T cell expansion method and use

Abstract

The present invention provides a method for expanding anti-tumor T cells, comprising the following steps: a) providing phagocytic particles having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutated amino acid known or suspected to be associated with a subject's cancer, or a mutated or non-mutated amino acid sequence known or suspected to be expressed in a subject's cancer cells; b) providing live antigen-presenting cells; c) contacting the particles with the antigen-presenting cells in vitro under conditions that allow the antigen-presenting cells to phagocytose the particles; d) providing a T cell sample containing live T cells from the subject; e) contacting the T cell sample with the antigen-presenting cells contacted with the particles in vitro under conditions that allow the anti-tumor T cells to be specifically activated in response to antigens presented by the antigen-presenting cells. The present invention also provides a tumor neoantigen construct as defined in the specification.

Description

Technical Field

[0001] The present invention relates to a method for expanding anti-tumor T cells, as well as a composition and use of the anti-tumor T cells produced by the method according to the present invention, in particular, use in treating cancer. Background Art

[0002] The adaptive immune system constitutes a branch of the immune system. Unlike the innate immune system, which responds in a more general manner, the adaptive immune system can adapt and respond specifically to different pathogens or cell-damaging challenges encountered by an organism. The adaptive system includes humoral immunity, namely antibodies secreted by B cells, and T cell-mediated immunity. The specificity of the adaptive immune system lies in the B cell receptors and T cell receptors expressed on B cells and T cells. Through a complex system of mixed gene fragments, the body produces a nearly infinite variety of B cells and T cells, each expressing a specific receptor for a particular protein or peptide.

[0003] T cells are lymphocytes that form an integral part of the adaptive immune system and play a central role in cell-mediated immunity. Defined T cell receptors (TCRs) are expressed on the cell surface, and each receptor recognizes antigen-derived peptides presented in the context of MHC (major histocompatibility complex) molecules. There are several types of T cells, each with distinct functions in cellular immune responses.

[0004] There are two main types of T cells with different functions. CD8 positive cytotoxic T cells will bind to peptides presented on class I MHC receptors on cells (called class I human leukocyte antigens (HLA) in humans). All nucleated cells express class I HLA. If it is believed that the presented peptide is foreign (the most common sign of viral infection), the cytotoxic T cells will kill the cells through the proteins granzyme B or perforin. Helper T cells (Th) expressing the surface marker CD4 do not kill cells, but coordinate the immune response by secreting cytokines, which are proteins that amplify pro-inflammatory signals between cells, and sometimes amplify inhibitory signals. Another important function of T helper cells is to induce class switching of B cells, for example, turning B cells that secrete IgM into cells that secrete IgG, which will increase the humoral immune response to antigens.

[0005] T helper cells will bind to their corresponding peptides presented on MHC-II (called class II HLA in humans) through their TCR, i.e. receptors specifically expressed on so-called antigen presenting cells (APCs) or endothelial cells. T cell activation depends on the microenvironment in which Th-APC interactions occur and the types of so-called costimulatory molecules expressed on APCs. T helper cells can differentiate into different Th subsets, for example, proinflammatory Th1, Th2, Th17 cells, or inhibitory T helper cell types called regulatory T cells (Tregs). The latter subset is very important for controlling the immune response, because an unrestricted immune system is harmful and can lead to tissue damage and autoimmunity.

[0006] Immunological approaches for treating cancer are known. Monoclonal antibodies targeting immune activation checkpoints have been used with great success, for example, in the treatment of melanoma, lung cancer, bladder cancer, and gastrointestinal cancer. Although different monoclonal antibodies have different mechanisms of action, they all lead to the activation and expansion of anti-tumor (or "tumor-reactive") T cells.

[0007] Because T cells are often the ultimate effectors of immune-mediated cancer therapy, strategies have been developed to directly utilize anti-tumor T cells. One approach is adaptive cell transfer (ACT), in which T cells are expanded ex vivo and then reinfused in large numbers into a subject with cancer. This approach is discussed, for example, in Klebanoff et al., Nature Medicine, 2016, 22, 26–36.

[0008] There have been some successes in this area. For example, Karlsson et al., Ann Surg Oncol., 2010, 17(7): 1747-57 reported a study using an adoptive immunotherapy protocol to treat patients with advanced colon cancer. The basis of this treatment was the isolation and in vitro expansion of autologous tumor-reactive lymphocytes isolated intraoperatively from the first lymph node that naturally drains the tumor (sentinel lymph node). Lymphocytes obtained from the sentinel lymph node were collected against autologous tumor extracts, activated, expanded, and returned as a transfusion. No toxicity or other adverse reactions were observed. Complete or significant regression of the disease occurred in four patients with liver and lung metastases, and twelve patients showed partial regression or stable disease. A dose-dependent response was found, and stage IV patients showed a significantly increased survival of 2.6 years compared to 0.8 years in control patients. In conclusion, lymphocytes obtained from freshly isolated sentinel lymph nodes can be expanded and safely returned to patients without complications.

[0009] However, obtaining sufficient anti-tumor T cells for adaptive cell transfer is challenging using current methods: surgical resection of the cancer is required to obtain tumor-infiltrating lymphocytes to be expanded. This is an invasive procedure. Furthermore, the cells obtained are few and often unresponsive (anergic) due to immune suppressive mechanisms from the tumor. This can result in a long time (months) required to achieve sufficient expansion.

[0010] Genetically engineered T cells have been developed to overcome the limitations of adaptive cell transfer using tumor-infiltrating lymphocytes. By introducing antigen receptors into T cells or synthetic recognition structures called "chimeric antigen receptors" into T cells, genetically engineered T cells can be obtained by genetically redirecting T cell specificity to the patient's cancer. Although genetically engineered T cells have been successful in treating hematological cancers, the safety and selectivity of genetically engineered T cells for treating solid cancers still need to be improved. WO 2016 / 053339 (U.S. Department of Health and Human Services) discloses a method for isolating T cells that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. The method comprises: identifying one or more genes in the nucleic acid of a patient's cancer cells, each gene comprising a cancer-specific mutation encoding a mutated amino acid sequence; inducing the patient's autologous APCs to present the mutated amino acid sequence; co-culturing the patient's autologous T cells with the autologous APCs presenting the mutated amino acid sequence; and selecting the autologous T cells. The selection step entails selecting autologous T cells that (a) are co-cultured with autologous APCs presenting the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of the major histocompatibility complex (MHC) expressed by the patient to provide isolated T cells that have antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. It is disclosed that once those cells are selected, they are expanded by culturing with feeder PBMCs (e.g., irradiated allogeneic PBMCs), interleukin (IL)-2, and OKT3 antibodies.

[0011] In order to induce the patient's autologous APC to present the mutated amino acid sequence, WO2016 / 053339 teaches the use of a pulse with a free peptide or nucleotide construct to introduce a new antigen (or new antigen encoding material) into the cell. The pulse with the free peptide introduces the new antigen into the cytosol (intracellular fluid) of the cell.

[0012] Therefore, there is still a need for improved methods to provide anti-tumor T cells suitable for treating cancer that have better safety and selectivity and can be practically used clinically. Summary of the Invention

[0013] The present invention provides a method for amplifying anti-tumor T cells, comprising the following steps:

[0014] a) providing a phagocytosable particle having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutated amino acid known or suspected to be associated with a subject's cancer, or a mutated or non-mutated amino acid sequence known or suspected to be expressed in a cancer cell of the subject;

[0015] b) providing live antigen-presenting cells;

[0016] c) contacting the particles with antigen-presenting cells in vitro under conditions that allow phagocytosis of the particles by the antigen-presenting cells;

[0017] d) providing a T cell sample comprising live T cells from the subject;

[0018] e) contacting the T cell sample with the antigen-presenting cells contacted with the particles in vitro under conditions that allow for specific activation of the anti-tumor T cells in response to the antigen presented by the antigen-presenting cells.

[0019] The inventors surprisingly discovered that phagocytic particles with tumor neoantigen constructs tightly associated therewith can be internalized by antigen-presenting cells, whereupon the tumor neoantigen constructs can be presented on the surface of those cells and induce expansion of T cells cultured in the presence of the particle / APC mixture. The use of phagocytic particles resulted in unexpectedly high uptake and processing of tumor neoantigen constructs.

[0020] High-level expansion of anti-tumor T cells can be achieved by using phagocytosable particles with one or more tumor neoantigen constructs tightly associated therewith, binding to antigen-presenting cells, and exposing the particles / antigen-presenting cells to a T cell sample from a subject with cancer. Typically, multiple particles, e.g., 100 to 1 × 10 7 The particles are arranged in a pattern of particles (which may all be of the same kind, or may be different, as described in further detail below).

[0021] This approach is particularly effective because it allows for control over the sequence of the tumor neoantigen construct and also allows for the inclusion of more than one tumor neoantigen construct on the particle. For example, different tumor neoantigen constructs containing one or more different neoantigen epitopes can be attached to the particle. This multi-functionality of the particle allows for stimulation with multiple epitopes to maximize T cell expansion.

[0022] The use of phagocytic particles also allows large numbers of tumor neoantigen constructs to enter cells and be presented on the cell surface. A single phagocytic particle according to the present invention typically has 500,000 to 1,000,000 tumor neoantigen constructs associated with it. This results in high levels of surface expression of neoantigens on APCs that have internalized the phagocytic particle.

[0023] The use of phagocytic particles also surprisingly allows tumor neoantigen constructs to be presented to antigen presenting cells in a highly sterile and pure form. For example, phagocytic particles are particularly effective in removing contaminants such as pyrogens.

[0024] Without wishing to be bound by any particular theory, it is believed that phagocytic particles with tightly associated tumor neoantigen constructs are particularly effective because they are internalized by antigen presenting cells due to engulfment into phagosomes. The tumor neoantigen constructs are then cut from the particles in the phagosomes, and fragments of the tumor neoantigen constructs are presented to the surface of the antigen presenting cells via the major histocompatibility (MHC) class II pathway and presented to the cell surface through MHC class II molecules. It is also believed that this is not the only process by which antigens are presented on APCs, and some fragments of the tumor neoantigen constructs can also be presented to the surface of antigen presenting cells through the major histocompatibility (MHC) class I pathway in a process called cross-presentation and presented to the cell surface through MHC class I molecules. Therefore, although fragments of the tumor neoantigen constructs are expected to be presented to APCs primarily through the MHC class II pathway, some are expected to be presented through the MHC class I pathway, and therefore the present invention utilizes these two pathways to varying degrees.

[0025] When antigen is presented by MHC class II molecules, they usually activate helper T cells (also referred to as CD4+ T cells), which do not directly kill other cell types, but coordinate immune response by secreting cytokines, thereby inducing the class conversion of B cells to help B cells manufacture antibodies and stimulate the amplification of other T cell types. This means that the T cell colony activated according to the method of the present invention has the following advantages when amplifying and being applied to patients: immune response starts slowly (therefore causing few side effects), has long-lasting effect, and can be targeted to cancer in a variety of different ways by utilizing the entire immune system (for example, rather than only activating the CD8 positive cytotoxic T cells that can only directly attack tumor cells). This is contrary to the situation expected to occur when antigen is provided with free peptides or the nucleotide construct expressing the peptide. It is expected that this antigen will be absorbed into the cytosol of APC, which will cause neoantigens to be presented to cell surface only via MHC class I pathways by MHC class I molecules. This and then mainly causes the activation of CD8 positive cytotoxic T cells.

[0026] In the treatment method of the present invention, the patient's own immune system is utilized and stimulated. This means that chemically induced depletion of endogenous T cells is not required before administering the T cells. Therefore, this method has fewer side effects than some comparable alternative methods.

[0027] Furthermore, after initial targeting, memory T cells derived from T helper cells remain in circulation, enabling a rapid and effective secondary immune response as long as the cancer cells expressing the neoantigen remain in the body or if the same cancer recurs.

[0028] Therefore, when a T cell population expanded according to the method of the present invention is expanded and administered to a patient, it will continue to function even after the death of the initially administered T cells due to the memory T cells derived from the initially activated T cells.

