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. Despite an increased progression-free survival upon therapy, 40–50% of patients develop relapse/refractory disease, therefore there remains an important medical need. 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. 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 ( 57 , 60 , 99 , 105 – 111 ). Additional chemoattraction and activation of T cells already present in the tumor is also likely ( 60 ).…”

“…Here, we utilized multi-parameter confocal imaging coupled with advanced spatial analysis using histocytometry and CytoMAP ( 1 , 25 ) 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 ( 50 , 57 , 78 , 79 ). 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 ( 50 , 79 ).…”

“…In addition to T cell infiltration, intercellular interactions directly within the tumor tissues have been linked with positive response outcomes (56)(57)(58). In particular, interferon gamma (IFNg) 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 (CXCL9, CXCL10, and interleukin-12 (IL-12), which in turn promote amplified recruitment, proliferation and differentiation of CD8 T cells (57,59,60). 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.…”

Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins

“…Glofitamab showed potent tumor cell killing and antitumor efficacy in preclinical in vitro, ex vivo and in vivo lymphoma models, as well as superiority over the respective conventional heterodimeric 1 + 1 TCB formats as a consequence of its head-to-tail orientation and bivalent binding to CD20, allowing pre-treatment with obinutuzumab, in this case as a strategy to reduce the incidence of cytokine-release syndrome by glofitamab. 86 , 141 , 142 Based on the clinical efficacy and safety in the Phase 1 clinical trial in relapsed/refractory non-Hodgkin lymphoma (NHL) patients and particularly the high rate of durable complete responses, 143 , 144 glofitamab is currently being evaluated in multiple clinical trials in lymphoma patients, including trials in patients relapsed after CAR-T cell therapy (NCT04703686) and in Phase 3 clinical trials in relapsed/refractory diffuse large B cell lymphoma patients (NCT04077723, NCT04408638). 145 No anti-drug antibodies recognizing glofitamab were detected in the Phase 1 clinical study.…”

T Cell Bispecific Antibodies: An Antibody-Based Delivery System for Inducing Antitumor Immunity

“…This issue is important because if the latter is true, it means that T-BsAbs could recruit peripheral T cells into a tumor even if the tumor contains no preexisting T cells. Although this question remains unanswered, another critical question, namely, whether resident intratumoral T cells must be present before T-BsAb treatment in order to further recruit peripheral T cells into the tumor, has been studied in a humanized mouse model [75]. Cremasco et al prepared two separately labeled T cell populations with the aim of distinguishing resident T cells, which were intradermally inoculated with cancer cells, from recruited T cells, which were intravenously injected as peripheral T cells.…”