KR20130075722A - Immunogenic compositions against human progastrin peptides - Google Patents

Abstract

Immunogens for human expansion-progastrin species are: (B) N-terminal and / or C-terminal processing species of progastrin or progastrin, coupled to an immunogenic carrier, ii) bound to a seven amino acid spacer. (A) mimetic peptide consisting of the amino acid sequence of. Exemplary mimetic peptide / spacer combinations are 21 amino acid peptides (SEQ ID NO: 1) and other, associated polypeptides (SEQ ID NOs: 2-5). Pharmaceutical compositions containing such immunogens exhibit improved immunological properties, including inducing effective antibody levels immediately after administration of the initial course of immunogen. The level of antibody so induced remains elevated for several months and can easily be raised to higher levels upon subsequent amplification by a single injection of the immunogen.

Description

Immunogenic compositions against human progastrin peptides

Cross-Reference to the Related Patent Application

This application claims priority to Chinese Patent Application No. 2010/10116229, filed March 3, 2010, the entire contents of which are incorporated herein by reference.

Gastrointestinal (GI) cancer is the most common cancer in humans worldwide and is the leading cause of death in most countries. Many growth factors are known to cause the proliferation of GI malignancies, among them in particular, a family of peptide hormones generated from the processing of preprogastrin. Maturation of preprogastrin in normal tissues involves multistep processing of intermediate progastrin, leading to the predominant amidated form of gastrin found in the circulation; These are gastrin-34 (G34) and gastrin-17 (G17), also known as "large" gastrins and "small" gastrins, respectively. These hormones regulate mucosal cell growth and secretion of gastric acid (post-meal), especially after food intake. Progastrins and gastrins, specifically the glycine-extended forms of G17 and G17 (gly-G17), have been reported to play a role in nourishing GI malignancies, both in vitro and in vivo. It supports the case for the targeted inhibition of these gastrins for therapeutic purposes.

Immunization against specific cancer-promoting growth factors and hormones is known to be useful for treating and preventing certain cancers, particularly breast cancer, lung cancer, and certain types of GI cancer. In addition, immunological approaches to treating and preventing diseases of ulceration in the gastro-esophageal and gastro-duodenum may also be effective in treating these chronic conditions.

Several therapeutic approaches have been successfully applied, in particular using targeted human or humanized monoclonal antibodies (huMAb). However, given the cost and difficulty of setting up the production and delivery of commercial huMAbs, a less expensive alternative strategy is needed, especially in developing countries.

For GI cancers and diseases, these immunological approaches involve the production of specific antibodies that neutralize the biological activity of disease promoting gastrointestinal peptide hormones. The required antibody must be specific for a particular growth factor or hormone, or hormone precursor. One or more factors or hormones may be selectively targeted to treat a particular disease.

For example, the human gastrointestinal hormone gastrin 17 (“huG17”) is involved in gastrointestinal disease processes, including gastro-esophageal reflux disease, because of its ability to stimulate acid and thus cause gastric and duodenal ulcers. In addition, huG17 has been shown to stimulate the growth of some GI cancers.

Specific anti-huG17 antibodies can neutralize the action of huG17 and thus have been used in clinical trials to treat diseases in which huG17 is involved. The anti-huG17 antibody may be administered to the patient, for example by passive immunization, or may be induced in the patient by active immunity.

Similarly, although amidated gastrins were considered to be the only biologically active form of gastrins in the circulation, the progastrin form of preprogastrin is a proliferative potential based on studies using human GI cancer patients and induced human cancer cell lines. There is currently considerable evidence of having sex. In fact, GI cancer cells are generally not effective at processing gastrin precursors such as preprogastrin and progastrin into their normal amidated form; Thus, serum levels in cancer patients show higher levels of precursor forms of these gastrins than normal amidated forms. It has been reported that plasma levels of progastrin but not amidated gastrin or glycine expanded gastrin are significantly increased in patients with colorectal polyps compared to patients with colorectal polyps (Siddheshwar et. al . , Gut 48: 47-52, 2001). Progastrin, amidated gastrin, total gastrin, and glycine-extended gastrin were also detected in 100%, 69%, 56%, and 44% of colorectal cancer (CRC) patient tumors, respectively (Ciccotosto et. al . , Gastroenterology 109: 1142-53, 1995), suggesting that the cancer is actually defective in fully processing gastrin.

