Design and molecular properties of BiTE and CiTE. (A) Schematic representation of a BiTE-like molecule, scFv domain, consists of 1 variable variable chain (V H ) joined by 1 variable light (V L ) domain, connected in one single polypeptide chain. One of the 2 distal (white) scFv domains is specific to CD3ε, and the other (gray) is specific to CD33 The black connecting line represents a flexible Gly4Ser link between domains. (B) Representation of CiTE molecular schematics. CiTE molecules are built by combining PD-1 ex into CD3 × CD33 BiTE. (C) The mechanism of the BiTE action. To induce activation and cytotoxic activity by T cells, the BiTE protein must involve T cells and tumor cells (AML blast) simultaneously. One cell that binds to T cells or target cells does not cause activation. Simultaneous binding of several BiTE molecules to T cells and tumor cells promotes immunological synapse formation leading to T cell activation, cytokine release (IFN-γ), and tumor cell cytotoxicity. This T-cell activation leads to increased regulation of checkpoint molecules such as PD-1 in T cells and PD-L1 in the AML explosion. PD-L1 interacts with PD-1 in T cells to suppress T-cell-mediated tumor cytotoxicity. (D) Mechanism of CiTE's actions. The blockade of interaction between PD-1 in T cells and PD-L1 tumors by the PD-1 ex domain of the CiTE prevents T-cell anergy and fatigue, leading to increased T-cell proliferation and killing of tumor cells.  Antibody antennas recruit T cells such as the BiTE consisting of a single chain variable fragment (scFvs) of at least 2 antibodies with different specificities, one for surface antigens associated with the tumor. and others for surface antigens in effector cells, such as CD3ε in T cells (see picture panel A). Through their dual specificity, BiTE carries tumor cells close to the T-cell effector independently of HLA to form artificial synapses. These synapses induce T cell activation followed by cytotoxic responses mediated through the release of perforin, granzim, cytokines, and expression of Fas ligands (see figure panel C). Various combinations of all antibodies and their fragments have produced> 60 different bispecific antibody formats that target AML. 2 At present, 7 different T cells direct bispecific antibodies that target CD33 (1945-1920) NCT02520427 [19659008NCT03224819 NCT03144245 and NCT03516760 ), CD123 ( NCT02152956 and NCT2730312 ), or CLL1 (MCLA 117; NCT03038230 ) are in early phase clinical trials for AML.
A mechanism that limits bispecific antibody activity seems is an anergy and fatigue T-cell driven by, among other things, PD-1 / PD-L1 axis (see picture panel C). In 2014, blinatumomab, a BiTE targeting CD3 and CD19, was approved for the treatment of B cell B (ALL-ALL) acute lymphoblastic leukemia. Correlative studies showed that PD-L1 expression was significantly higher in B-ALL cells from nonresponders to blinatumomab compared with respondents and controls. 3 Subsequent in vitro studies show that blockade PD1 / PD-L1 axis with mAb restores blinatumomab activity. Similar data have been described with the sister molecule of blinatumomab, AMG330, a BiTE molecule that recruits T cells that target CD33 in AML. AMG330 increases regulation of PD1 in T cells and PD-L1 in AML explosions in vitro. 4 5 Cytotoxic potential, T-cell activation, and proliferation are all greatly enhanced after blockade of PD Axis -1 / PD-L1 with mAbs. 4 5 Clinically, a child who does not respond to blinatumomab shows a remarkable reduction in bone marrow blasts from 45% to 1% after combination therapy with blinatumomab and pembrolizumab (anti-PD1 mAb). 3 In AML, we observed increased regulation of PD-1 in T cells and PD-L1 in explosions in patients treated with Flotetuzumab ( NCT02152956 ) 6
Molecular novels CiTE reported by Herrmann et al is unique because of its incorporation with the PD-1 domain ex  (low micromolar binding affinity for PD-L1) rather than anti-PP-L1 scFv (K D = 9 nM) into molecules such as CD33 × CD3 BiTE (see picture panel B). This new CiTE was engineered to bind with greater affinity for CD33 (K 19459013) D = 29 nM) from CD3 (K D 19459014 = 121 nM) or PD-L1 (low micromolar range) ordered to give preferential binding to CD33 + target cells. Pospoint inhibition mediated by novel CiTE depends on CD33 involvement in target cells. Furthermore, CiTE selectively targets CD33 + / PD-L1 + double positive cells over CD33 + / PD-L1 – and CD33 – / PD-L1 + single positive cell due to binding depends on the avidity of CD33 scFv and PD-1 domain ex . This increases the specificity for binding PD-1 ligands on CD33 + target cells in the CiTE molecule can provide significant clinical benefits compared to the administration of nontargeted PD-L1 blocking antibodies. The lack of tumor selectivity associated with PD-L1 blocking antibodies can currently induce indiscriminate reactivation of all experienced T cells — antigens, including autoreactive T cells that are functionally functional and potentially damaging, which cause immune-related side effects during and after treatment.  7
Herrmann et al. Compared PD-1 ex-containing CiTE domains to conventional CD3 × CD3 CD24 and one single chain (CD33 × CD3 × PD-L1) where the PD-1 module ex was replaced by anti-PD-L1 scrofit which had high affinity. All 3 molecules induced T-cell activation in a way that was restricted to CD33, giving efficient in vitro killing of CD33 + AML cells at very low concentrations (picomolar range), and cleaning human CD33 + Cell lines in murine xenograft models. CiTE significantly increased (1) interferon-γ (IFN-γ) and granzim B, (2) CD33 killings + PD-L1 + assassination targets, and (3) T cell proliferation in vitro compared to traditional CD24 CD3 × CD3 (see picture panel D). Importantly, CiTE exhibited less binding to the CD33 target – / PD-L1 + compared to triplebody one chain. The reduced binding of CiTE targets to CD33-negative may have reduced weight loss (possible immune-related side effects) associated with a single chain of triplebody in the murine xenograft model.
CD33 target antigens appear during the commitment of hematopoietic stem cells to the myelomonocytic lineage and expressed at ∼ 90% of AML myeloblasts. This is also stated in monocytes, myeloid dendritic cells, and less so, in macrophages and granulocytes. 2 Ligands for PD-1 and PD-1 ex. 1945914 domains included in the new one. CiTE is PD-L1 and PD-L2. Both ligands are constitutively expressed in B cells, dendritic cells, and macrophages, and proinflammatory signals are known to induce higher levels of both PD-L1 and PD-L2. 8 Taken together, these expression patterns indicate that the new CiTE can target a population of normal myeloid cells in addition to the AML explosion. Targeting this can worsen cytokine release syndrome, the main dose-limiting toxicity observed to date with bispecific antibody therapy. Future preclinical studies that examine new CiTE molecules in NSG mice that are manifested reshaped with human T cells and carry proven primary human AML tumors.
Early phase clinical trials that combine T-cell brand antibody constructs with PD antibodies -1 / PD-L1 is in progress. The increase in specificity provided by the new PD-1 ex CiTE-containing domain described by Herrmann et al. May be an important step forward if immune-related side effects limit in this initial trial.