Durable and dynamic hTERT immune responses following vaccination with the long-­peptide cancer vaccine UV1: long-­termfollow-­up of three phase I clinical trials

Espen Basmo Ellingsen  ⁜ ⁜ ,1,2,3 Elin Aamdal,2,4,5 Tormod Guren,4 Wolfgang Lilleby,4 Paal F Brunsvig,4 Sara M Mangsbo,6,7 Steinar Aamdal,3 Eivind Hovig,1,8

Nadia Mensali,9 Gustav Gaudernack,3 Else Marit Inderberg9

-1136/jitc.10 as published first Cancer: Immunother J

To cite: Ellingsen EB, Aamdal E, Guren T, et al . Durable and dynamic hTERT immune responses following vaccination with the long-­peptide cancer vaccine UV1: long-­termfollow-­ up of three phase I clinical trials. Journal for ImmunoTherapy

of Cancer 2022;10:e004345. doi:10.1136/jitc-2021-004345

© Author(s) (or their employer(s)) 2022. Re-­use permitted under CC BY-­NC. No commercial re-­use. See rights and permissions. Published by BMJ.

For numbered affiliations see end of article.

Dr Espen Basmo Ellingsen; espen.ellingsen@ultimovacs. com

Background  Therapeutic cancer vaccines represent a promising approach to improve clinical outcomes with immune checkpoint inhibition. UV1 is a second generation telomerase-­targeting therapeutic cancer vaccine being investigated across multiple indications. Although telomerase is a near-­universal tumor target, different treatment combinations applied across indications may affect the induced immune response. Three phase I/IIa clinical trials covering malignant melanoma, non-­small cell lung cancer, and prostate cancer have been completed, with patients in follow-­up for up to 8 years.

Methods  52 patients were enrolled across the three trials. UV1 was given as monotherapy in the lung cancer trial and concurrent with combined androgen blockade in the prostate cancer trial. In the melanoma study, patients initiated ipilimumab treatment 1 week after the first vaccine dose. Patients were followed for UV1-­specific immune responses at frequent intervals during vaccination, and every 6 months for up to 8 years in a follow-­up period. Phenotypic and functional characterizations were performed on patient-­derivedvaccine-­specific T cell responses.

Results  In total, 78.4% of treated patients mounted a measurable vaccine-­induced T cell response in blood. The immune responses in the malignant melanoma trial, where UV1 was combined with ipilimumab, occurred more rapidly and frequently than in the lung and prostate cancer trials. In several patients, immune responses peaked years after their last vaccination. An in-­depth characterization of the immune responses revealed polyfunctional CD4+ T cells producing interferon-γ and tumor necrosis factor-α on interaction with their antigen.

Conclusion  Long-­term immunomonitoring of patients showed highly dynamic and persistent telomerase peptide-­specific immune responses lasting up to 7.5 years after the initial vaccination, suggesting a plausible functional role of these T cells in long-­term survivors. The superior immune response kinetics observed in the melanoma study substantiate the rationale for future combinatorial treatment strategies with UV1 vaccination and checkpoint inhibition for rapid and frequent induction

WHAT IS ALREADY KNOWN ON THIS TOPIC

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE AND/OR POLICY

of anti-­telomerase immune responses in patients with cancer.

In the current era of immunotherapy, therapeutic cancer vaccines (TCVs) have earned interest for their potential to stimulate a patient's immune system against tumors. Numerous studies link a lack of response to checkpoint inhibition (CPI) to an insufficient T cell effector response owing to insufficient T cell priming, effector cell generation, or

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memory formation.1 TCVs do, therefore, hold promise as a potential next step to improve clinical outcomes through their combination with CPIs, providing an invigorated T cell response against relevant tumor antigens while simultaneously blocking T cell suppressor mecha- nisms. Despite past failures,2 3 TCVs targeting both shared tumor-­associated antigens and mutated neoepitopes have earned renewed enthusiasm, demonstrating promising clinical activity when combined with checkpoint inhibitors.4-6