As detailed below, in order to solve the shortcomings of the prior art, the present invention is in accordance with one of its aspects: A polypeptide immunogen comprising a (A) mimetic peptide whose C-terminus consists of the amino acid sequence of a species of processing progastrin and (B) an immunogenic carrier coupled to said mimetic peptide. According to one embodiment of the invention, the polypeptide immunogen described above is Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu- Mimetic peptides having the amino acid sequence of Asp-Glu-Asn (SEQ ID NO: 1). According to another embodiment, the mimetic peptide has an amino acid sequence selected from the group consisting of:

pGlu-Gly-Pro-Trp-β-isoVal-Glu-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2);

pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3);

pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4);

Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5); And

Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6).

According to the present invention, the immunogenic carrier can be chosen in particular from tetanus toxoid, diphtheria toxoid, pertussin toxoid, and tuberculin pure protein derivatives.

According to another aspect of the present invention, an immunogenic composition is provided comprising an effective amount of a polypeptide immunogen and a pharmaceutically acceptable vehicle for the immunogen. In a preferred embodiment, the pharmaceutically acceptable carrier comprises an aqueous phase in which an immunogen is present, and an emulsion of an oil phase. The oil phase comprises at least one biodegradable oil, such as squalene, squalane, sorbitan monooleate, polysorbate 40, and / or polysorbate 80. The oil phase may also include a separate emulsifier. In addition, either the oil phase or the aqueous phase may contain one or more adjuvants.

According to another aspect, the present invention provides a seven amino acid spacer peptide that is particularly suitable for linking with mimetic peptides, as described above. Exemplary such spacer peptides are in amino acid sequences selected from the group consisting of: Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7); Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8); Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9); And Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10).

Active immunity against growth factors or gastrointestinal peptide hormones is achieved by administering to the patient an immunogen containing a chemical structure that induces an antibody that binds to a targeted factor or hormone. These chemical structures are constructed as immunological peptide mimetics of a targeted factor or hormone, and they may be composed of any molecule that immunologically cross-reacts with the target or epitope of that target. These immunogens are immunological peptide mimetics (“immunomimics”) that may have intrinsic ability to induce antibodies, for example they may be immunogenic. However, immunomimetics are often intrinsically not immunogenic and therefore must be combined with a carrier having strong immunogenic properties (“immunogenic carrier”), thereby becoming complex immunogenic.

According to the present invention, any chemical structure is for example in Watson et al. al . , Expert Opin. Biol . 1: 309-17 (2001), and Watson meat al ., Cancer Res . 56: 880-85 (1996), which may act as an immunologically cross-reactive immuno mimetic with an epitope of huG17. In a preferred embodiment of the invention, the peptide mimetic is a portion of huG17 containing therein the amino terminal epitope of huG17, which is designed to stimulate human small gastrins as well as cross-reactive antibodies that will bind to glycine-extended gastrins. The spacer element of the immunogen acts as a binding point through which the immunomimetic (peptide mimetic + spacer) is attached to the carrier. The spacer may also affect the immune response to the epitope portion of the immunogen.

Immunization using the N-terminal nonapeptide portion of huG17 coupled to the diphtheria toxoid via a spacer has been reported to inhibit both experimental ulcers in rats and GI human cancer xenografts in immunocompromised mice. Many clinical trials using this immunogen, known as "G17DT" in patients with GI cancer, have shown significant results (see Watson et al. (2001)), but recently in Phase 3 clinical trials using this immunogen in the United States. The trial did not yield statistically significant results in the treatment of late pancreatic cancer.

Although some success has been shown in early clinical trials, the G17DT approach, particularly in GI malignant disease, has not considered the visible heterogeneity of gastrin gene expression. In normal gastric cells and tissues, the most abundant secreted / circulated gastrin species are G17 and G34. Numerous scientific reports, however, show that these latter species actually make up less than 50%, and in certain cases, make up as little as 10% -20% of the gastrin form in the circulation.

Many species are for example [Rehfeldmeat get .,Regulatory Peptides  120: 177-83 (2004), a processing intermediate known as “progastrin”. Progastrin has been shown to have substantial growth promoting activity independent of traditional gastrin receptors. Thus, the antibodies made by the G17DT immunogen, mentioned above, will only capture the G17 and C-terminal extended forms, and of the unprocessed / partially processed form of gastrin (progastrin), abundant in the circulatory system of GI cancer patients. The chief circulating species will not occupy.

