Targeted T cell immunotherapies using engineered T lymphocytes expressing tumor-directed chimeric antigen receptors (CARs) are designed to benefit patients with cancer. Although incorporation of costimulatory endodomains within these CARs increases the proliferation of CAR-redirected T lymphocytes, it has proven difficult to draw definitive conclusions about the specific effects of costimulatory endodomains on the expansion, persistence, and antitumor effectiveness of CAR-redirected T cells in human subjects, owing to the lack of side-by-side comparisons with T cells bearing only a single signaling domain. We therefore designed a study that allowed us to directly measure the consequences of adding a costimulatory endodomain to CAR-redirected T cells. Patients with B cell lymphomas were simultaneously infused with 2 autologous T cell products expressing CARs with the same specificity for the CD19 antigen, present on most B cell malignancies. One CAR encoded both the costimulatory CD28 and the ζ-endodomains, while the other encoded only the ζ-endodomain. CAR + T cells containing the CD28 endodomain showed strikingly enhanced expansion and persistence compared with CAR + T cells lacking this endodomain. These results demonstrate the superiority of CARs with dual signal domains and confirm a method of comparing CAR-modified T cells within individual patients, thereby avoiding patient-to-patient variability and accelerating the development of optimal T cell immunotherapies.

Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its predominant effect is preferential skipping of exon 12 during processing of pre-messenger RNA (mRNA). The lack of fulllength mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted. IntroductionThe last 5 years have been marked by an explosion of knowledge in the field of iron metabolism. These discoveries include identification of the proteins involved in iron absorption and trafficking; discovery of a small peptide, hepcidin, which appears to regulate iron uptake; and elucidation of the role of iron in gene expression. One of the newly identified proteins is divalent metal transporter 1 (DMT1), which is expressed at the brush border of enterocytes in the proximal duodenum where it is presumed to mediate pHdependent uptake of ferrous iron from the gut lumen. 1,2 In the erythroblast, the protein is present in the endosomal membrane, where it appears to function in transport of iron released from the transferrin-receptor complex toward the mitochondria, site of heme synthesis, 3 via a pathway that remains to be defined. In rodent models, mutation of a single nucleotide results in substitution of Arg for Gly at position 185 (G185R) of the DMT1 gene and leads to hypochromic/microcytic anemia and iron deficiency. 4,5 The predicted structure of the protein includes 12 transmembrane domains, asparagine-linked glycosylation signals in an extracytoplasmic loop, membrane targeting motifs, and a consensus transport motif that is common to homologous cation transport proteins found in other species. 6,7 Four major mammalian DMT1 isoforms, resulting from alternative splicing at the 5Ј and 3Ј ends of pre-messenger RNA (mRNA) are known. 8 Isoform I has an iron responsive element (IRE) in the 3Ј untranslated region, whereas isoform II lacks the IRE, and the C-terminal 18 amino acids are replaced by a novel 25-amino acid segment. 6 The duodenum expresses primarily isoform I, whereas erythroblasts express chiefly isoform II; o...

The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAMEspecific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02-restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFN␥ in response to PRAME peptides (between 113 ؎ 8 and 795 ؎ 23 spot forming cells/ 10 5 T cells) and lysed PRAME peptideloaded cells (45 ؎ 19% at an effector: target [E:T] ratio of 20:1) in a MHCrestricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02 ؉ /PRAME ؉ tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high ␣␤TCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML. (Blood. 2008;112: 1876-1885)