Bispecific T-Cell Engager (BiTE) Antibody Construct Blinatumomab for the Treatment of Patients With Relapsed/Refractory Non-Hodgkin Lymphoma: Final Results From a Phase I Study.

PURPOSE: Blinatumomab is a CD19/CD3 BiTE (bispecific T-cell engager) antibody construct for the treatment of Philadelphia chromosome-negative acute B-lymphoblastic leukemia. We evaluated blinatumomab in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: This 3 + 3 design, phase I dose-escalation study determined adverse events and the maximum tolerated dose (MTD) of continuous intravenous infusion blinatumomab in patients with relapsed/refractory NHL. Blinatumomab was administered over 4 or 8 weeks at seven different dose levels (0.5 to 90 µg/m(2)/day). End points were incidence of adverse events, pharmacokinetics, pharmacodynamics, and overall response rate. RESULTS: Between 2004 and 2011, 76 heavily pretreated patients with relapsed/refractory NHL, who included 14 with diffuse large B-cell lymphoma, were enrolled; 42 received treatment in the formal dose-escalation phase. Neurologic events were dose limiting, and 60 µg/m(2)/day was established as the MTD. Thirty-four additional patients were recruited to evaluate antilymphoma activity and strategies for mitigating neurologic events at a prespecified MTD. Stepwise dosing (5 to 60 µg/m(2)/day) plus pentosan polysulfate SP54 (n = 3) resulted in no treatment discontinuations; single-step (n = 5) and double-step (n = 24) dosing entailed two and seven treatment discontinuations due to neurologic events, respectively. Grade 3 neurologic events occurred in 22% of patients (no grade 4/5). Among patients treated at 60 µg/m(2)/day (target dose; n = 35), the overall response rate was 69% across NHL subtypes and 55% for diffuse large B-cell lymphoma (n = 11); median response duration was 404 days (95% CI, 207 to 1,129 days). CONCLUSION: In this phase I study of relapsed/refractory NHL, continuous infusion with CD19-targeted immunotherapy blinatumomab at various doses and schedules was feasible, with an MTD of 60 µg/m(2)/day. Single-agent blinatumomab showed antilymphoma activity.

Long-term survival and T-cell kinetics in relapsed/refractory ALL patients who achieved MRD response after blinatumomab treatment.

This long-term follow-up analysis evaluated overall survival (OS) and relapse-free survival (RFS) in a phase 2 study of the bispecific T-cell engager antibody construct blinatumomab in 36 adults with relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL). In the primary analysis, 25 (69%) patients with relapsed/refractory ALL achieved complete remission with full (CR) or partial (CRh) hematologic recovery of peripheral blood counts within the first 2 cycles. Twenty-five patients (69%) had a minimal residual disease (MRD) response (<10(-4) blasts), including 22 CR/CRh responders, 2 patients with hypocellular bone marrow, and 1 patient with normocellular bone marrow but low peripheral counts. Ten of the 36 patients (28%) were long-term survivors (OS ≥30 months). Median OS was 13.0 months (median follow-up, 32.6 months). MRD response was associated with significantly longer OS (Mantel-Byar P = .009). All 10 long-term survivors had an MRD response. Median RFS was 8.8 months (median follow-up, 28.9 months). A plateau for RFS was reached after ∼18 months. Six of the 10 long-term survivors remained relapse-free, including 4 who received allogeneic stem cell transplantation (allo-SCT) as consolidation for blinatumomab and 2 who received 3 additional cycles of blinatumomab instead of allo-SCT. Three long-term survivors had neurologic events or cytokine release syndrome, resulting in temporary blinatumomab discontinuation; all restarted blinatumomab successfully. Long-term survivors had more pronounced T-cell expansion than patients with OS <30 months.

Clinical overview of anti-CD19 BiTE(®) and ex vivo data from anti-CD33 BiTE(®) as examples for retargeting T cells in hematologic malignancies.