 In particular in the prior art, it is a disadvantage that the novel immunogen compositions of the present invention solve by targeting the gastrin and progastrin forms found in the circulation of cancer patients. In this regard, it has been found that certain peptide mimetics of huG17, when coupled with suitable spacer peptides, produce immunogens that result in unexpectedly improved immune responses when compared with conventional immunogens.

According to the present invention, we find that even though it is often possible to elicit cross-reactive antibodies to a much greater level than peptides containing native natural amino acids, the modified amino acids possess the "foreign" inherent properties of adjacent peptides. It has also been found that it can be used to enhance. More specifically, the processing by macrophages and monocytes of invading microorganisms and "foreign" antigens is known to result in halogenation reactions and oxidation and nitrosylation reactions. The resulting chemical modifications are therefore understood to increase the antigenicity and subsequent immunogenicity of proteins and peptides. Thus, the present invention includes the inclusion of one or more halogenated or nitrosylated amino acid residues, such as para-chloro or para-nitro-phenylalanine residues, to increase the immunogenicity of the peptide immunogen. This is why, for example, para-NO ₂ -phenylalanine (nPhe) is present in certain inventive immunogens (SEQ ID NOs: 1 and 6) described in detail below.

Thus, according to one embodiment of the present invention, the improved immunogen produces polyclonal antibodies against N-terminal and / or C-terminal processing species of progastrin and progastrin, respectively. Examples of such immunogens are (ii) Cys-pro-Pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser- coupled to an immunogenic carrier. It includes the peptide of the amino acid residue of Ala-Glu-Asp-Glu-Asn (SEQ ID NO: 1). (See below for the regulations applied to the sequence listings listed in this description.) Thus, this embodiment comprises 21 amino acids including seven amino acid spacers added to the progastrin component (residues 88-101 of preprogastrin). It constructs an immuno mimetic that is an amino acid (G21) peptide.

In this context, an immunogenic carrier is a protein or large (ie generally 6000) of sufficient molecular complexity that can elicit an immune response to any suitable, high molecular weight carrier, typically a heptene or peptide sequence covalently bound to the carrier. molecules larger than kD). Suitable types of immunogenic carriers include, but are not limited to, diphtheria toxoid (DT), tetanus toxoid (TT), pertuscin toxoid, and tuberculin pure protein derivative (PPD). Of these, tetanus toxoid is a preferred immunogenic carrier. This class includes [Fifis et al . , J. Immunol . 173: 3148-54 (2004) also include particulate carriers such as nano-beads, and commercially available dendrimers such as PAMAM dendrimers and MAP dendrimers. Aguilar et al . , J. Pept . Sci . 15: 78-88 (2009).

In this context, the phrase “pharmaceutically acceptable vehicle” refers to a medically safe, non-toxic material that will carry an immunogen without reducing its immunogenic effect. Suitable vehicles may be liquid emulsions, or further suitable particulate materials, such as pharmaceutically safe lyophilized powders or pharmaceutically acceptable silica gels or synthetic, non-infectious virus like particles (VLPs), as further described below. Can be. Fields Virology, Vol. 1, D.M. Knipe & P. Howley (eds.), Lippincott Williams & Wilkins (2007).

In the present invention, preferred forms of pharmaceutically acceptable vehicles are emulsions of an aqueous phase containing a polypeptide immunogen, and an oil phase. The oil phase includes at least one biodegradable oil, which is not mixed with the aqueous phase, which is non-toxic in the dosage range of the intended administration. Oils can be natural or synthetic, and there are a variety of oils available that are generally recognized to meet international regulatory standards for therapeutic use. Examples of such suitable oils are squalene, squalane, sorbitan monooleate, polysorbate 40, and polysorbate 80. Preferred oil phases include all five of these oils.

In addition, the oil phase may contain a separate emulsifier such as aluminum monostearate or an adjuvant-active saccharide oleate or saccharide stearate ester.

