AU2002301994B2 - Method for producing cytotoxic T-cells - Google Patents

Description

AUSTRALIA

Patents Act 1990 ALEXIS BIOTECH LIMITED COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Method for producing cytotoxic t-cells The following statement is a full description of this invention including the best method of performing it known to us:- METHOD FOR PRODUCING CYTOTOXIC T-CELLS.

Background of the Invention Existing technology for production of alloreactive peptide specific CTLs requires mixing donor cells with initially RMA cells (mouse cancer line) CIR or T2 cells (human tumour cell) and then Drosophila cells. This is both complicated and of low efficacy. These methods are also very 'dirty' which in this context means that they use cancer cells. It is undesirable to use cancer cells in the production of CTLs for administration to a subject.

Existing methods for producing CTLs in vitro are very labour intensive and expensive, involving culture of at least three different cell lines and complicated procedures of contacting the CTLs with each of these as part of the method(s). Is is an advantageous feature of the present invention that CTL production is simplified and is also cheaper and/or easier and/or faster than existing techniques.

Subjects can be known to tolerate their own tumours rather than mounting an immunological response to them. This is because the tumour can appear as 'own' or 'self to the immune system. Other people's immune systems (ie. alloreactive) can fight better. However, the drawback with this approach is that a dual response can be produced in that the ordinary tissues of the recipient can be attacked too. This is the so-called graft vs host disease. The actual host can be attacked which is problematic.

One of the advantages of the present invention is the alleviation of this problem with existing methods by provision of better CTLs which react with the target peptide in the appropriate context such as when complexed with HLA-A2.

A number of approaches to the generation of anti-tumour CTLs, including use of dendritic cells, DNA vaccines and peptide administration are in clinical development aiming to expand autologous tumour reactive CTLs. However producing autologous CTLs against certain targets is difficult or impossible due to immunological tolerance, and existing attempts at producing alloreactive CTLs have serious drawbacks as explained above (Sadovnikova 1998, Stauss 1999 Dutoit 2002).

Furthermore, some target peptides apparently cannot be targeted by autologous means.

Melan-A is an example of a peptide that can be targeted by autologous methods, but many others such as WT1 cannot be targeted by such methods. The present invention advantageously permits the targeting of a far wider range of peptides than conventional autologous methods.

Other techniques for production of CTLs have been described such as production using T2 cells. T2 cells are a genetically altered human cell line which is deficient in the genes encoding the transporter associated with antigen processing (TAP). This cell line therefore fails to properly load HLA-A2 Class I molecules with endogenous peptides. Therefore, exogenously added peptides can be made to bind a proportion of the HLA-A2 molecules on the surface of T2 cells. these other techniques for producing CTLs rely on the culture of T2 cells followed by peptide loading, cleaning of the cells and using them as a stimulatory cell to try to stimulate CTLs for example from PBMC samples. Even when this labour intensive method appears to work, the stimulatory cells have to be constantly prepared and replaced at regular intervals in a rolling maintenance program. The present invention advantageously avoids the use of such T2 cells and also greatly simplifies the procedure. Furthermore, use of T2 cells is limited to HLA-A2, whereas the methods of the present invention are also applicable to other HLA types.

The existing technologies employed to produce alloreactive CTLs are relatively inefficient and the choice of target HLA class I is restricted by the limited allotypes of the commonly used antigen presenting cells, which are themselves genetically altered artificial cell lines.

It is desirable to separate out the beneficial effects of alloreactive transplants 'graft versus leukaemia (tumour) effect' from the unwanted 'graft versus host disease'. Thus, the present invention relates to a method for the production of alloreactive peptide specific CTLs.

The production of alloreactive CTLs is advantageous over autologous CTLs because a subject may be tolerant to the autologous HLA peptide combination whilst tolerance in the alloreactive setting is much less likely. Thus the present invention relates to production of CTLs against a given HLA /peptide complex from a foreign donor (alloreactive) rather than autologous.

Without wishing to be bound by theory, it may be that some HLA types can produce better reactivity against certain other HLA types. For example, making an anti-HLA- A2 response may be easier for an HLA1 background but more difficult for an HLA4 background. The methods of the present invention may be advantageously applied to the study of this phenomenon.

Summary of the Invention It will be appreciated that the invention relates to the production of alloreactive antipeptide CTLs. Preferably the peptide is a tumour peptide or other antigenic peptide associated with a disorder such as cancer. The peptide may be a modified peptide such as a glycopeptide.

Briefly, in this aspect the invention relates to the treatment of a sample of CTLs in such a way as to promote the selection and expansion of CTLs having the desired reactivity. In this manner, alloreactive anti-peptide CTLs are advantageously produced.

In another aspect, the invention provides a method for producing antipeptide cytotoxic T lymphocytes (CTLs) comprising; providing a sample of peripheral blood lymphocytes (PBLs), attaching a complex of a class I HLA and a peptide to antigen presenting cell(s) (APCs) present in said PBLs, optionally removing excess class I HLA/peptide complex, and inubating with proliferative cytokine.

In another aspect, the invention provides a method as described above wherein the APC is a B-cell.

In another aspect, the invention provides a method as described above wherein the complex is attached to the APC by attachment means comprising a molecule capable of selective binding to a B cell.

In another aspect, the invention provides a method as described above wherein the attachment means comprises sfvSA to CD20 or CD19.

In another aspect, the invention provides a method as described above wherein said sample of PBLs is a sample of a class I HLA negative PBLs.

In another aspect, the invention provides a method as described above wherein the class I HLA is HLA-A2.

In another aspect, the invention provides a method as described above wherein the peptide is a tumour peptide.

In another aspect, the invention provides a method as described above wherein the tumour peptide is selected from the group consisting of Melan-A, WT1 and a telomerase.

In another aspect, the invention provides a method as described above wherein the proliferative cytokine is a combination of IL-7 and IL-2.

In another aspect, the invention provides a method as described above wherein the incubation is about 7 days.

In another aspect, the invention provides a method as described above wherein the incubation step is repeated for a further 7 days.

In another aspect, the invention provides a CTL obtainable by the methods described above. In another aspect, the invention provides a method of treating a subject comprising administering a CTL as described above to said subject.

In another aspect, the invention provides a CTL obtained by the methods described above. In another aspect, the invention provides a method of treating a subject comprising administering a CTL as described above to said subject.

In a preferred aspect, the production of alloreactive anti-tumour CTLs is accomplished in vitro. In a further preferred aspect, the present invention relates to a method for treating a subject comprising administering to said subject a sample of alloreactive CTLs according to the present invention.

Other technology for production of alloreactive peptide specific CTLs requires mixing donor PBLs with initially RMA cells (mouse cancer line) CIR cells (human tumour cell) and then Drosophila cells. This is both complicated and of low efficacy. These methods are also very 'dirty' which in this context means they use cancer cells. It is clearly undesirable to use cancer cells in the production of CTLs for administration to a subject. Thus, it is an advantage of the present invention that the use of cancer cells in the production of CTLs is avoided. In a preferred aspect of the invention, only PBLs and recombinant peptides/proteins are employed in said methods.

In one aspect, the present invention provides a method for producing antipeptide cytotoxic T lymphocytes (CTLs) comprising: providing a sample of peripheral blood lympocytes (PBLs); (ii) optionally removing excess class I HLA and a peptide to antigen presenting cell(s) (APCs) present in said PBLs; (iii) optionally removing excess class I HLA/peptide complex; and (iv) incubating with proliferative cytokine.

Further Detailed description of the Invention T cell receptor genes In addition to their direct use in therapy, the alloreactive CTLs of the present invention can also be used for cloning of their T cell receptor genes. This is preferably accomplished by methods commonly used in the art eg. PCR amplification and cloning of the TCR genes of the alloreactive CTLs of the present invention.

The T cell receptor genes obtained in accordance with the present invention find application in gene therapy, for example using TCR transfer technology, as well as for making recombinant TCR molecules for therapy, and in any other techniques making use of cloned TCR gene(s).

