Human antibodies for immunotherapy development generated via a human B cell hybridoma technology

Abstract

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes, humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however, this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g., affinity and titers) of mAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing.

Fig. 1.

Antigen panel ELISA for selection of antigen-specific human mAbs. Three GM-CSF-specific human mAbs, E10, G7, and E5, reacted with human GM-CSF and none of the other antigens in the panel. 215, murine mAb specific to human GM-CSF (hGM-CSF); mGM-CSF, murine GM-CSF.

Fig. 2.

Human mAbs highly specific to native human GM-CSF. (A) Murine hybridoma cells presenting cell surface Ig to human GM-CSF were loaded with (Bottom) or without (Top and Middle) soluble human GM-CSF. mAb E5 was subsequently added (Middle and Bottom) to the reaction, and its binding to human GM-CSF was measured by using FITC-conjugated goat anti-human Ig. E5 did not bind any of the surface proteins expressed by the murine hybridoma cells (Middle) but bound only soluble GM-CSF captured by the cell surface Ig (Bottom). (B) PE-labeled human GM-CSF (PE-GM) can react to Ig expressed on E10 cell surface (Middle). Excess of unlabeled GM-CSF competed for PE-GM binding (Bottom). No PE-GM binding was seen in absence of both PE-GM and GM-CSF (Top).

Fig. 3.

Human mAb highly specific to human mesothelin tumor antigen. (A) mAb C12 (10 μg/ml) was reacted to mesothelin-negative (−) or positive (+) cells followed by FITC-conjugated goat anti-human Ig. Bound Ig causing shifting of fluorescence intensity was detected only with cells expressing mesothelin. (B) Normal human IgM was reacted to same cells as in A. No fluorescence shift was observed with either cell type, demonstrating specificity of this assay.

Fig. 4.

Class-switched hybridoma cells secrete IgG, retaining antigen binding. Hybridoma E5 cells (Parent) were treated as described in Materials and Methods. Hybridoma clones that had class-switched (Switched) were identified by using an ELISPOT-based screening method. An ELISA measuring specific binding to human GM-CSF coated onto plates was carried out to assess binding of either IgM or IgG. Switched IgG mAbs exhibited binding to antigen comparable to that of the parental IgM.

Fig. 5.

Biological activity of human mAbs against GM-CSF. A cell-based assay was carried out to determine the level of GM-CSF neutralization (% of inhibition) mediated by G9 and E10 mAbs. Human GM-CSF-dependent TF1 cells were incubated in the presence of 0.1 ng/ml GM-CSF. No inhibition of growth was observed when normal isotype control IgG was included in the reaction, regardless of the concentration used. Both E10 and G9 mAbs neutralized GM-CSF activity.

Fig. 6.

Assessment of titers and stability of hybridomas secreting human mAbs. (A) The hybridoma E5-3D2 line was grown for 60 generations, and then stability of production was assessed by analyzing frequency of producing cells. Subclones (X1–X10) derived from 3D2 cells by means of limiting dilution were randomly chosen, and their Ig production was measured by using an ELISA-based assay. Absorbance at 405 nm was normalized for colony size by visual inspection of the cell-containing wells. (B) 3D2 cells were inoculated in a stirred bioreactor containing 1 liter of serum-free medium, and Ig production and number of viable cells were recorded on days 1–5. The specific productivity measured during the log phase was 24 pg per cell per day.

Fig. 7.

Genetic optimization of hybridoma-secreting human mAbs by MMR regulation. (A) Example of a single-nucleotide deletion in the BAT marker found in E5 hybridoma cells treated with morphogenics. Dotted lines crossing the central peak in the histogram represent the size of wild-type (wt) or contracted (−1 nt) fragment. (B) Parental and morphogenics-treated cells were seeded in microplates to yield 3,763 and 2,437 Ig-secreting clones (OD > 0.2), respectively. Ig concentrations were determined by ELISA, and the frequency of clones with OD values >1 was recorded and is expressed as percentage of total number of clones screened.