Ganglioside GM3-containing nanoparticles as immunomodulators

Abstract

The present invention describes a pharmaceutical composition whose active ingredient includes conjugates of membrane vesicles of Neisseria meningitidis and the GM3 ganglioside in a conjugation ratio in excess of proteins, has particular characteristics of size, surface charge and a morphology associated with nano-particulate systems that give it advantageous properties as an immunomodulator, because it induces a convenient and significant reduction of myeloid-derived suppressor cells that has an impact on the response of lymphocytes and on the survival of patients with tumors. The invention further discloses the use of the pharmaceutical composition disclosed in the treatment of cancer, particularly those cancers where the myeloid-derived suppressor cells (MDSCs) are high; as well as a method of treatment with said composition in cancer patients and a method to select those who will receive said treatment.

Claims

1. A pharmaceutical composition comprising nanoparticles characterized by hydrophobic conjugation of outer membrane protein complex (OMPC) of Neisseria meningitidis bacterium to a GM3 ganglioside, wherein the OMPC:GM3 ganglioside conjugation ratio ranges from 1.5: 1 to 10: 1.

2. The composition of claim 1, wherein the nanoparticles are characterized by a monomodal distribution of volume in a range from 15 to 25 nm particle size, polydispersity index of 0.230, and/or negative Z potential with nominal value in the range from 25 to 45 mV.

3. A method for treating a subject in need thereof comprising the administration of the pharmaceutical composition according to claim 1 by SC, intradermal, intramuscular, intratumoral routes or by direct application to mucosal surfaces with a weekly frequency for at least a total of four doses and subsequently fortnightly or monthly in maintenance doses for at least six months.

4. A pharmaceutical composition comprising nanoparticles, wherein each nanoparticle comprises outer membrane protein complex (OMPC) of Neisseria meningitidis bacterium hydrophobically conjugated to a GM3 ganglioside, wherein the protein-ganglioside conjugation ratio ranges from 1.5: 1 to 10: 1, and wherein the composition is characterized by a monomodal distribution of volume in a range from 15 to 25 nm particle size, polydispersity index of 0.230, negative Z potential with nominal value in the range from 25 to 45 mV.

5. A pharmaceutical composition comprising nanoparticles characterized by hydrophobic conjugation of outer membrane protein complex (OMPC) of Neisseria meningitidis bacterium to a GM3 ganglioside, wherein the nanoparticles are characterized by a monomodal distribution of volume in a range from 15 to 25 nm particle size, a polydispersity index of 0.230, and a negative Z potential with nominal value in the range from 25 to 45 mV.

6. A method of producing a nanopoarticle, the method comprising conjugating outer membrane protein complex (OMPC) of Neisseria meningitidis bacterium to a GM3 ganglioside, where the OMPC:GM3 ganglioside conjugation ratio ranges from 1.5:1 to 10:1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Evaluation by Photonic Correlation Spectroscopy of the diameter of VSSP-iMod particles.

(2) FIG. 2. Evaluation by Photonic Correlation Spectroscopy of Z Potential of VSSP-iMod particles.

(3) FIG. 3. Image of the Morphology of VSSP-iMod particles by Atomic Force Microscopy.

(4) FIG. 4. Evaluation by flow cytometry of the effect of treatment with VSSP-iMod in patients with metastatic renal cell carcinoma (mRCC) of a) the frequency of gMDSCs and b) percentage of patients with frequency of gMDSCs above and below the median.

(5) FIG. 5. Evaluation by flow cytometry of the effect of the treatment with VSSP-iMod on the capacity of the MDSCs to suppress the proliferation of: a) TCD4+lymphocytes and b) TCD8+lymphocytes.

(6) FIG. 6. Flow cytometric evaluation in patients with mammary carcinoma of the effect of treatment with VSSP-iMod on: a) frequency and % of patients with gMDSC frequency above and below the median, b) frequency and % of patients with mMDSC frequency above and below the median.

(7) FIG. 7. Determination by flow cytometry of the effect of treatment with VSSP-iMod on the absolute number of TCD8+lymphocytes in patients with mammary carcinoma.

EXAMPLES

Example 1. The VSSP-iMod has a Defined Size and Surface Charge

(8) The size in nanometers and Z potential of the particles that make up the VSSP-iMod were measured by Photonic Correlation Spectroscopy. Samples were evaluated in triplicate, size and potential Z values were obtained using the CONTIN and Smoluchowski algorithms respectively. As shown in FIG. 1, the VSSP-iMod showed a monomodal distribution in a range from 15 to 25 nm in volume distribution, with a polydispersity index (PDI) of 0.230, which means that we are in the presence of a heterogeneous formulation of particles. Additionally, the VSSP-iMod showed a negative Z potential and its nominal value was in the range between 25-45 mV as shown in FIG. 2.

Example 2. Nano-Particulate Morphology of VSSP-iMod

(9) The images of the VSSP-iMod were obtained in a Multimode Microscope of Atomic Force and a silicon cantilever was used. 504 of sample were applied to a mica previously functionalized with a 50 mol/L nickel chloride solution. A 1/10 dilution with Tris buffer solution 10 mmol/L pH 8.5 was performed to the VSSP-iMod, prior to the application to the mica. The image of FIG. 3 shows a heterogeneous formulation composed of nano-particulate structures of spherical nature in the order of tens of nanometers, which is in total correspondence with the result obtained by Photon Correlation Spectroscopy.

