Monoclonal Antibody B72 Research Articles

Site-directed spin-labeling of transmembrane domain VII and the 4B1 antibody epitope in the lactose permease of Escherichia coli.

Functional lactose permease mutants containing single Cys residues at positions 233-255 and a biotin acceptor domain at the C terminus were solubilized in dodecyl beta-d-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy (EPR). The EPR spectral line shapes and the influence of nonpolar O2 or polar potassium chromium oxalate relaxation agents on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to the relaxation agents, respectively. The results provide evidence that residues Ser233-Asn246 are within the hydrophobic core of the membrane and that Phe247 is at the lipid headgroup-solvent interface. Along with Phe247, Phe250 and Gly254 are also surface-exposed, as indicated by studies on the epitope for monoclonal antibody 4B1 [Sun, J., Wu, J., Carasco, N., and Kaback, H. R. (1996) Biochemistry 35, 990-998]. Furthermore, the nitroxide-labeled intramembrane Cys replacements exhibit variations in mobility and accessibility that are consistent with the conclusion that TM VII is an alpha-helix in contact with surrounding helices in the tertiary structure of the permease.

Comparison of synovial fluid cartilage marker concentrations and chondral damage assessed arthroscopically in acute knee injury

To examine the correlation between synovial fluid cartilage markers and degree of cartilage damage determined by arthroscopic evaluation in subjects with acute knee injury. Chondral damage was quantified using a validated arthroscopic scoring system in 20 subjects with effusive acute knee injuries of less then 4 months duration and no history or radiographic evidence of joint pathology. Levels of synovial fluid 3B3(-) neoepitope, 3B3(+) chondroitinase generated epitope of proteoglycan, keratan sulfate (KS) and hyaluronic acid (HA) were measured by competitive enzyme-linked immunosorbent assays using monoclonal antibodies 3B3 and 5D4. Total sulfated glycosaminoglycan (GAG) was measured by 1,9-dimethylmethylene blue colorimetric dye-binding assay. We found a dramatic decrease in levels of 3B3(-) (rs = -0.62, P = 0.004), and GAG (rs = -0.49, P = 0.03) with increasing chondral damage score; but no correlation of damage score with 3B3(+), KS or HA levels. These data reveal a change in cartilage metabolism within the first 4 months of symptomatic knee injury evinced by a significant inverse correlation of 3B3(-) and GAG levels to chondral lesion severity. These results suggest that serial measurement of these synovial fluid markers in the setting of acute knee injury could predict chondral lesion severity and aid in the decision to intervene surgically.

Generation and Characterization of an Anti-CD19 Single-Chain Fv Immunotoxin Composed of C-Terminal Disulfide-Linked dgRTA

Our laboratory utilized two methods to produce the anti-CD19 immunotoxin containing a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA). The first method produced the recombinant protein FVS191CDRTA from a fusing gene containing sequences encoding FVS191, catheptsin D proteinase digestion site (CD), and RTA. FVS191CDRTA did not show CD19 antigen binding and cytotoxic activity. The second method generated a disulfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an additional C-terminal cysteine, and a deglycosylated RTA (dgRTA). The formation of FVS191cys-dgRTA is efficient; up to 70% of the proteins participating in the reaction had formed FVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1. A competitive ELISA assay indicated that FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities. The protein synthesis inhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent than the intact antibody-conjugated B43-dgRTA, which had an IC50 = 2 x 10(-11) M. 125I-Labeled FVS191 and 125I-labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demonstrated by internalization studies; this result indicates that cross-linking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA is not due to the monovalent binding of CD19 by FVS191cys-dgRTA. Our study with anti-CD19 scFv immunotoxin indicates that the formation of a disulfide-linked scFv immunotoxin is an alternative to the recombinant method of producing scFv immunotoxin.

Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies.

We previously reported that the immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1 protects mice against disseminated candidiasis, whereas the IgM MAb B6 does not. Both MAbs are specific for an adhesin fraction isolated from the cell surface of Candida albicans, but their epitope specificities differ. In the present study, we examined the surface locations of both epitopes and obtained structural information regarding the B6.1 epitope. Immunofluorescence confocal microscopic analysis of C. albicans yeast forms showed that epitope B6.1 is displayed rather homogeneously over the entire cell surface, whereas epitope B6 appears to have a patchy distribution. Both antibodies were essentially nonreactive with the surfaces of mycelial forms of the fungus, indicating that neither epitope is expressed on the surfaces of these forms. For isolation of the B6.1 epitope, the adhesin fraction consisting of cell surface phosphomannan was subjected to mildly acidic (10 mM HCl) hydrolysis and was fractionated into acid-labile and acid-stable portions by size exclusion chromatography. Antibody blocking experiments showed that the B6.1 epitope is an acid-labile moiety of the phosphomannan and that the B6 epitope is located in the acid-stable fraction. The B6 epitope appeared to be mannan because it was stable to heat (boiling) and protease treatments but was destroyed by alpha-mannosidase digestion. The B6.1 epitope eluted from the size exclusion column in two fractions. Mass spectroscopic analyses showed that one fraction contained material with the size of a mannotriose and that the other was a mixture of mannotriose- and mannotetraose-size substances. Dose response inhibition tests of the fractions indicated that the B6.1 epitope is associated with the mannotriose. Nuclear magnetic resonance (NMR) spectroscopic analysis of the epitope yielded data consistent with a beta-(1-->2)-linked mannotriose. The fine structure of the B6 epitope is under investigation. Information derived from these investigations will be useful both in understanding protective versus nonprotective antibody responses to C. albicans and in improving anti-Candida vaccine formulations.

Evidence for clonal deletion and clonal anergy after intrathymic antigen injection in a transplantation model.

Intrathymic (IT) antigen injection has been shown to induce antigen-specific systemic tolerance in the rodent. To delineate the mechanisms responsible for the induction of tolerance, we used the 2C line of T cell receptor transgenic mice. The majority of T cells in 2C mice express an antigen receptor specific for the major histocompatibility complex class I alloantigen Ld and can be identified with the clonotypic monoclonal antibody 1B2. IT injection of lymphoid cells expressing Ld was found to induce a significant prolongation in BALB/c skin allograft survival. The allograft prolongation was associated with a marked reduction in the number of developing 1B2+ thymocytes (clonal deletion), which occurred primarily at the CD4+ CD8+ stage of thymocyte development, as well as a reduction in the number of mature CD8+ 1B2+ 2C T cells in peripheral lymphoid tissue. In addition, CD8+ 1B2+ 2C T cells that survive deletion have decreased CD8 expression levels and a significantly reduced in vitro proliferative response to specific alloantigen (clonal anergy). Exogenous recombinant interleukin 2 restores the capacity of 2C T cells to respond in vitro to alloantigen. Experiments involving separation of cells by fluorescence-activated cell sorter indicate that there is a precise correlation between the reduction in CD8 expression and anergy induction. Collectively, these data indicate that IT antigen injection can induce antigen-specific systemic tolerance by both clonal deletion and clonal anergy.

Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12.

Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.

Tumor-associated glycoprotein (TAG-72) in chronic submandibular sialadenitis and normal minor salivary glands defined by monoclonal antibody B72.3.

Formalin-fixed, paraffin-embedded samples of three normal minor salivary glands, 10 chronic submandibular sialadenitis, and three normal submandibular glands were studied immunohistochemically using the monoclonal antibody (Mab) B72.3 in order to have a better understanding of the distribution of tumor-associated glycoprotein (TAG-72). Diffuse expression of TAG-72 was observed in the mucous cells of normal minor salivary glands, and in the ducts with goblet cell metaplasia and/or hyperplasia of chronic submandibular sialadenitis (eight of 10). Focal expression of TAG-72 was seen in the acinar mucous cells of normal submandibular gland (three of three), and in the mucous cells of normal or atrophic acini of chronic submandibular sialadenitis (eight of 10). These results should be considered in the cytologic diagnosis of the mucoepidermoid carcinoma using the Mab B72.3 as a diagnostic aid, as well as in future studies for the radiation immunolocalization and immunotherapy of submandibular gland tumors.

Chemical and Immunological Assay of the Nonreducing Terminal Residues of Chondroitin Sulfate from Human Aggrecan

Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescence-based anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcAbeta1,3GalNAc4S and GlcAbeta1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were approximately 50% 4,6-disulfated in aggrecans from adults (22-72 years of age). Second, the terminal disaccharide GlcAbeta1,3GalNAc4S was on approximately 7% of chains on aggrecan from fetal through 15 years of age, but on only approximately 3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcAbeta1,3GalNAc6S, approximately 9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(-) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.

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.

