An Immune Atlas of Clear Cell Renal Cell Carcinoma.

Immune cells in the tumor microenvironment modulate cancer progression and are attractive therapeutic targets. Macrophages and T cells are key components of the microenvironment, yet their phenotypes and relationships in this ecosystem and to clinical outcomes are ill defined. We used mass cytometry with extensive antibody panels to perform in-depth immune profiling of samples from 73 clear cell renal cell carcinoma (ccRCC) patients and five healthy controls. In 3.5 million measured cells, we identified 17 tumor-associated macrophage phenotypes, 22 T cell phenotypes, and a distinct immune composition correlated with progression-free survival, thereby presenting an in-depth human atlas of the immune tumor microenvironment in this disease. This study revealed potential biomarkers and targets for immunotherapy development and validated tools that can be used for immune profiling of other tumor types.

PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells.

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.

Ibrutinib-based therapy reinvigorates CD8+ T cells compared to chemoimmunotherapy: immune monitoring from the E1912 trial.

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.

CD19-CD28: an affinity-optimized CD28 agonist for combination with glofitamab (CD20-TCB) as off-the-shelf immunotherapy.

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.

The PI3Kδ-Selective Inhibitor Idelalisib Minimally Interferes with Immune Effector Function Mediated by Rituximab or Obinutuzumab and Significantly Augments B Cell Depletion In Vivo.

Idelalisib is a highly selective oral inhibitor of PI3Kδ indicated for the treatment of patients with relapsed chronic lymphocytic leukemia in combination with rituximab. Despite additive clinical effects, previous studies have paradoxically demonstrated that targeted therapies potentially negatively affect anti-CD20 mAb effector mechanisms. To address these potential effects, we investigated the impact of PI3Kδ inhibition by idelalisib on the effector mechanisms of rituximab and obinutuzumab. At clinically relevant concentrations, idelalisib minimally influenced rituximab- and obinutuzumab-mediated Ab-dependent cellular cytotoxicity and phagocytosis on human lymphoma cell lines, while maintaining the superiority of obinutuzumab-mediated Ab-dependent cellular cytotoxicity. Consistent with this, idelalisib did not influence obinutuzumab-mediated B cell depletion in whole-blood B cell-depletion assays. Further, idelalisib significantly enhanced obinutuzumab-mediated direct cell death of chronic lymphocytic leukemia cells. In murine systems, in vivo inhibition of PI3Kδ minimally interfered with maximal rituximab- or obinutuzumab-mediated depletion of leukemic targets. In addition, the duration of rituximab- and obinutuzumab-mediated depletion of leukemia cells was extended by combination with PI3Kδ inhibition. Collectively, these data demonstrate that PI3Kδ inhibition does not significantly affect the effector mechanisms induced by rituximab or obinutuzumab and provides an effective in vivo therapeutic combination. Therefore, combinations of obinutuzumab and idelalisib are currently being assessed in clinical studies.

Engineering therapeutic bispecific antibodies using CrossMab technology.

Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct association of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technology that enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecific antibody. This versatile technology allows the generation of different bispecific antibody formats including asymmetric heterodimeric monovalent 1 + 1 bispecific antibodies and asymmetric heterodimeric bispecific antibodies with 2 + 1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technology as well as the generation of tetravalent symmetric bispecific antibodies with 2 + 2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale production of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technology entered clinical development, and additional CrossMabs are in late preclinical development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technology based bispecific antibodies as well as their therapeutic application.

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.

Antitumour activity of the glycoengineered type II anti-CD20 antibody obinutuzumab (GA101) in combination with the MDM2-selective antagonist idasanutlin (RG7388).

OBJECTIVES: To investigate whether the glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab (GA101) combined with the selective MDM2 antagonist idasanutlin (RG7388) offers superior efficacy to monotherapy in treating B-lymphoid malignancies in preclinical models. METHODS: The combined effect of obinutuzumab or rituximab plus idasanutlin on direct cell death/apoptosis induction and antibody-dependent cellular cytotoxicity (ADCC) was evaluated using p53 wild-type Z-138 and DoHH-2 lymphoma cells. Furthermore, whole blood B-cell depletion was analysed, and tumour growth inhibition was evaluated in subcutaneous xenograft models. RESULTS: Idasanutlin induced concentration-dependent death of Z-138 and DoHH-2 cells. At concentrations >10-100 nm, idasanutlin enhanced obinutuzumab-induced death of DoHH-2 and Z-138 cells without negatively impacting obinutuzumab-mediated ADCC, natural killer cell activation or whole blood B-cell depletion. In the Z-138 xenograft model, a suboptimal dose of obinutuzumab with idasanutlin yielded substantial tumour growth inhibition and prolonged survival in a time-to-event analysis. In the DoHH-2 model, idasanutlin plus obinutuzumab showed superior tumour growth inhibition to idasanutlin plus rituximab. CONCLUSIONS: Obinutuzumab plus idasanutlin enhanced cell death of p53 wild-type tumour cells vs. rituximab plus idasanutlin without affecting obinutuzumab-mediated ADCC or B-cell depletion and showed robust antitumour efficacy in xenograft models, strongly supporting the investigation of this combination in clinical trials.

Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity.

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids.

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.

Myeloid-T cell interplay and cell state transitions associated with checkpoint inhibitor response in melanoma.

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.

Securin and separase modulate membrane traffic by affecting endosomal acidification.

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.

Explainable machine learning for profiling the immunological synapse and functional characterization of therapeutic antibodies.

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.

Single-cell characterization of human GBM reveals regional differences in tumor-infiltrating leukocyte activation.

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.

Dissecting the Mechanisms Underlying the Cytokine Release Syndrome (CRS) Mediated by T-Cell Bispecific Antibodies.

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.

Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma.

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.

Novel strategies for the mitigation of cytokine release syndrome induced by T cell engaging therapies with a focus on the use of kinase inhibitors.

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.

Dissecting the mechanism of cytokine release induced by T-cell engagers highlights the contribution of neutrophils.

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.

The target antigen determines the mechanism of acquired resistance to T cell-based therapies.

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.