Locoregional immunotherapy of malignant effusion from colorectal cancer using the streptococcal preparation OK-432 plus interleukin-2: induction of autologous tumor-reactive CD4+ Th1 killer lymphocytes.

In total, 16 patients with cytologically proven malignant effusion from colorectal cancer were treated by locoregional administration of the streptococcal preparation OK-432 alone or OK-432 plus the T-cell growth factor interleukin (IL)-2, and the action mechanism of the treatment was studied. A positive clinical response, showing a cytologic disappearance of cancer cells and decrease of effusion, was observed in nine of 11 (82%) patients treated with OK-432 alone and in all five patients treated with OK-432 plus IL-2. Flow cytometric analysis revealed that OK-432 plus IL-2 locally induced acute inflammation-like responses, including serial cellular infiltrations of granulocyte migration within a matter of hours, and activation of macrophages and T lymphocyte involvement within the following days, and that a predominant expansion of CD3+CD4+ lymphocytes (CD: cluster of differentiation) was induced by in vitro stimulation with IL-2 of locoregional cells after the OK-432 administration (OK/IL-2AK cells). The OK/IL-2AK cells produced tumour necrosis factor-alpha and interferon-gamma, but these cells did not produce IL-4 and IL-6. The OK/IL-2AK cells expressed potent killing activity against autologous tumour cells. This activity was abrogated by treatment of the lymphocytes with anti-CD3, -CD4, -TCRalphabeta antibody, and by the treatment of target cells with anti-human leukocyte antigen (HLA)-DR antibody. The OK/IL-2AK cells expressed Fas-L gene, and flow cytometric analysis demonstrated HLA-DR expression in approximately 75% of CEA+ or cytokeratin+ effusion cells. TCRVbeta gene analysis of the OK/IL-2AK cells showed an oligoclonal usage of TCRbeta20, which was also involved in the cytotoxic mechanism of the OK/IL-2AK cells. Single-strand conformational polymorphism analysis demonstrated the clonotypes for the TCRVbeta20 gene, and the CDR3s of the gene were sequenced. The clonotypic PCR using the TCRVbeta20-CDR3 sequences could detect the CDR3-identical TCRs in effusion lymphocytes from the other patients. Taken together, it is suggested that locoregional administration of OK-432 plus IL-2 is highly effective for the management of malignant effusion from colorectal cancer. OK-432 plus IL-2 induces autologous tumour-reactive CD4+ Th1 killer lymphocytes, which recognise tumour antigen(s) presented with HLA class II molecules on effusion tumour cells by means of preferential usage of TCRVbeta20. The clonotypic PCR using the TCRVbeta20-CDR3 sequences may be informative for treating malignant effusion from colorectal cancer using OK-432 plus IL-2.