Genetic Engineering & Biotechnology News

JUL 2018

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Page 28 of 33 | Genetic Engineering & Biotechnology News | JULY 2018 | 27 ment, that is, an environment that can sup- press the activity of CAR T cells. To tackle these issues, Celyad engineered T cells expressing a CAR composed of the full-length human natural killer group 2D (NKG2D) receptor fused to the CD3ζ cyto- plasmic signaling domain. As its name sug- gests, the NKG2D receptor is derived from natural killer cells. The CD3ζ chain, which is found in most antibody-based CARs, pro- vides the primary stimulatory signal within the T cell when the NKG2D receptor binds to a ligand. The NKG2D receptor targets eight dif- ferent ligands that are normally expressed in cells after infection and other types of stress. Most important, as Dr. Sotiropoulou noted, these ligands are expressed by most types of cancer cells, but they are not expressed at all (or above very low levels) by healthy cells. The NKG2D-based CAR serves as the basis of Celyad's lead CAR T-cell oncology candidate, CYAD-01. (Celyad's NKG2D- CD3ζ CAR T cell was previously referred to as CAR T NKR-2.) NKG2D attracts a co- stimulatory adapter protein, DAP10, which is normally expressed in natural killer cells and T cells. Besides recognizing the formation of DAP10-stabilized NKG2D constructs, Celyad evaluated whether CYAD-01 would perform better if it incorporated an additional cyto- plasmic costimulatory domain. When Celyad tried adding CD28 or 4-1BB costimulatory domains, which are known to confer interest- ing properties to classical scFV-based CAR T cells, the company found that neither CD28 nor 4-1BB provided any advantages to NK- G2D-based CAR T cells in vitro. Unlike CD28 and 4-1BB, DAP10 does of- fer distinct advantages. "The overexpression of DAP10 allows T cells to express NKG2D- based CAR at the cell surface and tends to in- crease the secretion of proinflammatory cyto- kines," a Celyad poster paper indicated. "The increased secretion of these proinflammatory cytokines is of particular interest in the con- text of solid tumors." The advantages conferred by DAP10 were summarized by Dr. Sotiropoulou as fol- lows: "We have a first-generation CAR that acts like a second-generation CAR, provid- ing the first and second signal, rendering the cell effective to kill the tumor cell and pro- vide cytokines." Although the CAR T cells themselves don't seem to persist, a long-term antitu- mor activity was observed in mouse models. "These CAR T cells modulate the microenvi- ronment and boost the immune system of the host so that you can have an immunological memory," Dr. Sotiropoulou asserted. Hav- ing demonstrated some efficacy and a good safety profile in low-dose clinical trials for hematological cancers, CYAD-01 is currently in a dose escalation trial for several different solid and liquid tumors. Switching from CARs to TCRs In a panel discussion, Robert Hofmeis- ter, Ph.D., chief scientific officer of TCR2 Therapeutics, talked about the need to acces- sorize CAR T cells with more costimulatory molecules and to increase the expression of cytokines, "just to help the CARs over a cer- tain hurdle, to be active in the suppressive im- mune environment." Traditional CARs are artificial constructs, making use of only the ζ chain of the entire eight-subunit T-cell recep- tor (TCR) complex, and "that leads to inad- equate activation of the cell, which probably translates into a lack of persistence in patients and also a lack of activity." He sees the field pivoting away from CARs and toward the use of engineered TCRs to combat solid tumors. Yet TCRs are human leukocyte antigen (HLA)-restricted, mean- ing that they will recognize antigen only in the context of a given HLA—and different people carry different HLAs. "This is a major drawback when it comes to recruitment of pa- tients," Dr. Hofmeister insisted. TCR2 has developed a hybrid platform called TRuC that utilizes the entire TCR complex to drive T-cell activation, yet is inde- pendent of the HLA. "We tether an antibody- binding domain to one of the TCR subunits (in the lead program, we tether it to the ε chain) … which gets integrated into the natu- ral TCR complex," he explained. In animal studies, the TRuC cells showed maximal efficacy against tumors as well as long persistence. With TRuC cells, Dr. Hof- meister added, "we don't get the massive overshooting of cytokines [cytokine release syndrome] that you observe with CAR T cells." Engineering the Right T Cells Cytokine release syndrome and lack of durability in CAR T-cell therapy may due, at least in part, to too many of the wrong cells being active. "Groups using lentivirus to pro- duce CAR T cells are seeing anywhere from 5 to 30% of the T-cell product that they in- fuse actually express the CAR," said Devon Shedlock, Ph.D., vice president of preclinical development, Poseida Therapeutics. Although retroviruses enter effector memory T cells and also, to some extent, central memory T cells, they are effectively excluded from the early naïve T and stem cell memory T (T scm ) cells. Thus, many non- modified T cells are being introduced that also become activated and can potentially contribute to making cytokines and inflam- matory mediators. Poseida instead uses a transposon-based system, the PiggyBac DNA Modification Sys- tem, to produce their CAR T-cell products. PiggyBac has a large cargo capacity—enough even for several CARs and other genes to be introduced as a single transgene—allowing it to accommodate additional elements such as caspase-based safety switches as well as a drug-resistance genes for positive selection. PiggyBac seems to prefer early naïve T and T scm cells. After selection is carried out, virtu- ally 100% of the T cells express the CAR, and more than 70% are T scm cells. T scm cells are long-lived, multipotent, and self-renewing. "They first need to expand and differentiate into effector cells before they do their killing," Dr. Shedlock advised, adding that the slower kinetics "might ulti- mately give you enough buffer to better han- dle some of these acute toxicities that other groups have seen with a very differentiated, hot product." The first cohort of a clinical trial begun last year for multiple myeloma showed "a very rapid and marked response at this very low dose in all patients," noted Dr. Shedlock. "Simultaneously, we didn't see any evidence of cytokine release syndrome, and we think that's due to the T scm phenotype." Translational Medicine CAYMAN CONTRACT SERVICES Your Vision. Our Expertise. Accelerated Discovery. DISCOVER MORE Bioanalysis & Assay Development Analytical Chemistry Medicinal Chemistry & Structure-Based Drug Design Chemical Synthesis TCR2 Therapeutics has developed TRuC, which stands for T cell Receptor fUsion Construct. According to the company, TRuC is the first engineered T-cell platform to use the complete T-cell receptor (TCR) complex without the need for human leukocyte antigen (HLA) matching. By conjugating tumor antigen binder (for example, scFV, sdAb, Fab) to the TCR complex, the TRuC construct can recognize highly expressed surface antigens on tumor cells without HLA and employ the complete TCR machinery to drive the totality of T-cell functions required for tumor killing.

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