RESUMO
Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.
RESUMO
Chimeric antigen receptor (CAR)-T therapy has demonstrated remarkable outcomes for B cell malignancies, however, its application for T cell lymphoma, particularly cutaneous T cell lymphoma (CTCL), has been limited. Barriers to effective CAR-T cell therapy in treating CTCL include T cell aplasia in autologous transplants, CAR-T product contamination with leukemic T cells, CAR-T fratricide (when the target antigen is present on normal T cells), and tumor heterogeneity. To address these critical challenges, innovative CAR engineering by targeting multiple antigens to strike a balance between efficacy and safety of the therapy is necessary. In this review, we discuss the current obstacles to CAR-T cell therapy and highlight potential targets in treating CTCL. Looking forward, we propose strategies to develop more powerful dual CARs that are advancing towards the clinic in CTCL therapy.
Assuntos
Linfoma Cutâneo de Células T , Receptores de Antígenos Quiméricos , Neoplasias Cutâneas , Humanos , Imunoterapia Adotiva/efeitos adversos , Linfoma Cutâneo de Células T/terapia , Receptores de Antígenos Quiméricos/genética , Neoplasias Cutâneas/terapia , Linfócitos TRESUMO
Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células-Tronco Hematopoéticas/imunologia , Imunoterapia , Animais , Bovinos , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Citotoxicidade Imunológica , Sangue Fetal/citologia , Humanos , FenótipoRESUMO
Natural killer (NK) cells are potent innate immune system effector lymphocytes armed with multiple mechanisms for killing cancer cells. Given the dynamic roles of NK cells in tumor surveillance, they are fast becoming a next-generation tool for adoptive immunotherapy. Many strategies are being employed to increase their number and improve their ability to overcome cancer resistance and the immunosuppressive tumor microenvironment. These include the use of cytokines and synthetic compounds to bolster propagation and killing capacity, targeting immune-function checkpoints, addition of chimeric antigen receptors (CARs) to provide cancer specificity and genetic ablation of inhibitory molecules. The next generation of NK cell products will ideally be readily available as an "off-the-shelf" product and stem cell derived to enable potentially unlimited supply. However, several considerations regarding NK cell source, genetic modification and scale up first need addressing. Understanding NK cell biology and interaction within specific tumor contexts will help identify necessary NK cell modifications and relevant choice of NK cell source. Further enhancement of manufacturing processes will allow for off-the-shelf NK cell immunotherapies to become key components of multifaceted therapeutic strategies for cancer.
Assuntos
Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/terapia , Animais , Humanos , Neoplasias/imunologiaRESUMO
Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage "don't eat me" signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas.
RESUMO
Despite progress in developing cell therapies, such as T cell or stem cell therapies to treat diseases, immunoincompatibility remains a major barrier to clinical application. Given the fact that a host's immune system may reject allogeneic transplanted cells, methods have been developed to genetically modify patients' primary cells. To advance beyond this time-consuming and costly approach, recent research efforts focus on generating universal pluripotent stem cells to benefit a broader spectrum of patients. In this review, we first summarize current achievements to harness immunosuppressive mechanisms in cells to reduce immunogenicity. Then, we discuss several recent studies demonstrating the feasibility of genetically modifying pluripotent stem cells to escape immune attack and summarize the methods to evaluate hypoimmunogenicity. Although challenges remain, progress to develop genetically engineered universal pluripotent stem cells holds the promise of expediting their use in future gene and cell therapeutics and regenerative medicine.
RESUMO
In this issue of Cell Stem Cell, Xu et al. (2019) demonstrate that editing iPSCs' major histocompatibility antigens may potentially provide a small set of universally compatible stem cell lines for therapies. However, these modifications may result in patient minor histocompatibility responses and deficiencies in their T cell response repertoire to infection and cancer.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , HumanosRESUMO
This monograph, written by the pioneers of IVF and reproductive medicine, celebrates the history, achievements, and medical advancements made over the last 40 years in this rapidly growing field.