Blinatumomab, a bispecific antibody construct targeting CD19, is the most advanced member of bispecific T-cell engager (BiTE(®)) molecules. The clinical development program includes B-precursor acute lymphoblastic leukemia (ALL) and B-cell non-Hodgkin lymphoma (NHL). Minimal residual disease (MRD) response in patients with MRD-positive B-precursor ALL has translated into long-term clinical benefits as demonstrated by an estimated relapse-free survival (RFS) of 60% with sustained MRD negativity at a follow-up of 31 months. Remissions induced in pediatric and adult patients with relapsed/refractory B-precursor ALL have allowed for successful allogeneic hematopoietic stem cell transplantation (HSCT) in this setting. Blinatumomab has also induced durable responses in low-grade B-cell NHL. Blinatumomab recently gained approval in the United States by the U.S. Food and Drug Administration for treatment of Philadelphia chromosome-negative B-precursor relapsed/refractory acute lymphoblastic leukemia. AMG 330 is an investigational anti-CD33 BiTE(®) antibody construct. Targeting CD33 ex vivo in primary samples from patients with acute myeloid leukemia (AML) has shown AMG 330-mediated T-cell expansion and T-cell cytotoxicity against AML cells.

Long-term follow-up of hematologic relapse-free survival in a phase 2 study of blinatumomab in patients with MRD in B-lineage ALL.

Persistence or recurrence of minimal residual disease (MRD) after chemotherapy results in clinical relapse in patients with acute lymphoblastic leukemia (ALL). In a phase 2 trial of B-lineage ALL patients with persistent or relapsed MRD, a T cell-engaging bispecific Ab construct induced an 80% MRD response rate. In the present study, we show that after a median follow-up of 33 months, the hematologic relapse-free survival of the entire evaluable study cohort of 20 patients was 61% (Kaplan-Meier estimate). The hema-tologic relapse-free survival rate of a subgroup of 9 patients who received allogeneic hematopoietic stem cell transplantation after blinatumomab treatment was 65% (Kaplan-Meier estimate). Of the subgroup of 6 Philadelphia chromosome-negative MRD responders with no further therapy after blinatumomab, 4 are in ongoing hematologic and molecular remission. We conclude that blinatumomab can induce long-lasting complete remission in B-lineage ALL patients with persistent or recurrent MRD. The original study and this follow-up study are registered at www.clinicaltrials.gov as NCT00198991 and NCT00198978, respectively.

Crystal structure of Sol I 2: a major allergen from fire ant venom.

Sol i 2 is a potent allergen from the venom of red imported fire ant, which contains allergens Sol i 1, Sol i 2, Sol i 3, and Sol i 4 that are known to be powerful triggers of anaphylaxis. Sol i 2 causes IgE antibody production in about one-third of individuals stung by fire ants. Baculovirus recombinant dimeric Sol i 2 was crystallized as a native and selenomethionyl-derivatized protein, and its structure has been determined by single-wavelength anomalous dispersion at 2.6 Å resolution. The overall fold of each subunit consists of five helices that enclose a central hydrophobic cavity. The structure is stabilized by three intramolecular disulfide bridges and one intermolecular disulfide bridge. The nearest structural homologue is the sequence-unrelated odorant binding protein and pheromone binding protein LUSH of the fruit fly Drosophila, which may suggest a similar biological function. To test this hypothesis, we measured the reversible binding of various pheromones, plant odorants, and other ligands to Sol i 2 by the changes in N-phenyl-1-naphthylamine fluorescence emission upon binding of ligands that compete with N-phenyl-1-naphthylamine. The highest binding affinity was observed for hydrophobic ligands such as aphid alarm pheromone (E)-ß-farnesene, analogs of ant alarm pheromones, and plant volatiles decane, undecane, and ß-caryophyllene. Conceivably, Sol i 2 may play a role in capturing and/or transporting small hydrophobic ligands such as pheromones, odors, fatty acids, or short-living hydrophobic primers. Molecular surface analysis, in combination with sequence alignment, can explain the serological cross-reactivity observed between some ant species.

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.

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.

Mutational analysis of amino acid residues involved in IgE-binding to the Malassezia sympodialis allergen Mala s 11.

