GB2624871A - Process for preparing a population of dendritic cells and immunotherapy using the same - Google Patents
Process for preparing a population of dendritic cells and immunotherapy using the same Download PDFInfo
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- GB2624871A GB2624871A GB2217889.1A GB202217889A GB2624871A GB 2624871 A GB2624871 A GB 2624871A GB 202217889 A GB202217889 A GB 202217889A GB 2624871 A GB2624871 A GB 2624871A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A—HUMAN NECESSITIES
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Abstract
A method for the generation of functionally mature dendritic cells from a sample obtained from a non-human mammal. The method comprises obtaining a sample comprising dendritic cell precursors or immature dendritic cells, such as monocytes; incubating the sample in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 4 (IL-4) and a Bacillus Calmette-Guerin (BCG) composition. One or more cytokines, e.g. interferon gamma (IFN-γ) may also be included. The mature dendritic cells produced may be used as a vaccine for the treatment of cancer.
Description
Process for preparing a population of dendritic cells and immunotherapy using the same
Technical field of the invention
The present invention relates to an in vitro method for the generation of functionally mature dendritic cells from a sample obtained from a non-human mammal. In particular, the dendritic cells may be matured by incubation in a maturation mixture comprising BCG.
Background of the invention
Dendritic cells (DCs) are the most effective antigen-presenting cells in the immune system, playing an essential role in bridging the innate and adaptive immune response and have a key role in inducing antigen-specific T-cell responses. Further, DCs have an essential role in establishing immunologic memory. DCs present antigens to T cells and induce T cell activation and T helper cell polarization, by secreting differentiating cytokines and upregulating surface co-stimulatory molecules, thus ideally leading to induction of cytotoxic CDS T cells which secrete IFN-y and have enhanced memory capacity.
Therapeutic DC-based vaccines are an attractive vaccine strategy to induce an immune response against viral or cancerous disease. DCs are loaded with viral or tumor antigens either in the form of peptides (lysate, protein isolate, or specific peptides) or nucleic acids (mRNA or DNA). The antigens are then presented through their major histocompatibility complex (MI-IC) with the intent of interacting with antigen-specific T cells, which selectively have the capacity to eliminate cells expressing the viral or tumor antigen in question. Even though therapeutic DC-based vaccines are promising immunotherapeutic candidates, only a single DC vaccine (Sipuleucel-T) against human prostatic cancer has been approved by the U.S. Food and Drug Administration (FDA) in 2010. Despite this initial success, further DC vaccines have failed to deliver clinical benefits and various studies point toward inefficient DC culturing methods as being one out of numerous obstacles that need to be solved in order to generate effective DC-based vaccines.
Cancer has become one of the leading causes of death in dogs, resulting in significant emotional, physical, and economical burdens for both the dogs and their owners.
Despite the rapid rise of immunotherapies in human medicine, canine patients have limited immunotherapeutic options. This is mainly due to the lack of cross-reactivity with the commonly used immune checkpoint inhibitors, such as PD-1, PD-L1, CTLA-4 used in humans.
Some promising results have been seen when DC-based cancer vaccines have been applied to dogs with certain cancers, however, the studies are small, and their clinical benefit remains questionable. Knowledge from human trial cannot be directly extrapolated to non-human mammals.
Accordingly, it remains challenging and cumbersome to culture and mature a utologous non-human DCs ex vivo to generate a robust DC population that express a favourable phenotype and functionality suitable for use in a therapeutic DC-based vaccine for nonhuman mammals.
Hence, there is a need to develop immunotherapeutic cancer treatment options for non-human mammals, such as dogs, that achieve a durable anti-tumor response and increase the overall survival rate of the patients.
In particular, it would be advantageous to provide a method for producing a matured dendritic cell population from samples obtained from non-human mammal to generate a dendritic cell population with a favourable phenotype and increased functionality to be used in DC-based cancer vaccines for non-human mammals.
Summary of the invention
The present invention relates to an improved process for culturing and maturing autologous DCs from a sample obtained from a non-human mammal. The process includes exposure of DCs to a maturation mixture comprising a Bacillus CalmetteGuerin (BCG) composition. It has surprisingly been found that Mycobacterium bovis BCG works efficiently in stimulating DCs from a non-human mammal, and resulting DC populations display advantageous phenotypes and functionality. Accordingly, the findings described herein open up an avenue for effective immunotherapies of non-human mammals, such as dogs and other companion animals.
Thus, an object of the present invention relates to the provision of a method for efficiently stimulating and maturing a population of DCs from a non-human mammal.
In particular, it is an object of the present invention to provide a population of DCs that can be used in immunotherapy, such as cancer treatment, of a non-human mammal.
Thus, an aspect of the present invention relates to a process for producing a population of mature dendritic cells comprising the steps of: (i) providing a sample from a non-human mammal comprising dendritic cell precursors and/or immature dendritic cells; (ii) incubating the sample in presence of a maturation mixture comprising: -granulocyte-macrophage colony-stimulating factor (GM-CSF), -interleukin 4 (IL-4), and -a Bacillus Calmette-Guerin (BCG) composition; and (iii) recovering the sample comprising a population of mature dendritic cells.
Another aspect of the present invention relates to a population of mature dendritic cells obtainable by the process as described herein.
Yet another aspect of the present invention relates to a vaccine comprising the population of mature dendritic cells as described herein.
A further aspect of the present invention relates to a population of mature dendritic cells or a vaccine as described herein for use as a medicament.
Still, a further aspect of the present invention relates to a population of mature dendritic cells or a vaccine as described herein for use in treating, preventing or inhibiting a cancer in a non-human mammal.
Brief description of the figures
Figure 1 shows percentage of CD86 positive cells after 24 hours incubation in STD Cytokine Maturation Cocktail (STD Cytok.) or BCG + IFN-y. The CD86 positive population is calculated by gating the negative population on the isotype control. (A) Histogram plots of one representative experiment. (B) Results from three separate experiments with different donors.
Figure 2 shows IL-12 secretion by dendritic cells as measured by ELISA. (A) Amount of IL-12 secreted in the culture medium after 24 hours incubation in the following maturation conditions (from left to right): STD Cytokine Maturation Cocktail (STD Cytok.), IFN-y, BCG or BCG + IFN-y. (B) Stimulation of immature DCs (iDCs) or DCs matured with either STD Cytokine Maturation Cocktail (STD Cytok.) or a maturation mixture comprising BCG + IFN-y with CD4OL for 24 hours to mimic a putative in vivo response. (C) Stimulation of matured DCs with CD4OL for 24 hours to mimic a putative in vivo response of DCs matured with different maturation conditions (STD Cytok., IFNy, BCG or BCG + IFN-y).
The present invention will in the following be described in more detail.
