RESUMO
BACKGROUND: The treatment of melanoma, the deadliest form of skin cancer, has greatly benefited from immunotherapy. However, many patients do not show a durable response, which is only partially explained by known resistance mechanisms. METHODS: We performed single-cell RNA sequencing of tumor immune infiltrates and matched peripheral blood mononuclear cells of 22 checkpoint inhibitor (CPI)-naive stage III-IV metastatic melanoma patients. After sample collection, the same patients received CPI treatment, and their response was assessed. FINDINGS: CPI responders showed high levels of classical monocytes in peripheral blood, which preferentially transitioned toward CXCL9-expressing macrophages in tumors. Trajectories of tumor-infiltrating CD8+ T cells diverged at the level of effector memory/stem-like T cells, with non-responder cells progressing into a state characterized by cellular stress and apoptosis-related gene expression. Consistently, predicted non-responder-enriched myeloid-T/natural killer cell interactions were primarily immunosuppressive, while responder-enriched interactions were supportive of T cell priming and effector function. CONCLUSIONS: Our study illustrates that the tumor immune microenvironment prior to CPI treatment can be indicative of response. In perspective, modulating the myeloid and/or effector cell compartment by altering the described cell interactions and transitions could improve immunotherapy response. FUNDING: This research was funded by Roche Pharma Research and Early Development.
Assuntos
Inibidores de Checkpoint Imunológico , Melanoma , Neoplasias Cutâneas , Microambiente Tumoral , Humanos , Melanoma/tratamento farmacológico , Melanoma/imunologia , Melanoma/patologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Masculino , Feminino , Células Mieloides/imunologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/metabolismo , Pessoa de Meia-Idade , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacosRESUMO
ABSTRACT: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513.
Assuntos
Anticorpos Biespecíficos , Antígenos CD19 , Antígenos CD20 , Antígenos CD28 , Imunoterapia , Humanos , Antígenos CD28/imunologia , Antígenos CD28/agonistas , Animais , Camundongos , Anticorpos Biespecíficos/farmacologia , Antígenos CD19/imunologia , Antígenos CD20/imunologia , Imunoterapia/métodos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos NODRESUMO
ABSTRACT: Bruton tyrosine kinase inhibitors (BTKis) that target B-cell receptor signaling have led to a paradigm shift in chronic lymphocytic leukemia (CLL) treatment. BTKis have been shown to reduce abnormally high CLL-associated T-cell counts and the expression of immune checkpoint receptors concomitantly with tumor reduction. However, the impact of BTKi therapy on T-cell function has not been fully characterized. Here, we performed longitudinal immunophenotypic and functional analysis of pretreatment and on-treatment (6 and 12 months) peripheral blood samples from patients in the phase 3 E1912 trial comparing ibrutinib-rituximab with fludarabine, cyclophosphamide, and rituximab (FCR). Intriguingly, we report that despite reduced overall T-cell counts; higher numbers of T cells, including effector CD8+ subsets at baseline and at the 6-month time point, associated with no infections; and favorable progression-free survival in the ibrutinib-rituximab arm. Assays demonstrated enhanced anti-CLL T-cell killing function during ibrutinib-rituximab treatment, including a switch from predominantly CD4+ T-cell:CLL immune synapses at baseline to increased CD8+ lytic synapses on-therapy. Conversely, in the FCR arm, higher T-cell numbers correlated with adverse clinical responses and showed no functional improvement. We further demonstrate the potential of exploiting rejuvenated T-cell cytotoxicity during ibrutinib-rituximab treatment, using the bispecific antibody glofitamab, supporting combination immunotherapy approaches.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Rituximab , Monitorização Imunológica , Protocolos de Quimioterapia Combinada Antineoplásica , Ciclofosfamida , Imunoterapia , Linfócitos T CD8-PositivosRESUMO
Predicting the toxicity of cancer immunotherapies preclinically is challenging because models of tumours and healthy organs do not typically fully recapitulate the expression of relevant human antigens. Here we show that patient-derived intestinal organoids and tumouroids supplemented with immune cells can be used to study the on-target off-tumour toxicities of T-cell-engaging bispecific antibodies (TCBs), and to capture clinical toxicities not predicted by conventional tissue-based models as well as inter-patient variabilities in TCB responses. We analysed the mechanisms of T-cell-mediated damage of neoplastic and donor-matched healthy epithelia at a single-cell resolution using multiplexed immunofluorescence. We found that TCBs that target the epithelial cell-adhesion molecule led to apoptosis in healthy organoids in accordance with clinical observations, and that apoptosis is associated with T-cell activation, cytokine release and intra-epithelial T-cell infiltration. Conversely, tumour organoids were more resistant to damage, probably owing to a reduced efficiency of T-cell infiltration within the epithelium. Patient-derived intestinal organoids can aid the study of immune-epithelial interactions as well as the preclinical and clinical development of cancer immunotherapies.
