Phase 1 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 1 study (NCT 01417546) evaluated the safety profile, optimal immunologic/biologic dose (OID/OBD) and immunogenicity of the combination of two peptide B-cell epitope vaccines engineered to represent the trastuzumab and pertuzumab binding sites. While 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.

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 regimen. 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 (PR), 14 had stable disease (SD), and 19 had progressive disease (PD).

CONCLUSIONS:

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

INTRODUCTION

HER-2 is a transmembrane receptor that is overexpressed in multiple epithelial tumors, including subsets of breast, gastro-esophageal, esophageal, endometrial, uterine, ovarian, colorectal and lung cancers (1–6). HER-2 is associated with more aggressive forms of cancer (7), an increased risk of metastasis, increased tumor invasion, and decreased overall survival (8,9). Therefore, HER-2 is a key therapeutic target in several cancers. Trastuzumab (Herceptin; Genentech, San Francisco, CA) was the first humanized monoclonal antibody targeting HER-2 in combination with chemotherapy to be approved for clinical use in patients with metastatic HER-2 overexpressing breast cancer (10–17). Despite the benefit observed from trastuzumab, approximately one-third of patients with metastatic, HER-2 positive breast cancer experience primary resistance (18) and most responding patients eventually develop acquired resistance within one year of therapy (19). Since 2007, four additional HER-2-targeted therapies (lapatinib, neratinib, pertuzumab, and T-DM1) have been approved by the US Food and Drug Administration (FDA) for the treatment of breast cancer. Studies investigating novel agents and combination therapies with anti-HER-2 directed therapy, are also under investigation for solid tumor malignancies. A recent phase III clinical trial showed that the addition of pertuzumab, a recombinant humanized monoclonal antibody that blocks the heterodimerization of HER-2 with other HER family members, to docetaxel and trastuzumab in patients with untreated HER-2 overexpressing breast cancer, resulted in improvement in progression-free and overall survival from 12.4 months to 18.5 months and from 40.8 to 56.5 months, respectively (20–22)

To date, most HER-2 peptide cancer vaccine strategies have sought to induce a cellular antigen-specific T-cell response (23,24). CD8+ and CD4+ T cell vaccines are human leukocyte antigen (HLA)-restricted which limits their universal applicability, and therefore, they may need to be tailored to the specific subtype of cancer and tumor antigen expression level to achieve maximum effectiveness. Approaches aimed at stimulating humoral immunity specific to HER-2 may provide an advantage over T-cell directed therapies. For instance, in contrast to T-cell receptor/antigen interactions, epitopes to monoclonal antibodies are not required to be presented on specific isoforms of major histocompatibility complex (MHC), a process which can be impaired in some cancers (25). In addition, inducing B-cell mediated immunity could enable the host to generate antibodies potentially capable of functioning as “endogenous” trastuzumab and pertuzumab. However, unlike passive chimeric antibody therapy which is expensive, associated with clinically significant toxicities and requires repeated treatment, a peptide vaccine has the potential to result in sustained humoral immunity.

We have advanced a new paradigm in immunotherapy that focuses on humoral responses based on conformational B-cell epitope vaccines. These novel platforms elicit high affinity antipeptide antibodies against tumors that help circumvent intrinsic drug resistance. Importantly, this is hypothesized to provide durable treatment effects due to immunologic memory (26). We have previously identified the first generation of HER-2 B-cell peptide epitopes (628–647) and (316–339) through computer immunogenicity algorithms and extensive in vitro and in vivo preclinical studies (27,28). In a recently published phase 1 clinical trial, we showed that the combination of the two chimeric HER-2 vaccines in patients with metastatic solid tumors was safe, demonstrated activity (disease control rate of 24%), and elicited HER-2 specific humoral responses in 62.5% of patients (29).

Our research group has developed two novel B-cell epitope specific vaccines consisting of epitopes derived from the extracellular domain of the HER-2/neu molecule that are binding sites of trastuzumab and pertuzumab. Using the X-ray structures of the HER-2-trastuzumab and HER-2-pertuzumab complexes (30–32), we have rationally designed the trastuzumab-binding epitope (597–626) and the pertuzumab-binding epitope (266–296) (33,34). A series of six conformational peptides spanning residues 563–626 (trastuzumab binding site and 266–333 (pertuzumab-binding site) were engineered, synthesized and characterized to mimic the trastuzumab and pertuzumab binding sites and were tested for immunogenicity in mice and rabbits (33,34). The highest affinity and titer antibody responses were seen with epitope 266–296 located in domain II (pertuzumab binding site) and 597–626 located in domain IV (trastuzumab binding site). Thus, the vaccine was expected to stimulate patients’ immune systems to elicit a polyclonal antibody response to HER-2 and in particular to simulate trastuzumab and pertuzumab.

Our translational studies show that active immunotherapy with our peptide vaccine led to the generation of HER-2/neu specific B-lymphocytes that had the ability to inhibit HER-2 signaling while inducing potent anti-tumor humoral immune response against HER-2 positive cells, as confirmed in prior pre-clinical in vitro and in vivo experiments (26,28,35). The findings demonstrated that these peptides were recognized by trastuzumab and pertuzumab, elicited a strong sustained humoral and cell mediated immunity, interfered with HER-2 signaling, and led to anti-tumor effects in preclinical models (33,34). Additionally, these peptides inhibited multiple signaling pathways including HER-2 specific inhibition of cellular proliferation and cytoplasmic receptor domain phosphorylation. The peptide antibodies mediated antibody-dependent cellular cytotoxicity (ADCC). These vaccines had statistically reduced tumor onset in two transplantable tumor models (FVB/n and BALB/c) and led to significant reduction in tumor development in two transgenic mouse tumor models (BALB-neuT and VEGF^+/−Neu2–5^+/− (33,34)). Lastly, these 2 vaccines were capable of generating antibodies that exhibited properties similar to trastuzumab and pertuzumab, validating their use in this phase 1 clinical trial.

