Induction of CA125-specific B and T cell responses in patients injected with MAb-B43.13--evidence for antibody-mediated antigen-processing and presentation of CA125 in vivo.

The murine monoclonal anti-CA125 antibody MAb-B43.13 has previously been administered as an immunoscintigraphic agent in order to monitor recurrence of ovarian cancer in patients, and a long-term follow-up demonstrated a survival benefit for these patients. The clinical benefit was initially attributed to the activation of the idiotypic network. The objective of this study was to investigate the role of CA125-MAb-B43.13 immune complex formation on the induction of CA125-specific immune responses. Analysis of patient serum samples from pharmacokinetic studies demonstrated that the antibody forms immune complexes with CA125 in circulation within 30 minutes of injection. Induction of humoral and cellular anti-CA125 responses correlated with the amount of circulating CA125 antigen present at time of antibody injection. Subsequent to the injection of MAb-B43.13, the patients generated anti-CA125 antibodies that were directed against various epitopes on the antigen and were not restricted to the specific epitope recognized by MAb-B43.13. The generation of CA125-specific B and T cell responses after MAb-B43.13 injection correlated with improved survival. The influence of circulating CA125 for the induction of CA125-specific immune responses and the multi-epitopic nature of the human anti-CA125 antibodies suggest that the majority of these antibodies were not induced via the idiotypic network but by the autologous antigen itself. Since antibody and T cell responses to CA125 were not present before injection of MAb-B43.13, it is hypothesized that complex formation of MAb-B43.13 with circulating antigen triggers the induction of CA125-specific immune responses.

Immunotherapy of human ovarian carcinoma with OvaRex MAb-B43.13 in a human-PBL-SCID/BG mouse model.

The monoclonal antibody (MAb) B43.13, binding to the ovarian cancer-associated antigen CA125, has been injected into more than 200 patients with ovarian cancer to detect recurrence of the disease. The follow-up of the patients revealed surprisingly long survival spans for several patients despite high CA125 levels. To investigate the therapeutic effectiveness of OvaRex MAb-B43.13 (AltaRex, Edmonton, Canada) under well-controlled conditions, the antibody was tested in a human-PBL-SCID/BG mouse model with CA125 positive human ovarian cancer cells. Mice were reconstituted with human peripheral blood lymphocytes (PBL, normal donors) by intraperitoneal (IP) injection of 2 to 3 x 10(7) PBL/mouse. OvaRex MAb-B43.13 was administered at 100 microg/mouse in phosphate buffered saline (PBS), in three different experimental set-ups. An isotype-matched control antibody (MOPC21 or MAb-170) and PBS injection served as controls. The ovarian cancer cell line NIH:OVCAR-NU-3 was injected IP at 1 x 10(6) cells/mouse or subcutaneously (SC) at 4 x 10(6) cells/mouse. Human-PBL-SCID/BG mice were either immunized before injection of tumor cells, along with tumor cells or after small tumors were established (2 weeks after transplantation). Antibody injections were repeated twice in 2-week intervals. Functional and cellular characterization of serum and PBL from these mice demonstrated the successful engraftment of a human immune system in those mice. All three experiments showed that OvaRex MAb-B43.13 treatment could (a) delay or prevent development of tumors; (b) reduce the size of small established tumors (SC tumor injection) or suppress ascites formation; (c) delay tumor growth when injected prior to tumor implantation; and (d) prolong the survival of the mice (i.p. tumor injection).

OVAREX MAb-B43.13:IFN-gamma could improve the ovarian tumor cell sensitivity to CA125-specific allogenic cytotoxic T cells.

Various immunological parameters were studied in 100 ovarian cancer patients injected with the OVAREX therapeutic vaccine (the functional component of which is anti-CA125 MAb-B43.13) to explain the serendipitous observation of prolonged survival after such treatment. In addition to CA125-specific humoral and cellular responses, interferon-gamma (IFN-gamma) was also found to be induced in those patients receiving the vaccine. In vitro studies indicated that the expression of MHC I, MHC II, and ICAM I in ovarian tumor cells were upregulated in response to IFN-gamma. Such tumor cells were also found to be more sensitive to CA125-specific cytotoxic T cells compared to cells that were not incubated with IFN-gamma.

A pilot phase 2 study of oregovomab murine monoclonal antibody to CA125 as an immunotherapeutic agent for recurrent ovarian cancer.

