Bispecific antibodies (bs-Ab), designed and manufactured through genetic recombination, chemical conjugation, or quadromas to contain two target-binding units in one antibody-based molecule, could target two independent epitopes or antigens. Upon sequential or simultaneous binding, bs-Ab acts as a biophysical bridge between two antigens with multiple modes of action (MoA) in vivo to achieve specific effects. Nowadays, many bs-Abs are being tested in clinical trials for treating a variety of diseases, mostly cancer. Here, we provide scientific animal models and systematic bioassays for the preclinical study of antitumor bs-Ab, including target validation, safety profiles, efficacy evaluation, etc. 

METHODS

We have established a state-of-the-art pharmacology platform for supporting the application of investigational antibodies. For the preclinical study of bispecific antibodies, we have developed different animal models for fitting customized requirements, including syngeneic models, xenograft models, target-humanized models, and human immune system reconstitution models. As for in vitro study, our flow cytometry platform and cell base platform support a series of in-vitro studies in a quick, reliable, and reproducible way to evaluate the function and MOA of bispecific antibodies, including target validation, intracellular signal transduction, antibody Fc effects (ADCC, ADCP, CDC) and safety evaluation, etc 
RESULETS
Efficacy Evaluation of Bispecific Antibody Based on Humanized Mice Models 

Fig 1. Humanized mice models and efficacy evaluation of bispecific antibody. The humanized model was validated for the efficacy evaluation of bispecific antibodies. A-B: Strain-specific CD3E expression analysis in B-hCD3E and B-hCD3EDG mice by flow cytometry. Mouse CD3E was exclusively detectable in WT mice; human CD3E was exclusively detectable in homozygous B-hCD3E or B-hCD3EDG but not WT mice. C: Strain-specific PD1 and PD-L1 expression analysis in homozygous B-hPD-1/hPD-L1 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1 (H/H) mice stimulated with anti-CD3ε in vivo. Mouse PD-1 and PD-L1 were exclusively detectable in WT mice, while human PD-1 and PD-L1 were exclusively detectable in homozygous B-hPD-1/hPD-L1 mice but not in WT mice. D-E: Efficacy evaluation of CD3-based bispecific antibodies in humanized models. A high dose of Blinatumomab or BsAb X significantly inhibited tumor growth in B-hCD3E mice models inoculated with hCD19-MC38 cells (C) or in BhCD3EDG mice models inoculated with target-humanized-MC38 cells (D). F: B-hPD1/hPD-L1 humanized mice were subcutaneously implanted with MC38-hPD-L1 cells (5×105). Tested PD-1/PD-L1 BsAbs or IBI318 were inhibited significantly tumor growth to varying degrees .

Efficacy Evaluation of Bispecific Antibody Based on Human Immune System Reconstitution Models

Fig 2. Efficacy evaluation of bispecific antibody on human immune system reconstitution models.A: Human immune system reconstitution models for CD3-based bispecific antibody study. NUGC4 cells (5×106) were subcutaneously implanted after human PBMCs (5×106) were intravenously implanted into B-NDG mice. Anti-human CD3×Claudin18.2 bispecific antibody (AMG 910 analog) inhibited significantly NUGC4 tumor growth in human PBMC reconstituted B-NDG mice. B: B-NDG B2m mice plus reconstituted with PBMCs were used for anti-PD-1×PD-L1 bispecific antibody efficacy studies. Human RKO cells (5×106) were subcutaneously implanted after human PBMCs (5×106) were implanted into B-NDG B2m mice. A high dose of IBI318 (antihuman PD-1×PD-L1 bispecific antibody) inhibited significantly RKO tumor growth.

In vitro studies of bispecific antibody

Fig 3. Cell-based function study for CD3-based bispecific antibody.A: Blinatumomab significantly promoted the proliferation of T cells co-cultured with Daudi cells in a dose-dependent manner. B: Blinatumomab showed significantly cytotoxic efficacy in a dose-dependent manner at a 10:1 ratio of Daudi to T cells. C-D: Blinatumomab increased significantly the secretion of IFN-γ and IL-2 in a dose-dependent manner.

Optional Humanized Animal and Cell Models for Bispecific Antibody Study