[0029] The methods of the present invention also enable very rapid generation of expanded T cells, as phagocytable particles with one or more tumor neoantigen constructs tightly associated therewith can be rapidly prepared in a sterile and pure form, and expansion can then be rapidly initiated and completed. This is very important, as the success of treatment relies on the subject's cancer being essentially the same at the time the T cells are administered as when the sample was removed from the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Effect of particle size on T cell activation. Proliferation assay of ovalbumin-sensitized mouse splenocytes (thymidine incorporation). Comparison of ovalbumin conjugated to particles of varying sizes, 5.6 μm, 1 μm, and 0.2 μm in diameter. P values ​​were determined using the Student's T test, and values ​​were reported when p < 0.05 was found. Staples represent SD.

[0031] Figure 2. Expansion of T cells by stimulation with the neoantigenic peptide NA1-9. Figure 2A The number of cells in culture over time is shown. Figure 2B % CD4+ / total T cells are shown. Figure 2C T-bet expression in CD4 is shown. Figure 2D Shows the expression of granzyme B and perforin in CD8+ T cells.

[0032] Figure 3 Figure 3. Expansion of T cells by stimulation with neoantigen constructs. The percentages of T cells (small squares) and total CD4+ T cells (large squares) are shown, as well as proliferating CD4+ cells (circles).

[0033] Figure 4. Expansion of T cells by stimulation with neoantigen constructs. Figure 4A Shown are the number of cells in culture over time (red line: personalized peptides; pink and orange lines: predicted peptides) for control cultures, which have the same protein but with the predicted mutations (NA1, 3, 4, and 5). Figure 4B%CD4+ / total T cells are shown. Figure 4C visualizes the analysis of CD4 T cells using the Barnes-Hut Stochastic Neighbor Embedding (BH-SNE) algorithm, in which all cells in the sample are clustered on a 2D map based on the similarity of expression intensity of a set of selected markers, here CD28, CD57, T-bet, GATA-3, perforin, granzyme B (GZB), Ki-67, and PD-1.

[0034] Figure 5A Confocal microscopy images of PBMCs intracellularly phagocytosed antigen-coupled particles of three sizes (4.5 μm, 2.8 μm, or 1 μm) after incubation with antigen-coupled particles for 18 h at 37°C.

[0035] Figure 5B The figure shows the uptake of antigen-coupled particles of two sizes (4.5 μm or 2.8 μm) in PBMCs assessed by manual counting after 18 hours of incubation at 37°C.

[0036] Figure 5C The figure shows the uptake of antigen-coupled particles of three sizes (4.5 μm, 2.8 μm or 1 μm) in PBMCs assessed by volume calculation (*p<0.05, **p<0.01, ***p<0.001, calculated using Student's T test) after 18 hours of incubation at 37°C.

[0037] Figure 6A .The figure shows the relative increase in IFNγ production levels in PBMCs of CMV-susceptible healthy donors (n=2) stimulated with antigen-coupled particles of three sizes (4.5 μm, 2.8 μm or 1 μm) relative to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0038] Figure 6B .The figure shows the relative increase in IL22 production levels in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with antigen-coupled particles of three sizes (4.5 μm, 2.8 μm or 1 μm) relative to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0039] Figure 6C .The figure shows the relative increase in IL17 production levels in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with antigen-coupled particles of three sizes (4.5 μm, 2.8 μm or 1 μm) relative to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0040] Figure 6D.The figure shows the relative increase in dual cytokine production of IFNγ and IL17 in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with antigen-coupled particles of three sizes (4.5 μm, 2.8 μm or 1 μm) relative to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0041] Figure 6E .The figure shows the relative increase in dual cytokine production of IL22 and IL17 in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with antigen-coupled particles of three sizes (4.5 μm, 2.8 μm or 1 μm) relative to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv). DETAILED DESCRIPTION

[0042] definition

[0043] Endotoxins, such as lipopolysaccharide (LPS), consist of covalently linked lipid and polysaccharide subunits found on the outer cell wall of Gram-negative bacteria such as Escherichia coli.

[0044] CD4+ T cells (or T helper cells or CD4+ helper T cells) are cells that coordinate the immune response through cytokine secretion. They can inhibit or enhance other immune cells, for example, stimulating antibody class switching of B cells, the expansion of cytotoxic T cells, or enhancing phagocytes. They are activated by antigen presentation via MHC class II on APCs, and they express T cell receptors (TCRs) that are specific for fragments of approximately 15 amino acids within a specific antigen (so-called T cell epitopes).

[0045] CD8+ T cells (or cytotoxic T cells) are cells that kill tumor cells, infected cells, or other damaged cells. Unlike CD4+ T cells, they do not require APCs for activation. Their T cell receptors recognize antigen-derived peptides (approximately 7-10, for example, 8 amino acids long) presented by MHC class I (a protein expressed on all nucleated cells).

[0046] Antigen-specific T cell activation is a process that requires interaction between the TCR and defined peptides presented on MHC (HLA) molecules combined with costimulation.

[0047] Antigen-presenting cells (APCs), typically dendritic cells (DCs), B cells, or macrophages, are cells that phagocytose or internalize extracellular organisms or proteins, known as antigens, and after processing, present antigen-derived peptides on MHC class II to CD4+ T cells. Monocytes are the most abundant antigen-presenting cells in the blood.

[0048] Phagocytic particles are defined as particles that are capable of being phagocytosed by cells of the immune system, particularly monocytes.

[0049] Peripheral blood mononuclear cells (PBMCs) are a fraction of human blood prepared by density gradient centrifugation of whole blood. The PBMC fraction is primarily composed of lymphocytes (70-90%) and monocytes (10-30%), while red blood cells, granulocytes, and plasma have been removed. In some cases, monocytes may comprise 10% to 20%, e.g., 10% to 15%, of the cell count in a PBMC sample.

[0050] Tumor neoantigens are tumor-induced changes in the amino acid sequence of peptides or proteins that can be recognized as foreign by the immune system.

[0051] An amino acid sequence comprising a mutated amino acid is an amino acid sequence of a peptide or protein in a tumor cell of a subject that is altered compared to the sequence in a non-tumor cell of the subject. The mutated amino acid can be a point mutation: for example, an insertion or deletion of an amino acid in a protein or peptide sequence, or a substitution of a single amino acid with a different amino acid. In some cases, two or more mutated amino acids can be present consecutively. The mutation can be a frameshift mutation.

[0052] Detailed description

[0053] The present invention provides methods and means for expanding anti-tumor T cells, as well as compositions and uses of the anti-tumor T cells produced according to the methods of the present invention, as disclosed in more detail below.

[0054] Phagocytic particles and their properties:

[0055] The particles can be phagocytosed by antigen-presenting cells (APCs). APCs can phagocytose particles of many different materials and shapes. Therefore, the size of the particles needs to be selected to allow phagocytosis. Particles that are too small may not trigger phagocytosis by a specific APC. Particles that are too large may not be phagocytosed by a specific APC because they may not fit inside the cell.

[0056] Preferably, the maximum dimension of the particles used in the method of the present invention may be less than 5.6 μm, preferably less than 4 μm, more preferably less than 3 μm, for example less than 2.5 μm, less than 2 μm or less than 1.5 μm. Preferably, the maximum dimension of the particles used in the method of the present invention may be greater than 0.001 μm, preferably greater than 0.005 μm, preferably greater than 0.01 μm, preferably greater than 0.05 μm, preferably greater than 0.1 μm, more preferably greater than 0.2 μm, even more preferably greater than 0.5 μm. Preferably, the maximum dimension of the particles of the present invention is in the range of 0.1-5.6 μm, preferably 0.2-5.6 μm, preferably 0.5-5.6 μm, preferably 0.1-4 μm, preferably 0.5-4 μm, more preferably 0.1-3 μm, more preferably 0.5-3 μm, even more preferably 0.1-2.5 μm, even more preferably 0.5-2.5 μm, even more preferably 0.2-2 μm, even more preferably 0.5-2 μm, more preferably 1-2 μm, or for example about 1 μm, about 1.5 μm or about 2 μm (preferably about 1 μm). The particles may be substantially spherical, in which case the size refers to the diameter. Preferably, the particles are substantially spherical.

[0057] The preferred particle size according to the present invention is one that is large enough to enter the cell via phagocytosis, but small enough that more than one particle can enter the same cell via phagocytosis. Having more than one phagocytic particle means that the APC can simultaneously acquire peptides from multiple particles in different phagosomes, thereby maximizing the presentation of the neoantigen on the cell surface via the MHC class II pathway.

[0058] Generally, a size similar to that of bacteria facilitates complete engulfment by APCs. Complete engulfment can lead to better antigen degradation by APCs and subsequent better presentation to T cells via MHC class II. The present inventors have investigated the optimal size (see Examples 3a and 3b, and Figure 1 、 5A -C and 6A-E).

[0059] In certain preferred embodiments, the particles are paramagnetic, or more preferably, superparamagnetic. Paramagnetic or superparamagnetic particles can be completely separated from T cells using a magnet. Importantly, APCs containing internalized particles can also be separated from T cells using a magnet. This allows for rapid isolation of T cells from the incubation mixture. The T cells can then be administered to the patient after all particles have been removed. Thus, the present invention provides a particularly safe method for expanding T cells.

[0060] Paramagnetic particles also aid in the sterilization and / or denaturation of particles with associated tumor neoantigen constructs. Washing is discussed in further detail below. During washing, the particles can be collected and / or held in place by a magnet. Washing can also be performed by other means, such as by holding the particles (whether paramagnetic or not) in a column, or by allowing the particles to settle by gravity or centrifugation.

[0061] Examples of superparamagnetic particles are Dynabeads TM (Invitrogen). They are available in various functionalized formats, such as Dynabeads M-270 Carboxylic Acid, Dynabeads M-270 Amine, and Dynabeads MyOne Carboxylic Acid. Dynabeads MyOne Carboxylic Acid are uniform, single-sized superparamagnetic particles composed of highly cross-linked polystyrene with a uniform distribution of magnetic material. The particles are further coated with a hydrophilic layer of glycidyl ether, which conceals the iron oxide within the particle. Carboxylic acid groups are then introduced onto the particle surface.

[0062] Other examples of superparamagnetic particles include encapsulated carboxylated Superparamagnetic microspheres (Merck Chimie SAS) and Sera-Mag SpeedBeads (hydrophilic) carboxylate-modified magnetic particles (GE Healthcare UK Limited). Superparamagnetic microspheres are made of a core-shell structure. The superparamagnetic iron oxide material (~40%) is encapsulated by a polystyrene membrane, which does not interfere with components on the surface. Sera-Mag SpeedBeads (hydrophilic) carboxylate-modified magnetic particles are uniformly sized magnetic particles with a second layer of magnetite (two layers totaling ~60%). As a result, Sera-Mag SpeedBeads respond very quickly to magnetic fields, allowing for rapid and complete separation from suspensions. They have a cauliflower-like surface that increases the particle's total surface area and binding capacity.

[0063] Association of antigenic peptides with particles

[0064] The tumor neoantigen constructs are associated with the particles in a manner that allows sterilization and denaturing washes as described below without dissociating the tumor neoantigen constructs from the particles.

[0065] One way to associate a polypeptide with a particle is shown in Example 1. However, the precise manner of association is not critical to the methods of the invention. Preferably, the polypeptide is covalently linked to the particle (e.g., via an amide bond between an amine or carboxylic acid group of the polypeptide and a carboxylic acid or amine group on the particle surface). Alternatively, the candidate antigen polypeptide may be linked to the particle via a metal chelate. For example, particles linked to a metal chelating ligand such as iminodiacetic acid can bind metal ions such as Cu. 2+ 、Zn 2+ , Ca 2+ 、Co 2+ or Fe 3+ These metal chelates can in turn bind with high strength to proteins and peptides containing, for example, histidine or cysteine. Therefore, particles with metal chelates can non-covalently adsorb peptides / proteins in a manner that allows rigorous washing to reduce the amount of LPS and other contaminating components in the bound peptides / proteins.