Assuntos
Fertilização in vitro/história , Fertilização in vitro/tendências , Medicina Reprodutiva/história , Medicina Reprodutiva/tendências , Feminino , Fertilização in vitro/métodos , História do Século XX , História do Século XXI , Humanos , Recém-Nascido , Masculino , Indução da Ovulação/história , Indução da Ovulação/métodos , Indução da Ovulação/tendências , Gravidez , Medicina Reprodutiva/métodosRESUMO
In vitro fertilization (IVF) began in Melbourne in 1970 when Carl Wood founded a research group at the Queen Victoria Hospital. The group reported the first biochemical pregnancy from a transferred IVF embryo in 1973. The group included the Royal Women's Hospital Melbourne, and they were the first to report confirmation of the British group's pregnancies with the use of IVF in natural cycles in 1980. The group then split, and the Monash group pursued fertility drug-induced multiple follicle growth in controlled ovulatory cycles and demonstrated for the first time that they could achieve multiple pregnancies in 1980-1981. This became the basis of a sustainable procedure for treating infertile patients. Successful embryo freezing and thawing methods resulted in pregnancies for the first time and were adopted to cryopreserve excess embryos produced after superovulation. Embryo donation methods were devised for anovulatory patients and were the first reported use of oocyte in vitro maturation techniques (IVM) for polycystic ovarian syndrome patients. Sperm microinjection techniques were pioneered for enabling fertilization for severely infertile men, and micromanipulative techniques were published for embryo biopsy for potential use in preimplantation genetic diagnosis (PGD) for patients with inheritable genetic diseases. The latter research programs were hampered by creation of restrictive embryo research laws in the State of Victoria, handicapping their timely clinical applications. Work on cryopreservation of ovarian tissue for cancer patients enabled clinical application of this for patients at risk of loss of fertility. Vitrification was developed as an alternative to freezing for oocytes and embryos, and this has now replaced the original slow cooling methods. Blastocyst culture systems were devised and optimized to improve IVF success and PGD.
Assuntos
Fertilização in vitro/história , Austrália , Técnicas de Cultura Embrionária/história , Técnicas de Cultura Embrionária/métodos , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/tendências , História do Século XX , História do Século XXI , Humanos , Técnicas de Maturação in Vitro de Oócitos/história , Técnicas de Maturação in Vitro de Oócitos/métodos , Recém-Nascido , Masculino , GravidezRESUMO
The clinical translation of promising products, technologies and interventions from the disciplines of nanomedicine and cell therapy has been slow and inefficient. In part, translation has been hampered by suboptimal research practices that propagate biases and hinder reproducibility. These include the publication of small and underpowered preclinical studies, suboptimal study design (in particular, biased allocation of experimental groups, experimenter bias and lack of necessary controls), the use of uncharacterized or poorly characterized materials, poor understanding of the relevant biology and mechanisms, poor use of statistics, large between-model heterogeneity, absence of replication, lack of interdisciplinarity, poor scientific training in study design and methods, a culture that does not incentivize transparency and sharing, poor or selective reporting, misaligned incentives and rewards, high costs of materials and protocols, and complexity of the developed products, technologies and interventions. In this Perspective, we discuss special manifestations of these problems in nanomedicine and in cell therapy, and describe mitigating strategies. Progress on reducing bias and enhancing reproducibility early on ought to enhance the translational potential of biomedical findings and technologies.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Avaliação Pré-Clínica de Medicamentos , Nanomedicina , Projetos de Pesquisa , Ado-Trastuzumab Emtansina , Animais , Neoplasias da Mama/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Masculino , Maitansina/análogos & derivados , Maitansina/uso terapêutico , Neoplasias da Próstata/terapia , Reprodutibilidade dos Testes , Extratos de Tecidos/uso terapêutico , Trastuzumab/uso terapêuticoRESUMO
