Re-Directed Immunotherapy

Abstract

The invention provides an agent for preventing or treating a condition characterised by the presence of unwanted cells, the agent comprising: (i) a targeting moiety that is capable of targeting to the unwanted cells; and (ii) a T cell antigen, wherein the T cell antigen can be released from the targeting moiety by selective cleavage of a cleavage site in the agent in the vicinity of the unwanted cells.

Claims

1.-38. (canceled)

39. An agent for retargeting T cells to solid tumor cells, the agent comprising: (i) a targeting moiety that is capable of targeting to the solid tumor cells, wherein the targeting moiety is an antibody or antigen binding fragment thereof that binds a solid tumor antigen; and (ii) more than one viral T cell epitope that each elicit an existing immune response in the subject and that each bind to a HLA molecule on the surface of the cancer cell of the human subject and has a HLA matched to the subject, (iii) more than one peptide linker each comprising a peptide cleavage site cleavable by a tumor associated protease and wherein each linker can be selectively cleaved by a tumor associated protease to release each T cell epitope in the vicinity of, and outside of, the solid tumor cells, wherein each peptide linker is separately attached to the targeting moiety, wherein each linker attaches a single T cell epitope to the targeting moiety, and wherein the solid tumor is not a lymphoma.

40. The agent according to claim 39, wherein at least some of the peptide cleavage sites are the same.

41. The agent according to claim 39, wherein at least some of the peptide cleavage sites are not the same.

42. The agent according to claim 39, wherein the tumor associated protease is Cathepsin B, Cathepsin L, Cathepsin S, Cathepsin D, Cathepsin E, Cathepsin A, Cathepsin G, Thrombin, Plasmin, Urokinase, Tissue Plasminogen Activator, Metalloproteinase 1 (MMP1), MMP2, MMP3, MMP4, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP20, MMP21, MMP23, MMP24, MMP25, MMP26, MMP28, ADAM, ADAMTS, CD10 (CALLA), or prostate specific antigen.

43. The agent according to claim 39, wherein the more than one T cell epitopes are the same.

44. The agent according to claim 39, wherein the more than one T cell epitopes are not the same.

45. The agent according to claim 39, wherein one or more T cell epitope is from any of Varicella Zoster virus, Herpes simplex virus, cytomegalovirus, Epstein Barr virus, adenovirus, rhinovirus, or influenza virus.

46. The agent according to claim 39, wherein one or more T cell epitope is an MI-IC Class I restricted antigen, an MI-IC Class II restricted antigen, or an antigen that is capable of binding to a group I CD1 molecule.

47. The agent of claim 39, wherein one or more viral T cell epitope is one to which a number of T cells in the subject are already sensitized to.

48. The agent of claim 39, wherein one or more viral T cell epitope is one that elicits an existing immune response, wherein the existing immune response has been generated by prior vaccination against an infectious agent.

49. The agent of claim 39, wherein one or more viral T cell epitope is one that elicits an existing immune response, wherein the existing immune response has been generated by exposing the human subject's T cells to the epitope in vitro.

50. The agent of claim 39, wherein the solid tumor is chosen from an epithelial tumor, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, or osteosarcoma tumor.

51. The agent according to claim 39, wherein selective cleavage of each cleavage site enables release of each T cell epitope at or near to the cell surface of the solid tumor cells.

52. The agent according to claim 39, wherein the antibody or antigen binding fragment thereof is specific for any of Her2; CD22; EpCAM (CD326); EGFR; PSMA; CD30; CD20; CD33; membrane IgE; IgE Receptor (CD23), CD80; CD86; CD2; CA125; Carbonic Anhydrase IX; CD70; CD74; CD56; CD40; CD19; c-met/HGFR; TRAIL-R1; DR5; PD-1; PD1L; IGF-1R; VEGF-R2; Prostate stem cell antigen (PSCA); MUC1; CanAg; Mesothelin; P-cadherin; Myostatin(GDF8); Cripto (TDGF1); ACVRL1/ALK1; MUC5AC; CEACAM; SLC44A4; CD2/CS1; CD137; CXCR4; Neuropilin 1; Glypican; PDGFRa, EphA2, CD22, E-cadherin, FGFR3, and CD138.

53. The agent according to claim 39, wherein the antibody or antigen binding fragment thereof is an anti-epidermal growth factor receptor antibody, an anti-Her2 antibody, an anti-MUC1 antibody, an anti-P-cadherin antibody, an anti-EpCAM antibody, an anti-E-cadherin antibody, an anti-CEA antibody, or an anti-FGFR3 antibody.

54. The agent according to claim 53, wherein the antibody or antigen binding fragment thereof is an anti-CEA antibody.

55. The agent according to claim 39, wherein the antibody or antigen binding fragment thereof is specific for CEACAM.

56. The agent according to claim 39, wherein the antibody is Rituximab or Cetuximab.

57. A pharmaceutical composition, comprising an agent according to claim 39, and a pharmaceutically acceptable carrier, diluent or excipient.