[0066] The particles may comprise a polymer, glass or ceramic material (e.g., the particles may be polymer particles, glass particles or ceramic particles). Preferably, the particles comprise a synthetic aromatic polymer, such as polystyrene, or another polymer, such as polyethylene. The particles may comprise a polysaccharide polymer, such as agarose. Preferably, the particles of the present invention do not comprise a polysaccharide polymer (and preferably are not polysaccharide polymer particles or carbohydrate-based particles). Preferably, the particles of the present invention are polystyrene or polyethylene particles, and more preferably are polystyrene particles. Preferably, the particles of the present invention are monodisperse particles (i.e., particles of uniform size).

[0067] Preferably, the particles are paramagnetic, more preferably superparamagnetic.

[0068] Tumor neoantigen constructs

[0069] A tumor neoantigen construct is a polypeptide comprising more than one tumor neoantigen and / or at least one amino acid sequence known or suspected to be expressed in a subject's cancer cells. Preferably, a tumor neoantigen construct comprises more than one tumor neoantigen and / or a mutated amino acid sequence known or suspected to be expressed in a subject's cancer cells. A tumor neoantigen construct comprises one or more covalently linked peptides, wherein one or more covalently linked peptides preferably include an amino acid sequence containing at least one mutated amino acid known or suspected to be associated with a subject's cancer. In certain embodiments, one or more covalently linked peptides include an amino acid sequence containing at least one amino acid sequence known or suspected to be expressed in a subject's cancer cells. The amino acid sequence known or suspected to be expressed in a subject's cancer cells may be an amino acid sequence known or suspected to be expressed by a tissue or organ in which a tumor exists. The amino acid sequence may be mutated or non-mutated. Preferably, it is mutated. An amino acid sequence comprising at least one mutated amino acid known or suspected to be associated with a subject's cancer (or comprising an amino acid sequence containing at least one amino acid sequence known or suspected to be expressed in a subject's cancer cells, whether or not it is mutated) may also be referred to as a "neoantigen epitope."

[0070] Preferably, the mutated amino acid is a substituted amino acid (i.e., an amino acid in a protein or peptide in non-tumor cells is substituted with a different amino acid in tumor cells). Alternatively, the mutated amino acid may be an amino acid that has been deleted from the sequence (i.e., is deleted from the protein / peptide in tumor cells compared to non-tumor cells); or the mutated amino acid may be an amino acid that has been inserted into the sequence (i.e., a new amino acid has been added to the protein / peptide in tumor cells compared to non-tumor cells).

[0071] Neoantigen epitopes of tumor neoantigen constructs can be designed by selecting a mutant protein / peptide (or mutant gene) that is known or suspected to be associated with the subject's cancer; positioning the mutated amino acid in the sequence of the protein or peptide (or in the protein / peptide produced by gene expression); and selecting a portion of the protein / peptide containing the mutated amino acid, plus many flanking amino acids at the C-terminus and N-terminus of the mutated amino acid. In certain preferred embodiments, there is a single mutated amino acid in the sequence of the protein or peptide compared to the protein or peptide sequence in non-tumor cells. More preferably, there is a single substituted mutated amino acid in the sequence of the protein or peptide compared to the protein or peptide sequence in non-tumor cells.

[0072] Preferably, the number of flanking amino acids at the C-terminus of the mutated amino acid is at least 5, preferably at least 8, such as 8, 9, 10, 11 or 12 amino acids. Preferably, the number of flanking amino acids at the N-terminus of the mutated amino acid is at least 5, preferably at least 8, such as 8, 9, 10, 11 or 12 amino acids. The number of flanking amino acids at the C-terminus and N-terminus of the mutated amino acid may be the same or different, preferably, the number is the same.

[0073] In certain embodiments, the one or more covalently linked peptides include an amino acid sequence comprising at least one amino acid sequence known or suspected to be expressed in a subject's cancer cells. The amino acid sequence known or suspected to be expressed in a subject's cancer cells can be an amino acid sequence known or suspected to be expressed by a tissue or organ in which a tumor is present. The amino acid sequence can be mutated or non-mutated. Preferably, the amino acid sequence is mutated.

[0074] In embodiments where the tumor neoantigen construct comprises two or more covalently linked peptides, preferably, each covalently linked peptide comprises an amino acid sequence comprising at least one mutant amino acid (i.e., a neoantigen epitope) known or suspected to be associated with the subject's cancer. Alternatively, in embodiments where the tumor neoantigen construct comprises two or more covalently linked peptides, preferably, the tumor neoantigen construct comprises: at least one peptide comprising an amino acid sequence comprising at least one mutant amino acid (i.e., a neoantigen epitope) known or suspected to be associated with the subject's cancer; and at least one peptide comprising an amino acid sequence comprising an amino acid sequence known or suspected to be expressed in the subject's cancer cells.

[0075] In embodiments in which the tumor neoantigen construct comprises two or more covalently linked peptides, the peptides can be directly linked or linked via a spacer. The spacer can be a short sequence of amino acids, such as 1 to 15 amino acids, preferably 1 to 10 amino acids, and more preferably 1 to 5 amino acids (e.g., 1, 2, 3, 4, or 5 amino acids). Preferably, the spacer comprises the motif VVR. In one embodiment, the spacer has the sequence VVR. In another optional embodiment, the spacer does not comprise the motif VVR, such as the spacer does not have the sequence VVR. In another embodiment, the spacer comprises the motif GGS, such as the spacer has the sequence GGS. In another embodiment, the peptides are directly linked (i.e., without a spacer, such as without an amino acid spacer).

[0076] In a preferred embodiment, the tumor neoantigen construct comprises two or more covalently linked peptides, more preferably three or more covalently linked peptides (e.g., three, four, five, six, seven, eight, nine, or ten covalently linked peptides, preferably three, four, five, or six covalently linked peptides, more preferably three, four, or five covalently linked peptides). Preferably, the linked peptides are each linked via a spacer moiety. Each linker moiety may be the same or may be different. It has been found that three covalently linked peptides, each comprising an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with a subject's cancer, are particularly effective.

[0077] The structure of a tumor neoantigen construct comprising two linked peptides can be as follows:

[0078] -[epitope sequence 1]-[linker portion]-[epitope sequence 2].

[0079] The structure of a tumor neoantigen construct comprising three linked peptides can be as follows:

[0080] -[epitope sequence 1]-[linker portion]-[epitope sequence 2]-[linker portion]-[epitope sequence 3].

[0081] The structure of a tumor neoantigen construct comprising four linked peptides can be as follows:

[0082] -[epitope sequence 1]-[linker portion]-[epitope sequence 2]-[linker portion]-[epitope sequence 3]-[linker portion]-[epitope sequence 4].

[0083] The structure of a tumor neoantigen construct comprising five linked peptides can be as follows:

[0084] -[epitope sequence 1]-[linker portion]-[epitope sequence 2]-[linker portion]-[epitope sequence 3]-[linker portion]-[epitope sequence 5].

[0085] The structure of a tumor neoantigen construct comprising n linked peptides can be as follows:

[0086] -[epitope sequence 1]-[linker portion]-[epitope sequence 2]-[linker portion]-[epitope sequence 3]...-[linker portion]-[epitope sequence n].

[0087] In a preferred embodiment, each peptide of the tumor neoantigen construct comprises 5 to 50 amino acids, more preferably 5 to 30 amino acids, more preferably 8 to 30 amino acids, and most preferably 10 to 25 amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. In a particularly preferred embodiment, each peptide of the tumor neoantigen construct comprises 17 to 25 amino acids, for example, it comprises a mutant amino acid and 8 to 12 amino acids (preferably 10 amino acids) at the C-terminus and N-terminus of the mutant amino acid.

[0088] In embodiments where the particle is associated with two or more tumor neoantigen constructs, each tumor neoantigen construct can have the same polypeptide sequence or can have different polypeptide sequences. For example, a first tumor neoantigen construct can comprise one or more covalently linked peptides (e.g., one or more neoantigen epitopes), and a second tumor neoantigen construct can comprise one or more different covalently linked peptides (e.g., one or more neoantigen epitopes different from the neoantigen epitopes of the first tumor neoantigen construct).

[0089] The tumor neoantigen construct may comprise at least 20 amino acids, more preferably at least 30 amino acids, more preferably at least 40 amino acids, more preferably at least 50 amino acids, and even more preferably at least 60 amino acids. The tumor neoantigen construct may comprise less than 150 amino acids, preferably less than 130 amino acids, more preferably less than 100 amino acids, even more preferably less than 85 amino acids, and even more preferably less than 75 amino acids.

[0090] The present invention allows for more than one tumor neoantigen construct to be associated with the particle. Tumor neoantigen constructs can contain one or more neoantigen epitopes. Using multiple neoantigen epitopes on the particle increases the chances of expanding anti-cancer T cells. Antigen polypeptides larger than the fragment presented by APCs also ensure that a variety of epitopes for each antigen will be presented.

[0091] The present invention allows for the association of a single tumor neoantigen construct with a given particle, or allows for the association of more than one different neoantigen construct with a given particle. Particles associated with the same tumor neoantigen construct are considered to be of the same particle type. Particles associated with different tumor neoantigen constructs are considered to be different particle types.

[0092] Typically, 100 to 1x10 9 , for example 100 to 1x10 8 , for example 100 to 1x10 7 particles, for example, 1000 to 1x10 7For example, the ratio of particle concentration to APC cell concentration is in the range of 1000:1 to 1:10. The ratio can be optimized based on the size of the particles. For example, for particles with a cross-section of 1 μm, the ratio can be in the range of 50:1 to 2:1, such as 25:1 to 5:1, such as 15:1 to 7:1, such as 10:1.

[0093] The particles can be a homogenous population, i.e., all particles are of the same particle species. Alternatively, the particles can be a heterogeneous population, i.e., the particle population includes more than one particle species. For example, step a) can further include: providing a second phagocytic particle having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with the subject's cancer, or a mutant or non-mutated amino acid sequence known or suspected to be expressed in the subject's cancer cells, and wherein the sequence of the tumor neoantigen construct associated with the second particle is different from the sequence of the tumor neoantigen construct associated with the first particle. Optionally a third species, and optionally a fourth species, and optionally a fifth species, and optionally a sixth species, and optionally a seventh species, and optionally an eighth species, and optionally an nth species (where n can be an integer up to 50) of phagocytable particles can be provided, each of which has one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with a subject's cancer, or a mutant or non-mutated amino acid sequence known or suspected to be expressed in a subject's cancer cells, and wherein the sequence of the tumor neoantigen construct associated with the nth species of particle is different from the sequence of the tumor neoantigen constructs associated with particles of other species.

[0094] Therefore, in the methods of the present invention, a first particle associated with a first tumor neoantigen construct (a first type of particle) and a second particle associated with a second tumor neoantigen construct (a second type of particle) can be used in combination, wherein the first and second tumor neoantigen constructs have different sequences (for example, wherein the first tumor neoantigen construct comprises one or more different neoantigen epitopes compared to the second tumor neoantigen construct; and preferably, the first tumor neoantigen construct comprises different neoantigen epitopes compared to the second tumor neoantigen construct).

[0095] Other particle types may also be used, for example, 3 or more particle types may be used (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 25, 30, 40, or 50). Preferably, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 particle types may be used, and more preferably, 2, 3, 4, 5, 6, 7, or 8 particle types may be used (e.g., 4, 5, or 6 particle types). In embodiments where more than one particle species is used, each particle species has associated therewith a tumor neoantigen construct having a sequence different from that of the tumor neoantigen constructs associated with other particle species (e.g., each particle species has a tumor neoantigen construct comprising one or more neoantigen epitopes different from those of the tumor neoantigen constructs associated with other particle species; and preferably, each particle species has associated therewith a tumor neoantigen construct comprising all neoantigen epitopes different from those of the tumor neoantigen constructs associated with other particle species). In embodiments where more than one particle species is used, the tumor neoantigen construct on each particle is a construct as described above, comprising one or more neoantigen epitopes as described above. Preferably, the tumor neoantigen construct associated with each particle type has two or more covalently linked peptides, more preferably three or more covalently linked peptides (e.g., three, four, five, six, seven, eight, nine, or ten covalently linked peptides, preferably three, four, five, or six covalently linked peptides, more preferably three, four, or five covalently linked peptides), each peptide comprising an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with the subject's cancer (more preferably each being a neoantigen epitope). Preferably, the linked peptides are each linked via a spacer moiety. Each linker moiety may be the same or different.

