Combination CD200R/PD-1 blockade in a humanised mouse model

There is an increasing number of immune-checkpoint inhibitors being developed and approved for cancer immunotherapy. Most of the new therapies aim to reactivate tumour-infiltrating T cells, which are responsible for tumour killing. However, in many tumours, the most abundant infiltrating immune cells are macrophages and myeloid cells, which can be tumour-promoting as well as tumouricidal. CD200R was initially identified as a myeloid-restricted, inhibitory immune receptor, but was subsequently also found to be expressed within the lymphoid lineage. Using a mouse model humanised for CD200R and PD-1, we investigated the potential of a combination therapy comprising nivolumab, a clinically approved PD-1 blocking antibody, and OX108, a CD200R antagonist. We produced nivolumab as a murine IgG1 antibody and validated its binding activity in vitro as well as ex vivo. We then tested the combination therapy in the immunogenic colorectal cancer model MC38 as well as the PD-1 blockade-resistant lung cancer model LLC1, which is characterised by a large number of infiltrating myeloid cells, making it an attractive target for CD200R blockade. No significant improvement of overall survival was found in either model, compared to nivolumab mIgG1 monotherapy. There was a trend for more complete responses in the MC38 model, but investigation of the infiltrating immune cells failed to account for this. Importantly, MC38 cells expressed low levels of CD200, whereas LLC1 cells were CD200-negative. Further investigation of CD200R-blocking antibodies in tumours expressing high levels of CD200 could be warranted

Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti–PD-1 antibody that bound membrane-proximally excluded CD45, triggered SHP2 phosphatase recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti–PD-1 blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer

A structure-function analysis shows SARS-CoV-2 BA.2.86 balances antibody escape and ACE2 affinity.

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility

Interplay between LARP1 and MYC - a two-way regulation?

There is increasing evidence that several steps of carcinogenesis including initiation, progression and chemotherapy resistance are regulated by post-transcriptional mechanisms. One such example is the RNA-binding protein LARP1 which has been shown to drive tumorigenesis and is highly expressed in several epithelial cancers, where it correlates with poor prognosis. Interestingly, there is huge overlap between the transcriptome of MYC and the interactome of LARP1. In this project, I investigated whether there was co-regulation between MYC and LARP1, which could explain the mechanism through which LARP1 promotes cancer progression. In order to determine whether MYC drives the transcription of LARP1, siRNA knockdown of MYC was conducted in the cisplatin-resistant ovarian cancer cell lines OVCAR-3 and OVCAR- 8. While there are numerous ChIP- and RNA-seq datasets available suggesting a strong regulatory mechanism of MYC in various cell lines, only a modest change in LARP1 levels was observed after MYC depletion. To investigate whether MYC directly binds to the LARP1 promoter, chromatin immunoprecipitation was performed and analysed by qPCR. Indeed, MYC strongly binds the long isoform of LARP1, compared to IgG control. The knockdown experiment was repeated with a LARP1 targeting siRNA to analyse a potential stabilisation of MYC by LARP1. Upon LARP1 depletion, there was no significant effect on MYC levels or localisation, assessed by western blot and immunocytochemistry. While MYC had a modest impact on LARP1 expression, this thesis did not demonstrate a direct regulation of MYC by LARP1. Future work will elaborate on the idea that LARP1 regulates the translation of MYC target genes and is therefore a regulator of the ‘MYC signal’. Should this prove to be the case, this could evolve as another option to target the – currently – ‘undruggable’ MYC. Finally, this interaction would implicate LARP1 as a promising target for rational drug design in MYC driven cancers.</p

Interplay between LARP1 and MYC - a two-way regulation?

There is increasing evidence that several steps of carcinogenesis including initiation, progression and chemotherapy resistance are regulated by post-transcriptional mechanisms. One such example is the RNA-binding protein LARP1 which has been shown to drive tumorigenesis and is highly expressed in several epithelial cancers, where it correlates with poor prognosis. Interestingly, there is huge overlap between the transcriptome of MYC and the interactome of LARP1. In this project, I investigated whether there was co-regulation between MYC and LARP1, which could explain the mechanism through which LARP1 promotes cancer progression. In order to determine whether MYC drives the transcription of LARP1, siRNA knockdown of MYC was conducted in the cisplatin-resistant ovarian cancer cell lines OVCAR-3 and OVCAR- 8. While there are numerous ChIP- and RNA-seq datasets available suggesting a strong regulatory mechanism of MYC in various cell lines, only a modest change in LARP1 levels was observed after MYC depletion. To investigate whether MYC directly binds to the LARP1 promoter, chromatin immunoprecipitation was performed and analysed by qPCR. Indeed, MYC strongly binds the long isoform of LARP1, compared to IgG control. The knockdown experiment was repeated with a LARP1 targeting siRNA to analyse a potential stabilisation of MYC by LARP1. Upon LARP1 depletion, there was no significant effect on MYC levels or localisation, assessed by western blot and immunocytochemistry. While MYC had a modest impact on LARP1 expression, this thesis did not demonstrate a direct regulation of MYC by LARP1. Future work will elaborate on the idea that LARP1 regulates the translation of MYC target genes and is therefore a regulator of the âMYC signalâ. Should this prove to be the case, this could evolve as another option to target the â currently â âundruggableâ MYC. Finally, this interaction would implicate LARP1 as a promising target for rational drug design in MYC driven cancers.</p

New approaches to treating cancer using immune-checkpoint blockade

While immune-checkpoint blockade has truly revolutionised cancer treatment in many aspects, the benefits remain limited to certain tumour types. Furthermore, within those tumours that are likely to improve upon treatment, only a fraction of patients actually respond, which highlights the need for novel approaches to enhance this therapy. The work outlined in this thesis aimed to address this issue by employing two distinct strategies to improve on PD-1 blockade using the clinically-approved antibody nivolumab. As anti-PD-1 antibodies mostly act on T cells, the first approach trialled a new combination therapy comprising nivolumab mIgG1 and OX108, a CD200R blocking antibody. CD200R is an inhibitory immune receptor preferentially expressed on myeloid cells and, therefore, blocking PD-1 and CD200R should reactivate T cells and myeloid cells simultaneously. While the PD-1 blockade resistant model LLC1 did not exhibit any improvements upon combination therapy, the sensitive model MC38 produced a trend towards more complete responses, which might be linked to the expression of CD200 on these cells. The second strategy focused on improving the design of monoclonal antibodies used for immune-checkpoint blockade in order to enhance their efficacy. Based on the kinetic segregation model for receptor triggering, a potential agonistic effect of nivolumab mIgG1 on PD-1 was investigated, a phenomenon that should be reduced upon increasing the size of the receptor/antibody/FcγR complex. While nivolumab mIgG1 indeed exhibited FcγR-dependent agonistic activity in vitro, this could be diminished by (i) employing two different approaches to extend nivolumab mIgG1 or (ii) mutation of the FcγR-binding domain (nivolumab mIgG1 D265A). Intriguingly, whereas the extended antibodies produced a significantly decreased enrichment of PD-1 in ‘close contacts’ compared to nivolumab mIgG1 D265A, this did not translate into an increased T-cell activation. Finally, nivolumab mIgG1 D265A exhibited increased efficacy in the MC38 colorectal cancer model compared to nivolumab mIgG1, in accordance with previous studies investigating the confounding effects of FcγR binding on anti-PD-1 antibodies

SOX2 is essential for in vivo reprogramming of seminoma-like TCam-2 cells to an embryonal carcinoma-like fate

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