Humoral immune responses in patients vaccinated with 1–146 HER2 protein complexed with cholesteryl pullulan nanogel

Abstract

The CHP‐HER2 vaccine, comprising truncated 146HER2 protein complexed with nanogels of cholesteryl pullulan (CHP), is a novel protein antigen vaccine that elicits 146HER2‐specific CD8⁺ and CD4⁺ T‐cell immune responses in patients with HER2‐expressing tumors. We analyzed the humoral responses in patients vaccinated with CHP‐HER2 and those with CHP‐HER2 plus granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). The vaccine was injected subcutaneously at a dose of 300 µg protein. Nine patients received the vaccine alone over the first four injections, followed by CHP‐HER2 with GM‐CSF or OK‐432, whereas six received CHP‐HER2 plus GM‐CSF from the first cycle. 146HER2‐specific IgG antibodies were induced in 14 patients, who were negative at baseline. The antibodies became detectable after the second or third vaccination and reached plateau levels after the third or fourth cycle in patients vaccinated with CHP‐HER2 plus GM‐CSF. In contrast, the antibodies appeared only after the third to sixth vaccination and the plateau appeared after the fourth to eighth cycle in patients vaccinated with the CHP‐HER2 vaccine alone over the first four cycles. The antibodies induced by the vaccine were not reactive with HER2 antigen expressed on the cell surface in any of the patients. Epitope analysis using overlapping peptides revealed a single region in the 146HER2 protein, amino acids 127–146, in eight patients who were initially vaccinated with CHP‐HER2 alone. Similarly, the same HER2 region was recognized dominantly in patients vaccinated with GM‐CSF. Our results indicate that CHP‐HER2 induced HER2‐specific humoral responses in patients with HER2‐expressing tumors and that GM‐CSF seems to accelerate the responses. (Cancer Sci 2008; 99: 601–607)

Nanogels of cholesteryl pullulan (CHP) complexed with 146HER2 soluble protein, the CHP‐HER2 complex vaccine, has been demonstrated in a mouse model to elicit efficient CD8⁺ and CD4⁺ T‐cell responses and to produce higher titers of antibodies against 146HER2 protein, compared to vaccination with HER2 protein alone.^{( 1 )} Our previous clinical trial demonstrated that vaccination with CHP‐HER2 induced HER2‐specific CD8⁺ and CD4⁺ T‐cell immune responses in five out of nine patients.^{( 2 )} Another CHP protein vaccine using NY‐ESO‐1 protein, a cancer testis antigen, was formulated and used as the cancer vaccine CHP‐NY‐ESO‐1. CHP‐NY‐ESO‐1 has been reported to be a potent inducer not only of antigen‐specific humoral immune responses but also of specific T cells, including CD8⁺ and CD4⁺ T cells.^{( 3} , ^{4 )}

As the CHP antigen vaccine can boost the activity of CD4⁺ T cells, it is important to analyze 146HER2‐specific antibody responses following CHP‐HER2 vaccination in order to determine the immune responses of 146HER2‐specific helper CD4⁺ T cells and B lymphocytes. Because the IgG titer measured by enzyme‐linked immunosorbent assay (ELISA) can be assessed quantitatively in real time, it is possible to monitor vaccine‐specific immune status in vaccinated patients. In the present study, we describe 146HER2‐specific IgG antibody responses in patients vaccinated with CHP‐HER2 alone over the first four cycles, or with CHP‐HER2 plus recombinant human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), to explore the potential immunoadjuvant activity of GM‐CSF for the CHP‐HER2 vaccine.

Patients and Methods

CHP‐HER2 vaccine.  The recombinant 146HER2 protein used for the vaccine was prepared from Escherichia coli (Asahi Glass, Tokyo, Japan). CHP was synthesized by the reaction of pullulan with cholesterol isocyanate in pyridine and dimethylsulfoxide (Nippon Oil and Fat Corporation, Tokyo, Japan). The CHP‐HER2 complex was prepared as described previously.^{( 2 )} All processes were carried out under cGMP conditions. The vaccine material was assessed for its safety using animal models and its biochemical and biological stability was examined regularly in our laboratories.