Overall, immunotherapy using cancer vaccines, is an exciting and rapidly evolving field in oncology that leverages patients’ immune systems to target cancer. Chimeric B-cell epitope peptide vaccines incorporating a “promiscuous” T cell epitope offer an attractive immunotherapeutic option in the treatment of cancer, with considerable advantages in their safety ease of manufacture and administration. Additional advantages of B-cell cancer vaccines are exquisite specificity the potential for a durable treatment effect due to immunologic memory, and provides universal coverage bypassing HLA restriction. Herein, we report the results from the first-in-human, dose escalation portion of the phase 1 study testing the combination of two peptide B-cell epitope vaccines MVF-HER-2 (597–626) and MVF-HER-2 (266–296) incorporating a promiscuous measles virus (MVF) T cell epitope.

MATERIALS AND METHODS

Objectives

The primary objectives were to assess the safety and clinical toxicity of immunization, determine the optimum immunologic/biologically dose (OID/OBD) of combination HER-2 vaccines, measure both humoral and cellular immune responses including the specificity, class and kinetics of anti-HER-2 peptide, and evaluate whether the combination of HER-2 vaccines demonstrate therapeutic benefit, provide synergistic and/or additive effects, and to enumerate mechanisms of action. Secondary objectives were to collect and analyze post-immune sera and peripheral blood cells for an additional 6 months following the last injection and document clinical responses.

Patients

Eligible patients were required to have the following: (1) metastatic, incurable solid tumor malignancy, at least 3 weeks past any prior surgery, cytotoxic chemotherapy, other immunotherapy, hormonal therapy, or radiation therapy (2) adequate end organ function and (3) Eastern Cooperative Oncology Group (ECOG) performance status of 0–2. Subjects with history of properly treated and stable brain metastases were also eligible as long as there was no evidence of CNS progression within 3 months prior to the first dose of the study vaccine. Patients were excluded if they had a significant concurrent illness; left ventricular ejection fraction (LVEF) of <50%, uncontrolled or severe cardiac disease; active viral hepatitis, HIV, or other active infections requiring use of antibiotics; active autoimmune disease; or corticosteroid requirement. Baseline disease assessment was performed by physical examination, medical history, chest x-ray, and computed tomography (CT) scans.

The clinical trial was conducted under an investigational new drug application (IND# 14633, PKaumaya) approved by the FDA. The study protocol (2010C0075) was approved by The Ohio State University Cancer Institutional Review Board. All patients gave written informed consent prior to participation in the study (NCT01376505). The study was conducted in accordance with ethical principles founded in The Common Rule, the Belmont Report, Good Clinical Practice guidelines, and applicable local laws.

Peptide selection, manufacturing and vaccine preparation

The rationale, selection, design, synthesis and characterization of the two peptide constructs were originally described by the Kaumaya laboratory (33,34). The GMP peptides were purchased from Peptisynthia (Torrance, CA) and acquired by Solway Group (Zug, Switzerland). N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine (nor-MDP) was purchased from Penninsula Labs, (Torrance, CA). The GMP peptides met all the FDA and US Pharmacopeia requirements for sterility (ie, bacterial/fungal), endotoxins, and potency. The bulk peptides were supplied to University of IOWA Pharmaceuticals manufacturing facility (Iowa City, Iowa) for sterile vialing in 3 mg lots. Endotoxin levels of these peptides were tested and determined to be within acceptable levels as Good Manufacturing Practice (GMP) grade. The vehicle Montanide ISA™ 720 was purchased from SEPPIC (Fairfield, NJ), and it had an approval certificate of analyses for toxicity, emulsifying property, and sterility. The immunogenicity of each individual peptide and combination was verified in pairs of New Zealand rabbits. The combination vaccine was prepared by mixing the two chimeric MVF-B-cell epitope peptides that correspond to amino acid sequences 266–296 (LHCPALVTYNTDTFESMPNPEGRYTPGASCV: pertuzumab binding site) and 597–626 (VARCPSGVKPDLSYMPIWKFPDEEGACQPL: trastuzumab binding site). The chimeric constructs were synthesized with the MVF sequence KLLSLIKGVIVHRLEGVE at the N-terminus via a linker consisting of GPSL. On the day of vaccination for each dose level, the appropriate peptide concentration was prepared by dissolving with n-MDP (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine) adjuvant (0.025mg) in a total volume of 1.0 ml. This solution was emulsified with 1.0 ml saline-oil phase vehicle (Montanide ISA 720; SEPPIC Inc, a subsidiary of Air Liquid group, Paris, France).

Immunization schedule

All eligible patients underwent a skin test prior to the first dose of the study vaccine. Approximately 0.1 mL of each peptide was injected intra-dermally in two separate areas of the forearm. Patients were subsequently examined for delayed-type hypersensitivity (DTH) to the test dose approximately 20 minutes after administration (>20 mm erythema or development of a wheel). Patients who did not develop hypersensitivity reaction to the test dose, were to receive 3 intramuscular vaccinations, each given 21 days apart (days 1, 22 and 43). The vaccine was injected into the gluteus maximus muscle, with subsequent injections given in contralateral muscle to the prior vaccine administration site. No premedications were required but treating physicians had an option of ordering them per their discretion. Following three doses, patients with responding or stable disease had an option of returning to receive booster vaccinations at 6 month intervals.

