Littérature scientifique sur le sujet « Adoptive cell therapy, telomerase, leukemia »

BackgroundThe development of immunotherapies holds great promise for the treatment of refractory infections and cancer. Current approaches, although effective in many settings, have limitations that prevent their widespread use. Hence, several aspects require improvements, including the re-wiring of T-cell fates and function. T-cell dysfunction is central to the persistence of several chronic viral infections and the progression of malignancies. Upon activation, T cells can follow several paths of differentiation, leading to terminal effector differentiation and/or exhaustion which are widely recognized as dysfunctional features limiting human immune competence. Furthermore, dysfunctional features induced during laboratory-based manipulations of T-cell products prior to adoptive cell transfer has a determining effect on outcomes. Similarly, repeated antigen encounters after transfer in vivo favors the development of T-cell dysfunction. However, the nature and underlying mechanisms of T-cell dysfunction are still incompletely understood.MethodsCombining genomics, phenotypic and functional analyses in various physiologically and clinically relevant settings, we investigated the key factors leading to T-cell dysfunction. Specifically, we evaluated the impact of repeated stimulations using CD3/CD28-coated beads or antigen-loaded dendritic cells in human T-cell long term cultures, and BCMA-expressing cells for anti-BCMA CAR T cells. We also examined mouse antigen-specific T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection as well as datasets obtained from circulating T cells from acute myeloid leukemia (AML) patients.ResultsWe identified telomere-independent cellular senescence as a central aspect of exhausted PD-1-expressing T cells following repeated stimulations. Mechanistically, it is associated the induction of p16INK4a. Additionally, we found that cellular senescence features are partly regulated by the activation of caspase-8, through a non-apoptotic function of this molecule not previously described in T cells.ConclusionsWe thus conclude that caspase-8 may regulate the balance between apoptosis and proliferation by protecting T cells from cellular senescence. Senescence-associated mechanisms may be seen as key players in T-cell dysfunction occurring following repeated stimulations and as such should be considered as novel immune checkpoints impeding the success of T-cell adoptive immunotherapy in humans.Ethics ApprovalThis study was approved by the local Maisonneuve-Rosemont Hospital research ethics authorities and participants’ informed consent was obtained (CÉR2020-2141 and CÉR13030).

Abstract [Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab. [Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014). [Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface. Disclosures Okamoto: CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.

BackgroundImmunosenescence refers to the age-associated decline of the adaptive immune system, which results in increased incidence and severity of infections, cancers, and autoimmunity. The elderly show reduced numbers of naïve T cells, skewed CD4:CD8 ratio, reduced proliferative and functional capabilities, and increased expression of senescence markers. These phenomena have strong repercussion in adoptive immunotherapy.Notably, the ex vivo manufacturing process of CAR-T cells per se induces senescence extremely quickly; 15 days of T cell expansion age cells 30 years, as measured by telomere length, T cells differentiation and CDKN2a mRNA levels.To circumvent this problem, we here propose the modulation of USP16, an epigenetic regulator of stem cells and senescence in multiple tissues. Downregulation of USP16 rejuvenates T cells, offering a powerful tool to dramatically improve the efficacy of CAR-T treatments.MethodsDuring ex vivo CAR-T cell manufacture, cells age very rapidly, strongly decreasing T cell fitness. Importantly, we observed that cellular senescence is an early event that precedes T cell exhaustion upon CD3/CD28 T cell stimulation, making it a very interesting pathway to target. In line with this hypothesis, we demonstrated that reducing cellular senescence increases CAR-T cell functions both in vitro and in vivo.ResultsWe identified an epigenetic regulator, USP16, whose mRNA levels increase during T cell expansion and correlate with the expression of the aging marker par excellence, CDKN2a. Genetic modulation of USP16 in CD19 and GD2 CAR-expressing T cells not only reduces senescence markers but also expands the naive (CD45RA+CD62L+) population and enhances cell self-renewal, without negative effects on T cell expansion. USP16 modulation also results in increased killing, polyfunctionality, and expansion upon in vitro stimulation with tumor cells. Notably, the delay of cellular senescence induces long-lasting cellular fitness (figure 1) as T cells are less exhausted upon multiple tumor challenges. In vivo, T cells rejuvenated by USP16 modulation, are 60% more efficient in controlling tumor growth in a mouse model of leukemia (NALM-6) and neuroblastoma (CHLA-255).Abstract 830 Figure 1Effect of USP16 modulation in T cell agingThe schematic shows the relation between cell functionality, exhaustion and cellular senescence in normal T cell aging (top) and when USP16 is inhibited (bottom). USP16 modulation reduces T cell aging, increasing cell functionality and delaying exhaustion and cellular senescence.ConclusionsWe demonstrated that modulation of USP16 prevents cellular senescence and increases self-renewal in T cells. This approach can significantly improve CAR-T therapy in multiple diseases, including leukemias and solid tumors. Development of small molecules against USP16 could offer a viable solution to improve T cell fitness during manufacturing.Ethics ApprovalThe study was approved by Institutional Animal Care and Use Committees (IACUC), approval number CR-0104.

