
==== Front
Clin Cancer Res
Clin Cancer Res
Clinical Cancer Research
1078-0432
1557-3265
American Association for Cancer Research

39028916
CCR-24-0742
10.1158/1078-0432.CCR-24-0742
Version of Record
Gastrointestinal Cancers
Esophageal Cancer
Gastrointestinal Cancers
Gastrointestinal Cancers
Stomach Cancer
Clinical Trials: Immunotherapy
Phase II Trial of HER-Vaxx, a B-cell Peptide-Based Vaccine, in HER2-Overexpressing Advanced Gastric Cancer Patients Under Platinum-Based Chemotherapy (HERIZON)
Clinical Outcome and Vaccine-Induced Immune Responses
https://orcid.org/0000-0002-0961-5306
Tobias Joshua 1
https://orcid.org/0009-0008-0679-6499
Maglakelidze Marina 2
https://orcid.org/0000-0002-1495-2729
Andrić Zoran 3
https://orcid.org/0000-0002-7483-6228
Ryspayeva Dinara 4
https://orcid.org/0009-0008-2211-622X
Bulat Iurie 5
https://orcid.org/0000-0003-3023-6703
Nikolić Ivan 6
https://orcid.org/0009-0005-0288-3630
Petrović Zoran 7
https://orcid.org/0000-0003-3729-536X
Chawla Tanuj 8
https://orcid.org/0000-0002-8045-842X
Nagarkar Rajnish 9
https://orcid.org/0000-0002-5283-0458
Garner-Spitzer Erika 1
https://orcid.org/0000-0002-1440-9013
Zielinski Christoph C. 10
https://orcid.org/0009-0006-6795-4417
Chong Leslie Mi Ok 11
https://orcid.org/0009-0005-9166-325X
Nixon Bonnie 11
https://orcid.org/0000-0002-1394-1585
Ede Nicholas J. 11
https://orcid.org/0009-0000-6681-9762
Yavrom Sharon 11*
https://orcid.org/0000-0002-2707-3213
Kundi Michael 1
https://orcid.org/0000-0002-1302-3223
Wiedermann Ursula 1*
1 Medical University of Vienna, Vienna, Austria.
2 ARENSIA Exploratory Medicine LLC, Tbilisi, Georgia.
3 Clinical Hospital Center Bezanijska Kosa, Belgrade, Serbia.
4 ARENSIA Exploratory Medicine LLC, Kyiv, Ukraine.
5 ARENSIA Exploratory Medicine Research Unit, Institute of Oncology, Chisinau, Republic of Moldova.
6 Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia.
7 Military Medical Academy, Belgrade, Serbia.
8 Tata Medical Centre, Kolkata, India.
9 CG Manavata Cancer Centre, Nashik, India.
10 Central European Cancer Center, Wiener Privatklinik, Central European Cooperative Oncology Group (CECOG), Vienna, Australia.
11 Imugene Limited, Sydney, Australia.
* Corresponding Authors: Ursula Wiedermann, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Kinderspitalgasse 15, Vienna 1090, Austria. Email: ursula.wiedermann@meduniwien.ac.at; and Sharon Yavrom, Imugene Limited, Suite 804, Level 8, 37 Bligh Street, Sydney, New South Wales 2000, Australia. E-mail: syavrom@imugene.com
Clin Cancer Res 2024;30:4044–54

13 9 2024
19 7 2024
30 18 40444054
14 3 2024
01 5 2024
17 7 2024
©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
https://creativecommons.org/licenses/by-nc-nd/4.0/ This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.

Abstract

Purpose:

A multicenter, randomized, open-label, phase II study (HERIZON; NCT02795988) was conducted to evaluate the clinical and immunologic efficacy of HER-Vaxx (IMU-131), a B-cell, peptide-based vaccine targeting HER2 overexpressed in 6% to 30% of gastroesophageal adenocarcinomas (GEA).

Patients and Methods:

Patients (n = 36) with GEA were treated with standard-of-care chemotherapy (n = 17) or HER-Vaxx plus chemotherapy (n = 19), using the recommended phase 2 dose for the vaccine. Overall survival (OS; primary endpoint), safety, progression-free survival (PFS), clinical response (secondary endpoints), and vaccine-induced HER2-specific antibody levels in serum and correlation with tumor response rates (exploratory endpoints) were investigated.

Results:

A 40% OS benefit [HR, 0.60; median OS, 13.9 months; 80% confidence interval (CI), 7.52–14.32] for patients treated with HER-Vaxx plus chemotherapy compared with OS of 8.31 months (80% CI, 6.01–9.59) in patients that received chemotherapy alone. A 20% PFS difference was obtained for the vaccination arm (HR, 0.80; 80% CI, 0.47, 1.38). No additional toxicity due to HER-Vaxx was observed. The vaccine-induced high levels of HER2-specific total IgG and IgG1 antibodies (P < 0.001 vs. controls) that significantly correlated with tumor reduction (IgG, P = 0.001; IgG1, P = 0.016), had a significant capacity in inhibiting phosphorylation of the intracellular HER2-signaling pathways, mediated antibody-dependent cellular cytotoxicity, and decreased immunosuppressive FOXP3+ regulatory T cells.

Conclusions:

HER-Vaxx plus standard chemotherapy exhibits an excellent safety profile and improves OS. Furthermore, vaccine-induced immune response was significantly associated with reduced tumor size compared with standard-of-care chemotherapy. The presented vaccination approach may substitute for treatment with trastuzumab, upon unavailability or toxicity, based on further evidence of equivalent treatment efficacy.
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pmcTranslational Relevance

We have constructed a vaccine compound, HER-Vaxx (IMU-131), harboring three B-cell peptides including the binding epitope of trastuzumab (Herceptin). Vaccination with HER-Vaxx induced a strong polyclonal HER2-specific humoral and cellular response and antitumor effects in several preclinical evaluations. In the previous dose-escalating phase Ib trial, the optimal vaccine dose was linked to progression-free survival and tumor regression in patients with gastric/gastroesophageal junction adenocarcinoma. In the present open-label, phase II study, conducted based on the recommended phase II dose (50 µg), patients with gastric/gastroesophageal junction adenocarcinoma were randomly assigned to either standard-of-care chemotherapy alone or HER-Vaxx plus chemotherapy. Compared with chemotherapy alone, vaccination with HER-Vaxx was associated with a 40% overall survival benefit (primary endpoint), with no observed additive vaccine-associated toxicity. Furthermore, HER-Vaxx induced HER2-specific antibodies, mediating antibody-dependent cell cytotoxicity, inhibiting intracellular HER2 phosphorylation, and correlating with tumor reduction. These results provide proof of concept for a first-in-class HER2 B-cell, peptide-based immunotherapy.