OBJECTIVE: To develop and validate a new strategy to distinguish between balanced/euploid carrier and noncarrier embryos in preimplantation genetic diagnosis (PGD) cycles for reciprocal translocations and to successfully achieve a live birth after selective transfer of a noncarrier embryo. DESIGN: Retrospective and prospective study. SETTING: In vitro fertilization (IVF) units. PATIENT(S): Eleven patients undergoing mate pair sequencing for identification of translocation breakpoints, followed by clinical PGD cycles. INTERVENTION(S): Embryo biopsy with 24-chromosome testing to determine carrier status of balanced/euploid embryos. MAIN OUTCOME MEASURE(S): Definition of translocation breakpoints and polymerase chain reaction (PCR) diagnostic primers, correct diagnosis of euploid embryos for carrier status, and a live birth with a normal karyotype after transfer of a noncarrier embryo. RESULT(S): In 9 of 11 patients (82%), translocation breakpoints were successfully identified. In four patients with a term PGD pregnancy established with a balanced/euploid embryo of unknown carrier status, the correct carrier status was retrospectively determined, matching with the cytogenetic karyotype of the resulting newborns. In a prospective PGD cycle undertaken by a patient with a 46,XY,t(7;14)(q22;q24.3) translocation, the four balanced/euploid embryos identified comprised three carriers and one noncarrier. Transfer of the noncarrier embryo resulted in birth of a healthy girl who was subsequently confirmed with a normal 46,XX karyotype. CONCLUSION(S): The combination of mate pair sequencing and PCR breakpoint analysis of balanced reciprocal translocation derivatives is a novel, reliable, and accurate strategy for distinguishing between carrier and noncarrier balanced/euploid embryos. The method has potential application in clinical PGD cycles for patients with reciprocal translocations or other structural rearrangements.
Assuntos
Transferência Embrionária/métodos , Fertilização in vitro , Triagem de Portadores Genéticos/métodos , Diagnóstico Pré-Implantação/métodos , Translocação Genética , Adulto , Feminino , Fertilização in vitro/métodos , Humanos , Recém-Nascido , Cariotipagem , Masculino , Ploidias , Reação em Cadeia da Polimerase/métodos , Gravidez , Diagnóstico Pré-Natal/métodos , Estudos RetrospectivosAssuntos
Genes p53 , Mutação , Células-Tronco Pluripotentes , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/transplante , Proteína Supressora de Tumor p53/genéticaRESUMO
Merging iPSC models and human genetic research has opened up new avenues in understanding disease mechanisms and target biology, which facilitate exciting translation of this research to many areas of drug development. We highlight recent applications of these combined disciplines and discuss remaining challenges and potential solutions.
Assuntos
Descoberta de Drogas , Genética Humana , Células-Tronco Pluripotentes Induzidas/metabolismo , HumanosRESUMO
Basic experimental stem cell research has opened up the possibility of many diverse clinical applications; however, translation to clinical trials has been restricted to only a few diseases. To broaden this clinical scope, pluripotent stem cell derivatives provide a uniquely scalable source of functional differentiated cells that can potentially repair damaged or diseased tissues to treat a wide spectrum of diseases and injuries. However, gathering sound data on their distribution, longevity, function and mechanisms of action in host tissues is imperative to realizing their clinical benefit. The large-scale availability of treatments involving pluripotent stem cells remains some years away, because of the long and demanding regulatory pathway that is needed to ensure their safety.
