JP2003533181A - Mucin-1 derived antigen and its use in immunotherapy - Google Patents

Abstract

  (57) [Summary] A peptide or polypeptide capable of inducing an immune response, wherein said peptide or polypeptide comprises an amino acid sequence substantially corresponding to an amino acid sequence of an epitope of a non-VNTR, non-leader region of a mucin. Alternatively, there is provided a polypeptide or a fusion protein comprising the peptide or polypeptide and an appropriate carrier protein. Also provided is a method of inducing a cell-mediated immune response to mucin, comprising administering to a patient, optionally in combination with an adjuvant and / or a pharmaceutically acceptable carrier.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

  The present invention relates to immunotherapy of diseases, such as immunotherapy of carcinoma.

[0002]

[Prior art]

Cancer is the leading cause of death and serious trauma in modern society. Cancer is not limited to one group; young, old, male, female, and people of all races can have cancer, but cancer is relatively rare in children, except perhaps in childhood leukemia. Colon and lung cancers are the major diseases in the western world. Breast cancer is the most common form of cancer in women.

Many cancers are associated with overproduction of human mucin. Mucin is a highly glycosylated protein (greater than about 100 Kd) produced by many epithelial cells and tumors (1). The mucins found in cancer cells, unlike those found in normal epithelial cells, some mucins lack their carbohydrate coating and leave the protein core exposed (2). Known human mucins include MUC1 to MUC1
There are 12 forms called 2 (see, for example, 3, 4, 26, 27). MUC
1 is most widespread. All of the various mucins have very similar properties,
That is, they are transmembrane glycoproteins, all having varying numbers of repeating amino acid sequences with high contents of serine, threonine and proline. Overproduction of aberrantly glycosylated mucin (non-glycosylated or lacking glycosylation) is characteristic of tumors of the breast, ovary, pancreas, colon, lung, prostate and other secretory tissues. The cDNA sequences of the human mucin MUC1 to MUC7 protein cores have been cloned and characterized and were found to contain a highly repetitive central portion consisting of varying numbers of repeats of a particular amino acid motif (VNTR). known as's).

Surgery for tumor removal is traumatic to the patient, often disfiguring and expensive. Performed in place of or in combination with surgical procedures,
Established chemotherapies and radiation treatments for tumor therapy are often debilitating and are associated with serious side effects. Therefore, there is an urgent need for therapeutic compounds and methods for the prevention / treatment of tumors.

The prior art relating to mucins is primarily concerned with the use of VNTRs as a potential treatment or prevention for cancer. In one case, it was reported to use the leader sequence of MUC1 to induce cytotoxic T cells in vitro (7
1). However, studies on this peptide LLLLTVLTV (SEQ ID NO: 1) have been carried out in vitro and do not necessarily suggest how this peptide behaves in vivo. Moreover, this epitope is not always MUC.
Lack of such a predominance, but not one of the dominant T cell epitopes, can result in cytotoxic T lymphocytes (CTL) against other epitopes that are not LLLLTVLTV (SEQ ID NO: 1).

In research leading to the present invention, the present inventors have surprisingly found that HMFG (total MUC1
It was found that the non-VNTR, non-leader region of MUC1 can be selectively antigenic when the mannan conjugate of (1) is used for immunization. This is a non-MUC1
Cytotoxic T cells against the VNTR region are the first case documented in mice immunized with whole MUC1. This means that non-VNTR peptides may have high affinity for major histocompatibility complex (MHC) class 1.
This is surprising in that the VNTR peptide shows a low affinity for MHC class 1. Our work was performed in vivo.

[0007]

[Problems to be Solved by the Invention and Means for Solving the Problems]

Accordingly, in a first aspect, the invention provides a peptide or polypeptide capable of eliciting an immune response, wherein said peptide or polypeptide is substantially a epitope of the non-VNTR, non-leader region of mucin. The amino acid sequence corresponding to

It is also to be understood that the term “polypeptide” used above and below does not include the full length mucin protein.

Preferably, the peptide or polypeptide is completely mucin non-VNTR.
, Consisting of an amino acid sequence derived from the non-leader region (and this includes certain epitopes). However, the peptide or polypeptide may include additional amino acid sequences derived from other regions of mucin, including VNTRs and / or leader regions. This peptide or polypeptide is VN
Epitopes in the TR and / or leader regions can also be included. Further, the peptide or polypeptide may include additional amino acid sequences derived from other natural or artificial sources (eg, the peptide or polypeptide may include a heterologous leader and / or signal sequence). , Or an amino acid sequence substantially corresponding to an epitope from an antigen of any tumor morphology or other source that expresses MUC1). Examples of particular tumor antigens are the carcinoembryonic antigen (CEA) of colon and other cancers, or an antigen extracted from any tumor expressing MUC1.

Preferably, the immune response elicited by this peptide or polypeptide is a cell-mediated immune response, particularly one involving activation of cytotoxic T cells against cells expressing aberrantly glycosylated mucin (eg, Characteristics of breast, ovary, pancreas, colon, lung and prostate neoplastic cells).

The term “substantially corresponding” as used herein with respect to amino acid sequences is meant to include small changes in a particular amino acid sequence that do not substantially alter the biological activity of the particular amino acid sequence. Think For example, with respect to the amino acid sequence of an epitope of the non-VNTR, non-leader region of mucin, the term "substantially corresponding" means that the epitope activity is not substantially altered, ie the epitope variant still produces a substantially equivalent immune response. Includes sequence variations that can be derived (the variations can also be found in naturally occurring variant sequences or otherwise). Such mutations can include conservative amino acid substitutions. Possible conservative amino acid substitutions are: G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Nα. -It is an alkyl amino acid.

The peptides or polypeptides of the present invention can be derived from natural sources, synthetic by standard techniques, or recombinantly produced. Peptide synthesis is about 1
Used for polypeptides containing up to 00 amino acids. Generally, for polypeptides containing about 20 or more amino acids, the preferred means of production is recombinant expression in host cells. Methods for expressing recombinant proteins in prokaryotic and eukaryotic host cells are well established, see eg Sambrook et al. (7).

The peptide or polypeptide may be part of a fusion protein. Methods for expressing fusion proteins in prokaryotic and eukaryotic host cells are well established, see, eg, Sambrook et al. (7). The fusion protein utilizes this peptide or polypeptide with glutathione-S-transferase, β-galactosidase, or any other protein or portion thereof, particularly other affinity features for purifying the binding or resulting fusion protein. And may be fused to a carrier protein selected from those that allow for affinity purification. The fusion protein may comprise a fusion of a peptide or polypeptide according to the invention to the C-terminus or N-terminus of a carrier protein. The exact nature of the fusion protein depends on the vector system in which it is produced. Examples of bacterial expression vectors can be used to produce fusion proteins consisting of the peptide, polypeptide or protein of interest and glutathione-S-transferase.
It is GEX. The carrier protein may or may not be cleaved from the peptide or polypeptide of the invention after expression. This fusion protein may be treated with mild periodate oxidation.

As mentioned above, expression of a peptide or polypeptide, or a fusion protein comprising a peptide or polypeptide, may be performed in a host cell, eg, prokaryotic (eg E. coli or B. subtilis) or eukaryotic (baculovirus, CHO cell). , COS cells or yeast) host cell expression systems. In some of these systems, such as baculovirus or yeast, glycosylation of peptides, polypeptides or fusion proteins can be achieved by introducing known glycosylation motifs.

Similarly, the peptide or polypeptide is simply attached to a suitable carrier protein (eg, keyhole limpet hemocyanin) using any method established in the art (eg, glutaraldehyde treatment). Good.

Preferably, the peptide or polypeptide according to the present invention comprises an amino acid sequence derived from human mucin 1. More preferably, the peptide or polypeptide comprises an amino acid sequence derived from human milk fat globule membrane antigen (HMFG). More preferably, the peptide or polypeptide is an amino acid sequence derived from the non-leader, non-VNTR region extracellular or intracellular region of human MUC1 (eg, from amino acid 22 of human MUC1 of NCBI database No. M61170). 131 or amino acids 402 to 473 (see also Figure 1)), but human MU
The amino acid sequence of the transmembrane region of the non-leader, non-VNTR region of C1 may also be suitable. More preferably, the peptide or polypeptide comprises an amino acid sequence that substantially corresponds to one of the following amino acid sequences or immunogenic fragments thereof. TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: 2) RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3) SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATW
(SEQ ID NO: 4) SAPDNRPAL (SEQ ID NO: 6) NSSLEDPSTDYYQELQRDISE (SEQ ID NO: 7) TQFNQYKTEAASRVNL (SEQ ID NO: 8) AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: 9) YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10)

In a second aspect, the invention provides a compound comprising a conjugate of the peptide or polypeptide of the first aspect and a carbohydrate polymer.

