Preclinical targeting of human T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-engineered T cells.

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.

Preclinical and clinical evaluation of 5-fluorouracil biochemical modulation with folinic acid and hydroxyurea for patients with colorectal carcinoma.

BACKGROUND: Approximately 140,000 new cases of colorectal carcinoma will be diagnosed in 1995 in the United States, and more than one-third of these patients will die from progressive disease. Despite the modest improvement in response rate with chemotherapy, little improvement in patient survival has been noted. Consequently, the evaluation of new agents, modalities, and combinations is needed. METHODS: Two cell lines, HCT 116 and COLO 320 HSR, were treated with various concentrations of 5-fluorouracil (5-FU), folinic acid (FA), and hydroxyurea (HU). Subsequently, 41 patients with advanced, measurable metastatic colorectal carcinoma were enrolled in the study. Patients were treated with oral doses of HU (500 mg) every 8 hours on Days 1 and 2, 5-FU (400-500 mg/m2) intravenously Day 2 and FA (100 mg/m2) intravenously on Day 2 of every week for 6 consecutive weeks, followed by a 2-week rest period. All patients were evaluable for toxicity, and 40 were evaluable for response. RESULTS: In both cell lines, the combination of 5-FU/FA/HU consistently produced the best cytotoxic effect. Clinically, the maximum tolerated dose of 5-FU was established at a level of 500 mg/m2 (450 mg/m2 for patients older than 70 years of age). Ten patients experienced Grade 3 or 4 toxicity, consisting mainly of diarrhea. Eleven of 40 evaluable patients responded (three complete responses, eight partial responses), with a median survival of 12+ months and time to progression of 8.5+ months. CONCLUSION: The biochemical modulation of 5-FU with FA and HU were significantly effective in treating patients with metastatic colorectal carcinoma. Overall, this regimen was well tolerated with only moderate toxicity. Further studies incorporating intravenous HU as well as a randomized Phase III study of 5-FU/FA/HU versus 5-FU/FA are recommended.

ERG gene is translocated in an Ewing's sarcoma cell line.

Ewing's sarcoma (ES) and related neoplasias are characterized by the reciprocal translocation, t(11;22)(q24;q12). The translocation has been reported to generate a fusion gene between the EWS (a previously undescribed gene on chromosome 22) and FLI1 genes. We report a similar translocation of EWS and FLI1 in an Askin's tumor cell line (SK-NM-C). Further, we describe an alternative translocation in an ES cell line (#5838) in which the 5' end of the EWS gene is juxtaposed to the 3' end of the ERG gene. The ERG gene is on chromosome 21, but no microscopically visible changes in chromosome 21 were observed. Elevated steady state levels of the EWS/ERG fusion gene transcript were detected in the #5838 cell line. This is the first report of a structural alteration of ERG in human cancer. Also, it confirms a general mechanism of generating putative oncogenic fusion genes by placing an ETS DNA binding domain in direct proximity to the carboxy terminus domain (CTD) related region of the EWS gene.

Transformation of diploid human lung fibroblasts with oncogene ras increases the frequency of abnormal mitoses.

The human oncogene ras p21 was transfected into human lung fibroblast WI-38 diploid cells. All of the clones that were isolated (n = 36), exhibited rapid growth and transformed morphology which was ascertained by both transmission and scanning electron microscopy as extensive formations of microvilli on the plasma membrane, marked distortion of the nuclear membrane and increased number of pinocytotic vesicles in the cortical cytoplasm. The frequency of abnormal metaphases rose from 15% in parental WI-38 cells to 35.5-48.4% in all ten clones examined. Lagging chromosomes in prometaphase represented 42.1-69.4% of the total abnormal mitoses followed in frequency by 3-group metaphase and C-metaphase. All transformed cells were aneuploid. These data provide evidence for the association between cellular transformation with oncogenic ras and elevated abnormal mitoses in human cells.

c-src structure in human cancers with elevated pp60c-src activity.

We used RNAase protection and restriction fragment length polymorphism assays to detect activating mutations of c-src in a spectrum of human tumours. No mutations were detected at codons 98, 381, 444, and 530. We conclude that mutational activation is not the mechanism of enhancement of pp60c-src-specific kinase activity found in a number of human cancer types.

Molecular cloning of proteinase-encoding genes from cancer cells by zymogen assay and direct sequencing.

A method that permits the in vitro cloning and identification of proteolytic enzyme genes from cDNA expression libraries is described. The method can detect positive proteinase genes within 30 min following the transfer of plaques to nitrocellulose membrane filters. The method is based on the functional expression of fusion lac Z-proteinase protein in lambda gt11 infected Y1090 bacteria and does not require prior knowledge of either the sequence of the cDNA insert or a monoclonal antibody to its encoded antigen. This strategy when coupled with polymerase chain reaction of the cDNA insert using lac Z primer sequences that are flanking the EcoR1 cloning site in gt11 phage permits direct sequencing of the amplified DNA. With this method we have isolated 10 genes expressing protease activity in the human small-cell carcinoma of the lung. The same procedure could be applied to isolate unknown proteinases from cDNA libraries of virtually any eukaryotic cell.

High expression of ras p21 correlates with increased rate of abnormal mitosis in NIH3T3 cells.

The modulation of responsive genes by hormonal stimulation is an attractive in vitro model system for the study of a wide variety of biological processes. Using this methodology we have investigated the effect of the human oncogene protein p21ras on mitosis using mouse mammary tumor virus long terminal repeat (MMTV-LTR)-directed gene expression. Following the induction of p21 protein, abnormal mitotic figures were scored in metaphase and anaphase. Elevated expression of p21 was associated with marked increase in the proportion of abnormal mitoses most significantly during the metaphase. Concomitant with a three fold increase in p21 levels, abnormal mitosis rose from 14.0% to 27.25%. The increase in abnormal mitosis corresponded to a 225% increase in abnormal metaphase. The p21-induced mitotic abnormalities were exhibited as lagging chromosomes in prometaphase, 3 group metaphase and C-metaphase. In addition, high expression of p21 was accompanied by significant changes in the cell morphology and fine ultrastructure, e.g. disorganization of actin, the extensive formation of microvilli on the plasma membrane and marked dilatation of the rough endoplasmic reticulum. The mitotic and structural changes were reversible upon removal of dexamethasone and decline of p21 production to its basal levels. Our results identify an important biological effect of ras p21 during mitosis and the early stages of neoplastic transformation.

Activation of cancer cell proteases and cytotoxicity by EGF and PDGF growth factors.

The biological effects of EGF and PDGF growth factors on A172 and hEGFr-3T3 cell lines were studied using RBC induced cytolysis and polyacrylamide-gelatin gel electrophoresis assays. The authors report that growth factor-induced cytotoxicity in these cells is mediated by proteolytic enzymes. Treatment of A172 cells with either EGF or PDGF resulted in marked increase of their cytotoxicity (Release Index = 150%). Similarly, RBC induced release index by hEGFr-3T3 cells was elevated to 420% in the presence of 3.4 pM of EGF. However, in A172 cells, PDGF did not have a significant effect on DNA and protein synthesis indicating that stimulation of proteolytic activity is independent of the growth factor signaling pathway. Growth factor induced cytotoxicity was significantly reduced by protease inhibitors in both cell lines. Using EDTA and leupeptin several proteolytic species were identified and localized to cellular membranes as evidenced by polyacrylamide-gelatin electrophoresis assay. These data suggest that growth factors regulate the activation or secretion of proteolytic enzymes in cancer cells and may mediate the invasive and metastatic behavior of these cells.

Role of epidermal growth factor in the expression of A431 cancer cell protease and red blood cell cytotoxicity.

The role of epidermal growth factor in the regulation of the proteolytic and RBC cytolytic activity of the A431 cancer cell line has been evaluated using our previously described gelatin/polyacrylamide electrophoretic assay and tumor-induced RBC cytolysis assay, respectively. A431 cells maintained in 10% fetal bovine serum were actively cytolytic for RBC (release index, 53.0 +/- 2.9%), whereas serum-starved cells maintained in serum-free medium were not cytolytic for RBC. RBC cytotoxicity was restored by adding as little as 3.4 pM epidermal growth factor to the serum-deprived cells. The RBC cytolytic stimulating activity of epidermal growth factor could be mimicked by the metal chelating agent 1,10-phenanthroline, suggesting a possible role for calcium ions in the action of epidermal growth factor and proteases. An enriched cell membrane preparation of A431 cells was also cytolytic for RBC but was unaffected by metal chelating agents. RBC-induced cytotoxicity was inhibited by the protease inhibitor leupeptin. Gelatin substrate gels of enriched A431 cell membrane preparations and serum-free supernatants revealed a pattern of high- and low-molecular-weight proteases that were stimulated by metal chelators and inhibited by leupeptin. The activity of these proteases appears to be regulated by epidermal growth factor by a process that may involve divalent cations.

