Tumour antigen-targeted immunotherapy for chronic myeloid leukaemia: is it still viable?

Abstract

In haematological cancers, malignant cells circulate in the blood and lymphatic system. This may make leukaemic cells easier to target by immunotherapy than in other types of cancer. Various immunotherapy strategies have been trialled in several leukaemias including chronic myeloid leukaemia (CML) and in general, these have been aimed at targeting tumour-associated antigens (TAA). There are numerous TAA expressed by CML patients including WT1, proteinase 3, BCR-ABL and HAGE amongst others. The immunogenicity of the CML-specific tumour antigen, BCR-ABL, has been the subject of much debate and its role in the development of the disease and its unique sequence spanning the breakpoint region make it an ideal target for immunotherapy. However, there are a limited number of immunogenic epitopes across the junctional region, which are restricted to only a few HLA types, namely A2, A3 and B7 (Clark et al. in Blood 98:2887-2893, 2001). The second CML-associated antigen is the helicase antigen HAGE, a cancer-testis antigen found to be over-expressed in more than 50% of myeloid leukaemias (Adams et al. in Leukaemia 16:2238-2242, 2002). Very little is known about the function of this antigen and its significance to CML. However, its membership of the DEAD-box family of ATP-dependent RNA helicases and the involvement of other members of this family in tumour cell proliferation (Eberle et al. in Br J Cancer 86:1957-1962, 2002; Yang et al. in Cell Signal 17:1495-504, 2005) suggest a crucial role in the RNA metabolism of tumour cells. For these reasons, HAGE also seems to be a good target for immunotherapy as it would be applicable for the majority of patients with CML. This review aims to discuss the potential of immunotherapy for the treatment of leukaemia, in particular CML, and the prospect of targeting three CML associated antigens: BCR, ABL and HAGE. During his career, Prof. Tony Dodi made a significant contribution in this area of leukaemia research, confirming the identity of immunogenic HLA-A3 and B7-restricted peptides as targets for CTL. Published, as a highlighted paper in Clark et al. (Blood 98:2887-2893, 2001), this study demonstrated the expression of MHC-peptide complexes on the surface of CML cells and the presence of tetramer-positive CTL activity in CML patients positive for these two HLA alleles. His drive and dedication for research excellence will be remembered by all who knew and worked with him.

Fig. 1

Schematic diagram showing the effects of increased tyrosine kinase activity of BCR-ABL. BCR-ABL subverts normal cellular processes in which normal BCR and c-ABL play a role in control of cellular growth and apoptosis. It can cause production of reactive oxygen species (ROS), which are known to be involved in causing mutations leading to a transformed phenotype

Fig. 2

Representative results generated from HHDII mice immunised with FLN, MLT and LYG epitopes. At the top ratio of 100:1 effectors to targets, there is approximately 80% cytotoxicity in response to these peptides. These peptides can be classed as immunogenic as a significant level of cytotoxicity was detected. The graphs c and d are using CTL generated from HHDII mice immunised with the LYG peptide and then cultured with T2 cells pulsed with the LYG peptide c or the FLN peptide d. As can be seen a strong cytotoxic response could be generated against both these epitopes. This could be due to the fact that the 12-mer epitope could be acting as both a MHC class I and class II epitope stimulating CD8 + and CD4 + T cells. ***P < 0.001. See Table 1 for total number of mice tested

Fig. 3

Results from ELISPOT assay carried out on PBMC stimulated with either a LYG, b MLT or c FLN epitopes. T cells were generated from PBMC by re-stimulating in vitro with LYG, MLT and FLN peptides and following this, the level of IFN-γ releasing cells was measured by ELISPOT assay. A significant response was detected in patient sample 6 T2 (LYG 12-mer a), patient samples 17 and 6 T2 in response to the MLT peptide b, patient samples 4, 10, 19 T1, 19 T 2 and 19 T3 after one stimulation with FLN peptide c. *P < 0.05, **P < 0.01 and ***P < 0.001

Fig. 4

Partial sequence of BCR-ABL sequence across the junctional region showing the underlined selected peptides. The amino acid sequence above refers to a portion of the 210 kDa BCR-ABL protein. The sequence in capitals is from the BCR sequence and following this, is the ABL sequence, apart from the unique lysine (K) residue highlighted in bold. The two 9-mers and the 12-mer (see Table 2) used in this study are, respectively, underlined and framed above and were from across the BCR part of the sequence

Fig. 5

Expression of HAGE and BCR-ABL in chronic myeloid leukaemia. Real time PCR analysis was carried out on 20 CML samples and normal total blood cells (TBC). In a 10 CML samples from patients with high BCR-ABL transcript levels and in b 10 CML samples with low BCR-ABL transcript levels. The experiment was carried out once and data are expressed relative to the mRNA level of TBC, arbitrarily set as 1

Fig. 6

Therapy studies using gene gun immunisation in DR1/HHDII mice. Three days after injecting 6 × 10⁵ ALC/HAGE cells to two groups of 10 mice, mice were immunised three times with gold particles coated with either pBudCE4.1/(−) or pBudCE4.1/HAGE at 7-day intervals and monitored for tumour growth a and survival b. Results shown are representative of two independent experiments. *P < 0.05 are the statistical differences between HAGE DNA immunisation and control DNA immunisation determined by unpaired Student t test. Down arrow indicates timepoints of immunisation, dagger indicates termination