Periodic Reporting for period 1 - ARGONAUT (ARGONAUT: from the synthesis of gAnglioside tumouR antiGens to a platfOrm for caNcer Active immUnoTherapy)
Okres sprawozdawczy: 2019-08-01 do 2021-07-31
In 2015, in the EU-28, more than 1.3 million people died from cancer, which equated to 26.7% of the total number of deaths. For 2020, the European Cancer Information System estimates 2.7 million new cases of cancer (excluding non-melanoma skin cancers) and over 1.3 million cancer-related deaths in the EU-27. While traditional approaches including surgery, chemotherapy, and/or radiotherapy have greatly contributed to the treatment of this disease, they are known to suffer from adverse effects, due to lack of selectivity for malignant cells over healthy ones and from the inability to clear metastasis. Immunotherapy is currently revolutionizing cancer therapy by harnessing the power of the innate and adaptive immune system against cancer cells, thus providing a more tumor-selective approach in assistance to traditional treatments. The identification of tumor-associated carbohydrate antigens (TACAs), aberrant types of glycans decorating the surface of tumor cells, has paved the way for the development of cancer vaccines. However, while significant progress has been made in this area of research, TACA-based cancer vaccines have not yet reached the clinic.
In this context, the project addressed some limitations that characterize classical approaches in carbohydrate cancer vaccine development, while focusing at understanding how to exploit glycans as both tumor-associated epitopes and vaccine adjuvants to access novel and more effective synthetic candidates. The overall research objectives included the preparation of novel vaccine candidates, based on ganglioside-type TACAs (GM3, (Neu5Gc)GM3, GD2, and GD3 which are expressed in melanoma, neuroblastoma, and small cell lung cancer) conjugated with the iNKT cell adjuvant α-galactosylceramide (αGalCer), the design of a versatile formulation platform based on liposomes, and the testing of the vaccine candidates in vivo through mice immunization studies. Employing a carbohydrate chemistry approach, this proposal explored its application as a tool to contribute to current efforts in cancer research in a multidisciplinary and intersectoral environment. Moreover, through a systematic vaccine screening approach the developed research generated useful information in terms of carbohydrate-based cancer vaccine efficacy, enabling the definition of which parameters in carbohydrate vaccine design and formulation are essential and can be tailored to improve the immune response.
In this context, the project addressed some limitations that characterize classical approaches in carbohydrate cancer vaccine development, while focusing at understanding how to exploit glycans as both tumor-associated epitopes and vaccine adjuvants to access novel and more effective synthetic candidates. The overall research objectives included the preparation of novel vaccine candidates, based on ganglioside-type TACAs (GM3, (Neu5Gc)GM3, GD2, and GD3 which are expressed in melanoma, neuroblastoma, and small cell lung cancer) conjugated with the iNKT cell adjuvant α-galactosylceramide (αGalCer), the design of a versatile formulation platform based on liposomes, and the testing of the vaccine candidates in vivo through mice immunization studies. Employing a carbohydrate chemistry approach, this proposal explored its application as a tool to contribute to current efforts in cancer research in a multidisciplinary and intersectoral environment. Moreover, through a systematic vaccine screening approach the developed research generated useful information in terms of carbohydrate-based cancer vaccine efficacy, enabling the definition of which parameters in carbohydrate vaccine design and formulation are essential and can be tailored to improve the immune response.
The project ran between August 2019 and July 2021. Due to the SARS-CoV-2 pandemic the experimental activities were paused in the period March 2020 ‒ June 2020 and restrictions to university/laboratory access were also in place in the period November 2020 ‒ March 2021.
The first part of the project included the chemical synthesis of the GM3 and (Neu5Gc)GM3 tumor-associated carbohydrate antigens (TACAs) and of the iNKT cells agonist α-galactostylceramide (αGalCer). Following the synthesis of the components of the planned vaccine constructs, more synthetic efforts were dedicated to the conjugation of the TACAs to αGalCer. In the meantime, the preparation of protein conjugates (HSA) of GM3 and (Neu5Gc)GM3 antigens was also achieved.
The synthetic work towards the larger structures (GD2‒αGalCer and GD3‒αGalCer) was also commenced and it is ongoing. The primary focus of this part of the work was the construction of the Neu5Ac-α-(2→8)-Neu5Ac disaccharide, the characteristic moiety of GD2 and GD3 TACAs, and extensive synthetic efforts have been dedicated to access the difficult α-(2→8) disialyl linkage.
Moving forward in the project workflow, with the GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer constructs in hand, efforts were dedicated to the formulation in liposomes of the two vaccine constructs. Size-defined liposomes containing either GM3‒αGalCer, (Neu5Gc)GM3‒αGalCer, equimolar amounts of GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer, or equimolar amounts of the commercial GM3 ganglioside and αGalCer were prepared by lipid extrusion with an approximate size of 200 nm.
The prepared liposomes of the vaccine constructs were then evaluated in vitro and in vivo. The potency of the liposomes was investigated in vitro demonstrating their ability to activate iNKT cells. Similarly the same liposomes were able to induce cytokine production in vivo following mice immunization. Mice immunization with the prepared liposomes was capable of stimulating B cells to produce anti-GM3 or anti-(Neu5Gc)GM3 antibodies: carbohydrate-specific IgM and IgG antibodies were produced. Additionally, all vaccine candidates produced antibodies that exhibited strong binding to B16F10 cell line (melanoma cell line) and efficient activation of the complement system on their surface. However, mice immunized with GM3‒HSA and (Neu5Gc)GM3‒HSA produced the highest levels of IgM and IgG, thus outperforming the synthetic liposomal candidates.
