Pharmacokinetic Developability and Disposition Profiles of Bispecific Antibodies: A Case Study with Two Molecules

Bispecific antibodies (BsAb) that engage multiple pathways are a promising therapeutic strategy to improve and prolong the efficacy of biologics in complex diseases. In the early stages of discovery, BsAbs often exhibit a broad range of pharmacokinetic (PK) behavior. Optimization of the neonatal Fc receptor (FcRn) interactions and removal of undesirable physiochemical properties have been used to improve the ‘pharmacokinetic developability’ for various monoclonal antibody (mAb) therapeutics, yet there is a sparsity of such information for BsAbs. The present work evaluated the influence of FcRn interactions and inherent physiochemical properties on the PK of two related single chain variable fragment (scFv)-based BsAbs. Despite their close relation, the two BsAbs exhibit disparate PK in cynomolgus monkeys with BsAb-1 having an aberrant clearance of ~2 mL/h/kg and BsAb-2 displaying a an ~10-fold slower clearance (~0.2 mL/h/kg). Evaluation of the physiochemical characteristics of the molecules, including charge, non-specific binding, thermal stability, and hydrophobic properties, as well as FcRn interactions showed some differences. In-depth drug disposition results revealed that a substantial disparity in the complete release from FcRn at a neutral pH is a primary factor contributing to the rapid clearance of the BsAb-1 while other biophysical characteristics were largely comparable between molecules.

Expression, purification and characterisation of a human anti-CDK4 single-chain variable fragment antibody

Background Cyclin-dependent kinase 4 (CDK4) when hyperactivated drives development and maintenance of most tumour types, thus prompting its use as an essential cancer treatment target and a diagnostic tool. Target-binding molecules, such as single-chain variable fragment (scFv) antibodies, hold tremendous potential for use in a wide range of cancer diagnostic and therapeutic applications. Results A human anti-CDK4 scFv antibody (AK2) derived from a human phage display library was expressed in soluble form in Escherichia coli and shown to be secreted into the culture supernatant. Next, soluble AK2 within culture supernatant was successfully purified using affinity chromatography then was shown, using enzyme-linked immunosorbent assays, to bind to recombinant human CDK4 with high affinity and specificity. Further analyses of AK2 interactions with intracellular components demonstrated that AK2 recognised and interacted specifically with endogenous CDK4 and thus could be useful for detection of CDK4 within tumour cells. Conclusions A novel anti-CDK4 scFv antibody that can recognise and interact specifically with recombinant human CDK4 and endogenous CDK4 in tumour cells was expressed and purified successfully. These results suggest that the anti-CDK4 scFv antibody may serve as a new and promising tool for achieving CDK4-targeted diagnosis, prognosis and treatment of numerous types of cancers.

An engineered oncolytic vaccinia virus encoding a single-chain variable fragment against TIGIT induces effective antitumor immunity and synergizes with PD-1 or LAG-3 blockade

BackgroundIn addition to directly lysing tumors, oncolytic viruses also induce antitumor immunity by recruiting and activating immune cells in the local tumor microenvironment. However, the activation of the immune cells induced by oncolytic viruses is always accompanied by high-level expression of immune checkpoints in these cells, which may reduce the efficacy of the oncolytic viruses. The aim of this study is to arm the oncolytic vaccinia virus (VV) with immune checkpoint blockade to enhance its antitumor efficacy.MethodsThrough homologous recombination with the parental VV, an engineered VV-scFv-TIGIT was produced, which encodes a single-chain variable fragment (scFv) targeting T-cell immunoglobulin and ITIM domain (TIGIT). The antitumor efficacy of the VV-scFv-TIGIT was explored in several subcutaneous and ascites tumor models. The antitumor efficacy of VV-scFv-TIGIT combined with programmed cell death 1 (PD-1) or lymphocyte-activation gene 3 (LAG-3) blockade was also investigated.ResultsThe VV-scFv-TIGIT effectively replicated in tumor cells and lysed them, and prompt the infected tumor cells to secret the functional scFv-TIGIT. Compared with control VV, intratumoral injection of VV-scFv-TIGIT in several mouse subcutaneous tumor models showed superior antitumor efficacy, accompanied by more T cell infiltration and a higher degree of CD8+ T cells activation. Intraperitoneal injection of VV-scFv-TIGIT in a mouse model of malignant ascites also significantly improved T cell infiltration and CD8+ T cell activation, resulting in more than 90% of the tumor-bearing mice being cured. Furthermore, the antitumor immune response induced by VV-scFv-TIGIT was dependent on CD8+ T cells which mediated a long-term immunological memory and a systemic antitumor immunity against the same tumor. Finally, the additional combination of PD-1 or LAG-3 blockade further enhanced the antitumor efficacy of VV-scFv-TIGIT, increasing the complete response rate of tumor-bearing mice.ConclusionsOncolytic virotherapy using engineered VV-scFv-TIGIT was an effective strategy for cancer immunotherapy. Administration of VV-scFv-TIGIT caused a profound reshaping of the suppressive tumor microenvironment from ‘cold’ to ‘hot’ status. VV-scFv-TIGIT also synergized with PD-1 or LAG-3 blockade to achieve a complete response to tumors with poor response to VV or immune checkpoint blockade monotherapy.

Expression of a Tagless Single-Chain Variable Fragment (scFv) of Anti-TNF-α by a Salt Inducible System and its Purification and Characterization

Background: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases such as rheumatoid arthritis and Crohn’s disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb’s auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α. Objective: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer. Methods: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out Results: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ∼28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry. Conclusion: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.