mAb generation & engineering

mAb generation & engineering

Making the right Ab format for every application


Starting from a given antigen, MI-mAbs can generate, select and optimize antibodies to better suit their intended application.

Starting from different antigen material (cells, recombinant proteins, peptides, etc.), MI-mAbs 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, MI-mAbs choses to rapidly sequence and clone antibodies into mammalian expression vectors. MI-mAbs has developed a collection of versatile vectors for the production of different formats. They can be used for cloning antibodies generated at MI-mAbs 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

MI-mAbs 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:

Reference

Antibody format

Description

Purpose

GD

Mouse IgG1

Heavy chain mutation:

D265A

Fc silent, deglycosylated antibody

GE

Human IgG1

Heavy chain mutation:

E345K

mAb multimerization

GH

Human hybrid

CH1 (IgG2)

CH2-CH3 (IgG1)

Heavy chain mutation:

C219S

Enhanced mAb internalisation

M1

Human IgG1

Heavy chain mutations:

L235V, F243L, R292P,

Y300L, P396L

Enhanced ADCC response

S1

Human IgG1

Heavy chain mutation:

N297S

Deglycosylated antibody

2 BTG coupling sites

S2

Human or mouse IgG1

Heavy chain mutation:

N297Q

Deglycosylated antibody

4 BTG coupling sites

V1

Human IgG1

Heavy chain mutations:

E233D, G237D, P238D,

H268D, P271G, A330R

Enhanced binding to the Fc-g-RIIB receptor

Humanization is important to prevent an immune response against rodent antibodies in patients. MI-mAbs 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

The output of the humanization process consists of 9 to 16 humanized variants that contain backmutations in the VH and VL chains and can be tested downstream for antigen binding and specificity.

 

1 Kabat EA et al, NIH Publication 1991

2 Lefranc MP et al, Dev Comp Immunol 2003

3 Foote et Winter, J Mol Biol 1992

Nanobodies are small, heavy chain-only antibodies that derive from a type of antibody present in camelidae (camels, llamas…), and whose antigen-binding site consists of one single domain, known as VHH. Nanobodies retain full antigen binding properties, are very stable and preferentially bind buried antigens typically inaccessible to conventional antibodies. MI-mAbs has established a partnership with the Nanobody platform at the CRCM (Centre de Recherche en Cancérologie de Marseille) to produce high-quality custom-made nanobodies, starting from llama immunizations or llama phage-display libraries generated in-house.

For more information visit the CRCM website.