Patent Application
20210214686
The present invention relates to a method for producing a T-cell product containing tumor uber reactive immune cells (TURICs) and a composition containing at least one T-cell product with TURICs for use in treatment of a cancer patient.
The World Health Organization (WHO) reported that pancreatic cancer claimed more than 330,000 lives in 2012, with 68% of deaths occurring in countries with a high to very high human development index (HDI) (WHO, 2014). Since most patients present with metastatic disease at diagnosis, the 5-year survival rate is a meagre 5%. These statistics commensurate with limited treatment options for patients with pancreatic cancer, which include classical surgery (only 10-20% of patients qualify for this option) or chemotherapy.
A relatively new approach called adoptive cell therapy (ACT) is a progressively expanding discipline within modern oncology. This type of cell therapy relies on the transfusion of the body's own lymphocytes, thereby stimulating the patient's immune system with the intent of promoting an antigen specific anti-tumor effect. Durable clinical responses in patients with advanced cancers have been achieved using T-cells directed against tumors (tumor reactive T-cells). These approaches usually rely on the harvesting of T-cells from peripheral blood, e.g. peripheral blood mononuclear cells (PBMCs), or tumor infiltrating lymphocytes (TILs) from tumor lesions.
Since the first results of immune-based treatment of metastatic melanoma, using interleukin (IL) 2-conditioned autologous lymphocytic cells from patients' blood in the 1980s (Lotze et al, 1986; Rosenberg et al, 1985; Rosenberg et al, 1988; Topalian & Rosenberg, 1987) ACT has been further developed and has been applied to various types of cancer. Previously, it has been reported that T-cells can be reliably and successfully isolated from pancreatic cancer lesions and expanded in vitro using a cocktail of IL-2, IL-15 and IL-21 (Meng et al, 2016). Several other T-cell-based approaches to treat pancreatic cancer are currently pursued. The NCI is currently carrying out a clinical study of IL-2-stimulated TIL-infusion in patients with metastatic pancreatic cancer (ClinicalTrials.gov identifier: NCT01174121). Another study is evaluating the safety and efficacy of EGFR-directed bispecific antibody-expressing T-cells (BATs) in patients with locally advanced or metastatic pancreatic cancer who have already undergone 1-2 rounds of chemotherapy (ClinicalTrials.gov identifier: NCT03269526), although the T-cells themselves will be harvested from blood. Specialized T-cell-based therapies targeting private mutations in patients with metastatic cancers have resulted in remarkable clinical responses (Tran et al, 2015; Tran et al, 2016; Tran et al, 2014). In line with this, future clinical studies are likely to benefit translational data linking anti-tumor T-cells in pancreatic cancer to recognition of specific private mutations to improve survival as it was recently shown for a patient with metastatic breast cancer (Zacharakis et al, 2018). The activation of T-cell populations with T-cell receptors (TCRs), which specifically recognize mutated host molecules, i.e. neoepitopes, represent a clinically significant step towards refining T-cell-based immunotherapies and cancer vaccines.
Thus, it is an object of the present invention to improve and further develop personal ACTs for treatment of tumor diseases.
The inventors identified the concept of TURICs having improved tumor reactive properties. Populations of unstimulated T-cells that reside in a precursor T-cell pool exist in body samples, which are used as common T-cell sources. These unstimulated T-cells are so low in frequency that they have not been able to proliferate in large numbers and do not exhibit classical anti-tumor activity, e.g. cytokine production, in detectable amounts after stimulation of the freshly isolated cells with tumor specific peptides or alternatively, with synthetic peptides representing the tumor mutations. The inventors have further identified that these unstimulated precursor T-cells can readily cultured and selected by the method of the invention. As shown in the Examples, when co-cultured in the presence of tumor specific peptides or autologous tumor cells, highly focused immune cells (TURICs) are generated that exhibit strong anti-tumor activity directed to these particular type of tumor cells or tumor specific peptides. The so produced TURICs exhibit a highly specific tumor reactivity resulting in anti-tumor responses above the average of T-cells, which can be isolated and proliferated with known methods.
Thus, in a first aspect, the present invention provides a method for producing a T-cell product containing tumor uber reactive immune cells (TURICs) comprising the steps of
In a second aspect, the present invention also provides a composition containing at least one T-cell product with TURICs for use in treatment of a cancer patient, comprising performing the method according to the first aspect of the invention to obtain a T-cell product with TURICs and administering the T-cell product with TURICs to the patient.
The term “tumor disease” according to the invention refers to a type of abnormal and excessive growth of tissue. The term as used herein includes primary tumors and secondary tumors as well as metastasis.
A “primary tumor” according to the present application is a tumor growing at the anatomical site where tumor progression began and proceeded to yield a cancerous mass.
A “metastasis” according to the invention refers to tumors that develop at their primary site but then metastasize or spread to other parts of the body. These further tumors are also called “secondary tumors”.
A “peptide” as used herein may be composed of any number of amino acids of any type, preferably naturally occurring amino acids, which, preferably, are linked by peptide bonds. In particular, a peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9 amino acids. Furthermore, there is no upper limit for the length of a peptide. However, preferably, a peptide according to the invention does not exceed a length of 100 amino acids, more preferably, it does not exceed a length of 75 amino acids; even more preferably, it is not longer than 50 amino acids.
Thus, the term “peptide” includes “oligopeptides”, which usually refer to peptides with a length of 2 to 10 amino acids, and “polypeptides” which usually refer to peptides with a length of more than 10 amino acids.
A “tumor specific peptide” as used herein refers to a peptide, which is expressed only by tumor cells and thus are found in and/or on the tumor cells, but not in and/or on cells of healthy tissue. When a tumor specific peptide is only present on the surface of the tumor cell, it is also referred to as “tumor specific antigen”. Tumor specific peptides according to the invention contain an amino acid sequence with or without a mutation as found in tumor cells but not in cell of healthy tissue.
Accordingly, a tumor specific peptide as used herein is referred to as mutated or non-mutated tumor specific peptide.
As used herein an “antigen” is any structural substance, which serves as a target for the receptors of an adaptive immune response, T-cell receptor, or antibody, respectively. Antigens are in particular proteins, polysaccharides, lipids, and substructures thereof such as peptides. Lipids and nucleic acids are in particular antigenic when combined with proteins or polysaccharides.
“Disease associated antigens” are antigens involved in a disease. Accordingly, clinically relevant antigens can be tumor-associated antigens (TAA).
