MXPA00003819A - Anti-idiotype monoclonal antibodies, their use in active immunotherapy of malignant tumors and compositions containing them - Google Patents

Abstract

Monoclonal anti-idiotype antibody which is specific for murine antibodies against gangliosides carrying N-glycolylated sialic acid particularly against those gangliosides which are expressed by tumoral cells. The monoclonal antibody is useful as immunomodulator for the treatment of cancer. Specifically, the monoclonal anti-idiotype antibody of the present invention is capable of inducing a response which is predominantly anti-idiotypic in xenogenic models. The monoclonal anti-idiotype antibody also exerts a protector effect against malignant tumors in animals

Description

ANTI-IDIOTICO MONOCLONAL ANTIBODIES, THEIR USE IN THE ACTIVE IMMUNOTHERAPY OF MA IGNOS TUMORS, AND COMPOSITIONS THAT CONTAIN THEM TECHNICAL SECTOR The present invention relates generally to anti-idiotype monoclonal antibodies and their use as immunomodulators. More particularly, the present invention relates to a murine monoclonal antibody, which was developed against a murine monoclonal antibody reactive with N-glycolylated sialic acid bearing gangliosides and with antigens expressed on tumor cells, and its inhibitory effect on tumor growth.

Prior Art One of the strategies for the treatment of cancer has been the use of active immunotherapy, a treatment modality that aims to activate the natural potential of the host immune system against the tumor. Since Niels Jerne in 1974 (Jerne, N. K. 1974. Ann Immunol 125C, 373-339) proposed his theory of the idiotypic network, new possibilities opened up in the study of effective therapies against cancer. Jerne's theory presented for the first time, the immune system as a network of antibodies that can interact with each other and with a large number of natural epitopes, through their variable regions or idiotypes (Id). This complex set of Idiotype-anti-Idiotype interactions operates to regulate immune responses to antigens. These antibodies developed in response to the original antigen, named Abl, convert themselves into antigens and cause the production of a second set of antibodies, called anti-idiotype antibodies (anti-Id or Ab2), which can also be regulated by other antibodies. referred to as anti-anti-Idiotype (Ab3). Jerne's original theory continues to be reviewed, and it has been reported that the result of the interaction of Ab2 with Abl-bearing lymphocytes does not necessarily have to be the suppression of the immune response, but may be the stimulation of said response. In addition, Jerne limited his theory to B lymphocytes and antibodies, but it is now clear that T cells play an important role in regulation through the idiotypes of T cell receptors (Teitelbau, D. et al, 1984 J. Immunol 132, 1282-1285; Zanetti, M. et al (1986) J. Immunol 137, 3140-3146; Powell, J. et al (1988) 140, 3266-3272; Baskin, JG et al (1990) J Immunol 145,202-208; Furuyama, A. et al (1992) Anticancer Res 12, 27-32; Raychaudhuri, S. et al., J. Immunol 131, 271-278; Raychaudhuri et al (1987) J. Immunol 139, 3902-3910; Durrant et al (1994) Cancer Res. 54, 4837-4840). An idiotype is immunologically defined by its reactivity with more than one anti-Id that recognizes an idiotypic or idiotope determinant within a given idiotype. Therefore, since a particular Abl expresses multiple idiotopes, when this Abl is administered to syngeneic animals, a heterogeneous population of anti-Id antibodies is obtained. The classification of anti-Id antibodies is based on their junctions within the antigen binding site or to some other region of Id. If the binding of Ab2 with Abl is inhibited by the relevant antigen and if Ab2 is also able to induce an antibody response of the same specificity as that of Abl, it mimics the natural antigen and is classified as Ab2ß; This type of Ab2 is referred to as an anti-Id internal image and they can act as substitutes for the antigens. The anti-Id that are not inhibited by the antigen are designated Ab2a, these Ab2 react with Abl idiotopes that are not structurally related to the antigen-binding site. In 1984 Bqna and Kóhler proposed a third type of anti-Id (Ab2?) Antibodies, which are