JP5583648B2 - Monoclonal antibody against gastrin hormone - Google Patents

Description

Detailed Description of the Invention

(Related application)
[0001] This application claims rights under US Provisional Application No. 60 / 557,759 filed concurrently on March 29, 2004, with US Patent Application No. 10/8133636 entitled "Gastrin Hormone Immunoassays". The specification of each application is incorporated herein by reference in its entirety.
(Field of Invention)

[0002] The present invention relates to antibodies to specific regions of gastrin hormone and the various types of gastrin hormone present in living organisms of animals, particularly humans. The present invention is further directed to the application of these monoclonal antibodies (MAbs) to the detection and diagnosis and monitoring of gastrin mediated diseases and conditions, and to prevent and treat gastrin mediated diseases and conditions. And a method of using MAb.
(Background of the Invention)

[0003] Although gastrin hormone was first identified 100 years ago and purified in the 1960s, the effects of normal and diseased tissues on various tissues have not yet been fully elucidated. One of the main reasons for such a gap in the knowledge of the gastrin system is that it was difficult to detect and quantify several types of gastrin hormone separately.

[0004] There are several types of peptide hormones gastrin in mammals, based on the number of amino acid residues in the peptide chain, two of which are "small" gastrin and "large" gastrin. Classified into major size classes. “Small” gastrin types include mature gastrin 17 (G17) and G17 (G17-Gly) with glycine extension, and “large” gastrins include gastrin 34 (G34) and G34 with glycine extension. (G34-Gly) is included. The mature form of G17 is a major effector of gastric acid secretion and it is estimated that this role is approximately 6 times more potent than G34. Various types of gastrin are produced in vivo by cleaving from the precursor peptide progastrin, or optionally by modifying the truncated form. Human G34 has the entire 17 amino acid sequence of G17 at its C-terminus and, as expected, immunologically cross-reacts with G17.

[0005] Mature G17 is modified at both the amino and carboxy terminal residues. N-terminal glutamic acid is cyclized to form pyroglutamic acid (pGlu), and the free carboxyl group of the C-terminal phenylalanine residue is amidated by an enzyme called peptidyl α-amidated monooxygenase (PAM) to form C forming the terminal Phe-NH _2. Mature G34 is similarly amidated at the C-terminus to form the C-terminal Phe-NH ₂ (see Dockray et al., Ann. Rev. Physiol. (2001), 63: 119-139).

[0006] Mature G17 is the major “small” gastrin type in humans and has the amino acid sequence pEGPWLEEEEEEYGWMDF-NH2 (SEQ ID NO: 1). G17-Gly is an incompletely processed form of gastrin found as a minor component of “small” gastrin in healthy human individuals and has the amino acid sequence pegpwlEeeeeeegwmddf (SEQ ID NO: 2).

[0007] Gastrin 34 is the major, “large” gastrin type in humans, has the amino acid sequence pELGPQGPPPHLVADPSKKEGPWLEEEEEEYGWMDF-NH2 (SEQ ID NO: 3), and glycine extended gastrin 34 (G34-Gly) has an additional C-terminus. It has a glycine residue and has the amino acid sequence pELGPQGPPPHLVADPSKKEGPWLEEEEEEAYGWMDFG (SEQ ID NO: 4).

[0008] Gastrin is secreted by gastric antral G cells in response to gastrin releasing peptide (GRP), and the paracrine action of gastric acid and several peptide hormones, most notably somatostatin, Is suppressed by. Although gastrin peptides have traditionally been recognized to function to stimulate acid secretion in the stomach of healthy individuals, these peptides proliferate, differentiate, and mature in various cells of the gastrointestinal (GI) system. It is not until recently that the process is controlled.

[0009] In addition to local activity in the GI system, G17, and to a lesser extent G17-Gly, is released into the bloodstream and suffers from gastrointestinal disorders and diseases such as gastric cancer, colorectal cancer and pancreatic cancer It has been found to increase in the patient's serum. More recently it has been found that these gastrin species are also associated with other diseases not related to the gastrointestinal tract, including small cell lung cancer (SCLC) and liver metastatic tumors. For example, “Gastrin and Colon Cancer: a unifying hypothesis”; N. Joshi et al., Digestive Diseases (1996), 14: 334-344; and “Gastrin and Colorectal Cancer”, Smith, A. et al. M.M. And Watson, S .; A. , Alignmental Pharmacology and Therapeutics (2000), 14 (10): 1231-1247.

[0010] Antibodies have become major reagents in numerous assays used in medicine, veterinary medicine, and other fields. Such tests include a number of commonly used immunoassays such as, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC) assay, immunofluorescence (IF) assay, and the like. .

[0011] Monoclonal antibodies (MAbs) are in many respects suitable for many of the above assays compared to polyclonal antisera and antibodies purified from polyclonal antisera due to their inherent properties. Its attributes include single-determinant specificity for the target antigen (ie specificity for a single epitope), invariant specificity between different antibody preparations, and invariant affinity over time and Chemical composition is included. Furthermore, MAbs can be produced indefinitely and in unlimited amounts by in vitro methods. These properties are clearly different from those of polyclonal antibodies. Polyclonal antibodies require in vivo immunization and inevitably involve biological variability and limited antibody production capacity that limits the life of the immunized animal.

[0012] While having these advantages, there are differences between individual MAbs, and the differences exist even when different MAbs are specific for the same epitope. For example, there may be differences between any or all of the following properties between MAbs induced by immunization with a single antigenic epitope region. That is, 1) fine specificity with respect to the molecular composition and tertiary structure of the epitope, 2) antibody idiotype, 3) antibody affinity, 4) antibody allotype, and 5) antibody isotype. These differences in properties can affect the behavior of MAbs in a particular immunoassay so that different MAb isolates produced against the same antigenic region can behave differently in a given assay. May be indicated. Thus, some MAbs will be superior to others that bind to the same epitope when used as reagents in certain immunoassays.

[0013] The immunoassay may be an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), an immunodiffusion assay, or an immunodetection assay such as ELISPOT, slot blot, western blot. General guidelines for such methods include, for example, Ausubel et al. (Edit) (1987) “Current Protocols in Molecular Biology”, John Wiley and Sons, New York, N .; Y. checking ... Alternatively, the immunoassay may be an immunohistochemistry (IHC) staining or immunofluorescence (IF) method that visualizes one type of gastrin hormone in a tissue sample. For example, “Principles and Practice of Immunoassay” (1991), Christopher P. et al. Price and David J.M. Neoman (editor), Stockton Press, New York, N .; Y. checking ...

[0014] Monoclonal antibodies selective for the N-terminal region and C-terminal region of G17 have been described in the literature. For example, Azuma et al., Gastroenterolologica Japan (1986), 21 (4): 319-324; Ohning et al., Peptides (1994), 15 (3): 417-423; Fuerle et al., Pancreas (1995), 10 ( 3): 281-286; Kovacs et al., Peptides (1996), 17 (4): 583-587; Ohning et al., Am J. et al. Physiol (1996), 271 (3 Pt1): G470-476; Shipponen et al. (2002), Scan J. et al. Gastroenterol. 37 (7): 785-791. However, any of these antibodies, alone or in combination, can be identified and quantified from several types of gastrin hormones in normal and diseased biological fluids. It has not been.

[0015] It has been shown that anti-gastrin polyclonal antibodies are effective in suppressing gastrin activity ("Inhibition of gastrinity by the antibodies to the C-terminal tetrapeptide of gas, 19 years"). 65 (4): 633-639), non-human anti-gastrin polyclonal antibodies have been applied to treat patients suffering from Zollinger-Ellison syndrome. Zollinger-Ellison syndrome is a pathological condition in which excess gastrin is produced without stimulation by diet. See Hughes et al., “Therapy with Gastrin Antibody in the Zollinger-Ellison Syndrome”, Digestive Diseases (1976), 21 (3): 201-204. However, these rabbit anti-gastrin antibodies only had “short-term effects on this patient at best” (Hughes, p. 204). US Pat. Nos. 5,886,128 and 5,785,970 disclose methods of treating ulcers or tumors that grow or are stimulated depending on gastrin hormone by immunization with a gastrin hormone peptide conjugate.

[0016] Recently, the ratio of amidated gastrin hormone to nonamidated form in serum provides an indication of an individual's risk profile for developing duodenal ulcer disease or gastric atrophy. It was suggested. T. T. et al. C. See Wang, US Patent Application Publication No. 2003/0049689, entitled “Diagnostics and Treatment of Gastrointestinal Disease”. Another group has used G17 at fasting as a basis for assessing the risk of gastric acid related diseases by comparison with the “cut-off value” for G17 at fasting, along with the levels of markers for pepsinogen I / II and Helicobacter pylori. A method involving measuring levels was used. Please refer to the pamphlet of International Publication No. 0423148 issued on March 18, 2004.

[0017] Until now, no MAb has been available that can detect and accurately identify each of the G17, G17-Gly, G34, and G34-Gly types of gastrin hormone. . Furthermore, prior to the present invention, it was not possible to accurately measure the amount of each of the above types of gastrin hormone in a biological fluid sample. Use of the MAbs of the present invention in clinical trial assays will provide a more accurate indication of the biology of gastrin hormone in normal and disease states. The use of MAbs of the present invention also provides pharmaceutical MAb compositions and methods for preventing and treating diseases and conditions associated with gastrin.
(Summary of Invention)

[0018] The present invention is an epitope within the amino acid sequence pEGPWLE (corresponding to amino acids 1-6 in G17; SEQ ID NO: 5) and is selective for the N-terminus of gastrin 17 (G17) or glycine extension G17 (G17-Gly). Monoclonal antibodies (MAbs) that bind to Also provided are hybridomas that produce MAbs that selectively bind to the N-terminus of gastrin 17 (G17) or Gl7-Gly at an epitope within the amino acid sequence pEGPWLE (SEQ ID NO: 5).

[0019] The present invention also provides a MAb that selectively binds to the C-terminus of gastrin 17 (G17) or gastrin 34 (G34) at an epitope within the amino acid sequence EEAYWMDF-NH2 (SEQ ID NO: 6). Also provided is a hybridoma that produces an MAb that selectively binds to the C-terminus of gastrin 17 (G17) or gastrin 34 (G34) with an epitope within the amino acid sequence EEAYGWMDF-NH2 (SEQ ID NO: 6).

