Patent Application
20250171849
The present invention relates to a method of identifying one or more T cells, as well as method of isolating one or more T cells. Further, the invention relates to a method of identifying one or more TCR, a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR identified by the methods of the invention. The invention also relates to a method of generating one or more immune cells. The invention also relates to a host cell comprising a nucleic molecule encoding a TCR identified by the invention or generated by a method of the invention. The invention also relates to a pharmaceutical composition comprising a cell of the invention, or a cell of the invention for use in therapy. Finally, the invention also relates to a method of diagnosing cancer.
Tumor antigen-specific T cells are pivotal for the control of cancer (Schreiber, R. D., Old, L. J. & Smyth, M. J. Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion. Science 331, 1565-70 (2011)). This knowledge has been therapeutically harnessed by the use of tumor-infiltrating lymphocytes (TILs), donor lymphocyte infusions or, more recently, T cells with a transgenic tumor-specific T cell receptor (Stadtmauer, E. A. et al. Long-term safety and activity of NY-ESO-1 SPEAR T cells after autologous stem cell transplant for myeloma. Blood Adv. 3, 2022-2034 (2019)). The ability to engineer T cells with a defined T cell receptor (TCR) is now possible in a highly precise fashion (Schober, K. et al. Orthotopic replacement of T-cell receptor α- and β-chains with preservation of near-physiological T-cell function. Nat. Biomed. Eng. 3, 974-984 (2019)). This opens up new avenues for personalized medicine, but in turn also illustrates the relevance of understanding how tumor-specific T cells are programmed by their TCR. A central question is how tumor antigen-specific T cells with protective (high functionality) TCRs can be identified at different tissue sites. Deeper insights into the TCR driven spatiotemporal fate of T cells will thereby make the behavior of T cell products for adoptive cell therapy and the use of checkpoint inhibitory therapy more predictable, and guide de novo identification of protective TCRs.
During tumor disease, the composition of the T cell receptor (TCR) repertoire of tumor-specific CD8+ T cells changes in space and over time. Although TCR affinity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells, experimental evidence for this is still scarce. The effects of TCR affinity on the spatiotemporal fate of T cells and on their protective capacity against tumors remain poorly understood. Importantly, previous studies concentrated on tumor antigen specificity, and did not take the affinity of a given tumor-specific TCR into account. This is particularly noteworthy since—beyond mere antigen specificity—TCR affinity to its ligand is widely assumed to be one of the most important determinants of a T cell response (Tscharke, D. C., Croft, N. P., Doherty, P. C. & La Gruta, N. L. Sizing up the key determinants of the CD8(+) T cell response. Nat. Rev. Immunol. 15, 705-16 (2015)). Most studies find that high-affinity T cells are better at tumor infiltration than low-affinity T cells. Perhaps for this reason, high-affinity tumor-specific T cells have also been reported to preferentially undergo T cell exhaustion although the opposite has likewise been shown. Furthermore, it is unclear whether the prognostic value and the expression levels of PD-1 on tumor antigen-specific T cells (within or outside the tumor) depends on TCR affinity. A major technical difficulty to address these questions is the lack of methods that can identify TCR sequences and characterize their affinity at the same time. Many of the studies that did investigate the role of affinity in anti-tumor T cell immunity used human antigen mouse models examined responses against a self-antigen followed T cell fate after monoclonal transfers, adoptively transferred high cell numbers and/or included immunization schemes in addition to cell transfers. Such approaches are highly valuable for translational research on TCR candidates targeting human tumor antigens, but are limited in reflecting physiological anti-tumor T cell immunity since antigen-specific T cell populations develop from small numbers of precursor cells (Alanio, C., Lemaitre, F., Law, H. K. W., Hasan, M. & Albert, M. L. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Blood 115, 3718-25 (2010)) and are polyclonal (Sims, J. S. et al. Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire. Proc. Natl. Acad. Sci. 201601012 (2016) doi:10.1073/pnas.1601012113). Tracking TCR affinity-dependent T cell fate is therefore challenging, which renders surrogate markers for TCR affinity a particular relevance.
The technical problem underlying the present application is to comply with this need. The solution to said technical problem is the provision of means and methods as reflected in the claims, described herein, illustrated in the Figures and exemplified in the Examples of the present application.
The present invention relates to a method of identifying one or more T cells comprising: a) determining in a peripheral blood sample derived from a subject the expression level of PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, in a population of T cells; and b) identifying one or more T cells that have an expression level of PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, that is above the median expression of PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, in the population of T cells.
The invention also relates to a method of identifying one or more T cells comprising: a) determining in a peripheral blood sample the mRNA expression level of one or more markers selected from the group consisting of GZMB, CCL3, UNC13D, BTG1, BTG2, CLU, IRS1, and BCL2L in a population of T cells; and b) identifying one or more T cells that fulfill at least one of the following features: increased GZMB expression, increased CCL3 expression, increased UNC13D expression, increased BTG1 expression, increased BTG2 expression, increased CLU expression, increased IRS1 expression, and/or increased BCL2L expression.
The present invention further envisages a method of isolating one or more T cell, comprising isolating one or more T cells identified according to a method of the invention.
The present invention also relates to a method of identifying one or more TCR comprising determining the sequence of the TCR of the one or more T cells identified by a method of the invention.
The present invention further envisages a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR identified by a method of the invention or encoding a TCR comprised in a T cell identified by a method of the invention.
The present invention relates to a method of generating one or more immune cells comprising introducing one or more nucleic acid molecules encoding one or more TCRs or antigen-binding fragments thereof, identified by a method of the invention into one or more host cells.
The present invention relates to a host cell comprising a nucleic acid molecule of the invention or generated by a method of the invention.
The present invention envisages a pharmaceutical composition comprising a T cell identified by a method of the invention or isolated by a method of the invention, an immune cell generated by a method of the invention, or a host cell of the invention.
The present invention also relates to a T cell identified by a method of the invention or isolated by a method of the invention, an immune cell generated by a method of the invention, or a host cell of the invention for use in therapy.
The present invention also relates to a method of diagnosing cancer in a subject comprising measuring the PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, expression level in a peripheral blood sample obtained from the subject, wherein an elevated PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, expression level as compared to a reference blood sample from a healthy control indicates the presence of T cells having high affinity TCRs.
The inventors here set out to develop methods to isolate T cell preferably having high affinity TCRs for tumor antigens. Such high affinity TCR are believed to be correlated with high tumor protection. As used herein, the term “TCR” refers to a T cell receptor. The T-cell receptor (TCR) is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains (that is, it is a hetero dimer). In humans, in 95% of T cells the TCR consists of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (γ/δ) chains (encoded by TRG and TRD, respectively). This ratio changes during ontogeny and in diseased states (such as leukemia). It also differs between species. Orthologues of the 4 loci have been mapped in various species. Each locus can produce a variety of polypeptides with constant and variable regions. When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction
The inventors surprisingly found that high PD-1 expression levels within T cells populations found in peripheral blood samples, and general signatures of recent T cell activation within T cells populations found in peripheral blood samples can therefore serve as a surrogate marker for highly protective TCRs (i.e TCR with high affinity and displaying high tumor protection, as defined herein). Similarly, the inventors surprisingly found that high CD226, CD82, CD39 or ICOS expression levels within T cells populations found in peripheral blood samples, and general signatures of recent T cell activation within T cells populations found in peripheral blood samples can therefore serve as a surrogate marker for highly protective TCRs (i.e TCR with high affinity and displaying high tumor protection, as defined herein). This finding is important since it allows to correlate the phenotype of T cell clones in easily accessible compartments (e.g. peripheral blood) with actual TCR avidity/functionality. Previous studies have not tracked TCRs with different affinity against the same epitope as part of one polyclonal population reacting against a tumor. That TCR affinity correlates with PD-1 expression levels outside the tumor is surprising because based on previous studies, it could be assumed that all antigen specific T cells (irrespective of affinity) would become PD-1 positive upon antigen encounter and not be different in terms of expression PD-1 levels (as is the case in the tumor, only on a higher absolute expression level). Therefore, the finding of the present invention is of importance because it allows to identify T cells or TCRs with high functionality based on a simple biomarker in a compartment that is easily accessible, such as peripheral blood.