Telomerase is activated in 85%-90% of all cancers and is thus a potential near pan-­tumor antigen for immuno- therapy.7 8 Telomerase activation provides unlimited cell proliferation and increases metastatic potential, serving crucial functions for the tumor.9-11 Its high activity level is a negative prognostic factor across several malignan- cies.12-14 Conversely, spontaneous immune responses (IRs) against telomerase reverse transcriptase (hTERT) confer positive prognostic value in non-­small cell lung cancer, renal cell carcinoma, and anal squamous cell

carcinoma, and are associated with increased response to CPI in melanoma.15-17 The tumor telomerase reliance and

consequent continuous activation provide relevancy for an anti-­hTERT IR along the cancer disease continuum. TCVs designed to cover the active site of hTERT are potentially broadly applicable and may serve as an 'off-­the-­shelf' approach to treat cancer.18 Theoretically, there should be limited opportunities for resistance mutations to develop, as molecular alterations in the hTERT T cell epitopes would likely negatively affect telomerase activity leading to hampered tumor growth. Characterization of IRs induced by first-­generation hTERT vaccines led to the identification of a now clinically validated immunogenic region derived from the active site of hTERT.19 20 IRs against this region were robust CD4+ T helper 1 (Th1) responses associated with long-­term survival. Three highly immunogenic peptides covering the identified region were selected to develop a second-­generationtelomerase-­ targeting vaccine, UV1.

Utilizing synthetic long peptides that require intra- cellular processing facilitates antigen presentation on human leukocyte antigen (HLA) class II, and HLA class I by cross-­presentation, leading to induction of CD4+ and CD8+ T cell responses, respectively.21 Cyto- toxic CD8+ T cells have historically been the focus for describing immune-­mediated antitumor capacities. Recently, however, the importance of the CD4+ component of the adaptive immune system is becoming more established. Released tumor antigens can be engulfed either in situ or in the draining lymph node by tissue and lymph node-­residentantigen-­presenting cells (APCs), respectively, and be presented on HLA class II to CD4+ T cells.22 Activated CD4+ T cells serve as orchestrators of an IR by direct and indirect mechanisms.23 First, CD40L on the CD4+ T cells binds CD40 on the dendritic cells (DCs) initiating heightened antigen presentation and expression of cytokines and the co-­stimulatory molecules CD80 and CD86 by the DCs. These co-­stimulatory

molecules provide signal 2 for the CD8+ T cells, which in conjunction with the cytokines promote differentiation, effector function, and survival. Second, CD4 +T cells also secrete inflammatory cytokines, such as interleukin (IL)-­2 and interferon (IFN)-γ, directly supporting the CD8+ T cells. This inflammatory response promoted by the CD4+ T cells may thus reshape the tumor microenvironment and result in epitope spreading.24 Cancer cells can also express HLA class II on IFN-γ stimulation,25 providing a more direct target for CD4+ T cell-­mediated cytotox- icity in immunogenic tumors.26 27 A recent publication from Dillard et al further supports this concept, demonstrating antitumor efficacy of an HLA class II-­restrictedhTERT-­specific T cell receptor in an animal model.28 In vitro studies have also shown recognition of a melanoma cell line by a CD4+ UV1-­peptide specific T cell clone,19 supporting target antigen detection at endogenous levels.

UV1 has been investigated in three completed phase I/ IIa clinical trials covering malignant melanoma (MM),29 non-­small cell lung cancer (NSCLC),30 and prostate cancer (PC).31 In total, 52 patients have been treated in these studies. The long follow-­up time and longitudinal immunomonitoring allow for in-­depth characterization of the IR dynamics observed across these indications and treatment combinations. Herein, we provide a comprehensive overview of the IRs induced by UV1 vaccination and demonstrate its correlation with clinical outcomes.

Three phase I/IIa clinical trials evaluating UV1 have been completed, enrolling 52 patients with MM (NCT02275416),29 NSCLC (NCT01789099),30 or PC (NCT01784913).31 All trials were conducted at Oslo University Hospital, Oslo, Norway, and patients were treated between 2013 and 2015. All trials enrolled patients with advanced disease; stage IV melanoma (n=12), locally advanced or metastatic NSCLC with stable disease after chemotherapy alone or combined with radiotherapy (n=18), and newly diagnosed PC with non-­visceral metastases (n=22). All studies were open-­label,single-­armed,single-­center studies, with the primary objective to assess the safety and tolerability of UV1 and the secondary objective of evaluating IRs to the UV1 peptides.