According to another aspect of the invention, one of the oil or aqueous phases of the emulsion as described above contains at least one adjuvant which is distinguished from the immunogenic carrier component of the polypeptide immunogen. There are a wide variety of known auxiliaries, any one or more of which may be considered for use in the present invention. Examples of such known auxiliaries are Nor-MDP, imiquimod, cyclic diguanylate, threonyl-N-acetyl-muramil-L-alanyl-D-isoglutamine, isoprinosine, trehalose dimicholate, QS -21, alpha-galactosyl ceramide (α-GalCer), and alpha glucosyl ceramide (α-GluCer). In addition, for this adjuvant role, the present invention typically includes the use of substances that are nevertheless immunostimulatory, if not regarded as the adjuvant itself. Examples of such materials are ergamisol, cimetidine, praziquantel, uric acid, mannan and derivatives of mannan, and vitamin E.

According to a further aspect of the invention, the improved immunogen produces polyclonal antibodies against the amino terminal epitopes of huG17 and gly-huG17. Examples of these immunogens are, as described above, (ii) pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser coupled to an immunogenic carrier It includes the peptide of the sequence of -Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2). As in other parts of the present disclosure, “isoVal” refers to a beta amino acid mimetic of Leu, which may generally be used in place of Leu, and “pGlu” is an amino acid found at the bioactive N-terminus of the huG17 hormone. Pyroglutamate, which is a derivative. Tetanus toxoids are also preferred immunogenic carriers in this embodiment, wherein a seven amino acid spacer is added to the N-terminal huG17 (residues 76-86 of preprogastrin) to form an immuno mimetic that is a G-18 peptide.

A further embodiment of the invention is as described above: (ii) pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Thr- coupled to an immunogenic carrier An immuno mimetic comprising a peptide of the sequence of Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3). Tetanus toxoid is also a preferred immunogenic carrier in an embodiment of the invention, which is a modification of the N-terminal huG17 (modified residues 76-86 of preprogastrin) with the addition of a seven amino acid spacer to the immuno mimetics of huG17. Constitutes the G-18 LV peptide. As an alternative, according to this embodiment, the sequence pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro The peptide of -Cys is used as peptide (i) in constructing the homologous immuno mimetics of huG17.

Another embodiment of the invention is as described above: (ii) Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu coupled with an immunogenic carrier An immuno mimetic comprising a peptide of the sequence Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5). Thus, this embodiment is the glycine-expanded immuno mimetic of huG17, which adds seven amino acid spacers to residues 79-93 of preprogastrin to constitute a G-20 peptide.

Embodiments of the invention also provide for (i) Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu- coupled with an immunogenic carrier, as described above. An immuno mimetic comprising a peptide of the sequence Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6). Thus, this embodiment is an altered glycine-extended immuno mimetic of huG17, which adds seven amino acid spacers to residues 79-93 of preprogastrin to constitute a G-20 YF peptide.

The remainder of the peptide sequences listed below (SEQ ID NOS: 7-10) are spacers containing one or more D-isomer prolyl amino acids, a feature that we have found to be important for presenting adjacent immunogenic peptides on a carrier at a suitable location. In this context, D-amino acid isomers not only enable the proper morphology but also enhance the persistence of the immunogen for APC presentation, resulting in higher titers of antibodies.

The peptide identified as SEQ ID NO: 7 in the list below is a spacer peptide Ser-Ser-Pro-Pro-Pro-pro-Cys. In addition, SEQ ID NO: 8 is a spacer peptide Ser-Ser-pro-pro-pro-pro-Cys, SEQ ID NO: 9 is a spacer peptide Thr-Thr-Pro-Pro-Pro-pro-Cys, and SEQ ID NO: 10 is a spacer Peptide Thr-Thr-pro-pro-pro-pro-Cys.

In the prior art, the induction of an effective antibody response by immunization with an immunomimetic-carrier complex typically requires the administration of two or more immunogens and takes weeks or months to raise antibody titers to the desired level. . In contrast, the improved immunogens of the present invention induce effective antibody levels immediately after administration of the initial course of immunogen. The level of antibody so induced remains elevated for several months and is easily elevated to higher levels upon subsequent amplification by a single injection of immunogen.

