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Novel biotherapeutic glycoproteins, like recombinant monoclonal antibodies (mAbs) are widely used for the treatment of numerous diseases. The N-glycans attached to the constant region of an antibody have been demonstrated to be crucial for the biological efficacy. Even minor modifications of the N-glycan structure can dictate the potency of IgG effector functions such as the antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Here, we present the development of a glycoengineered CHO-K1 host cell line (HCL), stably expressing β1,4-N-Acetylglucoseaminyltransferase III (GnT-III) and α-mannosidase II (Man-II), for the expression of a-fucosylated antibodies with enhanced Fc-mediated effector function. Glycoengineered HCLs were generated in a two-step strategy, starting with generating parental HCLs by stable transfection of CHO-K1 cells with GnT-III and Man-II. In a second step, parental HCLs were stably transfected a second time with these two transgenes to increase their copy number in the genetic background. Generated glycoengineered CHO-K1 cell lines expressing two different mAbs deliver antibody products with a content of more than 60% a-fucosylated glycans. In-depth analysis of the N-glycan structure revealed that the majority of the Fc-attached glycans of the obtained mAbs were of complex bisected type. Furthermore, we showed the efficient use of FcγRIIIa affinity chromatography as a novel method for the fast assessment of the mAbs a-fucosylation level. By testing different cultivation conditions for the pre-glycoengineered recombinant CHO-K1 clones, we identified key components essential for the production of a-fucosylated mAbs. The prevalent effect could be attributed to the trace element manganese, which leads to a strong increase of a-fucosylated complex- and hybrid-type glycans. In conclusion, the novel pre-glycoengineered CHO-K1 HCL can be used for the production of antibodies with high ratios of a-fucosylated Fc-attached N-glycans. Application of our newly developed FcγRIIIa affinity chromatography method during cell line development and use of optimized cultivation conditions can ultimately support the efficient development of a-fucosylated mAbs.

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3+ T cell/myeloma cell crosslinking, followed by CD4+/CD8+ T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA+ cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.

The effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein. Therefore several approaches have been developed to rationally manipulate these glycans and improve the biological functions of the antibody. Overexpression of recombinant beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in production cell lines leads to antibodies enriched in bisected oligosaccharides. Moreover, GnT-III overexpression leads to increases in non-fucosylated and hybrid oligosaccharides. Such antibody glycovariants have increased antibody-dependent cellular cytotoxicity (ADCC). To explore a further variable besides overexpression of GnT-III, we exchanged the localization domain of GnT-III with that of other Golgi-resident enzymes. Our results indicate that chimeric GnT-III can compete even more efficiently against the endogenous core alpha1,6-fucosyltransferase (alpha1,6-FucT) and Golgi alpha-mannosidase II (ManII) leading to higher proportions of bisected non-fucosylated hybrid glycans ("Glyco-1" antibody). The co-expression of GnT-III and ManII led to a similar degree of non-fucosylation as that obtained for Glyco-1, but the majority of the oligosaccharides linked to this antibody ("Glyco-2") are of the complex type. These glycovariants feature strongly increased ADCC activity compared to the unmodified antibody, while Glyco-1 (hybrid-rich) features reduced complement-dependent cytotoxicity (CDC) compared to Glyco-2 or unmodified antibody. We show that apart from GnT-III overexpression, engineering of GnT-III localization is a versatile tool to modulate the biological activities of antibodies relevant for their therapeutic application.

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

Background On the basis of the compatible streptogramin- and tetracycline-responsive expression systems, a series of dual-regulated expression systems have been established for use in sophisticated biopharmaceutical manufacturing, advanced gene therapy, and tissue engineering. Methods Dual-regulated expression concepts enable streptogramin- and tetracycline-responsive control of two different (sets of) transgenes (multiregulated multigene metabolic engineering), dual-autoregulated expression configurations for one-step chromosomal integration of two antibiotic-adjustable expression units, and artificial regulatory cascades for multi-level regulation of transgenes and optimized integration of molecular interventions into mammalian regulatory networks. Results This report describes the construction and testing of a family of dual-regulated expression vectors which are compatible with the pTRIDENT vector construction kit, and, in some cases, adapted for retroviral expression technology enabling straightforward transduction of difficult-to-transfect cell lines such as primary cells and stem cells. Conclusions Dual-regulated expression technology will probably become of prime interest for a variety of therapeutic applications, including biopharmaceutical manufacturing, gene therapy, and tissue engineering. Copyright © 2001 John Wiley & Sons, Ltd.

Regulated expression of a cloned gene often provides much higher final expression of the gene product. Also, regulated expression of an activity can enable optional metabolic engineering and simplify functional genomic research. We constructed di-, tri-, and quattrocistronic mammalian expression vectors which allow the simultaneous, coordinated, and adjustable expression of up to two product genes. A single, tetracycline-regulatable promoter, PhCMV*-1, drives high-level expression of a multicistronic expression unit, containing the product gene(s), the gene for tetracycline-responsive transactivator (tTA), and, in the case of pQuattro-tTA, also the neomycin resistance gene. This autoregulatory genetic configuration retains a very low basal transcription activity in the presence of tetracycline, thereby reducing or eliminating possible toxic effects of tTA expression. However, upon withdrawal of tetracycline, a positive feedback regulation loop is activated which leads to higher levels of tTA expression and consequently also to higher expression levels of all other cistrons encoded on the multicistronic expression unit. Since such multicistronic expression vectors combine all genetic elements necessary for high-level expression as well as regulation in a single multicistronic expression unit, they alleviate limitations of previously reported tetracycline-regulatable vector systems and allow straightforward, one-step genetic engineering of eucaryotic cells to give an adjustable phenotype under strict control of the external stimulus, here tetracycline. Because the expression vectors described here were used for the expression for several heterologous product genes such as the green fluorescent protein and the tumor suppressor gene p21 in several cell lines (CHO-K1, BHK-21, and HeLa), we expect these multicistronic, positive feedback regulation vectors to function in a wide variety of eucaryotic cells and to be useful for basic as well as for applied research applications. Other vectors based upon the same autoregulation and multicistronic expression concepts can be constructed using other regulator gene-regulated promoter elements.

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.