Introduction

HER2 is a member of the EGFR family, one of the most important factors in human malignancies (1). The receptor is overexpressed in up to 30% of gastric cancers (2–6), with the highest reported rates of HER2 overexpression in the gastroesophageal junction (GEJ) or stomach cardia tumors compared with tumors arising more distally in the stomach (7). The application of the monoclonal antibody (mAb) trastuzumab (Herceptin), which binds to the extracellular domain IV of HER2, inducing antibody-dependent cellular cytotoxicity (ADCC) and blocking HER2 downstream signaling, has substantially improved the survival rate of patients with HER2-overexpressing carcinomas (8, 9). Based on the results from the phase III Trastuzumab for Gastric Cancer (ToGA) trial (2), cisplatin/fluoropyrimidine plus trastuzumab has become a standard first-line treatment for patients with HER2-overexpressing advanced gastric cancers (2). However, despite the tremendous success of therapeutic mAb, frequent administration for prolonged periods due to the lack of immunologic memory induction (10), as well as induced toxicity (11) and financial inconvenience (12), pose notable disadvantages. Furthermore, resistance to trastuzumab restricts its therapeutic efficacy (13). Conversely, anticancer vaccines in the category of active immunotherapy engage the host immune system and lead to the formation of immunologic memory (14, 15).

The success of passive immunotherapy with trastuzumab has paved the way for the identification of the mAb’s B-cell peptides/binding epitopes for use in active immunization (16). Using a computer algorithm, we identified and examined three single peptides (P4, P6, and P7), including trastuzumab’s binding site on HER2 (17). The initially formulated vaccine evaluated in a phase I study in patients with advanced breast cancer (18) proved the immunogenicity and safety of the compounds. The formulation was improved by fusing the single peptides into a hybrid peptide called P467, conjugating it to the carrier protein CRM197, with T-cell epitopes, and combining it with Montanide ISA51 (ISA 51), a Th1/Th2-driving adjuvant (17, 19). The vaccine activates B cells in both a T-cell–dependent and -independent manner, due to the T-cell epitope in the vaccine and the carrier protein. This feature results in the induction of both humoral and cellular immune responses to HER-Vaxx (IMU-131; refs. 19, 20).

The first clinical evaluation of HER-Vaxx was recently completed in a multicenter, phase Ib trial (NCT02795988) involving 14 patients grouped into three dose-escalated arms receiving 10, 30, or 50 µg of the vaccine (P467 equivalent; ref. 21). The results showed substantial HER2 immunoglobulin G (IgG)–specific antibodies in patients who had received the highest dose. Prolonged progression-free survival (PFS) following vaccination with the highest dose correlated with vaccine-specific humoral and cellular responses in patients receiving the vaccine plus chemotherapy (21).

Based on the success of the phase Ib trial, the highest dose (50 µg) of the vaccine was selected as the recommended phase II dose (RP2D) for this phase II, randomized, controlled trial to compare the effect of the HER-Vaxx plus chemotherapy versus chemotherapy alone. Here, we report the results of the phase II trial conducted in patients with HER2-overexpressing metastatic or advanced gastric/GEJ adenocarcinoma, randomized to chemotherapy alone (n = 17) or HER-Vaxx vaccine plus chemotherapy (n = 19).

Patients and Methods

Study design and treatment

Between 2019 and 2022, 36 patients (≥20 years of age) with metastatic gastric or GEJ adenocarcinoma (i.e., advanced cancer of the stomach) were enrolled in the study (Supplementary Fig. S1). These patients had no prior treatment (chemotherapy or radiotherapy), and their cancer was not amenable to surgical resection for stage III/IV gastric cancer. All patients were HER2+ or 3+ (overexpression levels) confirmed by FISH, or had HER2 overexpression confirmed by chromogenic in situ hybridization. Each patient had at least one measurable lesion as defined by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) criteria. This enrollment took place in Eastern Europe (the Republic of Georgia, the Republic of Moldova, the Republic of Serbia, and Ukraine) and the Republic of India, where treatment with trastuzumab was not widely available as a first-line treatment for these indications. Any previous treatment with trastuzumab or other HER2-targeting antibody or agent was the primary exclusion criterion. The complete inclusion and exclusion criteria are listed in Supplementary Table S1A and S1B, respectively. The background information on the cancer under study and the representativeness of the study population are provided in the “Representativeness of Study Participants” Supplementary Table S2.

The study was conducted in three parts, including enrollment, treatment, and posttreatment follow-up. During the treatment phase, subjects were randomized to receive either HER-Vaxx plus chemotherapy or chemotherapy as the only anticancer therapies. After disease progression occurred, study treatments were discontinued, and subjects began the posttreatment follow-up to collect information about the patients’ survival and any anticancer therapies received. The eligible patients (n = 36) were randomized to two arms of either treatment with chemotherapy alone (n = 17) or HER-Vaxx (refs. 19, 21; 50-µg dose, n = 19) plus chemotherapy (Supplementary Fig. S1). Written informed consent was obtained from each subject included in the study cohorts. Furthermore, the human investigations were performed after approval by an institutional review board and in accordance with an assurance filed with and approved by the U.S. Department of Health and Human Services.

The patients received the HER-Vaxx vaccine on days 0, 14, 35, 77, and every 63 days thereafter until disease progression, and platinum-based chemotherapy was started on day 0 and repeated every 21 days for a maximum of six cycles (Fig. 1). The preparation of and treatment with the vaccine and the standard-of-care (SOC) chemotherapy are described in Supplementary Materials and Methods S1.

Figure 1. Study arms and treatment schedule. The timepoints for the patients’ initial screening and the assessment of their tumors are indicated by the arrow boxes. CC, chemotherapy cycle; D, day; Vac, HER-Vaxx administration; W, week.

