HK1113374B - Immunotherapeutic formulations with interleukin-2-neutralising capacity - Google Patents

Description

Immunotherapeutic formulations with interleukin-2 neutralizing capacity

Technical Field

The present invention relates to pharmaceutical preparations capable of enhancing the immune response against interleukin-2 (IL-2) and producing autoantibodies which block the binding to receptors and which can be used for the treatment of tumours.

Description of the Prior Art

The discovery of the ability of the immune system, and in particular T cells, to recognize tumor antigens is one of the fundamental pillars in the development of strategies for controlling the immune system in order to treat cancer patients.

Therefore, in order to develop a method for recovering specific T cells infiltrating the tumor stroma, known as tumor-infiltrating lymphocytes (TILs), or specific T cells from the peripheral blood of untreated individuals or of individuals after the use of a therapeutic cancer vaccine, the main effort has been to stimulate said cells in order to enhance their anti-tumor effector capacity in vivo.

Thus, the main strategy is directed to enhancing their specific cytotoxic activity against a variety of Tumor Associated Antigens (TAAs). The main therapeutic approaches have focused on the in vitro use of interleukin-2 (IL-2) to activate and expand TIL from tumor-bearing individuals, and then reinfusion of these cells back into the individual (Rosenberg, S.A. et al (1986) Science 233, 1318-. However, despite the demonstration that stimulation of cellular immune responses is achieved in vitro, the intervention has limited therapeutic outcomes. This has led to the evaluation of therapeutic modalities based on the application of active specific immunization protocols using therapeutic cancer vaccines, and vaccine vectors comprising tumor antigens that bind to IL-2 were designed to facilitate the induction of an effective cellular immune response in vivo, however, the results of the above approach are poor (Rosenberg, s.a., et al (1998) nat. med.4, 321-.

Currently, the main clinical strategy has turned to the development of a means of tumor antigen based adaptive transfer of reactive T cells from a patient to itself. The cells were stimulated and expanded in vitro with anti-CD 3 monoclonal antibody (mAb) and IL-2, and after reinfusion back into the bloodstream, an IL-2 parenteral was provided. This approach constitutes one of the major therapeutic interventions designed to treat cancer patients, although the outcome of the treatment remains prudent (Dudley, M.E., et al (2002) Science 298, 850 & 854; Rosenberg, S.A. et al (2004) Proc Natl Acadsi USA.101Suppl 2, 14639-45).

The rational design of all these therapeutic strategies is based on the use of interleukin-2 as an essential molecule for the cellular activation of the antitumor immune response (US 6,060,068 and US 5,830,452).

The background of the role of IL-2 in immunity is based on experiments performed in vitro. From its discovery, IL-2 was recognized as its ability to stimulate T cell proliferation (hence, the synonym for IL-2 is T cell growth factor). This finding was further confirmed by the demonstration that T cell proliferation and function can be inhibited in vitro using anti-IL-2 or anti-IL-2 receptors (Smith, KA. Immunol Rev 51: 337-Bu 357, 1980).

Recently, it has been experimentally confirmed that human tumors can attenuate the immune system response by producing T cells with suppressive capacity against tumor immunity. The cells have been characterized in animal models and patients displaying different differentiation markers, although their correlation differs from the experimental model (Bach, J.F. (2003) Nat Rev Immunol 3, 189-.

Cluster of differentiation 25(CD25) constitutes the alpha chain of the IL-2 receptor. In addition, the structure of receptors for such cytokines includes the β (CD122) and γ (CD132) chains. They are constitutively expressed in resting T lymphocytes and activation of the cells induces alpha chain synthesis, formation of high affinity heterotrimeric receptors and IL-2 secretion. CD25 is constitutively expressed in 5-10% of CD4+ T lymphocytes and expressed in less than 1% of peripheral CD8+ T lymphocytes. The cells were anergic and exhibited inhibitory activity in vitro (Shevach, E.M. (2002) Nat Rev Immunol 2, 389-400).

It has recently been shown that passive administration of anti-CD 25mAb induces anti-tumor responses in some experimental tumors, although other tumors are resistant to this treatment (Onzuka, S. et al (1999) Cancer Res 59, 3128-3133).