Patients who had left the studies due to progression in May 2017 and those progressing thereafter were asked to participate in an IR and survival follow-­up study with the aim of monitoring UV1 vaccine responses in long-­term surviving patients. The follow-­up study encompassed 6-­month intervals for assessment of survival only or immunomonitoring and survival (up to 8 years). All but 6 patients alive enrolled (n=25), and 12 patients agreed to be followed for survival and peripheral blood mono- nuclear cell (PBMC) sampling. The remaining patients agreed to be followed for survival only (online supplemental figure 2 and table 2). The original clinical trials and the follow-­up study were approved by the competent

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regulatory authority and ethical committee, and patients provided written informed consent before enrollment.

UV1 (Ultimovacs ASA, Oslo, Norway) consists of three peptides, one 30-­mer(p719-­20) and two 15-­mers (p725 and p728), and is produced as a sterile aqueous solu- tion, lyophilized, and stored at −20° C before reconstitution in water for injection. Three doses of UV1 (100 µg, 300 µg, and 700 µg) were administered in the NSCLC and PC trials, whereas only the 300 µg dose was administered in the MM trial. UV1 vaccinations were administered intradermally in the lower abdomen. The vaccine adjuvant granulocyte-macrophagecolony-stimulating factor (sargramostim 75 µg) (lyophilized Leukine, Sanofi Aventis, Bridgewater, New Jersey, USA), was administered intradermally at the same injection site 10-15 min prior to UV1.

In the NSCLC trial, patients received UV1 mono- therapy. In the PC trial, patients received upfront and concomitantly combined androgen blockade (goserelin

10.8 mg subcutaneously every third month and bicalut- amide 50 mg per day). In the MM trial, patients received up to four infusions of ipilimumab (3 mg/kg) starting 1 week after the first UV1 vaccinations (see online supple- mental figure 1 for vaccination and biological sampling schedule).

PBMCs were prepared from whole blood samples (50 mL in acid citrate dextrose tubes) collected at scheduled intervals prior, during, and after UV1 vaccinations (up to 5 years for the MM and NSCLC studies and 8 years for the PC study) (online supplemental figure 1). The samples from the three studies were processed similarly and at the same laboratory facility. PBMCs were isolated and frozen as previously described.31 Thawed PBMCs were pre-­stimulated once in vitro with UV1 vaccine peptides for 10-12 days before the T cell assays below, as previously described.31

The T cell proliferation assay (3 H-thymidine incorpora- tion) was used to assess vaccine-­specific IR, as previously described.31 Briefly, after 10-12 days pre-­stimulation, PBMCs were re-­stimulated with 10 µM of the UV1 vaccine peptides (peptide 725, hTERT 691-705 (RTFVL- RVRAQDPPPE); peptide 719-20, hTERT 660-689 (​ ALFSVLNYERARRPGLLGASVLGLDDIHRA); peptide 728, hTERT 651-665 (AERLTSRVKALFSVL) (Bachem AG, Switzerland) and irradiated, autologous PBMCs as APCs, and tested in triplicates for proliferation by 3 H-thy- midine incorporation. A stimulation index (SI) (ratio of mean counts in wells with T cells stimulated with or without vaccine peptides) above or equal to three for any of the three peptides or a mix was considered positive. Doubling of the SI was required in patients with a spontaneous IR against UV1. Patients with a positive SI during

the treatment period were considered a vaccine immune responder. The treatment period was defined as the time from the first vaccine dose to 16 weeks after the last dose. Staphylococcus aureus enterotoxin C3 (SEC3) was used as a positive control to determine immunocompetence. Samples were considered evaluable for IR if SEC3 SI was ≥3

Generation of vaccine-specific T cell clones

Samples containing T cells highly reactive to vaccine peptide stimulation were further utilized to isolate vaccine-­specific T cells. T cells were cloned by limiting dilution seeding in Terasaki plates, essentially as previously described.19 Briefly, T cell lines from immune responders were co-­cultured in CellGro DC medium (Cellgenix, Freiburg, Germany) supplemented with 5% human serum (TCS BioSciences, Botolph Claydon, UK) with irradiated allogeneic PBMCs (30 Gy) used as feeder cells. IL-­2 and IL-­7 were added at 20 U/mL and 2200 U/mL, respectively. Phytohemagglutinin (PHA) was added at 2 µg/mL for polyclonal stimulation. After poly- clonal stimulation and when sufficient cell numbers were reached, the clones were tested in proliferation assays against the individual peptides using autologous Epstein-­ Barr Virus-­transformed B lymphoblastoid cell lines (EBV-­ LCL) as APCs. Unique clonotypes of T cell clone samples were verified by complementarity-­determining region three sequencing performed by the laboratory of Dr Mascha Binder, Martin‐Luther‐University Halle‐Witten- berg, Germany (online supplemental table 1).

