A viral vaccine encoding prostate-specific antigen induces antigen spreading to a common set of self-proteins in prostate cancer patients

Abstract

Purpose: We previously reported a randomized phase II clinical trial combining a poxvirus-based vaccine encoding prostate-specific antigen (PSA) with radiotherapy in patients with localized prostate cancer. Here, we investigate whether vaccination against PSA induced immune responses to additional tumor-associated antigens and how this influenced clinical outcome.

Experimental design: Pretreatment and posttreatment serum samples from patients treated with vaccine + external beam radiation therapy (EBRT) versus EBRT alone were evaluated by Western blot and serologic screening of a prostate cancer cDNA expression library (SEREX) to assess the development of treatment-associated autoantibody responses.

Results: Western blotting revealed treatment-associated autoantibody responses in 15 of 33 (45.5%) patients treated with vaccine + EBRT versus 1 of 8 (12.5%) treated with EBRT alone. SEREX screening identified 18 antigens, which were assembled on an antigen array with 16 previously identified antigens. Antigen array screening revealed that 7 of 33 patients (21.2%) treated with vaccine + EBRT showed a vaccine-associated autoantibody response to four ubiquitously expressed self-antigens: DIRC2, NDUFS1, MRFAP1, and MATN2. These responses were not seen in patients treated with EBRT alone, or other control groups. Patients with autoantibody responses to this panel of antigens had a trend toward decreased biochemical-free survival.

Conclusions: Vaccine + EBRT induced antigen spreading in a large proportion of patients. A subset of patients developed autoantibodies to a panel of four self-antigens and showed a trend toward inferior outcomes. Thus, cancer vaccines directed against tumor-specific antigens can trigger autoantibody responses to self-proteins, which may influence the efficacy of vaccination.

(c) 2010 AACR.

Figures

Figure 1

Treatment with vaccine + EBRT or EBRT without vaccine induces autoantibody responses to a variety of tumor antigens. Western blot analysis of serum from six patients probed against LNCaP (panels A-C) or PC3 (panel D) cell lysates. A, Treatment with vaccine + EBRT in patient NIH-33 is associated with the development of autoantibody responses against antigens of approximately 65 and 28 kDa. B, Vaccine + EBRT treatment in patients NIH-43, NIH-03 & NIH-04 is associated with the development of autoantibody responses against a ~55 kDa antigen in all three patients (arrows). C, Vaccine + EBRT treatment in patient NIH-32 is associated with development of autoantibody responses to two antigens of ~90 and 28 kDa. D, Patient NIH-02, treated with EBRT without vaccine, developed an autoantibody response to a ~53 kDa antigen.

Figure 2

An example of SEREX antigen array analysis of pre- and post-treatment serum from patient NIH-35, treated with vaccine + ERBT, reveals treatment-associated autoantibody responses to a common set of tumor antigens. A, Map of the antigen array containing the 18 antigens identified by SEREX screening (using serum from 6 patients with vaccine-associated autoantibody responses) plus 16 antigens derived from previous screens of prostate or ovarian cancer cDNA expression libraries. Note that several antigens are duplicated within the array. B, Sera collected from patient NIH-35 at three serial time points were used to screen the antigen array: pre-treatment; post 3 cycles of vaccine (prior to EBRT) and post 8 cycles of vaccine (after completion of EBRT). This revealed baseline autoantibody responses to two antigens (DLD and SON). In addition, autoantibody responses to four antigens (DIRC2, NDUFS1, MRFAP1 and MATN2) appeared only after treatment was initiated (arrows in post 3 and post 8 panels). These responses can be attributed to the vaccine as they are present in the post 3 serum sample, which was taken prior to the initiation of EBRT.

Figure 3

Summary of all treatment-associated autoantibody responses detected by antigen array. Black circles denote positive autoantibody responses against the indicated antigen. Of 33 patients treated with vaccine + EBRT, seven patients (NIH-35; NIH-03; NIH-43; NIH-006; NIH-7; NIH-11 and NIH-28) developed autoantibody responses to a common set of four antigens (DIRC2, NDUFS1, MRFAP1 and MATN2). Patients NIH-43 and NIH-28 showed an additional treatment-induced response to BRD9 and DLD, respectively. Responses to these antigens can be attributed to the vaccine, as they appeared after 3 cycles of vaccine but prior to initiation of EBRT. Additionally, they are not seen in patients treated with ERBT without vaccine (n = 8), ADT + EBRT (n = 15), watchful waiting (WW, n = 9) nor in cancer-free controls (n = 15).

Figure 4

Kaplan-Meier analysis of biochemical-free survival in relation to the development of autoantiobdy responses. A, Patients were stratified according to the presence or absence of treatment-associated autoantibody responses to any antigen seen by Western blot or antigen array. The outcomes of the two groups were similar (p=0.9537). B, Patients were stratified according to the presence or absence of treatment-associated autoantibody responses to the panel of four antigens (DIRC2, NDUFS1, MRFAP1 and MATN2) as assessed by antigen arrays. There is a trend towards decreased survival in autoantibody-positive patients (p=0.1136).