The ability of the immune system to induce a response against self molecules is limited, particularly against soluble molecules such as growth factors. However, active immunization with these factors conjugated to a carrier protein and emulsified in adjuvant can promote induction of an immune response against the molecule (u.s.5.984.018). Specific autoantibodies raised against self or heterologous molecules can inhibit their binding to their receptors and prevent the mechanisms of proliferation triggered by such binding.

From the above results, the dependence of the antitumor response on the presence of IL-2 can be regarded as the prior art. We therefore decided to characterize the effect of anti-IL-2 autoantibodies induced by active immunization with IL-2 conjugated to a carrier molecule in adjuvant on tumor evolution in vivo.

Surprisingly, the induction of autoantibodies that block the binding of IL-2 to its receptor can promote the attenuation of tumor growth, even tumors that are resistant to the anti-tumor effects induced by passive administration of anti-CD 25 mAb. In addition, the presence of the autoantibodies does not affect the immune response to the cancer vaccine in the subject being treated.

Detailed description of the invention:

the present invention relates to therapeutic agents that have an effect on the treatment of tumors for which the effect of the immune system of a subject is important. In particular, the invention includes the preparation of immunotherapeutic formulations capable of producing autoantibodies which block the binding of interleukin-2 to its receptor and inhibit tumor growth.

The present invention is directed to a therapeutic agent capable of inhibiting the binding of IL-2 to its receptor, useful for treating a patient with cancer, wherein the agent comprises IL-2 or a peptide thereof bound to a carrier protein; in addition, it contains suitable adjuvants. In particular, the therapeutic formulation of the invention comprises a carrier protein P64k from neisseria meningitidis and the adjuvant is selected from aluminium hydroxide and montanide isa 51.

In one embodiment of the invention, the therapeutic agent comprises IL-2 conjugated to P64k by chemical conjugation. In another embodiment of the invention, the formulation comprises IL-2 or a peptide thereof in the form of a fusion protein with P64 k.

The invention also includes therapeutic agents that inhibit the binding of IL-2 to its receptor, useful for treating individuals with cancer, and which include monoclonal antibodies specific for human IL-2 (hIL-2).

Another object of the invention is a method of treating a cancer patient in need of blocking IL-2 binding to its receptor in order to trigger a suitable immune response against the tumor, comprising administering a vaccine composition comprising IL-2 or an anti-IL-2 antibody.

In another aspect of the invention, therapeutic combinations of IL-2-based vaccines and other cancer vaccines based on specific tumor antigens or tumor growth factors are disclosed, as well as chemotherapeutic agents or radiation therapies in practical routine use.

The term "therapeutic combination" means a physical combination (i.e., in the form of a mixture) with respect to the association of two as distinct and separate constituent tissues (e.g., in the form of a reagent kit) when they are used in combination to treat a patient. Thus, a pharmaceutical combination is a useful combination in a therapeutic regimen involving the administration of two compounds, or by physical combination, as separate doses administered to the same patient during the course of treatment.

The term "cancer vaccine" means a useful agent in active immunotherapy to elicit an immune response in a subject receiving treatment that recognizes an antigen used in the vaccine and can be measured.

1. -obtaining an immunogenic composition.

The vaccine composition of the present invention comprises human recombinant interleukin-2 (hIL-2r) as an active ingredient conjugated to a carrier protein, preferably protein P64k (0474313 EPA2 and u.s.5.286.484) from the outer membrane complex of neisseria meningitidis. In addition, the vaccine composition includes a suitable adjuvant. The vaccine composition of the present invention preferably uses Montanide ISA 51 as an adjuvant.

Conjugation between hIL-2r and the carrier protein can be by chemical conjugation, or alternatively, construction of a fusion protein obtained by genetic engineering techniques.

-obtaining a vaccine composition comprising hIL-2r conjugated to P64k protein by chemical conjugation.

To obtain protein conjugation between hIL-2r and P64k proteins, the two components were mixed in a variable ratio of 20: 1 to 5: 1 (the ratio of moles of hIL-2r to moles of P64k protein), preferably 10: 1 (the ratio of moles of hIL-2r to moles of P64k protein). Glutaraldehyde is added to the mixture to a final concentration of 0.02-0.1%, preferably 0.5%, and incubated at Room Temperature (RT) for 1-5 hours. Finally, extensive dialysis was performed with Phosphate Buffered Saline (PBS) solution. The conjugation reaction was verified by 10% SDS polyacrylamide gel electrophoresis (Laemmli UK (1970) Nature 277, 680-685).

