Construction and purification of a covalently linked divalent tandem single-chain Fv antibody against placental alkaline phosphatase

Multivalency is a recognized means to increase the functional affinity of single-chain antibody fragments (scFvs) for optimizing tumor uptake at radioimmunotargeting. A unique divalent tandem single-chain Fv antibody [sc(Fv)2], based on the variable regions of the monoclonal antibody (MAb) H7, has now been generated. The antibody differs from other dimeric single-chain constructs in that a linker sequence (L) is introduced between the repeats of VL and VH domains (VL-L-VH-L-VL-L-VH). This construct was expressed as a His-tagged TrxA fusion protein in the Escherichia coli strain Origami B. Following cleavage with AcTev protease, the antibody was obtained in soluble and active form in E. coli and could be purified by Ni-NTA and cation-exchange chromatography. Purity and immunochemical properties were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), Western blot, and Biacore analyses. The [sc(Fv)2] displayed proper stability and could be purified to homogeneity. This antibody also maintained immunoreactivity at 42 degrees C with only slight decrease at 52 degrees C. The high affinity displayed by the original antibody H7, 6.7 x 10(9) M(-1), was only slightly decreased to 1.2 x 10(9) M(-1) as determined by Biacore. The generation of such a divalent single-chain Fv with a molecular weight around 60 kd would be of value for clinical applications such as radioimmunolocalization and radioimmunotherapy.     

Construction, expression and characterisation of a single chain anti-tumour antibody (scFv)-EL-2 fusion protein

We describe the production and preliminary characterisationof a fusion protein between interleukin-2 and a single-chainFv version of the H17E2 anti-placental alkaline phosphatase(PLAP) antibody. This molecule could be used to target interleukin-2to PLAP-expressing tumours cytokines, fusion protein, interleukin-2, monoclonal antibodies, single-chain antibodies (scFv), targeted immunotherapy     

Immunohistochemical localization of placental alkaline phosphatase, carcinoembryonic antigen, and cancer antigen 125 in normal and neoplastic human lung

Human placental alkaline phosphatase (HPLAP), carcinoembryonic antigen (CEA), and cancer antigen 125 (CA 125) were localized immunohistochemically in paraffin sections of normal lung tissue from 16 patients, using monoclonal antibodies and an indirect avidin-biotin-peroxidase staining procedure. HPLAP and CEA were present in epithelial cells of respiratory bronchioli and alveolar type I pneumocytes. CEA was also observed in the tracheal, bronchial, and bronchiolar epithelium. CA 125 was present in the tracheal, bronchial, bronchiolar, and terminal bronchiolar epithelium; in the tracheal and bronchial glands; and in the pleural mesothelium. Normal and hyperplastic type II pneumocytes were negative for HPLAP, CEA, and CA 125 but were histochemically positive for nonspecific alkaline phosphatase. Fetal lung tissue between 11 and 15 weeks of gestation was negative for HPLAP, CEA, and CA 125. The fetal tracheal and bronchial epithelium, tracheal glands, and pleural mesothelium were positive for CA 125. For ten malignant pulmonary tumors investigated, HPLAP staining was observed in five, CEA in nine, and CA 125 in seven. The localization of HPLAP, CEA, and CA 125 in apparently normal constituents of all pulmonary specimens is in disagreement with the concept that the expression of these substances in the lung is indicative of abnormal cellular activity.     

Radioimmunodetection of human choriocarcinoma xenografts by monoclonal antibody to placental alkaline phosphatase

Placental alkaline phosphatase (PLAP)-specific monoclonal antibody (MAb) 11-D-10, which did not react with other isoenzymes of alkaline phosphatase (AP), was raised by a hybridoma technique. MAb 11-D-10 was radiolabeled and administered to athymic mice bearing human choriocarcinoma containing PLAP. This antibody was found to be more specifically localized in tumor tissue as compared to normal tissues. The tissue-to-blood ratio (T/B ratio) of MAb 11-D-10 in tumor tissue increased from 1.38 at 2 days to 2.51 at 5 days after administration. On the other hand, the T/B ratios of isotype control non-immunized IgM in tumor tissue were 0.72 and 0.87 at 2 days and 5 days after administration, respectively. 131I-labeled MAb 11-D-10 was administered to athymic mice bearing choriocarcinomas of various sizes and various PLAP contents to examine the effect on the radioimage of the differences in tumor size and PLAP content. Tumors less than 0.3 cm in diameter could be imaged clearly by gamma-scintigraphy without blood pool image subtraction. The strength of the radioimage correlated fairly well with PLAP content.     