“Tumor associated antigens” or “TAA” according to the invention are antigens that are presented by MHC I or MHC II molecules or non-classical MHC molecules on the surface of tumor cells. As used herein, TAA includes “tumor-specific antigens”.
An “epitope” according to the invention is a portion of an antigen that is capable of stimulating an immune response. An epitope is the part of the antigen that binds to a specific antigen receptor, e.g. on the surface of an immune cell. It is possible for two or more different antigens to have an epitope in common. In these cases, the respective immune receptors are able to react with all antigens carrying the same epitope. Such antigens are known as cross-reacting antigens. “Neoepitopes”, as used herein, are newly identified epitopes, in particular epitopes of tumor associated proteins. Since each tumor carries individual mutations, a neoepitopes may only be present in one patient (individual Thutanome), giving rise to a highly personalized, antigen signature.
The terms “stimulation” or “stimulating” as used herein refer to the in vitro activation of clinically relevant lymphocytes, e.g. T-cells, by one or more stimulating agents.
Such stimulating agent may be, for example, stimulating peptides. Activation of clinically relevant lymphocytes means the onset of anti-tumor responses of these cells.
A “stimulating peptide” as used herein relates to a peptide, which is used for stimulation of T-cells. A stimulating peptide may be, for example, a tumor specific peptide, epitopes of known TAAs or neoepitopes.
“Expansion” or “clonal expansion” as used herein means production of daughter cells all arising originally from a single cell. In a clonal expansion of T-cells, all progeny share the same antigen specificity.
In agreement with the general understanding in the art “T-cell” or “T-lymphocyte”, is a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T-cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor on the cell surface. They are called T-cells because they mature in the thymus from thymocytes.
“PBMCs” as used herein refers to peripheral blood mononuclear cells, which can be obtained from peripheral blood. PBMCs mainly consist of lymphocytes, i.e. T-cells, B cells, and NK cells, and monocytes. “PBMCs” also relate to predecessor peripheral blood mononuclear cell and genetically modified cells.
“TILs” according to the invention refers to tumor infiltrating lymphocytes. These are lymphocytes, in particular T-cells predominantly found in a tumor. A lymphocyte sample derived from tumor is also referred as TIL. TILs also relate to any kind of lymphocyte that is located in, on or around a tumor or to lymphocytes that have contacted tumor tissue or tumor cells, respectively. TIL also relate to predecessor TILs and genetically modified TILs.
A “T-cell product” as used herein refers to a population of T-cells for use in immunotherapy. The “T-cell product” can be obtained by (clonal) expansion of T-cells. The T-cells can be autologous, allogeneic, or genetically modified T-cells.
The term “autologous” means that both the donor and the recipient are the same person. The term “allogenic” means that the donor and the recipient are different persons.
IL-2, IL-15 and IL-21 are members of the cytokine family each of which has a four alpha helix bundle. As used herein, “interleukin 2” or “IL-2” refers to human IL-2 and functional equivalents thereof. Functional equivalents of IL-2 include relevant substructures or fusion proteins of IL-2 that remain the functions of IL-2. Similarly, “interleukin 15” or “IL-15” refer to human IL-15 and functional equivalents thereof. Functional equivalents of IL-15 include relevant substructures or fusion proteins of IL-15 that remain the functions of IL-15. “Interleukin 21” or “IL-21” refer to human IL-21 and functional equivalents thereof. Functional equivalents of IL-21 include relevant substructures or fusion proteins of IL-21 that remain the functions of IL-21.
The term “tumor reactivity” as used herein relates to the ability of a T-cell to provide at least one of the following: containment of tumor cells, destruction of tumor cells, prevention of metastasis, stop of proliferation, stop of cellular activity, stop of progress of cells to malignant transformation, prevention of metastases and/or tumor relapse, including reprogramming of malignant cells to their non-malignant state; prevention and/or stop of negative clinical factors associated with cancer, such as malnourishment or immune suppression, stop of accumulation of mutations leading to immune escape and disease progression, including epigenetic changes, induction of long-term immune memory preventing spread of the disease or future malignant transformation affecting the target (potential tumor cells), including connective tissue and non-transformed cells that would favor tumor disease. Tumor reactive T-cells are of particular clinical/biological relevance for ACT. In contrast, T-cells, which do not provide one of the above-mentioned abilities are non-reactive.
The expressions “Tumor uber reactive immune cells” and “TURICs”, as used herein, refer to immune cells, in particular T-cells, which were specifically expanded from unstimulated precursor T-cells. TURICs recognize tumor specific peptides but not targets from healthy tissue. TURICs show stronger T-cell reactivity to a mutant peptide as compared to the corresponding non-mutated peptide. They harbor high affinity T-cell receptors and thus exhibit more exquisite tumor specificity, which also results in strongly increased anti-tumor activity compared to other T-cells from the same source or T-cell products, which can be obtained by methods known in the art.
A “reactivity factor” as used herein refers to a value obtained by assessing the tumor reactivity of T-cells either by directly measuring cytotoxic effects of the T-cells or indirectly by measuring typical T-cell responses upon treatment with tumor cells/peptides.
The transitional term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim, except for impurities ordinarily associated therewith. As subject matter defined by “comprising” may contain but not necessarily contains additional, unrecited elements or method steps, any subject matter defined herein by “comprising” may be limited to “consisting of”.
Method for Producing a T-Cell Product Containing TURICs
The method according to the first aspect of the invention provides a protocol for the production of TURICs. The inventors could show that the resulting lymphocyte population after cultivation with the cytokine cocktail of IL-2, IL-15 and IL-21 in the presence of tumor specific peptides or autologous tumor cells contains a composition of lymphocytes that is advantageous for clinical application.
According to a first aspect, the invention provides a method for producing a T-cell product containing tumor uber reactive immune cells (TURICS) comprising the steps of
Depending on the low number of unstimulated T-cells in the body sample it may be necessary to repeat the stimulation step c) for several times to increase the tumor reactivity of TURICs the initial stimulating step c) may be repeated several times, to enrich for The method according to claim 1, wherein an step c) is carried out up to three times before determining the reactivity factor in step d), preferably step c) is performed two times.
When a stimulating peptide or a group of stimulating peptides is used in the method of the invention it means that one particular type of stimulating peptide or a group of stimulating peptides of different types is applied. This does not limit the stimulating peptide to a particular number of molecules. The exact number of the applied peptide can be calculated from the given concentration. Thus, in one embodiment of the invention, the culture medium in steps c), f), and g) comprises multiple copies of a type of stimulating peptide. An increased number of copies of a stimulating peptide leads to increased expansion rate of T-cells.
