CN1360595A - Immunogenic peptides derived from MAGE and use thereof - Google Patents
Immunogenic peptides derived from MAGE and use thereof Download PDFInfo
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- CN1360595A CN1360595A CN00809230A CN00809230A CN1360595A CN 1360595 A CN1360595 A CN 1360595A CN 00809230 A CN00809230 A CN 00809230A CN 00809230 A CN00809230 A CN 00809230A CN 1360595 A CN1360595 A CN 1360595A
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- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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Abstract
Peptides derived from proteins of the MAGE family and the use thereof as immunogenic agents in the treatment of tumors, the compositions containing them, a method for inducing a cytotoxic response against tumor cells, a melanoma cell line not expressing histocompatibility antigens and the applications thereof.
Description
The present invention relates to derive from the peptide of MAGE family protein, also relate to these peptides as the purposes of immunogen in tumor prevention and treatment.
In the past few years, use tumor-specific cytotoxicity T lymphocyte (CTLs) to be separated to the gene (Van den Eynde, B.J. etc., Curr.Opin.Immunol., 9:684-693,1997) of several encoding tumour antigen.According to the expression pattern in tumour and healthy tissues, these antigens can be divided into four classes according to tumour-specific and clinical correlation.The first kind is included in the antigen of the genes encoding of expressing in the various tumours of histological types (histotype) but not expressing in all healthy tissuess except that the testis sexual cell, for example: MAGE (melanoma associated antigen), GAGE and BAGE.Second class is represented differentiation antigen, only expresses in melanoma and melanophore, for example tyrosine oxidase, Melan-A/MART-1, gp100, TRP-1 and TRP-2.The antigen that belongs to the 3rd class is produced through point mutation by the gene of generally expressing.The 4th class antigen only just is being defined recently, and its representative is TRP-2-INT2, in a kind of melanoma common exist and the antigen of in the normal cell of melanocyte pedigree, not expressing.
The tumour antigen that particularly belongs to MAGE family causes sizable interest, because in them six, MAGE-1,2,3,4,6 and 12 is by quite a few former and metastatic tumor (comprising melanoma, lung cancer, bladder cancer, ovarian cancer and mammary cancer) selective expression (van der Bruggen, P., Deng, Science, 254:1643-1647,1991).
Derive from MAGE-1 albumen (van der Bruggen, P., etc., Science, 254:1643-1647,1991) and the interior nine amino acid peptide of processing of the proteic cell of MAGE-3, length is 8-10 amino acid, and is open and be considered to immunogenic substance (WO 95/19783).
Based on the vaccine inoculation clinical experiment of using MAGE-1 and MAGE-3 coding for antigens peptide, in melanoma and other tumour patient make progress on one's body (Machand, M. etc., Int.J.Cancer80:219-230,1999).Up to now, in fact MAGE-2,4 and 6 antigens can not be considered to tumour antigen, so they can not be as the target of tumour-specific immunotherapy.Reason is to be much to seek to discern the cytotoxic T lymphocyte that derives from MAGE-2,4 and 6 peptide.
Limitation based on the treatment plan that uses peptide is that the CTL epi-position that has characterized (that is to say, tumor antigen peptide and suitable HLA-I class allelotrope) be limited quantity, and produced in the body and lost antigenic varient, it can escape the immune response (Restifo that is caused by monoclonal antibody originality vaccine, N.P., etc., J.Natl.Cancer.Inst., 88:100-108,1996).Particularly, lose the generation of antigenic varient, be a major cause of immune programme for children failure, found that this is owing to select the antigenic tumour cell of not expressing by the identification of tumor response lymphocyte, for example as the result of molecular defect, cause allelic disappearance of HLA-I class or downward modulation (Garrido, F., etc., Immunol.Today, 14:491-499,1993).Yet in some cases, tumour loses or has changed the antigenic gene of codes for tumor (Jager, Int.J.Cancer such as E., 71:142-147,1997), escapes immune control with another kind of mechanism.
Usually, the clinical protocol of tumour-specific immunity only has been applied to carry and has expressed the patient who knows tumour antigen and the allelic tumour of definite HLA.Unfortunately, most of tumour patients do not satisfy these standards.Therefore, be necessary to identify new antigenic determinant to be used for antineoplastic immunization protocol, those of " wide spectrum " preferably, promptly expressed by greater amt tumor tissues type or in the various ways of same tumour, express.
Have now found that, and this also is a first aspect of the present invention,, differentiates epi-position into new HLA-B*3701 restriction by one group of peptide of MAGE-1,2,3,4,6 and 12 dna homolog regional codes.