Assuntos
Anticorpos Biespecíficos , Apoptose , Organoides , Linfócitos T , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Humanos , Organoides/imunologia , Linfócitos T/imunologia , Intestinos/imunologia , Imunoterapia/métodos , Molécula de Adesão da Célula Epitelial/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Feminino , Mucosa Intestinal/imunologiaRESUMO
Glioblastoma (GBM) harbors a highly immunosuppressive tumor microenvironment (TME) which influences glioma growth. Major efforts have been undertaken to describe the TME on a single-cell level. However, human data on regional differences within the TME remain scarce. Here, we performed high-depth single-cell RNA sequencing (scRNAseq) on paired biopsies from the tumor center, peripheral infiltration zone and blood of five primary GBM patients. Through analysis of >45,000 cells, we revealed a regionally distinct transcription profile of microglia (MG) and monocyte-derived macrophages (MdMs) and an impaired activation signature in the tumor-peripheral cytotoxic-cell compartment. Comparing tumor-infiltrating CD8+ T cells with circulating cells identified CX3CR1high and CX3CR1int CD8+ T cells with effector and memory phenotype, respectively, enriched in blood but absent in the TME. Tumor CD8+ T cells displayed a tissue-resident memory phenotype with dysfunctional features. Our analysis provides a regionally resolved mapping of transcriptional states in GBM-associated leukocytes, serving as an additional asset in the effort towards novel therapeutic strategies to combat this fatal disease.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Linfócitos T CD8-Positivos , Macrófagos/patologia , Glioma/genética , Leucócitos/patologia , Microambiente Tumoral/genética , Neoplasias Encefálicas/patologiaRESUMO
Therapeutic antibodies are widely used to treat severe diseases. Most of them alter immune cells and act within the immunological synapse; an essential cell-to-cell interaction to direct the humoral immune response. Although many antibody designs are generated and evaluated, a high-throughput tool for systematic antibody characterization and prediction of function is lacking. Here, we introduce the first comprehensive open-source framework, scifAI (single-cell imaging flow cytometry AI), for preprocessing, feature engineering, and explainable, predictive machine learning on imaging flow cytometry (IFC) data. Additionally, we generate the largest publicly available IFC dataset of the human immunological synapse containing over 2.8 million images. Using scifAI, we analyze class frequency and morphological changes under different immune stimulation. T cell cytokine production across multiple donors and therapeutic antibodies is quantitatively predicted in vitro, linking morphological features with function and demonstrating the potential to significantly impact antibody design. scifAI is universally applicable to IFC data. Given its modular architecture, it is straightforward to incorporate into existing workflows and analysis pipelines, e.g., for rapid antibody screening and functional characterization.
Assuntos
Comunicação Celular , Sinapses Imunológicas , Humanos , Fluxo de Trabalho , Aprendizado de MáquinaRESUMO
PURPOSE: Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. EXPERIMENTAL DESIGN: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single-cell RNA-sequencing of whole blood treated with CD20-TCB together with bulk RNA-sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity. RESULTS: Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1ß, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1ß, MCP-1, MIP-1α, MIP-1ß, and IP-10 release. Endothelial cells contribute to IL6 and IL1ß release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1ß). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB-mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity. CONCLUSIONS: Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs. See related commentary by Luri-Rey et al., p. 4320.