In another embodiment of the invention, the culture medium in steps c), f), and g) comprises a group of stimulating peptides. The use of one or more stimulating peptides leads to a diverse set of lymphocytes, in particular T-cells reactive against the nominal clinically relevant antigen.
In the method according to the invention, either one type of stimulating peptide or a group of different types of stimulating peptides can be applied to stimulate the T-cells or for being co-cultivated with T-cells. However, when increasing the number of different types of stimulating peptides this also increases the risk of a so-called “clonal inflation”, which can lead to undesired side effects such as reduced anti-tumor activity of the resulting T-cell product. The inventors have found that in the method according to the invention up to 20 different types of stimulating peptides can be applied at the same time without significantly affecting the outcome of the method. Accordingly, in one embodiment of the present invention the group of stimulating peptides consists of up to 20 different stimulating peptides, preferably up to 10 different stimulating peptides, more preferably up to five different stimulating peptides. The group of stimulating peptides may consist of, for example, two, three, four, or five different stimulating peptides.
The T-cells/T-cell product, which are used in the method according to the first aspect, show focused recognition of several target peptides, which are identified from tumor tissue. Interestingly, the recognition of mutated tumor specific peptides triggers more pronounced anti-tumor responses than the recognition of the non-mutated peptides. Accordingly, in one embodiment of the invention, the stimulating peptides are mutated or non-mutated tumor-specific peptides, wherein the mutated tumor specific peptides contain an amino acid sequence with a mutation found in tumor cells of the patient but not in cells of healthy tissue of the patient.
Besides using tumor specific peptides or the respective tumor specific peptide epitopes as peptides alone, i.e. neoepitopes, the stimulation peptide can be represented to the T-cells in various ways. Such ways include, but are not limited to, presenting the epitopes on artificial scaffolds, as peptides with or without costimulatory molecules, with or without cytokine production of the tumor cells or other antigen-presenting cells, or autologous or allogeneic non-professional or professional cells that present the tumor epitope as a transgene or upon pulsing.
The method according to the invention uses tumor-specific peptides for stimulation of T-cells, resulting in expansion and enrichment of immune cells directed to this specific peptide. Consequently, this leads to a more precise and focused anti-tumor activity of the resulting T-cell product to tumor cells presenting said peptides. There is apparently no limitation of the type of tumor disease. Thus, the method of the invention can be used for producing a T-cell product containing TURICs directed to a huge variety of tumor diseases since peptides specific for a variety of tumors can be used, such as brain cancer, pancreas cancer, tumors derived from the neural crest, e.g. neuroblastoma, ganglioneuroma, ganglioneuroblastoma, and pheochromocytoma, epithelial, e.g. skin, lung, pancreas, colon, or breast, and mesenchymal origin, e.g. adipocytic, cartilaginous, fibrous, fibroblastic, myofibroblastic, osseous, or vascular, as well as hematopoietic tumors, e.g. blood, bone marrow, lymph, or lymphatic system.
According to an embodiment of the invention, the tumor disease is selected from brain cancer, pancreas cancer, hematopoietic tumors, tumors derived from the neural crest, and tumors of epithelial or mesenchymal origin.
Stimulation of T-cells either can be performed directly on the body samples or isolated T-cells with one or more stimulating peptides. It may be beneficial to isolate the T-cells prior to stimulation in order to provide a more selective tumor response, because of the absence of potential residual autologous stimulating agents.
Several approaches of epitope identification are currently in use, which can be used to identify new tumor associated antigens, e.g. neoepitopes. Mass spectrometry-based sequencing of peptides eluted from human leukocyte antigen (HLA) molecules derived from tumor cells aims to decipher naturally processed and presented peptides. Moreover, screening of cDNA libraries encoding TAAs has been used extensively. This peptide-based screening approach identifies mutations through whole-exome sequencing followed by in sllico analysis, based on an algorithm that predicts the peptide binding capacity of the major histocompatibility complex (MHC)-peptide complex. A further method is the tandem minigene (TMG) approach, where the patients' ‘private’ mutations are identified using whole-exome sequencing in order to subsequently construct a personalized library of gene sequences encoding mutated epitopes, e.g. neoepitopes. In the TMG setting, only a small portion of the gene around the mutation is synthesized, and its reaction with autologous T-cells is tested to identify whether the predicted neoepitopes are naturally processed and presented to the immune system based on the assumption that the surrogate antigen-presenting cells process and present the neoepitopes in a similar fashion as compared to tumor cells. The tumor specific peptides as described herein can be identified by any of the aforesaid methods or combinations thereof, e.g. as described in example 2.
Tumor antigens are mainly presented on the surface of the tumor cell via the MHC I or II complex. Since, MHC class II peptides mainly consists of 15 amino acids and most MHC class I peptides consist of 9 amino acids, rarely 8 amino acids or 10 amino acids, peptides length are chosen allowing the immune cells to decide to trim the peptide to the needed length. Accordingly, in one embodiment of the invention, the stimulating peptides have a length in the range of from 5 to 31 amino acids. The length of the stimulating peptides may be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 amino acids. In a further embodiment of the invention, the stimulating peptides have a length in the range from 7 to 25 amino acids. Preferably, the stimulating peptides have a length in the range from 9 to 21 amino acids.
In one embodiment of the invention, the tumor specific mutation is located in the middle of the peptide. In order to achieve equal trimming from both ends of the peptide, the peptide carrying the mutation in the middle preferably consists of an odd number of amino acids. Accordingly, the length of the stimulating peptides may be, for example, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, or 31 amino acids.
In the stimulation and cultivation steps, the cells may be additionally incubated with feeder cells and/or an antibody against CD3. A co-cultivation with feeder cells and the antibody against CD3 has been described in the state of the art. It is believed that feeder cells lead to an improvement of cell growth. Feeder cells are irradiated cells that do not proliferate or proliferate only to a small extent. The feeder cells increase the number of cell contacts in the culture and additionally feed the proliferating and expanding cell culture.
The antibody against CD3 is preferably the antibody defined as OKT3. OKT3 is a murine monoclonal antibody of the immunoglobulin IgG2a isotype. The target of OKT3, CD3, is a multi-molecular complex found only on mature T-cells. An interaction between T-cells, OKT3 and monocytes causes T-cell activation in vitro.