[0020] The present invention also provides a MAb that selectively binds to the N-terminus of human gastrin 34 (G34) at an epitope within the amino acid sequence pELGPQG (SEQ ID NO: 7). Also provided are hybridomas that produce MAbs that selectively bind to the N-terminus of human gastrin 34 (G34) at an epitope within the amino acid sequence pELGPQG (SEQ ID NO: 7).

[0021] The present invention further provides an MAb that selectively binds to the C-terminus of glycine-extended gastrin 17 (G17-Gly) and glycine-extended gastrin 34 (G34-Gly) with an epitope within the amino acid sequence ygwmddf (SEQ ID NO: 8). I will provide a. Also provided is a hybridoma that produces an MAb that selectively binds to the C-terminus of glycine-extended gastrin 17 (G17-Gly) and glycine-extended gastrin 34 (G34-Gly) with an epitope within the amino acid sequence YGWMDFG (SEQ ID NO: 8). To do.

[0022] A combination of two or more of the antibodies of the present invention is selectively bound to the N-terminus or C-terminus of each of the G17, G17-Gly, G34, and G34-Gly gastrin hormones. It can also be used in the form of a MAb panel.

[0023] (1) An epitope within the amino acid sequence pEGPWLE (corresponding to amino acids 1 to 6 in G17; SEQ ID NO: 5), selectively at the N-terminus of gastrin 17 (G17) or glycine extension G17 (G17-Gly) MAb binding, (2) MAb selectively binding to the C-terminus of gastrin 17 (G17) or gastrin 34 (G34) at an epitope within amino acid sequence EEAYWMDF-NH2 (SEQ ID NO: 6), (3) amino acid sequence pELGPQG MAb that selectively binds to the N-terminus of human gastrin 34 (G34) with an epitope within (SEQ ID NO: 7), or (4) an epitope within amino acid sequence YGWMDFG (SEQ ID NO: 8) with glycine-extended gastrin 17 ( G17-Gly) and glycine extended gastrin 34 (G34-Gly) MAb to the C-terminus of selectively binding, also of a pharmaceutical composition, is provided in combination with a pharmaceutically acceptable carrier.

[0024] A gastrin-mediated disease or condition in a patient is measured by measuring the level of gastrin hormone type in a sample of biological fluid obtained from the patient, and the level of gastrin hormone type in the sample. Diagnosis can be made by comparing with the normal level of the gastrin hormone type in the sample of biological fluid obtained from the above.

[0025] Such a gastrin mediated disease or condition is (1) an epitope within the amino acid sequence pEGPWLE (corresponding to amino acids 1-6 in G17; SEQ ID NO: 5), with gastrin 17 (G17) or glycine stretch G17 MAb that selectively binds to the N-terminus of (G17-Gly), (2) an epitope within the amino acid sequence EEAYGWMDF-NH2 (SEQ ID NO: 6), selected at the C-terminus of gastrin 17 (G17) or gastrin 34 (G34) (3) an MAb that selectively binds to the N-terminus of human gastrin 34 (G34) with an epitope within the amino acid sequence pELGPQG (SEQ ID NO: 7), or (4) the amino acid sequence YGWMDFG (SEQ ID NO: 8) ) With the glycine-extended gastrin 17 (G17-Gly) and C-terminal to selectively bind to MAb lysine extended gastrin 34 (G34-Gly), a pharmaceutical composition comprising, by administering to a patient in need thereof, may be prevented or treated.

[0026] A method for monitoring the progress of a gastrin mediated disease or condition in a patient is also provided. The method comprises: measuring at a first time the level of gastrin hormone type in a sample of biological fluid obtained from a patient suffering from or at risk of a gastrin-mediated disease or condition; Measuring at different time points the level of the gastrin hormone type in one or more samples of biological fluid obtained from, thereby monitoring the course of a gastrin-mediated disease or condition .

[0027] The present invention also provides a method for assessing gastrin hormone blockade therapy in patients suffering from a gastrin hormone mediated disease or condition. This method includes the following steps a) to j).
a) obtaining a first sample of biological fluid from a patient prior to or at the beginning of treatment b) measuring the level of gastrin hormone in the first sample by immunoassay c) the disease or condition to be treated, And diagnosing based on the level of gastrin hormone in the first sample d) a first agent that binds to the gastrin hormone and modulates the binding of the gastrin hormone to the target receptor in vivo; or E) the step of providing the patient with a treatment comprising a substance that produces a drug e) after obtaining an appropriate amount of time for which the therapeutic effect is expected to occur, obtaining a second sample of biological fluid from the patient f) the second sample Measuring the level of total gastrin hormone, including bound and free gastrin hormone, in the first aliquot of the sample by immunoassay,
A second agent that replaces a first aliquot of a second sample with (i) any gastrin hormone bound by the first agent; and (ii) an immobilized anti-gastrin hormone antibody that does not bind to the second agent; Incubate with,
Washing away the second drug,
Adding a detectable antibody that binds to gastrin hormone but does not compete with the immobilized antibody;
Forming an immune complex comprising an immobilized antibody bound to gastrin hormone, wherein the gastrin hormone is bound to the detectable antibody; g) detecting the amount of detectable antibody in the immune complex, thereby Measuring the amount of total gastrin hormone in the two samples h) measuring the level of free gastrin hormone by repeating steps f) and g) with a second aliquot of the second sample Because
Performing the incubation in step f) without the second agent j) comparing the measured amount of free gastrin hormone in the first sample with the amount of free gastrin hormone and total gastrin hormone in the second sample; Determining the effectiveness of gastrin hormone block therapy in a patient

[0028] The present invention further provides a kit for performing an immunoassay comprising an anti-gastrin hormone MAb and a suitable container. The anti-gastrin MAb is preferably selected from the group consisting of MAbs 400-1, 400-2, 400-3, 400-4, 401-2, 455-1, 455-2, and 458-1.
(Detailed description of the invention)

[0032] Definitions of terms used in the present specification are shown below.

[0033] As used interchangeably herein, "gastrin hormone" or "gastrin hormone form" refers to any gastrin hormone peptide having biological activity and / or immunological cross-reactivity. means. The major types of gastrin hormones are gastrin 17 (G17) that is amidated at the C-terminus or has a free C-terminus; glycine-extended gastrin 17 (G17-Gly); C-terminal amidated and free C-terminal types Including, but not limited to, gastrin 34 (G34); glycine-extended gastrin 34 (G34-Gly); and progastrin.

[0034] As used herein, "total amount" of gastrin hormone type in a sample means the sum of the amount of free (unbound) gastrin hormone type and the amount of complex (bound) gastrin hormone type. The complex gastrin may be bound to the antibody in the sample or may be bound to other binding moieties.

[0035] As used herein, "biological fluid" means any liquid containing a biologically-derived substance. Suitable biological fluids for use in the present invention are those containing body fluids of animals, particularly mammals, preferably human individuals. The bodily fluid can be any bodily fluid, including but not limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF), and the like.

[0036] As used herein, a "preservative" is any drug, supplement or additive that reduces the time-dependent degradation of gastrin in a sample of biological fluid, or a liquid sample containing biological components. means. Preservatives useful in the practice of the present invention may be any of a number of preservatives well known in the art, such as sodium azide, EDTA, protease inhibitors (eg, PMSF (phenylmethylsulfonyl fluoride), aprotinin (traj General chemical preservatives such as roll)) or biological preservatives such as heparin, but are not limited thereto.

[0037] (New anti-gastrin monoclonal antibody)
[0038] The selection of the optimal monoclonal antibody (MAb) for use in a particular application is preferably done by evaluating the performance of individual candidate MAbs in the intended application. Therefore, testing candidate MAbs for optimal functionality in the intended application becomes part of the selection process to obtain the optimal MAb for the intended use. This selection step is performed in addition to the selection steps normally performed to obtain MAbs, including binding to the target antigen and sequential cloning of the hybridoma producing the MAb, and provides for sustained cell growth and cell division. As well as the stability of the essential properties of the hybridoma cell line, including consistent and unlimited antibody production over an infinite period of time.

[0039] As used herein, the term "selective" for a particular type of gastrin hormone means that the antibody is specific for a particular target epitope present in a particular type of gastrin hormone, while the target epitope is It means binding to each of the multiple types of gastrin hormones it contains. For example, the C-terminus of mature (amidated) G17 is common to mature G17 and G34. Thus, MAbs specific for the target C-terminal epitope present at the C-terminus of mature G17 are selective for G17 (and G34).

[0040] In particular, the present invention relates to biologically active gastrin hormone forms, namely amidated gastrin 17 (G17), amidated gastrin 34 (G34), glycine extended gastrin 17 (G17- Gly), glycine-extended gastrin 34 (G34-Gly), and methods for identifying MAbs with superior properties selective for the N-terminus and C-terminus of progastrin are disclosed. These MAbs are immunoenzyme assays designed to be able to measure specific types of gastrin hormones in biological fluids (commonly referred to as “ELISA” or enzyme-linked immunosorbent assays). Particularly suitable for use in. The MAbs of the present invention are suitable for detecting and / or quantifying gastrin hormone in, for example, immunodetection assays such as ELISPOT, radioimmunoassay, antibody-based sandwich capture assay, dot blot, slot blot, western blot assay, etc. Yes.

[0041] In one aspect, the invention provides a MAb that selectively binds to the N-terminus of gastrin 17 (G17) at an epitope within the amino acid sequence pEGPWLE (SEQ ID NO: 5). Binding of these MAbs selective for the N-terminus (G17) of gastrin 17 to the BSA conjugate of peptide pEGPWLEEEE (SEQ ID NO: 11) is inhibited by human G17, horse G17, or human G17-Gly.

[0042] In another aspect, the invention provides a MAb that selectively binds to the C-terminus of gastrin 17 (G17) or gastrin 34 (G34) at an epitope within the amino acid sequence EEAYGWMDF-NH2 (SEQ ID NO: 6). To do.