This prospective, open-label, pilot phase 2 study examined the clinical and immunologic effects of oregovomab (OvaRex) in heavily pretreated patients with recurrent ovarian cancer (OC). Thirteen women were administered intravenous oregovomab (2 mg) at weeks 0, 2, 4, 8, and 12, followed by quarterly doses for up to 2 years or disease progression. Concomitant chemotherapy was not permitted. Eligibility criteria included recurrence after one or more platinum-based chemotherapy regimens, CA125 >35 U/mL, evaluable or measurable disease. Tumor burden was evaluated by physical or radiologic methods pretreatment, weeks 12, 24, and every 24 weeks thereafter. Immune responses, including antibodies and T cells to oregovomab and CA125, were demonstrated in over half the patients. Stabilization of disease and survival >2 years was observed in 3 of 13 patients and coincided with robust immune responses. Shrinkage of marker lesions was not observed; however, four patients showed decreases in CA125 levels. Treatment was well tolerated without serious adverse events or discontinuations due to therapy. This pilot study supports immunologic activity and safety of oregovomab in recurrent OC. Further study of this agent in the consolidation and adjuvant setting is ongoing to establish its clinical utility.

Homogeneous preparation of human thymidine-5'-triphosphatase by electroelution from SDS/PAGE with subsequent renaturation.

This paper describes a rapid and inexpensive method for homogeneous enzyme preparation from SDS/polyacrylamide gels with subsequent renaturation. The method was optimized for an enzyme of pyrimidine metabolism, thymidine-5'-triphosphatase (dTTPase), present in human serum in small amounts. After gel electrophoresis, the enzyme was eluted from gel pieces in an elution chamber based on a tube gel electrophoresis system. Renaturation conditions were optimized in preliminary tests. The best results were obtained with an initial acetone precipitation to remove sodium dodecyl sulfate. The precipitate was then dissolved in 8 M guanidine hydrochloride and diluted 50-fold for renaturation. Adding 1.5 mg/ml lauryl maltoside to the renaturation buffer, followed by subsequent dialysis of the renaturating samples, improved the renaturation yield up to 95%. This method was used to purify dTTPase to homogeneity from a partially purified sample, and to determine the molecular mass of the subunits. The procedure can also be applied to other enzymes and could give rise to a general strategy for enzyme purification.

Purification and characterization of two different thymidine-5'-triphosphosphate-hydrolysing enzymes in human serum.

Two different enzymes capable of hydrolysing dTTP to the corresponding diphosphate were purified from human serum in order to investigate their enzymatic properties. A specific dTTPase was purified to apparent homogeneity with a purification factor of ca. 10,000 and showed a molecular mass of 46,000 Da, consisting of two identical subunits. This enzyme revealed an isoelectric point of 5.8 and a Km value of 38 microM. The other enzyme showed substrate specificity for dTTP and dCTP and was purified with a factor of ca. 5,000. It seems to be a multifunctional enzyme of one subunit (96,000 Da) with two different catalytic sites for dTTP and dCTP. The isoelectric point was 5.2, the Km values were 20 microM for dTTP and 17 microM for dCTP, respectively. Both enzymes were sensitive to inorganic phosphate, but the dTTPase to a minor extent. In contrast to the dCTPase-dTTPase, the dTTPase was strongly inhibited by ZnSO4. Physico-chemical and biochemical data suggest the purification of two different enzymes.

Anti-idiotype induction therapy: anti-CA125 antibodies (Ab3) mediated tumor killing in patients treated with Ovarex mAb B43.13 (Ab1).

Intravenous injection of the murine monoclonal anti-CA125 antibody B43.13 (Ovarex: Ab1) into ovarian cancer patients led to the induction of an idiotypic network. Of the 75 patients who received one to ten injections of a 2-mg dose of the antibody, 48 developed anti-(mAb B43.13) antibodies (Ab2); 18 of these patients also had elevated levels of anti-[anti-(mAb B43.13)] antibodies (Ab3; = anti-CA125 antibodies) compared to pre-injection values. Characterization of these antibodies revealed that the binding to CA125 could be inhibited by mAb B43.13 in most samples. Human anti-CA125 antibodies or Ab3 purified from patient serum samples specifically recognized human ovarian tumor cells and tissues expressing CA125. In addition, these anti-CA125 antibodies were able to conduct Fc-mediated tumor cell killing (antibody-dependent cell-mediated cytotoxicity). This raises the possibility of using an Ab1 for anti-idiotype induction immunotherapy of cancer.

Characterization of an anti-MUC1 monoclonal antibody with potential as a cancer vaccine.

The monoclonal antibody (MAb) AR20.5 is a murine MAb, generated against the tandem repeat protein backbone of the tumor-associated antigen MUC1. MAb AR20.5 reacts strongly with either the soluble form or the cell surface epitope of MUC1 on many human cancer cell lines. It also reacts with a 23-amino acid MUC1 peptide, E23, which includes the core tandem repeat sequence. Epitope mapping confirmed that MAb AR20.5 recognizes a minimum of six residues with the sequence DTRPAP. Inhibition of glycosylation of MUC1 resulted in decreased binding of MAb AR20.5 to cell surface MUC1, suggesting that MAb AR20.5 binding is carbohydrate dependent. The antibody was studied in a human PBL-SCID/beige mouse model to evaluate its effect on progression of NIH:OVARCAR-3 tumors. Tumor reduction was observed in mice injected with MAb AR20.5, but not in mice treated with control murine antibody or PBS (p < 0.001 and p < 0.05, respectively). An anti-tumor effect could also be demonstrated in a CB6F1 mouse model with the MUC1 transfectoma 413BCR.