inhibited by the antigen due to steric interference, this type of anti-Id reacts with idiotopos structurally associated with the binding site. antigen, but they do not mimic the antigenic epitope recognized by the Abl (Bona and Kohler 1984, In Monoclonal and anti-idiotypic antibodies: Probes for receptor structure and function, Venter JC, Frasser CM, Lindstrom J (eds), NY, Alan R. Liss, pp. 141-149, 1984). Based on Jerne's theory, two main approaches have been developed in the search for vaccines for a large number of antigens, including tumor-associated antigens. The first approach is based on the presentation of epitopes in a different molecular environment, using Ab2ß. Vaccines containing this type of anti-Id antibodies have been able to induce protective responses against viruses; bacteria and parasites (Kennedy et al (1986) 232, 220-223; McNamara et al (1985) Science 226, 1325-1326). Also, Ab2ß have been used to induce immune responses to tumor-associated antigens and positive results have been obtained in animal models and in clinical trials (Raychauhuri et al (1986) J. I munol 37, 1743-1749; Raychauhuri et al ( 1987) J. Immunol 139, 3902-3910, Bhattacharya-Chatterj ee et al (1987) J. Immunol 139, 1354-1360, Bhattacharya-Chatterjee et al (1988) J. Immunol 141, 1398-1403, Herlyn, D. et al (1989) Intern Rev. Immunol 4, 347-357; Chen, ZJ et al., Cell Imm. Immunother, Cancer (1990) 351-359; Herlyn, D. et al (1991) In Vivo 5, 615- 624; Furuya et al (1992) Anticancer Res 12, 27-32; Mittelman, A. et al (1992) Proc. Nati, Acad. Sci. USA 89,466-470; Durrant, LG. Et al. (1994) Cancer Res. 54, 4837-4840; Mittelman, A. et al (1994) Cancer Res. 54, 415-421; Schmitt, H. et al. (1994) Hybridoma 13, 389-396; Chakrobarty, M. et al (1995) J., Immunother. 18, 95-103; Chakrobarty, M. et al (1995) Cancer Res. 55, 1525-1530; Foon K. A. et al (1995) Clin. Cancer Res. 1, 1285-1294; Herlyn, D. et al (1995) Hybridoma 14, 159-166; Sclebusch, H. et al (1995) Hybridoma 14, 167-174; Herlyn, D. et al (1996) Cancer Immunol. Immunother. 43, 65-76). However, it has been shown that the ß character of an Ab2 is not sufficient to predict the biological effect that. could induce said Ab2 (Raychauhuri et al, 1986, J. Immunol 137, 1743-1749, Raychauhuri et al, 1987, J. Immunol 139, 231-278, Maruyama et al, 1996, mt J Cancer 65, 547-553) . The second approach is based on the manipulation of the network by regulatory idiotopes which are not linked to antigen binding, but involve idiotopos shared with other antibodies or T cells. Evidence has accumulated that these anti-Id antibodies are also capable to produce immune responses and protective effects (Paul, E. and Bona, C. (1982) I munology Today 3, 230-234; McNamara MK et al (1985) Science 226, 1325-1326; Kohler and cois (1992) Proc. 8th Inter Cong Immunol, Budapest, pp. 619). Gangliosides are glycosphingolipids that contain sialic acid and are expressed in most of the membranes of mammalian cells. Although these antigens are present in normal tissues, they can be found in much greater amounts and expressed in a different conformation and organization on the surface of malignant cells (Hakomori, S. (1985), Cancer Res. 45, 2405-2415; Miraldi, F. (1989) Seminars in Nuclear Medicine XIX, 282-294, Hamilton et al (1993) mt J Cancer 53, 1-81). Although gangliosides may be useful targets against which to direct immune responses, their immunogenicity is extremely poor, due to their saccharide nature and their own antigen status (Livingston, P. et al, (1995) Seminars in Cancer Biology 6, 357 -366). The N-glycolylated variant of sialic acid is expressed in normal tissues of most mammals, but it is very difficult to detect it in normal tissues of humans (Watarai, S. et al (1995) J. Biochem 117, 1062-1069). On the other hand, the presence of these antigens has been reported in colon cancer, melanoma, retinoblastoma and breast cancer, among others (Higachi, H et al (1984) Jpn J Cancer Res (Gann) 75, 1025-1029; , H et al (1985) Cancer Res. 45, 3796-3802; Hirabayashi, I. Et al (1987) Jpn J Cancer Res (Gann) 78, 1614-1620; Higachi, H. et al (1988) Jpn J Cancer Res (Gann) 79, 952-956; Miyake, M. et al (1990) Cancer 65, 499-505; Devine, PL et al (1991) Cancer Res. 51, 