ImmunoRec is a spin-off company that officially started its works in July 2021 and aims to exploit the novel strategy of ex vivo immune regulation technology, given the names of “personalized implantable vaccines”, “vaccine-on-chip”, “organ-on-chip”.
Goal: cancer immunotherapy.
- “Personalized implant against cancer” patent (ΟΒΙ 20190100297) (venture capital)
- “Personalized vaccine against SARS-CoV-2” patent (OBI 245-0004327167, under evaluation)
ImmunoRec is a spin-off company of the University of Crete aiming to enter phase-I clinical trial on cancer vaccine personalized therapy (patent ΟΒΙ 20190100297). The novel technology is stated by the National Medicinal Organization as Cancer immunotherapy with active substance autologous macrophages, pre-activated against autologous tumor antigens, absorbed on a silicon surface and is categorized as combined Advanced Therapy Medicinal Product. The company is to be built in an “Ex vivo Immunization Center” (EVIC), where patients will be receiving the personalized, self-tumor activated implant and monitored in collaboration with their oncologists. No side effects, No need to stop other treatments.
ImmunoRec is the offspring of a 10-year laboratory work and collaboration of the Immunology Laboratory of the Department of Biology of the University of Crete with the Ultrafast Laser Micro- and Nano- processing Laboratory of the Institute of Electronic Structure and Laser of the Foundation for Research and Technology-Hellas.
It was a late afternoon brainstorming meeting back in 2009, where the Imunology Lab people were puzzled with the safety and effectiveness of vaccines, while the Ultrafast Laser Micro-processing Lab people were seeking for some application of their biomimetic materials. And the idea of “personalized implantable vaccines” was born.
Since 2009, many people from both sides have worked on the development, expansion and optimization of the arising innovative technology:
Ultrafast Laser Processing Laboratory
Head Researcher: Prof. Irene Athanassakis
Head Researcher: Dr. Emmanuel Stratakis
2009: Anastasia Tselekidou
2009-2015: Dr. Chara Simitzi
2010-today: Dr. Ioanna Zerva
2016: Despina Aggelaki
2013-2014: Eleni Katsoni
2017-2019: Christina Lanara
2014-2015: Despina Karayianni
2020-today: Anna Karayiannaki
2016-2017: Tasos Kouimtzidis
2018-today: Vasiliki Pateraki
2018-2019: Chara Lardou
2019: Nikoleta Kokkou
2020-2021: Nefeli Paraskevopoulou
2021: Maria Papageorgiou
Why use the technology of “personalized implantable vaccines”
Cancer immunotherapy based on the isolation or engineering of tumor antigen-specific autologous T cells has been a promising effort to fight intrinsically malignant cells. Such adaptive cell transfer (ACT) therapies include the use of tumor infiltrating lymphocytes (TILs), the engineering of patients T cells to express specific T cell receptors (TCRs) or fragments of synthetic antibodies that recognize tumor epitopes linked to various co-stimulators on the cell surface, known as chimeric antibody receptors (CARs) .
TILs include both B and T lymphocytes found inside and around the tumor displaying anti-tumor activity, with T cells expressing tumor antigen-specific TCRs and exerting cytotoxic activity through their interaction with the tumor antigenic epitopes complexed with major histocompatibility complex (MHC) class I molecules . This approach, although providing some promising results to certain types of tumors, requires the presence of pre-existing TILs with cytotoxic activity, which is not always the case.
The limitations of TILS prompted the development of engineered T cells expressing either specific TCRs or CARs. Specificities have varied over the years from common antigens characterizing tumors to neoantigens in a personalized-type immunotherapy . In the case of CAR engineering scfv antibody constructs have been linked to various co-stimulator domains, including CD28, CD137, CD3ζ [4-6].