Detailed description of the invention
Definitions Prior to outlining the present invention in more details, a set of terms and conventions is first defined: Dendritic cell precursors In the present context, the term "dendritic cell precursors" refers to a cell subset, such as monocytes that may be isolated from body fluid or tissue samples. Upon stimulation, the dendritic cell precursors may differentiate into immature dendritic cells.
Immature dendritic cells In the present context, the term "immature dendritic cells" refers to a cell subset derived from monocytes which possesses a phagocytic capacity. Pathogens and cell debris is captured by immature dendritic cells and degraded into small protein fragments. Upon stimulation, the immature dendritic cells mature into mature dendritic cells, capable of presenting the protein fragments on their cell surface.
Mature dendritic cells In the present context, the term "mature dendritic cells" refers to a cell subset with antigen-presenting capacity. Processed protein fragments or antigens derived from pathogens and cell debris will be transported to the cell surface and presented to immune cells using MHC molecules. This presentation of antigen to the immune cell leads to activation of the adaptive immune system, leading to long lasting memory of the presented antigen. Mature dendritic cells also express cell surface receptors that act as co-stimulatory receptors in T cell activation, such as CD86 and CD80.
Multiplicity of infection (MOI) In the present context, the term "MOI" refers to multiplicity of infection which is equivalent to the number of bacteria infecting one cell, e.g. MOI 1 is equivalent to 1 bacteria/cell.
Differentiation mixture In the present context, the term "differentiation mixture" refers to the culture media comprising stimulatory components provided to the dendritic cell precursors to differentiate these into immature dendritic cells.
Maturation mixture In the present context, the term "maturation mixture" refers to the culture media comprising stimulatory components provided to the immature dendritic cells to mature these into mature dendritic cells. About
Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, concentrations, weights, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by + 1 0 % , for example ± 5°/s and preferably ± 2/a (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 100/0' may mean ±100/0 about the number 10, which is anything between 9°/s and 110/0).
Method for preparation of a population of mature dendritic cells The inventors of the present invention have succeeded in developing a process for producing a population of mature dendritic cells with superior phenotypic and functional capacity, suitable to be used in DC-based vaccines as an immunotherapeutic treatment of cancer in non-human mammals, such as dogs.
In particular, it was surprisingly found that BCG is a crucial component in the process of generating a matured dendritic cell population with a favourable phenotype and functionality.
Thus, an aspect of the present invention relates to a process for producing a population of mature dendritic cells comprising the steps of: (i) providing a sample from a non-human mammal comprising dendritic cell precursors and/or immature dendritic cells; (ii) incubating the sample in presence of a maturation mixture comprising: - granulocyte-macrophage colony-stimulating factor (GM-05F), -interleukin 4 (IL-4), and - a Bacillus Calmette-Guerin (BCG) composition; and (Hi) recovering the sample comprising a population of mature dendritic cells.
Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is a live-attenuated vaccine, initially established to protect against childhood meningitis and disseminated tuberculosis (TB). Without being bound by theory, BCG may have the potential to increase the capacity of the immune system to combat other pathogens than TB, by boosting non-specific responses in both T-cell mediated adaptive responses and innate immune responses. Accordingly, BCG could serve as an immune stimulating component in culturing autologous DCs to generate a more efficient mature DC population that can be used in DC-based vaccine strategies against viral and carcinogenic disease.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.
Another embodiment of the present invention relates to the process as described herein, wherein the attenuated live Mycobacterium bovis BCG is Danish strain 1331 or an equivalent strain.
Adjuvants and excipients are key components in vaccines, with the important role of activating the immune system and paving the way for the vaccine to be effective and generate a long lasting memory response against the antigens/targets presented in the vaccine formulation.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the BCG composition comprises one or more excipients and/or adjuvants.
Another embodiment of the present invention relates to the process as described herein, wherein the BCG composition comprises one or more excipients and/or adjuvants selected from phorbol 12-myristate 13-acetate (PMA) and/or monophosphoryl-Lipid A (MPLA).
Still, another embodiment of the present invention relates to the process as described herein, wherein the one or more excipients and/or adjuvants are selected from the group consisting of sodium glutamate, magnesium sulphate heptahydrate, dipotassium phosphate, citric acid monohydrate, L-asparagine monohydrate, ferric ammonium citrate, and glycerol.
A further embodiment of the present invention relates to the process as described herein, wherein the BCG composition is a BCG vaccine.
For the BCG component to be efficient in inducing the phenotype and functional of the matured dendritic cell population observed in the present invention, BCG is preferably provided in a suitable concentration within the maturation mixture to avoid understimulation as well as overstimulation of the dendritic cells.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the concentration of the BCG composition in the maturation mixture is in the range of about MOI 1 to about MOI 10, such as about MOI 1, such as about MOI 2, such as about MOI 3, such as about MOI 4, such as about MOI 5, such as about MOI 6, such as about MOI 7, such as about MOI 8, such as about MOI 9, such as about MOI 10.
Another embodiment of the present invention relates to the process as described herein, wherein the concentration of the BCG composition in the maturation mixture is in the range of about MOI 1 to about MOI 10, such as about MOI 1.5 to about MOI 8, such as about MOI 2 to about MOI 6, such as about MOI 2.5 to about MOI 5, such as about MOI 3 to about MOI 4.
Yet another embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a maturation mixture is performed at a temperature in the range of about 36°C to about 38°C, preferably at about 37°C.
A further embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a maturation mixture is performed for a period of at least about 30 min, such as at least about 60 min, such as at least about 2 hours, such as at least about 4 hours, such as at least about 6 hours, such as at least about 8 hours, such as at least about 10 hours, such as at least about 12 hours, such as at least about 18 hours, such as at least about 24 hours.
B
A still further embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a maturation mixture is performed for a period of time in the range of about 30 min to about 24 hours.
Another embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a maturation mixture is performed for about 24 hours.
An even further embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a maturation mixture is performed in the presence of CO2.
Another embodiment of the present invention relates to the process as described herein, wherein the concentration of CO2 in said incubation of the sample in the presence of a maturation mixture is in the range of about 1% to about 10%, such as about 2°/s to about 8%, such as about 40/o to about 6 °/o, preferably about 5°/o CO2.
The differentiation of dendritic cell precursors into mature dendritic cells is driven by multiple signals. In order for the dendritic cell precursors to firstly differentiate into immature dendritic cells and hereafter mature into a mature dendritic cell population, a combination of cytokines may preferably be provided to stimulate the differentiation of precursor cells to the dendritic cell lineage.
Therefore, an embodiment of the present invention relates to the process as described herein further comprising a step, after step (i) and before step (H), of incubating the sample in the presence of a differentiation mixture comprising GM-CSF and IL-4.
