Strategies for modifying the chimeric antigen receptor (CAR) to improve safety and reduce toxicity in CAR T cell therapy for cancer.

Immune cell therapy with chimeric antigen receptor (CAR) T cells, which has shown promising efficacy in patients with some hematologic malignancies, has introduced several successfully approved CAR T cell therapy products. Nevertheless, despite significant advances, treatment with these products has major challenges regarding potential toxicity and sometimes fatal adverse effects for patients. These toxicities can result from cytokine release or on-target off-tumor toxicity that targets healthy host tissue following CAR T cell therapy. The present study focuses on the unexpected side effects of targeting normal host tissues with off-target toxicity. Also, recent safety strategies such as replacing or adding different components to CARs and redesigning CAR structures to eliminate the toxic impact of CAR T cells, including T cell antigen coupler (TAC), switch molecules, suicide genes, and humanized monoclonal antibodies in the design of CARs, are discussed in this review.

Characterization of CAR T Cells Manufactured Using Genetically Engineered Artificial Antigen Presenting Cells.

OBJECTIVE: Chimeric antigen receptor (CAR) T cell therapy has recently emerged as a promising approach for the treatment of different types of cancer. Improving CAR T cell manufacturing in terms of costs and product quality is an important concern for expanding the accessibility of this therapy. One proposed strategy for improving T cell expansion is to use genetically engineered artificial antigen presenting cells (aAPC) expressing a membrane-bound anti-CD3 for T cell activation. The aim of this study was to characterize CAR T cells generated using this aAPC-mediated approach in terms of expansion efficiency, immunophenotype, and cytotoxicity. MATERIALS AND METHODS: In this experimental study, we generated an aAPC line by engineering K562 cells to express a membrane-bound anti-CD3 (mOKT3). T cell activation was performed by co-culturing PBMCs with either mitomycin C-treated aAPCs or surface-immobilized anti-CD3 and anti-CD28 antibodies. Untransduced and CD19-CARtransduced T cells were characterized in terms of expansion, activation markers, interferon gamma (IFN-γ) secretion, CD4/CD8 ratio, memory phenotype, and exhaustion markers. Cytotoxicity of CD19-CAR T cells generated by aAPCs and antibodies were also investigated using a bioluminescence-based co-culture assay. RESULTS: Our findings showed that the engineered aAPC line has the potential to expand CAR T cells similar to that using the antibody-based method. Although activation with aAPCs leads to a higher ratio of CD8+ and effector memory T cells in the final product, we did not observe a significant difference in IFN-γ secretion, cytotoxic activity or exhaustion between CAR T cells generated with aAPC or antibodies. CONCLUSION: Our results show that despite the differences in the immunophenotypes of aAPC and antibody-based CAR T cells, both methods can be used to manufacture potent CAR T cells. These findings are instrumental for the improvement of the CAR T cell manufacturing process and future applications of aAPC-mediated expansion of CAR T cells.

Generation and Functional Characterization of PLAP CAR-T Cells against Cervical Cancer Cells.

Chimeric antigen receptor (CAR) T-cell therapy is one of the cancer treatment modalities that has recently shown promising results in treating hematopoietic malignancies. However, one of the obstacles that need to be addressed in solid tumors is the on-target and off-tumor cytotoxicity due to the lack of specific tumor antigens with low expression in healthy cells. Placental alkaline phosphatase (PLAP) is a shared placenta- and tumor-associated antigen (TAA) that is expressed in ovarian, cervical, colorectal, and prostate cancers and is negligible in normal cells. In this study, we constructed second-generation CAR T cells with a fully human scFv against PLAP antigen andthen evaluated the characteristics of PLAP CAR T cells in terms of tonic signaling and differentiation in comparison with ΔPLAP CAR T cells and CD19 CAR T cells. In addition, by co-culturing PLAP CAR T cells with HeLa and CaSki cells, we analyzed the tumor-killing functions and the secretion of anti-tumor molecules. Results showed that PLAP CAR T cells not only proliferated during co-culture with cancer cells but also eliminated them in vitro. We also observed increased secretion of IL-2, granzyme A, and IFN-γ by PLAP CAR T cells upon exposure to the target cells. In conclusion, PLAP CAR T cells are potential candidates for further investigation in cervical cancer and, potentially, other solid tumors.

CRISPRi-mediated knock-down of PRDM1/BLIMP1 programs central memory differentiation in ex vivo-expanded human T cells.

Introduction: B lymphocyte-induced maturation protein 1 (BLIMP1) encoded by the positive regulatory domain 1 gene (PRDM1), is a key regulator in T cell differentiation in mouse models. BLIMP1-deficiency results in a lower effector phenotype and a higher memory phenotype. Methods: In this study, we aimed to determine the role of transcription factor BLIMP1 in human T cell differentiation. Specifically, we investigated the role of BLIMP1 in memory differentiation and exhaustion of human T cells. We used CRISPR interference (CRISPRi) to knock-down BLIMP1 and investigated the differential expressions of T cell memory and exhaustion markers in BLIMP1-deficient T cells in comparison with BLIMP1-sufficient ex vivo expanded human T cells. Results: BLIMP1-deficiency caused an increase in central memory (CM) T cells and a decrease in effector memory (EM) T cells. There was a decrease in the amount of TIM3 exhaustion marker expression in BLIMP1-deficient T cells; however, there was an increase in PD1 exhaustion marker expression in BLIMP1-deficient T cells compared with BLIMP1-sufficient T cells. Conclusion: Our study provides the first functional evidence of the impact of BLIMP1 on the regulation of human T cell memory and exhaustion phenotype. These findings suggest that BLIMP1 may be a promising target to improve the immune response in adoptive T cell therapy settings.