According to their source, the peptide among the present invention is named as MAGE-1,2,3,4,6 and 12
127-136, and in SEQ ID NO.1-3, reported its sequence.MAGE-1,2,3 and 6 has identical sequence, and as described in SEQ ID NO.1, and the sequence of MAGE-4 and 12 is respectively SEQ ID NO.2 and 3.
Vitro cytotoxicity test has proved on cytotoxic T cell recognition expression MAGE-1,2,3,4, the 6 different antigenic various clones with 12 peptide of the present invention in conjunction with HLA-B*3701 histocompatibility allelotrope (allele), and is activated when being exposed to them.In these trials, the peptide with SEQ ID NO:1 sequence shows high reactivity, is preferred therefore.
Differentiate that the different antigenic common epitopes of MAGE family have superiority very much, can partly overcome above-mentioned shortcoming at least because it makes.The more important thing is, use this " many antigenicities " epi-position (pluriantigenic), that is to say, the epi-position that the more antigens of MAGE family are total, make and to overcome above-mentioned antigenic loss, because therefore common one above MAGE gene of tumour coexpression of determining very can not lose the immune response that peptide of the present invention induces.This just can improve patient's quantity that the clinical tumor immunity test can be successful.
Should point out emphatically, the immunogenicity potentiality of people MAGE-2 and MAGE-6 never were proved to be, even known 12% oophoroma lacks MAGE-1 and MAGE-3 and expresses MAGE-2 and/or MAGE-6.
The enough conventional art preparations of Toplink among the present invention, preferably preparation synthetically, for example according to Merrifield, (1986) science (Science) 232:341-347 and Barany and Merrifield, peptide (The Peptides), Gross and Meienhofer compiles, (N.Y., Academic Press), the described method of 1-284 page or leaf (1979) is carried out.Synthetic can carry out (Stewart and Young, Solid Phase PeptideSynthesis, the 2nd edition, Rockford Ill., Pierce Chemical Co. (1984)) in liquid phase or solid phase or with automatic DNA synthesizer DNA.Perhaps, peptide also can begin preparation with recombinant DNA technology or from containing required segmental natural precursor protein.Amino-acid residue can for example be attached on the fat by chemically modified, glycosylation and with other peptide coupling, to obtain how favourable characteristics, for example higher with HLA molecule avidity, immunogenicity is stronger, and it is higher to induce the immunne response selectivity, or higher bioavailability is arranged after using.Peptide also can induce the epi-position coupling that T " assists " cell response with other known MHC molecule by identical or different type.
The present invention further also relates to the pharmaceutical composition of the described peptide here that contains significant quantity.According to embodiment preferred, said composition is a vaccine, particularly is suitable for cancer susceptibility patient's preventative immunoprophylaxis or the therapeutic immunoprophylaxis in tumour patient.Except that active ingredient, said composition will comprise pharmaceutically acceptable vehicle." significant quantity " refers to the amount that is enough to excite the effective CTL at tumour to reply.This amount depends on employed peptide, route of administration and type, pathology severity to be treated and patient's generalized case, and usually should amount at 100-300 μ g, 1 month or shorter time are with 3 times or more times administration at interval.Usually carry out twice subcutaneous vaccination and twice intradermal inoculation, dosage is that each subcutaneous vaccination is 4/10 of a total dose, and each intradermal inoculation is 1/10 of total dose.Change inoculation position when inoculating as far as possible at every turn.The preparation of vaccine and use technology are known by these those skilled in the art, also be described in for example Paul, FundamentalImmunology (basic immunology), Raven Press, New York (New York) (1989) or Cryz, S.J., immunotherapy and vaccine (Immunotherapy and Vaccines), VCHVerlagsgesselschaft (1991).Vaccine is prepared into the form of injectable suspension or solution usually, but they also can use with the form of solid-state or liposome.Immunogenic components can with acceptable mixed with excipients on the pharmacology, for example adjuvant of emulsifying agent, buffer reagent and enhancing vaccine effect.Vaccine can be by single dose or multiple dose administration.Under the multiple doses situation, 1 to 10 independently dosage is provided, each contains 1 μ g-1000 μ g antigen, and at interval certain hour is kept or booster immunization reacts necessary other booster immunization subsequently, and if desired, and immunity is once again after the some months.Under every kind of situation, processing scheme will depend on by the process of treatment reaction, its general situation and tumour.