Clinical trial.  A phase I clinical trial of the CHP‐HER2 complex vaccine was designed to evaluate the safety and immune response to 146HER2. Participating patients were to have advanced cancers with HER2‐expressing tumor cells (score ≥1 by immunohistochemistry; HercepTest, Dako Corporation, Carpinteria, CA, USA). The CHP‐HER2 vaccine was injected subcutaneously every 2 weeks at a dose of 300 µg of 146HER2 protein. Toxicity was evaluated according to the Common Terminology Criteria for Adverse Events v.3.0. The trial was approved by the Human Ethics Committees of both Mie and Nagasaki Universities, and it was conducted in full conformity with the current revision of the Declaration of Helsinki. Written informed consent was obtained from each patient enrolled in the trial.

The trial consisted of two parts. In the first part, patients were assigned to receive CHP‐HER2 vaccine alone for the first four cycles (CHP‐HER2 alone), and in the second part patients were to be given CHP‐HER2 vaccine in combination with GM‐CSF from the first cycle (CHP‐HER2 combined with GM‐CSF). Fifteen patients were enrolled in the trial (Table 1). Nine were injected with CHP‐HER2 alone. Following four cycles of the vaccine alone and evaluation of its safety, injections of CHP‐HER2 were continued with an adjuvant of GM‐CSF (Leukine, Berlex, Richmond, CA, USA) or OK‐432, an immunomodulator (Chugai Pharmaceuticals, Tokyo, Japan).^{( 5 )} GM‐CSF was administered at the same site of CHP‐HER2 injection at a dose of 75 µg for three consecutive days starting from the day before vaccination. OK‐432 was injected at a dose of 0.02 mg on the day of vaccination. Injections of GM‐CSF or OK‐432 commenced from the fifth CHP‐HER2 vaccination. The other six patients received CHP‐HER2 combined with GM‐CSF, using the same aforementioned schedule. To analyze the immune reactions, serum samples were collected before the vaccination and 2 weeks after each vaccination. Sera were stored at –80°C until use.

Table 1.

Patients’ characteristics

Characteristic	CHP‐HER2	CHP‐HER2 plus GM‐CSF
No. patients	9	6
Sex (male/female)	4/5	1/5
Age(years)
Median	55	53
Range	42–66	48–63
Type of cancer
Breast cancer	4	3
NSCLC	2	0
Ovarian cancer	0	2
Miscellaneous	3	1
HER2 expression
1+, 2+, 3+	3, 4, 2	2, 1, 3
No. vaccinations
Median	13	12
Range	6–38	6–28

CHP, cholesteryl pullulan; GM‐CSF, granulocyte‐macrophage colony‐stimulating factor; NSCLC, non‐small‐cell lung cancer.

Enzyme‐linked immunosorbent assay.  The ELISA was carried out as described previously.^{( 6 )} In brief, 146HER2 protein was absorbed onto immunoplates (442404; GNunc, Roskilde, Denmark) at a concentration of 10 ng/50 µL/well at 4°C. The collected serum sample was diluted from 1:100 to 1:312 500. After washing and blocking the plate, the serum was added and incubated for 10 h. After further washing, goat anti‐human IgG(H + L chain)‐peroxidase (MBL, Nagoya, Japan) was added. With TMB substrate (Pierce, Rockford, IL, USA), the plate was read using a Microplate Reader (model 550; Bio‐Rad, Hercules, CA, USA). An optical density (OD)₄₅₀ absorption value of 0.5 or more was judged as a positive reaction in a serum dilution of 1:500.

Recombinant protein and overlapping peptides.  The recombinant 146HER2 protein used for assays was prepared using HER2 cDNA. Truncated HER2 cDNA encoding an amino acid terminal portion (amino acids 1–146) was polymerase chain reaction‐amplified and inserted into pET15b (Novagen, Madison, WI, USA). E. coli strain JM109 was transformed with the resulting plasmid and induced by isopropyl‐β‐D‐thiogalactopyranoside (IPTG) to produce 6His‐tagged truncated 1–146 HER2 protein. The recombinant protein of 146HER2 was produced stably by E. coli (Asahi Glass). Twenty‐eight types of 20‐mer peptides, which overlapped by 13 amino acids, were prepared, including 1–20, 8–27, 15–34, 22–41, 29–48, 36–55, 43–62, 50–59, 57–66, 64–73, 71–90, 78–96, 85–104, 92–111, 99–128, 106–125, 113–132, 120–139, 127–146, 134–153, 141–160, 148–167, 155–174, 162–181, 169–187, 176–185, 183–202, and 190–219 (Greiner Bio‐One, Monroe, NC, USA).