Study design and treatment

The dose escalation part of this clinical trial used a 3+3 schema. Initially, 3 patients were to be treated at each dose level (DL) and observed for a minimum of 4 weeks. If 0–1 patients experienced a dose-limiting toxicity (DLT), an additional 3 patients were entered at that DL and were observed for a minimum of 4 weeks. Dose escalation could proceed only after at least 6 evaluable patients in a cohort were observed for a minimum of 4 weeks, and if only 0–1 of the 6 patients in that DL experienced a DLT. If 2 or more of the 6 patients at a DL experienced a DLT, additional enrollment at that DL was terminated, and the previous DL was defined as the maximum tolerable dose (MTD).

At least 6 patients at each dose level were required to receive a total of 3 consecutive inoculations of the combination vaccines at 3-week intervals in order to better evaluate for the OID/OBD. The DLs were 1.0 mg (DL1), 1.5 mg (DL2), 2.0 mg (DL3) and 2.5 mg (DL4) of each peptide. Each dose contained 0.025 mg of n-MDP.

Definition of dose limiting toxicities (DLT)

All toxicities were graded based on Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The DLT period was defined as the first 4 weeks after the first administration of the study vaccine. In order to be evaluable for DLTs, patients were required to receive at least 2 doses of the study vaccine. DLTs were adverse events that were at least possibly related to study therapy and defined as follows: (1) Any grade 3 or greater toxicity including flu-like symptoms, (2) any grade 3–4 neutropenia lasting more than 5 days or accompanied by ≥ grade 2 fever, or any grade 4 thrombocytopenia; (3) clinical inability (due to toxicity) to start next cycle of treatment within 3 weeks of planned start date; (4) any grade 3 injection site reaction, defined as an abscess formation or cellulitis requiring antibiotics or surgical procedure such as incision and drainage or injection site reaction severe enough to require narcotic analgesia. Any grade 2 allergic reaction of asymptomatic bronchospasm or generalized uticaria resulted in cessation of vaccination in that individual and was considered to be dose-limiting only if serious allergic reactions have also been reported at that dose cohort.

Assessment of toxicity and response

Physical exam was performed at baseline and prior to each vaccine administration and on day 71 (4 weeks post 3^rd immunization). Laboratory studies, such as complete blood counts with differential, complete metabolic panel and urinalysis were also obtained. Cardiac function was assessed by history and physical exam and by Multigated Acquisition Scan (MUGA) at baseline and prior to booster vaccinations. Troponin I levels were checked prior to and following every vaccination to monitor for theoretical potential cardiotoxic effect of the vaccine and to ensure that vaccination did not cause cardiotoxicity.

Tumor response was measured by radiologic assessment with computed tomography (CT) or magnetic resonance imaging (MRI) (same measure was used serially) pre-treatment, day 71 and subsequently at the 6 months boost. Radiologic assessment was required for patients removed from study, unless patients withdrew further participation. Response was measured according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 criteria.

Procurement of patient plasma

Blood was drawn from patients using standard phlebotomy techniques. Peripheral blood for humoral and anti-tumor immune correlative studies were drawn from study patients into heparinized tubes prior to (40mL) and 4h after (10mL) each vaccination as well as on day 71. Blood samples were also obtained monthly for patients who received a 6 months booster vaccine to monitor levels of antibodies in their blood samples.

Cell Lines, antibodies, and protein

Cell culture medium, fetal calf serum and supplements were purchased from Invitrogen Life Technologies (Carlsbad, CA). The human breast tumor cell line BT-474 (American Type Culture Collection, Manassas, VA) was maintained according to the supplier’s guidelines and all tests were done within 6 months of receipt. The American Type Culture Collection authenticates cell lines by observations of recovery along with morphological appearance, testing viability by trypan blue exclusion. Isoenzymology and/or Cytochrome C subunit I (COI) by PCR is performed to confirm species of samples. Human Cell lines are tested by STR analyses to determine identity. Lines are also tested for mycoplasma contamination by PCR. AG825, a selective HER-2/neu kinase inhibitor (Calbiochem, San Diego, CA), human recombinant HER-2 protein (100ug, Acrobiosystem, Newark, DE and Herceptin (Genentech) was used as a control.

Patient sera purification

3ml crude sera 1:1 diluted with Protein A/G Binding Buffer (Thermo Scientific, Rockford, Illinois) was applied to equilibrated protein A/G column with 10mL of Binding Buffer followed by wash with 20mL of Binding Buffer. 10ml elution buffer (Thermo Scientific, Rockford, Illinois) was used to elute bound antibodies and separately collect initial and last 1ml eluate and middle 8ml eluate. 8ml eluate was then concentrated and exchanged with PBS and final volume was adjusted to 3ml. The protein concentration was determined with a calibration factor 1.5 using Bradford reagent.

Enzyme-linked immunosorbent assay

Levels of anti-HER-2 antibody were determined prior to immunization, during immunizations, and monthly after the 3rd and last immunizations. Although the induction of antibody against the immunizing peptide is an important observation relative to the approach in general, the key determination is whether the immunogen elicits an antibody that binds HER-2 protein. Because tissue distribution is essential for activity, it is also important to determine whether the antibody class elicited is IgG. The presence of antibodies specific for the HER-2 peptide vaccine in patient serum was directly assessed by using enzyme-linked immunosorbent assay (ELISA), as described previously (27).