On the other hand, the invention provides a kind of method, to induce at the cytotoxic reaction of expressing the antigenic tumour cell of one or more MAGE, this method comprises makes peptide of the present invention contact under the condition that is suitable for the activating cytotoxic T cell with the T lymphocyte.Directly exposing the T lymphocyte when being included in cultivation for the conditions suitable that obtains the ideal cytotoxic effect gives described peptide or makes described peptide in conjunction with HLA I quasi-molecule in advance, preferred HLA-B*3701, or prophylactically make described peptide in conjunction with the antigen presenting cell (APC) of expressing described HLA molecule, then be exposed to the T lymphocyte.Proper A PC cell is peripheral blood self mononuclearcell (PBMC) or dendritic cell, scavenger cell and activatory B cell.The APC that give to cultivate adds the sufficiently long time of peptide, forming peptide/APC combination, and adds subsequently and contains the lymphocytic cell mass of T and make it activation and propagation.Lymphocyte can be taken from subject patient, and the activation back feeds back to same patient.Can strengthen peptide/APC combination by preventative " peeling off " (" stripping ") of the histocompatibility molecule that exists on the APC cell.Selectively, the APC cell can be transformed into and express HLA-B*3701 histocompatibility allelotrope.And can comprise one or more in the substratum and help to improve CD8
+Precursor activatory cytokine.Feeding back to before the patient, lymphocyte can be by for example ligands specific affinity column purifying.
The invention still further relates to specific recognition by HLA I quasi-molecule, preferred HLA-B*3701 molecule and be selected from the cytotoxic T cell system of the mixture that the peptide of SEQ ID1-6 forms.Cytotoxic T cell system can be by obtaining by the activatory cell when being exposed to the tumour cell that contains antigenic determinant described herein from the B lymphocyte repertoire screening.
According to a further aspect, the invention provides and induce immunoreactive cell, for example APC, dendritic cell etc., the carrier of the encoded peptide of the present invention of described cell (for example virus or retroviral vector, for example derive from adenovirus or slow virus or MLV) transformation, peptide of the present invention is taked the fusion rotein form with the fusion of suitable carrier alternatively, expresses effectively in cell, and processes and be exposed to cell surface.In this case, the DNA of code book disclosure of the Invention epi-position is inserted in the suitable expression vector, make under its control that is in suitable promotor, the example of described promotor is a viral promotors, for example CMV and SV40 (if desired very high level expression), or the inducible promoter inducible promoter of moulting hormone (ecdisone) control for example.In this case, described epi-position is by the zone (Genbank of MAGE-1 cDNA corresponding to the 127-136 amino acids, N.M77481) other antigenic homologous region coding and in the MAGE family: amino acid MAGE-1 aminoacid sequence Nucleotide (cDNA) 127-136 REPVTKAEML 562-590
According to a further aspect, the present invention relates to a melanoma cell series, MSR3-mel by name also is kept at CBA-joint laboratory clone preservation center (Interlab Cell line Collection)-(Genua, Italy), be numbered N.PD99001, the histocompatibility antigen that its expression can not detection level.In embodiment 1 more detailed description is arranged, this clone can be used any allelic cDNA transfection of coding histocompatibility system, subsequently the tumour antigen that it is not expressed with coding or the cDNA transfection or the transduction of its fragment or virus antigen.And then be used for externally inducing the antigen-specific effector thing or being fed back in patient's body according to the application of active immunity treatment in the body.In preferred embodiments, tumour antigen is a melanoma antigen, and the histocompatibility molecule is HLA I class B*3701.Cytotoxic T lymphocyte stripped that MSR3-mel clone can be advantageously used in antigen-specific induces or increases being used for adoptive immunotherapy, and/or is used to identify new antigenic determinant.And, use the transformed MSR3-mel clone of gene genetic of coding HLA molecule and/or tumour antigen can be used as vaccine.
According to a further aspect, the present invention relates to antibody, their fragment or derivative at above-mentioned peptide.The general method that produces antibody is well-known and is described in Kohler and Milstein, nature (Nature) 256 (1975), 494 or J.G.R.Hurrel, mono-clonal hybridoma antibody: technology and application (Monoclonal Hybridoma Antibodies:Techniques andApplications), CRC Press Inc., Boco Raron, FL (1982).Antibody comprises mono-clonal (mAb) or polyclonal, and is preferably monoclonal, and their fragment can be F (ab ') 2, Fab, Fv or scFv.
Marginal data
Fig. 1: MSR3-mel and MSR3-B37 express HLA I quasi-molecule.Tumour cell hatches, cleans and use the sheep anti mouse Ig antibody labeling of coupling fluorescein with mAbW6/32 (anti-HLA-I class) or isotype contrast.Before HLA-B*3701 transfection MSR3-mel and analyze afterwards.
The antigen of Fig. 2: CTLs 337 identification HLA-B*3701 restrictions.Assess the cytotoxic activity of CTLs337 with allogeneic melanoma ETl from autologous melanoma MSR3mel and MSR3-B37 and different effect thing/target (E/T) ratio.
The evaluation of the tumour antigen of Fig. 3: CTLs 337 identifications.Separately with HLA-B*-3701 or with the cDNA cotransfection Cos-7 cell of coding MAGE-1,2,3,6 and 12 genes.After 48 hours, the IFN-that adds CTLs 337 and after 24 hours, discharge according to " materials and methods " described measurement.MSR3-B37 is as positive control.
