mAb generation & engineering

Starting from different antigen material (cells, recombinant proteins, peptides, etc.), MImAbs rapidly generates and automatically screens thousands of hybridoma clones.

Immunization

• Of mice (BALB/c and C57Bl/6 KO) or rats (Wistar) with repeated injections.
• Monitored via serum titers by ELISA or FACS.

Hybridoma fusion and cloning

• Performed between splenocytes and a mouse myeloma cell line (X63-Ag8-656) on semi-solid medium (methylcellulose) for growth of individual colonies.
• The clones are automatically screened and picked to ensure clonality and good antibody production using Molecular Devices’ ClonePix™ 2 Mammalian Colony Picker (size, shape, distance to neighbouring clones, IgG secretion).

Antigen-specificity screen

Automatic isolation of hybridoma clones is followed by two rounds of selection for the ones that produce antigen-specific antibodies, using:

• ELISA – for soluble antigens.
• High-throughput FACS – for antigens expressed at the cell surface

Because hybridomas are unstable, MImAbs choses to rapidly sequence and clone antibodies into mammalian expression vectors. MImAbs has developed a collection of versatile vectors for the production of different formats. They can be used for cloning antibodies generated at MImAbs or using sequences provided by clients.

Sequencing and cloning processes have been fully automated using the Tecan EVO200 robot to ensure a rapid, error-free process.

Antibody sequencing

Messenger RNA from hybridoma clones is reverse-transcribed into cDNA and amplified before sequencing the heavy VH and light VL chains of the antibody. Several sequencing reactions are performed to control for possible PCR and sequencing mistakes.

Antibody format choice

MImAbs can generate human and mouse antibodies in different standard or customized formats (IgG, Fab, Fab’, Fab’2…). Although other formats can be considered, those that are ready and available in-house include:

Humanization is important to prevent an immune response against rodent antibodies in patients. MImAbs operates by CDR-grafting following three key steps:

Determination of the most appropriate human framework sequence

Human VH and VL immunoglobulin gene sequences are analysed using two in silico tools: IMGT and IGBLAST. Those immunoglobulins that are phylogenetically closest to the parental rodent antibody are used as framework (FR) for the humanized antibody.

CDR definition

The three complementarity-determining regions (CDRs) of the variable domains are determined on the parental antibody following the Kabat¹ and IMGT² nomenclature systems, and are introduced in the human FR sequence.

Backmutations

Additional residues from the rodent mAb are re-introduced into the human framework regions to preserve antibody structure and antigen specificity:

• Residues responsible for CDR anchoring
• Residue from the Vernier zone which are directly under the CDRs and are important for proper CDR conformation³
• Residues involved in the interaction between the heavy and light chains