RESUMO
Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.
RESUMO
ABSTRACT: In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.
Assuntos
Anticorpos Monoclonais Humanizados , Imunoterapia , Índice Terapêutico , Antígenos CD19 , Imunoterapia Adotiva/métodosRESUMO
Background: Despite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects. Methods: Antibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Results: Three different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model. Conclusion: This study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo.
Assuntos
Linfoma de Burkitt , Linfoma Difuso de Grandes Células B , Camundongos , Animais , Camundongos Endogâmicos NOD , Camundongos SCID , Rituximab/farmacologia , Rituximab/uso terapêutico , Leucócitos Mononucleares , Anticorpos Monoclonais Humanizados , Linfoma de Burkitt/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológicoRESUMO
BACKGROUND: Acute myeloid leukaemia (AML) stem cells (LSCs) cause disease relapse. The CD47 "don't eat me signal" is upregulated on LSCs and contributes to immune evasion by inhibiting phagocytosis through interacting with myeloid-specific signal regulatory protein alpha (SIRPα). Activation of macrophages by blocking CD47 has been successful, but the ubiquitous expression of CD47 on healthy cells poses potential limitations for such therapies. In contrast, CD123 is a well-known LSC-specific surface marker utilized as a therapeutic target. Here, we report the development of SIRPα-αCD123 fusion antibodies that localize the disruption of CD47/SIRPα signalling to AML while specifically enhancing LSC clearance. METHODS: SIRPα-αCD123 antibodies were generated by fusing the extracellular domain of SIRPα to an αCD123 antibody. The binding properties of the antibodies were analysed by flow cytometry and surface plasmon resonance. The functional characteristics of the fusion antibodies were determined by antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity assays using primary AML patient cells. Finally, an in vivo engraftment assay was utilized to assess LSC targeting. RESULTS: SIRPα-αCD123 fusion antibodies exhibited increased binding and preferential targeting of CD123+ CD47+ AML cells even in the presence of CD47+ healthy cells. Furthermore, SIRPα-αCD123 fusion antibodies confined disruption of the CD47-SIRPα axis locally to AML cells. In vitro experiments demonstrated that SIRPα-αCD123 antibodies greatly enhanced AML cell phagocytosis mediated by allogeneic and autologous macrophages. Moreover, SIRPα-αCD123 fusion antibodies efficiently targeted LSCs with in vivo engraftment potential. CONCLUSIONS: SIRPα-αCD123 antibodies combine local CD47 blockade with specific LSC targeting in a single molecule, minimize the risk of targeting healthy cells and efficiently eliminate AML LSCs. These results validate SIRPα-αCD123 antibodies as promising therapeutic interventions for AML.
Assuntos
Antígenos de Diferenciação/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Antígeno CD47/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Imunológicos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Mieloide Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/imunologiaRESUMO
Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Peptídeos/uso terapêutico , Proteínas WT1/imunologia , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Antígeno HLA-A2/imunologia , Humanos , Leucemia Mieloide Aguda/imunologia , Camundongos , Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Células Tumorais CultivadasRESUMO
Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.
Assuntos
Imunoterapia Adotiva/métodos , Leucemia Experimental/terapia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Feminino , Humanos , Leucemia Experimental/imunologia , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: Innovative post-remission therapies are needed to eliminate residual AML cells. DC vaccination is a promising strategy to induce anti-leukaemic immune responses. METHODS: We conducted a first-in-human phase I study using TLR7/8-matured DCs transfected with RNA encoding the two AML-associated antigens WT1 and PRAME as well as CMVpp65. AML patients in CR at high risk of relapse were vaccinated 10× over 26 weeks. RESULTS: Despite heavy pretreatment, DCs of sufficient number and quality were generated from a single leukapheresis in 11/12 cases, and 10 patients were vaccinated. Administration was safe and resulted in local inflammatory responses with dense T-cell infiltration. In peripheral blood, increased antigen-specific CD8+ T cells were seen for WT1 (2/10), PRAME (4/10) and CMVpp65 (9/10). For CMVpp65, increased CD4+ T cells were detected in 4/7 patients, and an antibody response was induced in 3/7 initially seronegative patients. Median OS was not reached after 1057 days; median RFS was 1084 days. A positive correlation was observed between clinical benefit and younger age as well as mounting of antigen-specific immune responses. CONCLUSIONS: Administration of TLR7/8-matured DCs to AML patients in CR at high risk of relapse was feasible and safe and resulted in induction of antigen-specific immune responses. Clinical benefit appeared to occur more likely in patients <65 and in patients mounting an immune response. Our observations need to be validated in a larger patient cohort. We hypothesise that TLR7/8 DC vaccination strategies should be combined with hypomethylating agents or checkpoint inhibition to augment immune responses. TRIAL REGISTRATION: The study was registered at https://clinicaltrials.gov on 17 October 2012 (NCT01734304) and at https://www.clinicaltrialsregister.eu (EudraCT-Number 2010-022446-24) on 10 October 2013.
RESUMO
Immune checkpoint inhibition has been shown to successfully reactivate endogenous T cell responses directed against tumor-associated antigens, resulting in significantly prolonged overall survival in patients with various tumor entities. For malignancies with low endogenous immune responses, this approach has not shown a clear clinical benefit so far. Therapeutic vaccination, particularly dendritic cell (DC) vaccination, is a strategy to induce T cell responses. Interaction of DCs and T cells is dependent on receptor-ligand interactions of various immune checkpoints. In this study, we analyzed the influence of blocking antibodies targeting programmed cell death protein 1 (PD-1), HVEM, CD244, TIM-3, and lymphocyte activation gene 3 (LAG-3) on the proliferation and cytokine secretion of T cells after stimulation with autologous TLR-matured DCs. In this context, we found that LAG-3 blockade resulted in superior T cell activation compared to inhibition of other pathways, including PD-1/PD-L1. This result was consistent across different methods to measure T cell stimulation (proliferation, IFN-γ secretion), various stimulatory antigens (viral and bacterial peptide pool, specific viral antigen, specific tumor antigen), and seen for both CD4+ and CD8+ T cells. Only under conditions with a weak antigenic stimulus, particularly when combining antigen presentation by peripheral blood mononuclear cells with low concentrations of peptides, we observed the highest T cell stimulation with dual blockade of LAG-3 and PD-1 blockade. We conclude that priming of novel immune responses can be strongly enhanced by blockade of LAG-3 or dual blockade of LAG-3 and PD-1, depending on the strength of the antigenic stimulus.
Assuntos
Anticorpos Bloqueadores/farmacologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Receptor de Morte Celular Programada 1/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Receptores Toll-Like/metabolismo , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
RNA polymerase (Pol) II transcribes protein-coding genes in the nucleus of eukaryotic cells and consists of 12 polypeptide subunits. It is unknown how Pol II is imported into the nucleus. Here we show that Pol II nuclear import requires the protein Iwr1 and provide evidence for cyclic Iwr1 function. Iwr1 binds Pol II in the active center cleft between the two largest subunits, maybe facilitating or sensing complete Pol II assembly in the cytoplasm. Iwr1 then uses an N-terminal bipartite nuclear localization signal that is recognized by karyopherin α to direct Pol II nuclear import. In the nucleus, Iwr1 is displaced from Pol II by transcription initiation factors and nucleic acids, enabling its export and recycling. Iwr1 function is Pol II specific, transcription independent, and apparently conserved from yeast to human.