Oncogene ras p21- and v-src pp60-transformed cells exhibit altered expression of proteases.

To evaluate the proteolytic activities of oncogene-transfected 3T3 cells, we have developed a copolymerized substrate electrophoretic assay that permits the detection of picogram quantities of proteases produced by cells in culture. Our assay involves a gelatin substrate copolymerized in a polyacrylamide gel. Purified cell membrane preparations were run on gels and various protease activities were detected by amido black. Ras-transfected 3T3 cells appear to have a soluble metalloprotease that may be transiently membrane bound and responsible for destruction of red blood cells (RBC). Oncogene-transfected NIH-3T3 cells have been demonstrated to have RBC cytolytic activity. We have previously shown that v-src-transfected 3T3 cells and their cell membranes cause RBC cytolysis which is inhibited by the protease inhibitor leupeptin. Here we report that both H-ras- and K-ras-transfected 3T3 cells and their cell membranes are cytolytic for RBC, but are inhibited by the metalloprotease inhibitor ethylene diamine tetraacetic acid. Using the gelatin substrate gel assay, we determined that some of the proteases were intrinsic to the oncogene expressing cells, while other proteases were secreted into the culture growth medium.

Microinjection of ras p21 induces a rapid rise in intracellular pH.

Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (approximately 0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.

Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21.

The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.

Isolation and characterization of a cyclic AMP receptor protein from luminous Vibrio harveyi cells.

A cAMP receptor protein (CRP) species was purified from the luminous Vibrio harveyi cells to apparent homogeneity. This protein had a dimeric structure with a molecular weight of 23,000 per subunit. Among all eight nucleotides tested, only cAMP (Kd = 3 to 4 microM at 0 degrees C and 52 microM at 23 degrees C) and cGMP (Kd = 6 to 10 microM at 0 degrees C and 67 microM at 23 degrees C) bound to this protein. Its binding to poly(dI-dC), poly(dA-dT), and DNA fragments isolated from V. harveyi cells were all significantly enhanced by the addition of cAMP. Based on patterns of limited proteolysis by trypsin, this CRP assumes different conformations in the absence and presence of cAMP. Also consistent with this conclusion is the finding that the binding of cAMP to CRP induced about 50% quenching of the CRP fluorescence with a concomitant 3-nm blue shift from the original 336-nm emission peak. The binding of cGMP resulted in similar fluorescence changes but had no apparent effect on the pattern of proteolysis by trypsin. Using an in vitro transcription system known to be dependent on cAMP and Escherichia coli CRP, the synthesis of a run-off transcript product was also significantly enhanced by cAMP and this V. harveyi CRP.

Resonance energy transfer between cysteine-34, tryptophan-214, and tyrosine-411 of human serum albumin.

Reaction of p-nitrophenyl anthranilate with human serum albumin at pH 8.0 results in esterification of a single anthraniloyl moiety with the hydroxyl group of tyrosine-411. The absorption spectrum of the anthraniloyl group overlaps the fluorescence emission of the single tryptophan residue at position 214. This study complements that of the preceding paper [Suzukida, M., Le, H. P., Shahid, F., McPherson, R. A., Birnbaum, E.R., & Darnall, D. W. (1983) Biochemistry (preceding paper in this issue)] where an azomercurial group was introduced at cysteine-34. Anthraniloyl fluorescence was also quenched by the azomercurial absorption at Cys-34. Thus measurement of resonance energy transfer between these three sites allowed distances to be measured between Cys-34 in domain I, Trp-214 in domain II, and Tyr-411 in domain III of human serum albumin. At pH 7.4 in 0.1 M phosphate the Trp-214 leads to Tyr-411, Tyr-411 leads to Cys-34, and Trp-214 leads to Cys-34 distances were found to be 25.2 +/- 0.6, 25.2 +/- 2.1, and 31.8 +/- 0.8 A, respectively.