Preferably, the carbohydrate polymer is glucose, galactose, mannose,
Xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-o-methyl 1-D-galactose, 2-o-acetyl-β-D-xylose,
N-acetyl-glucosamine, iduronate, gulurona
te), mannuronate, methylgalacturonate, α-D-galactopyranose 6-sulfate, fructose and α abequose, their conformers and configurational isomers, or two or more different monomer units Is a polymer of carbohydrates selected from the group consisting of carbohydrates formed from The number of repeating monomer units in the polymer is not critical, but generally a carbohydrate polymer comprises at least 20 monomer units, preferably more than 100 monomer units, more preferably more than 1000 monomer units, and Preferably more than 10,000 monomer units. The carbohydrate polymer may be a mixture of polysaccharide chains of various molecular weights. More preferably, the carbohydrate polymer is
It is a polymer of mannose or a carbohydrate polymer containing mannose units. Most preferably, the carbohydrate polymer is a polymer of oxidized mannose.

The peptide or polypeptide according to the first aspect is prepared according to standard techniques well known in the art of carbohydrate chemistry of the reaction and derivatization of polysaccharides and monosaccharides.
It may be conjugated to a carbohydrate polymer. Carbohydrates may be oxidised using conventional oxidising reagents such as sodium periodate to give polyaldehydes which are then reacted directly with the peptides or polypeptides to give amino functionalities of the peptide chain. A group (eg, the ε-amino group of lysine) may be reacted with an aldehyde group and further reduced to form a Schiff base. The polysaccharide chain may be first activated with cyanogen bromide and then the activated polysaccharide is reacted with a diamine and then conjugated to a peptide or polypeptide to form a conjugate that may be optionally oxidized later. You may. The carbohydrate and the polysaccharide may be derivatized with a bifunctional reagent to crosslink the carbohydrate and the polysaccharide. Commonly used cross-linking agents are 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, 3,3′-dithiobis (
Homobifunctional imide esters including disuccinimidyl esters such as succinimidyl-propionate) and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatives such as methyl-3-[(p-azidophenyl) dithio] propioimidate yield photoactivatable intermediates that are crosslinkable in the presence of light. The oxidised carbohydrate may be reacted with a hydrazine derivative of the antigen to give a conjugate. Alternatively, the carbohydrate may be reacted with a reagent such as carbonyldiimidazole followed by reaction with the antigen, which after oxidation gives the desired conjugate.

Coupling a peptide or polypeptide to a carbohydrate converts any or all of the functional groups of the carbohydrate to reactive groups, followed by conversion of the carbohydrate reactive groups to the polypeptide reactive groups. Including reacting. Carbohydrate polymers contain hydroxide groups, and in some cases carboxyl groups (eg, idruionate).
onate)), an ester group (for example, methylgalacturonate) and the like are sufficiently contained. These groups may be activated according to standard chemical methods. For example,
Hydroxyl groups may be reacted with hydrogen halides such as hydrogen iodide, hydrogen bromide and hydrogen chloride to yield reactive halogenated polysaccharides. Hydroxyl groups may be activated with phosphorus trihalides, active metals such as sodium ethoxide, aluminum isopropoxide and potassium tert-butoxide, and esterification (using groups such as tosyl chloride or acetic acid). To form a reactive group, which can then be reacted with a reactive group of a polypeptide to form one or more bonds. Apart from the hydroxyl group, other functional groups on the carbohydrate
The reactive group may be activated to obtain the reactive group according to standard techniques.

The carbohydrate polymer may be purified from natural sources or may be synthesized according to standard techniques. Carbohydrates are commercially available from many sources.

Preferably, the carbohydrate polymer is conjugated to the peptide or polypeptide in any amount that allows the peptide or polypeptide to elicit a cell-mediated immune response in humans or other animals. Such amount can be, for example, in the range of about 0.1-10 mg per mg of peptide or polypeptide.

The peptide or polypeptide linked to the fusion protein and the appropriate carrier protein described above may be linked to a carbohydrate polymer, especially oxidized mannose. Similarly, preferably, the carbohydrate polymer is conjugated to the fusion protein in an amount such that the fusion protein can elicit a cell-mediated immune response in humans or other animals. In this case, however, the amount can be, for example, in the range of about 1-10 mg / mg fusion protein, more preferably about 5-8 mg / mg fusion protein.

In a third aspect, the invention provides a vaccine against a disease, in particular a human disease characterized by tumor cells expressing mucin or subunits thereof, wherein the peptide or poly of the first aspect of the invention is used. There is provided a vaccine comprising a peptide, or a fusion protein comprising the peptide or polypeptide of the first aspect of the invention, and optionally an adjuvant and / or a pharmaceutically acceptable carrier.

In a fourth aspect the invention provides a vaccine against a disease, in particular a human disease characterized by tumor cells expressing mucin or subunits thereof, wherein the conjugate compound of the second aspect of the invention, And a vaccine optionally comprising an adjuvant and / or a pharmaceutically acceptable carrier is provided.

A suitable adjuvant for use in the vaccine of the third or fourth aspect is Qu
il A. QS-21 Iscoms, including everything known in the art, such as liposomes, alum, salts, oils, emulsions and the like.

The vaccine of the third or fourth aspect is for protecting against a variety of disease states including cancer cell proliferation, especially tumors of secretory tissue, for example tumors of breast, colon, lung, pancreas, prostate, etc. , Can be administered to human patients. Patients may be immunized with the vaccine to protect the secretory tissue from tumorigenesis. Alternatively, tumor patients can be immunized with a vaccine as part of a therapeutic regimen for tumor treatment. For example, to protect a woman from breast cancer, the woman can be immunized with the vaccine before or after puberty, with one or more injections, preferably several months to several years apart, after the first immunization, One or more booster injections may be given. The route of immunization is the same as for normal human vaccine administration. Therefore, the vaccine of the third or fourth aspect can be administered subcutaneously, intramuscularly, orally, intravenously and the like.

The amount of peptide or polypeptide of the invention (which may be conjugated to a suitable carrier protein), fusion protein, or conjugated compound administered to a patient is
It is not definitive or limiting. However, a peptide or polypeptide according to the invention (which may be linked to a suitable carrier protein), a fusion protein,
Alternatively, an effective amount of the conjugate compound is one that stimulates an immune response. In this regard,
An effective amount can be used according to the patient's immune status (ie, depending on whether the patient is immunosuppressed or immunostimulated), that vaccine will prevent or treat the disease state or prevent tumor formation It can vary according to the diagnosis of the attending physician or veterinarian, whether or not the vaccine can be used to treat existing tumors. By way of example, the patient may be a peptide or polypeptide according to the invention (which may be linked to a suitable carrier protein), a fusion protein,
Alternatively, 1 μg to 10000 μg, more preferably 50 μg to 5000 μg, more preferably 100 μg to 1000 μg, and more preferably 100 μg to 500 μg of the conjugate compound may be administered. Adjuvants are generally not required. However, adjuvants can be used for immunization.

A peptide or polypeptide of the present invention (which may be conjugated to a suitable carrier protein), a fusion protein, or a conjugated compound is added to a cytokine or other immunomodulatory factor (eg, one or more GM-CSF, G -CSF, M-CSF, TN
Fα or β, interferon α or γ, any of IL1 to IL13,
Or any of the other cytokines). The immunomodulator is administered at the same time or at a different time as the peptide or polypeptide of the invention (which may be conjugated to a suitable carrier protein), fusion protein, or conjugated compound, optionally as part of a multi-component dosage form. May be.

