The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity

Background Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. Methods CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. Results CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. Conclusions CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

Harnessing iNKT cells to improve in situ vaccination for cancer therapy

<p>Toll-like receptor (TLR) agonism in combination with the activation of type I NKT (iNKT) cells through systemic administration of their respective agonists has been shown to have a cooperative effect on activating antigen-presenting cells, stimulating cytokine production, and inducing adaptive immune responses to co-administered antigens. Here, it was hypothesised that it might be possible to harness these activities to treat solid tumours locally via intratumoural treatment to combat tumour growth while reducing toxicity to other organs. An intratumoural treatment model combining the stimulatory activity of unmethylated DNA oligonucleotides consisting of synthetic cytosine-guanine motifs (CpG), a TLR9 agonist, with activation of iNKT cells through administration of the CD1d-binding iNKT agonist α-galactosylceramide (α-GalCer) intratumourally was shown to have significant anti-tumour activity. The treatment regimen showed superior efficacy to that achieved with either agent alone in several in vivo models representing different types of cancer. In some models, the combination of α-GalCer and CpG was effective at inducing the complete rejection of both treated and untreated tumours through the induction of a systemic adaptive immune response. Post tumour rejection, a memory response protected against rechallenge with the same, or similar, tumours. Intratumoural administration of the agents was associated with increases in IFN-α in the tumour (rather than the serum), and blockade or removal of the IFN-α receptor abrogated the anti-tumour response. The importance of the draining lymph node and spleen in anti-tumour activity (as shown by the excision of these organs), and liver enzyme responses, suggested that some of the agonists/antigens may have dispersed into the lymphoid organs and liver to support the response. Nonetheless, the anti-tumour effect was dependent on local effects of the intratumoural administration on the tumour microenvironment, as subcutaneous and peritumoural routes of administration only minimally affected tumour growth despite the reagents potentially having greater exposure to lymphoid organs. Through the use of various techniques including knockout mice, neutralising monoclonal antibodies, confocal microscopy and flow cytometry, it was shown that the combination of α-GalCer and CpG was dependent on the effector activity of CD8+ cells. However, optimal activity was associated with changes in other immune cell types, notably recruitment of iNKT cells into the tumour bed, and was also associated with induction of serum antibodies that could transfer some protection to naïve hosts. Induction of a successful response was dependent on conventional dendritic cells (DCs) of the “cDC1” phenotype, which are known to be effective at antigen cross-presentation to CD8+ T cells, while full tumour rejection also required the activity of plasmacytoid DCs, which are significant producers of IFN-α. In less immunogenic tumour models, the addition of relevant tumour associated antigens (TAAs) improved the anti-tumour response. The TAAs could be added as part of an admix, but improved responses were obtained when TAAs were chemically conjugated to α-GalCer via an enzymatically cleavable linker. Alternatively, intratumoural administration of α-GalCer and CpG as free agents could be combined effectively with low dose systemic chemotherapy to induce curative responses, potentially through a mechanism involving immunogenic cell death to improve the immunogenicity of TAAs in situ.</p>

