Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds.

Seed-based expression system is an attractive platform for the production of recombinant proteins in molecular farming. Despite the many advantages of molecular farming, little is known about the effect of the different subcellular accumulation of recombinant proteins on the endoplasmic reticulum (ER) quality control system in host plants. In this study, we analyzed the expression of anti-CD20 antibody fragments in seeds of Arabidopsis thaliana (ecotype Columbia) and corresponding glycosylation mutants, and evaluated the influence of three different signal sequences on the expression levels of scFv-Fc of C2B8. The highest protein accumulation level, with a maximum of 6.12 % total soluble proteins, was observed upon fusing proteins to the signal peptide of Arabidopsis seed storage albumin 2. The ER stress responses in developing seeds at 13 days post-anthesis were also compared across different transgenic lines under normal and heat shock conditions. Based on the gene expression profiles of ER stress transducers, our results suggest that accumulation of antibody fragments in the ER exerts more stress on ER homeostasis. In addition, quantitative PCR results also implicate enhanced activation of ER-associated degradation in transgenic lines. Last but not the least, we also demonstrate the anti-tumor potency of plant-derived proteins by showing the anti-tumor activity of purified scFv-Fc proteins against Daudi cells. Together, our data implies that better understanding of the interaction between exogenous protein production and the cellular quality control system of the host plant is necessary for the development of an optimal expression strategy that will be especially beneficial to commercial protein manufacturing.

Human monoclonal antibody fragments targeting matrilin-3 in growth plate cartilage.

PURPOSE: Many genetic disorders, including chondrodysplasias, and acquired disorders impair growth plate function, resulting in short and sometimes malformed bones. There are multiple endocrine and paracrine factors that promote chondrogenesis at the growth plate, which could potentially be used to treat these disorders. Targeting these growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues. METHODS: Using yeast display technology, we selected single-chain variable antibody fragments that bound to human and mouse matrilin-3, an extracellular matrix protein specifically expressed in cartilage tissue. The ability of the selected antibody fragments to bind matrilin-3 and to bind cartilage tissue in vitro and in vivo was assessed by ELISA and immunohistochemistry. RESULTS: We identified antibody fragments that bound matrilin-3 with high affinity and also bound with high tissue specificity to cartilage homogenates and to cartilage structures in mouse embryo sections. When injected intravenously in mice, the antibody fragments specifically homed to cartilage. CONCLUSIONS: Yeast display successfully selected antibody fragments that are able to target cartilage tissue in vivo. Coupling these antibodies to chondrogenic endocrine and paracrine signaling molecules has the potential to open up new pharmacological approaches to treat childhood skeletal growth disorders.

In vitro effects of antivascular endothelial growth factors on cultured human trabecular meshwork cells.

PURPOSE: To investigate the effects of a neutralizing vascular endothelial growth factor antibody and antibody fragment as well as vehicle components on primary cultures of human trabecular meshwork (TM) cells. METHODS: Assays of cellular metabolism were performed using the diphenyl tetrazolium bromide assay in confluent cultures of cells. Proliferative effects were determined by measuring 5'-bromo-2'-deoxyuridine uptake in subconfluent cultures of cells. RESULTS: Twenty-four-hour treatment with 4 mg/mL bevacizumab reduced TM metabolism to 34.4 ± 12.4% (mean ± SD) as compared with human immunoglobulin G controls (P<0.0001). 4 mg/mL bevacizumab also reduced TM cell proliferation to 62.7 ± 9.2% of controls (P<0.0001). No significant decrease was seen at 2 mg/mL bevacizumab, or with molar equivalents of the related anti-vascular endothelial growth factor agent ranibizumab. Exposure of TM cells to the components of bevacizumab and ranibizumab vehicle did not lead to significant antimetabolic effects. CONCLUSIONS: Our data reveal that high concentrations of bevacizumab are harmful to TM cells in vitro whereas no such effect was noted with human immunoglobulin G controls or ranibizumab. Further studies are needed to better understand the antimetabolic effects of higher concentrations of bevacizumab on intraocular cell lines and whether smaller concentrations may have a similar effect on TM cells after repeated exposures.

