Purpose: Passive immunotherapy with antitumor antibodies has the potential to induce active tumor immunity via the opsonic enhancement of immunogenicity of tumor antigen. We have assessed whether immune sensitization to the HER-2/neu tumor antigen occurs during treatment with the anti-HER-2/neu monoclonal antibody trastuzumab.

Experimental Design: Twenty-seven patients treated with trastuzumab and chemotherapy were assessed for the induction of HER-2/neu–specific immunity. Sera and peripheral blood mononuclear cells obtained before and after trastuzumab therapy were compared for the presence of anti-HER-2/neu endogenous Igλ antibodies and HER-2/neu–specific CD4 responses by ELISA and enzyme-linked immunospot, respectively.

Results: Anti-HER-2/neu antibodies were detectable in 8 of 27 (29%) patients before trastuzumab treatment and in 15 of 27 (56%) patients during trastuzumab treatment. In the overall study population, anti-HER-2/neu humoral responses significantly increased during therapy (P < 0.001) and were not associated with development of an anti-idiotypic response. In 10 evaluable individuals, 6 showed augmented HER-2/neu–specific CD4 T-cell responses during therapy. Of the 22 individuals treated for metastatic disease, those patients showing objective clinical responses exhibited more frequent (P = 0.004) and larger (P = 0.006) treatment-associated anti-HER-2/neu humoral responses.

Conclusion: Humoral immune sensitization occurs during treatment with chemotherapy and trastuzumab. Further studies are warranted to investigate whether augmented anti-HER-2/neu humoral and cellular immunity contributes mechanistically to clinical outcome.

Antitumor monoclonal antibodies (mAb) represent a major advance in the therapy of cancer. In the past decade, six “naked” antitumor mAbs have been approved and many others are in preclinical and clinical development. Although the clinical experience is still immature, the potency of this class of therapeutics as single agents in front-line therapy ranges from 15% to 50% (15). Combination therapies of antitumor antibodies and active cytotoxic agents are more effective and have increased median survival in CD20+ lymphoma (6, 7). In the treatment of HER-2+ breast cancer, the HER-2/neu mAb trastuzumab in combination with paclitaxel increases survival in the metastatic setting (8) and its addition to chemotherapy enhances disease-free survival in the adjuvant setting (9). Although multiple underlying synergistic mechanisms may contribute, the ability of epidermal growth factor receptor family member–targeted antibodies (1012) and small-molecule protein tyrosine kinase inhibitors (13, 14) to render tumor cells more susceptible to chemotherapy-induced cellular apoptosis has suggested that chemosensitization is central to clinical response (15). However, antitumor antibodies, as opposed to protein tyrosine kinase inhibitors, contain Fc domains that may also mechanistically contribute via the induction of an innate and/or adaptive antitumor immune response.

Recent genetic studies have provided strong evidence for the importance of the Fc domain in the efficacy of antitumor antibodies; in murine systems, Fcγ receptor (FcγR) engagement was required for efficacy of antitumor antibodies in several tumor antigen models, including HER-2 (16). Furthermore, four clinical studies have shown a positive correlation between the presence of favorable FcγR polymorphic alleles with higher affinities for IgG and improved clinical outcomes in rituximab-treated patients (1720). These studies have established that Fc-FcγR interactions are critical to antitumor antibody efficacy in the mouse and are correlative with clinical outcome in patients. Indeed, natural killer cells are recruited to tumor sites in patients during therapy with trastuzumab and chemotherapy but not with chemotherapy alone (21), providing supportive evidence for the potential involvement of antibody-dependent tumor cell cytotoxicity by FcγR-bearing effectors in situ.

In addition in their roles as opsonins, antitumor antibodies are predicted to enhance dendritic cell internalization and antigen presentation of tumor antigen via endocytosis and phagocytosis of tumor antigen–containing immune complexes and antibody-opsonized tumor target cells, respectively (22, 23). Although murine studies are supportive of the concept of immune-complex–mediated induction of tumor immunity (22, 24), evidence for the enhancement of immunity in antitumor antibody-treated patients is lacking. We hypothesized that tumor responses in patients treated with combination chemotherapy and trastuzumab would be accompanied by alterations in antitumor immunity and therefore have investigated the HER-2/neu immunologic response in these patients.

Study design. The study was reviewed and received prior approval by the institutional review boards of Columbia University Medical Center, University of California at Los Angeles School of Medicine, and the Mayo Clinic. Informed consent was obtained from nonpregnant adults with HER-2–positive solid tumors who were to receive trastuzumab with chemotherapy as clinically indicated. Thirty-five patients were recruited, of which 34 patients had adenocarcinoma of the breast and 1 patient had metastatic bladder cancer (Table 1). Eight patients did not complete study due to either death (three patients) or self-removal (five patients). Patients receiving paclitaxel were also administered dexamethasone as a premedication. Patients with signs of significant myelosuppression (i.e., absolute neutrophil count <1,000, absolute lymphocyte count <400, hemoglobin <8.0, platelets <90,000) were excluded. HER-2/neu overexpression was documented in 19 patients who were HercepTest 3+ (Dako Corp.) or HercepTest 2+ and confirmed by fluorescence in situ hybridization gene amplification (8 patients). Blood samples were collected before treatment and after ≥8 weeks of weekly treatment with trastuzumab. Sera were prepared from clotted tubes and stored at −80°C. Peripheral blood mononuclear cells (PBMC) were obtained from EDTA blood tubes using Hypaque-Ficoll centrifugation. Buffy coat samples were washed in PBS and counted before storage in liquid nitrogen in human AB serum/10% DMSO at 1 × 106/mL.

Table 1.

