An Fc‐optimized NKG2D‐immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia

Abstract

Recruitment of Fc-receptor-bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor-associated expression of NKG2DL for targeting leukemic cells by NKG2D-immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D-Fc containing a wild-type Fc part (NKG2D-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D-Fc-KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT already enhanced antileukemia reactivity by inducing antibody-dependent cellular cytotoxicity (ADCC) with NKG2D-Fc-ADCC mediating significantly stronger effects. Parallel application of NKG2D-Fc-ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumorassociated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D-Fc-ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.For more than a decade, chimeric or humanized secondgeneration monoclonal antibodies have been successfully used in cancer therapy. Introduction, for example, of Rituximab and Herceptin in therapeutic regimes applied to patients with B-cell Non-Hodgkin's lymphoma and Her2-positive breast cancer, respectively, lead to considerably improved outcomes. 1,2 Nevertheless, the "antibody success story" has its shortcomings: some patients do not respond at all, others for a limited time only. Thus, numerous strategies are presently being evaluated to increase the efficacy of antitumor antibodies. 3,4 To this end, improving the ability to recruit Fcreceptor (FcR)-bearing immune cells, one of the most important antibody functions constitutes a highly promising approach. 5,6 Currently, three antibodies, directed to CD19, CD30 and CD40, carrying the amino acid modifications S239D and I332E (SDIE modification) 7-9 and a CD20 antibody with improved, low-fucose glycosylation (GA101) 10 are in early clinical development. These reagents mediate markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) against tumor cells, and it appears that their

NKG2D‐ligands: Putting everything under the same umbrella can be misleading

“…NKG2D‐ligands mark cellular stress and they are expressed by many types of tumour cells that, as a consequence, can become targets for NKG2D. Indeed, the use of effector lymphocytes engineered to express NKG2D as therapy in cancer patients has already been translated into clinical trials and others have shown that Fc‐optimised NKG2D‐Fc constructs can induce NK cell antibody‐dependent cellular cytotoxicity against breast cancer and leukemic cells . However, although NKG2D can initiate cytotoxicity against a cell displaying NKG2D‐ligands at the surface, as we will discuss later, tumours can evade the immune response by releasing the ligand proteins.…”

“…Indeed, the use of effector lymphocytes engineered to express NKG2D as therapy in cancer patients has already been translated into clinical trials and others have shown that Fc-optimised NKG2D-Fc constructs can induce NK cell antibody-dependent cellular cytotoxicity against breast cancer and leukemic cells. 30,31 However, although NKG2D can initiate cytotoxicity against a cell displaying NKG2D-ligands at the surface, as we will discuss later, tumours can evade the immune response by releasing the ligand proteins. So, it is also important to study the dynamics and functional capacities of NKG2Dligands in order to understand properly this complex system as a whole, and to manipulate it for the benefit of patients.…”

Natural killer (NK) cells and anti-tumor therapeutic mAb: unexplored interactions

“…For instance, some of the novel or modified anti-CD20 mAb show an increased capability to trigger ADCC [9, 10, 15, 16, 20, 26, 39-46, 117, 118, 128, 160-162]. In addition, a NKG2D-Fc fusion protein was shown to trigger efficiently NK cell ADCC against NKG2D ligand-expressing tumor cells [168,169]. In addition, a NKG2D-Fc fusion protein was shown to trigger efficiently NK cell ADCC against NKG2D ligand-expressing tumor cells [168,169].…”

“…Along this line, bispecific and trispecific antibodies that bridge CD16 and tumor antigens represent a NK-oriented evolution of tumor-targeting mAb [3,42,[163][164][165][166][167]. In addition, a NKG2D-Fc fusion protein was shown to trigger efficiently NK cell ADCC against NKG2D ligand-expressing tumor cells [168,169]. Promising strategies that exploit the NKG2D activating receptor are represented by bispecific mAb directed against NKG2D/tumor antigen or by fusion proteins that link NKG2D ligands to an anti-tumor antigen mAb [95,96,135,170,171].…”

Designing multivalent proteins based on natural killer cell receptors and their ligands as immunotherapy for cancer

“…NKG2D can be involved in anti-tumor responses induced via IL-2 and IL-12 therapy, and also through CTLA-4 inhibitory receptor blockade [45–47]. A NKG2D-Fc fusion protein was shown to efficiently trigger NK cell ADCC against NKG2D ligand-expressing tumor cells [48, 49]. Novel strategies that exploit the NKG2D activating receptor are represented by bispecific mAbs directed against an NKG2D-tumor-associated antigen or by fusion proteins that link NKG2D ligands to an anti-tumor antigen Fv region to bring NKG2D+ effector cells to tumor cells [50–52].…”