Isotyping antibody identification of patients vaccinated with peptide based HER-2 vaccine

To identify what subclass of IgG antibodies patients were generating against the HER-2 peptides, we used an isotyping ELISA. Plates were washed with PBT and incubated at room temperature for 1h with (i) mouse anti-human antibodies of different isotypes (anti-IgA, anti-IgD, anti-IgE, anti-IgG, anti-IgM) (Southern Biotech, Birgmongham, AL), (ii) anti-human isotyping IgG (type 1,2,3 and 4) antibodies conjugated to horseradish peroxidase (HRP). Absorbance at 410nm was read in an ELISA reader. The percentage of isotype antibodies in sera were represented by their respective absorbance relative to the total absorbance by all 5 isotype antibodies. For IgG subtype analysis, mouse anti-human antibodies of different IgG subtypes (anti-IgG1, anti-IgG2, anti-IgG3, anti-IgG4) acted as the probe for bound sera antibodies

Flow cytometry

Immunofluorescence staining of cells to measure binding of human antibodies after immunization was evaluated as previously described (29). Human HER-2 overexpressing breast cancer BT-474 (1 × 10⁶) cells were incubated with patient serum in 100 μL of 2% FCS/0.1% NaN₃ in phosphate-buffered saline (PBS) for 2h at 4°C. Pre-immune sera were used as a negative control, and trastuzumab was used as a positive control. Unbound antibodies were removed with PBS, and the cells were incubated with fluorescein isothiocyanate–conjugated antihuman antibody for 30 minutes at 4°C in 100 μL of 2% FCS in PBS. Cells were washed in PBS and were fixed in 1% formaldehyde before they were analyzed by Coulter ELITE flow cytometer (Coulter, Hialeah, FL). A total of 10,000 cells were gated by light-scatter assessment before single-parameter histograms were drawn and smoothed.

MTT Cell proliferation assay

The proliferation assay was performed as previously described (33,34) with BT-474 cells (2 × 10⁴ per well) in 96-well, flat-bottom plates overnight. Inhibition percentage was calculated as previously described, and trastuzumab was used as a positive control while pre-immune sera served as negative controls (29).

HER-2 receptor phosphorylation assay

The HER-2 phosphorylation assay was performed by using BT-474 cells (1 × 10⁶ per well), as described previously in 6-well plates, which were incubated at 37°C overnight (33,34). Supernatants were collected, and protein concentration of each sample was measured by Coomassie plus protein assay reagent kit and lysates were stored at −80°C. Phosphorylation was determined as previously described (34) by using the Duoset IC for human phosphor-ErbB2 (R&D systems, MN) according to the manufacturer’s directions.

Antibody-dependent cellular cytotoxicity (ADCC)

ADCC was measured by using the bioluminescence non-radioactive cytotoxicity assay (aCella-TOX kit; Cell Technology, Mountain View, CA) according to the manufacturer’s recommendations as previously described (29). Briefly, BT-474 target cells (1 × 10⁴/well) were placed in a 96 well plate and patient antibodies (50μg) and trastuzumab (50μg) as positive control were added to the wells containing the BT-474 target cells. The plate was incubated at 37°C for 15min to allow opsonization of the patient antibodies to occur. Human peripheral-blood mononuclear cells (hPBMCs from Red Cross) as effector cells were then added to the wells at three different effector to target (E:T) ratios (20:1, 10:1 and 5:1) and the plate incubated at 37°C for 3h. Normal human IgGs served as negative controls. 10μl of lytic agent was added to the control wells for maximum lysis and incubated for 15min at room temperature followed by addition of 100μl of the Enzyme Assay reagent containing G3P to all wells. The detection reagent was added, and the plate was immediately read using an illuminometer.

Caspase activity assay for apoptosis

Apoptosis was measured using the caspase activity assay (Caspase-GLO; Promega, Madison, WI) per manufacturer’s instructions as previously described (46). Apoptosis was evaluated by measuring the amount of luminescence (as readout of caspase activity) using a luminometer. The percentage of increased release of caspases was calculated using the formula (ODUNTREATED - ODTREATED)/ODUNTREATED x 100. All experimental treatments were performed in triplicate.

Assessment of optimum immunologic/biologically dose

For the purpose of this study, the OID/OBD was defined as the dose of the vaccine that is capable of inducing strong immunogenic response in 5 of 6 treated patients in the given dose level. Strong immunogenic response was defined when the optical density (ELISA) was consistently greater than 1.5 across the various time points after the 3rd vaccination; that is where the observed immunogenicity (antibody titers) reaches a maximum response (titers have plateaued). Analysis of anti-tumor responses provided additional criteria for defining the OID/OBD.

Statistical analysis

Summary statistics were calculated for patient demographics and clinical characteristics. The maximum grade for each type of toxicity was recorded for each patient, and frequency tables were provided. P values < 0.05 were considered statistically significant. All analyses were conducted in SAS version 9.4 (SAS Institute, Cary, North Carolina).

RESULTS

Patients and demographics

Between July 2011 and February 2016, 56 patients were screened. Forty-nine patients were determined to be eligible for the study and received at least 1 dose of study vaccine (N=8, 9, 20, 12 in Cohorts 1–4, respectively; see Supplemental Table 1). The study population consisted of subjects with a wide range of different malignancies (see Supplemental Table 2 for demographics). The median age of patients was 59 (range 35–81) with a majority of patients having received ≥4 prior lines of therapy (see Table 1)

Table 1.

Summary of patients by cohort.