Preferably when the recombinant TCR molecules are used in therapy, the Avidex technology is employed to produce soluble TCR molecules (see below).

Furthermore, the dismantling of the alloreactive T cell receptor, co-receptor, and binding site on appropriate cells into their component parts is advantageously carried out. These can then be modified, for example to discover new small molecule drug candidates.

Techniques are now available to produce stable, soluble, reproducible T cell proteins that provide the same range of opportunities as monoclonal antibodies did for the humoral immune system; new protein drugs, diagnostics, reagents of importance to the pharmaceutical industry, and improvement of existing drugs by site-directed targeting are therefore within the scope of the present invention through the alloreactive CTLs provided herein.

Thus, the alloreactive T cells of the present invention may be used for the production of T cell proteins, a key component of the body's immune system, as stable compounds suitable for a broad range of clinical applications. One way in which they may be so used is through recombinant DNA technology to clone the T cell receptors and produce them in vitro. This is preferably accomplished following the techniques from the teachings of Bent Jakobsen et al (Institute of Molecular Medicine, University of Oxford), and the technologies stemming from and commercially available from Avidex Limited, Abingdon, Oxfordshire, UK.

Further applications of the Alloreactive CTLs In addition to their direct use in therapy, the alloreactive CTLs of the present invention can also be used in vitro, for example in purging cell preparations of tumour cells such as bone marrow cells. Advantageously this may be accomplished in accordance with the methods of Gao et al (Gao et al 2000 Blood95:2198-203).

The BQ cells described herein that recognize all A2/peptide complexes are also embraced by the present invention. These cells and their TCRs are useful for example in preventing organ transplant rejection as soluble TCRs that block all anti-HLA-A2 cells binding transplant organ cells. They may also be used for therapy of patients who develop allogeneic tumours after organ transplant, an unusual but acute medical problem.

HLA/peptide complex The HLA/peptide complex for use in production of alloreactive CTLs may be any HLA/peptide complex that is of immunological interest. In a preferred embodiment, the HLA used in the complex is the same HLA that the PBLs (ie. the CTLs) are negative for. Preferably, the HLA is a class I HLA. Preferably the class I HLA comprises one or more of HLA-Al, HLA-A2, HLA-A3 or HLA-B7. In a preferred embodiment, the HLA is HLA-A2. Thus, in a highly preferred embodiment, the CTLs are HLA-A2 negative and the HLA in the complex is HLA-A2. Specific examples of HLA/peptide complexes include HLA class I/telomerase (pan tumor) HLA-A2/melan A (melanoma) 9 HLA-A2/ WT1 (leukaemia) or any other peptides of interest.

Further examples of peptides of interest are given in tables 1-7.

Table 1 Class I HLA-restricted cancer/testis antigens. All these antigens were found to be expressed by normal spermatocytes and/or spermatogonia of testis.

Occasionally MAGE-3, MAGE-4 and the GAGE genes were found to be expressed also in placenta [26, 24]. The NY-ESO-1 antigen was found to be expressed in normal ovary cells [18].

Gene HLA allele Peptide epitope Author [Ref] Tissue distribution among tumorsa MAGE-Al Al EADPTGHSY Traversari et al., 1992 [119] Melanoma, breast carcinoma, SCLC [27, 29, 125] MAGE-Al A3 SLFRAVITK Chaux et al., 1999a [16] sarcoma, NSCLC [27, 29] thyroid medullary carcinoma MAGE-A A24 NYKHCFPEI Fujie et al., 1999 [37] [125] colon carcinoma[27] laryngeal tumors [29] MAGE-Al A28 EVYDGREHSA Chaux et al., 1999a [16] MAGE-Al, -A2 B37 REPVTKAEML Tanzarella et al., 1999 [113] Melanoma, colon and breast carcinomas, SCLC [27, 29, -A3, -A6 125] sarcoma, NSCLC [27, 29] thyroid medullary carcinoma, H/N tumors, bronchial SCC [125] laryngeal tumors [29] leukemias [27] Melanoma, breast carcinoma, SCLC [27, 29, 125] sarcoma, colon carcinoma, NSCLC [27, 29] thyroid medullary carcinoma [125] MAGE-A1 MAGE-Al MAGE-Al MAGE-Al MAGE-A2 MAGE-A2 B53 Cw2 Cw3 Cwl6 A2 A2

DPARYEFLW

SAFPTTINF

SAYGEPRKL

SAYGEPRKL

KMVELVHFL

YLQLVFGIEV

Chaux et al., 1999a [16] Chaux et al., 1999a [16] Chaux et al., 1999a [16] van der Bruggen et al., 1994b [127] Visseren et al., 1997 [128] Visseren et al., 1997 [128] Melanoma, colon and breast carcinomaa, SCLC [27, 29, 124] sarcoma, NSCLC [27, 29] thyroid medullary MAGE-A2 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A3 MAGE-A4 A24 Al A2 A24 A24 B44 B52 A2

EYLQLVFGI

EVDPIGHLY

FLWGPRALV

TFPDLESEF

IMPKAGLLI

MEVDPIGHLY

WQYFFPVIF

GVYDGREHTV

Tahara et al., 1999 [110] carcinoma [125] laryngeal tumors [77] leukemias [27] Gaugler et al., 1994 [40] Melanoma, colon and breast carcinomas [27, 125] H/N van der Bruggen et al., 1994a [126] tumors [18] bronchial SCC, thyroid medullary and Oiso et al., 1999 [89] bladder carcinoma, sarcomas, SCLC, NSCLC [125] Tanaka et al., 1997 [111] leukemias [29] Herman et al., 1996 [48], Fleischhauer et al., 1996 Russo et al. 2000 [103] Duffour et al., 1999 [33] Melanoma, NSCLC, sarcomas, esophageal, colon and MA GE-A 6 MAGE-A12 A34 A2 Cw7

MVKISGGPR

GLYDGMEHL

VRIGHLYIL

Zorn and Hercend, 1999b [147] Huang et al., 1999 [52] Panelli et al., 2000 [91], Heidecker et al., 2000 [47] breast carcinomas [27] Melanoma, NSCLC, colon carcinoma, leukemias [27] Not defined Melanoma, myeloma, brain tumors, sarcoma, leukemias, SCLC, NSCLC, H/N tumors, bladder, lung, esophageal, breast, prostate and colorectal carcinoma [27] Melanoma, bladder and mammary carcinomas, H/N SCC, NSCLC, sarcoma Melanoma,skin tumors,mammary and ovarian carcinomas BAGE Cwl6 AARAVFLAL Boel et al., 1995 [6] DAM-6, -10 A2 FLWGPRAYA Fleischhauer et al., 1998 [36] [77] lung carcinoma [25, 77] seminomas GAGE-I, -8 Cw6 YRPRPRRY Van den Eynde et al., 1995 [123] Melanoma, sarcoma, NSCLC, SCLC, mesothelioma, De Backer et al. 1999 [26] sarcoma, seminoma, leukemias, lymphomas, H/N tumors, bladder,esophageal, mammary,colon, prostate carcinomas GAGE-3, A29 YYWPRPRRY De Backer et al. 1999 [26] Melanomas, H/N tumors, leukemias, esophageal, lung and -7B bladder carcinomas NA88-A B13 MTQGQHFLQKV Moreau-Aubry et al., 2000 [82] Melanoma NY-ESO-1 A2 SLLMWITQCFL Jager et al., 1998 [54] Melanoma, sarcoma, B-lymphomas, hepatoma, H/N A2 SLLMWITQC Jager et al., 1998 [54] tumors, bladder, lung, prostate, ovarian, thyroid and A2 QLSLLMWIT Jager et al., 1998 [54] breast carcinoma [18] NY-ESO-la (CAG-3) A31 ASGPGGGAPR Wang et al., 1998b [134] a Tissue distribution among tumors as described in the given references when different from the paper first reporting the sequence of the epitope.