Tumor markers in mammary carcinoma. An evaluation of carcinoembryonic antigen, placental alkaline phosphatase, pseudouridine and CA-50

In 104 patients with breast cancer, carcinoembryonic antigen (CEA), placental alkaline phosphatase (PLAP) and the carbohydrate antigen CA-50 were analysed in serum. Excretion of the modified nucleoside, pseudouridine, was analysed in urine. The patients were subdivided in three different clinical stages according to disease manifestations. Levels of CEA and pseudouridine correlated to clinical stage and 58 per cent of the patients with distant metastases had elevated levels of CEA, compared with 36 per cent for pseudouridine. For PLAP and CA-50, the levels did not show any clear correlation to clinical stage. Increased activity of PLAP correlated strongly to tobacco smoking. A decrease in the level of CEA was observed following radical mastectomy. Increase in CEA levels predicted relapse in 5 out of 14 patients within about 3 to 6 months. In patients with tumor manifestations, elevated CEA levels predicted an inferior prognosis compared to those with ordinary levels.     

Monoclonal antibody assay of serum placental alkaline phosphatase in the monitoring of testicular tumours

A monoclonal antibody (H17E2) recognising both placental alkaline phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase was incorporated in a solid phase immunoassay. This was used to measure levels of PLAP in 257 sera from 148 patients with germ cell neoplasms of the testis. High levels of PLAP were found in all patients with active seminomas (mean 0.85 O.D.) compared to those in clinical remission (mean 0.20 O.D.) (P less than 0.0001). More importantly, changing levels of PLAP correlated with the course of disease in 79 samples from 33 patients with seminoma (P less than 0.0001). Elevated PLAP levels were also noted in patients in remission who were smokers (mean 0.32 O.D.) compared to non-smokers (mean 0.15 O.D.) (P less than 0.001). These data demonstrate that determination of PLAP levels using this sensitive immunoassay is an important new adjunct in the monitoring of the response to treatment in patients with seminoma.     

Expression on cultured human tumour cells of placental trophoblast membrane antigens and placental alkaline phosphatase defined by monoclonal antibodies

The cellular reactivity of six monoclonal antibodies (McAbs) produced to isolated human placental syncytiotrophoblast microvillous plasma membranes has been examined using a variety of normal and malignant cell types. Two McAbs reacted with antigenic determinants common to most normal human cells. Two other McAbs (H310 and H316) reacted predominantly with normal placental trophoblast and with lymphocytic cells, as well as with most transformed or neoplastic cultured cell lines. Two further McAbs (H315 and H317) identified foetal differentiation antigens expressed only on the membranes of normal placental trophoblast and of certain tumor cell lines. H317 has been shown to be specific for the heatstable L-phenylalanine-inhibitable placental-type alkaline phosphatase isoenzyme. These latter McAbs (H315 and H317) may prove useful in monitoring of some human cancers.     

Expression of placental alkaline phosphatase in gastric and colorectal cancers. An immunohistochemical study using the prepared monoclonal antibody

The authors developed monoclonal antibodies (MoAb) against human placental alkaline phosphatase (PLAP). Four specific MoAb reacting only with PLAP and two nonspecific MoAb reacting equally with isozymes of alkaline phosphatase (hepatic, intestinal, and placental) were obtained. Immunohistochemical staining with the specific MoAb showed that the cell membrane and cytoplasm of cancer cells were stained in gastric and colorectal carcinoma. The incidence of PLAP positivity was 23% (25 of 107) of all gastric carcinomas. Among gastric carcinomas, the 42% (13 of 31) positivity of highly differentiated carcinoma (papillary adenocarcinoma and well-differentiated tubular adenocarcinoma) was a significantly higher rate than that found in poorly differentiated carcinoma (poorly differentiated adenocarcinoma and signet-ring cell carcinoma, five of 41, 12%). The incidence of PLAP positivity was 11% (four of 35) in colorectal carcinoma. In contrast, gastric adenoma, intestinal metaplasia, and noncancerous tissue adjacent to cancer did not show staining. These results indicated that expression of PLAP was apt to occur in more highly differentiated gastric carcinoma and was highly specific for carcinoma in the gastrointestinal tract, although its incidence was not high.     

