Hematopoietic growth factor depletion vaccine composition for the treatment of inflammatory diseases

Abstract

The present invention relates to the fields of biotechnology and medicine. In particular, it describes a therapeutic vaccine composition capable of generating an autoimmune response against hematopoietic growth factors (such as G-SCF and / or GM-CSF) linked to other molecules or fragments thereof by chemical conjugation or fusion. Such a vaccine composition is useful in particular for the treatment of inflammatory diseases in which a pathological increase in circulating neutrophils occurs.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and pharmaceuticals. In particular, it describes a vaccine composition which is a hematopoietic growth factor, where the antigen can be granulocyte colony-stimulating factor and / or granulocyte macrophage colony-stimulating factor.

Background Art

[0002] Inflammation is a complex process, and effector proteins derived from white blood cells and plasma are mobilized to specific tissue sites to drive a local immune response (Newton K, Dixit VM. (2012) Cold Spring Harb Perspect Biol. 4(3):a006049). This is an effective approach to limit infection and initiate tissue remodeling, but it must be regulated to avoid the accompanying tissue damage. Short-term acute inflammation prevents infection or injury and enables wound healing, while long-term chronic inflammation (resulting from activation of cytokine-producing cells and granulocytes to increase cytokine production and generate a positive feedback loop) can cause increased local tissue damage and a globally regulated immunity (especially T cell response).

[0003] Chronic inflammation, for various reasons, lacks a complete healing phase and never truly ends. These reasons include prolonged contact with infection or irritants, and the continuous presence of cells secreting inflammatory mediators. Immunosuppression and immune tolerance begin when inflammatory stimuli become persistent. In this case, elevated levels of cytokines such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) (granulocyte-macrophage colony-stimulating factor), though below clinical levels, are associated with immune tolerance, while significantly elevated levels are associated with inflammatory exacerbations (Rogovskii V. (2020) Front Immunol. 11:2061). Indeed, inflammatory cytokines (G-CSF and GM-CSF, etc.) can mediate increased immune tolerance. The cost of increased immune tolerance is increased susceptibility to tumors (Stape T (2018) Asia Pac J Oncol Nurs 5:40-2).

[0004] G-CSF and GM-CSF are major cytokines in the formation and differentiation of normal granulocyte precursors in the bone marrow. Their physiological effects are mediated by binding to specific cell surface receptors. Plasma concentrations of G-CSF, GM-CSF, and their receptors are known to alter in inflammatory diseases exhibiting neutrophilia, fever, inflammation, and tissue destruction, and in some cases, in shock and death (Watari K et al. (1989) Blood. 73(1):117-22; Hamilton JA (2020) J Exp Med. 217(1):e20190945).

[0005] When infection occurs, the release of G-CSF and GM-CSF naturally increases because several components of infectious pathogens stimulate their production. Neutrophils originating from this chain of reactions then attack the infectious pathogens and assist in their destruction (Eyles JL et al. (2006) Nat. Clin. Pract. Rheumatol. 2(9):500-510; Ahandideh B et al. (2020) Hum Immunol. 81(5):206-217). G-CSF and GM-CSF also play important roles in the onset, progression, and metastasis of cancer (Do H et al. (2020) Cancers. 12(2):287). The combined action of cytokines produced by neoplastic cells modulates the cellular response of the host immune system. High levels of inflammatory cytokines have been associated with disease progression and poor prognosis in several types of cancer (Silva EM et al. (2017) PLoS ONE 12(7):e0181125.; Lippitz, BE (2013).; The Lancet Oncology, 14(6), e218-e228).

[0006] Neutrophils are known to play a crucial role in various pathologies characterized by chronic inflammation, particularly tumor development, chronic obstructive pulmonary disease, uveitis, arthritis, ankylosing spondylitis, lupus erythematosus, asthma, and cytokine release syndrome. The neutrophil / lymphocyte ratio is a prognostic indicator in some of these conditions (Lee HN et al. (2019) Rheumatol Int. 39(5):859-868).

[0007] In clinical practice, G-CSF and GM-CSF have been widely used to treat neutropenia associated with chemotherapy and to mobilize hematopoietic stem cells for transplantation (Roberts, AW (2005) Growth Factors 23(1):33-4; Mehta HM et al. (2015) J.Immunol. 195(4):1341-1349), and no precedents to the contrary have been found.

[0008] Monoclonal antibodies (Abs) have been described as potentially playing a diagnostic role or as passive therapies to combat tumors that highly express the G-CSF receptor (G-CSFR). Targeted humanized neutralizing monoclonal antibodies against G-CSFR have been reported to be well-tolerated in primates and without causing neutropenia. In addition, mouse anti-G-CSFR monoclonal antibodies suppressed arthritis and significantly inhibited neutrophil accumulation in the joints, and these effects occurred without neutropenia, suggesting that G-CSFR blockade affects neutrophil localization to inflammatory sites (Campbell IK (2016). J.Immunol. 197:4392-4402). However, evidence for antiproliferative and antitumor therapies has not been found. Since G-CSF is an important ligand in neutrophil regulation and function, the use of preparations that inhibit the activation of this cytokine is useful and important as a therapeutic indication in patients with this type of disease.