As a step beyond these laborious, but expensive, time consuming, and of limiting effectiveness strategies, ImmunoRec concentrates on the application of the personalized implantable vaccine technology . According to this strategy, the founders have been using a biomimetic platform to attach autologous macrophages and allow antigen presentation to take place in vitro, using controllable antigen doses. Such procedure allows natural antigen uptake, processing and loading to MHC molecules. Indeed, antigenic epitope editing is a complex and not yet well understood process, since it is directly related to the MHC haplotype and genetic background of the, individual.
Background and Rationale
The development of an immune response lies on the successful antigen presentation by antigen presenting cells (APCs). The traditional view of antigen presentation states that intracellularly synthesized antigens (viral or tumor antigens) are presented by MHC class I molecules and activate CD8+ cytotoxic T (Tc) cells, whereas extracellular antigens are presented by professional APCs with MHC class II molecules to CD4+ T helper (TH) cells. Intracellular antigens are generally being degraded by the immunoproteasome, transported to the endoplasmic reticulum, loaded to MHC class I molecules and transported to the cell membrane for recognition by antigen-specific Tc cells . Extracellular antigens are internalized into phagosomes or endosomes that subsequently mature and undergo a series of molecular changes, such as acidification and fusion with organelles containing degrading enzymes, in particular lysosomes. The generated antigenic peptides are loaded onto MHC class II molecules and transported to the cell surface for presentation to CD4+ TH cells . Activation of TH cells represents one of the most important activities of the immune system since it leads to humoral or cellular immunity as well as tolerance. Thus, TH will stimulate B cells for specific antibody production or Tc for target cell killing, while the expression of negative surface markers will suppress both types of immunity to ensure homeostasis of the immune system.
Any substance can activate an immune response. Low molecular weight substances (<2000 d), haptens, induce specific immune response when covalently linked to a bigger immunogen molecule, the carrier. In such cases, the simultaneous antigen non-specific stimulation is necessary and is succeeded with emulsification of the antigen into adjuvants.
Tolerance is the immune phenomenon characterized by memory and specificity and represents the absence or suppression of immunity. One of the major mechanisms attributing to the maintenance of self-tolerance is the development of immunosuppression. This term includes the acquisition of specific cell populations that exert negative regulatory effects to cellular targets either upon cell contact or through soluble products. The appearance of a novel antigen to the organisms may result in immune stimulation or suppression depending on the nature, localization, concentration of the antigen as well as the age of the organism. The administration of antigen along with adjuvant induces immunity instead of tolerance.
The development of effective and free of side effects immunization against cancer is a puzzling and open issue in research. One of the major problems is the weakness of immune responses against tumor-associated antigens (TAAs) which are usually recognized by the immune system as self-antigens. However, tumor cells express multiple neoantigens, which are specific for each tumor and most of the times specific for each individual. Neoepitopes develop in cancer cells through point mutations, insertions/deletions, amplifications/deletions, translocations, inversions etc . Since neoepitopes are exempted from central tolerance, the development of specific high-affinity T cells could be achieved. Once a neoepitope is selected, the patient’s individual vaccine could be manufactured. Indeed, mutation-based vaccination could indentify multiple shared neoantigens, including mutRas, mutP53, mutVHL, mutEGFR, or mutIDH1 , which could be used for diagnosis and amendment of therapeutic vaccination protocols. Yet, the introduction of next generation sequencing technologies (NGS) revealed that human cancers were much more complicated, carrying thousands of mutations which could provide potential MHC class I neo-epitopes [11,12]. Mapping the entire somatic mutations of an individual tumor, referred as “mutanome”, could allow the selection of neoepitopes. However, population common neoantigens have been unsuccessfully used in vaccine technologies. In such case, vaccine failure lies on neoepitope, carrier and adjuvant selection. According to the technology of second generation vaccines, neoepitopes are linked to a carrier and administered with adjuvant or cross-linked to variable biodegradable materials theoretically designed to target the tumor. However, none of these approaches has been successfully used to treat cancer. Reasons for such failure include: 1) Neoepitopes: their choice is hard to make, time consumable and doubtful as to the effectiveness for mounting an appropriate immune response, 2) Carriers: most of them favor humoral versus cellular immunity, are highly immumogenic themselves, misleading immunity, 3) Adjuvants: only a few with serious side effects are prescribed for human use and 4)Targeted bio-degradable material: may be useful as research goes by, but specific non-biased targeting is still an elusive goal. In addition, another parameter that has to be taken under consideration is the existence of tumor-related immunosuppressive mechanisms including tolerance-related inhibition of T cells  as well as development of granulocyte/macrophage-derived suppressor cells (MDSCs; ), which could cancel the neoepitope-specific response.
