ANTITUMOR CELL COMPRISING A CHARGE MODIFIED GLOBIN

Abstract

There is provided an antitumour cell, liposome or micelle, comprising at least one charge-modified globin associated with the membrane of the cell, liposome or micelle, and methods of making and using the same.

Claims

1. An antitumour cell, liposome or micelle, comprising at least one charge-modified globin associated with the membrane of the cell, liposome or micelle.

2. A cell according to claim 1, wherein the cell is an immune cell, preferably a tumour-infiltrating immune cell, more preferably a lymphocyte, neutrophil, dendritic cell or macrophage.

3. A cell according to claim 1 or 2, wherein the cell is a cytotoxic T cell, natural killer T cell or natural killer cell.

4. A cell according to any preceding claim, wherein the cell is a T cell.

5. A liposome or micelle according to claim 1, wherein the liposome or micelle comprises a therapeutic agent, preferably wherein the therapeutic agent is a checkpoint inhibitor, immunotherapeutic or chemotherapeutic agent.

6. A cell, liposome or micelle according to any preceding claim, wherein the globin is haemoglobin, myoglobin, neuroglobin, or cytoglobin, preferably myoglobin.

7. A cell, liposome or micelle according to any preceding claim, wherein the globin is linked to a secondary antitumour molecule, or a reactive functional group for linking to a secondary antitumour molecule, preferably wherein the secondary antitumour molecule is any one of an antibody, lectin, integrin or adhesion molecule; and/or preferably wherein the secondary antitumour molecule is any one of: (1) a tumour cell binding molecule; (2) a checkpoint inhibitor; (3) an enzyme that remodels the extracellular matrix of a tumour; or (4) an enzyme that metabolises tumour-associated compounds.

8. A cell, liposome or micelle according to claim 7, comprising a fusion protein comprising the globin and the secondary anti-cancer molecule.

9. A cell, liposome or micelle according to any preceding claim, wherein the globin is a cationised or anionised globin.

10. A cell, liposome or micelle according to any preceding claim, wherein the globin comprises a polymer surfactant coating.

11. A pharmaceutical composition comprising the antitumour cell, liposome or micelle according to any preceding claim, further comprising a pharmaceutically acceptable carrier, diluent or vehicle.

12. A cell, liposome or micelle according to any of claims 1-10, or the pharmaceutical composition according to claim 11, for use in the treatment of cancer.

13. A method of making the antitumour cell, liposome or micelle according to any of claims 1-10, comprising a) providing a charge-modified globin; and b) contacting the antitumour cell, liposome or micelle with the globin.

14. The method of claim 13, wherein step (a) comprises providing a charge-modified globin and a polymer surfactant under conditions which enable electrostatic conjugation of the polymer surfactant with the globin.

15. The method of claim 13 or 14, wherein a globin is converted to the charge-modified globin by a method comprising: i) mixing a solution of globin with a pH-neutralised solution of N,N′-dimethyl-1,3-propanediamine (DMPA) or analogue thereof and optionally adjusting the mixture to pH 5-7; ii) subsequently or concurrently adding a carbodiimide such as N-(3-dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC) and adjusting the mixture to pH 4-7; iii) agitating the mixture from (ii) for 1-30 hours at pH 4-7, at a temperature of 0-25° C.; iv) dialysing the protein in the mixture from (iii) against water or buffer for at least 4 hours at pH 6.5-8.5; v) if necessary, adjusting the pH of the mixture from (iv) to pH 6.5-8.5.

16. The method of claim 13 or 14, wherein the charge-modified globin is obtained by a method comprising expression of a recombinant DNA sequence encoding for the charge-modified globin.

17. A method of treating cancer, comprising administration of the cell, liposome or micelle according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11, to a patient in need thereof.

18. The use according to claim 12, or the method according to claim 17, wherein the cancer is a solid tumour cancer.

19. The use according to claim 12 or 18, or the method according to claim 17 or 18, wherein the cancer is selected from: breast, colorectal, prostate, lung, stomach, liver, oesophageal, cervical, or pancreatic cancer.

20. A polypeptide comprising the charge modified globin sequence of any of SEQ ID NOs: 1-14 or a functional variant of any of these having at least about 60% sequence identity with the non-variant globin sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0111] FIG. 1 shows the results of a fluorescence activated cell sorting (FACS) analysis applied to human Jurkat T cells (a and c) or murine T cells (b and d) that have undergone labelling procedures with no protein, native myoglobin (nMyo), cationised myoglobin (Cat Myo), conjugated cationised myoglobin (Con Myo), supercharged GFP (scGFP) and conjugated supercharged GFP (Con scGFP). Jurkat T cells were gated on their expression of CD3 and then the percentage of cells staining for surface presentation of His-Tag present on the myoglobin and GFP (a) or GFP fluorescence (c). Murine T cells were gated on their expression of CD8 and the percentage of cells staining for surface presentation of His-Tag present on the myoglobin and GFP (b) or GFP fluorescence (d). All data are presented as representative data plot (inset) and by chart (showing mean %+/−SD, n=4).

[0112] FIG. 2 shows the results of a FACS analysis applied to human Jurkat T cells (a) and murine T cells (b) (that have undergone labelling procedures as described for FIG. 1) that were stained with Aqua cell viability dye, which were gated on their expression of CD3 (a) or CD8 (b) and the percentage of Aqua negative live cells was calculated as mean %+/−SD, n=4; representative data plot in inset, processed data shown by chart.

[0113] FIG. 3 shows the results of a FACS analysis applied to T cell receptor transgenic human Jurkat T cells (a and b) that have undergone labelling procedures with (a) no protein, native myoglobin, cationised myoglobin, and conjugated cationised myoglobin, or (b) no protein, scGFP, and conjugated scGFP, that were analysed for the upregulation of CD69 in response to C1R antigen presenting cells pulsed with 0 μM, 1 μM or 10 μM cognate peptide.

[0114] FIG. 4 shows the results of murine T cells (that have undergone labelling procedures as described for FIG. 1) stained with cell trace violet (CTV) and then activated for 4 days in the presence of anti-CD3, anti-CD28 and IL-2 under normoxic (squares) or hypoxic (circles) conditions (a and b), and the results of murine T cells activated under normoxic (squares) or hypoxic (circles) conditions tested for expression of high levels of CD44 (c and d), separated by T cells of the CD4 expressing subtype (a and c) and the CD8 expressing subtype (b and d), presented as representative plot (inset) and by chart (showing mean %+/−SD, n=4).

[0115] FIG. 5 shows (a) the labelling efficiency of surfactant-conjugated supercharged myoglobin (Myo14[S]) on Jurkat T-cells, the viability of Myo14[S]-coated (b) Jurkat T-cells and (c) activated CD8+ murine T-cells, (d) the cell counts of murine T-cells 3 and 5 days post-coating versus an uncoated control (U/T), (e) the percentage of divided CD8+ murine T-cells 3 and 5 days post-coating, and (f) the level of CD69 activation of coated Jurkat T-cells versus an uncoated control (Untreated).

[0116] FIG. 6 shows (a) the levels of exhaustion markers on surfactant-conjugated supercharged myoglobin (Myo14[S])-coated CD8+ murine T-cells versus uncoated controls (U/T), (b) the change in fluorescence over time of a hypoxia-sensitive dye at 0.5% 02 for Myo14[S]-coated and uncoated Jurkat T-cells (untreated), and (c) the viability and levels of markers of inflammation of human mesenchymal stem cells with or without Myo14[S] and interferon gamma.

EXAMPLES

[0117] Materials and Methods

[0118] Myo14 Expression

[0119] Myo14 was obtained by expression in BL21(DE3) E. coli cells transformed with a plasmid containing the appropriate Myo14 gene by electroporation using routine methods. Briefly, a single colony was picked and placed in 10 mL of LB media for a starter culture, and incubated overnight at 37° C., shaken at 180 RPM. The starter culture was then used to inoculate 1 L of TB media supplemented with 0.02% glucose and 50 mg.Math.L.sup.−1 carbenicillin in a 2.5 L culture flask. The culture flask is then incubated at 37° C., 200 RPM. Once the TB reaches an optical density at 600 nm of 0.7-1.0, protein expression is induced with 1 mM IPTG. After 4 hours, the expression cultures are centrifuged at 4500×g for 20 minutes at 4° C. to pellet the cells. The supernatant is discarded, and the pellet can be frozen for storage or used immediately.

[0120] Myo14 Purification

[0121] Lysis buffer containing 20 mM HEPES, 1 M NaCl, at pH 7.0 was added to the Myo14 pellets, lysed using pulse sonication, and clarified in a centrifuge, using routine methods. The Myo14 is then purified using maltose binding protein affinity chromatography, and the maltose binding protein is then cleaved with TEV protease overnight at room temperature in a fully anoxic environment. Finally the resulting cleaved product is polished with size exclusion chromatography, using routine methods.

[0122] Preparation of Constructs

[0123] The chemically charge-modified myoglobin, termed cationised myoglobin, was prepared by covalent modification of acid residues with N,N-dimethyl-1,3-propanediamine (DMPA) via a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) mediated reaction. A myoglobin solution in 2-(N-morpholino)ethanesulfonic acid (MES) buffer was added to a pH-neutralised solution of DMPA at a ratio of 300 moles DMPA per mole of acidic residues on the myoglobin. The solution was pH adjusted to pH 5.5-6.0. EDC was then added at a ratio of 50 moles EDC per mole of acidic residues on the myoglobin, and left to stir at 4° C. overnight. The resulting solution was then desalted to remove by-products via desalting columns, spin-concentrating and diluting into fresh buffer to achieve a minimum 1 million-fold dilution, or dialysing against fresh buffer.

[0124] Surfactant-conjugated cationised-myoglobin, termed conjugated myoglobin, was prepared by adding anionic surfactant to a solution of cationised myoglobin. The surfactant was an oxidised form of glycolic acid ethoxylate 4-nonylphenyl ether (51783, as described above), and was added at a ratio of 1 mole of surfactant per mole of positively charged residues on the myoglobin. The surfactant may be added in solid form, or pre-dissolved in an appropriate buffer.

[0125] The recombinantly charge-modified (“supercharged”) myoglobins were prepared by altering the amino acid sequence of myoglobin, before ordering the corresponding DNA from commercial sources. The 1st mutant (SEQ ID NO 1) was modified to incorporate lysine residues at positions that were found by proteomic analysis to be modified by the addition of DMPA in the cationised myoglobin. The Max mutant (SEQ ID NO 2) was modified to swap all acid residues for lysine residues. The polarised mutant (SEQ ID NO 3) was modified to incorporate lysine residues at the surface of the myoglobin except within a 10 A radius of the C-terminus in which positive residues were modified to glutamic acids. The ROSETTA mutant (SEQ ID NO 4) was modified to incorporate mutations suggested by the ROSETTA algorithm on the ROSIE web service (rosie.graylab.jhu.edu). The AVNAPSA mutant (SEQ ID NO 5) was modified to incorporate mutations suggested by the AVNAPSA algorithm on the ROSIE web service. The TaB mutant (SEQ ID NO 6) was modified to incorporate either lysine or arginine residues at surface accessible positions without hydrogen bonds or electrostatic interactions and with a B-factor above the mean B-factor plus one standard deviation from the crystal structure of PDBID 3RGK. The consensus B model (SEQ ID NO 7) was designed at the TaB mutant, but excluded mutations to residues that were not found to be mutated in any homologues. Myo7 (SEQ ID NO 8) was modified to include positively charged residues found in several homologues from closely-related species, and was used as a base for the remaining sequences. Myo14 (SEQ ID NO 9) was modified to include residues determined to be surface accessible by structure inspection. cMyo9 (SEQ ID NO 10), cMyo14 (SEQ ID NO 11) and cMyo15 (SEQ ID NO 12) were modified to include successively more mutations than Myo7 (SEQ ID NO 8) that were either lower frequency and/or from more distantly related species.

[0126] TCR transgenic Jurkat T cells (jTc) or CD3+ T cells purified from murine spleen and lymph nodes (mTc) were incubated for 20 mins with cationised, conjugated or native myoglobin or GFP or conjugated super charged GFP. Cells were washed three times and analysed, in three different experiments, for viability, activation and proliferation.

[0127] To test T cells with Myo14[S] TCR transgenic Jurkat T cells (jTc) or CD3+ T cells were used, purified from murine spleen and lymph nodes (mTc). T cells were incubated for 30 mins at 37° C. with Myo14[S] or untreated (U/T). Cells were washed two times and analysed for Myo14[S] coating, viability, phenotype, activation, resistance to hypoxia and proliferation.

[0128] Conjugate Coating and Viability Assay

[0129] jTc were harvested from culture and washed once with PBS then incubated in PBS with no protein or 3 μM conjugated super charged GFP (con-scGFP), 3 μM super charged GFP (scGFP), 10 μM of native myoglobin (nMyo), 10 μM of cationised myoglobin (Cat Myo) or 10 μM conjugated myoglobin (Con Myo) for 20 mins at 37° C. Coated jTc were washed three times in PBS and resuspended in PBS at 1×10.sup.6 jTc in 100 μl per well and stained with αCD3-AF594 (Biolegend, Cat No. 300446), αHIS-TAG-APC (Biolegend, Cat No. 362605) and Aqua (Invitrogen, Cat No. L34957) for 30 min at 4° C. Stained cells were washed twice with PBS and analysed using a Fortessa X20 Cytometer.

[0130] Whole T cells (mTc) were purified from the spleen and lymph nodes of one Balb-c female mouse using a Dyna-bead negative selection kit (ThermoFisher. Cat No. 11413D) as per the manufacturer's instructions. mTc were washed once with PBS then incubated in PBS with no protein or 3 μM conjugated super charged GFP (Con scGFP), 3 μM super charged GFP (scGFP), 10 μM of native myoglobin (nMyo), 10 μM of cationised myoglobin (Cat Myo) or 10 μM conjugated myoglobin (Con Myo) for 20 mins at 37° C. Coated mTc were washed three times in PBS and resuspended in PBS at 1×10.sup.6 mTc in 100 μl per well and stained with αCD3-AF700 (Biolegend, Cat No. 152316), αCD4-BV785 (Biolegend, Cat No. 100552), αCD8-APCef780 (Invitrogen, Cat No. 47008182), αHIS-TAG-APC (Biolegend, Cat No. 362605) and Aqua (Invitrogen, Cat No. L34957) for 30 min at 4° C. Stained cells were washed twice with PBS and analysed using a Fortessa X20 Cytometer.

[0131] Jurkat T Cell Activation Assay

[0132] TCR transgenic Jurkat T cells (jTc) are Jurkat T cells transduced with a T cell receptor (TCR) that recognises a cognate peptide when presented by MHC Class I HLA-A2. When stimulated by their cognate peptide the expression of CD69 increases and can be detected by FACS as a surrogate marker for T cell activation.

[0133] jTc and C1R were maintained in RPMI 1640 supplemented with 10% v/v FCS, 2 μM glutamine, 50IU/ml Penicillin, 50 μg/ml Streptomycin, 50 μM 2-β-mercaptoethanol and 10 mM HEPES (R10) at 37° C.

[0134] C1R-HLA-A2 were washed twice with PBS and then resuspended in PBS with either 0 μM, 1 μM or 10 μM cognate peptide for 50 mins at 37° C. Cells were washed twice in R10 and plated at 1×10.sup.5 cells in 100 μl per well in a 96 U-bottomed plate.

[0135] jTc were harvested from culture and washed once with PBS then incubated in PBS with no protein or 3 μM conjugated super charged GFP (con-scGFP), 3 μM super charged GFP (scGFP), 10 μM of native myoglobin (nMyo), 10 μM of cationised myoglobin (Cat Myo) or 10 μM conjugated myoglobin (Con Myo) for 20 mins at 37° C. Coated jTc were washed once in PBS and resuspended in R10 at 5×10.sup.4 jTc in 100 μl per well and were mixed with C1R cells.

[0136] Identical plates with coated jTc and peptide pulsed C1R cells were co-cultured for 6 hours at 37° C. in incubators with environmental oxygen (21%) levels or hypoxic oxygen level (5%).

[0137] After 6 hours plates were washed once with PBS, cells were then incubated with αCD69-APC (Biolegend, Cat No. 310910), αCD3-AF594 (Biolegend, Cat No. 300446) and the live cell stain, Aqua (Invitrogen, Cat No. L34957) at 4° C. for 30 mins. Cells were then washed twice with PBS and fixed with 2% Paraformaldehyde (PFA) overnight. Cells were washed twice in PBS to remove the fixative and were run on the High throughput module of a Fortessa X20 Cytometer.

[0138] Murine T cell Proliferation Assay

[0139] 96 U bottomed plates were coated with 1 μg/ml αCD3 and 5 μg/ml αCD28 diluted in PBS for 1 hour at 37° C. PBS was removed.

[0140] Whole T cells (Tc) were purified from the spleen and lymph nodes of one Balb-c female mouse using a Dyna-bead negative selection kit (ThermoFisher. Cat No. 11413D) as per the manufacturer's instructions.

[0141] Purified Tc were washed once with PBS then incubated in PBS with no protein or 3 μM conjugated super charged GFP (Con scGFP), 3 μM super charged GFP (scGFP), 10 μM of native myoglobin (nMyo), 10 μM of cationised myoglobin (Cat Myo) or 10 μM conjugated myoglobin (Con Myo) for 20 mins at 37° C.

[0142] Coated Tc were washed twice in PBS and resuspended in PBS with 10 μM Cell Trace Violet (ThermoFisher. Cat No. C34557) at RT for 20 mins. CTV stained cells were washed in R10 and transferred at 4×10.sup.5 Tc per well in 200 μl R10 with 20 U/ml IL-2 to αCD3 and αCD28 pre-coated plates.

[0143] Identical plates with coated Tc were cultured for 4 days at 37° C. in incubators with environmental oxygen (21%) levels or hypoxic oxygen levels (5%).

[0144] After 4 days, plates were centrifuged at 500×g for 5 mins and washed once with PBS, cells were then incubated with two combinations of antibodies at 4° C. for 45 mins.

[0145] Combination 1: αCD3-AF700 (Biolegend, Cat No. 152316), αCD4-BV785 (Biolegend, Cat No. 100552), αCD8-APCef780 (Invitrogen, Cat No. 47008182), αCD44-BV605 (Biolegend, Cat No. 103047), αHIS-TAG-APC (Biolegend, Cat No. 362605) and Aqua (Invitrogen, Cat No. L34957).

[0146] Combination 2: αCD3-AF700, αCD4-BV785, αCD8-APCef780, αPDL-1-PE (Invitrogen, Cat No. 125982-82), Isotype Control-BV605 (Biolegend, Cat No. 400434), Isotype Control-APC (Invitrogen, Cat No. 17432181) and Aqua.

[0147] Cells were then washed twice with PBS and fixed with 2% Paraformaldehyde (PFA; FisherScientific, Cat No. 11586711) for 1 hr at 4° C. Cells were washed twice in PBS to remove the fixative and were run on the High throughput module of a Fortessa X20 Cytometer.

[0148] Tc were maintained in RPMI 1640 (ThermoFisher Scientific, Cat No. 21875-034) supplemented with 10% v/v FCS, 2 μM glutamine, 50 IU/ml Penicillin, 50 μg/ml Streptomycin, 50 μM 2-β-mercaptoethanol and 10 mM HEPES (R10) at 37° C.

[0149] Labelling Efficiency

[0150] Myo14 was labelled with FITC as per the manufacturer's instructions prior to conjugation. jTc were untreated or coated with 14 μM Myo14[S]-FITC and stained with αCD3-AF594 then analysed by flow cytometry. The mean proportion of FITC+CD3+Tc is presented with representative FACS plots (+/−SD, n=3).

[0151] FACS-Based Cell Viability Analysis

[0152] Live jTc coated with varying concentrations of Myo14[S] were stained for Aqua Live/dead cell stain and CD3-AF594. Tc were gated on Singlets and CD3+ Tc and then the mean percentage of live cells was calculated. Naive murine Tc were purified and activated in vitro with αCD3/CD28 and IL-2 for 1 day. On day 1 activated Tc were harvested and coated with Myo14[S] at varying concentrations. Coated, activated Tc were added back to culture with αCD3/CD28 and IL-2 for 1 day. Cells were harvested and stained with anti-CD8 and Aqua Live dead cell stain. Cells were gated on Singlets and CD8+ cells then the mean percentage of live cells was calculated.

[0153] FACS-Based Cell Proliferation Analysis

[0154] Whole Tc were purified from spleen and lymph nodes of one Balb-c mouse. Tc were untreated or coated with 2.5 μM Myo14[S]. Tc were activated with αCD3/CD28. Activated Tc were harvested at day 3 and day 5 and stained with trypan blue to exclude dead cells then counted. The mean cell number (+/−SD, n=3) is presented.

[0155] Purified whole Tc were stained with Cell Trace Violet then left untreated (U/T) or coated with 2.5 μM Myo14[S]. Tc were activated with αCD3/CD28 and IL2. Activated mTc were harvested at day 3 and day 5 and stained with αCD4 and αCD8. Cell were analysed by flow cytometry. Cells were gated on CD8 cells and the mean of all CTV diluted cells were included as ‘Divided CD8 Tc’ (+/−SD, n=3).

[0156] Antigen-Specific Activation

[0157] TCR transgenic jTc were untreated (U/T) or coated with 10 μM [Myo14-MBP][S]. jTc were cultured for 6 hours with C1R-HLA-A2+ cells peptide pulsed with the stated concentrations of cognate peptide. jTc were stained with αCD3-AF594, αCD69-APC and Aqua Live/Dead stain, the mean MFI of the activation marker CD69 was calculated on live, CD3+ jTc (+/−SD, n=3).

[0158] Hypoxia-Dye Assay

[0159] Image-iT Green hypoxia reagent (Thermo Fisher) was diluted to 5 mM by adding DMSO and mixing well. This stock was then added to jTc to a final concentration of 101 μM in R10 media, and incubated at 37° C., 20% O.sub.2, 5% CO.sub.2 for 40 minutes. The cells were then centrifuged at 500×g for 5 minutes, the supernatant poured away and replaced with fresh growth medium. The cells are then plated in a 96-well plate at 5×10.sup.5 cell per well and read on a fluorescence plate reader over time with the oxygen concentration fixed at the desired value, for example 0.5% 02.

[0160] hMSC Inflammation Assay

[0161] Human mesenchymal stem cells, isolated from four patients, were untreated or coated with 2.5 μM Myo14[S] and cultured for 7 days. As a positive control, cells were cultured with IFNγ. At day 7 cells were harvested, counted and stained for Aqua Live/Dead stain and the lineage markers, CD105 and CD73 and the inflammatory markers HLA-DR, HLA-ABC and PDL1. Cells were gated on singlets, live cells and CD105+CD73+ cells, then the mean percentage of cells expressing each marker was calculated.

[0162] 2D In Vitro Tumour Killing Assays

[0163] Antigen-specific killing of tumour cells, such as IGR-Heu lung carcinoma cells, SKBR3, BT20, MCF7 breast cancer cells, SKOV3 ovarian cancer cells or HeLa-CD19 cells, are carried out using human cytotoxic T cells derived from either PBMCs or isolation of tumour-infiltrating lymphocytes, or CAR-T cells.

