M36 EXECUTIVE PUBLISHABLE SUMMARY
1.1. Summary description of the project context and objectives
Based on the broad immuno-suppression and strong tumor promotion (proliferation and dissemination) exerted by adenosine, therapeutic strategies aimed at inhibiting adenosine are highly promising.
However, given involvement of the four known specific adenosine receptors, the use of mAbs neutralizing the enzymatic function of CD73, the sole 5’ ecto-nucleotidase expressed at surface membrane, thus preventing dephosphorylation of AMP into adenosine, represents an attractive, potent and safe strategy for the development of innovative immunotherapy.
The TumAdoR project aims at paving the way to clinical trials of potent neutralizing anti-CD73 mAb candidates in cancer patients. It should validate a novel cancer immunotherapy approach that would be applicable to a wide range of cancers and will enable the development of specific anti-tumour immune response inhibiting cancer progression and dissemination and preventing relapse through restoration of anti-tumour immune memory.
1.2. Description of the work performed since the beginning of the project and main results achieved
Target expression: Retrospective tumor collections with clinical annotations have been defined in different partners’ hospital institutions and IHC studies are currently on-going. This includes Melanomas, Colon, Lung, Breast, Ovarian, Bladder, Prostate, Head and Neck, Pancreatic carcinoma and Lymphoma.
CD73 biology: Focusing on understanding CD73 biology within T cell populations, it was demonstrated that in contrast to murine cells, CD39 and CD73 are never co-expressed within human T cell subsets. CD39 is selectively expressed by tumor infiltrating Treg and on activated T cells whereas CD73 is expressed on a discrete CD4 memory subset and naïve CD8 T cells. On CD4 cells, CD73 characterizes a population of memory/effector T cells of Th1/Th17 profile producing both IFNg and IL17 at high level. CD73 renders this CD4 population highly sensitive to suppression by AMP and by ATP in presence of CD39+ Treg. Our current model is that CD73 expression on this potentially damaging CD4 effector limits its reactivity in an inflammatory environment rich in ATP and Tregs. The effect of the crosstalk between CD39 and CD73 on adenosine generation and immunosuppression is under characterization. The connection between tumor progression, EMT process and CD73 induction on tumor cells has been documented.
Neutralizing anti-CD73 mAbs: This WP has represented a major collective effort. Monoclonal Abs were produced, purified and evaluated in various in vitro efficacy assays, using different cellular models. In vivo efficacy experiments are underway. Epitope mapping and mechanism of action exploration are also on-going. Humanized mAb candidates are under investigation for lead selection.
Preclinical models for in vivo therapeutic evaluation: A major investment has been made to develop mouse models for in vivo evaluation of mAbs recognizing hCD73 : i) syngeneic mouse tumor models expressing CD73, ii) transplantable syngeneic mouse tumor models expressing hCD73 (hCD73 transfection in mCD73 KO ID8 and B16 cells); iii) generation of hCD73 KI mouse strain; iv) transplantable xenogeneic human tumor (hCD73 wt and hCD73 KO); v) transplantable xenogeneic hCD73 human tumor in immuno-deficient mice reconstituted with human immune cells. hCD73 KI mouse strain has now been generated, and is available for in vivo experiments.
Mouse xenogeneic CD73+ and CD73 KO tumor models for in vivo evaluation have been validated and some selected mAbs are currently tested. Syngeneic murine tumor models with mCD73 and hCD73 are still under validation.
Assays for toxicity evaluation: Assays to evaluate toxicity on blood vessel integrity, vascular leakage and immune response to test antigens have been established. CD73 expression on frozen normal human tissues using existing anti-hCD73 has been performed.
Specific diagnostic tools are in development to assess target expression and to monitor relevant immune parameters that will be evaluated in patients before and during the treatment. Antibody panels for immuno-monitoring have been developed to assess by multicolor flow cytometry on-target and off-target effects of neutralizing anti-hCD73 Abs. In particular, we developed and validated fluorescently labeled antibody panels to evaluate the differentiation and polarization of T cells, B cells, NK cells, DCs, monocytes and their activation, exhaustion, cytotoxic and cytokine profiles in combination with CD73 and CD39 expression by flow cytometry. The analyses allowed a comprehensive evaluation of CD73 and CD39 expression by human PBMCs of healthy donors. Immuno-monitoring tools are crucial in the evaluation of immunotherapy interventions to identify relevant efficacy and safety predictive biomarkers.