Cytotoxic tumor targeting with scFv antibody-modified liposomes.

Specific targeting of liposome-formulated cytotoxic drugs or antigens to receptors expressed selectively on target cells represents an effective strategy for increasing the pharmacological efficacy of the delivered molecules. We have developed a feasible technique to selectively attach antibodies and fragments thereof, but also small-mol-wt ligands such as peptides, carbohydrates, or any molecules that recognize and bind target antigens or receptors to the surface of small unilamellar liposomes. Our concept is based on the site-specific functionalization of the ligands to be attached to the liposomes by thiol groups. These thiol groups can easily be introduced to antibodies or peptides by addition of cysteines, preferably at sites that do not interfere with the receptor binding domains. Optimally, the site-specific modification is introduced at the C-terminal end of the ligand, separated by an inert spacer sequence located between the thiols and the specific part of the ligand. The thiol-reactive molecules on the liposome surface are maleimides that are linked to phospholipids composing the liposome bilayer membrane. We illustrate the coupling method of a functionalized single-chain antibody fragment with binding specificity to ED-B fibronectin, an isoform of fibronectin exclusively expressed in tumor tissues, to long circulating small unilamellar poly(ethylene glycol) liposomes.

Targeting her-2/neu with antirat Neu virosomes for cancer therapy.

HER-2/neu (p185(HER2)) oncogene represents an attractive target for antibody-mediated immunotherapy. The major problem of combining chemotherapy and immunotherapy is the severe side effects that limit the use of doxorubicin (Doxo) as a cytotoxic drug. We have used virosomes (Vir; reconstituted fusion-active viral envelopes) as a new drug delivery system and have shown that Vir are capable of binding and penetrating into tumor cells, delivering cytotoxic drugs. We have additionally demonstrated that conjugating Fab' fragments of an antirat Neu (anti-rNeu) monoclonal antibody to Vir selectively and efficiently inhibits tumor progression of established rNeu-overexpressing breast tumors. Fab'-Doxo-Vir combine the antiproliferative properties of the monoclonal antibody and the cytotoxic effect of Doxo in vivo. Furthermore, Fab'-Doxo-Vir significantly inhibit tumor formation at a tumor load representing metastatic spread. These results indicate that Vir conjugated with an antibody against a tumor antigen are a promising new selective drug delivery system for the treatment of tumors expressing a specific tumor antigen.

Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.

Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.

Synergistic interaction between an anti-p185HER-2 pseudomonas exotoxin fusion protein [scFv(FRP5)-ETA] and ionizing radiation for inhibiting growth of ovarian cancer cells that overexpress HER-2.

OBJECTIVES: The HER-2 proto-oncogene is overexpressed in 15-30% of ovarian cancers, providing a target for therapy in a fraction of patients. We previously described the ability of a recombinant single-chain anti-p185HER-2-Pseudomonas exotoxin A fusion protein, scFv(FRP5)-ETA, to inhibit growth of cancer cells in culture and tumor xenografts in vivo. We have also described synergistic interaction between an anti-p185HER-2-ricin A chain immunotoxin and ionizing radiation for inhibiting growth of ovarian and breast cancer cells that overexpress HER-2. Our objective in this report was to evaluate the in vitro and in vivo effects of scFv(FRP5)-ETA against SKOv3 ovarian cancer cells that have been transfected to express >10(6) p185HER-2 receptors per cell (clone-9002-18). METHODS: Inhibition of clonogenic growth was measured in vitro by limiting dilution analysis. Inhibition of tumor growth was measured in vivo using heterografts established with the same ovarian cancer cell lines. RESULTS: Clone-9002-18 cells were substantially more sensitive than parental SKOv3 cells to the cytotoxic effects of scFv(FRP5)-ETA. Exotoxin fusion protein induced apoptosis in clone-9002-18 cells, but did not affect parental SKOv3 cells. Treatment of clone-9002-18 cells with 200- to 2000-cGy external beam irradiation in combination with scFv(FRP5)-ETA produced synergistic elimination of clonogenic tumor cells in culture. In vivo, subcutaneous or intraperitoneal growth of tumor xenografts in nu/nu mice was significantly inhibited (P = 0.004) by treatment for 10 days with scFv(FRP5)-ETA or with 131I-labeled-520C9 anti-p185HER-2 radionuclide conjugate, but additive effects were not observed with combined treatment. CONCLUSION: ScFv(FRP5)-ETA deserves further evaluation for intraperitoneal therapy of ovarian cancers that overexpress p185HER-2.