Demonstration of placental alkaline phosphatase in human breast cancer.

The screening of a series of 11 metastatic breast tumors for the presence of the placental isoenzyme of alkaline phosphatase (EC3.1.3.1) by RIA revealed one strong producer. The alkaline phosphatase of this tumor was characterized with respect to its immunochemical cross-reactivity, inhibition by L-phenylalanine and levamisole, subunit molecular weight (Mr) and isoelectric point (pl) in two-dimensional electrophoresis, and one-dimensional peptide map. In all parameters of the characterization, the tumor alkaline phosphatase, except for subunit molecular weight which was slightly lower (60,000 versus 64,000 for the placental isoenzyme). No strong placental alkaline phosphatase producers were found among 16 primary tumors examined by RIA. The screening of patients' sera for the placental alkaline phosphatase using RIA indicated elevated levels over post-menopausal controls in 20% of the metastatic patients. Only 3% of the primary patients had elevated serum levels. These results suggest that the placental isoenzyme of alkaline phosphatase may be a useful tumor marker for recurrent breast cancer.     

A simple radioimmunoassay of human placental alkaline phosphatase (Regan isoenzyme) using specific antibody polymers.

Rabbit antiserum against highly purified high-molecular-weight B-variant of human placental alkaline phosphatase (M.W. 200,000) was rendered monospecific by absorption with polymerized pooled male serum proteins; the absorbed antiserum was then polymerized with ethyl chloroformate and used in radioimmunoassay as a stable solid-phase immunoabsorbent. Homogeneous preparation of the enzyme, with a specific activity of 477 mumoles phenol per mg per min, was also obtained by absorbing the chromatographically purified enzyme with polymerized rabbit antiserum directed to whole human serum proteins; the pure enzyme was then labeled with 125-I as the tracer retaining at least 80% of its antigenicity. Only a minute quantity of the polymerized antibody particles is required for each assay in admixture with the labeled and unlabeled enzyme. By adding a small amount of starch-gel particles before low-speed centrifugation, complete phase separation was achieved. The radioimmunoassay could detect 0.4 to 0.8 ng enzyme protein per tube, which is comparable to the sensitivity achieved by enzymic assays. However, radioimmunoassay is advantageous over the enzymic assay in being direct, specific (no interference by the nonplacental-type alkaline posphatase), and capable of detecting both catalytically active and inactive forms of the enzyme. Native variants of placental-type alkaline phosphatase including Regan isoenzyme and Nagao isoenzyme (D-phenotype of normal placental alkaline phosphatase), could thus be directly determined by this procedure in the clinical specimens.     

Placental alkaline phosphatase as a tumour marker in seminoma using the H17 E2 monoclonal antibody assay

Serum samples from 62 patients with seminoma were assayed for placental alkaline phosphatase-like activity using the monoclonal antibody H17 E2, in order to evaluate its utility as a serum tumour marker. Fifteen of 16 patients (94%) with active seminoma had elevated serum PLAP levels. Sixteen of 46 (35%) of patients considered to be in remission had elevated PLAP levels (false positive rate 35%). Fifteen false positive results were considered attributable to concomitant smoking, and if these patients are excluded, only one false positive case was detected. In 7 out of 7 patients sequential PLAP assays reflected clinical response to treatment.     