1 shows mice measured by ELISA for three immunizations with immunogens comprising respective conjugates hG18-TT, hG18-DT, hG21-TT and hG21-DT (a, b, c and d, respectively) The antibody reaction in is shown.
Figure 2 shows the antibody response in rabbits as measured by ELISA with a single administration of the conjugates constructed using peptide 1 (hG18-TT) and peptide 2 (hG21-TT) of Examples 1 and 2, respectively, below. To follow.
3 shows inhibition of human gastric cancer cells (BCG-823 cells) in mice processed with immunogens G18-TT and G21-TT. On day 42 animals were transplanted using sterile, sutured, hollow fiber tubules containing human gastric cancer cells. As shown, some animals were administered with the immunogen plus conventional gastric cancer chemotherapy drugs.
4 shows the results obtained by mice immunized with the different immunogen conjugates of the present invention compared to the results obtained with the G21 immunogen.

The invention is further described with reference to the following examples, which are illustrative and do not limit the invention.

Example  One

Peptides were prepared by standard solid state synthesis methods. Each peptide was characterized for amino acid content and purity.

Thus, peptides having the amino acid sequences listed below were synthesized. As in other parts of the present disclosure, in these sequences, amino acids starting with an uppercase letter are L-isomers, while amino acids starting with a lowercase letter are D-isomers.

(1) Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn (SEQ ID NO: 1), Represented by "G-21"

(2) pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2), "G-18" Represented by

(3) pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3), "G-18 LV Represented as "(human G17 homologue)

(4) pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4), represented by "G18 LI"

(5) Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5), "G Represented by -20 "

(6) Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6), "G Represented by -20 "

(7) Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7), a 7 amino acid peptide seryl-prolyl spacer

(8) Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8), a seryl-all D-proyl spacer, a seven amino acid peptide

(9) Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9), a 7 amino acid peptide, threonyl-proylyl spacer

(10) Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10), a 7 amino acid peptide threonyl-all D-proyl spacer

Peptide 1 (SEQ ID NO: 1) is preceded by a carboxy terminal spacer-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7) attached to 88-101 on the preprogastrin residue N-terminal side, Para-nitrophenylalanine-modified amino terminal immuno mimetics of progastrin (-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn). Peptide 2 (SEQ ID NO: 2) is an 11-amino acid mimetic of huG17 (pGlu-Gly-Pro-Trp-Leu-isoVal-Glu-Glu-Glu-Glu-), where Leu is a homologue of huG17 substituted with isoVal Ala-), followed by the spacer -Ser-Ser-Pro-Pro-Pro-D-Pro-Cys (SEQ ID NO: 7) attached to amino acid 86 of the preprogastrin residue of the huG17 immuno mimetibody. Peptide 3 (SEQ ID NO: 3) is an 11 amino acid immuno mimetic homologue identical to peptide 2 except Val replaces isoVal, -Ser attached to amino acid 86 of the preprogastrin residue as described above -Ser-pro-pro-pro-pro-Cys; (SEQ ID NO: 8). Peptide 4 (SEQ ID NO: 4) is pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu, an 11 amino acid immuno mimetic of huG17 similar to peptide 2, except that isoVal is substituted with Ile -Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly followed by a spacer attached to 79 of the preprogastrin C-terminal residue of the immuno mimetic of huG17 -Ser-Ser-Pro-Pro-Pro-pro Include -Cys (SEQ ID NO: 7). Peptide 5 (SEQ ID NO: 5) comprises a spacer -Ser-Ser-Pro-Pro-Pro-pro-Cys; attached to the preprogastrin N-terminus of residue 79 of the glycine-extended immuno mimetic of huG17; Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly, a 15 amino acid immuno mimetic of gly-huG17, preceded by 7). Peptide 6 (SEQ ID NO: 6) is a spacer-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7 Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe, a para-nitrophenylalanine-modified 15 amino acid immuno mimetic of gly-huG17, preceded by Contains Gly. Peptide 9 (SEQ ID NO: 1) is preceded by a carboxy terminal spacer -Ser-Ser-pro-pro-pro-pro-pro-Cys, (SEQ ID NO: 8) attached to a preprogastrin N-terminal residue 88-101 peptide, It contains the amino terminal immuno mimetics of progastrin, -Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn, and forms the human peptide mimetics of progastrin. .

According to a preferred aspect of the invention, each of these peptides is conjugated to an amino group present on the tetanus toxoid (TT) immunogenic carrier. Binding was done via terminal peptide cysteine residues and heterobifunctional binders containing succinimidyl esters at one end and maleimide at the other end of the binder were used. To achieve binding between one of the peptides 1 and 2 and the carrier, the cysteine of the peptide was first reduced. The dry peptide was dissolved in 1.0 M sodium phosphate buffer with more than 5-50 moles of dithiothreitol, with a pH of 7-9. Peptides were lyophilized and stored under vacuum until use.