-obtaining a vaccine composition comprising a fusion protein between hIL-2r and P64k protein.

The gene encoding human IL-2 (447pb) was amplified by Polymerase Chain Reaction (PCR) using specific primers. The resulting DNA fragments are digested and ligated into specific binding sites of an expression vector, in which the gene encoding the carrier protein is cloned, so that the resulting protein comprises one or more copies of both molecules. Any expression vector from mammalian cells and from bacteria or yeast can be used. The vector may also include six histidines at the N-terminus of the carrier protein. The resulting plasmid was verified by the following method: restriction analysis was performed in agarose gel electrophoresis, using the enzyme Sequenace 2.0(Amersham-USB) for DNA sequence analysis, and finally, by means of "Western blot" techniques, using specific anti-hIL-2 monoclonal antibodies, for the production of the fusion protein in any expression strain of E.coli. To obtain the protein, the cell wall is disrupted using a strong disruption method, and then the protein is purified by a combination of a differential precipitation method using ammonium sulfate and a chromatography method. Finally, the protein is filtered under sterile conditions and stored at-20 ℃ or lyophilized and stored at 4 ℃ until use.

-obtaining a vaccine composition comprising a peptide from hIL-2r chemically conjugated to P64k protein or bound as a fusion protein.

Peptides derived from the amino acid sequence of IL-2 obtained by chemical synthesis can be bound to the P64k protein by chemical conjugation as described in u.s.5,984,018. In addition, the peptide derived from hIL-2r and P64k protein fusion protein was obtained substantially according to the same method as described in the previous section, and examples may include peptides derived from the following regions:

1) peptide N³³-A⁵⁰

Number of amino acids: 18

The sequence is as follows: NPKLTRMLTFKFYMPKKA

2) Peptide T¹¹³-T¹³³

Number of amino acids: 21

The sequence is as follows: TIVEFLNRWITFCQSIISTLT

Electrophoresis of chemically conjugated hIL-2r-P64 k.

Electrophoresis can be performed on a 10% SDS polyacrylamide gel (Laemmli U.K. (1970) Nature 277, 680-685). Mu.g of sample was added to each well and stained with Coomassie dye.

2. Characterization of the effects produced by the vaccine composition comprising hIL-2r and protein P64 k. Preclinical studies.

-immunogenicity of the vaccine composition.

To study the immunogenicity of the vaccine formulations of the invention in animals, female BALB/c mice, 8-12 weeks old, weighing 18-20g, were used. During the experiment, mice were housed under standard conditions of rearing and handling established under the Standard Operating Procedures (SOP) of Goodpractices of Care of and Use of the Experimental Animals.

Different immunization protocols can be followed:

regimen A, four doses of 0.1mL of 4 μ g equivalent doses of hIL-2r conjugated in the form of hIL-2r-P64k/Montanide ISA 51 vaccine, alternating the vaccination site of the limb, administered by intramuscular route every 2 weeks.

Scheme B, four doses of 0.1mL of 10 μ g equivalent doses of hIL-2r conjugated in the form of hIL-2r-P64k/Montanide ISA 51 vaccine, administered by intramuscular route. The first two doses were administered simultaneously at separate immunization sites on two limbs, and two weeks later, two doses were administered on the other two limbs.

-assessing the antibody response against hIL-2r induced by hIL-2r-P64k/Montanide ISA 51 vaccine.