For cytokine production and other anti-tumor reactivity assays, T-cells, are stimulated in defined cell densities. It has been reported that short-term stimulation at high cell densities, such as 102 to 108 cells/μg peptide renders human T-cells from PBMCs fully reactive to soluble tumor peptides. In contrast, stimulation of T-cells in a culture with low cell density below 102 often failed to stimulate T-cells. Thus, in one embodiment of the invention, the stimulation of the T-cells and/or the T-cell product is performed, for example, on 102 to 108 cells. The stimulation may be performed on 1×102 cells, 5×102 cells, 1×103 cells, 5×103 cells, 1×104 cells, 5×104 cells, 1×105 cells, 5×105 cells, 1×106 cells, 5×106 cells, 1×107 cells, 5×107 cells, or 1×108 cells. According to another embodiment of the invention, the stimulation is performed on 103 to 106 cells, preferably on 104 to 105 cells.
The time of stimulation and/or cultivation of the T-cells or the T-cell product is in the range from 6 hours to 180 days. The large range of time is due to the fact that samples from different donors may behave very differently. It was shown that the lymphocytes from different body samples have very different growth rates. For example, lymphocytes derived directly from the tumor of a glioblastoma or a pancreas cancer grow very differently. From pancreas cancer derived lymphocytes are already detectable within two to five days. Lymphocytes derived from glioblastoma are only detectable after one to two weeks. Accordingly, lymphocytes from other body samples may take even longer to become detectable.
As the stimulating step(s) are performed for assessing the tumor reactivity of the T-cells, it is not required that the cells proliferate for a long period. Instead, to obtain reliable results for the analysis of tumor reactivity, a minimal duration of stimulation in the presence of the stimulating peptide(s) is required. This minimum stimulation time is dependent on the method used for determining the tumor reactivity and can vary between a few hours and several days. Similarly, the maximal time T-cells are stimulate is highly variable. It has been observed that reliable results for the determination of tumor reactivity of T-cells can be achieved from 1 hour to 10 days, depending of the determination method. Thus, according to one embodiment of the invention, the T-cells and/or the T-cell product are stimulated for 1 hour to 10 days days. For example, the cell stimulation may be carried out for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. In one embodiment of the invention, the T-cells and/or the T-cell product are stimulated for 3 hours to 5 days. In another embodiment of the invention, the T-cells and/or the T-cell product are stimulated for 1 day to 3 days.
The method of the invention comprises a culturing step in culture medium either comprising autologous tumor cells or stimulating peptides, e.g. tumor specific peptides.
Due to the co-cultivation with autologous tumor cells, tumor specific T-cells are more effectively expanded. Although the culturing step can be rather short, it leads to significant improvement in the yield of expanded clinically relevant T-cells, in particular for clinically relevant T-cells expanded from peripheral blood.
It was shown that with peripheral blood cells, cultivation times about 7 days are particularly beneficial for the outcome of other cultivations. However, as mentioned above, depending on the sample an expansion of only 4 days may be enough. Given the low number of T-cells in the body sample, about 10 days or more may be necessary for co-cultivation. Thus, the culturing step is performed for 1 to 10 days. In one embodiment of the invention, the non-reactive T-cell sample is cultured with the autologous tumor cells or the stimulating peptide(s) for 1 to 10 days. The T-cell sample may be cultured, for example, for one days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. Preferably, the non-reactive T-cell sample is cultured for 3 to 9 days, more preferably the non-reactive T-cell sample is cultured for 6 to 8 days.
In the stimulation and/or the cultivation step of the method according to the invention, the cells may undergo several rounds of expansions to expand more cells and/or to outcompete other cells with other reactivity. It has been observed that already one single round of expansion is sufficient to stimulate and/or expand the T-cells. However, up to 5 rounds of expansion have been identified to be suitable to produce T-cells for reliable stimulation/cultivation results. Thus, in one embodiment of the invention, in steps c), f) and/or g), cells undergo 1 to 5 rounds of expansion. The cells may be expanded for, for example, 1 round, 2 rounds, 3 rounds, 4 rounds, or 5 rounds.
The T-cells and the T-cell product are cultivated and/or stimulated in the presence of IL-2, IL-15, and IL-21. According to a further embodiment of the invention, the concentration of IL-2 in the liquid composition is in the range of from 10 to 6000 U/ml. The International Unit (U) is the standard measure for an amount or IL-2. It is determined by its ability to induce the proliferation of CTLL-2 cells. The concentration of IL-2 is preferably in the range from 500 to 2000 U/ml. More preferably, the concentration of IL-2 is in the range from 800 to 1100 U/ml. According to one embodiment the concentration of IL-15 is in the range of 0.1 to 100 ng/ml. preferably, the concentration of IL-15 is in the range from 2 to 50 ng/ml, more preferably in the range from 5 to 20 ng/ml. The most preferred concentration is about 10 ng/ml. In a further embodiment of the invention, the concentration of IL-21 is in the range from 0.1 ng/ml, preferably in the range from 2 to 50 ng/ml, more preferably in the range from 5 to 20 ng/ml.
A high concentration of peptides, e.g. 5 μg peptide/well/105 cells, results in detectable anti-tumor responses to mutant and wild type peptides (see
Thus, in one embodiment, in steps c), f), and/or g) of the method, the stimulating peptide or each peptide of the group of stimulating peptides is present in a concentration of from 1 pg/105 cells to 1 mg/105 cells. Thus, the stimulating peptide(s) may be present, for example, in a concentration of 1 pg/105 cells, 10 pg/105 cells, 100 pg/105 cells, 1 ng/105 cells, 10 ng/105 cells, 100 ng/105 cells, 1 μg/105 cells, 10 μg/105 cells, 100 μg/105 cells, or 1 mg/105 cells. In another embodiment of the invention, the stimulating peptide(s) is/are in a concentration of from 1 ng/105 cells to 100 μg/105 cells, preferably in a concentration of 1 μg/105 cells to 10 μg/105 cells.
As one tumor cell is sufficient to stimulate multiple T-cells during co-cultivation, the number of autologous tumor cells in comparison to the number of T-cells is rather low. However, in order to allow for contacting all T-cells with tumor cells in the culture it also may be advantageous to use a high number of tumor cells compared to the number of T-cells. In particular, the ratio of T-cells to autologous tumor cells is in the range of from 1000:1 to 1:1000. It is found that the best results are achieved if the ratio of T-cells to autologous cancer cells is in the range of from 10:1 to 1:10. The ratio may be, for example, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 1:1, 1:2, 1:3; 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. Preferred is a ratio of from 7:1 to 3:1. Thus, in one embodiment of the invention, the non-reactive T-cell sample and the autologous tumor cells are cultured in a ratio ranging from of 1000:1 to 1:1000, preferably in a ratio ranging from of 10:1 to 1:10, more preferably in a ratio of from 7:1 to 3:1.