Various concentrations of the cytokines may be used in the maturation mixture and/ or differentiation mixture of the present invention.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the concentration of GM-CSF in the maturation mixture and/or the differentiation mixture is in the range of about 5 ng/mL to about 1000 ng/mL, such as about 10 ng/mL to about 500 ng/mL, about 10 ng/mL to about 250 ng/mL, such as about 15 ng/mL to about 100 ng/mL, such as about 15 ng/mL to about 50 ng/mL, such as about 15 ng/mL to about 35 ng/mL, such as about 20 ng/mL to about 30 ng/mL, such as about 25 ng/mL.
Another embodiment of the present invention relates to the process as described herein, wherein the concentration of IL-4 in the maturation mixture and/or the differentiation mixture is in the range of about 5 ng/mL to about 1000 ng/mL, such as about 5 ng/mL to about 500 ng/mL, about 5 ng/mL to about 250 ng/mL, such as about 5 ng/mL to about 100 ng/mL, such as about 5 ng/mL to about 50 ng/mL, such as about ng/mL to about 25 ng/mL, such as about 5 ng/mL to about 15 ng/mL, such as about 10 ng/mL.
A further embodiment of the present invention relates to the process as described herein, wherein the maturation mixture and/or the differentiation mixture comprises a concentration of GM-CSF of about 25 ng/mL and a concentration of IL-4 of about 10 ng/mL.
The conditions for differentiating the dendritic cell precursors into immature dendritic cells may be similar to those for maturing the immature dendritic cells into mature dendritic cells. This differentiation step comprising addition of the differentiation mixture may be included prior to step (ii).
Accordingly, an embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a differentiation mixture is performed at a temperature in the range of about 36°C to about 38°C, preferably at about 37°C.
A further embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a differentiation mixture is performed for a period of at least about 12 hours, such as at least about 18 hours, such as at least about 24 hours, such as at least about 36 hours, such as at least about 48 hours.
A still further embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a differentiation mixture is performed for a period of time in the range of about 24 hours to about 48 hours.
Another embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a differentiation mixture is performed for about 48 hours.
An even further embodiment of the present invention relates to the process as described herein, wherein said incubation of the sample in the presence of a differentiation mixture is performed in the presence of CO2.
Another embodiment of the present invention relates to the process as described herein, wherein the concentration of CO2 in said incubation of the sample in the presence of a differentiation mixture is in the range of about 1°A, to about 100k, such as about 2010 to about 8 °/o, such as about 40/c to about 6 °/o, preferably about 5% CO2.
-k) A variety of cytokines may be used in the maturation mixture of the present invention to drive the maturation of immature dendritic cells into mature dendritic cells.
A further embodiment of the present invention relates to the process as described herein, wherein the maturation mixture further comprises one or more cytokines selected from the group consisting of interleukins (IL), chemokines, interferons (IFN), colony-stimulating factor (CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF), and tumor necrosis factor (TNF), and combinations thereof.
In particular, IFN-y has been found to work synergistically with BCG to promote differentiation of the immature dendritic cells into mature dendritic cells. Herein are provided evidence that demonstrate a significant and surprising induction of maturation of dendritic cells when the maturation mixture comprises both BCG and IFN-y.
Thus, a preferred embodiment of the present invention relates to the process as described herein, wherein the maturation mixture further comprises IFN-y.
Another embodiment of the present invention relates to the process as described herein, wherein the concentration of IFN-y is in the range of about 25 ng/mL to about 1000 ng/mL, such as about 30 ng/mL to about 500 ng/mL, about 35 ng/mL to about 250 ng/mL, such as about 40 ng/mL to about 100 ng/mL, such as about 45 ng/mL to about 75 ng/mL, such as about 50 ng/mL.
Still, a further embodiment of the present invention relates to the process as described herein, wherein the maturation mixture further comprises one or more selected from the group consisting of Prostaglandin E2 (PGE2), Fms-like tyrosine kinase 3-ligand (F1t3L), stem cell factor (SCF), keyhole limpet hemocyanin (KLH), and combinations thereof.
Yet another embodiment of the present Invention relates to the process as described herein, wherein the maturation mixture further comprises one or more cytokines selected from the group consisting of IL-6, IL-15, and TNF-o, and combinations thereof.
Various media may be used in the maturation mixture and the differentiation mixture of the present invention.
Accordingly, a further embodiment of the present invention relates to the process as described herein, wherein the maturation mixture and/or the differentiation mixture further comprises Roswell Park Memorial Institute (RPMI) medium-1640, AIM-V or X-Vivo 15.
A still further embodiment of the present invention relates to the process as described herein, wherein the maturation mixture and/or the differentiation mixture further comprises Roswell Park Memorial Institute (RPMI) medium-1640.
In order to select the dendritic cell precursors to be used in the process as described in the present invention, a number of selection methods may be used. The selection methods are not limited to any particular type of selection, and includes any selection method known to a person skilled in the art.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the dendritic cell precursors prior to incubation with the differentiation mixture and/or maturation mixture are selected by a method selected from the group consisting of plastic adhesion, negative selection, counterflow centrifugation, expression of CD34, expression of CD14, and combinations thereof.
Another embodiment of the present invention relates to the process as described herein, wherein the dendritic cell precursors are selected for expression of CD14 prior to incubation with the differentiation mixture and/or maturation mixture.
In order to determine whether differentiation of the dendritic cell precursors into mature dendritic cells has been successful, a number of expression markers may be used for selecting cells with a mature dendritic cell profile.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the matured dendritic cell population is selected for CD86, CD80 and/or CD40 expression.
Another embodiment of the present invention relates to the process as described herein, wherein the matured dendritic cell population is selected for CD86 expression.
It is advantageous to have a mature dendritic cell population with a high percentage of the cells expressing CD86 on the cell surface, since this is a phenotype marker for io mature dendritic cells.
Another embodiment of the present invention relates to the process as described herein, wherein the matured dendritic cell population is enriched for cells having increased levels of CD86 expression, as compared to a reference dendritic cell population cultured with a maturation mixture without BCG.
In the present context, the amount of CD86 expression is measured by fluorescence-labelled antibody staining and analysed by flow cytometry. These results are presented in Example 2.
It is advantageous to have a mature dendritic cell population with a high percentage of the cells secreting IL-12 in response to antigen challenge, since this is a functionality marker of the mature dendritic cells.
Another embodiment of the present invention relates to the process as described herein, wherein the matured dendritic cell population is enriched for cells having increased levels of IL-12 secretion, as compared to a reference dendritic cell population cultured with a maturation mixture without BCG.
In the present context, the amount of IL-12 secretion is measured by ELISA. These results are presented in Example 2 and 3.