[0096] Most preferably, the tumor neoantigen construct associated with each particle species has three covalently linked peptides, each comprising an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with the subject's cancer (more preferably, each being a neoantigen epitope). In such an embodiment, it is preferred to use 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 particle species, and more preferably 4, 5, or 6 particle species, and even more preferably 5 particle species.

[0097] Antigen degradation by APCs is not a uniform process. This ensures that a wide variety of epitopes will be presented for each antigen. Therefore, when tumor neoantigen constructs are degraded by APCs, this further increases the diversity of epitopes that will be presented. By utilizing the phagocytic pathway of APCs, combined with the possibility of having one or more neoantigen epitopes per tumor neoantigen construct, and the possibility of using one or more tumor neoantigen constructs on the particle, improved expansion of T cells is achieved. This is because the possibility of presenting epitopes to a larger number of anti-tumor T cells is increased.

[0098] The peptides of the neoantigen constructs comprising an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with the subject's cancer can be mutant sequences known to be present in the subject's tumor. For example, mutation data (whole exome and hotspot analysis) or the antigen spectrum of a cancer type may be known; for example, mutation data for certain cancers are contained in databases such as the COSMIC database (accessible from the following website: http: / / cancer.sanger.ac.uk / cosmic). The mutant antigens in the antigen spectrum can then be targeted, that is, one or more peptides in the tumor neoantigen construct can be designed to have an amino acid sequence comprising at least one mutant amino acid sequence in the antigen spectrum.

[0099] The peptides of the new antigen constructs that include an amino acid sequence containing at least one mutated amino acid known or suspected to be associated with the subject's cancer can be sequences known to be expressed by tumors or by tissues in which tumors are present. In this way, antigens that are specific to a particular cancer or to a particular tissue or organ in which the cancer is located can be selected. There are databases available, such as the EBI expression profile (https: / / www.ebi.ac.uk / gxa / home), which provide information about proteins / peptides expressed in restricted tissues or organs. Peptides unique to proteins expressed in specific tissues can then be targeted.

[0100] Alternatively (or additionally), the DNA sequence of the cancer cell can be established and the mutations in the cancer cell can be deduced from this. The mutated sequence can then be targeted, i.e., one or more peptides in the tumor neoantigen construct can be designed to have an amino acid sequence that includes the mutated amino acid sequence in the cancer cell.

[0101] In certain preferred embodiments, the T cell sample is from a subject undergoing treatment, and the sequence of one or more peptides of each tumor neoantigen construct is derived from the subject (particularly from the tumor antigen spectrum and / or DNA sequence of the subject's cancer cells). Preferably, APCs are also derived from the subject. In another embodiment, the T cell sample can be from a subject, the APCs are from a subject, and the sequence of each peptide of each tumor neoantigen construct can be derived from a library of mutant sequences of the same type of cancer suffered by the subject.

[0102] Antigen presenting cells (APC) and T cell samples

[0103] In the context of the present invention, APCs are professional antigen-presenting cells, such as monocytes / macrophages or dendritic cells. APCs can be primary cells or immortalized cells. Preferably, the antigen-presenting cells are phagocytes, more preferably, they are monocytes or dendritic cells.

[0104] The APCs must be compatible with the T cells of the T cell sample so that they can present antigens to the T cells under conditions of antigen specificity to which the T cells can react (MHC restricted). The APCs and T cell sample are preferably derived from the same species and are donor matched with respect to MHC receptors. However, it is also contemplated to use genetically engineered APCs from different species. More preferably, the APCs and T cell sample are obtained from the same subject. If the antigen presenting cells and the T cell sample are from the same subject, the possibility of any mismatch between the APCs and the T cells can be avoided.

[0105] Antigen presenting cells and T cell samples can be derived from the same blood sample, which is advantageous from a practical point of view. Antigen presenting cells and T cell samples can be derived from PBMC samples of the same subject. Obtaining PBMC from a peripheral blood sample is a conventional approach that provides a convenient source of APCs and T cells simultaneously and from the same subject. PBMC samples can be used fresh or frozen. From a logistical transport perspective, the possibility of using frozen cells has great practical advantages.

[0106] The T cell sample may be derived from the tumor, preferably from lymphatic vessels in the tumor. Even more preferably, the T cell sample may be derived from the sentinel lymph nodes (ie, tumor-draining lymph nodes).

[0107] The T cell sample may comprise whole PBMCs, purified T cell populations, or PBMCs depleted of specific T cell populations, including both CD4+ and CD8+ T cells. Preferably, the T cell sample comprises both CD4+ and CD8+ T cells.

[0108] Preferably, the anti-tumor T cells expanded in the methods of the present invention are CD4+ helper T cells and / or CD8+ T cells. Anti-tumor CD4+ helper T cells can coordinate the anti-tumor response. CD8+ T cells can attack tumors. Therefore, a mixed T cell response can be beneficial. In the examples described herein, there is some evidence that the majority of anti-tumor T cells expanded in the methods of the present invention are CD4+ helper T cells.

[0109] Details of the method steps

[0110] The present invention provides a method for expanding anti-tumor T cells. The method of the present invention is performed in vitro. Preferably, the subject is a mammal, and more preferably, a human. Step (e) preferably lasts from 1 day to 8 weeks, preferably from 3 days to 8 weeks, preferably from 1 week to 6 weeks, and more preferably from 1 week to 4 weeks. The method of the present invention may also include a step (a') before step (a): providing phagocytic particles and tightly associating one or more tumor neoantigen constructs with the phagocytic particles.

[0111] In one embodiment of the method of the present invention, particles having one or more tumor neoantigen constructs tightly associated therewith, antigen-presenting cells, and a T cell sample can be contacted simultaneously in the same container. In another embodiment, particles having one or more tumor neoantigen constructs tightly associated therewith and antigen-presenting cells are first contacted, and then the T cell sample is added. In another embodiment, the antigen-presenting cells and the T cell sample are in a single sample, and particles having one or more tumor neoantigen constructs tightly associated therewith are added to the T cell / antigen-presenting cell sample.

[0112] In one embodiment of the inventive method, the step (g) of contacting the T cell sample with the antigen-presenting cells contacted with the particles also includes adding a low dose of IL-2 to the T cell sample, for example, greater than 1.25U / ml (for example 1.25U / ml, 2.5U / ml, 5U / ml or 50U / ml), preferably greater than 2.5U / ml, 5U / ml or 50U / ml. When there is IL-2 simultaneously, antigen-specific T cell amplification can occur in the presence of antigen-presenting cells. IL-2 promotes T cell differentiation into effector T cells and memory T cells. After the antigen-specific T cell amplification, antigen-presenting cells can be removed, for example, by magnetic separation from the T cell colony of amplification.

[0113] In another embodiment of the method of the present invention, step (g) of contacting the T cell sample with the antigen-presenting cells contacted with the particles may further comprise adding IL-2 and / or IL-7 and / or IL-15 to the T cell sample, for example, adding a low dose of IL-2 to the T cell sample, for example, greater than 1.25 U / ml (e.g., 1.25 U / ml, 2.5 U / ml, 5 U / ml or 50 U / ml), preferably greater than 2.5 U / ml, 5 U / ml or 50 U / ml of IL-2, and optionally adding IL-7 and / or IL-15.

[0114] In one embodiment of the method of the present invention, in each step (i.e., in steps (a) to (e), and, if present, in any other step), no anti-CD3 antibody and / or anti-CD28 antibody is added in any step. In particular, no anti-CD3 antibody and / or anti-CD28 antibody is added in step (d) or (e) (or, if present, in steps (e1), (f), (g) or (h)). In one embodiment of the method of the present invention, in each step (i.e., in steps (a) to (e), and, if present, in any other step), the conditions of each step do not contain anti-CD3 antibody and / or anti-CD28 antibody. In particular, in steps (d) and (e), and, if present, in steps (e1), (f), (g) or (h), the T cell sample in each step does not contain anti-CD3 antibody and / or anti-CD28 antibody. Anti-CD3 and anti-CD28 antibodies can stimulate T cells indiscriminately, leading to the expansion of T cells that are nonspecific for the target antigen as well as other T cells.

[0115] In a preferred embodiment of the present invention, the method does not include any step of separating or isolating antigen-specific T cells during or after step (e) (or step (e1)). More specifically, there is no step of separating or isolating T cells activated in response to antigens presented by antigen-presenting cells in the T cell sample from unactivated T cells in the T cell sample. This is because the method of the present invention only results in the expansion of the desired T cells specific for the presented antigen. In step (e) of the method of the present invention, T cells that are not activated in response to antigens presented by antigen-presenting cells in the T cell sample will die in the process. In this way, no separation or isolation step is required.

[0116] washing

[0117] Preferably, the conditions in the step of contacting the T cell sample with the antigen-presenting cells in contact with the particles with the associated peptides are aseptic, and more preferably sterile. The use of particles allows the particles with the associated peptides to be sterilized, and more particularly allows the use of harsh sterilization conditions so that all pathogens can be removed without losing or destroying the neoantigen constructs associated with the particles.

[0118] Thus, in certain preferred embodiments, the methods of the present invention may include prior to step (a) (and, where applicable, after step (a')): (a'") the tumor neoantigen construct tightly associated with the particles has been subjected to a sterilization wash, thereby resulting in the removal of substances harmful to the subject. The sterilization wash solution may also be a denaturing wash.

[0119] In certain preferred embodiments, the method of the present invention may further comprise, prior to step (a) (and, where applicable, after step (a')): (a") subjecting the tumor neoantigen construct tightly associated with the particles to a denaturing wash resulting in sufficiently low endotoxin levels that do not interfere with subsequent steps (i.e., steps (a) to (e), or, if present, steps (a) to (f); (a) to (g) or (a) to (h)).

[0120] In such embodiments comprising step (a") and / or step (a'"), the sterilization wash or denaturing wash may involve subjecting the particles with the associated polypeptide construct to a high pH, ​​e.g., at least pH 12, preferably at least pH 13, more preferably at least pH 14, and most preferably at least pH 14.3. Alternatively, the denaturing wash may involve subjecting the particles with the associated polypeptide construct to a low pH, e.g., less than pH 3, more preferably less than pH 2, and most preferably less than pH 1.

[0121] In such embodiments comprising step (a") and / or step (a'"), the sterilization wash or denaturation wash may additionally or alternatively involve subjecting the particles with the associated polypeptide construct to an elevated temperature, e.g., at least 90°C, preferably at least 92°C, more preferably at least 95°C, e.g., at least 100°C or at least 110°C. The sterilization wash or denaturation wash may also additionally or alternatively involve subjecting the particles with the associated polypeptide construct to a denaturing agent, e.g., urea or guanidine hydrochloride, at a sufficient concentration, e.g., at least 5M, 6M, 7M or 8M.

[0122] The sterilization wash may comprise subjecting the particles and associated polypeptide constructs to a base, preferably a strong base, such as a base of at least 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M. In certain preferred embodiments, the sterilization wash may comprise subjecting the particles and associated polypeptide constructs to a base of at least 1 M sodium hydroxide (NaOH), preferably at least 2 M NaOH. Other bases that may be used include, but are not limited to, lithium hydroxide (LiOH), potassium hydroxide (KOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), magnesium hydroxide (Mg(OH)2), calcium hydroxide (Ca(OH)2), strontium hydroxide (Sr(OH)2), and barium hydroxide (Ba(OH)2).

[0123] The sterilization wash may comprise subjecting the particles and associated polypeptide constructs to an acid, preferably a strong acid, such as an acid of at least 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M. In certain preferred embodiments, the sterilization wash may comprise subjecting the particles and associated polypeptide constructs to a hydrochloric acid (HCl) of at least 1 M, preferably at least 2 M. Other acids that may be used include, but are not limited to, hydroiodic acid (HI), hydrobromic acid (HBr), perchloric acid (HClO 4 ), nitric acid (HNO 3 ), and sulfuric acid (H 2 SO 4 ).

[0124] In a preferred embodiment, the sterilization washing results in the particles and associated polypeptide construct being pathogen-free, and more preferably, the particles and associated polypeptide construct being sterile. Pathogen-free as defined herein is free of pathogenic bacteria or other living microorganisms. Sterile is defined herein as free of bacteria or other living microorganisms.

[0125] In a preferred embodiment, the amount of endotoxin obtained by denaturation washing is such that the final concentration of endotoxin is less than 100 pg / ml, preferably less than 50 pg / ml, more preferably less than 25 pg / ml, and most preferably less than 10 pg / ml.

[0126] If the preparation is contaminated with live microorganisms, the subsequent steps of cell culture may be affected. Thus, in a preferred method of the present invention, the particles with the associated polypeptide constructs of step (a) are sterile washed, resulting in sufficiently low levels of microorganisms and endotoxins that do not interfere with subsequent steps.

[0127] In the context of the present invention, the specific manner of sterilization washing is not critical. For example, sterilization washing may comprise subjecting the particles and associated polypeptide constructs to high pH, ​​low pH, high temperature, a sterilizing / denaturing agent, or a combination thereof. Preferably, sterilization washing comprises subjecting the particles and associated polypeptide constructs to high pH or to a strong sterilizing / denaturing agent, such as 8M urea or 6M guanidine-HCl. Most preferably, sterilization washing comprises subjecting the particles and associated polypeptide constructs to a high pH of at least 13.0, more preferably at least 14.0, and most preferably at least 14.3. Preferably, denaturation may comprise washing the particles and associated polypeptide constructs with a strong base, such as NaOH or KOH, preferably NaOH, at a concentration of 1-5M, preferably 1-3M, more preferably 1.5-2.5M, and most preferably 2M.

[0128] A particular advantage of the sterilization wash is that conditions can be selected such that the formulation having the particles and associated polypeptide construct is simultaneously sterilized and denatured in a single step. In particular, a high pH wash (e.g., pH > 14) can conveniently and simultaneously sterilize the formulation and achieve a denaturing wash that effectively eliminates endotoxins.

[0129] The sterilization wash may comprise a single wash or several repeated washes, for example 2, 3, 4 or 5 washes. The denaturation wash may comprise a single wash or several repeated washes, for example 2, 3, 4 or 5 washes.

[0130] Determine the extent of T cell activation

[0131] After step (e), the method of the present invention may further comprise step (f): determining the extent of anti-tumor T cell activation in the T cell sample, for example, by comparing the extent of anti-tumor T cell activation with a relevant reference, whereby a higher degree of anti-tumor T cell activation in the sample compared to the reference indicates the following conclusion: one or more tumor neoantigen determinants cause anti-tumor T cell activation in the sample. Determining the extent of anti-tumor T cell activation in the T cell sample may comprise determining the fraction of activated T cells in the sample. The extent of anti-tumor T cell activation in the T cell sample may be determined using ELISpot or FluoroSpot technology.

[0132] The method may include a step of comparing the degree of T cell activation with a relevant reference, whereby a higher degree of T cell activation in the sample compared to the reference leads to the conclusion that the candidate antigen causes activation of antigen-specific T cells in the sample. This reference is important for defining "activation," and therefore the degree of activation is preferably defined relative to a reference sample. When analyzing tumor or tumor-draining lymph node samples, the reference sample may be, for example, a sample from normal tissue. The reference sample is used to set a threshold for the diagnostic / prognostic conclusion and to determine whether the antigen is specifically positive (i.e., activated) or negative (i.e., downregulated).

[0133] Determining the degree of T cell activation in a T cell sample can include determining the fraction of activated T cells in the sample, that is, relative to the total T cells in the sample, the number of activated T cells. Relative to the fraction of activated cells in the reference sample, the fraction of activated cells will provide a measure of the degree of T cell activation. Preferably, in each analysis, in a sample incubated in the absence of antigen, i.e., in a negative control, the spontaneous activation level ("background activation level") in each sample is determined. Background activation can be compensated in the analysis; In other words, the activation score can be calculated on a net value, wherein the activation score to the negative control is subtracted.

[0134] Several methods are available for determining antigen-specific T cell activation, including ELISpot, Fluorospot, intracellular staining for cytokines by flow cytometry, FASCIA, proliferation assays (e.g., thymidine incorporation, CFSE, or BrdU staining), specific TCR detection using MHC-I or II tetramers, and ELISA or Luminex analysis of secreted cytokines.

[0135] With the help of ELISpot technology, cells are directly detected to release specific cytokines in response to stimulation. Cells are seeded on a membrane and the number of cytokine-secreting cells is measured. Depending on the cytokine to be measured, different variables can be measured, such as macrophage or T cell activation. FluoroSpot is based on ELISpot technology, allowing the simultaneous reading of several different secreted cytokines. This allows for a more accurate and subtle estimate of cell activation. Both methods provide a quick and easy way to measure T cell activation. Preferably, the degree of T cell activation in a T cell sample can be determined using ELISpot / FluoroSpot technology or proliferation assay (i.e., incorporation of thymidine). Determining the degree of T cell activation in a T cell sample can include, for example, determining by ELISpot or FluoroSpot assay the fraction of T cells that contact antigen-presenting cells that respond to the secretion of interferon gamma (IFN-γ), interleukin 17 (IL-17), interleukin 22 (IL-22), or a combination thereof. Other related analytes (combinations of cytokines) can be used to analyze specific diseases or conditions, such as T-reg cytokines.

[0136] Optional further activation step

[0137] In certain embodiments of the present invention, the method further comprises, after step (e) (e.g., immediately after step (e), or immediately after step (f), or immediately after step (g); preferably immediately after step (e) or immediately after step (f)):

[0138] e1) contacting the T cell sample with the second antigen-presenting cells contacted in vitro with the second particles under conditions that allow specific activation of the anti-tumor T cells in response to the antigen presented by the antigen-presenting cells.

[0139] Step (e1) advantageously serves as a second round of activation and stimulation ("re-stimulation") of T cells. Thus, step (e1) can be referred to as a "re-stimulation" step. Step (e1) can, for example, last from 1 to 4 weeks, and more preferably from 2 to 3 weeks.

[0140] Preferably, in certain embodiments of the present invention, the method further comprises, after step (e) (e.g., immediately after step (e), or immediately after step (f), or immediately after step (g); preferably immediately after step (e) or immediately after step (f)):

[0141] a1) providing a second phagocytic particle having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutated amino acid known or suspected to be associated with the subject's cancer, or a mutated or non-mutated amino acid sequence known or suspected to be expressed in the subject's cancer cells;

[0142] b1) providing a second live antigen-presenting cell;

[0143] c1) contacting the second particle with a second antigen-presenting cell in vitro under conditions that allow phagocytosis of the particle by the antigen-presenting cell; and

[0144] e1) contacting the T cell sample with the second antigen-presenting cells contacted in vitro with the second particles under conditions that allow specific activation of the anti-tumor T cells in response to the antigen presented by the antigen-presenting cells.

[0145] Preferably, step (e1) is performed after steps (e) and / or (f) and / or (g) when the expansion rate of the T cell sample decreases (or, if present, steps (a1) to (e1) are performed). Preferably, step (e) lasts for 2 to 4 weeks (which is the time during which the expansion rate of the T cell sample is expected to decrease during step (e)), and step (e1) is performed after step (e) (or, if present, steps (a1) to (e1) are performed).

[0146] In an embodiment in which step (e1) is followed by step (f), the method may further comprise (f1): determining the extent of anti-tumor T cell activation in the T cell sample, for example by comparing the extent of anti-tumor T cell activation with a relevant reference, whereby a higher degree of anti-tumor T cell activation in the sample compared to the reference indicates the following conclusion: one or more tumor neoantigen determinants lead to anti-tumor T cell activation in the sample.

[0147] In embodiments where step (e1) follows step (g), the method may further comprise (gl): removing particles and antigen-presenting cells from the expanded T cell sample.

[0148] The second phagocytic particle with one or more tumor neoantigen constructs tightly associated therewith can be the same as the phagocytic particle with one or more tumor neoantigen constructs tightly associated therewith of step (a), or can be different. Preferably, they are the same.

[0149] The second live antigen-presenting cells may be the same as or different from the live antigen-presenting cells in step (b), but are preferably the same.

[0150] Removal of particles

[0151] After step (e) (and where applicable, after steps (f) and / or (el)), the method of the present invention may further comprise: the steps of (g) removing particles from the T cell sample; and / or (h) removing particles and antigen-presenting cells from the T cell sample.

[0152] In embodiments of the present invention where the particle is paramagnetic or superparamagnetic, advantageously, for example, before the amplified T cell sample is returned to the subject, a magnet or magnetic field can be used to completely remove the particle and any antigen-presenting cells with internalized particles from the T cell sample. In this way, the T cell sample at the end of the inventive method only comprises the amplified T cells and does not have any antigen-presenting cells that are engulfed by the particles. Therefore, in some preferred embodiments of the present invention, the particle is paramagnetic or superparamagnetic, and step (g) comprises removing the particle from the T cell sample by means of a magnet or magnetic field. Removing the particle in step (g) also removes the antigen-presenting cells with internalized particles. Typically, when removing the particle (and the antigen-presenting cells with internalized particles) in step (g) or (h), the antigen-presenting cells with internalized particles will die.

[0153] treat

[0154] The methods of the present invention provide anti-tumor T cells. The anti-tumor T cells can be used to treat cancer in a subject. The present invention provides a method for treating cancer, wherein T cells obtained using the methods of the present invention are administered to a subject. The T cells are obtained after step (e) (or, where applicable, after steps (f) or (g)). The expanded T cells can be administered intravenously, intra-arterially, intrathecally, or intraperitoneally.

[0155] Cancer can be any form of solid cancer. Solid cancers according to the present invention are abnormal tissue masses that originate in an organ. Solid cancers typically do not contain cysts or areas of fluid. Solid cancers can be malignant. Different types of solid cancers are named for the type of cells that form them. Types of solid cancers include sarcomas, carcinomas, and lymphomas.

[0156] The present invention provides a composition comprising anti-tumor T cells produced by the methods of the present invention. For example, the composition can comprise CD4+ helper T cells and / or CD8+ T cells, such as a mixture of CD4+ helper T cells and CD8+ T cells. For example, in one embodiment, the mixture can primarily comprise CD4+ helper T cells and CD8+ T cells.

[0157] The present invention also provides a composition comprising anti-tumor T cells produced by the method of the present invention, for use in treating cancer in a subject.

[0158] The present invention also provides a method of treating cancer in a subject, comprising administering the anti-tumor T cells produced by the method of the present invention to the subject.

[0159] The present invention also provides a use of the anti-tumor T cells produced by the method of the present invention for treating cancer in a patient.

[0160] Examples of solid cancers include adrenal cancer, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, B-cell lymphoma, bile duct cancer, bladder cancer, brain / CNS tumors, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, intravascular large B-cell lymphoma, renal cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer (non-small cell and small cell), lung carcinoid tumor lymphoma Granulomatosis, malignant mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, lymph node marginal zone B cell lymphoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, primary effusion lymphoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (basal and squamous cell, melanoma and Merkel cell), small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia and Wilms' tumor. The T cells of the present invention are particularly effective in the treatment of solid cancers. Thus, the subject of the present invention can suffer from solid cancers. The T cells of the present invention are particularly effective in treating solid cancers selected from the group consisting of anal cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, liver cancer, lung cancer (non-small cell carcinoma and small cell), lung carcinoid tumors, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, gastric cancer, testicular cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and are particularly useful in treating breast cancer, colon cancer, liver cancer, lung cancer (non-small cell and small cell), lung carcinoid tumors, pancreatic cancer, prostate cancer, ovarian cancer, and bladder cancer.

[0161] The T cells of the present invention are also particularly effective in the treatment of metastatic solid cancers. Metastatic cancer is cancer that has spread from its original site to one or more different areas of the body.