Repeated exposure to particular antigenic targets enrich for certain T-cell populations capable of durable anti-tumor responses (see Examples). This is based on the differential recognition of mutated peptides (as opposed to the wild type/native form), arising from important driver mutations. Accordingly, in one embodiment of the invention, step f) is carried out up to five times. Step f) may be, for example carried out once, twice, three times, four times, or five times. Preferably, step f) is performed four times, more preferably, step f) is performed three times.
Since co-culturing the T-cells to produce a T-cell product in the presence of either autologous tumor cells or stimulating peptides also triggers particular anti-tumor responses of the T-cells it is possible to directly measure anti-tumor activity of the cells after the co-culturing step of the method according to the invention. This direct measurement allows omitting an additional stimulating step before determination of the reactivity factor in step h) of the method, which reduces the overall time consumed by the method to identify and select TURICs. However, performing a stimulating step on the obtained T-cell product directly after the co-culturing step may lead to a more focused tumor reactivity and thus a more precise and consequently more reliable readout when determining the reactivity factor.
Accordingly, the method of the invention can be easily adapted to the needs of the practitioner.
Determination of parameters indicative for the presence of clinically relevant lymphocytes are known in the art and exemplified in the examples. According to one embodiment the reactivity factor is selected from T-cell proliferation, cytokine production, cytotoxicity, e.g. killing of the cells of the diseased body sample, degranulation, in particular defined by CD107a positivity, maturation and or differentiation, in particular defined by the combination of CD45RA and CCR7, expression of a T-cell activation marker in particular selected from CD25, CD56, CD69 of MHC class II molecules; an exhaustion and/or activation markers selected from Foxp3, LAG-3, TIM-3, 4-1BB, PD-1, CD127 (IL-7R), the IL-21 receptor or T-cell signaling, in particular selected from the zeta chain phosphorylation.
The testing of these parameters can be combined with flow cytometry and cell sorting. Accordingly, it is also possible to isolate the clinically relevant lymphocyte population, in particular the TURIC population from the expanded lymphocyte population. The isolated TURICs may further be cultured or directly used for immunotherapy.
One well established method to determine anti-tumor activity of immune cells is the measurement of cytokine production after stimulation with the respective stimulation peptide. Typical cytokines produced by immune cells after stimulation are e.g. IFN-γ, TNFα, IL-2, IL-17, IL-4, IL-5, GM-CSF, release of granzyme B, perforine, upregulation of activation markers, e.g. CD25, HLA-DR, CD69, 4-1BB. T-cells may produce more than one type of cytokine after stimulation simultaneously, which can be measured separately of as a whole to determine tumor reactivity of the cells. Preferred parameters indicative for the presence of clinically relevant lymphocytes are for example the production of one or more cytokines, in particular IFN-γ or TNFα production. Thus, in one embodiment of the invention, the reactivity factor is the IFN-γ concentration and the reactivity factor is positive if the concentration of IFN-γ is above a predefined IFN-γ threshold. The IFN-γ threshold used is highly variable since it depends on the experimental set-up and the culturing conditions. The threshold reflects biological relevance of the cytokine production, which—as measured ex vivo—has to be compared to a particular control experiment, e.g. medium control with or without stimulation peptides.
In one embodiment, the IFN-γ threshold is between 10 pg per 105 T-cells that were stimulated with 1 μg peptide, to 150 pg/105 T-cells/1 μg peptide. Accordingly, a threshold may be defined as, for example, 10 pg/105 T-cells/1 μg peptide, 20 pg/105 T-cells/1 μg peptide, 30 pg/105 T-cells/1 μg peptide, 40 pg/105 T-cells/1 μg peptide, 50 pg/105 T-cells/1 μg peptide, 60 pg/105 T-cells/1 μg peptide, 70 pg/105 T-cells/1 μg peptide, 80 pg/105 T-cells/1 μg peptide, 90 pg/105 T-cells/1 μg peptide, 100 pg/105 T-cells/1 μg peptide, 110 pg/105 T-cells/1 μg peptide, 120 pg/105 T-cells/1 μg peptide, 130 pg/105 T-cells/1 μg peptide, 140 pg/105 T-cells/1 μg peptide, or 150 pg/105 T-cells/1 μg peptide.
In one embodiment, the reactivity factor is the CD107a positivity and the reactivity factor is positive if a T-cell is CD107a positive.
In another embodiment, the reactivity factor is T-cell proliferation, which is considered positive, when proliferation is more than two times the standard deviation of the medium control.
The direct measurement of the ability of T-cells to actively kill and destroy (autologous) tumor cells represents the most reliable assay to determine if T-cells exhibit anti-tumor activity. Thus, in another embodiment, the reactivity factor is the ability of killing tumor cells, which can be determined via a Chromium-51 release assay.
In order to test whether T-cells/T-cell products are reactive to only personal mutations of tumor specific peptides or do recognize also the non-mutated tumor specific peptide and thus may be represent a general tumor specific target, the anti-tumor activity is also tested with the non-mutated sequence peptides corresponding to the mutated tumor specific peptide, a so-called comparative peptide. Accordingly, in one embodiment of the invention, steps c) and d) are additionally carried out with a comparative peptide or a group of comparative peptides as the stimulating peptide or the group of stimulating peptides, wherein the comparative peptide contains the non-mutated sequence corresponding to the tumor specific peptide sequence.
In one embodiment of the invention, steps g) and h) are additionally carried out with a comparative peptide or a group of comparative peptides as the stimulating peptide or the group of stimulating peptides, wherein the comparative peptide contains the non-mutated amino acid sequence corresponding to the mutated tumor specific peptide sequence and wherein the T-cell product is deselected in case the reactivity factor for stimulating the T-cell product with the comparative peptide or the group of comparative peptides is equal to or higher than the reactivity factor for stimulating the T-cell product with the mutated tumor specific peptide or the group of tumor specific peptides.
Since the concentration of the stimulating peptide, e.g. the mutated tumor specific peptide or the corresponding non-mutated peptide, plays a critical role for the read out of the assays used for determining the tumor reactivity (see
The body sample can be taken from any part of the body that contains T-cells, such as primary tumor tissue, metastasis, and peripheral blood, e.g. PBMCs. The availability of body samples for the purpose of the method of the invention can be scarce, since surgery is mostly performed for patients who present without metastasis at diagnosis. Thus, the use of PBMCs to screen for neoepitope recognition is also a viable approach for developing personalized cellular therapies. Accordingly, in one preferred embodiment of the invention, the body sample is whole blood, in particular the body sample are PBMCs.