The sample provided from the non-human mammal, wherefrom the dendritic cell precursors are isolated may be obtained from any suitable source including, but not limited to, body fluid and tissues.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the sample is a body fluid and/or a biopsy.
Another embodiment of the present invention relates to the process as described herein, wherein the sample is selected from the group consisting of blood, tumor, bone marrow, tissue, tears, saliva, mucous, sputum, urine, and faeces, and combinations thereof.
A further embodiment of the present invention relates to the process as described herein, wherein the sample is blood.
A still further embodiment of the present invention relates to the process as described herein, wherein the sample is from a tumor.
When using dendritic cells in DC-based vaccines as a treatment strategy for cancer, the dendritic cells are pulsed with the target antigens of interest with the purpose of presenting these antigens to the T cells in vivo, thereby activating the T cells and initiating an antigen-specific T cell response against the target antigens. This type of immunotherapeutic cancer treatment is capable of achieving durable anti-tumor responses, and may be used subsequent to tumor resection surgery. In order for the dendritic cells to facilitate a specific antitumor T cell response, it is advantageous to pulse the dendritic cells with tumor material from the patient.
Therefore, an embodiment of the present invention relates to the process as described herein further comprising a step of pulsing the sample with a pulsing agent obtained from said non-human mammal.
Another embodiment of the present invention relates to the process as described herein, wherein the pulsing agent is selected from the group consisting of tumor lysate, patient derived exosomes, protein extract, protein isolate, recombinant protein, peptide, tumor derived mRNA, tumor associated antigen coding mRNA or DNA, tumor specific antigen coding mRNA or DNA, and combinations thereof.
Yet another embodiment of the present Invention relates to the process as described herein, wherein the pulsing agent is a tumor lysate.
A further embodiment of the present invention relates to the process as described herein, wherein said tumor lysate is prepared from a tumor biopsy from said nonhuman mammal.
A still further embodiment of the present invention relates to the process as described herein, wherein the pulsing step is performed before, during, or after the incubation of the sample with the maturation mixture, preferably before incubation of the sample with the maturation mixture.
The sample provided in the process of the present invention may be obtained from various non-human mammals including, but not limited to, companion animals. Preferably, the non-human mammal is a dog.
Thus, an embodiment of the present invention relates to the process as described herein, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pigs, horse.
A preferred embodiment of the present invention relates to the process as described herein, wherein the non-human mammal is a dog.
The population of mature dendritic cells resulting from the process disclosed herein is suitable for use as a vaccine, preferably a therapeutic vaccine. Accordingly, the population of mature dendritic cells may be provided as part of a composition zo comprising also other components beneficial for generating an effective immune response.
Therefore, an aspect of the present invention relates to a population of mature dendritic cells obtainable by the process as described herein.
Another aspect of the present invention relates to a vaccine comprising the population of mature dendritic cells as described herein.
An embodiment of the present invention relates to the vaccine as described herein, wherein the vaccine further comprises one or more adjuvants.
Another embodiment of the present invention relates to the vaccine as described herein, wherein the one or more adjuvants are selected from the group consisting of Freund's adjuvants, oil-in-water emulsions, aluminium salts, muramyldipeptide, murabutide, CL429, lipopolysaccharides, and combinations thereof.
Antibodies can be used in immunotherapeutic treatment strategies in combination with cancer vaccines to further strengthen the antitumor response. In particular, monoclonal antibodies such as checkpoint inhibitors can inhibit crucial regulation mechanisms on immune cells to take the breaks off the immune system and thereby increase the response. It may therefore be advantageous to combine the vaccine composition as described herein with one or more monoclonal antibodies to increase the immune response.
Accordingly, an embodiment of the present invention relates to the vaccine as described herein further comprising a monoclonal antibody.
A further embodiment of the present invention relates to the vaccine as described herein, wherein the monoclonal antibody is selected from the group consisting of PD-1 antibody, PD-Li antibody, CTLA-4 antibody, LAG-4 antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRPa antibody, CD123 antibody and combinations thereof.
A still further embodiment of the present invention relates to the vaccine as described herein, wherein the vaccine further comprises a pharmaceutically acceptable carrier.
An obstacle for the success rate of DC-based cancer vaccines is the varying quality of the matured dendritic cell population used in the vaccine, due to the culturing conditions. Results from the present invention has surprisingly shown that using BCG as a component in the maturation mixture is advantageous, since BCG stimulation substantially increases the expression of co-stimulatory signals on the cell surface of the matured dendritic cells, as well as increases the capacity to secrete interleukins in response to antigen challenge. This functional profile is advantageous in vivo when the matured dendritic cell population is used in a DC-based cancer vaccine to overcome the immunosuppressive barrier created by the tumor and to effectively activate T cells to mount a robust antitumor T cell response. These results are presented in Example 1-3.
Thus, an aspect of the present invention relates to a population of mature dendritic cells or a vaccine as described herein for use as a medicament.
Another aspect of the present invention relates to a population of mature dendritic cells or a vaccine as described herein for use in treating, preventing or inhibiting a cancer in a non-human mammal.
The population of mature dendritic cells or vaccine of the present invention may activate a wider range of antigen-specific T cells, capable of recognising antigens originating from both solid and liquid tumors. The population of mature dendritic cells or vaccine of the present invention may therefore be applied in the treatment or prevention of a broad range of cancer types, arising from either solid or liquid tumors.
Thus, an embodiment of the present invention relates to the population of mature dendritic cells or vaccine for use as described herein, wherein the cancer is a solid tumor or liquid tumor.
Another embodiment of the present invention relates to the population of mature dendritic cells or vaccine for use as described herein, wherein the cancer is selected from the group consisting of skin cancer, mast cell cancer, melanoma, bone cancer, osteosarcoma, lymphoma, oral cavity cancer, nasal adenocarcinoma, nasopharyngeal cancer, lung cancer, breast cancer, soft tissue sarcoma, histiocytic sarcoma, hemangiosarcoma, cervical cancer, ovarian cancer, colon cancer, bladder cancer, prostatic cancer, testicular cancer, renal cancer, liver cancer, pancreatic cancer, brain cancer and combinations thereof.
The population of mature dendritic cells or vaccine of the present invention may be administered through various routes, depending on the location of the tumor. This is practical, since it allows for initiating a local antitumor response on the tumor site, which may be beneficial for the success rate of the treatment.
Therefore, an embodiment of the present invention relates to the population of mature dendritic cells or vaccine for use as described herein, wherein the population of mature dendritic cells or vaccine is administered via a route selected from the group consisting of intratumorally, subcutaneously, intravenously, intradermally, intranodal, intraperitoneally and intrathoracically.
The population of mature dendritic cells or vaccine of the present invention may be applied in non-human mammals, such as companion animals.