Fig. 4: A) CTLs 337 identification polypeptide MAGE.
127-136MSR3-EBV and the peptide MAGE. that begins three times of dilutions from 10mM
127-136Hatch and in the standard cell lines toxicity test, be used as target cell.E/T was than stuck-at-0: 1.Half peptide amount that needs of maximum cracking is expressed as ED
50B) with competitive trials assessment peptide M4.
127-136And M12.1
127-136Combination to HLA-B*3701.The competition peptide comprises M4.
127-136Peptide KELVTKAEML and M12.1
127-136Peptide REPFTKAEML.Can not be in conjunction with the M3.A1 of HLA-B*3701 molecule (M3. just
271-279) peptide is as negative control.The cracking of not competing peptide is 52%.
Fig. 5: CTLs 337 identification MAGE-6 male melanoma cell series.The HLAB*3701 feminine gender be the Me 14932 and the HLA-B*3701 positive be Me 14932-LB37 with or need not 16 μ M peptide MAGE.
127-136Stimulate, and in standard cell lines poison test, be used as target cell according to specified E/T ratio.
Materials and methods
Synthetic peptide
Synthetic peptide is available from PRIMM (Milan, Italy). Peptide is MAGE.127-136(REPVTKAEML), By MAGE-1,2,3 and 6 gene 127-136 bit codons coding; M4.127-136(KELVTKAEML) and M12.127-136(REPFTKAEML), correspond respectively to of MAGE-4 and MAGE-12 gene code The 127-136 amino acids. Peptide is dissolved into 10mM in DMSO, and further rare with 0.9%NaCl Release.
The allelic subclone of HLA-B*3701
With RNeasy Total RNA Kit (QIAGEN, Hilden, Germany) from MSR3 PBLs Prepare total RNA. With the oligo-dT primer with there is not the Moloney muroid leukaemia of RNase-H activity The reverse transcriptase of viral source (MMLVRT RNase-H-Superscript; Gibco BRL, Gaithersburg, MD) carry out strand cDNA according to manufacturer's suggestion from the total RNA of 2 μ g and close Become. With being suitable for a pair of of HLA-B allele full length coding region specific amplification and directed cloning The primer PCR amplification is corresponding to the cDNA of the total RNA of 300ng. 1.1kb the PCR-product by subclone Advance carrier for expression of eukaryon pcDNA3.1 (Invitrogen Corporation, Oxon, U.K.). With diagnostic Restriction Enzyme identification code HLA-B*3701 or the B*52011 (HLA-B37 of patient MSR3 With B5 allele) plasmid clone. Again to this HLA-B*3701 gene sequencing to confirm and to deliver The dna sequence dna correspondence. This plasmid is known as pcDNA3.1/B*3701.
The transfection of melanoma cell series
Also use with pcDNA3.1/HLA-B*3701 transfection melanoma cell series with the calcium phosphate precipitation technology G418 selects. Demonstrate,prove with HLA-A, B and C monoclonal antibody specific W6/32 at flow cytometer The expression of the HLA-B*3701 molecule of transfection in the tangible stable transfectant.
External evoked cytotoxic T lymphocyte is 337 (CTLs 337)
According to former other people scheme of describing (Van den Eynde, etc., Int.J.Cancer., 44:634-640,1989) to obtain CTL be 337 in a little change. In brief, use the Ficoll ladder Degree separates the PBLs of patient MSR3, and has replenished 10% human serum (HS), glutamine at 2ml With MSR3-B37 melanoma cells (0.5-1 * 10 of using among the antibiotic IMDM from the radiation of body5/ hole) cultivates (1-2 * 106/ hole). Cultivate after three days, adding 10U/ml IL-2 (Chiron, Milan, Italian) and 5ng/ml IL-7 (Genzyme Corp.). Lymphocyte uses weekly 0.5 * 105The MSR3-B37 cell of radiation stimulates again, and measures in cell toxicity test after stimulating three times. After stimulating again for the 5th time, add 2 * 106The LG2-EBV of LG2-EBV radiation is thin as feeder layer Born of the same parents and IL-2 bring up to 50U/ml.
The test of lysis activity and peptide binding
By aforementioned (Fleischhauer, K., etc., Cancer Res., 58:2969-2972,1998) Chromium-release test detects the lytic activity of cytotoxic T cell system. Use the chromium-release test detection of peptides:51Cr-The target cell of mark is cultivated at 96-hole trace before adding the effector cell with fixed effect thing/target ratio In the plate with variable concentrations peptide incubated at room 1 hour. By described in the past (Herman, J., etc., Immunogenetics, 43:377-384,1996) carry out peptide M4. with competitive trials127-136And M12.127-136Combination to the HLA-B*3701 molecule. As standard peptide, we have used CTLs 337 The peptide MAGE of identification127-136(300nM). E/T ratio by 30: 1 uses CTLs.