Moreover, it was found that some stimulating peptides are recognized by both, T-cells from PBMCs and T-cells from TILs, but there are also tumor specific peptides, which are either exclusively recognized by PBMC or TIL T-cells (see
Thus, in one embodiment of the invention, the method can be performed on actually an unlimited number of body samples. Preferably, the method can be performed on at least 2, at least 3, at least 4, at least 5, or at least 6 body samples. In particular, the method can be performed on 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 body samples.
One advantage of the present invention is that there is no need to provide T-cells from tumor tissue. This is in particular useful, because it enables the practitioner to obtain T-cells from non-tumor samples. These body samples can be easily obtained without performing surgery and thus preventing risks associated with such medical interventions. Accordingly, in one embodiment of the invention, the body sample is not a tumor a sample.
In a further embodiment, the body sample is selected from whole blood, serum, plasma, urine, tears, sperm, saliva, synovial fluid, umbilical cord, placenta tissue, bone marrow, exhaled air, lavage material, such as bronchoalveolar lavage, cerebrospinal fluid (CSF), primary, secondary lymphoid tissues, samples from the gut lumen, samples from peritoneal cavity, transplanted material, transplanted cells, transplanted tissue(s) or organ(s).
Methods for obtaining T-cells are known in the art. For example, T-cells can be isolated during surgical interventions such as biopsies. T-cells can also be isolated by aspiration of single cells from tissues and/or organs.
T-cells can be stimulated in the presence of IL-2, IL-15, and IL-21 directly after isolation from the body sample. Moreover, it is also possible to store the freshly isolated T-cells or the obtained T-cell product until use, e.g. by freezing.
Composition for Treatment
The inventors observed that the T-cell product obtained by the method according to the first aspect exhibit elevated therapeutic effects. Thus, in a second aspect, the invention provides a composition containing at least one T-cell product with TURICs for use in treatment of a cancer patient, comprising performing the method according to the first aspect to obtain a T-cell product with TURICs and administering the T-cell product with TURICs to the patient.
Since TURICs are considered immune cells with a tightly focused anti-tumor activity and, thus, are not limited to any specific type of cancer, they can be used in the treatment of literally any tumor disease. As a result, the composition according to the second aspect, can be used for the treatment of diverse tumor diseases such as brain cancer, pancreas cancer, tumors derived from the neural crest, e.g. neuroblastoma, ganglioneuroma, ganglioneuroblastoma, and pheochromocytoma, epithelial, e.g. skin, colon, or breast, and mesenchymal origin, e.g. adipocytic, cartilaginous, fibrous, fibroblastic, myofibroblastic, osseous, or vascular, as well as hematopoietic tumors, e.g. blood, bone marrow, lymph, or lymphatic system.
According to an embodiment of the invention, the tumor disease is selected from brain cancer, pancreas cancer, hematopoietic tumors, tumors derived from the neural crest, and tumors of epithelial or mesenchymal origin.
The T-cell product has a low percentage of regulatory T-cells. Regulatory T-cells are known to suppress the therapeutic function of the population of lymphocytes. According to one embodiment of the second aspect the T-cell product the percentage of Treg based on the total number of T-cells is below 5%, preferably below 3%.
An effective amount of the T-cell product containing TURICs, or compositions thereof, can be administered to the patient in need of the treatment via a suitable route, such as, for example, intravenous administration. The cells may be introduced by injection, catheter, or the like. If desired, additional drugs (e.g., cytokines) may also be co-introduced or introduced sequentially. Any of the cells or compositions thereof may be administered to a subject in an effective amount. As used herein, an effective amount refers to the amount of the respective agent (e.g., the cells or compositions thereof) that upon administration confers a desirable therapeutic effect on the subject. Determination of whether an amount of the cells or compositions described herein achieved the desired therapeutic effect would be evident to one of skill in the art.
The composition containing the T-cell product can be delivered by using administration routes known in the art. Suitable administrations routes are, for example, intravenous administration, subcutaneous administration, intra-arterial administration, intradermal administration, intrathecal administration.
Preferably, the composition containing the T-cell product is administered via the intravenous route, intra-arterial route, intrathecal route, or intraperitoneal route, or directly into the tissue, directly in the bone marrow, or into the cerebrospinal fluid via a catheter.
The person skilled in the art is aware of different formulations of the composition containing the T-cell product to be administered. As such, exemplary formulations may contain polyethylene glycol (PEG) or other substances supporting and/or facilitating the administration of the composition.
Moreover, the compounds administered can be obtained by well-known methods. Such methods may be, for example, production of proteins by recombinant means. Additionally, recombinant proteins can be produced in a variety of cell types that have been adapted to the production of recombinant proteins. Those cells can be transfected with the genetic construct of the respective protein to be produced by methods known in the art, e.g. retroviral, non-retroviral vectors, or CRISP-Cas9 based methods.
According to an embodiment of the present invention, the patient is administered with a dose of TURICs of from 107 to 108 cells per kg body weight. The dose of TURICs may be for example 1×107, 1.5×107, 2×107, 2.5×107, 3×107, 3.5×107, 4×107, 4.5×107, 5×107, 5.5×107, 6×107, 6.5×107, 7×107, 7.5×107, 8×107, 8.5×107, 9×107, 9.5×107, or 1×108 of cells per kilogram body weight.
The invention is further defined by the following non-limiting examples.
Pancreatic cancer TILs and autologous tumor cell lines were obtained from three patients with pancreatic cancer.
1.1 Generation of TILs
Individual single tumor fragments (1-2 mm3) were placed in each well of a 24-well tissue culture plate along with TIL medium, i.e. Cellgro GMP Serum-free medium (CellGenix, Freiburg, Germany), with 5% human AB serum (Innovative Research, Michigan, USA), supplemented with IL-2 1000 IU/ml, IL-15 10 ng/ml, IL-21 10 ng/ml, (Prospec, Ness-Ziona, Israel). Irradiated (55 Gry) feeder cells (allogeneic PBMCs) at the ratio of 1 (feeder cells):10 (TILs) was added on day 7. TILs were transferred into six-well plates; as they covered >70% of the 24-well surface, they were further expanded in G-Rex flasks (Wilson Wolf, Catalog Number: 800400S) using 30 ng OKT3/mL and irradiated (55 Gry) allogeneic feeder cells at the ratio of 1 (feeder cells):5 (TILs).