In a fifth aspect, the invention provides a method of inducing a cell-mediated immune response against mucins, comprising: administering to a patient an effective amount of a peptide or polypeptide of the first aspect (to a suitable carrier protein). Or a fusion protein comprising the peptide or polypeptide of the first aspect, optionally in combination with an adjuvant and / or a pharmaceutically acceptable carrier.

In a sixth aspect, the invention provides a method of inducing a cell-mediated immune response against mucin, wherein a patient is treated with an effective amount of a conjugate compound of the second aspect, optionally with an adjuvant and / or Alternatively, a method is provided that comprises administering in combination with a pharmaceutically acceptable carrier.

Administration of a peptide or polypeptide of the present invention (which may be conjugated to a suitable carrier protein), a fusion protein, or a conjugated compound to human and veterinary patients may be performed on cells that express mucin. It can cause an enhanced cellular response of injurious activated T lymphocytes. A potential advantage of the present invention is that the peptides or polypeptides of the present invention (which may be conjugated to a suitable carrier protein),
Humans and animals can be protected from cancer prior to tumor growth because the fusion protein, or the conjugated compound, provokes a cellular immune response against cytotoxic T cells that kill the mucin-expressing tumor cells. can get. The present invention is applicable to immunization against tumors of secretory tissues, such as adenocarcinomas, especially tumors of the breast, ovary, pancreas, colon, lung, prostate, etc.

The peptides or polypeptides of the invention (which may be conjugated to a suitable carrier protein), fusion proteins, or conjugated compounds may also be used as part of an overall treatment for the eradication or reduction of cancer. It can also be used as a therapeutic agent for the treatment of cancer patients or as a component thereof. Thus, a peptide or polypeptide of the present invention (which may be conjugated to a suitable carrier protein), fusion protein, or conjugated compound may be administered to a cancer patient before or after surgery to remove the tumor. Preferably, the peptide or polypeptide according to the present invention (
A fusion protein, which may be coupled to a suitable carrier protein), or a conjugated compound is administered as part of chemotherapy following tumor removal. In such a situation, the peptide or polypeptide of the present invention (which may be coupled to a suitable carrier protein), fusion protein, or conjugated compound may be used as standard chemotherapy for the administration of cytotoxic compounds used in tumor treatment. The appropriate dose is administered.

The peptides or polypeptides according to the invention (which may be conjugated to a suitable carrier protein), fusion proteins or conjugated compounds have the advantage of being substantially harmless upon administration to humans or animals, As a result, it appears to be well tolerated.

In a fifth aspect, the invention relates to the treatment of adenocarcinomas, in particular breast cancer, with a peptide or polypeptide according to the invention (which may be linked to a suitable carrier protein), a fusion protein or a conjugate compound. Regarding use.

In a further aspect, the invention provides a peptide or polypeptide according to the invention (
A fusion protein, which may be coupled to a suitable carrier protein), or a conjugate compound, for pulsing dendritic cells for in vivo introduction and for use as a vaccine.

In a further aspect, the invention encodes a peptide or polypeptide of the first aspect (which may be conjugated to a suitable carrier protein) or a fusion protein comprising the peptide or polypeptide of the first aspect. Provided is an isolated nucleic acid molecule that comprises a nucleotide sequence.

This nucleic acid molecule, even when incorporated into a transfer or expression vector, can
A vaccine may be used. Such nucleic acid molecules are described, for example, in Sambrook et al. (7).
It may be produced according to standard techniques by cloning or synthesis as described in.

In yet another aspect, the invention includes a conjugate of a MUC1 with a carbohydrate polymer as described above, such that the conjugate is capable of inducing a cell-mediated immune response in humans or other animals. A compound is provided. Preferably MUC1
Is a human MUC1 (eg HMFG) and the carbohydrate polymer is a polymer of mannose, especially oxidized mannose, or oxidized mannan. The carbohydrate polymer may be, for example, about 1-10 mg / mg MUC, preferably MU.
About 5-8 mg / mg C1, more preferably about 7 m / mg MUC1
It may be bonded to MUC1 in an amount within the range of g. This conjugate compound may be used in a vaccine or as a therapeutic agent similar to the above.

Throughout this specification the term “comprising” or variations thereof is meant to include the recited ingredient, whole or step, or ingredient, whole or group of steps, but other ingredients, whole or steps, It is also understood that it does not exclude any of the components, the whole or the group of steps.

Any discussion of publications, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. Any or all of these should not be considered part of the prior art or are common general knowledge in the technical field relating to the present invention in Australia prior to the priority date of the present application.

[0042]   The present invention will now be described with reference to the following non-limiting examples and the accompanying drawings.

[0043] Abbreviation:   The following abbreviations are used in the examples. ELISA: enzyme immunoassay DTH: Delayed type hypersensitivity FP: fusion protein GST: glutathione-S-transferase HMFG: human milk fat globule Kd: kilodalton KLH: Keyhole limpet hemocyanin PAGE: Polyacrylamide gel electrophoresis PBS: Phosphate buffered saline SDS: sodium dodecyl sulfate Tc: Cytotoxic T lymphocyte

[0044]

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 1. Induction The immunotherapeutic approach to treating breast cancer has involved the use of monoclonal antibodies and the production of cytotoxic lymphocytes (CTL) (29-34). The identification of target antigens, the availability of recombinant proteins and cytokines has fueled immunotherapy. Thus, there are new ways to generate effective cytotoxic T cell responses against MUC1-expressing carcinomas of milk and other tissues (35). MUC1 is a particularly attractive target for the production of CTLs: it is immunogenic in mice with respect to the production of antibodies, and we have recently described CD8 ⁺ CTLs, which are MHC class
The I H-2 and HLA-A ^* 0201 binding peptides were mapped to VNTR (36
-39). Moreover, there is a 100-fold increase in the amount of mucin in cancer cells (40),
There is a significant amount of MUC1 peptide bound by class I molecules. VNT
The reason to focus on the R peptide is clear: this is when whole tumor cells or mucin extract (HMFG) was used to immunize mice for antibody production,
This is because it is the most immunogenic region of MUC1 (40).

In this regard and the finding that non-HLA restricted CTLs are also directed to VNTRs,
Almost all MUC1 interest in CTL induction was focused on the VNTR peptide (37, 41, 42). In contrast, this example demonstrates that extracellular and intracellular MUC1 identified by immunizing mice with natural mucin obtained from human milk (HMFG) or with the peptides described herein. It relates to the induction of CTL against a partial non-VNTR epitope.

2. Materials and methods Mouse and tumor cells BALB / c (H-2 ^d ), C57BL / 6 (H-2 ^b ), human MUC1 transgenic mouse (obtained from B.Acres (Transgene, Strasbourg, France)), transgenic HLA -A ^* 0201 / K ^b mice ^{(H-2 b) (The} Scripps Cli
nic and Research Foundation, La Jolla, CA.), and double transgenic mice (A2K ^b MUC1) from The Austin Research Institute (ARI
). Human MUC1 of MUC1 transgenic mice (DBA / 2 backcross) is under the control of the human MUC1 promoter; MUC1
It is expressed in bronchioles of the lung, beta islets of the pancreas, renal tubules and stomach (43). HLA-A ^* 0201 / K ^b mice, expressing a transgene consisting of α1 and α2 domains of HLA-A ^* 0201, α3 includes the transmembrane and cytoplasmic domains of H-2K ^b (44). Double transgenic mice were subjected to HLA-A ^* 020 by flow cytometry using antibodies against HLA-A ^* 0201 and MUC1.
Screened for expression of 1 / K ^b and human MUC1 transgene. RMA-MUC1 was transfected with MUC1 (C57BL / 6 (H
2 ^b )) Lymphoma cell line (45). Tm211 is B.Acres (Transgene,
MUC1-transfected P815 mastocytoma (DBA / 2 origin; H-2 ^d ) obtained from Strasbourg, France). All mouse cell lines were
U / ml penicillin, 100 μg / ml streptomycin, and 10%
Fetal bovine serum (all of these are Commonwealth Serum Laboratories (CSL), Melbo
Dulbecco's modified Eagle's medium (DMEM) (obtained from urne) and human cell lines in RPMI with the same additives at 37 ° C. in a CO ₂ incubator with 7% humidity. BALB / c, C57BL / 6 and double transgenic A2K ^b MUC1 mice were immunized on day 0, 10, 17 with 5 μg of mannan-HMFG or HMFG by three intraperitoneal injections, while HLA-. A ^* 0201 / ^Kb mouse was injected once.

