Cross-Linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

Abstract

Diffuse large B cell lymphomas (DLBCL) are a highly heterogeneous subtype of Non Hodgkin Lymphoma (NHL), accounting for about 25% of NHL [1]. Despite an increased progression-free survival upon therapy, 40-50% of patients develop relapse/refractory disease, therefore there remains an important medical need [2]. T cell recruiting therapies, such as the CD20xCD3 T cell bi-specific antibody CD20-TCB (RG6026 or glofitamab), represent a novel approach to target all stages of DLBCL, especially those that fail to respond to multiple lines of treatment [3, 4]. We aimed for a better understanding of the molecular features related to the mode of action (MoA) of CD20-TCB in inducing Target/T cell synapse formation and human T cell recruitment to the tumor. To directly evaluate the correlation between synapse, cytokine production and anti-tumor efficacy using CD20-TCB, we developed an innovative preclinical human DLBCL in vivo model that allowed tracking in vivo human T cell dynamics by multiphoton intravital microscopy (MP-IVM). By ex vivo and in vivo approaches, we revealed that CD20-TCB is inducing strong and stable synapses between human T cell and tumor cells, which are dependent on the dose of CD20-TCB and on LFA-1 activity but not on FAS-L. Moreover, despite CD20-TCB being a large molecule (194.342 kDa), we observed that intra-tumor CD20-TCB-mediated human T cell-tumor cell synapses occur within 1 hour upon CD20-TCB administration. These tight interactions, observed for at least 72 hours post TCB administration, result in tumor cell cytotoxicity, resident T cell proliferation and peripheral blood T cell recruitment into tumor. By blocking the IFNγ-CXCL10 axis, the recruitment of peripheral T cells was abrogated, partially affecting the efficacy of CD20-TCB treatment which rely only on resident T cell proliferation. Altogether these data reveal that CD20-TCB’s anti-tumor activity relies on a triple effect: i) fast formation of stable T cell-tumor cell synapses which induce tumor cytotoxicity and cytokine production, ii) resident T cell proliferation and iii) recruitment of fresh peripheral T cells to the tumor core to allow a positive enhancement of the anti-tumor effect.

Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated with Anti-Tumor Immunity

“…In turn, this could lead to continued localized seeding of tumors with the generated effector T cells. Inflammatory signals produced by the activated T cells could also lead to further activation of neighboring myeloid and endothelial cells, promoting recruitment of additional resource and effector T cells from the vasculature, which has been observed after both checkpoint blockade and CEA-TCB therapies (Joyce and Fearon, 2015; Spitzer et al, 2017; Huang et al, 2018; Munn and Jain, 2019; Chow et al, 2019; Wu et al, 2020; Zhang et al, 2021; Cremasco et al, 2021; Pauken et al, 2021; Connolly et al, 2021). Additional chemoattraction and activation of T cells already present in the tumor is also likely (Chow et al, 2019).…”

“…In addition to T cell infiltration, intercellular interactions directly within the tumor tissues have been linked with positive response outcomes (Böttcher et al, 2018; Cremasco et al, 2021; Ozga et al, 2021). In particular, interferon gamma (IFN□) produced by CD8 T cells in response to checkpoint blockade, as well as with TCB immunotherapy, has been shown to enhance maturation of intra-tumoral DCs, leading to increased production of chemokine CXC ligand (CXCL)9, CXCL10, and interleukin-12 (IL-12), which in turn promote amplified recruitment, proliferation and differentiation of CD8 T cells (Garris et al, 2018; Chow et al, 2019; Cremasco et al, 2021). Such positive feedback between DCs and CD8 T cells requires direct cell-cell crosstalk, suggesting the need for extensive communication between innate and adaptive immune cells within tumors.…”

“…Here, we utilized multi-parameter confocal imaging coupled with advanced spatial analysis using histocytometry and CytoMAP (Gerner et al, 2012; Stoltzfus et al, 2020a) to examine the complexity of immune cell organization within partially and poorly infiltrated tumors during immunotherapy. To this end, mice bearing MC38 colon carcinomas or KPC pancreatic adenocarcinomas engineered to express human carcinoembryonic antigen (CEA) were treated with CEA-TCB murine surrogate antibody and / or with anti-PD-L1 checkpoint inhibitor, both of which can synergize to promote enhanced CD8 T cell immunity (Bacac et al, 2016a, 2016b; Sam et al, 2020; Cremasco et al, 2021). As expected, CEA-TCB monotherapy and CEA-TCB plus anti-PD-L1 combination therapy led to increased CD8 T cell numbers and decreased tumor burden, indicating control of tumor growth by the immune system (Bacac et al, 2016a; Sam et al, 2020).…”