Preclinical evaluation of novel immunotherapeutic strategies targeting the tumour microenvironment

Abstract

Although monoclonal antibodies (mAbs) have become one of the main pillars of cancer treatment, targeting solid malignancies has been challenging mainly due to the lack of tumour-specific targets and the absence of T-cell infiltrate. Immunomodulatory approaches targeting the tumour microenvironment (TME) could potentially mediate anti-tumour immune responses either by allowing T-cell infiltration and/or activating T-cells, overcoming these obstacles. Tumour Endothelial Marker 8 (TEM8) and Lymphotoxin β receptor (LtβR) are two receptors expressed within the TME that so far have caught little attention in immunomodulatory strategies. The overall goal of this thesis is to evaluate different antibody-based approaches to target TEM8 and LtβR in cancer. As TEM8 is a tumour specific marker, anti-TEM8 antibodies were generated in different formats and characterised. First, an anti-TEM8 x anti-CD3 bispecific antibody (bsAb) was generated as a novel way to mediate a tumour-specific T-cell stimulation. Although in vitro assays confirmed a TEM8-specific CD8+ T-cell activation, the bsAb presented with unacceptable toxicities in vivo. On the other hand, treatment with anti-TEM8 (clone L2) mAbs has been promising, presenting tumour growth inhibition in xenograft models. However, further evaluation was required to optimise their mechanism of action. The anti-TEM8 mAbs were designed in our facility in different isotypes (mIgG1, mIgG2a, mIgG2a N297A). Anti-TEM8 mAbs presented tumour specificity in vivo, delaying tumour growth and prolonging mouse survival in syngeneic mouse models, presenting a reduction in tumour-associated vasculature. Treatment with Fc-inert mIgG2a N297A or in Gamma KO mice revealed a strong dependency of the antibody anti-tumour efficacy on Fc-mediated effector mechanisms. The anti-tumour efficacy of the anti-TEM8 mAbs may involve multiple pathways, ranging from Fc-mediated effector functions to the control of the TME through inhibition of tumour vascularisation. In addition, anti-TEM8 combination treatment with anti-4-1BB antibodies effectively enhanced the effect of monotherapies, providing an alternative combination treatment approach. An alternative therapeutic target for solid tumours is LtβR that has a pivotal role in the formation of high endothelial venules (HEV) in lymphoid structures and therefore is indirectly involved in T-cell infiltration and activation. Different formats of the anti-LtβR agonistic antibodies (clone 4H8) were designed (mIgG1, mIgG1 tetravalent and their Fc-inert counterparts containing the N297Q mutation) to define the best approach for optimal LtβR agonism. In vitro and in vivo evaluation of the antibodies revealed that tetravalency enhances the agonistic effect of the antibodies. The anti-tumour effect of anti-LtβR antibodies was highly dependent on CD8+ T-cells but other immune cells could also be involved. Finally, a novel anti-LtβR x anti-B7-H3 bsAb was generated as a way to localise LtβR treatment in the TME. Preliminary evidence confirmed the vitro functional activity of the antibody and its capability for HEV formation. Overall, the therapeutic efficacy of anti-TEM8 and anti-LtβR antibodies was confirmed in vitro and in vivo, providing further investigation of their underlying mechanism of action and presenting such approaches as potential therapies for patients with solid malignancies.

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Identifiers

ORCID for Anastasia Pakidi: orcid.org/0000-0001-7048-5734

ORCID for Aymen Al-Shamkhani: orcid.org/0000-0003-0727-4189

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