| B lineage acute lymphoblastic leukemia (ALL) is the most common type of hematopoietic malignancies in children. The current primary treatment of childhood ALL is combined chemotherapy. Although the present therapies on childhood ALL are effective, severe toxicity and side effects are still the major complications due to the poor treatment selection of the chemotherapeutic drugs. Since 1975 when the technology of hybridoma and monoclonal antibody was developed, many investigators have been paying close attention to antibody targeting therapy for patients with cancer. As compared to conventional chemotherapy, antibody target therapy on leukemia cells (biotherapy) is well selective. It only kills leukemia cells expressing target molecules while leaving those without target molecules unaffected. Therefore targeting therapy can greatly reduce the opportunity of nonspecific toxicity and side effects on normal tissues.A good targeting reagent depends on the directing carrier and its ligand toxin conjugated. Use of monoclonal antibody (Mab) recognizing leukocyte differentiation antigens as directing carrier has an overwhelming advantage over any other targeting molecules. Mabs can specifically bind to the antigens existing on the leukemic cell surface and carry the toxin to the surface of malignant cells resulting in killing. The preliminary application of some of the reagents in clinical practice has showed the promising results.There are many mouse anti-human CD 19 Mabs reported in the literature, such as B4, B43, HD37, ZCH-4-2E8 (2E8), SJ25C1 and FMC63, etc. among which only B4, B43 and HD37 have been conjugated with toxins to form immunotoxins, such as B43-Genistein, B43-Pokeweed antiviral protein (B43-PAP), HD37-deglycosylated ricin A chain(HD37-dgRTA) and B4-blocked ricin (B4-bR); the effectiveness of these immunotoxins has showed promising in treatment of B precursor leukemias in both animal models and in clinical patients. According to the literature found, no CD 19 immunotoxin has been documented at home while no report at al has been published on the study using ZCH-4-2E8 as directing carrier to generate CD 19 imunotoxin both at home and abroad.ZCH-4-2E8, a new clone of CD 19 Mab was generated in this lab and assigned into CD 19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996.Many toxins have been used to conjugate directing antibodies so far, such as ricin A chain, pokeweed antiviral protein (PAP), exotoxin of pseudomonas aeruginosa (ETA), genistein (Gen) etc. The latter is a tyrosine kinase inhibitor which can effectively kill the leukemia cells when it linked to certain directing antibodies. Uckun et al have reported that their B43-Gen can kill 99.999% of the CD 19+ Nalm-6 cells in vitro and showed promising treatment results in patients with B precursor ALL with minimal toxicity.In this study, the new clone of CD 19, 2E8 was used to conjugate with Genistein (Gen) via a light sensitive agent Sulfo-SANPAH to generate 2E8-Gen immunotoxin. By using this immunotoxin, target killing of CD 19+ leukemia cells was carried out and the killing mechanism was studied. The results of this study have provided a better fundamentals for generating second generation of 2E8 immunotoxin such as single chain fragment of variable region immunotoxin (ScFv immunotoxin), human-mouse chimeric antibody or humanized antibody for clinical purpose.Material and Methods1. Generation and purification of the 2E8 antibodyBefore generation of large quantity of 2E8 antibody, three subcloning were performed to avoid the possible mutation of the hybridoma cells in which some none-secreting cells might exist. A large quantity of 2E8 ascites was made using the method previously described in the literature. Purification of the antibody was carried out using the Bouvet method with modification and followed by chromatography. The purity of antibody was checked with SDS-PAGE.2. Preparation of 2E8-Gen immunotoxin2E8-Gen was prepared to conjugate...
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