5826-5386; Vazquez, AM et al (1995) ) Hybridoma 14, 551-556; Marquina, G. et al (1996) Cancer Res. 56, 5165-5171). Immunization with vaccines containing gangliosides has resulted in the prolongation of survival of melanoma patients who developed anti-ganglioside antibodies (Livingston, P. et al (1987) Proc Nati Acad Sci USA 84, 2911-2915; Livingston, P. et al (1989) Cancer Res 49, 7045-7050; Livingston, P. (1995) Immunological Reviews 145, 147-166). However, the problems of obtaining large amounts of the antigen together with its poor immunogenicity have made the use of anti-Id antibodies an attractive alternative for active immunotherapy in this antigenic model. A murine anti-Id monoclonal antibody (mAb) (4C10) was generated against a human IgM mAb (L612) which recognizes GM3 on human melanoma. Sera from mice immunized with this anti-Id mAb coupled with KLH reacted strongly with an antigen-positive melanoma cell line and with purified GM3 which suggested that this mAb anti-Id carrying the internal image of GM3 (Ab2ß) can be an effective tool for active specific immunotherapy in melanoma patients (Yamamoto, S. et al (1990) J. Nati, Cancer, Inst. 82, 1757-1760; Irie, RE Patent Number US 5,208,146). The variable regions of the heavy and light chains (VL and VH) of this anti-Id mAb were cloned, sequenced and expressed as a mouse chimeric antibody / human IgGl (Hastings, A. et al (1992) Cancer Res 52, 1681-1686). In addition, an alpha-type Anti-Idiotype mAb was generated against human mAb L612 useful for immunodiagnostic procedures (Irie, R. F. Patent Number US 5,208,146). The BEC-2 mAb is a murine anti-Id mAb raised against a mouse mAb that recognizes ganglioside GD3 (AcMR24), which can mimic GD3 and consequently can induce antibodies against this ganglioside despite the expression of GD3 in normal rabbit tissue (Chapman, PE and Houghton, AN (1991) 88, 186-192). The results of a pilot study showed that said BEC-2 mAb plus BCG as adjuvant significantly increased the survival of patients with small cell lung cancer (Scrip Magazine (1996) pp 56-59; 33rd American Society of Clinical Oncology annual meeting (1997). In the immunization of rats with a murine mAb specific against GD2 (3F8), anti-Id mAbs were generated that when tested as immunogens in mice, could stimulate antibodies that reacted with ganglioside GD3. This suggested that these anti-Id mAbs may be useful in the construction of vaccines (Cheung, N-K.V. et al (1993) Int. J. Cancer 54, 499-505). Also, a human anti-Id mAb was generated using peripheral blood mononuclear cells obtained from a patient treated with murine mAb 14G2, which is specific against GD2. Immunization of rabbits with this human anti-Id mAb induces the production of anti-GD2 antibodies and a DTH response for antigen-positive tumor cells. This suggested that this antibody could potentially be used as an anti-Id vaccine in patients with malignant melanomas (Saleh, M. N. et al (1993) J. Immunol 151, 3390-3398). In the article "Ganglioside vaccine: anti-idiotypic monoclonal antibodies as antigen surrogates (Iglesias, E., et al Applied Biotechnology, Vol. 14, No. 1, 1997, page 50, XP002091466) seven anti-idiotype monoclonal antibodies are described IgGl (mAb) which reacted strongly with the P3 mAb and no reactivity was observed with the remaining anti-ganglioside mAbs tested.The seven anti-idiotype MAbs showed the ability to block the binding of P3 to NGcGM3 in a concentration range between 1 and 10 μg / ml Five of these anti-idiotype antibodies were able to induce a humoral response against NGcGM3, being classified as anti-idiotype Ab2ß and Ab2a monoclonal antibodies, on the other hand, Pérez et al. ("hat gangliosides show us about idiotypic networks. "Applied Biotechnology, Vol. 14, No. 1, 1997, page 42, XP-002091467) also describe that anti-ganglioside P3 antibody raised strong anti-idiot responses otypic IgG (Ab2, titers between 1/10 000 to 1/50 000) in Balb / c mice. The majority of these specific Ab2 clones obtained were able to block the binding of P3 mAb to GM3 (NeuGc). These antibodies, as in the previous case, were able to induce a natural auto-antibody response for the gangliosides, being also classified as Ab2ß anti-idiotype antibodies.

