RESUMEN
Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.
Asunto(s)
Antígenos CD28 , Receptores Quiméricos de Antígenos , Animales , Antígenos CD19/genética , Antígenos CD28/genética , Antígenos CD28/metabolismo , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva/métodos , RatonesRESUMEN
Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.
Asunto(s)
Antígenos CD28/metabolismo , Membrana Celular/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Integrinas/metabolismo , Linfoma/inmunología , Ratones Endogámicos NOD , Ratones SCID , Mucina-1/metabolismo , Multimerización de Proteína , Receptores del Factor Estimulante de Colonias/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.
Asunto(s)
Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células de la Médula Ósea/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Medio de Cultivo Libre de Suero/farmacología , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Activación de Linfocitos , Ratones SCID , PronósticoRESUMEN
Chimeric antigen receptor (CAR) T-cell immunotherapy has achieved unprecedented efficacy in the treatment of chemotherapy-resistant or refractory B-cell malignancies. Promising results from pivotal anti-CD19 CAR T-cell phase II trials have led to landmark approvals of two CD19-specific CAR T-cell products by the United States Food and Drug Administration and European Medicines Agency. However, several issues associated with CAR T-cell treatment remain unresolved, such as the management of severe toxicities and the frequent occurrence of both antigen-positive and antigen-negative relapse. Nonetheless, pre-clinical research is advancing at an unprecedented pace to develop innovative solutions to address these issues. Herein, we summarise recent clinical developments and outcomes of CD19-targeted CAR T-cell immunotherapy and discuss emerging strategies that may further improve the success, safety and broadened applicability of this approach.
RESUMEN
Despite the unprecedented clinical success of chimeric antigen receptors (CAR) T-cells against haematological malignancy, solid tumors impose a far greater challenge to success. Largely, this stems from an inadequate capacity of CAR T-cells that can traffic and maintain function within a hostile microenvironment. To enhance tumor-directed T-cell trafficking, we have engineered CAR T-cells to acquire heightened responsiveness to interleukin (IL)-8. Circulating IL-8 levels correlate with disease burden and prognosis in multiple solid tumors in which it exerts diverse pathological functions including angiogenesis, support of cancer stem cell survival, and recruitment of immunosuppressive myeloid cells. To harness tumor-derived IL-8 for therapeutic benefit, we have co-expressed either of its cognate receptors (CXCR1 or CXCR2) in CAR T-cells that target the tumor-associated αvß6 integrin. We demonstrate here that CXCR2-expressing CAR T-cells migrate more efficiently towards IL-8 and towards tumor conditioned media that contains this cytokine. As a result, these CAR T-cells elicit superior anti-tumor activity against established αvß6-expressing ovarian or pancreatic tumor xenografts, with a more favorable toxicity profile. These data support the further engineering of CAR T-cells to acquire responsiveness to cancer-derived chemokines in order to improve their therapeutic activity against solid tumors.
RESUMEN
Following the landmark approvals by the United States Food and Drug Administration, the adoptive transfer of CD19-directed chimeric antigen receptor (CAR) T-cells has now entered mainstream clinical practice for patients with chemotherapy-resistant or refractory B-cell malignancies. These approvals have followed on from a prolonged period of pre-clinical evaluation, informing the design of clinical trials that have demonstrated unprecedented efficacy in this difficult to treat patient population. However, the delivery of autologous CAR-engineered T-cell therapy is complex, costly and not without significant risk. Here we summarize the key themes of CAR T-cell preclinical development and highlight a number of innovative strategies designed to further address toxicity and improve efficacy. In concert with the emerging promise of precision genome editing, it is hoped these next generation products will increase the repertoire of clinical applications of CAR T-cell therapy in malignant and perhaps other disease settings.
Asunto(s)
Linfocitos B , Neoplasias Hematológicas , Inmunoterapia Adoptiva , Linfoma de Células B , Receptores Quiméricos de Antígenos/uso terapéutico , Linfocitos B/inmunología , Linfocitos B/patología , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Humanos , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Linfoma de Células B/terapia , Receptores Quiméricos de Antígenos/inmunologíaRESUMEN
Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance, but they can also play a detrimental role by preventing antitumor responses. Here, we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution, focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently, Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall, our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival, higher migratory capacity, and selective T-effector suppressive ability.