The primary endpoint of this randomized, open-label, phase II trial was to evaluate the clinical efficacy of HER-Vaxx based on overall survival (OS). The secondary endpoints included incidence of treatment-emergent adverse events (TEAE), adverse events (AE), PFS, duration of objective response (DOR), overall response rate (ORR), and change in tumor size. Exploratory endpoints included the evaluation of vaccine-induced, HER2-specific antibody levels in serum and tumor response rates according to radiographic assessments.

The trial was conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects (Council for International Organizations of Medical Sciences 2002), the Guidelines for Good Clinical Practice (International Council for Harmonisation 1996), and the Declaration of Helsinki (World Medical Association 1996, 2008) and registered at ClinicalTrials.gov (NCT02795988).

Safety assessment

The safety and tolerability of HER-Vaxx were evaluated by AE, laboratory measurements, physical examinations, 12-lead electrocardiograms, and measures of left ventricular ejection fraction. AE and laboratory abnormalities were graded by Common Terminology Criteria for Adverse Events Version 4.03. Physical examinations were performed at the screening visit: on days 0, 21, 42, 63, 84, 105, and 126 and at the end of treatment.

Evaluation of clinical responses

Radiographic assessment was performed for all patients at screening (for eligibility assessment), on days 56 and 98 and at all long-term maintenance visits until disease progression. CT scans of the chest, abdomen, and pelvis with contrast were conducted to visualize and measure tumor lesions; alternatively, MRI of the abdomen and pelvis with contrast and noncontrast CT of the chest were conducted in patients with iodine contrast dye allergy or lesions not visible on CT scan. The sum of the diameters of the respective lesions was calculated, and tumors were assessed according to RECIST 1.1. A sensitivity analysis was performed to censor subjects whose cause of death was considered unrelated to the underlying disease, including two patients in the HER-Vaxx plus chemotherapy arm (stroke and COVID-19) and one patient in the chemotherapy-alone arm (pulmonary thromboembolism).

Translational studies

Vaccine-specific immunologic evaluations by assessment of HER2-specific and P467-specific total IgG or its isotypes, immunophenotyping of peripheral blood mononuclear cells (PBMC) for evaluation of FOXP3+ regulatory T cells (Treg) by flow-cytometric analysis, and vaccine-induced antibody biologic functionality and mechanism of action, such as inhibiting intracellular HER2 and Akt downstream signaling pathway proteins, mediating ADCC, and binding to HER2-overexpressing cells, by ELISA, were carried out as described in Supplementary Materials and Methods S1. Trastuzumab, as a positive control and reference standard, and serum samples from a healthy individual as negative control, were included in the assays.

Sample size calculation

The primary endpoint of OS was measured from randomization to death due to any cause. The treatment arms were compared with respect to OS following the intent-to-treat (ITT) principle using the log-rank test.

The following specifications were made to compute the required number of OS events:• Alpha (false-positive probability) specified as one-sided 0.10.

• Power goal specified as 90% with no actionable interim analysis specified.

• The hypothesized HR specified as 0.35 (experimental arm HR divided by chemotherapy-alone control arm HR).

• The survival time distribution assumed to follow the exponential distribution.

• The randomization ratio specified as 1:1.

• These specifications led to the computation of a requirement for 24 OS events for the OS analysis.

• The critical HR was 0.60 for these specifications.

It is assumed that the control arm median survival time is 11.1 months from the ToGA trial (22). Based on this specification of the control arm median survival, the median survival in the experimental arm is hypothesized to be 31.7 months, and the critical median survival is 18.7 months.

The number of randomized patients for phase II is specified to be 36 to realize the required 24 OS events.

Vaccine-specific immunologic evaluation

The humoral immunity was assessed in patients’ sera obtained from whole blood samples taken at various time points.

Detection of HER2-specific total IgG and IgG isotypes

To evaluate the levels of HER2-specific total IgG and IgG isotype antibodies, ELISA were carried out as described previously (19, 21), using 96-well microtiter plates (Nunc-Maxisorp Plate, Nalge Nunc International, Denmark) coated with 0.2 µg/well human HER2 conjugated to HIS-tag, (Sino Biological Inc. Beijing, China). The bound total IgG antibodies and the IgG1/2/3/4 isotypes were detected using mouse antihuman IgG (cat. No. SA1-35470, RRID: AB_1075966), IgG1 (cat. No. A10648, RRID: AB_2534051), IgG2 (cat. No. 05-0520, RRID: AB_2532974), IgG3 (cat. No. MH1732, RRID: AB_2539713), and IgG4 (cat. No. A10654, RRID: AB_2534054; Thermo Scientific, USA), respectively. Trastuzumab, as standard at different concentrations, was used in each ELISA for total IgG, to measure the concentration (ng/mL) of the evaluated HER2-specific IgG. Each experiment was performed at least three times, and the reproducibility of the repeated experiments was ensured by including a positive serum from a patient enrolled in the phase Ib trial (TH02001; ref. 21) and a serum from a patient with an unrelated disease.

Detection of peptide P467-specific IgG

The peptide (P467)-specific IgG levels were measured similarly as above, with slight modifications. The 96-well microtiter plates (Nunc-Maxisorp Plate, Nalge Nunc International, Denmark) were coated with 0.5 µg/well P467 peptide (PiChem, Graz, Austria) in 100-mmol/L bicarbonate buffer, pH 9.6 overnight at 4°C. The detection of the P467-specific total IgG was carried out as described previously. The peptide-specific IgG levels were shown as arbitrary units based on the optical density (OD) values.

Cell lines and culture conditions

For the in vitro binding and phosphorylation inhibition evaluations, the HER2-expressing human gastric carcinoma cell line NCI-N87 (RRID: CVCL_1603; ATCC CRL5822; ref. 21), as well as the HER2-negative human melanoma cell line 518A2 (RRID: CVCL_UM77; originally provided from B. Jansen, Department of Dermatology, University of Vienna, Austria; refs. 17, 23), were used. All cell lines were grown and cultured in a humidified chamber with an atmosphere containing 5% CO2 at 37°C, as described previously (21, 23, 24). The humidified chamber used for the incubation of the cells was routinely examined for detection of Mycoplasma. Microscopic evaluation for morphology and flow-cytometric analysis for expression of HER2 (cell line NCI-N87) or lack of HER2 expression (cell line 518A2) were carried out for cell authentication.