Peripheral blood sample is a widely accessible source in patients, which can be derived with non-invasive procedures. In particular, while obtaining T cells directly from tumor tissue (i.e. Tumor infiltrating Lympocytes or TILs) requires invasive intervention (e.g. biopsy), using peripheral blood as sample allows the use of non-invasive techniques which are both easy to perform, and inexpensive. Therefore, in the context of the invention peripheral blood represents an easily obtainable biomaterial. The term “easily obtainable biomaterial” can interchangeably be used with the terms “easily accessible biomaterial” or “easily available biomaterial”. “Easily obtainable biomaterial” means in this respect that said biomaterial can be taken or achieved from a subject without the use of risky invasive methodologies or interventions, such as biopsies, in particular organ biopsies. Hence, such “easily obtainable biomaterial” can be quickly derived from a subject or patient. “Obtained or obtainable” means in this respect that the biomaterial is derived from said subject using any methods or means known to the person skilled in the art that allow to take a sample from said subject. Preferably, for obtaining whole blood from a subject, tools like syringes or lancets are applied.
When referring to PD-1 expression levels, extratumoral T cell populations are said to have lower levels of PD-1 expression in comparison with the levels of PD-1 expression levels found inside a solid tumor (in particular, in tumor infiltrating lymphocytes (TIL)). Compared to expression levels of PD-1 in TIL, PD-1 protein levels which are found in the overall population of extratumoral T cells (i.e T cells found outside a solid tumor, in particular in draining Lymph nodes (dLN), non-draining Lymph nodes (ndLN), and peripheral blood samples may be considered as intermediate, wherein peripheral blood samples comprise T cells with the lowest PD-1 expression when compared to dLN, ndLN and TILs). Accordingly, a T cell from peripheral blood that has high PD-1 expression as compared to other T cells from peripheral blood, may only have an intermediate PD-1 expression as compared to TILs.
Accordingly, the present invention relates to a method of identifying one or more T cells comprising: a) determining in a peripheral blood sample derived from a subject the expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, in a population of T cells; and b) identifying one or more T cells that have an expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, that is above the median expression of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, in the population of T cells found in said peripheral blood sample. PD-1 (Programmed cell death protein 1, also known as CD279 (cluster of differentiation 279), is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1 has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). The PD-1 protein in humans is encoded by the PDCD1 gene.
The term “identifying” when referred to the one or more T cells according to the invention may therefore refer to the selection of T-cells via any method that can be used to measure (or determine) the expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, in a population of T-cells. In particular, according to the disclosure the selected T-cells are characterized by having high affinity TCRs (defined elsewhere herein). The term “determining” as used herein, when referring to the expression levels of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, may comprise any technique useful to determine the expression levels of PD-1, CD226, CD82, CD39 or ICOS (protein), with PD-1 being preferred, in a population of T-cells, for example, expression levels of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, may be determined by flow cytometry, as described in details in the examples section. In the context of the present invention, the expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, therefore preferably refers to the amount of PD-1, CD226, CD82, CD39 or ICOS protein present in the T cells of the T cell population, with PD-1 being preferred. Accordingly, the amount of PD-1, CD226, CD82, CD39 or ICOS protein (or protein levels), with PD-1 being preferred, in the T cells of the invention can be measured by methods known to the skilled artisan, in particular, the amount of PD-1, CD226, CD82, CD39 or ICOS protein expressed by the cells of the invention, with PD-1 being preferred, is preferably measured using FACS. Various techniques to measure protein expression levels are known in the art, one of such techniques is Flow cytometry. Flow cytometry is a technique known to the skilled artisan, used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. Cells are often labelled with fluorescent markers so light is absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer. In particular, the inventors determined the expression levels of PD-1 by determining PD-1 MFI (mean fluorescence intensity) as described in Examples 1 and 5 and show in
As discussed herein, the disclosed methods envisage that after determining the PD-1 expression levels (step a), one or more T cells having PD-1 expression levels above the median expression of PD-1 in the population of T cells derived from a peripheral blood sample are identified (i.e. selected). In particular, the selected one or more T cells are one or more T cells obtained from a peripheral blood sample that have an expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, that is the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 being preferred, found within the about top 20%, preferably the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 being preferred, found within the about top 10%, preferably the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 being preferred, found within the about top 5%, preferably the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 being preferred, found within in the about top 4% of the population of T cells obtained from said peripheral blood sample. As the inventors surprisingly found, in the population of PD-1, CD226, CD82, CD39 or ICOS positive T cells, with PD-1 being preferred, found in blood sample, preferably the about top 20%, preferably the about top 10%, preferably the about top 5%, preferably the about top 4% PD-1, CD226, CD82, CD39 or ICOS expressing cells, with PD-1 being preferred, are comprising T cells with high affinity TCR, defined elsewhere herein. In preferred embodiments, the one or more T cells identified (i.e. selected) are T-cells that are the top PD-1, CD226, CD82, CD39 or ICOS expressing T cells in a peripheral blood sample, with PD-1 being preferred, preferably, the about top 4% PD-1, CD226, CD82, CD39 or ICOS expressing T-cells in a peripheral blood sample, with PD-1 being preferred, and, surprisingly, these T cells are also expressing high affinity TCRs (see Example 5 and
According to the invention a method to identify one or more T cells may comprise a) determining in a peripheral blood sample the mRNA expression level of one or more markers, selected from the group consisting of GZMB, CCL3, UNC13D, BTG1, BTG2, CLU, IRS1, and BCL2L in a population of T cells; and b) identifying one or more T cells that fulfill at least one of the following features: increased GZMB expression, increased CCL3 expression, increased UNC13D expression, increased BTG1 expression, increased BTG2 expression, increased CLU expression, increased IRS1 expression, increased BCL2L expression. For example, the one or more T cells may be identified, if at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the aforementioned group of markers are increased. An increase may be at least about 2-fold, at least about 5-fold, or at least about 10-fold, preferably in comparison with a reference value. The reference value is preferably determined in endogenous T cells from the same donor (mean values). Increased expression of one or more of said markers represent the “activation signature” of the T cells. According to the invention a method to identify one or more T cells may comprise a) determining in a peripheral blood sample the mRNA expression level of one or more markers indicative for a activation signature of the T cell, which can be selected from the group consisting of GZMB, CCL3, UNC13D, BTG1, BTG2, CLU, IRS1, and BCL2L in a population of T cells; and b) identifying one or more T cells that display an activation signature by an altered expression of said one or more makers, which can be the fulfilment of at least one of the following features: increased GZMB expression, increased CCL3 expression, increased UNC13D expression, increased BTG1 expression, increased BTG2 expression, increased CLU expression, increased IRS1 expression, increased BCL2L expression. Increased expression of one or more of said markers represent the “activation signature” of the T cells. As used herein said activation signature is characteristic of activation of the T cells of the invention, namely T cells with high affinity TCRs as defined herein. The term “T cell activation” is known in the art refers to the integration of three distinct signals delivered in sequence: 1) antigen recognition, 2) costimulation (i.e. simultaneous engagement of the T-cell receptor and a co-stimulatory molecule like CD28, or ICOS, on the T cell by the major histocompatibility complex MHCII), and 3) cytokine-mediated differentiation and expansion, which leads to an effective immune response. In order to identify one or more T cells based on said activation signature, prior to determining the mRNA expression level of the markers in the peripheral blood sample, the peripheral blood sample is subjected to standard methods, defined herein, for the isolation of the PBMCs (peripheral blood mononuclear cells) present in the peripheral blood sample as defined herein. Subsequently a single-cell suspension is generated, after which the T cells are stained with fluorochrome-conjugated antibodies for flow cytometric analysis—potentially pMHC multimer staining may be included to detect antigen-specific T cells —. After this, cell sorting is performed to identify T cell receptor sequences and investigate cellular transcriptome subsequently (e.g. via single-cell RNA sequencing). Accordingly the mRNA expression levels of the gene markers defined herein may be achieved by Single-Cell RNA-Sequencing, a technique which provides transcriptional profiling of thousands of individual cells. This level of throughput analysis enables to understand at the single-cell level what genes are expressed, in what quantities, and how they differ across thousands of cells within a heterogeneous sample. In this context, the term “identifying” when referred to the one or more T cells according to the invention may therefore refer to a selection of T-cells with high affinity TCRs (defined elsewhere herein), via any method that can be used to measure (or determine) the mRNA expression level of given genes in a population of T-cells.