Assessment of a mouse model of neutropenia and the effect of an anti-candidiasis monoclonal antibody in these animals.

As previously reported, monoclonal antibody (MAb) B6.1 increases resistance to hematogenous disseminated candidiasis in normal and SCID mice. In this study, MAb B6.1 was examined in a mouse model of neutropenia. The neutropenia was induced for a short period of time by a single dose of the anti-neutrophil antibody, MAb RB6-8C5, or for a protracted period by doses of MAb RB6-8C5 every other day. At low doses (< or = 25 microg/mouse), neutrophils were primarily affected, but at high doses (> or = 50 microg/mouse), lymphocytes were also depleted. Mice given either single or multiple doses of MAb RB6-8C5 were more resistant to experimental hematogenous disseminated candidiasis if they received MAb B6.1 before and after challenge with Candida albicans yeast cells intravenously. These results show the utility of MAb RB6-8C5 for induction of a protracted neutropenia in mice and demonstrate that MAb B6.1 can enhance resistance against candidiasis under neutropenic conditions.

Immunoreactivity of canine mammary neoplasms with monoclonal antibody CC49.

Monoclonal antibody (MAb) CC49 binds to human tumour-associated glycoprotein termed TAG-72. CC49 is a second-generation MAb with higher affinity to TAG-72 than the original MAb B72.3. CC49 was applied to 42 samples from different canine mammary tumours, belonging to seven different histopathological types. Immunoreactivity was detected by the use of an avidin-biotin complex immunoperoxidase method. Most sections from all types of mammary neoplasm reacted with this MAb. Normal tissue did not stain or stained only weakly. The results of this study suggest CC49 has selective immunoreactivity for a variety of canine mammary tumours, which seems superior to that reported with MAb 72.3. These results support the proposal for further study of diagnostic and therapeutic uses of CC49 in the management of canine mammary tumours.

Biochemical, histochemical, and immunohistochemical characterization of distal tibial osteochondrosis in horses

Abstract Objective To compare the biochemical, histochemical, and immunohistochemical profiles of articular cartilage from horses with naturally acquired distal tibial osteochondrosis (OC) with cartilage from a similar location in clinically normal horses. Animals 9 affected horses (group 1, 16 OC lesions) and 4 control horses (group 2, 8 normal osteochondral specimens). Procedure OC specimens were collected during arthroscopic removal of the fragment, and control specimens were collected by aseptic osteotomy. Uronic acid, total protein, total glycosaminoglycan (GAG), chondroitin sulfate (CS), and keratan sulfate (KS) contents were determined. Histomorphologic, histochemical, and immunohistochemical examinations were performed on specimens after snap freezing at −80 C and cryosectioning. Monoclonal antibodies (MAB) 3B3 and 5D4 were applied for location of epitopes of CS and KS, respectively. Results OC lesions had significantly lower quantity of uronic acid, total GAG, and CS, compared with normal cartilage. OC cartilage had significantly less intense staining with toluidine blue, along with irregular cellularity and tidemark characteristics, compared with normal cartilage. Monoclonal antibodies 3B3 and 5D4 stained OC cartilage, whereas MAB 5D4 did not stain control cartilage. Additionally, MAB 3B3 and 5D4 stained the fibrous tissue that was found firmly attached to the OC lesion located between the parent distal portion of the tibia and OC fragment. Conclusion OC cartilage lesions of the distal intermediate ridge of the tibia in horses are biochemically, histochemically, and immunohistochemically distinct from normal cartilage from the same location. Results may reflect the inability of the chondrocyte of the developing joint to alter matrix components that would allow proper maturation and differentiation into bone. (Am J Vet Res 1997;58:89–98)

Pharmacokinetics of iodine-125 CC49 monoclonal antibody in patients with colon cancer.

Radioimmunoguided Surgery techniques which use radiolabeled tumor specific markers and an intraoperative detector in an attempt to improve therapy and survival in patients with cancer have been under development for over fifteen years. Monoclonal antibody (MAb) CC49 is a second-generation murine IgG1 which has improved localization properties over its predecessor, MAb B72.3, and has been studied in a number of patients. In order to determine the pharmacokinetics of iodine-125 (125I) CC49 MAb, size-exclusion, high-performance liquid chromatography (HPLC) was used to assess radioactive components in serum and urine following administration of the drug to colon cancer patients. Five patients received an intravenous infusion of 10 mg of MAb CC49 labeled with 2 mCi 125I. Following infusion, serum and urine specimens were collected from patients at predetermined time intervals prior to surgery. HPLC analysis of these specimens was completed to determine the radioactive species in each sample. Serum and urine specimens showed that serum levels of CC49 decrease exponentially and become unmeasurable by day 14 (half-life 1.89 days, +/- 0.19), with a steady, low-level of free 125I measurable in postinjection serum until day 21 after infusion. There was no evidence of MAb fragmentation or antibody:antigen (Ab:Ag) complex formation in serum, and no evidence of whole MAb, F(ab')2, or Fab fragment excretion in urine. Preinjection sera with MAb added in vitro also failed to demonstrate Ab:Ag complex formation. Analysis of urine showed low level excretion of free 125I which peaked by day 1 and declined exponentially through day 21, with a very low molecular weight (< 1 kDa) MAb fragment excreted in urine between 1 and 21 days. Radioiodinated 125I CC49 MAb remains in serum of cancer patients approximately 14 days, and tissue radioactivity beyond this time may reflect tissue sequestered MAb and/or free 125I and not "bloo pool" radioactivity. CC49 MAb appears to be deiodinated in small but significant quantities before it is metabolized, and clearance of radioactivity is mainly in free 125I form in urine. Measurable quantities of a < 1 Kda MAb fragment in urine and not serum may suggest a renal mechanism of MAb metabolism, but may also represent a metabolic end product of MAb metabolism with a very short serum half-life (T1/2) which accumulates in urine.

Serum and tissue measurements of CA72-4 in patients with endometrial carcinoma.

To evaluate the clinical usefulness of CA72-4 as a serum tumour marker for endometrial carcinoma and to investigate its immunohistochemical localisation in endometrial carcinoma cells. Serum concentrations of CA72-4 were determined in 72 patients with endometrial carcinoma. Immunohistochemical localisation of CA72-4 was investigated using the streptavidin-biotin method, using monoclonal antibodies B72.3 and CC49. Serum CA72-4 was increased above the cut off value in 31.9% of the patients with endometrial carcinoma. Serum CA72-4 positivity was correlated with depth of myometrial invasion, adnexal metastasis, lymphovascular space involvement, and pelvic and para-aortic lymph node metastasis. Multivariate analysis showed a significant correlation between serum CA72-4 positivity and adnexal metastasis. The serum concentrations of CA125 and CA19-9, which could be tumour markers for endometrial carcinoma, were measured at the same time. In seven of 72 patients increased concentrations of serum CA72-4 were found while those for CA125 and CA19-9 were within the normal ranges; in four of the seven patients the disease had spread beyond the uterus. Immunohistochemical positivity for CA72-4 antigen was 76.9% and occurred in the tumour cell membrane and cytoplasm. There was no significant difference in immunohistochemical positivity between patients with increased CA72-4 and those with normal CA72-4 values. The measurement of serum concentrations of CA 72-4 could be useful for predicting and monitoring the progress of disease-for example, extracorporeal spread.

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.

Detection of pseudomyxoma peritonei by radioimmunohistochemistry and radioimmunoscintigraphy.

Pseudomyxoma peritonei (PP) is a local slowly progressing disease with typical abdominal swelling. Treatment is uneffective and the long-term prognosis is poor. Conventional radiology provides usually only a delineation of low density area relating gelatinous masses accumulating in the peritoneal cavity. In this study, immunohistochemistry based on digital quantitative autoradiography utilizing radiolabelled monoclonal antibody B72.3 (MoAb) recognizing TAG-72 antigen on epithelial carcinomas was used for diagnosis of pseudomyxoma (7 patients). The PP patients were studied with radioiodinated I-131-labeled MoAb after intravenous (2 patients) and intraperitoneal (7 patients) injections. Radioactivities of MoAbs varied considerably in the tumors. Both intra- and extracellular staining pattern was observed by immunohistochemistry. Gamma imaging at 1, 3, 7 and 14 days after i.v. injection (2 patients) revealed targeting of all known lesions. The intraperitoneally injected MoAb (7 patients) retained long time in the peritoneal cavity, specific tumour targeting was seen up to 16 days by an antibody-SPECT, while maximum blood radioactivity was measured between 8-12 hrs. Radiolabelled B72.3 MoAb recognizing TAG-72 antigen is also present within pseudomyxoma cells. It can be used for radioimmunohistochemistry of PP. Accurate imaging of PP is possible by MoAb suggesting earlier diagnosis and more accurate location of residual disease after operations, and evaluating treatment response. Estimated tumour dose for intraperitoneal tumour (MIRD formalism) was 13 mGy/MBq. This indicates that the radioiodinated B72.3 antibody can be used for in vivo targeting and therapeutic applications of intraperitoneal pseudomyxoma.