HLA restriction of vaccine-­specific T cell clones (TCCs) was determined by HLA Class II blocking antibodies used in the proliferation assay. Antibodies blocking HLA-­ DR, HLA-­DQ, and HLA-­DP were used (produced in the immune monitoring group of Department of Cellular Therapy, Oslo University Hospital, Norway, as described in.32 HLA class II restriction analysis of TCCs was refined using a panel of EBV-­LCLs as APCs, homozygous for patient HLA alleles.33 The EBV-­LCLs were irradiated (100 Gy), loaded with UV1 peptides, and co-­incubated with TCCs at a 1:1 ratio in proliferation assays (3H-­thymidine incorporation) as described for testing vaccine-­specific IRs.

Phenotypic characterization of vaccine-specific T cells

After T cell expansion, the vaccine-­specific T cells were phenotypically characterized by staining for surface markers. Briefly, cells were suspended in round-­bottom test tubes (5 mL, Falcon, Corning Life Sciences, Corning, USA) in staining buffer (Dulbecco's phosphate-­buffered saline (Lonza BioWhittaker) with 2% fetal calf serum (Thermo Fisher Scientific)). Aggregated γ-globulin (10 mg/mL) (Sigma-­Aldrich Norway AS) was added before staining to block Fc receptors. The cells were stained for viability (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, Invitrogen) and surface markers as follows (antibody clones and manufacturer in parenthesis); CD3 (UCHT1, Invitrogen), CD8 (RPA-­T8, eBioscience), CD4 (OKT4,

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BioLegend), CD45RO (UCHL1, BioLegend), CD197 (3D12, eBioscience), ICOS (ISA-­3, Invitrogen), PD-­1 (MIH4, eBioscience), LAG3 (11C3C65, eBioscience), TIM3 (F38-­2E2, eBioscience), TIGIT (MBSA43, eBiosci- ences), and incubated in the dark on room temperature for 20 min. The acquisition was performed on FACSCanto IVD 10 color configuration (Becton Dickinson, New Jersey, USA) and analyzed using FlowJo V.10.8 (Becton Dickinson, New Jersey, USA)

For intracellular staining of cytokines, vaccine-­specific TCCs were first stimulated with vaccine peptides. Autol- ogous EBV-­LCLs were used as APCs and loaded with vaccine peptides at 10 µM for 12 hours. The TCCs were co-­cultured with vaccine-­peptide-­loadedEBV-­LCL cells at a 1:2 ratio in a 96-­well plate for 9 hours. CD107a (H4A3, BD BioSciences) and Golgi plug and stop (BD BioSci- ences) were added to the culture. TCCs stimulated with phorbol myristate acetate and ionomycin (50 ng/mL and 1 µM, respectively) were used as a positive control. TCCs stimulated with a non-­cognate vaccine peptide were used as a negative control. The cells were harvested and stained with CD4 (OKT4, BioLegend) and, after fixation and permeabilization (FIX & PERM Cell Permeabilization Kit, Invitrogen), stained for IFN-γ (4S.B3, Invitrogen), and tumor necrosis factor-α(TNF-α) (Mab11, BD BioSci- ences). Acquisition was performed on FACSCanto IVD 10 color configuration (Becton Dickinson, New Jersey, USA) and analyzed using FlowJo V.10.8 software (Becton Dick- inson, New Jersey, USA)

The TCCs were co-­cultured with vaccine-­peptide-­loadedEBV-­LCL cells at a 1:2 ratio in a 96-­well plate for 24 hours, using the same positive and negative controls as above. A multiplex cytokine assay (Bio-­Plex Pro Human Cytokine Th1/T helper 2 (Th2) Assay, Bio-­Rad Labo- ratories, Hercules, USA) was performed on the culture supernatant. Th1 and Th2 cytokine concentrations were assessed following the manufacturer's protocol on the Bio-­Plex 200 instrument (Bio-­Rad Laboratories, Hercules, USA). Supernatants were analyzed in trip- licates. Concentrations for the negative control were subtracted from concentrations in supernatants from peptide-­stimulated TCCs. Z-­scores were calculated by subtracting the mean of all cytokine concentrations from the respective cytokine concentration and dividing by the SD.

