Abstract

Aim: Despite advances in therapy, such as stem cell transplantation and new drugs such as the proteosome inhibitors, multiple myeloma (MM) remains incurable for most patients. Immune therapy, which uses dendritic cells (DC) to generate cytotoxic T lymphocyte (CTL) responses targeting residual disease is a promising, potentially curative strategy, compatible with other therapies. We have shown that blood DC can be isolated from MM patients and that they induce CTL responses (

J. Immunotherapy.
2005
;
28
:
322
). The CMRF-56 monoclonal antibody (mAb) detects an antigen expressed on blood DC after a short period of in vitro culture. To create a GMP compatible DC purification platform for clinical investigation of DC immunotherapy, we developed a method for single step CMRF-56 mAb positive immunoselection of blood DC using magnetic beads.

Methods and Results: CMRF-56 mAb immunoselection produced a blood DC enriched population containing 33.2±3.5% CD11c+ blood DC (including CD1c+, CD141+ and CD16+ subsets) and 0.6±0.2% CD123hi plasmacytoid blood DC (n=6). Additional CMRF-56 positive B cells and monocytes comprised 14.7±5.8% and 32.4±3.4% of the preparations respectively (n=6). CMRF-56 selected cells stimulated CTL responses against defined tumour associated antigens, including MART-1 peptide and the MM associated antigen, HM1.24. Furthermore they generated strong MM specific CTL responses after incubation with MM cell lysates. Brief DC activation with GM-CSF enhanced these responses, improving MM-specific CTL activity (Cr release) in cultures primed with MM lysate loaded CMRF-56 selected cells from 18.9±7.4% to 44.1±8.0% (n=3, p=0.051). We have now developed and validated a procedure for isolation of CMRF-56+ blood DC using the CliniMACS device. Apheresed mononuclear cell preparations were cultured, labelled with antibody and blood DC immunoselected in a closed system, free of human or animal serum supplements, in a manner compatible with regulatory requirements for cell therapy. Simultaneously, our team has developed a bioprocess for production of the murine biotinylated CMRF-56 mAb, suitable for the large scale synthesis required for multi-centre clinical trials. It incorporates components that remove contaminating DNA, host cell proteins, viral particles and endotoxin. The first batch of clinical grade CMRF-56 mAb has been produced and used for successful laboratory scale blood DC isolation. We have also completed a number of clinical scale blood DC preparations from healthy donors (n=10) and MM patients (n=2). There were sufficient blood DC isolated from both the healthy donors and MM patients for the planned vaccination schedule. The average blood DC purity was 38% in healthy donors and 29% in MM patients, while the average yields were 35 and 44%, respectively.

Conclusions: When additional studies on MM patients are completed these studies will provide the necessary pre-clinical validation data to commence a Phase I Clinical Trial.

Disclosure: No relevant conflicts of interest to declare.

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