Western blot analysis

Extranular membrane proteins were prepared from C170HM2 xenograft from 2/3 experiments. These were analyzed by Western blotting analysis using GRP1 antisera. In FIG. 23 is a photograph of a Western blot showing xenotransplant treated with normal rabbit serum with two immunoreactive bands of 74 and 50 kDa, with the first band showing the strongest immunoreactivity. Two immunoreactive bands were identified in GRT1-treated xenografts along with an intermediate band that was not observed in control xenografts or in cells cultured under in vitro conditions. The 50 kDa band showed the strongest immunoreactivity, indicating that a greater proportion of CCK-B / gastrin receptors may be present in the internalized form in xenografts treated with GRP1 antiserum.

Histological analysis of C 170HM2 xenografts

Giant. 24 shows a microscopic view of a C170HM2 xenograft invading the liver of a nude mouse. This tumor is generally formed by a necrotic center with viable cell margins that suppress hepatocytes upon invasion of liver tissue. In the periphery of viable C170HM2 tumor cells, the degree of apoptosis was measured by the Tunnel method with visualization of positive cells by in situ hybridization. In FIG. 25, it is shown that apoptotic cells were present in viable xenograft tumor cells treated with GRP1 antiserum, but not in tumors treated with normal rabbit serum.

The results show that antisera against the amino-terminal epitope of the CCK-B / gastrin receptor selectively take up in liver invasive C170HM2 tumors. Neutralization of the GRP1 epitope leads to a significant effect on tumor attachment and tumor burden of an already established tumor. This tumor-inhibiting effect may be due to a) a general cytostatic effect induced by blockade of the CCK-B receptor / gastrin and / or b) an indirect effect of targeting the antibody to the nucleus of the cell, which may lead to its apoptosis.

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# 9 »9 9 9

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Watson, S.A., Steele, R. Gastrin receptors on gastrointestinal tumor cells, In: Gastrin receptors in GI Tumors. R.G. Landes Co., Austin TX: CRC 1993, p. 2033.

• • • •

Sequence protocol

1) General information:

(i) Applicant: Michaeli, Dov

Caplin, Martyn E.

Watson, Susan A.

Grimes, Stephen (v) Title of the Invention: Immunogenic CCK-B / Gastrin Receptor Preparations and Tumor Treatment Methods

(vi) Number sequences: 3 (vii) Mailing Address: (AND) Addressee: Dimitros T. Drivas, White & Time (B) Street: 1155 Avenue of the Americas (C) City: New York (D) State: NY (E) Country: USA (F) Postcode: 10036-2787 (xiii) To configure your computer: (AND) Media Type: Floppy Disk (B) Computer: compatible with personal computers counting IBM (C) Operating system / software: PC-DOS / MS-DOS (D) Software: PatentIn Release # 1.0 , version J # 1,30 (x) Data about the present invention (D) Application number: (E) Date of completion: (F) Classification (viii) Information about the patent office (AND) Name: Drivas, Dimitros T. (B) Registration number: 32218 (C) Registration / file number: 1102865- 0032

(ix) Connection:

(A) (B)

Phone: (212) 819-8200 Fax: (212) 3548113 · 9 9 9 9999 9999 9 999 «999

9999 «98 99 9

4f) 999999 9999

999 »9 99 99 99 99«

(8) Information concerning sequence number 1:

(i) Sequence characteristics:

(A) Length: 17 amino acids (B) Type: from amino acids (C) Fiber type: irrelevant (D) Topology: linear (ii) Molecule type: peptide (iii) Hypothetical molecule: No (iv) Anti-sense structure: No (v) Fragment type: N-terminal (xi) Sequence description: sequence number 1:

Lys Leu Asn Arg Ser, Val Gin Gly Thr Gly Pro Gly Pro Gly Ala Ser Leu

(8) Sequence information with identification number 2: (i) Sequence characteristics:

(E) (F) (G) (H) Length: 15 amino acids Type: from amino acids Fiber type: not relevant Topology: linear (ii) Type molecule: peptide (iii ) Hypothetical molecule: No. (iv) Type fragment: internal (xi) Description sequence: sequence identification number 2 Gly Pro Gly Ala His Arg. Ala Leu Ser Gly Ala Pro Ile

11) Sequence information with identification number 3: (i) Sequence characteristics:

(A) Length: 7 amino acids (B) Type: amino acid (C) Fiber type: irrelevant (D) Topology: linear (ii) Molecule type: peptide (xi) Sequence description: sequence number 3: Ser Ser Pro Pro Pro Pro Cys

Claims (2)

An immunogen consisting of a peptide from a CCKB / gastrin receptor molecule that is conjugated to an immunogenic carrier. The immunogen of claim 1, wherein the peptide has the amino acid sequence KLNRSVQGTGPGPGASL (SEQ ID NO: 1 in the Sequence Protocol), or GPGAHRALSGAPISF (SEQ ID NO: 2 in the Sequence Protocol). The immunogen of claim 1 or 2, further comprising a spacer peptide sequence. The immunogen of claim 3, wherein the spacer peptide sequence is SSPPPPC (Sequence Protocol Identification Number 3). The immunogen of claim 1, wherein the immunogenic carrier is selected from the group consisting of diphtheria toxoid, tetanus toxoid, and bovine serum albumin. Method of treatment of malignant disease caused by growth of gastrin-dependent malignant cells. The method comprises administering to an animal in need of such treatment an effective amount of an immunogen against the CCK-B / gastrin receptor. The method defined in claim 6, wherein the immunogen is a peptide from a CCK-B / gastrin receptor molecule. The method as defined in claim 7, wherein the peptide has the amino acid sequence KLNRSVQGTGPGPGASL (Sequence ID 1) or GPGAHRALSGAPISF (Sequence ID 2). The method defined in claim 8, wherein the immunogen is further comprised of a spacer peptide sequence. The method defined in claim 9, wherein the spacer peptide sequence is SSPPPPC (Sequence Protocol Identification Number 3). The method as defined in claim 10, further comprising an immunogenic carrier selected from the group consisting of an immunogenic carrier selected from the group consisting of: ·· · «· · · · 4ífe. Μ · 5L5. 1Γ Η. 16ŤS ?. η. > §. 4f Ν. fl, X). άο · 2xL. 2%. diphtheria toxoid, tetanus toxoid, and bovine serum albumin. Method of treatment of gastrin-dependent tumors. The method consists of administering to an animal in need of such treatment an effective amount of antibodies against the CCK-B / gastrin receptor that recognize and bind to CCK-B / gastrin receptors on tumor cells. The method defined in claim 12, wherein the antibodies are selected from the group consisting of chimeric, monoclonal and humanized antibodies. The method defined in claim 12, wherein the antibodies against the CCK-B / gastrin receptor recognize and bind the amino acid sequence KLNRSVQGTGPGPGASL (Sequence ID: 1 in the Sequence Protocol), or GPGAHRALSGAPISF (Sequence ID 2 in the Sequence Protocol). The method defined in claim 12 or 14, wherein the antibodies are further conjugated to a cytotoxic molecule. The method defined in claim 15, wherein the cytotoxic molecule is a toxin or a radioactive molecule. The method defined in claim 16, wherein the toxin is a cholera toxin. The method defined in claim 16, wherein the radioactive molecule is labeled with ¹²⁵ I or ¹³¹ I. Method for detecting gastrin-responsive tumors containing CCK-B / gastrin receptors. The method consists of exposing the anti-gastrin receptor antibody to cells isolated from a tumor biopsy sample and detecting the CCKB / gastrin receptor in the sample. The method defined in claim 19, wherein the anti-gastrin receptor antibody is specific for the amino-terminal CCK-B / gastrin receptor peptide. The method defined in claim 20, wherein the peptide originates from the amino-terminal end of the CCKB / gastrin receptor and consists of amino acid residues 5-21. • · 4 4 · · · · 4 4 · · 4 4 ·· · 4 · · · 44 · · · An immunogenic preparation consisting of an immunogen against the CCK-B receptor / gastrin. 2SL Preparation, which is subject patent claim 22, where immunogen is formed peptide from receptor molecules for CCK- . B / gastrin 2-S. Preparation, which is subject patent claim 22, where immunogen is formed anti-receptor antibodies for CCK- B / gastrin. . A method of diagnosing gastrin-dependent tumor by administering radiolabelled antibodies against the CCK-B / gastrin receptor to a patient with colorectal cancer and scintigraphic imaging the tumor. 2 <7. The method of claim 25, wherein the antibodies against the CCK-B / gastrin receptor are labeled with ¹¹¹ Indium or ⁹⁰ Ytrium radioisotopes.