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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 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.

// Gijs G. Zom 1 , Marij J.P. Welters 2 , Nikki M. Loof 2 , Renske Goedemans 2 , Sinead Lougheed 3 , Rob R.P.M. Valentijn 4 , Maarten L. Zandvliet 4 , Nico J. Meeuwenoord 5 , Cornelis J.M. Melief 1, 6 , Tanja D. de Gruijl 3 , Gijsbert A. Van der Marel 5 , Dmitri V. Filippov 5 , Ferry Ossendorp 1, * , Sjoerd H. Van der Burg 2, * 1 Department of Immunohematology and Blood Transfusion, Section Tumorimmunology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands 3 Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands 5 Leiden Institute of Chemistry, University of Leiden, Leiden, The Netherlands 6 ISA Pharmaceuticals BV, Leiden, The Netherlands * These authors have contributed equally to this work Correspondence to: Ferry Ossendorp, email: f.a.ossendorp@lumc.nl Sjoerd H. Van der Burg, email: shvdburg@lumc.nl Keywords: Toll-like receptor ligand, conjugate, synthetic long peptide, cancer vaccine, cervical cancer Received: April 19, 2016 Accepted: July 10, 2016 Published: August 23, 2016 ABSTRACT The potency of human papillomavirus type 16 (HPV16)-encoded synthetic long peptides (SLP), conjugated to an optimized Toll-like receptor 2 ligand (TLR2-L), was assessed in ex vivo activation of HPV16 + cancer patient-derived T cells. Two highly immunogenic SLP sequences derived from the oncogenic E6 protein of HPV16 were selected and conjugated to a Pam3CSK4-based TLR2-L under GMP conditions. Both conjugates were able to mature human DCs in vitro and to activate human skin-derived antigen-presenting cells (APCs) upon intradermal injection in an ex vivo skin model, associated with induction of a favorable chemokine profile to attract and activate T cells. The conjugated SLPs were efficiently processed by APCs, since HPV16-specific CD4 + and CD8 + T-cell clones isolated from HPV16+ cervical tumors proliferated in response to both conjugates. The TLR2-L SLP conjugates significantly enhanced ex vivo T helper type 1 T-cell activation in cell suspensions obtained from tumor-draining lymph nodes (LN) resected during hysterectomy of HPV16 + cervical cancer patients. These results show that TLR2-L SLP conjugates can activate circulating or LN-derived tumor-specific T cells, a promising outcome for studying these two conjugates in a phase I/II clinical safety and immunogenicity trial.

We have previously identified a natural B cell epitope derived from tetanus toxin named Minimal Tetanus Toxin Epitope (MTTE)[1]. Most healthy individuals have IgG1 antibodies to this peptide sequence but no IgM antibodies. We have assessed the possibility to apply this MTTE sequence in drug development with the intention to create immune complexes and use them as vehicles to carry antigen-material into dendritic cells. Thru a conjugation method three MTTE sequences and a longer synthetic peptide stretch harboring T cell epitopes are linked together. As immune complexes are efficient carriers of antigen material and can promote cross-presentation the aim is to use these conjugates for therapeutic purposes in diseases where a strong cellular immune response should be induced. To further evaluate the MTTE peptide conjugates in drug development we have investigated how a diphtheria, tetanus, pertussis (DTP) vaccination induces antibody titers against the linear tetanus sequence. Herein we have studied the MTTE location in the tertiary structure of the protein, how cancer patients respond with antibody titers against MTTE along with a subsequent T cell response and in vivo anti-tumor responses (mice). In addition we have studied safety in terms of cytokine release assessment pre and post a DTP vaccination. We found that the MTTE sequence is part of an alpha-helix that is externally oriented and thereby accessible for peptide binding. We found that not helper epitope is part of the MTTE sequence and that healthy individuals as well as cancer patients can mount IgG titers against the epitope, this also result in more efficient induction of recall responses post a DTP booster than pre boost. Mice seropositive to the MTTE sequence can mount anti-tumor responses, while this is not seen in seronegative animals. In addition, a cytokine release assessment using a modified chandler loop model with intact complement does not display a broad immediate cytokine release pre or post a DTP booster. We conclude that it is efficacious and safe to make use of a peptide conjugate in a drug development project with the aim to use it as a vaccine strategy. [1] Mangsbo et al. In press. Molecular Immunology 2017 Citation Format: Jan Wouter Drijfhout, Erika Fletcher, Justyna Leja-Jarblad, Iliana Kerzeli, Robert Cordfunke, Gunilla Tornqvist, Frida Lindqvist, Greta Hultqvist, Rob Valentijn, Sam Ladjervardi, Michael Haggman, Gustav Ullenhag, Sara Mangsbo. A tetanus-way of improving synthetic long peptide tumor vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5638.

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.