Targeted Erythropoietin Selectively Stimulates Red Blood Cell Expansion in Vivo
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Targeted erythropoietin selectively stimulates red blood cell expansion in vivo Devin R. Burrilla, Andyna Verneta, James J. Collinsa,b,c,d, Pamela A. Silvera,e,1, and Jeffrey C. Waya aWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; bSynthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139; cInstitute for Medical Engineering & Science, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; dBroad Institute of MIT and Harvard, Cambridge, MA 02139; and eDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115 Edited by Ronald A. DePinho, University of Texas MD Anderson Cancer Center, Houston, TX, and approved March 30, 2016 (received for review December 23, 2015) The design of cell-targeted protein therapeutics can be informed peptide linker that permits simultaneous binding of both elements by natural protein–protein interactions that use cooperative phys- to the same cell surface. The targeting element anchors the mu- ical contacts to achieve cell type specificity. Here we applied this tated activity element to the desired cell surface (Fig. 1A, Middle), approach in vivo to the anemia drug erythropoietin (EPO), to direct thereby creating a high local concentration and driving receptor its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) pre- binding despite the mutation (Fig. 1A, Bottom). Off-target sig- cursors and prevent interaction with EPO-Rs on nonerythroid cells, naling should be minimal (Fig. 1B) and should decrease in pro- such as platelets. Our engineered EPO molecule was mutated to portion to the mutation strength. weaken its affinity for EPO-R, but its avidity for RBC precursors Here we tested the chimeric activator strategy in vivo using was rescued via tethering to an antibody fragment that specifi- erythropoietin (EPO) as the drug to be targeted. EPO is a cally binds the human RBC marker glycophorin A (huGYPA). We pleiotropic hormone that signals in diverse cell types (13). EPO systematically tested the impact of these engineering steps on promotes the differentiation of RBC precursors, and recombi- in vivo markers of efficacy, side effects, and pharmacokinetics. nant EPO is used to treat anemia due to chronic kidney disease huGYPA transgenic mice dosed with targeted EPO exhibited ele- vated RBC levels, with only minimal platelet effects. This in vivo and myelosuppressive cancer chemotherapy (13); however, EPO selectivity depended on the weakening EPO mutation, fusion to also signals on megakaryocytes, capillary endothelial cells, and the RBC-specific antibody, and expression of huGYPA. The termi- tumor cells, which may promote the thrombosis and tumor nal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice progression that have been documented in clinical trials and vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on ma- have led to the inclusion of “black box” warnings on EPO-based ture RBCs acted as a significant drug sink but did not inhibit effi- products (13–16). The goal of the present work was to engineer a cacy. In a therapeutic context, our targeting approach may allow form of EPO that, by analogy to CNTF, depends on a non- higher restorative doses of EPO without platelet-mediated side signaling surface marker restricted to RBC precursors and can- effects, and also may improve drug pharmacokinetics. These results not activate cells that may cause EPO side effects. demonstrate how rational drug design can improve in vivo specific- Drug targeting schemes demonstrated in vitro often fail owing ity, with potential application to diverse protein therapeutics. to the demands of in vivo function. Targeted surface receptors might not be restricted to desired tissues; in particular, no known protein engineering | drug targeting | platelet | scFv surface proteins are expressed solely on RBC precursors. In vivo expression of targeted receptors may differ from that on im- n ongoing challenge in therapeutic protein design is speci- mortalized cells used in in vitro experiments. Finally, undesired Aficity of action. The receptor for a protein drug may exist on pharmacokinetics, distribution, and elimination can prevent tar- SCIENCES diverse cell types, resulting in undesired signaling. Numerous geted therapies from reaching desired tissues in adequate levels APPLIED BIOLOGICAL engineering strategies have been used to minimize side effects (17). We addressed these issues by systematically testing the design (1–7). One approach is to tether a protein drug to a cell-specific antibody or antibody fragment. This method can still produce Significance unwanted signaling, however. Even when fused to an antibody, a ligand can still bind to its receptor on cells that cause side effects (8). Erythropoietin is used to treat anemia but has prothrombotic Natural signaling systems often use multicomponent receptor side effects that limit its use. We have demonstrated in vivo the complexes to enable ligands to distinguish between the same ability to target erythropoietin to red