 TT was activated by treatment with heterobifunctional binder epsilon-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS) in a ratio sufficient to reach activation of approximately 25 free amino groups per 105 molecular weight of TT.

Preparation of Purified Tetanus Toxoid: TT was purified by ultrafiltration. The final concentration of recovered purified TT was expected to be 5-40 mg / ml. Purity was determined by chromatography (SEC HPLC), protein concentration (Lowry), and free-amino group (ninhydrin).

 Peptides were obtained commercially (Biosyn Corp, USA) and reducing peptides with known purity and content were used for conjugation. The peptide was reduced with tris (2-carboxyethyl) -phosphine-HCl (TCEP) and the mixture was used in conjugation. Free sulfhydral groups can be measured using an Elllmans assay.

Activation of tetanus toxoid: Purified TT was diluted to 5-50 mg / ml in activation buffer. Desired amounts of TT were transferred to glass vials containing Teflon-coated stir bars and EMCS (50-90 mg / ml DMF) was added to the TT solution. The molar ratio of EMCS / DT determines the level of activation. In the final concentration step, the total volume was reduced to give> -5-50 mg TT / ml. TT solutions were measured by SEC HPLC, and protein concentrations were determined by Lowry and activity levels by Elman analysis.

Conjugation of Peptide-TT: After calculating the amount of peptide to react with maleimido-TT, the peptide was added to the M-TT solution. Peptide-TT conjugates were purified by ultrafiltration apparatus.

The conjugates of peptides G18 and G21 were bound to TT via EMCS and separated from the other components of the mixture by low pressure chromatography at 4 ° C. equilibrated with 0.1-0.5 M ammonium bicarbonate on a G50 Sephadex column. In each case the conjugate was eluted in column pore volume, lyophilized and dried and stored at 4-0 ° C. until use.

Conjugates can be characterized for immunomimetic peptide content by a number of methods known to those skilled in the art, such as weight gain, amino acid analysis, and the like. The conjugates of TT with peptides G18 and G21 prepared by these methods had 10-30 moles of peptide per 104-106 MW of TT and were determined by amino acid analysis and all are suitable as immunogens for immunization of tested animals. Was considered. Similarly, DT conjugates of G18 and G21 were prepared in the same manner to measure ELISA titers using huG17 as substrate for analysis of antibody binding.

Example  2

The peptide-TT conjugate of Example 1 was administered in an eletric phase of aqueous and oily phase components prepared as follows. The conjugate and adjuvant were dissolved in phosphate buffered saline (PBS) to prepare an aqueous phase. The aqueous phase is prepared such that the concentration of the conjugate is twice the concentration that these components will have in the final enamel. To prepare the immunogen used in Example 4 below, the conjugate was dissolved in PBS, pH 6.5-8.0, at a concentration of 5-12 mg / ml. (It may exceed this wide range and can be used to adjust the degree of carrier desired to be conjugated)

The aqueous phase was combined 1: 1 (volume: volume) with the oil vehicle phase to produce an emulsion that constitutes the final immunogen formulation. One such vehicle is 20-60 parts squalene, 70-30 parts squalane, 2-12 parts sorbitan monooleate, 0.6-2.0 parts aluminum monostearate, 0.1-1 parts polysorbate 80, and 0.2-1.2 parts It is a mixture of polysorbate 40. The aqueous phase and oil phase vehicles can be mixed by any known method for forming a stably emulsified mixture. The emulsion must be stable upon storage, ie, during the minimum storage time of several weeks to several months, the emulsion must not be separated to a significant extent on aqueous and vehicle. The emulsion must also be of a concentration that can be easily injected through a hypodermic needle of acceptable size.

The aqueous phase containing the immunogen was enameled 1: 1 (volume: volume) with an oil vehicle mixture of two solutions through an 18 gauge double coupled needle between two glass syringes. The mixture was compressed 50 times through a needle. The emulsified mixture was then drawn up into a disposable syringe for injection into the animal. The final immunogen concentration in the emulsion, for Example 4, was conjugate: hG18TT in a concentration range of about 1 to about 5 mg per milliliter.