[0043] In yet another aspect, the present invention provides MAbs that selectively bind to the N-terminus of human gastrin 34 (hG34) at an epitope within the amino acid sequence pELGPQG (SEQ ID NO: 7).

[0044] In yet another aspect, the present invention is an epitope within the amino acid sequence ygwmdfg (SEQ ID NO: 8) that selects for the C-terminus of glycine extended gastrin 17 (G17-Gly) and glycine extended gastrin 34 (G34-Gly). MAb that binds automatically.

[0045] In yet another aspect, the present invention provides MAbs that selectively bind progastrin. This MAb binds to progastrin but not to the processed gastrin hormone type, ie, G17, G34, G17-Gly, or G34-Gly. MAbs of the present invention that are selective for progastrin include MAbs that bind to the C-terminus of human progastrin. The MAb is thought to bind to preprogastrin. Preprogastrin consists of a 101 amino acid peptide chain, which is processed to produce progastrin and gastrin in sequence. However, preprogastrin processing is rapid and takes place in the endoplasmic reticulum (ER) where its synthesis takes place. The MAbs of the invention that bind to progastrin are useful for detecting and quantifying progastrin in a sample in the assays described herein.

[0046] The MAb of the present invention has an association constant (Ka) of about 10 ⁶ to about 10 ⁷ LM ⁻¹ , preferably about 10 ⁷ to about 10 ⁸ LM ⁻¹ , more preferably about 10 ⁸ to about 10 ⁹ LM ^{−. 1} , more preferably about 10 ⁹ to about 10 ¹⁰ LM ^-1 , more preferably 10 ¹⁰ to 10 ¹¹ LM ^-1 , most preferably about 10 ¹¹ to 10 ¹² LM ^-1 binding to gastrin type It is preferable to do.

[0047] (Anti-gastrin monoclonal antibody panel)
[0048] The present invention provides for the first time an anti-gastrin hormone MAb panel that allows unambiguous identification and quantification of one or more of the G17, G17-Gly, G34, and G34-Gly forms of gastrin hormone. To do. For example, a MAb panel comprising a MAb selective for the N-terminus of G34 type of gastrin hormone and a MAb selective for the C-terminus of G17 / G34 (the C-terminus of G34 is identical to the C-terminus of G17) is Allows specific identification and quantification of G34 in a sample by any one of a number of immunoassays routinely performed in the art. Common immunoassays that can use the MAbs of the present invention include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIAs), immunofluorescence assays (IF), immunohistochemical assays (IHC), immunodiffusion assays, and the like But are not limited to these. Examples of such conventional diagnostic assays are described in, for example, “Synthetic peptides in human papilloma virus 1, 5, 6, 8, 11, 16, 18, 31, 33 and 56 useful in immunoassay for Dillner et al. See the specification of US Pat. No. 5,932,412 entitled “diagnostic purposes”.

[0049] Supplementing the MAb panel with one or more additional MAbs of the present invention provides the ability to specifically identify and quantify additional gastrin hormone species in the sample. For example, by adding a MAb selective to the N-terminus of G17 type to the antibody panel described above, the specificity of free G17 hormone and total (bound plus free) G17 hormone in the sample according to the method of the invention described below. Further identification and quantification is possible.

[0050] Similarly, a MAb panel comprising a MAb selective for the N-terminus of G34 and a MAb selective for the C-terminus of glycine extension G34 (which is identical to the C-terminus of glycine extension G17) is , Allowing specific identification and quantification of glycine elongation G34 in the sample. Furthermore, by adding to the panel a selective MAb at the N-terminus of G17, as described herein, the identification and quantification of free glycine extension G17 and total (bound plus free) glycine extension G17 in the sample can be achieved. It becomes possible.

[0051] Other combinations of MAb pairs selected from MAbs of the present invention useful as MAb panels for the identification, quantification, and monitoring of other types of gastrin hormone will be apparent to those skilled in the art. The invention also encompasses such MAb pairs of the invention, combinations of MAb pairs of the invention, and collections of MAb pairs of the invention.

[0052] The MAbs of the present invention are for assessing the predisposition to becoming gastrin hormone mediated diseases and conditions, and for detecting and diagnosing such diseases and conditions in patients suffering from them. A means of accurately measuring the amount and ratio of gastrin hormone type is provided. For example, the anti-gastrin of the present invention may be used in an ELISA assay for any one or all of the G17, G34, G17-Gly, and G34-Gly gastrin hormone types for large-scale screening of patient serum or other biological fluids. MAbs can be incorporated.

[0053] MAbs of the present invention, combinations of MAb pairs selected from MAbs of the present invention, and MAb panels of the present invention are particularly useful when applied to high throughput methods. Such methods include microchip and microarray methods for detecting gastrin hormone antigens, thereby allowing multiple samples on other assay substrates such as microplates, slides, or plates with virtual wells. Can be tested (eg, as described in US Pat. No. 6,565,813 issued to Garyantes et al.). Binding detection may be performed by any of the most advanced detection systems available. The detection of the binding can also be performed using a change in surface plasmon resistance caused by a specific biomolecular reaction such as antigen-antibody binding. For the application of this technique to enzyme assays, see, for example, US Pat. No. 5,981,167 issued to Talemi et al. This method can be applied in a continuous flow mode and also detects antibody binding to peptides or proteins (such as gastrin hormone) immobilized on the surface, or detects gastrin antibody complexes. It can also be applied. The latter complex may be detected by binding to an antibody specific for an epitope of the gastrin hormone type (G17, G34, G17-Gly, or G34-Gly) immobilized on the surface, An epitope is one that is not sterically hindered by the antibody of the complex. Furthermore, this technique has the advantage of allowing high throughput applicability and high sensitivity without the need for radioactive labels.

[0054] The MAbs of the present invention are also useful in immunohistochemistry (IHC) and immunofluorescence (IF) assays of tissue samples, such as those obtained from biopsy materials. Such an analysis can be used to detect abnormal levels of individual gastrin hormone types, thereby diagnosing gastrin hormone-mediated diseases and conditions.

[0055] The MAbs of the present invention can be humanized according to established methods well known in the art. For example, the specification of US Pat. No. 6,689,869 entitled “Labeled humanized anti-CD-18 antibodies and fragments and kits” issued to Waldman et al. And “Method for umbo diving” issued to Carter et al. U.S. Pat. No. 6,639,055, entitled "". The humanized antibody can be reconstituted to better match the binding affinity of the original mouse Mab. See, for example, US Pat. No. 6,699,974, entitled “Re-shaped human anti-HM1.24 antibody” issued to Ono et al.

[0056] The MAbs of the present invention are useful for the prevention and treatment of gastrin hormone mediated diseases and conditions. The anti-gastrin MAb of the present invention can be formulated as a pharmaceutical composition for passive immunization against a specific gastrin hormone type. See, for example, the specification of US Pat. No. 6,391,299 (hereinafter referred to as the '299 patent) issued to Blackburn et al., Entitled “Anti-factor IX / IXa antibodies”. Functional fragments of the MAb of the invention (eg, Fab fragment, F (ab ') 2 fragment, and any fragment that retains the ability to bind to the gastrin hormone type of interest. See the' 299 patent for fragments) Can also be incorporated into pharmaceutical compositions and applied therapeutically. See the '299 patent for useful pharmaceutical compositions. Suitable routes for administering the pharmaceutical composition of the present invention include parenteral routes of administration, such as subcutaneous, intramuscular, intravenous routes and the like. Alternatively, the pharmaceutical composition can be delivered by the intranasal route. For the prevention or treatment of such a disease or condition in a patient who is likely to become a gastrin hormone mediated disease or condition, or for the treatment of a patient already suffering from such a disease or condition. The pharmaceutical composition is particularly useful when administered in an effective amount.

[0057] An effective amount of a pharmaceutical composition containing a complete anti-gastrin MAb or functional fragment thereof of the present invention, particularly a humanized anti-gastrin MAb, to treat a gastrin-mediated disease or condition is the disease. Or an amount that prevents the onset of progression of the condition or reduces its rate, but is more preferably an amount that stabilizes the disease or condition, more preferably a regression of the disease or condition. An amount that causes complete healing of the disease or condition.

[0058] Furthermore, the MAbs of the present invention can be applied to immunoassays for monitoring the progression of gastrin hormone mediated diseases and conditions, wherein a specific gastrin hormone type, or a free gastrin type or binding The level or amount of gastrin type, or total gastrin type, provides an indication of the success or progression of treatment of a gastrin hormone mediated disease or condition.

[0059] Furthermore, the MAbs of the present invention are useful in methods of assessing gastrin hormone blocking therapy in patients suffering from a gastrin hormone mediated disease or condition. This method
a) obtaining a first sample of biological fluid from a patient prior to or at an early stage of treatment;
b) measuring the level of gastrin hormone in the first sample by immunoassay;
c) making a diagnosis based on the disease or condition to be treated and the level of gastrin hormone in the first sample;
d) administering to the patient a treatment comprising a first agent that binds to gastrin hormone and modulates the binding of the gastrin hormone to a target receptor in vivo, or a substance that produces the first agent;
e) obtaining a second sample of biological fluid from the patient after an appropriate time during which a therapeutic effect is believed to occur;
f) measuring the level of total gastrin hormone, including bound and free gastrin hormone, in the first aliquot of the second sample by immunoassay,
A second agent that replaces a first aliquot of a second sample with (i) any gastrin hormone bound by the first agent; and (ii) an immobilized anti-gastrin hormone antibody that does not bind to the second agent; Incubate with,
Washing away the second drug,
Adding a detectable antibody that binds to gastrin hormone but does not compete with the immobilized antibody;
Forming an immune complex comprising an immobilized antibody bound to gastrin hormone, wherein the gastrin hormone is bound to a detectable antibody;
g) detecting the amount of detectable antibody in the immune complex, thereby measuring the amount of total gastrin hormone in the second sample;
h) measuring the level of free gastrin hormone by repeating steps f) and g) with a second aliquot of the second sample,
Performing the incubation in step f) without a second agent;
j) comparing the measured amount of free gastrin hormone in the first sample with the amount of free gastrin hormone and total gastrin hormone in the second sample to determine the effectiveness of the patient's gastrin hormone blocker treatment; ,
including.