The generation of MAGE-1 subfragrnent
With Bgl II and EcoRI digestion MAGE-1 cDNA obtain MAGE-1 gene subfragment (495 and 1,072bp).Behind the sepharose purifying, fragment is cloned into pcDNA3.1 plasmid (Invitrogen).Separating clone, extracting plasmid DNA and with HLA-B*3701 gene transfection Cos-7 cell.
The transfection of Cos-7 cell and γ-IFN release test
Carry out Cos-7 cell transfecting (Coulie, P.G. is etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.), 91:6458-6463,1994) with DEAE-dextran-chloroquine method.In brief, contain the expression vector transfection 1.5 * 10 of gene M AGE-1,2,3,4, one of 6 and 12 cDNA with 100ng pcDNA3.1/B*3701 plasmid and 100ng
4The Cos-7 cell.After 48 hours the Cos-7 cell of transfection is carried out γ-IFN and test detection: 5000 reactor CTLs were added among the 150 μ l IMDM/10%HS that replenished 25U/ml IL-2 after stimulation on the 5th day.37 ℃ after following 24 hours, gather in the crops 100 μ l supernatants and (Genzyme MA) advises measuring γ-IFN concentration according to the manufacturer with γ-IFN release reagent box.
Me14392 is advanced in retroviral vector mediation HLA-B*3701 transgenosis
By described in the past (Fleischhauer, K., etc., J.Immunol, 159:2513-2521,1997) made up the retroviral vector B37-CSM of the HLA-B*3701 molecule of coding patient MSR3.In brief, the full-length cDNA s of coding HLA-B*3701 molecule is cloned under the viral LTR control, and people's low-affinity nerve growth factor receptor of clipped form (
LNGFR) under the control of Sv40 promotor.Parent preferendum mouse fibroblast cell be GP+E86 with 30 μ g retrovirus structures by standard calcium phosphate method transient transfection.48 hours culture supernatant of GP+E86 cell with transfection when 8 μ g/ml Polybrene exist infected two preferendum mouse package cell line GP+env Am 124 hours.With wrapping by LNGFR-monoclonal antibody specific 20.4 (American type culture collection (American Type Culture collection), Rockville, magnetic bead MD) (Dynabeads M-450, Dynal A.S., Oslo, Norway) packing cell that infects is carried out
The immunoselection that LNGFR expresses.Cultivate with transduction Me 14932 with containing retroviral supernatant when existing at polybrene (8 μ g/ml).Carry out 5 or 6 and take turns at least 4 hours infection.Carry out immunofluorescence analysis to estimate the validity that infects with LNGFR monoclonal antibody specific 20.4 and HLA-Bw4-monoclonal antibody specific.
RT-PCR analyzes
Detect MAGE-1,2,3,4,6,12 and β by pcr amplification
2-microglobulin (β
2M) cDNAs.Reaction mixture comprises 5 μ l cDNA suspensions, 4 μ l 10mM dNTPs mixtures (containing each 2.5mM of every kind of dNTP), 5 μ l, 10 * dna polymerase buffer liquid (Finnzymes Oy, Espoo, Finland), 2U DynaZyme archaeal dna polymerase (Finnzymes Oy) and sterilized water are mended 50 μ l total reaction volume.Oligonucleolide primers sequence and pcr amplification program are seen (Weynants such as Weynants, P., etc., Int.J.Cancer, 56:826-829,1994) (MAGE-1,2 and 3), (De Plaen, E. such as De Plaen, Deng, J.Immunol., 159:2513-2521,1997) (MAGE-4,6 and 12).With adopted primer Beta5 ' (5 '-AAC CAC GTG ACT TTGTCA CAG C-3 ') and antisense primer Beta 3 ' (5 '-CTG CTC AGA TAC ATC AAA CAT G-3 ') amplification β is arranged
2M cDNA; Pcr amplification carries out 30 circulations (94 ℃ 1 minute, 56 ℃ 30 seconds and 72 ℃ 2 minutes); β
2The expection length of m amplified production is 230bp.With beta-actin specific oligonucleotide primer carry out PCR reaction detection RNA integrity (De Smet, C., etc., Immnogenetics, 39:121-129,1994).The sample of seeing suitable size band when ethidium bromide exists on agarose gel is judged to be the positive.
The present invention will do more detailed description in the following embodiments.
