Induction of tumor-cell lysis by bi-specific monoclonal antibodies recognizing renal-cell carcinoma and CD3 antigen

Bi-specific monoclonal antibodies (MAbs) were developed by somatic hybridization of 2 mouse hybridomas, one producing MAb against the G250 renal-cell carcinoma (RCC)-associated antigen and the other against the T-cell antigen CD3 (OKT3). The dual specificity of the hybrid MAb produced by these so-called quadromas was analyzed by immunohistochemistry on tissue sections and by cytotoxicity assays with relevant target and effector cells. The bi-specific MAb could induce TCR alpha beta/CD3+ and TCR gamma delta/CD3+ cloned lymphocytes to kill RCC cells. A noteworthy finding was that the TCR alpha beta and gamma delta lymphocyte clones showed different triggering abilities. The specificity of target-cell lysis by the cytotoxic T cells (CTL) was dictated by the specificity of the G250 MAb. Control bi-specific MAb, recognizing a cell-surface structure not involved in T-cell activation, did not induce lysis. Several IgG subclass switch variants of the G250 hybridoma, i.e., IgG1, 2a, 2b and IgE, were used for somatic hybridization with the OKT3 hybridoma (IgG2a). Except for IgE, all IgG subclass combinations could equally induce cytolysis. Induction of cytolysis was inhibited only by excess OKT3 MAb. Comparison of 2 bi-specific MAb preparations of the same combination (IgG2a/1), produced by 2 quadromas derived from the same parental hybridomas after identical purification procedures, produced different amounts of bispecific MAb.     

Expression of GD3 ganglioside in childhood T-cell lymphoblastic malignancies

Lymphoblasts from seven children with T-cell lymphoblastic malignancies and three children with non-T, non-B acute lymphoblastic leukemia (ALL) were analyzed for ganglioside content. Nonmalignant T-cells from thymus served as controls. Both ganglioside and glycoprotein sialic acid were increased approximately 3-3.5-fold in T-cell disease compared to thymic tissue when expressed on a per cell basis, but not on a per milligram protein basis. Thin-layer chromatography of the isolated ganglioside fraction from T-cell lymphoblasts revealed two major resorcinol-positive bands. One ganglioside comigrated with II3-alpha-N-acetylneuraminosyllactosylceramide (GM3), the major ganglioside in normal lymphoid tissue, and the other ganglioside comigrated with authentic II3-alpha-N-acetylneuraminosyl-alpha 2----8-N-acetylneuraminosyllactosylceramide (GD3) in three different solvent systems. Neuraminidase treatment of the latter ganglioside yielded GM3 and lactosyl ceramide, hydrolysis products of GD3. Scanning densitometry revealed that whereas thymus cells contained 97% GM3 and 3% GD3, T-cell lymphoblasts contained from 22 to 86% GD3 and a corresponding decrease in GM3. The shift to increased GD3 was observed in the blasts from all seven T-cell patients, but not in the blasts from the non-T, non-B patients studied. Only trace quantities of GD3 were detected from two continuous T-ALL cell lines, HSB2 and RPMI 8402. The results demonstrate a consistently significant increase in ganglioside GD3 in uncultured, patient-derived T-cell ALL lymphoblasts when compared to non-T-cell ALL and normal lymphoid tissue. Therefore, GD3 may represent a tumor-associated antigen for the T-cell subclass of childhood lymphoblastic malignancy.     

Sequential immunization of melanoma patients with GD3 ganglioside vaccine and anti-idiotypic monoclonal antibody that mimics GD3 ganglioside.