[0009] There are Phase I trials in healthy volunteers using monoclonal anti-G-CSFR antibodies for the treatment of hidradenitis suppurativa and palmoplantar pustulosis (WO2019 / 178645 and WO2020 / 113270), in which the circulating neutrophil count does not decrease to lower, dangerous levels.

[0010] Blocking the GM-CSF pathway with directional monoclonal antibodies against cytokines or their receptors themselves is described in WO2010 / 124163, and ongoing clinical trials are underway in patients with refractory rheumatoid arthritis (https: / / clinicaltrials.gov / ct2 / show / NCT04333147 (accessed November 23, 2020); ClinicalTrials.gov.https: / / clinicaltrials.gov / ct2 / show / NCT04134728 (accessed November 23, 2020)) and COVID-19 patients (https: / / clinicaltrials.gov / ct2 / show / NCT04376684 (accessed November 23, 2020)). The use of antibodies against GM-CSF in the treatment of toxicity induced by ACT-type immunotherapy is also described (WO2019 / 070680).

[0011] The concentrations of G-CSF and GM-CSF cytokines detected in patients are high, making it impossible to completely neutralize their activity even with high doses of antibodies. Since these molecules are found in the body under normal conditions, the disruption of tolerance and the generation of immunogenicity are not easy processes for these molecules. To date, no aggressive immunotherapy has been proposed to inhibit the growth of G-CSF-dependent or GM-CSF-dependent tumors.

[0012] For the first time, this invention demonstrates an immune response that depletes these hematopoietic growth factors, and evidence of the therapeutic effect of this response is shown in experimental models of inflammation and cancer. The compositions produce increased titers of anti-GCSF antibodies and anti-GM-CSF antibodies, decreased circulating neutrophil count, antiproliferative anti-inflammatory effects, and high antitumor effects. In addition, toxicity is reduced by requiring low doses of the active ingredients, so they can be used in chronic conditions.

[0013] The effects exhibited by the vaccine compositions described herein are unexpected and unforeseen. Due to the complex nature of the cytokine network and its multifaceted effects, as well as the redundancy of the inflammation-generating mechanisms, it is not clear that the antibodies produced by these vaccine compositions can selectively suppress neutrophil populations, much less that this depletion translates into anti-inflammatory and antitumor effects. [Overview of the project]

[0014] In a single procedural act, the subject of the present invention is a therapeutic vaccine composition that induces an immune response to hematopoietic growth factors, comprising a carrier protein, an adjuvant, and at least one antigen which may be rG-CSF or rGM-CSF.

[0015] The carrier proteins used in these vaccine compositions include cholera toxin B, tetanus toxoid, KLH, Neisseria meningitides P64k, diphtheria toxoid, peptides capable of presenting T epitopes against G-CSF and GM-CSF, immunoglobulin G, immunoglobulin M, the Fc region of antibodies, variable fragments of antibodies, and proteins from bacteria, yeast, or mammals. These carrier proteins can be attached to antigens by chemical conjugation or fusion methods.

[0016] Adjuvants that can be used in the vaccine composition of the present invention include incomplete Freund's adjuvants, complete Freund's adjuvants, squalene-based adjuvants, synthetic adjuvants, mineral-derived adjuvants, plant-derived adjuvants, animal protein adjuvants, particulate protein adjuvants, proteoliposome-type adjuvants, liposomes, and any mixture of the above.

[0017] In a specific procedural action, the present invention relates to the use of the vaccine composition described herein in the treatment of inflammatory diseases selected from the group including cancer, chronic obstructive pulmonary disease, uveitis, rheumatoid arthritis, ankylosing spondylitis, lupus erythematosus, Crohn's disease, asthma, dermatitis, cytokine release syndrome, and diseases in which cell degranulation plays a significant role.

[0018] A method for treating a subject in need is described, comprising administering a therapeutically effective dose of the vaccine composition of the present invention in the range of 0.01 to 10 mg / kg body weight in a separate procedural action. In particular, an immune response induction stage is performed, in which doses between 1 and 6 are administered at least once a week, and another stage for maintaining the immune response is performed, in which doses between 1 and 6 are administered at least once a week. This method includes administration of the vaccine composition via an intramuscular, subcutaneous, or intratumoral route. [Modes for carrying out the invention]

[0019] Vaccine composition The present invention includes obtaining a vaccine preparation that induces an immune response to hematopoietic growth factors G-CSF and GM-CSF, and produces a reduction in circulating neutrophil count, an antiproliferative effect, and a high antitumor effect in vivo.

[0020] The active component of the vaccine composition of the present invention is a protein conjugate in which an antigen (G-CSF and / or GM-CSF) is bound to a carrier protein. The carrier protein is selected from, but is not limited to, cholera toxin B, tetanus toxoid, KLH, P64k from Neisseria meningitidis; diphtheria toxoid, and peptides capable of presenting a T epitope to G-CSF and GM-CSF. In the above conjugate, the protein may be fused to the Fc region of immunoglobulin G, immunoglobulin M, or an antibody, whether from human or another animal species. The Fc region may consist of IgG1 mutated in region Cγ2 by mutations L234A and L235A, and may be a variant that binds exclusively to the Fc receptor. In addition, the conjugate may be fused to a variable fragment of an antibody, a bacterial protein, a yeast protein, or a mammalian protein.

[0021] Furthermore, the protein conjugates described above include adjuvants that promote or complement the anti-G-CSF and / or anti-GM-CSF Ab response. These adjuvants may be incomplete Freund's adjuvants, complete Freund's adjuvants, squalene-based adjuvants, synthetic adjuvants, mineral-derived adjuvants, plant-derived adjuvants, animal-derived adjuvants, microparticle protein adjuvants, proteoliposome-type adjuvants, liposomes, or mixtures of any of the above.

[0022] The anti-G-CSF and / or anti-GM-CSF systems, in preferred uses of the present invention, have contained pharmaceutically acceptable excipients. These include, but are not limited to, water for injection, sodium chloride, phosphorus salts and potassium salts, calcium chloride, sodium hydroxide and sodium citrate, and EDTA. Patients may be administered parenterally at protein concentrations of 0.01–10 mg / mL and doses of 10–100 μL / kg, or 10–100 μg of total protein per kilogram, or up to 5 mg of total protein (10–60 μg / kg is more recommended).

[0023] The therapeutic composition can be stored in liquid form at a temperature of -80°C to 8°C, or at a temperature of 2 to 8°C after a freeze-drying process.

[0024] Acquisition of chemical conjugates between carrier proteins and recombinant G-CSF (rG-CSF) and / or recombinant G-CSF (rGM-CSF) From the evaluation and optimization of conditions for the chemical conjugation reaction between rGCSF protein and / or rGM-CSF protein and some of the carrier proteins mentioned above, a chemical conjugation method has been developed that requires a high molar ratio of self-protein to ensure high conjugation efficiency between the two during conjugation. The excess amount of self-protein required during conjugation is then removed through an ultrafiltration membrane purification method, thereby enabling the acquisition of a vaccine preparation with high homogeneity.

[0025] The procedure described in the present invention ensures proper chemical conjugation between both proteins and consists of a single step. The procedure starts by mixing the rGCSF protein and / or rGM-CSF protein pre-concentrated in the range of 0.1 - 1 mg / mL and the molecule it will be conjugated to in a conjugation reactor at a range of 0.6 - 20 mg / mL. Subsequently, a solution of PBS / MgCl2 (pH 6.0 - 7.2) and a glutaraldehyde conjugation solution in the range of 0.1 - 0.8% are added to this protein mixture. The mixture is maintained at a temperature of 20 - 24 °C ± 2 °C with continuous stirring for 15 minutes to 4 hours. The total protein concentration during the conjugation reaction is 1 - 20 mg / mL.

[0026] Subsequently, purification consisting of two stages is performed using an ultrafiltration membrane in the range of 50 - 100 kDa. In the initial stage, continuous exchange of the buffer solution (diafiltration) is carried out to remove glutaraldehyde, remove excess self-protein, and either free or conjugate only the rGCSF protein or only the rGM-CSF protein of different sizes. During this stage, 3 - 15 exchanges of the buffer solution are carried out. The second stage is the concentration of the purified chemical conjugate.

[0027] The therapeutic preparation obtained by membrane purification is characterized by a chemical conjugation ratio between the rGCSF protein and the molecule it is conjugated to in the range of 5:1 - 20:1 and the absence of glutaraldehyde.

[0028] Obtaining a Therapeutic Composition by a Method for Expressing a Fusion Protein The vaccine composition of the present invention can also be obtained by designing genetic constructs based on the rG-CSF gene and the rGM-CSF gene, which are cloned into an expression vector (preferably PCMX, but not limited thereto) and fused to the gene of any of the aforementioned carrier proteins. The cells used in transfection can be HEK-293T, HEK-293-GE, Expi 293, HEK-293, or CHOk1. The supernatant of the transfected cells is collected after culturing for 6 to 10 days. Then, the recombinant protein is purified by protein A affinity chromatography or metal ion affinity chromatography.

[0029] Treatment method The system described above is effective in maintaining the anti-G-CSF immune response and / or the anti-GM-CSF immune response by pre-inducing antibodies generated by the system described in the present invention. When G-CSF and GM-CSF play related roles, the system is useful in the treatment of inflammatory diseases. These diseases include, but are not limited to, cancer (especially tumors dependent on G-CSF or GM-CSF), chronic obstructive pulmonary disease, meningitis, arthritis, ankylosing spondylitis, lupus erythematosus, Crohn's disease, asthma, dermatitis, cytokine release syndrome, and diseases in which cell degranulation plays an important role.

[0030] The dosage approved for use in humans or animals is administered in induction doses of 1 to 6 administered at least weekly, and then, in the stage of maintaining the immune response, administered until toxicity that limits use occurs at 1 dose, and is administered not only at least weekly, but also every other week, monthly, quarterly, or annually, intramuscularly, subcutaneously, or intratumorally.

[0031] The present invention will be described in more detail by the following examples and drawings. However, these examples should not be construed as limiting the scope of the present invention.

