NZ262435A

NZ262435A - Kit for diagnosing an hcg fragment secreting cancer and immunotherapeutical means therefor

Info

Publication number: NZ262435A
Application number: NZ262435A
Authority: NZ
Inventors: Dominique Bellet; Frederic Troalen; Nathalie Rouas-Freiss; Jean-Michel Bidart
Original assignee: Lafon Labor
Priority date: 1993-03-11
Filing date: 1994-03-10
Publication date: 1997-05-26
Also published as: WO1994020859A1; JPH08507604A; CA2157868A1; AU6211294A; EP0688432A1

Description

<div class="application article clearfix" id="description"> New Zealand No. 262435 International No. PCT/FR94/00265 Priority D«le(s):....ll|s.|3S Sjiate Comply® Specification Fll»d: Claw: (6) I.Ha«jay:.. P.O. Journal No: J.yd.Jf?..,... NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Kit for diagnosing an HCG- or HCG fragment-secreting cancer and immunotherapeutical means therefor K'amo, address and nationality of applicant(s) as in international application form: LABORATOIRE L. LAFON, of 19, avenue du Professeur Cadiot, F-94701 Maisons Alfort, France WO 94/20859 1 - PCT/FR94/0(^^5^ 4 3 5 Diagnostic kit for a cancer secreting hCG or fragments of hCG and means intended for the immunotherapy of such a cancer. The present invention relates to a method for 5 treating a cancer secreting hCG or fragments of hCG and to a means for implementing this method. Human chorionic gonadotropin hormone (hCG) is an oligomeric glycoprotein consisting of the noncovalent association of two subunits a(hCG-a) and £(hCG-£). it 10 belongs to the family of gonadothyreotropic hormones also comprising the follicle-stimulating hormone (FSH), the luteotropic hormone (LH) and the thyrotropic hormone (TSH) . The hCG is secreted physiologically by the tropho-blast from the beginning of pregnancy and stimulates the 15 Corpus leuteum in order to maintain the synthesis of steroid hormones which is essential for the implantation of the embryo. Moreover, hCG and/or its subunits are also secreted by a majority of trophoblastic tumours and some nontrophoblastic tumours, thus constituting biological 20 markers which can help in the diagnosis or in the prognosis and in the monitoring of these tumours (Bellet et al. Rev. Prat. 40:1677-1990). The amino acid sequence of hCG has been described by Morgan F.J. et al., The amino acid sequence of Human 25 chorionic gonadotropin, J. Biol. Chem. 250, 5247, 1975. Within the normal population, very low serum levels of dimeric hCG are found in men (<0.05 ng/ml corresponding to the detection limit of the method used) and in nonmenopausal women (<0.1 ng/ml). The hCG is 30 mainly detectable 30% of women over 45 years old (from 0.1 to 1 ng/ml). Several studies suggest that the hCG present in the serum of healthy subjects might be produced by hypophyseal cells. The a-hCG is detectable in healthy men (<1 ng/ml) and in nonmenopausal women over 45 years old (<3 ng/ml) . In contrast, /S-hCG is not detectable the normal population. In the case of trophoblastic tumours, it has been shown that benign (hydatidiform moles) and malignant (choriocarcinomas) placental tumours are characterized by 262 435 - 2 - a high serum hCG//S-hCG ratio: the percentage of free hCG which is 0.05 to 1% during normal pregnancies increases to 1-5% in the event of mole and exceeds 5% in choriocarcinomas. This serum hCG//3-hCG ratio is used aB 5 control for the progression of testicular tumours: a percentage of /3-hCG which becomes less than 5% is often characteristic of a favourable progression of the tumour during the treatment. This ratio will rapidly increase in the event of an unfavourable progression or of relapse. 10 In the case of non trophoblastic tumours, Marcillac et al., Cancer Res. 52:3901, 1992 have shown that jS-hCG, which is nondetectable . in healthy individuals, 'is found in numerous patients suffering from certain nontrophoblastio tumours (tumours of the bladder, 15 of the pancreas, of the neck of the womb, tumours without an established primary*cause# tumours of the endometrium and of the lungs). Moreover, WO-A 90/02759 describes a product 20 termed UGP which is a mixture of a combination of three peptide fragments of which one is fi-hCG and the other two are said to be different from the peptides present in /3-hCG. Under a very general definition, this document 25 defines diagnostic agents which may be constituted by UGP, whereas in practice it is the antibodies which are used. EP-A-0,323,769 describes peptides comprising essentially a /3-hCG sequence coupled to an a-hCG sequence 30 in order to constitute an antifertility vaccine. These peptides are intended to induce the formation of antibodies . US-A-4,201,770 also essentially describes an antifertility vaccine. This vaccine comprises a hormone 35 (such as CG) , its subunits or fragments of these subunits which have been modified by coupling with a nonendo-geneous material. WQ-A-92/07272 simply describes a method for N.Z. PATFNT OFFICE 13 MAS 1597 262 435 - 2a - detecting, inter alia, a cancer and which consists in searching for the presence of "serum gonadotropin peptide complex" after dissociation. The subject o£ the present invention is a method 5 for treating a cancer secreting hCG or fragments of hCG which consists in - identifying, in the serum of a patient with the aid of a set of polypeptides covering at least part of the primary sequence of the hCG, a state of immunization 10 against one of these polypeptides, - administering to the patient at least one polypeptide corresponding to the antigen responsible for the state of immunization. 