2.2 Synthetic peptide A peptide (Table 1) was synthesized by ARI; the purity of this peptide (> 95%) was
It was measured by mass spectrometry.

2.3 Conjugation of HMFG to Mannan HMFG was isolated from human milk (49) and bound to mannan. Mannan (1 ml, 14 mg / ml) in phosphate buffer (0.1 M, pH 6.0) was treated with sodium periodate (100 μl, 0.1 M) and incubated at 4 ° C. for 30 minutes (48). The reaction was stopped by addition of ethanediol (10 μl) at 4 ° for 30 minutes and the mixture was passed through a PD10 column (Pharmacia Biotech, Sweden) equilibrated with bicarbonate buffer (0.2 M, pH 9.0). The oxidized mannan fraction was mixed with 1 mg HMFG overnight at room temperature to yield mannan-HMFG.

2.4 T Cell Epitope Prediction There are several CTL epitope prediction algorithms available, and in this study a program developed by Dr. Kenneth Parker (bimas.dcrt.nih.gov) available on the internet. / molbio / hla_bind /) was used to identify potential T cell epitopes. This program is based on the scores given to the amino acids at each of positions 1-9 from the input sequence by comparison with the reported database (49, 50). Higher numbers for the 9-mer predict a high likelihood of a T cell epitope. For example, the T cell epitope for ovalbumin (K ^b , SIINFEKL, SEQ ID NO: 11) and papillomavirus-16 E7 protein (D ^b , RAHYNIVTF; SEQ ID NO: 12) are:
Scores of 17 and 6 are given respectively.

2.5 Cytotoxic T Cells and Cytotoxic T Cell Precursor (CTLp) Frequency Assay CTL assays were performed as described (37, 39, 48). Briefly, 7-10 days after the final immunization, splenocytes were harvested, washed, resuspended in growth medium and serially diluted in 96-well microtiter plates. A standard 3 hr ⁵¹ Cr release assay was performed with 1 × 10 ⁴ peptide-pulsed or untreated P815 or RMA cells as targets at various effector: target ratios. Peptide-pulsed P815 or RMA target cells were prepared by overnight incubation with 9-mer peptide (25 μg / ml) (37). In the CTL assay using the A2K ^b MUC1 double transgenic effector, MCF7 (MUC1 ⁺ HLA-A ^* 02
01 ⁺ ) and BT20 (MUC1 ⁺ HLA-A ^* 0201 ⁻ ) breast cancer cell lines or ME272 (MUC1 ⁻ HLA-A ^* 0201 ⁺ ) melanoma cell lines were used as targets. All of these human tumor cell lines are susceptible to cell-mediated lysis (39,51,52). CTLp frequencies were examined from a minimum of 32 repeats (1 × 10 ³ −1.28 × 10 ⁵ ) for at least 6 effector cell numbers. U
10% fetal bovine serum, 5 μM various MUC1 in a V-shaped microtiter tray
5 × 10 ⁵ mitomycin C treated BALB / c (H-2 ^d ), C in DMEM supplemented with peptides (Table 1) or HMFG and 10 U / ml rhIL-2
Cells were cultured with 57BL / 6 (H-2 ^b ) or HLA-A ^* 0201 / K ^b spleen cells. After several days, 100 μl of culture medium was added to 10 ^{4 51} Cr-labeled T.
M211 100 containing (H-2 ^d), the ^{RMA-MUC1 (H-2 b} ) tumor or EBV-transformed human B cells (HLA-A ^* 0201) or MCF7 targeting
Each microculture was assayed for cytotoxicity by displacing μl of target cell suspension. As a specificity control, non-MUC1 expressing P815
(H-2 ^d ) or RMA (H-2 ^b ) cells were used. Cytotoxic activity was observed in each well when ⁵¹ Cr release was 3 standard deviations higher than the mean isotope release from 10 ⁴ effectors cultured with stimulator alone or from stimulated cells stimulated with peptide alone or rhIL2 alone. Thought to exist. Linear relationship (0.9
87 ≦ r ² ≦ 1) was present between the number of responder cells on a linear scale and the frequency of negative wells on a logarithmic scale. CTLp frequency was examined as the reciprocal of the dose of responder cells required to generate 37% negative wells (53-55). When the CTLp frequency assay was performed in triplicate, individual frequencies did not differ from the mean by 20%. However, it should be noted that CTLp frequency in immunized mice is directly associated with tumor protection (28).
).

2.6 Inhibition ELISA Antibody inhibition ELISA was performed to compare the activity of HMFG before and after conjugation with mannan. Polyvinyl chloride plates were loaded with 10 μg / ml HMFG in bicarbonate buffer (0.2M, pH 9.0) overnight at 4 ° C or 1 hour at 37 ° C.
Coated with 70 μl of H2O and non-specific binding was blocked with 2% bovine serum albumin (BSA). Anti-MU with various concentrations of HMFG or mannan-HMFG
Incubated with C1 antibody (VA2 (57), 1/200 supernatant) for 3 hours and added 100 μl to HMFG coated PVC microtiter well plates. After washing with phosphate buffered saline (PBS) containing 0.05% Tween 20, 50 μl of sheep anti-mouse immunoglobulin conjugated to horseradish peroxidase (Amersham, UK) was added and further for 1 hour at RT. Incubated. After washing with PBS / Tween20, the plate was stained with the chromogenic material 2,2 ''-azino-diene.
Color was developed with (3-ethylbenzthiazoline) sulfonate (ABTS) (Amersham, UK) and absorption at 405 nm recorded.

2.7 Radioimmunoassay A sandwich radioimmunoassay was performed to confirm that mannan was covalently linked to HMFG. Microtiter plates were coated overnight with serial dilutions of anti-MUC1 antibody (BC2 (58)) in bicarbonate buffer to block non-specific binding as described above. HMFG or Mannan-HMFG was added to the wells, incubated for 1 hour at RT and washed extensively with PBS containing 0.05% Tween20. Then 50 μl of radiolabeled concanavalin A (
Which specifically binds to mannan but not HMFG) was added and the plates were incubated for an additional hour and washed with PBS / Tween 20. Microsci
nt-O (120 μl) was added to the wells and the plates were counted in a β-scintillation counter.

3. Results 3.1 Preparation and characterization of mannan-HMFG The activity of HMFG after conjugation to mannan was measured by inhibition ELISA;
The 50% inhibitory concentration of HMFG was 22 μg / ml, while mannan-H
It was 20 μg / ml for MFG (FIG. 1a). That is, HMFG retained all reactivity after conjugation to mannan. The integrity of the mannan-HMFG complex was demonstrated by a sandwich radioimmunoassay with plate-bound anti-MUC1 antibody and ¹²⁵ I-labeled Con-A for reading (FIG. 1b). Unconjugated HMFG does not bind to ¹²⁵ I-Con-A, but mannan-HM
FG is bound, indicating that mannan is bound to HMFG.

3.2 CTL response to mannan-HMFG in BALB / c mice Spleen cells of BALB / c mice immunized with mannan-HMFG were derived from various peptides (both VNTR and non-VNTR regions, Table 1). In vitro
CTLp was examined by stimulation with and tested on target cells expressing native MUC1 (Table 2). CT immunization with mannan-HMFG reacts with epitopes derived from the entire MUC1, ie both VNTR and non-VNTR regions
It was clear to induce L. This reaction is a) HMFG. A CTLp frequency of 1/9700 was obtained when the total MUC1 (HMFG) protein was used as the source of stimulatory peptides. Apparently, HMFG is immunogenic for CTL production in BALB / c mice and can be engineered to yield peptides presented by class I molecules. b) VNTR. 1/7000 (Cp13-32) and 1/13200 (p1-3) when stimulated with the VNTR peptides Cp13-32 and p1-30.
A CTLp frequency of 0) was obtained, which is similar to the results previously found by immunizing with HMFG to produce anti-VNTR CTL and immunizing with mannan-conjugated VNTR peptide. (47). This is the first description of CTL obtained by immunization with native mucin that is glycosylated. c) Extracellular region. In vitro stimulation results in amino acids 31-55, 51-70, 33-1
When the peptide containing 03,344-364 was used, CTL was 1/1950.
0 (31-55); 1/10000 (51-70); 1/20150 (33-1)
03) and 1/36800 (344-364). Thus, CTL can be produced against the non-VNTR region of the extracellular region;
This is the first description regarding L. d) Intracellular area. Three different non-overlapping intracellular peptides containing amino acids 408-423, 471-493, 507-526 were examined using the above approach. 1
/ 30000 (408-423), 1/12500 (471-493) and 1
A CTLp frequency of / 22500 (507-526) was obtained. Amino acids 471-49
3 was most effective in restimulating cytolytic cells.