Western blot analysis.  The 146HER2 protein was loaded into a 15% gel and separated by gel electrophoresis using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to nitrocellulose membranes. After blocking, the membranes were immunoblotted by adding diluted serum from the patients or 146HER2‐immunized rabbit. After rinsing, biotinylated goat anti‐human IgG (H + L chain) (Vector Laboratories, Burlingame, CA, USA) was added and incubated. The membranes were stained using the ELITE universal kit (Vector Laboratories).

Cell lines.  SKBR3, a human breast carcinoma cell line, and KATOIII, a human gastric carcinoma cell line, were purchased from the American Type Culture Collection (Manassas, VA, USA).

Flow cytometric analysis and antibody‐dependent cellular cytotoxity assay.  HER2‐expressing tumor cells, SKBR3, and HER2‐negative tumor cells, KATOIII, were incubated at 37°C for 30 min with various concentrations of the serum collected from the patients, followed by incubation with fluoroscein‐5‐isothiocyanate (FITC)‐conjugated antihuman IgG (BD Biosciences, San Jose, CA, USA) on ice for 15 min. After washing, the cells were analyzed by flow cytometry (FACS Calibur; Becton Dickinson, Mountain View, CA, USA). Trastuzumab (10 µg/mL Herceptin; Chugai, Tokyo, Japan) and rituximab (10 µg/mL Rituxan; Chugai) were used as positive and negative controls, respectively.

The antibody‐dependent cellular cytotoxity (ADCC) assay was done using a ⁵¹Cr‐release method as reported previously.^{( 7 )} Briefly, after labeling target cells with 3.7 MBq ⁵¹Cr for 60 min and washing three times, the target cells (5 × 10³/well) and effecter cells (healthy donors’ peripheral blood mononuclear cells [PBMC]) at effector : target (E : T) ratios of 50:1, 25:1, and 12.5:1 were coincubated in 200 µL RPMI‐1640 in 96‐well U‐bottomed plates in duplicate for 6 h at 37°C, with patients’ serum at various dilutions. Trastuzumab (2 µg/well Herceptin) and rituximab (2 µg/well Rituxan) were used as positive and negative controls, respectively. The radioactivity of the supernatant (50 µL) was measured with a γ‐counter. The mean percentage of specific lysis was calculated as follows:

% specific lysis = ([c.p.m. experimental release – c.p.m. spontaneous release]/[c.p.m. detergent release – c.p.m. spontaneous release]) × 100.

Results

Safety of CHP‐HER2 combined with GM‐CSF.  Table 1 lists the clinicopathological characteristics of the enrolled patients. The two vaccine schedules (CHP‐HER2 alone, CHP‐HER2 combined with GM‐CSF) were well tolerated even after more than 13 and 12 vaccinations, respectively. All patients developed mild transient skin reactions at the sites of vaccine injection. No serious adverse effects were noted over more than 12 vaccinations. There were no differences between the toxicity profiles of patients vaccinated with vaccine alone and those vaccinated in combination with GM‐CSF (data not shown).

HER2‐specific antibody responses in CHP‐HER2‐vaccinated patients.  Serum samples were collected from each patient and the specific antibody titers were measured by ELISA. IgG antibody to 146HER2 protein was not detected in any of the 15 patients before vaccination (Table 2). However, 146HER2‐binding antibodies appeared in all patients after vaccinations regardless of GM‐CSF coadministration, except one patient with non‐small cell lung cancer (patient no. 8) (Table 2). Fig. 1 shows two representative patients who received CHP‐HER2 vaccination with and without GM‐CSF. In patient no. 1, who received CHP‐HER2 alone for the first four vaccinations (Fig. 1a, left panel), 146HER2‐specific antibodies became detectable after the sixth vaccination. The antibody titer reached a plateau after the ninth vaccination. In patient no. 10, who received GM‐CSF from the first vaccination (Fig. 1b, left panel), the antibodies became detectable after the second vaccination and reached a plateau after the third vaccination. Subtype analysis showed dominance of IgG₁ in both patients (Fig. 1, right panels), and a slight increase in IgG₃ in patient no. 10. Analysis of the serum samples by western blot assay further confirmed the specific reactivity of the antibody to 146HER2 protein (Fig. 2). The sera collected from the patients who were seroconverted to 146HER2 protein were assayed for binding activity to the cell surface antigen of HER2‐expressing tumor cells by the use of flow cytometric analysis and ADCC assay. Fig. 3a shows that trastuzumab binds to SKBR3 HER2‐expressing tumor cells. None of the patients’ sera showed binding activity with flow cytometric analysis (data not shown). In the ADCC assay, none of the sera revealed cytolytic functions to SKBR3, whereas trastuzumab showed the activity. An example of the experiments is shown in Fig. 3b and a summary is given in Table 2.