[0060] The above-described methods applied to assess gastrin hormone block therapy in patients are particularly beneficial in clinical practice. In clinical practice, the time to decide whether to proceed with one treatment regimen or another can be critical to the patient's prognosis. The method of the present invention provides the information on which these vital decisions are based. This method comprises measurement of gastrin hormone prior to or at an early stage of treatment (eg, immediately after immunization with a gastrin hormone peptide-binding vaccine, as described in US Pat. No. 5,622,702) and the effect of treatment is It comprises one or more measurements of total gastrin hormone and / or free gastrin hormone after a period of time expected to begin to occur.

[0061] The gastrin hormone blocking therapy may be active immunization, in which case an immunogen that produces antibodies to gastrin is administered to the patient as described above. Alternatively, a gastrin hormone blocking substance may be passively administered to the patient. The gastrin hormone blocking substance may be any gastrin hormone blocking substance and is not particularly limited, and examples thereof include anti-gastrin hormone antibodies, particularly humanized monoclonal anti-gastrin hormone antibodies. Alternatively, the gastrin hormone blocking substance may be a gastrin hormone receptor or a gastrin hormone receptor mimic. A gastrin hormone receptor mimic is, for example, a soluble gastrin hormone receptor, or a soluble gastrin hormone receptor fragment, but in addition to any other molecule that has the function of binding to gastrin hormone, gastrin hormone Any molecule that mimics the binding of the gastrin hormone receptor to the.

[0062] The present invention also provides compositions, methods, and kits for screening samples suspected of containing gastrin hormone. Such screening can be performed on patient samples or laboratory samples suspected of containing or producing such polypeptides. The kit can contain the antibody of the present invention. This kit may contain a reagent for detecting the interaction between the sample and the antibody of the present invention. The provided reagents may be radiolabeled, fluorescently labeled, or enzymatically labeled. The kit may contain a known radiolabeled substance capable of binding or interacting with the antibody of the present invention.

[0063] The reagents of the kit can be provided as a liquid solution, bound to a solid support, or provided as a dry powder. When the reagent is provided as a liquid solution, the liquid solution is preferably an aqueous solution. When the provided reagent is bound to a solid support, the solid support is preferably a chromatography medium, a test plate having a plurality of wells, or a microscope slide. If the reagent provided is a dry powder, the powder can be reconstituted by adding a suitable solvent, but may be provided in a reconstituted form.

[0064] The kit of the present invention is provided in a container, and the container usually includes a vial containing an antibody, an antigen, or a detection reagent, preferably a vial into which they are appropriately dispensed. It is. The kit of the present invention will usually also include means for housing the antibody container, antigen container, and reagent container for commercial use. Such containers include plastic containers that hold the desired vials, one or more necessary chemicals (eg, chromatographic materials, solvents, eluents), as well as test tubes for detection reactions, surfactants , Antibodies and chemical substances.

[0065] In yet another embodiment, the present invention relates to immunodetection methods and related kits. A gastrin hormone or peptide fragment thereof can be used to detect antibodies reactive therewith, or prepared according to the present invention to detect a gastrin hormone or a gastrin hormone-mediated epitope-containing peptide. It has been proposed that antibodies be available. Usually, these methods involve first obtaining a sample suspected of containing such a hormone, peptide or antibody, and subjecting the sample to conditions under conditions effective to form an immune complex. Contacting with an antibody or peptide according to the invention and then detecting the presence of an immune complex.

[0066] In general, detection of immune complex formation is well known in the art and can be achieved through the application of numerous methods. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (eg, dot blot, slot blot, western blot, etc.), indirect immunofluorescence, and the like. Usually, immune complex formation will be detected by the use of labels such as radioactive labels, enzyme tags (eg, alkaline phosphatase, horseradish peroxidase, or the like). Additional advantages may arise from using secondary binding ligands (eg, secondary antibodies, biotin / avidin ligand binding agents) according to methods well known in the art.

[0067] (Example 1: Production of monoclonal antibody against C-terminus of human G17)
[0068] CSSEEAYGWMDF-NH2 (SEQ ID NO: 10), a linker spacer (-Cys-Ser-Ser-) sequence, followed by an amino acid sequence (-EEAYGWMDF-NH2) containing the C-terminal epitope of human G17 and G34 Peptides containing SEQ ID NO: 6) were purchased by standard solid phase peptide synthesis methods.

[0069] This peptide was incorporated into the immunogen and antibodies against the C-terminus of G17 / G34 were induced as follows. Initially, the peptide was covalently linked to diphtheria toxoid (“DT”) to produce a peptide-carrier bond. The number of peptide units substituted on each DT carrier was measured and finally this conjugate was formulated as an immunogen. The method used was as described in US Pat. No. 5,622,702.

[0070] Briefly, chemical coupling of the peptide to the carrier was performed using ε-maleimidocaproic acid N-hydroxysuccinimide (ε-MCS), a heterobifunctional cross-linking material. The conjugate was purified by dialysis against 0.1 M sodium phosphate buffered saline, pH 7.3 (PBS), and protein concentration was determined by the Raleigh assay. The substitution level of the peptide on DT was determined in molar amounts by amino acid analysis of the conjugate. Subsequently, the dissolved conjugate is mixed with Montanide ISA by mixing the conjugate solution with Montanide ISA 703 (SEPPIC, France) oil at a ratio of 30/70 (wt / wt ratio of conjugate / adjuvant). Formulated as an immunogen with 703 as an adjuvant. Mixing was accomplished by drawing the appropriate volume of each liquid into the syringe and then quickly passing the solution back and forth through the connecting hub to and from the second syringe.

[0071] Immunization of mice was performed by first administering 0.1 mg of peptide DT conjugate immunogen / Maxtanide ISA 703 in a volume of 0.1 mL. p. Injected. A second injection was administered at the same dose 3 weeks after the first injection.

[0072] To generate hybridomas that produce selective MAbs at the C-terminus of G17 / G34, splenocytes obtained from immunized mice were prepared by standard methods well known to those skilled in the art. Fused to mouse myeloma fusion partner cell line. These methods are described in numerous reviews and experimental handbooks. For example, U.S. Pat. No. 4,196,265, “Method of producing antigens” issued to Kaprowski et al .; “Selected Methods in Cellular Immunology.” H. Freeman and Co., San Francisco, 1980); Harlowe and Lane, "Antibodies; A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988; Zola, Mon dies: A Manual of Techniques ", CRC Press, Inc. See, Boca Raton, FL, 1987. Booster immunization of the immunized mice was performed 4 days prior to harvesting their spleen cells for cell fusion using a 0.1 mg PBS solution of the peptide-DT conjugate described above i. p. This was done by injection. Initial selection of hybrid cells was performed using hypoxanthine-aminopterin-thymidine supplemented media as described in Mishell and Shigi. This fusion was named F458.

[0073] The initial selection step for isolating hybridomas producing MAbs against the C-terminus of G17 involved cell selection for antibody production against the target peptide and the stability of the hybrid cell line. Selection of cells producing antibodies was performed by screening cell culture media obtained from tissue culture wells containing a single clone for antibodies to the C-terminus of G17 / 34. The screening targets a conjugate containing a synthetic amidated peptide (amino acids 16-34-NH2 of G34) linked to the immune carrier bovine serum albumin (BSA) via cysteine with ricin-16 It was carried out by the ELISA used. Suitable ELISA methods are known to those skilled in the art, some examples of which are described in detail below. Stable cell lines were obtained by cloning twice each hybrid that produced an antibody that bound to the hG34 (16-34) NH2-BSA conjugate in an ELISA test. By these methods, 15 hybrid cell lines that produce MAbs against the C-terminus common to G17 and G34 were obtained.

[0074] (Example 2: Selection of monoclonal antibodies showing superior performance in immunoenzyme assay of total (bound plus free) G17)
[0075] A method has been developed to measure the total amount of G17 in a biological fluid sample, such as human plasma, that may contain an anti-gastrin antibody and is filed on March 29, 2004, US patent application Ser. No. 8133336. Briefly, this method may be bound via an N-terminal epitope to a G17 N-terminal epitope-specific antibody that is present in a biological fluid test sample and may also be present in the test sample. Adding an excess of a peptide containing human G17 amino acids 1-8 (human G17 (1-8) substituted peptide) to the biological fluid test sample to replace any gastrin hormone of . After incubation for a period of time, the sample mixture containing the replacement peptide is added to a 96 well ELISA plate coated with a capture antibody produced against the C-terminus of G17. After incubation, the plate is washed to remove the replacement peptide, followed by detection and quantification of bound G17 by adding an enzyme-linked antibody that binds to the N-terminal epitope of G17. To do. The removal of unbound enzyme-bound antibody requires another series of washing steps, adding a chromogenic substrate or other substrate that produces a detectable product by the action of the enzyme linked to the antibody. Generates a detectable signal. For example, when the enzyme is horseradish peroxidase (HRP), the substrate is tetramethylbenzidine sulfonate (TMBS). When alkaline phosphatase is the enzyme used for detection, p-nitrophenol phosphate can be used as a chromogenic substrate that produces the colored compound p-nitrophenol. The degree of color development is measured in absorbance units (AU, measured at 405 nm for p-nitrophenol and 450 nm for TNBS), which indicates the amount of G17 present in the test sample, The actual concentration is determined by measuring the absorbance of the test sample and contrasting it with a calibration curve generated using a known concentration of G17.

[0076] Preliminary testing was performed on this assay using plasma samples from human patients. A predetermined concentration of G17 was added to the plasma sample, and an immunoassay was performed using a polyclonal rabbit antibody against the C-terminal epitope of G17 as a capture antibody coated on the well of the test plate. The results and data obtained from these assays were inconsistent and did not give an acceptable level of sensitivity. Therefore, a selective MAb at the C-terminus was used to coat the test plate and tested as a capture antibody in this assay.

