Bispecific T‐cell engagers (BiTEs) are a new class of immunotherapeutic molecules intended for the treatment of cancer. These molecules enhance the patient's immune response to tumors by retargeting T cells to tumor cells. BiTEs are constructed of two single‐chain variable fragments (scFv) connected in tandem by a flexible linker. One scFv binds to a T‐cell‐specific molecule, usually CD3, whereas the second scFv binds to a tumor‐associated antigen. This structure and specificity allows a BiTE to physically link a T cell to a tumor cell, ultimately stimulating T‐cell activation, tumor killing and cytokine production. BiTEs have been developed, which target several tumor‐associated antigens, for a variety of both hematological and solid tumors. Several BiTEs are currently in clinical trials for their therapeutic efficacy and safety. This review examines the salient structural and functional features of BiTEs, as well as the current state of their clinical and preclinical development.

Uterine NK (uNK) cells express a unique set of markers compared with blood NK cells. However, recent studies suggest that uNK cells may be derived from the recruitment of blood NK cells into the endometrium. In this study, we used an in vitro organ culture system to demonstrate that estradiol induces expression of chemokines CXCL10 and/or CXCL11 within human endometrium in 85% of patient samples tested. The average increase in gene expression after 10−9 M estradiol treatment was 8.5-fold for CXCL10 and 7.7-fold for CXCL11 compared with medium alone. We observed that a specific estrogen receptor antagonist (ICI182780) was able to prevent chemokine gene induction, indicating that the effect of estradiol was receptor mediated. Moreover, our study showed that progesterone induced CXCL10 and CXCL11 expression in 83% of endometrial samples tested. We have also found that uNK cells and blood NK cells express the receptor for CXCL10 and CXCL11, CXCR3, with the highest expression found on uNK cells and CD56bright blood NK cells. These data indicate that sex hormones induce specific chemokines in nonpregnant human endometrium that can activate NK cell migration, and suggest that this mechanism may account for the increased NK cell numbers in endometrium during the menstrual cycle.

NKG2D is an activating cell-surface receptor expressed on natural killer (NK) cells and some T-cell subsets. Its ligands are primarily expressed on tumor cells. The aim of this study was to determine whether chimeric NK-receptor—bearing T cells would directly kill tumor cells and lead to induction of host immunity against tumors. Chimeric NK receptors were produced by linking NKG2D or DNAX activating protein of 10 kDa (Dap10) to the cytoplasmic portion of the CD3ζ chain. Our results showed that chimeric (ch) NKG2D-bearing T cells responded to NKG2D-ligand–bearing tumor cells (RMA/Rae-1β, EG7) but not to wild-type tumor cells (RMA). This response was dependent upon ligand expression on the target cells but not on expression of major histocompatibility complex (MHC) molecules, and the response could be blocked by anti-NKG2D antibodies. These T cells produced large amounts of T-helper 1 (Th1) cytokines and proinflammatory chemokines and killed ligand–expressing tumor cells. Adoptive transfer of chNKG2D-bearing T cells inhibited RMA/Rae-1β tumor growth in vivo. Moreover, mice that had remained tumor-free were resistant to subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor antigens. Taken together, our findings indicate that modification of T cells with chimeric NKG2D receptors represents a promising approach for immunotherapy against cancer.

Natural killer (NK) cells are a major population of lymphocytes in the human endometrium (EM), and NK cells can be a significant source of cytokines that alter local immune responses. The aim of this study was to determine the expression of NK cell receptors in situ and to test whether uterine NK (uNK) cells produce cytokines and how this activity may be regulated by transforming growth factor-β (TGF-β). We observed that human uNK cells were CD56+, CD3−, CD57−, CD9+, CD94+, killer inhibitory receptor+, and CD16+/− in situ by confocal microscopy. We examined cytokine production by uNK cells and uNK cell clones derived from human EM. Stimulation of uNK cells with interleukin (IL)-12 and IL-15, both of which are expressed in the human EM, induced interferon-γ (IFN-γ) and IL-10 production. IFN-γ production by uNK cell clones was completely inhibited by TGF-β1 in a dose-dependent manner with an inhibitory concentration 50% value of 20 pg/ml. IL-10 secretion by uNK cell clones was also inhibited by TGF-β1 at similar concentrations. Furthermore, blocking endogenous TGF-β in fresh human endometrial cell cultures increased the production of IFN-γ by uNK cells. These data indicate that uNK cells have a unique phenotype that is distinct from blood NK cells. Further, data demonstrate that uNK cells can produce immunoregulatory cytokines and that inhibition of uNK cells by locally produced TGF-β1 is a likely mechanism to regulate NK cell function in the human EM.

NKp30 is a natural cytotoxicity receptor that is expressed on NK cells and recognizes B7-H6, which is expressed on several types of tumors but few normal cells. To target effector T cells against B7-H6⁺ tumors, we developed several chimeric AgRs (CARs) based on NKp30, which contain the CD28- and/or CD3ζ-signaling domains with the transmembrane domains from CD3ζ, CD28, or CD8α. The data show that chimeric NKp30-expressing T cells responded to B7-H6⁺ tumor cells. The NKp30 CAR-expressing T cells produced IFN-γ and killed B7-H6 ligand-expressing tumor cells; this response was dependent upon ligand expression on target cells but not on MHC expression. PBMC-derived dendritic cells also express NKp30 ligands, including immature dendritic cells, and they can stimulate NKp30 CAR-bearing T cells to produce IFN-γ, but to a lesser extent. The addition of a CD28-signaling domain significantly enhanced the activity of the NKp30 CAR in a PI3K-dependent manner. Adoptive transfer of T cells expressing a chimeric NKp30 receptor containing a CD28-signaling domain inhibited the growth of a B7-H6–expressing murine lymphoma (RMA/B7-H6) in vivo. Moreover, mice that remained tumor-free were resistant to a subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor Ags. Overall, this study demonstrates the specificity and therapeutic potential of adoptive immunotherapy with NKp30 CAR-expressing T cells against B7-H6⁺ tumor cells in vivo.