Haptenized whole tumor cell vaccines for cancer immunotherapy

Abstract

This disclosure concerns methods of preparing haptenized cells using squaric esters. Also disclosed herein are immunogenic compositions containing the cells, and methods of preventing and treating cancer using the immunogenic compositions.

Description

[0001] Haptenized Whole Tumor Cell Vaccines for

[0002] Cancer Immunotherapy

[0003] Technical field

[0004] The invention relates generally to the field of immunology. In particular, the specification teaches a method of preparing squarate-haptenized cells using squaric esters, immunogenic compositions containing the cells, and methods of preventing and treating cancer using the immunogenic compositions.

[0005] Background

[0006] Tumor vaccines hold significant potential for transforming oncological therapies by harnessing the body’s immune system. However, their progress has been impeded by challenges such as tumor antigen heterogeneity, inadequate immunogenic epitopes, and mutagenic characteristics. Whole tumor cell vaccines (WTCVs), which encompass a diverse array of tumor antigens, have emerged as promising candidates that can activate innate immunity and subsequently elicit antigen- specific adaptive responses. Despite this promise, clinical trials have shown that WTCVs yield limited objective response rates and only modest improvements in patient survival.

[0007] This limited efficacy is primarily attributed to the ability of tumor cells to evade immune detection through mechanisms such as by downregulating tumor-associated or tumorspecific antigens and blocking antigen presentation. Efforts to improve the immunogenicity of WTCVs have focused on increasing antigen availability and enhancing antigen presentation. Techniques such as CRISPR-mediated genome editing, along with the incorporation of peptide -based, mRNA-based, and DNA-based neoantigens, have been employed to improve antigen display. Additional strategies to enhance antigen presentation involve inducing immunogenic tumor cell death, incorporating pathogen-associated molecular patterns, relying on synergistic interactions with the stimulator of interferon genes (STING) pathway, and promoting endogenous adjuvant generation. Despite these advances, the preparation of WTCVs remains complex and time-consuming (often exceeding 24 h),and frequently requires materials with uncertain side effects (e.g., inorganic nanoparticles, metal-organic frameworks, lentiviral vectors). Furthermore, additional steps are often required to eliminate tumorigenic potential (e.g., freeze-thaw, cryogenic freezing, phototherapy, UV / X-ray irradiation). Consequently, there remains a strong need to develop straightforward, safe, and innovative vaccines that improve antigen recognition and address deficiencies in antigen presentation and sustain antigen- specific immunity.

[0008] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0009] Summary

[0010] Disclosed herein is a method of preparing a squarate-haptenized cell, the method comprising contacting the cell with a squaric ester to immobilise and / or conjugate the squaric ester to the cell.

[0011] Disclosed herein is a cell obtainable according to a method as defined herein.

[0012] Disclosed herein is a cell that is conjugated to a squaric ester.

[0013] Disclosed herein is an immunogenic composition comprising a cell as defined herein and a pharmaceutically acceptable adjuvant.

[0014] Disclosed herein is a cell as defined herein, or an immunogenic composition as defined herein, for use as a medicament.

[0015] Disclosed herein is a method of modulating an immune response in a subject, the method comprising administering a cell as defined herein or an immunogenic composition as defined herein to the subject.

[0016] Disclosed herein is a method of preventing or treating cancer in a subject, the method comprising administering a cell as defined herein or an immunogenic composition as defined herein to the subject.Disclosed herein is a method of preventing or treating cancer in a subject, the method comprising administering a cell as defined herein or an immunogenic composition as defined herein in combination with a chemotherapy regime to the subject.

[0017] Disclosed herein is a cell as defined herein or an immunogenic composition as defined herein for use in preventing or treating cancer in a subject.

[0018] Disclosed herein is the use of a cell as defined herein or an immunogenic composition as defined herein in the manufacture of a medicament for preventing or treating cancer in a subject.

[0019] Brief description of the drawings

[0020] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0021] Figure 1. Fabrication and characterization of squaric esters haptenized WTCVs (SE-Vs) a Schematic illustration of the reaction between squaric esters and amino groups under mild conditions, b Schematic depiction of SE-Vs fabrication, c Diameter measurements obtained from bright- field images of BC, SME- Vs, SEE- Vs, SBE-Vs, and PFA-treated B16F10 cells (n = 100). d SEM images illustrating native B16F10 cells, SME-Vs, SEE-Vs, SBE-Vs, and PFA-treated B16F10 cells following a gradual dehydration process, e Flow cytometry analysis of cells stained with PI post various treatments, f-g In vitro proliferation capacity of native B16F10 cells, SME-Vs, SEE-Vs, and SBE-Vs investigated by (f) MTS and (g) CFSE assay, h In vivo proliferation of 5 x 105luciferase tagged native B16F10 cells, SME-Vs, SEE-Vs, and SBE-Vs indicated by the bioluminescence signals (n = 5). i Quantified the bioluminescence of tumor burden over time, j Survival of mice after various treatments (n = 5). Data are presented as mean ± standard deviation (SD). Statistical significance in (c) was determined using one-way analysis of variance (ANOVA) followed by a Tukey post-hoc test.

[0022] Figure 2. Proteogenomics analysis of SE-Vs. a Schematic illustration of the proteogenomics analysis process, b Venn diagram of the identified proteins, c Differential protein expression analysis showing up- and down-regulated proteins across BC group incomparison to SME- Vs, SEE- Vs, and SBE-Vs group. An adjusted p-value < 0.05 is indicated in red, while an adjusted -valuc > 0.05 is indicated in black, d Relative abundance of selected genes related to the TAAs, TSAs, and DAMPs from BC, SME-Vs, SEE- Vs, and SBE-Vs group, e Relative abundance of Calr in BC, SME-Vs, and SEE- Vs groups collected from the proteogenomics analysis, f Confocal laser scanning microscopy (CLSM) images showing Calr exposure in B16F10 tumor cells following various treatments, g Mean fluorescence intensity of Calr levels after various treatments. Statistical significance in (d) was calculated via a two-tailed Student’s / -test. Statistical significance in (f) was determined using ANOVA followed by a Tukey post-hoc test.

[0023] Figure 3. In vitro studies of antigen-presenting cell activation, a CLSM images and flow cytometry analysis showing the internalization of SE-Vs following incubation with BMDCs. b Quantitative analysis of DC maturation induced by different treatments (n = 3). c TNF-a and d IFN-y content in DC suspensions after different treatments (n = 3). e Quantitative analysis of DC maturation induced by different treatments following lyophilization, storage for seven days, and subsequently rehydration in PBS solution (n = 3). f Flow cytometry analysis of the phagocytic property of macrophages for SME-Vs, SEE-Vs, and SBE-Vs. g Quantitative analysis of macrophage polarization (CD86+CD80+) induced by different treatments (n = 3). h Young’s modulus of native BC, SME-Vs, SEE-Vs, and SBE-Vs (n = 20). i Schematic depiction of the enhanced innate immune response facilitated by SBE-Vs, highlighting their increased hydrophobicity and rigidity. Statistical significance in (b), (c), (d), and (e) was determined using ANOVA followed by a Tukey post-hoc test. Statistical significance in (g) was calculated via a two-tailed Student’s z-test.

[0024] Figure 4. In vivo preventive efficacy of the SE-Vs. a Schematic illustration of SE-Vs mediated CHS response for eliciting an effective immune response, b The size change of nodules after SE-Vs injection, c The ear weights (6 mm diameter) through the central portion of both ears following different treatments, d t-SNE map of immune cells in the nodules, e The relative content of DCs, macrophages, and granulocytes in the nodules (n = 5). f Treatment timeline of SE-Vs mediated preventive treatment, g Tumor growth curves of the tumors after different treatments, h Representative bioluminescence images of tumor in vivo after injection with different formulas, i Tumor growth kinetics in different treatment groups indicated by bioluminescence intensity (n = 5). Statistical significance in (b) and (c) was determined using ANOVA followed by a Tukey post-hoc test.Figure 5. In vivo immunologic mechanism of SE-Vs. a Schematic illustration of the immunologic mechanism of SE-Vs. b Quantification of mature DCs in the spleen after different treatments (n = 3). c Quantification of Ml -type macrophages (gated on F4 / 80+iNOS+) in the tumor after different treatments (n = 3). d Presentative immunofluorescent images of LNs in mice vaccinated with SE-Vs three times, e Representative quantification of spleen-infiltrating CD4+and CD8+T lymphocytes following various treatments (n = 3). f Representative flow cytometric plots and g quantification of memory T lymphocytes (CD44hlghCD62Lhlgh) in the spleen after different treatments (n = 3). ELISA analysis of h TNF-a and i IFN-y after different treatments (n = 3). Statistical significance in (c), (e), (h), and (i) was determined using ANOVA followed by a Tukey post-hoc test. Statistical significance in (b) and (g) was calculated via a two-tailed Student’s z-test.

[0025] Figure. 6 In vivo therapeutic efficacy of SBE-Vs. a Treatment timeline of SBE-Vs mediated therapeutic treatment, b Tumor growth curves of the tumors after different treatments, c Individual tumor growth curves of each group after different treatments, d Representative bioluminescence images of tumor in vivo after injection with living B 16F10-Luc cells on day 4. e Representative bioluminescence images of tumor after vaccination with different formulas, f Survival rates of mice in different groups (n = 5). Statistical significance in (b) was calculated via a two-tailed Student’s z-test.

[0026] Figure 7. Optimized biologically compatible haptenization renders cancer cells intact and discrete, a Bright-field images of B16F10 cells following different treatments as indicated, b Bright-field images of B16F10 cells following haptenization using variable concentrations of SADME, SADEE, and SADBE.

[0027] Figure 8. Cell haptenization prevents hypotonic swelling and osmotic lysis, a Bright-field images of different pH or PFA-treated B16F10 cells following incubation in water for 24 h. b Bright-field images of haptenized B16F10 cells following incubation in water for 24 h.

[0028] Figure 9. Size distribution of SE-Vs. Size distribution of a BC, b SME-Vs, c SEE- Vs, d SBE-Vs, or e PFA-fixed B16F10 cells collected from Figure 7 (n = 100 / condition). f Sizedistribution of native BC, SME-Vs, SEE-Vs, SBE-Vs, and PFA-treated B16F10 cells calculated from bright- field images.

[0029] Figure 10. CLSM images of SDB-Vs doping with 1% SBECy.

[0030] Figure 11. Bright- field images of SE-Vs following incubation in water for variable lengths of time as indicated.

[0031] Figure 12. Cell viability of different concentrations of SADME, SADEE, and SADBE-treated B 16F10 cells (pH 9.0, 154 mM NaCl) for 2 h.

[0032] Figure 13. Bright- field images of SE-Vs underwent initial dehydration through lyophilization followed by rehydration in phosphate-buffered saline (PBS).

[0033] Detailed description

[0034] The present specification provides squaric esters haptenized WTCVs (SE-Vs), compositions comprising SE-Vs and methods of treating or preventing cancer. Provided herein is a cell that is conjugated to a squaric ester. Provided herein is a cell that has been modified by a squaric ester.

[0035] Without being bound by theory, the inventors have found that the SE-Vs as referred to herein are safer than existing WTCVs, due to the use of FDA-approved drugs. The preparation time is shortened to about 2 hours and storage time is maintained for up to 7 days. Moreover, the establishment of an immune niche and the ability to recruit macrophages improved antigen presentation efficiency, resulting in enhanced immunotherapy.

[0036] Method of preparation

[0037] Disclosed herein is a method of preparing a squarate-haptenized cell, the method comprising contacting the cell with a squaric ester to immobilise and / or conjugate the squaric ester to the cell.In some embodiments, the method comprises conjugating a cell to the squaric ester, and isolating the cell that has been conjugated to the squaric ester.

[0038] In one embodiment, the squaric ester is represented by the following structure:

[0039]

[0040] wherein Ri and R2 are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl group.

[0041] In one embodiment, Ri and R2 are independently selected from:

[0042]

[0043] In one embodiment, Ri and R2 are independently selected from:

[0044]

[0045] In one embodiment, the squaric ester is a compound selected from:

[0046] >

[0047]

[0048] In one embodiment, the squaric ester is a compound of Formula (III) (squaric acid dimethyl ester, SADME), Formula (IV) (squaric acid diethyl ester (SADEE), or Formula (VI) (squaric acid dibutyl ester, SADBE).

[0049] In one embodiment, the squaric ester is provided at a concentration of about 0.1 mM to about 5 mM. The squaric ester may be provided at a concentration of about 0.1 mM to about 1 mM or any value or sub-range within this range. The squaric ester may be provided at a concentration of about 0.1 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, or about 5 mM.

[0050] In one embodiment, the contacting is performed at a pH of about 7 to about 10. The contacting may be performed at a pH of about 7, about 8, about 9, or about 10.In one embodiment, the cell is contacted with the squaric ester for duration of at least 1 hr. For example, the cell may be contacted with the squaric ester for duration of about 1 hr, about 2 hrs, or about 3 hrs.

[0051] Types of Cells

[0052] In one embodiment, the cell is a tumor cell (such as a mammalian tumor cell), immune cell (such as a mammalian immune cell), or bacteria cell. The cell can be a stem cell. The cell may be a live cell, attenuated cell, dead or inactivated cell. In one embodiment, the cell is a whole cell or intact cell. In one embodiment, the cell is a whole tumor cell.

[0053] The methods as defined herein may comprise selection of isolation or selection of the appropriate cells. For example, the methods as defined herein may comprise isolation or selection of a whole or intact cell. The isolation and selection of cells may be performed using techniques that are well known in the art (such as using Fluorescence- Activated Cell Sorting (FACS) or Magnetic-Activated Cell Sorting (MACS)).

[0054] Tumor cells used for vaccination could be sourced from the patient’s tumors (i.e., autologous tumor cells), other patient’s tumors (i.e. allogeneic tumor cells), orthotopically implanted tumors, or laboratory-cultured cell lines.

[0055] The term “tumor,” as used herein, refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term “precancerous” refers to a condition or a growth that typically precedes or develops into a cancer. By “non-metastatic” is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is a Stage 0, 1, or II cancer, and occasionally a Stage III cancer. By “early stage cancer” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, 1, or II cancer. The term “late stage cancer” generally refers to a Stage III or Stage IV cancer, but can alsorefer to a Stage II cancer or a substage of a Stage II cancer. One skilled in the art will appreciate that the classification of a Stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer.

[0056] In one embodiment, the tumor cell is from a tumor selected from the group consisting of melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, pancreatic cancer, glioblastoma, astrocytoma, renal cell carcinoma, esophageal cancer, ovarian cancer, bladder cancer and thyroid cancer.

[0057] The tumor cell types represent melanoma (B16F10, A375, SK-MEL-28, WM266-4); breast cancer (MCF-7, T47D, MDA-MB-231, 4T1); lung cancer (A549, H1299, H460, LLC); colorectal cancer (HCT116, SW480, HT29, CT26); prostate cancer (PC-3, LNCaP, DU145); liver cancer: (HepG2, Huh7, Hep3B); pancreatic cancer (PANC-1, MiaPaCa-2, BxPC-3); glioblastoma (U87MG, U251, LN229); astrocytoma (U373, A172); renal cell carcinoma (Ishikawa, HEC-l-A, KLE); esophageal cancer (KYSE-30, TE-1, OE33, FLO-1); ovarian cancer (SKOV-3, OVCAR-3, A2780); bladder cancer (T24, RT4, 5637); thyroid cancer (BCPAP, TPC-1, SW1736, C643).

[0058] Immune cells represent dendritic cells; macrophages; monocytes; B cells; T cells; NK cells; neutrophils; mast cells; innate lymphoid cells; and plasma cells. In one embodiment, the immune cell is selected from the group consisting of a dendritic cell, a macrophage, a monocyte, a B cell, a T cell, an NK cell, a neutrophil, a mast cell, an innate lymphoid cell and a plasma cell.

[0059] Bacteria can also be used as carriers to deliver epitopes to a subject. Typically the bacteria used are mutant or recombinant. The bacterium is optionally attenuated. For instance, a number of bacterial species have been developed for use as vaccines and can be used in the present invention, including, but not limited to, Shigella flexneri, E. coli, Listeria monocytogenes, Yersinia enterocolitica, Salmonella typhimurium, Salmonellatyphi or mycobacterium. The bacterial vector used in the immunogenic composition may be a facultative, intracellular bacterial vector. The bacterium may be used to deliver a polypeptide described herein to antigen-presenting cells in the host organism.

[0060] Modified Cells

[0061] In one embodiment, the cell is a modified cell. The cell may be modified to encode or present one or more tumor antigen proteins or tumor associated proteins. The tumor antigen proteins or tumor associated proteins may be capable of modulating an immune response in a subject.

[0062] In one embodiment, the modified cell comprises the following:

[0063] >

[0064]

[0065] wherein Pi and / or P2 is a cell surface protein (such as an integral membrane protein or peripheral membrane protein) that is present on the outer surface of a cell, and wherein R is as defined above.

[0066] The integral membrane protein may, for example, refer to a transmembrane protein or a monotopic protein. The peripheral membrane protein may be a protein that is loosely attached to the cell membrane’s surface.