According to the invention, the population of T cells wherein the PD-1, CD226, CD82, CD39 or ICOS levels, with PD-1 being preferred, is to be determined, or wherein the mRNA expression level of one or more of the gene markers disclosed herein is to be determined, is obtained from a peripheral blood sample derived from a subject. The term “subject” as used herein, also addressed as an individual, refers to a living mammalian organism. In some embodiment the mammal is a mouse. A subject also includes human and veterinary patients. Preferably, the term “subject” as used herein refers to a human subject. According to some embodiments, the subject from which the peripheral blood sample is obtained is a patient not yet diagnosed to suffer from cancer but is a subject suspected to be affected by cancer. According to some other embodiments, the subject is a cancer patient, meaning a human subject affected by cancer. Where the subject is a living human who may receive treatment for a disease or condition as described herein, it is also addressed as a “patient”. Those in need of treatment include those already suffering from the disease.
Accordingly, a population of T cells as described herein may refer to the population of T cells found in a sample of peripheral blood. In particular, the population of T cells may be a population of PBMCs (peripheral blood mononuclear cells). The “peripheral blood sample” according to the invention may be venous peripheral blood or capillary blood. The term “venous peripheral blood” as used herein can be equivalently substituted by “whole venous blood”, “whole venous peripheral blood” or “peripheral blood” and refers to the blood pool circulating throughout the body and not sequestered within the lymphatic system, spleen, liver, or bone marrow. According to the present invention, whole blood can be used for the purification (or isolation) of PMBCs. A “peripheral blood mononuclear cell” (PBMC) as described herein is any peripheral blood cell having a round nucleus. These cells consist of lymphocytes (T cells, B cells, natural killer cells) and monocytes, as opposed to erythrocytes and platelets that have no nuclei, and granulocytes which have multi-lobed nuclei. According to the present invention, said PBMCs are preferably derived from venous peripheral blood, which can be collected in 8×9 ml ethylene diamine tetra-acetic acid (EDTA) containing monovettes, which are part of the basic equipment found in medical practices and hospitals. Said PBMCs may be contained in capillary blood. The skilled person is aware of means and methods to prepare PBMCS from whole blood, such as venous peripheral blood or capillary blood obtained from a subject. To obtain a sufficient number of PBMC, withdrawal of several milliliters of blood is necessary. A population of peripheral blood mononuclear cells (PBMC) can be obtained by standard isolation methods such as Ficoll gradient of blood cells. Accordingly, single cell suspensions may be generated using Ficoll density gradient centrifugation to isolate cells, followed by washing steps. The cell population comprised in the sample may however also be in purified form and might have been isolated using a reversible cell staining/isolation technology as described patent in U.S. Pat. No. 7,776,562, US patent 8,298.782, International Patent application WO02/054065 or International Patent Application WO2013/011011 which are incorporated herein by reference. Alternatively, the population of cells can also be obtained by cell sorting via negative magnetic immunoadherence as described in U.S. Pat. No. 6,352,694 B1 or European Patent EP 0 700 430 B1 which are incorporated herein by reference. If an isolation method described here is used in basic research, the sample might be cells of in vitro cell culture experiments. From the blood samples, the PBMCs are therefore isolated (purified) and used for determining the expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, in a population of T cells. Accordingly, the isolated (or purified) PBMCs are used to generate a single cell suspension. The skilled artisan is aware of methods for generating a single cell suspension. In this context, a “single cell suspension” refers to a a type of cell culture in which single cells (i.e cells that do not form aggregates between themselves) are allowed to function and multiply in an agitated growth medium, thus forming a suspension. After the single cell suspension of PBMCs is generated, the T cells are stained with fluorochrome-conjugated antibodies for flow cytometric analysis. In preferred examples, the T cells are stained with fluorochrome-conjugated antibodies specific for PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred,. A complete list of antibodies used for flow cytometric analysis of T cells is listed in Table 1. The methods used by the inventors for identifying the T cells according to the invention are described in details in the examples and in the Materials and method section of the specification. Optionally, the T cells are also stained for pMHC multimer to detect antigen-specific T cells, as disclosed elsewhere herein and described in details in the examples and materials and methods. Peptide-MHC (pMHC) multimers have become the “gold standard” for the detection and isolation of antigen-specific T-cells. The term “staining”, as used herein, may refer to the use of dyes, or the use of antibodies in order to identify the presence of a specific compound in a cell or in a population of cells. In general, “staining”, as it will be understood by the skilled artisan, refers to adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound.
The methods disclosed herein, further envision that after the one or more T cells are selected (either by having an expression level of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, that is above the median expression of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, in the population of T cells, or by having increased expression of the gene markers as disclosed herein), said T cells are subjected to further analysis such as expansion, RNA sequencing, isolation. Preferably, said T cells are PB-T cells, that is, T cells found in a peripheral blood sample, as disclosed elsewhere herein. Said PB-T cells may be therefore comprised in PBMCs as defined elsewhere herein. For example, the identified (selected) T cells may undergo two alternative approaches: in one approach the one or more identified T cells are isolated and/or expanded directly to be subsequently used for clinical adoptive T cell therapy. The skilled artisan is aware of many techniques that can be used in order to isolate T cells. T cells isolation can be done, for example, via MACS or FACS. —Magnetic-activated cell sorting (MACS) MACS® (Miltenyi Biotec, Bergisch Gladbach, Germany) is a cell separation technology based on the use of monoclonal antibody-conjugated magnetic beads—. In a second approach the one or more identified T cells of interest are characterized by single-cell RNA sequencing to identify the TCR (and the transcriptomic phenotype) and then the most promising TCRs are, in a separate step, engineered into donor T cells for clinical adoptive T cell therapy. The skilled artisan is aware of different techniques for engineering TCRs into donor (i.e. host) cells. For example, the TCR may be engineered into host cells via transfection using CRISPR/Cas9 technology.
As used herein, “Adoptive cell therapy” also known as cellular immunotherapy refers to a form of treatment that uses the cells of the human immune system to eliminate cancer. Some of these approaches involve directly isolating patient's own immune cells and simply expanding their numbers, whereas others involve genetically modifying the immune cells to enhance their cancer-fighting capabilities. The adoptive cell therapy as used herein makes use of the cells of the invention.
