Fully Human Anti-CD19 CAR T Cells Derived from Systemic Lupus Erythematosus Patients Exhibit Cytotoxicity with Reduced Inflammatory Cytokine Production.

KYV-101 is an autologous anti-CD19 chimeric antigen receptor (CAR)-T cell therapy under investigation for patients with B-cell driven autoimmune diseases. Hu19-CD828Z is a fully human anti-CD19 CAR designed and demonstrated to have a favorable clinical safety profile. Since anti-CD19 CAR T cells target and kill B cells in both circulation and tissues, the treatment with Hu19-CD828Z CAR T cells offers great potential in depleting autoreactive B cells. Demonstrate that Hu19-CD828Z CAR T cells manufactured from cryopreserved leukaphereses from patients with systemic lupus erythematosus (SLE) exhibit CAR-mediated and CD19-dependent cytokine release, proliferation and cytotoxicity when co-cultured with autologous primary B cells. T cells were enriched from cryopreserved leukaphereses from SLE patients or healthy donors (HD). CAR T cells were generated by transducing these cells with a lentiviral vector encoding Hu19-CD828Z. CAR-mediated and CD19-dependent activity was monitored in vitro in a set of cytotoxicity, cytokine release, and proliferation studies, in response to autologous primary CD19+ B cells, a CD19+ cell line (NALM-6), or a CD19- cell line (U937). Hu19-CD828Z CAR T cells produced from SLE patients or HD induced greater proliferation and dose-dependent cytotoxicity against both autologous primary B cells and the CD19+ NALM-6 cells than nontransduced control T cells or co-cultures with a CD19- cell line. Interestingly, there was lower inflammatory cytokine production from SLE patient-derived CAR T cells compared to HD donor-derived CAR T cells with either CD19+ cells or primary B cells. Hu19-CD828Z CAR T cells generated from SLE patient lymphocytes demonstrate CAR-mediated and CD19-dependent activity against autologous primary B cells with reduced inflammatory cytokine production supporting KYV-101 as a novel potential therapy for the depletion of pathogenic B cells in SLE patients.

Design and Evaluation of Synthetic Terminators for Regulating Mammalian Cell Transgene Expression.

Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements ( i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.

Transcriptomics-Guided Design of Synthetic Promoters for a Mammalian System.

Despite recent advances in improving titers for therapeutic proteins such as antibodies to the 10 g/L scale, these high yields can only be achieved in select mammalian hosts. Regardless of the host or product, strong promoters are required to obtain high levels of transgene expression. However, the promoters employed to drive this expression are rather limited in variety and are usually either viral-derived or screened empirically during vector design. To begin to move away from viral parts, we employed a more systematic approach to identify and design new synthetic promoters using endogenous elements. To do so, we established a workflow to design these elements by (1) analyzing the transcriptomics profile of a specific cell line under a desired, representative cell culture condition, (2) identifying key genetic motifs using bioinformatics that can be used to rationally construct synthetic promoters, (3) building synthetic promoters using conventional DNA synthesis and molecular biology techniques, and (4) evaluating the performance of these synthetic promoters using model proteins. The resulting promoters perform comparably to the hCMV IE promoter variants tested, but with endogenous components. During this design-build-test cycle we also investigated the underlying design rules for transcription factor binding site arrangement in synthetic promoters. Overall, this approach of using an "omics-guided" workflow for designing synthetic promoters facilitates the construction of high expression vectors for immediate use in current production hosts.

Identifying and retargeting transcriptional hot spots in the human genome.

Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site-specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single-integrant, recombinant human cell lines that exhibit stable, high-level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non-protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12(th) exon-intron region of the GRIK1 gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4-fold. The information identified here along with recent advances in site-specific genomic editing techniques can lead to expedited cell line development.

The genome editing toolbox: a spectrum of approaches for targeted modification.

The increase in quality, quantity, and complexity of recombinant products heavily drives the need to predictably engineer model and complex (mammalian) cell systems. However, until recently, limited tools offered the ability to precisely manipulate their genomes, thus impeding the full potential of rational cell line development processes. Targeted genome editing can combine the advances in synthetic and systems biology with current cellular hosts to further push productivity and expand the product repertoire. This review highlights recent advances in targeted genome editing techniques, discussing some of their capabilities and limitations and their potential to aid advances in pharmaceutical biotechnology.