Efficient Generation of Bispecific Murine Antibodies for Pre-Clinical Investigations in Syngeneic Rodent Models

Abstract

Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.

Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering

“…This has led to a wide range of described mutants which can be used to increase affinity for activating or inhibitory FcRs (33)(34)(35), ablate Fc-effector functions (36), or recruit complement (37,38). Moreover, amino acid substitutions can also be used to control antibody half-life (39) or to construct bispecific antibodies (40,41). All of these mutant Fc variants can be implemented in our versatile single step CRISPR/HDR platform.…”

Perspective: Designing T-Cell Engagers With Better Therapeutic Windows

“…In addition, when including an Fc to increase half-life of TCEs, a critical consideration is the elimination of Fc receptor interactions. Significant off-target toxicities (20,21) and CRS that can arise from inadvertent cross-linking of standard Fccontaining bispecifics through adjacent Fc receptor-expressing cells (22), and active Fcs can potentially negatively impact in vivo efficacy (23). Arguably, when considering the aforementioned variables impacting TCE safety and efficacy, the failure of many early TCE therapeutic molecules may be a consequence of combining binding domains that were individually optimized but were not optimized to work together.…”

Blockade of ErbB2 and PD-L1 using a bispecific antibody to improve targeted anti-ErbB2 therapy

“…Bispecific antibodies were made as described previously. [30][31][32] Antibody heavy-chain expression vectors were constructed by de novo synthesis (Geneart) of codon-optimized VH coding regions of antibodies rHer2-7.16.4 and PDL1-MDPL3280A, genetically fused to the heavy-chain constant coding regions of mouse mmIgG2a (V00825) and inserted into expression vector pcDNA3.3 (Invitrogen). Likewise, separate light-chain expression vectors were constructed by inserting the appropriate VL coding regions in frame with the CL coding regions of the mouse (V00807) kappa light chain into expression vector pcDNA3.3.…”

“…The mixtures were typically incubated for 5 h at 31°C. To remove 2-MEA, the mixtures were buffer-exchanged against PBS using PD-10 desalting columns (5 kDa molecular weight cut-off; GE Healthcare) or dialysis using Slide-A-Lyzer cassettes (10 kDa molecular weight cut-off 30 ). Samples were stored overnight at 4°C to allow for the re-oxidation of the disulfide bonds.…”