Therefore, an embodiment of the present invention relates to the population of mature dendritic cells or vaccine for use as described herein, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pig, and horse.
A further embodiment of the present invention relates tothe population of mature dendritic cells or vaccine for use as described herein, wherein the non-human mammal is a dog or a cat.
Still, another and preferred embodiment of the present invention relates to the population of mature dendritic cells or vaccine for use as described herein, wherein the non-human mammal is a dog.
The listing or discussion of an apparently prior published document in this specification io should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method for preparing the matured dendritic cell population and all its features, which may readily be part of the matured dendritic cell population obtained by the method and compositions comprising the same as described herein.
Embodiments and features of the present invention are also outlined in the following items. Items
Xl. A process for producing a population of mature dendritic cells comprising the steps Of: (i) providing a sample from a non-human mammal comprising dendritic cell precursors and/or immature dendritic cells; (ii) incubating the sample in presence of a maturation mixture comprising: granulocyte-macrophage colony-stimulating factor (GM-CSF), -interleukin 4 (IL-4), and -a Bacillus Calmette-Guerin (BCG) composition; and (Hi) recovering the sample comprising a population of mature dendritic cells.
X2. The process according to item Xl, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.
X3. The process according to item X2, wherein the attenuated live Mycobacterium bovis BCG is Danish strain 1331 or an equivalent strain.
X4. The process according to any one of the preceding items, wherein the BCG composition comprises one or more excipients and/or adjuvants.
X5. The process according to item X4, wherein the one or more excipients and/or adjuvants are selected from the group consisting of sodium glutamate, magnesium sulphate hepta hydrate, dipotassium phosphate, citric acid monohydrate, L-aspa rag ine monohydrate, ferric ammonium citrate, and glycerol.
io X6. The process according to any one of the preceding items, wherein the BCG composition is a BCG vaccine.
X7. The process according to any one of the proceeding items, wherein the concentration of the BCG composition in the maturation mixture is in the range of about MOI 1 to about MOI 10, such as about MOI 1, such as about MOI 2, such as about MOI 3, such as about MOI 4, such as about MOI 5, such as about MOI 6, such as about MOI 7, such as about MOI 8, such as about MOI 9, such as about MOI 10.
X8. The process according to any one of the preceding items further comprising a step, after step (i) and before step (H), of incubating the sample in the presence of a differentiation mixture comprising GM-CSF and IL-4.
X9. The process according to any one of the proceeding items, wherein the concentration of GM-CSF in the maturation mixture and/or the differentiation mixture is in the range of about 5 ng/mL to about 1000 ng/mL, such as about 10 ng/mL to about 500 ng/mL, about 10 ng/mL to about 250 ng/mL, such as about 15 ng/mL to about 100 ng/mL, such as about 15 ng/mL to about 50 ng/mL, such as about 15 ng/mL to about 35 ng/mL, such as about 20 ng/mL to about 30 ng/mL, such as about 25 ng/mL.
X10. The process according to any one of the proceeding items, wherein the concentration of IL-4 in the maturation mixture and/or the differentiation mixture is in the range of about 5 ng/mL to about 1000 ng/mL, such as about 5 ng/mL to about 500 ng/mL, about 5 ng/mL to about 250 ng/mL, such as about 5 ng/mL to about 100 ng/mL, such as about 5 ng/mL to about 50 ng/mL, such as about 5 ng/mL to about 25 ng/mL, such as about 5 ng/mL to about 15 ng/mL, such as about 10 ng/mL.
X11. The process according to any one of the preceding items, wherein the maturation mixture further comprises one or more cytokines selected from the group consisting of interleukins (IL), chemokines, interferons (IFN), colony-stimulating factor (CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF), and tumor necrosis factor (TNF), and combinations thereof.
X12. The process according to any one of the preceding items, wherein the maturation mixture further comprises IFN-y.
X13. The process according to item X12, wherein the concentration of IFN-y is in the range of about 25 ng/mL to about 1000 ng/mL, such as about 30 ng/mL to about 500 ng/mL, about 35 ng/mL to about 250 ng/mL, such as about 40 ng/mL to about 100 ng/mL, such as about 45 ng/mL to about 75 ng/mL, such as about 50 ng/mL.
X14. The process according any one of the proceeding items, wherein the maturation mixture further comprises one or more cytokines selected from the group consisting of IL-6, IL-1p, and TNF-a, and combinations thereof.
X15. The process according to any one of the preceding items, wherein the maturation mixture and/or the differentiation mixture further comprises Roswell Park Memorial Institute (RPMI) medium-1640, AIM-V or X-Vivo 15.
X16. The process according to any one of the preceding items, wherein the maturation mixture and/or the differentiation mixture further comprises Roswell Park Memorial X17. The process according to any one of the preceding items, wherein the dendritic cell precursors prior to incubation with the differentiation mixture and/or maturation mixture are selected by a method selected from the group consisting of plastic adhesion, negative selection, counterflow centrifugation, expression of CD34, expression of CD14, and combinations thereof.
X18. The process according to any one of preceding items, wherein the dendritic cell precursors are selected for expression of CD14 prior to incubation with the differentiation mixture and/or maturation mixture.
X19. The process according to any one of the preceding items, wherein the sample is a body fluid and/or a biopsy.
X20. The process according to any one of the proceeding items, wherein the sample is selected from the group consisting of blood, tumor, bone marrow, tissue, tears, saliva, mucous, sputum, urine, and faeces, and combinations thereof.
X21. The process according to any one of the proceeding items, wherein the sample is blood.
X22. The process according to any one of the preceding items further comprising a ID step of pulsing the sample with a pulsing agent obtained from said non-human mammal.
X23. The process according to item X22, wherein the pulsing agent is selected from the group consisting of tumor lysate, patient derived exosomes, protein extract, protein isolate, recombinant protein, peptide, tumor derived mRNA, tumor associated antigen coding mRNA or DNA, tumor specific antigen coding mRNA or DNA, and combinations thereof.
X24. The process according to any one of items X22 or X23, wherein the pulsing agent is a tumor Iysate.
X25. The process according to item X25, wherein said tumor Iysate is prepared from a tumor biopsy from said non-human mammal.
X26. The process according to any one of items X22-X25, wherein the pulsing step is performed before, during, or after the incubation of the sample with the maturation mixture, preferably before incubation of the sample with the maturation mixture.
X27. The process according to any one of the proceeding items, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pig, and horse.
X28. The process according to any of the proceeding items, wherein the non-human mammal is a dog.
A population of mature dendritic cells obtainable by the process according to any one of items X1-X28.
Ul. A vaccine comprising the population of mature dendritic cells according to item Yl.