Embodiment
The immune response of MSR3-B37 inducing antigen-specific
Set up melanoma cell series MSR3 from the metastatic carcinoma of skin of patient MSR3 excision.HLA I class allelotrope is expressed specificity mAb W6/32 with HLA-A, B and C and is carried out immunofluorescence analysis and estimate on the tumour cell.
But observed detection does not go out or just the surface molecular of detection level (Fig. 1) as if deficiency so that immune effector cell is given in antigen presentation.In fact, MSR3 clone can not induce cytotoxic reaction from the PBLs from body.It is not because the β of damage that MSR3-mel lacks I class cell surface expression
2M is synthetic to be caused, because can detect β by enough RT-PCR
2The specific mRNA of m.
For determining whether HLA I class antigen presentation can recover, the MSR3-mel cell cDNA stable transfection of coding from body HLA-B*3701 molecule.After G418 selected, the MSR3-B37 clone that flow cytometry analysis shows transfection was by W6/32 monoclonal antibody dye (Fig. 1).
For estimating on the MSR3-B37 clone surface whether have tumour specific antigen, detected that melanoma cells induces tumor specific cytotoxicity effector cell's ability and they are by these CTLs cracked susceptibilities.According to " material and method " described PBLs with MSR3-B37 stimulated in vitro patient.After stimulating three-wheel, the polyclone cytotoxic T cell is 337 (CTLs 337) specificity cracking MSR3B37 clone, but the MSR3-mel of not cracking untransfected (Fig. 2).Be not identified from the MSR3-EBV of body cell and PHA-activated T parent cell, show that the epi-position of these CTLs identifications is that melanoma/melanocyte is special.In fact, except from the melanoma cells of body, CTLs 337 is cracking HLA-B*3701 male melanoma cell series ET1 (Fig. 2) also, shows the one or more total melanoma antigens of identification.
These data show that HLA I class is expressed can be recovered by the transfection of MSR3 melanoma cells, and transfection the melanoma cell series of HLA-B*3701 molecule can induce the cytotoxic T cell reaction of tumour-specific.
Identify CTLs 337 identified epitope
Be to identify the antigen of CTLs 337 identifications, in transfection the γ IFN release of evaluate CT Ls 337 in the presence of plasmid pcDNA3.1/B*3701 and coding MAGE 6 members' of family (MAGE-1,2,3,4,6 and 12) the Cos-7 cell of cDNA (some is expressed by MSR3-mel and ET1).The transfection of CTLs337 specific recognition MAGE-1,2,3 and 6 Cos-7 cell, the epi-position target that shows CTLs 337 is a common in four different antigens, and the identification of perhaps few clone (oligoclonal) T clone heterogeneity derives from the peptide of four MAGE-gene products.When existing, the Cos-7 cell of MAGE-4 and MAGE-12 transfection detects low-level γ-IFN (Fig. 3).
For identifying the sequence of the coding for antigens peptide that CTLs 337 discerns, it approximately is 495 and 1 that the cDNA of coding MAGE-1 obtains 2 with Bgl II and EcoRI digestion, the subfragment of 072bp.These fragments are cloned into plasmid pcDNA3.1 and are advanced the Cos-7 cell with the transfection of HLA-B*3701 molecule.495 and 1, there are the initiator codon that meets single open reading frame in segmental 202bp of 072bp and 707bp place, have guaranteed the expression of these two subfragments in the transfectional cell respectively.In transfection being on close level of can discharging with the Cos-7 cell of whole M AGE-1 gene transfection of the level of the segmental Cos-7 cell of 495bp γ-IFN that CTLs 337 discharges when existing, show that antigen peptide is in this regional code.Screen the peptide that carries the HLA-B*3701 binding motif in this 495bp fragment encode amino acid sequence (Rammensee, H.G., etc., Immnogenetics, 41:178-228,1995).Identify 5 peptides: in the position 2 places be aspartic acid or L-glutamic acid and in the position 9/10 place is Isoleucine or leucine.One of them peptide, REPVTKAEML also is present in MAGE-2, MAGE-3 and the MAGE-6 amino acid sequence coded.This peptide, called after MAGE.
127-136, in titration experiments, be used to make MSR3-EBV clone sensitization with by CTLs 337 cracking.The MSR3-EBV cell also is used as target cell again with peptide REPVTKAEML stimulation in cytotoxicity experiment (Fig. 4 a).The 90nM peptide reaches the cracking of half maximum.Do not observe with the MSR3EBV that can stimulate cracking (Fig. 4 b) is arranged in conjunction with the irrelevant peptide of HLA-B*3701.
CTLs 337 discharges low-level γ-IFN (Fig. 3) when the Cos-7 cell of expressing MAGE-4 and MAGE-12 exists.For confirming that whether this release can be used the MSR3-EBV that stimulates with two peptides as target cell by the identification owing to 127-136 bit codon encoded peptide among the MAGE-4, carries out peptide in conjunction with research.Peptide M4.