[0164] Upon isolation from PBMCs, T cells are activated using stimulating agents, such as irradiated autologous tumour cells, HLA-A2-binding HER-2/neu p 369-377 peptide or CD3/CD28 Dynabeads, and growth factors, such as IL-2.

[0165] Prior to co-culture with tumour target cells, T cells are coated with Myo14 or Myo14[S]. Uncoated T cells are used as a control for baseline killing activity. Tumour cell lines are either transfected for the expression of a reporter gene or incubated with dyes such as CFSE to be fluorescently labelled. During co-culture of activated T cells and tumour target cells, decrease in fluorescence is monitored via imaging, FACS or by using a plate reader to determine rate of tumour killing.

[0166] As positive control for tumour killing, an anti-cancer drug (i.e. staurosporine) is added to culture media on tumour cells alone.

[0167] 3D In Vitro Tumour Killing Assays

[0168] Target tumour cells are transfected to express GFP and grown into 3D spheroids. Isolated T cells are added to tumour spheroids, after being coated with Myo14 or Myo14[S]. Uncoated T cells are used as a control for baseline killing activity. During co-culture, decrease in fluorescence is monitored via imaging to determine rate of tumour spheroid cell death.

[0169] To monitor T cell infiltration in tumour spheroids, T cells are fluorescently labelled with a different dye and co-cultures are imaged.

[0170] As positive control for tumour killing, an anti-cancer drug (i.e. staurosporine) is added to culture media on tumour spheroids alone.

[0171] In Vivo Tumour Killing Assays

[0172] Myo14 based constructs are tested in vitro and in vivo in multiple models to further demonstrate functionality and efficacy. For in vivo efficacy, and to demonstrate that Myo14 based constructs are suitable for multiple oncological applications, Myo14[S] and [Myo14-PD1][S] are coated onto Chimeric Antigen Receptor T cells (CAR-T) or transgenic Clone 4 T cells and adoptively transferred into mice bearing MDA-MB-231 human breast cancer tumour or RencaHA tumours, respectively. Each experiment is described in more detail below.

[0173] For the CAR-T/MDA-MB-231 experiments, 32 NSG mice are inoculated with subcutaneous MDA-MB-231. When the tumour reaches the pre-determined time point the mice are split into 4 groups. Group 1 is left untreated, Group 2 is treated with i.v. CAR-T, Group 3 is treated with i.v. Myo14[S] coated CAR-T and Group 4 is treated with i.v. [Myo14-PD1][S] coated CAR-T. The experiment is repeated at least once.

[0174] Tumour size is measured regularly with callipers as well as bioluminescent imaging of luciferase transduced MDA-MB-231 tumour cells. Flow cytometric analysis of serial tail bleeds is performed to determine the persistence and expansion of infused Tc.

[0175] Sections from treated tumour tissues are analysed by multiplex staining (multicolour Vectra Automated Quantitative Pathology Imaging System and quantitatively analysed using Definiens software) available through the Cancer Research UK Birmingham Cancer Centre. Tumours are stained with the following antibody panel:

[0176] Anti-human CD3, pan-cytokeratin (tumour marker), CD34 (CAR marker), carbonic anhydrase IX (hypoxia marker), human annexin V (apoptosis marker) and human IFN gamma (functional marker) human PD1 and human PDL1 (plus DAPI nuclear stain). This enables visualisation, analysis, quantification and phenotyping of immune cells and identification of cell-to-cell interactions within a single tumour tissue section.

[0177] Secondary lymphoid tissues and tumour are analysed by FACS for the presence of CAR-T cells. The phenotype of adoptively transferred CAR-T cells is analysed for surface expression of PD-1, CD45 and CD62L and intracellular cytokine staining for IFNγ and TNFα. The phenotype of treated tumour cells is analysed by surface expression of PDL-1, MHC Class I and MHC Class II.

[0178] In an additional in vivo tumour model, Myo14[S] and [Myo14-PD1][S] are coated onto transgenic Clone 4 T cells purified from 6-8 week old CL4 TCR-transgenic Thy1.1+ BALB/c mice. 20×106 CL4 TCR-Transgenic Tc are injected i.v. to 8 randomly selected female Balti-c mice that have been previously injected s.c. with 1×10.sup.6 RencaHa tumour cells. The growth of the RencaHa tumour is measured in 4 groups which are untreated, treated with uncoated Tc, treated with Myo14[S] coated Tc and [Myo14-PD1][S] coated Tc. The scientific and humane end point of the experiment is set at 27 days based on previous work. Tumour diameter in two dimensions is measured every other day using callipers and the rate of tumour progression and the final tumour volume calculated. Flow cytometric analysis of serial tail bleeds is performed to determine the persistence and expansion of infused Tc.

[0179] Adoptively transferred Tc with the Thy1.1 congenic marker are identified in tumours, spleen, tumour draining and tumour non-draining lymph nodes, lungs, brain and liver by fluorescent immuno-histochemistry to enumerate the distribution and tumour specific migration and retention of treated and untreated Tc. Furthermore, sections from tumour samples are stained with markers for vasculature (CD31/MECA-32) and hypoxia (carbonic anhydrase IX8) to identify hypoxic regions. These stains, together with Tc identification, enable identification of Myo[S] treated Tc migration to, and persistence in, areas of low oxygen.

[0180] Parts of tumours, spleens, tumour non-draining and tumour draining lymph nodes are harvested and processed for FACS analysis. Tc purified from the tumour are analysed for their viability, ability to kill tumour cells in vitro and expression of CD44, CD62L, PD1, Annexin V, IFNγ and TNFα. Additionally, the phenotype of the tumour cells is analysed for MHC Class I and Class II as well as PDL1.

Examples—Results and Discussion

[0181] Percentage of Cells Labelled with Construct

[0182] Coated live cells were immediately stained with fluorescently conjugated antibodies and viability dyes as described above and then analysed, by flow cytometry, for viability and presence of construct. Both GFP and HIS-TAG were detectable on the surface of Tc after 3 washes suggesting that they had bound tightly to the cell surface (FIG. 1a-d). The percentage of jTc that had bound cationised myoglobin (83.5%+/−0.4) and conjugated myoglobin (79.4%+/−0.8) were equivalent (FIG. 1a) however there was a significant decrease in the percentage of mTc that bound to conjugated myoglobin (6.7%+/−0.8), compared to cationised myoglobin (47.7%+/−1.4)(FIG. 1c). Without wishing to be bound by theory, the reasons for the differential binding of cationised and conjugated myoglobin to jTc and mTc may be due to variable glycosylation on the cell membrane or differences in the ability to detect HIS-Tag labelled proteins. In support of the latter hypothesis GFP labelling of both jTc and mTc is greater than 87% with both supercharged and conjugated GFP (FIGS. 1b and d) however the equivalent HIS-Tag labelling varies from 20% to 91% suggesting that HIS-Tag labelling is not detecting all GFP labelling and needs further consideration. Without wishing to be bound by theory, it is possible that the low HIS-Tag readout and high GFP signal may be caused by internalisation of the construct, or by binding to the mTc cell in an orientation that masks the HIS-Tag, both of which would not affect the GFP signal but would prevent anti-HIS-Tag antibody binding.

[0183] Cell Viability

[0184] The viability of jTc incubated without myoglobin or with native, cationised or conjugated myoglobin was above 94%, however viability of jTc with scGFP and conjugated GFP was 47.75% and 63.95% respectively (FIG. 2a) suggesting that GFP but not myoglobin is toxic to jTc at the concentrations tested. The viability of mTc without myoglobin (92.3%+/−1.14) and with native myoglobin (94.65%+/−0.31) was significantly higher than mTc coated with cationised myoglobin (76.78%+/−0.50) and conjugated myoglobin (79.7%+/−0.4) suggesting that cationised and conjugated myoglobin were causing some cell death at the concentrations tested. There was even more cell death in mTc coated with scGFP (63.0%+/−1.9) and conjugated GFP (41.1%+/−1.12) (FIG. 2b).

[0185] Together, these results demonstrate good cell surface binding of cationised myoglobin to both jTc and mTc, with minimal cytotoxicity to jTc in particular. Further experiments with mTc are required to optimise the binding of conjugated myoglobin whilst improving cell viability. It is likely that the differential viability of mTc and jTc in response to identical concentrations of cationised and conjugated myoglobin is due to the inherent robustness of the cells, with the jTc cell line being more robust than primary naïve murine Tc.

[0186] To ascertain the optimal concentration of surfactant-conjugated supercharged myoglobin (Myo14[S]) for T cell treatment the viability of jTc and primary mTc were tested after coating with varying concentrations of Myo14[S]. The viability of jTc was above 90% at all concentrations tested up to a maximum of 40 μM (FIG. 5b). The viability of primary murine T cells, 1 day after coating with Myo14[S] was 90% or above at all concentrations tested up to 10 μM (FIG. 5c).

[0187] Activity—to Cognate Peptide

[0188] In order to test whether coating jTc with constructs inhibits the engagement of T cell receptors (TCR) with their cognate peptide presented by the major histocompatibility complex (MHC), TCR transgenic Jurkat T cells were utilised, which upregulate CD69 when they recognise cognate peptide presented by C1R-HLA-A2 (C1R) B cells. jTc were coated with myoglobin or GFP constructs and incubated, for 6 hours, with C1R cells that had been previously pulsed with 0 μM, 1 μM or 10 μM of the cognate peptide. The upregulation of CD69, an activation marker, was analysed by flow cytometry. The addition of 1 μM or 10 μM of peptide increased the percentage of cells expressing CD69 from 0.92% (+/−0.06) for untreated jTc to 9.32% (+/−1.16) for 1 μM of peptide and 20.97% (+/−1.80) for 10 μM of peptide (FIGS. 3a and b). There was no significant difference in the increase of CD69 expression after coating with cationised or conjugated myoglobin (FIG. 3a) or supercharged or conjugated GFP (FIG. 3b). The results show that binding of TCR transgenic jTc with the constructs does not inhibit T cell activation which suggests that there is no steric interference caused by the constructs between the TCR-MHC activation complex.

[0189] Proliferation and Activity—Under Hypoxic Conditions

[0190] mTc were activated under normoxic (22% oxygen) and hypoxic (5% oxygen) conditions after coating with myoglobin constructs. mTc were incubated for 4 days in the presence of anti-CD3 and anti-CD28 antibodies. Together, anti-CD3 and anti-CD28 bind to the TCR and the co-stimulatory molecule, CD28, and cause polyclonal activation of Tc. On day 4, mTc were harvested and analysed by flow cytometry. mTc were gated on CD4 or CD8, to analyse these two subtypes of T cells. Each subtype was analysed for the dilution of cell trace violet, (CTV) a marker of proliferation, and high expression of CD44, a marker of activation.

[0191] In untreated cells, hypoxia increased the percentage of divided CD4 mTc from 66.23% (+/−2.54) to 72.1% (+/−3.54) and CD8 mTc from 70.75% (+/−2.63) to 78.2% (+/−5.15) suggesting that the hypoxic environment increases Tc proliferation (FIGS. 4a and b). Under normoxic conditions, CD4 and CD8 mTc coated with cationised or conjugated myoglobin all had a significantly lower percentage of divided cells than their untreated or native myoglobin treated counterparts. This observation can be rationally explained by the decreased viability of these cells after coating with the myoglobin constructs (FIG. 2b) before activation. However, when coated CD4 and CD8 Tc were cultured under hypoxic conditions there was a higher percentage of divided mTc when compared to normoxic conditions suggesting that the presence of myoglobin and/or hypoxia was rescuing the proliferative defects in these cells.

[0192] In untreated CD4 Tc, when compared to normoxia, hypoxia increased the percentage of activated CD4 CD44hi Tc from 45.9% (+/−2.86) to 50.83% (+/−2.16). CD4 Tc coated with cationised or conjugated myoglobin and activated under normoxic conditions had a significantly lower percentage of CD4 CD44hi Tc than untreated or native myoglobin treated Tc, however the same cells activated under hypoxic conditions had an equivalent percentage of CD4 CD44hi Tc (FIG. 4c) when compared to untreated and native myoglobin treated mTc.

[0193] In untreated CD8 Tc, when compared to normoxia, hypoxia significantly (p=0.001) decreased the percentage of CD8 CD44hi Tc from 61.25% (+/−1.93) to 52.33% (+/−2.27). The addition of conjugated (49.83%+/−2.21), but not cationised (58.08%+/−1.34), myoglobin resulted in a reduction in the percentage of CD8 CD44hi Tc under normoxic conditions, when compared to untreated controls (61.25%+/−1.93). However, the addition of cationised or conjugated myoglobin, under hypoxic conditions, reversed the hypoxia induced reduction in CD8 Tc activation. In fact, the addition of both forms of myoglobin significantly increased the percentage of CD8 CD44hi Tc from 52.33% (+/−2.27) for untreated CD8 Tc to 71.5% (+/−2.50) for cationised myoglobin coated CD8 Tc and 63.88% (+/−3.67) for conjugated myoglobin coated CD8 Tc (FIG. 4d). It is unclear why myoglobin constructs cause a decrease in CD44hi Tc under normoxic, but not hypoxic conditions. It is possible that the original decrease in the viability of Tc may be involved. It is also possible that the constructs may be interfering with T cell signalling, to some extent, through the TCR or CD28 in a way that is only important under normoxic conditions. However, the demonstration that treatment with myoglobin rescues the activated phenotype of CD8 Tc under normoxic conditions is greatly encouraging as CD8 Tc are an important target of further development in both CAR-T cell and TIL therapies.

[0194] Proliferation

[0195] The proliferation and viability of in vitro activated mTc, 3 and 5 days after staining with Cell Trace Violet and coating with 2.5 μM Myo14[S] was evaluated. There was no significant difference in the number of live, activated T cells at day 3 or day 5 after coating with 2.5 μM Myo14[S] when compared to untreated (U/T) controls (FIG. 5d). Moreover, in the same experiment, the percentage of mTc that had undergone one or more divisions was not significantly different in Myo14[S] treated and untreated controls (FIG. 5e).

[0196] Antigen-Specific Activation

[0197] The ability of T cell receptor (TCR) transgenic Jurkat T cell to become activated upon recognition of their cognate MHC-peptide complex was tested in a CD69 activation assay. [Myo14-MBP][S] was used in this assay because the Maltose binding protein (MBP) was used as a surrogate for any other protein that can be linked to Myo14, including, but not restricted to, Cytokines such as IL-2, IL-15 and IL-17, T cell receptors such as PD-1, CTLA-4, TIM-3 or enzymes such as metalloproteinases. The upregulation of CD69 on jTc coated with [Myo14-MBP][S], in response to increasing peptide concentrations was not significantly different from untreated controls. These results demonstrate that coating with Myo14[S] linked to another protein (in this case MBP) does not interfere with normal jTc to target cell protein interactions and thus should not interfere with normal T cell signalling (FIG. 5f).

[0198] Exhaustion-Marker Profiling

[0199] In order to demonstrate that naïve mTc did not become stressed in the presence of surfactant-conjugated supercharged myoglobin (Myo14[S]), purified mTc were coated with 2.5 μM Myo14[S] and stimulated with αCD3/CD28 and IL-2 for 3 days. On day 3, activated mTc were harvested and analysed for their expression of the activation marker CD44 and exhaustion/activation markers LAG3, PD1 and TIGIT. There was no significant difference between untreated and Myo14[S] treated cells for any of the markers tested (FIG. 6a).

[0200] Hypoxia Dye Assay

[0201] Surfactant-conjugated supercharged myoglobin (Myo14[S]) is an oxygen carrying molecule and so the inventors sought to determine whether oxygen carried by Myo14[S] could be supplied to Tc. jTc were stained with Image-iT Green hypoxia reagent and then were left untreated, or were treated with 10 μM Myo14[S]. Stained and coated jTc were transferred to a Cytation plate reader and fluorescence was monitored for 22 hr at 5% Carbon dioxide and 0.5% Oxygen. The relative fluorescence and therefore the internal hypoxic state of the untreated jTc increases at a greater rate and reaches a higher level than the Myo14[S] treated jTc suggesting that Myo14[S] is supplying oxygen to the jTc (FIG. 6b).

[0202] hMSC Inflammation Assay

[0203] The technology described herein can be applied to numerous cell types and disease indications. The inventors have demonstrated that surfactant-conjugated supercharged myoglobin (Myo14[S]) can be coated onto human mesenchymal stem cells (hMSC). hMSC were coated with 5 μM Myo14[S] and then grown in vitro for 7 days. On day 7 hMSC were harvested, live cells were counted and then cells were stained with antibodies for lineage markers CD73 and CD105 and inflammatory markers HLA-ABC, HLA-DR and PD-L1. As a control for the upregulation of inflammatory markers cells were also cultured with the inflammatory cytokine IFNγ. There was no significant difference between the cell number or percentage of cells expressing HLA-ABC, HLA-DR and PD-L1 in untreated and Myo14[S] treated hMSC (FIG. 6c).