GD3 ganglioside is an attractive target for immunotherapy of melanoma because it is abundantly expressed on all melanomas but not expressed on most normal tissues. Although GD3 has proven to be one of the least immunogenic gangliosides, our recent studies showed that anti-GD3 antibodies can be induced in patients immunized either with GD3-lactone-KLH (GD3-L-KLH) plus QS-21 adjuvant or with BEC2 anti-idiotypic monoclonal antibody vaccine, which mimics GD3, plus Bacillus Calmette-Guérin. We compared the immunogenicity of these two vaccines and tested whether one vaccine could prime an antibody response to the other. This is the first clinical trial immunizing patients with both antigen and anti-idiotypic monoclonal antibody vaccine. Twenty-four melanoma patients were randomized to be immunized with either BEC2 followed by GD3-L-KLH or in the opposite order. Our prior study suggested that a 25-microg dose of BEC2 was more immunogenic than our standard dose of 2.5 mg and therefore was used in this trial. Overall, 10 of 24 patients (42%) developed anti-GD3 antibodies detectable by ELISA, five in each cohort. All antibody responses were in response to the GD3-L-KLH vaccine. We found no evidence of priming by either vaccine. Antibody responses did not correlate with survival outcomes. Cellular responses were detected by enzyme-linked immunospot against BEC2, Bacillus Calmette-Guérin, and KLH, but not against GD3. We confirmed that GD3-L-KLH vaccine induces anti-GD3 antibodies, but we were unable to confirm our previous finding that a 25-microg dose of BEC2 is immunogenic. Future multivalent ganglioside vaccines should include the GD3-L-KLH vaccine.     

DNA vaccine expressing the mimotope of GD2 ganglioside induces protective GD2 cross-reactive antibody responses

The GD2 ganglioside expressed on neuroectodermally derived tumors, including neuroblastoma and melanoma, is weakly immunogenic in tumor-bearing patients and induces predominantly immunoglobulin (Ig)-M antibody responses in the immunized host. Here, we investigated whether interconversion of GD2 into a peptide mimetic form would induce GD2 cross-reactive IgG antibody responses in mice. Screening of the X(15) phage display peptide library with the anti-GD2 monoclonal antibody (mAb) 14G2a led to isolation of mimetic peptide 47, which inhibited the binding of 14G2a antibody to GD2-positive tumor cells. The peptide was also recognized by GD2-specific serum antibodies from a patient with neuroblastoma, suggesting that it bears an internal image of GD2 ganglioside expressed on the tumor cells. The molecular basis for antigenicity of the GD2 mimetic peptide, established by molecular modeling and mutagenesis studies, led to the generation of a 47-LDA mutant with an increased mimicry to GD2. Immunization of mice with peptide 47-LDA-encoded plasmid DNA elicited GD2 cross-reactive IgG antibody responses, which were increased on subsequent boost with GD2 ganglioside. The vaccine-induced antibodies recognized GD2-positive tumor cells, mediated complement-dependent cytotoxicity, and exhibited protection against s.c. human GD2-positive melanoma growth in the severe combined immunodeficient mouse xenograft model. The results from our studies provide insights into approaches for boosting GD2 cross-reactive IgG antibody responses by minigene vaccination with a protective epitope of GD2 ganglioside.     

Shedding of GD2 ganglioside by human neuroblastoma

Substantial concentrations of the cell-surface glycosphingolipid, the disialoganglioside GD2, are uniformly present in human neuroblastoma tumors. This ganglioside can also be detected in the plasma of patients with neuroblastoma by direct thin-layer chromatographic analysis. Among 32 neuroblastoma patients in all clinical stages studied prior to the initiation of treatment, 27 (84%) showed measurably elevated plasma concentrations of GD2 (greater than or equal to 50 pmol/ml). The mean level (545 +/- 108 pmol/ml) was more than 50 times the normal plasma GD2 concentration of less than or equal to 10 pmol/ml. Circulating GD2 was not detected in the plasma of patients with the related, more differentiated tumors, ganglioneuroblastoma and ganglioneuroma, indicating an association of the shedding of this ganglioside with the undifferentiated phenotype. Circulating GD2 diminished in patients in response to therapy, and reappeared in patients whose disease recurred. The results suggest that the sequential determination of circulating GD2 will be of value in monitoring individual patients with neuroblastoma.     