Patient characteristics

PatientTreatment regimenSites of diseaseTime to progressionSites of progression
Adjuvant patients 13 AC→T + H 2/11 + LN >3 y — 
 19 AC→T + H 14/16 + LN >3 y — 
 22 AC→T→H 2/9 + LN >3 y — 
 34 AC→T→H 8/22 + LN >1 y — 
 35 AC→T + H 4/9 + LN >2 y — 
Patients with objective responses Trastuzumab alone Bone marrow 4 y Mediastinal LN 
 Vinorelbine + trastuzumab Bone and ST 20 mo Brain 
 11 Paclitaxel + trastuzumab Lung, liver, and ST 15 mo Brain 
 12 Paclitaxel + trastuzumab Bone and LN 3 mo Brain 
 14 Vinorelbine + trastuzumab Mediastinal LN >3 y — 
 15 Paclitaxel + trastuzumab Liver, LN, bone, and brain >22 mo — 
 32 Vinorelbine + trastuzumab Liver, bone, and ST >22 mo — 
 33 Vinorelbine + trastuzumab LN and bone >2 y — 
Patients with progressive disease Paclitaxel + trastuzumab LN and lung 2 mo Lung 
 Paclitaxel + trastuzumab Bone, liver, and ST 2 mo Liver 
 10 Paclitaxel + trastuzumab LN 2 mo Lung and liver 
 18 Vinorelbine + trastuzumab Bone 2 mo Bone 
 21 Vinorelbine + trastuzumab Lung and ST 2 mo Lung 
 27 Vinorelbine + trastuzumab Bone and ST 2 mo Bone 
 28 Vinorelbine + trastuzumab Inflammatory breast 2 mo Breast 
 30 Vinorelbine + trastuzumab Lung 2 mo Lung 
Patients with mixed response or stable disease 2* Paclitaxel + trastuzumab Liver, LN, and bone 4 mo Bone 
 Paclitaxel + trastuzumab LN and lung 4 mo Lung 
 20 Vinorelbine + trastuzumab ST 6 mo ST 
 24 Vinorelbine + trastuzumab Bone, LN, and lung 5 mo Bronchial mass 
 29 Vinorelbine + trastuzumab Bone and lung 4 mo Brain 
 31 Vinorelbine + trastuzumab Liver, lung, and bone 3 mo Brain 
PatientTreatment regimenSites of diseaseTime to progressionSites of progression
Adjuvant patients 13 AC→T + H 2/11 + LN >3 y — 
 19 AC→T + H 14/16 + LN >3 y — 
 22 AC→T→H 2/9 + LN >3 y — 
 34 AC→T→H 8/22 + LN >1 y — 
 35 AC→T + H 4/9 + LN >2 y — 
Patients with objective responses Trastuzumab alone Bone marrow 4 y Mediastinal LN 
 Vinorelbine + trastuzumab Bone and ST 20 mo Brain 
 11 Paclitaxel + trastuzumab Lung, liver, and ST 15 mo Brain 
 12 Paclitaxel + trastuzumab Bone and LN 3 mo Brain 
 14 Vinorelbine + trastuzumab Mediastinal LN >3 y — 
 15 Paclitaxel + trastuzumab Liver, LN, bone, and brain >22 mo — 
 32 Vinorelbine + trastuzumab Liver, bone, and ST >22 mo — 
 33 Vinorelbine + trastuzumab LN and bone >2 y — 
Patients with progressive disease Paclitaxel + trastuzumab LN and lung 2 mo Lung 
 Paclitaxel + trastuzumab Bone, liver, and ST 2 mo Liver 
 10 Paclitaxel + trastuzumab LN 2 mo Lung and liver 
 18 Vinorelbine + trastuzumab Bone 2 mo Bone 
 21 Vinorelbine + trastuzumab Lung and ST 2 mo Lung 
 27 Vinorelbine + trastuzumab Bone and ST 2 mo Bone 
 28 Vinorelbine + trastuzumab Inflammatory breast 2 mo Breast 
 30 Vinorelbine + trastuzumab Lung 2 mo Lung 
Patients with mixed response or stable disease 2* Paclitaxel + trastuzumab Liver, LN, and bone 4 mo Bone 
 Paclitaxel + trastuzumab LN and lung 4 mo Lung 
 20 Vinorelbine + trastuzumab ST 6 mo ST 
 24 Vinorelbine + trastuzumab Bone, LN, and lung 5 mo Bronchial mass 
 29 Vinorelbine + trastuzumab Bone and lung 4 mo Brain 
 31 Vinorelbine + trastuzumab Liver, lung, and bone 3 mo Brain 

NOTE: Adjuvant patients were treated on study NCCTG N-9831 and received either four cycles of Adriamycin and cytoxan followed by either concurrent Taxol and trastuzumab or sequential Taxol and trastuzumab (AC→T + H or AC→T→H). All patients in the adjuvant setting had pathologic evidence of regional nodal spread with the number of positive nodes provided. Patients in the metastatic setting were treated with weekly trastuzumab concurrently with either paclitaxel or vinorelbine.

Abbreviations: LN, lymph node; ST, soft tissue.

*

All patients had primary or metastatic adenocarcinoma of the breast, except for patient 2, who had metastatic bladder cancer.

Clinical assessment. Patients with metastatic disease were restaged after 8 weeks of therapy, or sooner if clinical progression was suspected, as part of their routine clinical care. All sites of known metastatic disease were reassessed by physical exam, bone scan, computed tomography, positron emission tomography, or magnetic resonance imaging as clinically indicated. Objective responses, partial responses, mixed responses, stable disease, and progressive disease were defined according to WHO criteria (25, 26). None of the patients treated in the adjuvant setting showed evidence of clinical recurrence during the observation period.

HER-2 ELISAs. ELISA plates were coated with 5 μg/mL HER-2 extracellular domain protein in PBS. HER-2/neu protein was purified by trastuzumab affinity chromatography from culture supernatants of baby hamster kidney cell–produced extracellular domain (27). Control plates were coated with tetanus toxoid (10 plaque-forming units/mL; Aventis Pharmaceuticals) diluted 1:100 in PBS. Plates were blocked with either 0.5% pig gelatin (Sigma) or 1% bovine serum albumin (Sigma) in PBS for HER-2/neu–coated and tetanus-coated plates, respectively, before diluted serum samples were added at room temperature for 2 h. Plates were subsequently washed four times in PBS/0.05% Tween 20. Igλ antibodies were detected with 1 μg/mL biotinylated anti-human Igλ (BD PharMingen) for determination of patient-derived anti-HER-2 responses. Serum trastuzumab concentrations were determined with 1 μg/mL anti-human Igκ antibody (BD PharMingen). ELISAs were developed with streptavidin-horseradish peroxidase (diluted 1:10,000; Southern Biotech), and A450 values were compared with a trastuzumab standard curve.

All samples were assayed in triplicate and the data are presented as the relative A450 calculated as follows: [serum sample mean A450 anti-HER-2/neu (sample on HER-2/neu or tetanus antigen-coated wells) − mean A450 background (sample on uncoated/blocked wells)] / [mean A450 (anti-HER-2/neu–positive standard sera on HER-2/neu or tetanus-coated wells) − mean A450 (positive standard on uncoated blocked wells)]. Normalization in this manner to the internal standard reference of pooled patient sera from trastuzumab-treated patients was done on every ELISA plate and served to eliminate plate-to-plate variation. All serologic assays were repeated at least twice for each individual patient. A humoral response was considered positive by a relative A450 index of >0.2 or a titer <1/100.

Igκ depletion. To deplete serum of Igκ antibodies, 850 μL of 1:25 diluted sera were applied in PBS to 1 mL of anti-Igκ beads [0.5 mg anti-Igκ mAb coupled to N-hydroxysuccinimide–activated agarose beads (Amersham)]. After 30 min, the unbound fraction was applied two subsequent times to eluted, regenerated anti-Igκ agarose beads. Each round of depletion resulted in >90% quantitative reduction in Igκ levels as determined by ELISA. κ-Depleted serum and nondepleted serum dilutions were equalized for total protein concentration based on results of Bradford assays. Antibody titers after κ depletion were assayed in the HER-2/neu and tetanus ELISAs as above.