Example  3

We constructed conjugates comprising the respective G18 and G21 peptides listed in Example 1 linked to TT and DT, as described in Examples 1 and 2. We then immunized 6 mice using the peptide G18 immunogen (FIG. 1A, b) and 6 mice using the peptide G21 immunogen (FIG. 1C, d).

Example  4

We constructed a conjugate comprising the respective G18 and G21 peptides described above linked to TT and DT, as described in Examples 1 and 2. We then immunized four rabbits using the G18 immunogen (FIG. 2A) and four rabbits using the G21 immunogen (FIG. 2B).

As shown in Figures 1 and 2, the results of these ELISA tests showed that immunogens 1 and 2 (of Examples 1 and 2) both induced their responses to both their potency and their antibody induction in many animal species, as well as induced antibody responses. It was also effective in the persistence of .

Example  5

We constructed a conjugate comprising the respective G18 and G21 peptides linked to TT and DT, as described above. We then immunized 6 mice using the G18TT immunogen and G21TT immunogen. At the highest titers (day 42) all mice were injected with sterile, intraperitoneal hollow fiber implants containing 50,000 human gastric cancer cells / 1-2 cm tube (BCG-823) for 5 days. Hollow fiber customs allow penetration of molecules less than 500 KD but not penetration of CTL or NK cells, which allows the survival of human cancer cells in immune compromised mice. On the last 5 days the implants of each mouse were removed and the viable cells counted by MTT assay and compared to control implants in non-immunized mice. As indicated, some animals also contain 10 mg / kg of cisplatin (CP) or 20 mg / kg of 5-fluorouracil (5-FU) or 10 mg / mg of CP + 5-FU (FUP), respectively. Treatment was with a single dose of the combination.

The results of this test are shown in FIG. 3. Immunogens 1 and 2 (of Examples 1 and 2) using one of anti-gastrin / progastrin (G18 and G21 immunogens) are not only effective in inhibiting the growth of human gastric cancer cells, but also conventional gastric It can be seen that it is also effective in inducing sufficient antibody responses that were effective in the presence of cancer chemotherapy.

Example  6

We constructed a conjugate comprising each of the above-mentioned G18-a, G-18-b, G20, and G20-a peptides (SEQ ID NOs: 3-6, respectively) and assigned them to G21 (SEQ ID NO: 1). ). As described in Examples 1 and 2, the conjugates were all linked to TT. The inventors then immunized 6 mice using these immunogens (see FIGS. 4A, b, c and d) and 6 mice using the G21 immunogen for comparison.

The improvements thus demonstrated are derived from the modifications specified in the immuno mimetics according to the invention and from the unique spacer regions of the immunogenic peptides. The peptide immunogens of the invention were tested against immunogens that did not incorporate any of the above-described immunogenic mimetics and spacers, the latter being found to be less effective. Thus, conventional immunogens have been improved by modifying our immuno mimetics and / or their spacers in accordance with the present invention by the inventors.

