89Zr-ImmunoPET Shows Therapeutic Efficacy of Anti-CD20-IFNα Fusion Protein in a Murine B-cell Lymphoma Model.

Antibody-mediated tumor delivery of cytokines can overcome limitations of systemic administration (toxicity, short half-lives). Previous work showed improved antitumor potency of anti-CD20-IFNα fusion proteins in preclinical mouse models of B-cell lymphoma. Although tumor targeting is mediated by the antibody part of the fusion protein, the cytokine component might strongly influence biodistribution and pharmacokinetics, as a result of its affinity, size, valency, and receptor distribution. Here, we used immunoPET to study the in vivo biodistribution and tumor targeting of the anti-CD20 rituximab-murine IFNα1 fusion protein (Rit-mIFNα) and compared it with the parental mAb (rituximab, Rit). Rit-mIFNα and Rit were radiolabeled with zirconium-89 (89Zr, t1/2 78.4 hours) and injected into C3H mice bearing syngeneic B-cell lymphomas (38C13-hCD20). Dynamic [(2 hours post injection (p.i.)] and static (4, 24, and 72 hours) PET scans were acquired. Ex vivo biodistribution was performed after the final scan. Both 89Zr-Rit-mIFNα and 89Zr-Rit specifically target hCD20-expressing B-cell lymphoma in vivo. 89Zr-Rit-mIFNα showed specific uptake in tumors (7.6 ± 1.0 %ID/g at 75 hours p.i.), which was significantly lower than 89Zr-Rit (38.4 ± 9.9 %ID/g, P < 0.0001). ImmunoPET studies also revealed differences in the biodistribution, 89Zr-Rit-mIFNα showed rapid blood clearance and high accumulation in the liver compared with 89Zr-Rit. Importantly, immunoPET clearly revealed a therapeutic effect of the single 89Zr-Rit-mIFNα dose, resulting in smaller tumors and fewer lymph node metastases compared with mice receiving 89Zr-Rit. Mice receiving 89Zr-Rit-mIFNα had enlarged spleens, suggesting that systemic immune activation contributes to therapeutic efficacy in addition to the direct antitumoral activity of IFNα. In conclusion, immunoPET allows the noninvasive tracking and quantification of the antibody-cytokine fusion protein and helps understand the in vivo behavior and therapeutic efficacy.

An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1-HK2+ Multiple Myeloma.

Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.

Characterization of IgA and IgM binding and internalization by surface-expressed human Fcα/µ receptor.

The Fcα/µ receptor (Fcα/µR) is an unusual Fc receptor in that it binds to two different antibody isotypes, IgA and IgM. This receptor is of interest because it is thought to be involved in the capture of IgA- and IgM-immune complexes and antigen presentation. To further characterize this receptor, we were able to stably express human Fcα/µR on the surface of the 293T cell line. Using this system, we determined the affinity of the interactions of the receptor with IgA and IgM, which led to novel insights including the important finding that IgM polymers can bind to human Fcα/µR in the absence of J chain. This is in contrast to the polymeric immunoglobulin receptor (pIgR), which requires the presence of J chain to bind to polymeric IgA and IgM. The dissociation constants (K(d)) of all of the different human IgA isotypes and allotypes for human Fcα/µR were determined, and we show that the N-linked glycans on IgA1 are not required for binding to the receptor. In addition, we demonstrate that IgA can be rapidly internalized by human Fcα/µR in the presence of cross-linking antibody.

Variable region domain exchange in human IgGs promotes antibody complex formation with accompanying structural changes and altered effector functions.

Variable region domain exchanged IgG, or "inside-out (io)," molecules, were produced to investigate the effects of domain interactions on antibody structure and function. Studies using ultracentrifugation and electron microscopy showed that variable region domain exchange induces non-covalent multimerization through Fab domains. Surprisingly, variable region exchange also affected Fc-associated functions such as serum half-life and binding to protein G and FcgammaRI. These alterations were not merely a consequence of IgG aggregation. Both the extent of multimerization and alterations in Fc-associated properties depended on the IgG isotype.

Cleavage of the human immunoglobulin A1 (IgA1) hinge region by IgA1 proteases requires structures in the Fc region of IgA.

Secretory immunoglobulin A (IgA) protects the mucosal surfaces against inhaled and ingested pathogens. Many pathogenic bacteria produce IgA1 proteases that cleave in the hinge of IgA1, thus separating the Fab region from the Fc region and making IgA ineffective. Here, we show that Haemophilus influenzae type 1 and Neisseria gonorrhoeae type 2 IgA1 proteases cleave the IgA1 hinge in the context of the constant region of IgA1 or IgA2m(1) but not in the context of IgG2. Both C(alpha)2 and C(alpha)3 but not C(alpha)1 are required for the cleavage of the IgA1 hinge by H. influenzae and N. gonorrhoeae proteases. While there was no difference in the cleavage kinetics between wild-type IgA1 and IgA1 containing only the first GalNAc residue of the O-linked glycans, the absence of N-linked glycans in the Fc increased the ability of the N. gonorrhoeae protease to cleave the IgA1 hinge. Taken together, these results suggest that, in addition to the IgA1 hinge, structures in the Fc region of IgA are required for the recognition and cleavage of IgA1 by the H. influenzae and N. gonorrhoeae proteases.

Biological activity of a mouse-human chimeric immunoglobulin G2 antibody to Cryptococcus neoformans polysaccharide.

The variable regions of the heavy and light chains of the protective murine monoclonal antibody (MAb) 2H1 (m2H1) were expressed with the human constant region genes for immunoglobulin G2 (IgG2) and kappa, respectively, to construct a chimeric antibody (ch2H1). ch2H1 retains the specificity of the parent MAb, exhibits biological activity, and lacks the toxicity of the parent murine IgG1 in chronically infected mice.