Induction of antibodies against GD3 ganglioside in melanoma patients by vaccination with GD3-lactone-KLH conjugate plus immunological adjuvant QS-21

The gangliosides GD3, GD2 and GM2 are expressed on the cell surface of malignant melanomas, GD3 being the most abundant. We have shown that immunization of melanoma patients with GM2 adherent to Bacillus Calmette-Guerin (GM2/BCG) induced an IgM antibody response. Vaccines containing GM2-keyhole limpet hemocyanin (KLH) conjugate and the immunological adjuvant QS-21 induced a higher titer IgM response and consistent IgG antibodies. Patients with antibodies against GM2 survived longer than patients without antibody. On the other hand, our previous trials with GD3/BCG, GD3 derivatives including GD3-lactone (GD3-L)/BCG failed to induce antibodies against GD3. In our continuing efforts to induce antibody against GD3, we have immunized groups of 6 melanoma patients with GD3-KLH or GD3-L-KLH conjugates containing 30 microg of ganglioside plus 100 microg of QS-21 at 0, 1, 2, 3, 7 and 19 weeks. Prior to vaccination, no serological reactivity against GD3 or GD3-L was detected. After immunization, IgM and IgG antibodies were detected against both GD3 and GD3-L in the GD3-L group exclusively. The GD3-L-KLH vaccine induced IgM titers against GD3-L of 1:40-1/1,280 in all patients and IgG titers of 1/160-1/1,280 in 4 patients. These antibodies also strongly cross-reacted with GD3. ELISA reactivity was confirmed by immune thin-layer chromatography on GD3 and melanoma extracts. Sera obtained from 4 of these 6 patients showed cell surface reactivity by FACS and from 2 showed strong cell surface reactivity by immune adherence (IA) assay and complement lysis against the GD3 positive cell line SK-Mel-28.     

Induction of apoptosis in head and neck tumor-cell lines by anti-fas antibody

Programmed cell death, currently termed apoptosis plays a key role in the maintenance of the steady state in continuously renewing tissues. Since little is known of apoptosis in head and neck tumors, we studied morphological changes in head and neck tumor-derived cell lines (KB, KBrc, HSC-2,3,4), induced by anti-Fas antibody. Light and electron microscopic examinations were carried out after culturing these cell lines with the antibody for 1-2 days. The antitumor effect of anti-Fas antibody on tumor cells differed with the cell lines. Most of the cell lines that were sensitive to anti-Pas antibody showed evidence of enhanced apoptosis when the cells were pretreated with interferon-gamma. The results suggest that the strategy of induction of apoptosis by anti-Fas antibody may be considered in treatment of some tumors of head and neck.     

Analysis of production,purification, and cytolytic potential of bi-specific antibodies reactive with ovarian-carcinoma-associated antigens and the T-cell antigen CD3

OV-TL 3 and MOvl8 MAbs, due to their restricted specificity, have been successfully used to visualize ovarian cancer in patients and might therefore be used to develop therapies for ovarian cancer. The bi-specific MAbs αT3/OC2 and αOC/TR (both being combinations of MOvl8 and aCD3) have been shown to lyse ovarian tumor cells in vitro. To evaluate the relative merits of MOvl8/CD3 and OV-TL 3/CD3, the present study was undertaken in which the bi-specific MAbs αT3/OC2 and αOC/TR, and a newly developed bi-specific MAb, OV-TL 3/CD3, were highly purified and compared for specificity, stability, purification and cytolytic potential. The dual specificity of the hybrid-hybridoma supernatants was analyzed by immunohistochemistry, and by testing bi-specific MAb-mediated cytotoxicity against relevant target cells in the presence of effector cells. Stability testing of bi-specific MAb-producing hybrid-hybridomas showed that, after sub-cloning, clones stably produced up to 40% bi-specific MAb even after prolonged in vitro culture. The purification of the bi-specific fractions was performed with protein A and by ion-exchange high-pressure liquid chromatography, depending on the sub-class combination of the bi-specific MAb. The purified bi-specific MAbs were tested for their ability to mediate target-cell lysis with the use of cytotoxic T-cell clones and activated peripheral-blood lymphocytes. The purified αT3/OC2, αOC/TR, and OV-TL 3/CD3 were all able to mediate highly specific lysis of various ovarian-carcinoma cell lines. No correlation was found between the level of antigen expression and bi-specific MAb-mediated cytolysis. © 1993 Wiley-Liss, Inc.     