Example 3. VSSP Decreases the Frequency and Suppressive Activity of MDSCs in Patients with mRCC

(10) The effect of VSSP-iMod on MDSCs in patients with mRCC was evaluated. To this purpose, fifteen patients with this diagnosis were treated with 400 μg of VSSP-iMod administered by SC route in the deltoid region. A total of four doses of VSSP-iMod were administered with a weekly frequency followed by a monthly maintenance doses until completing 6 months of treatment. In this assay the frequency of gMDSCs was evaluated by flow cytometry. To this purpose, a total of 200,000 cells were analyzed and the percentages of gMDSCs were determined by measuring the CD11b.sup.+/CD66b.sup.+/CD14.sup.− phenotype within the total PBMC. As a control, the frequency of gMDSCs in 15 healthy donors matched by age and gender was evaluated. As seen in FIG. 4a, the VSSP-iMod decreased the frequency of circulating gMDSCs in patients after 21 days or three doses after treatment initiation. The state of the art teaches that patients who present gMDSCs below the median established for patients of a certain location, have a significantly higher survival than those who have levels below it (Shipp C. et al (2016) Cell, Mol. Life, Sci. 73 (21): 4043-61). The analysis of the percentage of patients with gMDSC frequency above and below the median is shown in FIG. 4b. As can be observed after treatment with VSSP-iMod, only about 20% of the treated patients keep the MDSCs high. This result was maintained on day 147 or after the fifth month, which indicates that this effect of VSSP-iMod is maintained throughout the treatment.

(11) In these patients, the consequences for the response of lymphocytes of the effect of VSSP-iMod on the MDSCs was also evaluated in a proliferation experiment by flow cytometry. A total of 40×10.sup.6 cells from the PBMC of said patients were used as starting material. The CD11b+ cells were purified by the use of magnetic beads conjugated to a CD11b specific Ab. The CD11b.sup.− negative fraction was labeled with CFSE and cultured alone or with CD11b+ cells at a 5:1 ratio for 96 hours. FIG. 5 shows the relative proliferation percentage of T lymphocytes on day 0 and on day 21 after the administration of VSSP-iMod in RCC patients. As seen in FIG. 5a, the proliferation of TCD4+lymphocytes increases on day 21, an effect similar to that observed in TCD8+lymphocytes (FIG. 5b), which means that the VSSP-iMod is able to modulate the MDSCs-mediated suppression of T cell proliferation in the RCC patients.

(12) The majority of the patients enrolled in the trial showed good quality of life at the end of their treatment, as established in the protocol, it was decided that they continue with monthly immunizations. Treatment prolongation maintained the gMDSCs below the median at day zero and the survival median of the total patients in this trial was 37.5 months (Table 1). This value is much higher than the historical median of 6.6 months reported for similar patients treated with interferon, which is the current standard of treatment in Cuba. Additionally, the clinical practice guidelines for mRCC of the National Comprehensive Cancer Network (NCCN) classify patients according to the models of the Memorial Sloan Kettering Cancer Center (MSKCC) as having favorable, intermediate and poor prognosis. These guidelines state that patients with mRCC treated with therapies against the vascular endothelial growth factor have a median survival of 27 months in the case of those diagnosed as having intermediate prognosis, while 75% of those diagnosed as having a favorable prognosis are alive at 24 months. The relative comparison of the values stated in the guidelines against those obtained with VSSP-iMod, indicates that the effect of VSSP-iMod on the gMDSCs also produced a survival higher than the standard formed in the NCCN guidelines. In the VSSP-iMod trial, 100% of patients with favorable prognosis were alive at 36 months and those with an intermediate prognosis had a median survival of 42 months.

(13) TABLE-US-00001 TABLE 1 Survival of mRCC patients treated with VSSP-iMod. Prognosis Survival from according to Total number of inclusion in MSKCC at Patient doses received trial (months) inclusion RCC 01 38 64.47 Favorable RCC 02 8 5.2 Poor RCC 03 38 64.47 Intermediate RCC 04 5 2.57 Poor RCC 05 8 30.07 Intermediate RCC 06 12 37 Intermediate RCC 07 2 5.9 Poor RCC 08 16 59.37 Intermediate RCC 09 16 54.07 Favorable RCC 10 37 48 Intermediate RCC 11 16 43.6 Favorable RCC 12 41 36.5 Favorable RCC 13 24 36.5 Intermediate RCC 14 4 2.17 Poor RCC 15 12 10.3 Poor

Example 4. VSSP Decreases the Frequency of MDSCs of Monocytic and Granulocytic Phenotype in Patients with Breast Carcinoma

(14) The effect of VSSP-iMod on MDSCs was also evaluated in patients with breast carcinoma. To this purpose, a Phase 0 Window-of-Opportunity trial was designed, in which patients received 400 μg of VSSP-iMod with a weekly frequency for three weeks by SC route in the deltoid region. This treatment was administered in the conventional time established between the diagnosis and the start of the standard treatment of surgery or chemotherapy indicated by the physician. In this trial the frequency of G-MDSCs, mMDSCs and CD8 T cells was determined by flow cytometry. A total of 200,000 cells were analyzed and the percentages of gMDSCs and mMDSCs were determined using the phenotypes CD11b.sup.+/CD66b.sup.+/CD14y CD11b.sup.+/CD14.sup.+/HLA-DR.sup.low/neg, within the total of PBMC, respectively. As seen in FIG. 6a, the VSSP-iMod decreased the frequency of circulating gMDSCs and this same behavior was observed in the mMDSCs (FIG. 6b) circulating in the patients, after 21 days of treatment. Additionally, the analysis of the percentage of patients with frequency of gMDSCs and mMDSCs above and below the median shows that, after treatment with VSSP-iMod, only 15% and 0% of treated patients maintained the gMDSCs and mMDSCs high respectively. This treatment also increased the frequency of CD8+ T cells in the blood of patients (FIG. 7).