Accordingly, the one or more T cells of the present invention are tumor-reactive T cells. By “tumor reactive T cells” as used herein it is meant T cells able to recognize and bind to tumor specific antigens, therefore, tumor reactive T cells as disclosed herein comprise but are not limited to: cytotoxic T cells and CD4 T cells. As the skilled artisan knows, a cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell) is a T lymphocyte (a type of white blood cell) able to eliminate cancer cells, cells that are infected (particularly with viruses), or cells that are damaged in other ways. CD8+ T-cells are therefore T cells expressing on their surface CD8. CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co-receptor for the T-cell receptor (TCR). The CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 molecule is a marker for cytotoxic T cell population. The tumor reactive T cells as disclosed herein may also encompass CD4+ T cells. CD4 (cluster of differentiation 4) is a glycoprotein that serves as a co-receptor for the T-cell receptor (TCR). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. Such T cells expressing CD4 on their surface are referred to as CD4+ T cells.
The one or more T cells identified by the methods of the invention preferably comprise a high affinity TCR. An important parameter to describe T cell functionality is the affinity of their TCRs for a given antigen peptides bound to major histocompatibility complex (MHC) molecules. T cells with high affinity TCRs could be shown to be superior in clearing viral infections or inducing tumor regression, in this sense, high affinity TCR positively correlates with high tumor protection (also referred to as “high protective capacity” as used herein) as shown for example in Example 5. In this context, by tumor protection it is meant the ability of T cells of the invention to cause tumor regression. Tumor protection can be measured with many techniques known to the skilled artisan starting from preclinical mouse models wherein survival of animals is measured (as described in Example 4 and
Accordingly, the invention also relates to a method of isolating one or more T cell as defined herein, comprising isolating one or more T cells identified by the methods of the invention. Isolation of said T cell (population) can be performed by any method know in the art, for example by using a reversible cell staining/isolation technology as described patent in U.S. Pat. Nos. 7,776,562, 8,298,782, International Patent application WO02/054065 or International Patent Application WO2013/011011 which are incorporated herein by reference. After their isolation, the one or more T cells, may be further expanded, in order to generate a population of T cells. In this context “expansion” of one or more T cells refers to protocols wherein the T cells are allowed to proliferate, thereby forming a population of T cells. Various protocols of T cells expansion are known to the skilled artisan. Said protocols may be protocols useful for in vitro expansion of T cells, for example, expansion of one or more T cells isolated by the methods of the invention that might be directly expanded after their isolation. Alternatively, the T cells isolated by the method of the invention may be subjected to further analysis such as single-cell RNA sequencing (to identify the TCR sequences and the transcriptomic phenotype), after which the most promising TCRs thereby individuated are subsequently engineered into donor T cells, (or host cells, as defined herein)
In addition to the method of identifying one or more T cells disclosed herein, the inventors also developed a method of identifying one or more TCRs, comprising determining the sequence of the TCR of the one or more T cells. Accordingly, after identifying one or more T cells by the method of the invention (i.e. based on PD-1, CD226, CD82, CD39 or ICOS expression levels and/or the activation signature, with PD-1 being preferred), these T cells may be subjected to further analysis in order to determine the sequence of their TCR. Said further analysis may comprise any method known to the skilled artisan, which allows determining the nucleotide sequence encoding for any given TCR and/or determining the amino acid sequence of any given TCR. For example, the TCR sequences may be determined by single cell RNA sequencing (scRNA seq) as used in Example 6 and shown in
Accordingly, the invention also comprises a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR identified by the methods of the invention. A nucleic acid of the invention may be any nucleic acid in any possible configuration, such as single stranded, double stranded or a combination thereof. Nucleic acids include for instance DNA molecules, RNA molecules, analogues of the DNA or RNA generated using nucleotide analogues or using nucleic acid chemistry, locked nucleic acid molecules (LNA), and protein nucleic acids molecules (PNA). DNA or RNA may be of genomic or synthetic origin and may be single or double stranded. Such nucleic acid can be e.g. mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, a copolymer of DNA and RNA, oligonucleotides, etc. A respective nucleic acid may furthermore contain non-natural nucleotide analogues and/or be linked to an affinity tag or a label. A nucleic acid molecule comprising a nuclei acid sequence encoding the TCR according to the invention may be included in a vector such as a plasmid.
A nucleic acid molecule encoding TCR according to the invention can be expressed using any suitable expression system, for example in a suitable host cell or in a cell-free system. The obtained TCR is enriched by means of selection and/or isolation. Thus, the nucleic acid molecule of the present invention may be comprised in a host cell or the vector comprising the nucleic acid molecule of the present invention may be comprised in a host cell.
Therefore, the invention also encompasses a method of generating one or more immune cells as described herein, comprising introducing one or more nucleic acid molecules encoding one or more TCRs, identified by the methods of the invention, into one or more host cells. In this context, the generated immune cell is preferably an immune cell suitable for adoptive T cell therapy as defined herein. The one or more nucleic acid molecules encoding one or more TCR may be introduced into one or more host cells via transduction or transfection. The term “transfection” refers to the process of introducing naked or purified nucleic acids into eukaryotic cells. Said naked or purified nucleic acids can be in the form of a vector. Accordingly, in the context of the invention, oligonucleotides encoding the heterologous T-cell receptors (TCRs) are transfected into said host cells. As disclosed herein, the TCR may be engineered into host cells via transfection using CRISPR/Cas9 technology. Alternatively, a host cell of the invention can be modified by transduction. The term “transduction” refers to the process by which foreign DNA is introduced into a cell by a virus or viral vector. Viral vectors suitable for said transfection are known to the skilled artisan.
Accordingly, suitable host cells for the expression of the isolated TCRs may be preferably eukaryotic, preferably human host cells which are suitable to be transferred into a subject as defined herein for adoptive T cell therapy. Suitable host cells may by immune effector cells, preferably human immune effector cells, i.e. cells from the human body that have differentiated into a form capable of modulating or effecting an immune response. The host cells of the invention may be heterologous or autologous cells. In this context the term “heterologous” when applied to the host cell of the invention refers to a cell which is not derived from the subject as defined herein, while the term “autologous” when applied to the host cell of the invention refers to a cell which is derived from the subject as defined herein. Additionally, the host cell of the invention may be immortalized. An “immortalized” cell, or cell line, is a cell or a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells can therefore be grown for prolonged periods in vitro. The mutations required for immortality can occur naturally or be intentionally induced for experimental purposes. Examples of immortalized cells include HeLa cells—a widely used human cell line isolated from cervical cancer patient Henrietta Lacks; HEK 293 cells—derived from human fetal cells; Jurkat cells—a human T lymphocyte cell line isolated from a case of leukemia; OK cells—derived from female North American opossum kidney cells; Ptk2 cells—derived from male long-nosed potoroo epithelial kidney cells; Vero cells—a monkey kidney cell line that arose by spontaneous immortalization. Preferred immortalized cells are Jurkat cells. The host cell of the invention may also be an iPSC (induced Pluripotent Stem Cell). iPSCs are known to the skilled artisan, and they are a type of pluripotent stem cell that can be generated directly from a somatic cell. Preferably the host cell of the invention is an immune cell, preferably a human immune cell. Additionally, the host cell of the invention may be a T cell. In preferred embodiments the cell of the human immune system is a CD8+ T cell or a CD4+ T cell. The host cell according to the invention may also be a gamma-delta T cell, or a NK-T cell. Gamma delta T cells (γδ T cells) are T cells that have a distinctive T-cell receptor (TCR) on their surface. Most T cells are αβ (alpha beta) T cells with TCR composed of two glycoprotein chains called α (alpha) and β (beta) TCR chains. In contrast, gamma delta (γδ) T cells have a TCR that is made up of one γ (gamma) chain and one δ (delta) chain. This group of T cells is usually less common than αβ T cells. Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. They constitute only approximately 1% of all peripheral blood T cells. Natural killer T cells should neither be confused with natural killer cells nor killer T cells (cytotoxic T cells).