Expression of GD2 ganglioside by untreated primary human neuroblastomas

Primary neuroblastomas obtained before therapy from 36 patients were studied to determine the frequency of tumors expressing a specific glycosphingolipid, GD2 ganglioside. Total tissue gangliosides were purified by a new partition method, quantitated, and analyzed by high-performance thin-layer chromatography. All 36 neuroblastoma tumors, representing all clinical stages, contained GD2 ganglioside. The mean relative and absolute concentrations of GD2 were substantial (12% of the total tissue gangliosides and 50 nmol/g of tissue) and were independent of the clinical stage of the tumor. In contrast, 6 samples of related but more differentiated tumors (ganglioneuroblastoma and ganglioneuroma) had little or no detectable GD2 (less than or equal to 1.5% of total gangliosides and less than or equal to 4 nmol/g of tissue). These results suggest that GD2 is a sensitive marker for neuroblastoma tissue and may be an excellent target antigen for immunotherapy of this tumor.     

Effect of a chimeric anti-ganglioside GD2 antibody on cell-mediated lysis of human neuroblastoma cells

An anti-GD2 ganglioside human/mouse chimeric monoclonal antibody, ch14.18, like its murine counterpart, 14.G2a, was shown to bind to human neuroblastoma cells. This chimeric antibody proved to be more effective than 14.G2a in mediating the lysis of neuroblastoma cells with human effector cells, such as granulocytes and natural killer cells within the peripheral blood mononuclear cell population. A comparison of these two effector cell populations isolated from the same donor revealed granulocytes to be more effective than peripheral blood mononuclear cells in lysing neuroblastoma cells, which were coated with monoclonal antibody ch14.18. Addition of recombinant human granulocyte-macrophage colony-stimulatory factor increased ch14.18-mediated lysis of neuroblastoma cells by granulocytes but not by peripheral blood mononuclear cells. In fact, granulocytes were effective in mediating lysis of neuroblastoma cells coated with ch14.18 irrespective of whether they were obtained from normal adults or from neuroblastoma patients.     

Consistent antibody response against ganglioside GD2 induced in patients with melanoma by a GD2 lactone-keyhole limpet hemocyanin conjugate vaccine plus immunological adjuvant QS-21.

PURPOSE: Melanomas, sarcomas, and neuroblastomas abundantly express the ganglioside GD2 on the cell surface where it is susceptible to immune attack by antibodies. Overexpression of GD2 on these tumors is striking, as is the frequency of clinical responses after treatment of neuroblastoma with monoclonal antibodies against GD2. In addition, preclinical models have demonstrated the ability of a GD2-keyhole limpet hemocyanin (KLH) conjugate vaccine to induce antibodies that eliminate micrometastases. However, vaccination of patients with GD2-KLH has previously failed to induce a consistent relevant antibody response. We test here whether the use of GD2 lactone-KLH can overcome the low immunogenicity of GD2-KLH. EXPERIMENTAL DESIGN: Eighteen patients with melanoma were vaccinated s.c. in the adjuvant setting on weeks 0, 1, 2, 3, 10, and 24. Groups of 6 patients were entered at three dose levels (3, 10, or 30 micro g) of GD2 lactone (GD2L) in vaccines containing GD2L-KLH plus the immunological adjuvant QS-21. Blood was drawn at regular intervals to assess the antibody response. RESULTS: The vaccine was well tolerated. The majority of patients in all three dose levels produced anti-GD2 antibodies detectable by ELISA assay. Specificity for GD2 was also confirmed by immune thin-layer chromatography. Although there was no statistical difference in terms of titers between the three groups, patients at the 30- micro g dose level had higher titers and longer lasting antibody responses overall by ELISA (median IgM/IgG peak titer 1:640/1:80) and generated the strongest cell surface reactivity by fluorescence-activated cell sorting (median IgM peak percentage positive cells/mean fluorescence intensity for pre- and postvaccination sera is 10%/63 and 70%/135). Patients vaccinated with the 30- micro g GD2 dose also had the most potent complement dependent cytotoxicity using human complement, with 5 of 6 patients showing strong cell surface reactivity by fluorescence-activated cell sorting and >30% cytotoxicity by chromium release with a serum dilution of 1/100. CONCLUSIONS: GD2L-KLH conjugate vaccine plus adjuvant QS-21 induces antibodies against GD2 that bind to the cell surface and induce complement-dependent cytotoxicity in the majority of patients with melanoma.     