ID#	Cohort	# of Prior Therapies	Primary Cancer	Vaccinations (# of cycles + # of boosts)	HER-2/EGFR Status	Previous treatment type	Best Response
01–01	1	7	Colon	1	−ve/−ve	MC	PD
01–02		2	Anal	2	Unknown	MC	PD
01–03		1	Cartilage	3 +1	1A/ −ve/−ve	MC	SD
01–04		7	Lung	3 +1	1B/ −ve/−ve	MC	SD
01–05		1	Breast	3 +1	1C/ +ve/+ve	MC+Trastuzumab	PR
01–06		3	Bladder	3	1D/Unknown	MC	PD
01–07		11	Ovarian	3	1E/ −ve/+ve	MC	PR
01–08		4	Colon	2	1F/ −ve/+ve	MC	SD
02–01	2	3	Anal	1	Unknown	MC	PD
02–02		5	Colon	2	−ve/+ve	MC	PD
02–03		3	Bladder	1	−ve/+ve	MC	SD
02–04		2	Anus	3 +1	2A/ −ve/+ve	MC	SD
02–05		3	Colon	2	2B/−ve/+ve	MC	PD
02–06		0	Parotid	3 +7	2C/ +ve	Radiation only	NE
02–07		3	Colon	3 +1	2D/ −ve/+ve	MC	SD
02–08		3	Breast	3	2E/ +ve/+ve	MC+Trastuzumab	PD
02–09		8	Colon	3	2F/ Unknown	MC	SD
03–01	3	5	Rectal	3	3A/ −ve	MC	SD
03–02		5	Breast	2	+ve	MC+ Trastuzumab	NE
03–03		2	Colon	3	Unknown	MC	PD
03–04		4	Colon	3	−ve/+ve	MC	SD
03–05		5	Ovarian	1	Unknown	MC	NE
03–06		5	Lung	2	Unknown	MC	SD
03–07		4	Lung	2	Unknown	MC	SD
03–08		4	Peritoneal	3	3B/+ve/−ve	MC+Trastuzumab; Perjeta	SD
03–09		2	Ovarian	2	−ve	MC	NE
03–10		8	Colon	3	3C/ Unknown	MC	PD
03–11		5	Cervical	2	Unknown	MC	NE
03–13		4	Colon	2	Unknown	MC	PD
03–14		5	Lung	3	Not tested	MC	NE
03–15		3	Esophageal	1	−ve	MC	PD
03–16		8	Ovarian	2	−ve/+ve	Trastuzumab	NE
03–18		2	Colon	3	Unknown	MC	NE
03–19		6	Rectal	3	3D/−ve	MC	SD
03–20		7	Breast	3	3E/ +ve	MC +cetuximab	SD
03–24		7	Rectal	3	−ve/−ve	MC	PD
03–26		5	Breast	2	+ve	MC/Trastuzumab; Perjeta/Kadcyla	NE
04–01	4	3	Breast	3	+ve	MC+Trastuzumab; Perjeta	PD
04–02		2	Ovarian	1	unknown	MC	NE
04–03		3	Esophageal	3	4A/ +ve/U	MC+Trastuzumab	PD
04–04		5	Ovarian	3	4B/ −ve	MC	PD
04–06		7	Ovarian	2	Unknown	MC	NE
04–07		1	Lung	3	4D/U/−ve	MC	PD
04–08		9	Ovarian	3	U	MC	NE
04–10		8	Colon	3	4E/−ve/−ve	MC	PD
04–12		4	Rectum	2	4C/−ve	MC	PD
04–13		1	Breast	3	Unknown	MC	PD
04–14		4	Rectal	2	Unknown	MC	NE
04–15		1	Breast	3	4F/−ve	MC	NE

MC=multiple chemotherapy treatments; PR= partial response; PD=progressive disease; NE=non-evaluable; SD=stable disease; −ve=negative; +ve=positive. We have utilized standard RECIST 1.1 criteria to define PR, PD and SD (37).

Study treatment

We utilized the Cohorts-of-3 rule (see Materials and Methods) to simultaneously evaluate tolerability and evidence of biological activity at each dose level (36). Twenty-eight of the 49 patients (57%) completed the required 3 vaccinations (N=5, 5, 10, 8 in Cohorts 1–4, respectively). Of those, 6 patients received 1 booster at 6 months due to clinical benefit (Table 1), with one patient with parotid cancer in dose level 2 receiving a total of 7 booster vaccinations. He subsequently developed pulmonary nodules which were suspicious for disease progression and was removed from the study after receiving study treatment for over 3 years. Common reasons for discontinuation of therapy in the evaluable patients were disease progression, grade 3 injection site reaction, and patient or physician preference. Patients (N=21) that did not receive the required 3 vaccinations were not evaluable for dose-limiting toxicities (DLTs). The percent of patients who discontinued due to disease progression includes patients who received any number of the vaccinations (including patient who had less than 3 vaccinations).

Safety and tolerability

The vaccine was well-tolerated overall, with minimal or no toxicities in most patients. No DLTs were observed. The most common toxicities at least possibly related to study treatment were injection site reactions (Grade 1–2 in 24% of patients). Grade 2 buttock hematoma developed at the site of the injection in 1 (2%) patient. Additionally, 2% of patients developed grade 2 systemic allergic reaction (mild hypotension and diaphoresis). Grade 1 rash also developed in 2% of patients. Common adverse events that were considered at least possibly related to study therapy are listed in Table 2.

Table 2.

Treatment-related toxicities in all 49 patients.