Assuntos
Anticorpos Biespecíficos , Fator de Necrose Tumoral alfa , Humanos , Camundongos , Animais , Quimiocina CCL3 , Quimiocina CCL4 , Anticorpos Biespecíficos/farmacologia , Interleucina-8 , Quimiocina CXCL10 , Interleucina-6 , Síndrome da Liberação de Citocina , Células Endoteliais , Inflamassomos , Citocinas , Linfócitos T , Dexametasona/farmacologia , RNARESUMO
Recent transcriptomic-based analysis of diffuse large B cell lymphoma (DLBCL) has highlighted the clinical relevance of LN fibroblast and tumor-infiltrating lymphocyte (TIL) signatures within the tumor microenvironment (TME). However, the immunomodulatory role of fibroblasts in lymphoma remains unclear. Here, by studying human and mouse DLBCL-LNs, we identified the presence of an aberrantly remodeled fibroblastic reticular cell (FRC) network expressing elevated fibroblast-activated protein (FAP). RNA-Seq analyses revealed that exposure to DLBCL reprogrammed key immunoregulatory pathways in FRCs, including a switch from homeostatic to inflammatory chemokine expression and elevated antigen-presentation molecules. Functional assays showed that DLBCL-activated FRCs (DLBCL-FRCs) hindered optimal TIL and chimeric antigen receptor (CAR) T cell migration. Moreover, DLBCL-FRCs inhibited CD8+ TIL cytotoxicity in an antigen-specific manner. Notably, the interrogation of patient LNs with imaging mass cytometry identified distinct environments differing in their CD8+ TIL-FRC composition and spatial organization that associated with survival outcomes. We further demonstrated the potential to target inhibitory FRCs to rejuvenate interacting TILs. Cotreating organotypic cultures with FAP-targeted immunostimulatory drugs and a bispecific antibody (glofitamab) augmented antilymphoma TIL cytotoxicity. Our study reveals an immunosuppressive role of FRCs in DLBCL, with implications for immune evasion, disease pathogenesis, and optimizing immunotherapy for patients.
Assuntos
Linfoma Difuso de Grandes Células B , Linfócitos T , Humanos , Camundongos , Animais , Linfoma Difuso de Grandes Células B/patologia , Fibroblastos/metabolismo , Linfonodos , Microambiente TumoralRESUMO
Despite the revolution of immunotherapy in cancer treatment, patients eventually progress due to the emergence of resistance. In this scenario, the selection of the tumor antigen can be decisive in the success of the clinical response. T cell bispecific antibodies (TCBs) are engineered molecules that include binding sites to the T cell receptor and to a tumor antigen. Using gastric CEA+/HER2+ MKN45 cells and TCBs directed against CEA or HER2, we show that the mechanism of resistance to a TCB is dependent on the tumor antigen. Acquired resistant models to a high-affinity-CEA-targeted TCB exhibit a reduction of CEA levels due to transcriptional silencing, which is reversible upon 5-AZA treatment. In contrast, a HER2-TCB resistant model maintains HER2 levels and exhibit a disruption of the interferon-gamma signaling. These results will help in the design of combinatorial strategies to increase the efficacy of cancer immunotherapies and to anticipate and overcome resistances.
Assuntos
Anticorpos Biespecíficos , Humanos , Anticorpos Biespecíficos/uso terapêutico , Antígeno Carcinoembrionário , Interferon gama/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T , Linhagem Celular TumoralRESUMO
Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.