Flow-cytometric analyses

IgG binding assays to HER2-overexpressing gastric cancer cells

Human gastric carcinoma cell line NCI-N87 (passage 27) overexpressing HER2, as well as HER2 negative human melanoma cell line 518.A2 (passage 53), were used to show specific binding of isolated and purified patient IgG to native HER2, as described previously (21).

Immunophenotyping of PBMC for evaluation of FOXP3+ Treg

Patients’ frozen PBMC in liquid nitrogen were intracellularly stained for transcription factor FOXP3 for characterization of Treg, as described previously (21).

Detection of phosphorylation inhibition of intracellular HER2 and the downstream signaling pathway proteins Akt and MAPK

For the detection of intracellular HER2 and signaling pathway kinases Akt and MAPK phosphorylation, human gastric carcinoma cell line NCI-N87 was used. The examined patients’ serum samples were subjected to affinity purification by applying NAb Protein A/G Spin columns (Thermo Scientific, USA; cat. No. 89950) for isolation of total IgG fractions. The purified and isolated total IgG fractions were desalted by buffer exchanging to PBS using Zeba Spin Desalting Columns (Thermo Scientific, USA; cat. No. 89882).

The cells (passage 27) were seeded into a 12-well plate and incubated in a humidified chamber (95% air, 5% CO2) at 37°C overnight. The cell culture medium was removed, the cell monolayer was washed, and the culture medium containing the examined patients’ sera was added to the respective wells. The plate was incubated overnight at 37°C; thereafter, cells were lysed using the Cell Lysis Buffer according to the manufacturer’s instructions (Cell Signaling Technology, USA; cat. No. 9803). The level of intracellular HER2 phosphorylation in the lysates of the treated cells was evaluated by ELISA, using capture mouse antihuman HER2 (R&D Systems, USA; cat. No. MAB1129, RRID:AB_357477), phospho-HER2/ErbB2 (Tyr1221/1222; 6B12) rabbit mAb (Cell Signaling Technology, cat. No. 2243, RRID:AB_490899), and HRP-conjugated goat antirabbit IgG (Sigma-Aldrich; cat. No. 8275, RRID:AB_258382). Following TMB staining and stopping the reactions with H2SO4, the OD values were measured spectrophotometrically at wavelength 450 versus 630 nm using a Tecan Spark microplate reader (Tecan, Switzerland). For the evaluation of the phosphorylated signaling pathway kinases Akt and MAPK, the PathScan Phopsho-Akt (Thr308) and PathScan Phopsho-MAPK (T202/Y204) sandwich ELISA kits (Cell Signaling Technology, USA; cat. No. 7252CA and 7177CA, respectively) were used, and the assay was performed according to the manufacturer’s instructions.

The inhibition levels of intracellular HER2 and Phopsho-Akt (Thr308) and PathScan Phopsho-MAPK (T202/Y204) phosphorylation were calculated as follows: % inhibition = [(100 − (OD (cells treated with the examined serum sample)/OD (untreated cells)) × 100)]. The level of phosphorylation inhibition in cells treated with trastuzumab was used as a positive control.

Antibody-dependent cellular cytotoxicity

The colorimetric “CytoTox 96 Nonradioactive Cytotoxicity Assay” (Promega, USA, cat. No. G178) was applied to measure the capacity of the patients’ isolated and purified IgG antibodies in mediating ADCC. The HER2-overexpressing cell line NCI-N87, as target cells (T), and human PBMC from a healthy individual, as effector cells (E), at the ratio of 10:1 (E:T), were used. Target cells, 5,000 cells/reaction, in a phenol red-free culture medium HBSS (supplemented with 1% FBS, and 1% streptomycin and penicillin), were added to wells of V-bottom microtiter plates, and incubated with the examined antibodies (50 µg) or trastuzumab (5 μg), as a positive control, for 1 hour at 37°C, 5% CO2 to allow opsonization of the patient antibodies. Based on the ratio of 10:1 (E:T), 50,000 PBMC were added to the treated target cell wells. The assay was carried out based on the manufacturer’s instructions, and the levels of ADCC were calculated based on the formula: % Cytotoxicity (ADCC) = (Experimental − Effector Spontaneous − Target Spontaneous)/(Target Maximum − Target Spontaneous) × 100. The ADCC (% cytotoxicity) values are normalized to that of the positive control (trastuzumab).

Statistical analysis

Percent change in tumor diameters compared with baseline diameters, expressed as fold changes. If the tumor was no longer visible, the tumor diameter was arbitrarily set to 1 cm. Distribution analyses revealed for IgG and IgG subclasses a log-normal distribution and were log-transformed for graphical representation. In statistical analyses, a log-normal distribution was applied. A generalized estimating equations model was applied to account for the course of antibody and tumor assessment within patients. Weeks from baseline were chosen as the within-patient variable. For correlations of tumor size with immunologic data, the next tumor assessment at least 2 weeks later was chosen as the relevant size or fold change from baseline. Independent variables of interest were the number of vaccinations and the group assignment (chemotherapy alone or HER-Vaxx plus chemotherapy). For this purpose, the patients in the chemotherapy-alone arm were assigned the number of vaccinations they would have received at the respective week of observation if they had been in the HER-Vaxx plus chemotherapy arm. Kaplan–Meier curves were computed for OS and PFS up to January 24, 2024, on the ITT population, and a 1-sided log-rank test was performed—based on this analysis, a one-sided P value less than 0.1 meets statistical significance. A Cox regression analysis for time to event was performed, and HR and 80% confidence intervals (CI) were computed. Except for the Kaplan–Meier estimates of OS and PFS and Cox regression, all analyses were performed using Stata 17.0 software (StataCorp; RRID:SCR_012763) applying two-sided tests at a significance level of 0.05. Graphs were produced using Excel (Microsoft) or Statistica 14.0.0 (Cloud Software Group).

Data availability

The data presented here are not publicly available because the information could compromise patient privacy or consent. The data are available upon request from the first (J.T.) and/or corresponding author (U.W.).