Toxicity (number of patients %)	Grade 1–2	Grade 3–4	Total
Lymph node pain	1 (2%)	0 (0%)	1 (2%)
Fatigue	4 (8%)	0 (0%)	4 (8%)
Fever	2 (4%)	0 (0%)	2 (4%)
Flu like symptoms	1 (2%)	0 (0%)	1 (2%)
Injection site reaction	12 (24%)	0 (0%)	12 (24%)
Allergic reaction	1 (2%)	0 (0%)	1 (2%)
Alanine transaminase elevation	1 (2%)	0 (0%)	1 (2%)
Alkaline phasphagase elevation	1 (2%)	0 (0%)	1 (2%)
Lymphopenia	2 (4%)	0 (0%)	2 (4%)
Leukopenia	1 (2%)	0 (0%)	1 (2%)
Hypoalbminemia	2 (4%)	0 (0%)	2 (4%)
Hyponatremia	2 (4%)	0 (0%)	2 (4%)
Hypophosphatemia	0 (0%)	1 (2%)	1 (2%)
Buttock pain	1 (2%)	0 (0%)	1 (2%)
Myalgia	2 (4%)	0 (0%)	2 (4%)
Dry skin	1 (2%)	0 (0%)	1 (2%)
Pain of skin	1 (2%)	0 (0%)	1 (2%)
Pruritus	1 (2%)	0 (0%)	1 (2%)
Maculopapular Rash	1 (2%)	0 (0%)	1 (2%)
Skin and subcutaneous tissue disorder	3 (6%)	0 (0%)	3 (6%)
Skin ulceration	1 (2%)	0 (0%)	1 (2%)
Hematoma	1 (2%)	0 (0%)	1 (2%)

Response to treatment

Figure 1A displays radiographic response rates: The waterfall plot illustrates the maximum percentage of tumor reduction for target lesions in patients with measurable disease, and Figure 1B shows the swimmers plot of time to response and time to off-study for all 49 patients. Progressive disease (PD) at the time of the first re-staging scans developed in 19 (54%) evaluable patients. Partial response (PR) as the best response was observed in 2 (6%) evaluable patients. Stable disease (SD) was observed in 14 (40%) of evaluable patients (Supplemental Table 3). We have utilized standard response evaluation criteria in solid tumors (RECIST) 1.1 criteria to define PR, PD and SD (37).

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.

Antibody response to peptide vaccine and recombinant HER-2 protein.

Antibody responses to the combination vaccines were assessed by ELISA against MVF-HER-2 (266–296), MVF-HER-2 (597–626) and recombinant HER-2 protein. We monitored patient immune responses over the course of the trial (data not shown). Patient sera were purified using protein A/G column. Figure 2 shows individual patient purified antibodies in Cohort 2 at 4 weeks post third vaccination (5 patients in this cohort completed the 3 vaccination regimen). As shown in Figure 2, Panel 7, similar antibody responses were elicited in Cohort 2 patients (2A-F) against the individual vaccine immunogen and recombinant HER-2 in the range of 0.3–0.4 absorbance. Two patients (2A and 2F) had an increased response to the 597–626 epitope. Overall, Cohort 2 patients had more durable responses. Antibody responses in the other cohorts were variable: in Cohort 1 the antibody responses were quite low; in Cohort 3, there were 3 patients who responded with reasonable titers, 2 did not respond and 2 had moderate response; in Cohort 4 at the highest dose, 1 patient had high response and the others had low responses (Supplemental Table 4).

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 3^rd 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.

Reactivity of peptide antibodies with the native HER-2 receptor.

Antibodies elicited by peptide vaccination are only effective if they recognize the native receptor. Direct binding of purified patient antibodies to the native HER-2 receptor was evaluated by immunofluorescence staining and cytofluorimetric analysis (FACS) using HER-2 overexpressing cells BT-474. As shown in Figure 2 Panels 1–6, antibodies on day 71 (3Y+4weeks) for all 6 patients in Cohort 2 (2A-2F) showed significant fluorescent shift in binding relative to normal human IgG. Pre-immune antibodies were also used as negative control (data not shown). This observation is similar to the positive control, trastuzumab, although the fluorescent strength is weaker than the latter. This result demonstrated that patient antibody raised against the vaccine can recognize HER-2 receptor over-expressed tumor cells. Variable results were obtained for Cohorts 1, 3 and 4 with less pronounced fluorescent shifts (Supplemental Figures 1–3).

Patient 2C received seven 6 months booster vaccinations.

Patient 2C travelled to Columbus, Ohio from Seattle, Washington to receive his first vaccine on 05/30/2012 and two boosters at 3 weeks apart. The patient returned every 6 months through 12/10/2015 to receive the 7 booster vaccinations, spanning 3.5 years. Prior to enrollment in our study, this patient was diagnosed with T2N2bM0 poorly differentiated carcinoma of the right parotid gland and underwent radical parotidectomy and selective right neck dissection. The surgical pathology revealed 25 lymph nodes positive for metastatic disease. Interestingly, the tumor also had “patchy foci” of 3+ Her2/neu staining based on immunohistochemistry on a background of equivocal 2+ positivity. Recurrence was discovered in the right axillary lymph nodes about 2 years later and the patient underwent right axillary lymph node dissection which revealed that four of 46 lymph nodes were positive. He was subsequently enrolled in our study. The exceptional response in this patient may also be due to the fact that this patient did not receive any chemotherapy prior to enrollment. Thus, his immune system was very much intact and likely more responsive to vaccine treatment. This is an important fact as the vaccine therapy should be more effective in such patients

Figure 3, Panel F shows the immune responses of purified antibodies at various intervals. Significant antibody levels were detected to recombinant HER-2, MVF-HER-2 (266–296) and MVF-HER-2 (597–626) after the first boost (3 weeks post second vaccination, 2Y+3) and subsequent 6 months boost. These results demonstrate that Patient 2C was able to elicit antipeptide antibodies that recognized the recombinant HER-2 protein. In parallel studies, immunofluorescence staining and fluorescence cytofluorimmetric analysis were used to confirm the relative binding affinities of the patient sample antibodies (Figure 3, Panel A-E) using BT-474 cancer cells.

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.