To show that CTLs are specific to the MUC1 sequence and not due to non-specific killing by NK cells or other cells, P815 target cells were treated with a non-MUC1 peptide as a pulsed antigen, When used with T4N1, no CTLp was detected or the frequency was ≤ 1/200000,
It was considered negative (not shown). Three of the different regions were equivalent immunogenicity (using CTLp frequency for measurement): extracellular (51-70) = VNTR (
Cp13-32) = intracellular (471-493), all of which are ~ 1/100
It showed a high frequency of 00.

In contrast, BALB / c mice were immunized with unconjugated HMFG and V
When stimulated with the NTR peptide Cp13-32, the CTLp frequency is 1/805.
It was 00. This frequency was obtained with mannan conjugated to a recombinant bacterial fusion protein containing 5 repeats of the MUC1 VNTR of 1 / 95,000 C.
It is similar to the TLp frequency (47) and thus the conjugation of HMFG to mannan is required to give rise to strong CTLp frequencies in mice.

3.3 CTL response to mannan-HMFG in C57BL / 6 mice C57BL / 6 was immunized with mannan-HMFG and stimulated in vitro with the same antigens used for BALB / c mice (Table 2 ). 1/13500 for total HMFG and 1/125 for VNTR region peptide p1-30
There was a CTLp frequency of 00 (Table 2). For non-VNTR extracellular peptides,
CTL was detected at a frequency of 1/24500 against only one extracellular peptide (344-364). CTL was not detected for any of the intracellular peptides. Also, the specificity of CTL was determined by stimulating non-MUC1 peptide, T4N1
And using a non-MUC1 transfected parental RMA cell line as a target. Thus, C57BL / 6 mice show VNTR and non-VNTR
It was able to respond to both peptides but not some of the peptides that BALB / c mice reacted.

3.4 Mannan in transgenic HLA-A ^* 0201 / K ^b mice-
Cellular Immune Response to HMFG Transgenic HLA-A ^* 0201 / ^Kb mice were immunized once with mannan-HMFG (not x3 as described above) and HMFG, VNTR peptide (p.
1-30) or one of the extracellular peptides (31-55)
I was stimulated by itro. CTLp was measured in human EBV HLA-A ^* 0201 ⁺ cells (see below), with frequencies of 1/39000 (HMFG) and 1/33000 (VNTR).
p1-30), which was immunized with the mannan-VNTR peptide (1 /
48000), that is, all HMFGs are VNTRs.
It is as immunogenic as (Table 2). Furthermore, extracellular peptides (31-55)
, The CTLp frequency is 1/40000, that is, VNTR
Was the same as that observed in. Thus, HLA-A ^* 0201 can display extracellular and VNTR peptides. If the target cells are EBV-transformed B cells that express HLA-A ^* 0201 but not H- ^2b class I molecules (expressed by immunized mice), the detected CTL is MU.
It should be noted that it is restricted to HLA-A ^* 0201 which represents the C1 peptide.

3.5 Mannan-H in A2K ^b MUC1 Double Transgenic Mice
Cellular immune response to MFG To confirm the ability of MUC1 CTL to lyse MUC1 positive breast cancer cells,
A2K ^b MUC1 double transgenic mice injected with mannan HMFG three times were treated with HMFG, VNTR peptide (p1-30) and extracellular peptide (31-).
55, 344-364), or intracellular peptides (408-423, 471-4).
93, 507-526) and stimulated in vitro (Table 2). All H
The CTLp frequency was 1/2000 in MFG and 1/8000 in VNTR region peptide p1-30. CTL is 1/2000 and 1/11000 respectively
Of the extracellular peptides 31-55 and 344-364.
In intracellular peptides, CTLs were found to have peptide 408-42 at a frequency of 1/20000.
Only 3 was detected.

The spleens of immunized mice were used in a direct CTL assay for anti-MUC1 CT.
The specificity of L was confirmed. As shown in FIG. 2, the MUC1 CTL has E
55% MUC1 + MCF7 (HLA-A ^* 0201) breast cancer cells with a 12: 1: T ratio were lysed and reduced to 17% when incubated in the presence of cold K562 target. MUC1 CTL is MUC1 ⁺ BT20 (HLA-
A1) HLA restricted so that no lysis was detected when using the breast cancer cell line. This MUC1 CTL is a MUC1-ve melanoma cell line ME
272 did not dissolve.

Thus, immunization of A2K ^b MUC1 mice with mannan-HMFG results in specific class I-restricted CTLs capable of lysing native MUC1-expressing tumor cells, and further anti-MUC1 CTLs. Is a human MU expressed extracellularly
It can be produced in mice in the presence of C1.

3.6 T Cell Epitope Prediction and Mapping In order to accurately map the T cell epitopes involved in CTLp generation, a large number of overlapping 9-mer peptides need to be synthesized and used in CTL assays. Indeed, the CTL epitope prediction program was used to select putative immunogenic peptides, and these were synthesized to examine their antigenicity.

Deduced H-2 ^d restriction peptide (intracellular region MUC1) Several peptides (NYGQLDIFP (K ^d ) SEQ ID NO: 13; YGQLDIFPA (D ^d ) SEQ ID NO: 14; KNYGQLDIF (L ^d ) SEQ ID NO: : 15) is contained in 471-493 (C
TLp frequency = 1/12500), predicted to be 6, 6 and 10 respectively (Table 3
). To confirm whether this predicted 9-mer is presented by class I molecules, a cytotoxic T cell assay was performed using mannan-HMFG as an effector.
Spleen cells from immunized mice were used and P815 target cells were pulsed with synthetic peptides. These are NYGQLDIFP (K ^d ) (SEQ ID NO: 13); YGQLDIFPA (D ^d ).
(SEQ ID NO: 14); KNYGQLDIF (L ^d ) (SEQ ID NO: 15). The pulsed cells were not lysed by mannan-HMFG-induced CTL in BALB / c mice (Fig. 3a). That is, the CTL epitope was not accurately predicted by the algorithm. Previously identified as a CTL epitope in the VNTR region (3
8) MUC1 VNTR peptides SAPDTRPAP (D ^d ) (SEQ ID NO: 16) and APDTR
PAPG (L ^d ) (SEQ ID NO: 17) was used as a positive control, 50: 1
A dissolution rate of 62% and 50% was obtained in the E: T ratio of. Listeriolysin K ^d peptide (GYKDGNEYI; SEQ ID NO: 18) and H used as negative controls
The IV D ^d peptide (RKSIRIQRGPGRAFVTIGKGKGKGY; SEQ ID NO: 19) gave no lysis (Fig. 3a).