Dissemination and IPR: The results obtained in the course of TumAdoR have been presented at several scientific events since M30:
- Nicolas Gourdin et al – “CD39+ Treg cooperate with a CD73-expressing Th1/Th17 subset for Adenosine-mediated immunosuppression in human breast tumors” – Oral presentation at CLARA, Lyon, France, March 2016.
- Nicolas Gourdin et al – “CD39+ Treg cooperate with a CD73-expressing Th1/Th17 subset for Adenosine-mediated immunosuppression in human breast tumors” – Poster presented at AACR, New Orleans, USA, March 2016.
- Ivan Perrot et al – “Discovery and characterization of new original blocking antibodies targeting the CD73 immune checkpoint for cancer” – Poster presented at AACR, New Orleans, USA, March 2016.
- Selena Vigano et al – “Clinical immunomonitoring strategies defining biomarkers of anti-CD73 mAbs safety and efficacy. The TumAdoR collaborative project.” – Poster presented at CIMT, Mainz, Germany, May 2016.
- Selena Vigano et al – “Clinical immunomonitoring strategies assessing on-target and off-target effects of anti-CD73 mAbs - The TumAdoR collaborative project.” – Poster presented in Lausanne, Switzerland, May 2016.
1.3. Expected final results and their potential impact and use
Target expression analysis will generate knowledge on CD73 distribution and its impact on tumor progression and patient survival allowing selecting the pathology for a first clinical trial. It is expected that from the mAbs developed, a companion diagnostic for anti-hCD73 treatment could be developed.
CD73 biology: We will pursue the study of CD73 biology through analyzing i) the biology of CD73+ CD4 T cells, and on naïve CD8 T cells, ii) the cooperation with other CD39 expressing cells, iii) the determinants of CD73 regulation on T cells. From this work it is expected to generate new knowledge on CD73 as an immune check point blocker.
Neutralizing anti-CD73 mAbs: It is expected that the overall strategy will allow obtaining potent anti-hCD73 mAbs neutralizing CD73 activity with different MOA.
Preclinical models for in vivo therapeutic evaluation: The preferred model to evaluate the selected candidate mAbs will be the syngeneic models with hCD73 expressed on both tumor cells (hCD73-ID8 or hCD73-B16) and immune cells (hCD73 KI mice). A major investment in the development of mouse models allowing targeting the human molecules in mice was done and enables an optimal evaluation of the therapeutic potential of the drug candidates.
Assays for toxicity evaluation: The hCD73 KI mice will be used to evaluate in vivo toxicity assessment of anti-CD73 mAb candidates. For ethical consideration, equipment for in vivo imaging of tumor progression has been acquired allowing minimizing animal number for in vivo evaluation. All the tools and assays will be in place to perform efficient preliminary toxicology studies.
Immuno-monitoring tools: The immuno-monitoring assays selected for the anti-hCD73 mAb clinical trial will be validated across the consortium, harmonized and standardized following the international models. These original tools will allow the monitoring of relevant parameters during future clinical trials using neutralizing anti-hCD73.
Dissemination and IPR: Efficient rules and communication tools have been developed. External communication has been reinforced these last six months. TumAdoR consortium is also organized to efficiently validate disseminating actions. Importantly a lay-out of the Plan for Use and Dissemination of Foreground (PUDF) has been determined and its implementation has started.
At conclusion of the TumAdoR project, thanks to the complementary expertise and objectives of the partners we expect to have generated:
- New knowledge on CD73 as an immune check point blocker,
- New preclinical models to evaluate drug candidates blocking CD73 pathway and assess safety of this strategy,
- Humanized efficient neutralizing anti-hCD73 mAbs,
- Companion diagnostic to assess CD73 expression,
- Immuno-monitoring tools to follow relevant immune parameters in patients to assess biological efficacy