HLA genotyping was performed on PBMCs from patients with available samples (n=50). The HLA typing was performed by ProImmune (Oxford, UK) utilizing PCR sequence-­specific oligonucleotides. Major allele groups were resolved to four digits (eg, HLA-­A*23:01). For patient 802, HLA genotyping resolved to two digits for class I and four digits for class II were performed by Oslo University Hospital, Rikshospitalet, Oslo, Norway.

The study was designed to be analyzed descriptively, and no formal statistical plan was pre-­defined. Sample sizes

Patients, treatment, and clinical outcomes

Between 2013 and 2015, a total of 52 patients were enrolled across the three studies. The median age was 65 (range 44-78), and 37 (71.2%) patients were men (male dominance due to PC study). Most patients were Eastern Cooperative Oncology Group performance status 0 (88.5%). The median time from initial diagnosis until trial enrollment was 6.2 months (range 1.1-207.1). All patients in the MM and PC studies had stage IV disease. In the NSCLC study, 6, 5, and 7 patients had stage III, III/ IV, and IV disease, respectively. Patients were treated with a mean of 5.5 (range, 3-9), 12.4 (range, 9-18), and 13.5 (range, 7-18) UV1 vaccine doses in the MM, NSCLC, and PC studies, respectively. Patients have been followed for survival for a median of 55.7 months (range, 3.5-79.5),

Patient characteristics and IR development

PBMCs were collected for longitudinal IR assessment across the three trials. One patient in the MM trial was not evaluable for IR analysis due to a lack of post-­treatment

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Table 1  Patient characteristics and associations with immune response

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Demographics of immune response (IR) evaluable patients in each trial, and pooled IR negative and positive populations. The p values represent demographic differences between IR positive and negative populations.

*Levels of LDH and lymphocytes at baseline.

ECOG, eastern cooperative oncology group; LDH, lactate dehydrogenase; MM, malignant melanoma ; NSCLC, non-­small cell lung cancer ; PC, prostate cancer.

PBMC samples. Patients with samples having a SI≥3 in the proliferation assay were considered immune responders. In evaluable patients (n=51), the IR rate was 90.9%, 66.6%, and 81.8% for the MM, NSCLC, and PC trials, respectively (figure 1A ), providing a pooled IR rate of 78.4%. Spontaneous anti-­UV1 IRs were observed in six patients at baseline, all of whom developed an increase in SI after vaccination (figure 1B ). We found no associations between patient baseline characteristics and the development of vaccine-­induced IRs (table 1 )

UV1 induced IRs occurred earlier and more frequently when combined with ipilimumab

In the treatment period, the median highest SI observed in individual patients was 11.5 (range 2.3-60.0) in the

NSCLC, and PC trials, respectively, and 6.5 weeks (range, 1-40) for all studies combined (figure 1C ). The median number of vaccinations received until the first detectable IR was 5 (range 3-6) in the MM study, 6 (range 5-13) in the NSCLC study, and 8 (range 3-13) in the PC study, and 6 (range 3-13) for all studies combined (figure 1D ) (online supplemental figure 1).

UV1 vaccination induces persisting IRs

After the treatment period, patients were followed for long-­term immunomonitoring every 6 months for up to 8 years. Robust IR peaks occurred in many patients several years after the last vaccination, often exceeding SIs detected during the treatment period, particularly in the PC study (figure 2A-C ). Of note, several patients who mounted IRs against select peptides within UV1 during the treatment period displayed immune responses towards more peptides during the late peaks (patients N03 and N11 in the MM study, and patients 805, 816,

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Ultimovacs ASA published this content on 25 May 2022 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 26 May 2022 12:20:35 UTC.