Phagocytic particles for treating or preventing cancer

Abstract

The present invention provides phagocytic particles for treating or preventing cancer in a subject, wherein the phagocytic particle comprises a core and a neoantigen construct tightly associated with the core, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence corresponding to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by a cancer cell in the subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid. The present invention also relates to an injectable pharmaceutical composition for treating or preventing cancer.

Description

Technical Field

[0001] The present invention relates to phagocytic particles comprising a new antigen construct tightly bound to the nucleus, which are used for treating or preventing cancer. The present invention also relates to an injectable pharmaceutical composition for treating or preventing cancer. Background Art

[0002] There are various approaches to modulating a subject's immune system to treat cancer; this approach is often referred to as "immunotherapy." Examples of immunotherapy include immune checkpoint inhibitors, adoptive cell transfer (ACT) therapy, and cancer vaccines.

[0003] There has been considerable success in treating cancer using immune checkpoint inhibitors, such as monoclonal antibodies that target checkpoints that interact with cells and are important for immune activation. Immune checkpoint inhibitors have been used to treat various cancers, such as melanoma, lung cancer, bladder cancer, and gastrointestinal cancer.

[0004] Adoptive cell transfer (ACT) has also achieved some success. 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 is the isolation and in vitro expansion of autologous tumor-reactive lymphocytes isolated from the first lymph node that naturally drains the tumor (sentinel lymph node) during surgery. Lymphocytes obtained from the sentinel lymph node are collected, activated, amplified against autologous tumor extracts, and then returned to the patient via blood transfusion. No toxic 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.

[0005] A significant limitation of ACT is the need to prepare sufficient amounts of anti-cancer T cells, such as tumor-infiltrating lymphocytes (TILs), to administer to a subject. For example, current methods often require the use of invasive surgery to remove one or more cancer cells from a subject to obtain anti-cancer T cells. In addition, due to the immunosuppressive mechanisms of cancer, the cells obtained are few and often unresponsive (anergic). This can lead to slow in vitro expansion, which in turn means that it takes sufficient time to obtain a sufficient number of anti-cancer T cells for treatment.

[0006] To overcome some of the limitations of ACT, genetically engineered T cells have been developed. These cells can be genetically redirected to target a patient's cancer by introducing antigen receptors or synthetic recognition structures called chimeric antigen receptors into the T cells. While genetically engineered T cells have been successful in treating hematologic cancers, the safety and selectivity of genetically engineered T cells for solid cancers still need improvement.

[0007] Another approach to immune checkpoint inhibitors and ACT is to administer cancer antigens to subjects to elicit an anti-cancer immune response. Compositions that elicit anti-cancer immune responses are often referred to as "cancer vaccines." Cancer vaccines often contain cancer antigens, such as tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs). TAAs are abnormally expressed by cancer cells. For example, TAAs can be proteins or peptides that can be expressed by both normal cells and cancer cells, but can be expressed by cancer cells at significantly higher levels. TSAs are antigens expressed by cancer cells rather than normal cells. A specific example of a tumor-specific antigen is a new antigen. New antigens are mutant proteins or peptides expressed by cancer cells rather than normal cells that can bind to immune system molecules, such as antibodies or T cell receptors (TCRs) of T cells. New antigen regions that contain one or more cancer-specific amino acid mutations and are known or suspected of directly binding to immune system molecules are often referred to as new epitopes. Therapeutic agents or vaccines targeting TSAs (such as new antigens) are expected to provide more effective and safer cancer treatment methods.

[0008] Despite the interest in immunotherapies for treating cancer, their success to date has been limited due to ineffective regulation or induction of anti-cancer immune responses and challenges associated with the safety and selectivity of immunotherapies.

[0009] Therefore, there remains a need for improved immunotherapies for cancer treatment that can elicit potent and targeted anti-cancer immune responses while also being suitable for use in clinical settings. Summary of the Invention

[0010] The present invention provides a phagocytic particle for treating or preventing cancer in a subject, wherein the phagocytic particle comprises a core and a neo-antigen construct tightly associated with the core, and wherein the neo-antigen construct comprises a neo-epitope peptide having an amino acid sequence that corresponds to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by a cancer cell in the subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid.

[0011] The present invention also provides a method for treating or preventing cancer, the method comprising the following steps: administering to a subject a phagocytic particle, wherein the phagocytic particle comprises a core and a neoantigen construct tightly associated with the core, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence corresponding to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by cancer cells in the subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid.

[0012] The present invention also provides the use of phagocytic particles in the preparation of a medicament for treating or preventing cancer, wherein the phagocytic particle comprises a core and a neoantigen construct tightly associated with the core, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence corresponding to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by cancer cells in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid.

[0013] The present invention also provides an injectable pharmaceutical composition comprising a phagocytosable particle, wherein the phagocytosable particle comprises a core and a neoantigen construct tightly associated with the core, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence that corresponds to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by a cancer cell in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid.

[0014] The present invention further provides a phagocytosable particle of the present invention for treating or preventing cancer in a subject, or an injectable pharmaceutical composition of the present invention for treating or preventing cancer in a subject, or a method of the present invention for treating or preventing cancer in a subject, wherein the treating or preventing cancer further comprises the following steps:

[0015] One or more subsequent doses of the phagocytosable particles or injectable pharmaceutical composition are administered to a subject to which the subject has previously been administered a dose of the phagocytosable particles or injectable pharmaceutical composition sufficient to elicit an immune response against cancer cells in the subject.

[0016] The present invention further provides a phagocytosable particle of the present invention for treating or preventing cancer in a subject, or an injectable pharmaceutical composition of the present invention for treating or preventing cancer in a subject, or a method of the present invention for treating or preventing cancer in a subject, wherein the treating or preventing cancer further comprises the following steps:

[0017] (a) after administering the phagocytosable particles to the subject, harvesting APCs and anti-cancer T cells from the subject;

[0018] (b) expanding anti-cancer T cells obtained from the subject; and

[0019] (c) administering a therapeutic dose of the expanded anti-cancer T cells to the subject.

[0020] The inventors have found that after administration to a subject, the phagocytic particles described herein are internalized by antigen presenting cells (APCs). The bound neoantigen constructs are then presented on the surface of the APCs and cause activation and amplification of anti-cancer T cells in the subject. The use of phagocytic particles results in surprisingly high uptake of neoantigen constructs, and subsequently presents a variety of new epitopes on the surface of the APCs. The inventors have shown in a mouse model that phagocytic particles containing two types of neoantigen constructs, injected into the inguinal lymph nodes or subcutaneously, resulted in a dose-dependent increase in anti-neo-epitope antibodies in serum samples obtained from mice. The same mice were subsequently injected with melanoma cancer cells (B16F10). Advantageously, the inventors found that administering phagocytic particles to mice before the injection of cancer cells produced a dose-dependent preventive effect on tumor growth in mice. Therefore, the inventors found that by administering phagocytic particles as described herein to a subject, a strong anti-cancer immune response can be induced in the subject, and the phagocytic particles of the present invention can be successfully used as a preventive vaccination for cancer to reduce cancer growth.

[0021] In addition, the inventors have also shown in a mouse xenograft model of colorectal cancer that administration of a composition of phagocytic particles containing six neoantigen constructs before and after colon cancer cell (MC-38 cell line) transplantation resulted in a robust anti-cancer immune response that inhibited tumor growth in mice. Thus, the inventors have demonstrated the therapeutic and preventive potential of the phagocytic particles of the present invention in two mouse models of cancer.

[0022] The present inventors have also found that the core of the phagocytic particles (e.g., polymeric particles) described herein acts as a very effective carrier of the neoantigen construct. Without wishing to be bound by any particular theory, it is believed that the phagocytic particles defined herein are particularly effective because the entire phagocytic particle, including the core and the tightly bound neoantigen construct, is internalized into a phagosome by the APC through phagocytosis. The neoantigen construct is then cut from the core of the particle and processed in the phagosome. Fragments of the neoantigen construct are then presented on the surface of the APC via the class II major histocompatibility (MHC) pathway and on the cell surface via class II MHC molecules. It is also believed that this is not the only way to present the neoepitope on the surface of the APC, and some fragments of the neoantigen construct can also be presented on the surface of the APC via the class I major histocompatibility (MHC) pathway and on the cell surface via class I MHC molecules in a process known as cross-presentation. Therefore, although fragments of the neoantigen construct are expected to be presented on the APC primarily via the class II MHC pathway, some are expected to be presented via the class I MHC pathway, and therefore the present invention utilizes these two pathways to varying degrees.

[0023] When antigens are presented by class II MHC molecules, they typically activate helper T cells (also known as CD4+ T cells), which coordinate the immune response primarily by secreting cytokines, inducing class switching of B cells to assist B cells in producing antibodies and stimulating the activation and expansion of other T cell types, particularly cytotoxic T cells (e.g., CD8+ T cells) and memory T cells (e.g., CD8+ memory T cells). In addition, CD4+ T cells can directly kill other cell types (Borst et al., Nat Rev Immunol, 2018, 18(10), 635-647). This means that by administering phagocytic particles as defined herein to a subject, multiple types of immune cells can be activated, which results in a slow-onset anti-cancer immune response (and therefore few side effects), has a long-lasting effect, and can target cancer in a variety of different ways by utilizing the entire immune system (rather than activating only CD8+ T cells that can only directly attack tumor cells). This contrasts with what is expected to happen when an antigen (e.g., a neoantigen) is provided as a free peptide or a nucleotide construct expressing the peptide. This antigen is expected to be taken up into the cytoplasm of APCs, resulting in the neoantigen being presented on the cell surface exclusively by class I MHC molecules via the class I MHC pathway. This, in turn, primarily leads to the activation of CD8+ T cells. Furthermore, after the induction of an anti-cancer immune response, memory T cells derived from anti-cancer T helper cells remain in circulation and can quickly and effectively mount a secondary immune response as long as the cancer cells expressing the neo-epitope remain in the body or if the same cancer recurs.

[0024] The present inventors have also discovered that the phagocytic particles described herein can be effectively purified and sterilized to remove contaminants, such as pathogens (e.g., bacteria, fungi, and viruses), endotoxins, and other antigenic contaminants from the phagocytic particles prior to administration to a subject. This is particularly advantageous because removing contaminants from the phagocytic particles described herein reduces the nonspecific immune response of the subject after administration and thus improves the safety and efficacy of the phagocytic particles.

[0025] The inventors also appreciate that the phagocytosable particles are well tolerated in subjects following administration, in part due to the inert nature of the core and the high sterility of the phagocytosable particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of phagocytic particle size on T cell activation is shown. A proliferation assay (thymidine incorporation) was used to assess the number of splenocytes obtained from ovalbumin-sensitized mice. A comparison of ovalbumin coupled to phagocytic particles of varying sizes, 5.6 μm, 1 μm, and 0.2 μm in diameter, is shown. P values ​​were determined using a Student's T-test, and are indicated where p < 0.05 is found. Staples represent SD.

[0027] Figure 2 Stimulation of T cell expansion by the neo-epitope NA1-9 is shown. Figure 2 A shows the number of cells in culture over time.

[0028] Figure 2 B shows % CD4+ / total T cells. Figure 2 C shows T-bet expression in CD4+ T cells. Figure 2 D shows the expression of granzyme B and perforin in CD8+ T cells.

[0029] Figure 3 Expansion of T cells by stimulation with neoantigen constructs is shown. The percentage (small squares) and total number (large squares) of CD4+ T cells in T cells as well as proliferating CD4+ cells (circles) are shown.

[0030] Figure 4 Expansion of anti-cancer T cells by stimulation with neoantigen constructs was shown. Figure 4 A shows the cell number in PBMC culture over time (days). PBMC culture contains a variety of cell types, including APCs and T cells. Figure 4 The top row of A (Pat2 personalized NA) shows the TM carboxylic acid ) and the number of cells in PBMC culture after incubation with phagocytic particles of a personalized neoantigen construct (SEQ ID NO: 3) tightly bound to the nucleus. Figure 4 The bottom two rows of A (Pat2 NA 1+3 and Pat 2NA 4+5) show the results of the experiments with a polystyrene core (MyOne TM carboxylic acid ) in two separate PBMC cultures after incubation with phagocytosable particles of predicted neoantigen constructs tightly associated with the nucleus. Figure 4 B shows % CD4+ / total T cells. Figure 4 C visualizes the analysis of CD4+ T cells using the Barnes-Hut Stochastic Neighbor Embedding (BH-SNE) algorithm, in which all cells in a sample are clustered on a 2D plot based on the similarity of their expression intensities according to a set of selected markers: CD28, CD57, T-bet, GATA-3, perforin, granzyme B (GZB), Ki-67, and PD-1.

[0031] Figure 5 A shows a confocal microscopy image of PBMCs with intracellular phagocytosed particles. After incubation of PBMCs with phagocytosed particles at 37° C. for 18 h, three sizes of phagocytosed particles (4.5 μm, 2.8 μm, or 1 μm) are shown.

[0032] Figure 5 B shows the cellular uptake of phagocytable particles of two sizes (4.5 μm or 2.8 μm) assessed by manual counting after incubation with PBMCs for 18 h at 37°C.

[0033] Figure 5 C shows the cellular uptake of phagocytable particles of three sizes (4.5 μm, 2.8 μm or 1 μm), assessed by volume calculation (*p<0.05**p<0.01***p<0.001, calculated using Student's T-test) after 18 h incubation at 37°C in PBMCs.

[0034] Figure 6 A shows the relative increase in IFNγ production levels in PBMCs from CMV-susceptible healthy donors (n=2) stimulated with phagocytic particles of three sizes (4.5 μm, 2.8 μm or 1 μm) compared to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0035] Figure 6 B shows the relative increase in IL22 production levels in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with phagocytic particles of three sizes (4.5 μm, 2.8 μm or 1 μm) compared to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0036] Figure 6 C shows the relative increase in IL17 production levels in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with phagocytic particles of three sizes (4.5 μm, 2.8 μm or 1 μm) compared to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0037] Figure 6 D shows the relative increase in dual cytokine production levels of IFNγ and IL-17 in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with phagocytic particles of three sizes (4.5 μm, 2.8 μm, or 1 μm) compared to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0038] Figure 6 E shows the relative increase in dual cytokine production levels of IL22 and IL17 in PBMCs of a CMV-susceptible healthy donor (n=1) stimulated with phagocytic particles of three sizes (4.5 μm, 2.8 μm, or 1 μm) compared to unstimulated cells, as assessed in the FluoroSpot assay of Example 3a(iv).

[0039] Figure 7A、 7B Figures 7C and 7D show the proportion of anti-neoepitope antibodies in serum samples obtained from mice after low or high doses of two phagocytic particles were administered to the inguinal lymph nodes or subcutaneously (n=3 for each dose and each route of administration). The two phagocytic particles were: 1) polystyrene particles tightly bound to the neoantigen construct M272120 (SEQ ID NO: 1); and 2) polystyrene particles tightly bound to the neoantigen construct M304748 (SEQ ID NO: 2). Figure 7A and 7B shows the expression of anti-neoepitope antibodies to the neoantigen construct M272120 (SEQ ID NO: 1) in sera harvested from mice 22 days after the first dose of phagocytosable particles. Figure 7A ) or the neoantigen construct M304748 (SEQ ID NO: 2) ( Figure 7B ) ratio; and Figure 7C and 7D Shown are anti-neoepitope antibodies to the neoantigen construct M272120 (SEQ ID NO: 1) in serum harvested from mice 23 days after a second dose of phagocytosable particles approximately one month after the first dose of phagocytosable particles ( Figure 7C ) or the neoantigen construct M304748 (SEQ ID NO: 2) ( Figure 7D As a control, serum samples obtained from naive mice (n=3, not given any dose of phagocytic particles) were also analyzed. Mice that received one or two high-dose doses of phagocytic particles (via inguinal lymph node or subcutaneous administration) had higher rates of anti-neoepidermal antibodies in their serum compared to mice that received one or two low-dose doses of phagocytic particles by the same route, as well as mice that did not receive a dose of phagocytic particles (i.e., naive mice).

[0040] Figure 8 Figure 2 shows the change in tumor volume over time (days, D) after the melanoma cancer cell line B16F10 was injected into mice that had previously been given two doses of phagocytic particles (polystyrene particles tightly bound to M272120 and polystyrene particles tightly bound to M304748). Figure 2 shows the change in tumor volume over time in mice (n=3) that had previously been given the following doses of phagocytic particles: two low doses of phagocytic particles injected into the inguinal lymph nodes (squares, ■); two high doses of phagocytic particles injected into the inguinal lymph nodes (triangles, ); two low-dose phagocytic particles (triangle, ▲); two high-dose phagocytic particles (diamond, ◆) were injected subcutaneously. Administration of phagocytic particles showed a dose-dependent preventive effect on tumor volume.

[0041] Figure 9 Figure 2 shows the tumor volume of mice (n=5) after administration of the first and second doses of a composition of phagocytic particles comprising six different groups of phagocytic particles, each group comprising a core coupled to a different MC38 neoantigen construct (SEQ ID NOs. 13-18). The first dose was administered at day -5 (time point A) and the second dose was administered at day 13 (time point C). On day 0 (time point B), MC38 tumor cells were implanted in mice. Tumor progression was compared to an unvaccinated group (negative control group, n=5). The difference in tumor volume at the end of the experiment was calculated by Student's t-test. ***p<0.001. DETAILED DESCRIPTION

[0042] Neoantigen constructs and neoepitope peptides

[0043] The phagocytosable particles used in the present invention comprise a neoantigen construct tightly bound to the nucleus. The neoantigen construct of the present invention comprises a neoepitope peptide.

[0044] The new epitope peptide used in the present invention is a new epitope peptide having an amino acid sequence that corresponds to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by a cancer cell in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid. The "somatically mutated amino acid" of the new epitope peptide is an amino acid that is different or absent in the portion of the protein or peptide corresponding to the amino acid sequence of the new epitope peptide when the portion of the protein or peptide is expressed by a non-cancerous cell (e.g., a somatic cell). For example, the "somatically mutated amino acid" of the new epitope peptide can be a deletion (i.e., an amino acid that has been deleted), an addition (i.e., an amino acid that has been added), or a substitution (i.e., an amino acid that has been replaced by a different amino acid). Such somatically mutated amino acids can also be referred to as "cancer-specific somatically mutated amino acids" because the somatically mutated amino acids are present in cancer cells but not in normal cells (e.g., somatic cells). Preferably, the somatically mutated amino acids of the new epitope peptide are substitutions (i.e., one or more amino acids have been substituted by a different amino acid).

[0045] Mutated somatic amino acids in proteins or peptides expressed by cells can occur due to unfaithful DNA replication that occurs in each cell division that produces nucleotide substitutions, deletions, or insertions into the cell's DNA. Nucleotide substitutions can result in different encoded amino acids compared to the amino acids encoded by the somatic non-mutated nucleic acid sequence, thereby producing different amino acids in the protein / peptide compared to proteins / peptides in normal non-cancerous cells (e.g., somatic cells). Nucleotide insertions and / or deletions can result in reading frame errors (i.e., "frameshift mutations"), resulting in new amino acid sequences at the protein level (i.e., nucleotide insertions or deletions change the reading frame of the DNA compared to normal cells (e.g., somatic cells), thereby changing most or all of the amino acids encoded by the DNA after the mutation). Additionally or alternatively, insertions and / or deletions can result in the introduction of stop codons, thereby resulting in truncated proteins at the protein level. Nucleotide substitutions can individually change (one or more) codons and result in amino acid substitutions and / or the introduction of stop codons at the protein level, thereby resulting in truncated proteins at the protein level.

[0046] Neo-epitope peptides for use in the present invention are peptides having an amino acid sequence that corresponds to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by cancer cells in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid (e.g., 1, 2, 3, 4, or 5 or more somatically mutated amino acids).

[0047] A mutated protein or peptide known or suspected to be expressed by cancer cells in a subject may also be referred to as a “cancer-specific mutated protein or peptide.” This is because the mutated protein or peptide is known or suspected to be expressed in cancer cells but not in normal cells (eg, somatic cells).

[0048] A variety of techniques can be used to identify cancer-specific mutated proteins or peptides, as well as the amino acid sequences to which the neo-epitope peptide amino acid sequences can correspond. For example, cancer-specific mutant proteins or peptides and their amino acid sequences, including the mutated amino acids of their cancer-specific somatic cells, can be identified from publicly available protein databases, such as the COSMIC database (Forbes et al. Nucleic Acids Res, 45 (D1), D777-D783, which can be accessed at http: / / cancer.sanger.ac.uk / cosmic). The amino acid sequences of somatic mutations identified from the COSMIC database or similar databases are referred to herein as “predicted neo-epitope peptides”. A neoantigen construct consisting of one or more “predicted neo-epitope peptides” is referred to herein as a “predicted neoantigen construct”.

[0049] In an alternative or additional method for identifying cancer-specific mutated proteins or peptides and amino acid sequences to which the amino acid sequences of new epitope peptides may correspond, the genome, exome transcriptome and / or proteome of cancer cells obtained from a cancer in a subject can be established, and mutations in the cancer cells can be inferred therefrom. For example, this can be accomplished by comparing proteome, genome, exome or transcriptome derived data with a reference nucleotide sequence or amino acid sequence. Suitable reference sequences can be obtained from the genome, exome or transcriptome of non-cancerous cells (e.g., somatic cells) obtained from a subject, or from publicly available nucleotide or protein databases, such as ( https: / / www.uniprot.org / ) and EBI Expression Atlas ( https: / / www.ebi.ac.uk / gxa / home ), which provides information about proteins and polypeptides expressed in tissues and cancer cell lines. In an alternative or additional method, the amino acid sequence to which the cancer-specific mutant protein or peptide and its neo-epitope peptide amino acid sequence may correspond may be previously identified by analyzing the genome, exome, transcriptome and / or proteome of the subject's tumor. Suitable techniques for sequencing the genome, exome or transcriptome of cancer cells or normal cells are known in the art and include, for example, Sanger sequencing and next-generation sequencing. Suitable techniques for obtaining proteomic data include multiple reaction monitoring (MRM) mass spectrometry. The amino acid sequence of somatic mutations identified from the genome, exome, transcriptome or proteomic data obtained from the subject is referred to herein as a "personalized neo-epitope peptide". A neoantigen construct composed of one or more "personalized neo-epitope peptides" is referred to herein as a "personalized neoantigen construct".

[0050] The new epitope peptides used in the present invention have one or more somatically mutated amino acids. For example, it may have one somatically mutated amino acid, or more than one somatically mutated amino acid, that is, two to all amino acids in this part of the protein or peptide may be mutated. For example, the new epitope peptides of the present invention may have 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) somatically mutated amino acids, more preferably 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) mutated amino acids; or, for example, 1 to 6 (e.g., 1, 2, 3, 4, 5 or 6) mutated amino acids; or, for example, 1 to 5 (e.g., 1, 2, 3, 4 or 5) somatically mutated amino acids; or, for example, 1 to 4 (e.g., 1, 2, 3 or 4) somatically mutated amino acids. In a preferred embodiment of the present invention, the new epitope peptides of the present invention have 1, 2 or 3 somatically mutated amino acids. In a preferred embodiment, the new epitope peptides of the present invention have 1 or 2 somatically mutated amino acids. Even more preferably, the neo-epitope peptides of the present invention have one somatically mutated amino acid.

[0051] In a preferred embodiment, the neoepitope peptides of the present invention have most or all somatically mutated amino acids. Even more preferably, the neoepitope peptides of the present invention have all somatically mutated amino acids. Such neoepitope peptides having most or all somatically mutated amino acids may correspond to a portion of a protein or peptide resulting from a frameshift mutation in cellular DNA.

[0052] One or more amino acid mutations of the new epitope peptides of the present invention can be located at any amino acid position within the amino acid sequence of the new epitope peptide. In a preferred embodiment, at least one somatically mutated amino acid (or the one somatically mutated amino acid in the embodiment where there is only one somatically mutated amino acid in the new epitope peptide) is located in the central portion of the new epitope peptide. For example, when the length of the new epitope peptide amino acid sequence is at least 3 amino acids (e.g., a length of at least 5 amino acids or a length of at least 7 amino acids), the central portion of the new epitope peptide is the central 1 amino acid of the sequence when the new epitope peptide has an odd number of amino acids in its sequence; or the central 2 amino acids when the new epitope peptide has an even number of amino acids in its sequence. For example, when the length of the new epitope peptide amino acid sequence is at least 9 amino acids, the central portion of the new epitope peptide is the central 3 amino acids of the sequence when the new epitope peptide has an odd number of amino acids in its sequence (preferably 1 central amino acid); or the central 4 amino acids when the new epitope peptide has an even number of amino acids in its sequence (preferably 2 central amino acids). For example, when the length of the neoepitope peptide amino acid sequence is at least 11 amino acids, the central portion of the neoepitope peptide is the central 5 amino acids of the sequence (preferably 1 central amino acid) when the neoepitope peptide has an odd number of amino acids in its sequence; or the central 6 amino acids when the neoepitope peptide has an even number of amino acids in its sequence. More preferably, when the length of the neoepitope peptide amino acid sequence is at least 11 amino acids, the central portion of the neoepitope peptide is the central 3 amino acids of the sequence (preferably 1 central amino acid) when the neoepitope peptide has an odd number of amino acids in its sequence; or the central 4 amino acids (preferably 2 central amino acids) when the neoepitope peptide has an even number of amino acids in its sequence.

[0053] In a preferred embodiment, at least one somatically mutated amino acid of the neo-epitope peptide (or one somatically mutated amino acid in the embodiment where only one somatically mutated amino acid is present in the neo-epitope peptide) is located at a central position of the neo-epitope peptide when the neo-epitope peptide has an odd number of amino acids in its sequence, or at one of the two most central positions of the amino acid sequence when the neo-epitope peptide has an even number of amino acids in its sequence.

[0054] In certain embodiments of the present invention, most or all of the amino acids in the neo-epitope peptide are somatically mutated amino acids. In such embodiments, somatically mutated amino acids in a protein or peptide expressed by a cancer cell may occur due to an error in the reading frame of the encoding DNA (i.e., due to a frameshift mutation), resulting in all or most of the amino acids in a portion of the protein or peptide being different from that portion of the protein or peptide expressed in a normal non-cancerous cell.

[0055] The neo-epitope peptides of the present invention can have an amino acid sequence of 3 to 200 amino acids in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175 or 200 amino acids in length). Preferably, the new epitope peptide of the present invention may have an amino acid sequence of 3 to 50 amino acids in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids in length), more preferably 3 to 30 amino acids in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length), more preferably 3 to 25 amino acids in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length), more preferably 5 to 25 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length), more preferably 8 to 25 amino acids in length. In a preferred embodiment, the neoepitope peptides of the present invention are 3 to 25 amino acids in length, 5 to 25 amino acids in length, 10 to 25 amino acids in length, 11 to 25 amino acids in length, 12 to 25 amino acids in length, 13 to 25 amino acids in length, 15 to 25 amino acids in length, 17 to 25 amino acids in length, 19 to 25 amino acids in length, 20 to 25 amino acids in length or 21 to 25 amino acids in length. In another preferred embodiment, the length of the neoepitope peptide of the present invention is 3 to 23 amino acids, 5 to 23 amino acids, 10 to 23 amino acids, 11 to 23 amino acids, 12 to 23 amino acids, 13 to 23 amino acids, 15 to 23 amino acids, 17 to 23 amino acids, 19 to 23 amino acids, 20 to 23 amino acids or 21 to 23 amino acids.In another preferred embodiment, the length of the neoepitope peptide of the present invention is 3 to 21 amino acids, 5 to 21 amino acids, 10 to 21 amino acids, 11 to 21 amino acids, 12 to 21 amino acids, 13 to 21 amino acids, 15 to 21 amino acids, 17 to 21 amino acids or 19 to 21 amino acids. In another preferred embodiment, the length of the neoepitope peptide of the present invention is 3 to 19 amino acids, 5 to 19 amino acids, 10 to 19 amino acids, 11 to 19 amino acids, 12 to 21 amino acids, 13 to 21 amino acids, 15 to 21 amino acids or 17 to 19 amino acids. In another preferred embodiment, the length of the neoepitope peptide of the present invention is 3 to 17 amino acids, 5 to 17 amino acids, 10 to 17 amino acids, 11 to 17 amino acids, 12 to 17 amino acids, 13 to 17 amino acids or 15 to 17 amino acids. In another preferred embodiment, the length of the neoepitope peptides of the present invention is 3 to 15 amino acids, 3 to 15 amino acids, 10 to 15 amino acids, 11 to 15 amino acids, 12 to 15 amino acids or 13 to 15 amino acids. In another preferred embodiment, the length of the neoepitope peptides of the present invention is 3 to 19 amino acids, 5 to 17 amino acids, 5 to 15 amino acids, 5 to 13 amino acids or 5 to 10 amino acids. In another preferred embodiment, the length of the neoepitope peptides of the present invention is 3 to 19 amino acids, 5 to 17 amino acids, 3 to 15 amino acids, 3 to 10 amino acids or 5 to 10 amino acids. In another preferred embodiment, the length of the neoepitope peptides of the present invention is 3 to 19 amino acids, 3 to 17 amino acids, 3 to 13 amino acids, 3 to 10 amino acids or 3 to 7 amino acids.

[0056] In a preferred embodiment of the present invention, the neo-epitope peptide is 10 to 25 amino acids, 10 to 23 amino acids, 10 to 21 amino acids, 10 to 19 amino acids, 10 to 17 amino acids, 10 to 15 amino acids and comprises 1, 2, 3, 4 or 5 or all somatically mutated amino acids. Preferably, the neo-epitope peptide is 10 to 25 amino acids, 10 to 23 amino acids, 10 to 21 amino acids, 10 to 19 amino acids, 10 to 17 amino acids, 10 to 15 amino acids in length and comprises 1, 2 or 3 somatically or all mutated amino acids. More preferably, the neo-epitope peptide is 10 to 25 amino acids, 10 to 23 amino acids, 10 to 21 amino acids, 10 to 19 amino acids, 10 to 17 amino acids, 10 to 15 amino acids in length and comprises 1 or 2 somatically or all mutated amino acids. Even more preferably, the neo-epitope peptide is 10 to 25 amino acids, 10 to 23 amino acids, 10 to 21 amino acids, 10 to 19 amino acids, 10 to 17 amino acids, 10 to 15 amino acids in length and comprises one or all somatically mutated amino acids. Even more preferably, the neo-epitope peptide is 10 to 25 amino acids, 10 to 23 amino acids, 10 to 21 amino acids, 10 to 19 amino acids, 10 to 17 amino acids, 10 to 15 amino acids in length and comprises one somatically mutated amino acid.

[0057] In another preferred embodiment of the present invention, the length of the neoepitope peptide is 3 to 25 amino acids, 3 to 17 amino acids, 3 to 15 amino acids, 3 to 10 amino acids or 5 to 10 amino acids, and comprises 1, 2, 3, 4 or 5 or all somatic mutations of amino acids. Preferably, the length of the neoepitope peptide is 3 to 25 amino acids, 3 to 17 amino acids, 3 to 15 amino acids, 3 to 10 amino acids or 5 to 10 amino acids, and comprises 1, 2 or 3 or all somatic mutations of amino acids. More preferably, the length of the neoepitope peptide is 3 to 25 amino acids, 3 to 17 amino acids, 3 to 15 amino acids, 3 to 10 amino acids or 5 to 10 amino acids, and comprises 1 or 2 or all somatic mutations of amino acids. Even more preferably, the length of the neoepitope peptide is 3 to 25 amino acids, 3 to 17 amino acids, 3 to 15 amino acids, 3 to 10 amino acids or 5 to 10 amino acids, and comprises one or all somatic mutations of amino acids. Even more preferably, the neo-epitope peptide is 3 to 25 amino acids, 3 to 17 amino acids, 3 to 15 amino acids, 3 to 10 amino acids or 5 to 10 amino acids in length and comprises one or all of the somatically mutated amino acids.

[0058] The present inventors have advantageously discovered that the neo-epitopes of the present invention, particularly those having an amino acid sequence corresponding to a portion of a cancer-specific protein or peptide of 3 to 25 amino acids in length and comprising one or more somatically mutated amino acids, are particularly effective in eliciting an anti-cancer immune response in a subject without eliciting an autoimmune or non-cancer specific immune response in the subject.

[0059] The neoantigen construct can comprise one neoepitope peptide, or can comprise more than one neoepitope peptide. For example, the neoantigen construct can comprise 1 to 50 neoepitope peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 neoepitope peptides). Preferably, the neoantigen construct comprises 1 to 20 neoepitope peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 neoepitope peptides), more preferably 1 to 15 neoepitope peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 neoepitope peptides), more preferably 1 to 10 neoepitope peptides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), more preferably 1 to 8 neoepitope peptides (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), more preferably 1 to 6 neoepitope peptides (e.g., 1, 2, 3, 4, 5 or 6), even more preferably 1 to 5 neoepitope peptides (e.g., 1, 2, 3, 4 or 5). It is particularly preferred that the neoantigen construct comprises 1 to 5 neoepitope peptides (e.g. 1, 2, 3 or 4), even more preferably 3 to 5 neoepitope peptides, such as 3, 4 or 5 neoepitope peptides.

[0060] In one embodiment of the present invention, the neoantigen construct comprises two or more neoepitope peptides. For example, the neoantigen construct can comprise 2 to 50 neoepitope peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 neoepitope peptides). Preferably, the neoantigen construct may comprise 2 to 20 neoepitope peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 neoepitope peptides), more preferably the neoantigen construct may comprise 2 to 15 neoepitope peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 neoepitope peptides), more preferably 2 to 10 neoepitope peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), more preferably 2 to 8 neoepitope peptides (e.g., 2, 3, 4, 5, 6, 7 or 8), more preferably 2 to 6 neoepitope peptides (e.g., 2, 3, 4, 5 or 6), even more preferably 2 to 5 neoepitope peptides (e.g., 2, 3, 4 or 5). Particularly preferably, the neoantigen construct comprises 2 to 5 neoepitope peptides (e.g. 2, 3, 4 or 5), even more preferably 3 to 5 neoepitope peptides, e.g. 3, 4 or 5 neoepitope peptides.

[0061] In another embodiment of the present invention, the neoantigen construct comprises three or more neoepitope peptides. For example, the neoantigen construct can comprise 3 to 50 neoepitope peptides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 neoepitope peptides). Preferably, the neoantigen construct may comprise 3 to 20 neoepitope peptides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 neoepitope peptides), more preferably, the neoantigen construct may comprise 3 to 15 neoepitope peptides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 neoepitope peptides), more preferably 3 to 10 neoepitope peptides (e.g., 3, 4, 5, 6, 7, 8, 9 or 10), more preferably 3 to 8 neoepitope peptides (e.g., 3, 4, 5, 6, 7 or 8), more preferably 3 to 6 neoepitope peptides (e.g., 3, 4, 5 or 6), even more preferably 3 to 5 neoepitope peptides (e.g., 3, 4 or 5). It is particularly preferred that the neoantigen construct comprises 3 to 5 neoepitope peptides (eg 3, 4 or 5), even more preferably 5 neoepitope peptides.

[0062] In another embodiment of the present invention, the neoantigen construct comprises four or more neoepitope peptides (e.g., 4 neoepitope peptides), five or more neoepitope peptides (e.g., 5 neoepitope peptides), or six or more neoepitope peptides (e.g., 6 neoepitope peptides).

[0063] For the avoidance of doubt, in embodiments where a neo-epitope construct may comprise more than one neo-epitope peptide (e.g., one or more neo-epitope peptides, two or more neo-epitope peptides, or three or more neo-epitope peptides), each neo-epitope peptide is a neo-epitope peptide as described herein for use in the present invention (i.e., the amino acid sequence of each neo-epitope peptide of the neo-epitope construct corresponds to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by a cancer cell in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid). In such embodiments, each neo-epitope peptide may independently possess any of the properties and / or characteristics of the neo-epitope peptides described herein.

[0064] In embodiments where the neo-epitope construct may comprise more than one neo-epitope peptide (e.g., one or more neo-epitope peptides, two or more neo-epitope peptides, or three or more neo-epitope peptides), each neo-epitope peptide may have the same amino acid sequence, or the neo-epitope peptides may have different amino acid sequences (i.e., some of the neo-epitope peptides of the neo-antigen construct may have different amino acid sequences, or all of the neo-epitope peptides of the neo-antigen construct may have different amino acid sequences).

[0065] In certain preferred embodiments where the neoantigen construct comprises more than one neoepitope peptide, some or all of the neoepitope peptides have different amino acid sequences, more preferably, all of the neoepitope peptides have different amino acid sequences. In another embodiment where the neoantigen construct comprises more than one neoepitope peptide, each neoepitope peptide has the same amino acid sequence.

[0066] In embodiments where the neo-antigen construct may comprise more than one neo-epitope peptide (e.g., one or more neo-epitope peptides, two or more neo-epitope peptides, or three or more neo-epitope peptides) and some of the neo-epitope peptides have different amino acid sequences or all of the neo-epitope peptides have different amino acid sequences, the amino acid sequences may differ because:

[0067] The protein or peptide expressed by the cancer cell is known or suspected to be different in the subject; or

[0068] The protein or peptide known or suspected to be expressed by cancer cells in the subject is identical, but the portion corresponding to the amino acid sequence of the neoepitope peptide in the protein or peptide known or suspected to be expressed by cancer cells in the subject is different. If the protein or peptide known or suspected to be expressed by cancer cells in the subject is identical, but the portion corresponding to the amino acid sequence of the neoepitope peptide in the protein or peptide known or suspected to be expressed by cancer cells in the subject is different, the portion may be different due to one or more of the following reasons:

[0069] a longer portion having at least one somatically mutated amino acid in common,

[0070] a shorter portion having at least one somatically mutated amino acid in common, and / or

[0071] a portion having the same at least one somatically mutated amino acid, but with different positions of the mutated amino acid relative to the C-terminus and the N-terminus;

[0072] Different portions of the same protein or peptide having at least one different somatically mutated amino acid (e.g., the protein or peptide has a frameshift mutation, and the different portions are different portions of the frameshift mutated sequence of the protein or peptide).

[0073] In a preferred embodiment, the amino acid sequences are different because the protein or peptide known or suspected to be expressed by cancer cells in the subject is different for each neo-epitope peptide.

[0074] In a preferred embodiment, the amino acid sequences are different because the protein or peptide known or suspected to be expressed by the cancer cells in the subject is the same, but the corresponding portions of the amino acid sequence of the neo-epitope peptide in the protein or peptide known or suspected to be expressed by the cancer cells in the subject are different because they are each a different portion of the frameshift sequence of the protein or peptide. In embodiments of the present invention where the neo-antigen construct comprises more than one neo-epitope peptide (e.g., two or more neo-epitope peptides or three or more neo-epitope peptides), the neo-epitope peptides can be linked directly or through a spacer portion.

[0075] In certain preferred embodiments where the neoantigen construct comprises more than one neoepitope peptide (e.g., one or more neoepitope peptides, two or more neoepitope peptides, or three or more neoepitope peptides), the neoepitope peptides are covalently linked.

[0076] Neoantigen constructs of the present invention comprising more than one neoepitope peptide (e.g., two or more neoepitope peptides or three or more neoepitope peptides) may comprise directly linked neoepitope peptides and / or neoepitopes linked via spacer moieties. If more than one spacer moiety is present in the neoantigen construct, the spacer moieties of the neoantigen construct may all be the same or they may be different.

[0077] In certain preferred embodiments where the neoantigen construct comprises more than one neoepitope peptide (e.g., one or more neoepitope peptides, two or more neoepitope peptides, or three or more neoepitope peptides), the neoepitope peptides are each linked by a spacer moiety.

[0078] The spacer portion can be a short sequence of amino acids, for example, 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). The spacer portion can comprise a random combination of amino acids; or a synthetic amino acid sequence, for example, a polylysine, polyarginine, polyglycine, polyalanine, or polyhistidine amino acid sequence. Preferably, the spacer portion comprises the motif VVR and / or GGS.

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

[0080] -[the first neo-epitope peptide]-[spacer portion]-[the second neo-epitope peptide].

[0081] The structure of a neoantigen construct comprising three linked neo-epitopes can be as follows:

[0082] -[the first neo-epitope peptide]-[spacer portion]-[the second neo-epitope peptide]-[spacer portion]-[the third neo-epitope peptide].

[0083] The structure of a neoantigen construct comprising four linked neo-epitopes can be as follows:

[0084] -[the first neo-epitope peptide]-[spacer portion]-[the second neo-epitope peptide]-[spacer portion]-[the third neo-epitope peptide]-[spacer portion]-[the fourth neo-epitope peptide].

[0085] The structure of a neoantigen construct comprising five linked neo-epitopes can be as follows:

[0086] -[the first neo-epitope peptide]-[spacer portion]-[the second neo-epitope peptide]-[spacer portion]-[the third neo-epitope peptide]-[spacer portion]-[the fourth neo-epitope peptide]-[spacer portion]-[the fifth neo-epitope peptide].

[0087] The structure of a neoantigen construct comprising n linked neoepitope peptides can be as follows:

[0088] -[the first neo-epitope peptide]-[spacer portion]-[the second neo-epitope peptide]-[spacer portion]-[the third neo-epitope peptide]…–[spacer portion]–[the nth neo-epitope peptide].

[0089] When the neoantigen construct comprises more than one neoepitope peptide, the neoantigen construct may be composed of the neoepitope peptide and any optional spacer portion. Alternatively, when the neoantigen construct comprises more than one neoepitope peptide, the neoantigen construct may comprise the neoepitope peptide, any optional spacer portion and an additional amino acid sequence. This additional amino acid sequence may, for example, be a random combination of amino acids (particularly a random sequence with a high proportion of histidine and / or cysteine ​​residues); or a synthetic amino acid sequence, such as polylysine, polyarginine, polyglycine, polyalanine, polyhistidine or polycysteine ​​amino acid sequence (and preferably polyhistidine or polycysteine). Such additional amino acid sequences can be used as, for example, a linker and / or spacer between the core of a phagocytic particle and the neoepitope peptide of a neoantigen construct tightly bound thereto, or for tightly binding the neoantigen construct to phagocytic particles. For example, metal chelates can bind to proteins and peptides containing histidine or cysteine ​​with high strength. Thus, the core with the metal chelate can be non-covalently bound to a neoantigen construct comprising a polyhistidine or polycysteine ​​synthetic amino acid sequence and / or a random combination of amino acids with a high ratio of histidine and / or cysteine ​​residues as a further amino acid sequence. The neoantigen constructs described herein may also comprise an albumin binding domain (ABD).

[0090] In an embodiment of the present invention, when the neoantigen construct comprises a neoepitope peptide, the neoantigen construct may be composed of a neoepitope peptide. Alternatively, when the neoantigen construct comprises a neoepitope peptide, the neoantigen construct may comprise a neoepitope peptide and another amino acid sequence. This additional amino acid sequence may, for example, be a random combination of amino acids (particularly a random sequence with a high proportion of histidine and / or cysteine ​​residues); or a synthetic amino acid sequence, such as a polylysine, polyarginine, polyglycine, polyalanine, polyhistidine or polycysteine ​​amino acid sequence (and preferably polyhistidine or polycysteine). Such additional amino acid sequences can be used as, for example, a linker and / or spacer between the core of a phagocytic particle and the neoepitope peptide of a neoantigen construct tightly bound thereto, or for tightly binding the neoantigen construct to phagocytic particles. For example, metal chelates can bind to proteins and peptides containing histidine or cysteine ​​with high strength. Thus, the core with the metal chelate can be non-covalently bound to a neoantigen construct comprising a polyhistidine or polycysteine ​​synthetic amino acid sequence and / or a random combination of amino acids with a high ratio of histidine and / or cysteine ​​residues as a further amino acid sequence.

[0091] The length of the neoantigen construct used in the present invention depends on, for example, the number of neoepitope peptides in the neoantigen construct, the length of each neoepitope peptide in the neoantigen construct, the length of any spacer moiety that may be present if more than one neoepitope peptide is present, and the length of any other amino acid sequence that may be present. In certain preferred embodiments, the length of the amino acid sequence of the neoantigen construct of the present invention may be 3 to 300 amino acids, preferably 10 to 250 amino acids, more preferably 10 to 200 amino acids, and even more preferably 10 to 180 amino acids. For example, the length of the neoantigen constructs of the present invention can comprise 11 to 150 amino acids, 11 to 140 amino acids, 33 to 120 amino acids, 42 to 140 amino acids, 11 to 112 amino acids, 33 to 112 amino acids, 42 to 112 amino acids, 11 to 100 amino acids, 33 to 100 amino acids, 42 to 100 amino acids, 11 to 84 amino acids, 33 to 84 amino acids, 42 to 84 amino acids, 11 to 60 amino acids, 33 to 60 amino acids, or 42 to 60 amino acids, 11 to 50 amino acids, 33 to 50 amino acids, or 42 to 50 amino acids.

[0092] The new antigen constructs used in the present invention can be recombinantly prepared (e.g., in E. coli, mammalian cells or insect cells), synthetically prepared (e.g., using standard organic chemistry techniques, such as solution or solid phase peptide synthesis), or they can be prepared from polypeptides isolated from natural proteins or peptides derived from animal sources, such as human sources. Preferably, the new antigen constructs used in the present invention are recombinantly prepared (e.g., in E. coli, mammalian cells or insect cells). More preferably, the new antigen constructs used in the present invention are recombinantly prepared in E. coli.

[0093] Phagocytic particles and phagocytic particle cores

[0094] The phagocytic particles of the present invention are particles that can be engulfed by cells of the immune system. However, it should be understood that the phagocytic particles of the present invention can be internalized by cells of the immune system through different pathways (e.g., pinocytosis, clathrin-mediated endocytosis, and non-clathrin-mediated endocytosis). Preferably, the phagocytic particles can be engulfed by APCs, such as monocytes, dendritic cells, B cells, or macrophages, or other cells that engulf or endocytose extracellular molecules (e.g., antigens), and present antigen-derived peptides on class II MHC and / or class I MHC molecules to CD4+ T cells and / or CD8+ T cells. Antigens internalized into APCs via the engulfment pathway are degraded in an uneven manner, which subsequently results in APCs presenting a wider variety of antigen-derived peptides. Without wishing to be bound by theory, it is believed that the phagocytic particles used in the present invention further improve the activation and amplification of anti-cancer T cells because the neoantigen constructs are engulfed by APCs, which subsequently results in a wider variety of neoantigen construct-derived peptides being presented by APCs, and thus activating and expanding anti-cancer T cells to a greater extent.

[0095] For a particle to be phagocytosed by cells of the immune system, such as APCs, the particle needs to be within a size range suitable for phagocytosis. For example, particles that are too small may not trigger phagocytosis by a particular APC, or particles that are too large may not be phagocytosed by a particular APC. Complete phagocytosis results in good antigen degradation by the APC and subsequent good presentation to T cells via the class II MHC pathway. The present inventors have investigated the optimal size (see Examples 3a and 3b, and Figure 1 、 5 AC and 6A-E).

[0096] The phagocytic particles of the present invention comprise a core and a neoantigen construct tightly bound to the core. Therefore, the size of the core must be within a range such that when the core is tightly bound to the neoantigen construct, the core and the tightly bound neoantigen construct can be phagocytosed by immune system cells (particularly APCs). Preferably, the size of the core is within a range such that when the core is tightly bound to the neoantigen construct, the core and the tightly bound neoantigen construct are small enough that more than one phagocytic particle can enter the same APC by phagocytosis. Having more than one phagocytosed particle in an APC can maximize the presentation of new epitopes on the cell surface through the class II MHC pathway. In addition, it allows particles with different neoantigen constructs (particularly neoantigen constructs comprising different neoepitope peptides) to enter the APC, which means that the APC can present different neoepitopes from several particles in different phagosomes at the same time.

[0097] Thus, in a preferred embodiment, the maximum dimension of the core is less than 6 μm, less than 5.6 μm, less than 4 μm, less than 3 μm, less than 2.5 μm, less than 2 μm, or less than 1.5 μm. More preferably, the maximum dimension of the core is less than 1.5 μm. In another preferred embodiment, the maximum dimension of the core may be greater than 0.001 μm, greater than 0.005 μm, greater than 0.01 μm, greater than 0.05 μm, greater than 0.1 μm, greater than 0.2 μm, or greater than 0.5 μm. More preferably, the maximum dimension of the core is greater than 0.5 μm.

[0098] In a particularly preferred embodiment, the maximum dimension of the core is in the range of 0.1 to 6 μm, for example, 0.1 to 5.6 μm, 0.2 to 5.6 μm, 0.5 to 5.6 μm, 0.1 to 4 μm, or 0.5 to 4 μm. More preferably, the maximum dimension of the core is in the range of 0.1 to 3 μm, for example, 0.5 to 3 μm, 0.2 to 2.5 μm, 0.5 to 2.5 μm, 0.2 to 2 μm, 0.5 to 2 μm, or 1 to 2 μm. Even more preferably, the maximum dimension of the core is about 1 μm, about 1.5 μm, or about 2 μm. In a very preferred embodiment of the present invention, the core is about 1 μm.

[0099] The core of the phagocytic particle of the present invention takes the form of any three-dimensional shape, such as any regular or irregular three-dimensional shape. Preferably, the phagocytic particle is substantially spherical, in which case the size of the phagocytic particle refers to the diameter.

[0100] The core of the phagocytosable particle can include a polymer, glass, or ceramic material (e.g., the core can be a polymer particle, a glass particle, or a ceramic particle). The core material can be a biodegradable and / or biocompatible material (e.g., the particle can be a biodegradable and / or biocompatible particle).

[0101] Preferably, the core includes polymer (for example, core is polymer particles). If the core includes polymer, it can be selected from synthetic aromatic polymers (for example polystyrene, for example, core is polystyrene particles), synthetic non-aromatic polymers (for example polyethylene, polylactic acid, lactic acid / glycolic acid copolymer and polycaprolactone, for example, core is polyethylene particles, polylactic acid particles, lactic acid / glycolic acid copolymer particles or polycaprolactone particles), naturally occurring polymers (for example collagen, gelatin, protein (for example virus-like particles), lipid or albumin, for example, core is collagen particles, gelatin particles or albumin particles), polymeric carbohydrate molecules (for example polysaccharides, for example, agarose, alginate, chitosan or zymosan, for example, core is agarose particles, alginate particles, chitosan particles or zymosan particles).

[0102] In a preferred embodiment, the core comprises polystyrene or polyethylene, and more preferably comprises polystyrene (eg, the core is a polystyrene particle).Such polymers are biocompatible.

[0103] The present inventors have discovered that polystyrene particles are particularly useful cores for the engulfable particles of the present invention because they are non-toxic and widely commercially available in a variety of sizes and functional forms. Furthermore, the present inventors have discovered that engulfable particles comprising a polystyrene core, such as polystyrene particles, can withstand rigorous sterilization procedures to prepare the particles for administration to a subject. Such sterilization procedures may include repeated washing with acid or alkaline solutions and / or exposure to high temperatures.

[0104] In a very preferred embodiment of the present invention, the core is a polystyrene particle having a maximum dimension of less than 6 μm, preferably about 1 μm to about 3 μm, and more preferably about 1 μm. Phagocytic particles comprising a polystyrene particle core having a size of about 1 μm to about 3 μm, especially about 1 μm, are efficiently phagocytosed by APCs and are also able to withstand rigorous sterilization procedures to remove pathogens (e.g., bacteria, fungi, and viruses) and antigenic contaminants such as pyrogens (e.g., endotoxins) that may bind to the core or neoantigen construct.

[0105] In one embodiment of the present invention, the core has magnetic properties. For example, the core may have paramagnetism or superparamagnetism. Preferably, the core has superparamagnetism.

[0106] An example of a superparamagnetic core suitable for use in the present invention is Dynabeads TM (Invitrogen). Dynabeads TM Available in various functional forms, such as Dynabeads M-270 Carboxylic Acid, Dynabeads M-270 Amine, and Dynabeads MyOne Carboxylic Acid. TMIt is a single-sized superparamagnetic particle composed of highly cross-linked polystyrene and a uniform distribution of magnetic material. The magnetic material can be iron oxide. Other examples of magnetic cores, especially superparamagnetic cores, include encapsulated carboxylated Superparamagnetic microspheres (Merck Chimie SAS) and Sera-MagSpeedBeads (hydrophilic) carboxylate-modified magnetic particles (GE Healthcare UK Limited). The superparamagnetic microspheres are made of a core-shell structure that encapsulates an iron oxide core.

[0107] The phagocytic particles of the present invention comprise a neoantigen construct that is tightly bound to the core. A variety of means can be used to tightly bind the neoantigen construct to the core. For example, the neoantigen construct can be attached to the core via a covalent bond, such as an amide bond between an amine group or carboxylic acid group of the neoantigen construct and a carboxylic acid group or an amine group on the surface of the core. Alternatively, the neoantigen construct can be attached to the core via a metal chelate. For example, a core attached 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. Thus, the core with the metal chelate can be non-covalently bound to the neoantigen construct. Preferably, the neoantigen construct is covalently attached to the core. An example of binding a neoantigen construct to a core is shown in Example 1.

[0108] The phagocytic particles of the present invention comprise a core and a neoantigen construct tightly bound to the core. The phagocytic particles of the present invention may comprise one or more neoantigen constructs bound to the core. For example, the phagocytic particles of the present invention may comprise 1 to 3 million neoantigen constructs, preferably 1 to 2 million neoantigen constructs, more preferably 1 to 1 million neoantigen constructs, such as 1 to 800,000, 1 to 500,000, 1 to 100,000, 1 to 10,000, 1 to 1000, 1 to 100 or 1 to 10 neoantigen constructs; or for example 10 to 1 million, 100 to 1 million, 1000 to 1 million, 10,000 to 1 million, 100,000 to 1 million or 500,000 to 1 million. Preferably, the phagocytic particles of the present invention may comprise 500,000 to 1 million neoantigen constructs.

[0109] In preferred embodiments, to maximize delivery of the neoantigen construct to the APC (which can then be cleaved from the phagocytic particle and processed by the APC, thereby resulting in the presentation of a variety of neo-epitope-derived peptides on the surface of the APC), the phagocytic particles of the invention may comprise more than one (i.e., two or more, e.g., two to three million) neoantigen constructs associated with the core. For example, the phagocytic particles of the invention may comprise 2 to 1 million neoantigen constructs tightly associated with the core (e.g., 2 to 800,000, 2 to 500,000, 2 to 100,000, 2 to 10,000, 2 to 1000, 2 to 100, or 2 to 10 neoantigen constructs tightly associated with the core). Preferably, the phagocytic particles of the present invention comprise 10 or more neoantigen constructs tightly bound to the nucleus, for example, 10 to 1 million neoantigen constructs tightly bound to the nucleus (e.g., 10 to 800,000, 10 to 500,000, 10 to 100,000, 10 to 10,000, 10 to 1000, or 10 to 100 neoantigen constructs tightly bound to the nucleus). More preferably, the phagocytic particles of the present invention comprise 100 or more neoantigen constructs tightly bound to the nucleus, for example, 100 to 1 million neoantigen constructs tightly bound to the nucleus (e.g., 100 to 800,000, 100 to 500,000, 100 to 100,000, 100 to 10,000, or 100 to 1000 neoantigen constructs tightly bound to the nucleus). In certain embodiments, the phagocytic particles of the invention comprise 1000 or more gene constructs tightly bound to the nucleus, for example, 1000 to 1 million neoantigen constructs tightly bound to the nucleus (e.g., 1000 to 800,000, 1000 to 500,000, 1000 to 100,000, or 1000 to 10,000 neoantigen constructs tightly bound to the nucleus). In certain embodiments, the phagocytic particles of the invention comprise 10,000 or more neoantigen constructs tightly bound to the nucleus, for example, 10,000 to 1 million neoantigen constructs tightly bound to the nucleus (e.g., 10,000 to 800,000, 10,000 to 500,000, or 10,000 to 100,000 neoantigen constructs tightly bound to the nucleus). In certain embodiments, the phagocytic particles of the invention comprise 100,000 or more neo-antigen constructs tightly associated with the nucleus, e.g., 100,000 to 1 million neo-antigen constructs tightly associated with the nucleus (e.g., 100,000 to 800,000 or 100,000 to 500,000 neo-antigen constructs tightly associated with the nucleus).In a very preferred embodiment, the phagocytic particles of the present invention comprise 500,000 or more neoantigen constructs tightly associated with the nucleus, such as 500,000 to 1 million neoantigen constructs tightly associated with the nucleus, or 500,000 to 2 million neoantigen constructs tightly associated with the nucleus, or 500,000 to 3 million neoantigen constructs tightly associated with the nucleus. In another embodiment, the phagocytic particles of the present invention may comprise more than 1 million neoantigen constructs tightly associated with the nucleus, such as 1 million to 3 million or 1 million to 2 million neoantigen constructs tightly associated with the nucleus.

[0110] In embodiments of the invention, wherein the phagocytosable particles of the invention may comprise more than one neoantigen construct bound to the core (e.g., 2 or more, 10 or more, 100 or more, 1000 or more, 10,000 or more, 100,000 or more, or 500,000 or more neoantigen constructs bound to the core), the neoantigen constructs bound to the core may be the same, or may be different (i.e., some or all of the neoantigen constructs bound to the core may be different). They may be different by comprising different neoepitope peptide sequences; or they may be different by comprising different combinations of neoepitope peptides. If such portions and sequences are present, they may alternatively or additionally differ by comprising one or more different spacer portions or additional amino acid sequences. Different neoantigen constructs may be referred to as "different types" of neoantigen constructs. The same neoantigen construct may be referred to as "the same type" of neoantigen constructs. Cancer cells typically induce multiple amino acid mutations in multiple proteins or peptides expressed by cancer cells. The present inventors have found that phagocytic particles containing two or more different types of neoantigen constructs can deliver multiple neo-epitopes to APCs, thereby increasing the diversity of neo-epitope-derived peptides presented by APCs. The present inventors have found that this significantly improves the activation and expansion of anti-cancer T cells capable of targeting cancer cells.

[0111] The phagocytic particles of the present invention comprising more than one neoantigen construct bound to the core (e.g., 2 or more, 10 or more, 100 or more, 1000 or more, 10,000 or more, 100,000 or more, or 500,000 or more neoantigen constructs bound to the core) can comprise one type of neoantigen construct tightly bound to the core (i.e., all neoantigen constructs tightly bound to the core are identical). In one embodiment of the present invention, the phagocytic particles comprise 100,000 to 1 million neoantigen constructs tightly bound to the core, wherein the 100,000 to 1 million neoantigen constructs are of the same type.

[0112] In alternative embodiments of the invention, the phagocytosable particle comprising more than one neo-antigen construct associated with the core (e.g., 2 or more, 10 or more, 100 or more, 1000 or more, 10,000 or more, 100,000 or more, or 500,000 or more neo-antigen constructs associated with the core) may comprise two different types of neo-antigen constructs tightly associated with the core.

[0113] In another embodiment of the invention, phagocytic particles comprising more than 10 neoantigen constructs bound to the core (e.g., 100 or more, 1000 or more, 10,000 or more, 100,000 or more, or 500,000 or more neoantigen constructs bound to the core) may include two or more different neoantigen construct types tightly bound to the core. For example, such phagocytic particles may include 2 to 10 different neoantigen construct types (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 species) tightly bound to the core. Preferably, such phagocytic particles may include 2 to 6 different neoantigen construct types (e.g., 2, 3, 4, 5 or 6 species). In one embodiment of the invention, phagocytic particles include 100,000 to 1 million neoantigen constructs tightly bound to the core, wherein 100,000 to 1 million neoantigen constructs include two or more different neoantigen construct types. For example, 2 to 6 different neoantigen construct types (eg, 2, 3, 4, 5 or 6).

[0114] In one embodiment of the present invention, the phagocytic particles further comprise an adjuvant that is tightly bound to the nucleus. As used herein, the term "adjuvant" is understood to mean any substance that enhances the immune response to an antigen. Specific examples of adjuvants include dsRNA analogs, such as polyinosinic:polycytidylic acid, incomplete Freund's adjuvant, cytokines (e.g., IL-2, IL-4, IL-17, and IL-15), CD40, keyhole limpet hemocyanin, Toll-like receptors, CpG oligodeoxynucleotides, saponins, colloidal alum, and analogs of lipid A of lipopolysaccharide. The adjuvant can be tightly bound to the nucleus in the same manner as described herein for tightly binding the neoantigen construct to the nucleus.

[0115] Sterilization of phagocytic particles

[0116] The inventors have advantageously discovered that the phagocytic particles comprising a core and a neoantigen construct tightly bound to the core can be effectively washed and sterilized before administration to a subject. This is particularly advantageous because the washed and sterilized phagocytic particles contain lower levels of pathogens (e.g., bacteria, fungi, and viruses) and contaminants, such as endotoxins (e.g., lipopolysaccharides) and other antigenic contaminants. Such contaminants can cause nonspecific immune responses in subjects. Therefore, washing and sterilizing the phagocytic particles of the present invention before administration can improve their safety and efficacy.

[0117] In one embodiment of the invention, the phagocytic particles include a magnetic core, such as a paramagnetic or superparamagnetic core. The phagocytic particles comprising the magnetic core can be collected and / or held in place by a magnet. Washing can also be performed in other ways, such as by holding the phagocytic particles (whether paramagnetic or not) in a column, or by gravity or centrifugation to allow the particles to settle.

[0118] In the context of the present invention, the specific manner of washing is not critical. For example, washing may comprise subjecting the phagocytosable particles to high pH, ​​low pH, high temperature, sterilizing / denaturing agents, or a combination thereof.

[0119] Washing can involve subjecting the phagocytic particles to a base, preferably a strong base, such as 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 base. Preferably, washing can include subjecting the phagocytic particles to at least 1 M sodium hydroxide (NaOH), such as at least 2 M NaOH. Preferably, washing comprises subjecting the phagocytic particles to a high pH of at least 13.0, more preferably at least 14.0, and most preferably at least 14.3. Other bases that can 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). Preferably, washing comprises subjecting the phagocytic particles to a high pH of at least 13.0, more preferably at least 14.0, and most preferably at least 14.3.

[0120] Washing can also include subjecting the engulfable particles to an acid, preferably a strong acid, such as 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 acid. Preferably, washing can include subjecting the engulfable particles to at least 1 M hydrochloric acid (HCl), such as at least 2 M HCl. Other acids that can be used include, but are not limited to, hydroiodic acid (HI), hydrobromic acid (HBr), perchloric acid (HClO4), nitric acid (HNO3), and sulfuric acid (H2SO4).

[0121] Washing may also include subjecting the phagocytosable particles to further sterilizing / denaturing agents, such as urea and / or guanidine-HCl.

[0122] Preferably, washing results in the phagocytic particles being sterile and / or sterilized. More preferably, washing results in the phagocytic particles being sterilized. Sterile as defined herein is free of disease-causing microorganisms and viruses. Sterile as defined herein is free of all biological contaminants.

[0123] Preferably, washing also removes antigenic contaminants, such as pyrogens (e.g., endotoxins), from the phagocytosable particles. Preferably, washing reduces the endotoxin contamination of the phagocytosable particles to 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.

[0124] Thus, in a preferred embodiment of the invention, the phagocytosable particles are sterilized and have less than 100 pg / ml of endotoxin contamination.

[0125] A particular advantage of washing is that conditions can be selected so that the phagocytic particles are both sterilized and denatured in a single step. In particular, high pH washing (e.g., pH>14) can conveniently, simultaneously, and rapidly sterilize the phagocytic particles and eliminate sufficient amounts of endotoxins and other antigenic contaminants.

[0126] Washing may comprise a single wash or several repeated washes, for example 2, 3, 4 or 5 washes. Additionally, or alternatively, the phagocytosable particles may be subjected to elevated temperatures, for example at least 90°C, preferably at least 92°C, more preferably at least 95°C, for example at least 100°C or at least 110°C.

[0127] The present inventors have advantageously discovered that when the neoantigen construct is covalently bound to the core, the phagocytic particles can be subjected to rigorous sterilization and washing procedures that reduce the amount of antigenic contaminants, such as pyrogens (e.g., endotoxins), that may be bound to the neoantigen construct or core. This means that the phagocytic particles described herein are particularly suitable for treating or preventing cancer.

[0128] Injectable compositions

[0129] The present invention provides injectable compositions comprising the phagocytosable particles of the present invention.

[0130] The injectable compositions of the present invention comprising phagocytic particles as described herein may comprise one or more phagocytic particles. Preferably, they comprise more than one particle. In such embodiments, the phagocytic particles may be the same or different. The phagocytic particles may differ by core and / or by comprising different types of neoantigen constructs that are tightly bound to the core.

[0131] Preferably, in compositions comprising phagocytic particles of the invention in which the phagocytic particles differ, the phagocytic particles have the same core and differ in that the particles comprise different types of neo-antigen constructs tightly associated with the core.

[0132] Phagocytic particles having different cores (e.g., cores having different sizes and / or comprising different materials / polymers as described herein) are referred to herein as "different sets" of phagocytic particles. Phagocytic particles having the same core (e.g., cores having the same size and comprising the same material / polymer) may be referred to herein as "the same set" of phagocytic particles.

[0133] Phagocytic particles having the same core (i.e., they are of the same phagocytic particle group) but differing in having different types of neoantigen constructs tightly associated with the core are referred to herein as "different groups" of phagocytic particles. Phagocytic particles having the same core and the same type of neoantigen construct tightly associated with the core are referred to herein as "the same group" of phagocytic particles.

[0134] In one embodiment, the injectable composition of the present invention comprises a collection of phagocytic particles consisting of a group of phagocytic particles (i.e., all phagocytic particles in the composition are identical). Alternatively, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising two or more different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, or 50 or more groups of phagocytic particles). In one embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 50 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 40, or 50 groups of phagocytic particles). In one embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 30 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, or 30 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 20 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 20 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 15 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 10 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 8 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, or 8 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise a collection of phagocytic particles comprising 2 to 6 different groups of phagocytic particles (e.g., 2, 3, 4, 5, or 6 groups of phagocytic particles).

[0135] In one embodiment, the injectable composition of the present invention may comprise two or more different phagocytic particle collections (i.e., each collection has a different core, for example, each collection has a core of a different size and / or comprises different materials as described herein), and each collection may consist of one phagocytic particle group. For example, the injectable composition of the present invention may comprise 2, 3, 4, or 5 phagocytic particle collections, and each collection may consist of one phagocytic particle group. Preferably, the injectable composition of the present invention may comprise 2 or 3 phagocytic particle collections, and each collection may consist of one phagocytic particle group.

[0136] In another embodiment, the injectable composition of the present invention may comprise two or more different phagocytic particle collections (e.g., 2, 3, or 4 phagocytic particle collections), and each collection may comprise two or more different phagocytic particle groups (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or 30 phagocytic particle groups). In one embodiment, the injectable composition of the present invention may comprise two or more different phagocytic particle collections (e.g., 2, 3, or 4 phagocytic particle collections), and each collection may comprise 2 to 30 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, or 30 phagocytic particle groups). In another embodiment, the injectable composition of the present invention may comprise two or more different sets of phagocytic particles (e.g., 2, 3, or 4 sets of phagocytic particles), and each set may comprise 2 to 20 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 20 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise two or more different sets of phagocytic particles (e.g., 2, 3, or 4 sets of phagocytic particles), and each set may comprise 2 to 15 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise two or more different sets of phagocytic particles (e.g., 2, 3, or 4 sets of phagocytic particles), and each set may comprise 2 to 10 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 groups of phagocytic particles). In another embodiment, the injectable composition of the present invention may comprise two or more different phagocytic particle collections (e.g., 2, 3, or 4 phagocytic particle collections), and each collection may comprise 2 to 8 different groups of phagocytic particles (e.g., 2, 3, 4, 5, 6, 7, or 8 phagocytic particle groups). In another embodiment, the injectable composition of the present invention may comprise two or more different phagocytic particle collections (e.g., 2, 3, or 4 phagocytic particle collections), and each collection may comprise 2 to 6 different groups of phagocytic particles (e.g., 2, 3, 4, 5, or 6 phagocytic particle groups).

[0137] For the avoidance of doubt, in embodiments having more than one set of phagocytic particles and more than one collection of phagocytic particles, each set of phagocytic particles is independent of each set of another set of phagocytic particles. Thus, a set from one collection of phagocytic particles can have the same type of neoantigen construct as a set from another collection of phagocytic particles. Alternatively, a set from one collection of phagocytic particles can have a different type of neoantigen construct than a set from another collection of phagocytic particles.

[0138] Cancer cells often induce multiple amino acid mutations in various proteins or peptides expressed by cancer cells. Administration of an injectable composition containing two or more sets of phagocytic particles allows for the delivery of multiple neo-epitopes to APCs, thereby increasing the diversity of neo-epitope-derived peptides presented by APCs. This increased variety of neo-epitope-derived peptides presented by APCs can significantly improve the activation and expansion of anti-cancer T cells.

[0139] Pharmaceutically acceptable injectable formulations useful according to the present invention include those suitable for parenteral administration, including subcutaneous, intradermal, intramuscular, intravenous (bolus or infusion), intraarticular, and intralymphatic. The most appropriate route may depend, for example, on the condition and disorder of the recipient. Preferably, the pharmaceutical composition is suitable for intravenous and / or intralymphatic administration. Thus, the present invention provides an injectable composition comprising the phagocytic particles of the present invention.

[0140] Preferably, the injectable composition of the present invention is an injectable pharmaceutical composition. The injectable composition of the present invention includes compositions suitable for subcutaneous, intradermal, intramuscular, intravenous (bolus or infusion), intratumoral, intraarticular, and intralymphatic administration, although the most appropriate route may depend, for example, on the type of cancer or tumor present in the subject. Preferably, the injectable composition is suitable for intravenous and / or intralymphatic administration.

[0141] The pharmaceutically acceptable injectable formulations and injectable compositions of the present invention may include aqueous and non-aqueous sterile injectable solutions (e.g., saline, such as PBS), which may contain antioxidants, buffers (e.g., sodium phosphate, potassium phosphate, TRIS, and TEA), bacteriostats, surfactants (e.g., poloxamers, polysorbates, CHAPS, and Titon X-100), and solutes that render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The compositions may be present in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in freeze-dried (lyophilized) conditions requiring only the addition of a sterile liquid carrier (e.g., saline or water for injection) immediately prior to use. Exemplary injection solutions and suspensions for parenteral administration include injectable solutions or suspensions that may contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents including synthetic mono- or diglycerides and fatty acids including oleic acid or Cremaphor.

[0142] The pharmaceutically acceptable formulations of the present invention and the injectable compositions of the present invention may further comprise an adjuvant. As used herein, the term "adjuvant" is understood to mean any substance that enhances the immune response to an antigen. Examples of adjuvants for use in the present invention include dsRNA analogs, such as polyinosinic acid:polycytidylic acid, incomplete Freund's adjuvant, cytokines (e.g., interleukins), CD40, keyhole limpet hemocyanin, Toll-like receptors, CpG oligodeoxynucleotides, saponins, colloidal alum, and analogs of lipid A of lipopolysaccharides. Thus, the injectable compositions of the present invention may comprise dsRNA analogs, such as polyinosinic acid:polycytidylic acid, incomplete Freund's adjuvant, cytokines (e.g., IL-2, IL-4, IL-17, and IL-15), CD40, keyhole limpet hemocyanin, Toll-like receptors, CpG oligodeoxynucleotides, saponins, colloidal alum, and analogs of lipid A of lipopolysaccharides.

[0143] Preferred unit doses of the pharmaceutically acceptable formulations of the present invention and the injectable compositions of the present invention are those containing a therapeutic dose (i.e., a dose suitable for eliciting a primary immune response to cancer) or a booster dose (i.e., a dose suitable for inducing a secondary immune response to cancer) or an appropriate fraction thereof of phagocytic particles. The unit dose of phagocytic particles may be 1 μg to 4000 μg, 10 μg to 3000 μg, or 10 μg to 2000 μg. For example, a unit dose can be 10 μg to 1000 μg, 10 μg to 750 μg, 10 μg to 500 μg, 20 to 400 μg, 25 μg to 300 μg, or 30 μg to 200 μg, 50 μg to 1000 μg, 50 μg to 750 μg, 50 μg to 500 μg, 50 μg to 400 μg, 50 μg to 300 μg or 50 μg to 200 μg, 100 μg to 1000 μg, 100 μg to 750 μg, 100 μg to 50 0μg, 100μg to 400μg, 100μg to 300μg or 100μg to 200μg, 200μg to 1000μg, 200μg to 750μg, 200μg to 500μg, 200μg to 400μg, 200μg to 300μg, 400μg to 1000μg, 400μg to 750μg, 400μg to 500μg, 500μg to 1000μg, 500μg to 750μg or 750μg to 1000μg. For example, the unit dose of the phagocytosable particles can be 1, 10, 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1500, 2000, 3000 or 4000 μg. Preferably, the unit dose is 100 to 750 μg, more preferably 200 to 750 μg, more preferably 300 to 750 μg, more preferably 400 to 750 μg, more preferably 500 to 750 μg, more preferably 600 to 750 μg, even more preferably 650 to 750 μg. For example, the unit dose of the phagocytosable particles can be 100, 200, 300, 400, 500, 600, 700 or 750 μg.

[0144] It should be understood that the pharmaceutically acceptable formulations of the present invention and the injectable compositions of the present invention may include, in addition to the ingredients particularly mentioned above, other agents conventional in the art with regard to the type of composition in question.

[0145] Although the phagocytic particles of the present invention used in various embodiments of the present invention can be used as the sole active ingredient, the phagocytic particles can also be used in combination with one or more additional active agents. Therefore, the present invention also provides phagocytic particles, which are used according to the present invention together with additional active agents to treat or prevent cancer in a subject, or in a method for treating or preventing cancer in a subject, for simultaneous, sequential or separate administration. Such additional active agents can be agents or other pharmaceutically active materials that can be used to treat cancer, but are preferably agents known to be used to treat cancer. Such agents are known in the art. Examples of other active agents include alkylating agents, antimetabolites, antitumor antibiotics, histone deacetylase inhibitors, immunomodulatory drugs, microtubule-interacting drugs, protein kinase inhibitors, steroids, topoisomerase inhibitors, cell cycle inhibitors and angiogenesis inhibitors.

[0146] Thus, the phagocytosable particles of the present invention for treating or preventing cancer in a subject, or for use in a method of treating or preventing cancer in a subject, can be administered with one or more compounds known to be useful for treating or preventing cancer, such as one or more alkylating agents, antimetabolites, antitumor antibiotics, histone deacetylase inhibitors, immunomodulatory drugs, microtubule-interacting drugs, protein kinase inhibitors, steroids, topoisomerase inhibitors, cell cycle inhibitors, or angiogenesis inhibitors.