Assuntos
Linfócitos T CD8-Positivos , Receptor de Morte Celular Programada 1 , Receptores de Interleucina-2 , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Infecções/tratamento farmacológico , Infecções/imunologia , Interleucina-2/imunologia , Interleucina-2/farmacologia , Interleucina-2/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/agonistas , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores de Interleucina-2/agonistasRESUMO
T-cell bispecific antibodies (TCB) are engineered molecules that bind both the T-cell receptor and tumor-specific antigens. Epidermal growth factor receptor variant III (EGFRvIII) mutation is a common event in glioblastoma (GBM) and is characterized by the deletion of exons 2-7, resulting in a constitutively active receptor that promotes cell proliferation, angiogenesis, and invasion. EGFRvIII is expressed on the surface of tumor cells and is not expressed in normal tissues, making EGFRvIII an ideal neoantigen target for TCBs. We designed and developed a novel 2+1 EGFRvIII-TCB with optimal pharmacologic characteristics and potent antitumor activity. EGFRvIII-TCB showed specificity for EGFRvIII and promoted tumor cell killing as well as T-cell activation and cytokine secretion only in patient-derived models expressing EGFRvIII. Moreover, EGFRvIII-TCB promoted T-cell recruitment into intracranial tumors. EGFRvIII-TCB induced tumor regression in GBM animal models, including humanized orthotopic GBM patient-derived xenograft models. Our results warrant the clinical testing of EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.
Assuntos
Anticorpos Biespecíficos , Neoplasias Encefálicas , Glioblastoma , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Citocinas , Receptores ErbB/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismoRESUMO
BACKGROUND: Dendritic cells (DCs) are professional antigen presenting cells that initiate immune defense to pathogens and tumor cells. Human tumors contain only few DCs that mostly display a non-activated phenotype. Hence, activation of tumor-associated DCs may improve efficacy of cancer immunotherapies. Toll-like receptor (TLR) agonists and interferons are known to promote DC maturation. However, it is unclear if DCs in human tumors respond to activation signals and which stimuli induce the optimal activation of human tumor DCs. METHODS: We first screened combinations of TLR agonists, a STING agonist and interferons (IFNs) for their ability to activate human conventional DCs (cDCs). Two combinations: TL8-506 (a TLR8 agonist)+IFN-γ and TL8-506+Poly(I:C) (a TLR3 agonist) were studied in more detail. cDC1s and cDC2s derived from cord blood stem cells, blood or patient tumor samples were stimulated with either TL8-506+IFN-γ or TL8-506+Poly(I:C). Different activation markers were analyzed by ELISA, flow cytometry, NanoString nCounter Technology or single-cell RNA-sequencing. T cell activation and migration assays were performed to assess functional consequences of cDC activation. RESULTS: We show that TL8-506 synergized with IFN-γ or Poly(I:C) to induce high expression of different chemokines and cytokines including interleukin (IL)-12p70 in human cord blood and blood cDC subsets in a combination-specific manner. Importantly, both combinations induced the activation of cDC subsets in patient tumor samples ex vivo. The expression of immunostimulatory genes important for anticancer responses including CD40, IFNB1, IFNL1, IL12A and IL12B were upregulated on stimulation. Furthermore, chemokines associated with CD8+ T cell recruitment were induced in tumor-derived cDCs in response to TL8-506 combinations. In vitro activation and migration assays confirmed that stimulated cDCs induce T cell activation and migration. CONCLUSIONS: Our data suggest that cord blood-derived and blood-derived cDCs are a good surrogate to study treatment responses in human tumor cDCs. While most cDCs in human tumors display a non-activated phenotype, TL8-506 combinations drive human tumor cDCs towards an immunostimulatory phenotype associated with Th1 responses on stimulation. Hence, TL8-506-based combinations may be promising candidates to initiate or boost antitumor responses in patients with cancer.
Assuntos
Neoplasias , Receptor 8 Toll-Like , Adjuvantes Imunológicos/farmacologia , Citocinas/metabolismo , Células Dendríticas , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Interleucina-12/metabolismo , Poli I-C/metabolismo , Poli I-C/farmacologiaRESUMO
T cell engaging therapies, like CAR-T cells and T cell engagers, redirect T cells toward tumor cells, facilitating the formation of a cytotoxic synapse and resulting in subsequent tumor cell killing. T cell receptor or CAR-T downstream signaling triggers a release of pro-inflammatory cytokines, which can induce a Cytokine Release Syndrome (CRS). The incidence of CRS is still hardly predictable among individuals and remains one of the major dose-limiting safety liabilities associated with on-target activity of T cell engaging therapies. This emphasizes the need to elaborate mitigation strategies, which reduce cytokine release while retaining efficacy. Here, we review pre-clinical and clinical approaches applied for the management of CRS symptoms in the context of T cell engaging therapies, highlighting the use of tyrosine kinase inhibitors as an emerging mitigation strategy. In particular, we focus on the effects of Bruton's tyrosine kinase (BTK), Src family including Lck, mammalian target of rapamycin (mTOR) and Janus tyrosine kinase (JAK) inhibitors on T cell functionality and cytokine release, to provide a rationale for their use as mitigation strategies against CRS in the context of T cell engaging therapies.
Assuntos
Síndrome da Liberação de Citocina , Receptores de Antígenos de Linfócitos T , Síndrome da Liberação de Citocina/etiologia , Citocinas , Humanos , Linfócitos TRESUMO
Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.
Assuntos
Anticorpos Biespecíficos , Antígeno Carcinoembrionário , Neoplasias do Colo , Linfócitos T , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Complexo CD3/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Humanos , Ativação Linfocitária , Organoides/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
T cell engagers represent a novel promising class of cancer-immunotherapies redirecting T cells to tumor cells and have some promising outcomes in the clinic. These molecules can be associated with a mode-of-action related risk of cytokine release syndrome (CRS) in patients. CRS is characterized by the rapid release of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6 and IL-1ß and immune cell activation eliciting clinical symptoms of fever, hypoxia and hypotension. In this work, we investigated the biological mechanisms triggering and amplifying cytokine release after treatment with T cell bispecific antibodies (TCBs) employing an in vitro co-culture assay of human PBMCs or total leukocytes (PBMCs + neutrophils) and corresponding target antigen-expressing cells with four different TCBs. We identified T cells as the triggers of the TCB-mediated cytokine cascade and monocytes and neutrophils as downstream amplifier cells. Furthermore, we assessed the chronology of events by neutralization of T-cell derived cytokines. For the first time, we demonstrate the contribution of neutrophils to TCB-mediated cytokine release and confirm these findings by single-cell RNA sequencing of human whole blood incubated with a B-cell depleting TCB. This work could contribute to the construction of mechanistic models of cytokine release and definition of more specific molecular and cellular biomarkers of CRS in the context of treatment with T-cell engagers. In addition, it provides insight for the elaboration of prophylactic mitigation strategies that can reduce the occurrence of CRS and increase the therapeutic index of TCBs.
Assuntos
Anticorpos Biespecíficos , Citocinas , Síndrome da Liberação de Citocina , Humanos , Neutrófilos , Linfócitos TRESUMO
BACKGROUND: T cell engaging therapies, like chimeric antigen receptor T cells and T cell bispecific antibodies (TCBs), efficiently redirect T cells towards tumor cells, facilitating the formation of a cytotoxic synapse and resulting in subsequent tumor cell killing, a process that is accompanied by the release of cytokines. Despite their promising efficacy in the clinic, treatment with TCBs is associated with a risk of cytokine release syndrome (CRS). The aim of this study was to identify small molecules able to mitigate cytokine release while retaining T cell-mediated tumor killing. METHODS: By screening a library of 52 Food and Drug Administration approved kinase inhibitors for their impact on T cell proliferation and cytokine release after CD3 stimulation, we identified mTOR, JAK and Src kinases inhibitors as potential candidates to modulate TCB-mediated cytokine release at pharmacologically active