Anti-idiotypic trastuzumab capture ELISAs. ELISA plates coated with 1.5 μg/mL trastuzumab in PBS were blocked with 0.5% pig gelatin in PBS. Diluted sera or goat anti-human F(ab′)2 IgG (standard) was incubated with trastuzumab-coated plates for 2 h at room temperature followed by washing in PBS/0.05% Tween 20. Trastuzumab-bound IgG was detected with addition of 250 ng/mL of biotinylated trastuzumab, washed, and then developed with streptavidin-horseradish peroxidase.

FcγR polymorph determination. FcγRIIA and FcγRIIIA polymorphic allele status was determined by genomic PCR approaches as described by Wu et al. (28), with two minor modifications; the FcγRIIIA PCRs were done at annealing temperatures of 58°C and 60°C for the T and G reaction, respectively.

HER-2/neu–specific CD4 IFN-γ enzyme-linked immunospots. Twelve HER-2/neu peptides, each known to bind to multiple HLA-DR molecules (29), were used to detect T-cell responses by the enzyme-linked immunospot (ELIspot) method (30, 31). Four of the HER-2/neu helper peptides, p98, p369, p927, and p776, have been previously described in detail (32, 33). The remaining seven peptides (all 15-mers), designated by the position of the first amino acid, p62, p77, p83, p88, p350, p783, and p976, are recently identified epitopes that exhibit high-affinity binding to a variety of HLA class II molecules.7

7

K. Knutson, unpublished observations.

Both phorbol 12-myristate 13-acetate/ionomycin and pooled cytomegalovirus, EBV, and Flu viral peptides (CEF) were used as positive controls. In brief, cryopreserved PBMCs were cultured at 2 × 105 per well in 96-well plates for 7 days in medium containing individual HER-2/neu class II peptides (each at 10 μg/mL) or in the absence of any antigens (no-antigen control wells). In some cases where patient material was lacking, the number of peptides was reduced to nine peptides to accommodate. In these cases, all of the time points were assessed with the same panels of peptides. Interleukin-2 (10 units/mL) was added at day 5, and on day 7, peptide and 2 × 105 per well irradiated autologous PBMCs were added as antigen-presenting cells. On day 8, the cells were gently transferred to the ELIspot plate for detection of spots (Mabtech AB). ELIspots were developed, dried, and read with an AID Immunospot ELIspot reader as previously described (30). Peptide-specific immune reactivity was determined by subtracting the background spots in the no-antigen–containing wells. A positive response was defined as the peptide-specific spots that were statistically higher (triplicates) than control wells using a two-tailed t test (P < 0.05). A zero response was assigned if the peptide-specific wells were not different than control (i.e., no peptide) wells. The counts for each peptide were summed and presented as the total HER-2/neu–specific T cells assessed at each time point. Note that, although the peptides are known to bind to multiple HLA-DR alleles, it is difficult to rule out that they could contain embedded peptides that could stimulate CD8 T cells; however, as previously reported, HER-2/neu–specific CD8 T-cell responses are typically lower by at least one order of magnitude even in vaccinated patients (30). Changes between preimmune and postimmune responses were considered positive if there was at least a doubling for increases and a halving for decreases.

Statistical analysis. The antibody response of a single patient (ON:33) deviated significantly from the norm and was identified, by box plot of change in λ anti-HER-2 (post-pre) response, as an outlier. This patient exhibited the highest preexisting anti-HER-2/neu binding activity in the cohort, which remained positive during therapy, but, in contrast to all other 26 individuals studied, decreased in absolute magnitude. Accordingly, P values are provided for statistical analysis that either includes these outlier data or, as indicated (§), excludes this outlier.

Anti-HER-2/neu humoral immunity is induced during trastuzumab therapy. To assess whether immunologic responses to HER-2/neu were altered during therapy with chemotherapy and trastuzumab, patients were enrolled in an observational study before initiation of therapy. Twenty-seven patients completed the study and provided pretreatment and 8-week posttreatment sera and PBMCS for collection and storage. Of these 27 subjects, 26 were breast cancer patients and 1 patient was treated for HER-2/neu–positive metastatic bladder cancer (Table 1). Of the 26 breast cancer patients, 21 (81%) were treated in the stage IV setting with weekly trastuzumab and either vinorelbine (13 patients), paclitaxel (8 patients) or no chemotherapy (1 patient) and 5 (19%) individuals were treated in the high-risk adjuvant setting on protocol NCCTG N-9831 (9) and received either paclitaxel followed by trastuzumab or concurrent paclitaxel and trastuzumab.

Sera were analyzed by ELISA for evidence of a humoral response to HER-2/neu. For these ELISA-based assays, plates were coated with tetanus toxoid as a control antigen or with human HER-2/neu extracellular domain protein. Although there was a range of anti-tetanus Ig activity between patients, activities of individual patients did not vary during therapy, indicating that overall specific Ig levels were not influenced by treatment. In contrast, anti-HER-2/neu Igλ responses were induced in several patients during therapy (Fig. 1; Table 1). The Ig response of the λ subclass was specifically addressed to prevent the spurious detection of trastuzumab, a human IgG1 κ mAb present at high concentrations in the sera of treated patients. As previously recognized, anti-HER-2/neu antibody responses were detected in 8 of 27 (29%) patients before the initiation of trastuzumab therapy, consistent with preexisting immune recognition of HER-2/neu (34). During therapy, anti-HER-2/neu Igλ responses were detectable in 15 of 27 (56%) patients. Anti-HER-2/neu responses increased across the entire population of 27 patients after 8 weeks of therapy (P = 0.002 and P < 0.001§), with increases evident in 12 of 27 (44%) treated individuals (Table 2).

Fig. 1.

Patients develop increased anti-HER-2/neu Igλ responses during trastuzumab therapy. ELISA analysis was done using patient sera before trastuzumab (pre) and compared with sera obtained after 8 wks of treatment. A, Igλ anti-tetanus ELISA. Detectable levels were observed in most patients that were not significantly different in pretreatment versus posttreatment samples. B, Igλ anti-HER-2/neu ELISA. Posttreatment HER-2–specific humoral responses were significantly increased across the study subjects (P = 0.002 and P < 0.001§). C, anti-HER-2 antibody responses are sustained. HER-2 ELISAs were done at early (12 wks) and at late time points (after ≥20 wks of treatment) on 10 patients exhibiting treatment-associated anti-HER-2 responses. Anti-HER-2 levels were not significantly different at early and late time points.

Fig. 1.

Patients develop increased anti-HER-2/neu Igλ responses during trastuzumab therapy. ELISA analysis was done using patient sera before trastuzumab (pre) and compared with sera obtained after 8 wks of treatment. A, Igλ anti-tetanus ELISA. Detectable levels were observed in most patients that were not significantly different in pretreatment versus posttreatment samples. B, Igλ anti-HER-2/neu ELISA. Posttreatment HER-2–specific humoral responses were significantly increased across the study subjects (P = 0.002 and P < 0.001§). C, anti-HER-2 antibody responses are sustained. HER-2 ELISAs were done at early (12 wks) and at late time points (after ≥20 wks of treatment) on 10 patients exhibiting treatment-associated anti-HER-2 responses. Anti-HER-2 levels were not significantly different at early and late time points.

