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.

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.

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.

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.

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).

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.

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.

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.

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.