58. A composition comprising (i) an agent according to claim 39 and (ii) a therapeutic agent suitable for treating a solid tumor.

Description

[0199] The invention will be described in further detail with the aid of the following Figures and Examples.

[0200] FIG. 1: MDA.MB.231 cells transduced with MMP14 stained with Cetuximab conjugated to NLVPMVATV (SEQ ID No: 21) containing MMP14 cleavage sequence (NLVPMVATVLPRSAKELRC (SEQ ID No: 280) linked to Cetuximab using Sulpho-SMCC).

[0201] FIG. 2: B-LCL cells stained with Rituximab conjugated to the HLA class-II peptide DYSNTHSTRYV (SEQ ID No: 55) containing a protease cleavage sequence (DDYSNTHSTRYVTIPVSLRSGGGGSGGGGSC) (SEQ ID No: 274).

[0202] FIG. 3: B-LCL cells stained with Rituximab conjugated to the HLA class-I peptide TPRVTGGGAM (SEQ ID No: 31) containing a protease cleavage sequence (KTPRVTGGGAMAIPVSLRSGGGGSGGGGSC) (SEQ ID No: 273) linked to Rituximab using Sulpho-SMCC.

[0203] FIGS. 4A-4B: (A) Schematic diagram of an exemplary embodiment of the invention. (B) Schematic showing design and mechanism of viral peptide delivery. (i) Preferred targeting molecule consisting of a monoclonal antibody able to target a tumour cell, covalently linked to a synthetic peptide containing a protease recognition domain and a T cell peptide antigen. (ii) The peptide-conjugate is delivered to the target cell via a specific antibody and the peptide is cleaved in the proximity of the tumour cell. The released smaller viral peptide is passively binds to empty to empty MHC class I or II molecules on the cell surface. The MHC-peptide complexes are then recognised and by specific circulating T-cells mediating tumour cell-lysis.

[0204] FIGS. 5A-5C: In vitro activity of redirected virus-specific T cells. (A) Recognition of a lymphoblastoid lymphoma cell lines by CD8.sup.+ cytomegalovirus-specific cytotoxic T lymphocytes through conjugation of the cognate antigen TPRVTGGGAM (SEQ ID No: 31) peptide to Rituximab (anti-CD20). Recognition is only present if the peptide is flanked by the MMP2 cleavage motif. Controls are tumour cells alone, CTLs alone and tumour cells pulsed with free TPR peptide (KTPRVTGGGAMAIPVSLRSGGGGSGGGGSC) (SEQ ID No: 273) linked to Rituximab using Sulpho-SMCC. (B) Redirection of CD4.sup.+ cytomegalovirus-specific T cells specific for HLA-DR7.sup.+ restricted epitope DYSNTHSTRYV (SEQ ID No: 55) (DYSN). Lymphoblastoid cell lines were incubated with Rituximab crosslinked with the DYSN peptide without a cleavage site (IPDDYSNTHSTRYVC) (SEQ ID No: 309), DYSN-Protease Cleavage Site which contains a protease cleavage site (DDYSNTHSTRYVTIPVSLRSGGGGSGGGGSC) (SEQ ID No: 274) or a control peptide and washed. Tumour cells were then incubated overnight with DYSN-specific CD4.sup.+ T cells and T cell recognition determined using IFN release by ELISA. The inclusion of a protease cleavage site adjacent to the DYSN peptide dramatically increases recognition of the tumour cells by CD4.sup.+ T cells. (C) Application toward breast carcinoma cell line MDA-MB231 using cytomegalovirus-specific CD8.sup.+ CTL specific for cytomegalovirus pp65 (NLVPMVATV) (SEQ ID No: 21) epitope. Conjugation of the peptide combined with a MMP14 cleavage site mediates killing of the cells (NLVPMVATVLPRSAKELRC; SEQ ID No: 280) linked to Cetuximab using Sulpho-SMCC. Further data not shown indicates that a peptide conjugate lacking the MMP14 cleavage site shows killing comparable to Cetuximab alone.

[0205] FIGS. 6A-6C: In vitro and in vivo targeting of tumour cell lines using APEC approach. (A) EGFR expression on two breast carcinoma cell lines MCF7 and MB-MDA231. (B) Successful targeting of EGFR+ cell line MDA-MB231 using NLVPMVATV (SEQ ID No: 21) peptide containing a protease cleavage site (CSGGGGSGGGGAIPVSLRANLVPMVATV; SEQ ID No: 276) conjugated to Cetuximab. Arrow denotes efficient recognition by T cells of APEC treated cells. (C) MCF-7 which does not express EGFR is not targeted by Cetuximab immunoconjugate. In both (B) and (C), the B-RPH is a HLA-mismatched peptide deigned to not be recognised by the T cells and the VLE-protease cleavage peptide is an HLA-matched peptide. This peptide is designed to be a control for the protease cleavage sequence to ensure that the T cells do not recognise a portion of the protease cleavage sequence. As the response to the VLE-protease cleavage peptide is negligible, the response seen with the NLV-protease cleavage peptide is borne against the NLVPMVATV (SEQ ID No: 21) sequence and not the protease cleavage sequence.