[0077] To test each of the C-terminal G17 selective MAbs from fusion 458 in a total G17 assay, 15 individual MAbs against the C-terminus of G17 were first subjected to protein G affinity chromatography. Purified. This was done by using a commercial kit (HiTrap Protein G HP, 1 mL, Amersham Biosciences) according to the manufacturer's instructions. Thereafter, the concentration of each MAb was determined from the absorbance (A280) at a wavelength of 280 nm. To obtain the concentration, A280 was divided by a concentration factor of 1.4 mL / mg. The concentration was adjusted to be in the range of 0.1 to 1.0 mg / mL. Subsequently, each solution was retested by ELISA (no dilution) to qualitatively confirm the binding of MAb to the C-terminus of G17.

[0078] Next, three mixtures of 15 Mabs, 1 negative control MAb, and purified Mab were used to immunize all G17 using human G17 (1-8) substituted peptides as described above. Tested to determine performance in enzyme assay. Each MAb was added to assay coating buffer (1 vial of Convol pH 8.0 concentrated buffer solution (BDH, product no. 18052 1U) in 2.5 L water and sodium azide (2.5 g) added. Was used to coat the wells of a 96-well ELISA plate (0.1 mL added per well) as described above in the method of measuring total G17. .

[0079] Aliquots of known concentrations of G17 were added to serum samples that were depleted of native G17 by incubating overnight at room temperature to completely digest existing G17 with endogenous serum proteases. In order to prepare a standard solution having a known concentration of G17, a diluted solution of this “G17 added” serum was prepared. The G17 concentration in the standard solution was 0, 4.1, 64, and 800 pM. Subsequently, human G17 (1-8) substituted peptides containing the N-terminus of G17 were added and these samples were treated as test samples in the assay. Each G17 solution was then added to plate wells coated with individual MAb preparations selective for the C-terminus of G17 and a total G17 assay was performed according to the procedure described above.

[0080] The results of these assays are shown in Table 1. The results are shown in A280 absorbance units obtained with each concentration of G17 in an assay using each of the 15 strains of monoclonal antibodies coated in the wells of the test plate as capture MAbs.

[0082] The optimal MAb was selected based on the results of testing the performance of each MAb in the assay. The criteria used to compare isolated MAbs included the following: That is,
1) Low absorbance value when 0.0 pM G17 is added (baseline value, preferably ≦ 0.1 AU),
2) The absorbance when adding 4.1 pM G17 is double the baseline,
3) The AU increase (slope) is the steepest between the primary operating range of the assay, 4.1 pM G17 and 64 pM G17, and 4) the AU value is at a G17 concentration of 800 pM. It is the most expensive.

[0083] Based on these criteria, the MAb that performed best was F458-3-8G 1H 3C. This antibody was renamed MAb 458-1 and was used in subsequent assays as the optimal MAb that selectively binds to the C-terminus of G17. These criteria and similar assays were also applied to the selection of MAbs produced against epitopes at the ends of gastrin hormone types G17, G34, G17-Gly, and G34-Gly, exemplified below.

[0084] By incorporating the use of a substituted peptide of the appropriate amino acid sequence to replace the bound hormone into other gastrin hormone peptide type assays, the amount of free hormone in the sample and total hormone (bound hormone) + Free hormone) and both amounts can be measured. The use of substitution peptides can be applied to assays for the total amount of any peptide hormone available for the amino acid sequence of the region to which the peptide binds.

[0085] (Example 3: Isolation and characterization of monoclonal antibodies against the N-terminus of human G34)
[0086] Hybridomas producing MAbs against the amino terminus of G34 were generated as described in Example 1 for the production of MAbs against the C-terminus of G17 and G34. However, the peptide used to immunize the spleen cells of donor mice against the epitope present at the N-terminus of G34 and the peptide used to select the Mab specific for the N-terminal epitope of G34. The composition was different. In order to induce an antibody response to the N-terminal epitope of G34, a peptide called pELGPQGRPPPPC (SEQ ID NO: 12) was conjugated to DT to form an immunogen. This peptide was similarly linked to BSA to form a target antigen for use in an ELISA to identify Mabs against the N-terminal epitope of G34. This fusion was numbered F401.

[0087] F401 produced MAb 401-2. Specificity for G34 was verified by an inhibition ELISA. This inhibition ELISA showed that binding of MAb 401-2 to the G34 N-terminal peptide immunomimic (SEQ ID NO: 12) was inhibited only by the G34 peptide as shown in Table 2.

[0089] Other types of gastrin, including G17, G17-Gly, and equine G17, as well as CCK8 (non-sulfated) and the negative control GnRH, did not inhibit 401-2 Mab binding (see Table 2). As shown).

[0090] (Example 4: Isolation and characterization of monoclonal antibodies against the N-terminus of G17)
[0091] Hybridomas producing MAbs against the amino terminus of G17 were generated as described in Example 1 for the production of MAbs against the C-terminus of G17 and G34. However, the peptide used to immunize the spleen cells of donor mice against the epitope present at the N-terminus of G17 and the peptide used to select the Mab specific for the N-terminal epitope of G17. The composition was different. In order to induce an antibody response against the N-terminal epitope of G17, a peptide called pEGPWLERPPPPC (SEQ ID NO: 5) was conjugated to DT to form an immunogen. This peptide was similarly linked to BSA to form a target antigen for use in an ELISA to identify Mabs against the N-terminal epitope of G17. In addition, a peptide called pEGPWLEEEEAAPPC (SEQ ID NO: 16) was ligated to BSA to generate an ELISA target antigen for the N-terminal epitope of G17. This fusion was numbered F400. F400 produced 4 strains of MAb against the N-terminal epitope of G17. These were assigned MAb numbers from 400-1 to -4.

[0092] MAbs were produced as ascites fluid in mice by standard methods. For use in the test, ascites fluid from each of the F400 MAbs was mixed in an equal volume to form the antibody pool. The anti-G17 MAb titer of this pool was measured by ELISA. They are shown in Table 3.

[0093] The affinity of each of the four strains of F400 MAb was measured by Scatchard analysis of an inhibitory radioimmunoassay. In this inhibition radioimmunoassay, standard radioimmunoassay methods known to those skilled in the art are used to inhibit the binding of each MAb to radioiodinated G17 while increasing its concentration with unlabeled G17. did. Table 4 shows the affinity (Ka) of each of MAbs 400-1 to -4. The specificity of G17 for the N-terminal epitope was verified by a suppression ELISA. In this inhibition ELISA, binding of MAb 400-1 to -4 to the N-terminal peptide immunomimetics of G17 (SEQ ID NO: 11) is inhibited only by G17, G17-Gly, and equine G17 peptides. On the other hand, G34 and CCK8 (non-sulfated) and the negative control GnRH were shown not to inhibit binding of MAbs 400-1 to -4 (as shown in Table 5).

[0094] (Anti-G17 MAb characterization)

[0098] (Example 5: Isolation and characterization of monoclonal antibodies against the C-terminus of glycine-extended G17 / G34)
[0099] Hybridomas producing MAbs against the carboxy-terminal epitope of G17-Gly were generated as described in Example 1 for production of MAbs against the C-terminus of G17 and G34. However, the peptide used to immunize donor mouse spleen cells against the carboxy-terminal epitope of G17-Gly, and the peptide used to select a MAb specific for the carboxy-terminal epitope of G17-Gly. The composition of was different. To induce an antibody response to the carboxy terminal epitope of G17-Gly, a peptide called CPPPSSYGWMDFG (SEQ ID NO: 14) was conjugated to DT to form an immunogen.

[0100] The peptide CGGSKKEGPWLEEEEEEYGWMDFG (SEQ ID NO: 15) was linked to BSA to form the target antigen used in the ELISA to identify Mabs against the carboxy-terminal epitope of G17-Gly. In order to select MAbs that bind to G17-Gly but not to G17 or G34, this fusion suppresses MAb in G17-Gly (SEQ ID NO: 2), while G17 (SEQ ID NO: 1 ) Used an additional selection step to demonstrate that it was not suppressed. This fusion was numbered F445.

[0101] F445 produced two strains of MAb specific for glycine extension G17. These were assigned MAb numbers 445-1 and 445-2. It was particularly difficult to make these MAbs. Approximately 14 fusions had to be performed before the inventors succeeded. Usually, a single fusion is sufficient to obtain MAbs against peptide hormones, such as other gastrin hormones described herein.

[0102] The specificity for G17-Gly was verified by an inhibition ELISA. In this inhibition ELISA, binding of MAbs 445-1 and 445-2 to the G17-Gly C-terminal epitope target peptide (SEQ ID NO: 14) BSA conjugate was determined by combining G17-Gly peptide (SEQ ID NO: 2) and CPPPPSSYGWMDFG (SEQ ID NO: 14). ), And other types of gastrin, including G17, G34, and equine G17, and CCK8 (non-sulfated) and the negative control GnRH are 445-1 and 445-2 MAbs. Was not inhibited (as shown in Table 6).

[0105] (Example 6: Isolation and characterization of monoclonal antibodies against the C-terminus of G34)
[0106] Human G34 and G17 have the same C-terminal epitope. The MAb produced with fusion number F458 described in Example 1 produced a MAb that binds to both C34 and G17 C-terminal epitopes. The MAbs produced with this fusion were numbered serially from 458-1 to -5.

[0107] The specificity of MAb 458-1 to -5 to the C-terminal epitope shared by G17 and G34 was verified by an inhibition ELISA. In this inhibition ELISA, the binding of MAb 458-1 to -5 to the G17 / 34 C-terminal epitope target peptide (SEQ ID NO: 11) BSA conjugate was changed to G17 peptide (SEQ ID NO: 1), G34 peptide (SEQ ID NO: 3), And the CCK8 peptide (SEQ ID NO: 13) (which also presents a C-terminal epitope) is suppressed, while G17-Gly and other types of gastrin containing the G17 (1-9) N-terminus and negative control GnRH was shown not to inhibit binding of MAbs from 458-1 to -5 (as shown in Table 7).

[0109] (Example 7: Demonstration of anti-tumor cell efficacy of F400 MAb in vitro against pancreatic cancer, gastric cancer, and colon cancer cells)
[0110] The pool of MAbs against the N-terminus of G17, shown in FIG. 3 of Example 4, was tested for the ability to suppress the growth of tumor cell lines obtained from human pancreatic cancer, gastric cancer, and colon cancer. In these in vitro studies, two cell lines from each organ source were tested. The six individual tumor cell lines tested were known to produce their own G17 hormone. This may lead to an autocrine effect, and neutralizing MAbs against G17 may suppress it.

