First Bispecific Antibody Immunocytokine (Anti-CD20/HLA-DR-Interferon-á2b) Is Highly Toxic for Human Lymphoma Cells in Vitro.
Authors: Nordstrom Diane L , Rossi Edmund A , Goldenberg David M. , Chang Chien-Hsing 
Published: October 2019

Abstract
Abstract 1695
Poster Board I-721

Background
IFN-á2 is indicated for the therapy of a variety of hematopoietic tumors. As with most cytokines, the short serum half-life and severe side effects of IFN-á2 are major factors affecting its dosing schedule and efficacy. Fusion or conjugation of IFN-á2 to a tumor-targeting IgG has the potential to enhance in vivo potency due to increased tumor localization and more favorable pharmacokinetics. We have recently demonstrated that 20-2b, a monospecific immunocytokine generated by the dock-and-lock (DNL) method to comprise tetrameric IFN-á2b covalently linked to veltuzumab, a humanized anti-CD20 mAb, exhibited very potent anti-tumor activity in vitro and in human lymphoma xenografts (Rossi et al., Blood, in press). However, lymphomas and leukemias that express little or no CD20 are expected to be resistant to therapy with 20-2b. HLA-DR is expressed on many hematopoietic tumors and some solid cancers. A bispecific immunocytokine that could target IFN-á to both CD20 and HLA-DR might be a more effective therapeutic against a wide variety of hematopoietic malignancies, including those that express CD20, HLA-DR, or both. Since each component of the multifunctional complex (veltuzumab, anti-HLA-DR F(ab)2, and IFN-á2b) has anti-tumor activity independently, we evaluated if the bispecific immunocytokine can potentially be even more potent than the monospecific immunocytokine, 20-2b.

Methods
One strategy of the modular DNL method is to fuse either the dimerization-and-docking domain (DDD) derived from protein kinase A, or the anchoring domain (AD) of a cognate A-kinase anchoring protein, to a biological entity, resulting in respective DDD- and AD-modules that are readily combined to quantitatively generate stably-tethered structures of defined composition with retained bioactivity. We have selectively combined recombinant DDD-modules of both IFN-á2b and anti-HLA-DR Fab (derived from humanized L243) together with a recombinant AD-module of anti-CD20 IgG (veltuzumab) to generate the first bispecific antibody-based immunocytokine, designated 20-C2-2b, which comprises two copies of IFN-á2b and a stabilized F(ab)2 of hL243 site-specifically linked to veltuzumab.

Results
Each of the three modules, veltuzumab-AD, hL243-Fab-DDD, and IFN-á2b-DDD, was produced recombinantly in separate myeloma cell cultures. Combining equimolar amounts of the three modules under mild redox conditions resulted in the formation of 20-C2-2b, which was purified by sequential chromatographic processes involving Protein A, IMAC and anion exchange chromatography to remove potential side-products such as 20-2b and 20-C2 (the hexavalent bispecific antibody comprising veltuzumab and four Fabs of hL243). Size-exclusion HPLC analysis indicated a major peak of a retention time consistent with a ∼310 kDa protein. Reducing SDS-PAGE of 20-C2-2b revealed the presence of all three constituents. The complex was immunoreactive with an anti-IFN-á2b, an anti-idiotype to hL243, as well as an anti-idiotype to veltuzumab, and showed increased binding to Raji lymphoma cells compared to either veltuzumab or hL243. More importantly, 20-C2-2b was found to be extremely cytotoxic to Daudi, an IFNá-sensitive Burkitt lymphoma cell line, having an IC50 = 0.035 pM, which was 100,000-fold more potent than hL243 IgG, 100-fold more potent than a combination of veltuzumab, hL243 IgG and a structural analog comprising an irrelevant IgG and IFN-á2b, and 5-fold more potent than 20-2b. In the same assay, we have also determined that 20-C2-2b was about 2-fold more potent than C2-2b, which comprises hL243 IgG linked to four molecules of IFN-á2b.

Conclusions
The DNL method provides a modular approach to enable the creation of novel multifunctional complexes. Based on our experience with 20-2b, the bispecific immunocytokine 20-C2-2b is expected to have greater in vivo potency than IFN-á due to improved pharmacokinetics and endowed targeting specificity, and may potentially be useful for therapy of a variety of hematopoietic tumors that express either CD20 or HLA-DR.

Disclosures
Nordstrom: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.