To overcome these problems the present trial takes advantage of previous work of the laboratory in designing implantable vaccines for cancer immunotherapy, which stimulate not only humoral but also cellular response against the specific tumor of each individual. The vision towards an antigen-specific immune stimulation in vivo would be to find a way to trigger TH cells without the need of adjuvants. During a natural infection the organism develops an immune response in an antigen-dependent manner. Antigens will be immunogenic, if they succeed to be processed and presented by APCs. Initial efforts towards this direction were to develop implantable biomaterial scaffolds with tunable morphology and chemistry and investigate whether pre-activated macrophages absorbed on them will be able to stimulate the development of a humoral immune response . Although such approach represents a personalized vaccination, it eliminates all side effects due to the non-specific stimulation of adjuvants.
Vaccine safety is a serious issue occupying the World Health Organization since vaccine technology was developed. Although neoepitopes are free of virulence and infectivity, adjuvants and/or carrier material are prone to side effects, which could only be evaluated upon massive use raising thus serious bioethical issues. The use of non-biodegradable implants avoids adjuvant-dependent side effects, while providing the necessary immune stimulation to the host . The application described herein allows recipient APCs to present tumor’s “mutanome”, ensuring thus best neoantigen presentation for each individual. In such case, antigenic neoepitope screening will be naturally performed by APCs, which upon implantation could stimulate T cells for specific immune response development. As already mentioned, tolerance depends on the nature, localization, concentration of the antigen as well as the age of the organism. On the other hand, in the context of immune escape, many tumors develop mechanisms to exhaust the immune system to their benefit, as in the case of programmed death ligand-1 (PDL-1) expression . Programmed Cell Death Protein 1 (PD-1) is naturally expressed on T cells during thymic education promoting self-tolerance by activating apoptosis of self antigen-specific T cells. However, such mechanisms are tightly related to the affinity and avidity of the T cell receptor (TCR) for the antigen/MHC complex and control the development of stimulatory versus suppressive mediators [16, 17, 18].
Implantable vaccine-on-chip technology for cancer immunotherapy
The solid biomaterial used to support cellular growth can be any non-biodegradable, non-toxic material capable to allow natural antigen-loading and presentation in vitro and further activation of the immune response in vivo. Based on previous studies, 3-dimensional laser micro-textured implantable Si-scaffolds supported mouse macrophage adherence, allowed natural seeding with human serum albumin (antigen) and specific antibody and inflammatory cytokine production in vitro. Implantation of these Si-scaffolds loaded with antigen-activated macrophages induced a mild inflammatory reaction along with antigen-specific antibody production in vivo, which could be detected even 7 months post implantation . This technology was originally developed using a classic protein antigen (human serum albumin) and was later applied with success to Salmonella Typhimurium and M. luteus-derived peptidoglycan .