As is evident from the above-mentioned background, up to now no anti-Id mAbs of gamma type have been generated against mAbs that recognize gangliosides containing N-glycolylated sialic acid. Which at the same time exert an anti-tumor effect in an animal model.

Disclosure of the Invention. The present invention relates generally to monoclonal antibodies (mAbs) anti-idiotypes (anti-Id) and their use as immunomodulators for the treatment of cancer. More particularly, the present invention relates to a murine anti-Id mAb that was developed against a murine mAb that reacts with gangliosides carrying N-glycolylated sialic acid and with antigens expressed on tumor cells. The hybridoma producing said antibody has been deposited, in correspondence with the Budapest Treaty, on November 29, 1997 under accession number '97112901, at the Center for Applied Microbiology (CAMR) & European Cell Culture Collection (ECACC), in the United Kingdom. According to the present invention, one of its objects is to provide a new anti-IdM mAb able to induce a predominant anti-idiotypic response in xenogeneic models and to produce a protective effect in mice or other animal species carrying malignant tumors.

DETAILED DESCRIPTION OF THE INVENTION Immunization procedure to obtain anti-idiotype antibody (Ab2) response to anti-ganglioside MAbs. Mice and other mammalian species are immunized with doses of 25-200 μg of purified anti-ganglioside MAbs, with or without adjuvant, and optionally coupled to a carrier protein. The animals received 2-6 doses of antiganglioside MAbs, at intervals of 14 to 30 days between doses. Possible routes of immunization are intraperitoneal, subcutaneous, intravenous or a combination of these. Before and during the immunization period, samples of the animal's blood serum are taken and the Ab2 antibody response levels are determined by any known immunoassay method. Dilutions of the animal serum are incubated with the anti-ganglioside mAb used as immunogen, or with other anti-ganglioside mAbs not used in the immunization protocol.

Experiments were also carried out to define the ability of the sera of the immunized animals to block the binding to their antigen, of the anti-ganglioside mAb used as an immunogen Production of anti-idiotype MAbs against anti-ganglioside MAbs. Mice with high Ab2 antibody titers received a reimmunization with the mAbs used as immunogen, three days before the production of the antibody producing cells. Spleen cells should preferably be used, although other antibody producing cells can also be selected. These cells are fused with myeloma cells to give hybrid cells or hybridomas the property of reproduction or proliferation. both "in vi tro" and "in vivo". The cell fusion can be carried out by any of the methods known for this purpose. The antibodies produced by the hybridomas are tested by immunoassay methods, preferably by an immunoenzymatic assay in which supernatants of the hybridomas are incubated with the mAb used as immunogen and with other anti-ganglioside mAbs not used in the immunizations.

The ability of the hybridoma supernatant to block the binding of anti-ganglioside mAb used as an immunogen to its antigen is determined by incubating said supernatant with appropriate dilutions of antiganglioside mAb, followed by incubation of said antibody with its antigen. The selected hybridomas are donated at least twice and the resulting mAbs are produced "in vi tro" and "in vi vo" as described above. The anti-idiotype mAbs obtained recognize the antiganglioside MAbs and possess the ability to block the binding of anti-ganglioside MAb to their antigen.

Immunization procedure to obtain anti-anti-idiotype antibody response (Ab3) to mAb Ab2 in xenogeneic models. Monkeys or other xenogeneic species are immunized with anti-idiotype MAbs. These MAbs can be administered with or without adjuvant and optionally be coupled to a carrier protein, before being used as immunogens. Each animal received 2 to 8 doses of 250 g to 2 mg of the anti-idiotype MAbs at time intervals of 7 to 30 days between doses.

Immunization routes can be intradermal, subcutaneous, intravenous, intraperitoneal or a combination of these. The blood samples. of the animals are performed before and during the immunization protocol and the presence of Ab3 antibody response is monitored using any of the known immunoassay methods. The administration to the animals of the anti-idiotype MAbs produced by immunization with anti-ganglioside mAbs, can induce a predominant antibody response against the idiotype of the anti-idiotype MAb used as an immunogen, without the induction of antigen-specific antibody response .

