Abstract
BACKGROUND: Interferon-α2b (IFN-α2b) is active alone and in combination with other agents in the therapy of a variety of cancers, including hairy cell leukemia, chronic myelocytic leukemia, follicular lymphoma, and malignant melanoma. As for most cytokines, the pharmacokinetics are a major factor affecting schedule and efficacy. The protein is rapidly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Commercially available IFNs that are pegylated, such as PEG-INTRON and PEGASYS, have increased serum half-life and reduced renal clearance, which augment their biological activity. For therapy of lymphoma and other cancers, fusing IFN-α2 to tumor-targeting antibodies could increase serum half-life and target the IFN-α2 to the tumor, conceivably allowing less frequent and lower dosing with improved therapeutic efficacy and reduced side effects.
METHODS: The modular DNL method exploits a pair of distinct protein domains involved in the natural binding between protein kinase A (PKA) and A-kinase anchoring proteins (AKAP), whereby the dimerization-and-docking domain (DDD) of PKA and the anchoring domain (AD) of an interactive AKAP are each fused 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 comprising IFN-α2b with recombinant AD-modules derived from the anti-CD20 humanized mAb, veltuzumab, and other humanized mAbs to generate complexes comprising four copies of IFN-α2b site-specifically linked to the bivalent IgG.
RESULTS: The IgG-AD2 and IFN-α2b-DDD2 modules were expressed in separate myeloma cell cultures and purified from culture broths by Protein A and IMAC, respectively. Combining an IgG-AD2 module with slightly more than 2 molar equivalents of the cytokine-DDD2 module under mild redox conditions resulted in the formation of a covalent complex comprising one IgG and 4 IFN-α2b via the docking of each of the two AD2 domains on IgG with a dimer of IFN-α2b-DDD2, and subsequent formation of disulfide bonds (locking) between DDD2 and AD2. The 255-kDa conjugates, which were purified by Protein A, were readily detected by size-exclusion HPLC and non-reducing SDS-PAGE, and retained the biological functions of IFN-α2b in vitro. The IgG-IFN-α2b constructs exhibited potent anti-viral activity in vitro, with specific activities approaching that of recombinant human IFN-α2b. Additionally, the constructs all showed highly potent in vitro cytotoxicity against Burkitt lymphoma cell lines. Notably, the CD20-targeted IFN-α2b construct (20-2b) was 30-fold more potent than a control, non-targeting IgG-IFN-α2b. The enhanced cytotoxicity of 20-2b was not reproduced when non-targeting IgG-IFN-α2b was used in combination with veltuzumab, suggesting that IFN-α2b must be physically linked to achieve maximal potency. IgG-IFN-α2b fusion proteins, including 20-2b, induced significantly more potent ADCC compared to their parental MAbs. The targeting properties of 20-2b were comparable to veltuzumab, and its serum half-life was significantly longer than PEG-INTRON and PEGASYS. In the human Daudi xenograft model, 20-2b showed superior anti-tumor efficacy compared to both veltuzumab and other IgG-IFN-α2 agents. The median survival time (MST) for mice treated with single-dose 170 ng 20-2b was 101.5 days, whereas those treated with an equivalent dose of veltuzumab and untreated mice survived 39 and 28 days, respectively (P<0.0005). Lower 20-2b doses of 80, 17 and 8 ng resulted in MST of 97.5, 56.5 and 48 days, respectively, with the lowest dose still significantly better than the highest dose of veltuzumab (P=0.0434). Using a single 170-ng dose, a CD22-targeting IFN-α2b (22-2b) also increased MST significantly to 47 days (P =0.0119), while a non-targeting IgG-IFN-α2b (734-2b) did not.
CONCLUSIONS: The DNL method provided an IFN-α2-targeting mAb fusion protein that showed improved anti-tumor efficacy over the mAb by itself, based on improved pharmacokinetics, ADCC, and tumor targeting, as well as reduced systemic toxicity. Thus, DNL provides a modular approach to efficiently tether cytokines to targeting antibodies, resulting in higher in vivo potency than the original cytokines or mAbs.
The M r 140,000 Intermediate Chain ofChlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring