Cooperative Armoring of CAR and TCR T Cells by T Cell-Restricted IL15 and IL21 Universally Enhances Solid Tumor Efficacy.

PURPOSE: Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells. EXPERIMENTAL DESIGN: We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration. RESULTS: We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both. CONCLUSIONS: The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431.

Epigenetic reprogramming sensitizes immunologically silent EBV+ lymphomas to virus-directed immunotherapy.

Despite advances in T-cell immunotherapy against Epstein-Barr virus (EBV)-infected lymphomas that express the full EBV latency III program, a critical barrier has been that most EBV+ lymphomas express the latency I program, in which the single Epstein-Barr nuclear antigen (EBNA1) is produced. EBNA1 is poorly immunogenic, enabling tumors to evade immune responses. Using a high-throughput screen, we identified decitabine as a potent inducer of immunogenic EBV antigens, including LMP1, EBNA2, and EBNA3C. Induction occurs at low doses and persists after removal of decitabine. Decitabine treatment of latency I EBV+ Burkitt lymphoma (BL) sensitized cells to lysis by EBV-specific cytotoxic T cells (EBV-CTLs). In latency I BL xenografts, decitabine followed by EBV-CTLs results in T-cell homing to tumors and inhibition of tumor growth. Collectively, these results identify key epigenetic factors required for latency restriction and highlight a novel therapeutic approach to sensitize EBV+ lymphomas to immunotherapy.

Deregulation of the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) gene in a B-cell chronic lymphocytic leukemia with a t(12;14)(q23;q32).

The t(12;14)(q23;q32) breakpoints in a case of B-cell chronic lymphocytic leukemia (B-CLL) were mapped by fluorescence in situ hybridization (FISH) and Southern blot analysis and cloned using an IGH switch-gamma probe. The translocation affected a productively rearranged IGH allele and the carbohydrate (chondroitin 4) sulfotransferase 11 (CHST11) locus at 12q23, with a reciprocal break in intron 2 of the CHST11 gene. CHST11 belongs to the HNK1 family of Golgi-associated sulfotransferases, a group of glycosaminoglycan-modifying enzymes, and is expressed mainly in the hematopoietic lineage. Northern Blot analysis of tumor RNA using CHST11-specific probes showed expression of two CHST11 forms of abnormal size. 5'- and 3'-Rapid Amplification of cDNA Ends (RACE) revealed IGH/CHST11 as well as CHST11/IGH fusion RNAs expressed from the der(14) and der(12) chromosomes. Both fusion species contained open reading frames making possible the translation of two truncated forms of CHST11 protein. The biological consequence of t(12;14)(q23;q32) in this case presumably is a disturbance of the cellular distribution of CHST11 leading to deregulation of a chondroitin-sulfate-dependent pathway specific to the hematopoietic lineage.

MUC-1 mucin protein expression in B-cell lymphomas.

We have recently shown that MUC1, mapped to the chromosomal band 1q21, is rearranged or amplified in 15% of B-cell lymphomas and that rearrangement led to over-expression of MUC-1 mucin in a case of diffuse large B-cell lymphoma (DLBCL). To determine the incidence of MUC-1 mucin expression and its clinical significance in B-cell lymphomas, we investigated a panel of 113 cases by immunohistochemistry (IHC). MUC-1 mucin expression was detected in the majority of cases (92.9%), with moderate to high levels noted in 50.4% of all histologic subsets comprising DLBCL (82 cases), follicular lymphoma (FL) (15 cases), FL with transformation to DLBCL (4 cases), and other B-cell lymphomas (12 cases). No statistically significant correlation was found between MUC-1 mucin expression and MUC1 genomic status (amplification/rearrangement) evaluated by Southern blot analysis, and 1q21 abnormality by karyotypic analysis. For all cases, MUC-1 mucin expression correlated with a previous history of lymphoma (p=0.003).

Molecular cytogenetic analysis of genomic instability at the 1q12-22 chromosomal site in B-cell non-Hodgkin lymphoma.

Abnormalities of chromosome arm 1q have frequently been reported in B-cell non-Hodgkin lymphoma (NHL), and correlated with poor outcome. Five genes mapped to this region (BCL9, MUC1, FCGR2B, IRTA1, and RTA2) have been shown to be deregulated by juxtaposition with the IG genes. However, abnormalities of the 1q21-22 region that are not involved in translocations with the IG genes have not been addressed. We performed a molecular cytogenetic analysis of 1q12-22 abnormalities in 24 B-cell NHL cases. The cases analyzed were in two groups: one, composed of 18 cases with the single break in the 1q12-22 region, and another, composed of six cases with multiple breaks in the 1q12-22 region. The involvement of heterochromatin and its vicinity was observed most frequently in the single-break cases (13 of 18 cases). In this group, the recurring partner region was 1q32, which resulted in dup(1)(q12-21q32) or trp(1)(q12q32) in 5 cases. The 6 cases with multiple breaks showed an unexpected level of instability along with complex combinations of abnormalities, especially sequential duplication and inversion, in the 1q12-22 region. The BCL9 locus was deleted by complex aberration in 2 of 6 cases. High-level amplification of the WI-16757 locus was found in 2 cases. Our studies demonstrate a high level of instability of the 1q12-22 region, possibly stemming from its chromatin organization. Chromosome arm 1q is gene-rich, and characterization of aberrations described in this study can be expected to lead to the discovery of additional functionally significant genetic changes.

BCL8 is a novel, evolutionarily conserved human gene family encoding proteins with presumptive protein kinase A anchoring function.

BCL8 is a novel human gene family initially identified through cloning of BCL8A, located at the t(14;15)(q32;q11-q13) translocation breakpoint, in a case of diffuse large B-cell lymphoma. Multiple copies of BCL8A map to the 1-Mb proximal duplicated region at 15q. We identified additional copies on human chromosomes 13 (BCL8B), 22 (BCL8C), 2 (BCL8D), and 10 (BCL8E) by cDNA cloning and sequence analysis. BCL8A, BCL8C, BCL8D, and BCL8E are truncated at the genomic level and are presumably pseudogenes or sterile transcripts. BCL8B is expressed predominantly in human brain and encodes a 327-kDa protein with extensive homology to the Drosophila melanogaster protein kinase A anchoring protein RG. LRBA, a human gene with a ubiquitous expression pattern mapping to 4q32, encodes a protein closely related to BCL8. The phylogenetically conserved BCL8 gene family evolved by transchromosomal and intrachromosomal duplications within the human genome.