The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivity in atopic eczema (AE) patients. The M. sympodialis allergen Mala s 11 has a high degree of amino acid sequence homology to manganese superoxide dismutase (MnSOD) from Homo sapiens (50%) and Aspergillus fumigatus (56%). Humoral and cell-mediated cross-reactivity between MnSOD from H. sapiens and A. fumigatus has been demonstrated. Taken together with the recent finding that human MnSOD (hMnSOD) can act as an autoallergen in AE patients sensitised to M. sympodialis, we hypothesized that cross-reactivity could also occur between hMnSOD and Mala s 11, endogenous hMnSOD thus being capable of stimulating an immune response through molecular mimicry. Herein we demonstrate that recombinant Mala s 11 (rMala s 11) is able to inhibit IgE-binding to recombinant hMnSOD and vice versa, indicating that these two homologues share common IgE-binding epitopes and providing an explanation at a molecular level for the autoreactivity to hMnSOD observed in AE patients sensitised to Mala s 11. Using molecular modelling and mapping of identical amino acids exposed on the surface of both Mala s 11 and hMnSOD we identified four regions each composed of 4-5 residues which are potentially involved in IgE-mediated cross-reactivity. Mutated rMala s 11 molecules were produced in which these residues were altered. Native-like folding was verified by enzymatic activity tests and circular dichroism. The rMala s 11 mutants displayed lower IgE-binding in comparison to wild-type rMala s 11 using plasma from AE patients. In particular, mutation of the residues E29, P30, E122 and K125 lowered the IgE-binding to Mala s 11. The results of this study provide new insights in the molecular basis underlying the cross-reactivity between Mala s 11 and hMnSOD.

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.

Tumor regression in cancer patients by very low doses of a T cell-engaging antibody.

Previous attempts have shown the potential of T cells in immunotherapy of cancer. Here, we report on the clinical activity of a bispecific antibody construct called blinatumomab, which has the potential to engage all cytotoxic T cells in patients for lysis of cancer cells. Doses as low as 0.005 milligrams per square meter per day in non-Hodgkin's lymphoma patients led to an elimination of target cells in blood. Partial and complete tumor regressions were first observed at a dose level of 0.015 milligrams, and all seven patients treated at a dose level of 0.06 milligrams experienced a tumor regression. Blinatumomab also led to clearance of tumor cells from bone marrow and liver. T cell-engaging antibodies appear to have therapeutic potential for the treatment of malignant diseases.

Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab.

The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy.

Sol i 1, the phospholipase allergen of imported fire ant venom.

BACKGROUND: Sol i 1, the venom phospholipase of imported fire ant venom is an important allergen and exhibits some cross-reactivity with IgE antibodies from patients sensitized to other Hymenoptera venoms. OBJECTIVE: To determine the primary structure of Sol i 1 and evaluate the roles of protein and carbohydrate epitopes in its cross-reactivity. METHODS: Sol i 1 was purified from venom, proteolytic peptides prepared and amino acid sequences obtained. The cDNA for Sol i 1 was cloned, sequenced, and compared with sequences of other wasp venom phospholipases. The role of carbohydrate epitopes in the cross-reactivity with other Hymenoptera venoms was studied by RAST inhibition. RESULTS: The sequence identified Sol i 1 as a lipase of the GX class, lipoprotein lipase superfamily, pancreatic lipase homologous family and RP2 subgroup phospholipases as are the vespid venom phospholipases. The 148 residues identified by amino acid sequencing represent about 48% of the translated cDNA sequence. Sol i 1 was 31-32% identical to yellow jacket phospholipases. The identical regions of sequence were clustered in the domain which forms the serine hydrolase active site. Mannosylated N-glycans could completely inhibit binding of IgE from honeybee venom sensitized patients to Sol i 1. Inhibition by glycan of IgE binding from yellow jacket venom sensitized patients was low or absent for three of eight sera and substantial, but not complete for five sera. CONCLUSIONS: Sol i 1 is related to wasp venom phospholipases. Cross-reactivity with honeybee venom is caused by carbohydrate, whereas cross-reactivity with yellow jacket venom involves reactivity with both carbohydrate determinants of hyaluronidase and high molecular weight proteins and phospholipase protein determinants.

Hymenoptera venom protease allergens.