[0111] G17 (SEQ ID NO: 12) linked to Sepharose (Sulfo-Link, Pierce) by the method provided with the Sulfo-Link kit to prepare a F400 MAb mixture for in vitro testing on cells. Affinity purification of the antibody was performed by chromatography on peptides presenting the N-terminal epitope. These MAbs were dialyzed against PBS and their concentrations were determined by A280 measurements.

[0112] The cells were cultured under standard conditions (37 ° C, 5% CO ₂ , humidified incubator). The medium consisted of complete RPMI 1640 medium (Gibco) containing 10% (v / v) heat-inactivated fetal bovine serum (FBS, Sigma).

[0113] To collect experimental cells, cells in semi-confluent monolayer culture were collected using 0.025% ethylenediaminetetraacetic acid (EDTA, Sigma). Cells were washed with media, resuspended in media at a density of 1 × 10 ⁵ viable cells / mL and plated at 0.1 mL / well in 96-well culture plates. After overnight incubation, the medium was removed by aspiration and replaced with fresh medium containing 500 μg / mL of either a mixture of F400 MAbs or normal mouse immunoglobulin (NMIg). Cells were then incubated for an additional 48 hours, followed by assessment of cell proliferation by the tetrazolium-based MTT assay commonly used to assess cell proliferation in mammalian cell in vitro cultures. For each test group (n = 5), the absorbance of each well obtained in the MTT assay was averaged. Subsequently, the percent that F400 MAb inhibited cell growth compared to growth in the presence of NMIg was calculated.

[0114] The results of these tests are shown in Table 8. Table 8 shows that the anti-G17 MAb mixture inhibited the growth of each tumor cell line tested. Inhibition ranged from 19.5% for pancreatic tumor cell lines to 52.0% for gastric cell lines.

[0115] Thus, it was shown that the MAb of the present invention has anti-proliferative therapeutic activity in vitro against tumors derived from three common gastrointestinal malignancies.

[0117] (Example 8: Demonstration of anti-tumor efficacy of F400 Mab in vivo against gastric cancer cells)
[0118] The MAb pool for the N-terminus of G17 contains an equal volume mixture of ascites fluid containing each of MAb 400-1 to -4, but is a tumor cell line obtained from human gastric cancer, MGLVA1 Their ability to inhibit the growth of was tested. MGLVA1 cells are known to produce their own G17 hormone, which can lead to an autocrine effect, and neutralizing MAbs against G17 may suppress it.

[0119] To prepare F400 MAb ascites fluid mixtures for in vivo testing, the ascites fluid was depleted of complement in the ascites fluid by heating at 56 ° C for 30 minutes. The negative control ascites fluid purchased from Sigma was treated similarly.

[0120] MGLVA1 gastric tumor cells were grown as subcutaneous tumors in female nude mice. To transplant the tumor into a test mouse, the tumor was surgically excised from the tumor-bearing mouse and cut into small pieces of about 1 mm ³ . These fragments are then implanted subcutaneously into the flank of nude mice used in the study and tumors are allowed to engraft. Tumor sites were observed and tumor growth was measured with calipers. When tumor engraftment was observed, mice were randomly assigned to groups treated with test MAb (F400 mixture) or with negative control ascites. There were 12 mice / group in this study.

[0121] In the first week, 0.2 mL of ascites fluid (F400 mixture or negative control) was injected intraperitoneally into mice twice a week. After the first week, the injection volume was reduced to 0.1 mL twice a week. Tumor measurements were taken three times a week. This study lasted 27 days. At the end of the study, the mice were sacrificed, the tumors excised and weighed.

[0122] The average weight of MGLVA1 gastric cancer tumors in mice treated with the F400 test MAb mixture was 0.75 g. On the other hand, the average weight of the tumors of mice carrying MGLVA1 gastric cancer tumors and treated with the negative control ascites fluid was 1.5 g. Thus, the anti-G17 MAb of the F400 test mixture showed a strong growth inhibitory effect on gastric cancer cells and reduced tumor weight by 50%.

[0123] (Example 9: ELISA for measuring antibody titer against C-terminal of G17 and G34)
[0124] The purpose of this analysis method is to measure the titer of anti-hG17 antibody in the test serum by ELISA. Briefly, the anti-hG17 antibody ELISA of the present invention comprises hG17 (1-9) -AAPPC-BSA conjugate (amino acids 1-9 of human gastrin hormone peptide linked to BSA via the linker of SEQ ID NO: 16). ) Based on the specific binding of an antibody (either Ab, polyclonal or monoclonal) to the hG17 epitope presented by

[0125] In the first step, the conjugate was bound to the wells of a 96-well ELISA plate. Unbound conjugate was removed by a washing step using a 96 well plate washer. Thereafter, test (or control) antiserum was added. Anti-hG17 Ab present in the test serum bound to the conjugate due to the presence of hG17 peptide epitope present on the antigen surface. The antibody was then detected by the addition of a species specific anti-IgG alkaline phosphatase reagent to the anti-hG17 antibody to be detected. For example, a rabbit anti-hGH17 antibody is detected using a goat anti-rabbit IgG alkaline phosphatase conjugate (“GAR-AP”) as an Ab detection reagent. GAR-AP binds to rabbit anti-hG17 Ab. Subsequently, the AP moiety of the anti-Ig-AP conjugate catalyzes the conversion of the substrate to a colored product (p-nitrophenol). Color development was measured as absorbance at 405 nm with an ELISA plate reader.

[0126] Standard sera containing pooled anti-hG17 sera obtained from the same animal species as the test sample or ascites fluid containing anti-hGI7 MAbs with specified reference titers were used as positive controls. Sera obtained from the same animal species as the test sample, such as normal serum, pre-immune serum, etc. was used as a negative control.

[0127] The degree of color development in the linear region was directly proportional to the amount of anti-hG17 Ab bound to the target antigen. To generate a standard curve, a plot of absorbance against serial dilutions of positive standard (anti-hG17) serum was used. Subsequently, the anti-hG17 Ab titer of the test sample was determined from the dilution rate that produced the same absorbance as the reference titer of the positive standard (eg, 1: 200000 dilution of rabbit anti-hG17 positive standard).

[0128] Reagent solutions: The amounts of reagents and solutions specified to be prepared in this analytical method are for convenience only. The actual amount can be adjusted as needed.

1. Carbonate buffer containing 0.02% NaN ₃ (“Carbonate buffer”): 1.59 g Na ₂ CO ₃ and 2.93 g NaHCO ₃ are dissolved in about 750 ml distilled water with a magnetic stirrer. To make. Add 4 ml of 5% NaN ₃ solution and stir. Adjust to 1.0 liter by adding water. Measure the pH. The pH should be 9.6 ± 0.2 (adjust the pH with 1.0 M NaOH or 1.0 M HCl as necessary). Store in refrigerator until needed.

2. FTA (PBS) containing 0.05% Tween-20 and 0.02% NaN ₃ (“FTA / Tween”): 9.23 g of FTA is dissolved in about 750 ml of purified water. Add 0.5 ml Tween-20 and 4 ml 5% NaN ₃ . Add water to adjust to 1.000 liters.

  3. FTA / Tween containing 1% BSA (“BSA / FTA / Tween”): 10 g BSA is dissolved in 1000 ml FTA / Tween.

4). Substrate buffer: 50 mg MgCl ₂ .6H ₂ 0 is dissolved in 448 ml purified water. Add 50 ml DEA and 2 ml 5% NaN ₃ . Adjust the pH to 9.8 with concentrated hydrochloric acid. Store at room temperature protected from light.

  5. PBS, pH 7.2: can be prepared from solid FTA (FTA hemagglutination buffer ("FTA") (Becton Dickenson Microbiology Systems, Cockeysville, MD).

[0129] ELISA procedure: Antigen coating: solution of 1 μg / ml hG17 (1-9) -AAPPC-BSA conjugate in carbonate buffer (human gastrin linked to BSA via linker of SEQ ID NO: 16 Prepare amino acids 1-9) of the hormone peptide. A minimum of 5.2 ml of antigen solution is required for coating each plate. Prepare the antigen solution by making a 1: 1000 dilution of 1 mg / ml conjugate stock solution in carbonate buffer. Plates include, for example, Microtiter® immunoassay plates, rigid styrene (eg, Immulon® 2 round bottom 96 well plates, Dynatech Laboratories, Inc., VA; flat bottom 96 well plates, polystyrene: eg, Microwell Plates Any plate suitable for ELISA assay, such as Nunc, VWR). Coat Immulon® 2 round bottom plate with antigen by adding 50 μl / well of antigen solution. To prevent the loss of moisture, the plate is stored in a moist chamber (eg, a sealed container with a paper towel containing moisture) and incubated overnight in a refrigerator (2 ° -8 ° C.).

[0130] Serum dilution preparation: Positive standards and negative controls, and 1 / 100.5 serial dilution series of test sera were prepared as shown in Table 9. Serum was diluted in a BSA / FTA / Tween solution in a flat-bottom 96-well plate (a 12-channel multipipettor allows simultaneous dilution of up to 12 points of serum).

[0132] Each serum dilution was prepared in a volume sufficient to bring the minimum working volume to 200 μl. Depending on the serum titer, make a dilution in row A starting with 1/100 dilution (for low titer serum) or 1/1000 dilution (for high titer serum) of each serum, then serial dilutions Proceed down row to row H (see Table 9), producing a total of 8 dilutions of each sample. Serial dilutions of the negative control were prepared starting from 1/100. Serial dilutions of positive standard serum and pre-bleed / negative control serum were performed in duplicate on each plate.

[0133] Washing of plates: Using a plate washer (eg, Ultrawash Plus; or DynaWasher II (Dynatech Laboratories, Inc., VA) or equivalent), each of the coated plates was FTA / tween 4 times. And then “pressed” the plate onto a paper towel to remove residual solution.

[0134] Antibody Binding: As shown in Table 10 below, after performing serial dilution of the sample plate, 50 μl / well of diluted serum was transferred to the corresponding wells in antigen-coated round bottom plates. Plates were incubated for 1 hour at room temperature in a humid chamber.

