Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

Tertiary Lymphoid Structures as a Predictive Biomarker of Response to Cancer Immunotherapies.

Tertiary lymphoid structures (TLS) are ectopic lymphoid formations which are formed under long-lasting inflammatory conditions, including tumours. TLS are composed predominantly of B cells, T cells and dendritic cells, and display various levels of organisation, from locally concentrated aggregates of immune cells, through clearly defined B cell follicles to mature follicles containing germinal centres. Their presence has been strongly associated with improved survival and clinical outcome upon cancer immunotherapies for patients with solid tumours, indicating potential for TLS to be used as a prognostic and predictive factor. Although signals involved in TLS generation and main cellular components of TLS have been extensively characterised, the exact mechanism by which TLS contribute to the anti-tumour response remain unclear. Here, we summarise the most recent development in our understanding of their role in cancer and in particular in the response to cancer immunotherapy. Deciphering the relationship between B cells and T cells found in TLS is a highly exciting field of investigation, with the potential to lead to novel, B-cell focused immunotherapies.

Plinabulin, a Distinct Microtubule-Targeting Chemotherapy, Promotes M1-Like Macrophage Polarization and Anti-tumor Immunity.

Reprogramming tumor infiltrating myeloid cells to elicit pro-inflammatory responses is an exciting therapeutic maneouver to improve anti-tumor responses. We recently demonstrated that a distinct microtubule-targeting drug, plinabulin-a clinical-stage novel agent-modulates dendritic cell maturation and enhances anti-tumor immunity. Here, we investigated the effects of plinabulin on macrophage polarization in vitro and in vivo. Plinabulin monotherapy induced significant tumor growth inhibition in mice bearing subcutaneous MC38 colon cancer. Importantly, the regressing tumors were characterized by an increase in M1-like/M2-like tumor-associated macrophages (TAM) ratio. The efficacy of plinabulin remained unaltered in T cell-deficient Rag2-/- mice, suggesting an important role of macrophages in driving the drug's anti-tumor effect. Exposure of murine and healthy human macrophages to plinabulin induced polarization toward the M1 phenotype, including increased expression of co-stimulatory molecules CD80, CD86 and pro-inflammatory cytokines IL-1ß, IL-6, and IL-12. M2-associated immunosuppressive cytokines IL-10 and IL-4 were reduced. This pro-inflammatory M1-like skewing of TAMs in response to plinabulin was dependent on the JNK pathway. Functionally, plinabulin-polarized human M1 macrophages directly killed HuT 78 tumor cells in vitro. Importantly, plinabulin induced a functional M1-like polarization of tumor infiltrating macrophages in murine tumors as well as in tumor samples from ovarian cancer patients, by preferentially triggering M1 proliferation. Our study uncovers a novel immunomodulatory effect of plinabulin in directly triggering M1 polarization and proliferation as well as promoting TAM anti-tumoral effector functions.

Fibroblast activation protein-targeted-4-1BB ligand agonist amplifies effector functions of intratumoral T cells in human cancer.

BACKGROUND: The costimulatory receptor 4-1BB (CD137, TNFRSF9) plays an important role in sustaining effective T cell immune responses and is investigated as target for cancer therapy. Systemic 4-1BB directed therapies elicit toxicity or low efficacy, which significantly hampered advancement of 4-1BB-based immunotherapy. Therefore, targeted delivery of 4-1BB agonist to the tumor side is needed for eliciting antitumor efficacy while avoiding systemic toxicity. METHODS: We analyzed the immunostimulatory properties of a fibroblast activation protein (FAP)-targeted 4-1BB agonist (FAP-4-1BBL) by assessing tumor-infiltrating lymphocytes' (TIL) activity from patients with non-small cell lung cancer and epithelial ovarian cancer. RESULTS: Combination treatment with FAP-4-1BBL and T cell receptor stimulation by either anti-CD3 or T cell bispecific antibodies significantly enhanced TIL activation and effector functions, including T cell proliferation, secretion of proinflammatory cytokines and cytotoxicity. Notably, costimulation with FAP-4-1BBL led to de novo secretion of interleukin (IL)-13. This was associated with cytokine-mediated tumor cell apoptosis, which was partially dependent on IL-13 alpha 1/2 receptors and STAT6 phosphorylation. CONCLUSIONS: Our study provides mechanistic insights into T cell stimulation induced by FAP-4-1BBL in primary human tumors and supports the investigation of FAP-4-1BBL compound in early clinical trials.

Heterogeneity of Phenotype and Function Reflects the Multistage Development of T Follicular Helper Cells.

T follicular helper cells (Tfh) provide crucial signals for germinal center (GC) formation, but Tfh populations are heterogeneous. While PD1hi Tfh are important in the GC response, the function of the PD1lo Tfh-like subset is unknown. We show that these cells, like the PD1hi GC-Tfh, depend upon B cells; however, their entry to follicles is independent of CXCR5 or cognate interactions with B cells. The differentiation into PD1hi Tfh is dependent on MHC class II interactions with B cells and requires CXCR5. Our data suggest a Tfh differentiation pathway that is initially B cell-independent, then dependent on non-cognate B cell interactions, and finally following cognate interaction with B cells and CXCR5-ligands allows the formation of GC-Tfh. The PD1lo Tfh-like cells make early cytokine responses and may represent precursors of CD4 memory cells.

Plasmacytoid Dendritic Cells Respond Directly to Apoptotic Cells by Secreting Immune Regulatory IL-10 or IFN-α.

Plasmacytoid dendritic cells (pDCs) play a pivotal role in driving the autoimmune disease systemic lupus erythematosus, via the secretion of IFN-α in response to nuclear self-antigens complexed with autoantibodies. Apoptotic cells, generated at sites of inflammation or secondary lymphoid organs, are exposed to activated pDCs and also express the same nuclear antigens on their cell surface. Here, we show that in the absence of autoantibodies, activated pDCs directly respond to apoptotic cell-expressed chromatin complexes by secreting IL-10 and IL-6, which also induces T cells to secrete IL-10. Conversely, when activated by the viral mimetic CpG-A, apoptotic cells enhance their secretion of IFN-α. This study demonstrates that activated pDCs respond directly to apoptotic cells and may maintain tolerance via IL-10, or promote inflammation through secretion of IFN-α, depending on the inflammatory context.