Toll-Like Receptor-Based Strategies for Cancer Immunotherapy.

Toll-like receptors (TLRs) are expressed and play multiple functional roles in a variety of immune cell types involved in tumor immunity. There are plenty of data on the pharmacological targeting of TLR signaling using agonist molecules that boost the antitumor immune response. A recent body of research has also demonstrated promising strategies for improving the cell-based immunotherapy methods by inducing TLR signaling. These strategies include systemic administration of TLR antagonist along with immune cell transfer and also genetic engineering of the immune cells using TLR signaling components to improve the function of genetically engineered immune cells such as chimeric antigen receptor-modified T cells. Here, we explore the current status of the cancer immunotherapy approaches based on manipulation of TLR signaling to provide a perspective of the underlying rationales and potential clinical applications. Altogether, reviewed publications suggest that TLRs make a potential target for the immunotherapy of cancer.

Regulatory Roles of the Notch Signaling Pathway in Liver Repair and Regeneration: A Novel Therapeutic Target.

The liver is one of the significant regenerative organs in the body. Nevertheless, underlying molecular mechanisms regulating liver repair and regeneration following resection or damage remain largely unknown. The Notch signaling pathway is a profoundly evolutionarily well-conserved cell signaling system that mostly involves developing multicellular organisms. Malfunctions in this pathway lead to the progression of several liver disorders, including hepatoblastoma (HB), cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC). The Notch pathway plays a fundamental role in cell fate during the embryonic stage's progression to the adult stage in liver tissue. Modulation of Notch signaling may be used in many patients who succumb to cirrhosis due to chronic hepatitis by virus infection. This review describes the underlying mechanisms of the Notch signaling pathway in liver development and regeneration briefly and discusses how this pathway leads to the betterment of liver disorders in the clinic.

An Easy and Fast Method for Production of Chinese Hamster Ovary Cell Line Expressing and Secreting Human Recombinant Activin A.

OBJECTIVE: Growth factors are key elements of embryonic stem cell (ESC) research. Cell line development in eukaryotes is a time-consuming procedure which usually takes 12-18 months. Here, we report an easy and fast method with which production of Chinese hamster ovary (CHO) cells that express and secrete recombinant Activin A, as a major growth factor in endo/mesoderm differentiation of embryonic stem cells is achieved within 3-4 weeks. MATERIALS AND METHODS: In this experimental study, we cloned human Activin A into the pDONR/Zeo gateway entry vector using the BP reaction. Activin A was subcloned next into the pLIX_403 and pLenti6.3/TO/V5-DEST destination vectors by the LR reaction. The result was the production of constructs with which 293T cells were finally transfected for virus production. CHO cells were transduced using viral particles to produce a cell line that secretes the His6- Activin A fusion protein. RESULTS: We developed a quick protocol which saves up to 3-4 weeks of time for producing recombinant proteins in CHO cells. The recombinant cell line produced 90 mg/L of functional Activin A measured in human ESC line Royan H5 (RH5), during in vitro differentiation into meso-endoderm and definitive endoderm. CONCLUSION: Our results showed no significant differences in functionality between commercial Activin A and the one produced using our novel protocol. This approach can be easily used for producing recombinant proteins in CHO.

Inclusion Body Expression and Refolding of Recombinant Bone Morphogenetic Protein-2.

BACKGROUND: Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes Escherichia coli (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems. METHODS: BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in Origami™ E. coli strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line. RESULTS: No difference was observed between oxidoreductase systems in improving the efficiency of protein refolding. However, comparisons between two refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein. CONCLUSION: A new method for production of biologically active dimeric form of BMP-2 in E. coli expression system was established in this study.

Expression and purification of neurotoxin-associated protein HA-33/A from Clostridium botulinum and evaluation of its antigenicity.

BACKGROUND: Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. METHODS: Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. RESULTS: The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. CONCLUSION: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice.

Cloning, expression, and functional characterization of in-house prepared human leukemia inhibitory factor.

OBJECTIVE: Leukemia inhibitory factor (LIF) plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF (hLIF) is an essential growth factor for the maintenance of mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a pluripotent, undifferentiated state. MATERIALS AND METHODS: In this experimental study, we cloned hLIF into the pENTR-D/ TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami™ 2(DE3) pLacI competent cells to produce the His6-hLIF fusion protein. RESULTS: This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. CONCLUSION: Our results showed no significant differences in function between laboratory produced and commercialized hLIF.