Predicted H-2 ^d Restricted Peptides (Extracellular Domain MUC1) Many 9-mer peptides in the extracellular domain are predicted to be CTL epitopes [
(AVSMTSSVL (K ^d ), SEQ ID NO: 20; TTQGQDVTL (K ^d ), SEQ ID NO: 21; NAVSMTSSV
(K ^d ), SEQ ID NO: 22; TSATQRSSV (K ^d ), SEQ ID NO: 23; SSTTQGQDV (K ^d ), SEQ ID NO: 24; SVPSSTEKN (D ^d ), SEQ ID NO: 25; EPASGSAAT (L ^d ), sequence Number: 26
SPGSGSSTT (L ^d ), SEQ ID NO: 27; VPSSTEKNA (L ^d ), SEQ ID NO: 28; TPGGEKET
S (L ^d ), SEQ ID NO: 29; TSATQRSSV (L ^d ), SEQ ID NO: 30; SSTTQGQDV (L ^d ), SEQ ID NO: 24], contained in peptide 33-103 (CTLp frequency = 1020150), These are 58, 40, 29, 10, 10, 2.9, 39, 39, respectively.
, 36, 30, 10, and 10. A subset of these peptides was also included in the 51-70 peptide (CTLp frequency = 1/10000) (Table 3). Four of these were prepared (AVSMTSSVL (K ^d ), SEQ ID NO: 20, NAVSM
TSSV (K ^d ), SEQ ID NO: 22, VPSSTEKNA (L ^d ), SEQ ID NO: 28; SVPSSTEKN (D ^d ),
SEQ ID NO: 25), tested. Three of these four peptides were certainly shown, but one was not. Synthetic peptide AVSMTSSVL (K ^d ), SEQ ID NO: 20, N
AVSMTSSV (K ^d ), SEQ ID NO: 22, VPSSTEKNA (L ^d ), SEQ ID NO: 28 sensitized P815 target cells at 77%, 80% and 78% with an E: T ratio of 50: 1, respectively. However, SVPSSTEKN (lowest predictive value) was inactive (Fig. 3b). Therefore, AVSMTSSVL, SEQ ID NO: 20; VPSSTEKNA, SEQ ID NO: 28, and NAVSMT
SSV, SEQ ID NO: 22 is a CTL on peptides 33-103 and 51-70
It is an epitope.

Predicted H-2 ^b Restricted Peptides There are a small number of identified peptide epitopes in C57BL / 6 mice, but with a low score, there are a large number of potential CTL epitopes in the peptide (Table 3). 9-mer CRRKNYGQL (D ^b , K ^b ), SEQ ID NO: 32 is 471-493 (C
No TLp detected) and had scores of 10 and 1.4. It weakly sensitized the RMA target and dissolved with Mannan-HMFG CTL, 50:
E: T of 1 dissolved 20% and E: T of 100: 1 dissolved 42% (Fig. 3c). M
UC1 VNTR peptide APGSTAPPA (D ^b ), SEQ ID NO: 33 and SAPDTRPAP (
^Kb ), SEQ ID NO: 16 was used as a positive specificity control, where 7
0% and 80% lysis was obtained, but ovalbumin ^Kb 9mer SIINFEKL, SEQ ID NO:
No lysis was detected for the 11 and adenovirus D ^b 9mers (
Used as a negative specificity control). 9-mer peptide STEKNAVSM (D ^b ).
, SEQ ID NO: 34; AVSMTSSVL (D ^b ), SEQ ID NO: 20 and AVSMTSSVL (K ^b ), SEQ ID NO: 20 are contained in peptides 33-103 and 51-70 and scored at 15, 10 and 1.2. Had. All three of these peptides weakly sensitized and dissolved the RMA target (~ 20% at 50: 1, ~ 4 at 100: 1 E: T).
0% dissolution) (Fig. 3d). C57BL / 6 mouse peptides 31-55 and 5
There was no CTL reactive with 1-70.

Two high-score CTL epitopes deduced from all MUC1 molecules, intracellular region (YYQELQRDI (K ^d ), SEQ ID NO: 35 score 2880) and extracellular region VNTR from the N-terminus (SAPDNRPAL (D ^b ), sequence No .: 36 score 4723) is 2880
RMA and P815 target cells were sensitized and lysed 50% with 50: 1 E: T (Fig. 3e), with a score of 4723 and 4723. Therefore, some T cell epitopes are present in the non-VNTR region of the MUC1 molecule and a 9-mer peptide can be presented by target cells to CTLs produced by mannan-HMFG immunization.

4. Discussion Previous immunological studies by the present inventors used a MUC1 fusion protein (MFP) containing five repeats of VNTR linked to mannan, which is IFN-γ, I.
L-12 gave rise to a strong cellular response to MUC1 characterized by the production of very little IgG _2a antibody and protection from tumor growth (36,48). Also, the immune response in humans shows promise for therapeutic use of the MUC1 antigen in Phase I clinical trials with MFPs, with 4 out of 15 patients producing proliferative responses and 1 out of 25
Three showed high levels of MUC1-specific serum antibodies and two out of ten developed CTL against MUC1 (59). However, in vitro peptide binding studies and in vivo studies with transgenic HLA-A ^* 0201 mice show that the VNTR sequence is only presented by HLA-A ^* 0201 and HLA-A ^* 1101. (39, 60) and thus the study is MUC1 VNTR
Concentrated on. It is because of its preferential immunogenicity in mice, at least with respect to antibodies, and evidence from humans that implicates VNTRs in immune responses. MUC
Another protein sequence of 1 was never examined for its cellular immunity. In the past, we sought monoclonal antibodies against the non-VNTR region in MUC1-immunized mice, but with no results, and no international studies found anything. By scanning the entire MUC1 sequence for potential T cell epitopes, many peptides not previously tested were deduced. We therefore immunize mice with mannan-conjugated HMFG and rely on natural antigen processing for its presentation of all possible M
It provided the UC1 epitope and showed that a cellular immune response against the non-VNTR region of MUC1 could be generated. This was as effective as that generated against VNTR, further suggesting that both HLA-A ^* 0201 and A2K ^b MUC1 transgenic mice were immunized and that humans could be immunized as well. There is.

Cellular response was determined by mannan-HMFG immunization BALB / c, C57BL / 6, H
Of LA-A ^* 0201 / K ^b and double transgenic A2K ^b MUC1 mouse, the extracellular region of MUC1, it could be detected in VNTR, and intracellular peptide. Immunized BALB / c mice are more non-VN than C57BL / 6 mice.
It generated CTLs that were more reactive to the TR CTL epitope. This is 3
The 44-364 peptide and SAPDNRPAL (SEQ ID NO: 36) were recognized by CTL (Table 2, Figure 3e).

Of the various peptides used for restimulation, several potential 9-mer epitope candidates could be deduced using the peptide motif search program (Table 3). In BALB / c mice, the precursor frequency for the 471-493 peptide was 1 / 12,500, but the putative epitope peptide NYGQLDIF
P (SEQ ID NO: 13), YGQLDIFPA (SEQ ID NO: 14) and KNYGQLDIF (SEQ ID NO: 15) were unable to sensitize the P815 target for lysis by mannan-HMFG CTL (Fig. 3a). Therefore, stimulating CTL epitopes are not correctly identified by the algorithm, and these synthetic peptides are not properly processed and presented by target cells. In contrast, 33-103 and 51-7
Some 9-mers present in the 0 sequence (AVSMTSSVL SEQ ID NO: 20; NAVSMTSSV SEQ ID NO: 22 and VPSSTEKNA SEQ ID NO: 28) were functional CT in the lysis assay.
It was identified as an L epitope (Fig. 3b).

In C57BL / 6 mice, CRRKNYGQ of 51-70 and 471-493 sequences.
The L (SEQ ID NO: 32), STEKNAVSM (SEQ ID NO: 34) and AVSMTSSVL (SEQ ID NO: 20) peptides sensitized RMA cells for lysis, but CTLp was not identified by restimulation with the larger peptide. . Such observations are MUC1 ⁺
It could be obtained from such three 9-mers which were not treated and displayed by the cells.

Mouse K ^d , D ^d , L ^d , K ^b , D ^b , K ^k and human HLA-A1, HLA-A ^* 0
Further analysis of the entire MUC1 sequence using the T cell epitope algorithm for the 201, HLA-A3 and HLA-A24 epitopes reveals some candidate 9-mers for presentation by mouse or human cells. Of these 9-mer peptides, SAPDNRPAL (D ^b ), (SEQ ID NO: 36) and YYQELRDI (K ^d ) (SEQ ID NO:
35) were both highly efficient in stimulating P815 or RMA cells for lysis by mannan-HMFG CTL (Fig. 3e).
. From this and other studies it is clear that the estimation of CTL epitopes is not always accurate. Comparison of putative and experimentally examined T cell epitopes for the VNTR region shows that lower scores do not necessarily predict presentation or lack of antigenicity (Table 4). For example, SAPDTR
Although the PAP (SEQ ID NO: 16) peptide was confirmed to be a ^Kb -restricted epitope by class I stabilization when incubated with TAP-deficient RMA-S cells and by lysis of peptide-pulsed RMA cells. (FIG. 3c), the estimated score is only 0.004 (38). Similarly, K ^k , L ^d and D ^d were not accurately estimated (38). The HLA-A ^* 0201 T cell epitope, STAPPAHGV (SEQ ID NO: 37), independently identified by epitope mapping (39), was predicted, although it had a low score. Although the predictive algorithm serves as a guide to the potential for antigen presentation, in vivo response is dependent on antigen processing, immunodominance, T
It is determined by cell repertoire, glycosylation and other unknown factors [61,62].