[0067] In one embodiment, the modified immune cell results in an increase in immune response as compared to an unmodified cell. The increase in immune response may be an increase in antigen internalization, cytokine secretion, DC maturation and / or macrophage polarization. In one embodiment, the modified cell elicits an increase in CHS response as compared to a non-modified cell. This may promote immune cell recruitment and improved antigen crosspresentation, thereby eliciting robust antigen- specific adaptive immune responses and establishing immune memory.

[0068] Plasmids and viral vectors, for example, can be used to express a tumor antigen protein in a host cell. The host cell may be any prokaryotic or eukaryotic cell. Thus, for example, anucleotide sequence derived from the cloning of the tumor antigen protein, encoding all or a selected portion of the full-length protein, can be used to produce a recombinant form of the tumor antigen protein via cellular processes. The coding sequence can be ligated into a vector and the loaded vector can be used to transform or transfect hosts, either mammalian or bacterial cells. Such techniques involve standard procedures which are well known in the art.

[0069] A “cancer antigen” or “tumor antigen” herein refers to an abnormally expressed antigen in a cancer or tumor cell. Antigens which are overexpressed or aberrantly expressed in cancer cells or tumors but which are also present in normal tissues are termed cancer-associated antigens or tumor-associated antigens (TAAs). Antigens which are uniquely expressed by cancer or tumor cells and are absent from normal tissues are known as cancer-specific antigens or tumor- specific antigens (TSAs). Cancer- specific antigens may be neoantigens arising from genetic mutations in cancer cells. Cancer-associated antigens and cancerspecific antigens may be recognised by the immune system and may serve as targets for cancer immunotherapies, vaccines, and diagnostic applications.

[0070] The cells as defined herein (such as a mammalian tumor cell or mammalian immune cell or bacterial cell) may be modified to express, for example, tumor-associated antigens (e.g., gplOO, MART-1, TRP1, TRP2, HER2 / neu, CEA, AFP, PSA, MUC1), cancer / testis antigens (e.g., NY-ESO-1, MAGE-A family members), tumor- specific neoantigens (e.g., mutant KRAS, mutant p53, EGFRvIII), or other immunogenic tumor proteins (e.g. survivin, WT1, or HPV E6 / E7).

[0071] The cells as defined herein (such as 4T1 cells) may be modified to express transcriptional regulators (such as C / EBP and PU.l). The cell as defined herein may be modified to express transcriptional regulators to induce expression of co-stimulatory molecules (such as CD40, CD80 / CD86) and MHC-I.

[0072] The cells as defined herein (such as bacterial cells including E. coli, attenuated Listeria, or Salmonella) may be modified to express tumor- associated antigens such as gplOO, TRP2, CEA, HER2, or neoantigens such as mutant KRAS or p53.Typically, expression vectors used for expressing a polypeptide, in vivo or in vitro contain a nucleic acid encoding an antigen polypeptide, operably linked to at least one transcriptional regulatory sequence. Regulatory sequences are art-recognized and can be selected to direct expression of the subject proteins in the desired fashion (time and / or place).

[0073] The term “polynucleotide” or “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0074] The terms “polypeptide,” “proteinaceous molecule”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

[0075] By “vector” is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0076] As used herein, the terms “encode”, “encoding” and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to “encode” a polypeptide if it can be transcribed and / or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and / or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms “encode”, “encoding” and the like include a RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting from transcription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0077] Compositions

[0078] Disclosed herein is a cell obtainable according to a method as disclosed herein. Also disclosed herein is a cell prepared using a method as disclosed herein. Also disclosed herein is an immunogenic composition comprising a cell that has been prepared using a method disclosed herein. The immunogenic composition may comprise a pharmaceutically acceptable adjuvant.

[0079] Disclosed herein is an immunogenic composition comprising a cell that has been conjugated to a squaric ester as defined herein. The immunogenic composition may comprise a pharmaceutically acceptable adjuvant. In one embodiment, the immunogenic composition is a vaccine composition. In one embodiment, the immunogenic composition is a whole tumor cell vaccine (WTCV) composition.Provided is a method of preparing an immunogenic composition as defined herein. The method may comprise a method of preparing a squarate-haptenised cell. The method may further comprise isolation or selection of a whole or intact cell. The isolation or selection step may be performed prior to or following the step of contacting the cell with a squaric ester.

[0080] The preparation of immunogenic compositions and vaccines is known to one skilled in the art. The immunogenic composition may comprise a pharmaceutically acceptable carrier or excipient. A pharmaceutically acceptable carrier or excipient according to the present invention can be any solvent or dispersing medium etc., commonly used in the formulation of pharmaceuticals and immunogenic compositions to enhance stability, sterility and deliverability of the active agent and which does not produce any secondary reaction, for example an allergic reaction, in a subject. The excipient is selected on the basis of the pharmaceutical form chosen, the method and the route of administration. Appropriate excipients, and requirements in relation to pharmaceutical formulation, are described in Remington’s Pharmaceutical Sciences (19thEdition, A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995)). For example, E proteins may be encapsulated in liposomes for administration.

[0081] An immunogenic composition of the present disclosure may optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris-(hydroxymethyl) aminomethane and / or urea. In addition, the immunogenic composition may optionally comprise pharmaceutically acceptable additives including, for example, diluents, binders, stabilisers, and preservatives.

[0082] As used herein, the term “adjuvant” refers to a compound that, when used in combination with a specific immunogen (such as a cell that has been conjugated to a squaric ester) in a formulation, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both humoral and cellular immune responses. Modification of the immuneresponse can also mean decreasing or suppressing certain antigen- specific immune responses.

[0083] The inclusion of any adjuvant described in Vogel et al., A Compendium of Vaccine Adjuvants and Excipients (2ndEdition), herein incorporated by reference in its entirety, is envisioned within the scope of this disclosure. Exemplary adjuvants include complete Freund’s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis'), incomplete Freund’s adjuvant and aluminum hydroxide. Other adjuvants may include GMCSP, BCG, muramyl dipeptide (MDP) compounds, such as thur-MDP and nor-MDP, muramyl tripeptide phosphatidylethanolamine (MTP-PE), lipid A, and monophosphoryl lipid A (MPE). Those skilled in the art will be able to select an adjuvant which is appropriate for the active immunological substance.

[0084] In one embodiment, the pharmaceutical acceptable adjuvant is selected from the group consisting of Bacille Calmette-Guerin, CpG oligodeoxynucleotides, interleukin-2, aluminum hydroxide, Freund’s adjuvant, polyinosinic:polycytidylic acid, monophosphoryl lipid A, granulocyte-macrophage colony- stimulating factor and lipopolysaccharide.

[0085] As appreciated by skilled persons, immunogenic compositions are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include, for instance, intramuscular, transcutaneous, subcutaneous, intradermal, intranasal, or oral. The immunogenic composition may be formulated as a sterile liquid for administration, or as a solid (such as a lyophilised powder or water-free concentrate) for reconstitution in a suitable solvent or dispersing medium.

[0086] Treatment

[0087] Disclosed herein is a cell as defined herein or an immunogenic composition as defined herein for use as a medicament.

[0088] These cells, compositions or vaccines as defined herein may be used as preventative and therapeutic agents for cancer immunotherapy.Disclosed herein is a method of modulating an immune response in a subject, the method comprising administering a cell as defined herein or an immunogenic composition as defined herein to the subject.

[0089] The term “subject” as used herein refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the present disclosure. A subject regardless of whether a human or nonhuman animal may be referred to as an individual, subject, animal, patient, host or recipient. For convenience, an “animal” specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as domestic animals, such as dogs and cats. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. In one embodiment, the subject is human. Subjects in need of treatment include, but are not limited to, individuals already having a particular infection or condition as well as individuals who are at risk of acquiring a particular infection or condition.

[0090] An “immune response” is a response of a cell of the immune system, such as a B cell, T cell, monocyte, macrophage, natural killer (NK) cell or dendritic cell to a stimulus. The stimulus may be a cell, antigen or cytokine. The immune response can be a B cell response, which results in the production of specific antibodies, such as antigen- specific neutralising antibodies. The immune response can also be a T cell response, such as a CD4+or a CD8+T cell response. In other embodiments, the immune response may be an effector cell response mediated by, for example, CD4+T cells, CD8+T cells, natural killer T (NKT) cells, y5 T cells or NK cells. In yet other embodiments, the immune response may be a memory cell response mediated by memory B or T cells. B cell and T cell responses are aspects of a “cellular” immune response. An immune response can also be a “humoral” immune response, which is mediated by antibodies. In some cases, the response is specific for a particular antigen (i.e., an “antigen- specific response”), and this specificity can include the production of antigen- specific antibodies and / or production of a cytokine such as interferon gamma (IFN y).

[0091] Immune response may be assessed using clinical endpoints, imaging, histopathology, biomarker analysis, or immunological assays. DC maturation can be assessed, for example,by assaying for the presence of DC maturation markers such as CD80, CD83, CD86, CCR7, and MHC II. Antigen- specific or IgG antibodies can be detected using immunological assays such as ELISA. Cytokine levels can be measured using a multiplex approach, such as using ELISpot assays. T cell activation and changes in lymphocyte populations can be measured by flow cytometry. Changes in immune infiltrate can be assessed by flow cytometry, immunohistochemistry and next-generation sequencing (NGS), or positron emission tomography (PET) scan of a subject.

[0092] In one embodiment, an effective amount of the or an immunogenic composition is administered to the subject.

[0093] By “effective amount,” in the context of treating or preventing a disease or condition (e.g., a cancer) is meant the administration of an amount of active agent to a subject, either in a single dose or as part of a series or slow release system, which is effective for the treatment or prevention of that disease or condition. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

[0094] Disclosed herein is a method of preventing or treating cancer in a subject, the method comprising administering a cell as defined herein or an immunogenic composition as defined herein to the subject.

[0095] Disclosed herein is a cell as defined herein or an immunogenic composition as defined herein, for use in preventing or treating cancer in a subject.

[0096] Disclosed herein is the use of a cell as defined herein or an immunogenic composition as defined herein in the manufacture of a medicament for preventing or treating cancer in a subject.

[0097] By “treatment”, “treat”, “treated” and the like is meant to include both prophylactic and therapeutic treatment, including but not limited to preventing, relieving, altering, reversing, affecting, inhibiting the development or progression of, ameliorating, or curing (1) a disease or condition associated with the presence or aberrant expression of a target antigen, or (2) asymptom of the disease or condition, or (3) a predisposition toward the disease or condition, including conferring protective immunity to a subject.

[0098] Disclosed herein is a method of preventing or treating cancer in a subject, the method comprising administering a cell as defined herein or an immunogenic composition as defined herein in combination with a chemotherapy regimen to the subject.

[0099] The chemotherapy regimen may comprise administering a chemotherapy drug to the subject, the chemotherapy drug is selected from the group consisting of doxorubicin, camptothecin, cisplatin, 5-fluorouracil, gemcitabine, paclitaxel, and ifosfamide.

[0100] As used herein, the term “about” when used in conjunction with a numerical value is meant to encompass numerical values within the range of the lower limit that is 5% less than the specified numerical value and the upper limit that is 5% greater than the specified numerical value.

[0101] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0102] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0103] Throughout this specification and the statements which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0104] Throughout this specification and the statements which follow, unless the context requires otherwise, the phrase “consisting essentially of’, and variations such as “consists essentially of’ will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additionalunspecified elements which would affect the basic and novel characteristics of the method defined.

[0105] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0106] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0108] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0109] EXAMPLES

[0110] Materials and Methods

[0111] Instrumentation. SEM images were collected using a JEOL JSM-7600F microscope (JEOL, Japan). AFM measurements were performed with an MFP 3D (Asylum Research, Oxford Instruments, Santa Barbara, USA). Confocal laser scanning microscopy images were acquired by ZEISS LSM 800 microscopy. Flow cytometry analysis was performed using a Fortessa X20 (BD Biosciences, America). The absorbance intensities of each well in a 96-well plate were measured using a SpectraMax M5 microplate reader. Proteogenomicsanalysis were performed using an UltiMate 3000 HPLC system and an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). In vivo animal bioluminescent images were captured using an IVIS imaging system (IVIS-CT machine, PerkinElmer).

[0112] Chemicals. Unless otherwise noted, all reagents were obtained from Sigma-Aldrich. SADME, SADEE, and SADBE were purchased from BLD Pharm (China). CFSE and crystal violet were purchased from Thermo Fisher Scientific (C34554). The BCA protein assay kit was purchased from Thermo Fisher Scientific. D-Luciferin potassium salt (ab 143655) was purchased from Abeam Inc. (Cambridge, CA, USA). Anti-calreticulin antibody (cat no. ab227444, dilution of 1:200) and Alexa Fluor 488 conjugated goat anti-rabbit IgG H&L (cat no. abl50077, dilution of 1: 1000) were purchased from Abeam Inc. (Cambridge, CA, USA). Anti-CD20, Anti-Ki 67, caspase-3 antibody, and the secondary antibody (Alexa Fluor 488-goat anti-rabbit IgG, CY3-goat anti-rabbit IgG) were purchased from Servicebio. Purified anti-mouse CD16 / 32, Alexa Fluor 700 anti-mouse CD45, APC anti-mouse CDllc, FITC anti-mouse CD80, PE anti-mouse CD86 antibodies, anti-mouse CD16 / CD32, PerCP / Cy5.5 anti-mouse CD45, BV711 anti-mouse Ly-6G, pacific blue anti-mouse CDllb, APC / Cy7 anti-mouse F4 / 80, FITC anti-mouse CD3, PE anti-mouse CD4, BV510 anti-mouse B220, PE / Cy7 anti-mouse CD49b, APC anti-mouse CD8, PE / DazzleTM 594 anti-mouse CDllc, and ELISA kits (TNF-a and IFN-y) were purchased from BioLegend. Deionized (DI) water was used for all aqueous experiments.

[0113] Preparation of SE-Vs. B16F10 tumor cells were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 cell culture medium supplemented with 10% FBS and 1% antibiotics (penicillin-streptomycin, 10,000 U / mL) at 37 °C and 5% CO2. B16F10 cells were harvested and dispersed in saline (pH 7.4 or pH 9.0) containing various concentrations of SADME, SADEE, or SADBE (0.1, 0.5, 1, and 5 mM) at a density of 5 x 105cells / mL. The cellular medium was subsequently submerged at 37 °C for 2 hours. Then the solution was centrifuged at 300 g for 5 min and washed three times with PBS. The pellets were dispersed in PBS buffer or DI water and they were observed by an optical microscope at different times. The sizes were determined using the Analyze-Measure feature in Image J. In the standard procedure, B16F10 tumor cells were suspended in the cell haptenization medium (5 mM squaric esters, pH 9.0, 154 mM NaCl) for 2 hours. For the blank cells (BC) group, B16F10 tumor cells were suspended in the medium (pH 9.0, 154 mM NaCl) for 2 hours, following the same procedures as those for SE-Vs.To observe the structure of the SE-Vs, different treatment groups were gradually dehydrated in a series of ethanol solutions (50.0%, 70.0%, and 90.0% for 10.0-15.0 min, followed by three changes of 100.0% ethanol for 15.0 min each time at room temperature). Then the samples were coated with gold and observed using a scanning electron microscope. Native B16F10 cells and PFA-treated B16F10 cells (4% PFA) were selected as the control group.

[0114] AFM analysis. AFM was used to detect the stiffness of BC, SME-Vs, SEE-Vs, and SBE-Vs. Briefly, Force-Distance curves were collected at a relative setpoint of 0.5v and a loading rate of 2pm / s using a triangular silicon nitride cantilever (NANOWORLD, PNP-TR-TL-20) functionalized with a 10-pm diameter silica bead with a spring constant of 50 pN / nm, and then obtained the global cell stiffness by fitting to the Sneddon model (indent 100 nm) analyzed by AtomicJ software.

[0115] Synthesis of CyNFh. Synthetic routes for SBECy are listed in Scheme SI. Briefly, hemicyanine dye CyNs was synthesized according to previous literature. To a solution of CyNs (290 mg, 1 mmol) and concentrated HC1 aqueous (3 mL) in MeOH (10 mL) was added dropwise SnCh (379.2 mg, 2 mmol) and stirred overnight at 80 °C under an N2 atmosphere. After completion of the reaction, the mixture was concentrated in vacuo to remove MeOH and extracted with saturated NaHCOs (3 x 200 mL) and dichloromethane (3 x 100 mL). The organic layer was collected and dried with anhydrous Na2SO4 to obtain the residue that was purified with HPLC using methanol / water to give a blue solid CyNH2 (432 mg, yield 78%).

[0116] 1H NMR (400 MHz, MeOD): 88.77 (d, J = 14.4 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 4.0 Hz, 2H), 7.47 (s, 1H), 7.43 - 7.39 (m, 2H), 6.88 - 6.85 (m, 2H), 6.45 (d, J = 14.4 Hz, 1H), 4.37 (t, J = 7.2 Hz, 2H), 2.99 (t, J = 7.6 Hz, 2H), 2.78 (t, J = 5.6 Hz, 2H), 2.72 (t, J = 6.0 Hz, 2H), 1.99 - 1.92 (m, 4H), 1.85 - 1.78 (m, 8H).13C NMR (101 MHz, MeOD): 8 178.4, 164.2, 163.8, 156.3, 146.9, 143.2, 142.97, 137.0, 130.6, 130.2, 128.0, 127.6, 123.8, 116.5, 116.1, 115.9, 113.4, 103.6, 102.9, 51.7, 45.2, 40.3, 29.9, 28.5, 26.0, 25.8, 25.1, 21.7. MS (ESI+): m / z = 441.4 [M]+.