111In-CYT-103 scanning in recurrent colorectal cancer--does it affect standard management?

In a blinded fashion, radiolabeled B72.3 was investigated in operative cases of recurrent colorectal cancer to determine if diagnostic accuracy would be improved to ultimately maximize curability and minimize interventional morbidities. Study patients underwent conventional evaluation including history, physical examination, abdominal/pelvic computed tomographic scan (CT), colon examination, and carcinoembryonic antigen (CEA) determination, with select magnetic resonance imaging and ultrasonographic imaging as indicated. Murine monoclonal antibody B72.3 was labeled with indium-111 (111In-CYT-103 provided by Cytogen) and scans obtained at 48 hours and, selectively, at 72 and 96 hours. Unlike previous studies, the operating surgeon was blinded to 111In-CYT-103 abdominal scan results until surgical exploration was complete. Of 15 study patients (10 male; 5 female), average age was 57 years, and average CEA was 10 ng/ml (with eight elevated CEA levels). A single patient did not undergo surgery because of presence of pulmonary metastases identified on CT scan but not identified on a 111In-CYT-103 scan. Laparotomies included resection and intraoperative radiation (10), resection alone (1), and biopsy only (3). CT and 111In-CYT-103 scans were compared with operative findings. CT scans had an accuracy and positive predictive value of 47 and 100 percent, respectively, whereas those of 111In-CYT-103 scan were 60 and 82 percent, respectively. Contribution of the scan to diagnosis and management was graded by the surgeon as no effect (67 percent), beneficial effect (13 percent), or negative effect (20 percent). 111In-CYT-103 was more accurate compared with CT scan, but when value of the scan was examined with respect to its potential contribution to patient management, it was beneficial in only 13 percent of patients. Further refinements may enhance the value of antibody imaging techniques.

Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation.

B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.

Monoclonal antibody 4B1 alters the pKa of a carboxylic acid at position 325 (helix X) of the lactose permease of Escherichia coli.

A carboxylic acid at position 325 in helix X is obligatory for lactose/H+ symport at a step corresponding to deprotonation of lactose permease [Carrasco, N. et al. (1989) Biochemistry 28, 2533-2539]. In this paper, pH profiles for active transport, efflux, and equilibrium exchange are analyzed for wild-type permease and mutant Glu325-->Asp. With respect to active transport and efflux down a concentration gradient, both of which involve net H+ translocation and are defective in the mutant, the wild-type and the mutant exhibit similar profiles, and at no pH is the mutant stimulated relative to the wild-type. Strikingly, exchange which does not involve H+ translocation is comparable in the wild-type and the Glu325-->Asp mutant below pH 7.5. Above pH 7.5, however, the exchange activity of the mutant is progressively and reversibly inhibited with a midpoint at about pH 8.5; while the exchange activity of wild-type permease is only mildly decreased above pH 9.5, and exchange by Glu325-->Ala or Glu325-->Gln permease is comparable to wild-type and unaffected by pH. The findings are consistent with the idea that translocation of the ternary complex between the permease, lactose, and H+ does not tolerate a negative charge at position 325. In wild-type permease, the electrostatic interaction between Glu325 (helix X) and Arg302 (helix IX) is sufficiently strong that the carboxylate is unaffected by pH. In contrast, with Asp at position 325, the electrostatic interaction is broken, the carboxylate becomes protonated, and the acid exhibits a pKa of about 8.5. Monoclonal antibody 4B1 binds to the periplasmic loop between helices VII and VIII of the permease [Sun, J. et al. (1996) Biochemistry 35, 990-998] and mimics the Glu325 mutants. Dramatically, 4B1 shifts the apparent pKa for exchange from about pH 8.5 to 7.5 in the Glu325-->Asp mutant with little or no effect on the wild-type or the Glu325-->Ala mutant. The findings are consistent with the conclusion that the uncoupling effect of 4B1 involves a conformational change in helix VII and/or VIII that secondarily alters the pKa of the essential carboxylic acid at position 325.

Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli.

Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance. Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants. Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism. Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation.