Steven L. Giardina

Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies

PURPOSE To evaluate the toxicity, pharmacokinetics, immunogenicity, and antitumor activity of anti-Tac(Fv)-PE38 (LMB-2), an anti-CD25 recombinant immunotoxin that contains an antibody Fv fragment fused to truncated Pseudomonas exotoxin. PATIENTS AND METHODS Patients with CD25(+) hematologic malignancies for whom standard and salvage therapies failed were treated with LMB-2 at dose levels that ranged from 2 to 63 microg/kg administered intravenously over 30 minutes on alternate days for three doses (QOD x 3). RESULTS LMB-2 was administered to 35 patients for a total of 59 cycles. Dose-limiting toxicity at the 63 microg/kg level was reversible and included transaminase elevations in one patient and diarrhea and cardiomyopathy in another. LMB-2 was well tolerated in nine patients at the maximum-tolerated dose (40 microg/kg QOD x 3); toxicity was transient and most commonly included transaminase elevations (eight patients) and fever (seven patients). Only six of 35 patients developed significant neutralizing antibodies after the first cycle. The median half-life was 4 hours. One hairy cell leukemia (HCL) patient achieved a complete remission, which is ongoing at 20 months. Seven partial responses were observed in cutaneous T-cell lymphoma (one patient), HCL (three patients), chronic lymphocytic leukemia (one patient), Hodgkins disease (one patient), and adult T-cell leukemia (one patient). Responding patients had 2 to 5 log reductions of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous masses and splenomegaly. All four patients with HCL responded to treatment. CONCLUSION LMB-2 has clinical activity in CD25(+) hematologic malignancies and is relatively nonimmunogenic. It is the first recombinant immunotoxin to induce major responses in cancer. LMB-2 and similar agents that target other cancer antigens merit further clinical development.

Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification.

A GMP‐compliant process is described for producing F5cys‐PEG‐lipid conjugate. This material fuses with preformed, drug‐loaded liposomes, to form ”immunoliposomes” that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drugapos;s therapeutic index. The soluble, single‐chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high‐density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein‐A resin robustly recovered F5cys from high‐pressure‐disrupted, whole‐cell homogenates. Two product‐related impurity classes were identified: F5cys with mid‐sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low‐pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C‐terminal cysteine for conjugation. Site‐directed conjugation, conducted at pH 5.9 ± 0.1 with reaction monitoring and cysteine quenching, yielded F5cys‐MP‐PEG(2000)‐DSPE. Low‐pressure size exclusion chromatography separated spontaneously formed, high‐molecular‐weight conjugate micelles from low‐molecular‐weight impurities. When formulated at 1–2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below −70 °C. Six scale‐up lots were compared. The largest 40‐L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product‐related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale‐up of immunoliposome‐encapsulated therapeutics.

Preclinical Manufacture of Anti‐HER2 Liposome‐Inserting, scFv‐PEG‐Lipid Conjugate. 2. Conjugate Micelle Identity, Purity, Stability, and Potency Analysis

Analytical methods optimized for micellar F5cys‐MP‐PEG(2000)‐DPSE protein‐lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 °C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2‐propanol (29.4 vol %) and n‐butanol (4.5 vol %) to buffers for salt‐gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well‐resolved peaks. This method was used to assess stability, identify contaminants, establish lot‐to‐lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER‐2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin‐encapsulating liposomes for antibody‐directed uptake of doxorubicin by HER2‐overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys‐MP‐PEG(2000)‐DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.

Evaluation of the compatibility of a second generation recombinant anthrax vaccine with aluminum-containing adjuvants

Recombinant protective antigen (rPA) is the active pharmaceutical ingredient in a second generation anthrax vaccine undergoing pre-clinical evaluation. This rPA vaccine differs from the currently licensed vaccine, anthrax vaccine adsorbed (AVA), in that the sole component is a recombinant form of protective antigen (PA). Unlike AVA the rPA vaccine contains no lethal factor (LF) or edema factor (EF), components of the two bipartite toxins, nor many other Bacillus anthracis-related contaminating proteins that are present in AVA. The proposed clinical protocol involves adsorption of the rPA to an aluminum-based adjuvant. The adsorptive characteristics of rPA and two aluminum-containing adjuvants were examined in a physiological buffer with and without EDTA. Based on the pI of rPA (pI=5.6) and the zero charge point of aluminum hydroxide adjuvant (11.5) and aluminum phosphate adjuvant (4.5), it was predicted and demonstrated that rPA bound in a more efficient manner to aluminum hydroxide adjuvant than to aluminum phosphate adjuvant in the physiological buffer. Binding of the rPA to the aluminum hydroxide adjuvant was decreased by increasing amounts of phosphate in the buffer. The adsorptive capacity for rPA onto aluminum hydroxide adjuvant in the physiological buffer and in water were calculated to be 0.46 mg rPA/mg aluminum in DPBS and 0.73 mg rPA/mg aluminum in water. This study also demonstrated that upon desorption from the aluminum hydroxide adjuvant the rPA was physically intact and free of detectable aggregates. Further, the eluted material was biologically active in an in vitro cytotoxicity assay. Desorption was only possible after an overnight incubation of 2-8 degrees C and not after a room temperature incubation reflecting increased contact with the aluminum hydroxide adjuvant over time. These data suggest that the interaction between rPA and aluminum hydroxide adjuvant is predominantly electrostatic in character.

Production and purification of refolded recombinant human IL-7 from inclusion bodies.

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.

Attenuation of Neurovirulence, Biodistribution, and Shedding of a Poliovirus:Rhinovirus Chimera after Intrathalamic Inoculation in Macaca fascicularis

ABSTRACT A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied.

Evaluation Of Ires-Mediated, Cell Type-Specific Cytotoxicity Of Poliovirus Using A Colorimetric Cell Proliferation Assay

PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and cell-type specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the VH or VL. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, VL and VH PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL‐15) has shown remarkable biological properties of promoting NK‐ and T‐cell activation and proliferation, as well as enhancing antitumor immunity of CD8+ T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin‐15 (rhIL‐15) for clinical manufacturing. Human IL‐15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21‐AI, E. coli BL21‐AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20‐L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL‐15. Stop codons of the expression construct were further investigated after 15–20% of the purified rhIL‐15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21‐AI cells containing the rhIL‐15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.

Strategies for the purification of recombinant proteins

Publisher Summary An understanding of the function of a gene requires that the investigator produce, isolate, and characterize the gene product. Proteins, unlike nucleic acids, are generally not amenable to a ‘‘one-size-fits-all’’ approach to purification. Recombinant proteins may be used for a variety of purposes ranging from laboratory reagents to therapeutics and vaccines intended for human or veterinary use. It is the intended use of the product, therefore, that will define what is meant by purity and the strategies that will lead to a successful outcome. No one wants to expend precious time and resources on what appears to be an unnecessary effort. However, focusing on short-term research needs without considering the possibility of future commercialization with its associated regulatory requirements can lead to costly mistakes and delays down the road. The requirements of the regulatory agencies begin at the time of discovery, and to ignore them is folly. The chapter focuses on current trends and methods for the purification of recombinant proteins and the regulatory concerns and issues.