NeoAb approach to therapeutic antibodies

Neoclone's hu-mAb platform (which has been marketed under the NeoAb process) offers certain advantages:

• The mAbs are fully human, having been developed from human immune cells.
• The approach does not require immunizing patients or access to convalescent blood, therefore, the platform is amenable for mAb development using de novo immunization against any therapeutic target.
• mAbs developed using this approach will likely have higher affinities than Abs generated by library-based methods because reactivation focuses on selection of affinity-matured cell targets that proliferate in very low Ag levels.
• hu-mAbs generated using the NeoAb process can have fully-human glycosylation patterns, a distinct advantage over human mAbs expressed in non-human cells (18).
• The process enables selection for or against certain secondary characteristics such as Ab-dependent cellular cytotoxicity or complement-dependent cytotoxicity (19).

The NeoClone human mAb process can be divided into four stages: immunization, reactivation, cloning, and expression (see Figure 1).

Reactivation: B cell culture and affinity screening. Activated B cells are enriched from the spleen and lymph nodes of immunized SCID-huPBL mice and then B cells are cultured in vitro with a mixture of cytokines. Antigen supernatants from the cultured B cells are tested for affinity and specificity using an Octet QK (ForteBio) which uses BioLayer interferometry to determine affinity binding constants (K_D) of purified Abs, as well as rank-order crude tissue culture supernatants by Kd estimates (26). Antigens are biotinylated and immobilized on a streptavidin Octet sensor using attachment methods recommended by the manufacturer (27). Reactivated culture supernatants are rank-ordered by affinity using crude supernatant samples. Generally, affinities from 10^-9 to 10^-11 M are considered an acceptable affinity range. Harvested cell cultures are then subjected to fluorescence activated cell sorting (FACS) analysis in which single B cells specific for the immunizing antigen are deposited in one cell per spot on a 48-spot AmpliGrid glass slide (Beckman).

Figure 2: Screenshot from a NanoDrop 8000 showing concentration and purity measurements of paired antibody heavy and light chain constructs cloned from single cells to be used for transient transfection.

Expression: protein expression and scale up. A NanoDrop 8000 spectrophotometer is used to quantitate purified preps of candidate hu-mAbs. Midiprep DNAs of paired Ig heavy and light chains cloned into antibody expression vectors are co-transfected into non-adherent mammalian cells using the TransIT reagent (Mirus Bio). Supernatants are screened for affinity and specificity as described above. Larger scale transfections can be performed on candidate hu-mAbs. Hu-mAbs can be purified by standard protocols and quantitated using the NanoDrop 8000 spectrophotometer for concentration and purity. Affinity constant (KD) determinations can be performed on purified antibodies. Additional assays to identify desired hu-mAb characteristics will be performed prior to stable cell line generation.