In this study, we demonstrate for the first time the capacity of ACE from HER-2/neu-overexpressing primary ovarian tumour cells to induce in vitro autologous CTL from MEAMNC with the ability to exert in vivo antitumour effects against HER-2/neu+ human tumour cell lines. Such CTL recognised peptides within ACE which were naturally processed and expressed on the surface of tumour cells in the context of the HLA-A2.1 allele. This was indicated by the following findings: first, ACE peptides induced stabilisation of HLA-A2.1 molecules on T2 cells and such ACE-pulsed T2 cells were efficiently lysed by our ACE-induced CTL effectors; second, HER-2/neu+, HLA-A2.1+ primary tumour cells as well as human tumour cell lines of breast and ovarian origin were lysed in an HLA-A2.1-restricted fashion since the cytotoxicity was to a great extent abrogated in the presence of the mAb BB7.2, and third, HER-2/neu-overexpressing but HLA-A2.1− human cell lines were not lysed.
By pulsing T2 cells with synthetic HER-2/neu CTL peptides, we could identify immunodominant nanomers that were recognised by our ACE-induced bulk CTL. These included peptides HER-2 (9369
), HER-2 (9435
) (both recognised by bulk CTL induced by ACE preparations from Ova-1, Ova-2 and Ova-3 primary HER-2/neu-overexpressing tumours), peptide HER-2 (9689
), which may be recognised by Ova-2 and Ova-3 induced CTL, and peptides HER-2 (9851
) and HER-2 (9665
), which were recognised by one of the three bulk CTL. These HER-2/neu peptides have been demonstrated to bind to HLA-A2.1 molecules with high affinities (Fisk et al 1995
; Rongcun et al, 1999
), thereby eliciting CTL activity among tumour-associated lymphocytes in patients with breast and ovarian cancer (Fisk et al, 1995
; Rongcun et al, 1999
; Baxevanis et al, 2002
). Moreover, we have recently demonstrated the capacity of these peptides to sensitise CTL for lysing their autologous tumour cells in patients with prostate, lung and colorectal cancer (Sotiropoulou et al, 2003a
). Thus, our data demonstrate the utility of ACE preparations as polyepitope carriers. Such carriers besides the HER-2/neu peptides identified, most likely contain others which remain to be identified by screening the entire HER-2/neu protein for sequences with HLA-A2.1-binding motifs, followed by peptide synthesis, loading onto T2 targets and testing in cytotoxicity assays with ACE-induced CTL effectors.
Although HLA-A2.1 is the most popular allele for presenting HER-2/neu CTL epitopes, there are also other MHC class I alleles functioning as restriction elements for HER-2/neu peptide presentations (Kiessling et al, 2002
). Accordingly, recognition of the HER-2/neu+
SKOV3 or SKBR3 cell lines by our ACE-induced bulk CTL effectors could also be accomplished through HLA- class I alleles other than HLA-A2.1. However, according to the serotyping, only the HLA-A2.1 subtype was shared between the primary HER-2/neu-overexpressing Ova-1, Ova-2 and Ova-3 tumours (which served as source for the ACE preparation) and the SKOV3 or SKBR3 tumour cell lines and therefore only HER-2/neu peptide-specific and HLA-A2.1-restricted CTL clones would have a chance to recognise them. Of course, recognition could also have been established through CTL clones specific for HLA-A2.1-restricted peptides other than those derived from the HER-2/neu oncoprotein. Although this is quite likely, still the contribution of such clones would not add much to overall killing of the HLA-A2.1+
tumour targets. This hypothesis is based on our findings demonstrating that CTL induced by ACE from the Ova-4 and Ova-5 HLA-A2.1+
primary tumours only weakly lysed their autologous tumour targets (i.e. the Ova-4 and Ova-5 primary tumours) as well as the HLA-A2.1+
SKOV3.A2 and MCF-7 tumour cell lines. Thus, it seems possible that peptides within ACE from the HER-2/neu−
primary ovarian tumours are weakly immunogenic most likely due to inefficient expression and presentation on the tumour cell surface. This is supported by the findings that both the Ova-4- and the Ova-5-ACE induced CTL, although exerting weak cytotoxic activity against the HLA-A2.1+
autologous primary tumours and against the HLA-A2.1+
tumour cell lines, still were capable of (i) efficiently lysing T2 targets pulsed with the same ACE (i.e. those prepared from Ova-4 and Ova-5 primary tumours), and (ii) producing substantial amounts of IFN-γ
after the restimulation phase. Both parameters were comparable to those of CTL induced by ACE from the HER-2/neu-overexpressing Ova-1, Ova-2 and Ova-3 primary tumours.
SCID mice were protected against inoculation with HER-2/neu+, HLA-A2.1+ human tumour cell lines when bulk CTL specific for ACE prepared from the HER-2/neu-overexpressing primary ovarian tumours were adoptively transferred. Independently of the HER-2/neu peptides recognised, Ova-1CTL (recognising HER-2 (9851), HER-2 (9435), HER-2 (9665) and HER-2 (9369)), Ova-2CTL (recognising HER-2 (9689), HER-2 (9435) and HER-2 (9369)) and Ova-3CTL (recognising HER-2 (9689), HER-2 (9435) and HER-2 (9369)) induced similar levels of protection demonstrating the immunodominance of these HER-2/neu epitopes, but not excluding, however, the possibility that also CTL clones specific for other HER-2/neu epitopes may be included in these bulk CTL populations.
There are several models described so far in the literature utilising different vehicles for vaccination studies with polyepitope constructs. These include attenuated virus vectors (Toes et al, 1997
; Schneider et al, 1998
) naked DNA (Thomson et al, 1998
) or transfected DC (Condon et al, 1996
). Acid cell extracts preparations from primary tumour cells may be advantageous over such polyepitope vaccines not solely due to the fact that these apparently contain a plethora of CTL epitopes, but also due to the simplicity of the method used for their preparation and administration. In addition, ACE derived from primary tumours may contain T helper epitopes as well (Baxevanis et al, 2000
), which will contribute to the enhancement of CTL-mediated antitumour responses. In contrast to other reports utilising tumour cell lysates or unfractionated tumour peptides in active immunisation studies (Nair et al, 1997
; Fields et al, 1998
; Gatza and Okada, 2002
; Schnurr et al, 2002
; Wen et al, 2002
; Gad et al, 2003
; Graner et al, 2003
; Vegh and Mazumder, 2003
), our data emphasise the role of such preparations in the cellular adoptive immunotherapy of cancer by sensitising in vitro
the effector CTL population.
Collectively, the studies presented herein provide evidence that pooled peptides from HER-2/neu-overexpressing ovarian tumours can be utilised in cellular adoptive immunotherapy of patients with HER-2/neu+ ovarian cancer. They also imply that vaccination with such multiepitope preparations may allow enhanced efficacy in the clinical treatment of ovarian cancer. Moreover, the identification of immunodominant HER-2/neu peptides within these unfractionated peptide extracts may help in the collection of peptides to be included in multipeptide vaccines.