Shedding of GD2 ganglioside in patients with retinoblastoma

Retinoblastoma is a rare tumor of the young child with an intraocular localization that leads to certain problems of diagnosis. With the aim of defining a biochemical marker—which is still lacking for this disease—the gangliosides of a pool of fresh retinoblastoma tumors were analyzed. The ganglioside pattern was shown to have GM3, GM2, GM1, GD3, GD2, GD1b and GT1b as the major components. The occurrence of a high concentration of GD2 in the tumors led us to investigate the possibility of changes in the level of GD2 in the sera of retinoblastoma patients, using quantitative immunostaining with GD2-specific mouse monoclonal antibodies (MAbs). In 9 out of 10 tumor-bearing patients, the serum level of GD2 ganglioside was significantly higher than the average value found in normal individuals. A 2-year follow-up of patients showed that successful treatment resulted in a rapid decrease in the serum level of GD2 down to the normal range, from which a subsequent elevation was seen only in relapsing patients. Although the clinical study needs further development, the results obtained to date suggest that GD2 is shed in the serum of tumor-bearing patients and that the level of GD2 could be a potential serum marker of human retinoblastoma.     

ICAM- melanoma cells are relatively resistant to CD3-mediated T-cell lysis

The primary activation pathway of T cells is via the T-cell receptor (TCR)/CD3 complex, which is functionally interrelated with various accessory molecules. We examined the contribution of the lymphocyte-function-associated antigen-I/intercellular adhesion molecule 1 (LFA-1/ICAM-1) interaction to CD3/TCR-mediated lysis by cytotoxic T lymphocytes (CTL). We used ICAM-I-or+ tumor cell lines as target cells and anti-CD3- or anti-LFA-1 containing hetero-cross-linked monoclonal antibody (MAb) to bridge CTL and target cells and simultaneously to activate CTL. The ICAM-1- melanoma-derived cell line IgR39 was relatively resistant to CD3-mediated lysis by both TCR alpha beta + and TCR gamma delta + CTL, when compared with ICAM-1+ cell lines. Induction of ICAM-1 on the membrane of IgR39 cells by tumor necrosis factor (TNF) rendered these cells more susceptible to CD3-mediated lysis. Anti-ICAM-1 MAb inhibited this TNF-enhanced susceptibility to lysis, directly demonstrating that the induction of ICAM-1 was critical in the TNF-induced increase in susceptibility to lysis of IgR39 cells. CTL formed less efficient conjugates with the ICAM-1- cells as compared to ICAM-1+ cells. Both spontaneous and CD3-induced conjugate formation as well as CD3-mediated lysis of ICAM-1- tumor cells by CTL were enhanced by the addition of anti-LFA-1 containing hetero-cross-linked MAb, thereby mimicking the LFA-1/ICAM-1 interaction between CTL and target cells. Soluble anti-CD18 MAb inhibited CD3-mediated lysis of ICAM-1- target cells by CTL without affecting their conjugate formation. Anti-LFA-1 MAb added after conjugate formation still inhibited lysis of both ICAM-1+or- tumor cells. Taken together, these findings suggest that the LFA-1/ICAM-1 interaction co-activates CD3/TCR-mediated lysis by CTL through both an enhanced CTL-target cell binding and the delivery of post-conjugate costimulatory signals.     

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.     

Inhibition of metastases of a human melanoma xenograft by monoclonal antibody to the GD2/GD3 gangliosides

A human melanoma variant cell line was obtained from a lung metastasis that arose spontaneously after we inoculated melanoma cells sc into a nude mouse. In this model, IgG2a monoclonal antibody (MAb) ME 36.1 defining the GD2/GD3 gangliosides inhibited melanoma growth at the primary site and metastatic spread of the cells, whereas an IgG1 variant of MAb ME 36.1 inhibited lung metastasis formation only. Possible mechanisms of antitumor effects of MAb ME 36.1 are discussed.     

