Purpose: Antibody-directed enzyme prodrug therapy is a two-stage treatment whereby a tumor-targeted antibody-enzyme complex localizes
in tumor for selective conversion of prodrug. The purpose of this ...study was to establish optimal variables for single administration
of MFECP1, a recombinant antibody-enzyme fusion protein of an anti–carcinoembryonic antigen single-chain Fv antibody and the
bacterial enzyme carboxypeptidase G2 followed by a bis-iodo phenol mustard prodrug. MFECP1 is manufactured in mannosylated
form to facilitate normal tissue elimination.
Experimental Design: Pharmacokinetic, biodistribution, and tumor localization studies were used to test the hypothesis that MFECP1 localizes in
tumor and clears from normal tissue via the liver. Firstly, safety of MFECP1 and a blood concentration of MFECP1 that would
avoid systemic prodrug activation were tested. Secondly, dose escalation of prodrug was done. Thirdly, the dose of MFECP1
and timing of prodrug administration were optimized.
Results: MFECP1 was safe and well tolerated, cleared rapidly via the liver, and was less immunogenic than previously used products.
Eighty-fold dose escalation from the starting dose of prodrug was carried out before dose-limiting toxicity occurred. Confirmation
of the presence of enzyme in tumor and DNA interstrand cross-links indicating prodrug activation were obtained for the optimal
dose and time point. A total of 28 of 31 patients was evaluable for response, the best response being a 10% reduction of tumor
diameter, and 11 of 28 patients had stable disease.
Conclusions: Optimal conditions for effective therapy were established. A study testing repeat treatment is currently being undertaken.
This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of ...protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.
Purpose: Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor.
MFE-CP, a multifunctional genetic fusion protein of antibody and ...enzyme, was designed to achieve this by two mechanisms. First
by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid
removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is
to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy.
Experimental Design: MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg
MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days)
was studied in established human colon carcinoma xenografts, LS174T, and SW1222.
Results: Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting
in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced
reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single
cycle in the LS174T xenografts ( P = 0.001) and produced regressions in the SW1222 xenografts ( P = 0.0001), with minimal toxicity.
Conclusions: MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential
for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.
MFECP1 is a mannosylated antibody-enzyme fusion protein used in antibody-directed enzyme prodrug therapy (ADEPT). The antibody selectively targets tumor cells and the targeted enzyme converts a ...prodrug into a toxic drug. MFECP1 is obtained from expression in the yeast Pichia pastoris and produced to clinical grade. The P. pastoris-derived mannosylation of the fusion protein aids rapid normal tissue clearance required for successful ADEPT. The work presented provides evidence that MFECP1 is cleared by the endocytic and phagocytic mannose receptor (MR), which is known to bind to mannose-terminating glycans. MR-transfected fibroblast cells internalize MFECP1 as revealed by flow cytometry and confocal microscopy. Immunofluorescence microscopy shows that in vivo clearance in mice occurs predominantly by MR on liver sinusoidal endothelial cells, although MR is also expressed on adjacent Kupffer cells. In the spleen, MFECP1 is taken up by MR-expressing macrophages residing in the red pulp and not by dendritic cells which are found in the marginal zone and white pulp. Clearance can be inhibited in vivo by the MR inhibitor mannan as shown by increased enzyme activities in blood. The work improves understanding of interactions of MFECP1 with normal tissue, shows that glycosylation can be exploited in the design of recombinant anticancer therapeutics and opens the ways for optimizing pharmacokinetics of mannosylated recombinant therapeutics.
Purpose: The efficacy of solid tumor radioimmunotherapy is reduced by heterogeneous tumor distribution of the radionuclide, with dose
mainly deposited in the normoxic region and by the relative ...radioresistance of hypoxic tumor cells. In an attempt to overcome
these challenges, radioimmunotherapy was combined with 2-deoxy- d -glucose (2DG), a hypoxia-selective cytotoxic inhibitor of glucose metabolism.
Experimental Design: In vitro toxicity of 2DG in LS174T cultures was tested using a colony-forming assay. The effect of combining 2DG with radioimmunotherapy
in vivo was tested by administering radiolabeled anti–carcinoembryonic antigen antibody ( 131 IA5B7 IgG1 whole monoclonal) to nude mice bearing s.c. LS174T tumors, followed by 10 daily injections of 2DG (2.0 g/kg).
Tumors were measured to assess therapeutic efficacy.
Results: Data from in vitro studies confirmed 2DG cytotoxicity in this cell line. Greater toxicity was observed under standard laboratory conditions
and in hypoxic cultures than at intermediate, physiologically relevant levels of glucose and oxygen. Alone, 2DG had no effect
on in vivo tumor growth ( P = 0.377 compared with saline-treated controls). Combination of radioimmunotherapy with 2DG reduced the therapeutic effect
of radioimmunotherapy (e.g., 150 μCi 131 I alone mean survival time, 48.33 ± 16.83 days; combined with 2DG, 30.67 ± 5.62 days, P = 0.038).
Conclusions: The combination investigated had a detrimental effect on survival. It is suggested that a cellular metabolic response to
more aggressive therapy, previously reported in vitro , caused this. The results of this study have implications for the clinical application of combined cancer therapies with
an antimetabolic modality component.