Autoantibody titers in serum can be detected, and specific antibodies or B cells determined by one of the methods currently available for determining blocking antibodies or for assessing specific immune responses in peripheral blood.

anti-hIL-2 r antibodies induced by the vaccine hIL-2r-P64k/Montanide ISA 51 can be evaluated by indirect ELISA (enzyme linked immunosorbent assay) using sera from immunized animals, see below:

a96-well flat-bottomed microtiter plate (Costar, HighBinding, USA) was coated with 50. mu.l/well of hIL-2r and prepared to a concentration of 10. mu.g/mL using a carbonate-bicarbonate 0.1M, pH 9.6 solution. The plate was incubated at 4 ℃ overnight and washed 2 times with 200. mu.l/well PBS containing 0.05% Tween 20 (PBS-Tween 20). After 1 hour incubation at 37 ℃ with 100. mu.l/well PBS containing 1% bovine serum albumin (PBS-BSA 1%), the plates were washed with 200 l/well PBS-Tween 20, and then 50. mu.l/well of serum samples at various dilutions in PBS-BSA 1% were added. After 1 hour incubation at 37 ℃, the plates were washed with PBS-Tween 20 and 50 μ Ι/well of sheep anti-mouse serum conjugated with alkaline phosphatase (jackson immunoresearch Laboratories), which was diluted with PBS-BSA at a ratio of 1/5000, was added and incubated for 1 hour at 37 ℃. The plates were washed and 50. mu.l/well of substrate solution consisting of 1. mu.g/mL of p-nitrophenyl phosphate (Sigma) in diethanolamine buffer at pH 9.8 was added. After incubating the plate at Room Temperature (RT) for 30 minutes, the absorbance of the reaction product was measured by an ELISA reader (Organon Teknika, Austria) at 405nm wavelength.

Evaluation of anti-hEGF antibody response induced by EGF-based vaccine

Autoantibody titers in serum can be detected, and specific antibodies or B cells measured by one of the methods currently available for assaying blocking antibodies or for assessing specific immune responses in peripheral blood.

anti-hEGF antibodies induced by EGF-based vaccines can be evaluated by indirect ELISA (enzyme-linked immunosorbent assay) using sera from immunized animals, see Gonzalez, G., et al (2002) Vaccine Research 5, 233-244.

-effect on proliferation of CTLL-2 cell line after incubation with serum from animals immunized with hIL-2-P64k/Montanide ISA 51 or with specific monoclonal antibodies (mAb) against hIL-2 r.

Rendering IL-2 dependentThe T-cell line CTLL-2 was maintained in RPMI-1640 medium containing 1U/mL of hIL-2h, in a continuously proliferating state. CTLL-2 was cultured in 25mL RPMI-1640 containing 8-20% Fetal Calf Serum (FCS) and 0.5X 10⁵-10⁶75cm of cells/mL of cell suspension²In cell culture flasks. The cells were used two days after expansion in vitro.

To perform the assay, cells were extracted from the in vitro culture and washed no less than four times with RPMI-1640 or PBS. In 96-well flat-bottom culture plates (Costar, High Binding, USA), 5X10 was inoculated³A cell. These cells were treated with serum dilutions from immunized animals immunized with hIL-2-P64k/Montanide ISA 51 vaccine with ELISA titers to IL-2 of 1: 10000, or cells were treated with anti-IL-2 specific mAb and 1U/mL hIL-2r was added. At 37 deg.C in a humid atmosphere containing 5% CO₂Was cultured in an incubator for 24 hours. By using the [ 2 ] gene for the last 18 to 24 hours of the culture³H]Thymidine (1. mu. Ci/well) was pulsed to measure proliferation. Thymidine incorporation was determined by liquid scintillation counting. All manipulations were performed under aseptic conditions.

The 3-hIL-2-P64k/Montanide ISA 51 vaccine has anti-tumor effect. Preclinical studies.

To study the antitumor effect of the vaccine formulations of the present invention in animals, female BALB/c mice weighing 18-20g at 8-12 weeks of age were used. Animals can be immunized with hIL-2-P64k/Montanide ISA 51 vaccine according to protocols A and B detailed above, and one week later, by orthotopic vaccination with a tumor cell line, such as breast cancer F3II, at 5X10⁴Cell/animal. Mice with palpable tumors were scored positive and tumor growth was measured using calipers, the maximum surface length (a) and its vertical width (b) were determined, and the tumor size was reported as a × b. Tumors were examined periodically.

Surprisingly and unexpectedly, the authors of the present invention have found that the neutralization of the binding of IL-2 to its receptor in subjects carrying malignant tumors enhances the immune response against the tumor and induces a reduction in tumor size. The prior art shows that this effect results in the suppression of the response of the individual's immune system to the tumor.