Assuntos
Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células-Tronco Pluripotentes Induzidas , Transplante de Células-Tronco , Animais , HumanosRESUMO
INTRODUCTION: Neural crest cells make up a transient migratory population of cells found in all vertebrate embryos. Great advances have been made over the past 20 years in clarifying the molecular basis of neural crest induction and, although much still remains unclear, it appears that it is a process involving several factors acting at different stages of embryogenesis. In the future, an understanding of the precise mechanisms involved in orofacial development, even at the earliest stages, may well be of use to all clinicians interested in the management of these tissues. AIM: The present study was designed to determine if the early addition of noggin (a bone morphogenetic protein lBMP) antagonist) and/or the late addition of BMP4 would increase the expression of the transcription factors: Msx-1, Snail, Slug and Pax-7. METHOD: This involved an assessment of the effects of early addition ( Days 0 to 3) of noggin and/or the late addition ( Days 4 to 7) of BMP4 on2the expression of the neural crest markers by human embryonic stem cells, co-cultured for eight days on a feeder layer of mouse PA6 cells. RESULTS AND CONCLUSIONS: The expression of the neural crest markers Pax-7, Msx-1, Slug, and Snail by human embryonic stem cells is likely to be affected by the addition of noggin and BMP4. Not all of these effects will necessarily be significant. The late addition of BMP4 is likely to significantly increase the expression of Pax-7 by human embryonic stem cells (hESCs), when compared with the effects of co-culturing with stromal cell-derived inducing activity, alone. The early addition of noggin and the late addition of BMP4 are likely to significantly increase the expression of Msx-1 by hESCs, when compared with the late addition of BMP4, alone. The hESC results support those from animal ESC studies that the late addition of BMP4, especially, may result in the differentiation of neural crest precursors.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Crista Neural/citologia , Animais , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Transporte/farmacologia , Células Cultivadas , Humanos , Fator de Transcrição MSX1/metabolismo , Camundongos , Fator de Transcrição PAX7/metabolismo , Fotografação , Reação em Cadeia da Polimerase , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Clinical investigations using stem cell products in regenerative medicine are addressing a wide spectrum of conditions using a variety of stem cell types. To date, there have been few reports of safety issues arising from autologous or allogeneic transplants. Many cells administered show transient presence for a few days with trophic influences on immune or inflammatory responses. Limbal stem cells have been registered as a product for eye burns in Europe and mesenchymal stem cells have been approved for pediatric graft versus host disease in Canada and New Zealand. Many other applications are progressing in trials, some with early benefits to patients.
Assuntos
Transplante de Células-Tronco/tendências , Ensaios Clínicos como Assunto , Células Endoteliais/transplante , Feminino , Humanos , Terapia de Imunossupressão , Limbo da Córnea/citologia , Hepatopatias/terapia , Transplante de Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Células-Tronco Neurais/transplante , Osteoartrite/terapia , Placenta/citologia , Células-Tronco Pluripotentes/transplante , Gravidez , Medicina Regenerativa , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/métodos , Acidente Vascular Cerebral/terapia , Falha de TratamentoRESUMO
The development of a California-based induced pluripotent stem cell (iPSC) bank based on human leukocyte antigen (HLA) haplotype matching represents a significant challenge and a valuable opportunity for the advancement of regenerative medicine. However, previously published models of iPSC banks have neither addressed the admixed nature of populations like that of California nor evaluated the benefit to the population as a whole. We developed a new model for evaluating an iPSC haplobank based on demographic and immunogenetic characteristics reflecting California. The model evaluates haplolines or cell lines from donors homozygous for a single HLA-A, HLA-B, HLA-DRB1 haplotype. We generated estimates of the percentage of the population matched under various combinations of haplolines derived from six ancestries (black/African American, American Indian, Asian/Pacific Islander, Hispanic, and white/not Hispanic) and data available from the U.S. Census Bureau, the California Institute for Regenerative Medicine, and the National Marrow Donor Program. The model included both cis (haplotype-level) and trans (genotype-level) matching between a modeled iPSC haplobank and the recipient population following resampling simulations. We showed that serving a majority (>50%) of a simulated California population through cis matching would require the creation, redundant storage, and maintenance of almost 207 different haplolines representing the top 60 most frequent haplotypes from each ancestry group. Allowances for trans matching reduced the haplobank to fewer than 141 haplolines found among the top 40 most frequent haplotypes. Finally, we showed that a model optimized, custom haplobank was able to serve a majority of the California population with fewer than 80 haplolines.