Multivalent and bispecific immunostimulatory antibodies for cancer immunotherapy

Abstract

Immunostimulatory antibodies (ISAs) targeting TNFRSF members have shown anti-tumour efficacy in pre-clinical cancer models, but this has not translated into clinical success, primarily due to lack of efficacy and dose-limiting toxicity. Antibody engineering can be utilised to overcome the issues with ISAs. Tetravalent anti-CD27 antibodies were developed by attaching an additional F(ab) fragments to the N-terminus of the of each IgG VH domain. This enhanced the potency of anti-CD27 ISAs through increased avidity and improved receptor clustering. The tetravalent anti-CD27 antibodies also provided greater T cell co-stimulation, enhancing proliferation and anti-tumour responses. Optimal activity of the tetravalent anti-CD27 antibodies was still dependent on FcγR-crosslinking, which could increase the risk of on-target, off-tumour toxicity. To overcome FcγR-dependency and localise the activity of ISAs to the tumour microenvironment, we produced a range of bispecific antibodies targeting m4-1BB and B7-H3. In vitro and in vivo assessment identified that a tetravalent bispecific antibody (2x2) format was the most effective. This format was then used to show that anti-m4-1BB x anti-B7-H3 2x2 bispecific antibodies provided robust anti-tumour immunity and explore whether there was an optimal T cell expressed TNFRSF co-stimulatory receptor to target. Co-stimulation through m4-1BB provided the best T cell co-stimulation, resulting in increased expression of granzyme B and anti-tumour activity in an MC38 hB7-H3 model, compared to 2x2 bispecific antibodies targeting CD27, GITR and OX40. Whilst the anti-m4-1BB x anti-B7-H3 2x2 bispecific antibody greatly enhanced anti-tumour immunity, it was not toxic so the effect of the bispecific format on tumour localisation and reducing dose-limiting toxicity could not be assessed. To assess toxicity, anti-hCD40 x anti-B7-H3 2x2 bispecific antibodies were produced. Anti-hCD40 bispecific antibodies showed that B7-H3 mediated crosslinking enhanced anti-hCD40 antibody activity, increasing NF-κB activation and B cell proliferation. However, anti-hCD40 x anti-B7-H3 bispecific antibodies did not result in decreased toxicity or enhanced anti-tumour activity. Engineered ISAs showed enhanced activity compared to the parental ISAs, with the tetravalent anti-CD27 ISAs and anti-hCD40 bispecific antibodies requiring further optimisation to induce robust anti-tumour immunity. However, the anti-m4-1BB x anti-B7-H3 2x2 bispecific antibody induced anti-tumour immunity in multiple tumour models and further exploration should look to identify the mechanism of action and potentially humanise the bispecific antibody for clinical translation.

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Identifiers

ORCID for Marcus Alexander Widdess: orcid.org/0000-0002-2063-8090

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

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

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