[0206] FIGS. 7A-7D: In vivo targeting of tumour cell lines using APEC approach. (A) in vivo xenograft data demonstrating that the human MDA-MB-231 breast cancer cell line can be eradicated by CMV-specific T cells when Cetuximab-peptide conjugates are given via intraperitoneal injection. Cetuximab-peptide complexes alone are unable to control the tumour growth (upper), whilst neither are CMV-specific T-cells (middle). However the combination of both cause eradication of this aggressive breast cancer tumour in vivo. (B) Successful targeting of colorectal cell line Colo205 using anti-Muc1 antibodies linked with CMV-specific T cell epitope and protease cleavage site. GP1.4 and SM3 are well-characterised anti-MUC1 specific antibodies. (C) This anti-Muc1 antibody-peptide conjugate also very efficiently targets pancreatic carcinoma cell line Panc1. MH1, SM3 and GP1.4 are all well-characterised MUC1-specific monoclonal antibodies. MH1 is specific for the cytoplasmic tail of MUC1 and therefore does not bind to intact tumour cells, whereas, SM3 and GP1.4 bind to the extracellular portion of MUC1 glycoprotein. For FIG. 7A, the peptide used is the NLV peptide using the reverse sequence (CSGGGGSGGGGAIPVSLRANLVPMVATV) (SEQ ID No: 276). In FIGS. 7B and C, the peptides are as follows NLVR (CSGGGGSGGGGAIPVSLRANLVPMVATV) (SEQ ID No: 276) containing the protease cleavage site, RNLV (RNLVPMVATVQIPVSLRSGGGGSGGGGSC) (SEQ ID No: 275) containing the same protease cleavage site as NLV-R and differing from NLV-R in the orientation of the viral epitope and the biotin peptide (Biotin-PFMRPHERNGFTVLC: SEQ ID No: 320) which does not contain the protease cleavage sequence and is used as a control peptide. (D) Single chain fragment V (scFv) protein construct encoding for the same peptide sequence as used in (A) but contained within the scFv single polypeptide chain demonstrates activity against MDA-MB-231 cell line in vitro. The protease recognition site within the ScFv construct is IPVSLRS (SEQ ID No: 310).

[0207] FIGS. 8A-8B: Plasma stability of the antibody-peptide conjugate and specific targeting of tumour B cells. (A) Recognition of a lymphoblastoid cell line or healthy B cells by CD8+ cytomegalovirus specific cytotoxic T cells after labelling cells with Rituximab conjugated with MHC class I peptides derived from cytomegalovirus. There is no recognition of healthy B cells whereas there is recognition of target cells only in the presence of the viral peptide the T cells are specific for. This data demonstrates the specificity of the APEC for malignant cells compared with healthy tissue. (B) The antibody peptide-epitope conjugate (APEC) was incubated at 37 C. in human plasma and assayed by ELISA to determine stability of the peptide conjugation. The half-life of the APEC is 50 minutes and is not altered by the addition of an acetyl group at the C terminus of the peptide. In FIG. 8, the peptide sequences used were NLV-Protease Cleavage-Reverse (CSGGGGSGGGGAIPVSLRANLVPMVATV) (SEQ ID No: 276) containing the protease cleavage site IPVSLRS (SEQ ID No: 310), VLE-Protease Cleavage (YVLEETSVMLIPVSLRSGGGGSGGGGSC) (SEQ ID No: 277) containing the protease cleavage site IPVSLRS (SEQ ID No: 310) and Biotin-RPH (Biotin-PFMRPHERNGFTVLC: SEQ ID No: 320) used as a control peptide without the protease cleavage sequence.

[0208] FIGS. 9A-9B: (A) Amino acid sequence of cytomegalovirus pp 65 protein (SEQ ID No: 318); (B) Amino acid sequence of some peptide constructs referenced in the Examples. Key: bold and underlined=T-cell epitope; boxed=flexible linker; italics=protease recognition site; boxed and bold=epitope extension reside as per parent pp 65; double underlined=coupling residue bearing a sulfhydryl.