127-136(KELVTKAEML) with peptide REPVTKAEML two amino acid different (position 1 place's arginine changes Methionin into, and 3 place's proline(Pro) change leucine in the position) are arranged, and peptide M12.
127-136(REPFTKAEML) 1 amino acid difference (position 4 place's Xie Ansuans change phenylalanine into) is only arranged.The result discloses two Toplink and combines with HLA-B*3701, because both amount increases can both suppress with peptide REPVTKAEML rather than (be M3. with irrelevant HLA-A1-binding peptide
271-279) cracking (Fig. 4 b) of the MSR3-EBV that stimulates.Yet, do not observe and use peptide M4.
127-136And M12.
127-136The identification of the EBV cell that stimulates.
In a word, these data show that CTLs 337 can discern MAGE-1,2,3 and 6 peptides of endogenous processing.
The product of CTL 337 specific recognition MAGE-2 and MAGE-6 gene
Up to now, do not exist people MAGE-2 and MAGE-6 proteins encoded that immunogenic evidence is arranged.In fact, although identified MAGE-1,3,4 and 12 encoded peptides that can form the antigenic complex of being discerned by various CTLs, also do not identify the peptide of MAGE-2 or MAGE-6 genes encoding in conjunction with HLA I quasi-molecule.
Can be from MAGE-2 and MAGE-6 gene processed and be and pass CTLs 337 in order to prove in melanoma cells peptide REPVTKAEML, once seek the melanoma cell series of expressing MAGE-2 or MAGE-6 and not expressing other MAGE gene.Unfortunately, the MAGE expression of gene is strict relevant in the melanoma, and most of melanoma is expressed the more than one composition of MAGE gene family.In fact, do not have to find optionally to express the melanoma cell series of MAGE-2, and found the melanoma cell series of a strain selective expression MAGE-6 that Me 14932.
With the specific oligonucleotide primer of each MAGE gene Me 14932 melanoma cell series are made RT-PCR and analyze, confirm that only MAGE-6 is positive, be low expression level.For determining whether the REPVTKAEML peptide is presented from the processing of MAGE-6 product endogenous and by HLA-B*3701, according to " material and method " described retroviral vector transduction Me14932 with a coding HLA-B*3701 molecule and a surface markers (Δ LNGFR).The cell of transduction, Me 14932-LB37 is with the expression of magnetic bead immunoselection Δ LNGFR.The immunofluorescence analysis of making of HLA-Bw4-specificity mAb shows, the low at least twice of the expression ratio MSR3-B37 melanoma cells of HLA-B*3701 on Me 14932 transducer cell surfaces.CTLs 337 can discern Me 14932-LB37 clone in cytotoxicity experiment, and exogenous (hexogenous) when adding peptide REPVTKAEML the cracking level improve, and stimulate and Me 14932 clones that do not stimulate not to be identified (Fig. 5).The melanomatous low cracking level of Me 14932-LB37 may with the low expression of MAGE-6 gene or relevant with the low expression of HLA-B*3701 surface molecular.
Comprise for estimating whether MAGE-2 and MAGE-6 can improve the ratio that can treat the patient in the target antigen list of tumour-specific immunotherapy, to have analyzed MAGE-1,2 in the fresh tumor sample of various types of organizations, 3 and 6 expression.Do not analyze melanoma because different MAGE expression of gene clearly be associated (Dalerba, P., etc., Int.J.Cancer, 77:200-204,1998).The result shows that 12% ovarian cancer and 5% colorectal carcinoma and mammary cancer are expressed MAGE-2 and/or MAGE-6, and lacks MAGE-1 and MAGE-3 (table).On the other hand, these four genes coexpression always in the bladder cancer of all researchs and the lung cancer.
In a word, the data of report show that MAGE-2 and MAGE-6 can be included in the possible target antigen list of tumour-specific immunotherapy in this research, and having improved can be from the benefited patient's quantity of this treatment.
Table
MAGE expression of gene in the fresh tumor sample
(a)The MAGE-1 MAGE-3 MAGE-2 MAGE-6 of types of organization only MAGE-2 or
MAGE-6 lung cancer (n 28)
(b)35 39 32 29 0 mammary cancer (n 20), 30 10 10 15 5 ovarian cancers (n 25), 24 20 32 20 12 bladder cancer (n 25), 28 28 20 24 0 colorectal carcinomas (n 17) 05555
(a)The reverse transcription-pcr analysis is determined.The result is expressed as the percentage ratio (%) of RT-PCR positive tumor
(b)The fresh tumor sample quantity of analyzing
Reference
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7. Coulie, P.G., Brichard, V., VanPel, A., Wolfel, T., Schneider, J., Traversari, C., Mattei, S., De Plaen, E., Lurwuin, C., Szikora, J.P., Renauld, J.C. and Boon, coding of T. is by the new gene of the differentiation antigen of the autogenous cell cracking performance T lymphocyte identification relevant with the in-vivo tumour repulsion.Institute of NAS newspaper (Proc.Natl.Acad.Sci.), 91:6458-6463,1994.