Anti-tumor effect of treatment with anti-idiotype MAbs. Anti-idiotype MAbs are administered in an effective amount with or without adjuvants and optionally stored on a carrier protein. The term effective amount should be understood as the amount of anti-idiotype mAb required to achieve an anti-tumor effect. The treatment with the anti-idiotype MAbs is carried out before or after the experimental administration of the malignant cells in the animals.

The animals received 1-5 doses of 10-200 g of the anti-idiotype MAbs, at time intervals of 7-30 days between doses. Immunization routes can be intradermal, intraperitoneal, subcutaneous, intravenous or a combination of these. After treatment with the anti-idiotype MAbs, tumor incidence and survival in the group treated with the anti-idiotype MAbs was compared with that of the control groups. This trial revealed that the treatment with the anti-idiotype MAb significantly increased the survival of the animals carrying tumors and decreased the pulmonary metastases in them.

EXAMPLES OF EMBODIMENT: Example 1: Generation of an anti-idiotype antibody against the P3 mAb in a syngeneic model. Balb / c female mice 6-8 weeks old, were immunized with two doses of 50 μg of the purified P3 mAb (antibody developed against gangliosides carrying N-glycolylated sialic acid, Accession No. ECACC 94113026, European patent application EP 657471 Al; Hybridoma (1995) 14, 551-556)) coupled to KLH, at intervals of 14 days between doses.

Three days after the last immunization, the spleens were extracted from the mice that showed high serum levels of Ab2 antibodies against the P3 mAb and a cell suspension was prepared by pressing the tissue through a stainless steel sieve or perfusion. spleen. The fusion was carried out by the method described by Kohler and Milstein (1975, Nature (Lond) 256, 495-497), with slight modifications. Spleen cells from mice were fused with the non-secreting murine myeloma cells P3 / X63 Ag8 6.5.3. Accession No. ECACC. 85011420, in a 10: 1 ratio, in 0.5 ml of the fusion medium containing 42% polyethylene glycol (PEG) in RPMI-1640 medium. After fusion, the cells are cultured in the selective medium HAT (hypoxanthine-aminopterin and thymidine) at 37 ° C in a humid 5% C02 atmosphere. Within ten to fifteen days after the fusion, the evaluation of the presence of the antibodies in the supernatants of the hybridomas was determined by means of an ELISA. ELISA plates (COSTAR) were incubated overnight at 4 ° C with 10 μg / ml of P3 mAb and with another anti-ganglioside IgM mAb, in carbonate-bicarbonate buffer pH 9.8.

The plates, after being washed with PBS containing 0.05% Tween 20, were blocked with the same buffer containing 1% BSA for one hour at 37 ° C. The washing step is repeated and 50 μl / well of the supernatants were added. After incubation for 2 hours, the plates are washed again and a ram antiserum is added against the murine IgG Fe region, conjugated to alkaline phosphatase. After washing, 100 μl / well of the substrate solution (1 mg / ml of p-nitrophenylphosphate diluted in diethanolamine buffer, pH 9.8) was added. Absorbance was measured at 405 nm in an ELISA reader. ~~ In addition, the ability of the supernatants to block the binding of P3 mAb to NeuGcGM3 was determined by an indirect ELISA carried out on activated polyvinyl chloride (ICN-FLOW) plates with Immobilized NeuGcGM3, according to the following method: Fifty microliters of the ganglioside in methanol (4 μg / ml) were added to each well. The methanol is evaporated by incubating the plates at 37 ° C for one hour. Then 150 μl / well of 0.05 M TRIS-HCL buffer pH 7.8 containing 1% BSA were added and the plates were incubated at 37 ° C for 30 minutes. The supernatants of the hybridomas were incubated with 4 μg / ml of the P3 mAb for 3 hours at 37 ° C and then, 50 μl / well of the mixtures were added and the plates were incubated at 37 ° C for 90 minutes. The wells were washed 4 times with 200 μl of PBS and 50 μl of an anti-immunoglobulin antiserum conjugated to alkaline phosphatase, properly diluted. After washing with PBS the wells were incubated with the substrate solution as described above. An anti-idiotype MAb, named 1E10, of subclass IgGl was obtained. This mAb 1E10 anti-IdP3 was found to be very specific for the P3 mAb used as an immunogen. The hybridoma producing said antibody has been deposited, in correspondence with the Budapest Treaty, on November 29, 1997 under accession number 97112901, at the Center for Microbiology Applied (CAMR) & European Cell Culture Collection (ECACC), in the United Kingdom. This selected anti-IdP3 mAb 1E10 does not react with other anti-ganglioside antibodies such as El, A3 and F6 (Figure 1) and is capable of inhibiting the binding of P3 mAb to NeuGcGM3 (Figure 2) and to the tumor cell line of breast P3 positive.

