Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice

Genetically modified CD8⁺ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer.     

Role of Chimeric Antigen Receptor T Cell Therapy in Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common primary malignant cancer of brain, which is extremely aggressive and carries a dreadful prognosis. Current treatment protocol runs around radiotherapy, surgical resection, and temozolomide with median overall survival of around 12–15 months. Due to its heterogeneity and mutational load, immunotherapy with chimeric antigen receptor (CAR) T cell therapy can be a promising treatment option for recurrent glioblastoma. Initial phase 1 studies have shown that this therapy is safe without dose-limiting side effects and it also has a better clinical outcome. Therefore, CAR T cell therapy can be a great future tool in our armamentarium to treat advanced GBM. In this article, we have explained the structure, mechanism of action, and rationale of CAR T cell therapy in GBM; we also discussed various antigenic targets and clinical outcome of initial studies of this novel therapy.     

Chimeric Virus as a Source of the Potato Leafroll Virus Antigen

Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.     

A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.     

K-ras as a target for cancer therapy

The central role K-, H- and N-Ras play in regulating diverse cellular pathways important for cell growth, differentiation and survival is well established. Dysregulation of Ras proteins by activating mutations, overexpression or upstream activation is common in human tumors. Of the Ras proteins, K-ras is the most frequently mutated and is therefore an attractive target for cancer therapy. The complexity of K-ras signaling presents many opportunities for therapeutic targeting. A number of different approaches aimed at abrogating K-ras activity have been explored in clinical trials. Several of the therapeutic agents tested have demonstrated clinical activity, supporting ongoing development of K-ras targeted therapies. However, many of the agents currently being evaluated have multiple targets and their antitumor effects may not be due to K-Ras inhibition. To date, no selective, specific inhibitor of K-ras is available for routine clinical use. In this review, we will summarize the structure and function of K-ras with attention to its role in tumorigenesis and discuss the successes and failures of the various strategies designed to therapeutically target this important oncogene.     

一种新的人源化抗CD19嵌合抗原受体NK细胞的制备和对体外肿瘤细胞杀伤作用

摘要 目的：构建人源化抗CD19嵌合抗原受体NK细胞（hCAR19-NK）,并且在体外证明其对CD19 阳性血液病肿瘤细胞杀伤作用。方法：构建人源化的第二代CD19 CAR的逆转录病毒载体,使用辐照的K562-4-1BBL-mIL21细胞刺激外周血来源的NK细胞,通过逆转录病毒转导NK细胞获得hCAR19-NK细胞;采用流式细胞术和Western blot检测转导效率;采用4 h荧光杀伤实验和ELISA法检测hCAR19-NK细胞对淋巴瘤细胞的杀伤能力和IFN-γ释放水平;采用CD107a脱颗粒实验评估淋巴瘤细胞对hCAR19-NK细胞的特异性激活;比较对照组（Mock）和hCAR19-NK组细胞扩增倍数。结果：流式细胞术和Western blot 证明构建的CAR可以成功转导外周血来源的NK细胞;4 h荧光杀伤实验证明随着效靶比例升高,hCAR19-NK对Raji-GL杀伤率增加,明显高于Mock组;ELISA法检测显示Raji和K562-CD19作为靶细胞时,hCAR19-NK细胞的IFN-γ释放明显高于Mock组（P<0.01）;CD19⁺细胞（Raji和K562-CD19）可以特异性刺激hCAR19-NK细胞表达CD107a,具有统计学意义（P<0.05）;Mock组和hCAR19-NK组细胞扩增倍数无显著差异。结论：成功构建了可以杀伤CD19⁺ 肿瘤的人源化scFv的第二代hCAR19-NK细胞。     

Exocytotic fusion pores as a target for therapy

Regulated exocytosis can be split into a sequence of steps ending with the formation and the dilation of a fusion pore, a neck-like connection between the vesicle and the plasma membrane. Each of these steps is precisely controlled to achieve the optimal spatial and temporal profile of the release of signalling molecules. At the level of the fusion pore, tuning of the exocytosis can be achieved by preventing its formation, by stabilizing the unproductive narrow fusion pore, by altering the speed of fusion pore expansion and by completely closing the fusion pore. The molecular structure and dynamics of fusion pores have become a major focus of cell research, especially as a promising target for therapeutic strategies. Electrophysiological, optical and electrochemical methods have been used extensively to illuminate how cells regulate secretion at the level of a single fusion pore. Here, we describe recent advances in the structure and mechanisms of the initial fusion pore formation and the progress in therapeutic strategies with the focus on exocytosis.     