[0117] Synthesis of SBECy. The mixture of CyNH2 (290 mg, 0.2 mmol), triethylamine (139 pl, 1 mmol), and SADBE (90 mg, 0.4 mmol) was dissolved in anhydrous EtOH (10 mL) and stirred overnight at room temperature under an N2 atmosphere. After completion of the reaction, the mixture was concentrated in vacuo to remove EtOH to obtain the residue whichwas purified by HPLC with HPLC using methanol / water to give a blue solid SBECy (432 mg, yield 83%).1H NMR (400 MHz, MeOD): 8 8.76 (d, J = 14.8 Hz, 1H), 7.64 (d, J = 7.2 Hz, 1H), 7.51 - 7.40 (m, 5H), 6.87 (d, J = 7.6 Hz, 2H), 6.43 (dd, J = 14.4, 5.6 Hz, 1H), 4.63 (t, J = 6.4 Hz, 2H), 4.36 (t, J = 7.2 Hz, 2H), 3.65 - 3.46 (m, 2H), 2.79 (t, J = 6.0 Hz, 2H), 2.70 (d, J = 4.4 Hz, 2H), 1.91 - 1.93 (m, 4H), 1.81 (s, 6H), 1.77 - 1.67 (m, 4H), 1.43 - 1.36 (m, 2H), 0.93 - 0.89 (m, 3H).13C NMR (101 MHz, MeOD): 8 156.3, 146.7, 143.1, 136.7, 130.6, 130.2, 128.0, 123.8, 121.4, 116.5, 116.0, 116.0, 115.8, 115.8, 113.5, 113.4, 103.0, 74.5, 51.7, 33.1, 29.9, 28.5, 25.6, 25.4, 25.1, 21.7, 19.7, 13.9. MS (ESI+): m / z = 593.4 [M]+.

[0118] Proliferation of SE-Vs. SE-Vs and native B16F10 cells were seeded in a 96-well plate at 5 x 105per well with complete culture medium, respectively. At 24 h (Day 1) and 48 h (Day 2), 10 pL 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) solution was added to each well and incubated for 4 h. The absorbance at 490 nm was measured using a microplate reader.

[0119] SE-Vs and native B16F10 cells were seeded into a 6-well plate at 2 x 105per well with complete culture medium, respectively. CFSE was added and labeled to the native B16F10 according to the protocol provided by the manufacturer. The CFSE fluorescence signal of the cells cultured for 0 and 48 h was measured by flow cytometry.

[0120] For in vivo proliferation assay of SE-Vs, C57BL / 6 mice were intraperitoneally (i.p.) administered with 5 xlO5SE-Vs and native B16F10-luc cells. Tumorigenesis and growth were monitored based on bioluminescence using IVIS spectrum imaging and quantified in photons per second (p s’1). The survival of mice was also recorded.

[0121] CRT detection of SE-Vs. B16F10 cells were seeded on an 8-well plate (1 x 104cells per well) and cultured overnight. SABME, SADEE, and SADBE (5 mM in pH 9.0, 154 mM NaCl solution) were added, respectively. Native B16F10 cells in pH 9.0, 154 mM NaCl solution were set as control. After incubation for 2 h, the cells were washed, fixed (4% paraformaldehyde for 15 min), and blocked (3% BSA for 1 h). Then anti-CRT antibody was incubated with the cells at 4 °C overnight. After washing with PBS three times, the cells were incubated with the corresponding secondary antibody at room temperature for 2 h. Subsequently, the nuclei were stained with Hoechst33342 and the samples were observed by CLSM.In vitro DC internalization of SE-Vs. BMDCs were freshly collected from C57BL / 6 mice (purchased from InVivos Pte Ltd., Singapore). For confocal laser scanning microscope (CLSM) observation, BMDC were seeded in 8-well plates and cultured overnight. SE-Vs stained with CFSE were added and co-incubated with BMDC. At designed time points, BMDC were labeled with anti-CDllc-APC and Hoechst 33342. Subsequently, the internalization and location of SE-Vs in BMDC were determined using CLSM. Moreover, the in vitro uptake was confirmed by Fortessa X20 (BD Biosciences).

[0122] In vitro DC maturation assay. Immature BMDCs were co-incubated with BC and SE-Vs for 6 h. Then BMDCs were collected and washed with PBS 3 times. After being stained with APC-CD11c, PE-CD86, and FITC-CD80 for 20 min at 4 °C. Stained cells were detected by Fortessa X20 (BD Biosciences). The populations of CD80+and CD86+(gated on CDllc+DCs) cells were analyzed by FlowJo software.

[0123] In vitro macrophage internalization of SE-Vs. For the macrophage internalization of SE-Vs, RAW264.7 cells were seeded in 24-well plates (1 x 105cells per well) and cultured overnight. SE-Vs stained with CFSE were added and co-incubated with RAW264.7 cells. At designed time points, RAW264.7 cells were labeled with anti-CDllb-pacific blue. Subsequently, the internalization of SE-Vs in RAW264.7 cells was determined using Fortessa X20 (BD Biosciences).

[0124] In vitro macrophage polarization assay. BMDMs were seeded on a six-well plate (5 x 105cells per well) and cultured overnight. After incubation with BC and SE-Vs for 6 h, BMDMs were collected and washed with PBS 3 times. After being stained with AF647-CD206, PE-CD86, and FITC-CD80 for 20 min. Stained cells were detected by Fortessa X20 (BD Biosciences). The populations of CD80+and CD86+cells were gated and analyzed by FlowJo software.

[0125] Proteogenomics analysis of SE-Vs. The prepared SE-Vs were suspended in 500 pL of the zwittergent solution, and whole-cell proteins were extracted using an ultrasonic cell pulverizer for an appropriate duration. Protein concentration was measured using the BCA assay. Subsequently, 50 pg of proteins were subjected to alkylated and enzymatic hydrolyzed.LC-MS DIA runs were analyzed using Spectronaut 18.0. In brief, the digested peptides were analyzed using an UltiMate 3000 HPLC system (Thermo Fisher Scientific). A spectral library was generated from pooled samples measured in DIA mode. The collected DIA spectra were searched against the UniProtKB (Swiss-Prot, Mus musculus) database using an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific). Data extraction of the DIAMS acquisitions was conducted using dDIA analyses in Spectronaut version 18.0. The analysis results were then imported into Spectronaut Pulsar version 18.0 for spectral library generation.

[0126] Contact hypersensitivity (CHS) analysis. C57BL / 6 mice were subcutaneously (s.c.) administrated with 4 xlO6BC and SE-Vs cells on days 0 and 2. The nodule size was recorded every other day. On day 7, SADME, SADEE, or SADBE dissolved in acetone (2%) was topically applied on the right ear of the mice with a volume of 10 pL. The left ears were treated with 10 pL acetone. On day 8, the mice were sacrificed, and the ear pieces were placed on the left and right ear symmetry by a 6 mm diameter puncher, and the mass of each mouse ear was weighed with a precision electronic balance.

[0127] On day 8, the nodules are collected and processed by mechanical destruction, then crushed, and passed through a 70 pM cell strainer. The isolated cells were first blocked with antimouse CD16 / CD32 and then incubated with PerCP / Cy5.5 anti-mouse CD45, BV711 antimouse Ly-6G, pacific blue anti-mouse CD 11b, APC / Cy7 anti-mouse F4 / 80, FITC antimouse CD3, PE anti-mouse CD4, BV510 anti-mouse B220, PE / Cy7 anti-mouse CD49b, APC anti-mouse CD8, PE / Dazzle™ 594 anti-mouse CD 11c. Stained cells were detected by Fortessa X20 (BD Biosciences).

[0128] In vivo preventive efficacy of the SE-Vs. C57BL / 6 mice were subcutaneously (s.c.) administrated with 4 xlO6BC and SE-Vs cells on days -21, -14, and -7. On day 0, 5xl05live B16F10-luc cells were intravenously injected into the mice. The tumor growth was monitored every other day via vernier caliper and bioluminescence intensity by IVIS after 10 min of the injection of D-Luciferin (150 mg / kg). The exposure time was 2 min.

[0129] In vivo immunologic mechanism of SE-Vs. C57BL / 6 mice were subcutaneously (s.c.) administered with 4 xlO6BC and SE-Vs cells on days -21, -14, and -7. On day 0, 5xl05liveB16F10-luc cells were intravenously injected into the mice. On day 14, mice were sacrificed and blood, tumors, lymph nodes, and spleens of mice were harvested. Blood was placed at room temperature for 2 h and then centrifuged at 300 x g for 5 min to obtain serum for cytokine detection. The tumors were collected for H&E, CD4, and CD8 immunofluorescence staining. At the same time, the tumors were collected and cut into small pieces and incubated with the solution (containing 1 mg mL-1collagenase I and IV, and 0.2 mg mL-1DNase I) at 37 °C for 2 h, and then the mixture was filtered through a 70 pM strainer to get single cell suspension. Thereafter, the cell suspensions were co-stained with Alexa Fluor®700 anti-mouse CD45, FITC-conjugated anti-mouse CD3, PE-conjugated antimouse CD4, and APC-conjugated anti-mouse CD8 for 20min, and followed by flow cytometry analysis. Spleen and tumor-draining lymph nodes were pressed gently and filtered through a 70 pM strainer to get a single-cell suspension. Then, the cell suspensions were stained for DC maturation (APC anti-mouse CD 11c, FITC-conjugated anti-mouse CD80, and PE-conjugated anti-mouse CD86), memory T cells detection (FITC-conjugated antimouse CD3, APC-conjugated anti-mouse CD8, PE-conjugated anti-mouse CD44, and BV510-conjugated anti-mouse CD62L) followed by flow cytometry analysis.

[0130] In vivo therapeutic efficacy of SBE-Vs. C57BL / 6 mice were subcutaneously (s.c.) administered with 5xl05live B16F10-luc cells on day 0. Then 4 xlO6BC and SBE-Vs were subcutaneously (s.c.) administered on days 3, 7, and 11. On day 2, DOX at a concentration of 2.5 mg kg1was intraperitoneally injected. The tumor growth was monitored every other day via vernier caliper and bioluminescence intensity by IVIS after 10 min of the injection of D-Luciferin (150 mg / kg). The exposure time was 2 min. The survival of mice was also recorded.

[0131] Statistical Analysis. GraphPad Prism 9.5.0 was used to perform statistical analysis. Results were expressed as mean ± SD. Shapiro-Wilk test was used to assess the normality of the data. Statistical comparisons were conducted by one-way ANOVA with a Tukey post-hoc test or two-tailed Student’s z-test. Kaplan-Meier analysis was used to plot survival curves, and the significance of differences was assessed using the log-rank test. For all results, p values less than 0.05 were considered statistically significant; n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Scheme SI. Synthetic routes for SADBE-conjugated hemicyanine dye (SBECy): (i) hemicyanine dye CyNs, SnCh, HC1, MeOH 80 °C, overnight, 78% yield; (ii) CyNHi, SADBE, triethylamine, anhydrous EtOH, room temperature, overnight, N2, 83% yield.

[0132]

[0133] Example 1

[0134] Fabrication of characterization of SE-Vs

[0135] Squaric esters are favored for their straightforward reaction protocols, high tolerance for diverse functional groups, and generally high conversion rates, which facilitate their reaction with amino groups for mono- or dual-ligation reactions on cell surface proteins or intracellular proteins (Fig. la). Here, squaric acid dimethyl ester (SADME), squaric acid diethyl ester (SADEE), and squaric acid dibutyl ester (SADBE), three commonly used squaric esters, were selected to modify and inactivate malignant melanoma (B 16F10) tumor cells for the fabrication of SE-Vs, termed SME-Vs, SEE-Vs, and SBE-Vs, respectively (Fig. lb). Various pH values and squaric ester concentrations were optimized to preserve cell integrity, facilitate fixation, and prevent cell aggregation (Figs. 7 and 8). Bright-fieldimaging demonstrated a marginal increase in the cellular size of SME- Vs, SEE-Vs, and SBE-Vs relative to blank cells (BC), which are native B16F10 cells suspended in a pH 9.0 solution and rendered non-viable via freeze-thaw treatment (Fig. 1c, Figs. 7 and 9). This increase was less pronounced than that observed in cells treated with paraformaldehyde (PFA). Additionally, doping SADBE with 1% SADBE-conjugated hemicyanine dye (SBECy) resulted in the detection of red fluorescence within the cells, indicating that squaric esters can readily permeate cell membranes and enter the cytoplasm (Fig. 10). Scanning electron microscopy (SEM) analysis revealed that native B16F10 cells underwent rupture during the gradual dehydration process with ethanol solutions (Fig. Id). Squaric ester treatment effectively preserved cell integrity, comparable to PFA fixation. Notably, osmotic resistance experiments demonstrated that the SE-Vs maintained cell integrity for up to 100 days after exposure to pure water, indicating high stability (Fig. 11).

[0136] To evaluate their safety, SADME, SADEE, and SADBE were incubated with tumor cells in the haptenization medium. After a 2-hour incubation, nearly all cells were nonviable, as determined by a 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)-tetrazolium (MTS) assay (Fig. 12). Additionally, intracellular propidium iodide (PI) staining confirmed the nonviability of these vaccines (Fig. le). All of SE-Vs demonstrated an absence of proliferative activity in comparison to live B16F10 cells, indicating the eradication of tumorigenic potential (Fig. If). To further assess cell proliferation, native B16F10 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), a membrane-permeable dye, prior to haptenization. CFSE covalently binds to intracellular proteins, enabling the even distribution of its fluorescence to daughter cells during cell division. As shown in Fig. 1g, the intensity of CFSE in the native cells exhibited a retarded division performance in a time-dependent manner, while it remained relatively stable in the SE-Vs groups, indicating stringent suppression of cell division. Furthermore, the absence of pathogenicity of SE-Vs was verified in vivo. Luciferase-positive B16F10 cells (B16F10-Luc) were administered intraperitoneally into mice. The native cells proliferated rapidly, resulting in a 100% mortality rate within 42 days. In contrast, mice treated with SE-Vs exhibited no bioluminescence signals, indicating an absence of tumor growth, and all mice in these groups survived for at least 80 days (Fig. Ih-j).

[0137] Proteogenomics analysis of SE-VsTo reveal the efficacy of SE-Vs in eliciting an immune response, a proteogenomics analysis was performed to assess whether the haptenization technique influences the immunogenicity of tumor cells (Fig. 2a). A total of 7,026 proteins were identified. The Venn diagram illustrated a substantial overlap in protein types among the BC, SME- Vs, SEE-Vs, and SBE-Vs groups (Fig. 2b). Further analysis of the 5,002 overlapping proteins revealed a significant upregulation of numerous proteins in SE-Vs groups compared to BC group, under a threshold with p-values < 0.05 (Fig. 2c). The investigation of tumor- associated antigens (TSAs) and tumor- specific antigens (TAAs) indicated elevated levels in SE-Vs groups, likely attributable to antigen loss during the preparation process of BC group (Fig. 2d). Further analysis of damage-associated molecular patterns (DAMPs) indicated increased levels in SE-Vs groups, particularly for immunologically potent mutations such as calreticulin (Calr), galectin (Lgalsl, Lgals3, Lgals8, Lgals9), integrin (Itga4, Itga5, Itga6, Itgav, Itgb, Itgb3), and heat shock protein transcripts (Hspbpl, Ahsal, Ahsa2, Hspal4, Hspdl) (Fig. 2d). This suggested that squaric esters may induce tumor cell death in an immunogenic manner. Calr exposure, a critical factor in the immunogenicity of cancer cell death, was significantly elevated in SE-Vs groups according to the proteogenomic analysis (Fig. 2e). Immunofluore scent staining demonstrated that Calr expression in B16F10 cells from SME-Vs, SEE-Vs, and SBE-Vs groups was 4.9-, 5.0-, and 4.4-fold higher, respectively, compared to BC group (Fig. 2f, g). These findings suggested that haptenization effectively induced immunogenic cell death and showed potential in capturing and preserving antigens within WTCVs to enhance their immunogenicity.

[0138] In vitro studies of antigen-presenting cell activation

[0139] Antigen-presenting cells (APCs), such as DCs and macrophages, play a crucial role in bridging innate and adaptive immune responses. To assess antigen uptake, a co-culture experiment was conducted using SE-Vs pre-labeled with green fluorescent CFSE prior to haptenization and bone marrow-derived dendritic cells (BMDCs) tagged with APC-CDllc. The presence of green fluorescence within BMDCs indicated successful internalization of SE-Vs (Fig. 3a). Flow cytometry analysis further demonstrated that SBE-Vs exhibited a marginally higher uptake compared to SME-Vs and SEE-Vs (Fig. 3a). The maturation rate of DCs (gated on CDllc+CD80+CD86+) increased following SE-Vs treatment. Specifically, SBE-Vs treatment enhanced DC maturation to 56.3%, which is 1.2-, 1.2-, and 1.4-fold higher compared to SME-Vs (45.4%), SEE-Vs (48.5%), and PBS group (40.0%), respectively (Fig.