Table 2.

HER2‐specific antibody responses and antibody‐dependent cellular cytotoxity (ADCC) activities in sera from cholesteryl pullulan‐HER2 vaccinated patients

Figure 1.

Antibodies against 146HER2 protein in two representative patients. Left panels: Antibody titers at OD₄₅₀ absorption in the enzyme‐linked immunosorbent assays at each vaccination cycle. Right panel: IgG subclass analysis. (a) Patient no. 1 who received cholesteryl pullulan (CHP)‐HER2 vaccination alone in the first four cycles. (b) Patient no. 10 who received vaccination of CHP‐HER2 plus granulocyte‐macrophage colony‐stimulating factor.

Figure 2.

Serum antibody binding to 146HER2 protein measured by western blot analysis in a representative patient (patient no. 1). Serum diluted at 1/100 was used. Data represent the results of examinations of serial serum samples. Serum from a 146HER2 protein‐immunized rabbit was used as a positive control.

Figure 3.

Binding and antibody‐dependent cellular cytotoxity (ADCC) of the seroconverted patients’ serum to HER2‐expressing tumor cells. (a) Trastuzumab, anti‐HER2 monoclonal antibody, binds to HER2‐expressing SKBR3 cells, whereas it does not react to HER2‐negative KATOIII cells. (b) Diluted serum samples of two demonstrative patients are assayed for ADCC activity against SKBR3. The cytolytic activity of trastuzumab to SKBR3 is shown as a positive control, and rituximab, anti‐CD20 monoclonal antibody, was used as a negative control.

Combination of CHP‐HER2 with GM‐CSF‐accelerated antibody responses.  In 14 patients who were seroconverted to 146HER2 antigen in response to the CHP‐HER2 vaccine, we assayed the serially collected sera for the IgG antibody level to 146HER2 protein. In six patients who were vaccinated with GM‐CSF from the first cycle (patient no. 10–15), specific IgG became detectable after the second or third vaccination (Table 2; Fig. 4), whereas in nine patients who received CHP‐HER2 alone over the first four cycles (patient no. 1–9), the antibodies appeared after the third to seventh vaccination (Table 2; Fig. 4). The peak of the antibody titers was noted after three to four cycles in the CHP‐HER2 combined with GM‐CSF group, compared with after four to nine cycles in the CHP‐HER2 alone group (Table 2; Fig. 4). The mean number of cycles in which the antibody became detectable and the titer reached a plateau was 4.5 and 6.1 in the CHP‐HER2 group, and 2.7 and 3.3 in the CHP‐HER2 combined with GM‐CSF group, respectively. The differences between the two groups were statistically significant, assessed using Mann–Whitney's U‐test (P < 0.05) (Table 2).

Figure 4.

HER2‐specific IgG responses in 14 seroconverted patients over the vaccinations with cholesteryl pullulan (CHP)‐HER2. (a) Upper panel indicates OD₄₅₀ absorptions in sera from eight patients vaccinated CHP‐HER2 alone for the first four cycles. Lower panel indicates those from six patients given CHP‐HER2 plus granulocyte‐macrophage colony‐stimulating factor from the first vaccine cycle. (b) Enzyme‐linked immunosorbent assay for specific IgG to 146HER2 protein in serially collected sera. Diluted serum as indicated in the figure was assayed.