Brief Description of Drawings

[0032] [Figure 1] Characterization of the rG-CSF-P64k system by SDS-PAGE electrophoresis. [Figure 2] Characterization of rG-CSF-Fc and rGM-CSF-Fc systems by SDS-PAGE electrophoresis. [Figure 3] A study of the dynamics of anti-G-CSF antibody titer generation after administration of the therapeutic composition rG-CSF-P64k alone or in combination with G-CSF. [Figure 4] A) Generation of anti-G-CSF antibody titers after administration of therapeutic composition rG-CSF-P64k at different doses. B) Dynamics of anti-G-CSF antibody titer generation after administration of therapeutic composition rG-CSF-P64k at different doses. [Figure 5] Evaluation of circulating neutrophil counts after administration of the therapeutic composition rG-CSF-P64k alone or in combination with G-CSF. [Figure 6] Inhibition of cell proliferation induced by serum obtained from mice immunized with the therapeutic composition rGCSF-P64k. [Figure 7] Evaluation of the anti-inflammatory effect of the therapeutic composition rGCSF-P64k after administration in an inflammation model induced by croton oil application. A) Decrease in circulating neutrophil count. B) Decrease in atrial edema. [Figure 8] A study of the dynamics of Ab anti-GCSF titer generation after administration of the therapeutic composition rG-CSF-Fc alone or in combination with GCSF. [Figure 9] A) Study on the generation of anti-G-CSF antibody titers after administration of therapeutic composition rG-CSF-Fc at different doses. B) Dynamics of anti-G-CSF antibody titer generation after administration of therapeutic composition rG-CSF-Fc at different doses. [Figure 10] A study of the dynamics of anti-G-CSF antibody titer generation after administration of the therapeutic composition rG-CSF-Fc in the Balb / c strain. [Figure 11] Evaluation of circulating neutrophil counts after administration of the therapeutic composition rG-CSF-Fc alone or in combination with G-CSF. [Figure 12]Inhibition of cell proliferation induced by serum obtained from mice immunized with the therapeutic composition rGCSF-Fc. [Figure 13] A study of the dynamics of anti-GM-CSF Ab titer generation after administration of the therapeutic composition rGM-CSF-Fc. [Figure 14] A) A study on the generation of anti-GM-CSF antibody titers after administration of therapeutic composition rGM-CSF-Fc at different doses. B) A study on the kinetics of anti-GM-CSF antibody titer generation after administration of therapeutic composition rGM-CSF-Fc at different doses. [Figure 15] Inhibition of cell proliferation induced by serum obtained from mice immunized with the therapeutic composition rGMCSF-Fc. [Examples]

[0033] Example 1. Acquisition of a chemical conjugate between recombinant protein rP64k and rG-CSF. To obtain a chemical conjugate between rGCSF and P64k proteins in a 20:1 ratio, 17.6 mg of GCSF protein and 3 mg of P64k carrier protein are added in a NaHCO3Na2CO3 buffer solution (0.01 M) and a 0.5% glutaraldehyde conjugation solution in a reactor with stirring for 1 hour. Subsequently, detail impurities and unconjugated proteins are removed via ultrafiltration. This process is carried out using a buffer containing polysorbate 80, sorbitol, sodium acetate, acetic acid, and water for injection. Seven buffer changes are performed during this operation. The remaining ultrafiltration solution is concentrated to adjust the concentration to a dosage of 1 mg / ml. It is then stored at 2-8°C.

[0034] The purity of the purified protein was assessed using a 10% SDS-PAGE gel and the mass of one μg of protein. Figure 1 shows that the obtained vaccine composition corresponds to a molecular weight of approximately 200 kD.

[0035] Example 2. Obtaining a therapeutic composition via a fusion protein expression method. Genetic constructs based on the G-CSF gene and another gene based on the GM-CSF gene were designed, fused to the Fc region of human immunoglobulin G1 and cloned into a PCMX expression vector. Expi 293 cells were transfected with polyethyleneimine mixed with the gene constructs to be evaluated. The supernatant from the transfected cells was collected after 6 days of culture. The recombinant proteins were purified by affinity to the protein A matrix. The purity of the purified proteins was evaluated on a 7.5% SDS-PAGE gel, with 2 μg of G-CSF-Fc used and 9 μg in the case of GM-CSF-Fc.

[0036] Figure 2 shows that the molecular weights of both compositions evaluated correspond to approximately 35 kD.

[0037] Example 3. The therapeutic composition rG-CSF-P64k induces anti-G-CSF antibodies. C57BL / 6 mice (n=5) were immunized intramuscularly on days 0, 7, 21, 35, and 42 with the formulation from Example 1, administered at 50 μg / kg body weight, with montanide as an adjuvant (V / V 1:1). G-CSF or PBS was also administered subcutaneously three times a week, starting on day 14.

