Functional Domain Order of an Anti-EGFR × Anti-CD16 Bispecific Diabody Involving NK Cell Activation

Abstract

Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.

Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

“…However, this might also lead to crosslinking of an effector cell bound by the Fab to a second effector cell bound to the Fc, resulting in target cell independent activity, as known from bivalent CD3-targeting antibodies ( 89 ). To circumvent this risk, many NK cell engagers in the literature bind to CD16a in monovalent fashion ( 14 , 17 , 18 ). One the other hand, a competent or a (glyco-)engineered Fc in combination with a 1 + 1 bsAb can effectively engage NK cells ( 90 ).…”

“…NK cells express CD16a, also known as FcγRIIIa, which binds with low affinity to the Fc parts of antibodies ( 12 , 13 ). Furthermore, engagement of CD16a is less demanding compared to CD3 engagement due to lower steric hindrances and additionally facilitated by the lack of accessory molecules ( 14 ).…”

“…Upon binding to its receptor, the epidermal growth factor (EGF) promotes cell proliferation and survival ( 21 ). Multiple bispecific diabodies targeting EGFR×CD16a have been engineered ( 14 , 22 ) and recently, AFM24, a tetravalent bispecific EGFR×CD16a targeting molecule, entered clinical testing in a phase I/II study (NCT04259450). Even though BiKEs and TriKEs exhibit extraordinary favorable properties, their therapeutic usage is limited by their short half-life.…”

The War Is on: The Immune System against Glioblastoma—How Can NK Cells Drive This Battle?

“…BiKEs and TriKEs have other advantages, such as providing a stronger interaction with anti-CD16 than the one observed in natural CD16-Fc antibody interactions [ 353 ], having reduced size (50–75 kDa) in comparison to bi- and tri-specific antibodies (300–450 kDa) [ 354 ], which leads to a better biodistribution needed for the treatment of solid tumors, in addition to being non-immunogenic [ 350 ]. BiKEs and TriKEs have already been developed and evaluated for a broad variety of antigens from different tumors, such as CD30 for Hodgkin’s lymphoma [ 355 , 356 ], HER2 for breast cancer [ 357 , 358 , 359 ], CD19 for non-Hodgkin’s lymphoma cells [ 360 , 361 , 362 ], CD33 or CD33 together with CD123 for acute myeloid leukemia [ 363 , 364 ] (NCT03214666), EpCAM for carcinomas [ 365 ], CD133 for colorectal cancer [ 366 ], EGFR for carcinomas [ 295 , 367 , 368 ], and B7-H3 for a variety of solid tumors [ 369 ]—but not yet for GBM. Further alternatives that have been already discussed in the literature include the blockage of immune checkpoint receptors via scFvs, and scFv blocking of TGF-β or ADAM-17— the later, a disintegrin and metalloproteinase involved in CD16 shedding [ 349 ].…”

Natural Killer Cells in Post-Transplant Lymphoproliferative Disorders

“…In another attempt to enhance the natural function of tumor-infiltrating NK cells, antibody constructs known as bispecific killer engagers (BiKE) have been developed, which bring these cells into contact with tumor cells in an antigen-specific manner via CD16 [ 78 , 79 , 80 , 81 , 82 ]. In addition to bridging contact, the juxtaposition of NK cells and targets facilitates other activating receptor interactions and other missing-self signals.…”