Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all... Show moreTherapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant (R) adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7(+) cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days. Show less
Speetjens, F.M.; Welters, M.J.P.; Slingerland, M.; Poelgeest, M.I.E. van; Steenwijk, P.J.D. van; Roozen, I.; ... ; Ossendorp, F. 2022
Background: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long... Show moreBackground: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP. Methods: A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-) malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 mu g per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays. Results: Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial. Conclusions: Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity. Show less
Speetjens, F.M.; Welters, M.J.P.; Slingerland, M.; Poelgeest, M.I.E. van; Steenwijk, P.J.D. van; Roozen, I.; ... ; Ossendorp, F. 2022
Background: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long... Show moreBackground: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP. Methods: A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-) malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 mu g per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays. Results: Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial. Conclusions: Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity. 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
Background: Therapeutic vaccines based on synthetic long peptides (SLPs) have a great potential for immunotherapy of cancer patients as these SLPs include both human leukocyte antigen (HLA) class I... Show moreBackground: Therapeutic vaccines based on synthetic long peptides (SLPs) have a great potential for immunotherapy of cancer patients as these SLPs include both human leukocyte antigen (HLA) class I and II epitopes and no patient selection for HLA types is required. The antigen-induced immune response can be strengthened with immune stimulating additives. Amplivant (AV) is a synthetic Toll-like receptor 2 ligand which can be directly conjugated to tumor peptide antigens. In preclinical studies, AV-conjugation to antigens led to both enhanced antigen presentation by dendritic cells and T-cell priming and caused superior induction of effective anti-tumor responses. Moreover, AV-conjugated SLPs showed a 100 times higher immune response compared to unconjugated SLP. The current study is a first-in-human trial to investigate safety and immunogenicity of AV-conjugated human papillomavirus (HPV)16-SLPs. Methods: A dose escalation phase I trial was performed in 24 patients with HPV16 positive (pre-) malignant lesions. AV was conjugated to two SLPs derived from the most immunodominant regions of the HPV16 E6 oncoprotein. Four dose groups (1, 5, 20 or 50 μg of each peptide) in 6 patients each were studied. The vaccine was injected three times intradermally in DMSO / water with a three-week interval. Adverse events (AE) were collected according to CTCAE v4.0 up to 26 weeks. Peptide-specific T-cell immune responses were determined in blood samples taken before and after vaccination using complementary immunological assays (proliferation assay, IFNγ-ELISPOT and cytokine bead array). Results: Toxicity after three AV-conjugated HPV16-SLP vaccinations was limited to CTCAE grade 1 or 2, with predominantly inflammation at the vaccination site and sometimes flu-like symptoms, which generally resolved within one day. Dose increase resulted from no AE in the lowest dose group to mild/moderate AE in all vaccinated persons in the highest dose group. In the lowest dose group, minor vaccine-induced T-cell responses were observed in three of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T-cell response after vaccination. The induced T-cell response against HPV16 lasted until the end of the trial. Conclusions: This first-in-human study showed that AV conjugated to SLPs can safely be used as an intradermal therapeutic vaccine. AV-conjugated HPV16-SLP was able to induce robust HPV16-specific T-cell immunity in patients treated for HPV16 positive (pre-) malignancies without any other vaccine adjuvant or formulation. Increase in dose resulted in both a higher number of mild adverse events as well as stronger T-cell immunity. Clinical trial information: NCT02821494.Show less
CD8(+) T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor... Show moreCD8(+) T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8(+) T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8(+) T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8(+) T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8(+) T-cell dysfunction. Show less
Saxena, M.; Burg, S.H. van der; Melief, C.J.M.; Bhardwaj, N. 2021
Therapeutic cancer vaccines have undergone a resurgence in the past decade. A better understanding of the breadth of tumour-associated antigens, the native immune response and development of novel... Show moreTherapeutic cancer vaccines have undergone a resurgence in the past decade. A better understanding of the breadth of tumour-associated antigens, the native immune response and development of novel technologies for antigen delivery has facilitated improved vaccine design. The goal of therapeutic cancer vaccines is to induce tumour regression, eradicate minimal residual disease, establish lasting antitumour memory and avoid non-specific or adverse reactions. However, tumour-induced immunosuppression and immunoresistance pose significant challenges to achieving this goal. In this Review, we deliberate on how to improve and expand the antigen repertoire for vaccines, consider developments in vaccine platforms and explore antigen-agnostic in situ vaccines. Furthermore, we summarize the reasons for failure of cancer vaccines in the past and provide an overview of various mechanisms of resistance posed by the tumour. Finally, we propose strategies for combining suitable vaccine platforms with novel immunomodulatory approaches and standard-of-care treatments for overcoming tumour resistance and enhancing clinical efficacy.This Review broadly discusses therapeutic cancer vaccines, covering resistance mechanisms and strategies to overcome these, how to improve the antigen repertoire for vaccines and vaccine platforms, and approaches for enhancing immunotherapy efficacy. Show less
Dou, Y.Y.; Jansen, D.T.S.L.; Bosch, A. van den; Man, R.A. de; Montfoort, N. van; Araman, C.; ... ; Buschow, S.I. 2020
Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core... Show moreSynthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4(+ )and CD8(+) T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV. Show less
Melief, C.J.M.; Welters, M.J.P.; Vergote, I.; Kroep, J.R.; Kenter, G.G.; Ottevanger, P.B.; ... ; Burg, S.H. van der 2020
Therapeutic cancer vaccines have effectively induced durable regressions of premalignant oncogenic human papilloma virus type 16 (HPV16)-induced anogenital lesions. However, the treatment of HPV16... Show moreTherapeutic cancer vaccines have effectively induced durable regressions of premalignant oncogenic human papilloma virus type 16 (HPV16)-induced anogenital lesions. However, the treatment of HPV16-induced cancers requires appropriate countermeasures to overcome cancer-induced immune suppression. We previously showed that standard-of-care carboplatin/paclitaxel chemotherapy can reduce abnormally high numbers of immunosuppressive myeloid cells in patients, allowing the development of much stronger therapeutic HPV16 vaccine (ISA101)-induced tumor immunity. We now show the clinical effects of ISA101 vaccination during chemotherapy in 77 patients with advanced, recurrent, or metastatic cervical cancer in a dose assessment study of ISA101. Tumor regressions were observed in 43% of 72 evaluable patients. The depletion of myeloid suppressive cells by carboplatin/paclitaxel was associated with detection of low frequency of spontaneous HPV16-specific immunity in 21 of 62 tested patients. Patients mounted type 1 T cell responses to the vaccine across all doses. The group of patients with higher than median vaccine-induced immune responses lived longer, with a flat tail on the survival curve. This demonstrates that chemoimmunotherapy can be exploited to the benefit of patients with advanced cancer based on a defined mode of action. Show less
Dou, Y.; Jansen, D.T.S.L.; Bosch, A. van den; Man, R.A. de; Montfoort, N. van; Araman, C.; ... ; Buschow, S.I. 2020
Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core... Show moreSynthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4+ and CD8+ T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV. 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
Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine... Show moreEnhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin–derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer. Show less
Dou, Y.Y.; Montfoort, N. van; Bosch, A. van den; Man, R.A. de; Zom, G.G.; Krebber, W.J.; ... ; Woltman, A.M. 2018
