Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - CHEMCHECK (CHECKPOINTS IN CHECK: Novel Chemical Toolbox for Local Cancer Immunotherapy)
Teaser
Cancer evades the immune system by generating an immunosuppressive tumour-microenvironment through various mechanisms to enable unhampered growth. Recent breakthroughs in blocking one of these mechanisms – the so called ‘immune checkpoints’ – put cancer immunotherapy...
Summary
Cancer evades the immune system by generating an immunosuppressive tumour-microenvironment through various mechanisms to enable unhampered growth. Recent breakthroughs in blocking one of these mechanisms – the so called ‘immune checkpoints’ – put cancer immunotherapy back in the spotlights. Although promising, clinical benefits of these checkpoint inhibitors as single treatment has been limited to a subset of patients and goes along with unwanted systemic autoimmune toxicity. I hypostasize, that attacking the tumour microenvironment from multiple immunological angles simultaneously by local, conditional, and multimodal immunomodulation will greatly improve success of cancer immunotherapy and patient wellbeing. To achieve this, I will develop a highly defined synergistic chemistry-based molecular therapeutic toolbox to specifically attack cancer, acting on effector T cells, macrophages as well as tumour cells simultaneously. In this highly multidisciplinary endeavour I will (i) generate novel multifunctional dendritic cell targeted anti-cancer vaccines to ‘educate’ the patient’s immune system to recognise the tumour, (ii) I will develop conditional, targeted immune checkpoint inhibitors to release the immunosuppressive break specifically within the tumour microenvironment without the risk of autoimmunity and (iii) I will generate chemical tools to locally eliminate the tumour-associated macrophages to tear down a major immunosuppressive barrier. I will do so utilizing the novel ModimAb technology which I developed to obtain functionalized antibody fragments. These individual therapeutic tools will allow me and my research team to explore uncharted tumour immunological territories in vitro as well as in vivo, greatly advancing the field of cancer immunotherapy. But above all, together they will form a highly dedicated symbiotic immunotherapeutic regime which will be extremely effective without systemic side effects, dramatically improving patient care.
Work performed
CHEMCHECK is largely evolving as described in the description of action. Major results achieved include the establishment of the ModimAb technology platform, allowing the generation of stable cell lines continuously producing site-specific modified and chemoenzymatically functionalizable antibody fragments (Fab’s) (WP1.1). Encouraged by these results we expanded this technological platform to genetically engineer hybridomas to stably produce chimeric isotype switched and chemoenzymatically functionalizable monoclonal antibodies (mAbs). Currently, (preclinical) antibody research with panels of recombinant functionally diverse mutant antibody variants is hampered and is largely restricted to specialized academic laboratories and biotech companies, which is therefore most often outsourced to contract research organizations. We believe this easy one-step hybridoma engineering method will enable the entire scientific community to start evaluating their antibodies in different functional formats early on in development, which has been shown to be crucial to determine in vivo efficacy.
Using our ModimAb technology site-specific modifiable mouse and human dendritic cell targeting antibody fragments (Fab’s) have been produced directed towards the endocytic receptors mDEC205, mCD40, mCD169, as well as hClec9a and hDC-SIGN. The first step in the synthesis of highly defined targeted multifunctional synthetic vaccines (WP1.1) is the conjugation of clickable antigenic peptides. Azidolysine-containing sortaggable antigen MHC I and MHC II epitopes have been synthesized for the model antigen ovalbumin, the tumor associated antigen TRP-2 and recently reported B16 melanoma neoantigens. After optimization, chemoenzymatic conjugates have been produced. We have obtained a panel of clickable immunostimulatory adjuvants, which are currently being conjugated to result ‘AAA-vaccines’(antibody-antigen-adjuvant conjugate vaccines). We will be able to, for the first time, systematically evaluate the influence of endocytic target, antigen, adjuvant and antibody format and isotype on targeted vaccine induced immunity.
To generate the toolbox for local and cell specific checkpoint inhibition (WP1.2 and 1.3) we used the ModimAb technology to produce site-specific modifiable checkpoint blocking Fab’s targeting mPD-L1, mPD-1 and mCTLA-4, as well as mCD28 and mCD3. We are investigating synthetic multimodal conjugates for local and conditional activation of effector functions. As a stepping stone towards in vivo application of these constructs we have generated multimodal fluorescent and SPECT immunoimaging Fab and mAb constructs targeting mPD-L1. We have established that these molecularly defined conjugates perform on par with the SPECT golden standard, with the added advantage of multimodality.
The first generation of tumor-associated macrophage (TAM) eliminating activity-based probes (WP1.4) have been synthesized. The cathepsin labeling potency of these compounds is lower than expected, probably due to poor solubility. We are currently developing photosensitizers with increased water solubility in an effort to optimize these cysteine cathepsin targeted photodynamic activity-based probes.
The be able to characterize the immunomodulatory capacities of the molecular entities described above, we have set up in vitro 3D immune cell and cancer spheroid cultures (WP2.1). We have been able to recapitulate specific macrophage labeling with cysteine cathepsin activity-based probes in these spheroid co-cultures. We have validated and established probe labeling as a bona fide non-invasive marker for pro-tumor, M2-like macrophages. We have used this knowhow to identify small molecule TLR agonists with the ability to repolarize pro-tumor macrophage towards an anti-tumor phenotype. We have evidence that proteolytic changes add significantly to the changes in immune status as anti-tumor mechanism. To enable ultimate in vivo validation we have optimized the pr
Final results
We have developed the ModimAb platform for easy one-step hybridoma engineering method which enables the entire scientific community to start evaluating their antibodies in different functional formats. We believe that this simple and effective “open access†method to obtain such antibody variants represents a major advancement of the field and will boost preclinical antibody research. Using this method we have generated a still growing toolbox of functionally diverse and modifiable antibodies and antibody fragments which form the basis for the synthesis of the immunomodulating molecules described in this project and beyond. We are building a complete set of ‘AAA-vaccines’ buildup from a defined set of different antibody formats targeting several endocytic targets, (neo)antigens and adjuvants to systematically evaluate their influence on targeted vaccine induced immunity. We hypothesize we will find different conjugate formats for optimal CD8 and CD4 T cell priming. Combining these formats in an optimized targeted AAA-vaccine cocktail will induce a robust anti-tumor immune response.
Our isotype and species switched chimeric multimodal fluorescent and SPECT immunoimaging Fab and mAb constructs targeting mPD-L1 will enable us to monitor the immunomodulatory changes in the tumor microenvironment in response to our interventions in longitudinal studies. An evolved opportunity is the translation of these multimodal imaging tools towards photodynamic theranostic tools enabling image guided photoimmunotherapy. We will continue our efforts to synthesize multimodal conjugates for local and conditional activation of checkpoint inhibition, as well as the ‘second generation’ cysteine cathepsin targeted photodynamic activity-based probes. All these tools will be evaluated in our 3D cancer immune cell co-culture system. We will further unravel the proteolytic changes during tumor-associated macrophage re-polarization in our 3D co-culture system and the significance thereof to the anti-tumor mechanism of this strategy. Ultimately, we will combine these strategies in the optimized preclinical models for in depth immunological characterization. We expect to indentify multiple novel molecular immunomodulators, which together will form a highly dedicated symbiotic immunotherapeutic regime.
Website & more info
More info: http://www.verdoeslab.com.