A rational mouse model to detect on-target, off-tumor CAR T cell toxicity.

Off-tumor targeting of human antigens is difficult to predict in preclinical animal studies and can lead to serious adverse effects in patients. To address this, we developed a mouse model with stable and tunable human Her2 (hHer2) expression on normal hepatic tissue and compared toxicity between affinity-tuned Her2 chimeric antigen receptor T cells (CARTs). In mice with hHer2-high livers, both the high-affinity (HA) and low-affinity (LA) CARTs caused lethal liver damage due to immunotoxicity. In mice with hHer2-low livers, LA-CARTs exhibited less liver damage and lower systemic levels of IFN-γ than HA-CARTs. We then compared affinity-tuned CARTs for their ability to control a hHer2-positive tumor xenograft in our model. Surprisingly, the LA-CARTs outperformed the HA-CARTs with superior antitumor efficacy in vivo. We hypothesized that this was due, in part, to T cell trafficking differences between LA and HA-CARTs and found that the LA-CARTs migrated out of the liver and infiltrated the tumor sooner than the HA-CARTs. These findings highlight the importance of T cell targeting in reducing toxicity of normal tissue and also in preventing off-tumor sequestration of CARTs, which reduces their therapeutic potency. Our model may be useful to evaluate various CARTs that have conditional expression of more than 1 single-chain variable fragment (scFv).

Driving cars to the clinic for solid tumors.

FDA approval of chimeric antigen receptor T cells (CART cells) is the culmination of several decades of technology development and interrogation of the properties of these gene therapies. CART cells exist as personalized "living drugs" and have demonstrated astounding anti-tumor efficacy in patients with leukemia and lymphoma. However, the future promise of CART efficacy for solid tumors, the greatest unmet burden, is met with a number of challenges that must be surmounted for effective immune responses. In this review, we discuss the next-generation developments of CARs to target solid tumors, including fine-tuned and combinational-targeting receptors. We consider the structural intricacies of the CAR molecules that influence optimal signaling and CART survival, and review pre-clinical cell-intrinsic and cell-extrinsic combinational therapy approaches.

Surveillance of the tumor mutanome by T cells during progression from primary to recurrent ovarian cancer.

PURPOSE: Cancers accumulate mutations over time, each of which brings the potential for recognition by the immune system. We evaluated T-cell recognition of the tumor mutanome in patients with ovarian cancer undergoing standard treatment. EXPERIMENTAL DESIGN: Tumor-associated T cells from 3 patients with ovarian cancer were assessed by ELISPOT for recognition of nonsynonymous mutations identified by whole exome sequencing of autologous tumor. The relative levels of mutations and responding T cells were monitored in serial tumor samples collected at primary surgery and first and second recurrence. RESULTS: The vast majority of mutations (78/79) were not recognized by tumor-associated T cells; however, a highly specific CD8(+) T-cell response to the mutation hydroxysteroid dehydrogenase-like protein 1 (HSDL1)(L25V) was detected in one patient. In the primary tumor, the HSDL1(L25V) mutation had low prevalence and expression, and a corresponding T-cell response was undetectable. At first recurrence, there was a striking increase in the abundance of the mutation and corresponding MHC class I epitope, and this was accompanied by the emergence of the HSDL1(L25V)-specific CD8(+) T-cell response. At second recurrence, the HSDL1(L25V) mutation and epitope continued to be expressed; however, the corresponding T-cell response was no longer detectable. CONCLUSION: The immune system can respond to the evolving ovarian cancer genome. However, the T-cell response detected here was rare, was transient, and ultimately failed to prevent disease progression. These findings reveal the limitations of spontaneous tumor immunity in the setting of standard treatments and suggest a high degree of ignorance of tumor mutations that could potentially be reversed by immunotherapy.

Sequence analysis of T-cell repertoires in health and disease.