262435 - 3 - correspond to sequences which overlap. There may be used for example a set of polypeptides corresponding to the following sequences: a-hCG 1-20 5 15-35 30-50 45-65 60-80 75-92 10 0-hCG 1-20 15-35 30-50 45-65 60-80 15 75-95 90-110 105-125 120-145 At least a set of polypeptides which covers the 2 0 primary sequence of the jS subunit of hCG or at least part of this sequence will preferably be used. It should be noted that the synthesis of the 0 subunit is dependent on a multigene group containing 7 genes (Talmadge et al., Nucl. Acids Res. 12, 8415, 25 1984) . Analysis of the nucleotide sequences shows that three of these genes (1, 3, 5) have an identical structure, another four of these genes (2, 6, 7 and 8) encode a variant in codon 117 with substitution of alanine for 3 0 an aspartic acid. A study of the expression of the genes (Bo et al., J. Biol. Chem. 267, 3179, 1992) shows that genes 1, 3 and 5 are expressed by the normal placenta. It is thought that in the tumorigenesis processes affecting the nontrophoblastic cells, the expression of 35 several genes leads both to the secretion of the normal protein and of a protein mutated in 117. In the present invention, /S subunit therefore designates both the normal protein and the protein mutated in 117. 262 435 "Polypeptide corresponding to the antigen responsible for the state of immunization designates a polypeptide which has the same sequence as one of the polypeptides responsible for the state of immunization or 5 a sequence resulting from the combination of at least partial sequences of the polypeptides responsible for the state of immunization. The state of immunization in relation to one of the polypeptides can be identified by a test of stimula-10 tion of the proliferation of the T lymphocytes by the polypeptides, or alternatively by a test of cytoxicity of the T lymphocytes in relation to the polypeptides. In the case of cytotoxic T lymphocytes, the polypeptides recognized are formed by a chain of at least 15 9 amino acids corresponding to part of the primary sequence of the hCG. In order to be recognized by the cytotoxic T lymphocytes, the polypeptides interact with the class I molecules of the patient's major histocompatibility complex. 2 0 Each class I molecule interacts with peptides containing precise units. There may be used for example the polypeptides corresponding to the following sequences of the jS-hCG: 25 30 35 3 - 12 4 - 12 8 - 16 10 - 18 11 - 20 17 - 25 23 - 1-3 24 - 33 28 - 37 32 - 41 40 - 48 40 - 49 44 - 52 47 - 55 48 - 56 51 - . 59 262435 - 5 - 55 - 62 61 - 69 68 - 76 71 - 80 5 72-80 75 - 84 77 - 86 78 - 86 83 - 92 10 100 - 108 119 - 128 125 - 134 134 - 143. The possible presence of serum antibodies speci-15 fic for hCG or its fragments can also be detected. These antibodies can be detected especially by an imsiuno-enzymatic method such as an ELISA method. The subject of the present invention is also a diagnostic kit for a cancer secreting hCG or fragments of 20 hCG comprising a set of polypeptides covering at least part of the primary sequence of hCG. The subject of the present invention is also a composition intended for the immunotherapy of cancers secreting hCG or fragments of hCG comprising an effective 25 dose of a polypeptide corresponding to at least part of the primary sequence of hCG and especially a polypeptide corresponding to at least part of the primary sequence of the § subunit of hCG and a pharmaceutically acceptable vehicle. 30 The subject of the present invention is also the use of a polypeptide corresponding to at least part of the primary sequence of hCG and especially a polypeptide corresponding to at least part of the primary sequence of the /3 subunit of hCG for the manufacture of a composition 35 intended for the immunotherapy of cancers secreting hCG or fragments of hCG. For therapy, the polypeptides can be used in free form or in a more immunogenic form, especially in a form in which the peptide is coupled to a polylysine matrix 262435 - 6 - according to the MAP technique (Posnett et al. J. Biol. Chem. 263, 17, 1988) . In addition, for therapy, the polypeptide is advantageously administered with an immunity adjuvant 5 agent. As examples of immunity adjuvants, there may be mentioned muramyldipeptide and SAF-1 (Allison et al., J. Immunol. Methods 