Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level.

We describe a mammalian expression construct (SPLICELECT™) that allows the redirection of a proportion of a secreted protein onto the cell surface using alternative splicing: whereas the majority of the RNA is spliced into a transcript encoding a secreted protein, a weak splice donor site yields a secondary transcript encoding, in addition, a C-terminal transmembrane domain. The different sequence elements can be modified in order to modulate the level of cell surface display and of secretion in an independent manner. In this work, we demonstrated that the cell surface display of stable cell lines is correlated with the level of the secreted protein of interest, but also with the level of heterodimerization in the case of a bispecific antibody. It was also shown that this construct may be useful for rapid screening of multiple antibody candidates in binding assays following transient transfection. Thus, the correlation of product quantity and quality of the secreted and of membrane-displayed product in combination with the flexibility of the construct with regards to cell surface display/secretion levels make SPLICELECT™ a valuable tool with many potential applications, not limited to industrial cell line development or antibody engineering.

Analytical assessment of clonal derivation of eukaryotic/CHO cell populations.

Cell populations that are exclusively composed by descendants from a defined single parental progenitor are referred to as "monoclonal", "clonal" or more correctly as "clonally derived cell population". Clonal derivation of cell lines used in the manufacturing of recombinant biologics is a regulatory requirement that aims to ensure a robust process and consistent quality throughout the life cycle of a product. Clonal derivation of cell lines is usually ensured by the process (e.g. two subsequent rounds of limiting dilution). Here we present an approach to analytically assess the probability of clonal derivation of existing cell populations. Using target locus amplification (TLA) followed by next generation sequencing (NGS), unique genetic features can be identified, for example the integration site of the plasmid in the host cell genome or plasmid-plasmid junctions of the plasmids used for expression of recombinant biologics. Whereas a direct assessment of clonal derivation using TLA/NGS data is challenging due to limitations in specificity, confirmed clonally derived populations generated from the cell line population can be analyzed by qPCR for the presence of the unique genetic features identified by TLA/NGS. In the present study, a statistical analysis allowed the demonstration that two independently generated CHO cell lines were clonally derived with an upper 95% confidence interval limit of a potentially present contaminating population of 1.3%.

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Optimization of culture conditions for the expansion of umbilical cord-derived mesenchymal stem or stromal cell-like cells using xeno-free culture conditions.

First isolated from bone marrow, mesenchymal stem or stromal cells (MSC) were shown to be present in several postnatal and extraembryonic tissues as well as in a large variety of fetal tissues (e.g., fatty tissue, dental pulp, placenta, umbilical cord blood, and tissue). In this study, an optimized protocol for the expansion of MSC-like cells from whole umbilical cord tissue under xeno-free culture conditions is proposed. Different fetal calf sera and human serum (HS) were compared with regard to cell proliferation and MSC marker stability in long-term expansion experiments, and HS was shown to support optimal growth conditions. Additionally, the optimal concentration of HS during the cultivation was determined. With regard to cell proliferative potential, apoptosis, colony-forming unit fibroblast frequency, and cell senescence, our findings suggest that an efficient expansion of the cells is carried out best in media supplemented with 10% HS. Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged and immunomodulatory properties, which were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester-labeled peripheral blood mononuclear cells. These findings may be of great value for the establishment of biotechnological protocols for the delivery of sufficient cell numbers of high quality for regenerative medicine purposes.

Growth and differentiation properties of mesenchymal stromal cell populations derived from whole human umbilical cord.

Up to 2.8 × 10(7) fibroblast-like cells displaying an abundant presence of mesenchymal stem cell (MSC) markers CD73, CD90, CD105 and a low level of HLA-I expression can be isolated from one whole human umbilical cord (UC) using a simple and highly reproducible explant culture approach. Cells derived from whole UC, similar to cells collected from separate compartments of UC, display a distinct chondrogenic and adipogenic potential. Therefore they are potential candidates for cartilage and adipose tissue engineering. Cell differentiation along the osteogenic pathway is, however, less efficient, even after the addition of 1.25-dihydroxyvitamin D3, a potent osteoinductive substance. Isolated cells are highly proliferative, tolerate cryopreservation with an average survival rate of about 75% and after thawing can be propagated further, at least over 20 population doublings before their proliferative activity begins to decline. More importantly, they synthesize numerous trophic factors including neurotrophins and factors which facilitate angiogenesis and hematopoiesis. In conclusion, cells isolated from whole UC satisfies all requirements essential for the generation of stem cell banks containing permanently available cell material for applications in the field of regenerative medicine. Nevertheless, further studies are needed to improve and adjust the methods which are already employed for adult MSC expansion and differentiation to specific properties and requirements of the primitive stem cells collected from UC. So, our data verify that the choice of individual parameters for cell propagation, such as duration of cell expansion and cell seeding density, has a substantial impact on the quality of UC-derived cell populations.

Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.

Mesenchymal stromal cells derived from human umbilical cord tissues: primitive cells with potential for clinical and tissue engineering applications.

Mesenchymal stem or stromal cells (MSCs) have a high potential for cell-based therapies as well as for tissue engineering applications. Since Friedenstein first isolated stem or precursor cells from the human bone marrow (BM) stroma that were capable of osteogenesis, BM is currently the most common source for MSCs. However, BM presents several disadvantages, namely low frequency of MSCs, high donor-dependent variations in quality, and painful invasive intervention. Thus, tremendous research efforts have been observed during recent years to find alternative sources for MSCs.In this context, the human umbilical cord (UC) has gained more and more attention. Since the UC is discarded after birth, the cells are easily accessible without ethical concerns. This postnatal organ was found to be rich in primitive stromal cells showing typical characteristics of bone-marrow MSCs (BMSCs), e.g., they grow as plastic-adherent cells with a fibroblastic morphology, express a set of typical surface markers, and can be directly differentiated at least along mesodermal lineages. Compared to BM, the UC tissue bears a higher frequency of stromal cells with a higher in vitro expansion potential. Furthermore, immune-privileged and immune-modulatory properties are reported for UC-derived cells, which open highly interesting perspectives for clinical applications.

Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord.

BACKGROUND: A variety of cell types can be identified in the adherent fraction of bone marrow mononuclear cells including more primitive and embryonic-like stem cells, mesenchymal stem cells (MSC), lineage-committed progenitors as well as mature cells such as osteoblasts and fibroblasts. Different methods are described for the isolation of single bone marrow stem cell subpopulations - beginning from ordinary size sieving, long term cultivation under specific conditions to FACS-based approaches. Besides bone marrow-derived subpopulations, also other tissues including human umbilical cord (UC) have been recently suggested to provide a potential source for MSC. Although of clinical importance, these UC-derived MSC populations remain to be characterized. It was thus the aim of the present study to identify possible subpopulations in cultures of MSC-like cells obtained from UC. We used counterflow centrifugal elutriation (CCE) as a novel strategy to successfully address this question. RESULTS: UC-derived primary cells were separated by CCE and revealed differentially-sized populations in the fractions. Thus, a subpopulation with an average diameter of about 11 mum and a small flat cell body was compared to a large sized subpopulation of about 19 mum average diameter. Flow cytometric analysis revealed the expression of certain MSC stem cell markers including CD44, CD73, CD90 and CD105, respectively, although these markers were expressed at higher levels in the small-sized population. Moreover, this small-sized subpopulation exhibited a higher proliferative capacity as compared to the total UC-derived primary cultures and the large-sized cells and demonstrated a reduced amount of aging cells. CONCLUSION: Using the CCE technique, we were the first to demonstrate a subpopulation of small-sized UC-derived primary cells carrying MSC-like characteristics according to the presence of various mesenchymal stem cell markers. This is also supported by the high proliferative capacity of these MSC-like cells as compared to whole primary culture or other UC-derived subpopulations. The accumulation of a self-renewing MSC-like subpopulation by CCE with low expression levels of the aging marker senescence-associated beta-galactosidase provides a valuable tool in the regenerative medicine and an alternative to bone-marrow-derived MSC.