Example 2: Generation of anti-anti-idiotype antibody (Ab3) response to the anti-Id mAb 1E10 in a xenogeneic model. The anti-IdP3 1E10 mAb precipitated in aluminum hydroxide used to immunize Cymomolgus monkeys intradermally at 14 days interval with a dose of 2 mg of MAb by injection. The animals received several doses of the MAb. Samples of the sera were taken before and after the immunizations. The presence of Ab3 antibody response in monkey sera was determined by ELISA. ELISA plates (COSTAR) were incubated overnight at 4 ° C with 10 ml of 1E10 MAb or its F (ab ') 2 fragments in carbonate-bicarbonate buffer, pH 9.8. The plates, after being washed with PBS containing 0.05% Tde ween 20, were blocked with the same buffer containing 1% BSA for one hour at 37 ° C. The washing step was repeated and 50 μl / well of the dilutions of the different sera were added. After incubation for 2 hours at 37 ° C, the plates were washed again and anti-human immunoglobulin, anti-IgM or anti-IgG, conjugated to alkaline phosphatase antisera were added. After washing, the substrate solution was added as described above. Blocking experiments were carried out to distinguish between anti-isotypic and anti-idiotypic antibody responses. Monkey sera were incubated overnight at 4 ° C with 500 μg / ml of an mAb with the same isotype as that of mAb 1E10, but with different specificity. Then, the remaining reactivity of the serum against mAb 1E10 was measured by ELISA, as described above. Monkey sera were checked for their ability to inhibit binding of biotinylated P3 mAb to the F (ab ') 2 fragments of mAb 1E10 in ELISA plates, using the ELISA method described above, but where after After incubating the plates with the dilutions of the sera, 50 μl / well of the biotinylated _A.cM P3 was added. After an incubation for one hour at 37 ° C, the plates were washed and 50 μl / well of the avidin-biotin-peroxidase complex were added and incubation of said plates was carried out for one hour at 37 ° C. After washing, the substrate buffer (8 mg of o-phenylenediamine in 12 ml of phosphate-citrate buffer, pH 5.0) was added. The absorbance at 492 nm was measured in an ELISA reader. Sera obtained from monkeys immunized with anti-IdP3 1E10 mAb reacted strongly with the F (ab ') 2 1E10 fragments (Fig. 3). The sera obtained from immunized monkeys specifically bound with the 1E10 mAb and with less reactivity than the unrelated mAb (ior C5). The presence of antibodies against the idiotype 1E10 was confirmed after the incubation of the sera of the animals with the unrelated MAb, due to a strong reactivity of the sera adsorbed with the anti-IdP3 mAb 1E10; surprisingly the induced anti-idiotypic antibody response was superior in comparison to the anti-isotypic antibody response generated (Fig. 4). Monkey sera also inhibited biotinylated P3 mAb binding to anti-Id mAb 1E10, indicating that Ab3 antibodies in monkeys serum share idiotopes with P3 mAb (Fig. 5). A predominant IgG antibody response was generated against the anti-IdP3 mAb 1E10 (Fig. 6). The sera obtained from the monkeys reacted with the F (ab ~> 2 1E10 fragments still 4 months after the animals received the last immunization (Fig. 7), as well as Ab3 antibodies with inhibitory capacity of the P3 mAb binding. at 1E10 were also detected at that time.No antibody was detected against NeuGcGM3 in the serum of the animals.This shows that the anti-idiotype mAb 1E10 is of the gamma type, since the anti-idiotype 1E10 is able to inhibit the binding of P3 mAb with its antigen and is not able to induce the production of antibodies similar to P3 in animals.