45, 157, 1986). The polypeptide can also be administered coupled to nanospheres or to nanoparticles such as those 10 described in EP 02 04 424. Finally, the polypeptide can be protected at its NHj- and COOH-terminal ends in order to reduce the risk of degradation. In the case of the treatment of certain cancers 15 and especially in the case of cancer of the bladder, BCG (Calmette-Guerin bacillus) vaccine consisting of attenuated live Btrains of tubercle bacillus can also be additionally administered concomitantly or as a mixture (see especially O. Lamm et al., New England Journal of 20 Medicine 325, 17, 1205, 1991). The subject of the present invention is therefore, in addition, a composition intended for the immunotherapy of cancers secreting hCG or fragments of hCG comprising an effective dose of a polypeptide correspond-25 ing to at least part of the primary sequence of hCG and an effective dose of BCG in a pharmaceutically acceptable vehicle. In practice, the immunotherapy can be carried out with polypeptide doses from 1 to 100 mg, administered 30 especially in the region of the tumour. For example, the polypeptides can be administered in the case of a cancer of the bladder, as is already done in the case of an immunotherapy using BCG, that is to say by intravesical instillation one week after the resection of the tumour, 3 5 then this administration is repeated each week for 6 weeks and additional intravesical administrations are performed 3, 6, 12, 18 and 24 months later. Administrations via the percutaneous route or via the general route are also possible. 262435 - 7 - Description of the figures Fig. 1 represents the proliferation of the lymphocytes obtained from a patient having cancer of the bladder in the presence of hCG, of the free a and /5 5 subunits and of different polypeptides. Fig. 2 represents the proliferation of the lymphocytes obtained from another patient having cancer of the bladder in the presence of the free a and /S sub-units and of different polypeptides. 10 Fig. 3A represents the proliferation of the lymphocytes obtained from a patient having cancer of the bladder in the presence of different polypeptides. Fig. 3B represents the proliferation of the lymphocytes obtained from a patient having cancer of the 15 testicle in the presence of different polypeptides. An account of the diagnostic tests will be given below. 1) Isolation of the mononuclear cells (PBMC) bv Ficoll gradient: 20 Heparinized whole blood collected from cancer patients is deposited on a Ficoll-hypaque solution (1.077 g/1) in a conical tube. The tube is centrifuged for 30 min and 900 g, 18-20°C. The layer corresponding to the mononuclear cells is recovered with the aid of a. 25 pipette. The cells are washed in HANKs buffer. 2) Test of lvmphocvte proliferation The PBMCs are cultured (2 x 10s cells per well) in RPMI 1640 supplemented with 10% human serum AB, 2 mM L-glut amine, 100 IU/ml of penicillin and 100 ^tg/ml of 30 streptomycin, in the presence of polypeptide at different concentrations. The cells are then incubated at 37°C with 5% C03 for 5 days. One fid of tritiated thymidine is then added to each well. The cells are collected 18 hours later and the incorporation of the radioactivity is 35 analysed by spectrometry in a scintillation liquid using a /3 counter. The results of proliferation of the lymphocytes obtained from a patient having cancer of the bladder in the presence 262435 8 - - of the culture medium (M) - of the hCG and its a and 0 subunits - of different polypeptides belonging to the a and £ subunits, 5 are given in Pig. 1. The results reveal a significant proliferation with the polypeptide corresponding to the sequence 95-115 of the hCG jS subunit as well as with the polypeptide corresponding to the sequence 25-59 of the hCG a subunit. 10 The polypeptides 95-115 j3-hCG and 25-59 a-hCG can be used for an immunotherapy in the patient tested. The same type of results on the lymphocytes from another patient having cancer of the bladder are given in Fig. 2. The results reveal a significant proliferation 15 with the polypeptide corresponding to the sequence 31-50 of the hCG /? subunit as well as with the polypeptide corresponding to the sequence 41-61 of the hCG a stibunit. In this patient, the polypeptides 31-50 of the /3-hCG and 41-61 of the a-hCG can be used for an immunotherapy. 20 The results of tests of proliferation of PBMC from another patient having cancer of the bladder secreting the hCG fi subunit in the presence of different polypeptides covering the entire hCG /5 subunit (50 fan) are given in Fig. 3A. 25 The results show that for this patient, the poly peptide corresponding to the /J region (2 0-47) of hCG is most effective for inducing proliferation of the T cells. Moreover, other tumours (pancreatic and testicular) were studied. The T cells of a patient, having 30 cancer of the testicles, secreting 0-hCG, were proliferated in the presence of the /S-hCG peptide (20-47) (Fig. 3B). These results show that the peptides corresponding to part of the /3-hCG sequence are immunogenic in 35 patients having a j8-hCG-producing tumour and can thus be used to develop an antitumour immunotherapy. In contrast, no response against hCG, its sub-units or peptides of analogous structure was observed in 21 pregnant women. 