Results

Patient characteristics

Patient median ages were 68 (range, 44–79 years) and 65 (range, 48–84 years), with a male:female ratio of 13:4 and 10:9, in the chemotherapy alone and HER-Vaxx plus chemotherapy and arms, respectively. Clinical characteristics of patients, that is, Eastern Cooperative Oncology Group (ECOG) Performance Status, initial tumor type diagnosis (adenocarcinoma of the gastroesophageal junction or the stomach), tumor stage at screening (stage IIIb or IV), and prior treatment (gastric cancer surgery, drug therapy, or radiotherapy) were balanced between the two treatment arms (Table 1).

Table 1. Patient demographics and baseline characteristicsa.

	Chemotherapy (n = 17)	HER-Vaxx + chemotherapy (n = 19)	
Age, median (range)	68 (44, 79)	65 (48, 84)	
 Male/female, n (%)	13 (77)/4 (23)	10 (53)/9 (47)	
ECOG performance grade, n (%)	
 Grade 0	8 (47)	8 (42)	
 Grade 1/2	9 (53)	11 (58)	
Initial tumor diagnosis type, n (%)	
 Adenocarcinoma of gastroesophageal junction	2 (12)	2 (10)	
 Adenocarcinoma of stomach	15 (88)	17 (90)	
Tumor stage at screening, n (%)	
 Stage IIIb	4 (24)	5 (26)	
 Stage V	13 (77)	14 (74)	
Prior treatment, n (%)	
 Prior gastric cancer surgery	7 (41)	10 (53)	
 Prior gastric cancer drug therapy	2 (12)	4 (21)	
 Prior gastric cancer radiotherapy	0	1 (5)	
a Two subjects received prior radiotherapy treatment. SB03201 (chemotherapy-alone arm) received radiotherapy to the spinal cord as palliative treatment. SB03202 (HER-Vaxx plus chemotherapy arm) received radiation therapy to the stomach as part of an adjuvant treatment.

Safety evaluations

As shown in Fig. 2, no significant difference in TEAE between the study arms was observed. The rates of any TEAE were 95% and 94% followed by any treatment-related TEAE (84% vs. 77%), any serious TEAE grade ≥3 (11% vs. 29%), and any TEAE leading to treatment reduction or interruption (45% vs. 32%) in the HER-Vaxx plus chemotherapy arm and the chemotherapy-alone arm, respectively.

Figure 2. TEAE associated with the respective treatments in the study arms. The values at the bottom of each bar indicate the number of patients who experienced the respective TEAE.

Evaluation of AE, either as grade 1/2 or ≥3 showed no significant difference between treatment arms (Supplementary Fig. S2). The most common AE occurring in ≥20% of patients in the HER-Vaxx plus chemotherapy arm include headache, decreased appetite, diarrhea, and nausea (Supplementary Fig. S2). Treatment-related AE include injection site reactions, peripheral swelling, malaise, and weight loss. These results indicate that the addition of HER-Vaxx did not add additional safety concerns to standard chemotherapy treatment.

Clinical responses to HER-Vaxx

Overall survival

An ITT analysis showed a median OS of 13.9 months (80% CI, 7.52–14.32) in patients receiving HER-Vaxx plus chemotherapy versus 8.31 months (80% CI, 6.01–9.59) in patients receiving chemotherapy alone. This translated to an OS benefit of 40% (ITT: HR, 0.60; 80% CI, 0.380–0.957; P value = 0.08, one-sided; Fig. 3). Overall, 12 patients, six in each treatment arm, received further antineoplastic therapy during the posttreatment follow-up period, including five patients receiving trastuzumab (three in the HER-Vaxx plus chemotherapy arm and two in the chemotherapy-alone arm). In these patients, trastuzumab was given after end-of-study treatment and thus could not affect OS, PFS, or any outcome of the immunologic analyses reported here. To verify this, a post hoc analysis was carried out, showing that the palliative trastuzumab treatment did not numerically affect the OS benefit observed between the two arms (Supplementary Fig. S3; Supplementary Table S3).

Figure 3. Kaplan–Meier estimate of OS based on ITT analysis. Censored patients are indicated by the “+”. Two patients in the HER-Vaxx plus chemotherapy arm were alive at the end of the study.

PFS

Based on the ITT data set, a 20% PFS was calculated (HR, 0.80; two-sided 80% CI, 0.467, 1.381) with a median PFS of 6.93 months (80% CI, 5.59–9.86) in the vaccinated patients compared with 6.01 months (80% CI, 2.17–8.31) in the chemotherapy-alone patients. The 25th and 75th percentiles were 4.7 and 11.07 months in the vaccinated patients versus 1.41 and 8.44 months in the chemotherapy-alone patients, respectively (Supplementary Fig. S4).

Disease objective responses and overall response rates

A Blinded Independent Central Review (BICR) analysis showed a median DOR of 7.1 months (80% CI, 2.79-not assessable value) and 4.4 months (80% CI, 4.07-not assessable value) in the HER-Vaxx plus chemotherapy arm and chemotherapy-alone arm, respectively. The investigator-assessed analysis of the DOR indicated a median DOR of 8.18 months (80% CI, 5.59–9.53) and 2.83 months (80% CI, 1.41–4.2) in the vaccinated and nonvaccinated patients, respectively.

Based on BICR, the ORR revealed the following outcomes among HER-Vaxx plus chemotherapy-treated patients: one complete response (5.3%), six partial responses (31.6%), seven cases of stable disease (36.8%), and two cases of progressive disease (10.5%). In comparison, among the chemotherapy-alone group, there were seven partial responses (41.2%), three cases of stable disease (17.6%), and four cases of progressive disease (23.5%) (Supplementary Table S4). This resulted in ORR of 37% and 41%, respectively.

Vaccine-induced antibody and clinical responses

HER-Vaxx-induced strong HER2-specific antibodies

To evaluate the correlation between the observed clinical responses and induced humoral responses in the vaccinated patients, available serum samples from baseline and weeks 6, 12, and 24 of the treatment schedule (Fig. 1) were examined by ELISA to determine the levels of HER2-specific IgG antibodies.

Initially, the levels of tumor-induced, HER2-specific antibodies in the patients from the chemotherapy-alone arm were measured. As shown in Fig. 4A; Supplementary Fig. S5A, the levels were below 100 ng/mL at all examined time points in all patients. This value (100 ng/mL) was assigned as the cutoff. Among the evaluated patients in the 50-µg HER-Vaxx arm, two patients (13.3%) responded strongly to the vaccine and induced high IgG antibody levels exceeding 100,000 ng/mL after four vaccinations (Fig. 4B; Supplementary Fig. S5A).