Abstract Introduction Radioimmunotherapy (RIT) has been shown to be more effective against solid tumor micrometastases, possibly due to an inverse relationship between tumor size and radiolabeled ...antibody uptake. In this study, the accretion of radiolabeled antibody in intrahepatic micrometastases in an experimental model was investigated using quantitative digital autoradiography, enabling the analysis of antibody uptake in microscopic tumors. Methods Mice bearing subcutaneous or intrahepatic metastatic models of LS174T colorectal cancer were injected with radiolabeled anti-carcinoembryonic antigen antibody (125 IA5B7). Tissues were taken to investigate distribution of radionuclide and tumor uptake. In a therapy study, mice bearing intrahepatic metastatic tumors were injected with 131 IA5B7. Results Subcutaneous tumors and large metastatic deposits had similar uptake (e.g., ∼15%ID/g at 24 h). Small metastatic deposits had higher uptake (e.g., ∼80%ID/g at 24 h) and prolonged retention at later time points. Small deposit uptake was significantly reduced by accompanying large deposits in the same liver. RIT resulted in increased survival time (untreated mean survival of 21.6±12.9 vs. treated mean survival of 39.1±30.8 days), but there was a large range of response within groups, presumably due to variation in pattern and extent of tumor as observed in the biodistribution study. Liver function tests and body weight did not change with tumor growth or therapy response, strongly supporting the use of in vivo imaging in metastatic tumor therapy studies. Conclusions Radioimmunodetection and therapy might be greatly influenced by the size and distribution of intrahepatic tumor deposits.
Purpose: Most radioimmunotherapy studies on radiolabeled antibody distribution are based on autoradiographic and radioluminographic
data, which provide a lack of detailed information due to low ...resolution. We used fluorescently labeled anti–carcinoembryonic
antigen (CEA) antibody (A5B7) to investigate quantitatively the kinetics and microdistribution of antibody in a clinically
relevant orthotopic colorectal cancer model (LS174T) using high-resolution digital microscopy.
Experimental Design: Nude mice bearing LS174T liver orthotopic tumors received a single i.v. injection of fluorescently labeled A5B7 and were
sacrificed at 10 minutes, 1 hour, or 24 hours postinjection. Before sacrifice, mice were injected with the perfusion marker
Hoechst 33342. An anti-CD31 antibody was used to detect blood vessel distribution. Cryostat sections were processed with immunofluorescence
procedures and analyzed with fluorescence microscopy and image analysis techniques. The fluorescence images were related to
morphologic images of the same or adjacent tumor sections.
Results: Fluorescently labeled antibody showed rapid, selective uptake into tumor deposits, with a strong negative correlation with
tumor size at 10 minutes and 1 hour ( P ≤ 0.01). By 24 hours, the correlation was no longer significant. The study showed movement of antibody across the tumor with
time and a tendency to localize more uniformly by later time points (24 hours). The rate of antibody motility was similar
in small and large tumor metastases, but small deposits showed more rapid antibody localization. Intratumoral vessels were
positively related to tumor size ( P ≤ 0.001).
Conclusion: The obtained data suggest that radioimmunotherapy can be highly efficient in an adjuvant or minimal residual disease setting.
Antibody-directed enzyme prodrug therapy (ADEPT) aims to restrict the cytotoxic action to tumour sites. The obstacles to achieve this were recognised at the outset, but time and experience have given ...these better definition. The development of fusion proteins has provided the means of making consistent antibody-enzyme constructs on an adequate scale, and glycosylation has provided the means to control the clearance of enzyme from non-tumour sites. Human enzymes have yet to be tested in a clinical setting, and there are pointers indicating that the immunological response to foreign enzymes can be overcome. The relatively small number of purpose-designed prodrugs tested so far leaves this an area ripe for further development. The ongoing iterative process between preclinical and clinical studies is critical to achieving the objective.
The alpha v beta 6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-beta1 and ...transforming growth factor-beta 3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumor cells. The viral protein 1 (VP1) coat protein of the O(1) British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for alpha v beta 6, and we recently reported that a peptide derived from VP1 exhibited alpha v beta 6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to alpha v beta 6. A 17-mer peptide of VP1 was inserted into the complementarity-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to alpha v beta 6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with alpha v beta 6 but not with alpha 5 beta 1, alpha v beta 3, alpha v beta 5, alpha v beta 8 or CEA. B6-2 was internalized into alpha v beta 6-expressing cells and inhibited alpha v beta 6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of alpha v beta 6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block alpha v beta 6-mediated cancer cell invasion or to deliver and internalize toxins specifically to alpha v beta 6-expressing tumors.
Combretastatin A-4 phosphate (CA4-P) is an antivascular agent which inhibits tumour blood flow. The effects of CA4-P were studied at 1 and 24
h in colorectal xenografts by the concomitant imaging of ...multiple physiological parameters (hypoxia, blood vessels and perfusion), selected to demonstrate changes related to vascular shut-down. Untreated tumours were viable, with perfused blood vessels throughout and only small areas of hypoxia. At 1
h post-treatment, although blood vessels remained throughout the tumour, perfused vessels were mainly restricted to the rim. However, hypoxia was widespread in both peripheral and central parts of the tumour. Quantitative analysis also revealed a significant decrease in perfusion and a maximum increase in hypoxia at this time-point. Conversely, at 24
h after treatment, when most of the tumour was necrotic, pathophysiological conditions in the surviving viable rim were already returning to normal: perfusion was increasing, and hypoxia was greatly reduced and restricted to regions bordering central necrosis. In conclusion, these data provide an insight into the actions by which CA4-P may exert its effects on solid tumours.
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