T-cell antigen receptor (TCR) variability enables the cellular immune system to discriminate between self and non-self. High-throughput TCR sequencing (TCR-seq) involves the use of next generation sequencing platforms to generate large numbers of short DNA sequences covering key regions of the TCR coding sequence, which enables quantification of T-cell diversity at unprecedented resolution. TCR-seq studies have provided new insights into the healthy human T-cell repertoire, such as revised estimates of repertoire size and the understanding that TCR specificities are shared among individuals more frequently than previously anticipated. In the context of disease, TCR-seq has been instrumental in characterizing the recovery of the immune repertoire after hematopoietic stem cell transplantation, and the method has been used to develop biomarkers and diagnostics for various infectious and neoplastic diseases. However, T-cell repertoire sequencing is still in its infancy. It is expected that maturation of the field will involve the introduction of improved, standardized tools for data handling, deposition and statistical analysis, as well as the emergence of new and equivalently large-scale technologies for T-cell functional analysis and antigen discovery. In this review, we introduce this nascent field and TCR-seq methodology, we discuss recent insights into healthy and diseased TCR repertoires, and we examine the applications and challenges for TCR-seq in the clinic.

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice.

BACKGROUND: The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome') strategy to expand our understanding of human gene regulation in vivo. RESULTS: In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. CONCLUSIONS: We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

Clonal evolution of high-grade serous ovarian carcinoma from primary to recurrent disease.

High-grade serous carcinoma (HGSC) is the most common and fatal form of ovarian cancer. While most tumours are highly sensitive to cytoreductive surgery and platinum- and taxane-based chemotherapy, the majority of patients experience recurrence of treatment-resistant tumours. The clonal origin and mutational adaptations associated with recurrent disease are poorly understood. We performed whole exome sequencing on tumour cells harvested from ascites at three time points (primary, first recurrence, and second recurrence) for three HGSC patients receiving standard treatment. Somatic point mutations and small insertions and deletions were identified by comparison to constitutional DNA. The clonal structure and evolution of tumours were inferred from patterns of mutant allele frequencies. TP53 mutations were predominant in all patients at all time points, consistent with the known founder role of this gene. Tumours from all three patients also harboured mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control, and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumours were present in primary tumours. Our findings highlight both known and novel pathways that are commonly mutated in HGSC. Moreover, they provide the first evidence at single nucleotide resolution that recurrent HGSC arises from multiple clones present in the primary tumour with negligible accumulation of new mutations during standard treatment.

Derivation of HLA types from shotgun sequence datasets.

The human leukocyte antigen (HLA) is key to many aspects of human physiology and medicine. All current sequence-based HLA typing methodologies are targeted approaches requiring the amplification of specific HLA gene segments. Whole genome, exome and transcriptome shotgun sequencing can generate prodigious data but due to the complexity of HLA loci these data have not been immediately informative regarding HLA genotype. We describe HLAminer, a computational method for identifying HLA alleles directly from shotgun sequence datasets (http://www.bcgsc.ca/platform/bioinfo/software/hlaminer). This approach circumvents the additional time and cost of generating HLA-specific data and capitalizes on the increasing accessibility and affordability of massively parallel sequencing.

Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma.

An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. We screened colorectal carcinoma and matched normal tissue specimens using RNA-seq followed by host sequence subtraction and found marked over-representation of Fusobacterium nucleatum sequences in tumors relative to control specimens. F. nucleatum is an invasive anaerobe that has been linked previously to periodontitis and appendicitis, but not to cancer. Fusobacteria are rare constituents of the fecal microbiota, but have been cultured previously from biopsies of inflamed gut mucosa. We obtained a Fusobacterium isolate from a frozen tumor specimen; this showed highest sequence similarity to a known gut mucosa isolate and was confirmed to be invasive. We verified overabundance of Fusobacterium sequences in tumor versus matched normal control tissue by quantitative PCR analysis from a total of 99 subjects (p = 2.5 × 10(-6)), and we observed a positive association with lymph node metastasis.

