Engineered CD4 T cells for in vivo delivery of therapeutic proteins.

The CD4 T cell, when engineered with a chimeric antigen receptor (CAR) containing specific intracellular domains, has been transformed into a zero-order drug-delivery platform. This introduces the capability of prolonged, disease-specific engineered protein biologics production, at the disease site. Experimental findings demonstrate that CD4 T cells offer a solution when modified with a CAR that includes 4-1BB but excludes CD28 intracellular domain. In this configuration, they achieve ~3X transduction efficiency of CD8 T cells, ~2X expansion rates, generating ~5X more biologic, and exhibit minimal cytolytic activity. Cumulatively, this addresses two main hurdles in the translation of cell-based drug delivery: scaling the production of engineered T cell ex vivo and generating sufficient biologics in vivo. When programmed to induce IFNß upon engaging the target antigen, the CD4 T cells outperforms CD8 T cells, effectively suppressing cancer cell growth in vitro and in vivo. In summary, this platform enables precise targeting of disease sites with engineered protein-based therapeutics while minimizing healthy tissue exposure. Leveraging CD4 T cells' persistence could enhance disease management by reducing drug administration frequency, addressing critical challenges in cell-based therapy.

MACC1-the first decade of a key metastasis molecule from gene discovery to clinical translation.

Deciphering the paths to metastasis and identifying key molecules driving this process is one important issue for understanding and treatment of cancer. Such a key driver molecule is Metastasis Associated in Colon Cancer 1 (MACC1). A decade long research on this evolutionarily conserved molecule with features of a transcription factor as well as an adapter protein for versatile protein-protein interactions has shown that it has manifold properties driving tumors to their metastatic stage. MACC1 transcriptionally regulates genes involved in epithelial-mesenchymal transition (EMT), including those which are able to directly induce metastasis like c-MET, impacts tumor cell migration and invasion, and induces metastasis in solid cancers. MACC1 has proven as a valuable biomarker for prognosis of metastasis formation linked to patient survival and gives promise to also act as a predictive marker for individualized therapies in a broad variety of cancers. This review discusses the many features of MACC1 in the context of the hallmarks of cancer and the potential of this molecule as biomarker and novel therapeutic target for restriction and prevention of metastasis.

Primary T-cell-based delivery platform for in vivo synthesis of engineered proteins.

Primary T cell has been transformed into a cell-based delivery platform that synthesizes complex biologics at the disease site with spatiotemporal resolution. This broadly applicable technology can circumvent toxicities due to systemic administration of biologics that necessitates the use of high doses and may diffuse to the healthy tissues. Its clinical translation, however, has been impeded by manufacturing bottlenecks. In this work, a range of process parameters were investigated for increasing the production yield of the primary T cells engineered for delivery function. Compared to the common spinoculation-based method, the transduction yield was enhanced ~2.5-fold by restricting the transduction reaction volume for maximizing the lentivector-to-T-cell contact. Cell density and cytokines used in the expansion process were adjusted to achieve >100-fold expansion of the T-cell-based delivery platform in 14 days, and the function of these cells was validated in vivo using intraperitoneally implanted tumor cells. The primary T-cell-based delivery platform has human applications because it can be scaled and administrated to express a broad range of therapeutic proteins (e.g., cytokines, interferons, enzymes, agonists, and antagonists) at the disease site, obviating the need for systemic delivery of large doses of these proteins.

Electrically regulated cell-based intervention for viral infections.

This work reports on an engineered cell that-when electrically stimulated-synthesizes a desired protein, that is, ES-Biofactory. The platform has been used to express interferon (IFN)-ß as a universal antiviral protein. Compelling evidence indicates the inevitability of new pandemics and drives the need for a pan-viral intervention that may be quickly deployed while more specific vaccines are in development. Toward this goal, a fast-growing mammalian cell (Chassis) has been engineered with multiple synthetic elements. These include-(1) a voltage-gated Ca2+ channel (Voltage-Sensor) that, upon sensing the electric field, activates the (2) Ca2+-mediated signaling pathway (Actuator) to upregulate (3) IFN-ß, via an engineered antiviral transgene (Effector), that is, ES-Biofactory➔IFN-ß. The antiviral effects of the ES-Biofactory➔IFN-ß have been validated on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells. The irradiated ES-Biofactory, that does not exhibit oncogenic capacity, continues to exert antiviral effect. The resulting ES-Biofactory➔IFN-ß uses a novel signaling pathway that, unlike the natural IFN synthesis pathway, is not subject to viral interference. Once clinically validated, the ES-Biofactory will be a universal antiviral cell therapy that can be immediately deployed in the event of an outbreak. The platform may also be useful in treating other diseases including cancer and autoimmune disorders.

Genetically engineered pair of cells for serological testing and its application for SARS-CoV-2.

We have developed a serology test platform for identifying individuals with prior exposure to specific viral infections and provide data to help reduce public health risks. The serology test composed of a pair of cell lines engineered to express either a viral envelop protein (Target Cell) or a receptor to recognize the Fc region of an antibody (Reporter Cell), that is, Diagnostic-Cell-Complex (DxCell-Complex). The formation of an immune synapse, facilitated by the analyte antibody, resulted into a dual-reporter protein expression by the Reporter Cell. We validated it with human serum with confirmed history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. No signal amplification steps were necessary. The DxCell-Complex quantitatively detected the target-specific immunoglobulin G (IgG) within 1 h. Validation with clinical human serum containing SARS-CoV-2 IgG antibodies confirmed 97.04% sensitivity and 93.33% specificity. The platform can be redirected against other antibodies. Self-replication and activation-induced cell signaling, two attributes of the cell, will enable rapid and cost-effective manufacturing and its operation in healthcare facilities without requiring time-consuming signal amplification steps.

Cell-Based Platform for Antigen Testing and Its Application for SARS-CoV-2 Infection.

We have engineered a cell that can be used for diagnosing active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Isolation of individuals with active infections offers an effective solution for mitigating pandemics. However, the implementation of this practice requires robust infrastructure for rapid and intuitive testing, which is currently missing in our communities. To address this need, we engineered a fast-growing cell line into a cell-based antigen test platform for emerging viruses, i.e., DxCell, that can be rapidly deployed in decentralized health care facilities for continuous testing. The technology was characterized using cells engineered to present spike glycoprotein of SARS-CoV-2 (SARS-CoV-2-Sgp-cells) and Calu-3 host cells infected with competent SARS-CoV-2. Preclinical validation was conducted by directly incubating the DxCell with oropharyngeal swabs from mice infected with SARS-CoV-2. No sample preparation steps are necessary. The DxCell quantitatively detected the SARS-CoV-2-Sgp-cells within 1 h (P < 0.02). Reporter signal was proportional to the number of SARS-CoV-2-Sgp-cells, which represents the infection burden. The SARS-CoV-2 DxCell antigen test was benchmarked against quantitative PCR (qPCR) test and accurately differentiated between infected (n = 8) and control samples (n = 3) (P < 0.05). To demonstrate the broad applicability of the platform, we successfully redirected its specificity and tested its sensing function with cells engineered to present antigens from other viruses. In conclusion, we have developed an antigen test platform that capitalizes on the two innate functions of the cell, self-replication and activation-induced cell signaling. These provide the DxCell key advantages over existing technologies, e.g., label-free testing without sample processing, and will facilitate its implementation in decentralized health care facilities. IMPORTANCE Pandemic mitigation requires continuous testing of symptomatic or asymptomatic individuals with rapid turnaround time, and lack of this capability in our community has prolonged pandemic duration leading to obliteration of world economies. The DxCell platform is a cell-based self-replicative antigen test that detects molecular signatures of the target pathogen and can be distributed in small quantities to testing facilities for expansion on site to the desired volume. In this work, we directed this platform to target SARS-CoV-2. Unlike the PCR detection of viral mRNA that requires trained personnel, the DxCell does not require any sample preparation or signal amplification step and introduces an opportunity for a decentralized testing network.

Combinatorial treatment with statins and niclosamide prevents CRC dissemination by unhinging the MACC1-ß-catenin-S100A4 axis of metastasis.