4 65 - 9 - The results thus obtained will be given below. Cellular immune response against hCG or its subunits. Populations tested Total Responses Vesical tumours 21 5 /5-hCG+ tumours 14 5 /S-hCG- tumours 7 0 Pregnant women 21 0 Healthy subjects 13 0 10 3) Test of cytotoxicity The PBMCs isolated by Ficoll gradient are tested for their cytotoxic action against autologous targets (B lymphocytes from the patient which are immortalized by the Epstein-Barr virus) labelled with "chromium and 15 serving as the hCG polypeptide-presenting cells. The cytotoxicity is evaluated by counting the radioactivity due to the release of sxCr from the lysed target. 2624 - 10 - </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS I. Diagnostic kit for a cancer secreting hCG or fragments of hCG comprising a set of polypeptides covering at least part of the primary sequence of hCG./ 5 2. Diagnostic kit according to Claim 1, comprising a set of polypeptides which covers the primary sequence of the /3 subunit of hCG (/3-hCG or /3-hCG-Ala 117) . v 3. Diagnostic kit according to Claim 1, comprising a set of polypeptides which covers at least part of the 10 primary sequence of the /3 subunit of hCG (/3-hCG or jS-hCG-Ala 117) . / 4. Diagnostic kit according to any one of Claims 1 to 3, comprising polypeptides having from 9 to 40 amino acids. j 15 5. Diagnostic kit according to any one of Claims 1 to 4, comprising polypeptides whose sequences overlap, 6. Use of a polypeptide corresponding to at least part of the primary sequence of hCG for the manufacture of a composition intended for the immunotherapy of 2 0 cancers secreting hCG or fragments of hCG. / 7. Use according Co Claim 6, in which the polypeptide corresponds to at least part of the primary sequence of the /3 subunit of hCG (/5-hCG or /5-hCG-Ala 117)./ 25 8. Use according to Claim 6 or Claim 7, in which the polypeptide(s) have from 9 to 40 amino acids./ 9. Use according to any one of Claims 6 to 8/ in which the polypeptide is coupled to a polylysine template . J 3 0 10. Use according to any one of Claims 6 to 9, comprising in addition an immunity adjuvant agent.y II. Use according to any one of Claims 6 to 10, in which the polypeptide is protected at the NH2- and COOH-terminal ends. / 35 12. Composition intended for the immunotherapy of cancers secreting hCG or fragments of hCG comprising an effective dose of a polypeptide corresponding to at least part of the primary sequence of hCG and an effective dose AMENDED SHEET -ii- 262 435 in identifying, in the circulating blood of the patient with the aid of a set of polypeptides covering at least part of the primary sequence of hCG, a state of immunization in against one of these polypeptides. 5 14. Method according to Claim 13, in which the state of immunization in relation to one of the polypeptides is identified by a test of stimulation of the T lymphocytes by the polypeptides. 15. Method according to Claim 13, in which the pos-10 sible presence of serum antibodies specific for hCG or for its fragments is detected. 16. Method according to Claim 13, in which the state of immunization in relation to one of the polypeptides is identified by a test of cytotoxicity of the T lymphocytes 15 against the polypeptides. 17. Method according to claim 13, in which the state of immunisation against one of the polypeptides is identified by a test of cytotoxicity of the T lymphocytes in relation to the polypeptides. 20 18. A diagnostic kit as claimed in any one of claims 1-5, substantially as herein described and with reference to any one of the figures. 25 19. A use of a polypeptide, as claimed in any one of claims 6-11, substantially as herein described. 20. A composition as claimed in claim 12, substantially as herein described. 30 21. A method of in vitro diagnosis, as claimed in any one of claims 13-17, substantially as herein described. 35 262435 - 12 22. Method according to any one of Claims p. to 20, in which the possible presence of serum antibodies specific for hCG or for its fragments is detected. 23. Method according to Claim 13, in which the immune 5 response in relation to one of the polypeptides is identified by a test of cytotoxicity of the T lymphocytes in relation to the polypeptides. 24. Method according to any one of Claims 17 to 23, in which an immunity adjuvant agent ie administered with 10 the polypeptide .^/ 25. Method according to any one of Claims 17 to 24, in which BCG is administered concomitantly with the administration of the polypeptide or as a mixture with the polypeptide. ^ AMENDED SHEET </div>