Inhibition of TNF-alpha produced by Kupffer cells protects against the nonspecific liver toxicity of immunotoxin anti-Tac(Fv)-PE38, LMB-2.

LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas: exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-alpha by these cells. Following LMB-2 administration to mice, the levels of TNF-alpha in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-alpha in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-alpha produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.

Reduction of the nonspecific animal toxicity of anti-Tac(Fv)-PE38 by mutations in the framework regions of the Fv which lower the isoelectric point.

Anti-Tac(Fv)-PE38, also called LMB-2, is a very active recombinant immunotoxin that has produced eight responses, including a durable clinical complete remission in a recently completed phase I trial of leukemias and lymphomas. Dose escalation was limited by liver toxicity. We have noted that the Fv of anti-Tac has an isoelectric point (pI) of 10.2. We hypothesize that the overall positive charge on the Fv portion of anti-Tac(Fv)-PE38 contributes to nonspecific binding to liver cells and results in dose-limiting liver toxicity. We have used a mouse model to investigate the basis of this toxicity and found that lowering the pI of the Fv of anti-Tac from 10.2 to 6. 82 by selective mutation of surface residues causes a 3-fold decrease in animal toxicity and hepatic necrosis. This change in pI did not significantly alter the CD25 binding affinity, the cytotoxic activity toward target cells, or antitumor activity, resulting in a 3-fold improvement in the therapeutic index. If this decreased toxicity occurs in humans, it should greatly increase the clinical utility of this immunotoxin.

Renal dysfunction accounts for the dose limiting toxicity of DT390anti-CD3sFv, a potential new recombinant anti-GVHD immunotoxin.

The toxicity of a highly selective, recombinant fusion immunotoxin, DT390anti-CD3sFv, was examined in mice. The protein was expressed from a hybrid gene in which the single chain Fv of the anti-murine CD3 epsilon antibody cDNA was spliced to truncated diphtheria toxin cDNA. DT390anti-CD3sFv, previously shown to have significant anti-GVHD effects when administered to mice given transplants of allogeneic MHC-disparate donor T cells (Vallera et al., Blood 88, 2342-2353, 1996), preferentially localized to kidney and had profound renal toxicity as assessed by histology and serum levels of blood urea nitrogen (BUN), and creatinine. Kidney effects were more severe than liver effects at the maximum tolerated dose (MTD) of 10 microg/day BID given over a six day interval. Toxic injury was attributed in part to the toxin moiety since DT390 administered alone was more toxic than equivalent doses of DT390 given in the context of DT390anti-CD3sFv fusion protein. The presence of anti-CD3sFv ligand reduced toxicity since DT390anti-CD3sFv was twice as toxic to severe combined immunodeficiency disease (scid) mice which do not have CD3epsilon expressing T cells as compared to their normal counterparts. Together, these findings further our understanding of the toxicities limiting the in vivo administration of DT fusion immunotoxins in mice and provide a foundation for future genetic modifications which should be directed at reducing these effects.

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity.

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.

In vitro and in vivo activities of a doxorubicin prodrug in combination with monoclonal antibody beta-lactamase conjugates.