Harnessing iNKT cells to improve in situ vaccination for cancer therapy

<p>Toll-like receptor (TLR) agonism in combination with the activation of type I NKT (iNKT) cells through systemic administration of their respective agonists has been shown to have a cooperative effect on activating antigen-presenting cells, stimulating cytokine production, and inducing adaptive immune responses to co-administered antigens. Here, it was hypothesised that it might be possible to harness these activities to treat solid tumours locally via intratumoural treatment to combat tumour growth while reducing toxicity to other organs. An intratumoural treatment model combining the stimulatory activity of unmethylated DNA oligonucleotides consisting of synthetic cytosine-guanine motifs (CpG), a TLR9 agonist, with activation of iNKT cells through administration of the CD1d-binding iNKT agonist α-galactosylceramide (α-GalCer) intratumourally was shown to have significant anti-tumour activity. The treatment regimen showed superior efficacy to that achieved with either agent alone in several in vivo models representing different types of cancer. In some models, the combination of α-GalCer and CpG was effective at inducing the complete rejection of both treated and untreated tumours through the induction of a systemic adaptive immune response. Post tumour rejection, a memory response protected against rechallenge with the same, or similar, tumours. Intratumoural administration of the agents was associated with increases in IFN-α in the tumour (rather than the serum), and blockade or removal of the IFN-α receptor abrogated the anti-tumour response. The importance of the draining lymph node and spleen in anti-tumour activity (as shown by the excision of these organs), and liver enzyme responses, suggested that some of the agonists/antigens may have dispersed into the lymphoid organs and liver to support the response. Nonetheless, the anti-tumour effect was dependent on local effects of the intratumoural administration on the tumour microenvironment, as subcutaneous and peritumoural routes of administration only minimally affected tumour growth despite the reagents potentially having greater exposure to lymphoid organs. Through the use of various techniques including knockout mice, neutralising monoclonal antibodies, confocal microscopy and flow cytometry, it was shown that the combination of α-GalCer and CpG was dependent on the effector activity of CD8+ cells. However, optimal activity was associated with changes in other immune cell types, notably recruitment of iNKT cells into the tumour bed, and was also associated with induction of serum antibodies that could transfer some protection to naïve hosts. Induction of a successful response was dependent on conventional dendritic cells (DCs) of the “cDC1” phenotype, which are known to be effective at antigen cross-presentation to CD8+ T cells, while full tumour rejection also required the activity of plasmacytoid DCs, which are significant producers of IFN-α. In less immunogenic tumour models, the addition of relevant tumour associated antigens (TAAs) improved the anti-tumour response. The TAAs could be added as part of an admix, but improved responses were obtained when TAAs were chemically conjugated to α-GalCer via an enzymatically cleavable linker. Alternatively, intratumoural administration of α-GalCer and CpG as free agents could be combined effectively with low dose systemic chemotherapy to induce curative responses, potentially through a mechanism involving immunogenic cell death to improve the immunogenicity of TAAs in situ.</p>

Synthesis and Biological Evaluation of Trehalose Glycolipids

<p>Numerous α,α-trehalose diesters have been isolated from bacteria such as Mycobacteria and Corynebacteria, and more recently from Caenorhabditis elegans dauer larvae. Although these glycolipids are thought to confer protection to the bacteria and larvae against harsh environmental conditions, it is the biological activities of these compounds, including anti-tumour and adjuvant activities, which have been of major interest to scientists over recent years. In this thesis, three different aspects relating to the synthesis and testing of defined trehalose glycolipids will be presented. First, the synthesis of a variety of fatty acid trehalose diesters (TDEs) with varying lipid lengths was performed and the ability of these glycolipids to activate macrophages was studied. Two different synthetic strategies were employed to attain the TDEs of interest and it was observed that lipid lengths of more than 18 carbons were required for macrophage activation. Furthermore, the C22 fatty acid trehalose monoester (TME) and the C26 TME were also synthesised and interestingly they both showed macrophage activation abilities, with subsequent studies indicating that like TDEs, the TMEs were also ligands for mincle, a C-type lectin found on macrophages. This is the first time that TMEs have been tested for their ability to activate macrophages via Mincle. The cytotoxicity of these compounds and subsequent anti-tumour activity of a few selected compounds were also studied and although the TDEs and TMEs did not exhibit any significant cytotoxicity, in in vivo models the C10 TDE and C22 TDE both showed anti-tumour activity. This depicts that the mechanism for anti-tumour activity of these compounds is not due to cytotoxicity but due to as yet unidentified pathway. Methodology that can be applied to the synthesis of more complex trehalose glycolipids, such as trehalose dicorynomycolates (TDCMs, isolated from Corynebacteria) and trehalose dimycolates (TDMs, isolated from Mycobacteria) was also explored. One of the key steps frequently used in the synthesis of these glycolipids is the Fráter-Seebach alkylation. To improve the efficacy of this methodology allylic iodides, rather than alkyl iodides were used for theα-alkylation of β-hydroxy esters. Our results showed that for all substrates studied, the yield of the α-alkylation was greatly improved when the allylic, rather that the alkyl halide was used. The use of this methodology in the synthesis of trehalose monocorynomycolate (TMCM) was also investigated. The third aspect of this thesis focuses on the use of Affinity Based Proteome Profiling (AƒBPP) for elucidating the receptors that TDMs bind to upon interacting with host cell. AƒBPP focuses on using small molecules which mimic the natural substrate for a particular protein and through the use of ‘trap’ and ‘tag’ groups on the molecule the identity of the protein/receptors can be determined. The synthesis of a TDM probe containing a benzophenone ‘trap’ group and an alkyne ‘tag’ group will be discussed.</p>

Synthesis and Biological Evaluation of Trehalose Glycolipids

<p>Numerous α,α-trehalose diesters have been isolated from bacteria such as Mycobacteria and Corynebacteria, and more recently from Caenorhabditis elegans dauer larvae. Although these glycolipids are thought to confer protection to the bacteria and larvae against harsh environmental conditions, it is the biological activities of these compounds, including anti-tumour and adjuvant activities, which have been of major interest to scientists over recent years. In this thesis, three different aspects relating to the synthesis and testing of defined trehalose glycolipids will be presented. First, the synthesis of a variety of fatty acid trehalose diesters (TDEs) with varying lipid lengths was performed and the ability of these glycolipids to activate macrophages was studied. Two different synthetic strategies were employed to attain the TDEs of interest and it was observed that lipid lengths of more than 18 carbons were required for macrophage activation. Furthermore, the C22 fatty acid trehalose monoester (TME) and the C26 TME were also synthesised and interestingly they both showed macrophage activation abilities, with subsequent studies indicating that like TDEs, the TMEs were also ligands for mincle, a C-type lectin found on macrophages. This is the first time that TMEs have been tested for their ability to activate macrophages via Mincle. The cytotoxicity of these compounds and subsequent anti-tumour activity of a few selected compounds were also studied and although the TDEs and TMEs did not exhibit any significant cytotoxicity, in in vivo models the C10 TDE and C22 TDE both showed anti-tumour activity. This depicts that the mechanism for anti-tumour activity of these compounds is not due to cytotoxicity but due to as yet unidentified pathway. Methodology that can be applied to the synthesis of more complex trehalose glycolipids, such as trehalose dicorynomycolates (TDCMs, isolated from Corynebacteria) and trehalose dimycolates (TDMs, isolated from Mycobacteria) was also explored. One of the key steps frequently used in the synthesis of these glycolipids is the Fráter-Seebach alkylation. To improve the efficacy of this methodology allylic iodides, rather than alkyl iodides were used for theα-alkylation of β-hydroxy esters. Our results showed that for all substrates studied, the yield of the α-alkylation was greatly improved when the allylic, rather that the alkyl halide was used. The use of this methodology in the synthesis of trehalose monocorynomycolate (TMCM) was also investigated. The third aspect of this thesis focuses on the use of Affinity Based Proteome Profiling (AƒBPP) for elucidating the receptors that TDMs bind to upon interacting with host cell. AƒBPP focuses on using small molecules which mimic the natural substrate for a particular protein and through the use of ‘trap’ and ‘tag’ groups on the molecule the identity of the protein/receptors can be determined. The synthesis of a TDM probe containing a benzophenone ‘trap’ group and an alkyne ‘tag’ group will be discussed.</p>

Pharmacogenomics characterization of the MDM2 inhibitor MI-773 reveals candidate tumours and predictive biomarkers

MI-773 is a recently developed small-molecule inhibitor of the mouse double minute 2 (MDM2) proto-oncogene. Preclinical data on the anti-tumour activity of MI-773 are limited and indicate that tumour cell lines (CLs) with mutated TP53 are more resistant to MI-773 than wild type TP53. Here, we explored the compound’s therapeutic potential in vitro using a panel of 274 annotated CLs derived from a diversity of tumours. MI-773 exhibited a pronounced selectivity and moderate potency, with anti-tumour activity in the sub-micromolar range in about 15% of the CLs. The most sensitive tumour types were melanoma, sarcoma, renal and gastric cancers, leukaemia, and lymphoma. A COMPARE analysis showed that the profile of MI-773 was similar to that of Nutlin-3a, the first potent inhibitor of p53–MDM2 interactions, and, in addition, had a superior potency. In contrast, it poorly correlates with profiles of compounds targeting the p53 pathway with another mechanism of action. OMICS analyses confirmed that MI-773 was primarily active in CLs with wild type TP53. In silico biomarker investigations revealed that the TP53 mutation status plus the aggregated expression levels of 11 genes involved in the p53 signalling pathway predicted sensitivity or resistance of CLs to inhibitors of p53–MDM2 interactions reliably. The results obtained for MI-773 could help to refine the selection of cancer patients for therapy.