The implantable vaccine-on-chip technology has been successfully applied to cancer immunotherapy. The implantable scaffolds consist of 3-dimensional micro-textured Si-scaffolds, produced by ultrafast lasers . These are preferred over 2D surfaces since they offer more realistic micro- and local-environment for cell activity. Indeed, 2D surfaces have been shown to alter cell metabolism and gene expression patterns . In addition, it has been shown that 3D micro/nano topography and surface chemistry influence the differentiation and migration of macrophages . Ultrafast laser structuring presents distinct advantages as it (a) is versatile and material independent (b) is rapid, easily adaptable and scalable through parallel processing and (c) allows the unique possibility for controllable, high resolution features at both the micro- and nano- length scales. The latter is due to the limited size of the affected volume – very close to the diffraction-limited volume. Additional advantages of laser structuring include high fabrication rate, non-contact interaction, applicability to many types of materials and reproducibility. Furthermore lasers can be easily incorporated to computer-assisted fabrication systems for complex and customized 3D matrix structure design and manufacture. Such systems gave rise to a versatile class of scaffold production techniques which are laser-based solid-free-form (SFF) fabrication techniques. The SFF is essentially a rapid prototyping technique which allows control over macroscopic properties, such as scaffold shape and microscopic internal architecture.
Although neoepitopes are free of virulence and infectivity, adjuvants and/or carrier material are prone to side effects, which could only be evaluated upon massive use raising thus serious bioethical issues. The use of non-biodegradable implants avoids adjuvant-dependent side effects, while providing the necessary immune stimulation to the host . The present trial is designed to allow recipient APCs to present tumor’s “mutanome”, ensuring thus best neoantigen presentation for each individual. In such case, antigenic neoepitope screening will be naturally performed by APCs, which upon implantation could stimulate T cells for specific immune response development. The determination of the optimal antigen dose in vitro is a crucial parameter to define, since this will dictate the immune responsiveness. Small or very high antigen concentrations, as well as low affinity or very high affinity TCR-antigen-MHC interactions will drive to tolerance instead of immunity. Providing the whole “mutotome” to APCs, it is mandatory to define the conditions for the development of anti-tumor effector TH, Tc as well as B cells.
Strategy of the application
In cancer immunotherapy the application of the vaccine-on-chip technology includes 5 stages:
- Patient’s biopsy acquisition: No special intervention is needed, because all patients at some stage of the disease have been submitted to solid tumor sampling. No fresh biopsy is needed. Cell extracts are obtained without the use of chemicals or enzymes.
- Determining patient’s antigenicity to tumor extract: Blood collection from the patient for white cell isolation and testing. This step will determine the most effective dose of the tumor extract to be used for the patient. The tumor cell extract contains all neoantigens of the specific patient, against which the immune system will be able to mount a cellular and humoral immune reaction. Finding the right immunogenic dose of the extract is essential, because it should also outweigh the possible suppression that the patient may have developed against these neoantigens.
- Cell loading to the microstructured silicon scaffolds: Blood collection for isolation and culture of white blood cells on top of microstructured silicon scaffolds.
- Activation of the adherent cells: The immunogenic dose of tumor extract determined from step 2 is provided to the adherent cells of step 3. After the 24-hour incubation, the non-adherent cells are removed and the culture medium is replaced by fresh medium containing the appropriate dose of tumor cell extract, as defined in step 2.
- Subcutaneous implantation of the activated silicon scaffold to the patient and monitoring of efficacy. The effectiveness of the procedure is apparent one week after implantation.
Safety of the application
The design of the personalized implantable vaccines has been carried out within the context of maximal safety for the patient, absence of side effects and minimal burden for the body.
- The use of autologous peripheral blood cells ensures the absence of any graft rejection reactions.
- The use of the patient’s own biopsy ensures the specificity of the reaction against the specific tumor, while at the same time avoiding any allogeneic reactivity.
- The ex-vivo activation of the autologous cells with the tumor cell extracts allows determination of the optimal conditions for the desired response.
- The choice of a non-biodegradable material as a platform for creating a system resembling a secondary lymphatic organ on-chip, ensures the absence of by-products with unknown side effects.
Preclinical studies in experimental animals show that under the above conditions, the “activated” implant stimulates the organism to the desired degree, activates cytotoxic Tc cells against the tumor, and induces tumor specific IgG antibodies.