A cephalosporin derivative of doxorubicin (C-Dox) was evaluated as a prodrug for activation by mAb conjugates of the beta-lactamase from Enterobacter cloacae P99 (beta L; EC 3.5.2.6). The conjugates consisted of beta L and the F(ab') fragments of either of the mAbs L6, P1.17, or 96.5. L6 binds to antigens on a variety of carcinomas, including the two lung adenocarcinoma cell lines H2981 and H2987 used in this study. 96.5 binds to the melanoma-associated antigen p97, and P1.17 was used for the control conjugate. C-Dox was found to be less cytotoxic to three different tumor cell lines in vitro compared to the parent drug doxorubicin (Dox). Immunospecific activation took place when the cells were pretreated with beta L conjugates that could bind to antigens on the tumor cells. In vivo toxicity and pharmacokinetics studies in athymic female nu/nu mice revealed that C-Dox was at least 7-fold less toxic than Dox (on a molar basis), despite the fact that a > or = 320-fold greater area-under-the-curve (blood concentration versus time) of C-Dox compared to Dox was obtained 0-2 h after administration of the two agents. Pharmacokinetic studies at maximum tolerated doses in mice bearing xenografts of either H2981 or H2987 revealed that the intratumoral levels of Dox after treatment with L6-beta L in combination with C-Dox were higher than were obtained by either systemic treatment with Dox or a combination of P1.17-beta L and C-Dox. This finding suggested that the conversion of C-Dox to Dox was tumor specific and dependent on the presence of the targeted antigen. Furthermore, the best antitumor activity against both H2981 and H2987 tumors was obtained by treatment with L6-beta L and C-Dox compared to P1.17-beta L and C-Dox or Dox alone. Thus, higher levels of Dox corresponded to greater therapeutic effects in both of the tumor models studied.

Radioimmunotherapy of metastatic colorectal tumours with iodine-131-labelled antibody to carcinoembryonic antigen: phase I/II study with comparative biodistribution of intact and F(ab')2 antibodies.

Studies in animal tumour models of colorectal cancer suggest that F(ab')2 antibody fragments to carcinoembryonic antigen (CEA) labelled with iodine-131 give superior therapy compared with intact anti-CEA antibody. The purpose of this study was to investigate this hypothesis in patients. Ten patients received intact A5B7 IgG1 mouse monoclonal antibody (MAb) to CEA and nine patients received the F(ab')2 fragment of the same antibody. The biodistribution for each molecule was compared using quantitative single-photon emission computerised tomographic (SPECT) gamma-camera imaging. Tumour responses were seen in both groups and myelosuppression was the limiting toxicity. F(ab')2 localised more rapidly than intact antibody in tumour, giving a mean percentage injected activity per kg at 4.25 h after injection of 8.2% for F(ab')2 compared with 4.4% for intact antibody (P < 0.05). No significant difference in antibody clearance from, or cumulative dose per unit administered activity (cGy MBq-1) to, tumour was seen. Distribution in blood was similar for both the intact and fragment antibody. These findings are consistent with more rapid penetration of the smaller F(ab')2 into tumour masses. More efficient early uptake will give higher maximum dose rates to the tumour which is valuable for radioimmunotherapy (RIT) when low dose rates may limit effectiveness of treatment. F(ab')2 fragments may provide a substantially enhanced method of delivering RIT.

Digoxin-specific Fab fragments impair renal function in the rabbit.

A 2 mg kg-1 intravenous bolus dose of digoxin-specific Fab fragments produced a 28% reduction in creatinine clearance in rabbits after 24 h. Urine output was reduced, while plasma and urinary creatinine concentrations were unaffected and increased, respectively. By 5 days the creatinine clearance had returned to normal. The fractional excretion of Na+ was nearly halved, indicating that the tubular reabsorption of Na+ increased to compensate for the reduced glomerular filtration rate, suggesting that tubular (as opposed to glomerular) function was not impaired.