[0162] Alternatively, the cancer can be any form of hematologic malignancy. Hematologic malignancy according to the present invention is a form of cancer that originates from cells of blood-forming tissues, such as the bone marrow or lymphatic system. In many hematologic malignancies, the developmental process of normal blood cells is interrupted by the uncontrolled growth of abnormal types of blood cells. Examples of hematologic cancers include leukemias, lymphomas, myelomas, and myelodysplastic syndromes (lymphomas can be divided into solid cancers and hematologic malignancies).

[0163] Examples of hematologic malignancies include acute basophilic leukemia, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid leukemia, acute myeloid dendritic cell leukemia, acute promyelocytic leukemia, adult T-cell leukemia / lymphoma, aggressive NK-cell leukemia, anaplastic large cell lymphoma, and plasmacytoma, angioimmunoblastic T-cell lymphoma, B-cell chronic lymphocytic leukemia, and B-cell leukemia. Lymphoma, B-cell prolymphocytic leukemia, chronic idiopathic myelofibrosis, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, chronic neutrophilic leukemia, extramedullary hairy cell leukemia, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, intravascular large B-cell lymphoma, Kahler's disease, lymphomatoid granulomatosis, mast cell leukemia, multiple myeloma, myelomatosis, nodal marginal zone B-cell lymphoma, non-Hodgkin's lymphoma, plasma cell leukemia, primary effusion lymphoma, and Waldenstrom's macroglobulinemia.

[0164] In a preferred embodiment, when the T cells obtained using the method of the present invention are used to treat cancer in a subject, or for a method of treating cancer in a subject, the subject is not administered any chemotherapy that reduces the number of immune cells in the body 1 week prior to administration of the T cells obtained using the method of the present invention (preferably within the previous 4 weeks).

[0165] An advantage of the present invention is that repeated treatment with anti-tumor T cells generated by the methods of the present invention is only necessary if the memory T cells that recognize the neoantigens are exhausted, or if the cancer evolves such that there are neoantigens on the tumor that are different from the original neoantigens targeted by the T cells. If this is the case, and the cancer becomes resistant to the initial T cell treatment, the treatment can be performed a second time by administering another composition comprising anti-tumor T cells generated by the methods of the present invention, wherein in step (a), the phagocytic particles have a different tumor neoantigen construct tightly associated therewith than the tumor neoantigen construct used to generate the T cells of the previous treatment. For example, the neoantigen construct has a different sequence, for example, comprises one or more different neoantigen epitopes than the phagocytic particle tightly associated tumor neoantigen construct used to prepare the first anti-tumor T cell composition in the previous method; or the neoantigen construct comprises all different neoantigen epitopes than the phagocytic particle tightly associated tumor neoantigen construct used to prepare the first anti-tumor T cell composition in the previous method. For example, neo-epitopes in the second treatment are epitopes that were not present in the first treatment, for example because they had not yet developed in the tumor at that time.

[0166] The present invention also provides a composition comprising anti-tumor T cells produced by the methods of the present invention for use in treating cancer in a subject, wherein after administering the composition comprising anti-tumor T cells to the subject, the subject is monitored for the presence of neoantigen-specific T cells (e.g., memory T cells) derived from the anti-tumor T cells produced by the methods of the present invention on day X after administration of the composition, wherein X is about 7, 14, 21, 28, 30, 35, 42, 49, 56, 60, 90, 120, 150, 180, 300 and / or 365 days; and optionally, additional anti-tumor T cells produced by the methods of the present invention are administered to the subject.

[0167] The present invention also provides a method of treating cancer in a subject, comprising administering anti-tumor T cells produced by the methods of the present invention to the subject, and further comprising monitoring the presence of neoantigen-specific T cells (e.g., memory T cells) derived from the anti-tumor T cells produced by the methods of the present invention on day X after administration of the composition, wherein X is about 7, 14, 21, 28, 30, 35, 42, 49, 56, 60, 90, 120, 150, 180, 300 and / or 365 days; and optionally administering additional anti-tumor T cells produced by the methods of the present invention.

[0168] The present invention also provides a composition comprising anti-tumor T cells produced by the method of the present invention for treating recurrent cancer in a subject who has previously been treated with a first anti-tumor T cell composition produced according to the present invention, wherein the composition comprising anti-tumor T cells is produced by the method of the present invention, wherein in step (a), the tumor neoantigen construct tightly associated with the phagocytic particles has a sequence different from the tumor neoantigen construct tightly associated with the phagocytic particles used in the method for preparing the first anti-tumor T cell composition produced according to the present invention. For example, in step (a), the tumor neoantigen construct tightly associated with the phagocytic particles comprises one or more different neoantigen epitopes than the tumor neoantigen construct tightly associated with the phagocytic particles used in the method for preparing the first anti-tumor T cell composition produced according to the present invention; and preferably, in step (a), the tumor neoantigen construct tightly associated with the phagocytic particles comprises all different neoantigen epitopes than the tumor neoantigen construct tightly associated with the phagocytic particles used in the method for preparing the first anti-tumor T cell composition produced according to the present invention.

[0169] The present invention also provides a method for treating recurrent cancer in a subject who has previously been treated with a first anti-tumor T cell composition produced according to the present invention, the method comprising administering to the subject anti-tumor T cells produced by the method of the present invention, wherein in step (a), the tumor neoantigen construct tightly associated with the phagocytic particles has a sequence different from the tumor neoantigen construct tightly associated with the phagocytic particles used in the method for preparing the first anti-tumor T cell composition produced according to the present invention. For example, in step (a), the tumor neoantigen construct tightly associated with the phagocytic particles comprises one or more neoantigen epitopes that are different from the tumor neoantigen construct tightly associated with the phagocytic particles used in the method for preparing the first anti-tumor T cell composition produced according to the present invention; and preferably, in step (a), the tumor neoantigen construct tightly associated with the phagocytic particles comprises all different neoantigen epitopes from the tumor neoantigen construct tightly associated with the phagocytic particles used in the method for preparing the first anti-tumor T cell composition produced according to the present invention.

[0170] The particles of the present invention:

[0171] The present invention also provides a paramagnetic or superparamagnetic phagocytable particle having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with a subject's cancer.

[0172] Preferably, the maximum dimension of the particles of the present invention may be less than 5.6 μm, preferably less than 4 μm, more preferably less than 3 μm, for example less than 2.5 μm, less than 2 μm or less than 1.5 μm. Preferably, the maximum dimension of the particles used in the method of the present invention may be greater than 0.001 μm, preferably greater than 0.005 μm, preferably greater than 0.01 μm, preferably greater than 0.05 μm, preferably greater than 0.1 μm, more preferably greater than 0.2 μm, even more preferably greater than 0.5 μm. Preferably, the maximum dimension of the particles of the present invention is in the range of 0.1-5.6 μm, preferably 0.2-5.6 μm, preferably 0.5-5.6 μm, preferably 0.1-4 μm, preferably 0.5-4 μm, more preferably 0.1-3 μm, more preferably 0.5-3 μm, even more preferably 0.1-2.5 μm, even more preferably 0.5-2.5 μm, even more preferably 0.2-2 μm, even more preferably 0.5-2 μm, more preferably 1-2 μm, or for example about 1 μm, about 1.5 μm or about 2 μm (preferably about 1 μm). Preferably, the particles are substantially spherical.

[0173] Preferably, the tumor neoantigen construct is covalently linked to the particle of the present invention. For example, the polypeptide can be linked via an amide bond between an amine group or carboxylic acid group of the polypeptide and a carboxylic acid group or an amine group on the particle surface.

[0174] Alternatively, the candidate antigenic polypeptide may be linked to the particle via a metal chelate.

[0175] For example, particles linked to metal chelating ligands such as iminodiacetic acid can bind metal ions such as Cu 2 + 、Zn 2+ , Ca 2+ 、Co 2+ or Fe 3+ These metal chelates can in turn bind with high strength to proteins and peptides containing, for example, histidine or cysteine. Therefore, particles with metal chelates can non-covalently adsorb peptides / proteins in a manner that allows rigorous washing to reduce the amount of LPS and other contaminating components in the bound peptides / proteins.

[0176] The particles according to the present invention may comprise a polymer, glass or ceramic material (e.g., the particles may be polymer particles, glass particles or ceramic particles). Preferably, the particles comprise a synthetic aromatic polymer, such as polystyrene, or another polymer, such as polyethylene. The particles may comprise a polysaccharide polymer, such as agarose. Preferably, the particles of the present invention do not comprise a polysaccharide polymer (and preferably are not polysaccharide polymer particles). Preferably, the particles of the present invention are polystyrene or polyethylene particles, and more preferably are polystyrene particles. Preferably, the particles of the present invention are monodisperse particles (i.e., particles of uniform size).

[0177] Kit of the present invention

[0178] The present invention also provides a kit comprising the paramagnetic or superparamagnetic phagocytic particles of the present invention (i.e., paramagnetic or superparamagnetic phagocytic particles having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with a subject's cancer) and a reagent suitable for expanding a T cell population. The kit may also contain reagents for assisting in the expansion of the T cell population, such as IL-2. It may also optionally contain IL-7 and / or IL-15. The kit may also contain reagents for sterilizing the particles of the present invention.

[0179] Preferably, the kit does not comprise anti-CD3 antibodies and / or anti-CD28 antibodies.

[0180] The present invention also provides a kit comprising paramagnetic or superparamagnetic phagocytosable particles, a coupling reagent for coupling the peptide to the phagocytosable particles, and a reagent suitable for expanding a T cell population.

[0181] The kit may also include instructions for designing tumor neoantigen constructs. The kit may also include reagents for generating tumor neoantigen constructs, such as ready-to-use vectors adjusted for different cloning and expression conditions. The kit may also or additionally optionally include one or more tumor neoantigen constructs, for example, it may include a library of tumor neoantigen constructs for one or more specific cancers. The kit may include one or more neoantigen epitopes. For example, it may include a library of neoantigen epitopes for one or more specific cancers. The kit may include instructions and / or reagents for generating tumor neoantigen constructs from neoantigen epitopes (and optionally one or more spacer portions).

[0182] The kit may also contain reagents for assisting in the expansion of T cell populations, such as IL-2. It may also optionally contain IL-7 and / or IL-15. The kit may also contain reagents for sterilizing the particles of the invention.

[0183] Preferably, the kit does not contain anti-CD3 antibodies and / or anti-CD28 antibodies. Preferably, the kit includes instructions for not adding anti-CD3 antibodies and / or anti-CD28 antibodies when using the kit, for example, when using the kit in the methods of the present invention. The kit may also include instructions for not isolating activated T cells from the T cell sample prior to T cell expansion.

[0184] Example

[0185] Example 1: Coupling of polypeptides to particles

[0186] The peptide is covalently coupled to paramagnetic particles containing free carboxylic acid groups. MyOne TM Carboxylic acid (ThermoFischer Scientific) (spheres with a diameter of 1 μm) was used as particles. The coupling step was performed according to the manufacturer's protocol (two-step method using NHS (N-hydroxysuccinimide) and EDC (ethylcarbodiimide)):

[0187] The particles were washed twice with MES buffer (25 mM MES (2-(N-morpholino)ethanesulfonic acid, pH 6). The carboxylic acid groups were then activated by adding 50 mg / ml NHS (N-hydroxysuccinimide) and 50 mg / ml EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide) in MES buffer to the particles and incubated at room temperature for 30 minutes. The particles were collected with a magnet, the supernatant removed, and washed twice with MES buffer. The protein was diluted to a concentration of 1 mg / ml in MES buffer for a total of 100 μg, added to the particles, and incubated at room temperature for 1 hour. The particles were collected with a magnet, the supernatant removed, and stored for protein concentration measurement. Unreacted activated carboxylic acid groups were quenched with 50 mM Tris pH 7.4 for 15 minutes. The particles were then washed with PBS, pH 7.4, and then stored at -80°C.

[0188] To measure the amount of protein coupled to the particles, a BCA protein assay kit (Pierce BCA protein assay kit, ThermoFisher Scientific) was used to measure the concentration of protein before protein coupling and in the supernatant after coupling. The BCA assay was used according to the manufacturer's protocol.

[0189] The protein concentration before and after coupling was measured using a bicinchoninic acid (BCA) protein assay to ensure successful coupling. Several peptides were tested, with an average of 48.7 μg coupled per 1 mg of particles (mean: 48.7, SD: 20.5, N=10). According to the manufacturer's instructions, 50 μg of peptide can be coupled per 1 mg of particles, indicating that the coupling efficiency achieved in this example is very high.

[0190] Example 2: Washing

[0191] The particles were coupled to recombinant proteins produced in E. coli. The particles were washed in sterile water at room temperature with one of three different wash buffers: 2M NaOH pH 14.3, 8M urea, or 6M guanidine (guanidine-HCl), or incubated in PBS at 95°C. The particles were suspended in the buffer and shaken for 4 minutes, collected with a magnet, and the supernatant removed. This was repeated three times. The heat-treated particles were placed in PBS at pH 7.4 and placed in a heating block at 95°C for 5 minutes, then collected with a magnet and the supernatant removed. This was repeated three times. The particles were then washed three times with sterile PBS to remove any remaining wash buffer.

[0192] Four different washing conditions were tested: (a) high pH (2M NaOH pH 14.3), (b) heat (95°C), and sterilization / denaturing agent ((c) 8M urea and (d) 6M guanidine hydrochloride). After washing, the polypeptide associated with the particles remained associated with the particles.

[0193] Example 3a: Identification of a Suitable Particle Size for Phagocytic Particles

[0194] A thymidine-incorporating proliferation assay was used to test the effect of particle size on antigen-specific T cell activation. Splenocytes from ovalbumin (OVA)-immunized mice were stimulated with OVA-coupled particles of varying sizes to measure antigen-specific proliferation.

[0195] According to the protocol in Example 1, paramagnetic particles having diameters of 5.6 μm, 1 μm, and 0.2 μm and having carboxylic acids on their surface were coupled to ovalbumin or bovine serum albumin.

[0196] To test the effectiveness of particle stimulation of antigen-specific T cell activation, a proliferation assay (incorporation of 3H thymidine) was used. The particle concentration relative to the cell concentration was 1:1 for 5.6 μm particles, 10:1 for 1 μm particles, and 500:1 for 0.2 μm particles. The total protein concentration during incubation with cells was calculated to be 125 ng / ml, 160 ng / ml, and 160 ng / ml for 5.6 μm, 1 μm, and 0.2 μm, respectively. The proliferation assay was run as follows:

[0197] Proliferation assays were performed using splenocytes from ovalbumin-sensitized mice after thymidine incorporation. MyOne TM Mice were immunized with ovalbumin (Sigma Aldrich) and BSA (Sigma Aldrich) coupled to a 5-hydroxy-2-hydroxy-2-oxo-1 ...

[0198] Cells were incubated with ovalbumin-coupled or BSA-coupled beads (10 beads per cell) in cRPMI for 5 days. All cells were incubated at 37°C in a humidified atmosphere of 6% CO2 for 6 days. During the last 18 hours of incubation, 1 3 Ci / well of [3H]thymidine was added to the cell cultures. The mean counts per minute (cpm) obtained from triplicate stimulations was divided by the mean cpm value of unstimulated cells to express the stimulation index (SI). An SI value of ≥2.0 was generally considered positive.

[0199] like Figure 1As seen in Figure 3, cells incubated with OVA particles with a diameter of 0.2 μm showed increased proliferation, with a mean SI of 4.1 (95% CI 2.4-5.8, P = 0.007). Cells incubated with OVA particles with a diameter of 1 μm showed increased proliferation, with a mean SI of 8.4 (95% CI 6.1-10.6, P < 0.005). Cells incubated with OVA particles with a diameter of 5.6 μm failed to stimulate proliferation, with a mean SI of 1.1 (95% CI 0.4-2.7, P = 0.876).

[0200] These results show that antigens coupled to particles of varying sizes can stimulate cell proliferation. Particles with a diameter of approximately 1 μm appear to be the most efficient in terms of cell stimulation, but particles as small as 0.2 μm are still effective. It is reasonable to predict that particles larger than 1 μm will also be effective, although particles will not stimulate cells at all as the diameter approaches 5.6 μm. It is reasonable to assume that 1 μm is the optimal size because it is similar to the size of bacteria. Our immune system has evolved to engulf and react to microorganisms / particles of this size. Normal antigen-presenting cells are in the range of 10-15 μm in size.

[0201] Example 3b: Comparison of Antigen-Conjugated Particles of Different Sizes and Their Effectiveness in Activating and Expanding T Cells

[0202] (i) Preparation of antigen-coupled phagocytic particles:

[0203] Three different sizes of paramagnetic polystyrene phagocytic particles were used:

[0204] - 1 μm in diameter (Dynabeads MyOne Carboxylic Acid, ThermoFisher),

[0205] - 2.8 μm in diameter (Dynabeads M-270 Carboxylic Acid, ThermoFisher), and

[0206] - 4.5 μm in diameter (Dynabeads M-450 epoxy resin, ThermoFisher).

[0207] According to the manufacturer's instructions, the phagocytic particles were coupled to the antigen (cytomegalovirus protein PP65). To remove endotoxin, all phagocytic particles were washed five times with 0.75 M sodium hydroxide buffer and then resuspended in sterile PBS.

[0208] (ii) Incubation of antigen-coupled phagocytosable particles:

[0209] Peripheral blood mononuclear cells (PBMCs) from healthy donors, isolated by standard Ficoll-based density gradient centrifugation, were cultured with phagocytic particles coupled to antigens (hereinafter referred to as "antigen-coupled particles") at a concentration of 500,000 cells / well in a 48-well plate at 37° C., 5% CO 2 for 18 hours. The concentration of antigen-coupled particles was equalized based on total surface area (a surrogate marker of the amount of antigen because it is bound to the surface of the antigen-coupled particles). Based on the number of total PBMCs in the sample, this was equivalent to 10 antigen-coupled particles / PBMC for 1 μm particles, 1.4 antigen-coupled particles / PBMC for 2.8 μm particles, and 0.5 antigen-coupled particles / PBMC for 4.5 μm particles.

[0210] Table 1

[0211]

[0212] (iii) Uptake Assessment:

[0213] After incubation, the number of phagocytosed antigen-coupled particles was manually counted using confocal microscopy. Eight cells were counted to obtain the mean and standard deviation values. Figure 5A Figure 2 shows a confocal microscopy image of a representative cell with intracellular phagocytic antigen-coupled particles. The black dashed line indicates the cell outline. The white line shows the size of the total intracellular antigen-coupled particles.

[0214] This method is not suitable for 1 μm antigen-coupled particles because they are too small to be accurately counted. To estimate the amount of 1 μm antigen-coupled particles, the total volume of all phagocytosed antigen-coupled particles was measured, and the amount of individual antigen-coupled particles was back-calculated based on the total volume assuming a packing density of 60%. This method proved to be quite accurate for 2.8 μm antigen-coupled particles (manual count of 9.1 antigen-coupled particles / cell vs. an estimate of 11.9 antigen-coupled particles / cell) and 4.5 μm antigen-coupled particles (manual count of 3.1 antigen-coupled particles / cell vs. an estimate of 2.4 antigen-coupled particles / cell), and therefore it can be assumed that the amount of 1 μm antigen-coupled particles is also accurately estimated.

[0215] Uptake of antigen-coupled particles Figure 5B and 5C shown. Figure 5BDemonstrate the quantity (each bead type counts 8 cells) of the antigen-coupled particles of each cell uptake by manual counting assessment.Use manual counting method, find that for 4.5 μ m antigen-coupled particles, the quantity of the engulfed antigen-coupled particles of each cell is 3.1 (± 1.1).For 2.8 μ m antigen-coupled particles, it is 9.1 (± 2.2).Use this method can not count the quantity of 1 μ m antigen-coupled particles.

[0216] Figure 5C The quantity (each bead type measures 3 cells) of the antigen-coupled particles that demonstrate each cell uptake by volumetric calculation assessment (*p<0.05, **p<0.01, ***p<0.001, use Students T to check and calculate). Use volume calculation method, find that for 4.5 μ m antigen-coupled particles, the quantity of the phagocytic antigen-coupled particles of each cell is 2.4 (± 1.1). For 2.8 μ m antigen-coupled particles, it is 11.9 (± 3.2). For 1 μ m antigen-coupled particles, it is 203.7 (± 21.9).

[0217] The total surface area of ​​phagocytosis was calculated based on the number of antigen-coupled particles taken up by each cell, as assessed by volumetric calculations, and the total amount of antigen was calculated by extension. For 1 μm antigen-coupled particles, the surface area taken up was calculated to be 639.6 (±68.9) μm 2 , calculated for 2.8 μm antigen-coupled particles to be 293.1 (± 79.3) μm 2 , calculated for 4.5 μm antigen-coupled particles is 150.7 (± 67.0) μm 2 These data are shown in Table 2 below.

[0218] Table 2:

[0219]

[0220] (iv) Assessment of T cell stimulation

[0221] The ability of antigen-coupled particles to stimulate T cells and thus promote their expansion was assessed by measuring the release of IFNγ, IL22, and IL17A from PBMC using the FluoroSpot assay (Mabtech, Sweden). PBMCs (250,000 / well) from CMV-sensitive healthy donors (n=2) were stimulated with triplicate antigen-coupled particles. As previously described, the concentration of antigen-coupled particles was equalized based on total surface area: 1 μm antigen-coupled particles / cell was 10, 2.8 μm antigen-coupled particles / cell was 1.4, and 4.5 μm antigen-coupled particles / cell was 0.5.

[0222] Table 3:

[0223]

[0224] The cells were incubated for 44 hours at 37°C with 5% CO2. The plates were developed according to the manufacturer's instructions and read in an automated FluoroSpot reader. FluoroSpot reports indicate that the number of spots is higher when cells are stimulated with antigen-coupled particles than when they are not.

[0225] The levels of IFNγ production as assessed in the FluoroSpot assay are shown in Figure 6A As can be seen, there is almost no difference between the antigen particles.

[0226] The levels of IL22 and IL17 production as assessed in the FluoroSpot assay are shown in Figure 6B and 6C As can be seen, the 1 μm antigen-coupled particles elicited significantly higher IL22 and IL17 production in one individual compared to the larger antigen-coupled particles, with similar trends observed for IL22 in the other individuals.

[0227] The levels of dual cytokine production as assessed in the FluoroSpot assay are shown in Figure 6D and 6E As can be seen, when stimulated with 1 μm antigen-coupled particles, 1 μm antigen-coupled particles elicited significantly higher dual cytokine release (IFNγ+IL17 and IL22γ+IL17) from one healthy donor compared to stimulation with larger antigen-coupled particles.

[0228] Cytokine release in these experiments can be used as a proxy for T cell expansion. Typically, IFNγ is produced by CD4+ T cells (Th1 subset) and CD8+ T cells. IL17 and IL22 are primarily produced by pro-inflammatory Th17 CD4+ T cells. Such pro-inflammatory cells have been shown to contribute to tumor elimination. The data show that 1 μm beads activate and induce expansion of Th1 CD4+ T cells and CD8+ T cells to the same extent as other beads, with the added benefit of also activating and inducing expansion of additional pro-inflammatory Th17 CD4+ T cells and less pronounced but still pro-inflammatory dual cytokine-producing T cells.

[0229] Example 4: Preliminary studies

[0230] Preliminary studies utilizing the methods of the present invention have been conducted:

[0231] (i) Identification of neoantigen targets in bladder cancer

[0232] Bladder cancer displays a high mutation rate and therefore expresses a large number of neoantigens that may be recognized as non-self by the immune system. Thus, the present inventors investigated tumor polymorphisms that are suitable as neoantigens and T cell targets by mining a mutation database containing a large reservoir of potential neoantigens that can be used to expand T cells for immunotherapy.

[0233] The COSMIC database [27899578] contains mutation data for 4754 transitional cell carcinomas, whole exome sequencing and hotspot analysis. The inventors focused on bladder cancer (UBC) and selected 15 of the most common mutations that resulted in single amino acid changes, thus qualifying as neoantigens. The inventors also focused on polymorphisms in genes known to be associated with tumor pathogenesis, such as kinases, growth factor receptors and cell cycle proteins. The 15 mutations selected alone covered 73% of bladder cancer mutations found in COSMIC. The new peptides were designed to the mutated amino acid, with ten flanking amino acids added to the C-terminus and N-terminus of the mutated amino acid, resulting in a 21-amino acid peptide. To design the new antigen construct, the three new antigen sequences were linked to two VVR spacers, because the VVR motif is removed by cathepsin S in the lysosome after translation as a step in human leukocyte antigen presentation.

[0234] Alternatively, whole genome sequencing of tumors from patients with UBC can be performed to identify additional polymorphisms and new targets for immunotherapy. RNA sequencing of tumors can be performed to verify the presence of transcripts carrying polymorphisms. Multiple reaction monitoring (MRM) mass spectrometry can be used to rapidly scan a patient's most common neoantigens for expression at the protein level, thereby tailoring neoantigens for individual immunotherapy.

[0235] (ii) Generation of recombinant neoantigens

[0236] Efficient protocols have been developed for recombinant protein production. Synthetic genes are designed, purchased, and subcloned into ready-to-use vectors tailored for diverse cloning and expression conditions. Neoantigens are expressed in E. coli, purified by Ni-IMAC chromatography, and, after testing for purity and endotoxin levels, prepared for efficient antigen presentation by coupling to Dynabeads. Using established methodologies and platforms, recombinant protein cloning and expression can be performed within weeks.

[0237] (iii) Activation and expansion of T cells by neoantigens

[0238] As described above, nine neoantigens were identified through bioinformatics. The designed neoantigens were based on genes containing reported bladder cancer-associated mutations, such as FGFR3 and p53. In preliminary experiments using constructs containing three neoantigens, the inventors were able to identify IFN-γ-producing T cells from the blood of patients with bladder cancer using FluoroSpot, confirming the effectiveness of the neoantigen approach.

[0239] For the same patient with bladder cancer, T cell activation was performed using predicted neoantigenic peptides NA1-9. Proliferation was observed in response to NA1, 3, 5, 7, and 8. Figure 2A Shown are the cell numbers in culture over time (PB = peripheral blood). Figure 2A The arrows in the figure indicate the time of restimulation (i.e., the time when the T cell sample is contacted with a second batch of antigen-presenting cells contacted with phagocytosable particles under conditions that allow specific activation of anti-tumor T cells in response to antigens presented by the antigen-presenting cells). Figure 2B % CD4+ / total T cells are shown. Figure 2C showing T-bet expression in CD4, Figure 2D Shows the expression of granzyme B and perforin in CD8+ T cells.

[0240] The expanded T cells express the transcription factor Tbet and high levels of the effector molecules perforin and granzyme B (GZB).

[0241] The methods of the present invention have also been used on cells from a patient with sporadic colon cancer from whom sequenced tumor data was available. The patient exhibited two polymorphisms in p53, one known and one novel. The patient also exhibited a mutation in PIK3CA. Based on the specific mutations in the patient's tumor data, tumor neoantigen constructs were designed, expressed, and purified. The neoantigen constructs were used to amplify the cells, resulting in a neoantigen-specific response. Peripheral blood mononuclear cells (PBMCs) were used for culture. Figure 3 Shown are the percentages between the total number of T cells (small squares) and CD4+ T cells (large squares) in T samples before and during expansion, as well as proliferating CD4+ cells (circles). Figure 4A Shown are the number of cells in culture over time (the red line shows personalized peptides, the pink and orange lines show predicted peptides for control cultures, which have the same protein but with the predicted mutations (NA1, 3, 4, and 5)). Figure 4B % CD4+ / total T cells are shown. Figure 4A and 4BThe arrows in the figure indicate the time of restimulation (i.e., the time when the T cell sample was exposed to a second batch of antigen-presenting cells that were exposed to phagocytic particles under conditions that allow the specific activation of anti-tumor T cells in response to antigen-presenting cells). Figure 4C visualizes the analysis of CD4 T cells using the Barnes-Hut Stochastic Neighbor Embedding (BH-SNE) algorithm, in which all cells in the sample are clustered on a 2D map based on the similarity of the expression intensity of a set of selected markers, here CD28, CD57, T-bet, GATA-3, perforin, granzyme B (GZB), Ki-67, and PD-1.

[0242] Expression of the proliferation marker Ki67 and T cell numbers increased during expansion, while expression of important markers of antitumor activity, such as Tbet, perforin, and granzyme B, increased in both CD4+ and CD8+ cells over the 14-day culture period. The percentage of CD8+ T cells decreased to approximately 10% of CD4+ T cells, but the total number of CD8+ T cells increased.

[0243] The entire process, from receiving sequence data to analyzing expanded cells, can be completed within 4-5 weeks.

[0244] These results suggest the presence of a tailored Th1 neoantigen response.Thus, the inventors have demonstrated that predicted and sequence-verified neoantigens can be designed and used for T cell activation and expansion.

Claims

1. A method for expanding anti-tumor T cells from a cancer subject, comprising the following steps: a) providing phagocytic particles having a maximum dimension of 0.5-2 μm, said particles being paramagnetic and having one or more tumor neoantigen constructs tightly associated therewith, wherein said tumor neoantigen constructs comprise an amino acid sequence comprising at least one mutant amino acid known or suspected to be associated with said cancer of said subject, or a mutant amino acid sequence known or suspected to be expressed in cancer cells of said subject; b) providing live antigen-presenting cells; c) contacting the particles with the antigen-presenting cells in vitro under conditions that allow the antigen-presenting cells to phagocytose the particles; d) providing a T cell sample comprising live T cells from the subject; e) contacting the T cell sample with the antigen-presenting cells contacted with the particles in vitro under conditions that allow specific activation and expansion of anti-tumor T cells in response to antigens presented by the antigen-presenting cells; wherein the antigen presenting cell is a phagocyte; wherein, in each of steps a) to e), no anti-CD3 antibody and / or anti-CD28 antibody is added in each step; and The anti-tumor T cells expanded in the method are CD4+ helper T cells and CD8+ T cells.

2. The method according to claim 1, wherein the particles having the associated polypeptide construct of step (a) have been subjected to sterilization washing to produce sterile particles having the associated polypeptide construct.

3. The method according to claim 1, wherein the particles having the associated polypeptide construct in step (a) have been sterilized and washed to produce pathogen-free particles having the associated polypeptide construct.

4. The method according to claim 1, further comprising: (f) removing the particles from the T cell sample; and / or (g) removing the particles and antigen-presenting cells from the T cell sample.

5. The method of any one of the preceding claims, wherein the antigen-presenting cells are from the subject.

6. The method of claim 1, wherein the tumor neoantigen construct comprises one or more covalently linked peptides, wherein one or more of the covalently linked peptides comprises an amino acid sequence containing at least one mutated amino acid known or suspected to be associated with a cancer in the subject.

7. The method of claim 6, wherein the tumor neoantigen construct comprises one or more covalently linked peptides, wherein one or more of the covalently linked peptides comprises an amino acid sequence corresponding to a mutant amino acid sequence known or suspected to be expressed in a cancer cell of the subject.

8. The method of claim 6 or 7, wherein the tumor neoantigen construct comprises three or more covalently linked peptides.

9. The method of claim 6 or 7, wherein the tumor neoantigen construct comprises four or five covalently linked peptides.

10. The method according to claim 6 or 7, wherein each peptide of the tumor neoantigen construct comprises 10 to 25 amino acids.

11. The method of claim 1, wherein two or more tumor neoantigen constructs are tightly associated with the particle, and each tumor neoantigen construct may have the same polypeptide sequence or may have a different polypeptide sequence.

12. The method of claim 1, wherein the subject is a mammal.

13. The method of claim 1, wherein the subject is a human. The method of claim 1 , wherein the T cell sample comprises CD4+ helper T cells and / or CD8+ T cells.

15. The method of claim 1, wherein the T cell sample is derived from a tumor.

16. The method of claim 1, wherein the T cell sample is derived from lymphatic vessels in a tumor.

17. The method of claim 1, wherein the antigen-presenting cells are dendritic cells.

18. The method of claim 1, wherein the cancer is a solid cancer.

19. The method of claim 1, wherein the maximum dimension of the particles is 1 μm.

20. The method of claim 19, wherein the particles are spherical.

21. The method of claim 2, wherein the sterilization wash comprises subjecting the particles with associated polypeptides to a pH of at least pH 13; or wherein the sterilization wash comprises subjecting the particles with associated polypeptides to a pH of less than pH 2.

22. The method of claim 2, wherein the sterilization wash comprises subjecting the particles with associated polypeptides to a temperature of at least 90°C.

23. The method of claim 2 or 3, wherein the sterilization wash is a denaturing wash.

24. The method of claim 23, wherein the denaturing wash comprises subjecting the particles with associated polypeptides to a denaturing agent at a concentration of at least 5M.

25. The method of claim 23, wherein the denaturing wash comprises subjecting the particles with associated polypeptides to a denaturant at a concentration of at least 6M.

26. The method of claim 23, wherein the denaturing wash comprises subjecting the particles with associated polypeptides to a denaturant at a concentration of at least 7M.

27. The method of claim 23, wherein the denaturing wash comprises subjecting the particles with associated polypeptides to a denaturant at a concentration of at least 8M.

28. The method according to any one of claims 23 to 27, wherein the denaturing agent is urea or guanidine hydrochloride.

29. The method of claim 1, wherein the particles are polymeric particles.

30. The method of claim 29, wherein the particles are polystyrene particles.

31. The method of claim 1, wherein one or more tumor neoantigen constructs are covalently linked to the particle.

32. The method of claim 1, wherein one or more tumor neoantigen constructs are linked to the particle via a metal chelate.

33. The method of claim 1, wherein the antigen-presenting cells and the T cell sample are derived from the same subject.

34. The method of claim 1, wherein the antigen presenting cells and the T cell sample are derived from a PBMC sample of the same subject.

35. The method of claim 2, wherein the washed phagocytosable particles, the live antigen-presenting cells, and the T cell sample are contacted simultaneously.

36. The method of claim 1, wherein step (e) further comprises the steps of: The degree of anti-tumor T cell activation in the T cell sample is determined by comparing the degree of anti-tumor T cell activation with a relevant reference, whereby a higher degree of anti-tumor T cell activation in the sample compared to the reference indicates the conclusion that one or more tumor neoantigen constructs cause anti-tumor T cell activation in the sample.

37. The method of claim 36, wherein determining the extent of anti-tumor T cell activation in the T cell sample comprises determining the fraction of activated T cells in the sample.

38. The method of claim 36 or 37, wherein the extent of anti-tumor T cell activation in the T cell sample is determined using ELISpot or FluoroSpot technology.

39. The method of claim 1, further comprising, immediately after step (e): a1) providing a second phagocytic particle having one or more tumor neoantigen constructs tightly associated therewith, wherein the tumor neoantigen construct comprises an amino acid sequence containing at least one mutant amino acid known or suspected to be associated with a subject's cancer, or a mutant amino acid sequence known or suspected to be expressed in cancer cells of the subject; b1) providing a second live antigen-presenting cell; c1) contacting the second phagocytosable particle with the second live antigen-presenting cell in vitro under conditions that allow the second live antigen-presenting cell to phagocytose the second phagocytosable particle; and e1) contacting the T cell sample with the second live antigen-presenting cells contacted in vitro with the second phagocytosable particles under conditions that allow specific activation of anti-tumor T cells in response to antigens presented by the second live antigen-presenting cells.

40. The method of claim 39, wherein the second phagocytic particles having one or more tumor neoantigen constructs tightly associated therewith of step (a1) are the same as the phagocytic particles having one or more tumor neoantigen constructs tightly associated therewith of step (a); and / or the second live antigen-presenting cells of step (b1) are the same as the live antigen-presenting cells of step (b).

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