BCA101 Is a Tumor-Targeted Bifunctional Fusion Antibody That Simultaneously Inhibits EGFR and TGFß Signaling to Durably Suppress Tumor Growth.

The EGFR and TGFß signaling pathways are important mediators of tumorigenesis, and cross-talk between them contributes to cancer progression and drug resistance. Therapies capable of simultaneously targeting EGFR and TGFß could help improve patient outcomes across various cancer types. Here, we developed BCA101, an anti-EGFR IgG1 mAb linked to an extracellular domain of human TGFßRII. The TGFß "trap" fused to the light chain in BCA101 did not sterically interfere with its ability to bind EGFR, inhibit cell proliferation, or mediate antibody-dependent cellular cytotoxicity. Functional neutralization of TGFß by BCA101 was demonstrated by several in vitro assays. BCA101 increased production of proinflammatory cytokines and key markers associated with T-cell and natural killer-cell activation, while suppressing VEGF secretion. In addition, BCA101 inhibited differentiation of naïve CD4+ T cells to inducible regulatory T cells (iTreg) more strongly than the anti-EGFR antibody cetuximab. BCA101 localized to tumor tissues in xenograft mouse models with comparable kinetics to cetuximab, both having better tumor tissue retention over TGFß "trap." TGFß in tumors was neutralized by approximately 90% in animals dosed with 10 mg/kg of BCA101 compared with 54% in animals dosed with equimolar TGFßRII-Fc. In patient-derived xenograft mouse models of head and neck squamous cell carcinoma, BCA101 showed durable response after dose cessation. The combination of BCA101 and anti-PD1 antibody improved tumor inhibition in both B16-hEGFR-expressing syngeneic mouse models and in humanized HuNOG-EXL mice bearing human PC-3 xenografts. Together, these results support the clinical development of BCA101 as a monotherapy and in combination with immune checkpoint therapy. SIGNIFICANCE: The bifunctional mAb fusion design of BCA101 targets it to the tumor microenvironment where it inhibits EGFR and neutralizes TGFß to induce immune activation and to suppress tumor growth.

Identification, characterization and control of a sequence variant in monoclonal antibody drug product: a case study.

Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Early detection of specific sequence modifications, that can result in altered physicochemical and or biological properties, is therefore desirable in product manufacturing. Because of their low abundance, and finite resolving power of conventional analytical techniques, they are often overlooked in early drug development. Here, we present a case study where trace amount of a sequence variant is identified in a monoclonal antibody (mAb) based therapeutic protein by LC-MS/MS and the structural and functional features of the SV containing mAb is assessed using appropriate analytical techniques. Further, a very sensitive selected reaction monitoring (SRM) technique is developed to quantify the SV which revealed both prominent and inconspicuous nature of the variant in process chromatography. We present the extensive characterization of a sequence variant in protein biopharmaceutical and first report on control of sequence variants to < 0.05% in final drug product by utilizing SRM based mass spectrometry method during the purification steps.

Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris.

Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.