146HER2‐specific CD4⁺ T cells became detectable in four patients (patient no. 1, 2, 7, and 8) out of nine patients who received the vaccine alone in the first four cycles,^{( 2 )} and in two of six patients who received GM‐CSF from the first cycle (patient no. 10 and 11).

Long‐term follow up of a repeatedly vaccinated patient (patient no. 10) showed maintenance of specific IgG levels for up to 26 cycles (Fig. 5).

Figure 5.

Long‐term follow up of 146HER2‐specific IgG titers in a patient who received cholesteryl pullulan‐HER2 vaccine up to 26 cycles. Each curve shows the titer at OD₄₅₀ absorption at each vaccination cycle measured by enzyme‐linked immunosorbent assay.

Antibody epitope analysis using overlapping peptides.  Finally, we used a synthetic 20‐mer series of overlapping peptides to analyze HER2 antibody epitopes. As shown in Table 3, peptide 127–146 was recognized in all 14 seroconverted patients with CHP‐HER2 vaccine. Among those patients who received GM‐CSF from the first cycle, two additional peptides, 22–41 and 36–55, were recognized in two patients and one patient, respectively. These three peptides were not responsive to prevaccinated sera from all patients.

Table 3.

Reactivities of sera from patients vaccinated against HER2 overlapping peptides

Patient no.	1–20	8–27	15–34	22–41	29–48	36–55	43–62	50–69	57–76	64–83	71–90	78–97	85–104	92–111	99–118	106–125	113–122	120–139	127–146	134–153	141–160
1	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
2	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
3	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
4	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
5	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
6	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
7	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
9	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
10	–	–	–	+	–	+	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
11	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
12	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
13	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
14	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–
15	–	–	–	+	–	–	–	–	–	–	–	–	–	–	–	–	–	–	+	–	–

An OD value of >0.5 was judged as a positive reaction in serum diluted 1/500. Patients 1–9 were vaccinated with cholesteryl pullulan (CHP)‐HER2 alone in the first four cycles, followed by the combination of CHP‐HER2 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Patients 10–15 were vaccinated with CHP‐HER2 plus GM‐CSF from the first cycle.

Discussion

In this trial, we found efficient HER2‐specific IgG antibody responses in 14 of 15 (93%) patients, in whom no specific antibodies were detected before the vaccination. The antibody titers increased over repeated vaccinations, and all reached the plateau levels within nine vaccinations. These findings indicate that the CHP‐HER2 vaccine activates HER2‐specific CD4⁺ T cells and help HER2‐specific B cells to elicit humoral immune responses, as expected in the previous preclinical animal study.^{( 1 )} IgG₁ was the dominant isotype of the IgG antibodies induced in the vaccinated patients, which suggests that the HER2‐specific CD4⁺ T‐cell immune response could be shifted to T helper type‐1. In one patient who received 26 cycles of vaccinations (Fig. 5), the IgG levels were well maintained without any serious adverse events. These results highlight the safety of the CHP‐HER2 vaccine when used to induce sustained specific humoral immunity even after repeated vaccinations.

Previous studies reported the presence of HER2‐specific antibodies in 20–27% patients with HER2‐expressing breast and other cancers.^{( 8} , ⁹ , ^{10 )} The antibodies were detected mainly in earlier disease stages rather than advanced ones.^{( 10 )} The reason for the lack of pre‐exiting IgG for HER2 protein in our 15 patients is not clear at present. It could be largely due to the use of 1–146 truncated HER2 protein, which is denatured in urea, as targets for the detection of specific‐HER2 antibody by ELISA.

Several studies have indicated that HER2‐specific antibody plays an important role in immune‐mediated cytotoxicity against HER2‐expressing tumor cells by specific T cells.^{( 11} , ¹² , ¹³ , ^{14 )} In the tolerant stage of HER2‐transgenic mice, the collaboration of both humoral and cellular immune responses against HER2 is required for the complete eradication of HER2‐expressing tumors.^{( 11 )} In another study of HER2‐expressing recombinant adenoviral vaccine designed to protect against tumorigenesis, induction of anti‐HER2 antibody was both necessary and sufficient for protection.^{( 12 )} It would be more important to investigate the functional significance of the antibodies that were induced in almost all of the patients vaccinated with CHP‐HER2 vaccine. Most antibodies were reactive with the 127–146 region of HER2 sequences, and failed to bind to HER2 antigen expressed on the cell surface.

It has been reported that the immune complex of tumor antigen NY‐ESO‐1 protein and its antibody is incorporated efficiently into dendritic cells and that the antigen is cross presented to MHC class I.^{( 15} , ^{16 )} Valmori et al. reported that in patients vaccinated with NY‐ESO‐1 antigen protein, antigen‐specific CD4⁺ cells and antibody responses were induced in the early phase with repeated vaccination. Induction of CD8⁺ T cells developed later, where the antibody was able to promote in vitro cross‐presentation of the antigen by their autologous dendritic cells.^{( 17 )} These indicate the importance of specific antibody induction for cross‐priming to occur in response to the immunization with tumor antigen vaccine. Efficient HER2‐specific antibody induction in CHP‐HER2‐vaccinated patients might facilitate induction of HER2‐specific CD8⁺ T cells, in addition to delivery of HER2 molecules to the MHC class I pathway in antigen‐presenting cells (APC), as we reported previously.^{( 1 )}

In our patients, GM‐CSF seems to have accelerated the immune responses. GM‐CSF is known to play a role in augmentation of antigen presentation by growth of APC, such as dendritic cells and macrophages.^{( 18} , ¹⁹ , ^{20 )} It has also been shown that GM‐CSF increases the immunogenicity of tumors in animal models.^{( 18} , ^{19 )} Transfection of the GM‐CSF gene into tumor cells and subcutaneous injection of them resulted in production of GM‐CSF by the tumor cells themselves together with enhanced immunogenicity.^{( 18} , ^{19 )} The increased immune response is presumed to be mediated by the maturation and function of APC. In rat models, serial administration of GM‐CSF augmented antigen‐specific antibody and cellular responses both for non‐self antigen and self antigen HER2 peptides.^{( 21 )} In a previous clinical trial, Jäger et al. were the first group to report that coadministration of GM‐CSF with melanoma‐antigen peptide vaccine enhanced peptide‐specific immune responses and supported cytotoxic T lymphocyte (CTL)‐mediated tumor rejection.^{( 22 )} Moreover, a randomized phase II trial compared the response to vaccination using gp100 and tyrosinase peptides in an emulsion with GM‐CSF to that of peptide‐pulsed monocyte‐derived dendritic cells.^{( 23 )} The study concluded a high frequency of CTL responses and clinical tumor regression in the subjects who received GM‐CSF.^{( 23 )} With regard to humoral responses, GM‐CSF is also an efficient adjuvant for seroconverter in hepatitis B virus vaccination.^{( 24 )} In cancer vaccines using recombinant carcinoembryonic antigen (CEA) protein, GM‐CSF significantly augments the IgG titers and is associated with increased survival in vaccinated colorectal cancer patients.^{( 25 )} In our six patients who were vaccinated with GM‐CSF from the first cycle, specific IgG became detectable after only two vaccinations. In contrast, in the other eight patients who received the vaccine alone for the first four cycles, the specific IgG appeared after three to seven vaccinations. The peak of the antibody titers was noted after three to four vaccinations in the CHP‐HER2 combined with GM‐CSF group, whereas four to nine vaccinations were required in the CHP‐HER2 vaccine group. These findings imply that GM‐CSF potentially accelerated the immune responses in patients who were vaccinated from the first cycle over the repeated vaccinations.

As the clinical trials presented were not designed to directly compare CHP‐HER2 vaccine to GM‐CSF coadministration, clinical trials should be planned to compare the adjuvant use of GM‐CSF with the vaccine alone in a CHP‐based protein vaccine setting and to evaluate antigen‐specific antibody responses and possible clinical benefits.

Finally, in the first study in humans, we demonstrated that HER2‐specific CD8⁺ and CD4⁺ T‐cell immune responses were detected in five out of nine patients who were vaccinated with the CHP‐HER2 vaccine.^{( 2 )} As the T cells are presumed to be difficult to detect directly without in vitro stimulation, IgG titer measured by ELISA could act as a surrogate biomarker, and such a titer can be assessed quantitatively in real time to monitor the magnitude of specific immunity in CHP‐HER2‐vaccinated patients.