[0038] Blood samples were collected on day 0 (preimmunization), day 14, day 35, and day 56 and processed into serum. The titer of antibodies specific to G-CSF was determined by ELISA. For this purpose, plates were covered with 5 μg / mL G-CSF and incubated at 37°C for 1 hour. After corresponding blocking, serum dilutions (1 / 10, 1 / 100, 1 / 500, 1 / 1000, 1 / 5000, 1 / 10000, 1 / 20000) were added. The reaction was visualized using anti-mouse IgG antibody / alkaline phosphatase conjugate (Sigma) and the corresponding enzyme substrate. Absorbance was read at 405 nm. Preimmunization serum was used as a negative control. Antibody titer was expressed as ELISA optical density (OD) reading, and the maximum serum dilution was defined as exceeding 5 standard deviations of the mean OD obtained in the well containing preimmunization serum.

[0039] The conditions for an immune response were defined using the geometric mean of anti-G-CSF antibody titers, evaluated by ELISA in each experimental group. Immunized mice produced specific antibodies, reaching titers exceeding 1 / 10,000, indicating that the vaccine composition induced an immune response against G-CSF itself (Figure 3).

[0040] Example 4. The therapeutic composition rG-CSF-P64k at different concentrations induces anti-G-CSF antibodies. C57BL / 6 mice (n=5) were intramuscularly immunized with six doses of the formulation from Example 1 at 14-day intervals, using montanide as an adjuvant (V / Ver 1:1) at different concentrations of 50, 25, 12.5, 6.25, and 3.125 μg. From the second immunization onward, G-CSF or PBS was also administered subcutaneously twice a week.

[0041] Blood samples were collected on day 0 (pre-immunization) and 15 days after each immunization and processed into serum. The titer of antibodies specific to G-CSF was determined by ELISA. For this purpose, plates were covered with 5 ug / mL of G-CSF and incubated at 37°C for 1 hour. After corresponding blocking, serum dilutions (1 / 100, 1 / 1000, 1 / 10000, 1 / 100000, 1 / 1000000) were added. The procedure described in Example 3 was then followed.

[0042] Immunized mice produced specific antibodies, which reached titers exceeding 1 / 1000 at different concentrations of the vaccine composition, demonstrating their ability to induce an immune response to G-CSF. Significant differences were observed between titers obtained in mice immunized with multiple doses of the antigen (Figure 4A). This demonstrates dose-dependence between immunization groups with respect to the response, as measured by antibody titer.

[0043] Humoral immune responses were also evaluated at different time intervals corresponding to doses 3, 4, 5, and 6 of the vaccine composition. Antibody titers in all animals immunized with different concentrations of the vaccine preparation increased in a dose-dependent manner, reaching a plateau from the fifth immunization (Figure 4B).

[0044] Example 5. The therapeutic composition rG-CSF-P64k reduces the circulating neutrophil count in C57BL / 6 mice. C57BL / 6 mice (n=5) were immunized with the vaccine formulation of Example 1 (V / V 1:1) with montanide as an adjuvant, according to the immunization schedule of Example 3. G-CSF or PBS was also administered subcutaneously three times a week, starting on day 14. Peripheral blood was collected from the maxillary sinuses of the animals on day 0 (pre-immunization) and 56 days after the first immunization, and collected in vials containing EDTA (40 μl per 1 mL of blood). Carl Zeiss microscope.

[0045] Normality was verified by the Kolmogorov-Smirnov test, and homovariance was verified by Rubine's test. Paired Student's t-tests were performed between the values ​​of each animal before immunization and the values ​​of each animal at days 35 and 56. The significance level was p<0.05. In animals treated with the G-CSF-P64k conjugate, a statistically significant reduction in neutrophils was observed at day 56, suggesting that this pharmaceutical composition can induce neutropenia through the anti-G-CSF antibodies it generates (Figure 5). It should also be noted that neutropenia does not necessarily mean a decrease in mouse survival.

[0046] Example 6. The therapeutic composition rGCSF-P64k inhibits cell proliferation of the mouse myeloblast cell line NFS60. The effects of serum obtained on days 14 and 28 using the therapeutic composition of Example 1 and the immunotherapy scheme described in Example 3 were evaluated by cell proliferation assays in the mouse myeloblast cell line NFS60 (G-CSF dependent) after immunization of C57BL / 6 mice.

[0047] Cells were pre-thawed and maintained in culture for 48 hours to achieve exponential growth. Incubation conditions during the test were a temperature of 37°C and a 5% CO2 atmosphere. Cells were seeded in 96-well culture plates at a concentration of 10,000 cells per well in the presence of a vaccine formulation in which the active ingredient is an rP64k-rG-CSF protein conjugate at dilutions of 1 / 250; 1 / 500; and 1 / 1000. hG-CSF reference material (MRT(QFB)G-CSF / 1905) was used for these assays at the same concentration as a positive control, and cells in G-CSF-free medium were included as a negative control. After 48 hours of incubation, 20 μL of Alamar Blue was added per well and incubated for 6 hours. Plates were read at 540 nm and 620 nm. All samples were tested in duplicate.

[0048] Figure 6 shows that administration of the vaccine composition inhibits cell division and proliferation in a dilution-dependent manner (lower dilutions indicate greater effect) and time elapsed since the start of treatment. This is because a higher inhibitory effect was observed on day 28 than on day 14, indicating that the vaccine formulation inhibits the proliferative effect of G-CSF (which is important for granulocyte formation in the tumor strain being evaluated).