Figure 4. Detected levels of HER2-specific total IgG antibodies. The HER2-specific total IgG antibodies in patients treated with chemotherapy alone A, and 50 µg of HER-Vaxx plus chemotherapy B, measured by ELISA, are shown. The results are representative of at least two experiments.

ELISA was performed to assess the distribution of the IgG isotypes IgG1, IgG2, IgG3, and IgG4 in the vaccine-induced total IgG antibodies in patients vaccinated with HER-Vaxx. Predominantly high levels of IgG1 isotype were detected in the vaccinated patients, followed by IgG2, IgG3, and IgG4 isotypes (Supplementary Fig. S5B). Strong induction of the vaccine-induced IgG1 antibody responses against HER2 at all timepoints after three or more vaccinations was observed (Supplementary Fig. S5C; P < 0.001).

To evaluate whether higher doses of HER-Vaxx would further induce HER2-specific IgG antibodies, seven patients were enrolled in an extension study and treated with 100 µg of HER-Vaxx plus chemotherapy. As shown in Supplementary Fig. S6, both doses of the vaccine induced HER2-specific antibody levels of similar magnitude, suggesting that a plateau was reached with the 50-µg dose, which was the previous RP2D.

The B-cell peptide component in HER-Vaxx is the hybrid peptide P467. Thus, the serum samples from the HER-Vaxx arms of the study were also assessed by ELISA for the induced P467-specific IgG. Considering no difference between the HER-Vaxx-induced antibodies with the examined 50- and 100-µg doses, values from the higher dose were included for enlarging the sample number and statistical analysis. As shown in Supplementary Fig. S7, a significant correlation (P < 0.001) between the HER2-specific total IgG and P467-specific IgG was present.

HER-Vaxx–induced, HER2-specific total IgG and IgG1 antibodies promoted antitumor effects and cancer cell-specific ADCC

Based on the high levels of HER2-specific antibodies following vaccination with HER-Vaxx, we aimed to assess whether the induced IgG and IgG1 antibodies correlated with tumor responses. As shown in Fig. 5A, the HER-Vaxx-induced IgG antibodies strongly correlated with tumor regression, indicating the significant capacity of the antibodies to reduce tumor diameters (P = 0.001) in addition to chemotherapy. No significant correlation between the magnitude of the antibody response and tumor regression in the chemotherapy-alone arm (P = 0.0973) was observed.

Figure 5. Correlation between the induced HER2-specific antibody levels and antitumor effect and mediation of ADCC. The total IgG A, and IgG1 B, antibody levels in all subjects’ available sera after four vaccinations (week 12; Fig. 1) and the observed antitumor effect (as fold change compared with baseline) in each respective patient are shown. The correlation of the induced HER2-specific IgG C, or IgG1 D, antibody levels in representative sera of low, high, and very high responders [after four vaccinations (week 12) with 50- or 100-µg vaccine dose], and ADCC expressed in percent of the positive control (trastuzumab) are presented. The levels of correlation and significance are indicated in the boxes. The target cells NCI-N87 were incubated with effector cells (i.e., PBMC) after treatment with 50 μg of the examined patients' isolated and purified total IgG antibodies. Trastuzumab as a positive control and serum samples from a healthy individual as a negative control were included in the assays. The results represent at least two experiments.

Considering the more dominant induction of HER2-specific IgG1 antibodies, we also examined the correlation between this vaccine-induced isotype and reduction in tumor diameters. As shown in Fig. 5B, IgG1 antibodies were also significantly associated with fold change of tumor diameters in the respective patients (P = 0.016).

The key mechanism of action of trastuzumab and other mAb is ADCC directed at HER2-overexpressing tumor cells by specific binding of the mAb to the extracellular domain of the receptor and simultaneous interaction of the mAb’s Fc region and Fc receptor on effector cells in vivo (25). Based on the observation that HER-Vaxx–induced IgG strongly binds to HER2, we assessed the ability of isolated IgG from patients with varying levels of antibody responses to mediate ADCC in vitro. This was done using the NCI-N87 cell line as targets and PBMC from healthy human donors as effector cells. As shown in Fig. 5C, a significant correlation was observed between IgG from high-responder patients and the induction of ADCC (P = 0.003). Because the IgG isotype IgG1 has been strongly associated with mediated ADCC (17, 26, 27), the correlation between the IgG1 levels from the respective patients and the observed ADCC was examined, showing a significant correlation between the two (P = 0.05; Fig. 5D). These results indicate the immune mechanism of the HER-Vaxx-induced IgG, which mediated ADCC and thus reduced the respective patients’ tumor sizes.

Treg have been shown to promote cancer development and progression in several lines of preclinical and clinical investigations (28–30). In the phase Ib evaluation of HER-Vaxx, we have observed a correlation between tumor regression and a reduced number of Treg (CD4+/CD25+/FOXP3+). Thus, FACS analysis was carried out to examine the number of Treg in available PBMC from patients vaccinated with 50 µg or 100 µg of HER-Vaxx. As shown in Supplementary Fig. S8, the strong induction of HER-Vaxx-induced IgG antibodies after four vaccinations was significantly associated with a decreased number of Treg. The significant correlation was shown to be further enhanced after five vaccinations (Supplementary Fig. S8).

Overall, this significant association of the HER-Vaxx-induced IgG antibodies with decreased Treg numbers, although based on a limited number of available samples, points in the direction of successful vaccination leading to increased activation of tumor-associated/antigen-specific T cells that enhance antibody production and contribute to tumor regression, as shown in the phase Ib evaluation of HER-Vaxx (21).