Abstract In this issue of Blood, Ugel and colleagues provide evidence that, in murine models, telomerase is an efficient target for adoptive cell therapy against a variety of cancer cells, but also can elicit an autoimmune response against B cells.1

Abstract Telomerase reverse transcriptase (TERT) is a good candidate for cancer immunotherapy because it is overexpressed in 85% of all human tumors and implicated in maintenance of the transformed phenotype. TERT-based cancer vaccines have been shown to be safe, not inducing any immune-related pathology, but their impact on tumor progression is modest. Here we show that adoptive cell therapy with the use of high-avidity T lymphocytes reactive against telomerase can control the growth of different established tumors. Moreover, in transgenic adenocarcinoma mouse prostate mice, which develop prostate cancer, TERT-based adoptive cell therapy halted the progression to more aggressive and poorly differentiated tumors, significantly prolonging mouse survival. We also demonstrated that human tumors, including Burkitt lymphoma, and human cancer stem cells, are targeted in vivo by TERT-specific cytotoxic T lymphocytes. Effective therapy with T cells against telomerase, different from active vaccination, however, led to autoimmunity marked by a consistent, although transient, B-cell depletion in primary and secondary lymphoid organs, associated with alteration of the spleen cytoarchitecture. These results indicate B cells as an in vivo target of TERT-specific cytotoxic T lymphocytes during successful immunotherapy.

Key Points The efficacy and safety of a novel redirected T-cell–based adoptive immunotherapy targeting hTERT for patients with adult T-cell leukemia. hTERT-specific T-cell receptor gene-transduced CD8+ T cells lyse ATL cells, but not normal cells, both in vitro and in vivo.

