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

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.

Preclinical development of an anti-CD30/anti-CD16A bispecific tetravalent TandAb antibody for the treatment of Hodgkin lymphoma.

e18532 Background: CD30 is a validated tumor target that is expressed on both Hodgkin’s Lymphoma cells and some activated immune cells. To improve antibody-based therapies targeting CD30, Affimed developed an NK cell-recruiting bispecific antibody. Methods: To enhance the recruitment and activation of NK cells for tumor cell lysis, we isolated a human antibody that is highly specific for the A isoform of FcgRIII (CD16A). Its variable domain (Fv) was used to construct a tetravalent bispecific TandAb, consisting of four Fvs linked in tandem, with two binding sites for both the CD16A on NK cell and the CD30 target cell antigens. The resulting anti-CD30/anti-CD16A bispecific tetravalent TandAb AFM13 was compared to both an anti-CD30/anti-CD16A bispecific diabody and an Fc-enhanced anti-CD30 IgG in binding and cytotoxicity assays in vitro. Pharmacokinetics and safety of the TandAb were investigated in Cynomolgus monkeys. Results: AFM13 is retained longer on NK cells than either the anti-CD30 IgG or the bispecific diabody due to its bivalency for CD16A; it binds both CD16A alleles (158F, 158V) equally well indicating the potential for broader patient response than that achieved with current therapeutic mAbs. AFM13 did not induce systemic activation of NK cells. It displays higher potency (EC50) and higher efficacy (maximal lysis) than either the anti-VD30 IgGs or the bispecific diabody in a cytotoxicity assay. The cytotoxic activity of AFM13 is unaffected by physiological concentrations of polyclonal IgG, whereas that of anti-CD30 IgGs is dramatically reduced. Cynomolgus monkeys were identified as the only relevant species for toxicity testing. Pharmacokinetics studies reveal AFM13 half-lives of up to 22.9 h after repeated dosing. MTD was not reached in a single dose escalation study ranging to 100 mg/kg maximum. A 28 days toxicity study, with dosing up to 10 mg/kg every other day, revealed an excellent safety profile. No signs of immunotoxicity or systemic cytokine release were observed. Conclusions: AFM13 may be a more effective treatment for Hodgkin’s Lymphoma that is refractory to treatment with anti-CD30 IgGs and is under evaluation in a Phase I clinical trial.

Novel Anti-CD19/Idiotype Bispecific Diabody Vaccine for B-Cell Lymphoma.

Abstract 2712 Poster Board II-688 The unique immunoglobulin idiotype (Id) on B-cell lymphoma is an excellent target for a “custom-made” vaccine therapy for individual patients. Previous studies have shown that vaccine-induced anti-Id humoral responses are effective in protecting mice from the aggressive 38C13 lymphoma. Therefore, we explore a novel strategy of direct activation of Id-specific B cells using a bispecific diabody containing the Id variable fragments (Fv) of 38C13, and those of an anti-CD19 monoclonal antibody. This molecule is designed to cross-link the Id-specific B cell receptor to the CD19/CD21/CD81 co-stimulatory complex, an interaction that has been shown to lower the activation threshold of B cells and result in a dramatic increase in antibody production. The αCD19/Id diabody is a 52 kDa heterodimer of two single chain Fv's (scFv), αCD19VH-38C13VL and 38C13VH -αCD19VL, held together by non-covalent interactions between the cognate VH and VL domains. It was produced in an E. coli extract-based Cell-Free Protein Synthesis (CFPS) System developed by our group, which has been used successfully to rapidly produce cytokine-Id fusion vaccines in previous studies. Within 2 hours, the two diabody scFv's were co-expressed and self-assembled in vitro. The heterodimer product was purified by metal affinity chromatography. Correct folding was confirmed by a flow cytometry assay in which the diabody bound simultaneously to the CD19 on the surface of B cells and also to an anti-38C13 Id antibody labeled with FITC. Most importantly, as a vaccine the αCD19/Id diabody generated a robust anti-Id IgG response comparable to that induced by 38C13 IgM conjugated to the carrier protein keyhole limpet hemocyanin (KLH). Diabody vaccinated mice also showed improved survival compared to control groups when both were challenged with a lethal dose of tumor. Further optimization of the therapeutic efficacy and the development of improved methods for the rapid and economical production of αCD19/Id diabody by the CFPS technology may make it possible to use individualized diabody-based therapy as a frontline treatment, before the use of toxic chemotherapy and radiation therapy. Disclosures: No relevant conflicts of interest to declare.