The authors have found that this effect on tumor growth is obtained when subjects receive active treatment with the vaccine composition of the invention, with a significant reduction in circulating IL-2 levels, as is obtained with animal therapy with anti-IL-2 monoclonal antibodies.

To this end, the invention brings about undeniable advantages in the treatment of patients suffering from malignant tumors and provides a method of immunization with IL-2 which is effective, simple and more acceptable and less aggressive to the patient than the conventional treatments used in these cases.

The following examples include experimental details illustrating the immunological effects of the vaccine compositions of the present subject matter.

Example (b):

human recombinant IL-2(hIL-2r) for use in the vaccine compositions of the present invention is commercially available (U.S. 5.614.185). The protein P64k from neisseria meningitidis used in this vaccine was obtained by recombinant DNA technology, see EP 0474313a2 and u.s.5,286,484.

Example 1: antibody response induced by hIL-2r-P64k/Montanide ISA 51 vaccine.

To promote immunogenicity against human interleukin-2, it was chemically conjugated to the carrier protein P64k protein from neisseria meningitidis. The chemical conjugation can be achieved by the glutaraldehyde method (u.s.5.984.018). The efficiency of conjugation was verified by 10% SDS polyacrylamide gel electrophoresis, and samples of each component (hIL-2r, P64k) were added separately and compared to the chemical conjugate hIL-2r-P64k and standard molecular weight profiles. The conjugate was obtained as verified by the continued presence in the lane where hIL-2r-P64k was added (FIG. 1).

To evaluate the antibody response to hIL-2r induced by hIL-2r-P64k/Montanide ISA 51 vaccine, BALB/c mice were immunized with protocols A and B. Hyperimmune serum from animals responding to the hIL-2r-P64k/Montanide ISA 51 vaccine was used as a positive control, and preimmune serum was used as a negative control. It is defined as the antibody titer of the immunized animal, the optical density of the serum being greater than the average of the optical densities of the serum before immunization plus a greater dilution of five times the standard deviation. To determine titers in control animals, the same criteria as before were used except that the preimmune serum was replaced with PBS-BSA 1% as a negative control.

Induce antibody response to reach titer of 1: 100-1: 50000. This immunization protocol lasted approximately 52 days, which is why the protocol had to be modified in order to obtain similar or better antibody titers in a shorter time, so we used protocol B and obtained similar results, see figure 2.

Example 2: proliferation experiments of CTLL-2 cell lines treated with serum from animals immunized with hIL-2-P64k/Montanide ISA 51 vaccine

The in vitro IL-2 binding capacity of serum antibodies produced in animals immunized with hIL-2r-P64k/Montanide ISA 51 vaccine was assessed by culture with the IL-2-dependent T-cell line CTLL-2. CTLL-2 cells were seeded into culture plates in the presence of IL-2, allowed to grow, and different serum dilutions from animals with antibody titers of approximately 1: 10000 were added. A positive correlation between serum concentration and inhibition of proliferation of the CTLL-2 cell line was observed (FIG. 3). The in vitro IL-2 neutralizing capacity of sera from immunized animals was demonstrated.

Example 3: anti-tumor experiments in animals treated with hIL-2-P64k/Montanide ISA 51 vaccine.

Animals were immunized with hIL-2r-P64k/Montanide ISA 51 vaccine according to protocol B. After one week, 5X10⁴The F3II tumor cells challenged the animals. Use of hIL-2r-P64k/Monta compared to control group inoculated with PBS-P64k/Montanide ISA 51Tumor growth kinetics were slower in nide ISA 51 immunized animals (fig. 4 a). Statistically significant differences in tumor size between the two groups were observed.

Example 4: proliferation assay of CTLL-2 cell lines treated with anti-IL-2 specific mAb.

The in vitro IL-2 binding capacity of specific antibodies raised by the S4B6(ATCC # HB-8794) hybridoma against hIL-2r was assessed by culture with the IL-2 dependent T-cell line CTLL-2. CTLL-2 cells were seeded into culture plates in the presence of IL-2, allowed to grow, and different antibody dilutions were added. A positive correlation between antibody concentration and inhibition of proliferation of the CTLL-2 cell line was found (fig. 5). A dilution of 1: 100 antibody was found to inhibit over 80% of the proliferation of CTLL-2 cell lines, confirming the IL-2 neutralizing capacity of the monoclonal antibody in vitro.