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Table 2.

Anti-HER-2/neu CD4 frequency and Igλ-binding activities and titers

PatientHER-2 statusFcγR haplotype
Anti-HER-2/neu CD4 cells per 106 PBMCs
HER-2 Igλ-binding activity
Anti-HER-2 Igλ titer
Antibody response/T-cell response
FcγRIIA/FcγRIIIAPrePostPrePostPrePost
Adjuvant patients 13 3+ HR/VV ND ND 0.24 0.70 1/172 1/163 Yes 
 19 3+ RR/FF <10 10,348 0.41 0.67 1/251 1/468 Yes/yes 
 22 3+ HR/VV <10 14,704 0.24 0.52 1/142 1/1,058 Yes/yes 
 34 2+ HH/FF ND ND Und. Und. No 
 35 2+ HR/FF ND ND 0.36 Und. 1/335 Yes 
 Enhanced CD4 responses    2/2   Ab response rate  80% 
Patients with objective responses 3+ RR/VF ND ND 0.64 Und. 1/544 Yes 
 3+ HR/VV <10 555 0.47 Und. 1/260 Yes/yes 
 11 3+ HR/VF <10 615 0.03 0.44 Und. 1/219 Yes/yes 
 12 2+ ND ND ND Und. Und. No 
 14 3+ RR/VF <10 435 0.03 0.32 Und. 1/213 Yes/yes 
 15 3+ HH/VV ND ND 0.53 1.16 1/1,525 1/1,167 Yes 
 32 3+ RR/VF ND ND 0.25 Und. 1/244 Yes 
 33 3+ HR/FF 13,137 14,333 1.03 0.31 1/432 1/262 No/no 
 Enhanced CD4 responses    3/4   Ab response rate  75% 
Patients with progressive disease 3+ HH/VF 1,553 1,195 0.49 0.55 1/250 1/243 No/no 
 2+ HR/FF ND ND 0.23 Und. 1/163 Yes 
 10 3+ HR/VV ND ND Und. Und. No 
 18 3+ HR/VF ND ND 0.02 0.14 Und. 1/61 No 
 21 2+ HH/FF 1,040 <10 Und. Und. No/no 
 27 2+ HR/FF ND ND 0.04 Und. 1/77 No 
 28 3+ HH/VF ND ND 0.03 Und. Und. No 
 30 3+ HR/FF 1,195 265 0.30 0.18 1/231 1/103 No/no 
 Enhanced CD4 responses    0/3   Ab response rate  13% 
Patients with mixed response or stable disease 3+ HH/VF ND ND 0.56 Und. 1/550 Yes 
 2+ RR/VV ND ND Und. Und. No 
 20 3+ HR/FF ND ND 0.05 Und. Und. No 
 24 2+ HH/VV <10 600 0.13 Und. 1/90 No/yes 
 29 3+ HR/FF ND ND 0.23 0.25 1/419 1/156 No 
 31 3+ HR/VV ND ND 0.16 1/98 Und. No 
 Enhanced CD4 responses    1/1   Ab response rate  17% 
PatientHER-2 statusFcγR haplotype
Anti-HER-2/neu CD4 cells per 106 PBMCs
HER-2 Igλ-binding activity
Anti-HER-2 Igλ titer
Antibody response/T-cell response
FcγRIIA/FcγRIIIAPrePostPrePostPrePost
Adjuvant patients 13 3+ HR/VV ND ND 0.24 0.70 1/172 1/163 Yes 
 19 3+ RR/FF <10 10,348 0.41 0.67 1/251 1/468 Yes/yes 
 22 3+ HR/VV <10 14,704 0.24 0.52 1/142 1/1,058 Yes/yes 
 34 2+ HH/FF ND ND Und. Und. No 
 35 2+ HR/FF ND ND 0.36 Und. 1/335 Yes 
 Enhanced CD4 responses    2/2   Ab response rate  80% 
Patients with objective responses 3+ RR/VF ND ND 0.64 Und. 1/544 Yes 
 3+ HR/VV <10 555 0.47 Und. 1/260 Yes/yes 
 11 3+ HR/VF <10 615 0.03 0.44 Und. 1/219 Yes/yes 
 12 2+ ND ND ND Und. Und. No 
 14 3+ RR/VF <10 435 0.03 0.32 Und. 1/213 Yes/yes 
 15 3+ HH/VV ND ND 0.53 1.16 1/1,525 1/1,167 Yes 
 32 3+ RR/VF ND ND 0.25 Und. 1/244 Yes 
 33 3+ HR/FF 13,137 14,333 1.03 0.31 1/432 1/262 No/no 
 Enhanced CD4 responses    3/4   Ab response rate  75% 
Patients with progressive disease 3+ HH/VF 1,553 1,195 0.49 0.55 1/250 1/243 No/no 
 2+ HR/FF ND ND 0.23 Und. 1/163 Yes 
 10 3+ HR/VV ND ND Und. Und. No 
 18 3+ HR/VF ND ND 0.02 0.14 Und. 1/61 No 
 21 2+ HH/FF 1,040 <10 Und. Und. No/no 
 27 2+ HR/FF ND ND 0.04 Und. 1/77 No 
 28 3+ HH/VF ND ND 0.03 Und. Und. No 
 30 3+ HR/FF 1,195 265 0.30 0.18 1/231 1/103 No/no 
 Enhanced CD4 responses    0/3   Ab response rate  13% 
Patients with mixed response or stable disease 3+ HH/VF ND ND 0.56 Und. 1/550 Yes 
 2+ RR/VV ND ND Und. Und. No 
 20 3+ HR/FF ND ND 0.05 Und. Und. No 
 24 2+ HH/VV <10 600 0.13 Und. 1/90 No/yes 
 29 3+ HR/FF ND ND 0.23 0.25 1/419 1/156 No 
 31 3+ HR/VV ND ND 0.16 1/98 Und. No 
 Enhanced CD4 responses    1/1   Ab response rate  17% 

NOTE: Anti-HER-2/neu CD4 frequency and Igλ-binding activities and titers of individuals listed according to clinical category. A positive treatment-related antibody response was assigned in the last column as defined by an increase of binding activity of >0.2 between pretreatment and posttreatment samples. Note that all antibody and T-cell responses are concordant, except in the case of patient 24, in whom the antibody response was weak (<0.2 change in binding activity). Posttreatment CD4 frequencies are ≥15 wks after initiation of trastuzumab, except for patient 4, whose posttreatment CD4 frequency is provided at 10 wks. Anti-HER-2/neu Igλ responses occurred more frequently in the objective response group (P = 0.002§ and 0.004, χ2 analysis).

Abbreviations: ND, not determined; Und., undetected; Ab, antibody.