doses. Using an in vitro model of target cell killing by human peripheral blood mononuclear cells, we assessed the effects of mTOR, JAK and Src kinase inhibitors combined with 2+1 T cell bispecific antibodies (TCBs) including CEA-TCB and CD19-TCB on T cell activation, proliferation and target cell killing measured by flow cytometry and cytokine release measured by Luminex. The combination of mTOR, JAK and Src kinase inhibitors together with CD19-TCB was evaluated in vivo in non-tumor bearing stem cell humanized NSG mice in terms of B cell depletion and in a lymphoma patient-derived xenograft (PDX) model in humanized NSG mice in terms of antitumor efficacy. RESULTS: The effect of Src inhibitors differed from those of mTOR and JAK inhibitors with the suppression of CD19-TCB-induced tumor cell lysis in vitro, whereas mTOR and JAK inhibitors primarily affected TCB-mediated cytokine release. Importantly, we confirmed in vivo that Src, JAK and mTOR inhibitors strongly reduced CD19-TCB-induced cytokine release. In humanized NSG mice, continuous treatment with a Src inhibitor prevented CD19-TCB-mediated B cell depletion in contrast to mTOR and JAK inhibitors, which retained CD19-TCB efficacy. Ultimately, transient treatment with Src, mTOR and JAK inhibitors minimally interfered with antitumor efficacy in a lymphoma PDX model. CONCLUSIONS: Taken together, these data support further evaluation of the use of Src, JAK and mTOR inhibitors as prophylactic treatment to prevent occurrence of CRS.
Assuntos
Anticorpos Biespecíficos/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Imunoterapia/métodos , Inibidores de Janus Quinases/uso terapêutico , Inibidores de MTOR/uso terapêutico , Animais , Humanos , Inibidores de Janus Quinases/farmacologia , Inibidores de MTOR/farmacologia , CamundongosRESUMO
Glofitamab, a novel CD20xCD3, T-cell-engaging bispecific antibody, exhibited single-agent activity in Study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation, and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab's clinical activity, including pharmacodynamic profile, mode of action, and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in Study NP30179 received single-dose obinutuzumab pretreatment (1000 mg) 7 days before IV glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per the clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsy samples. Pharmacodynamic modulation was observed with glofitamab treatment, including dose-dependent induction of cytokines, and T-cell margination, proliferation, and activation in peripheral blood. Gene expression analysis of pretreatment tumor biopsy samples indicated that tumor cell intrinsic factors such as TP53 signaling are associated with resistance to glofitamab, but they may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence regarding glofitamab's mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.
Assuntos
Anticorpos Biespecíficos , Linfoma de Células B , Linfoma não Hodgkin , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD20/uso terapêutico , Humanos , Linfoma não Hodgkin/tratamento farmacológicoRESUMO
We present a novel approach for first-in-human (FIH) dose selection of the CD20xCD3 bispecific antibody, glofitamab, based on pharmacokinetic/pharmacodynamic (PKPD) assessment in cynomolgus monkeys to select a high, safe starting dose, with cytokine release (CR) as the PD endpoint. Glofitamab pharmacokinetics were studied in mice and cynomolgus monkeys; PKPD of IL-6, TNF-α and interferon-γ release following glofitamab, with/without obinutuzumab pretreatment (Gpt) was studied in cynomolgus monkeys. Potency differences for CR between cynomolgus monkeys and humans were determined by glofitamab incubation in whole blood of both species. The PKPD model for CR was translated to humans to project a starting dose that did not induce CR exceeding a clinically-predefined threshold. In cynomolgus monkeys, glofitamab showed a species-specific atypical high clearance, with and without B-cell debulking by Gpt. CR was related to glofitamab serum levels and B-cell counts. B-cell reduction by Gpt led to a marked decrease in CR. FIH starting dose (5 µg) was selected based on IL-6 release considering the markedly higher glofitamab in vitro potency in human vs monkey blood. This is a novel PKPD-based approach for selection of FIH starting dose for a CD20xCD3 bispecific antibody in B-cell lymphoma, evidenced in the glofitamab study, NP30179 (NCT03075696).
Assuntos
Anticorpos Biespecíficos , Linfoma de Células B , Animais , Citocinas , Humanos , Interleucina-6 , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Macaca fascicularis , CamundongosRESUMO
Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.