SARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal... Show moreSARS-CoV-2 is the third zoonotic coronavirus to cause a major outbreak in humans in recent years, and many more SARS-like coronaviruses with pandemic potential are circulating in several animal species. Vaccines inducing T cell immunity against broadly conserved viral antigens may protect against hospitalization and death caused by outbreaks of such viruses. We report the design and preclinical testing of 2 T cell–based pan-sarbecovirus vaccines, based on conserved regions within viral proteins of sarbecovirus isolates of human and other carrier animals, like bats and pangolins. One vaccine (CoVAX_ORF1ab) encoded antigens derived from nonstructural proteins, and the other (CoVAX_MNS) encoded antigens from structural proteins. Both multiantigen DNA vaccines contained a large set of antigens shared across sarbecoviruses and were rich in predicted and experimentally validated human T cell epitopes. In mice, the multiantigen vaccines generated both CD8+ and CD4+ T cell responses to shared epitopes. Upon encounter of full-length spike antigen, CoVAX_MNS-induced CD4+ T cells were responsible for accelerated CD8+ T cell and IgG Ab responses specific to the incoming spike, irrespective of its sarbecovirus origin. Finally, both vaccines elicited partial protection against a lethal SARS-CoV-2 challenge in human angiotensin-converting enzyme 2–transgenic mice. These results support clinical testing of these universal sarbecovirus vaccines for pandemic preparedness. Show less
Gu, Y.Z.; Zhang, Z.K.; Camps, M.G.M.; Ossendorp, F.; Wijdeven, R.H.; Dijke, P. ten 2023
Therapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient... Show moreTherapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient drug penetration and rapid drug clearance and toxicity can be improved by using a liposomal platform. Drug resistance for instance against pemetrexed, can be reduced by combination with docetaxel. Here, we developed a specific liposomal formulation to simultaneously deliver docetaxel and pemetrexed to enhance efficacy and safety. Hydrophobic docetaxel and hydrophilic pemetrexed were co-encapsulated into pH-sensitive liposomes using a thin-film hydration method with high efficiency. The physicochemical properties, toxicity, and immunological effects of liposomes were examined in vitro. Biodistribution, anti-tumor efficacy, and systemic immune response were evaluated in vivo in combination with PD-L1 immune checkpoint therapy using two murine colon cancer models. In cellular experiments, the liposomes exhibited strong cytotoxicity and induced immunogenic cell death. In vivo, the treatment with the liposome-based drug combination inhibited tumor development and stimulated immune responses. Liposomal encapsulation significantly reduced systemic toxicity compared to the delivery of the free drug. Tumor control was strongly enhanced when combined with anti-PDL1 immunotherapy in immunocompetent mice carrying syngeneic MC38 or CT26 colon tumors. We showed that treatment with liposome-mediated chemotherapy of docetaxel and pemetrexed combined with anti-PD-L1 immunotherapy is a promising strategy for the treatment of colon cancers. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of major histocompatibility complex (MHC) class II-eluted natural peptides. MHC class II -presented peptide sequences are identified by introducing the MHC class II transactivator (CIITA) in tumor cells that were originally MHC class II negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals oncoviral-and neo-epitopes as well as shared epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses, which helped tu-mor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes pre-sented by any MHC class II allele that would be otherwise very difficult to predict and reveals important tar-gets for cancer immunotherapy. Show less
Hos, B.J.; Tondini, E.; Camps, M.G.M.; Rademaker, W.; Bulk, J. van den; Ruano, D.; ... ; Ossendorp, F. 2022
We report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide... Show moreWe report an approach to identify tumor-specific CD4+ T cell neo-epitopes of both mouse and human cancer cells by analysis of MHC class II-eluted natural peptides. MHC class II-presented peptide sequences are identified by introducing the MHC class II transactivator CIITA in tumor cells that were originally MHC class II-negative. CIITA expression facilitates cell-surface expression of MHC class II molecules and the appropriate peptide-loading machinery. Peptide elution of purified MHC class II molecules and subsequent mass spectrometry reveals many novel oncoviral-, shared and neo-epitopes. Immunological relevance of these epitopes is shown by natural presentation by dendritic cells and immunogenicity. Synthetic peptide vaccination induced functional CD4+ T cell responses which helped tumor control in vivo. Thus, this CIITA transfection approach aids to identify relevant T helper epitopes presented by any MHC class II allele that would be otherwise very difficult to predict and reveals important new targets for cancer immunotherapy. Show less