La telomerasi (TERT) è la trascrittasi inversa implicata principalmente nell’allungamento dei telomeri nelle cellule di mammifero ed è considerato un antigene tumorale universale chiave, in quanto overespresso in più dell’85% delle cellule tumorali. Quando TERT viene riattivata nelle cellule tumorali, l'enzima viene processato e i suoi peptidi antigenici vengono presentati in associazione con le moleole del complesso maggiore di istocompatibilità di classe I, promuovendo il riconoscimento del tumore da parte di linfociti T citotossici specifici. Recentemente, il nostro gruppo ha dimostrato che il trasferimento adottivo di linfociti T citotossici murini specifici per il peptide TERT198-205 sono stati in grado di controllare la progressione di diversi modelli tumorali trapiantabili ma, allo stesso tempo, la somministrazione ripetuta ha indotto una deplezione autoimmune temporanea a carico dei linfociti B. Questi risultati rappresentano un punto di partenza per investigare un nuovo meccanismo basato sulla telomerasi per il trattamento di tumori maligni, come ad esempio la leucemia linfatica cronica. I livelli di espressione e di attività della telomerasi sono generalmente bassi o assenti nelle cellule normali, ad eccezione delle cellule germinali maschili, delle cellule embrionali e delle cellule staminali ematopoietiche, mentre sono solitamente elevati nelle cellule B maligne. Inoltre, i livelli di attività di TERT correlano con una peggiore prognosi in pazienti leucemici. Abbiamo quindi sviluppato un approccio immunoterapico passivo, basato sul trasferimento adottivo di linfociti T citotossici specifici per TERT, da un lato in un modello murino di leucemia B, dall’altro, in un contesto umanizzato in cui cellule leucemiche primarie umane sono state inoculate in topi immunodeficienti. Nel modello murino, abbiamo utilizato un tumore maligno B altamente aggressivo derivato da topi transgenici IgH.TE, che presenta caratteristiche comuni alla leucemia linfatica cronica umana, come l'espansione di una popolazione cellulare clonale CD19+/CD5+ esprimente alti livelli di attività telomerasica. I linfociti T citotossici specifici per mTERT198-205 sono stati in grado di riconoscere le suddette cellule B maligne sia in vitro che in vivo: il trasferimento adottivo di linfociti T citotossici specifici per mTERT198-205, in topi con tumore derivato dal modello IgH.TE, ha indotto la completa remissione in circa il 70% degli animali. Fiduciosi di questi dati, abbiamo traslato la nostra ipotesi di lavoro in ambito preclinico. Partendo da un clone da noi precedentemente isolato, abbiamo clonato le sequenze delle catene  e  del TCR specifico per il peptide hTERT865-873 che sono poi state inserito in un vettore di espressione retrovirale per poter infettare cellule mononucleate del sangue periferico, creando linfociti T citotossici specifici per il peptide hTERT865-873. Queste cellule T citotossiche erano in grado di riconoscere, sia in vitro che in vivo, sia neoplasie B immortalizzate, sia cellule maligne isolate da pazienti leucemici, in entrambi i casi in modo HLA-A2-ristretto. Inoltre, il trasferimento adottivo TERT specifico in topi immuno-deficienti NOG inoculati con una linea leucemica umana, ha portato ad una riduzione significativa della diffusione delle cellule neoplastiche negli organi secondari. Inoltre, cellule T specifiche per hTERT865-873 sono state in grado di controllare l'attecchimento di cellule B derivate da pazienti con leucemia linfatica cronica in topi NOG umanizzati, confermando non solo l'efficacia, ma anche la specificità e la sicurezza del nostro approccio immunoterapico. Questi risultati suggeriscono che il complesso hTERT865-873/HLA-A2 fisiologicamente processato ed espresso sulla superficie delle cellule neoplastiche sia sufficientemente immunogenico da essere riconosciuto da linfociti T citotossici specifici. Questi linfociti ingegnerizzati sono in grado di eliminare selettivamente le cellule maligne, ma non le cellule normali o i progenitori ematopoietici, in modo HLA-A02-ristretto sia in vitro che in vivo. Le nostre osservazioni sperimentali supportano quindi lo sviluppo di un nuovo approccio immunoterapeutico basato sul trasferimento adottivo di linfociti T ingegnerizzati specifici per il peptide hTERT865-873 per pazienti affetti da leucemia linfatica cronica a cellule B, che potrebbe essere potenzialmente traslato in clinica anche per il trattamento di tumori solidi di diversa istologia.
Telomerase reverse transcriptase (TERT) is considered a key universal tumor associated antigen, since it is overexpressed in more than 85% of tumor cells. When TERT is reactivated in tumor cells, the enzyme is processed and its antigenic peptides are presented in association with the class I molecules of the major histocompatibility complex, promoting tumor recognition by specific cytotoxic T-lymphocytes. Recently, our group demonstrated that the adoptive cell transfer of mouse TERT198-205-specific cytotoxic T-lymphocytes were able to control the progression of several transplantable tumor models but, at the same time, the repeated administration induced a temporary autoimmune depletion of B cells. We had therefore hypotized that these results could represent a promising background for the treatment of haematologic B cell malignancies, such as B-cell chronic lymphocytic leukemia. Telomerase expression and activity is generally low or absent in normal cells, except for male germline, embryonic and hematopoietic stem cells, while it is usually elevated in malignant B cells. Moreover TERT expression and functional activity is reported to directly correlate with a worst prognosis of leukemic patients. We developed a passive immunotherapeutic approach based on the adoptive transfer of specific TERT cytotoxic T-lymphocytes both in murine B cell leukemia cancer models but also in a humanized context in which immunodeficient mice were engrafted with human leukemic cells and treated with specific engineered anti h-TERT T lymphocytes. In murine setting, we took advantage of a highly aggressive B-cell malignancy derived by IgH.TE transgenic mice, which displayed common characteristics with human B-cell chronic lymphocytic leukemia. patients such as the expansion of a CD19+/CD5+ clonal cell population expressing high levels of active telomerase. mTERT198-205-specific cytotoxic T-lymphocytes could recognize these malignant B cells both in vitro and in vivo: ~70% of treated IgH.TE-derived B cell tumor-bearing mice with the adoptive transfer of mTERT specific cytotoxic T-lymphocytes achieved complete remission. Confident about these data, we translated our work hypothesis into human setting. We cloned the hTERT865-873-specific full-length rearranged TCR / genes from our previously isolated cytotoxic T-lymphocyte clone and inserted into a novel retroviral expression vector to infect naive peripheral blood mononucleated cells, creating selective hTERT865-873-specific CTLs. These cytotoxic T cells were able to recognize both immortalized B-cell malignancies and peripheral blood mononucleated cells from leukemic patients expressing hTERT865-873 in an HLA-A2-restricted manner, both in vitro and in vivo. In fact, TERT specific adoptive transfer in leukemic immune-deficient NOG mice led to a significant reduction in the spreading of neoplastic cells in secondary organs. Moreover, hTERT865-873 specific T cells controlled the engraftment of human primary B-cell chronic lymphocytic leukemia cells in humanized NOG mice, thus confirming not only the efficacy, but also the specificity and safety of our immunotherapeutic approach. These findings suggest that naturally processed hTERT865-873/HLA-A2+ complexes presented on the surface of B-malignancies are sufficiently immunogenic to be recognized by hTERT865-873-specific cytotoxic T cells. Gene-modified T cells successfully killed malignant B cell, but not normal cells and hematopoietic progenitors, in an HLA-restricted manner both in vitro and in vivo. Our experimental observations therefore support the development of a novel hTERT865-873-targeting redirected T cell-based immunotherapy for B-cell chronic lymphocytic leukemia patients, that can also be potentially translated in clinic to treat tumors with different histology.