Idiotypic cascades after injection of the monoclonal antibody OC125. A study in a mouse model.

In an animal model, we evaluated the possibility to induce antibodies directed against the tumor-associated antigen CA125 by immunization with the anti-CA125 antibody OC125 via activation of the idiotypic network. Our results show that Balb/c mice, immunized by repeated administrations of F(ab')2-fragments of the OC125 antibody (Ab1), produced anti-idiotypic antibodies (Ab2). The binding of these antibodies to the OC125 could be completely inhibited by the antigen CA125, suggesting that the anti-idiotypic antibodies imitate the original target antigen of the OC125. After induction of these paratope-binding anti-idiotypic antibodies (Ab2 beta), a murine IgG-anti-CA125 (Ab3) response arose in the same mice. The induction of idiotypic cascades offers the possibility of immunization against tumor-associated antigens without using the original antigen and breaking antitumor tolerance.

Idiotypic cascades after injection of the monoclonal antibody OC125: a study in a mouse model. Induction of antibodies against OC125 and CA125 after immunization with an anti-CA 125 (MAb OC125) monoclonal antibody by activation of the idiotypic network.

By immunization of mice with the anti-CA 125 monoclonal antibody OC125, we tried to induce antibodies directed against the tumour-associated antigen CA125, via activation of the idiotypic network. Mice immunized by repeated administrations of F(ab')2-fragments of the OC125 antibody produced anti-idiotypic antibodies, imitating the original target antigen of the OC125. After induction of these anti-idiotypic antibodies (Ab2 beta) a murine IgG-anti-CA125 (Ab3) response was detected. The induction of idiotypic cascades offers the possibility of immunization against tumour-associated antigens, without using the original antigen and breaking antitumour tolerance.

Phase I trial of a murine antibody to MUC1 in patients with metastatic cancer: evidence for the activation of humoral and cellular antitumor immunity.

BACKGROUND: BrevaRex mAb-AR20.5 is a murine anti-MUC1 monoclonal antibody generated to induce MUC1 antigen-specific immune responses through the formation of immune complexes with circulating MUC1 and/or MUC1-expressing tumor cells that may target these immune complexes (IC) to receptors on dendritic cells (DCs). PATIENTS AND METHODS: A phase I study focusing on safety and immunology evaluated 1, 2 and 4-mg doses. Seventeen patients with MUC1-positive cancers received intravenous infusions of the antibody over 30 min on weeks 1, 3, 5, 9, 13 and 17 of treatment. RESULTS: mAb-AR20.5 was well-tolerated, not associated with dose-limiting toxicity, and did not induce hypersensitivity reactions. Overall, five of 15 evaluable patients developed human anti-mouse antibodies (HAMA), five developed anti-idiotypic antibodies (Ab2) and seven developed anti-MUC1 antibodies. Immune responses were most prominent in the 2-mg dose cohort for all parameters tested, and treatment-emergent MUC1-specific T-cell responses were detected in five of 10 evaluable patients treated with mAb-AR20.5. CONCLUSIONS: The injection of a murine antibody to MUC1 induces MUC1-specific immune responses in advanced cancer patients. Anti-MUC1 antibody increases correlated with decrease or stabilization of CA15.3 levels (P=0.03). The 2-mg dose of mAb-AR20.5 showed strongest biological activity, and will be evaluated in future efficacy trials.

Summary report on the ISOBM TD-6 workshop: analysis of 20 monoclonal antibodies against Sialyl Lewisa and related antigens. Montreux, Switzerland, September 19-24, 1997.

The ISOBM TD-6 Workshop is the first international workshop on monoclonal antibodies against the Sialyl Lewisa (SLea) antigen. Eight research groups participated in a blind study to characterize the epitope binding, relative affinity and performance in immunoradiometric assays, of a panel of 20 monoclonal antibodies. The antibodies were tested against a diverse panel of neoglycoconjugates, purified antigens and human serum pools from gastrointestinal malignancies. Epitope specificities were determined for the majority of antibodies in the panel. Cross-reactivity with related saccharide structures was noted in several antibodies. Overall, the results of the TD-6 Workshop show further development of SLea immunoassays may yield yet more specific assays for the detection and management of gastrointestinal and other malignancies.