The “activated” implant provides the necessary immune memory to the host (patient) against the original antigen, which is detected even 7 months after application, allowing thus and suggesting implant removal 6 months post-imlantation.
The only foreign to the organism material used in this application is the Si-scaffold, which consists of single crystal n-type Silicon (1 0 0) wafers subjected to laser irradiation in a vacuum chamber evacuated down to a residual pressure of 10–2 mbar. Silicon dioxide (SiO2) has been proposed and used in clinical trials to coat metal stents or to construct drug delivery devices and implants [23, 24]. In this application Si-scaffold is used as a local mild adjuvant necessary to stimulate the immune system but avoiding generalized side effects.
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Grow into “Ex vivo Immunization Center” (EVIC)
Related founders’ publications:
- Zerva, I., Simitzi, C., Ranella, A., Stratakis, E., Fotakis, C., Athanassakis, I. 3-dimensional laser structured scaffolds improve macrophage adherence and antigen-specific response. Procedia Engineering, 59: 211-218, 2013. http://dx.doi.org/10.1016/j.proeng.2013.05.113
- Zerva, I., Simitzi, C., Siakouli-Galanopoulou, A., Ranella, A., Stratakis, E., Fotakis, C., Athanassakis I. Implantable vaccines: In vitro antigen presentation enables in vivo immune response. Vaccine 33: 3142–3149, 2015. http://dx.doi.org/10.1016/j.vaccine.2015.04.017
- Ζέρβα I., Λαναρά X., Στρατάκης E., Αθανασάκη E. Εμφυτεύσιμα εμβόλια με τη χρήση προενεργοποιημένων ικριωμάτων πυριτίου στη θεραπεία του καρκίνου. Aνοσία; 14, 3: 49 – 52, 2018
- Zerva, I., Katsoni, E., Simitzi, C., Stratakis, E., Athanassakis, I. Laser micro-structured Si scaffold implantable vaccines against Salmonella Typhimurium. Vaccine 37: 2249-2257 (2019) org/10.1016/j.vaccine.2019.02.080
- Zerva Ι, Simitzi C, Stratakis E and Athanasakis I. Personalized Implantable Vaccines with Antigen PreActivated Macrophages. Austin J Clin Immunol. 2019; 6(1):1038
- Zerva I, Pateraki V, Athanassakis I. Implantable vaccines: a solution for immune system manipulation to any antigenic stimulus. J Immunological Sci 2020; 4:5-11.
- Αθανασάκη E. Νέα στρατηγική ανάπτυξης εξατομικευμένων εμβολίων στη λοίμωξη COVID-19. Ανοσία; 16, 3: 56 – 58, 2020.
- Zerva I, Bakela K, Athanassakis I. Immunotherapy-on-chip against an experimental sepsis model. Inflammation 2021 (in press)
- Zerva, I., Simitzi, Ch., Ranella, A., Stratakis, E., Fotakis, C., Athanassakis, I. 3-dimensional laser structured scaffolds improve macrophage adherence and antigen-specific response. European Congress of Immunology, September 5-8, 2012, Glasgow, Scotland (travel grant award)
- Zerva, I., Simitzi C., Ranella A., Stratakis E., Fotakis C., Athanassakis I. 3-dimensional laser structured scaffolds improve macrophage adherence and antigen-specific response. ICTE2013, International Conference of Tissue Engineering, June 2013, Portugal.
- Zerva, I., Simitzi C., Ranella A., Stratakis E., Fotakis C., Athanassakis I. 3-dimensional laser structured scaffolds improve macrophage adherence and antigen-specific response. TERMIS-EU 2013 June 17-20 Istanbul.
- Ranella A., Zerva, I., Simitzi C., Fotakis C., Stratakis E., Athanassakis I. 3D micro laser textured transplantable scaffolds improve macrophage adherence and antigen-specific response. E-MRS 2013 FALL MEETING September 16-20, Warsaw University of Technology.