Regarding the dissemination of the project results, the activities that have been carried out and completed include i) the training of students (BSc, MSc, and Erasmus internships), ii) the circulation of a Project Newsletter among the collaborators (DTU HealthTech, SSI), iii) opening and updating of a Twitter account (@msca_argonaut) and creation of a project logo and infographics, iv) presentation of the project results as an invited oral communication at the European Glycoscience Community Webinar series, v) presentation of the project results with a poster in the online conference “Joint Warren and Beilstein Symposium on Glycosciences”, v) preparation of a manuscript summarizing the overall results of the project which is freely available as pre-print in ChemRxiv, vi) organization of the CNT Science Mixer event (online) and presentation of the project results.
Regarding exploitation of the results, the project has benefitted from collaborative research with DTU HealthTech. It has also led to the establishment of an additional collaborative research line with DTU Bioengineering investigating the mechanism of action of the prepared liposomes in human DC and iNKT cells. The DTU Office for Innovation has been recently involved for assessing whether the function of the platform of compounds will be potentially patentable and commercially exploitable.
The first part of the project included the chemical synthesis of the GM3 and (Neu5Gc)GM3 tumor-associated carbohydrate antigens (TACAs) and of the iNKT cells agonist α-galactostylceramide (αGalCer). Following the synthesis of the components of the planned vaccine constructs, more synthetic efforts were dedicated to the conjugation of the TACAs to αGalCer. In the meantime, the preparation of protein conjugates (HSA) of GM3 and (Neu5Gc)GM3 antigens was also achieved.
The synthetic work towards the larger structures (GD2‒αGalCer and GD3‒αGalCer) was also commenced and it is ongoing. The primary focus of this part of the work was the construction of the Neu5Ac-α-(2→8)-Neu5Ac disaccharide, the characteristic moiety of GD2 and GD3 TACAs, and extensive synthetic efforts have been dedicated to access the difficult α-(2→8) disialyl linkage.
Moving forward in the project workflow, with the GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer constructs in hand, efforts were dedicated to the formulation in liposomes of the two vaccine constructs. Size-defined liposomes containing either GM3‒αGalCer, (Neu5Gc)GM3‒αGalCer, equimolar amounts of GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer, or equimolar amounts of the commercial GM3 ganglioside and αGalCer were prepared by lipid extrusion with an approximate size of 200 nm.
The prepared liposomes of the vaccine constructs were then evaluated in vitro and in vivo. The potency of the liposomes was investigated in vitro demonstrating their ability to activate iNKT cells. Similarly the same liposomes were able to induce cytokine production in vivo following mice immunization. Mice immunization with the prepared liposomes was capable of stimulating B cells to produce anti-GM3 or anti-(Neu5Gc)GM3 antibodies: carbohydrate-specific IgM and IgG antibodies were produced. Additionally, all vaccine candidates produced antibodies that exhibited strong binding to B16F10 cell line (melanoma cell line) and efficient activation of the complement system on their surface. However, mice immunized with GM3‒HSA and (Neu5Gc)GM3‒HSA produced the highest levels of IgM and IgG, thus outperforming the synthetic liposomal candidates.
Regarding the dissemination of the project results, the activities that have been carried out and completed include i) the training of students (BSc, MSc, and Erasmus internships), ii) the circulation of a Project Newsletter among the collaborators (DTU HealthTech, SSI), iii) opening and updating of a Twitter account (@msca_argonaut) and creation of a project logo and infographics, iv) presentation of the project results as an invited oral communication at the European Glycoscience Community Webinar series, v) presentation of the project results with a poster in the online conference “Joint Warren and Beilstein Symposium on Glycosciences”, v) preparation of a manuscript summarizing the overall results of the project which is freely available as pre-print in ChemRxiv, vi) organization of the CNT Science Mixer event (online) and presentation of the project results.
Regarding exploitation of the results, the project has benefitted from collaborative research with DTU HealthTech. It has also led to the establishment of an additional collaborative research line with DTU Bioengineering investigating the mechanism of action of the prepared liposomes in human DC and iNKT cells. The DTU Office for Innovation has been recently involved for assessing whether the function of the platform of compounds will be potentially patentable and commercially exploitable.
Progress beyond the state of the art has been achieved in the following areas:
• Chemical synthesis of GM3 and (Neu5Gc)GM3 antigens has been expedited
• Chemical synthesis of αGalCer has been optimized and expedited
• The two constructs prepared, GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer, are novel
• The two constructs prepared, GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer, are effective vaccine candidates and their mixed formulation is a step beyond the state of the art, opening to the possibility of building a liposomal platform for the delivery of several TACAs
The project contributed to address current challenges in cancer research, with an approach aimed at filling the gap between basic research and the clinic and with the potential of impact on the field of cancer immunotherapy. Although still needing improvement, the prepared liposomes demonstrated activity as preliminary vaccine candidates, an important result considering the all-carbohydrate structure of the synthetic constructs. The findings of the project so far have consequences for future vaccine designs, thus strongly impacting the scientific area and society.
• Chemical synthesis of GM3 and (Neu5Gc)GM3 antigens has been expedited
• Chemical synthesis of αGalCer has been optimized and expedited
• The two constructs prepared, GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer, are novel
• The two constructs prepared, GM3‒αGalCer and (Neu5Gc)GM3‒αGalCer, are effective vaccine candidates and their mixed formulation is a step beyond the state of the art, opening to the possibility of building a liposomal platform for the delivery of several TACAs
The project contributed to address current challenges in cancer research, with an approach aimed at filling the gap between basic research and the clinic and with the potential of impact on the field of cancer immunotherapy. Although still needing improvement, the prepared liposomes demonstrated activity as preliminary vaccine candidates, an important result considering the all-carbohydrate structure of the synthetic constructs. The findings of the project so far have consequences for future vaccine designs, thus strongly impacting the scientific area and society.