1.2 Generation of Autologous Tumor Cells
After surgery or biopsy, tumor tissues were cut with surgical scissors and a scalpel. Single tumor fragments (1-2 mm3) were placed in 24-well tissue culture plates with 1 ml of tumor medium, i.e. RPMI 1640 with 20% FBS (Life technologies, CA, USA), Epidermal growth factor (20 ng/ml, ImmunoTools, Friesoythe, Germany) supplemented with antibiotics (penicillin, 100 IU/mL and streptomycin, 10 mg/mL) (Life Technologies, Carlsbad, USA) and Amphotericin B (2.5 mg/L, Sigma-Aldrich, MI; USA). Tumor cell lines were then cultured and passed without EGF. Tumor cells required for the cytotoxicity experiments were obtained during passage fifteen to twenty.
1.3 PBMC Isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood over a Ficoll-Hypaque gradient (GE Healthcare, Uppsala, Sweden) and washed twice in sterile PBS prior to use in experiments.
Isolation and purification of genomic DNA, library construction, exome capture of all coding genes as well as next generation sequencing of tumor tissue and control patient samples were performed as briefly described in the following. Genomic DNA from patient samples (tumor tissue and TILs) was fragmented for constructing an Illumina DNA library (Illumina, San Diego, Calif.). Regions of DNA corresponding to exons were captured in solution using the Agilent SureSelect 50 Mb kit Version 3 as per manufacturer's instructions (Agilent, Santa Clara, Calif.). Paired-end sequencing resulting in 100 bases from each end of every fragment was performed using a HiSeq 2000 Genome Analyser (Illumina, San Diego, Calif.). Results of the sequencing data were mapped to the reference human genome sequence. Alterations within the sequencing data were determined by comparing over 50 million bases of tumor DNA from non-malignant lesions. A high fraction of the sequences obtained for each sample was found to occur within the captured coding regions. More than 43 million bases of target DNA were analyzed in the tumor and normal samples; an average of 42 to 51 reads per base was obtained for both sample types. The tags were aligned to the human genome reference sequence (hg18) using the Eland algorithm of CASAVA 1.6 software (Illumina, San Diego, Calif.). The chastity filter of the BaseCall software of Illumina was used to select sequence reads for subsequent analysis. The ELANDv2 algorithm of CASAVA 1.6 software (Illumina, San Diego, Calif.) was applied for identifying point mutations, small insertions, deletions or stop codons in the sequences obtained. Mutation polymorphisms recorded in the Single Nucleotide Polymorphism Database (dbSNP) were excluded from analysis. Potential somatic mutations were filtered out as previously described, (Jones et al, 2010) while only non-synonymous single and dinucleotide substitutions, respectively were listed in an Excel spreadsheet for downstream work.
The filter criterion for selecting candidate peptides is that the expression level of mutated genes in tumor tissue surpasses 5%. Alternatively, spliced products or mutated sequences with stop codons may result in epitopes that are shorter than the standard 15-mer peptides that are used for screening immunogenicity. The length of the resulting peptide sequences was set at 15-mer to include all possible epitopes presented by HLA class I (8-10 amino acids) as well as HLA class II (11-20 amino acids) molecules.
After identification of mutations through whole-exome sequencing followed by in sllico analysis, the 15-mer peptides were constructed by placing the mutation at the centre position of the 15-amino acid sequence (Peptide & Elephants, Berlin, Germany). The corresponding wild type epitopes were also synthesized to compare the matched mutant and wild type sequences (peptide pairs) in immunological assays.
3.1 IFN-γ Production
T-cells (1.0×105 cells), e.g. from TILs or PBMCs, were cultured in 200 μl of T-cell medium with 1 μg of the individual wild type or mutated peptide in round-bottom 96-well microtiter plates. Negative controls contained assay medium alone while the positive control contained 30 ng/mL of the anti-human CD3 antibody clone OKT3 (Biolegend, San Diego, Calif.) for maximal TCR stimulation. Cells were incubated for 3 days at 37° C. with 5% CO2, after which supernatants were harvested for IFN-γ production using a standard sandwich enzyme-linked immunosorbent assay (ELISA) kit (Mabtech, Stockholm, Sweden). Values from the negative control (medium) were subtracted from epitope (peptide)-specific responses and the data reported to reflect the IFN-γ production (in pg/3 days/1.0×105 T-cells) representing the net IFN-γ production from T-cell populations. Where necessary, the mAbs w6/32 (anti-MHC class I, HLA-A, B and -C) and L243 (anti-HLA-DR) were used as blocking antibodies to assess MHC class I or—class I restriction.
3.2 CD107a Induction Assay
T-cells (2×105) were co-cultured with 4×104 autologous tumor cells for 5 hours at 37° C. (and 5% CO2) in a 96-well tissue culture plate containing 200 μl assay medium/well (RPMI 1640 with 10% FBS and penicillin/streptomycin; both from Thermo Fisher Scientific, Waltham, Mass.). During the incubation period, 1.3 μg/ml of monensin (Merck KGaA, Darmstadt, Germany), and 4 μl of the anti-human CD107a-Alexa Fluor 700 antibody (Clone H4A3; BD Biosciences, Franklin Lakes, N.J.) were added. PMA was used as the positive control and assay medium alone without tumor cells was used as negative control. After 5 hours of incubation, the cells were stained with anti-human CD3-PE/Cy7 (Clone HIT3A; BioLegend, San Diego, Calif.), anti-human CD4-V450 (Clone RPA-T4) and anti-human CD8-APC/Cy7 (Clone SK1) (both from BD Biosciences, Franklin Lakes, N.J.), and analyzed by flow cytometry.
3.3 Chromium-51 Release Assay
Specific cytotoxicity was determined in a standard Chromium-51 (Cr51) release assay. Autologous or control tumor cell lines (‘target cells’, T) were labeled with 100 μCi Na2 51CrO4 for 2 hours. T-cells were co-incubated with the autologous tumor cell line at a ratio of 12:1 (T-cell:tumor cells; represented as effector (E) to target (T) cell ratio). Parallel wells with the T-cell:tumor cell co-culture were incubated with either anti-HLA class-I antibody (clone W6/32) or anti-HLA class-II antibody (clone L243, anti-HLA-DR) to test for decreased tumor cell killing using the blocking antibody (interfering with the MHC Class I or MHC class II antigen presentation). Chromium-51 release was measured in the supernatant and specific cytotoxic activity was calculated by the standard method.
3.4 Repeated Stimulation with the Autologous Tumor Cell Line
For repeated stimulation with the autologous tumor cell line, T-cells were co-cultivated with the tumor cells in six-well tissue culture plates (5×106 TILs: 1×106 tumor cells) containing T-cell medium for seven days, after which TILs were stimulated with tumor cells two more times, thereby forming a T-cell product.
After the repeated stimulation with tumor cells, the resulting T-cell product was assessed for immunoreactivity either directly after co-cultivation with the tumor cells for 7 days or following the above stimulation in the presence of the mutated peptide.
4.1 Flow Cytometry and Analysis
All flow cytometry experiments were performed on a BD FACS Aria flow cytometer while data analysis was performed using FlowJo software version 7 (both from BD Biosciences, Franklin Lakes, N.J.).
4.2 T-Cell Phenotype
T-cells were stained with anti-CD3 Brilliant violet 570, anti-CD4 Brilliant violet 510, anti-CXCR3 FITC (all from Biolegend, San Diego, Calif.) and anti-CD8a APC-Cy7 (BD Biosciences, Franklin Lakes, N.J.). After 15 minutes, cells were washed in PBS-0.1% FBS, and analyzed by flow cytometry. Differentiation and maturation marker analysis based on CD45RA and CCR7 expressed was performed as described previously (Liu et al, 2016).
Results of Examples 1 to 4
To better facilitate presentation of data relevant to the present study, the results section has been organised to reflect the findings pertinent to each patient individually. The reliable expansion of CD4+ and CD8+ TILs from pancreatic cancer tissue using IL-2, IL-15 and IL-21, particularly within the central and effector memory compartments, is shown in
Patient Pan TT26
TILs and the corresponding tumor cell line was established from patient PanTT26. Flow cytometry analysis revealed that TILs before stimulation with autologous tumor cells (“young” TILs) comprised approximately 59% CD8+ T-cells and 22% CD4+ T-cells (
Using the W6/32 (anti-HLA-I) and L243 (anti-HLA-DR) antibodies in Cr51-release assays, it was found that TILs, prior to 3× stimulation with the autologous tumor cell line, displayed a dampened cytotoxic effect with HLA class II inhibition while the W6/32 (anti-MHC class I) antibody abrogated tumor recognition completely (
Whole-exome sequencing of the pancreatic tumor tissue from patient PanTT26 was performed to identify cancer-related mutations that may give rise to mutated antigens (neoantigens). The identified peptide sequences were synthesized along with the corresponding wild type sequences and tested for T-cell reactivity by measuring antigen-specific IFN-γ production. In total, 298 peptides (149 wild type and mutated, respectively) were identified and tested for both TILs before (“young TILs) and after 3× stimulation with the autologous tumor cells. It was found that more than 150 pg IFN-γ (per 105 T-cells/1 microgram peptide) was produced by young TIL or tumor cell-stimulated TIL in response to subsequent exposure to wild type or mutated peptides as it is shown in Table 1.
For example, increased IFN-γ production to the mutated KRAS peptide KLWVGAVGVGKSAL was observed after 3× stimulation with the autologous tumor cell-stimulated TILs compared to young TILs (Table 1). The clinical relevance of this finding is underlined by the established knowledge that oncogenic mutant KRAS commonly plays a crucial role in PDAC pathogenesis (Eser et al, 2014). The strongest IFN-γ response by both PanTT26 young TILs and tumor cell-stimulated TILs was observed after exposure to a mutated peptide from NCOR1 (KLKKKQVKVFA, mutation: N99K). Young TILs produced 327 pg IFN-γ/10e5 TILs in response to mutated NCOR1, while tumor cell-stimulated TILs showed 710 pg IFN-γ/10e5 TIL (Table 1).
PanTT26 TILs also showed strong IFN-γ responses to a mutated peptide derived from WDFY4 (RKFISLHKKALESDF), which is a protein likely associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. 17% of mutations (25/149 mutations) in PanTT26 are associated with zinc finger proteins (ZNF), which display diverse biological functions (Cassandri et al, 2017). The recognition of a ZNF730-derived peptide was pronounced following stimulation of PanTT26 TILs with autologous tumor cells, although four other wild type ZNF peptides were recognized (Table 1). It is plausible that a high number of wild type ZNF targets were obtained due to the filter that was applied for detecting mutations in the tumor samples (minimum of 5% mutation load). However, the function and immunological significance of ZNF as a target for cellular immune responses in pancreatic cancer therefore warrants further exploration.
Patient Pan TT39
TILs isolated from this patient were characterized by flow cytometry and found to contain exclusively CD4+ T-cells (>99%) (
G
RFLRGYRQ
Since the TILs from PanTT39 consisted exclusively of CD4+ T-cells and no CD8+ T-cells, the analysis was focused on the peptides that could bind HLA class II molecules. In Table 3, fourteen HLA class II-binding targets are shown that were identified using a predicted consensus rank of less than or equal to 1.0. It is important to mention that the mutational burden among HLA-DRB1 alleles in PanTT39 tumor was calculated as 8.8%. Peptides that would bind to HLA-DRB1 were nevertheless incorporated, assuming >90% chance that an adequate number of tumor cells would still be able to present antigen via HLA-DRB1. TILs from this patient were then screened for recognition of peptides in a three-day 96-well co-culture assay, as described for PanTT26 TILs.
R
HRLPL
I
HRLPL
Table 4 shows that PanTT39 TILs produced lower IFN-γ/105 TIL in response to mutated peptides as compared to PanTT26 TILs. It is considered the possibility that CD4+ T-cells in PanTT39 TILs could comprise a mixture of different T-cell subsets, e.g. Th1, Th2 and Th17.
In order to better define TIL PanTT39 reactivity, a T-cell product was obtained by cultivation of the TILs as described above. Flow cytometry analysis revealed that the the T-cell product was positive for TCR Vβ9+ (see
Patient Pan TT77
As shown in
Table 5 shows the IFN-γ production of PBMC T-cells co-cultured and stimulated with neoepitopes that trigger a response in TIL T-cells but not PBMCs in an initial stimulation step. PBMC T-cells were co-cultured one or two times one of the peptides in the presence or absence of OKT3 and then tested for IFN-γ production by stimulation using the respective peptide. As can be seen in Table 5, all six neoepitopes were recognized by the PBMC T-cells already after the first co-cultivation. Interestingly, a second co-cultivation step led to no recognition of the WT epitope and increased IFN-γ production upon stimulation with the mutated epitope in the absence of OKT3. The response increases further when co-cultivation was performed in the presence of OKT3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18186726.8 | Jul 2018 | EP | regional |
| 18195826.5 | Sep 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/070696 | 7/31/2019 | WO | 00 |