Pendrin as a novel target for diuretic therapy

The Cl(-)/HCO(3)(-) exchanger pendrin (SLC26A4, PDS) and the thiazide-sensitive NaCl cotransporter NCC (SLC12A3) are expressed on the apical membranes of distal nephron segments and mediate salt absorption, with pendrin working in tandem with the epithelial Na channel (ENaC) and NCC working by itself. Pendrin is expressed on the apical membrane of intercalated cells in late distal convoluted tubule (DCT), connecting tubule (CNT) and the cortical collecting duct (CCD) whereas the thiazide-sensitive NaCl cotransporter NCC is primarily detected on the apical membrane of DCT cells. Recent studies indicate that pendrin expression is increased in kidneys of NCC knockout mice, raising the possibility that pendrin and NCC can compensate for loss of the other by increasing their expression and activity. Current investigations in our laboratories demonstrate that pendrin plays an important role in compensatory salt absorption in response to the loop diuretics and the thiazide derivatives. These studies further demonstrate that whereas single deletion of pendrin or NCC does not cause salt wasting in mutant mice under baseline conditions, double knockout of pendrin and NCC causes profound polyuria and polydipsia, along with salt wasting under basal conditions. As a result, animals develop significant dehydration. We propose that pharmacologic inhibition of pendrin and NCC can provide a novel and strong diuretic regimen for patients with fluid overload, including those with congestive heart failure, nephrotic syndrome or renal failure.     

Multicellular organization in bacteria as a target for drug therapy

Antibiotic treatments are now reaching the limit of their efficiency, especially in hospitals where certain bacteria are resistant to all available drugs. The development of new drugs against which resistance would be slower to evolve is an important challenge. Recent advances have shown that a potential strategy is to target global properties of infections instead of harming each individual bacterium. Consider an analogy with multicellular organisms. In order to kill an animal two strategies are possible. One can kill each of its cells individually. This is what antibiotics do to get rid of bacterial infections. An alternate way, for instance, is to disorganize the hormonal system of animal's body, leading eventually to its death. This second strategy could also be employed against infections, in place of antibiotics. Bacteria are indeed often involved into coordinated activities within a group, and certain drugs are able to disorganize these activities by blocking bacterial communication. In other words, these drugs are able to target infections as a whole, rather than individuals within infections. The present paper aims at analysing the consequence of this peculiarity on the evolution of bacterial resistance. We use a mathematical model, based on branching process, to calculate the fixation probability of a mutant resistant to this type of drug, and finally to predict the speed of resistance evolution. We show that this evolution is several orders of magnitude slower than in the case of antibiotic resistance. The explanation is as follows. By targeting treatments against adaptive properties of groups instead of individuals, we shift one level up the relevant unit of organization generating resistance. Instead of facing billions of bacteria with a very rapid evolutionary rate, these alternate treatments face a reduced number of larger organisms with lower evolutionary potential. In conclusion, this result leads us to emphasize the strong potential of anti‐bacterial treatments aiming at disorganizing social traits of microbes rather than at killing every individual.     

Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will high-light the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.     

TWEAK as a target for therapy in systemic lupus erythematosus

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a recently identified proinflammatory cytokine of the TNF superfamily. Through activation of the fibroblast growth factor-inducible 14 (Fn14) receptor, TWEAK regulates cell proliferation, cell death and inflammation. The available evidences have indicated that TWEAK might be a target for therapeutic intervention in renal, vascular injury and neuropathy. Since renal, vascular and neuropsychiatric complications are frequently encountered in systemic lupus erythematosus (SLE)—a systemic autoimmune disease, TWEAK-Fn14 pathway may be implicated in the pathogenesis of SLE. In this review, we will discuss the TWEAK-Fn14 pathway and the therapeutic potential of modulating this pathway in SLE.     