HER-Vaxx-induced IgG antibodies inhibited the phosphorylation of HER2 and the signaling pathway protein kinases Akt and MAPK

Another mechanism of action of trastuzumab upon binding is inhibition of HER2 intracellular phosphorylation and consequent inhibition of the signaling pathway proteins Akt and MAPK (31, 32). Thus, total IgG from selected patients with different levels of HER-Vaxx-induced IgG antibodies were first isolated and then examined for their capacity to bind to HER2 and inhibit intracellular phosphorylation (Supplementary Fig. S9). High binding of the isolated IgG from higher responders to the HER2-overexpressing human gastric carcinoma cells NCI-N87 (Supplementary Fig. S9A and S9C), but not to the HER2-negative human melanoma cells 518.A2 (Supplementary Fig. S9B and S9C), was observed. Furthermore, the vaccine-induced, HER2-specific antibodies directly correlated with phosphorylation inhibition of HER2, Akt, and MAPK (Supplementary Fig. S9D–S9F) and inversely with reduced tumor diameters in the respective patients (Supplementary Fig. S9G–S9I). These results demonstrate the capacity of the HER-Vaxx-induced antibodies to promote anti-tumor effects, not only by mediating ADDC but also by inhibiting phosphorylation of HER2-associated signaling pathway proteins Akt and MAPK, as seen with trastuzumab.

Discussion

In the present HERIZON study, we show an OS benefit with no additional AE or increased safety concerns with the vaccination, which was associated with strong HER2-specific IgG and IgG1 antibodies with the capacity to elicit antitumor activity.

The study arms in this phase II trial included patients treated with either SOC chemotherapy alone or HER-Vaxx plus SOC chemotherapy. Routine vaccines against infections in patients with cancer are optimally given before or at the beginning of chemotherapeutic treatment for sufficient immune response (33–35). Accordingly, the patients in the HER-Vaxx arm received the first dose of the vaccine together with the SOC chemotherapy, whereas subsequent vaccinations were administered on different days than the chemotherapy treatments (Fig. 2).

A low evaluable number (n = 5) of patients received a higher dose of the vaccine (100 µg) in a dose-extension study. The higher dose showed a similar capacity as the 50-µg dose in inducing vaccine-specific antibody response. These results indicate that the 50-µg dose achieves a near-peak effect on humoral immune stimulation, which plateaus with further dose escalation, validating the RP2D.

In addition to an excellent safety profile, this phase II study showed a 40% survival benefit for patients treated with HER-Vaxx plus chemotherapy compared with chemotherapy alone, which translated to an OS HR of 0.6 (by BICR), although the observed tendency of the improved OS following the vaccination requires further evaluation in larger studies. PFS was the secondary endpoint of the study, showing a difference of 20% between the vaccination arm and the chemotherapy-alone arm, but the difference was not significant. In the phase Ib dose-escalation trial, the observed median PFS was ∼2 months (1.7–3 months), ∼3.8 months (2–3.8 months), and ∼9.3 months (6–16 months) for the examined 10‐, 30‐, and 50‐µg HER-Vaxx treatment groups, showing an improved PFS with higher doses of HER-Vaxx (21). In the present phase II study, which was based on using the recommended 50-µg dose of HER-Vaxx, the additional effect of HER-Vaxx to chemotherapy was compared with chemotherapy alone, thus more correctly mirroring the add-on effects of vaccination than in the phase Ib study. Furthermore, the phase II study, unlike the phase Ib trial, mandated concomitant treatment of HER-Vaxx and chemotherapy in the vaccinated arm commencing on Day 0 of the study. These factors may reflect the differences in the PFS levels observed in the two studies.

The data from the large phase III ToGA trial, which examined the effect of trastuzumab plus chemotherapy versus chemotherapy alone in advanced gastric cancer, and paved the way for approval of the combined treatment in patients with gastric cancer, had an OS HR of 0.74. Although all patients in the ToGA trial had HER2 overexpression by FISH analysis, the level of receptor overexpression by IHC ranged from 0 to 3+. In this study, patients were HER2+ by the FISH assay and showed +2 or +3 results (overexpression levels) by IHC testing. Thus, the distinct levels of HER2 overexpression in patients from the two studies may reflect the differences in observed OS HR values and suggest the potent therapeutic effect of HER-Vaxx due to polyclonal antibody and T-cell vaccine responses as well as immunologic memory, which is not able to be induced by passive administration of trastuzumab.

A decline of antitumor antibodies below the protective level can lead to tumor progression; thus, boosters are required to keep high antibody titers. In this study, the primary vaccinations were performed on days 0, 14, and 35, followed by a booster 42 days later (on day 77) and subsequent boosters every 63 days. A significantly higher HER2-specific IgG and IgG1 antibody response at all timepoints was induced, particularly after three or more vaccinations with HER-Vaxx. In the patients treated with chemotherapy alone, a background level of HER2-specific antibodies, with no increase at any examined timepoint, was observed and expected. However, vaccination significantly increased the level of antibodies.

An additional advantage of targeting HER2 by vaccination was observed with a significant reduction of FOXP3+ Treg. Reduction of FOXP3+ Treg is a prerequisite for successful vaccine response, which has been shown in preclinical cancer settings (36, 37) as well as in clinical settings with routine vaccines, such as the tick-borne encephalitis vaccine (38–40) and in the phase Ib study of our HER2 B-cell vaccine (21). The decreased levels of the examined CD4+/CD25+/FOXP3+ Treg possibly suggest a proportional expansion of vaccine-specific effector T cells rather than a reduction of the absolute number of Treg.

The mechanism of induced antibodies to reduce tumor growth is based on two modes of action: (i) inhibition of intracellular phosphorylation and (ii) mediation of ADCC. Dimerization of the HER2 receptor and its phosphorylation initiates signal transduction, subsequently leading to activation of MAPK (Ras/Raf/MEK/ERK) and phosphoinositide 3-kinase (PI3K)/Akt. These two major intracellular signaling pathways have a significant role in the invasion and metastasis of gastric cancers (41, 42). Studies have demonstrated that trastuzumab blocks tumor growth by reducing downstream signaling, inhibiting angiogenesis, and increasing immune activity (43, 44). Additionally, due to its IgG1 backbone, trastuzumab’s Fc domain interacts with the Fcγ receptor of effector cells, leading to their activation and consequent lysis of the tumor cells (31, 45). Based on our results, vaccine-induced antibodies showed the capacity to bind to HER2, subsequently resulting in phosphorylation inhibition of the receptor-associated signaling pathway proteins Akt and MAPK, and inversely correlated with the fold change of tumor diameters in the respective patients. In particular, a significant correlation of the levels of the HER-Vaxx-induced IgG1 subtype with BOR and mediated ADCC levels was observed. Studies have shown that in patients with primary operable HER2-overexpressing breast cancer, trastuzumab as a preoperative monotherapy does not inhibit HER2 downstream signaling (46, 47), suggesting ADCC as the major mechanism of mAb in vivo. Thus, HER-Vaxx-induced antibodies not only mediate ADCC but also inhibit intracellular phosphorylation, indicating the vaccine’s multiple modalities in inducing the demonstrated antitumor effect.