[0209] FIGS. 10A-10B: (A) The dependency of external proteolytic activity: target cells are lightly fixed in paraformaldehyde or not and then incubated with either peptide (NLVPMVATV) (SEQ ID No: 21) or Cetuximab conjugated with either an irrelevant peptide (VLE) or cognate peptide (NLVPMVATV: SEQ ID No: 21-protease cleavage site) and incubated with NLVPMVATV (SEQ ID No: 21) specific T-cells. Data demonstrate that fixed cells are able to process antibody-peptide conjugates and thus internalisation is not necessary for processing. (B) Data demonstrating that receptor peptide agonists (NLVPMVATVAIPVSLRSAAAFCDGFYACYMDV (SEQ ID No: 311)=Her2/neu agonist peptide [FCDGFYACYMDV] (SEQ ID No: 312) with NLVPMVATV (SEQ ID No: 21) and protease cleavage site AIPVSLR (SEQ ID No: 313); NLVPMVATVAIPVSLRSAAAYCRDYDYDGRYFDCY (SEQ ID No: 314)=EGFR agonist peptide (YCRDYDYDGRYFDCY) (SEQ ID No: 319) with the viral peptide NLVPMVATV (SEQ ID No: 21) and protease cleavage site AIPVSLR (SEQ ID No: 313)) can be used to re-direct T-cells against EGFR+ tumour cells) (Ponde et al, Bioorg Med Chem Lett 21(8): 2550-3).

[0210] FIGS. 11A-11B: (A) Demonstration that cytokines can be used as targeting moiety with IL-2 (A) and IL-4 (B) being conjugated with HLA-class II peptide (DR7-restricted) PDDYSNTHSTRYVC (SEQ ID No: 309) and using the Biotin-RPH (Biotin-PFMRPHERNGFTVLC; SEQ ID No: 320) as a control peptide. After labelling target cells with cytokine-peptide complex, and culturing with DYSN-specific CD4 T cells for either 4 or 24 hours, there is a strong T cell response towards target cells labelled with the DR7-restricted peptide (PDDYSN) and no response when using the control peptide (Biotin-RPH) after 4 and 24 hours (A) and 24 hours (B).

[0211] FIG. 12: T cell recognition by range of tumour cell lines. Eight HLA-A*0201 NCI-60 cell lines are all recognised by HLA-A*0201-restricted CMV-specific T-cells when pulsed with cognate antigen.

[0212] FIGS. 13A-13E: in vitro targeting of human carcinoma cell lines using antibody-peptide-epitope conjugate (APEC) approach. (A) Early data showing that Cetuximab-peptide conjugates could be used to target carcinoma cell lines. Positive control is cognate viral peptide pulsed tumoursas in FIG. 12. (B) The introduction of a protease cleavage site in the peptide is critical for effective targeting of tumour cells. Peptides not bearing a cleavage sequence (PC) are not cleaved and therefore not recognised by T-cells. (C) Using Cetuximab-MMP2-NLVPMVATV (SEQ ID No: 21) APEC we can successfully target 4 out of 7 HLA-A*0201 NCI-60 cell lines. NCI-H522 and Colo205 are also weakly recognised, whereas HCT-116 is not recognised. Caki-2 is not HLA-A*0201 and serves only as a control. (D) We have produced scFv protein based upon Cetuximab sequence and N-terminally linked MMP2-NLVPMVATV (SEQ ID No: 21). This agent is also able to target MDA-MB-231 tumour cells in vitro (shown in red). (E) Assessment of in vitro potency of Cetuximab APEC. These experiments suggest an EC50 of between 2-5 ug/ml (13-30 nM). In FIG. 13, when the peptide epitope is NLVPMVATV (SEQ ID No: 21), the peptides used were CSGGGGSGGGGAIPVSLRANLVPMVATV (SEQ ID No: 276) (NLVPMVATV-PC) that contains the protease cleavage sequence AIPVSLR (SEQ ID No: 313), and CSGGGGSGGGGANLVPMVATV (SEQ ID No: 315) (NLVPMVATV) that does not contain a protease cleavage site.

[0213] FIGS. 14A-14F: (A) Immunohistochemistry of 4 human adenocarcinomas showing CD8+ T cells by immunohistochemistry reveal abundant CD8+ T-cell infiltrate in human carcinomas. (B) Using Anti-Muc1(SM3)-(Protease Cleavage)-NLVPMVATV (SEQ ID No: 21) APEC we can successfully target the pancreatic carcinoma cell line Panc-1. (C) Using Rituximab-Protease Cleavage)-NLVPMVATV (SEQ ID No: 21) APEC we can differentially target a model tumour B cell line (LCLs) (black bars) and avoid targeting healthy B cells (white bars). (D) Using Rituximab-PC-DYSNTHSTRYV (SEQ ID No: 55) APEC we can successfully target a model tumour B cell line (LCLs) using HLA Class-II derived peptides eliciting a CD4+ T cell response. (E) Using Cetuximab-MMP2-NLVPMVATV (SEQ ID No: 21) APEC we can successfully target cells previously fixed (black bars) at a similar level to that seen in unfixed cells (white bars). (F) Using anti-CD138 antibody to target the myeloma cell line U266. PC=Protease Cleavage. U266+ NLVPMVATV (SEQ ID No: 21)+T cell corresponds to the native 9 amino acid peptide alone as a free peptide epitope (i.e. no linker etc), and serves as a positive control as it is essentially the maximum possible response. In FIG. 14, when the peptide epitope is NLVPMVATV (SEQ ID No: 21), the peptides used were CSGGGGSGGGGAIPVSLRANLVPMVATV (SEQ ID No: 276) (NLVPMVATV-PC) that contains the protease cleavage sequence AIPVSLR (SEQ ID No: 313), and CSGGGGSGGGGANLVPMVATV (SEQ ID No: 315) (NLVPMVATV) that does not contain a protease cleavage site.

EXAMPLE 1: STIMULATION OF T CELLS BY CETUXIMABNLVPMVATV (SEQ ID NO: 21) CONJUGATE

Summary

[0214] We contacted breast cancer cells with an agent comprising Cetuximab conjugated to a HLA-B7 peptide with and without a cleavage site. Subsequent exposure to T cells resulted in the generation of a T cell response when the breast cancer cells were contacted with the agent that contained the cleavage site.

Results

[0215] MDA.MB.231 cells, often used as a model for breast cancer, were transduced with the MMP14 gene to ensure expression of the MMP14 protein within the cell. After staining the target cells (110.sup.5) with Cetuximab either unconjugated (1) or conjugated to RPHERNGFTVL (SEQ ID No: 32), a HLA-B7 peptide (2), NLVPMVATV (SEQ ID No: 21) without the MMP14 cleavage sequence (3) or NLVPMVATV (SEQ ID No: 21) including the cleavage sequence (4), stained cells were incubated overnight. The following day, the cells were washed and NLV-specific T cells were added to the culture (110.sup.4) and incubated overnight. Supernatant was harvested and an ELISA used to determine the presence of IFN- in each culture, n=3. The results are shown in FIG. 1.

[0216] There was very little IFN- release from T cells cultured together with cells stained using Cetuximab alone and cetuximab conjugated with the mismatched HLA-peptide. T cells cultured with cells stained using cetuximab conjugated with the correct peptide but lacking the MMP14 cleavage site also produced very little IFN- whereas T cells cultured with the cells stained using cetuximab conjugated with the correct peptide containing the MMP14 cleavage site produced a large amount of IFN-.

EXAMPLE 2: STIMULATION OF CD4.SUP.+ T CELLS BY RITUXIMABDYSNTHSTRYV (SEQ ID NO: 55) CONJUGATE

Summary

[0217] We contacted B-lymphoblastoid cells (B-LCL) with an agent comprising Rituximab conjugated to a cytomegalovirus HLA Class-II restricted peptide DYSNTHSTRYV (SEQ ID No: 55) with and without a cleavage site. Subsequent exposure to CD4.sup.+ T cells resulted in the generation of a T cell response when the B-LCL cells were contacted with the agent that contained the cleavage site.

Results

[0218] After staining the B-LCL cells with Rituximab conjugated to an irrelevant mismatched peptide RPHERNGFTVL (SEQ ID No: 32), a HLA-B7 peptide, not containing the protease cleavage sequence (1), an irrelevant, mismatched HLA class-I peptide VLEEETSVML (SEQ ID No: 316), an HLAA-A2 peptide, with the protease cleavage sequence (2), the relevant peptide DYSNTHSTRYV (SEQ ID No: 55) without the protease cleavage sequence (3), or the relevant peptide DYSNTHSTRYV (SEQ ID No: 55) including the protease cleavage sequence (4), stained cells were incubated overnight. The following day, the cells were washed and DYSN-specific CD4.sup.+ T cells were added to the culture and incubated overnight. Supernatant was harvested to determine the presence of IFN- in each culture, n=3. There was very little IFN- release from CD4.sup.+ T cells cultured together with cells stained using Rituximab conjugated with the mismatched HLA-peptide without the protease cleavage site. CD4.sup.+ T cells cultured with cells stained using Rituximab conjugated with the correct peptide but lacking the protease cleavage site also produced very little IFN- whereas T cells cultured with the cells stained using Rituximab conjugated with the correct peptide containing the protease cleavage site produced a large amount of IFN-. However, when T cells were cultured with Rituximab conjugated with the HLA-mismatched peptide containing the protease cleavage site, there was no IFN- produced. The results are shown in FIG. 2.

[0219] A similar example showing stimulation of CD4.sup.+ T cells by Rituximab-TPRVTGGGAM conjugate is shown in FIG. 3.

EXAMPLE 3: STANDARD OPERATING PROCEDURE FOR CHEMICAL CONJUGATION OF CYSTEINYLATED PEPTIDE TO ANTIBODY

[0220] 1. Cysteinylated peptides dissolved in DMSO to final concentration of 5 mg/ml. [0221] 2. Weigh 1 mg Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) and dissolve in 500 l phosphate buffered saline (PBS). [0222] a. Other heterobifuctional cross-linkers could be used in place of Sulfo-SMCC e.g. Sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido) hexanoate (Sulfo-LC-SPDP) and N-[-Maleimidopropionic acid] hydrazide, trifluoroacetic acid salt (BMPH) amongst others. [0223] 3. Add 20 l antibody (1 mg/ml, 20 g antibody) to dissolved Sulfo-SMCC and incubate at room temperature for 1 hour. [0224] 4. Wash a Protein G column (GE Healthcare) by firstly spinning the column at 13,000 rpm for 30 seconds to remove the ethanol (storage buffer). [0225] 5. Add 500 l PBS and mix protein G beads well before spinning at 13,000 rpm for 30 seconds. Remove eluate and repeat a further two times. [0226] 6. Add antibody-SMCC to protein G column, mix well and incubate for 5 minutes. Centrifuge at 13,000 rpm for 30 seconds and remove eluate. [0227] 7. Wash antibody by adding 500 l PBS and mixing the beads well before spinning at 13,000 rpm for 30 seconds and removing eluate. Repeat this step a further two times. [0228] 8. To elute the bound antibody, add 125 l 0.1M acetic acid to the beads and incubate for 2 minutes at room temperature. Place column in a 1.5 ml eppendorf and spin at 13,000 rpm for 30 seconds and collect eluate. [0229] 9. Repeat step 8. [0230] 10. Add 250 l 0.2M Na.sub.2HCO.sub.3 and allow to stand at room temperature for 5 minutes. [0231] 11. Add 2 l peptide, previously dissolved in DMSO, to the SMCC-activated antibody and incubate at room temperature for 2 hours. [0232] 12. Repeat steps 4 to 10 to remove excess unbound peptide from the antibody. [0233] 13. After adding 250 l 0.2M Na.sub.2HCO.sub.3, add a further 500 l PBS before storage. Antibody can now be used to stain cells. [0234] 14. Store antibody at 4 C.

EXAMPLE 4: TREATMENT OF BREAST CANCER

[0235] An agent comprising Cetuximab that is attached to a peptide T cell antigen such as NLVPMVATV (SEQ ID No: 21), derived from a cytomegalovirus, via a linker comprising a PRSA-KELR (SEQ ID No: 321) protease cleavage site (cleavable by Matrix metalloproteinase 14 (MMP14)) is prepared. The agent is formulated with a pharmaceutically acceptable excipient and administered to patient with an epithelial malignancy such as breast cancer. The agent, Cetuximab, is targeted to breast cancer cells and upon binding comes into contact with MMP14. The cleavage of the protease cleavage site releases the T cell antigen, NLVPMVATV (SEQ ID No: 21), which subsequently binds to the HLA-A*0201 molecules on the surface of the breast cancer cell. The breast cancer cells expressing the T cell antigen is targeted by the host immune system for cytolysis by the effector CD8 T cells.

EXAMPLE 5: TREATMENT OF B-CELL LYMPHOMA (EG CHRONIC LYMPHOCYTIC LEUKAEMIA)

[0236] An agent comprising Rituximab that is attached to a HLA class-II peptide T cell antigen such as DYSNTHSTRYV (SEQ ID No: 55), derived from a cytomegalovirus, via a linker comprising a TIPV-SLRS (SEQ ID No: 317) protease cleavage site (cleavable by Matrix metalloproteinase 2 (MMP2)) is prepared. The agent is formulated with a pharmaceutically acceptable excipient and administered to patient with B cell lymphoma (eg chronic lymphocytic leukaemia). The agent, Rituximab, is targeted to B cells and upon binding comes into contact with a protease. The subsequent cleavage of the protease cleavage site releases the T cell antigen, DYSNTHSTRYV (SEQ ID No: 55), which subsequently binds to the HLA-DR*0107 molecules on the surface of the B cell. The B cells expressing the T cell antigen would then be targeted by the host immune system for cytolysis by the effector CD4 T cells.

EXAMPLE 6: TREATMENT OF BOWEL CANCER

[0237] An agent comprising Cetuximab that is attached to a peptide T cell antigen derived from a cytomegalovirus via a linker comprising a CPGR-VVGG (SEQ ID No: 254) protease cleavage site (cleavable by uPA) is prepared. The agent is formulated with a pharmaceutically acceptable excipient and administered to a bowel cancer patient.

EXAMPLE 7: TREATMENT OF B CELL LYMPHOMA (EG CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL))

[0238] An agent comprising Rituximab that is attached to a peptide T cell antigen derived from a cytomegalovirus via a linker comprising a PQG-IAGQ (SEQ ID No: 269) protease cleavage site (cleavable by MMP2) is prepared. The agent is formulated with a pharmaceutically acceptable excipient and administered to a B cell lymphoma (eg CLL) cancer patient.