[0147] When used in combination, the precise dosage of (one or more) additional active agents will vary depending on the dosing schedule, the oral efficacy of the particular agent selected, the age, size, sex and condition of the subject (typically a mammal or human), the nature and severity of the cancer, and other relevant medical and physical factors. Therefore, a precise therapeutic dose or booster dose cannot be specified in advance, and a caregiver or clinician can easily determine the precise therapeutic dose or booster dose. An appropriate amount can be determined by routine experimentation in animal models and human clinical studies. For humans, a person of ordinary skill in the art will know or otherwise determine a therapeutic or booster dose.

[0148] The individual components of such combinations may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms.The instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment.

[0149] When used in combination with the compounds useful in the present invention, the aforementioned additional active agent(s) may be used, for example, in amounts specified in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[0150] treat

[0151] The present invention provides phagocytic particles for treating or preventing cancer in a subject, wherein the phagocytic particles comprise a core and a neoantigen construct tightly bound to the core, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence corresponding to the amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by cancer cells in the subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid. The present invention also provides a method for treating or preventing cancer in a subject, comprising the step of administering the phagocytic particles of the present invention to the subject. The present invention also provides the use of the phagocytic particles of the present invention in the preparation of a medicament for treating or preventing cancer.

[0152] The inventors have found that the phagocytic particles used in the present invention are surprisingly effective in eliciting a powerful anti-cancer immune response against cancer cells in a subject. In general, immune responses can be divided into innate immune responses or adaptive immune responses. The innate immune response is an immune response that is not intrinsically affected by prior contact with the antigen. This response is typically characterized by the activation and expansion of naive T cells and B cells. In comparison, the adaptive immune response is an immune response that requires prior contact with the antigen. The adaptive immune response typically occurs shortly after the innate immune response and ultimately leads to immunological memory of the antigen. When the immune system first comes into contact with the antigen, the immune responses (innate and adaptive responses) that are triggered are generally referred to as "primary immune responses." The subsequent activation of memory T cells and B cells by the same antigen leads to a rapid specific immune response to the antigen, which is generally referred to as a "secondary immune response."

[0153] The present inventors have unexpectedly discovered that the phagocytosable particles of the present invention elicit a potent anti-cancer immune response by activating both the innate and adaptive immune responses of a subject.

[0154] The immune response caused by the phagocytic particles used in the present invention may include phagocytosis of the phagocytic particles by antigen presenting cells (APCs). APCs are typically dendritic cells (DCs), B cells, or macrophages, or cells that phagocytose or internalize extracellular organisms or proteins (i.e., antigens) and, after processing, present antigen-derived peptides on class II MHC and / or class I MHC molecules to CD4+ T cells and / or CD8+ T cells. In the blood, monocytes are the most abundant APCs, such as dendritic cells, macrophages, and B cells.

[0155] The immune response induced by the phagocytic particles used in the present invention can also induce the activation and expansion of naive or memory T cells. For example, the phagocytic particles of the present invention can induce the activation and expansion of CD4+ T cells (or T helper cells or CD4+ helper T cells) and / or CD8+ T cells (or cytotoxic T cells). CD4+ T cells are cells that coordinate the immune response through cytokine secretion. They can inhibit or enhance other immune cells, for example, stimulate antibody class switching in B cells, stimulate the activation and expansion of cytotoxic T cells, or enhance phagocytes. They are activated by antigen presentation by class II MHC on APCs, and they express T cell receptors (TCRs) that are specific for a stretch of approximately 15 amino acids within a specific antigen (so-called T cell epitopes). 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 class I MHC (a protein expressed on all nucleated cells).

[0156] The treatments of the invention may be used to treat or prevent any form of cancer, such as solid cancers, metastatic solid cancers, or hematological malignancies.

[0157] "Solid cancer" herein is an abnormal mass of tissue, such as that originating from an organ. Solid cancer may be malignant. Different types of solid cancers vary depending on the type of cell that forms them. Types of solid cancer include sarcomas, carcinomas, and lymphomas. 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 carcinoids, gastrointestinal stromal tumors (GISTs), gestational trophoblastic disease, hepatosplenic T-cell lymphoma, Hodgkin lymphoma, intravascular large B-cell lymphoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer (non-small cell and small cell carcinoma), lung carcinoid tumors, Lymphoma granuloma, malignant mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, lymph node marginal zone B cell lymphoma, non-Hodgkin 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 treatment of the present invention is particularly effective in the treatment of solid cancers. Thus, the subject of the present invention can suffer from solid cancers. The treatment of the present invention is 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 and small cell), lung carcinoid, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, stomach cancer, testicular cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and is particularly useful in treating breast cancer, colon cancer, liver cancer, lung cancer (non-small cell and small cell), lung carcinoid, pancreatic cancer, prostate cancer, ovarian cancer and bladder cancer.

[0158] The treatment of the present invention is 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.

[0159] Alternatively, the cancer can be any form of hematologic malignancy. Hematologic malignancies are a form of cancer that begins in the 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 blood cell types. Examples of hematologic cancers include leukemias, lymphomas, myelomas, and myelodysplastic syndromes (lymphomas can be divided into solid cancers and hematologic malignancies). 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, B-cell leukemia, B-cell B-cell lymphoma, B-cell lymphocytic 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 lymphoma, intravascular large B-cell lymphoma, Kahler disease, lymphomatoid granulomatosis, mast cell leukemia, multiple myeloma, myeloma, nodular marginal zone B-cell lymphoma, non-Hodgkin lymphoma, plasma cell leukemia, primary effusion lymphoma, and Waldenstrom's macroglobulinemia.

[0160] Administration and dosage regimen

[0161] A therapeutic dose of the phagocytosable particles or injectable compositions of the invention is a dose sufficient to elicit an immune response to cancer in a subject, for example, a primary immune response and / or a secondary immune response.

[0162] In certain embodiments of the present invention, the use of the phagocytic particles as described herein for treating or preventing cancer comprises administering a therapeutic dose of the phagocytic particles to a subject. In certain embodiments of the present invention, the methods of treating or preventing cancer comprise administering a therapeutic dose of the phagocytic particles to a subject. In certain embodiments of the present invention, the use of the phagocytic particles of the present invention or the methods of treatment of the present invention comprise administering a therapeutic dose of the injectable composition of the present invention to a subject.

[0163] The therapeutic dose of the phagocytic particles or the injectable composition of the present invention required to treat or prevent cancer in a subject will vary depending on the route of injection and the characteristics of the individual being treated, such as species, age, weight, sex, medical condition, specific cancer and its severity, and other relevant medical and physical factors. One of ordinary skill can readily determine and administer the effective amount of phagocytic particles required to treat or prevent cancer.

[0164] The therapeutic dose of phagocytosable particles can be 1 μg to 4000 μg, 10 μg to 3000 μg or 10 μg to 2000 μg. For example, the dose can be 10 μg to 1000 μg, 10 μg to 750 μg, 10 μg to 500 μg, 20 μg to 400 μg, 25 μg to 300 μg, 30 μg to 200 μg, 50 μg to 1000 μg, 50 μg to 750 μg, 50 μg to 500 μg, 50 μg to 400 μg, 50 μg to 300 μg or 50 μg to 200 μg, 100 μg to 1000 μg, 100 μg to 750 μg, 100 μg to 5 00μg, 100μg to 400μg, 100μg to 300μg or 100μg to 200μg, 200μg to 1000μg, 200μg to 750μg, 200μg to 500μg, 200μg to 400μg, 200μg to 300μg, 400μg to 1000μg, 400μg to 750μg, 400μg to 500μg, 500μg to 1000μg, 500μg to 750μg or 750μg to 1000μg. For example, the dose of phagocytic particles can be 1, 10, 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1500, 2000, 3000 or 4000 μg. Preferably, the dose is 100 to 750 μg, more preferably 200 to 750 μg, more preferably 300 to 750 μg, more preferably 400 to 750 μg, more preferably 500 to 750 μg, more preferably 600 to 750 μg, even more preferably 650 to 750 μg. For example, the dose of phagocytic particles can be 100, 200, 300, 400, 500, 600, 700 or 750 μg.

[0165] Alternatively, the therapeutic dose of phagocytosable particles may be determined based on the number of phagocytosable particles. For example, a dose may be approximately 10 4 to 10 10 , 10 5 to 10 9 , 10 5 to 10 8 , 10 5 to 10 7or 10 5 to 10 6 phagocytic particles (e.g. 10 4 , 5 5 , 10 5 , 5 6 , 10 6 , 5 7 , 10 7 , 5 8 , 10 8 , 5 9 , 10 9 , 5 10 or 10 10 Preferably, the dose may be about 10 5 to 10 8 , 10 5 to 10 7 or 10 5 to 10 6 phagocytic particles, for example, approximately 10 5 to 10 9 , 5x10 5 to 10 8 , 5x10 5 to 7.5x10 7 , 5x10 5 Up to 5x10 7 , 5x10 5 to 2.5x10 7 or 5x10 5 to 10 7 More preferably, the dosage may be about 10 7 to 10 9 , for example about 5x10 7 to 10 9 , 7.5x10 7 to 10 9 , 7.5x10 7 to 7.5x10 8 , 7.5x10 7 Up to 5x10 8 or 7.5x10 7 to 2.5x10 8 More preferably, the dose is about 7.5 x 10 7 Up to 5x10 8 phagocytosable particles, such as about 75 million, 100 million, 150 million, 200 million, or 250 million phagocytosable particles.

[0166] Alternatively, the therapeutic dose of phagocytosable particles can be determined based on the amount of neoantigen construct bound to the nucleus. For example, the dose can be 1 μg to 4000 μg, 10 μg to 3000 μg, or 10 μg to 2000 μg of the neoantigen construct. For example, 1 μg to 4000 μg, 10 μg to 3000 μg, or 10 μg to 2000 μg. For example, the dose of the neoantigen construct is 10 μg to 1000 μg, 10 μg to 750 μg, 10 μg to 500 μg, 10 μg to 400 μg, 10 μg to 300 μg, 10 μg to 200 μg, 10 μg to 100 μg or 10 to 50 μg, 10 to 25, 20 μg to 400 μg, 25 μg to 300 μg, 30 μg to 200 μg, 50 μg to 1000 μg, 50 μg to 750 μg, 50 μg to 500 μg, 50 μg to 400 μg, 50 μg to 300 μg or 50 μg to 200 μg, 10 0μg to 1000μg, 100μg to 750μg, 100μg to 500μg, 100μg to 400μg, 100μg to 300μg or 100μg to 200μg, 200μg to 1000μg, 200μg to 750μg, 200μg to 500μg, 200μg to 400μg, 200μg to 300μg, 400μg to 1000μg, 400μg to 750μg, 400μg to 500μg, 500μg to 1000μg, 500μg to 750μg or 750μg to 1000μg. For example, the dose of the neoantigen construct can be 1, 10, 15, 20, 25, 30, 40, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1500, 2000, 3000 or 4000 μg. Preferably, the dose can be 10 μg to 1000 μg, 10 μg to 750 μg, 10 μg to 500 μg, 10 μg to 400 μg, 10 μg to 300 μg, 10 μg to 200 μg, 10 μg to 100 μg or 10 to 50 μg of the neoantigen construct. Preferably, the dose is 10 to 100 μg of the neoantigen construct, such as 10 μg to 75 μg, 10 μg to 50 μg, 10 μg to 25 μg, 25 μg to 50 μg, or 50 μg to 75 μg of the neoantigen construct. For example, the dose of the phagocytosable particles can be 1, 10, 15, 20, 25, 30, 40, 50, 75, 100 μg of the neoantigen construct. Preferably, the dose is 10 to 50 μg of the neoantigen construct.

[0167] The therapeutic dose can be administered as a single unit dose comprising a therapeutic dose of phagocytosable particles, or as multiple unit doses when the unit dose comprises a fraction of a therapeutic dose.Preferably, a therapeutic dose of the phagocytosable particles of the invention is administered to a subject as a single dose.

[0168] A therapeutic dose of phagocytosable particles or an injectable composition comprising a therapeutic dose of phagocytosable particles of the invention can be administered to a subject once.

[0169] In certain preferred embodiments, a therapeutic dose of phagocytic particles or an injectable composition comprising a therapeutic dose of phagocytic particles is administered to a subject, and then at least one further (or "subsequent") therapeutic dose of phagocytic particles of the invention or an injectable composition comprising a therapeutic dose of phagocytic particles of the invention is administered to the subject. Further (or "subsequent") therapeutic doses of phagocytic particles can be administered daily, every second or third day, weekly, every second, third or fourth week, monthly, every second, third or fourth month, every six months, or annually. The number and frequency of further therapeutic doses of phagocytic particles of the invention or an injectable composition comprising a therapeutic dose of phagocytic particles of the invention will depend on the subject and the form and severity of the cancer being treated.

[0170] In one embodiment, the use of phagocytic particles for treating or preventing cancer comprises administering to a subject one or more subsequent therapeutic doses of phagocytic particles or an injectable composition comprising a therapeutic dose of phagocytic particles of the invention, wherein the subject is a subject who has previously been administered a therapeutic dose of phagocytic particles or an injectable composition comprising a therapeutic dose of phagocytic particles of the invention. Each of the one or more subsequent therapeutic doses is a dose sufficient to elicit an immune response (i.e., a primary immune response and / or a secondary immune response) against cancer cells in the subject.

[0171] In embodiments of the invention that include administering to a subject one or more subsequent therapeutic doses of phagocytic particles or injectable compositions comprising a therapeutic dose of phagocytic particles of the invention, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, or n subsequent therapeutic doses of phagocytic particles or injectable compositions are administered to the subject; wherein "n" is any number of doses greater than 10 doses (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 doses). Preferably, the number and frequency of subsequent therapeutic doses administered to the subject is sufficient to treat or prevent cancer in the subject.

[0172] The therapeutic dose of phagocytic particles or injectable compositions of the present invention can independently have any properties and / or characteristics of the therapeutic dose of phagocytic particles or injectable compositions as described herein. In a preferred embodiment of the present invention, the phagocytic particles administered to the subject as a subsequent therapeutic dose contain the same type of neo-antigen construct as the phagocytic particles previously administered to the subject as a therapeutic dose. The core of the phagocytic particles administered as a subsequent therapeutic dose can be the same or different from the core of the phagocytic particles previously administered to the subject as a therapeutic dose (e.g., the core can have a different size and / or contain different materials / polymers, as described herein). Preferably, the phagocytic particles administered as a subsequent therapeutic dose may contain the same type of neo-antigen construct and the same core as the phagocytic particles previously administered to the subject as a therapeutic dose (i.e., the phagocytic particles belong to the same set and are the same as the groups previously administered to the subject as a therapeutic dose).

[0173] In embodiments of the present invention comprising administering to a subject one or more subsequent therapeutic doses of phagocytosable particles or injectable compositions comprising a therapeutic dose of phagocytosable particles, preferably, the subject is administered one or more subsequent therapeutic doses at intervals of days, weeks, or months. For example, one or more subsequent therapeutic doses are administered to the subject every day, every second day, every third day, every fourth day, every fifth day, every sixth day. Alternatively, or in addition, one or more subsequent therapeutic doses are administered to the subject, for example, once a week, once every two weeks, once every three weeks, or once every four weeks. Alternatively, or in addition, one or more subsequent therapeutic doses are administered to the subject, for example, once a month, once every two months, once every three months, or once every sixth month. Alternatively, or in addition, one or more subsequent therapeutic doses are administered to the subject, for example, once a year.

[0174] In one embodiment of the invention, one or more subsequent treatment doses are administered to the subject once a year, twice a year, or three times a year. For example, one or more subsequent treatment doses may be administered to the subject once a year, twice a year, or three times a year over a period of 1 to 10 years, 1 to 20 years, 1 to 30 years, 1 to 40 years, or 1 to 60 years (e.g., a period of 1 to 10 years, 10 to 20 years, 20 to 30 years, 30 to 40 years, or 50 to 60 years).

[0175] The present inventors have found that by administering one or more subsequent treatment doses to a subject for a long time (i.e., more than one year or more), the number of anti-cancer memory B cells and memory T cells present in the subject can be increased. It is expected that by increasing the number of anti-cancer memory B cells and memory T cells in the subject, the anti-cancer immune memory in the subject is maintained, thereby preventing the growth (e.g., formation of a tumor) or recurrence of cancer.

[0176] In certain embodiments of the invention, a subject who has been successfully treated for cancer is given a booster dose (such as one or more therapeutic doses described herein) to prevent recurrence of the cancer.

[0177] The booster dose of phagocytic particles or injectable compositions of the present invention can independently have any properties and / or characteristics of the therapeutic dose of phagocytic particles or injectable compositions as described herein. In a preferred embodiment of the present invention, the phagocytic particles administered to the subject as a booster dose contain the same type of neoantigen construct as the phagocytic particles administered to the subject as a therapeutic dose. The core of the phagocytic particles can be the same or different from the core of the phagocytic particles administered to the subject as a therapeutic dose (e.g., the core can be of a different size and / or contain different materials / polymers, as described herein). Preferably, the booster dose includes phagocytic particles having the same type of neoantigen construct and the same core as the phagocytic particles administered to the subject as a therapeutic dose (i.e., the phagocytic particles belong to the same set and are the same as the groups previously administered to the subject as a therapeutic dose).

[0178] In one embodiment of the invention, one or more booster doses are administered to a subject, for example, once a week, once every two weeks, once every three weeks, or once every four weeks. Alternatively, or in addition, one or more booster doses are administered to a subject, for example, once a month, once every two months, once every three months, or once every six months. Alternatively, or in addition, one or more booster doses are administered to a subject, for example, once a year.

[0179] In another embodiment of the invention, one or more booster doses are administered to the subject annually, twice annually, or three times annually. For example, one or more booster doses may be administered to the subject annually, twice annually, or three times annually over a period of 1 to 10 years, 10 to 20 years, 20 to 30 years, 30 to 40 years, or 50 to 60 years.

[0180] Increasing the number of anti-cancer memory B cells and memory T cells in a subject by administering one or more booster doses is expected to maintain anti-cancer immunological memory in the subject, thereby preventing growth or recurrence of the cancer.

[0181] In vitro activation and expansion of anticancer T cells

[0182] The present invention also provides a therapeutic method of the present invention, which further comprises the following additional steps: a) harvesting APCs and anti-cancer T cells from a subject; b) expanding the anti-cancer T cells harvested from the subject; and c) administering a therapeutic dose of the expanded anti-cancer T cells to the subject.

[0183] The present invention also provides a method for treating or preventing cancer in a subject, comprising the following additional steps: a) harvesting APCs and anti-cancer T cells from the subject; b) expanding the anti-cancer T cells harvested from the subject; and c) administering a therapeutic dose of the expanded anti-cancer T cells to the subject.

[0184] Step a) harvesting APCs and anti-cancer T cells from the subject after administering the phagocytosable particles to the subject;

[0185] In one embodiment of the present invention, in step a), after administering a dose (preferably a therapeutic dose) of phagocytic particles or an injectable composition of the present invention to the subject, APCs and anti-cancer T cells are harvested from the subject. Alternatively or additionally, APCs and anti-cancer T cells can be harvested from the subject simultaneously with and / or before administering a dose (preferably a therapeutic dose) of phagocytic particles or an injectable composition of the present invention to the subject. Preferably, APCs and anti-cancer T cells are harvested from the subject after administering a dose (preferably a therapeutic dose) of phagocytic particles or an injectable composition of the present invention to the subject.

[0186] APCs must be compatible with anticancer T cells so that they can present antigens to anticancer T cells in an antigen-specific context (MHC restricted) to which the anticancer T cells can react. APCs and anticancer T cells are preferably obtained 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, APCs and anticancer T cells are obtained from the same subject. If the APCs and anticancer T cells are from the same subject, the possibility of any mismatch between the APCs and anticancer T cells is avoided.

[0187] APCs harvested from a subject may comprise phagocytes, monocytes, and / or dendritic cells. Anti-cancer T cells may comprise CD4+ and / or CD8+ T cells.

[0188] APCs and anti-cancer T cells can be harvested from a blood sample of a subject. Preferably, the blood sample is a peripheral blood mononuclear cell (PBMC) sample. PBMC is a portion of human blood prepared by whole blood density gradient centrifugation. PBMC 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 account for 10% to 20%, for example, 10% to 15%, of the cell count in a PBMC sample.

[0189] APCs and anti-cancer T cells can also be derived from a subject's tumor, for example, from a sample of lymphatic vessels in the tumor or lymph nodes draining the tumor (i.e., sentinel lymph nodes). Preferably, APCs and anti-cancer T cells are harvested from the same sample and / or the same tumor in the subject. Alternatively or additionally, APCs and anti-cancer T cells can be harvested from different samples derived from the subject. For example, APCs can be harvested from a blood sample, and anti-cancer T cells can be harvested from a tumor.

[0190] Preferably, APCs and anti-cancer T cells are harvested from a PBMC sample. For example, APCs and anti-cancer T cells can be harvested from the same PBMC sample or from different PBMC samples. Obtaining PBMCs from a peripheral blood sample is a common approach that provides a convenient source of both APCs and T cells from the same subject. PBMC samples can be used fresh or can be cryopreserved prior to use.

[0191] Step b): Expanding anti-cancer T cells obtained from the subject:

[0192] The APCs and anti-cancer T cells harvested from the subject in step a) can be used to prepare anti-cancer T cells suitable for treating or preventing cancer in the subject. The anti-cancer T cells suitable for use in the subject are prepared by in vitro activation and amplification of the anti-cancer T cells harvested from the subject. The in vitro activation and amplification method may include the following steps:

[0193] i) providing a phagocytosable particle comprising a core and a neoantigen construct tightly bound to the core, wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence corresponding to an amino acid sequence of a portion of a protein or peptide known or suspected to be expressed by a cancer cell in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid;

[0194] ii) provide APC;

[0195] iii) contacting the phagocytosable particles with APCs in vitro and under conditions that allow the APCs to phagocytose the phagocytosable particles;

[0196] iv) providing anti-cancer T cells harvested from the subject;

[0197] v) contacting the anti-cancer T cells with the APCs from step iii) in vitro and under conditions that allow for specific activation and expansion of the anti-cancer T cells in response to the neo-epitopes presented by the APCs.

[0198] In vitro expansion methods may include using APCs with 100 to 1 x 10 9 phagocytosable particles, for example, 100 to 1x10 8 For example, 100 to 1x107 phagocytosable particles, for example, 1000 to 1x10 7 phagocytosable particles. For example, the ratio of phagocytosable particles to APCs is in the range of 1000:1 to 1:10. This ratio can be optimized based on the size of the phagocytosable particles. For example, for phagocytosable particles with a maximum diameter of about 1 μm, the ratio can be in the range of 50:1 to 2:1, such as 25:1 to 5:1, 15:1 to 7:1, and 10:1.

[0199] For the avoidance of doubt, a phagocytosable particle suitable for contact with an APC may independently possess any property and / or characteristic of a phagocytosable particle for administration to a subject as a therapeutic dose or booster dose.

[0200] In certain embodiments of the present invention, APCs are contacted with phagocytic particles comprising the same type of neo-antigen construct as the phagocytic particles administered to the subject as a therapeutic dose. The core of the phagocytic particle can be the same or different from the core of the phagocytic particle administered to the subject as a therapeutic dose (e.g., the core can be of a different size and / or comprise a different material / polymer, as described herein). In a preferred embodiment of the present invention, APCs are contacted with phagocytic particles comprising the same type of neo-antigen construct and the same core as the phagocytic particles administered to the subject in a therapeutic dose.

[0201] In one embodiment of the present invention, the method for amplifying anti-cancer T cells includes adding a low dose of IL-2 to the anti-cancer T cell sample, for example, greater than 1.25U / ml (e.g., 1.25U / ml, 2.5U / ml, 5U / ml, or 50U / ml), preferably greater than 2.5U / ml, 5U / ml, or 50U / ml. When IL-2 is present at the same time, antigen-specific T cell amplification occurs in the presence of antigen-presenting cells. IL-2 promotes the differentiation of anti-cancer T cells into effector anti-cancer T cells and memory anti-cancer T cells. After the anti-cancer T cells are amplified, APCs can be removed from the amplified T cell population, for example, by magnetic separation.

[0202] In another embodiment of the present invention, the method of expanding anti-cancer T cells comprises adding IL-2 and / or IL-7 and / or IL-15 to an anti-cancer T cell sample, for example, adding a low dose of IL-2 to the anti-cancer 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, wherein IL-7 and / or IL-15 are optionally added. For example, low-dose IL-7, 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-7, is added to the anti-cancer T cell sample; and / or low-dose IL-15, 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-15, is added to the anti-cancer T cell sample.

[0203] In a preferred embodiment of the present invention, the method for activating and expanding anti-cancer T cells in vitro comprises the step of removing APCs that have internalized the phagocytic particles of the present invention from the anti-cancer T cells. In embodiments of the present invention in which the phagocytic particles comprise a magnetic core, the APCs are removed from the anti-cancer T cells by using magnetic separation.

[0204] After the anti-cancer T cells are contacted with the APC that has internalized the phagocytic particles of the present invention, the degree of anti-cancer T cell activation can be determined, for example, by comparing the degree of anti-cancer T cell activation with a relevant reference. Determining the degree of anti-cancer T cell activation can be performed using T cell activation assays known in the art, such as ELISpot, FluoroSpot, intracellular staining of cytokines using a flow cytometer, FASCIA (flow cytometry for activation of specific cell-mediated immune responses in whole blood), proliferation assays (e.g., thymidine incorporation, CFSE or BrdU staining), specific TCR-detection of MHC-I or II tetramers, and ELISA or Luminex analysis of secreted cytokines ELIS potassays. The method can include a step of comparing the degree of anti-cancer T cell activation with a relevant reference. Suitable references include, for example, T cell samples that do not contain anti-cancer T cells, or anti-cancer T cell samples that have not yet been contacted with APC that have internalized the phagocytic particles.

[0205] c) administering a therapeutic dose of the expanded anti-cancer T cells to the subject.

[0206] A therapeutic dose of the expanded anti-cancer T cells can be administered to a subject. Therefore, the present invention also provides anti-cancer T cells for treating or preventing cancer in a subject. The expanded anti-cancer T cells can be administered intravenously, intraarterially, intrathecally, or intraperitoneally to a subject.

[0207] The exact dosage of the expanded anti-cancer T cells will vary with the dosing schedule, the age, size, sex and condition of the subject (typically a mammal or human), the nature and severity of the condition, and other relevant medical and physical factors. Thus, a clinician can readily determine the precise therapeutically effective amount. An appropriate amount can be determined by routine experimentation in animal models and human clinical studies. For humans, one of ordinary skill in the art will know or otherwise be able to determine an effective dose.

[0208] Example

[0209] Example 1: General protocol for coupling neoantigen constructs or model peptides / proteins to magnetic cores

[0210] Conjugation of neoantigen constructs or model peptides / proteins to the nucleus:

[0211] use MyOne TM Carboxylic acid (ThermoFischer Scientific) (spheres with a diameter of 1 μm) served as cores.

[0212] MyOne TM Carboxylic acid particles are paramagnetic polystyrene particles containing iron oxide and functionalized with free carboxylic acid groups on the particle surface. The coupling steps were performed according to the manufacturer's protocol (two-step procedure using NHS (N-hydroxysuccinimide) and EDC (ethylcarbodiimide)):

[0213] Step 1): The polystyrene 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 polystyrene particles and incubated at room temperature (RT) for 30 min. The polystyrene particles were collected with a magnet, the supernatant was removed, and the particles were washed twice with MES-buffer.

[0214] Step 2): The neoantigen construct or model peptide / protein sample was diluted to a concentration of 1 mg / ml in MES buffer, totaling 100 μg, and then added to the polystyrene particles and incubated at room temperature for 1 hour. The polystyrene particles were collected with a magnet, and the supernatant was removed and saved for peptide concentration measurement. Unreacted activated carboxylic acid groups were quenched with 50 mM Tris pH 7.4 for 15 minutes. The polystyrene particles were then washed with PBS pH 7.4 and stored at -80°C.

[0215] A BCA (bicinchoninic acid) protein assay kit (Pierce BCA Protein Assay Kit, ThermoFisher Scientific) was used according to the manufacturer's protocol to measure the amount of peptide coupled to the polystyrene particles and to determine the peptide concentration of the neoantigen construct samples before coupling and the peptide concentration of the supernatant after coupling.

[0216] Several peptides were tested, and an estimated average of approximately 48.7 μg (mean: 48.7, SD: 20.5, N=10) of neoantigen constructs were coupled per 1 mg of polystyrene particles. According to the manufacturer's instructions, 50 μg of peptide could be coupled per 1 mg of particles, indicating that the coupling efficiency was high.

[0217] Example 2: Cleaning

[0218] According to the method described in Example 1, polystyrene particles were coupled to recombinant neoantigen constructs produced in Escherichia coli. After coupling, the polystyrene particles were washed with one of three different wash buffers: 2M NaOH pH 14.3, 8M urea, or 6M guanidine (guanidine-HCl), all washed in sterile water at room temperature, or incubated in PBS at 95°C. The polystyrene particles were suspended in the buffer and shaken for 4 minutes, and the supernatant was collected and removed with a magnet. Repeat 3 times. The heat-treated polystyrene particles were placed in PBS pH 7.4 and placed in a heating block at 95°C for 5 minutes, and the supernatant was then collected and removed with a magnet. Repeat 3 times. The particles were then washed 3 times with sterile PBS to remove any remaining wash buffer. Four different washing conditions were tested: (a) high pH (2M NaOH pH 14.3), (b) heating (95°C), and sterilization / denaturing agent ((c) 8M urea and (d) 6M guanidine hydrochloride). In each case, the neoantigen constructs bound to the polystyrene particles remained bound to the polystyrene particles.

[0219] Example 3a: Determining the appropriate size of phagocytosable particles

[0220] The effect of phagocytosable particle size on antigen-specific T cell activation was tested using a cell proliferation assay measuring thymidine incorporation. Splenocytes from ovalbumin (OVA) immunized mice were stimulated with OVA-conjugated polystyrene particles of varying sizes to measure antigen-specific proliferation.

[0221] According to the procedure in Example 1, the diameters of 5.6 μm, 1 μm and 0.2 μm were MyOne TM The carboxylic acid particles were coupled to either OVA (OVA particles) or bovine serum albumin (BSA particles).

[0222] To test the effectiveness of OVA particles in stimulating the activation of antigen-specific T cells, a proliferation assay ( 3 (H thymidine incorporation). The particle concentration relative to cell concentration was 1:1 for 5.6 μm particles, 10:1 for 1 μm particles, and 500:1 for 0.2 μm particles. During incubation with cells, total protein concentrations were calculated to be 125 ng / ml, 160 ng / ml, and 160 ng / ml for 5.6 μm, 1 μm, and 0.2 μm, respectively. Proliferation assays were performed as follows:

[0223] As a stimulus, use MyOne TM Ovalbumin (Sigma Aldrich) and BSA (Sigma Aldrich) coupled to carboxylic acid particles (OVA particles or BSA particles). Mice were immunized with ovalbumin by monthly injections of 100 μg of ovalbumin (Sigma) adsorbed on aluminum hydroxide. Three months after the first injection, mice were sacrificed and spleens harvested. Splenocytes were prepared by standard procedures as described by Thunberg et al., 2009, Allergy 64:919.

[0224] The cells were incubated with OVA particles or BSA particles (10 particles per cell) in cRPMI for 5 days. All cells were incubated at 37°C in a humidified atmosphere containing 6% CO2 for 6 days. During the last 18 hours of incubation, one μCu / well was added. 3 [H] Thymidine was added to the cell culture. The mean counts per minute (cpm) obtained from three replicates of stimulation were divided by the mean cpm value of unstimulated cells and expressed as the stimulation index (SI). An SI value ≥ 2.0 was generally considered positive.

[0225] like Figure 1As shown, cells incubated with OVA particles with a diameter of 0.2 μm showed an increase in proliferation, with an average 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 an increase in proliferation, with an average 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 an average SI of 1.1 (95% CI 0.4-2.7, P = 0.876).

[0226] These results demonstrate 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 effective in terms of cell stimulation, but particles as small as 0.2 μm can be used. It is reasonable to predict that particles larger than 1 μm will also be effective, although these particles are completely incapable of stimulating cells as their 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 10-15 μm range.

[0227] Example 3b: Comparison of Antigen-Conjugated Particles of Different Sizes and Their Effectiveness in Activating and Expanding T Cells

[0228] (i) Preparation of antigen-coupled phagocytic particles:

[0229] Three different sizes of paramagnetic polystyrene phagocytic particles were used:

[0230] - 1 μm in diameter (Dynabeads MyOne Carboxylic Acid, ThermoFisher),

[0231] - 2.8 μm in diameter (Dynabeads M-270 Carboxylic Acid, ThermoFisher), and

[0232] - 4.5 μm in diameter (Dynabeads M-450 epoxy resin, ThermoFisher).

[0233] The phagocytic particles were coupled to the model antigen cytomegalovirus (CMV) protein pp65 construct (SEQ ID NO: 19) according to the manufacturer's instructions. To remove endotoxin, the phagocytic particles were washed five times with 0.75 M sodium hydroxide buffer and then resuspended in sterile PBS.

[0234] (ii) Incubation of antigen-coupled phagocytosable particles:

[0235] Peripheral blood mononuclear cells (PBMC) separated from healthy donors sensitive to CMV by density gradient centrifugation based on ficoll of standard are together with the phagocytic particles (hereinafter referred to as " CMV particles ") of having coupled CMV constructs in 48-well plates with a concentration of 1,000,000 cells / ml at 37 ℃, 5% CO , 500,000 cells / well were cultivated for 18 hours. Based on total surface area (surrogate marker of CMV amount, because it is bound to the surface of CMV particles), the concentration of CMV particles is equalized. Based on the quantity of total PBMC in the sample, this is equal to 10 CMV particles / PBMC for the particle of 1 μm size, equals 1.4CMV particles / PBMC for the particle of 2.8 μm size, and equals 0.5CMV particles / PBMC for the particle of 4.5 μm size. The result of every kind of particle size is shown in Table 1.

[0236] Table 1:

[0237]

[0238] (iii) Assessment of learning:

[0239] After incubation, the number of phagocytosed CMV particles was counted manually using a confocal microscope. Eight cells were counted to obtain the mean and standard deviation values. Figure 5 In A, confocal microscopy images of representative cells with intracellular phagocytic CMV particles are shown. The black dashed line indicates the outline of the cell. The white line shows the size of the total intracellular CMV particles.

[0240] This method is not applicable to 1 μm CMV particles because they are too small to be accurately counted. To estimate the amount of 1 μm CMV particles, the total volume of all phagocytosed CMV particles was measured, and the amount of individual CMV particles was back-calculated based on the total volume, assuming a packing density of 60%. This method was reasonably accurate for 2.8 μm CMV particles (9.1 CMV particles / cell by manual count vs. an estimated 11.9 CMV particles / cell) and 4.5 μm CMV particles (3.1 CMV particles / cell by manual count vs. an estimated 2.4 CMV particles / cell), so it can also be assumed that this method accurately estimates the number of 1 μm CMV particles.

[0241] Figure 5 B and 5C show the uptake of CMV particles. Figure 5B shows the number of CMV particles absorbed by each cell assessed by manual counting (8 cells were counted for each bead type). Using the manual counting method, it was found that for 4.5 μm CMV particles, the number of phagocytic particles per cell was 3.1 (± 1.1). For 2.8 μm CMV particles, it was 9.1 (± 2.2). The number of 1 μm CMV particles could not be counted using this method.

[0242] Figure 5 C shows the number of CMV particles (each bead type measured 3 cells) that each cell absorbs by volume calculation assessment (*p<0.05**p<0.01***p<0.001, using Student's T test calculation). Using volume calculation method, it was found that for 4.5 μm CMV particles, the number of phagocytic particles of each cell was 2.4 (± 1.1). For the CMV particles of 2.8 μm, it was 11.9 (± 3.2). For the CMV particles of 1 μm, it was 203.7 (± 21.9).

[0243] The total phagocytic surface area was calculated based on the number of CMV particles taken up per cell, as assessed by volumetric calculations, and by extension, the total number of CMV. For 1 μm CMV particles, the taken-up surface area was calculated to be 639.6 (±68.9) μm. 2 , which was calculated to be 293.1 (± 79.3) μm for a 2.8 μm CMV particle 2 , which was calculated to be 150.7 (± 67.0) μm for a 4.5 μm CMV particle 2 These data are shown in Table 2 below.

[0244] Table 2:

[0245]

[0246] (iv) Assessment of T cell stimulation

[0247] The ability of the particles coupled to the antigen 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 CMV particles and repeated three times. As previously described, the concentration of the particles coupled to the antigen was normalized based on total surface area: 10x1 μm CMV particles / cell, 1.4x2.8 μm CMV particles / PBMC and 0.5x4.5 μm CMV particles / cell. For each particle size, the number of PBMCs in each well of the FluoroSpot assay is shown below (Table 3), as well as the estimated number of monocytes in each well (based on an estimated 20% monocyte content in the PBMC sample).

[0248] Table 3:

[0249] Particle size PBMC number per well Number of monocytes per well 1μm 250,000 50,000 2.8μm 250,000 50,000 4.5μm 250,000 50,000

[0250] PBMCs were incubated at 37°C, 5% CO2 for 44 hours. Plates were developed and read in an automated FluoroSpot reader according to the manufacturer's instructions. FluoroSpot data reported when cells were stimulated with CMV particles are spot numbers, which are greater than the spot numbers when not stimulated with CMV particles.

[0251] The IFNγ production levels assessed in the FluoroSpot assay were as Figure 6 A. As can be seen, there is little differentiation between the CMV particles.

[0252] The levels of IL22 and IL17 production, as assessed in FluoroSpot assays, were shown in Figure 6 B and 6C. As can be seen, 1 μm CMV particles elicited significantly higher production of IL22 and IL17 in one individual compared to larger CMV particles, with a similar trend observed for IL22 in the other individual.

[0253] The levels of dual cytokine production assessed in the FluoroSpot assay were as Figure 6 D and 6E. As can be seen, when stimulated with 1 μm antigen-coupled particles, 1 μm CMV particles induced significantly higher dual cytokine release (IFNγ+IL17 and IL22γ+IL17) compared to larger CMV particles from a healthy donor.

[0254] The cytokines released in these experiments can be used as surrogates 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. These cells have been shown to be pro-inflammatory and contribute to tumor eradication. The data suggest that 1 μm beads activated and caused the proliferation of Th1 CD4+ T cells and CD8+ T cells to the same extent as other beads, with the added benefit of activating and eliciting additional pro-inflammatory Th17 CD4+ T cells and less pronounced, but still pro-inflammatory, dual-cytokine T cells.

[0255] Example 4: Preventive Effect of Phagocytic Particles in Mouse Cancer Model:

[0256] Two sets of phagocytic particles were used in this experiment: 1) phagocytic particles comprising a polystyrene particle core and comprising the neoantigen construct M272120 (SEQ ID NO: 1) tightly bound to the core; and 2) phagocytic particles comprising a polystyrene particle core and the neoantigen construct M304748 (SEQ ID NO: 2) tightly bound to the core. The phagocytic particles were prepared using the methods described in Examples 1 and 2: the neoantigen constructs were coupled to 1 μm superparamagnetic beads (Sera-Mag SpeedBeads (hydrophilic) carboxylate-modified magnetic particles, GE Healthcare) according to the protocols outlined in Examples 1 and 2. The neoantigen construct design was based on previously published studies using the B16-F10 tumor model (Kreiter et al. (2015), Nature 520: 692 and Castle et al. (2012) Cancer Res 72: 1081). After preparation, the two sets of phagocytic particles were mixed (1:1 ratio), purified, and a stock solution of the particles in PBS was prepared using CpG oligodeoxynucleotide (ODN 1668, Enzo) as an adjuvant. The stock solution concentration was 10 million particles / μl.

[0257] Healthy mice (C57BL / 6 mice) were administered a low dose (1 μl of phagocytic particles in 9 μl PBS) or a high dose (10 μl of phagocytic particles) of phagocytic particles by injection into the inguinal lymph nodes or subcutaneously. Each dose and route of administration was evaluated in triplicate (n=3). The phagocytic particle stock samples used to prepare the low and high doses contained 10 million beads / μl. Therefore, the low dose contained approximately 10 million phagocytic particles, while the high dose contained approximately 100 million phagocytic particles. Each dose of phagocytic particles contained two different groups of phagocytic particles: 1) phagocytic particles comprising a polystyrene particle core and comprising the neoantigen construct M272120 (SEQ ID NO: 1) tightly bound to the core; and 2) phagocytic particles comprising a polystyrene particle core and the neoantigen construct M304748 (SEQ ID NO: 2) tightly bound to the core.

[0258] The mice were given a first dose of the phagocytic particles on day 1, and then a second dose was given to the same mice approximately one month later (33 days later). Blood samples were collected from the mice approximately three weeks after the first dose (22 days) and approximately three weeks after the second dose (23 days).

[0259] The health of mice that received two doses of phagocytic particles via inguinal lymph node injection was also evaluated. The inventors found that injecting 2 x 10 μl of phagocytic particles into the inguinal lymph nodes did not affect the health of the animals (weight and overall health). The inguinal lymph nodes and the mouse spleens also showed no macroscopic abnormalities, indicating that the mice tolerated the phagocytic particles well.

[0260] The collected blood samples were analyzed using an enzyme-linked immunosorbent assay (ELISA). The assay was performed in 96-well plates. The plates were coated with the neoantigen construct M272120 (SEQ ID NO: 1) or M304748 (SEQ ID NO: 2) by adding 100 μl of the neoantigen construct at a concentration of 5 μg / ml in PBS. The plates were incubated overnight before washing. The coated 96-well plates were then incubated with 100 μl of diluted (1: 1000) mouse serum harvested from the mice after administration of the first or second dose of phagocytic particles. The plates were then washed and then incubated with an anti-mouse IgG-HRP secondary antibody (Jackson Labs, 1: 8000 dilution). Finally, the plates were washed and then developed with TMB substrate solution (Sigma-Aldrich) and the absorbance was measured at 370 and 650 nm. The absorbance of each mouse serum sample for each neoantigen construct is shown, Figure 7A(serum harvested from mice after the first dose; neoantigen construct = M272120 (SEQ ID NO: 1)), Panel B (serum harvested from mice after the first dose; neoantigen construct = M304748 (SEQ ID NO: 2)), Figure 7C (Serum harvested from mice after the second dose; neoantigen construct = M272120 (SEQ ID NO: 1)), Figure 7D (Serum harvested from mice after the second dose; neoantigen construct = M304748 (SEQ ID NO: 2)). As control samples, serum samples obtained from naive mice (n = 3, not administered any dose of phagocytosable particles) were also analyzed. Figure 7A 、 7B , 7C, and 7D, mice that received a high dose of phagocytosable particles (administered via lymph nodes or subcutaneously) had a greater proportion of anti-neoepitope antibodies in serum samples compared to serum samples harvested from mice that received a low dose of phagocytosable particles by the same route and from mice that did not receive a dose of phagocytosable particles (i.e., naive mice).

[0261] Approximately 2 months (54 days) after the second dose of phagocytic particles, mice were injected subcutaneously with 500,000 cells of the melanoma cancer cell line B16-F10. Tumor volume was measured daily. Figure 8 Shown is the increase in tumor volume over time (days, D).

[0262] Example 5: Preliminary study on in vitro expansion of anti-cancer T cells using phagocytic particles:

[0263] (i) Identification of neoepitope targets in bladder cancer

[0264] Bladder cancer exhibits a high mutation rate and therefore expresses a large number of neoepitopes that may be recognized as non-self by the immune system. Thus, the present inventors investigated tumor polymorphisms suitable as neoepitope peptides and T cell targets by mining a mutation database containing a large reservoir of potential neoepitopes that could be used to expand T cells for immunotherapy.

[0265] The COSMIC database contains mutation data for 4754 transitional cell carcinomas, including whole exome sequencing and hotspot analysis. The inventors focused on bladder cancer (UBC) and selected the 15 most common mutations that result in single amino acid mutations, particularly substitution mutations, and therefore qualify as new epitopes. The inventors also focused on polymorphisms in genes known to be involved in tumor pathogenesis, such as kinases, growth factor receptors, and cell cycle proteins. Only the 15 mutations selected covered 73% of the bladder cancer mutations found in COSMIC. The new epitope peptides identified from the COSMIC database are referred to as "predicted new epitope peptides" in this example.

[0266] As an alternative, whole genome sequencing of tumors from UBC patients was performed to identify additional polymorphisms and new targets for immunotherapy. In RNA, tumors can be sequenced to verify the presence of transcripts carrying polymorphisms. Multiple reaction monitoring (MRM) mass spectrometry conversion can be used to rapidly scan patients for expression of the most common neoepitopes at the protein level, thereby customizing neoepitope peptides for individual immunotherapy. Neoepitope peptides identified using this method are referred to in this example as "personalized neoepitopes."

[0267] The neoantigen construct was designed as a 21-amino acid peptide, in which the somatically mutated amino acid was located in the center of the sequence (i.e., at amino acid position 11). To design the neoantigen construct, three neoepitope peptides were linked with two VVR spacers, because after translation, the VVR motif is cleaved in lysosomes by cathepsin S, which is a step in human leukocyte antigen presentation.

[0268] (iii) Neo-epitopes activate and expand T cells

[0269] As described above, nine new epitopes were identified by bioinformatics. The predicted new epitope peptides were based on genes with reported bladder cancer-associated mutations (such as FGFR3 and p53). In a pilot experiment using a new antigen construct containing three new epitope peptides, the inventors were able to identify IFN-γ-producing T cells from the blood of patients with bladder cancer by FluoroSpot, demonstrating the effectiveness of the new epitope peptide method.

[0270] For the same patient with bladder cancer, T cell activation was performed using predicted neo-epitope peptides NA1-9 (SEQ ID NOs: 4-12, see Table 4 below). Proliferation was observed in response to NA 1, 3, 5, 7, and 8 (SEQ ID NOs: 4, 6, 8, 10, and 11). Figure 2 A shows the time course of cell number in PBMC cultures (PB = peripheral blood) after incubation with APCs contacted with phagocytosable particles containing predicted neoepitope peptides (NA1-9) linked to polystyrene particles. Figure 2 The arrow in A indicates the time of restimulation (i.e., the time when the T cell sample was contacted with a second batch of APCs, which were contacted with phagocytable particles containing predicted neo-epitope peptides (NA1-9) under conditions that allow specific activation of anti-cancer T cells in response to the neo-epitopes presented by the APCs). Figure 2 B shows % CD4+ / total T cells. Figure 2 C shows T-bet expression in CD4, Figure 2 D and E show the expression of granzyme B and perforin in CD8 + T cells.

[0271] The expanded T cells express the transcription factor T-bet and high levels of the effector molecules perforin and granzyme B (GZB).

[0272] The methods of the present invention have also been used on cells from a patient with sporadic colon cancer from whom sequenced tumor data were 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, a personalized neoantigen construct comprising three neoantigen constructs and having an amino acid sequence according to SEQ ID NO: 3 was designed, expressed and purified, allowing the identification of personalized neo-epitopes. Following the procedures outlined in Examples 1 and 2, phagocytic particles were prepared by coupling the neoantigen constructs to polystyrene particles. The phagocytic particles comprising the personalized neoantigen construct (SEQ ID NO: 3) were used to expand cells, resulting in a neo-epitope-specific response. Peripheral blood mononuclear cells (PBMCs) were used for culture. Figure 3 Shown are the percentage (small squares) and total number (large squares) of CD4+ T cells in T cells, as well as proliferating CD4+ cells (circles) in T samples before and during expansion with phagocytosable particles consisting of a polystyrene particle core and a personalized neoantigen construct tightly associated with the core.

[0273] Figure 4 Figure A shows the change in cell number in PBMC cultures over time (days). The top row (Pat 2 personalized NA) shows the number of cells in PBMC cultures that were in contact with phagocytic particles containing personalized neoantigen constructs (SEQ ID NO: 3) after incubation. The bottom two rows of the graph (Pat 2 NA 1+3 and Pat 2 NA 4+5) show the number of cells in parallel PBMC cultures that were in contact with phagocytic particles of NA1 and NA3 or NA4 and NA5. Figure 4 B shows % CD4+ / total T cells. Figure 4 The top row in B is for the 14-day time point, followed by a Pat 2 personalized NA experiment. Figure 4 Arrows in A and 4B indicate the time of restimulation (i.e., the time at which the T cell sample was contacted with a second batch of APCs that were contacted with phagocytosable particles under conditions that allow specific activation of anticancer T cells in response to neoepitopes presented by the APCs). Figure 4 C visualizes the analysis of CD4+ T cells using the Barnes-Hut Stochastic Neighbor Embedding (BH-SNE) algorithm, where all cells in a sample are clustered on a 2D plot based on the similarity of their expression intensities according to a set of selected markers: here CD28, CD57, T-bet, GATA-3, perforin, granzyme B (GZB), Ki-67, and PD-1.

[0274] During the 14-day culture period, expression of the proliferation marker Ki67 and the number of T cells increased during expansion, while expression of important markers of antitumor activity, such as T-bet, perforin, and granzyme B, increased in both CD4+ and CD8+ cells. The percentage of CD8+ T cells decreased to approximately 10% of CD4+ T cells, but the total number of CD8+ T cells increased.

[0275] The entire process, from receiving sequence data to analyzing expanded cells, can be completed within 4-5 weeks.

[0276] These results indicate that there is a neo-epitope-specific T cell response. Thus, the inventors demonstrated that predicted and personalized neo-epitope peptides can be designed and used for T cell activation and expansion.

[0277] The T cell expansion method of this embodiment and the T cells expanded using the expansion method of this embodiment can be used in the uses and methods for treating and preventing cancer described herein.

[0278] Table 4:

[0279]

[0280] Example 6: Tumor Mouse Model Evaluating Tumor Growth Following Vaccination with Phagocytic Particles Conjugated to MC38 Colorectal Tumor-Specific Neoantigens

[0281] Materials and methods

[0282] In this study, an phagocytic particle composition comprising six different phagocytic particle sets (referred to herein as the "MC38 particle composition") was used. Each particle set comprised a polystyrene particle core (Sera-Mag SpeedBeads (Hydrophilic) Carboxylate-Modified Magnetic Particles, GE Healthcare) coupled to one of the following types of MC38 neoantigen constructs: 1) SEQ ID NO: 13, 2) SEQ ID NO: 14, 3) SEQ ID NO: 15, 4) SEQ ID NO: 16, 5) SEQ ID NO: 17, or 6) SEQ ID NO: 18 (see Table 5).

[0283] Table 4:

[0284]

[0285]

[0286] Each MC38 neoantigen construct contains six 20-23 amino acid neoantigen peptide sequences derived from the MC38 colon cancer cell line. MC38 neoantigen constructs were recombinantly expressed in E. coli and purified using column chromatography.

[0287] MC38 particles were prepared according to the protocol outlined in Example 1. Once prepared, the six sets of phagocytosable particles were mixed and then sterilized using the protocol described in Example 2. A stock solution of the MC38 particle composition was prepared at a concentration of 10 million particles / μl in PBS.

[0288] Test mice (n=5) received the first and second doses of the MC38-particle composition. The total volume of each dose was 10 μl (9.5 μl of MC38 particle composition stock solution and 0.5 μl of 0.05 nmol CpG oligodeoxynucleotide (ODN1668, Enzo) as an adjuvant). Control mice did not receive treatment (n=5, non-vaccinated group).

[0289] Five days before transplantation of MC38 tumor cells ( Figure 9 Time point A) injection of the first dose of phagocytic particles (MC38 particles). Figure 9 B) Implantation of MC38 tumor cells. 6 MC38 tumor cells were injected into each mouse to achieve transplantation. Thirteen days after transplantation, the mice were injected with a second dose ( Figure 9 The tumor volume of each mouse was measured during the study (see Figure 9 ).

[0290] result

[0291] The results are as follows Figure 9 As shown. Figure 9 As can be seen, mice that received MC38 particles ("vaccinated" mice) had significantly reduced tumor growth compared to unvaccinated mice ("negative control" mice). These results suggest that MC38 particles can effectively induce an anti-cancer immune response, which inhibits tumor growth in the MC38 colorectal tumor mouse model.

[0292] Example 7: Toxicity and biodistribution studies:

[0293] To assess the maximum tolerated dose of phagocytosable particles, the toxicity and biodistribution characteristics of phagocytosable particle cores were evaluated using a rat model.

[0294] Materials and methods

[0295] Particles used in the study: Sera-Mag SpeedBead carboxylate-modified magnetic particles (hydrophilic). These particles were provided by GE Healthcare Life Sciences (particle batch / lot number GE: 16807675, concentration: 2.3% solids (g / 100 g) (equivalent to 10 mg Fe / mL) in Dulbecco's phosphate buffered saline, pH 7.1). The particles were stored at 2-8°C until use.

[0296] Animal Details: Wistar rats. Upon arrival at the study site, rats weighed approximately 250 g. Rats were acclimated for at least 5 days prior to the start of the study. Rats were individually housed in ventilated cages (IVC type 4) at +22°C ± 3°C, 50% ± 20% humidity, and a 12-hour light / 12-hour dark cycle. Food and water were available ad libitum. Three rats were housed per cage.

[0297] Study 1):

[0298] Five female Wistar rats were used in the pilot study. Rat #1 was injected intravenously (IV) with the maximum feasible concentration of particles (50 mg / kg iron at a concentration equivalent to 5 mg / kg) and then placed in a computed tomography (CT) camera to acquire images. The intravenous injection should be performed as slowly as possible. If there is a significant toxic reaction, the particles are delivered to the remaining rats by slow infusion over 20 minutes (maximum 20 mL / kg). The concentration of particles delivered to the rats is titrated until the maximum tolerated dose is determined. Rats that did not receive a dose of particles serve as negative controls and are used to acquire background CT scans.

[0299] After intravenous injection / infusion of particles, rats were anesthetized with isoflurane and eye ointment was placed on the eyes to prevent dehydration. The rats were then placed on a heated bed of a CT instrument and maintained inhalation anesthesia. During the experiment, vital parameters (pulse and respiration) were monitored using a respiratory sensor and a rectal thermometer. The rats were inserted into a CT camera and images were acquired over a period of approximately 20 minutes. The health status after intravenous injection of particles was recorded to assess possible toxic reactions, and whole-body CT images were used to assess the visibility of the particles and to determine the position of the particles after injection. After acquiring the CT images, the rats were euthanized.

[0300] Study 2):

[0301] A further study was conducted using twelve rats to determine the rate of particle elimination. All rats received intravenous injections of particles at the dose determined in the pilot study. Immediately after particle administration, the rats were placed in a CT camera. Thereafter, the rats were monitored in the CT camera 24 hours, 3 days, 7 days, 14 days, and 1 month after administration. At each time point, two rats were euthanized and their organs removed for histopathological analysis. The health and weight changes of the rats were monitored 24 hours, 3 days, and 7 days after administration, and thereafter weekly.

[0302] Example 7: Example of a sterilization protocol for phagocytable particles using Bacillus subtilis.

[0303] The experiment was performed under sterile conditions in a laminar air flow (LAF) safety cabinet. The phagocytic particles containing the core (Sera-Mag SpeedBeads carboxylate-modified magnetic particles, GE Healthcare) connected to the neoantigen construct were washed four times with a high concentration of alkaline solution (2M to 5M NaOH). After the first wash, the phagocytic particles were transferred to a new sterile test tube, the supernatant was removed, and a second volume of alkaline solution was added. The phagocytic particles were sonicated in an ultrasonic bath for 10 minutes and then incubated in the same alkaline solution for 30 minutes with rotation upside down. The process was repeated two more times and then washed four times with sterile Dulbecco's modified PBS.

[0304] By spiking phagocytic particles (Sera-Mag SpeedBeads carboxylic acid-modified magnetic particles without attached neoantigens) with a high load (>1.2 CFU) of Bacillus subtilis sp. spizizenii ( 6633 TM Epower106 CFU) were then subjected to the above-described NaOH treatment protocol to evaluate the effectiveness of the NaOH treatment protocol. After NaOH treatment, the complete bead suspension and the supernatant of the untreated (positive control) samples relative to the 5M or 2M NaOH-treated samples were plated on nutrient agar without antibiotics and incubated at 37°C overnight (>16 hours).

[0305] result

[0306] Both 5M and 2M NaOH treatments effectively eliminated bacterial growth, while colonies of B. subtilis subsp. steierensis flourished without washing. In conclusion, 2M and 5M NaOH treatments were highly effective in removing artificially high bioburden from phagocytable particles. 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<210> 3 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> Neoantigen construct <400> 3 Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro 1 5 1-5 10 15 Ala Ala Pro Thr Pro Val Val Arg Gln Ser Gln His Met Thr Glu Val 20 25 30 Val Arg His Cys Pro His His Glu Arg Cys Ser Asp Ser Val Val Arg 35 40 45 Cys Ala Thr Tyr Val Asn Val Asn Ile Arg Asn Ile Asp Lys Ile Tyr 50 55 60 Val Arg Thr Gly 65 <210> 4 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> Neoantigen Constructs <400> 4 Ala Gln Thr Tyr Thr Leu Asp Val Leu Glu Arg Cys Pro His Arg Pro 1 5 10 15 Ile Leu Gln Ala Gly Leu Val Val Arg Ser Thr Arg Asp Pro Leu Ser 20 25 30 Glu Ile Thr Lys Gln Glu Lys Asp Phe Leu Trp Ser His Arg Val Val 35 40 45 Arg Leu Val Glu Ala Asp Glu Ala Gly Ser Val Cys Ala Gly Ile Leu 50 55 60 Ser Tyr Gly Val Gly Phe Gly Ser 65 70 <210> 5 <211> 72 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 5 Ala Lys Ala Ile Ser Thr Arg Asp Pro Leu Ser Lys Ile Thr Glu Gln 1 5 10 15 Glu Lys Asp Phe Leu Trp Val Val Arg Pro Tyr Asn Tyr Leu Ser Thr 20 25 30 Asp Val Gly Phe Cys Thr Leu Val Cys Pro Leu His Asn Gln Val Val 35 40 45 Arg Arg Gln Thr Tyr Thr Leu Asp Val Leu Glu Cys Ser Pro His Arg 50 55 60 Pro Ile Leu Gln Ala Gly Gly Ser 65 70 <210> 6 <211> 72 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 6 Ala Ala Leu Leu Ala Leu Trp Leu Cys Cys Ala Thr Pro Ala His Ala 1 5 10 15 Leu Gln Cys Arg Asp Gly Val Val Arg Val Lys Glu Gly Trp Leu His 20 25 30 Lys Arg Gly Lys Tyr Ile Lys Thr Trp Arg Pro Arg Tyr Phe Val Val 35 40 45 Arg Glu Tyr Phe Met Lys Gln Met Asn Asp Ala Arg His Gly Gly Trp 50 55 60 Thr Thr Lys Met Asp Trp Gly Ser 65 70 <210> 7 <211> 72 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 7 Ala Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Val Val Arg Glu Glu Glu Leu Val Glu Ala 20 25 30 Asp Glu Ala Cys Ser Val Tyr Ala Gly Ile Leu Ser Tyr Gly Val Val 35 40 45 Arg Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Lys Glu Glu Asn Leu 50 55 60 Arg Lys Lys Gly Glu Pro Gly Ser 65 70 <210> 8 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> Neoantigen Construct <400> 8 Ala Thr Glu Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Val Val Arg Phe Glu Val Arg Val Cys Ala 20 25 30 Cys Pro Gly Thr Asp Arg Arg Thr Glu Glu Glu Asn Leu Arg Val Val 35 40 45 Arg Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser 50 55 60 Ala Met Arg Asp Gln Tyr Gly Ser 65 70 <210> 9 <211> 50 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 9 Ala Met Ala Ser Ala Ala Ala Ala Glu Ala Glu Lys Gly Ser Pro Val 1 5 10 15 Val Val Gly Leu Leu Val Val Gly Asn Ile Ile Ile Leu Leu Ser Gly 20 25 30 Leu Ser Leu Phe Ala Glu Thr Ile Trp Val Thr Ala Asp Gln Tyr Arg 35 40 45 Gly Ser 50 <210> 10 <211> 76 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 10 Ala Cys Phe Gln Gly Leu Leu Ile Phe Gly Asn Val Ile Ile Gly Cys 1 5 10 15 Cys Gly Ile Ala Leu Thr Ala Glu Cys Ile Phe Phe Val Ser Asp Gln 20 25 30 His Ser Leu Tyr Pro Leu Leu Glu Ala Thr Asp Asn Asp Asp Ile Tyr 35 40 45 Gly Ala Ala Trp Ile Gly Ile Phe Val Gly Ile Cys Leu Phe Cys Leu 50 55 60 Ser Val Leu Gly Ile Val Gly Ile Met Lys Gly Ser 65 70 75 <210> 11 <211> 58 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 11 Ala Thr Leu Pro Leu Ile Leu Ile Leu Leu Ala Leu Leu Ser Pro Gly 1 5 10 15 Ala Ala Asp Phe Asn Ile Ser Ser Leu Ser Gly Leu Leu Ser Pro Ala 20 25 30 Leu Thr Glu Ser Leu Leu Val Ala Leu Pro Pro Cys His Leu Thr Gly 35 40 45 Gly Asn Ala Thr Leu Met Val Arg Gly Ser 50 55 <210> 12 <211> 62 <212> PRT <213> Artificial sequence <220> <223> Neoantigen Constructs <400> 12 Ala Phe Gly Ser Ala Val Asn Leu Gln Pro Gln Leu Ala Ser Val Thr 1 5 10 15 Phe Ala Thr Asn Asn Pro Thr Leu Thr Thr Val Ala Leu Glu Lys Pro 20 25 30 Leu Cys Met Phe Asp Ser Lys Glu Ala Leu Thr Gly Thr His Glu Val 35 40 45 Tyr Leu Tyr Val Leu Val Asp Ser Ala Ile Ser Arg Gly Ser 50 55 60 <210> 13 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Neoantigen Construct <400> 13 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu [[ID=4)]44]]85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Tyr Glu Gly Asp Gly 130 135 140 Gly Asp Ala Ser Arg Val Leu Glu Asp Ser Asn Ile Ser Tyr Gly Ser 145 150 155 160 Gly Gly Ser Arg Glu Pro Val Ala Ala Thr Trp Glu Ala Ser Trp Ser 165 170 175 Glu Gly Ser Lys Ser Leu Asp Ser Gly Gly Ser Lys Ala Thr Gly Ser 180 185 190 Pro Thr Pro Arg Ile Asn Trp Leu Lys Gly Gly Arg Pro Leu Ser Leu 195 200 205 Gly Gly Ser Thr Ser Trp Leu Met Leu Pro Asp Gly Ile Asn Val Glu 210 215 220 Val Ile Val Val Asn Gln Val Asn Gly Gly Ser Tyr Ile Leu Leu Val 225 230 235 240 Gly Tyr Pro Pro Phe Cys Asp Glu Asp Gln His Lys Leu Tyr Gln Gln 245 250 255 Gly Gly Ser Asp Gly Asn Asn Asn Leu Glu Asp Asp Ser Ile Val Ser 260 265 270 Glu Asp Leu Asp Val Asp Trp Ser Ser Gly 275 280 <210> 14 <211> 282 <212> PRT <213> Artificial sequence <220> <223> Neoantigen construct <400> 14 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu 85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Ser Gln Gly Glu Leu 130 135 140 Ile His Pro Lys Ala Phe Pro Leu Ile Val Gly Ala Gln Leu Ile His 145 150 155 160 Gly Gly Ser Lys Arg Lys Glu Gln Glu Ala Gln Glu Glu Lys Arg Arg 165 170 175 Lys Gln Arg Glu Ala Gln Ala Trp Gly Gly Ser Met Gly Pro Gly Ala 180 185 190 Gly Arg Pro Trp Pro Ser Pro Asn Ser Ala Asn Ser Ile Pro Tyr Ser 195 200 205 Gly Gly Ser Asp Arg Val Pro Asn Val Arg Val Leu Leu Thr Lys Thr 210 215 220 Leu Arg Gln Thr Leu Leu Glu Lys Gly Gly Ser Ile Ser Leu Ala Phe 225 230 235 240 Phe Glu Ala Ala Ser Ile Met Arg Gln Val Ser His Lys His Ile Val 245 250 255 Gly Gly Ser Ser Asn Tyr Gln Leu Gly Glu Leu Val Lys Leu Glu Asn 260 265 270 Tyr Pro Asp Val Ile Arg Leu Ile Ser Gly 275 280 <210> 15 <211> 281 <212> PRT <213> Artificial sequence <220> <223> Neoantigen construct <400> 15 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu 85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Lys Val His Ala Val 130 135 140 Phe Leu Asp Gly Val Lys Val Thr Leu Asn Trp His Leu Ser Ser Ser 145 150 155 160 Gly Gly Ser Met Met Leu Gly Pro Glu Gly Gly Glu Ser Tyr Val Val 165 170 175 Lys Leu Arg Gly Leu Pro Trp Gly Gly Ser Lys Gly Thr Ile Val Ala 180 185 190 Gln Val Asp Ser Ile Glu Ser Phe Gln Glu Phe Cys Ser Thr Ser Gly 195 200 205 Gly Ser Lys Tyr Met Cys Asn Ser Ser Cys Met Gly Val Met Asn Arg 210 215 220 Arg Pro Ile Leu Thr Ile Ile Gly Gly Ser Glu Glu Lys Gln Ala Ala 225 230 235 240 Lys Lys Arg Lys Leu Glu Glu Ser Val Glu Gln Lys Arg Ser Lys Gly 245 250 255 Gly Ser Thr Trp Ser Leu Ala Ser Ile Thr Tyr Trp Arg Pro Thr Cys 260 265 270 Ala Asn Thr Val Ser Asp Asn Ser Gly 275 280 <210> 16 <211> 282 <212> PRT <213> Artificial sequence <220> <223> Neoantigen construct <400> 16 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu 85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Arg Lys Arg Pro Tyr 130 135 140 Ser Ser Phe Ser Asn Cys Lys Asp His Arg Glu Trp Asp His Tyr Arg 145 150 155 160 Gly Gly Ser Arg Asn Val Met Cys Lys Lys Asp Ser Pro Leu Arg Thr 165 170 175 Thr Thr Ile Val Pro Pro Val Glu Gly Gly Ser His Asn Cys Leu Ser 180 185 190 Asp Pro Ala Asp His Arg Arg Leu Thr Glu His Val Ala Lys Ala Phe 195 200 205 Gly Gly Ser Ser Ala Gly Gly Trp Gly Thr Glu Ile Leu Trp Ser Thr ​​​​​​​​​​​​​​​​​​​​​​​​​​<220> <223> Neoantigen construct <400> 17 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu 85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Gly Glu Asp Asn Arg 130 135 140 Pro Gly Met Arg Gly Cys His Gln Met Val Ile Asp Val Gln Thr Glu 145 150 155 160 Gly Gly Ser Gly Gln Ile Gln Glu Glu Ser Glu Gly Ala Arg Phe Lys 165 170 175 Ala Pro Pro Asp Ser Thr Val Ser Gly Gly Ser Ser Val Ala Ala Ala 180 185 190 Ala Ala Ala Ala Val Ser Val Val Glu Ser Met Val Thr Ala Thr Glu 195 200 205 Gly Gly Ser Val Ser His Lys His Ile Val Tyr Leu Tyr Val Val Cys 210 215 220 Val Arg Asp Val Glu Asn Ile Met Gly Gly Ser Glu Tyr Leu Lys Leu[[ID=?]] 225 230 235 240 Leu His Ser Phe Val Tyr Ser Val Gly Phe Val Thr Ser Pro Phe Ser 245 250 255 Gly Gly Ser Asp Ala Val Ala Ser Phe Ala Asp Val Gly Phe Val Ala 260 265 270 Thr Glu Glu Gly Glu Cys Ser Ile Ser Gly 275 280 <210> 18 <211> 282 <212> PRT <213> Artificial Sequence <220> <223> Neoantigen Construct <400> 18 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu 85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Val Val Asp His Arg 130 135 140 Pro Lys Ala Leu Pro Val Gly Gly Phe Ile Glu Glu Glu Lys Asp Glu 145 150 155 160 Gly Gly Ser Lys Gln Asp Glu Tyr His Met Val His Leu Leu Cys Ala 165 170 175 Ser Arg Ser Pro Pro Ser Ser Pro Gly Gly Ser Gly Asp Thr Leu Glu 180 185 190 Glu Ala Phe Glu Gln Ser Ala Met Ala Met Phe Gly Tyr Met Thr Asp 195 200 205 Gly Gly Ser Ile Ser Met Ser Ser Ser Lys Leu Leu Leu Ser Ala Lys 210 215 220 Ala Leu Ser Thr Asp Pro Ala Ser Gly Gly Ser Arg Asp Leu Gly Asp 225 230 235 240 Glu Tyr Gly Trp Lys His Val His Gly Asp Val Phe Arg Pro Ser Ser 245 250 255 Gly Gly Ser Arg Val Ser Leu Ser His Ala Cys Lys Asn Thr Val Lys<000126\0>260 265 270 Thr Asp Ala Pro Pro Glu Ala Leu Ser Gly 275 280<00\01263><210> 19 <211> 257 <212> PRT <213> Artificial Sequence <220> <223> Neoantigen Construct <400> 19 Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Leu Ala 1 5 10 15 Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser 20 25 30 Asp Tyr His Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val 35 40 45 Lys Asp Leu Gln Ala Gln Val Val Glu Ser Ala Lys Lys Ala Arg Ile 50 55 60 Ser Glu Ala Thr Asp Gly Leu Ser Asp Phe Leu Lys Ser Gln Thr Pro 65 70 75 80 Ala Glu Asp Thr Val Lys Ser Ile Glu Leu Ala Glu Ala Lys Val Leu 85 90 95 Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn 100 105 110 Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp 115 120 125 Glu Ile Leu Ala Ala Leu Pro Gly Gly Ser Ala Glu Thr Arg Leu Leu 130 135 140 Gln Thr Gly Ile His Val Arg Val Ser Gln Pro Ser Leu Ile Leu Val 145 150 155 160 Gly Gly Ser Ile Ile Lys Pro Gly Lys Ile Ser His Ile Met Leu Asp 165 170 175 Val Ala Phe Thr Ser His Glu His Phe Gly Gly Ser Trp Pro Pro Trp 180 185 190 Gln Ala Gly Ile Leu Ala Arg Asn Leu Val Pro Met Val Ala Thr Val 195 200 205 Gln Gly Gly Ser Arg Gly Pro Gln Tyr Ser Glu His Pro Thr Phe Thr 210 215 220 Ser Gln Tyr Arg Ile Gln Gly Lys Leu Gly Gly Ser Gln Asn Leu Lys 225 230 235 240 Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp Ile Tyr Arg Ile Phe Ala 245 250 255 Glu

Claims

1. A use of an phagocytic particle in the manufacture of a medicament for treating cancer in a subject, wherein the phagocytic particle comprises a core and a neoantigen construct tightly associated with the core, wherein the core is a polymeric particle, wherein the phagocytic particle is substantially spherical and has a diameter of 0.5 to 2 μm, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence that is a portion of a protein or peptide known or suspected to be expressed by a cancer cell in the subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid.

2. The use according to claim 1, wherein the neoantigen construct comprises two or more covalently linked neoepitope peptides.

3. The use according to claim 2, wherein the neoantigen construct comprises three or more covalently linked neoepitope peptides.

4. The use according to claim 3, wherein the neoantigen construct comprises three, four or five covalently linked neoepitope peptides.

5. The use according to any one of claims 2 to 4, wherein the covalently linked neo-epitope peptide is covalently linked via a spacer moiety. The use according to claim 5 , wherein the spacer portion is a sequence of 1 to 15 amino acids.

7. The use according to claim 6, wherein the spacer portion is a sequence of 1 to 5 amino acids.

8. The use according to claim 6, wherein the spacer portion is a sequence comprising the amino acid sequence VVR and / or the amino acid sequence GGS.

9. The use according to any one of claims 2 to 4, wherein each of the covalently linked neo-epitope peptides is 3 to 25 amino acids in length.

10. The use according to any one of claims 1 to 4, wherein the neoantigen construct is covalently attached to the core.

11. The use according to any one of claims 1 to 4, wherein the phagocytosable particle comprises two or more different neoantigen constructs tightly associated with the core.

12. The use according to claim 11, wherein the phagocytosable particle comprises two, three, four or five neoantigen constructs tightly associated with the core.

13. The use according to claim 11, wherein each different neo-antigen construct comprises a different neo-epitope peptide sequence or a different combination of neo-epitope peptides.

14. The use according to any one of claims 1 to 4, wherein the phagocytosable particles have a diameter of about 1 μm.

15. Use according to any one of claims 1 to 4, wherein the core is paramagnetic or superparamagnetic.

16. The use according to claim 1, wherein the core comprises polystyrene.

17. The use according to any one of claims 1 to 4, wherein the phagocytosable particles are administered with an adjuvant, or comprise an adjuvant tightly associated with the core.

18. The use according to claim 17, wherein the adjuvant comprises IL-2, IL-15, IL-17 and IL-4.

19. The use according to any one of claims 1 to 4, wherein the cancer is a solid cancer.

20. The use according to claim 19, wherein the cancer is selected from breast cancer, colon cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid, pancreatic cancer, prostate cancer, ovarian cancer and bladder cancer.

21. An injectable pharmaceutical composition comprising a phagocytic particle comprising a core and a neoantigen construct tightly associated with the core, wherein the core is a polymeric particle, wherein the phagocytic particle is substantially spherical and has a diameter of 0.5-2 μm, and wherein the neoantigen construct comprises a neoepitope peptide having an amino acid sequence that is a portion of a protein or peptide known or suspected to be expressed by a cancer cell in a subject, wherein the portion of the protein or peptide has at least one somatically mutated amino acid.

22. An injectable pharmaceutical composition according to claim 21, wherein the phagocytosable particles are as defined in any one of claims 2 to 18.

23. Use of the injectable pharmaceutical composition according to claim 21 or 22 in the manufacture of a medicament for treating cancer in a subject.

24. The use according to claim 23, wherein the cancer is a solid cancer.

25. The use according to claim 24, wherein the cancer is selected from breast cancer, colon cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid, pancreatic cancer, prostate cancer, ovarian cancer and bladder cancer.

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