Production of monoclonal antibody alpha Pro3 recognizing a human prostatic carcinoma antigen

The monoclonal antibody alpha Pro3 recognizes an antigen concentrated in human primary prostatic carcinoma tissue removed surgically. Although the antigen was detectable in extracts of human normal and malignant nonprostatic tissue, as well as in benign prostate tissue, quantitative absorption analysis revealed a substantially greater quantity of the antigen in malignant prostate tissue. The antigen recognized by alpha Pro3 in primary prostatic carcinoma has an apparent nonreduced molecular weight of 175,000 and an apparent subunit molecular weight of 54,000. This antigen, p54, appears to be present on the surface of cultured prostatic tumor cells of the PC-3 cell line, but its location in vivo has not been defined. Successful competition between alpha Pro3 and prostatic carcinoma patient serum immunoglobulin for a Mr 54,000 antigen (reduced molecular weight) present in prostatic carcinoma tissue extract suggests that p54 may play a significant role in the immunobiology of prostatic carcinoma. alpha Pro3 has potential as a sensitive probe for an antigen relevant to human tumor biology.     

Characterization of BIS20x3, a bi-specific antibody activating and retargeting T-cells to CD20-positive B-cells

This paper describes a bi-specific antibody, which was called BIS20x3. It retargets CD3varepsilon-positive cells (T-cells) to CD20-positive cells and was obtained by hybrid-hybridoma fusion. BIS20x3 could be isolated readily from quadroma culture supernatant and retained all the signalling characteristics associated with both of its chains. Cross-linking of BIS20x3 on Ramos cells leads to DNA fragmentation percentages similar to those obtained after Rituximab-cross-linking. Cross-linking of BIS20x3 on T-cells using cross-linking F(ab')2-fragments induced T-cell activation. Indirect cross-linking of T-cell-bound BIS20x3 via Ramos cells hyper-activated the T-cells. Furthermore, it was demonstrated that BIS20x3 effectively re-targets T-cells to B-cells, leading to high B-cell cytotoxicity. The results presented in this paper show that BIS20x3 is fully functional in retargeting T-cells to B-cells and suggest that B-cell lymphomas may represent ideal targets for T-cell retargeting bi-specific antibodies, because the retargeted T-cell is maximally stimulated in the presence of B-cells. Additionally, since B-cells may up-regulate CD95/ Fas expression upon binding of CD20-directed antibodies, B-cells will become even more sensitive for T-cell mediated killing via CD95L/ Fas L, and therefore supports the intention to use T-cell retargeting bi-specific antibodies recognizing CD20 on B-cell malignancies as a treatment modality for these diseases.     

Role of MHC class-I antigens and the CD3 complex in the lysis of autologous human tumours by T-cell clones

Peripheral blood lymphocytes (PBL) of 4 patients with malignant effusions were stimulated for 6 days with purified autologous tumour cells, before isolation of the lymphoblasts and cloning by limiting dilution in interleukin-2 (IL-2). Forty-five clones were analyzed for cytotoxicity (CTX) against autologous, allogeneic tumour and erythromyeloid K562 cells of known status with respect to expression of major histocompatibility complex (MHC) antigens, estimated by reaction with the W6/32 (anti HLA, -A, -B, -C monomorphic) and TDR 31.1 (anti HLA-DR) monoclonal antibodies (MAb). All 45 clones were CD3+. Twenty-five (56%) of them were cytotoxic for at least one target; 24 were autoreactive (restricted in 7); 17 were alloreactive; 16 were K562 reactive. Under comparable conditions autoreactivity was partially blocked by W6/32 in 12/20 effector:target combinations; alloreactivity in 8/13 and K562 reactivity in 0/14. Modulation of effector cell surface CD3 antigens by OKT3 monitored by flow cytometry reduced autoreactivity in 9/14 combinations, alloreactivity in 2/6 and K562 reactivity in 0/4. W6/32 blocking and T3 modulation of cytotoxicity were almost invariably concordant against the same target. The data suggest that, to accomplish lysis of autologous and allogeneic tumour targets, certain clones require MHC recognition and a functional CD3 complex, while for others with similar target cell repertoires, there is no such requirement. It is possible that T-cell clones responding to a tumour-associated antigen (TAA) in the context of self MHC antigens can also respond to an allogeneic class-I product in the absence of TAA, and/or that aberrant class-I antigen expression on autologous tumours accounts for the alloreactivity.