. Although site-specific conjugation strategies have been developed that improve the yield and specificity of the conjugation site5,6, issues remain with the cost and intrinsic heterogeneity of the PEG itself7.

maximal hydrodynamic radius to shield its protein payload, we first excluded hydrophobic amino acids (F, I, L, M, V, W and Y), which typically contribute to compact structures and/or protein aggregation.

Renally cleared PEGylated proteins or their metabolites may accumu-late in the kidney, causing formation of PEG hydrates that interfere with normal glomerular filtration. In addition, animals and humans can be induced to make antibodies to PEG9,10

using an anti-CD3 scFv to an anti-TAA scFv via a short five residue peptide linker (GGGGS)

5 µg per square meter per day in relapsed NHL patients led to an elimination of target cells in blood

The absence of a need for co-signaling through CD28 for these BiTEs might also be explained by the observation that among all T cell subtypes, CD8+ effector memory cells CD45RO+ (TEM) and CD8+ effector memory CD45RA+ (TEMRA) contribute the most to BiTE activity, whereas naïve T cells do not contribute at all to the killing efficiency.50 It is believed that memory T cells do not require CD28 costimulation for expansion during secondary responses, which could explain the efficiency of BiTEs. However, this dogma has recently been challenged.5

In the case of molecules targeting CD19 on B cells, one cannot exclude a possible co-signal triggered by the interaction between CD28 and B7, known to be expressed on normal and malignant B cells. However, BiTE molecules targeting EpCAM expressed on a variety of solid tumor of epithelial origin that do not express B7 show similar efficacy.

short flexible glycine–serine linker connects the two scFv domains facilitating bending and/or twisting of the two domains against each other.

Blinatumomab is a murine BiTE® antibody construct combining dual specificities for the human pan-B-cell antigen CD19 and the human CD3ε chain of the TCR complex on a single polypeptide chain, with binding affinities of approximately 10−9 M and 10−7 M, respectively

classic T-cell activation by pMHC-TCR interaction and the BiTE® mode of action share several features: (i) Formation of BiTE®-mediated cytolytic synapses structurally resembles normal immunologic synapses. (ii) Induction of full-blown T-cell activation utilizes common signal transduction pathways. This leads to upregulation of activation markers, cytokine release, and proliferation, and is strictly target cell-dependent. (iii) Redirected lysis of target cells is mediated by induction of apoptosis as evidenced by perforin and granzyme B release, caspase 3/7 activation, PARP cleavage, DNA fragmentation, and membrane blebbing and perforation. (iv) T cells can adopt a serial lysis mode to sequentially kill multiple target cells.

(v) BiTE® antibody constructs engage any T cell irrespective of its TCR specificity. Thus, they induce a polyclonal T-cell response against tumor cells only relying on the surface expression of the respective target antigen. (vi) Except for activation of naive T cells, BiTE® antibody constructs do not require any costimulatory signals for efficient T-cell activation and redirected lysis. Cytotoxicity against target cells is mainly mediated by highly potent CD8+ and CD4+ TEM cells, but other T-cell subsets might further add to the large killing capacity observed with BiTE® antibody constructs. (vii) Formation of the BiTE®-induced cytolytic synapse is supposed to be much more efficient than that of the naturally occurring immunologic synapse which relies on the sparse availability of specific pMHC complexes. In contrast, as target antigen expression is expected to be much higher, both number and size of BiTE®-induced cytolytic synapses are assumed to be adjustable by a variable BiTE® concentration. This difference might further explain the exceptional cytotoxic potency associated with BiTE® antibody constructs.

high cytotoxic potency is linked to CD8+ effector memory T (TEM) and CD45RA+ TEM (TEMRA) cells, and to CD4+ TEM and central memory T (TCM)-cells due to their large number of preformed perforin and granzyme B-filled vesicles and their great proliferative capacity. Another notable T-cell subset in the context of immunotherapy are CD4+ regulatory T (TREG) cells. Their presence in the tumor microenvironment is implicated in the suppression of naturally occurring anti-tumor T-cell responses

efficiently mitigated by dexamethasone which does not influence BiTE®-induced T-cell activation and redirected lysis

Upon synapse formation, T cells not only release perforin and granzyme B but also release large amounts of cytokines, namely interleukin (IL)-6, IL-10, and interferon (IFN)-γ

10–15 of these interactions lead to the formation of a cytolytic synapse much like a pMHC-TCR interaction-based immunologic synapse between an antigen-presenting cell and a T cell [15]

BiTE®-mediated interaction between a target tumor cell and a T cell is completely independent of both pMHC expression and specific TCR; it exclusively relies on TAA expression and CD3ε.

non‐glycosylated protein that does not have an Fc domain, thus it does not undergo FcRn‐mediated recycling

short elimination half‐life of roughly 2 h and negligible renal clearance

Blinatumomab activates T cells at picomolar concentrations, mimicking a natural MHC class I/peptide interaction with the T‐cell receptor. Expression of MHC class I and the presence of T‐cell receptor are not required for the redirected lysis, indicating that the effect of blinatumomab is independent of peptide antigen presentation and the presence of a T‐cell receptor.66 An advantage of this approach is that engaged T cells will be less susceptible to major immune escape mechanisms of tumor cells, thereby improving the effectiveness of T‐cell‐mediated killing.