[0049] Example 7. The therapeutic composition rG-CSF-P64k has an anti-inflammatory effect. C57BL / 6 mice were subcutaneously immunized with the vaccine formulation from Example 1 (V:V 1:1) at a dose of 50 μg / kg body weight in complete Freund's adjuvant on days 0, 7, and 21. The remaining immunization was carried out in Freund's incomplete adjuvant. As a control group, mice were administered saline via the same route and frequency.

[0050] On days 0, 7, and 21, 10 μL of 0.4% croton oil was applied topically to each surface of the right auricle of the animals. An equal volume of saline solution was applied to the left auricle. Blood samples were taken from all animals for neutrophil counting on day 0 and 20 days prior to the start of croton oil administration. On day 35, 4 hours after the application of the irritant, the animals were sacrificed to determine the response to auricular edema. For these animals, tissue fragments with a diameter of 6 mm were taken from the auricle and weighed on an analytical balance. The percentage of inhibition of edema and inflammation was calculated from the weights obtained from both auricular discs of each animal, taking the following into consideration: Edema = Weight of the swollen auricle (right auricle) - Weight of the non-swollen auricle (left auricle) Inflammation inhibition % = [(Pc-Pt) / Pc] × 100 in the formula: Pc: Arithmetic mean of the change in weight in the control group. Pt: Arithmetic mean of weight variation in the treatment group.

[0051] The statistical package MINITAB (Minitab Inc., version 16.1.0.MINITAB, 2010) was used for data processing, and a 90% confidence level was established for interpreting the results. Data were compared between groups, and normality and homovariance were analyzed using the Kolmogorov-Smirnov (KS) test and the Rubine test, respectively. To determine whether there were significant differences between experimental groups, parametric Student's t-tests (neutrophil counts) and the Mann-Whitney U test for edema were performed.

[0052] As a result of the previous procedure, a statistically significant reduction in neutrophil counts (p<0.1) was detected in the GCSF-P64K conjugate-treated group compared to the control group on day 20 (Figure 7A). On day 35, inhibition of acute inflammation was detected in the GCSF-P64K conjugate-treated group compared to the control group, characterized by a statistically significant 53.85% reduction (p<0.1) in croton oil-induced auricular edema (Figure 7B), indicating that the vaccine preparation has an anti-inflammatory effect in a model of acute inflammation.

[0053] Example 8. The therapeutic composition rG-CSF-Fc induces an antibody response. A group of five C57BL / 6 mice was intramuscularly immunized on days 0, 14, 28, and 42 with the vaccine composition described in Example 2, consisting of G-CSF coupled to Fc as the antigen and Montanide as the adjuvant, at a concentration of 20 μg / kg. Blood was collected on days 14 and 56, and serum was processed to determine the specific antibody titer against G-CSF by ELISA. For this purpose, plates were coated with 5 μg / mL of G-CSFh, which has six histidine tails, at 4°C for 16-20 hours. The plates were blocked at 25°C for 1 hour with 4% skim milk powder diluted in phosphate-buffered saline. Serial dilutions (1 / 10 to 1 / 10) of serum and pre-immunized serum from immunized mice were used in the blocking solution. 7The mixture was then incubated at 25°C for 1 hour. Six washes were performed with 0.1% (V:V) Tween 20 solution. Anti-mouse IgG immunoglobulin-horradish peroxidase conjugate (Sigma, A2554-1 mL), diluted 1:35000 in blocking solution, was added and incubated at 25°C for 1 hour. Orthophenylenediamine (0.5 mg / mL) (Sigma, USA) was added with 0.015% hydrogen peroxide in a substrate buffer solution (200 mmol / L Na2HPO4, 100 mmol / L citrate, pH=5) and incubated at 25°C for 30 minutes for color development. The reaction was stopped with 10% H2SO4. Absorbance was determined at 490 nm using a Dialab GmbH ELISA reader, ELx808.

[0054] The highest dilution was determined when the absorbance was at least twice that of the preimmune serum from the same animal. The results shown in Figure 8 correspond to the titer measurements of serum obtained on days 14 and 56. The graph shows that immunity to G-CSFh in mice can generate a humoral response and high titer against the cytokines mentioned above.

[0055] Example 9. The therapeutic composition rG-CSF-Fc at different concentrations induces anti-G-CSF antibodies. C57BL / 6 mice (n=7) were immunized intramuscularly with six doses at 14-day intervals using the formulation from Example 2, which contained GCSF coupled to Fc as the antigen and montanide as an adjuvant (V / V 1:1), at different concentrations of 80, 40, 20, 10, and 5 μg. Serum was collected on day 0 (pre-immunization) and 14 days after each immunization for serum processing. The titer of antibodies specific to G-CSF was determined by ELISA, and then the procedure of Example 3 was followed.

[0056] The immunized mice produced specific antibodies, which reached titers exceeding 1 / 1000 without statistically significant differences between groups immunized with different concentrations of the vaccine composition, demonstrating that they induced a dose-independent immune response to G-CSF at the evaluated interval (Figure 9A).

[0057] Humoral immune responses were evaluated at different time intervals in animals immunized with 40 and 5 ug vaccine compositions, following the procedure of Example 4. Antibody titer dynamics remained in a plateau phase from the third immunization, with only small fluctuations after the fourth dose. Concentration independence was maintained in the development of the humoral response over time (Figure 9B).

[0058] Example 10. The therapeutic composition rG-CSF-Fc induces anti-G-CSF antibodies in the Balb / c lineage. Balb / c mice were isolated at 14-day intervals and subcutaneously immunized with 5 doses of the therapeutic composition from Example 2, which contained G-CSF fused to Fc as an antigen and montanide as an adjuvant. Blood samples were collected on day 0 (pre-immunization), day 42, day 70, and day 77.

[0059] Specific antibody titers against G-CSF were determined by ELISA for different serum extracts. For this purpose, plates were covered with 5 ug / mL of G-CSF and incubated at 37°C for 1 hour. After corresponding blocking, serum dilutions (1 / 100, 1 / 1000, 1 / 10,000, 1 / 100,000, 1 / 1,000,000) were added, and the process proceeded as described in Example 3.

[0060] Immunized Balb / c mice produced specific antibodies, reaching an average titer of 1 / 10,000, demonstrating that the vaccine composition induces a strong immune response against G-CSF (Figure 10).

[0061] Example 11. The therapeutic composition rG-CSF-Fc reduces the circulating neutrophil count in C57BL / 6 mice. C57BL / 6 mice (n=5) were administered 20 μg / kg of the vaccine composition described in Example 2, containing G-CSF coupled to Fc as an antigen and Montanide as an adjuvant, according to the immunization schedule described in Example 3. Neutrophil counts were assessed using a Carl Zeiss microscope. Peripheral blood was extracted from the maxillary sinuses of the animals on day 0 (pre-immunization) and on day 56 of the first immunization and collected in vials containing EDTA (40 μl per 1 mL of blood). Neutrophil counts were performed using a Carl Zeiss microscope.

[0062] Normality was verified by the Kolmogorov-Smirnov test and equal variances by Rubine's test. Paired Student's t-tests were performed between the values ​​of each animal before immunization and the values ​​of each animal at day 35 and day 56. The significance level is p<0.05.

[0063] A statistically significant reduction in neutrophils was observed at day 56 in animals treated with the GCSF-P64k conjugate, suggesting that this pharmaceutical composition can induce neutropenia through the anti-G-CSF antibodies it generates (Figure 11). It should also be noted that neutropenia does not necessarily mean a decrease in mouse survival.

[0064] Example 12. The therapeutic composition rGCSF-Fc inhibits cell proliferation of the mouse myeloblast cell line NFS60. C57BL / 6 mice were isolated at 14-day intervals and immunized intramuscularly with 5 doses of the therapeutic composition from Example 2, which contained GCSF fused to Fc as an antigen and used Montanide as an adjuvant, in doses of 40 and 5 µg.

[0065] The effects of serum obtained on day 0 (pre-immunization) and at the end of the immunization scheme were evaluated using a cell proliferation assay in the mouse myeloblast cell line NFS60 (G-CSF dependent), and the procedure is described in Example 5.

[0066] Figure 12 shows that administration of the vaccine composition inhibits cell division and proliferation, but this inhibition does not occur in the serum of unimmunized animals (pre-immunized). This indicates that the vaccine formulation inhibits the proliferation and mitotic effect of G-CSF, which is important for granulocyte formation in the NFS60 strain. The evaluated inhibition of cell division and proliferation in serum did not show dependence on the concentration of the vaccine composition.

[0067] Example 13. The therapeutic composition rGM-CSF-Fc induces an antibody response. A group of four BALB / c mice was subcutaneously immunized with 20 μg of human GM-CSF fused to the IgG1 Fc chain. Serum was collected from the mice two days before the first immunization and used as a pre-immunization serum control. Six immunizations were performed at 14-day intervals, and blood was collected seven days after the sixth dose. On day 0, immunization was performed with 20 μg of protein (V:V 1:1) emulsified in Freund's complete adjuvant. The remaining immunizations were performed in Freund's incomplete adjuvant.

[0068] Blood samples were collected on day 0, day 35, day 49, and day 77, and antibody titers against GM-CSF were determined in serum by ELISA. For this purpose, 96-well plates were coated with 5 μg / mL human GM-CSF dissolved in phosphate-buffered saline at 4°C for 16-20 hours. The plates were blocked at 25°C for 1 hour with 4% skim milk powder diluted in phosphate-buffered saline. Serial dilutions (1 / 10 to 1 / 107) of serum and pre-immunized serum from immunized mice were added to the blocking solution and incubated at 25°C for 1 hour. Six washes were performed with 0.1% (V:V) Tween 20 solution. Anti-mouse IgG-horradish peroxidase conjugate diluted to 1 / 35,000 was added to the blocking solution and incubated at 25°C for 1 hour. For color development, orthophenylenediamine (0.5 mg / mL) (Sigma, USA) was used with 0.015% hydrogen peroxide in a substrate buffer solution (200 mmol / L Na2HPO4, 100 mmol / L citrate, pH=5) at 25°C for 30 minutes. The reaction was stopped with 10% H2SO4. All samples were applied in double doses, and the highest dilution was considered to be when the antibody titer present during the formation of immunized mice showed an absorbance at least twice that of the pre-immunized serum of the same animal.