A regulatory toolbox of MiniPromoters to drive selective expression in the brain.

The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination "knockins" in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5' of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type-specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

The role of islet neogenesis-associated protein (INGAP) in islet neogenesis.

Islet Neogenesis-Associated Protein (INGAP) is a member of the Reg family of proteins implicated in various settings of endogenous pancreatic regeneration. The expression of INGAP and other RegIII proteins has also been linked temporally and spatially with the induction of islet neogenesis in animal models of disease and regeneration. Furthermore, administration of a peptide fragment of INGAP (INGAP peptide) has been demonstrated to reverse chemically induced diabetes as well as improve glycemic control and survival in an animal model of type 1 diabetes. Cultured human pancreatic tissue has also been shown to be responsive to INGAP peptide, producing islet-like structures with function, architecture and gene expression matching that of freshly isolated islets. Likewise, studies in normoglycemic animals show evidence of islet neogenesis. Finally, recent clinical studies suggest an effect of INGAP peptide to improve insulin production in type 1 diabetes and glycemic control in type 2 diabetes.

The identification and sequence analysis of a new Reg3gamma and Reg2 in the Syrian golden hamster.

The regenerating (Reg) genes are associated with tissue repair and have been directly implicated in pancreatic beta-cell regeneration. A hamster Reg3, Islet neogenesis associated protein (INGAP), has been shown to possess anti-diabetic properties in rodent models. Although several Reg3 proteins have been identified in other species, INGAP is the only Reg3 found in hamsters. To identify new Reg3 genes in the hamster pancreas we employed homology reverse transcription polymerase chain reaction (RT-PCR) using degenerate Reg3 primers, followed by rapid amplification of cDNA ends (RACE). We report here the discovery of a new hamster Reg3 gene of 765 nucleotides (nt) that encodes a 174-amino acid (aa) protein. This protein sequence was identified as a novel hamster Reg3gamma with 78% and 75% identity to the rat Reg3gamma and mouse Reg3gamma protein, respectively. We also fully sequenced the previously reported partial sequence of the hamster Reg1 gene coding region using RACE to yield a 756-nt transcript that encodes a deduced 173 aa protein. This protein was identified as hamster Reg2, rather than Reg1 as was initially reported, with an 81% identity to mouse Reg2. The spatial gene expression patterns of the hamster Reg genes, analyzed by RT-PCR, were similarly distributed with low level expression being found globally throughout the body. Mice and hamsters are the only species known to carry either of the functional INGAP or Reg2 genes. It remains to be determined whether these genes bestow mice and hamsters with special regenerative abilities in the pancreas.

Acinar plasticity: development of a novel in vitro model to study human acinar-to-duct-to-islet differentiation.

OBJECTIVES: The plasticity of pancreatic tissue is demonstrated in many pancreatic diseases. It has previously been shown that pancreatic islet-to-duct transformation and acinoductal metaplasia have been associated with both pancreatic regeneration and adenocarcinoma in various in vivo and in vitro settings. Understanding this inherent morphogenetic plasticity of the adult pancreas could lead to new therapeutic approaches to pancreatic disease. METHODS: Cadaveric human pancreases (n = 7) were digested, and purified acinar tissue, which was approximately 85% immunoreactive for amylase and approximately 15% immunoreactive for CK-19, was embedded in a type 1 collagen matrix and cultured in a differentiation medium (DM) consisting of Dulbecco modified Eagle/F12 medium supplemented with cholera toxin (100 ng/mL), epidermal growth factor (10 ng/mL), and insulin (24 mU/mL) for 8 days. After this initial period, the resulting tissues were cultured in DM without cholera toxin, supplemented with gastrin (50 nmol/L) and hepatocyte growth factor (HGF; 10 ng/mL), with islet neogenesis-associated protein (INGAP; 167 nmol/L) or with gastrin + HGF + INGAP for 6 days. Tissue samples were then analyzed for amylase, cytokeratin 19, pancreas duodenum homeobox 1, and endocrine hormone immunoreactivity as well as dithizione positivity. RESULTS: After 8 days of culture, approximately 90% of acini transformed into ductlike structures. This acinoductal transformation was characterized by a complete absence of amylase staining, with virtually all cells CK-19 immunoreactive. Addition of INGAP led to an approximately 18-fold increase in pancreas duodenum homeobox 1 immunoreactivity, although without an observed increase in insulin production as measured by dithizone positivity. However, when acinar-derived ductlike structures were cultured with gastrin + HGF + INGAP, the total incidence of dithizone-positive structures increased approximately 6-fold (10.9 +/- 2.9% vs 1.7 +/- 0.4%, P = 0.037). Treatment with gastrin + HGF alone led to no significant change in any of the measured parameters. CONCLUSIONS: We have developed a novel in vitro model of adult human acinoductal metaplasia that will aid not only in developing new methods of expanding beta-cell mass but also provide insights into pancreatic carcinogenesis.