Preexistent humoral immunity was more frequently observed (P < 0.05, χ2 test) in HercepTest 3+ patients (8 of 19 patients) than in HercepTest 2+ patients (0 of 8 patients), but there was no evidence for an association of preexistent humoral immunity and clinical outcome (Table 2). Most patients lacked detectable endogenous HER-2/neu Igλ antibodies before initiation of trastuzumab, and in this group, 9 of 19 developed detectable endogenous anti-HER-2/neu Igλ antibodies. Of the eight patients with circulating anti-HER-2/neu antibodies before receiving trastuzumab, the binding activities increased in three and were unchanged or decreased in the other six individuals. Overall, preexistent anti-HER-2 levels were not related to posttreatment anti-HER-2 antibody levels (Pearson r = 0.44).

To examine whether humoral responses were durable, anti-HER-2 levels were examined at later time points in 10 of the patients that exhibited increases in anti-HER-2 Igλ responses during initial therapy. Mean anti-HER-2 levels were not significantly different across the population at early and late time points (Fig. 1B). Individually, eight of the 10 patients showed persistent humoral immunity after more than 20 weeks of ongoing trastuzumab therapy, indicating that humoral immunity was sustained in most patients (data not shown).

Induced humoral immunity correlates with favorable clinical response. Although limited by the statistical power of this small patient population size, we have addressed whether induction of endogenous humoral responses during treatment occurred more frequently or was of greater magnitude in patients who responded clinically (Fig. 2; Tables 2 and 3). When restaged after 2 months of treatment, 8 of the 22 (36%) patients with metastatic disease exhibited an objective clinical response, 6 (27%) had stable disease or a mixed response, and 8 (36%) exhibited progressive disease. Based on a one-way ANOVA model, there was a significantly greater increase of Igλ anti-HER-2/neu in the objective response group than in the combined mixed response, partial response, and progressive disease group (P = 0.004 and 0.006§), and conversely, there was a marginally significantly smaller increase of Igλ anti-HER-2/neu in the progressive disease group than in the combined mixed response and objective response group (P = 0.056). Similarly, the frequency of occurrence of antibody responses in individuals was related statistically to clinical outcome. Among the clinical groups, treatment-associated increases in anti-HER-2/neu Igλ immunity occurred frequently in patients exhibiting objective responses (6 of 8 objective response patients, 75%) and significantly more often than in patients that did not show clinical objective responses (progressive disease, mixed response, and partial response groups) in whom rises in anti-HER-2/neu Ig responses occurred in just 2 of 14 (14%) patients [P = 0.004, χ2 test (P = 0.002§)]. Thus, induction of an endogenous anti-HER-2/neu humoral response during therapy with trastuzumab and chemotherapy is associated with clinical response. Administration of chemotherapy was not required for the induction of humoral immunity because enhanced anti-HER-2 Igλ levels were seen in two of the three patients that received trastuzumab without concomitant chemotherapy (patients 1, 22, and 34).

Fig. 2.

Anti-HER-2/neu humoral responses according to clinical category.

Fig. 2.

Anti-HER-2/neu humoral responses according to clinical category.

Close modal
Table 3.

Mean changes (pre-post) in Igλ anti-HER-2/neu activity in all patients and by clinical category

VariableMean ± SDP
Anti-tetanus   
    All patients (n = 27) 0.019 ± 0.124 0.485 
Anti-HER-2   
    All patients (n = 27) 0.210 ± 0.310 0.002 
 0.245 ± 0.252* <0.001* 
        OR (n = 8) 0.310 ± 0.465 0.004 
 0.457 ± 0.225* <0.001* 
        MR/SD (n = 6) 0.154 ± 0.291 NS 
        POD (n = 8) 0.100 ± 0.165 0.056 
        ADJ (n = 5) 0.281 ± 0.152 NS 
VariableMean ± SDP
Anti-tetanus   
    All patients (n = 27) 0.019 ± 0.124 0.485 
Anti-HER-2   
    All patients (n = 27) 0.210 ± 0.310 0.002 
 0.245 ± 0.252* <0.001* 
        OR (n = 8) 0.310 ± 0.465 0.004 
 0.457 ± 0.225* <0.001* 
        MR/SD (n = 6) 0.154 ± 0.291 NS 
        POD (n = 8) 0.100 ± 0.165 0.056 
        ADJ (n = 5) 0.281 ± 0.152 NS 

NOTE: Average increases (post-pre) of endogenous anti-HER-2 Igλ responses in all patients and according to clinical category. Anti-HER-2/neu Igλ responses significantly increased during treatment across the entire treated population (P = 0.002 and P < 0.001§). Based on a one-way ANOVA model, there is a significantly greater increase of Igλ anti-HER-2/neu in the objective response group than either the mixed response or the progressive disease group (P = 0.004 and 0.006§), and there is a marginally significantly smaller increase of Igλ anti-HER-2/neu in the progressive disease group than either the mixed response or the objective response group (P = 0.056). However, the difference in the increase of Igλ anti-HER-2/neu between the adjuvant and other three groups was not significant (P = 0.735).

Abbreviations: MR, mixed response; SD, stable disease; POD, progressive disease; OR, objective response; ADJ, adjuvant.

*

Outlier removed.

It has been noted that clinical responses occur more frequently in rituximab patients harboring either the FcγRIII V158 or the FcγRIIA R131 alleles (17, 18). Correlative studies in trastuzumab-treated patients have yet to be reported. FcγRIIA and FcγRIIIA polymorphic allelic status in this group of 27 trastuzumab-treated patients is reported in Table 1. Conclusions are limited, however, by the small sample size of the studied population about the association of the FcγRIIIA V158 or FcγRIIA R131 alleles with either antibody or clinical response.

Endogenous anti-HER-2/neu Igλ responses do not correlate with serum trastuzumab levels or the appearance of idiotypic antibodies. The presence of trastuzumab in sera obscured the specific detection of endogenous anti-HER-2/neu IgG/κ with available human IgG secondary reagents. Thus, to confirm the specificity of the anti-λ secondaries and to rule out the potential for this artifactual contribution of trastuzumab, serologic levels of trastuzumab were determined in all treated patient samples. Had trastuzumab contributed to the detection of Igλ antibodies, one would have expected a correlation between serum trastuzumab levels and anti-HER-2/neu Igλ levels. Importantly, serum trastuzumab levels and anti-HER-2/neu Igλ titers did not correlate significantly (Pearson r = 0.3806; Fig. 3A), discounting this potential source of artifact.

Fig. 3.