[0069] In Figure 13, the graph shows that immunity to human GM-CSF produced a high response with a maximum titer of 1 × 10⁷. The results indicate that immunity is capable of generating a high-titer humoral response to such cytokines in mice.

[0070] Example 14. The therapeutic composition rGM-CSF-Fc at different concentrations induces anti-GM-CSF antibodies. C57BL / 6 lineage mice (n=7) were immunized intramuscularly with six doses at 14-day intervals using the formulation from Example 2, which contained GMCSF coupled to Fc as the antigen and montanide as an adjuvant (V / V 1:1), at different concentrations of 40, 20, 10, 5, and 2.5 μg.

[0071] For serum processing, blood was collected on day 0 (pre-immunization) and 14 days after each immunization. The titer of antibodies specific to GM-CSF was determined by ELISA. To this end, plates were covered with 5 μg / mL of GM-CSF and incubated at 37°C for 1 hour, following the procedure of Example 4.

[0072] The conditions for an immune response were defined using the geometric mean of anti-GM-CSF antibody titers evaluated by ELISA in each experimental group. Immune mice produced specific antibodies that reached titers exceeding 1 / 1000 without statistically significant differences between groups immunized with different concentrations of the vaccine composition, demonstrating that an immune response to GM-CSF was induced in a concentration-independent manner within the tested dose range (Figure 14A).

[0073] Humoral immune responses were evaluated at different time intervals in animals immunized with 20 and 5 ug vaccine compositions, following the procedure of Example 4. The dynamics of antibody titers increased in correspondence with the number of doses. From the 5th dose onward, a plateau phase with no fluctuation in the response was observed. Independence from concentration was maintained in the development of the humoral response over time (Figure 14B).

[0074] Example 15. The therapeutic composition rGMCSF-Fc inhibits cell proliferation of the mouse myeloblast cell line NFS60. Balb / c mice were isolated at 14-day intervals and subcutaneously immunized with 5 doses of the therapeutic composition from Example 2, which contained GMCSF fused to Fc as an antigen and was adjuvanted with Montanide. Blood samples were collected on day 0 (pre-immunization) and 14 days after the final immunization.

[0075] The effects of serum obtained from immunotherapy were evaluated by a cell proliferation assay in the mouse myeloblast cell line NFS60 (G-CSF-dependent), following the same procedure described in Example 6.

[0076] Figure 15 shows that administration of the rGMCSF-Fc vaccine composition inhibits cell division and proliferation in immunized animals, but not in the serum of unimmunized animals (pre-immunized). This indicates that the vaccine formulation inhibits the proliferative effect of G-CSF, which is important for granulocyte formation in the NFS60 strain.

Claims

1. Carrier proteins, Adjuvants and, At least one antigen selected from the group consisting of recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) Includes, The aforementioned carrier protein, P64k of Neisseria meningitidis, and Antibody Fc region Selected from the group including, A therapeutic vaccine composition for inducing an immune response to granulocyte colony-stimulating factor (rG-CSF) or granulocyte-macrophage colony-stimulating factor (rGM-CSF), wherein the carrier protein is bound to the antigen.

2. The aforementioned carrier protein, Chemical conjugation, and fusion The vaccine composition according to claim 1, wherein the antigen is bound by any of the following.

3. The vaccine composition according to claim 1 or 2, wherein the antigen is rG-CSF.

4. The vaccine composition according to claim 1 or 2, wherein the antigen is rGM-CSF.

5. The aforementioned adjuvant, Incomplete Freund adjuvant, Complete Freund adjuvant, Squalene-based adjuvant, Synthetic adjuvants, Mineral-derived adjuvants, Plant-derived adjuvants, Animal-derived adjuvants, Microparticle protein adjuvants, Proteoliposome adjuvant, Liposomes, and The above adjuvant mixture A vaccine composition according to any one of claims 1 to 4, selected from the group including the following.

6. cancer, chronic obstructive pulmonary disease, Uveitis, Rheumatoid arthritis, ankylosing spondylitis, erythematous lupus, Crohn's disease, asthma, dermatitis, Cytokine release syndrome, and Diseases associated with cell degranulation A vaccine composition according to any one of claims 1 to 5, selected from the group comprising rG-CSF or rGM-CSF, for the treatment of inflammatory diseases associated with increased rG-CSF.

7. A vaccine composition according to any one of claims 1 to 6, administered in a therapeutically effective dose in the range of 0.01 to 10 mg / kg.

8. The vaccine composition according to claim 7, wherein the immune response induction stage is achieved by administering the vaccine composition in 1 to 6 doses at least weekly, and the maintenance stage is achieved by administering the vaccine composition weekly until toxicity occurs at a dose of at least 1 dose.

9. The vaccine composition according to claim 8, wherein the administration route of the vaccine composition is selected from the group including intramuscular, subcutaneous, and intratumor.

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