Example 3: Treatment of mice with the anti-Id mAb 1E10. CS7BL / 6 mice were immunized with five doses of 50 μg of anti-IdP3 1E10 mAb precipitated in aluminum hydroxide at 14 day intervals. One week later, the mice were injected subcutaneously with 5 x 103 B16 melanoma cells. Animals that were treated in the same way, but that did not receive mAb 1E10, were used as controls. The survival curves of Kaplan-Meyer are shown in Figure 8, which shows that the survival is significantly better in the group immunized with the anti-IdP3 mAb 1E10 than in the control group. C57BL / 6 mice were inoculated intravenously with 50 x 103 Lewis tumor cells (lung cancer). Fourteen days later, 10 μg of 1E10 was administered intravenously and 6 days later, the mice were sacrificed and the number of lung metastases was counted. Animals treated with an irrelevant IgM mAb or that received only PBS were used as controls. Seven of the 10 animals treated with anti-P3 1E10 mAb did not develop lung metastases, in the other 3 animals the maximum total number of metastases was 3. In contrast, all animals treated with irrelevant IgGl or those that received only PBS developed lung metastasis. These results indicate that treatment with this gamma-type anti-Id mAb produced a protective effect against tumors.

BRIEF DESCRIPTION OF THE FIGURES: Figure 1: Shows the reactivity of the anti-IdP3 mAb 1E10 against the anti-ganglioside mAbs P3, El, AS and F6. Figure 2: Shows the results of an inhibition assay where the MAb PS was incubated with the anti-IdPS MAb 1E10, and then the reactivity of the MAb MA to the NeuGcGMS is measured by ELISA. Figure 3: Shows the reactivity of sera from a monkey with the F (ab ') 2 fragments of mAb 1E10 measured by ELISA, after the animal received different doses of anti-Id mAb 1E10 precipitated in aluminum hydroxide. Figure: Shows the results of an inhibition assay where sera of a monkey were incubated or not with irrelevant mAbs of the same isotype as the mAb 1E10, and then the binding of serum antibodies to mAb 1E10 and irrelevant mAb was determined by a ELISA The arrows indicate immunization time and continuous lines the moment samples of the animal's serum are obtained. Figure 5: Shows the inhibition of the binding of AcM PS to mAb anti-Id 1E10 by the serum of a monkey immunized with mAb 1E10, determined by ELISA. The arrows indicate immunization time and the continuous lines the moment samples of the animal's serum are obtained. Figure 6: Shows the kinetics of the IgG and IgM antibody response against mAb 1E10 in the serum of a monkey immunized with the anti-idiotype MAb, determined by ELISA. The arrows indicate immunization time and continuous lines the moment samples of the animal's serum are obtained. Figure 7: Shows recognition of F (ab ') 2 fragments of mAb 1E10 by pre-immune monkey serum; by the serum obtained after the monkey received the last dose of anti-idiotype 1E10 mAb precipitated in aluminum hydroxide (indicated by the arrow), and by the serum obtained 4 months after the animal received the last immunization. Figure 8: Kaplan-Meyer survival curves in mice treated with the anti-IdPS mAb 1E10 and inoculated with B16 melanoma cells.

Claims (6)

CLAIMS:

1. Anti-idiotype gamma monoclonal antibody obtained against murine anti-ganglioside antibodies carrying N-glycolylated sialic acid.

2. A gamma-type anti-idiotype monoclonal antibody according to claim 1 wherein the N-glycolylated antiganglioside murine antibody used as immunogen is the monoclonal antibody designated as PS produced by the hybridoma deposited under accession number ECACC 94113026.

3. Anti-monoclonal antibody anti-idiotype. gamma type idiotype according to claims 1 and 2 capable of inducing a predominant anti-idiotypic response in xenogenic animals and exerting a protective effect against tumors.

4. producer of the monoclonal antibody ibridoma of claims 1 to 5 deposited on November 29, 1997 under accession number 97112901, in the Center for Applied Microbiology (CAMR) & European Cell Culture Collection (ECACC), ÜK.

5. A pharmaceutical composition containing an effective amount of the anti-idiotype monoclonal antibody according to claims 1 to 5, with an appropriate diluent, adjuvant or carrier molecule.

6. The use of the monoclonal antibody of claims 1 to 3 for the manufacture of a medicament useful in the treatment of malignancies.