EXAMPLE 8: STIMULATION OF T CELLS BY RITUXIMABTPRVTGGGAM (SEQ ID NO: 31) CONJUGATE

Summary

[0239] We contacted B-lymphoblastoid cells (B-LCL) with an agent comprising Rituximab conjugated to a cytomegalovirus peptide TPRVTGGGAM (SEQ ID No: 31) with and without a cleavage site. Subsequent exposure to T cells resulted in the generation of a T cell response when the B-LCL cells were contacted with the agent that contained the cleavage site.

Results

[0240] After staining the cells with Rituximab conjugated to RPHERNGFTVL (SEQ ID No: 32), a HLA-B7 peptide (1), an irrelevant, mis-matched HLA class-I peptide, VLEEETSVML (SEQ ID No: 316), (a HLA-A2 peptide) containing the protease cleavage sequence (2), the relevant peptide TPRVTGGGAM (SEQ ID No: 31) with the protease cleavage sequence (3) or the relevant peptide TPRVTGGGAM (SEQ ID No: 31) without the cleavage sequence (4), stained cells were incubated overnight at 37 C. The following day, the cells were washed and TPR-specific T cells were added to the culture and incubated overnight. Supernatant was harvested to determine the presence of IFN- in each culture, n=3. The results are shown in FIG. 3.

[0241] There was very little IFN- release from T cells cultured together with cells stained using Rituximab conjugated with the mismatched HLA-peptide with or without the protease cleavage site (1 & 2). T cells cultured with cells stained using Rituximab conjugated with the correct peptide but lacking the protease cleavage site also produced very little IFN- (4) whereas T cells cultured with the cells stained using Rituximab conjugated with the correct peptide containing the protease cleavage site (3) produced a large amount of IFN-.

EXAMPLE 9: TREATMENT OF B CELL LYMPHOMA (EG CHRONIC LYMPHOCYTIC LEUKAEMIA)

[0242] An agent comprising Rituximab that is attached to a HLA class-I peptide T cell antigen such as TPRVTGGGAM (SEQ ID No: 31), derived from a cytomegalovirus, via a linker comprising a TIPV-SLRS (SEQ ID No: 317) protease cleavage site (cleavable by Matrix metalloproteinase 2 (MMP2)) is prepared. The agent is formulated with a pharmaceutically acceptable excipient and administered to patients with B cell lymphoma (eg chronic lymphocytic leukaemia). The agent, Rituximab, is targeted to B cells and upon binding comes into contact with a protease. The cleavage of the protease cleavage site releases the T cell antigen, TPRVTGGGAM (SEQ ID No: 31), which subsequently binds to the HLA-B*0702 molecules on the surface of the B cell. The B cells expressing the T cell antigen would then be targeted by the host immune system for cytolysis by the effector CD8 T cells.

EXAMPLE 10: TUMOR TARGETING BY RE-DIRECTING ANTI-VIRAL IMMUNE RESPONSES IN VITRO AND IN VIVO

Summary

[0243] We have shown that antibodies can be engineered to deliver and release viral peptides at the tumor site by exploiting a tumor-associated proteolytic environment thus allowing resident anti-viral T cells to specifically kill tumor cells. We screened 15 HLA-A2.sup.+ tumor cell lines (THP-1 (acute monocytic leukemia cell line); A498 (renal cell carcinoma); MDA-MB-231 and MCF-7 (breast cell adenocarcinomas); NCI-H522 (non-small cell lung carcinoma); Ovcar-3 (ovarian adenocarcinoma); Colo205 and HCT-116 (colorectal carcinoma)) and showed that 100% are recognized and killed by human anti-viral T cells when pulsed with cognate viral peptides (FIG. 12). Furthermore we used both molecular and cellular approaches to demonstrate that immunodominant anti-viral CD8+ T cells are present in a variety of human tumors providing a rationale for such an approach.

Results

[0244] Antibody-peptide epitope conjugates (APEC) were generated, through covalently linking T cell epitope peptides with clinically available antibodies Cetuximab and Rituximab. Neither APEC in solution nor plate-bound were able to activate cognate T cells. Healthy CD20+ B cells bound Ritixumab-APEC (RPEC) but were unable to activate T cells in vitro. However, CD20+ lymphoma cell lines were able to be efficiently targeted by T cells when bound by RPEC in vitro through proteolytic release of bound peptide demonstrating differential tumor targeting (FIG. 5).

[0245] Using breast cancer as a solid tumor model, EGF receptor was targeted on the malignant cell line MDA-MB-231 using Cetuximab-APEC (OPEC). Results demonstrate T cell recognition when target cells were bound by OPEC (p<0.01) (FIG. 6). FIGS. 7B and 7C also demonstrate successful targeting of colorectal adenocarcinoma cell lines and pancreatic carcinoma cell lines using an anti-Muc1 antibody peptide conjugate.

[0246] In vivo data using a xenograft mouse model demonstrate significant efficacy in mice treated with the OPEC and T cells compared with mice treated with either T cells alone or with OPEC alone (see FIG. 7A). Because the conjugates are given by intraperitoneal injection and the tumour is given subcutaneously, these data also demonstrate plasma stability in vivo.

[0247] FIG. 7D demonstrates successful targeting of T cells using a protein that contains a viral peptide sequence as part of its polypeptide chain. A single chain fragment V (scFv) antibody-like construct was prepared, encoding a protease recognition domain and T cell epitope, and shown to efficiently target MDA-MB-231 cells in vitro.

[0248] FIG. 8A shows that the conjugates according to the invention may be used to selectively target cancer cells. Following labelling of lymphoblastoid cells or healthy B cells with Rituximab conjugated with viral peptide, there is no recognition of healthy B cells whereas there is recognition of lymphoblastoid cells only in the presence of the viral peptide that the T cells are specific for.

[0249] FIG. 8B demonstrates plasma and serum stability of conjugates according to the invention.

[0250] FIG. 10A demonstrates the mechanism of action of the Cetuximab-peptide complex occurring at the cell surface and not by way of internalisation of the whole complex. By chemical fixation of target cells, internalisation of the complex is inhibited and a positive IFN- response using fixed cells would demonstrate external processing of the APEC. The results show that target cells chemically fixed demonstrate a similar ability to induce IFN- production by T cells when they are incubated with Cetuximab-NLVPMVATV-Protease Cleavage as that seen when target cells have not been chemically fixed. Similarly, chemically fixed target cells cannot induce an IFN- response when labelled with an irrelevant APEC (Cetuximab-VLEETSVML-Protease Cleavage) similar to that seen in untreated target cells.

[0251] FIG. 10B demonstrates the ability to use a peptide as a targeting moiety in place of an antibody. Peptides which directly bind to either EGF receptor or Her2/neu receptor were synthesised containing a protease cleavage sequence and the viral epitope named receptor peptides (EGF-NLVPMVATV (NLVPMVATVAIPVSLRSAAAYCRDYDYDGRYFDCY) (SEQ ID No: 314) Ponde et al (2011) Bioorg Med Chem Lett, 21 or Her2-NLVPMVATV (NLVPMVATVAIPVSLRSAAAFCDGFYACYMDV) (SEQ ID No: 311) Park et al (2000) Nat Biotech 18). Target cells labelled with either peptide were able to induce an IFN- response from viral-specific CD8+ T cells similar to that seen when target cells were pulsed with exogenous viral peptide. When either receptor peptide is removed, the target cells were not recognised by the T cells.

[0252] FIG. 13 shows that antibody peptide epitope complexes (APEC) formed of anti-EGFR Cetuximab and peptides were able to re-target antigen specific T cells to target these malignant cell lines, but only when the peptide contained a protease cleavage site.

[0253] FIG. 14 shows that antigen specific T cells are at the tumour site and that other antibodies may be used in the approach (eg anti-MUC1 antibody SM3). The figure also demonstrates selective targeting of malignant cells compared to healthy cells. For example, the anti-CD20 Rituximab APEC efficiently targets malignant lymphoma cells in vitro but spares healthy B cells derived from peripheral blood (FIG. 14C). The figure also shows the applicability toward MHC Class-II restricted peptides using CD4+ cytotoxic T cells (FIG. 14D), and that the APEC approach does not require the antigen processing capacity of target cells (FIG. 14E) suggesting that peptide cleavage is occurring at the cell membrane.

Discussion

[0254] Targeting tumors in this way bypasses the requirement of an intact antigen processing system in the tumor cell. We believe that the processing of the protease cleavage site and subsequent loading of peptide onto MHC class I/II molecules occurs extracellularly, without the requirement of classical antigen processing components.

[0255] Furthermore, the results demonstrate that conjugating peptides to different antibodies allows targeting of many different malignancies including breast cancer, multiple myeloma, acute myeloid leukemia and pancreatic cancer. Therefore, the immunotherapeutic potential of this mechanism is far-reaching.

[0256] Xenograft Studies

[0257] Xenograft models use NOG mice (NOD Rag2.sup./c.sup./) (M. Ito et al., Blood 100, 3175 (2002)). Tumour cell lines are grown using standard laboratory tissue culture techniques and injected subcutaneously in Matrigel and left to engraft for 7 days. Human CD4+ and CD8+ T cells are cultured using standard techniques and cultured from healthy laboratory donors. 10.sup.6 T cells are infused into each study mouse intraperitoneally. Antibody or APEC is injected into the intraperitoneal cavity. Mice are injected with 120 mg/kg luciferin weekly and growth and metastatic dissemination of the cells monitored using IVIS Spectrum (Caliper Lifesciences). Quantitation of outgrowth kinetics is determined and metastasis quantified by measuring luminescent signal from each organ at the experimental endpoint.

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