U2. The vaccine according to item Ul, wherein the vaccine further comprises one or more adjuvants.
U3. The vaccine according to any one of items Ul or U2, wherein the one or more adjuvants are selected from the group consisting of Freund's adjuvants, oil-in-water emulsions, aluminium salts, muramyldipeptide, murabutide, CL429, lipopolysaccharides, and combinations thereof.
U4. The vaccine according to any one of items Ul-U3 further comprising a monoclonal antibody.
U5. The vaccine according to item U4, wherein the monoclonal antibody is selected from the group consisting of PD-1 antibody, PD-Li antibody, CTLA-4 antibody, LAG-4 antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRPa antibody, CD123 antibody and combinations thereof.
U6. The vaccine according to any one of items Ul-U5, wherein the vaccine further comprises a pharmaceutically acceptable carrier.
Vi. The population of mature dendritic cells according to item Y1 or the vaccine according to any one of items Ul-U6 for use as a medicament.
Wl. The population of mature dendritic cells according to item Y1 or the vaccine according to any one of items U1-U6 for use in treating, preventing or inhibiting a cancer in a non-human mammal.
W2. The population of mature dendritic cells or vaccine for use according to item Wl, wherein the cancer is a solid tumor or liquid tumor.
W3. The population of mature dendritic cells or vaccine for use according to any one of items W1 or W2, wherein the cancer is selected from the group consisting of skin cancer, mast cell cancer, melanoma, bone cancer, osteosarcoma, lymphoma, oral cavity cancer, nasal adenocarcinoma, nasopharyngeal cancer, lung cancer, breast cancer, soft tissue sarcoma, histiocytic sarcoma, hemangiosarcoma, cervical cancer, ovarian cancer, colon cancer, bladder cancer, prostatic cancer, testicular cancer, renal cancer, hver cancer, pancreatic cancer, brain cancer and combinations thereof.
W4. The population of mature dendritic cells or vaccine for use according to any one of items W1-W3, wherein the population of mature dendritic cells or vaccine is administered via a route selected from the group consisting of intratumorally, subcutaneously, intravenously, intradermally, intranodal, intraperitoneally and intrathoracically.
W5. The population of mature dendritic cells or vaccine for use according to any one of items W1-W4, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pigs, horse.
W6. The population of mature dendritic cells or vaccine for use according to any one of items W1-W5, wherein the non-human mammal is a dog.
II. A method of treating, preventing or inhibiting a cancer in a non-human mammal, the method comprising administering to the subject a therapeutically effective amount of the population of mature dendritic cells according to item Y1 or the vaccine according to any one of items Ul-U6.
Z2. The method according to item Z1, wherein the cancer is a solid tumor or liquid tumor.
Z3. The method according to item Z2, wherein the tumor burden in the non-human mammal is reduced or eradicated.
Z4. The method according to any one of items Z2 or Z3, wherein the tumor size is reduced and/or tumor growth rate is slowed.
Z5. The method according to any one of items Z1-Z4, wherein tumor metastases are prevented or inhibited.
Z6. The method according to any one of items Z1-Z5, wherein the cancer is selected from the group consisting of skin cancer, mast cell cancer, melanoma, bone cancer, osteosarcoma, lymphoma, oral cavity cancer, nasal adenocarcinoma, nasopharyngeal cancer, lung cancer, breast cancer, soft tissue sarcoma, histiocytic sarcoma, hemangiosarcoma, cervical cancer, ovarian cancer, colon cancer, bladder cancer, prostatic cancer, testicular cancer, renal cancer, liver cancer, pancreatic cancer, brain cancer.
Z7. The method according to any one of items Z1-Z6, wherein the matured dendritic cell population or vaccine is administered intratumorally, subcutaneously, intravenously, intradermally, intranodal, intraperitoneally or intrathoracically.
Z8. The method according to any one of items Z1-Z7, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pigs, horse.
ID Z9. The method according to any one of items Z1-Z8, wherein the non-human mammal is a dog.
P1. A method of treating, preventing or inhibiting a cancer in a non-human mammal, the method comprising administering to the non-human mammal a therapeutically effective amount of a population of mature dendritic cells obtained by the following steps: (i) providing a sample from a non-human mammal comprising dendritic cell precursors and/or immature dendritic cells; (ii) incubating the sample in presence of a maturation mixture comprising: -granulocyte-macrophage colony-stimulating factor (GM-05F), interleukin 4 (IL-4), and -a Bacillus Calmette-Guerin (BCG) composition; and (iii) recovering the sample comprising a population of mature dendritic cells.
P2. The method according to item P1, wherein the sample is body fluid and/or biopsy.
P3. The method according to any one of items P1 or P2, wherein the sample is selected from the group consisting of blood, tumor, bone marrow, tissue, tears, saliva, mucous, sputum, urine, and faeces, and combinations thereof.
P4. The method according to any one of items P1-P3, wherein the sample is blood.
P5. The method according to any one of items P1-P4, wherein the cancer is a solid tumor or liquid tumor.
P6. The method according to item P5, wherein the tumor burden in the non-human mammal is reduced or eradicated.
P7. The method according to any one of items P5 or P6, wherein the tumor size is reduced and/or tumor growth rate is slowed.
P8. The method according to any one of items P5-P7, wherein tumor metastases are prevented or inhibited.
P9. The method according to any one of items P1-P8, wherein the cancer is selected from the group consisting of skin cancer, mast cell cancer, melanoma, bone cancer, osteosarcoma, lymphoma, oral cavity cancer, nasal adenocarcinoma, nasopharyngeal cancer, lung cancer, breast cancer, soft tissue sarcoma, histiocytic sarcoma, hemangiosarcoma, cervical cancer, ovarian cancer, colon cancer, bladder cancer, prostatic cancer, testicular cancer, renal cancer, liver cancer, pancreatic cancer, brain cancer.
P10. The method according to any one of items P1-P9, wherein the matured dendritic cell population or vaccine is administered intratumorally, subcutaneously, intravenously, intradermally, intranodal, intraperitoneally or intrathoracically.
P11. The method according to any one of items P1-P10, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pigs, horse.
P12. The method according to any of items P1-P11, wherein the non-human mammal is a dog.
Examples
Example 1: Preparation of population of mature dendritic cells This example describes the protocols and samples used to assess maturation of dendritic cell populations.
Method PBMC Isolation: A sample of blood (60 ml) was collected from a dog in heparinized tubes and processed within 48 hrs. The blood was diluted with an equal volume of Dulbecco's Phosphate Buffered solution (DPBS) and loaded onto a Ficoll Paque solution in SepMate 50 mL conical tubes. The tubes were then centrifuged for 10 min at 1200 x g at room temperature (RT; 18-22°C). The upper layer containing the peripheral blood mononuclear cells (PBMCs) was transferred to a new tube and the volume was brought to 40 mL with DPBS to dilute the Ficoll solution. The tubes were centrifuged at 500 x g for 10 min at 4°C and the supernatant discarded to eliminate the Ficoll still in solution. The cell pellets were resuspended in DPBS, pulled together into one tube and the volume brought to 40mL with DPBS. As second centrifugation at 200 x g for 10 min at 4°C was performed to wash away residual Ficoll and to remove platelets. The cell pellet was then resuspended in 4-6 mL (depending on the amount of red blood cells, RBCs) of 1X Red Blood Cell Lysis buffer and incubated 5 min at RT to lysate the contaminating RBCs. 40 mL of DPBS were added to dilute the RBC lysis buffer and the tube was -P) centrifuged at 300 x g for 5 min at 4°C. The supernatant was discarded, the cell pellet was resuspended in 10 mL of DPBS, and an aliquot of this cell suspension was used for cell counting and viability assessment.
CD14+ Cell Enrichment The cell suspension obtained at the end of the PBMC isolation protocol was centrifuged at 300 x g for 10 min at 4°C, the pellet resuspended in 80 pL of MACS Buffer per 10"7 cells and 20 pL of anti-CD14 antibody-conjugated MicroBeads (hereafter called CD14beads) per 10"7 cells are added. The mixture was incubated for 15 min at 4°C to allow the binding of the CD14-beads to the CD14 positive monocytes monocytes and then resuspended in 1 mL of MACS Buffer per 10^7 cells. Excess CD14-beads that did not specifically bind to the CD14 positive monocytes, or unspecifically and loosely bound to non-target cells, were removed by centrifugating the sample for 10 min centrifugation at 300 x g at 4°C and discarding the supernatant containing the unbound CD14-beads. The pellet was resuspended in 500 pL of MACS Buffer and loaded on a LS column placed on a magnetic stand. The cells that did not bind to the CD14-beads, which constitute non target cells in this protocol, were then removed through three washes with 3 mL of MACS Buffer through the column, while the CD14+ cells bound to the magnetic beads remained on the column. The column was then removed from the magnet and placed on a 15 mL collection tube and the CD14+ cells were flushed into the tube by adding 5 mL of MACS buffer and pressing a plunger into the column. An aliquot of CD14+ cell suspension was used for counting and viability assessment.
Cell Culture and Differentiation mixture The CD14+ cells were centrifuged for 10 min at 300 x g at 4°C to remove the MACS Buffer and resuspended at a concentration of 4 x 10^6 per mL in RPMI 1640 medium supplemented with 2.5% autologous serum (previously heat inactivated at 55°C for 30 min), 25 ng/mL rcGM-CSF and 10 ng/mL rcIL-4 for 48 hours at 37°C, 5% CO2 to induce the differentiation of monocytes into immature DCs (iDCs).
Maturation mixtures After 48 hours in culture, the differentiation medium was removed, the cells were collected and washed by centrifugation and plated at a concentration of 1-4 x 10^6 per mL in RPMI 1640 medium supplemented with a maturation mixture.
The following maturation mixtures were tested: (1) 2.5% heat inactivated autologous serum, 25 ng/mL rcGM-CSF, 10 ng/mL rcIL-4, and BCG at 3 MOI. This maturation mixture is referred to as "BCG".
(2) 2.5% heat inactivated autologous serum, 25 ng/mL rcGM-CSF, 10 ng/mL rcIL-4, 50 ng/mL IFN-y, and BCG at 3 MOI. This maturation mixture is referred to as "BCG + IFN-y".
(3) 2.5% heat inactivated autologous serum, 25 ng/mL rcGM-CSF, 10 ng/mL rcIL-4 and 50 ng/mL IFN-y. This maturation mixture is referred to "IFN-y".
(4) 2.5% heat inactivated autologous serum, 25 ng/mL rcGM-CSF, 10 ng/mL rcIL-4, 15 ng/mL rcIL-6, 10 ng/mL rcTNF-a, and 10 ng/mL rcIL-lb. This maturation mixture is a standard cytokine maturation cocktail and is referred to as "STD Cytokines".
The cells were then cultured for 24 hours at 37°C, 5% CO2 to induce the maturation of iDCs into mature DCs (mDCs).
Results The differentiation of immature DCs from the monocyte precursors and their maturation, after stimulation with the BCG mixture, was monitored during the culture process by daily morphological assessment at the microscope. Over time, the DCs acquire the characteristic DC morphology of mature cells and at the end of the maturation protocol maturation markers were assessed (the result are described in
example 2 and 3).
Conclusion
The protocol described herein, including stimulation with BCG, efficiently transition immature DCs (iDCs) to a mature phenotype (mDCs).
Example 2: Evaluation of mature dendritic cells prepared with maturation mixture comprising BCG The purpose of this Example was to evaluate the impact of BCG in the maturation mixture and the effect of adding a further stimulation component to the maturation mixture, such as IFN-y. The effect was analysed by assessing the expression of CD86 surface marker and 1-12 secretion of the matured dendritic cell population prepared with the maturation mixture comprising BCG and compared to a dendritic cell population matured with the STD Cytokines.
Method The methods regarding PBMC isolation, CD14+ cell enrichment, cell culture, differentiation mixture, and maturation mixtures were the same as described in
Example 1.
Staining of Maturation Surface Markers and Flow Cytometry Analysis After 24 hours culture of the iDCs with the maturation mixture comprising either BCG + IFN-y or the STD cytokines, the medium was removed, the cells were collected and washed by centrifugation in 10 mL of DPBS for 10 min at 200 x g at 4°C. The supernatant was then removed, and the cells were resuspended in 100 pL of Fc blocker solution and incubated for 20 min at 4°C. After incubation, 100 pl of the antibody (Ab) working solution (Ab dilution according to manufacturer's instructions) were added and the cells were incubated for 30 min at 4°C protected from light. The antibodies used in this example were FITC-conjugated anti-CD86 (clone FUN-1) and FITC-conjugated IgGl,k isotype control. After incubation, the cells were washed twice by centrifugation in 200 pL of staining buffer. The supernatant was then removed, and the cells were either analyzed at the flow cytometer fresh or after fixation in PFA 4% and then analyzed within 48 hours.
Detection of 11-12 Secreted in the Culture Medium after Maturation -IL-12 ELISA After 24 hours culture of the iDCs with the maturation mixture comprising either BCG, IFN-y, BCG + IFN-y or the STD cytokines, the medium was collected and centrifuged for 20 min at 1000 x g at 4°C. The clear supernatant was analyzed by IL-12 ELISA according to manufacturer's instructions. Briefly, the supernatant was diluted 1:2 with sample dilution buffer, and 100 pL of sample were plated on the anti-IL-12 Ab pre-coated 96 well ELISA plate along with a standard curve and incubated for 90 min at 37 °C to allow the Ab to capture the 1-12 in solution. After incubation, the solution was discarded, the plate was washed three times with 200 pL of wash buffer and then 100 A of Biotin-Ab solution were added to all samples and standards and the plate was incubated again for 1 hour at 37°C. After incubation, the solution was discarded, the plate was washed three times with 200 pL of wash buffer and then 100 pL of HRP-Streptavidin Conjugate (SABC) solution were added to all samples and standards and the plate was incubated again for 30 min at 37 °C. After incubation, the solution was discarded, the plate was washed five times with 200 pL of wash buffer and then 90 pL of TMB substrate were added to all samples and standards and the plate was incubated again at 37 °C until the correct development of a colorimetric reaction in the standard curve (10-30 min). The reaction was immediately stopped by the addiction of 50 pL of stop solution in all samples and standards and the absorbance at 450 nm was immediately read at the microplate reader. The amount of IL-12 in each samples was calculated by the interpolation of the optical density (0.D.) values of the samples on the plotted standard curve and by multiplying this value for the dilution factor.
Results Generation of mature DCs with the maturation mixture comprising BCG + IFN-y induced a higher percentage of mature DCs (mDCs), assessed by the expression of CD86 maturation surface marker, compared to the STD Cytokines (Figure 1). The test performed to identify the impact of single components of the maturation mixture comprising BCG, where IL-12 secreted in the medium correlates with the amount of mature DCs, showed that BCG strongly induces the maturation of DCs and that the addition of IFN-y was boosting such effect, compared to the STD Cytokines or IFN-y alone (Figure 2A).
Conclusion
The generation of mature DCs with the maturation mixture comprising BCG induced a higher amount of mature DCs compared to the STD cytokine maturation cocktail, and the main component inducing such enhanced response was BCG, while an adjuvant, such as IFN-y, can further potentiate the BCG activity.
Example 3: Functional assessment of mature dendritic cells prepared with maturation mixture comprising BCG The purpose of this Example was to evaluate the functionality of the matured dendritic cell population. The functionality was evaluated by assessing newly secreted IL-12 in response to a new stimulus in the form of CD4OL.
Method The methods regarding PBMC isolation, CD14+ cell enrichment, cell culture, differentiation mixture, and maturation mixtures were the same as described in Example 1.
CD4OL Stimulation Assay and Analysis of Secreted IL-12 by ELISA After 24 hours culture of the iDCs with the maturation mixture comprising either BCG, IFN-y, BCG + IFN-y or the STD Cytokines, the medium was removed, the cells were collected and washed by centrifugation in 10 mL of DPBS for 10 min at 200 x g at 4°C and 1x10A5 cells were plated in a 96 well in 120p1 of RPMI supplemented with 1% of autologous serum. CD4OL was then added at a final concentration of 0.25 mg/mL for 24 hours at 37°C, 5% CO2, to stimulate the mDCs and mimic an in vivo response. After 24 hours the medium was collected and centrifuged for 20 min at 1000 x g at 4°C. The clear supernatant was analyzed by IL-12 ELISA according to manufacturer's io instructions and as previously described in Example 2.
Results In a CD4OL assay, where matured DCs were challenged with CD4OL to mimic one of the responses expected to take place in the animal after the injection of the DC vaccine, mDCs obtained with the maturation mixture comprising BCG + IFN-y showed a higher response, measured by IL-12 secreted in the medium, compared to immature DC (iDCs) and maturation with the STD Cytokines (Figure 28). The same assay showed that a maturation mixture comprising BCG alone can induce a functional mature phenotype (demonstrated by the secretion of IL-12 in response to stimulation with CD4OL) and that IFN-y can be used as adjuvant to potentiate the BCG effect (Figure 2C).
Conclusion
The results clearly demonstrates that a maturation mixture comprising BCG can drive the generation of mature DCs with high functional capacity. The addition of IFN-y to the maturation mixture comprising BCG can further strengthen this effect.
Claims (15)
- Claims 1. A process for producing a population of mature dendritic cells comprising the steps of: (i) providing a sample from a non-human mammal comprising dendritic cell precursors and/or immature dendritic cells; (ii) incubating the sample in presence of a maturation mixture comprising: granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and -a Bacillus Calmette-Guerin (BCG) composition; and (iii) recovering the sample comprising a population of mature dendritic cells.
- 2. The process according to claim 1, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.
- 3. The process according to any one of claims 1 or 2 further comprising a step, after step (i) and before step (ii), of incubating the sample in the presence of a differentiation mixture comprising GM-CSF and IL-4.
- 4. The process according to any one of the preceding claims, wherein the maturation mixture further comprises one or more cytokines selected from the group consisting of interleukins (IL), chemokines, interferons (IFN), colony-stimulating factor (CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor (TGF), and tumor necrosis factor (TNF), and combinations thereof.
- 5. The process according to any one of the preceding claims, wherein the maturation mixture further comprises IFN-y.
- 6. The process according to any one of the preceding claims, wherein the sample is a body fluid and/or a biopsy.
- 7. The process according to any one of the preceding claims further comprising a step of pulsing the sample with a pulsing agent obtained from said non-human mammal.
- 8. The process according to claim 7, wherein the pulsing agent is selected from the group consisting of tumor lysate, patient derived exosomes, protein extract, protein isolate, recombinant protein, peptide, tumor derived mRNA, tumor associated antigen coding mRNA or DNA, tumor specific antigen coding mRNA or DNA, and combinations thereof.
- 9. The process according to any one of claims 7 or 8, wherein the pulsing step is performed before, during, or after the incubation of the sample with the maturation mixture, preferably before incubation of the sample with the maturation mixture.
- 10. The process according to any one of the proceeding claims, wherein the non-human mammal is a companion animal selected from the group consisting of dog, cat, rabbit, hamster, ferret, guinea pigs, horse.
- 11. A population of mature dendritic cells obtainable by the process according to any one of claims 1-10.
- 12. A vaccine comprising the population of mature dendritic cells according to claim 11.
- 13. The population of mature dendritic cells according to claim 11 or the vaccine according to claim 12 for use as a medicament.
- 14. The population of mature dendritic cells according to claim 11 or the vaccine according to claim 12 for use in treating, preventing or inhibiting a cancer in a nonhuman mammal.
- 15. The population of mature dendritic cells or vaccine for use according to claim 14, wherein the cancer is a solid tumor or liquid tumor.
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Also Published As
| Publication number | Publication date |
|---|---|
| GB202217889D0 (en) | 2023-01-11 |
| WO2024115494A1 (en) | 2024-06-06 |
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