According to the invention, after introducing one or more nucleic acid molecules encoding one or more TCRs of the invention into the one or more immune cells, said cells are subjected to expansion. As defined elsewhere herein “expansion” of one or more T cells refers to protocols wherein the T cells are allowed to proliferate, thereby forming a population of T cells. In particular, in the context of the invention more than one TCR identified by the methods of the invention may be introduced into more than one T cell. In the context of the invention, when said more than one T cells are then subjected to expansion, they can generate a polyclonal population of T cells. A polyclonal population of T cells is obtained when said population of T cells is derived from expansion of more than one T cell (clone), wherein each clone presents a different TCR of the invention on its surface.
The present invention also relates to a pharmaceutical composition comprising the T cell identified by the method of the invention, the T cell isolated by the method of the invention, the population of T cells obtained by the method of the invention, the host cell of the invention, or one or more immune cells generated by the method of the invention. Accordingly, the term “pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient. Pharmaceutical compositions or formulations are usually in such a form as to allow the biological activity of the active ingredient (the T cells of the present invention) to be effective and may therefore be administered to a subject for therapeutic use as described herein. The pharmaceutical composition may be a composition for intraperitoneal, intravenous, intraarterial, subcutaneous, intramuscular, parenteral, transdermal, intraluminal, intrathecal and/or intranasal administration or for direct injection into tissue. Said composition may be administered to a patient via infusion or injection, preferably by injection. Administration of the suitable compositions is preferably intravenously, intraperitoneally, intraarterially, subcutaneously, intramuscularly. The pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. In the context of the present invention the pharmaceutical composition may comprise a therapeutically effective amount of the T cell isolated by the method of the invention, the population of T cells obtained by the method of the invention, the host cell of the invention, or one or more immune cells generated by the method of the invention. Accordingly, a therapeutic effective amount of T cells according to the invention may be less than 400 T cells.
The present invention therefore envisages the T cell identified by the methods described herein, the T cell isolated by the methods described herein, the population of T cells obtained by the methods described herein, the host cell described herein, or the one or more immune cells generated by the method described herein for use in therapy. Preferably, said therapy is adoptive T cell therapy, as defined elsewhere herein. Preferably, said therapy is adoptive T cell therapy is cancer therapy. Adoptive cell therapy can entail use of autologous or heterologous immune cells. For example, in the case of an autologous cell, T cells may be isolated according to the methods of the invention from a subject. The obtained tumor-reactive T cells are expanded and subsequently transferred back to the subject. In the case of heterologous T cells, one or more immune cells generated by the method of the invention may be used, for example a host cells as defined herein may be subjected to transfer of a TCR according to the invention, and transferred to a subject for adoptive T cell therapy.
As used herein, the term “treat”, “treating” or “treatment” means to reduce (slow down (lessen)), stabilize or inhibit or at least partially alleviate or abrogate the progression of the symptoms associated with the respective disease. Thus, it includes the administration of said T cell isolated by the method of the invention, the population of T cells obtained by the method of the invention, the host cell of the invention, or the one or more immune cells of the invention, preferably in the form of a medicament, to a subject, defined elsewhere herein. Those in need of treatment include those already suffering from the disease, here cancer. Preferably, a treatment reduces (slows down (lessens)), stabilizes, or inhibits or at least partially alleviates or abrogates progression of a symptom that is associated with the presence and/or progression of a disease or pathological condition. “Treat”, “treating”, or “treatment” refers thus to a therapeutic treatment. In particular, in the context of the present invention, treating or treatment refers to an improvement of the symptom that is associated with cancer, as defined elsewhere herein.
The present invention also relates to a method of diagnosis of cancer. The method of diagnosing accordingly may comprise measuring the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 expression level being preferred, in a blood sample as defined herein, and determining whether the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 expression level being preferred, in a population of T cells in the sample is above the expression of PD-1, CD226, CD82, CD39 or ICOS, with PD-1 being preferred, in the population of T cells found in a reference blood sample. Said levels of PD-1, CD226, CD82, CD39 or ICOS protein expression, with PD-1 being preferred, found in this particular type of sample would therefore indicate that the patient cells comprise T cells having high-affinity TCRs as defined herein. This finding would indicate that said patient could benefit more from checkpoint blockade with PD-1, CD226, CD82, CD39 or ICOS inhibitor antibodies, with PD-1 being preferred, compared to a reference blood sample. Said reference may be (a blood sample from) a healthy subject or another patient who doesn't have such high PD-1, CD226, CD82, CD39 or ICOS expressing T cells in peripheral blood samples, with PD-1 being preferred. The determination of whether the PD-1, CD226, CD82, CD39 or ICOS expression level, with PD-1 being preferred, in a test sample is above the PD-1, CD226, CD82, CD39 or ICOS expression, with PD-1 being preferred, in a reference sample can further be assisted, for example, by comparing different patients and including positive controls, i.e. samples after T cell stimulation. Accordingly, the method of diagnosing cancer may be a method of selecting a patient for treatment with an PD-1, CD226, CD82, CD39 or ICOS inhibitor, with PD-1 being preferred, such as a PD-1, CD226, CD82, CD39 or ICOS inhibiting antibody, with PD-1 being preferred. The disclosure thus also envisions an PD-1, CD226, CD82, CD39 or ICOS inhibitor, with PD-1 being preferred, such as a PD-1, CD226, CD82, CD39 or ICOS inhibiting antibody, with PD-1 being preferred, for use in the treatment of a patient selected by the methods disclosed herein.
The invention is further characterized by the following items.
Item 1. A method of identifying one or more T cells comprising: a. determining in a peripheral blood sample derived from a subject the expression level of PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, in a population of T cells; and b. identifying one or more T cells that have an expression level of PD-1 that is above the median expression of PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, in the population of T cells.
Item 2. The method of item 1 wherein PD-1, CD226, CD82, CD39 or ICOS expression is PD-1, CD226, CD82, CD39 or ICOS protein expression.
Item 3. The method of item 1 or 2, wherein the one or more T cells are identified if the one or more T cells have an expression level of PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, that is within about the top 20%, preferably within about the top 10%, within about the top 5%, preferably within about the top 4% of the PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, expression level found in said population of T cells.
Item 4. A method of identifying one or more T cells comprising: a. determining in a peripheral blood sample the mRNA expression level of one or more markers selected from the group consisting of GZMB, CCL3, UNC13D, BTG1, BTG2, CLU, IRS1, and BCL2L in a population of T cells; and b. identifying one or more T cells that fulfill at least one of the following features: i. increased GZMB expression; ii. increased CCL3 expression; iii. increased UNC13D expression; iv. increased BTG1 expression; v. increased BTG2 expression; vi. increased CLU expression; vii. increased IRS1 expression; viii. increased BCL2L expression.
Item 5. The method of item 4, wherein prior to determining the mRNA expression level in the peripheral blood sample, said peripheral blood sample is subjected to generation of single cell suspension, staining, and/or cell sorting.
Item 6. The method of any one of the preceding items, wherein the method is for identifying one or more tumor-antigen specific T cells.
Item 7. The method of any one of the preceding items, wherein the peripheral blood sample is derived from a human subject.
Item 8. The method of any one of the preceding items, wherein the subject has or is suspected to have cancer.
Item 9. The method of any one of the preceding items, wherein the peripheral blood sample is a peripheral blood mononuclear cell (PBMC) sample.
Item 10. The method of any one of the preceding items, further comprising selecting the one or more T cells identified in step b. for further analysis.
Item 11. The method of any one of the preceding items, further comprising isolating the one or more T cells identified in step b.
Item 12. The method of any one of the preceding items, further comprising sequencing the one or more T cells identified in step b.
Item 13. The method of any one of the preceding items, further comprising expanding the one or more T cells identified in step b.
Item 14. The method of any one of the preceding items, wherein the one or more T cells are peripheral blood T cells (PB-T cells).
Item 15. The method of any one of the preceding items, wherein the one or more T cells are tumor-reactive T cells.
Item 16. The method of any one of the preceding items, wherein the one or more T cells are cytotoxic T cells.
Item 17. The method of any one of the preceding items, wherein the one or more T cells comprise a high affinity TCR that is preferably specific for a tumor target.
Item 18. A method of isolating one or more T cell, comprising isolating one or more T cells identified according to a method of any one of items 1-17.
Item 19. The method of item 18, further comprising expanding the one or more T cells.
Item 20. A method of identifying one or more T cell receptors (TCR) comprising determining the sequence of the TCR of the one or more T cells identified by the method of any one of items 1-17.
Item 21. A nucleic acid molecule comprising a nucleic acid sequence encoding the TCR identified by the method of item 20, or encoding a TCR comprised in a T cell identified by the method of any one of items 1-17.
Item 22. A method of generating one or more immune cells comprising introducing one or more nucleic acid molecules encoding one or more TCRs or antigen-binding fragments thereof, identified by the method of item 20 into one or more host cells.
Item 23. The method of item 22, wherein the host cell is an immune cell.
Item 24. The method of item 22 or 23, wherein the host cell is an immune effector cell.
Item 25. The method of any one of items 22-24, wherein the host cell is a T cell.
Item 26. The method of any one of items 22-25, wherein the host cell is a cytotoxic T cell.
Item 27. The method of any one of items 22-25, wherein the host cell is a gamma-delta T cell.
Item 28. The method of any one of items 22-24, wherein the host cell is an NK-T cell.
Item 29. The method of item 22, wherein the host cell is a stem cell, preferably an induced pluripotent stem cell (iPSC).
Item 30. The method of any one of items 22-29, wherein the host cell is a heterologous cell.
Item 31. The method of any one of items 22-29, wherein the host cell is autologous cell.
Item 32. The method of any one of items 22-31 wherein the host cell is immortalized.
Item 33. The method of item 20-32 wherein the host cell is a human cell.
Item 34. The method of any one of items 22-33, further comprising expanding the one or more immune cells.
Item 35. The method of any one of items 22-23, wherein the method comprises introducing more than one TCR identified by the method of item 20 into more than one host cell.
Item 36. A host cell comprising a nucleic acid molecule of item 21 or generated by the method of any one of items 22-35.
Item 37. A pharmaceutical composition comprising a T cell identified by the method of any one of items 1-17 or isolated by the method of item 18 or 19, or an immune cell generated by the method of any one of items 22-35, or a host cell of item 36.
Item 38. A T cell identified by the method of any one of items 1-17 or isolated by the method of item 18 or 19, or an immune cell generated by the method of any one of items 22-35, or a host cell of item 36, for use in therapy.
Item 39. The T cell, immune cell, or host cell for the use of item 38, wherein the therapy is adoptive T cell therapy.
Item 40. The T cell, immune cell, or host cell for the use of item 38 or 39, wherein the use is in the treatment of cancer.
Item 41. A method of diagnosing cancer in a subject comprising measuring the PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, expression level in a peripheral blood sample obtained from the subject, wherein an elevated PD-1, CD226, CD82, CD39 or ICOS, preferably PD-1, expression level as compared to a reference peripheral blood sample from a healthy control indicates the presence of T cells having high affinity TCRs.
Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.
Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
It is to be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.
Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.
The term “and/or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.
The term “about” or “approximately” as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.
When used herein “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.
In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms.
It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.
Tracking TCR-transgenic T cells. The inventors first set out to examine if they can track low numbers of transferred T cells over space and time, and which phenotypic adaptation tumor-specific T cells undergo during the course of the anti-tumor immune response. To this end, the inventors performed total body irradiation (TBI) on C57BL/6 mice and transferred 5.000 naïve (CD44lo) CD8+ CD45.1+ OT-1 T cells i.p., the next day followed by s.c. inoculation of MC38 OVA tumor cells, thereby making use of the well characterized syngeneic MC38 OVA tumor model40-43. Despite being a transplantable tumor model, recent evidence demonstrates MC38 OVA-induced reversible changes on systemic immunity, and a tumor microenvironment that resembles human tumors particularly well (44). The OT-1 TCR recognizes the target H2kb/SIINFEKL—which is a neoepitope45 in this setting—with high affinity (46). On day 7, 12 and 17 after transfer, OT-1 T cells were readily detectable by flow cytometry in peripheral blood, draining (dLN) and non-draining (ndLN) lymph nodes, as well as among TILs (
Site-dependent PD-1 expression levels indicate differential phenotypes of tumor specific T Cells. T cell exhaustion markers are relevant in two ways. One the one hand, they indicate the cellular differentiation state of tumor-specific T cells, thereby contributing to a basic understanding on the evolution of tumor-specific T cell immunity (23 Bos, R., Marquardt, K. L., Cheung, J. & Sherman, L. A. Functional differences between low- and high-affinity CD8+ T cells in the tumor environment. Oncoimmunology 1, 1239-1247 (2014), 26. Martinez-Usatorre, A., Donda, A., Zehn, D. & Romero, P. PD-1 Blockade Unleashes Effector Potential of Both High- and Low-Affinity Tumor-Infiltrating T Cells. J. Immunol. ji1701644 (2018) doi:10.4049/jimmunol.1701644. 27. Caserta, S., Kleczkowska, J., Mondino, A. & Zamoyska, R. Reduced Functional Avidity Promotes Central and Effector Memory CD4 T Cell Responses to Tumor-Associated Antigens. J. Immunol. 185, 6545-6554 (2010)). On the other hand, exhaustion markers such as PD-1 represent potential surrogate markers for guiding the use of checkpoint inhibitor therapy12 and the identification of tumor antigen-specific T cells (13. Gros, A. et al. PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors. J. Clin. Invest. 124, 2246-59 (2014). 14. Gros, A. et al. Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat. Med. (2016) doi:10.1038/nm.4051). The inventors checked adoptively transferred OT-1 cells as well as endogenous CD8+ T cells for the expression of PD-1, TIM-3 and LAG-3 (
Ultra-low T cell numbers can mediate tumor protection. The inventors next tested whether the T cells for which they detected the described spatio-temporal phenotypic adaptations were of functional importance, or whether in immunocompetent C57BL/6 mice the addition of low numbers of high-affinity T cells to the endogenous response was irrelevant for tumor protection. To this end, they sorted with high purity and adoptively transferred 128, 320, 800, 2.000 or 5.000 naïve (CD44lo) CD8+ CD45.1+ OT-1 T cells, or—as negative controls—5.000 P14 T cells, which recognize the LCMV antigen gp33. 2.000 or 5.000 OT-I T cells conveyed almost perfect protection, whereas the endogenous immune response was unable to mediate any protection when only antigen-unspecific P14 cells were transferred in addition (
A SIINFEKL-specific TCR library provides TCRs with distinct profiles of in vitro and in vivo functionality. With this system at hand, the inventors aimed to investigate the impact of TCR affinity on tumor protection and spatiotemporal T cell fate. In the past, OT-I T cells have frequently been used for such purposes, often together with altered peptide ligands (APLs) of SIINFEKL to cover differential affinity TCR-epitope ligand interactions26,49. However, despite its wide usage, the OT-I TCR has some unconventional features50,51, which argues for the importance of studying different TCRs (best with a gradient of the parameter under investigation, e.g. TCR-pMHC affinity) before drawing general conclusions. Furthermore, the APL system is prone to biases through differences in peptide-pMHC affinity and/or stability52. Finally, most T cell studies use adoptive transfers of monoclonal populations. While this reflects the clinical situation of adoptive cell therapy, the translation of findings to physiological T cell responses, which are polyclonal, is questionable in this setting. For these reasons the inventors previously identified a repertoire of 15 unique H2kb/SIINFEKL-specific TCRs with differential structural affinity, and developed a polyclonal transfer system that allows in vivo monitoring of defined composite TCR repertoires during the course of an immune response37. To study TCR-dependent tumor-specific immunity, the inventors chose four TCRs—TCR16-30, TCR9, TCR28 and TCR39—which represented the entire affinity spectrum and first performed further in-depth TCR characterization experiments (
In polyclonal populations, PD-1 is a surrogate marker for TCRs with high protective capacity outside the tumor. Having established that TCR16-30 is a highly protective TCR, the inventors wondered whether T cells with this TCR would show distinct features in terms of T cell homing, abundance or phenotypic adaptation within polyclonal populations harboring also TCRs with gradually worse functionality, as it is the case in physiological T cell responses. To this end, the inventors generated retrogenic mice for each TCR (
High-functionality neoantigen-specific TCRs can be identified in peripheral blood of a human melanoma patient based on phenotypic traces of recent activation. The inventors next aimed to investigate whether their findings would hold true for human tumor-specific T cells in vivo. To this end, the inventors analyzed T cells specific for the HLA-A*01:01-restricted neoantigen ENTPD4P>L, in which the wildtype sequence ATDTNNPNVNY (SEQ ID NO: 3) is mutated to ATDTNNLNVNY (SEQ ID NO: 4), in a human melanoma patient57. This patient was a 44-year old woman with a stage M1b BRAF V600E mutation positive melanoma with lymphadenopathy and subcutaneous and soft tissue metastases. Her previous treatment included the CTLA-4 targeting checkpoint inhibitor ipilimumab. The patient received 7×1010 TIL with two doses of IL-2, which induced partial remission. Partial remission lasted for 6 months, and the patient survived in total for 16 months after TIL treatment. T cells reactive to ENTPD4P>L constituted 5.06% of CD8 T cells in the TIL infusion product and could also be identified in peripheral blood before (from here on referred to as ‘PB-pre’) and 212 days after (from here on referred to as ‘PB-post’) TIL treatment (constituting 0.74% and 0.26% of CD8 T cells, respectively;
Investigation of a second human melanoma patient additionally reveals surface proteins biomarkers expressed on tumor-specific TCR T cells in peripheral blood
Finally, we extended our analyses to a second human melanoma patient with a different neoepitope specificity, and investigated T cells reactive to the HLA-B*58:01-restricted RBM12S>L epitope from a male 46-years old patient with metastatic melanoma, whose previous treatment also included ipilimumab (“patient 3” in van den Berg et al. (J. H. van den Berg et al., J. Immunother. Cancer 8, e000848 (2020).)) (
Transcriptomic profiling (
With this low number of clonotypes, meaningful quantitative correlations of phenotype and functionality, as performed for ENTPD4P>L-specific T cells, were not possible. We therefore decided to integrate all clonotypes in our analysis (including highly functional single-cell clonotypes R8, R9, R122 as well as the non-functional hTCR R42), and check which clonotypes are enriched in subpopulations with different levels of phenotypic markers that are expressed as surface proteins. In fact, this approach most closely resembles the proposed clinical workflow, i.e. biomarker-based prospective identification of high-functionality TCRs from a polyclonal population with unknown clonotypic composition. In our mouse model, we had observed that filtering on peripheral blood T cells with highest PD-1 expression levels (which corresponded to intermediate absolute PD-1 expression levels) enriches for functional TCRs. Among RBM12S>L-specific T cells, PD-1 surface protein levels did not differ much between individual antigen-reactive TCRs, but clearly discriminated functional TCRs from the non-functional hTCR R42 (
In order to investigate enrichment of high-functionality TCRs among cells with high expression for a given surface marker in a systematic manner, we ranked all cells (encompassing all clonotypes) according to the surface expression of each marker and calculated the average functional avidity (EC50 of IFNγ release) for each rank. Lower surface expression ranks cumulatively entailed all functionalities from cells with higher ranks so that the highest ranked cell represented the functionality of only the cell with highest surface expression, the second highest ranked cell entailed the average functionality of the two cells with highest surface expression levels, and the lowest ranked cell encompassed the average functionality of all cells (as if no filtering on surface expression had been performed). For this reason, the lowest ranked cell for any surface marker always had the same average functionality, which corresponded to the average functionality of the overall population as defined through the relative share that each TCR contributes (see pie charts in
Cells with high expression of PD-1 were enriched for functional TCRs, whereas cells with high levels of CD3—which is downregulated upon T cell activation (S. Valitutti et al., J. Exp. Med. 185, 1859-64 (1997))—were enriched for the non-functional hTCR R42 (
C57Bl/6JOlaHsd (females, 6-8 weeks) were purchased from Envigo. 6-24 weeks old female SIINFEKL peptide-specific TCR transgenic OT-1 (C57Bl/6-Tg(TcraTcrb)1100Mjb/J) were originally obtained from The Jackson Laboratory and bred under specific pathogen-free conditions at our mouse facility at the Technical University of Munich. Different congenic marker backgrounds of CD45.1/.2 and CD90.1/.2 were derived from in-house breeding39. All animal experiments were approved by the district government of upper Bavaria (Department 5—Environment, Health and Consumer Protection).
Human blood samples. Written informed consent was obtained from the donors and use of the blood samples was approved according to national law by the Dutch (CCMO) or by the German (Ethikkommission der Medizinischen Fakultst der Technischen Universitst Munchen) Ethics Institutional Review Board, according with the Declaration of Helsinki and Good Clinical Practice. Further clinical information on the human melanoma patient has been previously described (van den Berg, J. H. et al. Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up. J. Immunother. Cancer 8, e000848 (2020). Listeria Infection with recombinant Listeria monocytogenes expressing Ovalbumin was performed by i.v. injection of 5000 CFU81. Tumor model Mice were injected subcutaneously in the right flank with 106 tumor cells. Tumors were measured every 2-3 days with a caliper. Tumor size was determined as product of two diameters (length×width). Upon reaching 225mm2 or ulceration of the tumor, mice were sacrificed. For in vivo transfer experiments, mice were irradiated with 5 Gy followed by intraperitoneal injection of T cells. Where indicated, tumors were excised, mechanically disaggregated and enzymatically digested with collagenase type VIII (125 CDU/mg, Sigma-Aldrich) and DNAse I (2000 U/mg, Sigma Aldrich) for 60 minutes at 37° C. Tumor cell suspensions were passed through a 70-μm cell strainer and lymphocytes were enriched in a Percoll (GE Healthcare) density gradient (40% vs. 80%) at 3600g for 20 minutes. Generation of T cell retrogenic mice Retrogenic mice were generated as described before37. In brief, bone marrow was harvested from congenically marked donor mice with a Rag-1−/− background. Sca-1 positive HSCs were retrovirally transduced with a given TCR and injected i.v. into irradiated C57BL/6 mice.
Flow cytometry. Tumor cell suspensions were produced as previously described. Axillary and inguinal lymph nodes were harvested and mashed through a 40-μm cell strainer to generate a single cell suspension. Blood was obtained bleeding from the facial vein. Spleens were harvested and mashed through 70-μm cell strainers. Following red blood cell lysis with Tris-Buffered Ammonium Chloride, cells were stained with the respective antibody panel for 30 min at 4° C. in the dark (for multimer staining see below). After washing with FACS buffer (PBS with 0.5% BSA and 2 mM EDTA), the cells were stained with PI for 5 min and washed again. Data were collected by flow cytometry on a CyAn ADP 9 color (Beckman Coulter) or Cytoflex S flow cytometer (Beckman Coulter). For analysis, FlowJo software (FlowJo LLC) was used.
Adoptive transfer and tracking of T cells from TCR retrogenic mice. For adoptive transfer experiments TCR retrogenic donors with TCRs of different affinities for H2Kb/SIINFEKL on a unique congenic marker background (CD45, CD90) were used. Blood from retrogenic mice was stained following red blood cell lysis with antibodies directed against CD8 and CD44, with PI staining for live/dead discrimination. Naïve T cells (living, CD8+, CD44low, congenic+) were sorted (MoFlo legacy; Beckman Coulter) into a 96-well v-bottom plate containing a cell pellet of 400.000 C57Bl/6 splenocytes in FCS. Cells were injected i.p. into WT recipients. Peripheral blood of recipient mice was analyzed via flow cytometry at different timepoints after transfer. The unique congenic marker backgrounds allowed tracking of individual TCRs over time.
Peptide-MHC (pMHC) multimer staining. Biotinylated pMHC molecules for the generation of pMHC multimers were refolded according to the protocol described in82. 0,4 μg of biotinylated pMHC I molecule, 0.5 μg of Streptavidin-PE or Streptavidin-APC and 50 μl of FACS buffer for every 5×106 cells, were preincubated for at least 30 min for multimerization. Cells were then incubated with the multimer mix for 30 min. 20 min before the end of the staining period antibodies for the staining of surface antigens were added. PI for live/dead staining was added 5 min before the end of the staining period.
Peptide titration and intracellular cytokine staining. Samples were split and incubated with different peptide concentrations (10-6-10-12 M) of the SIINFEKL-peptide, a negative (RP10+ only) and a positive control (PMA/lonomycin). Samples were then incubated at 37° C. for five hours, after one hour 2 μg/well of Golgi Plug was added without resuspension. Next EMA/Fc-block staining (EMA 1:1000, Fc-Block 1:400) and staining of surface antigens (CD8, CD19, CD45.1/CD90.1) were performed. After fixation and permeabilization using Cytofix/Cytoperm (BD) intracellular cytokine staining (IFNγγ, TNFαα, IL2) was performed. The maximal percentage of IFNγ+CD8+ cells was normalized to 100%, and a nonlinear curve was fitted into the normalized data.
In vivo L.m. OVA proliferation. Mice were injected with 100 retrogenic T cells for each TCR and infected with 5000 CFU L.m. OVA (both i.v.). On day 12 after infection, spleens were harvested, processed as described above and subsequently analyzed by flow cytometry.
Analysis of in vitro cytotoxicity. Experiments were performed using the xCELLigence technology (Roche/ACEA Biosciences, San Diego, CA) at 37° C. with 5% CO2. For measuring cytotoxic responses, 104 PancOVA cells were seeded on 96 well E-Plates (E-Plate® 96) with gold micro electrodes. Changes in impedance were monitored at 15-minute intervals for up to 120 hours. After 24 hours the supernatant was removed and T cells were added to the culture. All incubations were performed in volumes of 200 ul of cDMEM. Cell index (CI) values were analyzed by the RTCA Software 2.0 (Acea Bioscience, Inc.). For time point-independent and thus more unbiased analysis, the area-under-the-curve of cell index values was determined to indicate killing capacity.
Tissue culture. All cell lines were grown in cDMEM (DMEM (Life Technologies), supplemented with 10% FCS, 0.025% L-Glutamine, 0.1% HEPES, 0.001% gentamycin and 0.002% streptomycin).
Single cell RNA sequencing. After cells have been sorted, they were centrifuged and the supernatant was carefully removed. Cells were resuspended in the Mastermix+37.8 μl of water before 70 μl of the cell suspension were transferred to the chip. (Step 1.1 and 1.2 of the original protocol). After each step, the integrity of the pellet was checked under the microscope to ensure that all cells are loaded onto the chip. From here on, 10× experiments have been performed according to the manufacturer's protocol (Chromium next GEM Single Cell VDJ V1.1 with Feature Barcode, Rev D). QC has been performed with a High sensitivity DNA Kit (Agilent #5067-4626) on a Bioanalyzer 2100 as recommended in the protocol and libraries were quantified with the Qubit dsDNA hs assay kit (life technologies #Q32851). All steps have been performed using RPT filter tips (Starlab #S1183-1710, #SS1180-8710, #S1182-1730) and DNA LoBind tubes (Sigma #EP0030108051, #EP0030108078, #EP0030124359).
scRNA seq data analysis. References GRCh38-2020-A and vdj_GRCh38_alts_ensembl-5.0.0 were used for Transcriptome and VDJ annotation (CellRanger 5.0.0), respectively. Data analysis was performed with SCANPY (V1.4.3, Wolf et al 2018). The notebooks containing all steps of data processing and analysis can be found online (GitHub repository)
TCR DNA template design. DNA templates were designed in silico and synthesized by Twist Bioscience. DNA constructs for CRISPR/Cas9-mediated homology-directed repair (HDR) had the following structure: 5′ homology arm (300-400 bp), P2A, TCR β (including murine TRBC with additional cysteine bridge (Cohen C J, et al 2006, Cohen C J, et al 2007, Kuball J et al. 2007)), T2A, TCR α (including murine TRAC with additional cysteine bridge (Cohen C J, et al 2006, Cohen C J, et al 2007, Kuball J et al. 2007)), bGHpA tail, 3′ homology arm (300-400 bp). The homology arm sequences of the TRBC locus were derived from TRBC1 and are highly homologous to TRBC2.
CRISPR/Cas9-mediated KO and KI. Cas9 RNPs and HDR repair templates were generated as described before. Bulk PBMCs were activated for two days in RPMI with CD3/CD28 Expamer (Juno Therapeutics), 300 IU ml−1 IL-2, 5 ng ml−1 IL-7 and 5 ng ml−1 IL-15. Expamer stimulus was removed by incubation with 1 mM D-biotin. Cells were electroporated (pulse code EH100) with Cas9 ribonucleoprotein and DNA templates in Nucleofector Solution (20 μl per 1×106 T cells; Lonza) with a 4D Nucleofector X unit (Lonza). After electroporation, cells were cultured in RPMI with 180 IU ml-1 IL-2 until a first FACS analysis on day five after editing.
Antigen-specific activation and intracellular cytokine staining. On the day before co-culture with T cells, K562 cells (retrovirally transduced to express the human MHC class-I molecule of interest) were irradiated (80 Gy) and loaded with peptide (10−12M, 10−10M, 10−9 M, 10−8 M, 10−7 M, 10−6 M, 10−5 M, 10−4 M) overnight at 37° C. T cells were cocultured with peptide-loaded K562 cells and Golgi plug (BD Biosciences) in a 1:1 ratio for 4 h at 37° C. PMA (25 ng ml-1) and ionomycin (1 μg ml-1) were used for positive control. Surface marker antibody staining for CD3 (BV421, BD Biosciences), CD8 (PE, Invitrogen) and antimurine TCR β-chain (APC/Fire750, BioLegend) was followed by permeabilization using Cytofix/Cytoperm (BD Biosciences), and intracellular staining of IFNγ (FITC, BD Pharmingen), TNFα (PC7, eBioscience) and IL-2 (APC, BD Biosciences). Live/dead discrimination was performed with ethidium-monoazide-bromide (Invitrogen).
Statistical analyses. Statistical analyses were performed using the GraphPad PRISM software. Statistical tests were used as indicated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22155928.9 | Feb 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/053191 | 2/9/2023 | WO |