Vaccine elicits predominantly IgG (IgG3 and IgG4) antipeptide antibodies

Patients in Cohort 2 elicited predominantly antibodies of the IgG isotypes (Figure 4, A-B) to both vaccines MVF-HER-2 (266–296) and MVF-HER-2 (597–626). The amount of IgA, IgD and IgE were minimal; however there were some 20% IgM antibodies. The elicited IgG were further subtyped (Figure 5, C-D) into mostly IgG3 with variable amounts of IgG1, IgG2 and IgG4. Similar profiles were also obtained for Cohorts 1, 3 and 4 (see Supplemental Table 5, Panels A-B). These studies indicate that isotype switching occurred during the process of vaccination and boosting with activation of the T cells.

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.

Patient vaccine antibodies cause enhanced antiproliferative effects

The effects of the patient antibodies were tested using BT-474 in the presence of native HER-2 ligand, heregulin (HRG). Figure 5A shows that each of the patient sera in Cohort 2 was able to inhibit proliferation of the HER-2 overexpressing cells BT-474 as compared to pre-immune sera. The level of inhibition was comparable to trastuzumab, suggesting that our vaccine antibodies were equally effective. Results for Cohorts 1, 3 and 4 are shown in Supplemental Table 6, Panel A.

Patient vaccine antibodies inhibit HER-2 phosphorylation.

Phosphorylation of HER-2 plays a critical role in signaling pathway response to ligand binding. The main mode of action of pertuzumab is the interruption of HER-2/neu dimerization with other members of the ErbB receptor family. To determine whether our peptide antibodies disrupted dimerization and phosphorylation of the receptor cytoplasmic tyrosine kinase regions, we used a phospho-HER-2 ELISA. Sequence 266–296 represents a sequence that mimics the binding site of pertuzumab that has been shown to disrupt ligand-dependent receptor complexes independent of HER-2 expression. To evaluate if patient sera containing HER-2-specific antibodies were able to function as dimerization inhibitors, we used a total phospho-HER-2 ELISA. BT-474 cells were treated with patient serum antibodies, and HRG was used to activate HER-3 before cell lysates were captured with an anti-HER-2 monoclonal antibody and were probed with a phospho-HER-2 antibody. All patients at dose level 2 lowered the concentration of the phosphotyrosine on BT-474 cells by 25% to 35% compared with the HER-2 phosphorylation inhibitor AG825, which was used as the positive control. Overall, as shown in Figure 5B, our data suggest that the patient antibodies inhibited receptor phosphorylation. Results for Cohorts 1,3 and 4 are shown in Supplemental Table 6, Panel B.

Patient antibodies induce apoptosis

Targeting apoptotic regulatory pathways in cancer is a promising strategy for therapeutic agents. We next evaluated whether patients’ vaccine antibodies following vaccination were capable of inducing apoptosis of cancer cells by caspase activity assay. Using breast cancer cells BT-474 treated with the patient antibodies as inhibitors, we found that each of the patient antibodies (3Y+4; 4 weeks post third vaccine) demonstrated significant increases in the amount of caspase 3 and 7 activity in treated cells as compared to the negative controls (pre-immune sera) (Figure 5C). Vaccine antibodies increased caspase release more than 100-fold, clearly indicative of increased cell death similar to trastuzumab. Apoptosis results for Cohorts 1, 3 and 4 are shown in Supplemental Table 6, Panel C.

Patients’ vaccine antibodies induce antibody-dependent cellular cytotoxicity (ADCC) of cancer cells

One major mechanism of immunologic action of antibodies is to induce ADCC of cancer cells. It has been well-documented that in vivo, the Fc portions of Abs can be of foremost importance for efficacy against tumor targets (38). We determined the ability of the patient antibodies to mediate ADCC in vitro using BT-474 breast cancer cells as targets and peripheral-blood mononuclear cells (PBMC’s) from normal human donors as effector cells using a bioluminescense cytotoxicity assay kit (aCella-Tox™) as previosusly described (29). All patient antibodies demonstrated increased tumor cell lysis (40–45% at 20:1 effector to target ratio) in a dose-dependent manner similar to trastuzumab (Figure 5D). Normal human IgG was used as negative control. ADCC results for Cohorts 1,3 and 4 are shown in Supplemental Table 6, Panel D.

DISCUSSION

This phase 1 clinical trial evaluated the safety, tolerability, toxicity and immunogenicity of a combination of two peptide vaccines, as well as the OID/OBD and the maximum tolerable dose (MTD). The vaccine combination consisted of two B cell HER-2 epitopes: HER-2 (266–296) and HER-2 (597–626) fused to a Measles Virus Fusion protein (MVF) “promiscuous” T helper cell epitope. The peptides were combined with a potent adjuvant nor-muramyl dipeptide (n-MDP) and emulsified in Montanide ISA 720. Three vaccinations given at 3 week intervals with the chimeric peptides at four different doses were well-tolerated without any severe systemic adverse events, with the most common adverse events consisting of grade 1–2 injection site reactions. Dose level 2 was selected as the OID/OBD given its immunogenicity, ability to inhibit proliferation and phosphorylation of the chimeric vaccines, and its biological properties such as ADCC and apoptosis.

The vaccine generated sustained humoral response eliciting antibodies that recognized the HER-2 receptor in the majority of responding patients. Of note, the clinical benefit was observed across all dose levels, with few patients showing evidence of sustainable disease control (as shown in Figure 1). Also of note, these patients were a heavily pretreated cohort that had been exposed to an average of 4 prior therapies. There was preliminary evidence of clinical activity in six patients (21.4%) who received 6 months boost, including one patient who received seven 6 months booster vaccinations over 3.5 years without evidence of resistance to therapy. The number of patients who received 6 months boosts, including the one patient who received seven 6 month boots with our vaccine treatment, highlights the great potential of this therapy for circumventing the intrinsic drug resistance that limits currently available cancer therapies. The promising preliminary results suggest an important potential benefit of this vaccine compared to humanized monocolonal antibodies in which most patients develop secondary resistance. One needs to exercise caution when interpreting the efficacy results in this trial because of limited patient numbers and phase 1 study design which is focused on establishing safety and best dose of the vaccine for further clinical development.

