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).
Immunization: de novo immunization of human peripheral blood lymphocytes (PBL) in a SCID mouse model. NeoClone's current protocols for generating a human immune response to targeted antigens are based on published SCID-huPBL
engraftment models (20–25) coupled with modifications of Ag-pulsed dendritic cell (DC) immunizations and multimer-linked Ag
complex immunizations. The SCID-huPBL model repopulates immune deficient mice with human peripheral blood lymphocytes. SCID
mice cannot make mature, antigen-specific B and T cells. Engraftment of human PBLs repopulates secondary lymphoid organs
such as lymph nodes and spleen with human immune cells. Because of the enrichment process used, cells recovered from the immunized
SCID-huPBL mouse are human, and the Ig genes cloned out from these cells are therefore fully human (20, 21, 24). All antigens
are assayed for optimal concentration using a NanoDrop 8000 spectrophotometer (Thermo Scientific) before immunization. Antigen
integrity and concentration are confirmed by gel electrophoresis and densitometry. Antigens are required to pass quality control
by all three methods before they are formulated for immunization.
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 (KD) 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).
Cloning: recovery of Ig heavy and light chains, cloning, and sequencing. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) is performed on an AmpliGrid system (Beckman Coulter)
to capture the matched Ig heavy and light chain pairs from sorted cells. Ig heavy and light chains are cloned into antibody
expression vectors and sequenced (see Figure 2).
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.