<110> ONKOLOGIX LTD <120> IMMUNOGENIC COMPOSITIONS AGAINST HUMAN PROGASTRIN PEPTIDES <130> IPA120809 <150> CN 201010116229.3 <151> 2010-03-03 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (2) <223> D-Pro <220> <221> MOD_RES <12> <223> nPhe <400> 1 Cys Pro Pro Pro Ser Ser Gly Trp Met Asp Phe Gly Arg Arg Ser   1 5 10 15 Ala Glu Asp Glu Asn              20 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <220> <221> MOD_RES <222> (5) <223> Beta-isoVal <220> <221> MOD_RES <222> (18) <223> D-Pro <400> 2 Glu Gly Pro Trp Val Glu Glu Glu Glu Glu Glu Ala Ser Ser Pro Pro   1 5 10 15 Pro Pro Cys             <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <220> <221> MOD_RES <222> (17) <223> D-Pro <400> 3 Glu Gly Pro Trp Ile Glu Glu Glu Glu Glu Ala Ser Ser Pro Pro Pro   1 5 10 15 Pro cys         <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <220> <221> MOD_RES <222> (17) <223> D-Pro <400> 4 Glu Gly Pro Trp Val Glu Glu Glu Glu Glu Ala Ser Ser Pro Pro Pro   1 5 10 15 Pro cys         <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (2) <223> D-Pro <400> 5 Cys Pro Pro Pro Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp   1 5 10 15 Met Asp Phe Gly              20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (2) <223> D-Pro <220> <221> MOD_RES <222> (14) <223> nPhe <400> 6 Cys Pro Pro Pro Pro Trp Leu Glu Glu Glu Glu Glu Ala Phe Gly Trp   1 5 10 15 Met Asp Phe Gly              20 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (6) <223> D-Pro <400> 7 Ser Ser Pro Pro Pro Pro Cys   1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES (222) (3) .. (6) <223> D-Pro <400> 8 Ser Ser Pro Pro Pro Pro Cys   1 5 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (6) <223> D-Pro <400> 9 Thr Thr Pro Pro Pro Pro Cys   1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Thr Thr Pro Pro Pro Pro Cys   1 5 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <220> <221> MOD_RES <222> (5) <223> IsoVal <220> <221> MOD_RES <222> (17) <223> D-Pro <400> 11 Glu Gly Pro Trp Val Glu Glu Glu Glu Glu Ala Ser Ser Pro Pro Pro   1 5 10 15 Pro cys         <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <220> <221> MOD_RES <222> (17) <223> D-Pro <400> 12 Glu Gly Pro Trp Val Glu Glu Glu Glu Glu Ala Thr Thr Pro Pro Pro   1 5 10 15 Pro cys         <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (5) <223> nPhe <400> 13 Gly Trp Met Asp Phe Gly Arg Arg Ser Ala Glu Asp Glu Asn   1 5 10 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <220> <221> MOD_RES <222> (6) <223> IsoVal <400> 14 Glu Gly Pro Trp Leu Val Glu Glu Glu Glu Ala   1 5 10 <210> 15 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (1) P223 <400> 15 Glu Gly Pro Trp Ile Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp   1 5 10 15 Phe Gly         <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 Trp Ile Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp Phe Gly   1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MOD_RES <222> (9) <223> nPhe <400> 17 Trp Ile Glu Glu Glu Glu Glu Ala Phe Gly Trp Met Asp Phe Gly   1 5 10 15 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 18 Gly Trp Met Asp Phe Gly Arg Arg Ser Ala Glu Asp Glu Asn   1 5 10

Claims (13)

 (ii) a (i) mimetic peptide consisting of (i) an amino acid sequence of progastrin or an N-terminal and / or C-terminal processing species of progastrin bound to a seven amino acid spacer and coupled to said mimetic peptide (B) A polypeptide immunogen comprising an immunogenic carrier. The polypeptide immunogen of claim 1, wherein the mimetic peptide has the following amino acid sequence:
Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn (SEQ ID NO: 1). The polypeptide immunogen of claim 1, wherein the mimetic peptide has an amino acid sequence selected from the group consisting of:
pGlu-Gly-Pro-Trp-β-isoVal-Glu-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5); And
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6). The polypeptide immunogenic composition of claim 1, wherein said immunogenic carrier is selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussin toxoid, and tuberculin pure protein derivative. The polypeptide immunogenic composition of claim 4, wherein said immunogenic carrier is tetanus toxoid. An immunogenic composition comprising an effective amount of a polypeptide immunogen according to claim 1 and a pharmaceutically acceptable vehicle for said immunogen. The immunogenic composition of claim 6, wherein the pharmaceutically acceptable carrier comprises an emulsion in an aqueous phase and in an oil, in which the immunogen is present. 8. The immunogenic composition of claim 7, wherein said oil phase comprises at least one of squalene, squalane, sorbitan monooleate, polysorbate 40, and polysorbate 80. 8. An immunogenic composition according to claim 7, wherein said oil phase comprises an emulsifier. 8. An immunogenic composition according to claim 7, wherein one of said oil phase or said aqueous phase contains at least one adjuvant. The method of claim 10, wherein the adjuvant is Nor-MDP, imiquimod, cyclic diguanylate, threonyl-N-acetyl-muramil-L-alanyl-D-isoglutamine, isopinosine, trehalose dimicholate , QS-21, alpha-galactosylceramide, and alpha-glucosylceramide. The immunogenic composition of claim 10, wherein said adjuvant is selected from the group consisting of ergamisol, cimetidine, praziquantel, uric acid, mannan and mannan derivatives, and vitamin E. Spacer peptides selected from the group consisting of:
Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7);
Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8);
Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9); And
Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10).

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