Tumor necrosis factor-alpha production by human islets leads to postisolation cell death.

BACKGROUND: Recent successes in islet transplantation highlight the importance of islet isolation by experienced centers and minimization of cell injury as crucial to the achievement of insulin independence. Islet injury may manifest as cell death by apoptosis, shorter graft survival, and the need for retransplantation. Although an inflammatory cytokine response at the graft site is known to inhibit engraftment, recent evidence indicates that islet cells may contribute to this response. METHODS: Isolated human islets were cultured for up to one week in serum-free CMRL-1066 with 25 microM of tumor necrosis factor (TNF)alpha inhibitor RDP58. Gene expression was measured by reverse transcriptase polymerase chain reaction, apoptosis and TNFalpha secretion by enzyme-linked immunosorbent assay and enzyme-linked immunospot, and islet function by stimulated insulin secretion. RESULTS: Isolation induced a twofold increase in TNFalpha expression between days one and three (P<0.05), while TNFalpha secretion peaked at day one. RDP58 reduced TNFalpha secretion by 70.6% (P<0.02), though TNFalpha gene expression was unaffected. RDP58 reduced the frequency of TNFalpha-secreting islets by 64.4% (P<0.05) and reduced apoptotic levels by 26.4% within 24 hr postisolation (P<0.05). The reduction in apoptosis was maintained throughout the week (P<0.01), while apoptosis increased in control cultures. Finally, RDP58-treated islets displayed increased insulin secretion in response to both elevated glucose (1915.0+/-396.6 vs. 825.3+/-261.1 mU/L, P<0.01) and secretagogues (2294.3+/-529.5 vs. 939.8+/-333.7 mU/L, P<0.02). CONCLUSIONS: These data demonstrate that intraislet cytokine production should be considered as a factor leading to islet cell death postisolation and postengraftment, and strategies aimed at countering islet cytokine production represent a novel target for improving islet viability and function.

Islet neogenesis: a potential therapeutic tool in type 1 diabetes.

Current therapies for type 1 diabetes, including fastidious blood glucose monitoring and multiple daily insulin injections, are not sufficient to prevent complications of the disease. Though pancreas and possibly islet transplantation can prevent the progression of complications, the scarcity of donor organs limits widespread application of these approaches. Understanding the mechanisms of beta-cell mass expansion as well as the means to exploit these pathways has enabled researchers to develop new strategies to expand and maintain islet cell mass. Potential new therapeutic avenues include ex vivo islet expansion and improved viability of islets prior to implantation, as well as the endogenous expansion of beta-cell mass within the diabetic patient. Islet neogenesis, through stem cell activation and/or transdifferentiation of mature fully differentiated cells, has been proposed as a means of beta-cell mass expansion. Finally, any successful new therapy for type 1 diabetes via beta-cell mass expansion will require prevention of beta-cell death and maintenance of long-term endocrine function.