blood cell precursors and receptor on two different cells. For example, ciliary neurotrophic away from platelet precursors, thereby potentially avoiding factor (CNTF) first binds a nonsignaling receptor, CNTFRα, off-target effects. We have systematically determined the that is expressed solely by neurons, followed by a second binding protein design features required for in vivo success of the event with the LIFRβ/gp130 signaling receptor complex (9). engineered protein. Our results reveal how rational engineer- CNTF has poor affinity for LIFRβ/gp130 and will activate it only ing of protein drugs can be used to reduce side effects, with on cells that also express CNTFRα. CNTFRα serves to anchor broad implications for designers of therapeutic signaling systems. CNTF on a subset of cells, placing it in a high local concentration β near LIFR and gp130. Thus, CNTF and other cytokines reveal Author contributions: D.R.B. and J.C.W. designed research; D.R.B. and A.V. performed re- that cell specificity can be created through binding to a high- search; D.R.B. and J.C.W. analyzed data; and D.R.B., J.J.C., P.A.S., and J.C.W. wrote the paper. affinity, nonsignaling surface protein that positions the signaling Conflict of interest statement: D.R.B., P.A.S., and J.C.W. are listed as inventors on patent molecule in physical proximity with low-affinity signaling receptors. applications relating to targeted erythropoietin fusion proteins. Earlier work defined a class of engineered proteins, termed This article is a PNAS Direct Submission. “ ” chimeric activators, that can direct signaling to one cell type Data deposition: The sequences reported in this paper have been deposited in the Gen- in vitro (Fig. 1) (10–12). These fusion proteins contain a “tar- Bank database (accession nos. KX026660–KX026663). geting element” (e.g., an antibody fragment) that binds a cell- 1To whom correspondence should be addressed. Email: [email protected]. A Top specific surface marker (Fig. 1 , ) and is tethered to a mutated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. “activity element” (e.g., a hormone or cytokine) by a flexible 1073/pnas.1525388113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1525388113 PNAS | May 10, 2016 | vol. 113 | no. 19 | 5245–5250 Downloaded by guest on September 28, 2021 AB We inferred that huGYPA and EPO-R are coexpressed on RBC precursors in vivo (Fig. 1D). Late RBC precursors (BFU-E descendants) express huGYPA approximately 5 d before ma- turing into erythrocytes (24), whereas EPO-R binding events on cultured late-stage precursor cells (CFU-E) can be detected as late as 24 h before the cells mature into reticulocytes or RBCs (25). These results suggest that EPO-R and huGYPA are coex- pressed during late erythropoiesis, although this idea has not been previously addressed directly in vivo. C We tested 10F7-EPOR150A and control variants in huGYPA transgenic mice (26) because 10F7 does not cross-react with murine GYPA (27), and human EPO activates murine EPO-Rs (13). This animal model reflects the normal expression pattern of huGYPA (26). Transgenic RBCs express less huGYPA than D human RBCs (Fig. S2A); 1.6 μM huGYPA is exposed to plasma in transgenic mice vs. 2.8 μM in humans (Fig. S2B). Treatment of huGYPA transgenic mice with 10F7-EPOR150A should stimulate erythropoiesis, and this production should depend on huGYPA expression and a functional 10F7 element. In Vitro Characterization of Chimeric Activator Variants. Our ex- perimental strategy was designed to identify the chimeric acti- vator features required for the desired in vivo behavior: RBC expansion in the absence of platelet production. We compared 10F7-EPOR150A with variants in which EPO was not mutated (10F7-EPO), 10F7 was mutated to mitigate huGYPA binding Fig. 1. Targeting EPO to RBC precursors with a chimeric activator. (A, Top) (10F7W99G-EPOR150A),andEPOwasmutatedtoeliminateEPO-R Chimeric activator schematic illustrating a targeting element (light blue) affinity (10F7-EPOK45D). Darbepoetin alfa (Aranesp) was used tethered by a linker (black circles) to a mutated activity element (red, black A = as a nontargeted form of EPO (28). Fig. 2 shows engineered X mutation). The chimeric activator initially binds (bold arrow) a target protein schematics and verification of their size and N-linked cell receptor (dark blue) via the targeting element, whereas the mutated activity element has a low affinity (dashed arrow) for its own receptor (pink). glycosylation. (A, Middle) Increased local concentration of the activity element overcomes We quantified the effect of the R150A mutation on the in- its receptor-binding deficit and facilitates interaction (bold arrow). (A, Bottom) teraction between EPO and EPO-R (Fig. 2B). EPO and 10F7- This interaction results in signaling via the activity receptor (bold arrow). (B)On EPO had similar binding kinetics (KD = 5.4 and 7.2 nM, re- off-target cells lacking a receptor for the targeting element, the activity ele- spectively) in accordance with published values (29), indicating ment has little effect (dashed arrow), owing to its mutation.