Upconversion nanoparticles (UCNPs) represent a group of NPs that can convert near-infrared (NIR) light into ultraviolet and visible light, thus possess deep tissue penetration power with less... Show moreUpconversion nanoparticles (UCNPs) represent a group of NPs that can convert near-infrared (NIR) light into ultraviolet and visible light, thus possess deep tissue penetration power with less background fluorescence noise interference, and do not induce damage to biological tissues. Due to their unique optical properties and possibility for surface modification, UCNPs can be exploited for concomitant antigen delivery into dendritic cells (DCs) and monitoring by molecular imaging. In this study, we focus on the development of a nano-delivery platform targeting DCs for immunotherapy and simultaneous imaging. OVA 254-267 (OVA24) peptide antigen, harboring a CD8 T cell epitope, and Pam3CysSerLys4 (Pam3CSK4) adjuvant were chemically linked to the surface of UCNPs by amide condensation to stimulate DC maturation and antigen presentation. The OVA24-Pam3CSK4-UCNPs were thoroughly characterized and showed a homogeneous morphology and surface electronegativity, which promoted a good dispersion of the NPs. In vitro experiments demonstrated that OVA24-Pam3CSK4-UCNPs induced a strong immune response, including DC maturation, T cell activation, and proliferation, as well as interferon gamma (IFN-gamma) production. In vivo, highly sensitive upconversion luminescence (UCL) imaging of OVA24-Pam3CSK4-UCNPs allowed tracking of UCNPs from the periphery to lymph nodes. In summary, OVA24-Pam3CSK4-UCNPs represent an effective tool for DC-based immunotherapy. Show less
Photodynamic therapy (PDT) has shown impressive therapeutic effects on various types of cancers by reactive oxygen species (ROS) generation and induction of immune responses. However, under certain... Show morePhotodynamic therapy (PDT) has shown impressive therapeutic effects on various types of cancers by reactive oxygen species (ROS) generation and induction of immune responses. However, under certain conditions, the immune responses induced by PDT are not always sufficient to eradicate the remaining tumor cells. On the other hand, the photosensitizer indocyanine green (ICG) can mediate PDT under near-infrared (NIR) illumination, thereby enhancing the penetration depth of the excitation light into the tumor. We found that ICG is rapidly taken up in vitro by colorectal MC38 and CT26 tumor cells and it promotes PDT-mediated cell-killing effects. Our results furthermore revealed that ICG induces immunogenic cell death (ICD), as dendritic cells (DCs) were found to engulf ICG-PDT-treated tumor cells and undergo phenotypic maturation. ICG accumulated in tumors 2 h after administration, as measured by fluorescence and photoacoustic imaging. Considering the advantages of ICG as a photosensitizer, we sought to design a therapy that combines PDT and immune checkpoint blockade to maximize tumor control. To this end, a 25% thermosensitive polymer 407 hydrogel was included as a co-delivery platform for this treatment scheme. NIR-PDT under 808 nm irradiation in combination with cytotoxic T-lymphocyte-associated protein 4 (CTLA4)/programmed death-ligand 1 (PD-L1) checkpoint blockade prolonged survival rate of colorectal tumor-bearing mice by inducing a series of immune responses, like the phagocytosis of tumor debris by macrophages and DCs, and induction of acute inflammation, leukocyte infiltration, maturation and activation of DCs. Altogether, our work presents a NIR-triggered PDT strategy in combination with immune checkpoint blockade. Compared to a single treatment, the combination treatment increased efficiency to inhibit solid tumor growth and improved the survival rate of tumor-bearing mice. Show less
Pardieck, I.N.; Sluis, T.C. van der; Gracht, E.T.I. van der; Veerkamp, D.M.B.; Behr, F.M.; Duikeren, S. van; ... ; Arens, R. 2022
Vaccination regimens and the number of doses required for optimal immunity and protection are critical factors in the translation of vaccines. Here the authors show administration of a three dose... Show moreVaccination regimens and the number of doses required for optimal immunity and protection are critical factors in the translation of vaccines. Here the authors show administration of a three dose protocol of a single T cell epitope to the SARS-CoV-2 spike protein induces a robust CD8(+) T cell response and confers protection in a lethal murine challenge model of infection.Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8(+) T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8(+) T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8(+) effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8(+) T cell response leading to protection against otherwise lethal SARS-CoV-2 infection. Show less
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. Show less
Leeuwen, T. van; Araman, C.; Pournara, L.P.; Kampstra, A.S.B.; Bakkum, T.; Marqvorsen, M.H.S.; ... ; Kasteren, S.I. van 2021
Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and... Show moreProteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation. Show less
Ho, N.I.; Camps, M.G.M.; Verdoes, M.; Munz, C.; Ossendorp, F. 2021
Autophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in... Show moreAutophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross-presentation to CD8(+) T cells. In the current study, we investigated the role of autophagy in long-term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5(-/-)DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross-presentation. This augmented antigen cross-presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long-term MHCI cross-presentation. Show less
Hos, B.J.; Camps, M.G.M.; Bulk, J. van den; Tondini, E.; Ende, T.C. van den; Ruano, D.; ... ; Ossendorp, F. 2020
The murine MC-38 colorectal cancer model is a commonly used model for cancer with high mutational burden, which is sensitive for immune checkpoint immunotherapy. We set out to analyze endogenous... Show moreThe murine MC-38 colorectal cancer model is a commonly used model for cancer with high mutational burden, which is sensitive for immune checkpoint immunotherapy. We set out to analyze endogenous CD8(+) T cell responses to MC-38 neo-antigens in tumor-bearing mice and after anti-PD-L1 checkpoint therapy. Through combination of whole-exome sequencing analysis with mass spectrometry of MHC class I eluted peptides we could identify eight candidate epitopes. Of these, a neo-epitope encoded by a point-mutation in the sequence of the ribosomal protein L18 (Rpl18) strongly dominated the CD8(+) T cell response to our MC-38 cell-line in comparison to a previously described neo-epitope in the Adpgk protein. Therapeutic vaccination with synthetic peptides induced CD8(+) T cell responses against the mutated Rpl18 epitope, which controlled tumor growth in vivo. This immunologically dominant response to mutated Rpl18 is of great importance in the development and optimization of immunotherapeutic strategies with the MC-38 tumor model. Show less
Ligands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of... Show moreLigands for the Toll-like receptor (TLR) family can induce activation of cells of the innate immune system and are widely studied for their potential to enhance adaptive immunity. Conjugation of TLR2-ligand Pam3CSK4 to synthetic long peptides (SLPs) was shown to strongly enhance the induction of antitumor immunity. To further improve cancer vaccination, we have previously shown that the novel TLR2-L Amplivant (AV), a modified Pam3CSK4, potentiates the maturation effects on murine DCs. In the current study, we further assessed the immunological properties of AV.\nNaïve mice were vaccinated with a conjugate of either Pam3CSK4 or AV and an SLP to assess specific T cell priming efficiency in vivo. The potency of AV and Pam3CSK4, either as free compounds or conjugated to different SLPs, to mature murine DCs was compared by stimulating murine dendritic cells overnight followed by ELISA and flow cytometry analysis. Murine tumor experiments were carried out by vaccinating mice carrying established HPV16 E6 and E7-expressing tumors and subsequently analyzing myeloid and lymphoid cells infiltrating the tumor microenvironment. Furthermore, tumor outgrowth after vaccination was monitored to enable comparison of the efficiency to induce antitumor immunity by Pam3CSK-SLP and AV-SLP conjugates. To enhance therapeutic efficacy, AV-SLP conjugate vaccination was combined with ablative therapies to assess whether synergism between such therapies would occur.\nSLPs conjugated to AV induce stronger DC maturation, in vivo T cell priming and antitumor immunity compared to conjugates with Pam3CSK4. Interestingly, AV-SLP conjugates modulate the macrophage populations in the tumor microenvironment, correlating with a therapeutic effect in an aggressive murine tumor model. The potency of AV-SLP conjugates in cancer vaccination operates optimally in combination with chemotherapy or photodynamic therapy.\nThese data allow further optimization of vaccination-based immunotherapy of cancer by use of the improved TLR2-ligand Amplivant.\nBACKGROUND\nMETHODS\nRESULTS\nCONCLUSION Show less
Zom, G.G.; Willems, M.M.J.H.P.; Khan, S.; Sluis, T.C. van der; Kleinovink, J.W.; Camps, M.G.M.; ... ; Ossendorp, F. 2018
Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we... Show moreActivation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels–Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo. Show less
