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.

A new signal sequence for recombinant protein secretion in Pichia pastoris.

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro MATα sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins.

Sorbitol is used as a non-repressive carbon source to develop fermentation process for Mut(s) recombinant clones obtained using the AOX1 promoter in Pichia pastoris. Sorbitol dehydrogenase is an enzyme in the carbohydrate metabolism that catalyzes reduction of D-fructose into D-sorbitol in the presence of NADH. The small stretch of 211bps upstream region of sorbitol dehydrogenase coding gene has all the promoter elements like CAAT box, GC box, etc. It is able to promote protein production under repressive as well as non-repressive carbon sources. In this study, the strength of the sorbitol dehydrogenase promoter was evaluated by expression of two heterologous proteins: human serum albumin and erythrina trypsin inhibitor. Sorbitol dehydrogenase promoter allowed constitutive expression of recombinant proteins in all carbon sources that were tested to grow P. pastoris and showed activity similar to GAP promoter. The sorbitol dehydrogenase promoter was active in all the growth phases of the P. pastoris.