Although the purified form of the entire MUC1 protein has not been used to immunize mice to generate cell-mediated immunity, several other immunization methods have been used. The entire MUC1 protein is retained in vaccinia constructs [46, 63], as a component of DNA immunization [64], in transfected dendritic cells [65] and in transfected EBV-B cells [66]. Was there. The specificity of CTL was not confirmed in these studies. However, glycosylated MU
The importance of using C1 (HMFG) should be emphasized. Another study in mice and humans used non-glycosylated peptides that led to antibody production in both MUC1 transgenic mice [67] and humans [59, 68, 69]; these studies used B cells and sometimes T cell tolerance was overcome, but as for antibodies, the non-glycosylated peptides presented new antigens and the response was not considered surprising. However, in the study described here, native glycosylated mucin (HMFG) linked to mannan successfully induced CTL in several strains of mice, including A2K ^b MUC1 transgenic mice. Mannan-HMFG confers higher CTLp frequencies on A2K ^b MUC1 mice (1/2000) compared to BALB / c or C57B1 / 6 mice, which is different strains of mice or higher affinity HLA-A ^*. 020
This is due to the presence of the 1CTL epitope. In BALB / c mice, HMG
F gave a CTLp frequency of 1/80500. This is the non-glycosylated form of M
CTLp frequency was comparable in mice immunized with UC1 VNTR [47]. That is, both glycosylated and non-glycosylated forms of VNTR
It was equally immunogenic when presented with oxidized mannan. Apparently, the carbohydrate coating did not obscure the underlying peptide. Thus, mannan-HMFG can disrupt the tolerance of A2K ^b MUC1 transgenic mice by producing CTL for VNTR, extracellular and intracellular peptides in MUC1. These results reinforce the notion that MUC1 is a valuable target in therapy.

The use of mannan-HMFG in humans ensures some controversy that MUC1 is present in some normal cells such as the pancreas, kidney. Thus, an immune response can be generated in these tissues resulting in autoimmunity. Thus, in our clinical trial using MUC1 VNTR conjugated to mannan, no autoimmunity was detected, but careful expansion studies and monitoring are required [59].
. HMFG obtained directly from a donor is less preferred for use and recombinant material may be more suitable. However, high levels of glycosylation of HMFG should be sought using recombinant materials. Probably
Eukaryotic vectors will be needed. Third, we have recently shown that VNTR peptides can evade the immune response to antibodies by cross-reacting with existing natural human antibodies [70]. Such deviation can occur when using the entire MUC1.

Example 2 A non-VNTR peptide was coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde and then reacted with oxidized mannan as follows. 2 mg of peptides 471 to 507 were dissolved in 1.75 ml of phosphate buffer, mixed with 0.25 ml of KLH (2 mg / ml) and treated with 0.25% glutaraldehyde at room temperature in the dark. Mix overnight. The mixture was dialyzed overnight against phosphate buffer. The dialyzed mixture was mixed with 1 ml of oxidized mannan prepared as described in European patent application 94303817.4 and left overnight.

BALB / c mice (6-8 weeks) were immunized intraperitoneally with 5 micrograms of the mannan-peptide KLH on days 0, 10 and 17 and splenocyte (splenocyte).
) Activity in) was measured as described. Non-VNTR bound to mannan
Peptide is a positive control (VNTR peptide bound to mannan)
A positive reaction was shown in the CTL assay compared to (Figs. 4 and 5).

Example 3 Non-leader, non-VNTR peptides and polypeptides are also used for the preparation of DNA vaccines. This can be done using established techniques in DNA cloning and nucleic acid inoculation. For example, nucleic acid sequences encoding one or more non-leader, non-VNTR peptides and polypeptides with the necessary restriction enzyme sites at the 3'and 5'ends were synthesized on an automated DNA synthesizer, such as pcDNA3 or pSV3. It can be cloned into an appropriate vector [72].
This clone can be screened for nucleic acid sequence incorporation by restriction enzyme cleavage or protein expression. This DNA can be injected at various sites in humans and other animals for immunization.

“References”

Those skilled in the art can make numerous changes and / or modifications to the invention described in a particular embodiment without departing from the broadly described spirit or scope of the invention. You will understand. Therefore, the embodiments of the present invention are considered in all respects to be illustrative and not restrictive.

[0079]

[Table 1]

[0080]

[Table 2]

[0081]

[Table 3a]

[Table 3b]

[0082]

[Table 4]

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of human MUC1 protein (NCBI database
No.M61170).

FIG. 2. HMFG and mannan assay. (A) Inhibition of anti-MUC1 antibody binding to HMFG by preparing a competitor of HMFG (m) and mannan-HMFG (l). (B) Mannan-HMFG (l) and HMFG against anti-MUC1 antibody
ConA detected by binding of (m) and radioimmunoassay.

FIG. 3: Mannan-H of A2K ^b MUC1 double transgenic mice
Immunized with MFG and splenocytes were used for CTL assay. The cytotoxic activity of effector cells with the following components (n) or without cold K562 (l);
⁵¹ Cr-labeled MCF7 without BT20 (p) or without ME272 (m)
It was measured at.

FIG. 4: C57BL / 6 and BALB / c mice were loaded with mannan-HMF.
Immunization with G and splenocytes were used in the CTL assay. Intracellular peptide 47
P815 (a) or RMA pulsed with various 9-mer peptides 1-449
(C) Cell lysis; lysis of P815 (b) or RMA (d) cells pulsed with various 9-mer peptides of extracellular peptides 33-103 and 51-70, and (e) YYQELQRDI (SEQ ID NO: 35). Pulsed P815 cells and SAPDNRPAL
Lysis of RMA-MUC1 cells pulsed with (SEQ ID NO: 36). Known peptide antigens were used as controls for peptide pulsing and antigen-specific cell lysis. Shown on each panel and described in the text.

FIG. 5: Balb / c mice were immunized with mannan-507-KLH and splenocytes were immunized with C
Used for LT assay. Cp13-32 at various effector: target ratios
Alternatively,% lysis of ⁵¹ Cr-labeled P815 target cells unpulsed or pulsed with 507 peptide was measured.

FIG. 6: Balb / c mice were immunized with mannan-471-KLH and splenocytes were C-immunized.
Used for LT assay. Cp13-32 at various effector: target ratios
Alternatively,% lysis of unpulsed or pulsed ⁵¹ Cr-labeled P815 target cells with 471 peptide was measured.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 37/04 C07K 14/705 C07K 14/435 19/00 14/705 C12N 15/00 ZNAA 19/00 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, B A, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU , ZA, ZW (72) Inventor Joff A. Peters Australia, Victoria, 3088, Greensborough, Jeanvanna Street, 10 (72) Inventor, Basso, Apostropoulos, Australia, Victoria, 3021, Saint Albans Buham Street 14F Term (reference) 4B024 AA01 BA36 BA80 CA02 CA07 DA03 GA11 HA17 4C084 AA02 AA07 BA19 NA14 ZB26 4C085 AA03 BB01 BB11 CC23 4C086 AA01 AA04 EA16 MA01 MA04 NA14 ZB09 ZB10 A30 A10 A30 A10 A30 A10 A30 A11 A36 A50 CA50 DA86 EA20 EA50 FA20 FA74

Claims (47)

