Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy.

The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery

Chemotherapy induces canalization of cell state in childhood B-cell precursor acute lymphoblastic leukemia

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We therefore investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population, with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of the cell state accounts for a significant component of bottleneck selection during induction chemotherapy

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471

Issue Highlights - January 2013

To date, the diagnosis of acute myeloid leukemia (AML) with monocytic differentiation was limited by the lack of highly sensitive and specific monocytic markers. Over the last 2 decades, several immunophenotypic markers (lysozyme, elastase, UPA-R, GM-CSF-R, among others) were found to be preferentially expressed by cells belonging to the monocytic lineage, but unfortunately none of them allowed the easy recognition of the pure M5 FAB subvariety of AML.ILT3 is an immune inhibitory receptor expressed by myelomono- cytic cells and at high levels by tolerogenic dendritic cells, making it feasible to be incorporated into the initial diagnostic work-up and monitoring of patients with AML. However, the diagnostic usefulness of this pheno- typic marker for the recognition of the monocytic variant of AML needs to be validated in larger series of patients

Automated Software Transplantation

Automated program repair has excited researchers for more than a decade, yet it has yet to find full scale deployment in industry. We report our experience with SAPFIX: the first deployment of automated end-to-end fault fixing, from test case design through to deployed repairs in production code. We have used SAPFIX at Facebook to repair 6 production systems, each consisting of tens of millions of lines of code, and which are collectively used by hundreds of millions of people worldwide. In its first three months of operation, SAPFIX produced 55 repair candidates for 57 crashes reported to SAPFIX, of which 27 have been deem as correct by developers and 14 have been landed into production automatically by SAPFIX. SAPFIX has thus demonstrated the potential of the search-based repair research agenda by deploying, to hundreds of millions of users worldwide, software systems that have been automatically tested and repaired. Automated software transplantation (autotransplantation) is a form of automated software engineering, where we use search based software engineering to be able to automatically move a functionality of interest from a ‘donor‘ program that implements it into a ‘host‘ program that lacks it. Autotransplantation is a kind of automated program repair where we repair the ‘host‘ program by augmenting it with the missing functionality. Automated software transplantation would open many exciting avenues for software development: suppose we could autotransplant code from one system into another, entirely unrelated, system, potentially written in a different programming language. Being able to do so might greatly enhance the software engineering practice, while reducing the costs. Automated software transplantation manifests in two different flavors: monolingual, when the languages of the host and donor programs is the same, or multilingual when the languages differ. This thesis introduces a theory of automated software transplantation, and two algorithms implemented in two tools that achieve this: µSCALPEL for monolingual software transplantation and τSCALPEL for multilingual software transplantation. Leveraging lightweight annotation, program analysis identifies an organ (interesting behavior to transplant); testing validates that the organ exhibits the desired behavior during its extraction and after its implantation into a host. We report encouraging results: in 14 of 17 monolingual transplantation experiments involving 6 donors and 4 hosts, popular real-world systems, we successfully autotransplanted 6 new functionalities; and in 10 out of 10 multlingual transplantation experiments involving 10 donors and 10 hosts, popular real-world systems written in 4 different programming languages, we successfully autotransplanted 10 new functionalities. That is, we have passed all the test suites that validates the new functionalities behaviour and the fact that the initial program behaviour is preserved. Additionally, we have manually checked the behaviour exercised by the organ. Autotransplantation is also very useful: in just 26 hours computation time we successfully autotransplanted the H.264 video encoding functionality from the x264 system to the VLC media player, a task that is currently done manually by the developers of VLC, since 12 years ago. We autotransplanted call graph generation and indentation for C programs into Kate, (a popular KDE based test editor used as an IDE by a lot of C developers) two features currently missing from Kate, but requested by the users of Kate. Autotransplantation is also efficient: the total runtime across 15 monolingual transplants is 5 hours and a half; the total runtime across 10 multilingual transplants is 33 hours

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells

Clonogenic neural stem cells (NSCs) are self-renewing cells that maintain the capacity to differentiate into brain-specific cell types, and may also replace or repair diseased brain tissue. NSCs can be directly isolated from fetal or adult nervous tissue, or derived from embryonic stem cells. Here, we describe the efficient conversion of human adult bone marrow stromal cells (hMSC) into a neural stem cell-like population (hmNSC, for human marrow-derived NSC-like cells). These cells grow in neurosphere-like structures, express high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, MSl1 as well as otx1 and nestin, but lose the characteristics of mesodermal stromal cells. In the presence of selected growth factors, hmNSCs can be differentiated into the three main neural phenotypes: astroglia, oligodendroglia and neurons. Clonal analysis demonstrates that individual hmNSCs are multipotent and retain the capacity to generate both glia and neurons. Our cell culture system provides a powerful tool for investigating the molecular mechanisms of neural differentiation in adult human NSCs. hmNSCs may therefore ultimately help to treat acute and chronic neurodegenerative diseases