[0204] Table 4 provides the amino acid sequences of a series of charge-modified (supercharged) myoglobins generated using the above-described techniques:

TABLE-US-00004 TABLE 4 Amino acid sequences of charge-modified myoglobins and charge-modified GFP SEQ ID NO Description Sequence  1 1st mutant- MHHHHHHGSSGENLYFQGLSDGEWQLVLNVWGKVEADIPGHGQEVL mutations IRLFKGHPKTLKKFDRFKHLKSKKKMKASEKLKKHGATVLTALGGILKK informed by KGHHEAKIKPLAQSHATKHKIPVKYLKFISKAIIKVLQSKHPGDFGKKA chemical QGAMNKALKLFRKKMASNYKEL cationisation  2 Max-all acid MHHHHHHGSSGENLYFQGLSDGEWQLVLNVWGKVEAKIPGHGQEVL residues IRLFKGHPKTLKKFDRFKHLKSKKKMKASKKLKKHGATVLTALGGILKK modified to KGHHKAKIKPLAQSHATKHKIPVKYLKFISKAIIQVLQSKHPGKFGAKA lysines QGAMNKALKLFRKKMASNYKKL  3 Polarised- MHHHHHHGSSGENLYFQGLSKGKWQLVLNVWGKVKAKIPGHGQKV mutations LIRLFKGHPKTLKEFKRFKHLKSKKKMKASKKLKKHGATVLTALGGILK made to acids KKGHHEAKIEPLAQSHATEHEIPVEYLEFISKAIIQVLQSKHPGKFGAKA excluding 10 Å QGAMNKALKLFRKDMASNYEEL radius around C-terminal  4 Rosetta MHHHHHHGGGGSENLYFQGLSDGEWQLVLNVWGKVEADIPGHGQE VLIRLFKGHPETLKKFDRFKKLKSEDKMKKSEDLKKHGATVLKRLGGIL KKKGRHEAKIKPLAQRHAKKHKIPVKYLEFRSEAIIRVLRSKHPGDFGA DAQGAMNKALELFRKDMASNYKELGFQG  5 AVNAPSA MHHHHHHGGGGSENLYFQGLSKGEWKLVLNVWGKVEADIPGHGQE VLIRLFKGHPKTLKKFKRFKHLKSEKKMKASKDLKKHGATVLTALGGIL KKKGHHKAEIKPLAQSHATKHKIPVKYLEFISEAIIQVLQSKHPGKFGA KAQGAMNKALELFRKDMASNYKKLGFQG  6 TaB MHHHHHHGGGGSENLYFQGLSDGEWRLVLKVWGKVERDIPGHGQE VLIRLFKGHPETLKKFDRFKHLKSEREMKASKDLKKHGATVLTALGGIL KKKGHHEAEIKPLAKSHATKHKIPVKYLKFISKAIIQVLQSKHPGDFGA RAQGAMNKALELFRKDMARNYKKLGFQG  7 Consensus B- MHHHHHHGGGGSENLYGLSDGEWRLVLKVWGKVERDIPGHGQEVLI The same as RLFKGHPETLKKFDRFKHLKSRDEMKASEKLKKHGATVLTALGGILKK TaB, but KGHHEAEIKPLAKSHATKHKIPVKYLKFISKAIIQVLQSKHPGDFGADA adjusted to QGAMNKALELFRKDMASKYKKLGFQG exclude conserved domains  8 Myo7 MHHHHHHGSGGLSDGEWQLVLKVWGKVEADIPGHGQEVLIRLFKGH PETLEKFDRFKHLKSEDEMKASEDLKKHGATVLTALGKILKKKGHHEA EIKPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADAQGAMKK ALELFRKDMASKYKELGFQG  9 Myo14 MHHHHHHGSGGLSDGEWQLVLKVWGKVEADIPGHGQEVLIRLFKGH PETLKKFDRFKHLKSEDEMKASEDLKKHGATVLKKLGKILKKKGKHEA EIKPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADAQGAMKK ALKLFRKDMASKYKELGFQG 10 cMyo9 MHHHHHHGSGGLSDGEWQLVLKVWGKVEADIPGHGQEVLIRLFKGH PETLEKFDRFKHLKSEDEMKRSEDLKKHGATVLKALGKILKKKGHHEA EIKPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADAQGAMKK ALELFRKDMASKYKELGFQG 11 cMyo14 MHHHHHHGSGGLSDGEWQLVLKVWGKVEADIPGHGQEVLIRLFKGH PETLEKFDRFKKLKSEDEMKRSEDLKKHGATVLKKLGKILKKKGKHEA EIKPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADAQGAMKK ALKLFRKDMASKYKELGFQG 12 cMyo15 MHHHHHHGSGGLRDGEWQLVLKVWGKVEADIPGHGQEVLIRLFKGH PETLEKFDRFKKLKSEDEMKRSEDLKKHGATVLKKLGKILKKKGKHEA EIKPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADAQGAMKK ALKLFRKDMASKYKELGFQG 13 His_MBP_PD1_ MHHHHHHKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHP Myo14 DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDK LYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKE LKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNA GAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWS NIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN YLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMP NIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNN NNNLGENLYFQGSGWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVL NWYRMSPSNQTVKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVV RARRNDSGTYLCSAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSP GGGGSGGGGSGGGGSGLSDGEWQLVLKVWGKVEADIPGHGQEVLI RLFKGHPETLKKFDRFKHLKSEDEMKASEDLKKHGATVLKKLGKILKK KGKHEAEIKPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADA QGAMKKALKLFRKDMASKYKELGFQG 14 MBP_PD1_ MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF Myo14 PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKS ALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLT FLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKV NYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEG LEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMS AFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGE NLYFQGSGWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMS PSNQTVKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDS GTYLCSAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPGGGGSGG GGSGGGGSGLSDGEWQLVLKVWGKVEADIPGHGQEVLIRLFKGHPE TLKKFDRFKHLKSEDEMKASEDLKKHGATVLKKLGKILKKKGKHEAEI KPLAQSHATKHKIPVKYLKFISEAIIKVLQSKHPGDFGADAQGAMKKAL KLFRKDMASKYKELGFQG 15 scGFP MASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFI CTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQE RTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYN FNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGP VLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYK