Construction of Macaque Immune-Libraries

Abstract

Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (muromomab-CD3), the first limits of the therapeutic use of antibodies deriving from hybridoma technology appeared. Indeed, the nonhuman nature of these therapeutic antibodies makes them immunogenic when administrated to patients, which develop anti-drug antibodies (ADA). If repeated drug-administrations are needed, the immune response will accelerate the elimination of the drug, leading to a therapeutic failure, or in the worst case to an anaphylactic reaction against the murine protein. Several antibody generations were then developed to obtain better-tolerated molecules: chimeric, humanized, and fully human antibodies. The first antibody generation is fully based on cellular technology (mice hybridoma technology), but the next generations are improved by molecular engineering. Immune antibody phage-display libraries are one successful approach to isolating such engineered antibodies. One strategy to isolate high-affinity and well-tolerated antibodies when no immunized patients are available is based on the phage-display-screening of immune libraries deriving from immunized nonhuman primates, which are phylogenetically close to humans. This chapter presents the strategy for the construction of macaque antibody immune-libraries.

Antibodies against Anthrax Toxins: A Long Way from Benchlab to the Bedside

“…A pipeline for the isolation of recombinant antibodies was developed in our lab ( Figure 1 ) [ 13 ]. Because antibodies will be administrated in human, they have to be well-tolerated.…”

“…Because macaques are a model of choice for the development of human-like therapeutic antibodies, they were immunized with recombinant surface antigen or with non-toxic toxin (inactivated toxin or toxin subunit). Generally, three antigen administrations were realized at a 1-month interval [ 13 ]. After a resting period of at least 120 days after the last immunization, bone marrow was sampled, total RNA extracted and a RT-PCR was done to amplify the DNA region coding the antibody variable domains.…”

“…A final boost was then realized after this control and bone marrow was iteratively sampled with a 2-to-3-day frequency until a decrease in the immune response was observed. Polymerase Chain Reaction (PCR) products obtained when the immune response was optimal were pooled and used for the generation of a phage-display single chain Fragment variable (scFv) or Fragment antigen binding (Fab) library in pHAL14, pHAL32, or pComb3X phagemid vector [ 12 , 13 , 15 , 16 ]. The resulting libraries were screened against the recombinant protein or the whole pathogen of interest.…”

Single‐chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety

“…Based on this, the isolation of high-affinity scFv against each target antigen cannot be guaranteed (Farajnia et al, 2014;Kumar et al, 2019). Second, immune libraries, that are a heterogeneous pool of antibody genes, are typically derived from diseaseinfected individuals or different types of immunized animals, such as mice (Li, Hou, et al, 2021), rabbits (Nguyen et al, 2018), and macaques (Avril et al, 2018). Immune libraries contain a rich population of high-affinity antibodies that are based on specific antigens.…”

Construction of an equine antibody library in the single-chain-Fragment-variable format (scFv) to express equine immunoglobulins

“…The first immunised library consisted of the immunoglobulin repertoire of mice and was generated by PCR from immunised animals (88). Other examples of immunized libraries include a human scFv library (89), a nonhuman library derived from macaques, both in the scFv format (90), bovine scFv library (91), a rabbit immune library (92), a camel immune library (93) or chicken immune library in the scFv format (94). The immunization procedure prior to library generation conveys the disadvantages of being time consuming, and bears the risk of lack of efficacy of the vaccine or immune response and its unpredictability (86).…”