Example 5: anti-tumor effects of anti-IL-2 mAb and anti-CD 25mAb (ATCC-PC 61). Experimental model F3II (breast cancer).

Mice were treated daily for five consecutive days with a dose of 1mg of specific monoclonal antibody (anti-IL-2 mAb or anti-CD 25 mAb). After two days, use 5X10⁴Cell/mouse experimental breast cancer F3II mice were challenged by subcutaneous vertical injection. Its vertical width (b) of the maximum surface length (a) is measured periodically. The tumor size of the control group (treated with PBS) was larger than that of the group treated with anti-IL-2 mAb (FIG. 6). The tumor sizes between the two experimental groups were statistically different. However, the tumor size of the anti-CD 25mAb (ATCC-PC61) treated animals was similar to the control group. This result indicates that the IL-2 neutralizing antibody has a potent antitumor effect even on tumors in which elimination of CD25 regulatory cells has no effect.

Example 6: anti-tumor effects of anti-IL-2 mAb and anti-CD 25mAb (ATCC-PC 61). EL4 experimental model (lymphoma).

With 5x10⁴EL4 lymphoma from cells/mouse subcutaneously challenged C57BL/6 animals. Two days prior to tumor challenge, a dose of 1mg of anti-CD 25mAb (PC61) was administeredThe animals were treated intravenously, or with 1mg dose of anti-IL-2 mAb (S4B6) daily for five consecutive days, starting six days prior to tumor inoculation. The other groups were administered anti-CD 25 and anti-IL-2 mAbs simultaneously based on a similar protocol as previously described.

Tumor growth was recorded for each group of mice. After tumor challenge, tumor size was measured twice weekly in two perpendicular dimensions for each mouse. The statistical differences of EL4 tumors were 0.0232, 0.0039, and < 0.0001. Figure 7 shows that the combination of two monoclonal antibodies has a strong effect on tumor growth.

Example 7: induction of anti-IL-2 autoantibodies did not affect the response to EGF cancer vaccine.

To assess whether the antibody response induced by the vaccine hEGF-P64k/Montanide ISA 51 against hEGF was affected by prior immunization with hIL-2r (induction of autoantibodies), BALB/c animals were immunized according to protocol A with the prepared hIL-2r-P64k/Montanide ISA 51 vaccine and after 19 days with the prepared hEGF-P64k/Montanide ISA 51 or hEGF/Montanide ISA 51 vaccine. In all experiments, 4 μ g equivalent of hEGF conjugated in a vaccine prepared in a volume of 0.1mL was administered by the intramuscular route when vaccinated with the hEGF vaccine. Hyperimmune serum from animals responding to vaccine hEGF-P64k/Montanide ISA 51 was used as a positive control, and preimmune serum was used as a negative control. It is defined as the antibody titer of the immunized animal, the optical density of the serum being greater than the mean of the optical densities of the serum before immunization plus a greater dilution of 5 times the standard deviation. To determine the titer of animal controls, the same standard as before was used except that 1% PBS-BSA was used instead of preimmune serum as negative control. The titers of anti-EGF antibodies induced by immunization with EGF/Montanide ISA 51 or hEGF-P64k/Montanide ISA 51 were found not to be affected by the induction of anti-hIL-2 r autoantibodies, see FIG. 8.

Example 8: neutralization of interleukin-2 in vivo restores cytolytic activity, evaluated in lymph node cells of tumor-bearing hosts.

In the presence of a tumorThe effect of IL-2 neutralization on the immune response to the nominal antigen was evaluated in vivo in the host. Female C57BL/6J mice (H-2b) were maintained under standard housing conditions. For all experiments, 6-12 week old mice were used. Ovalbumin (OVA) and peptides: OVA grade VII (Sigma, st. louis, MO) is the model protein Ag used in these experiments. Dominant peptide OVA used_275-264(SIINFEKL) was > 90% pure. Proliferation test: by 10⁴The MB16F10 tumor or PBS cells were challenged subcutaneously in the left flank of C57BL/6 mice. Tumor diameters were measured periodically. Three weeks later, the mice were immunized subcutaneously with 1mg of OVA on day 0 and 100 g/mouse of poly-inosinic acid [ poly I: c](PIC) (Sigma, st. louis, MO), whereas PIC was administered only 2 days later. Meanwhile, animals were treated with a monoclonal antibody specific for hIL-2r (α IL-2) or PBS for five consecutive days. Splenocytes from naive mice differentially labeled with the fluorescent dye CFSE (Molecular Probes, Paisley, UK) were used to determine in vivo cytolytic activity. Cells labeled CFSEhigh were used as targets and pulsed with SIINFEKL (1. mu.M; 90 min, 37 ℃, 5% CO)₂) Whereas CFSElow labeled cells were not pulsed to serve as internal controls. The peptide-pulsed target cells were washed thoroughly to remove free peptide and then co-injected intravenously into previously immunized mice at a ratio of 1: 1. Sixteen hours later, lymph nodes and spleen were removed and the total events corresponding to both fluorescence intensities (CFSElow and CFSEhigh) were measured by flow cytometry. Calculating the ratio between the percentage of uncoated and SIINFEKL Coated (CFSE)^int/CFSEh^igh) In order to obtain a value of cytotoxicity. Lymph node cells evaluated in vivo in C57BL/6 mice immunized with OVA plus PIC produced the greatest response to the cytolytic activity of OVA peptide. The anti-IL-2 monoclonal antibody administered by intravenous injection at a dose of 1mg daily for 5 consecutive days did not affect the cytolytic response in these animals. C57BL/6 mice challenged with MB16F10 tumor were immunosuppressed and had reduced cytolytic activity against OVA. However, in vivo administration of monoclonal antibodies with IL-2 neutralizing capacity restored the immune response of lymph node cells (FIG. 9).

Example 9: neutralization of interleukin-2 in vivo restores cytolytic activity, assessed in splenocytes of tumor-bearing hosts.

The effect of IL-2 neutralization on the immune response to the nominal antigen was evaluated in tumor-bearing hosts. C57BL/6 mice were immunized with OVA and PIC and splenocytes were evaluated for cytolytic activity against OVA peptide in vivo, giving maximal response. Administration of a 1mg dose of anti-IL-2 monoclonal antibody by intravenous injection daily for 5 consecutive days did not affect the cytolytic response in the animals.

C57BL/6 mice challenged with MB16F10 tumor were immunosuppressed and had reduced cytolytic activity against OVA. However, in vivo administration of monoclonal antibodies with IL-2 neutralizing ability restored the immune response of the glandular cells (FIG. 10).

Example 10: blood cell counts of animals immunized with the prepared vaccine hIL-2r-P64k/Montanide ISA 51.

To assess whether immunization with the prepared vaccine hIL-2r-P64k/Montanide ISA 51 induced a change in blood cell number, red blood cells, white blood cells, and platelets were counted, and animals immunized with the prepared vaccine (according to protocol A) or non-immunized animals, up to 100 days after the first immunization. Referring to fig. 11, no difference in cell count was found between the analysis groups.

Brief description of the drawingsthe accompanying drawings:

FIG. 1-electrophoresis of chemically conjugated hIL-2r-P64 k. Bands from left to right correspond to standard patterns of P64k, hIL-2r, hIL-2r-P64k and molecular weight, respectively.

FIG. 2-titers of anti-hIL-2 r antibodies induced by immunization with hIL-2r-P64k/Montanide ISA 51, using the two immunization protocols previously disclosed. The y-axis represents the geometric mean of the anti-IL-2 antibody titers. The x-axis represents the group corresponding to animals immunized with:

comparison: p64k/Montanide ISA 51.

Sch A IL-2: hIL-2r-P64k/Montanide ISA 51, according to scheme A.

Sch B IL-2: hIL-2r-P64k/Montanide ISA 51, as per protocol B.

FIG. 3-proliferation of CTLL-2 cell line in the presence of hIL-2r and different dilutions of serum from animals immunized with the vaccine hIL-2r-P64 k/MontanideeISA 51 and with serum titers in the order of 1: 1000 to 1: 50000.

FIG. 4-tumor growth in animals immunized with vaccine hIL-2r-P64k/Montanide ISA 51 or P64k/Montanide ISA 51 (control group) and subsequently challenged with F3II tumor. The y-axis represents the tumor area, defined as the larger diameter multiplied by the smaller diameter selected vertically.

FIG. 5-proliferation of CTLL-2 cell line in the presence of different dilutions of hIL-2r and anti-IL-2 antibody.

FIG. 6-tumor growth in animals treated with anti-IL-2 mAb, anti-CD 25mAb or PBS (control) and challenged with the F3II tumor cell line. The y-axis represents the tumor area, defined as the larger diameter multiplied by the vertically selected smaller diameter.

Figure 7-tumor growth in animals treated with α IL-2mAb, anti-CD 25mAb or PBS (control group) and subsequently challenged with lymphoma EL 4. The y-axis represents the tumor area, defined as the larger diameter multiplied by the vertically selected smaller diameter.

FIG. 8-antibody titers against EGF induced by vaccination with hIL-2r-P64k/Montanide ISA 51 and hEGF-P64k/Montanide ISA 51. The y-axis represents the geometric mean of antibody titers against EGF. The x-axis represents the group immunized with:

hIL-2r：hIL-2r-P64k/Montanide ISA 51

hEGF：hEGF/Montanide ISA 51

hIL-2r+hEGF：hIL-2r-P64k/Montanide+hEGF/Montanide。

hEGF/P64k：hIL-2r-P64k/Montanide+hEGF/Montanide。

hIL-2r+hEGF/P64k：hIL-2r-P64k/Montanide+hEGF/P64k/Montanide。

FIG. 9-induced antibodies to interleukin-2 restore cytolytic activity, evaluated in lymph node cells of tumor-bearing hosts.

All mice were immunized subcutaneously on day 0 with 1mg of OVA and 100 μ g/mouse PIC and then administered PIC only 2 days later. Splenocytes from naive mice, differentially labeled with the fluorescent dye CFSE, were used to determine in vivo cytolytic activity. Cells labeled CFSEhigh were used as targets and pulsed with SIINFEKL, while cells labeled CFSElow were not pulsed and used as internal controls. The previously immunized mice were then co-injected intravenously in a 1: 1 ratio. Cytolytic activity was assessed in lymph node cells.

Control-animals treated with PBS, α IL-2-animals treated with IL-2 neutralizing monoclonal antibody, MB16F 10-animals similar to control but challenged with MB16F10, α IL-2+ MB16F 10-mice bearing MB16F10 tumor treated with IL-2 neutralizing monoclonal antibody.

FIG. 10-induced antibodies to interleukin-2 restore cytolytic activity, evaluated in tumor-bearing host splenocytes.

All mice were immunized subcutaneously on day 0 with 1mg of OVA and 100 μ g/mouse PIC and then administered PIC only 2 days later. In vivo cytolytic activity was determined using splenocytes from naive mice that were differentially labeled with the fluorescent dye CFSE. Cells labeled CFSEhigh were used as targets and pulsed with SIINFEKL, while cells labeled CFSElow were not pulsed, used as internal controls, and then co-injected intravenously into previously immunized mice at a 1: 1 ratio. Cytolytic activity was assessed in splenocytes.

Control-animals treated with PBS, α IL-2-animals treated with IL-2 neutralizing monoclonal antibody, MB16F 10-animals similar to control but challenged with MB16F10, α IL-2+ MB16F 10-mice bearing MB16F10 tumor treated with IL-2 neutralizing monoclonal antibody.

FIG. 11-comparison of blood cell counts (white blood cells, red blood cells, and platelets) for animals immunized with hIL-2r-P64k/Montanide ISA 51 vaccine with controls (P64k/Montanide ISA 51).

Claims (3)

1. Use of an anti-IL-2 monoclonal antibody in the manufacture of a therapeutic preparation for enhancing an immune response against a tumor, wherein the antibody is an IL-2 neutralizing antibody, and wherein the antibody reduces circulating IL-2 levels, thereby enhancing an immune response against the tumor in a subject in need thereof.

2. The use of claim 1, wherein said anti-IL-2 monoclonal antibody is anti-human IL-2.

3. The use of claim 1 or 2, wherein the therapeutic agent comprises an anti-IL-2 monoclonal antibody in combination with an EGF cancer vaccine.