[0136] Antibody detection reagent: Appropriate dilutions of anti-Ig-alkaline phosphatase conjugate were prepared with FTA / Tween. The assay required a minimum of 5.2 ml per plate. The plate was washed as described above. 50 μl / well GAR-AP solution (for testing anti-Ig-alkaline phosphatase conjugates, eg, rabbit anti-hG17 antibody, goat anti-rabbit IgG (H + L) -alkaline phosphatase (Antibodies Inc., Davis, Calif.)) Was added to all wells of the round bottom plate and incubated for 1 hour at room temperature in a humid chamber.

[0137] To detect anti-hG17 antibodies in sera obtained from species other than rabbits, an anti-Ig-AP conjugate specific for the species that produced the test serum must be used (eg, human anti-hG17). The antibody will be detected with the anti-human IgG-AP reagent used at the established dilution for each lot of reagent). Positive standard and negative control sera should be obtained from the same species as the test sera.

[0138] Substrate solution: p-NPP tablets (p-nitrophenyl phosphate, Phosphatase Substrate Tables, Sigma 104 ("p-NPP") (sold as Sigma Chemical Co., St. Louis, MO) from the freezer Removed and left to warm to room temperature. Immediately before use, a 1 mg / ml p-NPP solution was prepared by adding one p-NPP tablet to 5 ml DEA substrate buffer (room temperature). For one assay plate, each 5 ml volume of substrate solution was sufficient. The substrate solution was stored in the dark until use.

[0139] Substrate addition: Plates were washed as described above. To all wells starting in column 1, 50 μl / well of p-NPP solution was added simultaneously, starting with row A, using an 8 (or 12) channel multipipettor.

[0140] Reaction monitoring: Color development of the substrate solution was stopped when the absorbance of the positive standard dilution closest to the reference titer reached 10-30% of the maximum linear measurement of the ELISA plate reader. Monitor the absorbance of the positive standard to determine when the dilution corresponding to the reference titre reaches 10-30% of the reader's reading range (usually after a color development time of 10-30 minutes) An ELISA plate reader such as an MRX automatic plate reader; or MR580 MicroELISA automatic reader (Dynatech Laboratories, Inc., VA); or an equivalent thereof was used. The ELISA reader was set to measure at A405 nm for p-nitrophenol.

[0141] Stop reaction: 50 μl with 8 (or 12) channel pipettor when the absorbance of the dilution selected as above in the positive standard reaches 10-30% of the linear range of the reader The reaction was stopped by adding 1.0 M NaOH to each well. The NaOH solution was added to the wells in the same order and timing as the substrate solution was added. The reagents were gently mixed by carefully shaking the plate on the table.

[0142] Measurement of absorbance: The whole plate was measured with an ELISA reader.

[0143] Analysis of data: The titer of each serum was determined as follows. The absorbance obtained with the negative control serum was subtracted from the absorbance of the corresponding dilutions in the positive standard and test serum. (The average of each dilution of the positive standard and negative control was used.) For each serum containing the positive standard, the absorbance relative to (1 / dilution) on the abscissa (log scale) on the semi-log graph scale. Are plotted on the ordinate (linear scale). By plotting the reciprocal of the dilution, the titer could be read directly on the X axis. Occasionally, the absorbance was clearly deviated from the binding curve of a particular serum (abnormal value), but such a value was excluded from the curve. The titer of each serum is determined as the reciprocal of the dilution that produces the same absorbance as that produced by the reference titer of the positive standard (eg, 1: 200000 dilution of rabbit anti-hG17 positive standard). An example of data analysis is shown in FIG.

[0144] (Example 10: Measurement of antibody specificity by suppression ELISA)
[0145] Peptide inhibition ELISA was carried out in the same manner as in the above examples, except for the following points.

[0146] Preparation of inhibitory substance: Usually a suitable target hormone peptide, in this case hG17, is prepared as a working stock of 1 μmol / ml (1000 μM). Suppressive serial dilutions are prepared from working stock at dilutions from 1: 2 to 1:10, producing a total of 8 to 12 dilutions depending on the plate layout.

[0147] Preparation of sample dilution: To determine the dilution of the antibody sample at 50% maximum binding, a sample titration series is performed prior to inhibition analysis. Samples were then prepared at 2X with a 50% binding concentration for mixing with an equal volume of peptidic inhibitor and buffer as a control for inhibition analysis. The sample mixture was incubated for about 30 minutes in a humid chamber and then added to the coated and washed ELISA plate and incubated for about 1 hour in the humidity chamber.

[0148] Percent binding is a measure of absorbance by dividing the absorbance obtained from the sample in the presence of the inhibitor by the absorbance obtained from the sample control in the absence of the inhibitor and multiplying this value by 100. (Background value is subtracted). Finally, percent inhibition was determined by subtracting percent binding from 100%.

[0149] The test sample may be serum, MAb in cell culture supernatant, ascites fluid, or affinity purified antibody (Ab). For Ab against target antigens other than the amino terminus of G17, appropriate target hormone antigens and inhibitors are used. As a negative control, an irrelevant peptide should be included.

[0150] (ELISA: data analysis)
[0151] An example of data obtained by the above-described ELISA is shown in FIG. To obtain the net absorbance at each dilution, the average negative control serum absorbance was subtracted from the average positive standard and test serum values. Net absorbance values were plotted against titer (in this example, negative controls were also plotted to reveal typical values).

[0152] (Stability of gastrin 17)
[0153] The stability of gastrin at room temperature (about 22 ° C) was evaluated by the total gastrin assay described above. This was done by measuring total G17 immediately after preparing the sample to a known G17 concentration of 15, 100, and 600 pM and after standing on a desk at room temperature for 2 hours. The results are shown in Table 11 below. They demonstrate that the G17 concentration of each sample is substantially reduced.

[0155] (Example 11: Antiserum suppression radioimmunoassay (RIA) for HG17-Serum titer measurement and antigen suppression RIA for measuring antigen-binding ability (ABC) of anti-human gastrin 17 (hG17) antiserum)
[0156] Dilution buffer:
1. Phosphate buffered saline, pH 7.2 (PBS) + 0.02% sodium azide (NaN ₃ ). “FTA hemagglutination buffer”, a commercial preparation of soluble solids, can be dissolved in distilled water to produce PBS (9.23 g / l yields a solution of pH 7.2 ± 0.1) .
FTA containing 2.1% bovine serum albumin (BSA) and 0.02% NaN ₃ .
3. Supplemental calf serum (SCS; GIBCO), frozen and stored in an amount of 50 ml or less.
4). Prepared in PEG, MW 8000, 25% solution (250 g per liter, slowly dissolved). Store at 4 ° C.
5. Human gastrin 17 (15-Leu) (Research Plus, # 07-027-002); for single use, 5-10 μg / ml in FTA / 1% BSA / azide. Store at -70 ° C.
6). Human gastrin ^17- 125 I (NEN Co.).

[0157] (Method)
[0158] To establish the volume of each antiserum to be tested with inhibitory RIA, titrations were first performed against a set of hG17-125I with different amounts of test serum. Serum was then tested by inhibitory RIA and antigen binding capacity (ABC) was calculated by Scatchard analysis.

[0159] Titration RIA protocol:
1. For positive control antisera and all test antisera, duplicate tubes were prepared for each antiserum dilution to be assayed. Preferably, five 10-fold dilutions of serum were performed so that the final dilution in the assay tube was 1:40 to 1: 400000. This is equivalent to the addition of antisera ranging from 10 μl to 0.001 μl per tube.
2. 300 μl of dilution buffer was dispensed into two tubes. These functioned as reagent blanks. 200 μl of dilution buffer was added to all remaining tubes.
3. 100 μl of diluted antiserum was transferred to each of duplicate serum tubes (2 × 100 μl required for each dilution). For serial dilution, it was sufficient to start with 30 μl of antiserum (producing 300 μl of 1:10 dilution), even taking into account the loss during transfer.
4). As a control for non-specific binding, at least one dilution (1:40, lowest dilution of test serum) negative serum was included.
5. ¹²⁵ I-labeled antigen (Ag) diluted to approximately 10,000 cpm / 0.1 ml with RIA buffer. See dilution procedure below.
6). 100 μl of labeled gastrin was added to all tubes. To confirm the total count added, 100 μl of labeled hG17 was added to 10 scintillation vials or gamma counter tubes.
7). The contents of the tube were mixed by shaking or vortexing and covered with parafilm.
8). Tubes were incubated overnight at 4 ° C. for approximately 18 hours. This is the minimum incubation time and longer incubations can be used for titration RIA, but this is usually not necessary.
9. 100 μl of SCS was added to all tubes and the tubes were shaken.
10. 500 μl of 25% PEG (4 ° C. or RT) was added to all tubes and mixed by vortexing.
11. The tube was centrifuged at 2000 × g and 4-12 ° C. for 30 minutes.
12 The supernatant was aspirated from all tubes and discarded.
13. The precipitate in the assay tube was measured with a γ-ray counter, or prepared for scintillation counting as described below.

[0160] (Calculation)
[0161] The counts per minute (cpm) of the duplicate samples were averaged. Non-specific background binding subtraction was not performed. Data was plotted as% hG17-125I binding against the volume of serum added. The amount of each serum bound to 35% of the total cpm added per sample was selected for inhibitory RIA.

[0162] (Inhibition RIA)
1. For each inhibition series, one dilution of antiserum determined by titration RIA was used. The number of duplex tubes to be prepared is determined by the number of dilutions of inhibitor to be tested, including uninhibited controls. Normally, 8 anti-serum dilutions (16 tubes) and 2 non-suppressed tubes were used per antiserum.
2. As a zero counting blank, 300 μl of dilution buffer was dispensed into two tubes (to determine the natural background count).
3. As receiving the negative control serum (to obtain a non-specific binding background), 200 μl of buffer was dispensed into 6 tubes, 2 of which were tested at the end of the assay. 200 μl of buffer solution was dispensed into two tubes for each antiserum (for counting the total number of bound counts). No hG17 inhibitor was added to these tubes.
4). 100 μl of dilution buffer was added to all remaining tubes.
5. 100 μl of unlabeled hG17 (suppressor) diluted to obtain the appropriate final concentration (see below) was dispensed into duplicate tubes for each of the test and control antisera. These series established hG17 inhibition curves for each antiserum. The hG17 inhibitor was prepared by 1: 1 serial dilution in FTA / azide starting at 5120 pg / 0.1 ml.
8). The contents of the tube were mixed.
9. ¹²⁵ I-labeled antigen in RIA buffer was diluted to approximately 10,000 cpm / 0.1 ml.
10. To confirm the added total count, it was added all tubes in hG17- ¹²⁵ I 100μl containing 12 or more tubes that are located throughout the assay.
11. Finally, 100 μl of appropriately diluted anti-hG17 control serum, negative control serum, or test serum was added to each tube in the appropriate tube set.
12 The contents of the tube were mixed and covered (eg, using parafilm).