Selective cytotoxic effects of a ricin A chain immunotoxin made with the monoclonal antibody SWA11 recognising a human small cell lung cancer antigen

The potential of mouse monoclonal antibodies for recognising different antigens associated with human small cell lung cancer (SCLC) to form active immunotoxins was assessed by an indirect in vitro screening assay. The screening agent used was a conjugate made by linking ricin A chain to a sheep anti-mouse IgG Fab' fragment via a disulphide bond. The monoclonal antibodies SWA11 and SWA20 both mediated the toxic effects of ricin A chain against the HC12 classic SCLC cell line in dose-dependent fashion. The SWA11 antibody was the more effective; in combination with the screening agent at a concentration of 1 x 10(-7) M, it inhibited the incorporation of [3H] leucine into HC12 cells by 94% compared with only 44% inhibition in the case of SWA20. An immunotoxin made by the direct chemical conjugation of ricin A chain to SWA11 exhibited selective toxic effects upon HC12 cells in tissue culture inhibiting the incorporation of [3H] leucine by 50% at a concentration (IC50) of 6.2 x 10(-10) M and by 98% at 1 x 10(-7) M. SWA11-ricin A chain had an IC50 of 4.4 x 10(-10) M against the NCI-H69 classic SCLC cell line but showed no cytotoxic activity against the human lung adenocarcinoma cell line NCI-H23 at a concentration of 1 x 10(-8) M.     

Carcinoembryonic antigen, alpha-fetoprotein and hepatoma alkaline phosphatase in gastric carcinoma

Reported here is a 38-year-old woman who had a gastric cancer accompanied with liver metastasis. Abnormal serum levels of a carcinoembryonic antigen, alpha-fetoprotein, and an alkaline phosphatase isozyme were observed persistently after a gastrectomy. The properties of this alkaline phosphatase isoenzyme were identical to a hepatoma alkaline phosphatase type. Histologic findings of the stomach revealed a poorly differentiated adenocarcinoma. The patient died on the 180th postoperative day.     

Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase

Recombinant technologies to engineer ordinary hybridoma monoclonal antibodies (MAbs) to single-chain fragment variable (scFv) may cause loss of antibody affinity, increased tendency to aggregate, increased temperature sensitivity, and low yield of active protein. In the present investigation, the well-characterized MAb H7 against placental alkaline phosphatase (PLAP), used as a model antibody, was engineered to improve solubility and stability of scFv with retained high affinity. The original procedure to generate single-chain antibodies with a 10-amino acid linker between VH and VL yielded an almost insoluble product. By site-directed mutagenesis, four selective sequence substitutions were made in the VL fragment and one in the VH fragment to improve solubility. The importance of the linker length was investigated, and a 25/30 amino acid linker was found to improve solubility. In order to further increase the stability of the single-chain antibody, an additional covalent -S-S- bond was introduced between amino acid 100 in the VL fragment and amino acid 44 in the VH region, to make a single-chain disulphide stabilized variable fragment (scdsFv). Altogether five different antibody constructs were produced and compared in terms of solubility, stability, affinity, and production properties. Immunospecificity was tested by enzyme-linked immunosorbent assay (ELISA) against the target antigen, temperature sensitivity by exposing the purified scFv to higher temperatures. All the new constructs retained almost equal activity and high affinity for their target antigen, placental alkaline phosphatase (PLAP), compared to the intact MAb H7, up to +42 degrees C as evaluated by ELISA. The overall affinity K(A) > 10(9) (M(1)) of the new antibodies could be maintained in the same order of magnitude as the original one (H7), when evaluated by Biacore technology. The best final single-chain antibody was obtained by performing the specific site-directed mutations and introducing a linker of 30 amino acids, but not by additional stabilizing disulphide bonds. The yield of the final antibody was improved approximately 10-fold by the modifications. This antibody could easily be expressed in a bacterial system using the PET-32a TrxA vector and the Escherichia coli strain BL21 Origami B (DE3). Purified antibody, which could be kept at concentrations up to 0.8 mg/mL, was obtained, which is sufficient for clinical testing of therapeutic applications.     

Carcinoembryonic antigen, alpha-fetoprotein and carcinoplacental alkaline phosphatase in gastric carcinoma metastatic to the liver.

Carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and carcinoplacental alkaline phosphatase (CPALP) were detected simultaneously in the sera and body fluids of two male patients with gastric carcinoma matestatic to the liver. At autopsy, widely disseminated gastric cancer of Borrmann III type with liver metastases was revealed in both bases. Histologically, they were moderately differentiated tubular and papillary adenocarcinomas with marked cellular atypia and necrosis. In Case 1, the properties of CPALP were identical to Nagao type CPALP, and in Case 2 the Variant type CPALP. Using immunofluorescence, CEA and CPALP were demonstrated in both primary and metastatic cells. However, only in Case 2 was AFP observed in some of the primary tumor cells.     

Passive immunotherapy of mice bearing Ehrlich ascites tumor expressing human, membrane-bound placental alkaline phosphatase.

The objective of our study was to test if a tumor expressing a transgene coding for a membrane-bound protein is amenable to immunotherapy by antibodies to the same protein. To this end, we have established an Ehrlich ascites tumor (EAT) cell line, EAT-DAP, stably expressing human, membrane-bound placental alkaline phosphatase (PLAP) by infecting EAT cells (EATC) with the retroviral vector DAP and selecting neomycin-resistant cells. EATC and EAT-DAP cells grew at similar rates in vitro, and produced ascites tumor in Swiss-Webster mice with similar efficiency. We have treated mice bearing EAT-DAP ascites tumor with a mouse monoclonal antibody to human PLAP or with a monoclonal antibody to human C proteins of the heterogenous ribonucleoprotein complex (hnRNP). The average survival of mice treated with anti-hnRNP was 16.4 +/- 1.1 days (n = 8). Treatment with anti-PLAP prolonged the survival of mice; in 4 mice average survival was 23.3 +/- 5.7 days. Four animals, however, survived for 60 days when they were killed and had no visible signs of tumor. These data support the notion that passive immunotherapy using antibodies against a membrane protein, expressed in tumor cells transduced by a viral vector coding for that protein, may be effective in controlling tumor growth.     

A bispecific monoclonal antibody against methotrexate and a human tumour associated antigen augments cytotoxicity of methotrexate-carrier conjugate

A bispecific monoclonal antibody, reactive with methotrexate (MTX) and a tumour associated antigen (gp72) has been produced by fusing spleen cells from MTX immunised mice with 791T/36/3 (anti-gp72) hybridoma. The hybrid antibody was purified from anti-MTX and anti-gp72 antibodies present in the hybridoma culture supernatant by combinations of affinity chromatography on a MTX-agarose immunoabsorbent and stepwise acid elution from Sepharose-Protein A. A particular feature of the present antibody is that it reacts with conjugated MTX; this would allow in vivo targeting of conjugates, increasing many fold the number of molecules of drug carried or localising to pre-targeted antibody. Dual binding between tumour cell surface antigen and MTX was demonstrated by the ability of the hybrid antibody to bridge between tumour cells and MTX as MTX-HSA conjugate, reaction here being detected by flow cytofluorimetry. Purified hybrid antibody specifically enhanced the in vitro cytotoxicity of MTX-HSA for gp72 positive tumour cells.     

Examination of the oncogenic potential of a tumor-associated antigen, intestinal alkaline phosphatase, in HeLa x fibroblast cell hybrids.

An exclusive correlation exists between the ectopic expression of the cell-surface marker, intestinal alkaline phosphatase (IAP), and the tumorigenic phenotype of segregants derived from suppressed, nontumorigenic HeLa x fibroblast cell hybrids. This specific association suggests that loss of tumor suppressor function is closely linked to the re-expression of IAP and, therefore, that IAP may be a critical oncogenic factor in these cells. To address this directly, we have used a HeLa IAP cDNA expression vector (pHIAP) to introduce constitutive IAP expression into a nontumorigenic HeLa x fibroblast cell hybrid. Sequence analysis of the HeLa IAP cDNA revealed 5 separate nucleotide alterations when compared to the native IAP cDNA sequence; however, none of these resulted in amino acid substitutions. Four pHIAP transfectants were analyzed for the presence of the intact integrated IAP cDNA and their relative expression levels of exogenous IAP mRNA and protein. The functional integrity of the cDNA-derived IAP product was confirmed by demonstrating proper enzymatic activity and localization to the extracellular membrane. The tumorigenic potentials of the pHIAP transfectants were assayed by s.c. injection into athymic nude mice. No tumors were observed, even after an 11-week incubation in animals. Therefore, expression of IAP in the nontumorigenic HeLa x fibroblast cell hybrid is not sufficient to confer the tumorigenic phenotype. Although ectopic IAP expression is unlikely to be functionally relevant to tumorigenicity in these hybrids, the significance of IAP as a tumor marker is still evident from its apparent strong association with a tumor suppressor locus.