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Sequence table＜110〉GENERA S.P.A＜120〉from immunogenic peptide and application＜130 thereof of MAGE〉Genera＜140〉＜141＜160〉3＜170〉PatentIn Ver.2.1＜210〉1＜211〉10＜212〉PRT＜213〉people＜400〉1Arg Glu Pro Val Thr Lys Ala Glu Met Leu, 15 10＜210〉2＜211〉10＜212〉PRT＜213〉people＜400〉2Lys Glu Leu Val Thr Lys Ala Glu Met Leu, 15 10＜210〉3＜211〉10＜212〉PRT＜213〉people＜400〉3Arg Glu Pro Phe Thr Lys Ala Glu Met Leu 15 10
Claims (15)
1. in conjunction with the peptide of allelotrope HLA-B*3701, it is selected from:
a)REPVTKAEML
b)KELVTKAEML
c)REPFTKAEML
2. at the mono-clonal or the polyclonal antibody of the peptide of claim 1.
3. comprise the peptide of claim 1 of significant quantity and the pharmaceutical composition of pharmaceutically useful vehicle.
4. the composition of claim 3 is used as vaccine.
5. induce method, comprise the peptide that allows the T lymphocyte under the condition that is suitable for activated lymphocyte self, contact claim 1 at the cell-cytotoxic reaction of expressing MAGE-1,2,3,4,6,12 antigenic tumour cells.
6. the method for claim 5 wherein directly is exposed to described peptide with lymphocyte in cultivation.
7. the method for claim 5, wherein peptide is before in conjunction with HLA I quasi-molecule, preferred HLA-B*3701 allelotrope.
8. cytotoxic T cell is, it discerns the quasi-molecule by HLA I specifically, the mixture that the peptide of preferred HLA-B*3701 and claim 1 is formed.
9. the melanoma cell series of expression tissue compatibility antigen not is deposited in CBA-joint laboratory clone preservation center, and numbering is N.PD99001.
10. the clone of claim 9 was transformed with the gene genetic of coding HLA molecule and/or tumour antigen.
11. the clone of claim 10, wherein said HLA molecule is I class B*3701, and tumour antigen is melanomatous.
12. the clone of claim 9-11 exsomatize induce with the special cytotoxic T lymphocyte of expansion of antigen in purposes.
13. the purposes of the clone of claim 10 and 11 in the preparation vaccine.
14. the purposes of the peptide of claim 1 in the preparation antitumor drug.
15. the purposes of claim 14, wherein said medicine is a vaccine.
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| ITMI99A001121 | 1999-05-21 |
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| AU (1) | AU777474B2 (en) |
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| HK (1) | HK1047116A1 (en) |
| IL (1) | IL146605A0 (en) |
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| US6235525B1 (en) * | 1991-05-23 | 2001-05-22 | Ludwig Institute For Cancer Research | Isolated nucleic acid molecules coding for tumor rejection antigen precursor MAGE-3 and uses thereof |
| US5662907A (en) * | 1992-08-07 | 1997-09-02 | Cytel Corporation | Induction of anti-tumor cytotoxic T lymphocytes in humans using synthetic peptide epitopes |
| US5965535A (en) * | 1997-09-12 | 1999-10-12 | Ludwig Institute For Cancer Research | Mage-3 peptides presented by HLA class II molecules |
| US6291430B1 (en) * | 1997-09-12 | 2001-09-18 | Ludwig Institute For Cancer Research | Mage-3 peptides presented by HLA class II molecules |
| EP1124834B1 (en) * | 1998-10-29 | 2002-11-06 | The Dow Chemical Company | Preparation of titanium(ii) or zirconium(ii) complexes |
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| CN1227264C (en) | 2005-11-16 |
| HK1047116A1 (en) | 2003-02-07 |
| ITMI991121A1 (en) | 2000-11-21 |
| CA2374354A1 (en) | 2000-11-30 |
| AU777474B2 (en) | 2004-10-21 |
| IL146605A0 (en) | 2002-07-25 |
| JP2003502026A (en) | 2003-01-21 |
| KR20020011418A (en) | 2002-02-08 |
| WO2000071573A2 (en) | 2000-11-30 |
| EP1179019A2 (en) | 2002-02-13 |
| WO2000071573A3 (en) | 2001-04-05 |
| IT1312568B1 (en) | 2002-04-22 |
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