13. Tubes were incubated at 4 ° C. for approximately 42 hours (2 days).
14 100 μl of SCS was added to all tubes and mixed.
15. All tubes were mixed with 500 μl of 25% PEG.
16. The tube was centrifuged at 2000 × g for 30 minutes at 4 ° C.
17. The supernatant was aspirated from all tubes and discarded.
18. The precipitate in the assay tube was measured with a γ-ray counter or prepared for scintillation counting. For scintillation counting, 250 μl of dH ₂ O was added to all assay tubes. By heating the water to 90-100 ° C., the dissolution of the pellets is accelerated. It took 2-3 hours to dissolve the pellets. Thereafter, 3 ml of scintillation fluid was added to each of the scintillation vials. All of the dissolved pellets were transferred from one tube to one scintillation vial and placed in a rack for measurement.

[0164] (Calculation)
1. Duplicate samples cpm were averaged to subtract non-specific background binding (determined by the average of negative serum controls).
2. To confirm that the total count bound to the uninhibited anti-HG17 antibody was within the expected range, the total count was added and a baseline background control was used.
3. For each amount of inhibitory substance, the total counts added and the counts bound were used to calculate the ABC and affinity constants of the antisera, a Scatchard analysis (binding / release plots for bound antigen). Determined by. For each antiserum, the point that gave the best linear regression line was selected and the rest removed. This was done by looking at the plot and noting the regression coefficient. In general, the low value part of the plot was not used. ABC and affinity constants were automatically calculated using the spread set for this purpose.

[0165] (dilution ^{HG17- 125} I)
^[0166] hG17- ¹²⁵ I was purchased from NEN company. This radiolabeled hormone (15 μCi) had a specific activity of 2200 μCi / mmole at the time of shipment. The lyophilizate was diluted to 50 μCi / ml with dH ₂ O based on the days of disintegration according to the attached product instructions. After dissolution, a 50 μl aliquot was prepared and stored in a lead container at −70 ° C.

[0167] (dilution to 10000 CPM)
[0168] Approximately 10,000 cpm of labeled compound was placed in each assay tube (0.1 ml) (usually 10000-10400 cpm / tube). In determining the volume of dilution hG17- 125 ^I in need, a total count measurement, in consideration of the impairment due to the movement and foaming, giving an extra 3-4 ml.

[0169] Note: The test sample tested in this assay may be serum, MAb in cell culture supernatant, ascites fluid, or affinity purified Ab. Appropriate ¹²⁵ I-labeled target hormone antigens and inhibitors are used for antibodies to target antigens other than the amino terminus of G17. Unrelated peptides should be tested as negative controls.

[0170] (Example 12: Detection of CCK2 receptor on paraffin-embedded tissue using rabbit α-GRE11 antibody)
[0171] The tissue sections were deparaffinized by immersion in 3 separate xylene baths (5-6 up and down for each bath). It was then rehydrated by incubation in 100% industrial methylethanol (IMS) (5-6 up and down for each bath). The slide was rinsed in distilled water for 5 minutes. Endogenous alkaline phosphatase activity was blocked by incubating the slides in 15% acetic acid for 20 minutes. The slide was then rinsed in distilled water for 5 minutes. Slides placed in plastic slide racks 2 spaces apart and placed in pH 6 citrate buffer (2.1 g citrate monohydrate, 12.5 mL 2M NaOH per liter) for maximum output In a microwave oven (600 W) for 10 minutes. Care was taken to ensure that there was a sufficient amount of buffer to cover the slide during the entire process. The slide was then immediately transferred into a stream of cold distilled water and allowed to sit for 3-4 minutes, being careful not to let the slide dry.

[0172] The section was marked with a hydrophobic pen, placed in a wet chamber, pH 7.6 Tris-buffered saline (TBS) (0.66 g Tris- (hydroxymethyl) methylamine, 8.75 g NaCl, Soaking in approximately 4.15 mL HCL) at room temperature (“RT”) for 5 minutes. Nonspecific binding of the secondary antibody ("Ab") was blocked by incubating the slides with 10% normal goat serum in TBS for 20 minutes at RT. Liquid was removed from the slides, and primary antibody (200 μl / slide) was added to each slide and placed in a humid chamber at RT for 1 hour. The slides are washed by first gently rinsing with TBS (in a wash bottle; be careful not to direct water flow directly on the tissue section) and then immersed in buffer for 5 minutes. It was.

[0173] Alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (or an appropriate antibody targeted to the source of the test antibody) was added to the slide at 1/50 dilution in 200 μl / slide TBS. The slides were then incubated for 1 hour at RT and then washed in TBS for 5 minutes.

[0174] Fast Red substrate (Vector Red, Vector Labs / Fast Red, Sigma) was prepared just prior to use and added to each section for a maximum time of 20 minutes (Vector) or 30 minutes (Sigma). Incubation). Slides were rinsed with TBS and then distilled water, followed by counterstaining with Mayer's hematoxylin (time varying). After staining, slides were transferred into distilled water.

[0175] The slides were then immersed in 1% acid alcohol (10 mL concentrated hydrochloric acid, 700 mL IMS, 290 mL distilled water per liter) to remove excess counterstaining. However, when Fast Red substrate (Sigma) was used, it was transferred to distilled water instead. Slides were moved up and down several times in 0.5% sodium tetraborate solution (diluted with water). At this point, it is advisable to examine one of the sections under a microscope to see if the nucleus is blue rather than purple. The stained slides were then transferred to distilled water, followed by IMS and finally to xylene, and then embedded with DPX (xylene embedding agent).

[0176] Appropriate dilutions of test and control sera / purified antibodies were determined using serial dilutions. GI sections are best stained with an alkaline phosphatase reagent system. ABC is more specific but leads to a lot of non-specific staining due to its high sensitivity. Intestinal alkaline phosphatase can be blocked with levamisole (Vector labs), which is added to the substrate solution when the sections are colored. Vector red substrates are less problematic than Sigma substrate products. However, one drop of levamisole needs to be added to the prepared substrate solution.

[0177] The primary antibody (Ab) used in this immunohistochemical method may be serum, MAb, Ab in cell culture supernatant, ascites fluid, or affinity-purified Ab.

[0178] The procedures described herein above are applicable to the isolation of MAbs optimal for immunodetection and immunoenzyme assays of other peptides, particularly other hormone peptides including other gastrin hormone types. Will be readily recognized by those skilled in the art. The present invention contemplates the full range of MAbs taught and exemplified by the non-limiting examples described herein. All patents and publications cited herein are hereby incorporated by reference in their entirety.

[0179] (Deposit of hybridoma cell line)
[0180] The following hybridomas producing specific MAbs of the present invention were deposited with the ATCC (American Type Culture Collection, Manassas, VA) on March 25, 2004.
1. Accession number PTA-5889 was assigned to hybridoma 400-1 producing MAb 400-1.
2. Accession number PTA-5890 was assigned to hybridoma 400-2 producing MAb 400-2.
3. Accession number PTA-5891 was assigned to hybridoma 400-3 producing MAb 400-3.
4). Accession number PTA-5892 was assigned to hybridoma 400-4 producing MAb 400-4.
5. Accession number PTA-5893 was assigned to hybridoma 401-2 producing MAb 401-2.
6). Accession number PTA-5894 was assigned to hybridoma 445-1 producing MAb 445-1.
7). Accession number PTA-5895 was assigned to hybridoma 445-2 that produces MAb 445-2.
8). Accession number PTA-5896 was assigned to hybridoma 458-1 that produces MAb 458-1.

FIG. 6 shows an ELISA using a plate coated with hG17-BSA. It is a plot which shows the light absorbency (A405) of 405 nm with respect to the titer of serum. Squares represent test samples, diamonds represent pre-bleed samples, and triangles represent reference standards. The absorbance (2) obtained with a titer (1) of 2 × 10 ⁵ in the positive standard is determined. The point where the test sample curve intersects this absorbance indicates the titer (3) of the test sample. In this example, the test sample has a titer of 2.8 × 10 ⁴ . FIG. 3 is a diagram showing a typical calibration curve of total gastrin 17; Gastrin concentration is plotted in picomoles against absorbance at 450 nm (A450) after enzymatic color development using tetramethylbenzidine sulfonate (TMBS) chromogen. 2 is a diagram showing a typical calibration curve of free gastrin 17. FIG. Gastrin concentration is plotted in picomoles against absorbance at 450 nm (A450) after enzymatic color development using tetramethylbenzidine sulfonate (TMBS) chromogen.

Claims (2)

Hybridoma 400-1 (ATCC accession number PTA-5589), hybridoma 400-2 (ATCC accession number PTA-5890), hybridoma 400-3 (ATCC accession number PTA-5589) and hybridoma 400-4 (ATCC accession) and a number PTA-5892) carriers monoclonal antibody and a pharmaceutically acceptable produced by, pancreatic cancer pharmaceutical composition for the treatment. A pharmaceutical composition for treating pancreatic cancer comprising a humanized monoclonal antibody and a pharmaceutically acceptable carrier,
The humanized monoclonal antibodies include hybridoma 400-1 (ATCC accession number PTA-5890), hybridoma 400-2 (ATCC accession number PTA-5890), and hybridoma 400-3 (ATCC accession number PTA-5891). And a humanized antibody produced by hybridoma 400-4 (ATCC Accession No. PTA-5892),
A pharmaceutical composition for treating pancreatic cancer.

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