Rhodes, Elizabeth G H; (1993) The development and application of lectin/monoclonal antibody purging in the treatment of multiple myeloma. Doctoral thesis (M.D), University of London, Royal Free Hospital School of Medicine.

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Abstract

The aim of the work described in this thesis was to find a means of selectively removing neoplastic cells from myeloma bone marrow in vitro to be used in conjunction with autologous bone marrow transplantation. The inspiration for this work derived from the demonstration by Professor McElwain that tumour clearance could be attained in vivo by using very high doses of alkylating agents. It was soon apparent that autologous stem cell rescue was required to counteract the morbidity of such treatment, but that this entailed reinfusion of neoplastic cells mixed with the normal haemopoietic cells. Obstacles to successful in vitro purging were: a) uncertainty as to the identity of the malignant cell in myeloma and, b) lack of monoclonal antibodies reactive with the surface of plasma cells that could be used as purging agents. Studies were undertaken to develop a method for growing myeloma cells in vitro, as a means to identify target cells for purging. After unsuccessful attempts to reproduce several previously described techniques for growing myeloma colonies, rigorous criteria were established for assessing true in vitro myeloma colony formation. After experimenting with many nutrient media, 17 out of 32 consecutive myeloma bone marrow samples were observed to yield true colonies using one particular serum-free medium. Immunophenotyping of these colonies revealed 2 populations of light-chain restricted B-lineage cells, (CD19+ and RPD6+), and these were selected as target cells for purging. Next, studies with a panel of lectins led to the novel observation that peanut agglutinin (PNA) binds to bone marrow plasma cells but not to normal haemopoietic progenitor cells. Two different methods were then developed to separate PNA+ cells from bone marrow, one exploiting exposed Thomsen Friedenreich [T] antigen (galactose β1,3 N Acetyl galactosamine α-, the PNA ligand) on sialidase-treated normal red blood cells with PNA in a rosetting technique, and the other utilising magnetised microspheres for which a lectin coating technique was developed. Using the latter technique small scale experiments in normal and myeloma bone marrow samples confirmed the initial prediction that removal of PNA+ cells did not affect the in vitro growth of normal haemopoietic colonies. CD19-coated immunomagnetic depletion was then combined with PNA purging and shown to achieve satisfactory depletion of target cells with conservation of normal cells. The scaling-up procedure, well known to be the stumbling block of many successful small scale purging models, was achieved by trial and error using a variety of manual and automated techniques. Experiments were performed using normal bone marow spiked with radio-labelled tumour cell lines and myeloma bone marrow cells until a successful technique suitable for clinical use had been established. A clinical pilot study was then initiated to assess the feasibility of autologous bone marrow transplantation in myeloma using marrow that had been purged in vitro with PNA- and CD19-coated magnetic microspheres. At the time of writing 5 myeloma bone marrow harvests have been successfully purged and cryopreserved. Two of these have been used to rescue patients following high-dose chemoradiotherapy. Both purged autografts engrafted rapidly and the patients are in good health 21 and 24 months later, although 1 patient still has a serum paraprotein. This work has demonstrated the feasibility of autologous bone marrow transplantation in myeloma with combined lectin/monoclonal antibody purging of malignant cells. This will now allow further investigation of the potential benefits of high-dose chemoradiotherapy.

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