Margit Schmidt

Targeted Therapy With the T-Cell–Engaging Antibody Blinatumomab of Chemotherapy-Refractory Minimal Residual Disease in B-Lineage Acute Lymphoblastic Leukemia Patients Results in High Response Rate and Prolonged Leukemia-Free Survival

PURPOSE Blinatumomab, a bispecific single-chain antibody targeting the CD19 antigen, is a member of a novel class of antibodies that redirect T cells for selective lysis of tumor cells. In acute lymphoblastic leukemia (ALL), persistence or relapse of minimal residual disease (MRD) after chemotherapy indicates resistance to chemotherapy and results in hematologic relapse. A phase II clinical study was conducted to determine the efficacy of blinatumomab in MRD-positive B-lineage ALL. PATIENTS AND METHODS Patients with MRD persistence or relapse after induction and consolidation therapy were included. MRD was assessed by quantitative reverse transcriptase polymerase chain reaction for either rearrangements of immunoglobulin or T-cell receptor genes, or specific genetic aberrations. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dose of 15 μg/m2/24 hours. RESULTS Twenty-one patients were treated, of whom 16 patients became MRD negative. One patient was not evaluable due to a grade 3 adverse event leading to treatment discontinuation. Among the 16 responders, 12 patients had been molecularly refractory to previous chemotherapy. Probability for relapse-free survival is 78% at a median follow-up of 405 days. The most frequent grade 3 and 4 adverse event was lymphopenia, which was completely reversible like most other adverse events. CONCLUSION Blinatumomab is an efficacious and well-tolerated treatment in patients with MRD-positive B-lineage ALL after intensive chemotherapy. T cells engaged by blinatumomab seem capable of eradicating chemotherapy-resistant tumor cells that otherwise cause clinical relapse.

Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli ☆ ☆☆ ★ ★★

BACKGROUND Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described. OBJECTIVE We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity. METHODS In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells. RESULTS Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli. CONCLUSIONS Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.

cDNA analysis of the mite allergen Lep d 1 identifies two different isoallergens and variants.

For the first time the complete cDNA encoding two isoallergens of the non‐pyroglyphid dust mite Lepidoglyphus destructor, Lep d 1, allergen has been sequenced. In addition, one of the isoallergens was found to have two variants. Oligonucleotides were designed from known amino acid sequences. The cDNA sequences were obtained by hybridizing these primers to mRNA and enhancement by the RT‐PCR technique. To obtain the different complete encoding cDNA sequences and eliminate heteroduplex artifacts, we performed PCR + 1 reactions. Comparison of the amino acid sequence of the allergen shows leader sequences of 16 amino acids for both isoforms.

Expression systems for production of recombinant allergens

Recombinant allergenic proteins have been produced in a variety of different expression systems. This review gives examples of and compares prokaryotic expression systems, such as Escherichia coli, and eukaryotic systems including the yeasts, Saccharomyces cerevisiae and Pichia pastoris, baculovirus-insect cell systems, mammalian cell systems and plant systems. Important factors to consider in choosing an expression system are discussed and examples of applications are given.

Crystal Structure of the Major Allergen from Fire Ant Venom, Sol i 3

Fire ant venom is an extremely potent allergy-inducing agent containing four major allergens, Sol i 1 to Sol i 4, which are the most frequent cause of hypersensitivity reactions to hymenoptera in the southern USA. The crystal structure of recombinant (Baculovirus) major fire ant allergen Sol i 3 has been determined to a resolution of 3.1 A by the method of molecular replacement. The secondary-structure elements of Sol i 3 are arranged in an alpha-beta-alpha sandwich fold consisting of a central antiparallel beta-sheet surrounded on both sides by alpha helices. The overall structure is very similar to that of the homologous wasp venom allergen Ves v 5 with major differences occurring in the solvent-exposed loop regions that contain amino acid insertions. Consequently, the limited conservation of surface chemical properties and topology between Sol i 3 and Ves v 5 may explain the observed lack of relevant cross-reactivity. It is concluded that Sol i 3 recognizes immunoglobulin E antibodies with a distinct set of its own epitopes, which are different from those of Ves v 5. Indeed, the molecular area in Sol i 3 covered by non-conserved residues is large enough to accommodate four unique Sol i 3 epitopes.

Allergens in Hymenoptera venom. XXII. Comparison of venoms from two species of imported fire ants, Solenopsis invicta and richteri

Venoms were collected by electrical stimulation from the two major species of imported fire ants found in the United States, Solenopsis invicta (Sol i) and S. richteri (Sol r). Antigens similar to three of the four known Sol i venom proteins (I, II, III, and IV) were isolated from Sol r. The N-terminal amino acid sequences for the antigens III were identical; but those for the antigens II demonstrated only nine of 20 residues to be identical. Two monoclonal antibodies raised against Sol i II did not react to Sol r. No protein IV could be detected in Sol r by molecular weight, charge, or immunologically with either polyclonal mouse antibodies or five monoclonal antibodies. Both venoms were compared with a panel of 60 sera from Sol i-allergic individuals; mean bindings were similar with an r = 0.94 for linear regression. RAST inhibition was performed with 17 individual sera representing a variety of Sol i allergen specificities. Four sera were tested from patients resident in the Sol r endemic area and five sera from patients who experienced reactions to S. xyloni stings. All sera reacted comparably to both imported fire ant venoms. The two venoms appear to be allergenically similar, although antigen IV is absent from Sol r and the antigens II have significant sequence variation. Sol i venom appears to be sufficient for diagnostic purposes.

Immunologic characterization of the recombinant fire ant venom allergen Sol i 3.

Individuals sensitized to fire ant stings show immunoglobulin (Ig)E antibodies against the venom protein Sol i 3. We determined the full‐length complementary DNA (cDNA) sequence of this protein and expressed recombinant Sol i 3 in immunogenic form. The complete cDNA of Sol i 3 was obtained by reverse transcription polymerase chain reaction (RT‐PCR) and PCR + 1 reactions using gene‐specific oligonucleotides, and oligonucleotides designed from the amino acid sequence of this protein. The encoding cDNA is 705 bp in length corresponding to 235 amino acids. The first 22 amino acids are a leader sequence. The protein with an added C‐terminal hexahistidine tag was expressed in insect cells using a baculovirus system. The recombinant protein was secreted into the supernatant and affinity purified with a cobalt chelating resin. The recombinant fire ant venom allergen Sol i 3 showed similar IgE binding activity to the native protein in radioallergosorbent test (RAST) and RAST inhibition assays. It was produced in both a glycosylated and an unglycosylated form. A three‐dimensional reconstruction of Sol i 3 was compared with the experimentally determined structure of the related allergen Ves v 5. This model is supported by results of circular dichroism spectroscopy.

Production of a recombinant imported fire ant venom allergen, Sol i 2, in native and immunoreactive form

BACKGROUND The complementary DNA encoding for the important imported fire ant venom allergen, Sol i 2, has previously been cloned. The binding of human IgE antibodies to Sol i 2 has been demonstrated to be conformation-dependent. METHODS A couple cDNA clone encoding the Sol i 2 protein sequence and its natural signal sequence has been produced by polymerase chain reaction. The clone was ligated into a pBluebac III transfer vector (Invitrogen Corp., San Diego, Calif.), and the recombinant baculovirus was isolated by plaque purification. The recombinant baculovirus was grown in Sf9 and High-Five cells (Invitrogen Corp.) in serum-free media. The recombinant Sol i 2 was isolated and characterized. RESULTS Recombinant (r) Sol i 2 was produced in microgram/per milliliter amounts in Sf9 cells and at 30 micrograms/ml in High-Five cells. It was isolated by ultrafiltration and reverse-phase chromatography. The rSol i 2 demonstrated similar binding to natural-Sol i 2 in both a conformation-dependent ELISA assay and in RAST with sera from patients allergic to Sol i 2. The N-terminal sequence of the rSol i 2 was identical to that of the natural molecule. No significant increase in binding activity was found after treatment of rSol i 2 with protein disulfide isomerase. The binding of rSol i 2 to a conformation-dependent monoclonal antibody was lost by heating in sodium dodecylsulfate and reduction. CONCLUSION A recombinant Sol i 2 protein was produced at high yield in a baculovirus expression system by using serum-free medium with a sequence identical to that of the natural molecule. Conformation-dependent immunologic assays indicate that the recombinant protein is produced with the native conformation.

Allergenic crossreactivity between Lepidoglyphus destructor and Blomia tropicalis

Background In general, the non‐pyroglyphid mites Lepidoglyphus destructor and Blomia tropicalis show a different geographical distribution. Allergic sensitization to both species have been demonstrated in several investigations. However, whether this reflects crossreactivity or dual sensitization is so far not known.

Phase II Study of the Human Anti-Epithelial Cell Adhesion Molecule Antibody Adecatumumab in Prostate Cancer Patients with Increasing Serum Levels of Prostate-Specific Antigen after Radical Prostatectomy

Background: Rising serum levels of prostate-specific antigen (PSA) after radical prostatectomy are indicative of recurrent prostate cancer. This double-blind, placebo-controlled phase II study evaluated the anti-tumour activity of the anti-epithelial cell adhesion molecule (EpCAM) antibody adecatumumab in delaying biochemical disease progression. Patients and Methods: Prostate cancer patients with increasing serum PSA levels following radical prostatectomy were randomized to low- (2 mg/kg) or high-dose adecatumumab (6 mg/kg) or placebo. The primary efficacy endpoint was the mean change from baseline in total serum PSA at week 24. Secondary endpoints included PSA response rate, prolongation of serum PSA doubling time and time to biochemical disease progression. Results: The primary and secondary endpoints of the study were not met in the predefined analyses. In a retrospective analysis of patients with baseline PSA ≤1 ng/ml and a high EpCAM expression, both the mean increase in PSA from baseline to week 24 and the PSA doubling time at week 15 were significantly improved in the high-dose adecatumumab group compared with the placebo group. Most frequent treatment-related clinical adverse events were gastrointestinal (diarrhoea and nausea) or general events (chills), showing a dose dependency but no grade 3/4 intensity in any patient. Conclusion: In men with rising PSA levels after radical prostatectomy and no evidence of clinical relapse, adecatumumab delayed disease progression in a subgroup of patients with baseline PSA levels ≤1 ng/ml and high EpCAM-expressing tumours.