Monoclonal antibodies

Objectives

The establishment in 2006 of a structure dedicated to the production of monoclonal antibodies (MAbs) in the Center of Immunology INSERM-CNRS de Marseille-Luminy responded to the major developments in the use of these reagents as research or diagnostic tools and / or therapeutic purposes. Moreover, several arguments made the creation of this platform within the CIML relevant:

• The long experience in this area by several researchers of the Institute (M. Pierres, B. Malissen), who have widely used the technology since the late 1970s.
• The various research themes and experimental models developed in the CIML, allowing the identification and characterization of functional molecules involved in various aspects of the physiology of the immune system.
• The ability to identify animals in murine models of target molecules with homologous or orthologous human potential clinical development.
• The interfaces between the academic institutions (Centre d'Immunologie Marseille-Luminy, Centre anti-cancer Paoli-Calmettes, etc.), and some biotech firms (Innate Pharma, In-Cell-Art). .
• Finally, the methodologies currently available for the humanization of mAbs developed in other species.

The installation of the CIML MAb platform was carried out in this context in 2006. Its specific objectives can be summarized as follows:

• A structure to respond to requests from groups CIML, but also interfaced with other academic structures, regional, national & international.
• Optimization methodologies to improve the conditions for obtaining mAbs against poorly immunogenic targets [e.g. the use of diverse immunogens (peptide conjugates, transfected cells expressing a "tagged" transgene, etc.) The development of techniques for high flow conditions, etc.]
• The development of interfaces with the bio-technology
• Partnership with transfer facilities of INSERM (INSERM-Transfer) or university (Protisvalor)

Structure & operation

This structure was created in 2006 with logistics support from the CIML, Université Aix-Marseille II, ARC, CNRS and INSERM. Moreover, a close partnership has been associated - from its inception, with the cancéropôle PACA, which represents one of the platforms.

It occupies 3 modules in the CIML, one dedicated to cytofluorometry.

The staff is assigned 3 technical assistants [2 AI (CNRS and National Cancer Institute CDD), a technician (TN INSERM)], under the responsibility of a DR1 INSERM.

This platform provides advice [e.g. definition of operational strategies for each program (choice of species, type of immunogen, screening strategies, etc.)], and implementation (preparation of immunogens, immunization, cell fusion, screening, cloning, isotyping and purification of mAbs, etc.). Equipment approaches emphasize high-speed (two FACSArrays Becton-Dickinson custom lasers and two automated plate supports, allowing analysis by two-color flow cytometry of a large series of samples (about 4000 supernatants tested per program).

Activity.

Since its inception, the platform has produced 127 programs. 

Initially focused on internal CIML programs, the platform is currently in great demand by external academic laboratories or bio-industry. Achieving the latter is most often the subject of billing for the service, punctually or in a global context (contract research).

Immunization strategies

We optimized immunization strategies based on the immunogenicity of the target molecules predicted polymorphism (rat / mouse / human), localization (membrane or intracellular), and the use of mAbs desired in various research applications (FACS, biochemistry, in vivo depletion, immunocytochemistry, etc.). The distribution of the various approaches is summarize below: 

46 programs have involved commercially available recombinant proteins or those prepared locally.
A roughly comparable number (45) have been developed by approaches using peptide conjugates prepared by the platform [selection of areas based on interspecies protein polymorphisms, synthesis of peptides (Schafer, Copenhagen), coupling to a carrier protein (chicken ovalbumin) involving glutaraldehyde and the heterobifunctional agent SPDP), purification of peptide conjugates].

30 programs were carried out using transfected cells as a source of immunogen expressing a "tag". Finally, more recently we have demonstrated the feasibility of genetic immunization (DNA vectorized by carriers in the form of nanoparticles, with the company In-Cell-Art).

Screening strategies

Implementing the various programs has involved several approaches over the phases of primary and secondary screening of hybridoma supernatants. Distribution and various combinations are summarized in the Table below:

The choice of primary screening is dictated by the type of immunogen used (e.g. ELISA for immunization with conjugated peptide, or two-color FACS in the case of immunization with transfected cells expressing a "tagged" transgene).

Partnerships

One of the goals of the platform has been the rapprochement with the bio-industrial sector.

To date, 2 close collaborations involve 2 private firms, under research contracts negotiated with the help of Inserm-Transfert:

• Innate-Pharma: development of mAbs against targets of interest to that firm (e.g. effector molecules and regulatory responses to NK and innate immunity)
• In-Cell-Art: using innovative approaches to genetic immunization using nanocarriers for the development of mAbs without immunogenic protein. We validated this approach in the model huCD22.

Valorization

In addition to numerous contracts for the development of mAbs developed within the team that I led at the CIML, (see activity report attached), the platform is associated with several contracts for valorization of mAb specific for targets ABCA1, ABCA7, ABCA2, NKp46, Notch-1, Notch-2, Delta-like ligand 1, Delta-like ligand 4 and GFP. All these contracts are managed by Inserm-Transfert; some are in partnership with the Ludwig Cancer Research Center (New York).

Furthermore, the platform is associated with two patent applications describing the development and use of mAbs against the HIV transactivator Tat-1 (E. Loret, Marseille) and B7H6 (E. Vivier, Marseille).

Developments

The mAb proved central in various fields, especially concerning their use for diagnostic and / or therapeutic purposes.

The ability to validate functional targets with mAbs in a mouse model makes them particularly relevant to explore their potential uses in humans. The platform aims to develop future strategies appropriate to address the humanization of such reagents. Animal models currently being characterized to the CIML should also allow development of such reagents directly from humanized animals.