Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity.

Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) due to potential for deleterious recognition of normal cells. We sought to generate CAR(+) T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from nonmalignant cells.

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation.

Methotrexate (MTX) is an anti-folate that inhibits de novo purine and thymidine nucleotide synthesis. MTX induces death in rapidly replicating cells and is used in the treatment of multiple cancers. MTX inhibits thymidine synthesis by targeting dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The use of MTX to treat cancer also causes bone marrow suppression and inhibits the immune system. This has led to the development of an MTX-resistant DHFR, DHFR L22F, F31S (DHFR(FS)), to rescue healthy cells. 5-Fluorouracil-resistant TYMS T51S, G52S (TYMS(SS)) is resistant to MTX and improves MTX resistance of DHFR(FS) in primary T cells. Here we find that a known mechanism of MTX-induced increase in DHFR expression persists with DHFR(FS) and cis-expressed transgenes. We also find that TYMS(SS) expression of cis-expressed transgenes is similarly decreased in an MTX-inducible manner. MTX-inducible changes in DHFR(FS) and TYMS(SS) expression changes are lost when both genes are expressed together. In fact, expression of the DHFR(FS) and TYMS(SS) cis-expressed transgenes becomes correlated. These findings provide the basis for an unrecognized post-transcriptional mechanism that functionally links expression of DHFR and TYMS. These findings were made in genetically modified primary human T cells and have a clear potential for use in clinical applications where gene expression needs to be regulated by drug or maintained at a specific expression level. We demonstrate a potential application of this system in the controlled expression of systemically toxic cytokine IL-12.

Universal artificial antigen presenting cells to selectively propagate T cells expressing chimeric antigen receptor independent of specificity.

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to nonviral gene transfer in T cells uses ex vivo numeric expansion of CAR T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through coculture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed 1 ligand that could activate CAR T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated that CARL aAPC propagate CAR T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Using CARL enables 1 aAPC to numerically expand all CAR T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR T cells of any specificity.

RNA inhibition, microRNAs, and new therapeutic agents for cancer treatment.

Over the past few years, molecular oncology research has revealed that abnormalities in both protein-coding genes (PCGs) and noncoding RNAs (ncRNAs) can be identified in tumors and that the interplay between PCGs and ncRNAs is causally involved in the initiation, progression, and metastases of human cancers. MicroRNAs (miRNAs), which are among the most studied ncRNAs, are small 19- to 25-nucleotide genes involved in the regulation of PCGs and other ncRNAs. With the recent findings of miRNAs' involvement in cancer, RNA inhibition can be used to treat cancer patients in two ways: (1) by using RNA or DNA molecules as therapeutic drugs against messenger RNA of genes involved in the pathogenesis of cancers and (2) by directly targeting ncRNAs that participate in cancer pathogenesis. In this review, we focus on the possible use of miRNAs or compounds interacting with miRNAs as new therapeutic agents in cancer patients.

Litter environment affects behavior and brain metabolic activity of adult knockout mice.

In mammals, the formative environment for social and anxiety-related behaviors is the family unit; in the case of rodents, this is the litter and the mother-young bond. A deciding factor in this environment is the sex ratio of the litter and, in the case of mice lacking functional copies of gene(s), the ratio of the various genotypes in the litter. Both Sex and Genotype ratios of the litter affect the nature and quality of the individual's behavior later in adulthood, as well as metabolic activity in brain nuclei that underlie these behaviors. Mice were raised in litters reconstituted shortly after to birth to control for sex ratio and genotype ratio (wild type pups versus pups lacking a functional estrogen receptor alpha). In both males and females, the Sex and Genotype of siblings in the litter affected aggressive behaviors as well as patterns of metabolic activity in limbic nuclei in the social behavior network later in adulthood. Further, this pattern in males varied depending upon the Genotype of their brothers and sisters. Principal Components Analysis revealed two components comprised of several amygdalar and hypothalamic nuclei; the VMH showed strong correlations in both clusters, suggesting its pivotal nature in the organization of two neural networks.

Effect of incubation temperature and androgens on dopaminergic activity in the leopard gecko, Eublepharis macularius.

Male leopard geckos that hatch from eggs incubated at a female-biased temperature (Tf) behave differently when compared with males hatching at a temperature which produces a male-biased sex ratio (Tm). We investigated the effect of incubation temperature and androgen implantation on aspects of the dopaminergic system of Tf and Tm males. Our data suggest that more dopamine (DA) is stored in the nucleus accumbens of naive Tf males compared with naïve Tm males when they encounter a receptive female conspecific across a barrier. No difference was measured in the preoptic area and the ventral tegmental area (VTA). This difference in intracellular DA levels in a motivation-related brain nucleus might be correlated with differences in sociosexual behavior observed between the two morphs. There were no differences in tyrosine hydroxylase (TH) expressing cell numbers in the VTA of cholesterol (CH)-implanted naive castrated Tf and Tm males. Only Tf males implanted with testosterone had significantly higher TH immunopositive cell numbers in the VTA compared with CH- and dihydrotestosterone-implanted Tf males. These data indicate that both the embryonic environment as well as the circulating hormonal milieu can modulate neurochemistry, which might in turn be a basis for individual variation in behavior.