Bone Marrow–generated Dendritic Cells Pulsed with Tumor Extracts or Tumor RNA Induce Antitumor Immunity against Central Nervous System Tumors

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene–modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow–generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors

Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78–83%), high retention of cell viability (71–74%), high tumour cell enrichment against leukocytes (1.7–2 × 10^3), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4–0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research

Practical Fully Secure Three-Party Computation via Sublinear Distributed Zero-Knowledge Proofs

Secure multiparty computation enables a set of parties to securely carry out a joint computation on their private inputs without revealing anything but the output. A particularly motivated setting is that of three parties with a single corruption (hereafter denoted 3PC). This 3PC setting is particularly appealing for two main reasons: (1) it admits more efficient MPC protocols than in other standard settings; (2) it allows in principle to achieve full security (and fairness). Highly efficient protocols exist within this setting with security against a semi-honest adversary; however, a significant gap remains between these and protocols with stronger security against a malicious adversary. In this paper, we narrow this gap within concretely efficient protocols. More explicitly, we have the following contributions: * Concretely Efficient Malicious 3PC. We present an optimized 3PC protocol for arithmetic circuits over rings with (amortized) communication of 1 ring element per multiplication gate per party, matching the best semi-honest protocols. The protocol applies also to Boolean circuits, significantly improving over previous protocols even for small circuits. Our protocol builds on recent techniques of Boneh et al.\ (Crypto 2019) for sublinear zero-knowledge proofs on distributed data, together with an efficient semi-honest protocol based on replicated secret sharing (Araki et al., CCS 2016). We present a concrete analysis of communication and computation costs, including several optimizations. For example, for 40-bit statistical security, and Boolean circuit with a million (nonlinear) gates, the overhead on top of the semi-honest protocol can involve less than 0.5KB of communication {\em for the entire circuit}, while the computational overhead is dominated by roughly 30 multiplications per gate in the field $F_{2^{47}}$. In addition, we implemented and benchmarked the protocol for varied circuit sizes. * Full Security. We augment the 3PC protocol to further provide full security (with guaranteed output delivery) while maintaining amortized 1 ring element communication per party per multiplication gate, and with hardly any impact on concrete efficiency. This is contrasted with the best previous 3PC protocols from the literature, which allow a corrupt party to mount a denial-of-service attack without being detected

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches

CHAPTER 9 - Use of Retrovirus-Derived Vectors to Introduce and Express Genes in Mammalian Cells

This chapter discusses the usage of retrovirus-derived vectors to introduce and express genes in mammalian cells. There has been a surge in the development of experimental systems to introduce and express genes in mammalian cells. The main feature of such transduction systems is a vector, a defined DNA fragment carrying various properties affecting the propagation and/or expression of the transduced gene in the recipient cells. The first and most widely used mammalian transduction system was derived from the genome of SV40, a DNA virus of monkey origin. The chapter discusses a mammalian transduction system derived from the genome of a murine retrovirus, Moloney murine leukemia virus (M-MuLV). This experimental system is designed to introduce genes into the chromosome of mammalian cells at the rate of one or a few copies per genome and to permit expression of the transduced genetic information in a stable fashion, enabling the isolation and indefinite propagation of cells carrying the newly acquired gene

Induction of Tumor Immunity by Targeted Inhibition of Nonsense-Mediated mRNA Decay

Whereas tumor progression in cancer patients can elicit a weak immune response which keeps the tumor in check, albeit transiently, the weak antigenicity of the tumor provides the time and opportunity for the tumor to elaborate immune evasion mechanisms. Weak antigenicity is, therefore, the root cause why tumors escape immune control. We have recently described a way to activate the antitumor immune response that is fundamentally different from current strategies collectively referred to as “cancer vaccination,” because instead of stimulating immune responses against existing and mostly weak, tumor antigens, novel, and thereby potent, antigens are induced de novo in the disseminated tumor lesions of the patient. The approach is to inhibit a process in the tumor cells known as nonsense-mediated mRNA decay (NMD). The physiological role of NMD is to eliminate defective products generated in the cells, and therefore inhibiting the NMD process will lead to the accumulation of defective products, some of which will encode novel, and thereby potent, antigens to which the immune system has not been tolerized. Inhibition of NMD was accomplished using chemically synthesized siRNAs to downregulate key mediators of the NMD process such as Smg-1 or Upf-2. However, since NMD is a constitutive process that operates in all the somatic cells of the body, global inhibition of NMD could lead to system-wide autoimmune pathology. To obviate the risk of autoimmunity, NMD inhibition was limited to the disseminated tumor lesions by targeted delivery of siRNAs conjugated to oligonucleotide aptamer ligands that bind to receptors expressed preferentially, if not exclusively, on the tumor cells. We have shown that in subcutaneous and metastatic murine tumor models, the tumor-targeted delivery of NMD factor siRNAs led to significant inhibition of tumor growth which was superior to that of a gold standard “conventional” cancer vaccination protocol. Tumor-targeted NMD inhibition forms the basis of a simple, broadly useful, and clinically feasible approach to enhance the antigenicity of disseminated tumors leading to their immune recognition and rejection. The cell-free chemically synthesized oligonucleotide backbone of aptamer–siRNAs reduces the risk of immunogenicity and enhances the feasibility of generating reagents suitable for clinical use

Expression of new antigens on tumor cells by inhibiting nonsense-mediated mRNA decay

The main reason why tumors are not controlled by the immune system of the cancer patient is that tumors do not express potent tumor antigens that can be recognized by the immune system as "foreign." The current focus in developing immune-based modalities is to potentiate an immune response against the existing, albeit weak, antigens expressed in the tumor. An alternative approach is to express new, and hence potent, antigens in tumor cells in situ. To this end, we have developed an approach to generate new antigenic determinants in tumor cells using siRNA technology to inhibit nonsense-mediated mRNA decay (NMD), a surveillance mechanism which prevents the expression of mRNAs containing a premature termination codon. Targeting siRNA inhibition to tumor cells--an essential requisite because of the constitutive nature and physiological roles of the NMD process--is accomplished by using a novel targeting technology based on using oligonucleotide aptamer ligands. Aptamers or aptamer-targeted siRNA conjugates, unlike antibodies, can be synthesized in a chemical process providing a more straightforward and cost-effective manufacturing and regulatory approval process to generate clinical-grade reagents. In murine tumor models, the aptamer-targeted siRNA-mediated NMD inhibition in tumor cells led to significant inhibition of tumor growth, which was superior to best-in-class "conventional" cancer vaccination protocols. Tumor-targeted NMD inhibition forms the basis of a simple, broadly useful, and clinically feasible approach to enhance the antigenicity of disseminated tumors leading to their immune recognition and rejection. The cell-free chemically synthesized oligonucleotide backbone of aptamer-siRNAs reduces the risk of immunogenicity and enhances the feasibility of generating reagents suitable for clinical use

Retrovirus vectors and their uses in molecular biology

Retroviral vectors utilize the biochemical processes unique to retroviruses, to transfer genes with high efficiency into a wide variety of cell types in tissue culture and in living animals. With such vectors, the effect of newly introduced genes and the mechanism of gene expression can be studied in cell types so far refractory to other methods of transfer