13 Table 2 Class I HLA-restricted melanocyte differentiation antigens. These antigens can only be expressed in normal and neoplastic cells of the same lineage (namley melanocytes, skin, retina, peripheral ganglia) or in normal cells of the prostate gland Gene HLA allele Peptide epitope A uth ors [refJ MAR T-J/Melan-A' A2 AAGIGILTV Coulie et a. 1994 [22] Kawakcami et 1994a [58] A2 EAAGIGILTV Schneider eta!., 1998 [106] A2 ILTVILGVL Castelli et at., 1995 [14] AEEAAGIGIL Schneider et 1998 [106] AEEAAGIGILT Schneider et 1998 [106] MCJR A2 TILLGIFFL Salazar-Onfray et al., 1997 [104] A2 FLALIICNA Salazar-Onfray et at., 1997 [104] GpJOO A2 KTWGQYWQV Bakker et al., 1995 [3] A2 A2 A2

AMLGTHTMEV

MLGTHTMEV

SLADTNSLAV

ITDQVPFSV

LLDGTATLRL

YLEPGPVTA

VLYRYGSFSV

RLMKQDFSV

RLPRIFCSC

LIYRRRLMK

ALNFPGSQK

SLlYRRRLMK Tsai et 1997 [120] Tsai et 1997 [120] Tsai et 1997 [120] Kawakamni et 1995 [61] Kawakami et 1994b [59] Cox et 1994 [24] Kawakami et 1995 [61] Kawakami et al., 1998 [62] Kawakami et al., 1998 [62] Kawakamid et al., 1998 [62] Kawashima et 1998 Kawashima et 1998 A3 A3 A24 Cw8

ALLAVGATK

VYFFLPDHL

SNDGPTLI

Skipper et al., 1996 [108] Robbins et al., 1997 [99] Castelli et al., 1999 PSA Al VSHSFPHPLY Corman et al., 1998 A2 FLTPKKLQCV Correale et at., 1997 [21 A2 VISNDVCAQV Correale et 1997 [21 PSM Al HSTNGVTRIY Corman et at., 1998 Tyrosinase Al KCDICTDEY Kittlesen et at., 1998 [68] Al A2

SSDYVIPIGTY

YMDGTMSQV

MLLAVLYCL

AFLPWHIRLF

SEIWRDIDF

Kawakami et al., 1998 [62] W6lfel et 1994 [137] W6lfel et at., 1994 [137] Kaig et al., 1995 [57] Brichard et at., 1996 TRP-J (or gp7S) A31 MSLQRQFLR Wang et at., 1996b 132] TRP-2 A2 SVYDFFVWL Parkhurst et at., 1998 [92] A2 A31 A33 Cw8

TLDSQVMSL

LLGPGRPYR

LLGPGRPYR

AN"DPIFVVL

Noppen et al., 2000 [86] Wang et 1996a 131] Wang et at, 1998a [133] Castelli et al., 1999 a Two different groups simultaneously discovered this gene and gave it two different names, MART- I and Melan-A respectively.

Table 3 Class I HLA-restricted widely expressed antigens Gene HLA Peptide epitope Tissue distribution Reference allele Tumors Normal tissues ART-4 A24 AFLRHAAL SCC, SCLC, H/N tumors, leukemia, lung, Testis, placenta, fetal liver Kawano et al., 2000 [64] DYPSLSATDI esophageal, gastric, cervical, endometrial, ovarian and breast carcinomas CAMEL A2 MLMAQEALAFL Melanoma Testis, placenta, heart, skeletal muscle, Aarnoudse et al., 1999 [1] pancreas CEA A2 YLSGANLNL Melanoma Testis, placenta, heart, skeletal muscle, Tsang et al., 1995 [121] (CAP-1)a pancreas CEA A3 HLFGYSWYK Colon, rectum, pancreas, gastric, breast Gastrointestinal embryonic tissue Kawashima et al., 1999 [66] and lung carcinomas Cyp-B A24 KFHRVIKDF Lung adenocarcinoma, T cell leukemia, Ubiquitously expressed in normal Gomi et al., 1999 [42] DFMIQGGDF lymphosarcoma bladder, ovarian, uterine tissues.

and esophageal SCC HER2/neu A2 KIFGSLAFL Melanoma ovarian and breast carcinomas Epithelial cells Fisk et al., 1995 [34] HER2/neu A2 IISAVVGIL Melanoma, ovarian, pancreatic [96] b and Epithelial cells Peoples et al., 1995 breast carcinomas HER2/neu A2 RLLQETELV Melanoma, ovarian, gastric, pancreatic [96] Epithelial cells Kono et al., 1998 [71] and breast carcinomas HER2/neu A2 VVLGVVFGI Melanoma, ovarian, gastric, pancreatic [96] Epithelial cells Rongcun et al., 1999 [101] ILHNGAYSL and breast carcinomas

YMIMVKCWMI

HER2/neu A3 VLRENTSPK Melanoma, ovarian, gastric, pancreatic [96] Epithelial cells Kawashima et al., 1999 [66] and breast carcinomas hTER A2 ILAKFLHWL Lung and ovarian carcinomas multiple Hematopoietic stem cells and Vonderheide et al., 1999 myeloma, melanoma, sarcoma, acute progenitors; germinal center cells; basal [131] leukemias, non-Hodgkin's lymphomas keratinocytes; gonadal cells; certain proliferating epithelial cells hTR T A2 ILAKFLHWL Lung, prostate and ovarian carcinomas, Circulating B cells; germinal center B Minev et al., 2000 [81] RLVDDFLLV multiple myeloma, melanoma, sarcoma, cells; thymocytes; CD34+ progenitor acute leukemias, non-Hodgkin's lymphomas hemopoietic cells iCE B7 SPRWWPTCL RCC Kidney, colon, small intestine, liver, Ronsin et al., 1999 [102] heart, pituitary gland, adrenal gland, prostate, stomach MUC1 All STAPPAHGV Breast and ovarian carcinomas, multiple Noned Domenech et al. 1995 [31] myeloma, B-cell lymphoma MUC1 A2 STAPPVHNV Breast and ovarian carcinoma, multiple Noned Brossart et al., 1999 [11] myeloma, B-cell lymphoma MUC2 A2 LLNQLQVNL Ovary, pancreas and breast mucinous Colon, small intestine, bronchus, cervix B6hm et al., 1998 [7] MLWGWREHV tumors, colon carcinoma ofnon-mucinous and gall bladder type PRAME A24 LYVDSLFFL Melanoma, H/N and lung SCC, NSCLC Testis, endometrium, ovary, adrenals, Ikeda et al., 1997 [53] [122], RCC, adenocarcinoma, sarcoma, kidney, brain, skin leukemias [122] A24 AYGLDFYIL Melanoma Testis, spleen, thymus, liver, kidney, Robbins et al., 1995 [97] adrenal tissue, lung tissue, retinal tissue RU1 B51 VPYGSFKHV Melanoma, renal and bladder carcinomas Testis, kidney, heart, skin, brain, ovary, Morel et al., 2000 [83] liver, lung, lymphocytes, thymus, fibroblasts RU2 B7 LPRWPPPQL Melanoma, sarcomas, leukemia brain, Testis, kidney, liver, urinary bladder Van den Eynde et al. 1999 esophageal and H/N tumors renal, colon, [124] thyroid, mammary, bladder, prostatic and lung carcinomas SART-1 A24 EYRGFTQDF Esophageal, H/N and lung SCC Testis, fetal liver Kikuchi et al., 1999 [67] adenocarcinoma, uterine cancer SART-1 A*2601 KGSGKMKTE Esophageal, H/N and lung SCC, Testis, fetal liver Shichijo et al., 1998 [107] adenocarcinoma, uterine cancer SART-3 A24 VYDYNCHVDL H/N, esophageal and lung SCC, Lymphoid cells, fibroblasts, testis, fetal Yang et al., 1999 [139] AYIDFEMKI adenocarcinoma, leukemia, melanoma liver WTI A2 RMFPNAPYL Gastric, colon, lung, breast, ovary, uterine, Kidney, ovary, testis, spleen Oka et al., 2000 thyroid and hepatocellular carcinomas leukemia (including AML, ALL and CML) a CAP-1 is an alternative name of this peptide.

b Tissue distribution among tumors as described in the given references when different from the paper first reporting the sequence of the epitope.

c Telomerase is expressed in most human tumors: those listed were shown to be susceptible to lysis by cytotoxic T lymphocytes.

d All epithelial tissues express mucin like hyperglycosylated molecules.