Colorectal cancer (CRC) is the second-most common malignant disease worldwide, and metastasis is the main culprit of CRC-related death. Metachronous metastases remain to be an unpredictable, unpreventable, and fatal complication, and tracing the molecular chain of events that lead to metastasis would provide mechanistically linked biomarkers for the maintenance of remission in CRC patients after curative treatment. We hypothesized, that Metastasis-associated in colorectal cancer-1 (MACC1) induces a secretory phenotype to enforce metastasis in a paracrine manner, and found, that the cell-free culture medium of MACC1-expressing CRC cells induces migration. Stable isotope labeling by amino acids in cell culture mass spectrometry (SILAC-MS) of the medium revealed, that S100A4 is significantly enriched in the MACC1-specific secretome. Remarkably, both biomarkers correlate in expression data of independent cohorts as well as within CRC tumor sections. Furthermore, combined elevated transcript levels of the metastasis genes MACC1 and S100A4 in primary tumors and in blood plasma robustly identifies CRC patients at high risk for poor metastasis-free (MFS) and overall survival (OS). Mechanistically, MACC1 strengthens the interaction of ß-catenin with TCF4, thus inducing S100A4 synthesis transcriptionally, resulting in elevated secretion to enforce cell motility and metastasis. In cell motility assays, S100A4 was indispensable for MACC1-induced migration, as shown via knock-out and pharmacological inhibition of S100A4. The direct transcriptional and functional relationship of MACC1 and S100A4 was probed by combined targeting with repositioned drugs. In fact, the MACC1-ß-catenin-S100A4 axis by statins (MACC1) and niclosamide (S100A4) synergized in inhibiting cancer cell motility in vitro and metastasis in vivo. The MACC1-ß-catenin-S100A4 signaling axis is causal for CRC metastasis. Selectively repositioned drugs synergize in restricting MACC1/S100A4-driven metastasis with cross-entity potential.

NK-Cell Biofactory as an Off-the-Shelf Cell-based Vector for Targeted In Situ Synthesis of Engineered Proteins.

The NK-92MI, a fast-growing cytolytic cell line with a track record of exerting clinical efficacy, is transformed into a vector for synthesizing calibrated amounts of desired engineered proteins at our disease site, that is, NK-cell Biofactory. This provides an allogeneic option to the previously published T-cell-based living vector that is limited by high manufacturing cost and product variability. The modularity of this pathway, which combines a "target" receptor with an "effector" function, enables reprogramming of the NK-cell Biofactory to target diseases with specific molecular biomarkers, such as cancer, viral infections, or auto-immune disorders, and overcome barriers that may affect the advancement of NK-cell therapies.

Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production.

A process for maximizing the titer of lentivirus particles, deemed to be a necessity for transducing primary cells, is developed. Lentivirus particles, with a set of transgenes encoding an artificial cell-signaling pathway, are used to transform primary T cells as vectors for calibrated synthesis of desired proteins in situ, that is, T-cell biofactory cells. The process is also used to generate primary T cells expressing antigen-specific chimeric antigen receptors, that is, CAR T cells. The two differently engineered primary T cells are expanded and validated for their respective functions, that is, calibrated synthesis of desired proteins upon engaging the target cells, which is specific for the T-cell biofactory cells, and cytolysis of the target cells common to both types of cells. The process is compliant with current Good Manufacturing Practices and can be used to support the scale-up for clinical translation.

MACC1 regulates Fas mediated apoptosis through STAT1/3 - Mcl-1 signaling in solid cancers.

MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis.

Human adult stem cells maintain a constant phenotype profile irrespective of their origin, Basal media, and long term cultures.

The study aims to identify the phenotypic marker expressions of different human adult stem cells derived from, namely, bone marrow, subcutaneous fat, and omentum fat, cultured in different media, namely, DMEM-Low Glucose, Alpha-MEM, DMEM-F12 and DMEM-KO and under long term culture conditions (>P20). We characterized immunophenotype by using various hematopoietic, mesenchymal, endothelial markers, and cell adhesion molecules in the long term cultures (Passages-P1, P3, P5, P9, P12, P15, and P20.) Interestingly, data revealed similar marker expression profiles irrespective of source, basal media, and extensive culturing. This demonstrates that all adult stem cell sources mentioned in this study share similar phenotypic marker and all media seem appropriate for culturing these sources. However, a disparity was observed in the markers such as CD49d, CD54, CD117, CD29, and CD106, thereby warranting further research on these markers. Besides the aforesaid objective, it is understood from the study that immunophenotyping acts as a valuable tool to identify inherent property of each cell, thereby leading to a valuable cell based therapy.