RESUMO
Genetic modification of tumor-infiltrating lymphocytes (TILs) or circulating T cells has become an important avenue in cancer therapy. Here we describe a comprehensive method for establishing and expanding TIL cultures and genetically modifying them with a gene of interest (GOI) via retroviral transduction or mRNA transfection. The method includes all the important steps starting with TIL extraction from tumors through to the maintenance of the genetically modified TILs. The protocol includes instructions for retroviral transduction and mRNA transfection of circulating T cells or T-cell lines. The GOIs most commonly introduced into the target cells are chimeric antigen receptors (CARs); genetic adjuvants, such as membrane-bound interleukins; and antitumor T-cell receptors (TCRs).
Assuntos
Linfócitos do Interstício Tumoral , Linfócitos T , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos T/metabolismo , Transfecção , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linhagem Celular , Linfócitos T CD8-Positivos , Imunoterapia Adotiva/métodosRESUMO
Targeting solid tumors with absolute precision is a long-standing challenge in cancer immunotherapy. The identification of antigens, which are expressed by a large fraction of tumors of a given type and, preferably, across various types, but not by normal cells, holds the key to developing safe, off-the-shelf immunotherapies. Although the quest for widely shared, strictly tumor-specific antigens has been the focus of tremendous effort, only few such candidates have been implicated. Almost all antigens that are currently explored as targets for chimeric antigen receptor (CAR) or T cell receptor (TCR)-T cell therapy are also expressed by healthy cells and the risk of on-target off-tumor toxicity has remained a major concern. Recent studies suggest that this risk could be obviated by targeting instead combinations of two or more antigens, which are co-expressed by tumor but not normal cells and, as such, are tumor-specific. Moreover, the expression of a shared tumor antigen along with the lack of a second antigen that is expressed by normal tissues can also be exploited for precise recognition. Additional cues, antigenic or non-antigenic ones, which characterize the tumor microenvironment, could be harnessed to further increase precision. This review focuses on attempts to define the targetable signatures of tumors and assesses different strategies employing advanced synthetic biology for translating such information into safer modes of immunotherapy, implementing the principles of Boolean logic gates.
Assuntos
Imunoterapia Adotiva/métodos , Lógica , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Medicina de Precisão/métodos , Animais , HumanosRESUMO
The costimulatory domains incorporated into second-generation and third-generation chimeric antigen receptors (CARs) strongly influence CAR-T-cell function. Here, we explored second-generation and third-generation CARs harboring the signaling domain of the CD40 receptor as a new costimulatory element in comparison with similar CARs carrying the 4-1BB domain. In CARs of both generations, CD40 was more potent than 4-1BB in triggering the NF-κB signaling pathway. In human T cells from 2 donors, CD40 was comparable to 4-1BB in upregulating costimulatory and activation markers, inducing proinflammatory cytokine secretion and mediating target cell killing. Interestingly, differences in the response pattern of T cells from the 2 donors with respect to CD40 and 4-1BB were evident. We conclude that in human T cells, the CD40 signaling domain is a potent costimulatory element in both second-generation and third-generation CARs.
Assuntos
Antígenos CD40/imunologia , Domínios e Motivos de Interação entre Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Antígenos CD40/química , Antígenos CD40/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Engenharia Genética , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Plasmídeos/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/metabolismoRESUMO
Adoptive T cell therapy (ACT) holds great promise for cancer treatment. One approach, which has regained wide interest in recent years, employs antitumor T cells isolated from tumor lesions ("tumor-infiltrating lymphocytes" or TIL). It is now appreciated that a considerable proportion of anti-melanoma TIL recognize new HLA-binding peptides resulting from somatic mutations, which occurred during tumor progression. The clinical efficacy of TIL can potentially be improved via their genetic modification, designed to enhance their survival, homing capacity, resistance to suppression, tumor killing ability and additional properties of clinical relevance. Successful implementation of such gene-based strategies critically depends on efficient and reproducible protocols for gene delivery into clinical TIL preparations. Here we describe an optimized protocol for the retroviral transduction of TIL. As the experimental system we employed anti-melanoma TIL cultures prepared from four patients, recombinant retrovirus encoding an anti-CD19 chimeric antigen receptor (CAR) as a model gene of interest and CD19+ and CD19- human cell lines serving as target cells. Transduction on day 7 of the rapid expansion protocol (REP) resulted in 69 ± 8% CAR positive TIL. Transduced, but not untransduced TIL, from the four patients responded robustly to CD19+, but not CD19- cell lines, as judged by substantial secretion of IFN-γ following co-culture. In light of the rekindled interest in antitumor TIL, this protocol can be incorporated into a broad range of gene-based approaches for improving the in-vivo survival and functionality of TIL in the clinical setting.
Assuntos
Linfócitos do Interstício Tumoral/imunologia , Retroviridae/imunologia , Antígenos CD19/imunologia , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva/métodos , Interferon gama/imunologia , Células K562 , Ativação Linfocitária/imunologia , Melanoma/imunologia , Receptores de Antígenos Quiméricos/imunologiaRESUMO
Among the earliest protein products of most cellular genes are those synthesized during the pioneer round of translation (PRT), a key step in nonsense-mediated mRNA decay (NMD) that allows scanning of new transcripts for the presence of a premature termination codon (PTC). It has been demonstrated that at least some PRT degradation products can be targeted to major histocompatibility (MHC)-I presentation. To gain new insight into this putative PRT-to-MHC-I route, we have assembled 2 pairs of reporter genes so that the 2 genes in each pair encode an identical fusion protein between a model antigenic peptide and enhanced green fluorescent protein (EGFP), one of which harbors a PTC. We expressed these genes in different mouse and human cell lines and confirmed enhanced NMD activity for the PTC(+) gene in each pair by monitoring the effect of cycloheximide on the level of the respective mRNA. We then exploited several strategies for establishing the ratio between level of peptide presentation and total amount of protein product. We consistently observed significantly higher ratios for the PTC(+) mRNAs compared with the PTC(-) ones, pointing to correlation between the turnover of otherwise identical proteins and the fate of their template mRNA. Using confocal microscopy, we showed a clear link between NMD, the presence of misfolded EGFP polypeptides, and enhanced MHC-I peptide presentation. Altogether, these findings imply that identical full-length gene products differing only in 3' noncoding sequences can be differentially degraded and targeted to the MHC-I presentation pathway, suggesting a more general role for the PRT in establishing the MHC-I peptidome.-Weinstein-Marom, H., Hendel, L., Laron, E. A., Sharabi-Nov, A., Margalit, A., Gross, G. MHC-I presentation of peptides derived from intact protein products of the pioneer round of translation.
Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Códon sem Sentido/genética , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genéticaRESUMO
Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing antitumor TCRs or chimeric antigen receptors often yields a high rate of clinical response in several types of cancer. New approaches for enhancing the functional properties of antitumor T cells could improve the clinical outcome of these treatments. To this end, we created 3 classes of genes, each designed to operate autonomously upon expression in T cells. We recently reported on the enhancing effects of constitutively active toll-like receptor 4 (caTLR4), membrane (mem) interleukin-2, memIL-12, and memIL-15, and self-oligomerizing, constitutively active CD40 (caCD40). Here, we evaluated their combined effects on peripheral blood CD8 T cells and different antimelanoma TIL cultures following mRNA electroporation. Expression in CD8 T cells induced transient production of interferon-γ and prolonged and robust upregulation of CD25, CD69, 4-1BB, and OX40. The adjuvants enhanced cytolytic activity of TILs and production of interferon-γ and TNF-α in the presence of autologous, but not mismatched, melanoma for at least 3 days after electroporation. Expression of the 3 adjuvants in young TILs from different patients markedly increased the expression of CD25, OX40, 4-1BB, CD127, and CD28 and exhibited cooperative and, at times, synergistic effects. Furthermore, predefined mixtures of mRNA encoding these adjuvants markedly enhanced the specific antitumor response of selected TILs and killing of autologous melanoma cells by young TILs. Our findings suggest that combinations of these new genetic adjuvants can substantially improve the functional properties of antitumor T cells, offering a new tool of unique versatility in adoptive cell therapy.
Assuntos
Linfócitos T CD8-Positivos/transplante , Eletroporação , Técnicas de Transferência de Genes , Imunoterapia Adotiva , Linfócitos do Interstício Tumoral/transplante , RNA Mensageiro/administração & dosagem , Adjuvantes Imunológicos/administração & dosagem , Células Cultivadas , Humanos , Interferon gama , Melanoma/terapiaRESUMO
New strategies for augmenting the actual performance of therapeutic T cells in vivo are needed for improving clinical outcome of adoptive cell therapy. Cumulative findings suggest that CD40 plays an intrinsic role in T cell costimulation. Recently, we demonstrated the ability of truncated, auto-oligomerizing CD40 derivatives to induce strong activation of APCs in a ligand-independent manner. We reasoned that constitutively active CD40 (caCD40) can similarly exert enhancing effects on human antitumor T cells. To test this assumption, we transfected human T cells with in vitro-transcribed caCD40 mRNA. In polyclonal T cells, caCD40 triggered IFN-γ secretion and upregulated CD25 and 4-1BB. In antimelanoma tumor-infiltrating lymphocytes (TILs), caCD40 induced massive production of IFN-γ, exerting a pronounced synergistic effect when coexpressed with constitutively active TLR4 devoid of its extracellular ligand binding. In unselected "young" TILs, caCD40 reproducibly increased surface expression of CD25, OX40, 4-1BB, CD127, and CD28. Three days post-mRNA electroporation of CD8 TILs, caCD40 elevated IFN-γ and TNF-α production and cytolytic activity in the presence of autologous but not HLA-I-mismatched melanoma. Enhanced killing of autologous melanoma by young TILs was observed 4 d posttransfection. These findings suggest that caCD40 can function as a potent T cell adjuvant and provide essential guidelines for similar manipulation of other key members of the TNFR family.
Assuntos
Antígenos CD40/imunologia , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Humanos , Imunoterapia Adotiva/métodos , Melanoma/imunologia , RNA Mensageiro , Neoplasias Cutâneas/imunologia , Células Tumorais CultivadasRESUMO
Proinflammatory cytokines are widely explored in different adoptive cell therapy protocols for enhancing survival and function of the transferred T cells, but their systemic administration is often associated with severe toxicity which limits their clinical use. To confine cytokine availability to the therapeutic T cells, we expressed 3 key cytokines, IL-2, IL-12, and IL-15, as integral T-cell membrane proteins. To prevent permanent activation of growth signaling pathways, we delivered these genes to T cells through mRNA electroporation. The engineered cytokines could be detected on the surface of mRNA-transfected cells and binding to their cell-surface receptors mainly occurred in cis. The 3 human cytokines supported the ex vivo growth of activated human CD8 and CD4 T cells for at least 6 days posttransfection, comparably to high-dose soluble IL-2. Similarly, membrane IL-2, membrane IL-12, and, to a lesser extent, membrane IL-15, were comparable with their soluble counterparts in supporting proliferation of splenic mouse CD8 T cells. Following electroporation of human CD8 T cells and antimelanoma tumor-infiltrating lymphocytes, membrane cytokines synergized with constitutively active toll-like receptor 4 in inducing interferon-γ secretion. Efficient cooperation with TLR4 was also evident in the upregulation of the activation molecules CD25, CD69, CD137 (4-1BB), and CD134 (OX40). Taken together, membrane cytokines expressed through mRNA transfection emerge as effective tools for enhancing T-cell proliferation and function and may have potential use in adoptive T-cell therapy.
Assuntos
Membrana Celular/metabolismo , Citocinas/metabolismo , Imunoterapia Adotiva/métodos , Linfócitos T/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Eletroporação , Feminino , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Engenharia de Proteínas , RNA Mensageiro/genética , Linfócitos T/transplanteRESUMO
Most current methods for identifying peptides that are bound to a distinct MHC-I product in a given cell sample utilize detergent solubilization of membrane proteins followed by immunoaffinity purification. Since detergent traces and cell debris hamper subsequent peptide analysis, exceedingly large cell samples are often required. To avoid the use of detergents, truncated MHC-I heavy chains have recently been expressed by stable DNA transfection or retroviral transduction, resulting in the secretion of soluble MHC-I complexes to the growth medium. The electroporation of in vitro-transcribed mRNA achieves remarkable efficacy and uniformity of gene expression in numerous cell types, exhibiting exceedingly fast kinetics. We reasoned that mRNA transfection offers a simple, fast and widely applicable alternative to current gene delivery protocols for expressing secreted MHC-I products in cells of interest. To test this assumption we used mRNA to express soluble derivatives of HLA-A2 in the human AF10 B cell myeloma and 624mel melanoma and H-2K(d) in the mouse SP2/0 B cell myeloma. The level of MHC-I complexes secreted by these cells peaked within less than 24h post-transfection and they could be affinity-purified directly from the culture medium in considerably greater yields when compared to nonionic detergent lysates on a cell-to-cell basis. Mass-spectrometry analysis of eluted peptides revealed larger pools in the secreted material than in lysates with substantial overlap in composition. Our results introduce mRNA transfection as a powerful tool for determining the cell's MHC-I peptidome, which can be potentially applied to a broad range of cell types.