History
In 1998, Micromet GmbH of Martinsried, Germany, licensed from German academic institutions a CD19/CD3-bispecific antibody construct that was later called MT103, blinatumomab (INN), and AMG103 and received the trade name Blincyto(R).Micromet was a biotech start-up company founded in the mid-1990s inMunich, Germany, that went public in 2006 at NASDAQ(previous ticker symbol: MITI) with headquarters in Rockville, Maryland, and a research and development hub in Munich.
Blinatumomab is a non-glycosylated murine antibody construct composed of two tandemly arranged scFvs produced. Its scFv antibody binding domains were derived by linker technology from mouse antihuman CD16 mAb HD37 and from a sequence variant of mouse antihuman CD3 mAb OKT3. Figure 5.2 depicts the generation of blinatumomab from two mAbs and its particular domain arrangement vis-à-vis a mAb. One scFv is binding the B-cell antigen CD19 with an equilibrium dissociation constant (KD) of approximately 1 nM, and the other the invariant CD3 epsilon subunit of the TCR complex with a KD of approximately 100 nM. The anti-CD19 and anti-CD3 scFvs each use a (Gly4Ser)3 linker for connecting their variable domains, and a Gly4Ser linker is used for linkage of the two scFvs in tandem. The particular order of variable heavy (VH) and light chains (VL) domains on the single polypeptide BiTE chain is critical for the biological activity of blinatumomab. Out of the eight possible VH/VL arrangements, only few are suitable, and one got selected for blinatumomab. The C-terminus of the CD3-binding scFv is extended with a hexahistidine sequence (also called a His-tag) used for affinity purification and detection of the protein by anti-polyHis antibodies. The molecular weight of blinatumomab is approximately 55 kDa.
Production Methods
Small antibody fragments are typically produced in prokaryotic cell culture systems. Because several attempts failed to produce functional blinatumomab in bacteria, the standard production system for mAbs, a Chinese hamster ovary (CHO) cell clone,was chosen. In eukaryotic cells, recombinant secretory proteins are chaperoned and properly folded in the endoplasmic reticulum; otherwise they cannot exit into the cell culturemedium. Both functional monomeric and dimeric forms of blinatumomab are found secreted by CHOcells of which only monomer is purified and used therapeutically.Monomer is preferred over dimer because it is not prone to down-modulate surface expression of the TCR and to activate T cells in the absence of target cells. Because dimeric blinatumomab has a severalfold higher cytotoxic activity than the monomer, it was critical to find ways of keeping the dimer concentration below a certain threshold in the final drug product and to reliably detect dimer concentrations. For commercial-scale production, the manufacturing process was finally transferred to the contract manufacturer Lonza where it was adopted to production at a 2000 L scale. Productivity of blinatumomab by a producer CHO cell clone is >50-fold lower than typically achieved these days for mAbs. However, the high potency of the BiTE antibody translating into consumption of only low mg amounts for a treatment cycle well compensates for its low production yield.
Indications
The CD19/CD3-bispecific antibody construct blinatumomab (INN) is the first T-cell-engaging and first CD19-specific antibody approved by the Food andDrug Administration (FDA). Blinatumomab showed as single-agent treatment high clinical activity at extremely low dose levels in patients with refractory or relapsed B-cell precursor acute lymphocytic leukemia (r/r ALL) and r/r non-Hodgkin lymphoma (NHL).
Blinatumomab received conditional approval by the FDA on December 3, 2014, for treatment of Philadelphia chromosome-negative adult patients with r/r ALL. EU approval was obtained on November 24, 2015. Its conditional approval was on the basis of results from a single-arm phase II clinical trial with 185 r/r ALL patients showing a complete response rate of 41.6% and the obligation to conduct a phase III clinical trial in adult patients with r/r ALL (called TOWER) comparing blinatumomab against best standard of care. First results from the phase III study TOWER were announced by Amgen in a press release on February 5, 2016. An interim analysis of the open-label study showed a positive impact of blinatumomab on overall survival in r/r ALL patients, leading to a premature termination of the trial for ethical reasons. Clinical trials with blinatumomab monotherapy are ongoing in pediatric patients with r/r ALL, patients with Philadelphia chromosome-positive r/r ALL, and patients with diffuse large B-cell lymphoma (DLBCL), the most frequent form of NHL. Blinatumomab showed clinical activity to different extents in all B-cell malignancies investigated to date.
Side effects
So-called first-dose reactions are often seen with mAb therapies, for example, with anti-CD20 mAb rituximab. This is why infusion periods for therapeutic mAbs take several hours and steroid hormones are often proactively administered for suppression of immunological reactions upon start of infusion. A first-dose reaction is also observed for blinatumomab. In contrast to IgG1 mAb therapies, which engage Fc-gamma receptor-positive immune cells (mostly NK cells), blinatumomab uniquely engages T cells. Both kinds of immune cells will release pro-inflammatory cytokines upon stimulation, but the magnitude of response and composition of cytokine profiles may differ. Due to the enormous signal amplification mediated by the TCR complex, T cells will require much less antibody than Fc-gamma receptor-positive immune cells to elicit a strong cytokine release. This has already been evident for the T-cell-activating mouse antihuman CD3 mAb OKT-3 (muromab), where IV injection of only microgram amounts caused severe CRS. Robust cytokine release was also observed for blinatumomab at low microgram doses upon start of infusion. This may explain a number of its expected adverse reactions including chills, fever, nausea, hypotension, or headache. Overt cytokine release can lead to a “cytokine storm” requiring intensive care. Cell culture experiments suggest that the release of cytokines from T cells in response to blinatumomab is absolutely dependent on the presence of CD19+ target cells and that the magnitude of cytokine release strictly correlates with the number of accessible target cells. Cytokine release in patients in response to blinatumomab administration may therefore indicate that T cells have encountered target cells and that its intensity may relate to the number of accessible target cells, that is, the accessible tumor and normal target cell load.