This study has the following limitations: (i) Despite randomization, gender imbalance occurred. Additionally, the imbalance regarding the prior lines of therapy/surgery/radiation of 52% (chemotherapy alone) versus 80% (HER-Vaxx plus chemotherapy) may be due to differing local practices in the regions the study was conducted. (ii) As an exploratory analysis, we planned to test if HER-Vaxx treatment induced loss of HER2 expression in patient tissue. However, on-study biopsy was optional, resulting in a lack of tumor samples for the evaluation. (iii) Technical difficulties with the collection of viable PBMC prohibited the analyses of immunophenotyping. However, such cellular responses were analyzed in depth in the phase Ib evaluation of the vaccine (21). (iv) The evaluation of ADCC mediation by vaccine-induced antibodies was done on isolated IgG from only a few patients who were selected based on the magnitude of their IgG response.

This study included patients from countries where treatment with trastuzumab was not widely available as a first-line treatment for this indication. A study comparing passive immunization with trastuzumab and active immunization with HER-Vaxx would eventually indicate the vaccine’s clinical relevance. However, such comparison might be difficult due to the different kinetic of immunologic responses induced by the respective immunizations and was not the scope of this phase II study. Because a synergistic effect with a combination of targeting HER2 and PD1 was shown in a preclinical study to induce tumor growth inhibition by 90% (48), and ramucirumab plus paclitaxel is an approved second-line treatment for patients with gastric cancer or gastric/gastroesophageal junction adenocarcinoma who have failed first-line treatment chemotherapy or trastuzumab (49), the clinical benefit of adding HER-Vaxx to ramucirumab plus paclitaxel or the anti-PD1 immune checkpoint inhibitor pembrolizumab, following progression on trastuzumab is now being evaluated in a phase II study (nextHERIZON; ref. 50).

In conclusion, the results of this phase II trial indicate the safety and immunogenicity of our novel B-cell epitope vaccine, HER-Vaxx, in patients with HER2-overexpressing gastric cancer and its capacity to strongly induce HER2-specific antibodies correlating with tumor reduction, inhibition of intracellular phosphorylation, and mediation of ADDC. In this trial, HER-Vaxx not only induced HER2-specific humoral responses but also counteracted immune tolerance effects, thereby helping to improve the effects of SOC chemotherapy in vaccinated patients with gastric cancer.

Supplementary Material

Supplementary Materials and Methods S1 Supplementary Materials and Methods

Supplementary Figure S1 Supplementary Figure S1. Trial profile and patient disposition.

Supplementary Figure S2 Supplementary Figure S2. Adverse Events (AEs), with grade 1/2 or ≥3 associated with the respective treatments in the study arms.

Supplementary Figure S3 Supplementary Figure S3. The Kaplan-Meier estimate of OS.

Supplementary Figure S4 Supplementary Figure S4. The Kaplan-Meier estimate of PFS.

Supplementary Figure S5 Supplementary Figure S5. Levels of total IgG antibodies and IgG isotypes in the patients treated with chemotherapy alone or HER-Vaxx plus chemotherapy.

Supplementary Figure S6 Supplementary Figure S6. Comparison of HER2-specific total IgG antibody levels in the patients vaccinated with 50-µg and 100 µg of HER-Vaxx plus chemotherapy.

Supplementary Figure S7 Supplementary Figure S7. Correlation between the induced HER2-specific and P467-specific total IgG antibodies in each patient.

Supplementary Figure S8 Supplementary Figure S8. Association between decreased FOXP + Tregs and induced HER2-specific IgG antibody levels.

Supplementary Figure S9 Supplementary Figure S9. Correlation between the induced levels of HER2-specific IgG antibodies, and their binding to HER2-expressing cells and intracellular phosphorylation inhibition.

Supplementary Table S1 Supplementary Table S1. Inclusion and exclusion criteria.

Supplementary Table S2 Supplementary Table S2. Representativeness of Study Participants.

Supplementary Table S3 Supplementary Table S3. Post hoc analysis for mOS, with and without patients treated with trastuzumab.

Supplementary Table S4 Supplementary Table S4. Overall response rates (by BICR).

Acknowledgments

The authors thank the patients and their families for their contributions to this study, the principal investigators of the study, and the staff at the clinical sites for their help in collecting the clinical samples. The study was funded by Imugene Ltd.

Authors’ Disclosures

C. C. Zielinski reports consulting fees from Athenex, MSD, Imugene Limited (until September 2018), AstraZeneca, Servier, and Eli Lilly and institutional fees from Eli Lilly, BMS, MSD, Pfizer, AstraZeneca, Merck, Amgen, Servier, Takeda, Daiichi Roche, Boehringer, Celgene, and Halozyme. L. M. O. Chong reports leadership fees from Imugene Limited. B. Nixon reports employment from and is a shareholder/stockholder of Imugene Limited. N. J. Ede reports employment at Imugene Limited. S. Yavrom reports employment at Imugene Limited. M. Kundi reports consultation fees from BlueSky Immunotherapies and Pfizer (funding to institute). U. Wiedermann reports consulting fees from Imugene Limited (until September 2018) and funding to the institute from GSK, Pfizer, and Themis. No disclosures were reported by the other authors.

Authors’ Contributions

J. Tobias: Data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. M. Maglakelidze: Supervision. Z. Andrić: Supervision. D. Ryspayeva: Supervision, writing–review and editing. I. Bulat: Supervision. I. Nikolić: Supervision. Z. Petrovic: Supervision. T. Chawla: Supervision, writing–review and editing. R. Nagarkar: Supervision. E. Garner-Spitzer: Methodology. C.C. Zielinski: Conceptualization. L.M.O. Chong: Resources, project administration. B. Nixon: Supervision, project administration. N.J. Ede: Validation. S. Yavrom: Data curation, formal analysis, validation, project administration, writing–review and editing. M. Kundi: Formal analysis, validation, visualization, methodology, writing–review and editing. U. Wiedermann: Conceptualization, writing–review and editing.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
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