Cancer immunotherapy by targeting 4-1BB with immunostimulatory antibodies

Abstract

4-1BB (CD137) is a T cell activating receptor belonging to the TNF receptor superfamily that can betargeted by mAb to eradicate established tumours. 4-1BB is expressed on a multitude of hematopoietic cells, including CD8+, CD4+, regulatory (Treg) T cells, and dendritic cells (DCs). 4-1BB signalling promotes the proliferation, survival and effector function of conventional T cells and therapeutic 4-1BB antibodies are thought to mediate their effects through receptor agonism. 4-1BB functions as a DC survival factor, with 4-1BB ligation facilitating DC maturation, secretion of proinflammatory cytokines, and supporting the development of highly potent cytotoxic T lymphocytes (CTLs). CD11c+ DCs are critical for the response to anti-4-1BB therapy through cross-priming CTLs against tumour antigens. The specific role of targeting 4-1BB on DCs to generate anti-tumour immunity with anti-4-1BB mAb has not been established. Treg cells suppress anti-tumour immune responses with the presence of tumour-infiltrating Treg cells associated with poorer prognosis in many tumour types. Consequently, immunotherapeutic strategies have focused on targeting Treg cells by depleting mAb whilst promoting the activity of tumour-specific effector T cells (Teff). The role of 4-1BB signalling on Treg cells has not been established, with conflicting reports of the effect of 4-1BB ligation on Treg-mediated immunosuppression, expansion and survival. Tumour-infiltrating Treg cells highly express 4-1BB when compared with conventional T cells. This finding raised the possibility that anti-4-1BB mAb mediate their antitumour effects through depletion of intratumoural Treg cells. Moreover, precedence for a Treg cell dependent mechanism exists for mAb targeting the related receptors OX40 and GITR.

This thesis investigates the therapeutic activity of anti-4-1BB through its differential targeting of 4-1BB on DCs and Treg cells, and examines how antibody isotype selection impacts on this therapeutic mechanism of action. Antibody isotype is a critical determinant of the engagement of activatory and inhibitory FcγRs. Murine IgG1 and rat IgG1 mAb confer good agonistic potential through co-engagement of inhibitory FcγRs leading to receptor cross-linking and activation to promote CD8+ T cell mediated anti-tumour immunity. In contrast, murine IgG2a mAb have the potential to deplete target cells through activation of FcγR-expressing macrophages within the tumour microenvironment.

I generated novel mouse strains with conditional deletion of 4-1BB on CD11c+ cells (4-1BBCD11c-/-) and Foxp3+ Treg cells (4-1BBFoxp3-/-) to study the differential targeting of anti-4-1BB in murine tumour models. Agonist anti-4-1BB induces tumour growth delay in poorly immunogenic B16 tumours and the eradication of established EG7 tumours. Tumour-infiltrating lymphocyte (TILs) analysis demonstrates treatment with anti-4-1BB mIgG1 and anti-4-1BB parental rat IgG1 induces a highly polarised cytotoxic effector cell population which upregulate Eomesodermin, KLRG1 and Ki67 on intratumoural CD8+ T cells and increases the ratio of CD8+: Foxp3+ cells. EG7 tumours treated with anti-4-1BB mIgG2a show evidence of intratumoural Treg cell depletion whilst conserving agonist potential to enable CD8+ T cell proliferation.

4-1BBCD11c-/- and 4-1BB sufficient mice (4-1BBfl/fl) inoculated with B16 tumours and treated with anti-4-1BB plus Ova peptide demonstrated a clear difference in tumour protection between the two strains. The loss of 4-1BB on CD11c+cells reduced the efficacy of anti-4-1BB therapy highlighting the anti-cancer role of 4-1BB triggering on cDCs. Treatment of 4-1BBFoxp3-/- mice with anti-4-1BB mIgG2a was able to achieve complete tumour clearances, demonstrating that in the absence of 4-1BB ligation on Tregs, anti-4-1BB mIgG2a is able to generate responses through T cell costimulation. TIL analysis demonstrated that anti-4-1BB mIgG2a induced Treg cell depletion in 4-1BBfl/fl mice, which was abrogated in 4-1BBFoxp3-/- mice since the target was no longer present. Treg cell suppressionassays involving co-culture of purified CD4+ Foxp3- Teff and CD4+ Foxp3+ Treg cells showed anti-4-1BB is able topartially rescue Teff cells from the suppressive effect of Treg cells isolated from 4-1BBfl/fl mice but not from 4-1BBFoxp3-/-mice. This suggests that 4-1BB triggering on Treg cells by anti-4-1BB plays a role in modulating Tregmediated immunosuppression. This project provides mechanistic insights that have clinical relevance in optimising human anti-4-1BB mAb for cancer immunotherapy

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Identifiers

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

ORCID for Peter Johnson: orcid.org/0000-0003-2306-4974

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