[0140] 3b). Furthermore, the release of proinflammatory cytokines, including tumor necrosis factor-a (TNF-a) and interferon-^ (IFN-y), was detected in the supernatant of SE-Vs-treated DCs, further corroborating the maturation process (Fig. 3c-d). Specifically, maintaining vaccine efficacy and safety through appropriate storage conditions is pivotal for successful vaccine development. SE-Vs were subjected to lyophilization, stored for seven days, and subsequently rehydrated in PBS solution, which preserved their original morphology (Fig.

[0141] 13). Furthermore, the immunogenic efficacy of the stored vaccines, as evidenced by their ability to promote DC activation, was comparable to that of freshly prepared vaccines (Fig.

[0142] 3e). This finding underscored the potential of lyophilization as a viable storage method. Notably, the preservation of vaccine activity after seven days of lyophilization storage significantly extended the vaccine’s shelf life, exceeding the previously reported 18-hour viability for M-Vax™.

[0143] Macrophages, another vital component of the innate immune system, possess the ability to uptake, process, and present antigens. Specifically, squaric esters have been documented to facilitate the recruitment and induce Ml-like polarization of macrophages. Here all SE-Vs were internalized by murine bone marrow-derived macrophages (BMDMs) after 4 hours of incubation and resulted in an increased population of Ml macrophage phenotype compared to BC group (Fig. 3f,g). Among the various groups, SBE-Vs demonstrated the most effective results in promoting DC maturation, cytokines release, and Ml macrophage polarization. The capacity of pathogens to elicit an immune response is influenced by their physicochemical properties, encompassing surface characteristics such as hydrophobicity, nano-patteming, and charge, as well as physical parameters including size, shape, and rigidity. Typically, hydrophobicity enhances cellular uptake and the escape of antigens from endosomes and lysosomes, thereby markedly increasing their immunogenicity. The rigidity of pathogens constitutes another critical factor influencing the immune response, impacting processes such as distribution, cellular uptake, antigen presentation, and activation of APCs. Atomic force microscopy (AFM) studies on SE-Vs demonstrated an increased single-cell cortical stiffness relative to BC group (Fig. 3h). Specifically, SBE-Vs exhibited a 1.9-fold and 1.7-fold increase in stiffness compared to SME- Vs and SEE- Vs, respectively. This augmentation in stiffness is postulated to arise from the cross-linking of the squaric ester with cell membrane proteins, in conjunction with the insertion of the alkyl chain, which collectively diminishes membrane fluidity and enhances rigidity. Additionally, SADBE, characterized by a longer alkyl chain and increased hydrophobicity, shows potential in promoting the recruitment of APCs. Consequently, it is plausible to propose that theincreased hydrophobicity and rigidity of SBE-Vs facilitate their uptake by APCs, enhance antigen cross-presentation, and potentiate innate immune responses (Fig. 3i).

[0144] In vivo preventive efficacy of SE-Vs

[0145] To assess the in vivo efficacy of the vaccine, C57BL / 6 mice were subcutaneously immunized with SE-Vs, resulting in the formation of a multicellular immune niche and indicating the potential occurrence of CHS (Fig. 4a). The injection site reached its maximum size on day 8 and persisted for approximately two weeks (Fig. 4b). In the context of the CHS ear reaction, mice treated with SBE-Vs demonstrated an ear weight difference of 4.1 ± 0.057 mg, which was higher than that observed in SME- Vs, SEE-Vs, and BC treated groups (Fig.

[0146] 4c). Subsequent analysis utilizing hematoxylin and eosin (H&E) staining demonstrated a significant increase in ear diameters within the SBE-Vs group, accompanied by pronounced infiltration of granulocytes and lymphocytes, thereby indicating successful sensitization to SBE-Vs (results not shown). Additionally, flow cytometry combined with t-SNE dimensionality reduction revealed that the nodule recruited a diverse array of immune cells. This included a substantial number of APCs such as macrophages and DCs, as well as adaptive immune cells, including CD4+T cells, B220+B cells, and CD8+T cells (Fig. 4d). Notably, SBE-Vs group demonstrated the most significant enhancement in macrophages and DCs recruitment compared to the other groups, thereby facilitating antigen presentation (Fig.

[0147] 4e).

[0148] To assess the in vivo protective efficacy, C57BL / 6 mice were immunized with SE-Vs at intervals of 21, 14, and 7 days prior to being challenged with live B16F10-Luc cells (Fig.

[0149] 4f). Furthermore, CpG, a Toll-like receptor 9 agonist, was administered to facilitate macrophages in bypassing CD47, thereby eliciting robust cell-mediated immune responses. The findings indicated that SBE-Vs group exhibited the most pronounced delay in tumor onset and demonstrated the highest tumor inhibition rate of 87.0% compared to the other groups (Fig. 4g). Furthermore, the incorporation of CpG significantly enhanced tumor suppression, resulting in a tumor inhibition rate of 92.8% (Fig. 4g). Tumor burden was also assessed through bioluminescence using IVIS spectrum imaging, with quantification expressed in photons per second (p s’1; Fig. 4h). Consistent findings were observed in the bioluminescence data; quantitative analysis revealed that the tumor bioluminescence intensity in the group treated with SBE-Vs and CpG was substantially lower compared to the control groups, with 40% of the mice remaining tumor-free (Fig. 4i). Subsequent H&Eanalysis of the tumors demonstrated the highest level of cellular apoptosis in SBE-Vs + CpG group (results not shown). Immunofluorescence staining of the tumors indicated that the fluorescence intensity of the Cy3-labeled anti-caspase-3 antibody was significantly elevated in the SBE-Vs + CpG group relative to the other groups (results not shown).

[0150] During the treatment period, body weight remained stable across all treatment groups (results not shown). Histological analysis of major organs indicated an absence of detectable damage post-treatment (results not shown). Furthermore, serum biochemistry analyses of mice vaccinated with SE-Vs on day 7 revealed that all liver and kidney function parameters were within normal ranges when compared to healthy controls (results not shown), thereby affirming the high biosafety and excellent biocompatibility of the treatment

[0151] In vivo immunologic mechanism of the SE-Vs

[0152] Following vaccination, APCs primarily internalize and process the antigens, thereby stimulating innate immunity and subsequently leading to the development of adaptive immunity (Fig. 5a). To elucidate the underlying mechanisms of vaccination, mice were administered three doses of SE-Vs. Subsequently, serum, tumor-draining lymph nodes (LNs), spleens, and tumors were collected for analysis. The results indicated that SE-Vs treatment groups exhibited a significant upregulation of CD80+CD86+DCs in both the LNs and spleens (Fig. 5b). Furthermore, there was a notable increase in iNOS+macrophages within the tumors of SE-Vs group, suggesting a polarization towards the Ml -type macrophage (Fig. 5c). Among the three SE-Vs, SBE-Vs elicited the highest activation of APCs, which was further augmented by the incorporation of CpG. Specifically, in SBE-Vs + CpG group, the population of Ml-type macrophages within the tumors was 1.2-, 2.7-, and 1.7-fold higher compared to SBE-Vs, SEE-Vs, and SME-Vs groups, respectively (Fig. 5c). Following the effective initiation of antigen cross-presentation and the enhancement of innate immune responses, subsequent adaptive immune responses were evaluated. Given the critical role of germinal center (GC) reactions in antibody responses and immune memory, the potential of SE-Vs to activate GC B cells within the LNs was investigated. Immunofluorescent staining of the LNs revealed a substantial increase in both the size and number of GCs following three administrations of SE-Vs, suggesting enhanced B cell activation (Fig. 5d). Furthermore, a significant elevation in the populations of CD8+and CD4+T cells was observed in the spleens and tumors of mice post-SE-Vs injection (Fig. 5e). In mice treated with SBE-Vs + CpG, CD8+T cells and CD4+T cells were upregulated by10.3-fold and 25.0-fold in the tumor compared to the PBS group, and 1.3-fold higher in the spleen for both cell types. Concurrently, immunofluorescent staining of CD4+and CD8+T cells in tumor tissue indicated enhanced infiltration of cytotoxic T cells in SBE-Vs + CpG groups (results not shown). Furthermore, central memory T cells (TEM), which play a critical role in immune memory and disease prevention, were analyzed using flow cytometry. SE-Vs immunization facilitated the generation of central memory T cells (CD3+CD8+CD44hlghCD62Lhlgh), suggesting a robust activation of the memory immune response (Fig. 5f,g). In alignment with these findings, SBE-Vs + CpG exhibited the highest population of TEM cells, indicative of prompt immune protection. Furthermore, the concentrations of various cytokines, including TNF-a and IFN-y, were quantified in serum samples post-treatment using an enzyme-linked immunosorbent assay (ELISA) kit. In comparison to the moderate expression of pro-inflammatory factors observed in the PBS group, SBE-Vs + CpG treatment group exhibited significantly elevated concentrations of TNF-a and INF-y (Fig. 5h,i), suggesting a robust activation of the immune system. These findings collectively demonstrate that SE-Vs are capable of eliciting a potent immune response and can synergize with CpG to enhance antitumor immunity effectively.

[0153] In vivo therapeutic efficacy of the SBE-Vs

[0154] Given their superior performance relative to SME- Vs and SEE- Vs in both in vitro and in vivo studies, SBE-Vs were selected for combination with DOX-based chemotherapy to evaluate their therapeutic efficacy. This combination aims to enhance the clearance of established tumors, thereby advancing the clinical application of SBE-Vs. C57BL / 6 mice inoculated with B16F10-Luc cells on day 0 and received a 2.5 mg kg1dose of DOX via intraperitoneal injection on day 2, followed by subcutaneous injections of SBE-Vs on days 3, 7, and 11 (Fig. 6a). After 26 days, the SBE-Vs + DOX group demonstrated the most significant tumor growth inhibition, achieving a rate of 73.1%, which was 1.2-fold and 1.9-fold higher than that observed in the SBE-Vs and DOX groups, respectively (Fig. 6b, c). Tumor burden, assessed via bioluminescence, confirmed successful tumor establishment by day 4 (Fig. 6d). Notably, 20% of mice treated with SBE-Vs and 40% of those treated with SBE-Vs + DOX remained tumor-free 70 days post-tumor challenge, whereas all control mice succumbed by day 30 (Fig. 6e, f). During the treatment period, the body weight of the mice remained stable in the SBE-Vs-treated groups (results not shown), thereby corroborating the high biosafety profile of SBE-Vs. These results underscore the efficacy of SBE-Vs in cancer therapy, suggesting their potential for integration into clinical oncologytreatment protocols.

[0155] Example 2

[0156] Here, the inventors present a haptenized WTCV engineered to enhance tumor cell immunogenicity, recruit a multitude of APCs, and activate CHS to stimulate innate immunity and amplify vaccine responses. Squaric esters were introduced to haptenize WTCVs by reacting with amino groups under mild conditions within a 2-hour timeframe. This approach eliminated the necessity for additional inactivation steps and significantly reduced the overall vaccine preparation time (Fig. le-j). The resulting SE-Vs demonstrated increased stability and maintained structural integrity for up to 100 days in pure water (Fig. Id, Fig. 11). Additionally, the preservation of vaccine activity after 7 days of lyophilization storage significantly extended its shelf life, exceeding the reported 18-hour viability of M-Vax™ (Fig. 3e). Further proteogenomic analysis indicated that haptenization effectively induced immunogenic cell death and showed potential in capturing and preserving antigens within WTCVs to enhance their immunogenicity (Fig. 2). In vitro studies have shown that SE-Vs promoted antigen internalization, cytokine secretion, DC maturation, and macrophage polarization (Fig. 3). Among the three SE-Vs evaluated, SBE-Vs exhibited superior performance, probably attributed to their enhanced hydrophobicity and rigidity (Fig.

[0157] 3h,i).

[0158] Following vaccination, SBE-Vs elicited a CHS response, which promoted immune cell recruitment and improved antigen cross-presentation, thereby eliciting robust antigenspecific adaptive immune responses and establishing immune memory (Fig. 4a-e). As a prophylactic vaccine, SBE-Vs demonstrated synergistic effects with CpG, resulting in a tumor inhibition rate of 92.8% in healthy mice subjected to tumor challenges (Fig. 4). In a therapeutic setting, the combination of SBE-Vs with clinical DOX effectively suppressed tumor growth, achieving a suppression rate of 73.1% (Fig. 6).

[0159] The advantages of SE-Vs are further summarized below:

[0160] 1. Cost-effective: chemical costs were about 0.2 $ / mouse;

[0161] 2. Safe starting materials: using the Food and Drug Administration (FDA)-approved drugs; 3. Short preparation time: squaric esters induced immunogenic tumor cell death within 2 hours;4. Enhanced stability and rigidity: maintained efficacy for up to 7 days of storage;

[0162] 5. Elimination of tumorigenicity and exposure to danger signals: without the additional inactivation steps;

[0163] 6. Trigger a contact hypersensitivity: recruitment of macrophages to synergize with dendritic cells to promote antigen cross-presentation.

[0164] SE-Vs offer significant advantages over existing hapten-modified WTCVs, including superior long-term storage capabilities, the elimination of traditional inactivation methods, and enhanced recruitment and activation of APCs to bolster adaptive immune responses. Beyond the established B16F10 model, this approach shows considerable promise for the development of WTCVs tailored to a diverse array of tumor types, including those derived from patient specimens.

[0165] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of preparing a squarate-haptenised cell, the method comprising contacting the cell with a squaric ester to immobilise and / or conjugate the squaric ester to the cell.

2. The method of claim 1, wherein the squaric ester is a compound of Formula (I):wherein Ri and R2 are independently selected from H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocyclyl, optionally substituted aryl, and optionally substituted heteroaryl.

3. The metho d of claim 1 or 2, wherein Ri and R2 are independently selected from:

4. The method of any one of claims 1 to 3, wherein the squaric ester is a compound selected from:" "5. The method of any one of claims 1 to 4, wherein the squaric ester is a compound of Formula (VI), (III) or (IV).

6. The method of any one of claims 1 to 5, wherein the squaric ester is a compound of Formula (VI).

7. The method of any one of claims 1 to 6, wherein the squaric ester is provided at a concentration of about 0.1 mM to about 5 mM.

8. The method of any one of claims 1 to 7, wherein the contacting is performed at a pH of about 7 to about 10.

9. The method of any one of claims 1 to 8, wherein the cell is a tumor cell, immune cell or bacteria cell.

10. The method of claim 9, wherein the tumor cell is from a tumor selected from the group consisting of melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, pancreatic cancer, glioblastoma, astrocytoma, renal cell carcinoma, esophageal cancer, ovarian cancer, bladder cancer and thyroid cancer.

11. The method of claim 9, wherein the immune cell is selected from the group consisting of a dendritic cell, a macrophage, a monocyte, a B cell, a T cell, an NK cell, a neutrophil, a mast cell, an innate lymphoid cell and a plasma cell.

12. The method of any one of claim 1 to 11, wherein the cell is a modified cell.

13. The method of any one of claims 1 to 12, wherein the method comprises isolating or selecting for a whole or intact cell.

14. A cell obtainable according to a method of any one of claims 1 to 13.

15. A cell that is conjugated to a squaric ester.

16. The cell of claim 15, wherein the squaric ester is a compound of Formula (VI), (III) or (IV).

17. The cell of claim 16, wherein the squaric ester is a compound of Formula (VI).

18. An immunogenic composition comprising a cell of any one of claims 16 to 17 and a pharmaceutically acceptable adjuvant.

19. The immunogenic composition of claim 18, wherein the cell is a whole cell.

20. The immunogenic composition of claim 18 or 19, wherein the pharmaceutical acceptable adjuvant is selected from the group consisting of Bacille Calmette- Guerin, CpG oligodeoxynucleotides, interleukin-2, aluminum hydroxide, Freund’s adjuvant, polyinosinic:polycytidylic acid, monophosphoryl lipid A, granulocytemacrophage colony-stimulating factor and lipopolysaccharide.

21. A cell of any one of claims 14 to 17 or an immunogenic composition of any one of claims 18 to 20, for use as a medicament.

22. A method of modulating an immune response in a subject, the method comprising administering a cell of any one of claims 14 to 17 or an immunogenic composition of any one of claims 18 to 20 to the subject.

23. A method of preventing or treating cancer in a subject, the method comprising administering a cell of any one of claims 14 to 17 or an immunogenic composition of any one of claims 18 to 20 to the subject.

24. A method of preventing or treating cancer in a subject, the method comprising administering a cell of any one of claims 14 to 17 or an immunogenic composition of any one of claims 18 to 20 in combination with a chemotherapy regime to the subject.

25. The method of claim 24, wherein the chemotherapy regime comprises administering a chemotherapy drug to the subject, the chemotherapy drug is selected from the group consisting of doxorubicin, camptothecin, cisplatin, 5-fluorouracil, gemcitabine, paclitaxel and ifosfamide.

26. A cell of any one of claims 14 to 17 or an immunogenic composition of any one of claims 18 to 20, for use in preventing or treating cancer in a subject.

27. Use of a cell of any one of claims 14 to 17 or an immunogenic composition of any one of claims 18 to 20 in the manufacture of a medicament for preventing or treating cancer in a subject.

Images