Humanization and characterization of the anti-HLA-DR antibody 1D10

Abstract

1D10 is a previously described antibody that binds to cells from a majority of B-cell malignancies. The current studies were designed to further evaluate the antigen specificity of 1D10 and its potential as an immunotherapeutic agent. Studies with transfectants and immunoprecipitation demonstrated that 1D10 recognizes some, but not all, of the human HLA-DR ␤ chains. Both normal and malignant B cells can express the 1D10 antigen. A humanized version of 1D10 was produced using CDR grafting. The resulting antibody has an affinity that is similar to that of the parental murine antibody. In addition, the humanized antibody is capable of inducing complement-mediated cytotoxicity, antibody-dependent cell cytotoxicity, and direct apoptosis of 1D10-expressing B cells. Based on these in vitro anti-tumor activities, we conclude humanized 1D10 deserves further evaluation as an immunotherapeutic agent.

Apoptosis assays with lymphoma cell lines: problems and pitfalls

“…This factor appears to have been overlooked in some previous reports. For example, earlier studies of the effect of L243 (anti-HLA-DR) on Raji cells (identical to the model we used), which claimed that apoptosis occurred, did not mention the dramatic aggregation that occurs (Kostelny et al, 2001;Nagy et al, 2002). One consequence of aggregation, which we documented, is that it may be impossible to disperse the cells in tight aggregates without killing the cells.…”

Characterization of Humanized Antibodies Secreted byAspergillus niger

“…It has been shown that Hu1D10 produced in NS0 cells can induce the apoptosis of Raji cells through cross-linking of HLA-DR ␤ chains on the cell surface (15). The avidities of the Hu1D10 antibodies were compared by monitoring the level of apoptosis induced by different antibody samples in a population of Raji cells.…”

“…NS0-Hu1D10 can mediate the lysis of Raji cells through ADCC (15). It has been shown that the removal of carbohydrates attached at position 297 in the heavy chain abolishes the ADCC activity of human IgG1 antibodies (5).…”

“…The variable and constant regions were joined and inserted into the expression vectors by standard methods. cDNA clones expressing the entire mature antibody Hu1D10 and ␥ chains (15) were amplified by PCR and inserted into the expression vectors.…”

“…The human Burkitt's lymphoma-derived cell line Raji (ATCC, Manassas, Va.), which expresses the HLA-DR ␤-chain allotype recognized by antibody Hu1D10 (15), was maintained in RPMI 1640 (Gibco BRL, Grand Island, N.Y.) containing 10% FBS (HyClone, Logan, Utah) in a 7.5% CO 2 incubator. For the competition experiments, a mixture of fluorescein isothiocyanate (FITC)-labeled antibody NS0-Hu1D10 (0.25 g/test) and competitor antibody (serial twofold dilutions of NS0-Hu1D10 [Hu1D10 derived from a mouse myeloma NS0 cell culture] or Aspergillus-derived Hu1D10 starting at 6.25 g/ test) in fluorescence-activated cell sorting staining buffer (phosphate-buffered saline containing 1% bovine serum albumin [BSA] and 0.2% sodium azide) was added to Raji cells (5 ϫ 10 5 cells/test) in a final volume of 100 l. After incubation on ice for 30 min, the cells were washed three times with fluorescence-activated cell sorting staining buffer and analyzed by flow cytometry with a FACScan (Becton Dickinson, San Jose, Calif.).…”