In order to elicit high affinity antibodies (a prerequisite for an effective outbred vaccine), two criteria are essential: (i) the conformational epitopes must mimic the tertiary structure of the antigen; (ii) the peptides must include a “promiscuous” T-cell epitope such as one from the Measles Virus Fusion Protein (MVF) to ensure high immunogenicity and maintainence of sustained immune response by activating the Th2 helper T-lymphocyte subset. We also combined our vaccine with a potent adjuvant nor-muramyl dipeptide (n-MDP) and emulsified in Montanide ISA 720 (SEPPIC, Inc., Paris, France) to improve antigen presentation by formation of the depot of the vaccine peptides at the site of injection. In contrast to Incomplete Freund’s adjuvant, this solution is biodegradable and less cytotoxic (39,40). Previous studies have shown that trastuzumab blocks tumor growth through reducing downstream signaling, inhibiting angiogenesis, and increasing immune activity, primarily ADCC (19). Constitutive PI3K/Akt activation through PTEN downregulation or PIK3CA hyper-activating mutations significantly abrogates response to trastuzumab (41,42). The lack of effective ADCC immune response also promotes trastuzumab resistance (43–45).

The study vaccine bypasses the disadvantages of passive immunotherapy with monoclonal antibodies, i.e. high cost and the need for repeated treatments that can result in serious toxicities such as hypersensitivity reactions, cardiomyopathy and in rare cases, pneumonitis. Our peptide vaccine was not associated with an increased risk of cardiomyopathy, despite sustained production of endogenous, fully human antibodies that function very similarly to trastuzumab and pertuzumab. Peptide cancer vaccines are an attractive therapeutic option as they are safe and easily manufactured and administered. Additional advantages of peptide cancer vaccines are exquisite specificity, low toxicity, and the potential for a durable treatment effect due to immunologic memory. In addition, our vaccine avoided the limitations of T-cell specific cancer vaccines that require specific MHC isoforms in order to bind the peptide.

The trial had limitations that should be addressed. First, it was a single institutional study in a heterogeneous group of cancer patients many of whom were heavily pretreated. Such patients tend to have poor ability to mount an effective anti-tumor immunity because of tumor- and treatment-induced immunosuppression. While we found that such patients were still able to generate a sustained humoral response to the vaccine, patients who are less heavily pretreated or have a lower tumor burden have been able to mount a more effective, sustained immune response to the vaccine, potentially resulting in a greater observed clinical benefit. Notably, the patient that received multiple boosters did not receive any chemotherapy prior to enrollment. Thus, his immune system was very much intact and likely more responsive to vaccine treatment. This is an important fact as the vaccine therapy should be more effective in such patients. Additionally, it is possible that some patients who received 3 or more vaccinations may have more indolent malignancy and therefore receive more vaccinations before progression based on RECIST 1.1 criteria compared to patients who completed less than 3 vaccinations. However, the phase 1 studies are not designed to test efficacy of experimental therapy but to establish safety profile and best dose of the therapy. This trial also suffered from lack of randomization making it difficult to ascertain whether the vaccine is truly effective, but randomization in not commonly used in Phase 1 trials. Despite these limitations, our trial shows at least a signal of promising anti-tumor activity of the vaccine. We need to await the results of a future randomized study before definitive conclusions about the vaccine’s efficacy can be made.

Lastly, a limitation for the study is the absence of consistent measurement of HER-2 overexpression in patients enrolled in the dose escalation part. This is partly because the Phase 1 trial was opened to all patients irrespective of whether they overexpressed HER-2. Since this was a first-in-human trial focused on establishing the safety profile and recommended phase 2 dose of the vaccine, we did not collect information about HER-2 expression in this portion of the study. We therefore cannot make any conclusions that over-expression of the target protein was responsible for efficacy in the responding patients.

In conclusion, we demonstrate that the combination of HER-2 vaccines is well-tolerated and able to generate sustained anti-HER-2 immune response. The most common toxicity was an injection site reaction. A majority of the patient antibodies that were generated in response to the vaccine showed potent anti-tumor activity and defense mechanisms (induction of ADCC and apoptosis, inhibition of proliferation and phosphorylation). There were limited but few very meaningful clinical responses in this heavily pretreated patient population with a heterogeneous group malignancies. Given the initial promise, continuous development of the vaccine is ongoing at the suggested OBD in a less heavily pretreated patient population in breast and/or gastrointestinal malignancies (gastroesophageal and colorectal) with HER-2/EGFR overexpression.

Statement of Translational Relevance:

This is the first-in-human dose escalation study of combination of two HER-2 B-cell peptide vaccines to safely deliver curative and transformative cancer immunotherapies to advanced cancer patients. We have created and established a novel chimeric B-cell peptide vaccine eliciting antibodies with high immunogenicity that binds with high specificity to native human HER-2, demonstrates promising anti-tumor activity, shows preliminary indication that peptide vaccination may avoid therapeutic resistance and offer a promising safe alternative to monoclonal antibody therapies. Continuous development of the vaccine is ongoing in a Phase II trial at the suggested OBD in a less heavily pretreated patient population in breast and/or gastrointestinal malignancies with HER-2/EGFR overexpression. B-cell cancer vaccines have the advantage of producing specific immune responses that can potentially induce memory B & T cell responses.We need to await the results of a future randomized study before definitive conclusions about vaccine’s efficacy can be made.