Anti-HER-2/neu Igλ reactivity is not due to the presence of trastuzumab. A, lack of correlation between trastuzumab serum concentration and Igλ anti-HER-2/neu levels. Trastuzumab concentrations in posttreatment sera were plotted against Igλ anti-HER-2/neu levels from the same sera. Pearson correlation coefficient is shown. B, idiotypic anti-trastuzumab antibody responses are undetectable. Posttreatment sera were assessed by anti-trastuzumab sandwich ELISA. No anti-idiotypic antibodies were detected. A standard curve generated by the capture of goat anti-human Ig indicated the assay sensitivity at 20 ng/mL. C, Igκ depletion does not reduce Igλ anti-HER-2/neu levels. Posttreatment serum was depleted of trastuzumab using multiple anti-κ columns. Trastuzumab concentrations were reduced by 99.9% (data not shown). In contrast, Igλ anti-HER-2/neu concentrations were not reduced.

Fig. 3.

Anti-HER-2/neu Igλ reactivity is not due to the presence of trastuzumab. A, lack of correlation between trastuzumab serum concentration and Igλ anti-HER-2/neu levels. Trastuzumab concentrations in posttreatment sera were plotted against Igλ anti-HER-2/neu levels from the same sera. Pearson correlation coefficient is shown. B, idiotypic anti-trastuzumab antibody responses are undetectable. Posttreatment sera were assessed by anti-trastuzumab sandwich ELISA. No anti-idiotypic antibodies were detected. A standard curve generated by the capture of goat anti-human Ig indicated the assay sensitivity at 20 ng/mL. C, Igκ depletion does not reduce Igλ anti-HER-2/neu levels. Posttreatment serum was depleted of trastuzumab using multiple anti-κ columns. Trastuzumab concentrations were reduced by 99.9% (data not shown). In contrast, Igλ anti-HER-2/neu concentrations were not reduced.

Close modal

Further studies were done to eliminate the possibility that the ELISA detection of HER-2/neu–specific λ antibodies was due to indirect binding to HER-2/neu through trastuzumab, as might be expected, for instance, by the potential presence of idiotypic anti-trastuzumab antibodies. Therefore, the presence of high-titered anti-idiotypics was ruled out in all patients: no anti-idiotypic antibodies were detected in any patient using trastuzumab-coated plates in an assay whose sensitivity for detection was 20 ng/mL (Fig. 3B). In a second direct experimental approach, trastuzumab antibodies were removed from patient sera by three successive rounds of Igκ depletion, by anti-κ affinity chromatography. κ-Depleted sera were then reassayed for Igλ anti-HER-2/neu reactivity, which remained unchanged despite a >99% reduction in Igκ anti-HER-2/neu reactivity (data not shown; Fig. 3C). Thus, detection of anti-HER-2/neu antibodies is not confounded by the presence of trastuzumab antibodies in the sera.

Treatment with trastuzumab and chemotherapy augments HER-2/neu–specific CD4 T-cell immunity. PBMC samples were available for testing for pretreatment and at least one posttreatment CD4 T-cell responses from nine individuals. Eight early (i.e., <15 weeks) posttreatment samples showed that 50% of patients developed elevated HER-2–specific CD4 T cells early in the course of treatment (Fig. 4A). Nine late (i.e., ≥15 weeks) posttreatment samples showed that 78% of patients had elevated CD4 T cells (Fig. 4B). Figure 3C shows persistent immunity in four patients, all of whom showed clinical benefit. Overall, there was remarkable concordance between the enhancement of HER-2–specific antibody responses and the development of augmented CD4 T-cell immunity, consistent with the development of a T-dependent humoral response (Table 1). In the two patients evaluated in the adjuvant setting (patients 19 and 22), CD4 T-cell responses were of the greatest magnitude and were sustained for at least 4 months (Fig. 4C). HER-2–specific CD4 T-cell responses were detectable in all four evaluated patients who showed objective clinical response (patients 3, 11, 14, and 33). In contrast, of the four patients in whom objective responses were not evident (patients 4, 21, 24, and 30), only one showed a treatment-associated increase in HER-2/neu–specific CD4 frequency. Responses against the individual peptides are shown in Table 4. Three of the patients exhibited broad reactivity to several HER-2 epitopes.

Fig. 4.

Treatment with trastuzumab and chemotherapy augments HER-2/neu–specific CD4 immunity. A, pretreatment and early (≤15 wks) posttreatment HER-2/neu–specific CD4 T-cell levels in seven patients with evaluable T cells at both time points. Each point is the total CD4 T-cell frequency (per million PBMCs) calculated from ELIspots of HER-2/neu–derived MHC II binding peptides. Each peptide-specific cell precursor frequency was statistically significant (P < 0.05) from control wells. Inset arrow and fraction, number of patients that showed elevated HER-2/neu–specific immunity. All lines are labeled with the patient number. Patient 11 showed neither pretreatment nor posttreatment HER-2/neu–specific immunity. B, similar to (A), except that it compares late (15-30 wks) posttreatment HER-2/neu–specific CD4 T levels with pretreatment levels in nine evaluable patients. Seven of nine patients showed increased T cells. These data were repeated with similar results in a second set of restimulation/ELIspot assays done on 7 of the 10 patients for whom PBMCs were available for replicate testing. C, longitudinal responses from four patients that were assessed at multiple time points showing persisting HER-2/neu–specific T-cell immunity. D and E, columns, mean of spots (per million PBMC) specific for phorbol 12-myristate 13-acetate/ionomycin (PMA/IONO) or the CEF peptide pool, respectively, in the pretreatment (Pre) and posttreatment (Post) samples; bars, SE. The HLA-DQ and HLA-DR haplotypes of the patients exhibiting treatment-associated anti-HER-2 CD4 responses are as follows: patient 3, DQ5, DQ5/DR10, and DR16; patient 11, DQ5, DQ6/DR15, and DR16; patient 14, DQ2, DQ7/DR12, and DR17; patient 19, DQ5, DQ6/DR10, and DR15; patient 22, DQ6, DQ8/DR4, and DR13; and patient 24, DQ6, DQ6/DR13, and DR15.

Fig. 4.

Treatment with trastuzumab and chemotherapy augments HER-2/neu–specific CD4 immunity. A, pretreatment and early (≤15 wks) posttreatment HER-2/neu–specific CD4 T-cell levels in seven patients with evaluable T cells at both time points. Each point is the total CD4 T-cell frequency (per million PBMCs) calculated from ELIspots of HER-2/neu–derived MHC II binding peptides. Each peptide-specific cell precursor frequency was statistically significant (P < 0.05) from control wells. Inset arrow and fraction, number of patients that showed elevated HER-2/neu–specific immunity. All lines are labeled with the patient number. Patient 11 showed neither pretreatment nor posttreatment HER-2/neu–specific immunity. B, similar to (A), except that it compares late (15-30 wks) posttreatment HER-2/neu–specific CD4 T levels with pretreatment levels in nine evaluable patients. Seven of nine patients showed increased T cells. These data were repeated with similar results in a second set of restimulation/ELIspot assays done on 7 of the 10 patients for whom PBMCs were available for replicate testing. C, longitudinal responses from four patients that were assessed at multiple time points showing persisting HER-2/neu–specific T-cell immunity. D and E, columns, mean of spots (per million PBMC) specific for phorbol 12-myristate 13-acetate/ionomycin (PMA/IONO) or the CEF peptide pool, respectively, in the pretreatment (Pre) and posttreatment (Post) samples; bars, SE. The HLA-DQ and HLA-DR haplotypes of the patients exhibiting treatment-associated anti-HER-2 CD4 responses are as follows: patient 3, DQ5, DQ5/DR10, and DR16; patient 11, DQ5, DQ6/DR15, and DR16; patient 14, DQ2, DQ7/DR12, and DR17; patient 19, DQ5, DQ6/DR10, and DR15; patient 22, DQ6, DQ8/DR4, and DR13; and patient 24, DQ6, DQ6/DR13, and DR15.