- Zerva, I., Simitzi C., Ranella A., Stratakis E., Athanassakis I. Biomaterial-induced inflammatory reaction upon in vivo implantation in mice. E-MRS 2014 FALL MEETING, Warsaw University of Technology.
- Zerva, I., Simitzi, C., Stratakis, E., Ranella, A., Athanassakis, I. Transplantable immune modulation in response to autologous cancer cells. 4th European Congress of Immunology, September 6-9, 2015, Vienna
- Zerva I., E. Katsoni E., Simitzi C., Ranella A., Stratakis E., Athanassakis I. Differential behavior of laser micro-structured Si scaffolds according to the biological stimulant. 10th FRONTIERS IN IMMUNOLOGY RESEARCH INTERNATIONAL CONFERENCE Island of Crete, Greece Minoa Palace Resort & Spa, July 1-4, 2017
- Zerva, I., I.Athanassakis, I. «Εξατομικευμένα Εμφυτεύσιμα Εμβόλια για τον καρκίνο του μαστού» Συνέδριο Κλινικής και Μεταφραστικής Ογκολογίας, Ηράκλειο, Ελλάδα., 2017
- Zerva, I., Kouimtzidis, A., Pateraki,V., Lanara,C., Stratakis, E., Athanassakis,I «Implantable, pre-activated microconed-Si scaffold vaccines for cancer therapy». 5th European Congress of Immunology-ECI 2018, Άμστερνταμ, Ολλανδία, 2018.
- Πατεράκη B., Ζέρβα I., Λανaρά X., Στρατάκης, E., 2, Ε. Αθανασάκη E. ΕΞΑΤΟΜΙΚΕΥΜΕΝΑ ΑΝΟΣΟΕΜΦΥΤΕΥΜΑΤΑ ΙΚΡΙΩΜΑΤΩΝ ΠΥΡΙΤΙΟΥ ΣΤΗ ΘΕΡΑΠΕΙΑ ΤΟΥ ΚΑΡΚΙΝΟΥ, 11ο Πανελλήνιο Συνέδριο Ανοσολογίας, Αθήνα, Δεκέμβριος 2019.
- Αθανασάκη Ε. Νέα στρατηγική ανάπτυξης εξατομικευμένων εμβολίων στη λοίμωξη COVID-19. Ετήσιο Συνέδριο Ανοσολογίας 2020, Αθήνα, Νοέμβριος 2020. INVITED SPEAKER
- Bakela K, Zerva I, Athanassakis I. Immunotherapy-on-chip against an experimental sepsis model. 14thGlobal Summit on Immunology and Cell Biology, March 22-23, 2021
- Αθανασάκη Ε. Τεχνολογία εξατομικευμένων εμφυτεύσιμων εμβολίων: αποτελεσματικότητα και ασφάλεια. ΕΕΑ, Παγκόσμια Ημέρα Ανοσολογίας 2021, Μάϊος 2021. INVITED SPEAKER
- Zerva I, Bakela K, Athanassakis I. Immunotherapy-on-chip against an experimental sepsis model. 6th European Congress of Immunology – ECI 2021 which will take place virtually from September 1-4, 2021. (EFIS grant award-EFISGrantECI2021)
- Athanassakis, I., Zerva, I., Simitzi, C., Ranella, A., Stratakis E., Personalized implantable vaccines using antigen pre-activated monocytes (Εξατομικευμένα εμφυτεύσιμα εμβόλια με αντιγονικά διεγερμένα μονοκύτταρα). ΟΒΙ 20140100471, 19/9/2014 https://worldwide.espacenet.com/patent/search/family/056090633/publication/GR1008652B?q=20140100471
- Athanassakis I., Zerva, I., Stratakis E. Personalized implant against cancer. Εξατομικευμένο εμφύτευμα κατά του καρκίνου. ΟΒΙ 20190100297
- ImmunoRec IKE. Personalized vaccine against SARS-CoV-2 (OBI 245-0004327167, under evaluation)