Midkine as a novel target for antibody therapy in osteosarcoma

Osteosarcoma is a malignant tumor with poor prognosis, and lack of accurate prognostic factors is one of the reasons that make this tumor difficult to cure. The heparin-binding growth factor, midkine is involved in generation and progression of many types of tumors. However, the relationship between midkine and osteosarcoma has been unclear. We show here that midkine is overexpressed in osteosarcoma and the level of midkine expression is correlated with prognosis (P<0.05; log-rank test). Treatment with functional antibodies against midkine suppresses growth of osteosarcoma cell lines, 9N2, 3N1, Saos-2, and NOS-1, to 25-65% of untreated controls. Our results suggest that midkine is useful as a prognostic marker, and is a candidate therapeutic target for osetosarcomas.     

Wee1 kinase as a target for cancer therapy

Wee1, a protein kinase, regulates the G₂ checkpoint in response to DNA damage. Preclinical studies have elucidated the role of wee1 in DNA damage repair and the stabilization of replication forks, supporting the validity of wee1 inhibition as a viable therapeutic target in cancer. MK-1775, a selective and potent small-molecule inhibitor of wee1, is under clinical development as a potentiator of DNA damage caused by cytotoxic chemotherapies. We present a review of the role of wee1 in the cell cycle and DNA replication and summarize the clinical development to date of this novel class of anticancer agents.     

Integrins as a potential target for targeted anticancer therapy

The review briefly summarizes information of structure of integrins and their involvement in the development and malignant progression of tumors. Special attention is paid to approaches based on modification of functional properties of integrins that prevent/antagonize tumor growth and progression; these approaches developed in modern experimental biology have certain perspective in clinical application.     

靶向人源BCMA的嵌合抗原受体T细胞构建及体外抗肿瘤活性研究

摘要 目的：通过对CAR结构的ScFv单链可变区进行改造,构建并筛选具有更强杀伤肿瘤细胞功能的新型靶向人源B细胞成熟抗原（BCMA）的嵌合抗原受体 (CAR)-T细胞。方法：构建靶向人源BCMA的CAR分子,用逆转录病毒载体包装成功后转导健康志愿者的T细胞,制备Anti-BCMA-CAR-T细胞。将Anti-BCMA-CAR-T细胞作为观察组,普通T细胞作为对照组,将其与RPMI-8226细胞共培养,采用CFSE染色的T细胞增殖实验观察两组体外增殖能力。采用荧光素酶化学发光实验检测两组细胞在不同效靶比（1:8、1:4、1:2、1:1、2:1、4:1）对RPMI-8226细胞的杀伤效率,采用流式细胞术检测两组细胞在不同效靶比（1:4、1:2、1:1、2:1、4:1）对RPMI-8226细胞的杀伤效率。结果：CFSE检测结果显示,与对照组比较,观察组FITC信号明显左移,表明T细胞增殖能力越强。流式细胞术检测结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组（P均<0.05）;荧光素酶化学发光实验结果显示,相同效靶比时,观察组对RPMI-8226细胞的杀伤效率均高于对照组（P均<0.05）。在效靶比为4:1时,CAR170-T（未经改造的传统的ScFv）细胞和CAR174-T（经改造的ScFv）细胞的杀伤效率分别达到了88.5±0.3 %和98.5±0.7 %。结论：通过对CAR结构的ScFv单链可变区进行改造后成功构建出的新型靶向BCMA的CAR-T细胞,它能保持较强的增殖活性且具有更强的杀伤肿瘤细胞的能力。     

Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule

Under certain conditions, India ink particles can penetrate the capsule of the opportunistic pathogen Cryptococcus neoformans. India ink penetration gave two distinct patterns, one as a ring in the middle of the capsule, and another as a double spot located at opposite poles of the cells. These spots were perpendicularly orientated to the bud. This pattern suggests the existence of a localized structure deep in the capsule that can accumulate large insoluble particles. Although the mechanisms responsible for the assemblage and maintenance of ring-like channels are not understood, their existence deep within the capsule implies a new level of complexity for this enigmatic structure.