Close modal
Table 4.

Frequency of HER-2/neu epitope-specific T cells

PeptidePatient 3
Patient 4
Patient 11
PreEarlyLatePreEarlyLatePreEarlyLate
H2 100 ND ND 
p776 ND ND 
H3 ND ND 
p369 ND ND ND 420 ND ND 
H4 180 1,050 ND ND 
p927 ND ND ND 560 ND ND 
H6 ND ND 
p98 ND ND ND ND ND 
H10 120 ND ND 
H11 273 45 ND ND 
H14 ND ND 
H15 717 555 ND ND 615 
Totals 717 555 1,553 1,195 ND ND 615 
          
Peptide
 
Patient 14
 
  Patient 19
 
  Patient 21
 
  
 Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
H2 265 1,363 270 ND 
p776 395 190 ND 
H3 ND 
p369 170 ND ND ND ND 
H4 1,790 1,748 285 ND 
p927 ND ND ND ND 
H6 1,446 1,928 ND 
p98 ND ND ND ND 
H10 1,373 713 ND 
H11 2,875 2,548 ND 
H14 2,163 ND 
H15 1,501 295 ND 
Totals 435 10,348 9,495 1,040 ND 
          
Peptide
 
Patient 22
 
  Patient 23
 
  Patient 24
 
  
 Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
H2 1,787 2,018 
p776 1,260 
H3 992 1,797 1,330 
p369 ND ND ND ND ND ND 
H4 1,618 390 
p927 ND ND ND ND ND ND 
H6 2,590 658 
p98 ND ND ND ND ND ND 
H10 103 1,980 103 
H11 631 1,220 
H14 2,220 891 
H15 3,085 ND 
Totals 103 6,495 14,703 390 103 1,988 891 
          
Peptide Patient 30
 
  Patient 33
 
  
 Pre Early Late Pre Early Late 
H2 345 ND 110 1,388 2,326 
p776 ND 2,003 478 1,995 
H3 25 ND 1,135 433 1,755 
p369 ND ND ND ND 
H4 295 ND 1,801 648 1,398 
p927 ND ND ND ND 
H6 70 ND 155 1,245 1,748 
p98 95 ND ND ND ND 
H10 175 ND 1,400 1,016 2,065 
H11 85 ND 1,038 553 1,856 
H14 25 ND 1,680 695 1,612 
H15 80 ND 1,447 ND 1,652 
Totals 1,195 ND 265 13,137 3,823 16,407 
PeptidePatient 3
Patient 4
Patient 11
PreEarlyLatePreEarlyLatePreEarlyLate
H2 100 ND ND 
p776 ND ND 
H3 ND ND 
p369 ND ND ND 420 ND ND 
H4 180 1,050 ND ND 
p927 ND ND ND 560 ND ND 
H6 ND ND 
p98 ND ND ND ND ND 
H10 120 ND ND 
H11 273 45 ND ND 
H14 ND ND 
H15 717 555 ND ND 615 
Totals 717 555 1,553 1,195 ND ND 615 
          
Peptide
 
Patient 14
 
  Patient 19
 
  Patient 21
 
  
 Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
H2 265 1,363 270 ND 
p776 395 190 ND 
H3 ND 
p369 170 ND ND ND ND 
H4 1,790 1,748 285 ND 
p927 ND ND ND ND 
H6 1,446 1,928 ND 
p98 ND ND ND ND 
H10 1,373 713 ND 
H11 2,875 2,548 ND 
H14 2,163 ND 
H15 1,501 295 ND 
Totals 435 10,348 9,495 1,040 ND 
          
Peptide
 
Patient 22
 
  Patient 23
 
  Patient 24
 
  
 Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
Pre
 
Early
 
Late
 
H2 1,787 2,018 
p776 1,260 
H3 992 1,797 1,330 
p369 ND ND ND ND ND ND 
H4 1,618 390 
p927 ND ND ND ND ND ND 
H6 2,590 658 
p98 ND ND ND ND ND ND 
H10 103 1,980 103 
H11 631 1,220 
H14 2,220 891 
H15 3,085 ND 
Totals 103 6,495 14,703 390 103 1,988 891 
          
Peptide Patient 30
 
  Patient 33
 
  
 Pre Early Late Pre Early Late 
H2 345 ND 110 1,388 2,326 
p776 ND 2,003 478 1,995 
H3 25 ND 1,135 433 1,755 
p369 ND ND ND ND 
H4 295 ND 1,801 648 1,398 
p927 ND ND ND ND 
H6 70 ND 155 1,245 1,748 
p98 95 ND ND ND ND 
H10 175 ND 1,400 1,016 2,065 
H11 85 ND 1,038 553 1,856 
H14 25 ND 1,680 695 1,612 
H15 80 ND 1,447 ND 1,652 
Totals 1,195 ND 265 13,137 3,823 16,407 

NOTE: Shown are the frequencies of T cells to specific HER-2/neu–derived epitopes at pretreatment and at early (<15 wks) and late (≥15 wks) time points. Values are expressed as the number of cells per million PBMCs, calculated from triplicate determinations. All values are statistically higher than no-antigen–containing wells.

Responses to phorbol 12-myristate 13-acetate/ionomycin and the CEF peptide pool were compared between pretreatment and posttreatment samples to determine if there were differences in responses to noncancer stimuli. As shown in Fig. 4D, the responses to the control stimuli were not different between the two populations. The mean number of spots per million PBMC for the pretreatment samples in the phorbol 12-myristate 13-acetate/ionomycin–containing wells was 2,177 ± 634 (mean ± SE; n = 11), which was not statistically different than the response in the posttreatment samples (3,056 ± 323; n = 21; P = 0.18). The mean number of spots per million PBMC for the pretreatment donors in the CEF peptide–containing (Fig. 4E) wells was 424 ± 223, which also was not statistically different than the response in the posttreatment samples (95 ± 73; P = 0.1).

We provide evidence for the induction of a humoral and cellular HER-2/neu–specific immune response during the treatment of HER-2/neu–overexpressing malignancies with trastuzumab and chemotherapy in the high-risk adjuvant breast cancer and stage IV setting. Enhanced endogenous humoral responses were seen in 44% of treated patients and, interestingly, were of greater magnitude and more frequently observed in clinically responding patients. This association of endogenous anti-HER-2/neu humoral immunity with favorable clinical outcomes may simply be a marker of clinical response, possibly the consequence of immunomodulatory effects of enhanced tumor cell apoptosis/necrosis occurring in clinically responding patients receiving trastuzumab and chemotherapy. Alternatively, the association could be causal and indicate that augmented tumor immunity contributes to the efficacy of trastuzumab and chemotherapy.

There is substantial preclinical experimental support for the idea that antitumor antibodies in their roles as opsonins can promote the immunogenicity of both human and murine tumor antigens. Immunization of mice with dendritic cells pulsed with antibody-opsonized tumor antigens acquired via either FcγR-mediated endocytosis (22, 24) or phagocytosis (35) induces CD4- and CD8-mediated tumor immunity. Dhodapkar et al. (23) showed that cross-presentation mediated by FcγRs on human dendritic cells can enhance the presentation of multiple myeloma antigens to patient-derived T cells, thus suggesting that uptake of antibody-opsonized tumor cells and cellular fragments by antigen-presenting cells could lead to antigenic/epitope spreading and the induction of immunity to several tumor-associated antigens. Herein, we provide data supportive of this concept in patients by showing the first direct evidence for an induced immunologic response in antibody-treated cancer patients and further studies will be important to assess whether concomitant immunity to other tumor-associated antigens is also induced in trastuzumab-treated patients as predicted by an antigenic cascade. Anti-HER-2/neu antibodies enhance the potency of HER-2/neu–expressing whole-cell vaccines in mice, suggesting that mAbs may enhance priming of effective tumor immunity (36). In trastuzumab-treated patients, the opsonic enhancement of HER-2/neu immunogenicity may be the consequence of trastuzumab bound to either shed soluble HER-2/neu extracellular domain protein or to HER-2/neu bearing necrotic/apoptotic tumor or normal cells.

We cannot formally address whether the coadministered chemotherapy contributes to the occurrence of immunologic responses. However, chemotherapy is not an absolute requirement because three of the patients showing immunologic responses (patients 1, 22, and 34) received trastuzumab alone. Patient 1 received trastuzumab in the metastatic setting, whereas patients 22 and 34 received trastuzumab alone after completing adjuvant chemotherapy. Chemotherapy and/or dexamethasone (administered in patients as an antiemetic and to prevent paclitaxel and cremophor hypersensitivity responses) have been traditionally perceived as detrimental to the induction of immunity via myelosuppression and inhibition of lymphocyte and antigen-presenting cell function. However, a recent vaccine trial found no evidence for chemotherapy-associated impairment of T-cell responses in 28 patients randomized to receive either vaccine alone or vaccine concurrently with docetaxel/dexamethasone (37). Preclinical studies have shown that administration of chemotherapy enhances the immunostimulatory capacity of coadministered vaccines (38). Indeed, there is accumulating evidence that supports the notion that chemotherapy could enhance tumor antigen priming through multiple mechanisms, including (a) promotion of tumor cellular apoptosis/necrosis, thus increasing the antigenic load available for uptake by antigen-presenting cells, and (b) inhibition of regulatory T-cell function (39).

The assessment of the anti-HER-2/neu humoral response has been limited to the anti-HER-2/neu response of the Igλ subclass, as detection of IgG responses was complicated by the high serum concentrations of trastuzumab, an IgG1 κ antibody. Most patients who exhibited Igλ anti-HER-2/neu increases also showed an increase in anti-HER-2/neu IgM levels (data not shown). Determination of levels of class-switched antibodies of the IgG subclass has not been possible to date because a screen of several secondary reagents recognizing human IgG2, IgG3, and IgG4 subclasses has failed to identify a reagent specific enough in our assays to avoid detection of trastuzumab (present in the sera at 20-600 μg/mL). The concordance in 10 individuals of the presence of augmented HER-2/neu–specific CD4 cell responses with the presence of increased humoral responses, however, makes it likely that the humoral responses observed are the product of a T-dependent response. Further analysis of additional patients will be required to determine whether augmented CD4 immunity occurs significantly more frequently in clinically responding patients. HER-2/neu–specific IFN-γ–producing CD4 cells would be expected to contribute as effectors of antitumor inflammatory responses or through the provision of T-cell help for CD8 T-cell responses. Preliminary ELIspot assays of six HLA A0201+ patients in this cohort have not yet revealed induction of a CD8 anti-HER-2/neu response using two previously defined HER-2/neu immunodominant epitopes (369-377:KIFGSLAFL and RLLQETELV 689-697; refs. 40, 41).

What role could endogenous HER-2/neu antibodies play in treatment responses? The high levels of trastuzumab already present in treated patients might suggest that, for both antibody-dependent tumor cell cytotoxicity and inhibition of HER-2 signaling, growth-regulatory consequences are already saturated and optimized. However, endogenous antibodies might bind epitopes distinct from the trastuzumab-binding site on the juxtamembrane region of HER-2/neu. By binding distinct epitopes, endogenous antibodies could provide additive roles promoting trastuzumab-mediated antibody-dependent tumor cell cytotoxicity or furthering HER-2/neu signaling perturbation.

Recent clinical data have suggested that favorable clinical outcomes in patients treated with the anti-CD20 mAb rituximab occur more frequently in patients harboring allotypic FcγR alleles conveying higher affinity for IgG (1719). The data presented here of 22 patients treated in the metastatic setting lack the necessary statistical power to appropriately address this question in trastuzumab-treated patients. With regard to immune sensitization, activating FcγR haplotypes did not strongly segregate with the occurrence of endogenous humoral response, although again the strength of this conclusion is limited by sample size. Uptake of HER-2/neu immune complexes through activating FcγRs would be expected to enhance the HER-2/neu Thelper CD4 cell response (42), thus predicting that activating FcγR subtype would be contributory. However, immune complex uptake by other receptors, including the inhibitory FcγRIIB receptor and/or complement receptors, on antigen-presenting cells and B cells, respectively, could also positively regulate immune complex enhancement of activation of anti-HER-2/neu–specific B cells (43, 44).

The data provided here are the first to show immune sensitization during treatment with antitumor antibodies. The induction of CD4 and endogenous humoral immunity suggests that therapeutic antibodies not only provide passive immunotherapy through antibody-dependent tumor cell cytotoxicity but also can promote active immunity. Although these data do not prove causality, they nevertheless suggest that strategies aimed at promoting the vaccinal effect of opsonic antitumor antibodies would augment immunologic memory and thereby enhance durable clinical benefit.

Grant support: NIH/National Cancer Institute grants CA94037 (R. Clynes) and K01-CA100764 (K. Knutson). Also funded in part by NIH/NCCR Clinical and Translational Science Award, No. 1 UL1 RR024156, and by a Pilot Award of the Alexander and Margaret Stewart Trust (R. Clynes). Several peptides were synthesized and supplied by Epimmune, Inc. (San Diego, CA) under the auspices of NIH/National Cancer Institute STTR grant R41-CA107590-01 (K. Knutson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Chao Yu and Courtney Erskine for technical assistance.

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