[Claims] 1. A peptide or polypeptide capable of inducing an immune response, wherein the peptide or polypeptide is a mucin non-VNTR,
A peptide or polypeptide comprising an amino acid sequence substantially corresponding to the amino acid sequence of the epitope of the non-leader region. 2. The peptide or polypeptide according to claim 1, which comprises an amino acid sequence derived from the non-VNTR, non-leader region of mucin. 3. The peptide or polypeptide according to claim 1, wherein the epitope is derived from the extracellular region of the non-VNTR, non-leader region of mucin. 4. The peptide or polypeptide according to claim 1, wherein the epitope is derived from the intracellular region of the non-VNTR, non-leader region of mucin. 5. The peptide or polypeptide according to claim 1, wherein the epitope is derived from a non-VNTR, non-leader transmembrane region of mucin. 6. The peptide or polypeptide according to any one of claims 1 to 5, wherein the mucin is mucin 1 (MUC1). 7. The peptide or polypeptide according to claim 6, wherein the mucin 1 is human mucin 1. 8. The peptide or polypeptide according to claim 7, wherein the human mucin 1 is human milk fat globule membrane antigen (HMFG). 9. The epitope is AVSMTSSVL (SEQ ID NO: 20) or NAVSMTS.
The peptide or polypeptide according to claim 3, which has an amino acid sequence selected from SV (SEQ ID NO: 22), VPSSTEKNA (SEQ ID NO: 28) and SAPDNRPAL (SEQ ID NO: 36). 10. The peptide or polypeptide according to claim 4, wherein the epitope has the amino acid sequence: YYQELQRDI (SEQ ID NO: 35). 11. The following amino acid sequence: TGSGHASSTPGGEKETSATQRSSVP (SEQ ID NO: 2) RSSVPSSTEKNAVSMTSSVL (SEQ ID NO: 3) SGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATW.
(SEQ ID NO: 4) SAPDNRPAL (SEQ ID NO: 6) NSSLEDPSTDYYQELQRDISE (SEQ ID NO: 7) TQFNQYKTEAASRVNL (SEQ ID NO: 8) AVCQCRRKNYGQLDIFPARDTYH (SEQ ID NO: 9) and YVPPSSTDRSPYEKVSAGNG (SEQ ID NO: 10) or one of its immunogenic fragments 3. A peptide or polypeptide according to claim 1 or 2 comprising an amino acid sequence substantially corresponding to one. 12. A fusion protein comprising the peptide or polypeptide according to any one of claims 1 to 11 and a suitable carrier protein. 13. The fusion protein of claim 12 conjugated to a carbohydrate polymer. 14. The carbohydrate polymer is glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine,
Rhamnose, 6-o-methyl 1-D-galactose, 2-o-acetyl-β-D-xylose, N-acetyl-glucosamine, iduronate, gluronate, manuronate, methylgalacturonate, α-D-galactopyranose 6- Is a polymer of carbohydrate monomer units selected from the group consisting of sulphate, fructose, alpha abequeose, and their conformers and configurational isomers, or of carbohydrates composed of two or more different types of said carbohydrate monomer units. The fusion protein according to claim 13, which is a polymer. 15. The fusion protein of claim 14, wherein the carbohydrate polymer comprises at least 20 monomer units. 16. The fusion protein of claim 15, wherein the carbohydrate polymer comprises more than 1000 monomeric units. 17. The fusion protein of claim 16, wherein the carbohydrate polymer comprises more than 10,000 monomeric units. 18. The fusion protein according to any one of claims 13 to 17, wherein the carbohydrate polymer is a polymer of mannose or a carbohydrate polymer containing mannose. 19. The fusion protein according to any one of claims 13 to 17, wherein the carbohydrate polymer is a polymer of oxidized mannose or is oxidized mannan. 20. The method according to any one of claims 1 to 1, which is bound to a suitable carrier protein.
1. The peptide or polypeptide according to any one of 1. 21. The peptide or polypeptide according to claim 20, wherein the peptide or polypeptide and / or the carrier protein is conjugated to a carbohydrate polymer. 22. The carbohydrate polymer is glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-o-methyl 1-D-galactose, 2-o-acetyl-β-D-xylose. , N-acetyl-glucosamine, iduronate, gluronate, manuronate, methylgalacturonate, α-D-galactopyranose 6-sulfate,
A polymer of carbohydrate monomer units selected from the group consisting of fructose, α-avequaose, and their conformers and configurational isomers, or a polymer of carbohydrates consisting of two or more different types of said carbohydrate monomer units. 22. The peptide or polypeptide of claim 21, which is: 23. The peptide or polypeptide of claim 22, wherein the carbohydrate polymer comprises at least 20 monomer units. 24. The peptide or polypeptide of claim 23, wherein said carbohydrate polymer comprises more than 1000 monomeric units. 25. The peptide or polypeptide of claim 24, wherein the carbohydrate polymer comprises more than 10,000 monomeric units. 26. The peptide or polypeptide according to any one of claims 21 to 25, wherein the carbohydrate polymer is a polymer of mannose or a carbohydrate polymer containing mannose. 27. The peptide or polypeptide according to any one of claims 21 to 25, wherein the carbohydrate polymer is a polymer of oxidized mannose or is oxidized mannan. 28. A compound comprising a conjugate of a peptide or polypeptide according to any one of claims 1 to 11 and a carbohydrate polymer. 29. A compound comprising a conjugate of mucin 1 and a carbohydrate polymer, wherein said conjugate is capable of inducing a cell-mediated immune response in humans or other animals. 30. The compound of claim 29, wherein the mucin 1 is human mucin 1. 31. The compound of claim 30, wherein the human mucin 1 is human milk fat globule capsule antigen (HMFG). 32. The carbohydrate polymer is glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-o-methyl 1-D-galactose, 2-o-acetyl-β-D-xylose. , N-acetyl-glucosamine, iduronate, gluronate, manuronate, methylgalacturonate, α-D-galactopyranose 6-sulfate,
A polymer of carbohydrate monomer units selected from the group consisting of fructose, α-avequaose, and their conformers and configurational isomers, or a polymer of carbohydrates consisting of two or more different types of said carbohydrate monomer units. 32. A compound according to any one of claims 28 to 31. 33. The compound of claim 32, wherein the carbohydrate polymer comprises at least 20 monomer units. 34. The compound of claim 33, wherein the carbohydrate polymer comprises more than 1000 monomeric units. 35. The compound of claim 34, wherein the carbohydrate polymer comprises more than 10,000 monomeric units. 36. The compound according to any one of claims 28 to 35, wherein the carbohydrate polymer is a polymer of mannose or a carbohydrate polymer containing mannose. 37. The compound according to any one of claims 28 to 35, wherein the carbohydrate polymer is a polymer of oxidized mannose or is oxidized mannan. 38. A peptide or polypeptide according to any one of claims 1 to 11 or 20 to 27 or a fusion protein according to any one of claims 12 to 19 and optionally an adjuvant and And / or a vaccine or therapeutic agent containing a pharmaceutically acceptable carrier. 39. A vaccine or therapeutic agent comprising the conjugate compound according to any one of claims 28 to 37 and optionally an adjuvant and / or a pharmaceutically acceptable carrier. 40. An effective amount of the peptide or polypeptide according to any one of claims 1 to 11 or 20 to 27, or the fusion protein according to any one of claims 12 to 19, optionally. , A method of inducing a cell-mediated immune response against mucin comprising administering to a patient in combination with an adjuvant and / or a pharmaceutically acceptable carrier. 41. Administering to a patient an effective amount of a conjugate compound according to any one of claims 28 to 37, optionally in combination with an adjuvant and / or a pharmaceutically acceptable carrier. A method of inducing a cell-mediated immune response against mucin. 42. A method of preventing or treating carcinoma of a patient, comprising administering to said patient the vaccine or therapeutic agent of claim 38 or 39. 43. The method of claim 42, wherein the carcinoma is adenocarcinoma. 44. The method of claim 43, wherein the adenocarcinoma is breast cancer. 45. The peptide or polypeptide according to any one of claims 1 to 11 or 20 to 27, the fusion protein according to any one of claims 12 to 19, or the fusion protein according to any one of claims 28 to 37. Use of the conjugate compound according to any one of claims for pulsing dendritic cells for in vivo introduction and for use as a vaccine. 46. An isolated nucleic acid comprising a nucleotide sequence encoding a peptide or polypeptide according to any one of claims 1 to 11 or a fusion protein according to any one of claims 12 to 19. molecule. 47. A DNA vaccine comprising the nucleic acid molecule of claim 46.