Table 4 Class I HLA-restricted tumor specific antigens, including both unique (CDK-4, MUM-i, MUM-2, P-catenin, HLA-A2-R170I, ELF2m, myosin-m, caspase- 8, KIAAO2O5, HSP7O-2m) and shared (CAMEL, TRP-2/JNT2, GnT-V, G 250) antigens Gene HLA Pep tide epitope Tissue expression Reference allele Tumors Normal tissues AFP A2 GVALQTMKQ Hepatocellular carcinoma Fetal liver Butterfield et 1999 [12] f3-caten in/rn A24 SYLDSGIHF Melanoma None Robbins et 1996 [98] Caspase-8/m B35 FPSDSWCYF H/N tumors None Mandruzzato el al., 1997 [78] CDK-4/rn A2 ACDPHSGHFV Melanoma None Wolfel et al., 1995 [138] ELF2M A68 ETVSEQSN V Lung SCC None Hogan et al., 1998 GnT-V A2 VLPDVFIRC(V) a Melanoma, brain tumors, sarcoma Breast and brain Guilloux et al., 1996 (low expression) G250 Al HLSTAFARV RCC, colon, ovarian and cervical None Vissers et 1999 [129] carcinomas HA-A*020J-RJ7OI Al CVEWLRIYLENGK RCC None Brandle et 1996 [9] HSP7O-2M A2 SLFEGIDIY RCC, melanoma, neuroblastoma None Gaudin el al., 1999 [39] HST-2 A31 YSWMDISCWI Gastric signet cell carcinoma None Suzuki et al., 1999 [109] KIAA 0205 B44*03 AEPTNIQTV Bladder cancer None Gueguen et 1998 [44]

MUM-I

MUM-2 MUM-2 MUM-3 Myosin/m B44 B44 Cw6 A28 A3

EEKLIVVLF

SELFRSGLDY

FRSGLDSYV

EAFIQPITR

KINKNPKYK

Melanoma Melanoma Melanoma Melanoma Melanoma None None None None None Coulie et al., 1995 [23] Chiari et al., 1999 [19] Chiari et al., 1999 [19] Baurain et al., 2000 [4] Zorn and Hercend, 1999a [146] RAGE B7 SPSSNRIRNT Melanoma, sarcomas, mesotheliomas, Retina only Gaugler et al., 1996 [41] H/N tumors, bladder, renal, colon and mammary carcinomas SART-2 A24 DYSARWNEI H/N and lung SCC, lung None Nakao et al., 2000 AYDFLYNYL adenocarcinoma, RCC, melanoma, SYTRLFLIL brain tumors, esophageal and uterine cancers TRP-2/INT2 A68 EVISCKLIKR Melanoma None Lupetti et al., 1998 [76] 707-AP A2 RVAALARDA Melanoma None b Morioka et al., 1995 [84] a VLPDVFIRC(V) nonamer and decamer peptides are both recognized by CTLs.

b This antigen is not expressed in normal cells but, as the tissue of the testis wase not tested, it will not become clear to which category the antigen may belong until more information is available.

Table 5 Class 11 HLA-restricted antigens Gene HLA- Pep tide epitope Tissue expression Reference allele Tumors Normal tissues Epitopes from normal protein antigens Annexin II DRB*0401 DVPKWISIMTERSVPH Melanoma Not done Li et al., 1998 [73] GpJOO DRBl1*0401 WNRQLYPEWTEAQRLD Melanoma Melanocytes Li et at., 1998 [73] MA GE-i, -6 DRB*1301, LLKYRAREPVTKAE Melanoma, lung and breast Testis, placenta Chaux et 1999a 16] DRB* 1302 carcinomas, H/N SCC MAGE-3 DR*1 101 TSYVKVLHHMVKISG Melanoma, lung and breast Testis, placenta Manici et al., 1999 [79] carcinomas, H/N SCC MAGE-3 DRB*1301, AELVHFLLLKYRAR Melanoma, lung and breast Testis, placenta Chaux et 1999b 17] DRB* 1302 carcinomas, H/N SCC MART-J/Melan-A DRBL*0401 RNGYRALMDKSLHVGTQCALTRR Melanoma Melanocytes Zarour et al., 2000 [144] MUCJ DR3 PGSTAPPAHGVT Breast and ovarian cancers, None' Hiltbold et at., 1998 [49] multiple myeloma, B-cell lymphoma NIY-ESO- 1 DRB4*0 101 VLLKEFTVSG NY-ESO-J DRB4*01O 1-0103 PLPVPGVLLKEFTVSGNI

VLLKEFTVSGNILTIRLT

AADHRQLQLSISSCLQQL

Melanoma, B-lymphoma, hepatoma [18] b, sarcoma, H/N tumors, bladder, lung, prostate, ovarian, thyroid and breast carcinomas B-lymphoma, melanoma, sarcoma, 1-uN tumors, hepatoma [18] bladder, lung, prostate, ovarian, thyroid and breast carcinomas Prostate carcinoma Testis Zeng et al., 2000 [145] Testis Jager et al. 2000 PSA DR4 ILLGRMSLFMPEDTG

SLFHPEDTGQVFQ

QVFQVSHSFPHPLYD

NDLMLLRLSEPAELT

KKLQCVQLHVISM

GVLQGITSMGSEPCA

Prostate gland Corman et al., 1998 Tyrosinase DRB I*04Q 1 QNILLSNAPLGPQFP Melanoma Melanocytes Topalian et al., 1994 [117] DYSYLQDSDPDSFQD Topalian et al., 1996 [118]

SYLQDSDPDSFQD

Tyrosinase DRBI* 1501 RHRPLQEVYPEANAPIGI{NRE Melanoma Melanocytes Kobayashi et at., 1998a [69] Tyrosinase DRBL1*0405 EIWRDIDFAHE Melanoma Melanocytes Kobayashi et al., 1998b Epitopes from mutated protein antigens HPV-E7 DR*0401, LFMDTLSFVCPLC Cervical carcinoma None H~rn et 1999 [51] DR*0407 LFMDSLNFVCPWC CD C2 7/rn DRBL*0401 FSWAMDLDPKGA Melanoma None Wang et at., 1999a 135] TPI/m DRB1*0101 GELIGILNAAKVPAD Melanoma None Pieper et 1999 [96] a'All epithelial tissues express highly glycosilated mucins whereas tumor cells often show hypoglycosilated mucins with a normal protein sequence.

b Tissue distribution among tumors as described in the given references when different from the paper first reporting the sequence of the epitope.

Table 6 Epitopes derived from fusion proteins (fusion proteins are never found in normal tissues) Gene HLA allele Pep tide epitope Tissue distribution Reference among tumors HLA class I restricted epitopes bcr-abla A2 FMVELVEGA CML Buzyn et at., 1997 [13]

KLSEQESLL

MLTNSCVKL

bcr-ablp2JO(b3a2) A2 SSKALQRPV CMIL Yotnda eta!., 199 8a 141] bcr-abl (bWa2) A3 ATGFKQSSK CMEL Greco et at., 1996 [43]

KQSSKALQR

bcr-ablp2JO (bHa2) A3, All HSATGFKQSSK CML Bocchia et at., 1996 bcr-ablp2O(b3a2) A3 KQSSKALQR CML1 Norbury et al., 2000 [87] bcr-ablp2J 0(b3a2) B8 GFKQSSKAL CML Norbury et 2000 [87] ETV61AML A2 RIAECILGM ALL Yotnda et 1998b 142] HLA class 11 restricted epitopes bcr-ablp 190 (ela2) DRB 1 *1501 EGAFHGDAEALQRPVAS ALL Tanaka et 2000 [112] bcr-ablp2JO (b2a2) DRB5*0101 IPLTINKEEALQR.PVAS CMIL ten Bosch et 1999 [116] bcr-ablp2JO (b3a2) bcr-ablp2JO (b3a2) bcr-abl (b3a2) bcr-abl (b3a2) bcr-abl (b3a2) Dek-cain

LDLRIFUT

DRBI1*0401 DRBL* 1501 DRB 1*0901 DRBl 1101 DRIlI DRB4*0 103 DRBI*0101

ATGFKQSSKALQRPVAS

ATGFKQSSKALQRPVAS

ATGFKQSSKALQRPVAS

LIVVIVHSATGFKQSSKALQRPVA

IVHSATGFKQSSKALQRPVASDFEP

TMKQICKKEIRRLHQY

GGAPPVTWRRAPAPG

WRRAPAPGAKAMAPG

CML

CML

CML

CML

CML

AML

Melanoma ten Bosch et 1996 [115] ten Bosch et 1995 [114] Yasukawa et at., 1998 [140] Pawelec et 1996 [93] Bocchia et at., 1996 Ohmmnami et 1999 [88] Wang et al., 1999b 132] Pml/RARcx DRI 1 NSNHVASGAGEAAIETQSSSSEEIV [28] APL Gambacorti-Passerini et al., 1993 [38] p190 minor bcr-abl (ela2) DRB1I* 1501 EGAFHGDAEALQRPVAS AMvL Tanaka et at., 2000 [112] TEL/A ML 1 DP5, DP 17 IGRL4AECILGMNPSR AML Yun et al., 1999 [143] a These bcr-abl epitopes are not true fusion proteins generated-epitopes, because they derive from outside the bcr-abl junction.

Table 7 Frequency of epitopes recognised by a given HLA allele Antigen No. of HLA-A HLA-B HLA-C epitopes AdAGE-i, -10, -12 24 13 7 4 (17%) GAGE-], -7B, -8 8 5 0 3 (37.5%) MART-] 6 4 2 0 GplOO 12 11 0 Tyrosinase 6 5 1 0 Table 8 Donor (#cycles) HLA-A2/M1 IHLA-A2/M\elan-A HLA-A2/WT1 MG(x3) 5.2% 14.9% BQ (x5) 3.1 22.8% (x5) 0.74% PS (x4) 0.8% LH 0.5% 1 1.4% Treatment of a subject In one aspect, CTLs are produced in vitro from donor PBLs according to the present invention. They are then expanded as described herein. Expansion may also be accomplished by any other suitable method known to those skilled in the art either in combination with or instead of the expansion techniques disclosed herein. Preferably, the expansion techniques are as disclosed herein.

Alloreactive peptide specific CTLs may then be introduced/administered into a subject. This may be by any suitable technique such as by infusion.

It will be appreciated that advantageously alloreactive CTLs from a single donor may be used to treat many different subjects. Preferably such subjects are HLA-A2 positive and the CTLs are HLA-A2 negative.

Starting material The methods of making CTLs described herein preferably begin with a sample of peripheral blood lymphocytes (PBLs/PBMCs). In a preferred aspect, the methods of making CTLs described herein do not involve comixing of the PBLs with any further cell(s) in vitro.

In a preferred aspect of the invention, these PBLs are chosen so that they are complementary to the HLA type of the intended recipient. For example, if the intended recipient is HLA-A2, then preferably the PBLs are HLA-A2 negative so that the strongest alloreactive CTLs are produced.

It is an advantage of the present invention that the antigen presenting cell(s) (APCs) used are those APCs present in the starting material PBLs. In this way, the comixing of other cell(s) with the CTLs is advantageously avoided.

Attachment In this embodiment of the invention, the attachment means is capable of selectively binding to an antigen presenting cell (APC), and to the HLA/peptide complex.

Preferably the APC is a B cell. Preferably the complex is attached to the APC by attachment means comprising a molecule capable of selective binding to a B cell.

Preferably the attachment means comprises sfvSA to CD20 or CD19. Preferably the attachment means comprises sfvSA to CD20, such as the B9E9 moiety.

Expansion/proliferation of CTLs This is advantageously accomplised In this embodiment of the invention, the proliferative cytokine may be a single cytokine or a combination thereof. Preferably the proliferative cytokine is a combination of IL-7 and IL-2.

Typically, the incubation period for expansion is about 7 days. Optionally, the incubation step is repeated for about 7 further days, and may be advantageously repeated for further time periods if desired such as a further 7 days (eg. 21days in total) or even more.

It will be appreciated that the present invention relates to a CTL obtainable by the methods of the present invention. Thus the invention further relates to a method of treating a subject comprising administering a CTL obtainable by the methods of the present invention to said subject.

Preferably said CTL is directly obtained by the methods of the present invention. Thus the invention further relates to a method of treating a subject comprising administering a CTL obtained by the method of the present invention to said subject.

The invention is now illustrated by way of further examples which should not be regarded as limiting in scope. In the Examples, reference is made to the following figures and table: Figure 1 Shows six plots (Donor MG HLA-A2 negative), explained in more detail below.

Figure 2 Shows six plots (three HLA-A2/M1 and three HLA-A2/Melan-A) after 0, 1 and 2 rounds of stimulation (see example 7).

Figure 3 Schematic representation of the two step targeting system delivering HLA-A2/peptide complexes to the surface of B cells from HLA-A2 -ve donors. The presence of the immobilised complexes on the B cells leads to expansion of alloreactive CTL populations.

Figure 4 shows four plots (0,1,2 and 3) Figure shows four plots (Melan-A, Ml, Telo and E7) Figure 6a and Figure 6b Donor MG Tetramer analysis of PBMCs demonstrating the increasing numbers of tetramer positive cells with repeated cycles of stimulation with targeted HLA- A2/Melan-A (2a) or HLA-A2/M1 (2b) Figure 7 Donor PS Tetramer analysis of CTLs produced with targeted HLA- A2/Melan-A and HLA-A2/WT1 complexes. Staining of both PBMC groups with a panel of 5 HLA-A2 tetramers demonstrates significant staining only with the appropriate tetramer.

Figure 8 Donor BQ Tetramer analysis of CTLs produced with targeted HLA- A2/Melan-A and HLA-A2/WT1 complexes. Staining of the HLA-A2/Melan-A PBMCs with the panel of 5 HLA-A2 tetramers demonstrates significant staining only with the appropriate tetramer. Staining of the HLA-A2/WT1 PBMCs demonstrate similar levels of staining with all 5 tetramers.

Figure 9a and 9b Donor PS 4hr Chromiuim release assays using PBMCs targeted with 4 cycles of HLA- A2/Melan-A (5a) or 4 cycles of HLA-A2/WT1 (5b) tested against native and peptide pulsed T2 cells. Both groups of PBMCs show significant peptide specificity towards the immunising peptide complex.

Figure 10a and Donor BQ 4hr Chromiuim release assays using PBMCs targeted with 4 cycles of HLA-A2/Melan-A (5a) or 4 cycles of HLA-A2/WT1 (Sb) tested against native and peptide pulsed T2 cells. The cells from the HLA-A2/Melan-A PBMCs show a small degree of peptide specificity whilst the HLA-A2/WT1 PBMCs show no significant lysis of any of the T2 cells.

Table 8 Frequencies of tetramer positive alloreactive CTLs to HLA-A2/M1/Melan-A/WT1 before and after 1-4 cycles of stimulation with targeted HLA-A2/peptide complexes.

Example 1: Method for producing antipeptide cytotoxic T lvmphocvtes (CTLs) Method: Purify PBLs from donor (HLA-A2 -ve) Mix with sfvSA (to Add HLA-A2/peptide monomer Wash off excess Incubate for 7 days with IL-7 and 1L-2 Repeat after 7 days Results Figure 1 shows CTLs according to the present invention which are specific for HLA- A2/M1 (flu) and HLA-A2 Melan-A (melanoma) as measured after 0,1 or 2 rounds of stimulation.

Example 2: Method for producing antipeptide cytotoxic T lymphocytes (CTLs) In vitro method to produce alloreactive CTLs against a designated HLA class I/peptide combination is demonstrated.

In this Example, HLA-A2/Melan-A is used.

Figure 3 helps to illustrate this embodiment of the invention.

PBLs from healthy HLA-A2 -ve donors Purified by Histopaque centrifugation B9E9 scFvSA in PBS at 10ug/ml for 1 hour at RT then wash Biotinylated HLA-A2/Melan-A complex at 0.5ug/ml for 30 minutes at RT then wash 24 well plates at 3x 106 cells per well in RPMI with 10% AB serum IL-7 day 1 l0ng/ml IL-2 10U/ml on day 4 and every further 3 days 1, 2, 3 cycles of stimulation Read-out by Tetramer/CD8 staining and 51Cr release assay using peptide pulsed T2 cells.

Results Figures 4 and 5 illustrate the efficient production and specificity of CTLs produced according to the present invention.

Figure 4 shows the increasing population of CTLs through cycles of stimulation from 0.12% at 0 cycles to 17.3% after 3 cycles.

Figure 5 shows specificity of CTLs according to the present invention for the target HLA-A2/peptide complex.

Thus it can be seen that the methods of the present invention produce high outputs of CTLs. It further demonstrates that CTLs produced according to the present invention exhibit excellent reactivity with the target complex.

Example 3: Production of anti-tumour alloreactive T cells using antibody targeted HLA class I/peptide complexes Outline: The effective production of cytotoxic T lymphocytes (CTLs) that recognise clinically important HLA class I/peptide combinations is demonstrated herein.

Using recombinant HLA-A2 class I/peptide complexes targeted to autologous B cells of HLA-A2 +ve donors we have demonstrated that specific CD8 +ve CTLs can be made from peripheral blood mononuclear cells (PBMCs). Using this technology to deliver HLA-A2/peptide complexes to the surface of the B cells in PBMCs from HLA- A2 -ve donors, we have demonstrated the production of alloreactive CTLs against HLA-A2. Using PBMCs stimulated with targeted HLA-A2/Melan-A complexes tetramer staining demonstrated that the HLA-A2/Melan-A tetramer positive proportion of the CD8 +ve cells increased from 0.12% prior to stimulation to 15.1 after 3 weekly cycles of stimulation.

The specificity of alloreactive CTLs produced to HLA-A2/MelanA and HLA-A2/WT1 from two donors is demonstrated by staining with a panel of HLA-A2 tetramers.

From donor PS the CTLs produced to the HLA-A2/Melan-A and WT1 complexes only produced significant staining with the appropriate tetramer.

From donor BQ the CTLs produced against HLA-A2/Melan-A stained only with that tetramer, but in contrast the CTLs produced against HLA-A2/WT1 demonstrated comparable levels of binding to each of the panel of tetramers.

This difference in specificity was reflected in functional Cr release assays. Cells from donor PS showed a significant degree of specificity whilst the WT1 cells from donor BQ demonstrated no peptide specificity.

The ability to simply produce large numbers of alloreactive CTLs with targeted recombinant HLA class I/peptide complexes preferably to GMP standards offers a number of therapeutic and scientific possibilities. Thus the invention further relates to the production of alloreactive cells for adoptive immunotherapy, the cloning of T cell receptor genes from high affinity alloreactive CTLs, the screening of allograft donors for the ease and specificity of production of CTLs against designated targets and the investigation of the molecular mimicry between HLA class I/peptide complexes.

Background to Example 3 In comparison to normal cells malignant cells may have differences in the immunogenic peptides displayed within the peptide binding grove of their HLA class I molecules. These immunological differences give the potential for therapies based on enhancing specific T cell mediated immune responses to these cells (Pardoll 2000).

The increasing description of more tumour specific and tumour related HLA class I binding peptides is now further encouraging the development of T cell mediated immunotherapy directed against these defined targets (Rosenberg 1996).

In some malignancies the existence of pre-existing low levels of CTLs reactive with a number of tumour related HLA class I/peptide combinations has already been demonstrated and these epitopes may be the most suitable for autologous cancer vaccine studies (Pittet 1999). However, due to immunological tolerance, it is difficult to produce autologous high affinity CTLs to many other HLA class I/peptide combinations.

A therapeutic approach of the present invention aimed at overcoming tolerance, is to make high affinity alloreactive anti-tumour CTLs which can recognise these targets (Sadovnikova 1998). This is demonstrated herein.

These CTLs are expanded for use in adoptive immunotherapy. They are also useful in gene delivery mediated immunotherapy using the cloned T cell receptor. (Gao 2000, Stanislawski 2001, Kessels 2001).

The clinical value of alloreactive anti-tumour CTLs can already be seen in leukaemia patients who receive donor bone marrow transplants. Here the production of an alloreactive CTL mediated graft versus leukaemia activity within the graft versus host activity is associated with an enhanced prognosis (Molldrem 2000) and currently a number of projects are in progress which aim to separate these two immunological effects.

As explained above, existing methods of production of alloreactive CTLs frequently rely on the use of antigen presenting cells such as peptide pulsed T2 or HLA class I transfected drosophila cells (Moris 2001 Sadovnikova 1998, Dutoit 2002). These systems have a number of limitations including; modest immunostimulatory actions, potential HLA mismatches at more than one allele, the lack of GMP standard reagents and also a lack of flexibility to present other than HLA-A2. In the autologous setting, antibody targeted HLA class I complexes delivered to B cells are effective in the production of autologous CTLs against the targeted complex (Savage et al 2002). In this example we describe application of antibody targeted HLA class I complexes to produce allorereactive CTL responses to HLA-A2/peptide complexes from PBMCs from non-HLA-A2 donors (Fig 3).

Materials and methods Antibodies and cells The B9E9 anti-CD20 sfvscSA fusion protein (Schultz et al 2001) (NeoRx Corp, Seattle USA).

HLA-A2 +ve T2 (Salter 1985) cells were grown in RPMI 10% FCS supplemented with penicillin and streptomycin in a 37 0 C incubator with 5% CO 2 Venous blood was obtained from tissue typed healthy volunteers. (MG HLA Al, Al, B8, B44. PS HLA A3, A24, B35, B57. BQ HLA Al, A26, B7, B27). Unfractionated PBMCs were isolated by differential centrifugation using Histopaque (Sigma, Poole,

UK).

HLA-A2/peptide complex monomers and tetramers.

Recombinant HLA-A2 class I monomers and fluorescent tetramers were obtained from Prolmmune Ltd, (Oxford Science Park, Oxford UK). The peptides used in these experiments were Influenza virus Ml peptide GILGFVFTL (Gotch et al, 1987), the modified melanoma associated Melan A peptide ELAGIGILTV (Valmori et al 1998), the WT1 p126 peptide RMFPNAPYL (Oka et al 2000 the telomerase p540 eTRT peptide ILAKFHWL (Minev et al 2000) and the HPV16 E7 11-20 peptide (YMLDLQPETT) (Ressing et al 1995) In vitro immunisation protocol PBMCs were incubated with the B9E9 scFvSA (10ug/ml) diluted in PBS for 1 hour at room temperature. After washing cells were incubated with the biotinylated HLA class I/peptide complex (0.5ug/ml in PBS) for 30 minutes at room temperature. After washing, cells were placed into 24 well plates at 3 x 10 6 cells per well and cultured in RPMI with 10% human AB serum. IL-7 (R and D Systems, Minneapolis, MN) was added on day 1 at 10ng/ml and IL-2 (Chiron, Harefield, UK) was added at 10U/ml on day 4 and every further 3 days following the method described by Lalvani (Lalvani et al, 1997). For further stimulation cycles fresh PBMCs were obtained and treated as above. These new cells were then mixed with the existing culture at a 1:2 ratio and the culture continued for a further 7 days.

Flow cytometry and Tetramer analysis To stain CD8 +ve cells from the PBMC culture, approximately lx 10 6 cells were washed in PBS, resuspended and incubated with tetramer solution for 30 minutes at 37 0 C followed by FITC conjugated anti-CD8 for 20 minutes at 4 0 C. After incubation the cells were washed, resuspended in PBS and analysed by dual colour flow cytometry. The results of flow cytometry analysis of dual stained PBMCs are shown with anti-CD8 (Y axis) and HLA-A2/M1 tetramers (X axis). Percentage figures relate to the number of tetramer positive CD8 +ve cells from the total CD8 +ve population.

Chromium release assay T2 cells were labelled with 2uCi/uL of 51 Cr (Amersham Pharmacia, UK) for 1 hr at 37 0 C then washed and pulsed with the peptide of choice at a concentration of lOuM for lhr at 37 0 C. The target cells were plated at 3000 cells per well in U bottomed 96 well plates. PBMCs, media or 5% Triton X-100 were added to a final volume of 200ul.

Plates were incubated for 4 hours at 37 0 C in a 5% CO 2 atmosphere and 50ul of supernatant was collected and added to 150ul of scintillant.

The specific lysis was calculated as: lysis experimental cpm-spontaneous cpm x 100 maximum cpm-spontaneous cpm The spontaneous release was measured from the cells incubated in media alone, the maximum release was measured from the cells incubated in 5% Triton.

Example 3.1 Timecourse of alloreactive CTL production In Figs 6a and 6b the tetramer staining results of PBMCs from donor MG (HLA Al, Al, BO8, B44) targeted with HLA-A2/Melan-A and HLA-A2/M1 complexes are demonstrated. These show increases in the number of HLA-A2/Melan-A tetramer positive CD8+ve cells from 0.12% pre-stimulation, through to 15.1% after 3 cycles.

The results for HLA-A2/M1 are similar increasing from a background level of 0.13% through to 5.16% after 3 cycles.

In Table 8 the results from donors PS (HLA A3, A24, B35, B57) and BQ (HLA Al, A26, B7, B27) are included. These demonstrate similar levels of CTL production to the more stable HLA-A2/M1 and Melan-A complexes with an approximately 10 fold lower level of CTL production to the less stable HLA-A2/WT 1 complex.

Example 3.2 Specificity of tetramer staining of alloreactive CTLs from HLA-A2 ve donors.

The tetramer binding characteristics of the CTLs produced using the targeted HLA-A2 peptide/complexes were investigated with panel of 5 HLA-A2/peptide tetramers. The results from donor PS shown in Figure 24 demonstrate that cells produced against both HLA-A2/Melan-A and HLA-A2/WT1 show significant staining only against the appropriate tetramer. In contrast donor BQ gives a different result. Figure 25 shows that the CTLs produced from donor BQ with HLA-A2/Melan-A show significant staining with only that tetramer, whilst the CTLs produced with HLA-A2/WT1 show similar levels 0.90%) of staining with all 5 tetramers.

PBMCs stained prior to stimulation or after 3-4 cycles of cytokine treatment alone showed staining to all tetramers of 0.15% (data not shown).

Example 3.3: Functional activity of alloreactive CTLs from HLA-A2 -ve donors.

The functional activity of the alloreactive CTLs contained within the PBMCs stimulated with targeted HLA-A2/peptide complexes was investigated in standard 4 hour Cr release assays using native and peptide pulsed HLA-A2 +ve T2 target cells.

The results from donor PS are shown in Figs 9a and 9b. The cells produced with the HLA-A2/Melan-A complex demonstrated a degree of functional specificity producing, at an E:T ratio of 10:1, 62% lysis of the T2 cells pulsed with the Melan-A peptide and from the control T2 cells a maximum lysis of 27%. Similar results are seen with CTLs produced with HLA-A2/WT1 with 61% lysis of the WT1 pulsed cells with levels of 29-41% seen for control T2 cells.

Control mock targeted PBMCs treated with weekly cycles of the anti-CD20 sfvSA antibody, cytokines but without HLA-A2/peptide complexes showed approximately lysis of each of the peptide pulsed T2 cells at an E:T ratio of 10:1.

In Fig 10a and 10b the functional results of the alloreactive CTLs contained within the PBMCs from donor BQ are shown. The cells produced with HLA-A2/Melan-A show their highest activity of 28% against the Melan-A pulsed targets compared to a maximum of 24% with the other peptides. The cells produced with HLA-A2/WT1 demonstrate no peptide specificity with activity levels equivalent to the control mock targeted CTLs.

Summary of Example 3 The ability to generate CTLs that have specificity for tumour cells as demonstrated herein is of enormous potential value in haematology and cancer immunotherapy.

In this Example, HLA-A2/peptide complexes are targeted to the surface of B cells of HLA-A2 -ve donors producing a powerful alloreactive stimulus to a single designated HLA class I/peptide combination. The ability of this system to quickly and simply produce large numbers of alloreactive CTLs is demonstrated in Figures 6a and 6b.

Here for HLA-A2/Melan-A a background level of 0.12% prior to stimulation rising to 0.13%, 0.66%, and 15.1% after 1, 2 and 3 rounds of stimulation is demonstrated.

Similar results producing CTLs with the HLA-A2/M1 complex are demonstrated in Figure 6b with 5.16% of the CD8 +ve cells tetramer positive after 3 cycles of stimulation. These results compare positively with conventional T2 based technologies that produced 0.06% CD8+ve CTLs to HLA-A2/M1 after 10 days (Moris et al 2001).

In Table 8 the additional results of further tetramer analyses of CTLs produced to these epitopes and to HLA-A2/WT1 in two other donors are shown.

The stability of the HLA class I/peptide complex has been shown to be important in the induction of CTL in vitro (Valmori 1999, Micheletti 1999). The results here of CTL production via the alloreactive route suggest a similar effect with more rapid and higher levels of CTL production to the more stable complexes HLA-A2/M1 and HLA- A2/Melan-A, which score 30 and 28 on the SYFPEITHI database, compared to HLA- A2/WT1 which scores only 22 (Rammensee 1999). Preferably peptides used in the present invention have high stability as assessed by this technique.

The specificity characteristics of the alloreactive CTLs produced was investigated by staining with a panel of HLA-A2 tetramers. In Figure 8 the results demonstrate that the CTLs produced from donor PS using targeted HLA-A2/MelanA and HLA-A2/WT1 complexes showed only significant staining with the appropriate tetramer, with no increase in the staining with the other tetramers (HLA-A2/M1/Telo/E7). The results of the functional T2 cell chromium release assays parallel the tetramer staining with a significant degree of peptide specificity for the cells produced from donor PS to HLA- A2/MelanA (62% vs 18-27%) and HLA-A2/WT1 (61% vs 29-41%).

Similar tetramer results were shown with the CTLs from donor BQ using targeted HLA-A2/Melan-A, however with targeted HLA-A2/WT1 the PBMCs contain populations of CD8 +ve cells that stain with all 5 tetramers examined. From donor BQ a potential degree of functional specificity for the cells to HLA-A2/Melan-A is apparent (28% vs 15-24%), however the cells produced with HLA-A2/WT1 showed no significant increase in lysis of any of the T2 cells compared to the background level of mock targeted cells..

Without wishing to be bound by theory, the detailed interpretation of these functional Cr release assays may be limited by the advantageously simple nature of the in vitro stimulation procedure of the present invention which uses unfractionated PBMCs. As a result the bulk cell culture may contain other cell types in addition to CD8 ve CTLs and from the cytokine exposure some may possibly have some non-specific lytic activity. It is also theoretically possible that the bulk population even from donor PS may contain populations of alloreactive that can recognise A2 targets but may not bind specific tetramers.

This example demonstrates that the use of targeted HLA class I/peptide complexes according to the present invention is a simple and powerful method for generating alloreactive CTLs. From the three donors investigated in this example it is also apparent that targeted HLA class I/peptide complex can produce mixed populations of alloreactive CTLs including those that may be peptide specific, as judged by tetramer staining and functional assays, cells that may recognise HLA-A2 with any peptide and potentially a population of non-tetramer staining cells that may recognise HLA-A2 with any peptide. These results are in keeping with the previous observations of the width of the alloreactive response to single epitope stimulation (Sadovnikova 1998, Moris et al 2001, Dutoit 2002) This example demonstrates that alloreactive CTL induction with a single HLA class I/peptide complex advantageously produce either completely or predominantly peptide specific CTLs. However, from some donors there may possibly be produced a proportion of peptide non-specific cells. Without wishing to be bound by theory, it is possible that these in vitro results could be a parallel to the situations that occur in vivo in bone marrow allograft patients. Here these differing responses could represent the extremes of beneficial graft versus leukaemia as in the PS donor and broad nonspecific graft versus host disease as with the BQ donor response to HLA-A2/WT1.

Nevertheless, checking the quality and form of the response is easily accomplished as shown herein, so that only suitable CTLs are infused into subjects according to the present invention.

The systems of the present invention could serve as screening approaches to indicate which allograft donor recipient mismatches are likely to produce a predominant graft versus leukaemia effect with minimised graft versus host disease. Using the HLA class I/peptide targeted B cells in vivo post transplant may advantageously also lead to the selective expansion of CTLs which recognise leukaemic cells without producing

GVHD.

For tumour immunotherapy with either adoptive transfer of CTLs or TCR mediated gene therapy the requirement is the production of alloreactive CTLs that are peptide 41 specific. As this example demonstrates, the present invention provides such CTLs and methods for making them.

References Cited Dutoit V, Guillaume P, Romero P, Cerottini J-C, and Valmori D (2002) Functional analysis of HLA-A*0201/Melan-A peptide multimer+ CD8+ T cells isolated from an HLA-A*0201- donor: exploring tumor antigen allorestricted recognition Cancer Immunity 2;7-19.

Gao L, Bellantuono I, Elsasser A, Marley SB, Gordon MY, Goldman JM, Stauss HJ (2000) Selective elimination of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes specific for WT1. Blood 95:2198-203 Gotch F, Rothbard J, Howland K, Townsend A, McMichael A (1987) Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326: 881-882 Kessels HW, Wolkers MC, van den Boom MD, van der Valk MA, Schumacher TN (2001) Immunotherapy through TCR gene transfer. Nat Immunol 2: 957-61 Lalvani A, Dong L, Ogg G, Pathan AA, Newell H, Hill AVS, McMichael AJ and Rowland-Jones S (1997) Optimisation of a peptide-based protocol employing IL-7 for in vitro restimulation of human cytotoxic T lymphocyte precursors. J Imm Methods 210: 65-77 Micheletti F, Bazzaro M, Canella A, Marastoni M, Traniello S and Gavioli R (1999) The lifespan of major histocompatability complex calssl/peptide complexes determines the efficiency of cytotoxic T lymphocyte responses. Immunology 96: 411- 415 Minev B, Hipp J, Firat H, Schmidt JD, Langlade-Demoyen P, Zanetti M (2000).

Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc NatlAcadSci USA 97: 4796-801 Molldrem JJ, Lee PP, Wang C, Felio K, Kantarjian HM, Champlin RE, Davis MM (2000) Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 6:1018-23 Moris, Teichgraber, Gauthier, Buhring, H-J. and Rammensee H-G. (2001) Cutting edge: Characterization of Allorestricted and peptide-selective alloreactive T cells using HLA-tetramer selection. Joflmmunol 166: 4818-4821.

Oka Y, Udaka K, Tsuboi A, Elisseeva OA, Ogawa H, Aozasa K, Kishimoto T, Sugiyama H (2000). Cancer immunotherapy targeting Wilms' tumor gene WTI product Jlnmmunol 164: 1873-80 Pardoll DM (2000) Therapeutic vaccination for cancer. Clin Immunol 95: S44-62 Pittet M, Valmori D, Dunbar PR, Speiser DE, Lienard D, Lejeune F, Fleischhauer K, Cerundolo V, Cerottini J-C and Romero P (1999) High frequencies of naive Melan- A/Mart-1 specific CD8+ T cells in a large proportion of human histocompatability leukocyte antigen (HLA)-A2 individuals. JExp Med 190: 705-716 Rammensee H-G, Bachmann J, Emmerich NN, Bachor OA, Stevanovic S (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213- 219 (access via http://www.uni-tuebingen.de/uni/kxi/): Ressing ME, Sette A, Brandt RM, Ruppert J, Wentworth PA, Hartman M, Oseroff C, Grey HM, Melief CJ, Kast WM (1995). Human CTL epitopes encoded by human papillomavirus type 16 E6 and E7 identified through in vivo and in vitro immunogenicity studies ofHLA-A*0201-binding peptides. JImmunol 154: 5934-43 Rosenberg SA (1996) Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens. JNatl Cancer Inst 88: 1635-1644 Sadovnikova E, Jopling LA, Soo KS, Stauss HJ (1998). Generation of human tumorreactive cytotoxic T cells against peptides presented by non-self HLA class I molecules. Eur JImmunol 28: 193-200 Salter RD, Cresswell P (1986). Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J 5:943-9 Savage P, Cowburn P, Clayton A, Man S, McMichael A, Lemoine N, Epenetos A, Ogg G (2002). Induction of viral and tumour specific CTL responses using antibody targeted HLA class I peptide complexes. BrJCancer 86: 1336-42 Schultz J, Lin Y, Sanderson J, Zuo Y, Stone D, Mallett R, Wilbert S and Axworthy D (2000) A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy. Cancer Res 60: 6663-6669.

Stanislawski T, Voss RH, Lotz C, Sadovnikova E, Willemsen RA, Kuball J, Ruppert T, Bolhuis RL, Melief CJ, Huber C, Stauss HJ, Theobald M (2001). Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer. Nat Immunol 2: 962-70 Valmori D, Fonteneau J-F, Lizana CM, Gervois N, Lienard D, Rimoldi D, Jongeneel V, Jotereau F, Cerottini J-C and Romero P (1998). Enhanced generation of specific tumour reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. Jlmmunol 160: 1750-1758 All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in immunology, biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Claims (16)

1. A method for producing antipeptide cytotoxic T lymphocytes (CTLs) comprising: providing a sample of peripheral blood lymphocytes (PBLs); (ii) attaching a complex of a class I HLA and a peptide to antigen presenting cell(s) (APCs) present in said PBLs; (iii) optionally removing excess class I HLA/peptide complex; and (iv) incubating with proliferative cytokine.

2. A method according to claim 1 wherein the APC is a B-cell.

3. A method according to claim 1 or claim 2 wherein the complex is attached to the APC by attachment means comprising a molecule capable of selective binding to a B cell.

4. A method according to any one of the preceding claims wherein the attachment means comprises sfvSA to CD20 or CD19.

5. A method according to any one of the preceding claims wherein said sample of PBLs is a sample of a class I HLA negative PBLs.

6. A method according to any one of the preceding claims wherein the class I HLA is HLA-A2.

7. A method according to any one of the preceding claims wherein the peptide is a tumour peptide.

8. A method according to claim 7 wherein the tumour peptide is selected from the group consisting of Melan-A, WT1 and a telomerase. 00 4/ 0

9. A method according to any one of the preceding claims wherein the proliferative cytokine is a combination of IL-7 and IL-2.

10. A method according to any one of the preceding claims wherein the incubation is about 7 days.

11. A method according to claim 10 wherein the incubation step is repeated for a further 7 days.

12. A CTL obtained by the method of any one of the preceding claims.

13. A method of treating a subject comprising administering a CTL according to claim 12 to said subject.

14. A CTL obtained by the method of any one of claims 1 to 11 or 13. A method of treating a subject comprising administering a CTL according to claim 12 to said subject.

16. A method according to any one of claims 1 to 11 or 13 or 15 substantially as hereinbefore described with particular reference to the figures and/or examples.

17. A CTL according to claim 12 or 14 substantially as hereinbefore described with particular reference to the figures and/or examples.