Phase I Immunotherapy Trial with Two Chimeric HER-2 B-Cell Peptide Vaccines Emulsified in Montanide ISA 720VG and Nor-MDP Adjuvant in Patients with Advanced Solid Tumors

Abstract

Purpose: This first-in-human phase I study (NCT01417546) evaluated the safety profile, optimal immunologic/biological dose (OID/OBD), and immunogenicity of the combination of two peptide B-cell epitope vaccines engineered to represent the trastuzumab- and pertuzumab-binding sites. Although trastuzumab and pertuzumab have been approved for clinical use, patients often develop resistance to these therapies. We have advanced a new paradigm in immunotherapy that focuses on humoral responses based on conformational B-cell epitope vaccines.

Patients and methods: The vaccine is comprised of two chimeric HER-2 B-cell peptide vaccines incorporating a "promiscuous T-cell epitope." Patients were immunized with the vaccine constructs emulsified with nor-muramyl-dipeptide adjuvant in a water-in-oil Montanide ISA 720VG vehicle. Eligible patients with metastatic and/or recurrent solid tumors received three inoculations every 3 weeks.

Results: Forty-nine patients with a median of 4 prior lines of chemotherapy received at least 1 vaccination. Twenty-eight patients completed the 3 vaccination regimens. Six patients received 1 six-month boost after the regimen, and one patient received 7 six-month boosts. No serious adverse reactions or dose-limiting toxicities were observed. The vaccine was well tolerated with dose level 2 as the recommended phase II dose. The most common related toxicity in all patients was injection-site reactions (24%). Two patients had a partial response, 14 had stable disease, and 19 had progressive disease.

Conclusions: The study vaccine is safe, exhibits antitumor activity, and shows preliminary indication that peptide vaccination may avoid therapeutic resistance and offer a promising alternative to monoclonal antibody therapies.

Conflict of interest statement

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed by all authors.

Conflict of Interest statement: The authors declare no potential conflicts of interest

©2019 American Association for Cancer Research.

Figures

Figure 1:. Response rates for patients by dose level.

A) Radiographic response. The waterfall plot illustrates the maximum percentage of tumor reduction for target lesions in patients with evaluable disease for tumor response. Each bar represents an individual patient. The red dash line represents the cut-off for disease progression, and the black dashed line represents the cut-off for partial response measured by RECIST 1.1. B) The swimmers plot of time to response and time to off study for all patients. Duration of response (months) to treatment for the 2 patients who achieved partial response. Each individual bar represents one patient. Red triangle indicates patients who achieved a partial response. PR=partial response; SD=stable disease; PD=progressive disease.

Figure 2:. Cohort 2 antibody binding analyses.

Panels 1–6) Flow cytometry analysis of cohort 2 patient antibodies binding to BT-474 with overexpressed HER-2 (Cohort 2 patients 2A-2F) after the 3rd vaccination. 5×10 5 BT-474 cells were incubated with 500μg/ml patient antibodies, 500μg/ml control antibody (normal human IgG) and 50μg/ml commercial human anti-HER2 (Trastuzumab) in staining buffer (PBS+0.05%BSA+0.2% NaN3) respectively at 4°C for 2 hours and then with 1.6 μg/ml Alexa Fluor® 488 anti-human IgG at 4°C for 45min. The stained cells were analyzed on a FACS Caliber machine. Panel 7) Binding of Cohort 2 antibodies to MVF-HER-2 (266–296) (blue), MVF-HER-2 (597–626) (orange) and recombinant HER-2 (yellow) assessed using ELISA. Antibodies purified via protein A/G column from sera before or after immunization were diluted to 20μg/ml with PBS/HS and incubated with immobilized MVF-HER-2 vaccines and recombinant HER-2. Bound antibodies were probed using HRP conjugated anti-human IgG. IgG, normal human IgG.

Figure 3:. Patient 2C antibody binding analyses.

Panels A-E) Flow cytometry of patient 2C antibodies by multiple boosts binding to BT-474 with overexpressed HER-2. 5×10 5 BT-474 cells were incubated with 500μg/ml patient antibodies (blue), 500μg/ml control antibody (normal human IgG; red) and 50μg/ml commercial human anti-HER2 (Trastuzumab; yellow) in staining buffer (PBS+0.05%BSA+0.2% NaN3) respectively at 4°C for 2 hours and then with 1.6 μg/ml Alexa Fluor® 488 anti-human IgG at 4°C for 45min. The stained cells were analyzed on a FACS Caliber machine. Panel F) ELISA purified 2C antibodies binding to rhHER-2, MVF-HER-2 (266–296) and MVF-HER-2(597–626). Pre, pre-immunization; control, normal human IgG. 1Y+3w, blood drawn 3 weeks after the first immunization. 2Y+3w, blood drawn 3 weeks after the second immunization. Months (mts) and years (yr) denote the time of drawing blood after first immunization.

Figure 4:. Cohort 2 antibody isotype and IgG subtype identification.

A-B) Antibody isotype identification of cohort 2 patient sera by ELISA. After incubation of sera (1:16) with immobilized MVF-HER-2 (266–296) (A) and MVF-HER-2 (597–626) (B), isotypes of bound antibodies were identified using HRP conjugated different secondary antibodies. The percentage of isotype antibodies in sera was represented by their respective absorbance relative to the total absorbance by all five isotype antibodies. C-D) IgG subtype identification of cohort 2 patient sera by ELISA. After incubation of sera (1:16) with immobilized MVF-HER-2 (266–296) (C) and MVF-HER-2 (597–626) (D), IgG subtypes of bound antibodies were probed using HRP conjugated different secondary antibodies. The percentage of isotype antibodies in sera was represented by their respective absorbance relative to the total absorbance by all four isotype antibodies.

Figure 5:. Effects of Cohort 2 purified antibodies on proliferation, phosphorylation, apoptosis, and antibody dependent cell cytotoxicity.

A) BT-474 (10,000 cells per well) were treated for 72 hours before addition of MTT. After extraction with lysis buffer, plates were incubated overnight at 37 degrees and were read on an ELISA plate reader at 570nm. Results shown is an average of three different experiments with each treatment performed in triplicates for dose level 2. Cells were treated with medium alone or with pre-immunized, post-immunized antibodies and control respectively. B) BT-474 (1 million cells per well) in six well plate were incubated with patient antibodies and cells lysed in RIPA lysis buffer and then, spun at 13,000 X g and supernatants collected. Phosphorylation was determined by Duoset IC (R&D systems) for total human phosphoHER-2 according to manufacturer directions. Results shown is an average of three different experiments with each treatment performed in triplicates for dose level 2. C) BT-474 breast cancer cells were treated with the vaccine antibodies and caspase activity was measured after treatment and results show the activity levels after treatment. Data shows average of three different experiments following treatment with patient antibodies from dose level 2. D) Cohort 2 patients purified antibodies cause antibody-dependent cellular cytotoxicity ADCC. BT-474 target cells were incubated with different amounts of effector cells (PBMC) after treatment with 100μg patient antibodies 2A-2F in dose level 2. Trastuzumab was used as positive control, and normal human IgG was used as the negative control. Results shown are an average of three different experiments with each treatment performed in triplicates for dose level 2.