BACKGROUND: Recent studies have shown the presence of additional allergenic proteins in honeybee and paper wasp venoms. Both venoms contain serine protease enzymes. OBJECTIVE: We isolated and obtained complete sequences of honeybee and Mediterranean paper wasp venom proteases, both of which have significant IgE binding activity. The structures are compared with bumblebee venom protease. METHODS: Venom proteases were chromatographically isolated from venoms and partial amino acid sequences determined. RT-PCR and rapid amplification of cDNA ends methods were used to clone cDNA, and complete sequences were determined for honeybee and a paper wasp venom protease. RESULTS: The venom proteases are all serine proteases of the trypsin type. The honeybee protease contains a complement, embryonic sea urchin protein, bone morphogenetic protein interaction domain as well as a linker and propeptide sequence, and a unique methionine residue near the active site. It has IgE binding activity. The paper wasp protease is a single trypsin domain and is an important allergen. The framework residues are poorly conserved among honeybee, bumblebee, and paper wasp enzymes. CONCLUSIONS: The 3 venom serine proteases have significant IgE binding activities. The structures are poorly conserved even among the Apidae , suggesting little cross-reactivity among the protein portions. The paper wasp venom proteases are important allergens.

Malassezia sympodialis stimulation differently affects gene expression in dendritic cells from atopic dermatitis patients and healthy individuals.

It is known that 28-84% of patients with atopic dermatitis exhibit IgE and/or T-cell reactivity to the opportunistic yeast Malassezia sympodialis, which can be taken up by immature monocyte-derived dendritic cells (MDDCs), resulting in MDDC maturation. The aim of this study was to investigate whether MDDCs from patients with atopic dermatitis respond differently to M. sympodialis compared to MDDCs from healthy individuals. Immature MDDCs were stimulated with M. sympodialis and the gene expression profiles were analysed with cDNA arrays containing 406 genes. Our results show that M. sympodialis differently affected MDDCs from patients with atopic dermatitis, and more so in severely ill patients, compared with healthy individuals. Six genes were more than fivefold up-regulated in MDDCs from more than one patient with atopic dermatitis, coding for CD54, CD83, IL-8, monocyte-derived chemokine (MDC), BTG1 and IL-1R antagonist. In healthy individuals this was true only for BTG1. Up-regulations of IL-8 and MDC were confirmed at the protein level. Our findings might reflect an increased trafficking and stimulatory capacity in MDDCs from the patients, which is likely to result in a stronger inflammatory response to M. sympodialis.

Alternative splicing of major histocompatibility complex class II DXB transcripts in Xiphophorus fishes.

Classical MHC class II glycoproteins present peptides to T cells. In Xiphophorus fishes and in the guppy, Poecilia reticulata, a classical MHC class II B-like transcript has been identified, DAB, as well as a divergent MHC class II B-like transcript, DXB. In the two species of Xiphophorus fishes studied here, X. multilineatus and X. pygmaeus, alternative splicing of the DXB transcript was observed, but not of the classical type DAB transcripts. Two alternative splice patterns were found: a 16-codon deletion and a five-nucleotide deletion that leads to an extension of the transcript. A single DXB transcript that terminates before the transmembrane region was also observed. The alternative splice pattern and the divergence of DXB from DAB suggest that in fish, DXB may have an alternate function. Alternative splicing transcripts of DXB may allow for signaling and localization of DXB within the cell.

Cloning, expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase.

Malassezia sympodialis is an opportunistic yeast that colonizes human skin and may induce IgE and T cell reactivity in patients with atopic eczema/dermatitis syndrome (AEDS). Previously, we have cloned and expressed six recombinant allergens (rMala s 1 and rMala s 5 to rMala s 9) from this yeast. By combining high throughput screening and phage surface display techniques, 27 complete and partial IgE-binding clones of M. sympodialis have been identified. Here we enlarged the panel of recombinant M. sympodialis allergens by RACE-PCR, cloning and nucleotide sequencing to obtain the coding sequences of two new IgE-binding clones. The coding sequences of one of the clones showed similarity to the heat shock protein (HSP) family and the other to manganese superoxide dismutase (MnSOD), and both had a high degree of homology to human counterparts. The coding sequences were expressed in Escherichia coli as six-histidine tagged recombinant proteins and generated products with molecular masses of 86.1 kDa for HSP and 22.4 kDa for MnSOD. Their IgE-binding frequencies were shown to be 69% and 75%, respectively, to 28 sera from AEDS patients with serum IgE to M. sympodialis extract, indicating that HSP and MnSOD are major M. sympodialis allergens. In inhibition immunoblotting, M. sympodialis extract could inhibit the binding of serum IgE from AEDS patients to rHSP and rMnSOD in a concentration-dependent manner. The high frequency of sera from AEDS patients, showing IgE binding to both HSP and MnSOD, indicates that these allergens, designated Mala s 10 and Mala s 11, could play a role in AEDS.