Generation of CD4(+) and CD8(+) T lymphocyte responses by dendritic cells armed with PSA/anti-PSA (antigen/antibody) complexes.

Dendritic cells (DC) acquire antigens through a number of cell surface structures including receptors for the Fc portion of immunoglobulins and mannose. Little is known about the effects of antigen uptake via these receptors on antigen processing and presentation. We compared the capacity of DC to generate CD4(+) and CD8(+) T cell responses after exposure to prostate-specific antigen (PSA) alone, PSA targeted to the mannose receptor (mannosylated PSA (PSA-m)), or PSA targeted to Fc receptors by combining PSA with an anti-PSA antibody (AR47.47). Autologous CD3(+) T cells were added to monocyte-derived immature DC that had been cultured with GM-CSF/IL-4 for 4 days, exposed to antigen, and matured with CD40L or TNFalpha/IFN-alpha. After several rounds of stimulation, T cell responses were assessed by intracellular IFN-gamma production using flow cytometry. Both CD4(+) and CD8(+) T cell responses were observed after stimulation with DC exposed to the PSA/anti-PSA complexes, whereas CD4(+) predominated over CD8(+) T cell responses after stimulation with PSA-armed DC or PSA-m. These CD8(+) T cells responded when rechallenged with DC pulsed with HLA allele-restricted PSA peptides. These results indicate that PSA and PSA-m are processed primarily through pathways that favor HLA Class II presentation, while the PSA/anti-PSA immune complexes are processed through both Class I and Class II pathways in monocyte-derived DC. These findings have potential applications in designing more effective cancer vaccines for prostate cancer.

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.

Therapeutic antibodies for prostate cancer.

Twenty five years ago, monoclonal antibodies were envisioned as magic bullets capable of targeting radioisotopes, toxins or cytotoxic drugs to the tumor site. It was soon realized that the potential of therapeutic antibodies far exceeded their use as carrier molecules and that native antibodies could also act as effector molecules capable of triggering a wide variety of antitumor responses. Today, we recognize that the utility and versatility of antibody-based products are unlimited; at the same time we have also learned that many obstacles need to be addressed to make antibody therapy an effective treatment modality. Past experiences from clinical trials and new developments in the field of antibody technology have paved the way towards the creation of new strategies capable of circumventing or minimizing these difficulties. Antibody therapy is currently being tested in multiple trials for a variety of cancers, including prostate cancer. This review discusses the different antibody-based strategies currently under investigation for prostate cancer.

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).

Expression of a fusion protein of scFv-biotin mimetic peptide for immunoassay.

We constructed two fusion proteins of scFv linked to biotin mimetic sequence (BMS) via different linkers, and expressed them in the Pichia pastoris expression/secretion system. We found that both bi-functional scFv proteins exhibited their intrinsic binding activities to antigen CA125 determined in competitive radioimmunoassay experiments, but the fusion protein with a spacer between the scFv and BMS (scFv-spacer-BMS) showed higher binding activity of streptavidin than the one with c-Myc peptide as a linker.

Use of encapsulated single chain antibodies for induction of anti-idiotypic humoral and cellular immune responses.

The use of biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses has been investigated using a single chain antibody scFv-pDL10, which recognizes the human ovarian cancer antigen CA125. Immunization of mice with scFv-pDL10 encapsulated in PLGA microspheres resulted in enhanced humoral and cellular immune responses when compared to scFv-pDL10 alone. Induced anti-idiotypic antibodies (Ab2) which mimic the original antigen CA125 compete with CA125 for the epitope. A cellular response (T2 induction) was also observed. These results raise the possibility of anti-idiotypic antibody induction by a single chain antibody, encapsulated in biodegradeble microspheres, as a potential vaccine for ovarian carcinoma.

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres.

The use of biodegradable poly(DL-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(DL-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Abl can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer.

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.

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.

The effects of circulating antigen on the pharmacokinetics and radioimmunoscintigraphic properties of 99m Tc labelled monoclonal antibodies in cancer patients.

UNLABELLED: PURPOSE. This article reports the pharmacokinetics, radiation dosimetry and radioimmunoscintigraphy (RIS) of two (99m)Tc-labelled monoclonal antibodies (MAb) used to detect cancer. METHODS: The effects of circulating antigen in female cancer patients are explored and their effects on the ability of these MAbs to effectively perform as RIS agents noted. To illustrate the effects of circulating antigen, data using MAb B43.13 (OVAREX, AltaRex Corp., Waltham, MA, USA) from a Pilot study in ovarian cancer patients are presented. The results from a Phase II study of MAb 170H.82 (Tru-Scint AD, BIOMIRA INC., Edmonton, Alberta, Canada) in patients with primary and locally recurrent breast cancer were used to portray the biodistribution patterns when no circulating antigen is present. Data from planar gamma camera images were obtained for both groups and used for pharmacokinetic and radiation dosimetry analyses. RESULTS: A pharmacokinetic analysis indicated a shorter residence time and higher clearance of (99m)Tc-MAb-B43.13 that was ascribed in part to the circulating CA 125 antigen in this group of ovarian cancer patients. CONCLUSION: These clearance patterns resulted in acceptable, though higher radiation doses to the spleen and urinary bladder wall for these patients when compared to the MAb-170H.82 group. Both MAbs were found to produce acceptable radioimmunoscintigraphic images

Pharmacokinetics and radiation dosimetry of 99Tcm-labelled monoclonal antibody B43.13 in ovarian cancer patients.

OVAREX MAb B43.13 is a new radiopharmaceutical based on a monoclonal antibody (MAb-B43.13) known to recognize CA 125, a tumour antigen associated with epithelial ovarian cancer. This MAb is capable of facile radiolabelling with 99Tcm and has been shown previously to localize in the tumours of ovarian cancer patients. The present study was initiated to measure the pharmacokinetics of this MAb in the serum of 10 patients with primary or metastatic ovarian cancer. A two-compartment model was found to be best at representing the biodistribution of the 99Tcm-labelled MAb, yielding a 2.6 h distribution phase half-life and a 31.3 h elimination phase half-life. The serum and renal clearances for 99Tcm-MAb-B43.13 were 121 and 53 ml h-1 respectively. These parameters were compared with a similar model developed from the serum values of the MAb itself (determined using an ELISA detection method). Based on the serum pharmacokinetics of 99Tcm-MAb-B43.13 and whole-body planar gamma camera images, an estimate of the radiation dose from 99Tcm was calculated using standard MIRD schema. The organs demonstrating significant 99Tcm uptake included the liver, kidneys, heart and spleen. The whole-body dose was similar to other 99Tcm-labelled MAbs.

Radiolabeling of monoclonal antibody B43.13 with rhenium-188 for immunoradiotherapy.

In this study we report a novel method for direct radiolabeling of monoclonal antibody B43.13 (MAb-B43.13) with 188Re and have evaluated the product's radiochemical, biochemical, immunochemical and selected biological properties. 188Re-MAb-B43.13 was readily prepared by the addition of generator produced perrhenate to a preformulated antibody vial after an optimal amount of supplemental stannous ion, in the form of stannous tartrate, was added. The final radiolabeled product retained its biochemical purity (as determined by size-exclusion HPLC and R/NR-SDS-PAGE), its immunoreactivity (as determined by immunoassay) and presented with a typical stability (in the presence of serum and cysteine) and biodistribution (in tumored mice) profile. The evaluation of the product for immunoradiotherapy of ovarian cancer in a clinical setting requires further studies.

An engineered bivalent single-chain antibody fragment that increases antigen binding activity.

Bivalent single chain Fv (scFv) was constructed by fusing a polypeptide extension containing one or two cysteines to the COOH-terminus of an scFv antibody fragment. The scFv protein was expressed and secreted in a recombinant Pichia pastoris system as a dimer with a C-terminal disulfide bridge, as determined by Western blot analysis under non-reducing conditions. We found that the scFv construct with one cysteine in the C-extension (scFv-1Cys) exhibited a much higher dimer/monomer ratio than the two cysteine counterpart (scFv-2Cys). Binding activity measurements performed by means of a competitive radioimmunoassay showed that scFv-1Cys exhibited specific antigen binding activity, which was almost the same as that of the parental MAb, and approximately four- and fortyfold higher than those of the control scFv monomer and scFv-2Cys.

Role of transchelation in the uptake of 99mTc-MAb in liver and kidney.

High radioactivity in liver and kidney after administration of 99mTc-labeled antibodies is a major detriment to the use of radiolabeled antibodies for diagnosis and therapy. In the present study, the uptake mechanism of radioactivity by liver and kidney involving 99mTc moiety was investigated. The data of in vitro and in vivo thiol transchelation studies, biodistribution alteration of 99mTc-MAb after specific modulation of endogenous thiol containing compounds, and the finding of 99mTc-labeled cysteine and GSH in bile, urine and kidney after administration of 99mTc-MAb demonstrated that transchelation by thiols (cysteine and GSH) played an important role in the localization of radiotracer from 99mTc MAb in normal tissues such as liver and kidney.

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.

Epitope mapping of prostate-specific antigen with monoclonal antibodies.

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

Studies on metabolism of directly labeled 99mTc-antibody in mice.

The elucidation on the metabolic products of the 99mTc-antibody conjugates may provide insights and approaches that would reduce the undesirable deposition of radioactive species in normal tissues. In this investigation, the radiolabeled species in blood, urine, bile and extracts of liver and kidney obtained at different times after the injection of a model antibody, 99mTc-MAb170, into mice were analyzed with various chromatographic methods. Ninety-nine to 100% of the radioactivity in serum was associated with intact MAb170. The radioactivity in liver homogenate extract was strictly protein-bound to either intact MAb or low molecular weight species (LMW). In kidney extracts, the majority of the radioactivity was protein bound 99mTc, with less than 8% of the activity being non-protein bound 99mTc. Multiple 99mTc-containing protein and non-protein species were found in urine and bile. Evidence supporting the presence of 99mTc-cysteine and 99mTc-glutathione in bile, kidney and urine was also obtained. No evidence for the in vivo formation of 99mTc-pertechnetate in mouse blood, liver, kidney, bile and urine was observed.

Preparation and protein conjugation of a divinyl sulphone derivatized bifunctional chelating agent.

A new bifunctional chelating agent with a novel linking arm, 2-[p-¿N-benzyl-N-(2-vinylsulfoethyl)¿- (aminobenzyl)¿-1,3-propane-diamine-N,N,N',N'-tetraacetic acid (VS-PDTA) was synthesized and was conjugated to protein for the purpose of attaching radiometals to monoclonal antibodies (MAbs). The effect of various parameters such as ligand concentration, protein concentration, pH, temperature and reaction period on the conjugation have been examined using chromatographic (SE and TLC) analysis after labeling with 111In. The parameters and chemical variables studied have significant effects on the efficiency and rate of protein conjugation.

Generation of human monoclonal anti-idiotypic antibodies with specificity to the murine monoclonal anti-CA 125 antibody B43.13.

Two human monoclonal antibodies, HID-7E7 and ROB-6F2, were produced by EBV transformation of peripheral blood lymphocytes (PBL). PBL were obtained from a patient with ovarian cancer who had been exposed several times to a Tc-99m labeled murine monoclonal anti-CA 125 antibody (B43.13, Biomira, Edmonton) for immunoscintigraphy. The HID-7E7 and ROB-6F2 producing B-cells were cloned with a limiting dilution technique and have shown stable immunoglobulin secretion within a period of three years. The human monoclonal antibodies HID-7E7 and ROB-6F2 are of the IgG isotype, and bind with significant affinity to the murine monoclonal antibody B43.13, which was used for immunoscintigraphy. Binding affinity of ROB-6F2 to other murine antibodies could not be detected. Cross reactivity of HID-7E7 to a murine anti-CEA monoclonal antibody was observed. In order to verify the anti-idiotypic character of the generated human antibodies, the ability of HID-7E7 and ROB-6F2, respectively, to inhibit the formation of the CA125/B43.13 complex is demonstrated via an enzyme-linked immunosorbent assay. These human anti-idiotypic antibodies are possible candidates for immunotherapy of ovarian cancer in patients with a small tumor burden following surgery and/or chemotherapy.

An antibody-lectin sandwich assay for the determination of CA125 antigen in ovarian cancer patients.

A two-step forward sandwich assay was developed for the determination of the ovarian tumour associated glycoconjugate antigen CA125 with anti-CA125 Monoclonal antibody B27.1 on the solid phase and 125I-labelled wheat germ lectin as tracer in the solution phase. This Mab-lectin heterosandwich assay was optimized and the clinical utility was evaluated in sera from healthy volunteers and ovarian cancer patients. A correlation was established between Mab-lectin assay and the dual monoclonal antibody sandwich assay, TRUQUANTOV2 RIA, that uses the same MAb B27.1 on the solid phase and a second 125I-labelled B43.13 MAb in the solution phase. A potentially improved clinical utility is suggested for the Mab-lectin assay. The unique format seems to identify novel isoforms of CA125 with different carbohydrate side chains that would otherwise be undetectable in the MAb-MAb sandwich assay wherein the paratopes are likely directed to protein determinants.