Structural basis of γ chain family receptor sharing at the membrane level.

Common γ chain (γc) cytokine receptors, including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with a common γc receptor (CD132) by concomitant binding of their ectodomains to an interleukin. In this work, we find that direct interactions between the transmembrane domains (TMDs) of both the γc and the interleukin receptors (ILRs) are also required for receptor activation. Moreover, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences within the family. Heterodimer structures of γc TMD bound to IL-7 and IL-9 receptor TMDs-determined in a lipid bilayer-like environment by nuclear magnetic resonance spectroscopy-reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Thus, signaling in the γc receptor family requires specific heterotypic interactions of the TMDs.

Structural basis of γ -chain family receptor sharing at the membrane level.

The common γ-chain (γc) family of cytokine receptors, including interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors, are activated upon engagement with the common γc receptor in ligand dependent manner. Sharing of γc by the IL receptors (ILRs) is thought to be achieved by concomitant binding of γc and ILR ectodomains to a cytokine. Here, we found that direct interactions between the transmembrane domain (TMD) of γc and those of the ILRs are also required for receptor activation, and remarkably, the same γc TMD can specifically recognize multiple ILR TMDs of diverse sequences. Heterodimer structures of γc TMD bound to the TMDs of IL-7R and IL-9R, determined in near lipid bilayer environment, reveal a conserved knob-into-hole mechanism of recognition that mediates receptor sharing within the membrane. Functional mutagenesis data indicate the requirement of the heterotypic interactions of TMDs in signaling, which could explain disease mutations within the receptor TMDs. One-Sentence Summary: The transmembrane anchors of interleukin receptors of the gamma-chain family are critical for receptor sharing and activation.

Self-Assembled L-DNA Linkers for Rapid Construction of Multi-Specific Antibody-Drug Conjugates Library.

One of the key challenges of improving clinical outcomes of antibody drug conjugates (ADCs) is overcoming cancer resistance to the antibody and/or drug components of ADCs, and hence the need for ADC platforms with high combinatory flexibility. Here, we introduce the use of self-assembled left-handed DNA (L-DNA) oligonucleotides to link combinatory single-domain antibodies and toxin payloads for tunable and adaptive delivery of ADCs. We demonstrate that the method allows convenient construction of a library of ADCs with multi-specific targeting, multi-specific payloads, and exact drug-antibody ratio. The newly constructed ADCs with L-DNA scaffold showed favorable properties of in vitro cell cytotoxicity and in vivo suppression and eradication of solid tumors. Collectively, our data suggest that the L-DNA based modular ADC (MADC) platform is a viable option for generating therapeutic ADCs and for potentially expanding ADC therapeutic window via multi-specificity.

Autoinhibitory structure of preligand association state implicates a new strategy to attain effective DR5 receptor activation.

Members of the tumor necrosis factor receptor superfamily (TNFRSF) are important therapeutic targets that can be activated to induce death of cancer cells or stimulate proliferation of immune cells. Although it has long been implicated that these receptors assemble preligand associated states that are required for dominant interference in human disease, such states have so far eluded structural characterization. Here, we find that the ectodomain of death receptor 5 (DR5-ECD), a representative member of TNFRSF, can specifically self-associate when anchored to lipid bilayer, and we report this self-association structure determined by nuclear magnetic resonance (NMR). Unexpectedly, two non-overlapping interaction interfaces are identified that could propagate to higher-order clusters. Structure-guided mutagenesis indicates that the observed preligand association structure is represented on DR5-expressing cells. The DR5 preligand association serves an autoinhibitory role as single-domain antibodies (sdAbs) that partially dissociate the preligand cluster can sensitize the receptor to its ligand TRAIL and even induce substantial receptor signaling in the absence of TRAIL. Unlike most agonistic antibodies that require multivalent binding to aggregate receptors for activation, these agonistic sdAbs are monovalent and act specifically on an oligomeric, autoinhibitory configuration of the receptor. Our data indicate that receptors such as DR5 can form structurally defined preclusters incompatible with signaling and that true agonists should disrupt the preligand cluster while converting it to signaling-productive cluster. This mechanism enhances our understanding of a long-standing question in TNFRSF signaling and suggests a new opportunity for developing agonistic molecules by targeting receptor preligand clustering.

Structure-guided and phage-assisted evolution of a therapeutic anti-EGFR antibody to reverse acquired resistance.

Acquired resistance to cetuximab in colorectal cancers is partially mediated by the acquisition of mutations located in the cetuximab epitope in the epidermal growth factor receptor (EGFR) ectodomain and hinders the clinical application of cetuximab. We develop a structure-guided and phage-assisted evolution approach for cetuximab evolution to reverse EGFRS492R- or EGFRG465R-driven resistance without altering the binding epitope or undermining antibody efficacy. Two evolved cetuximab variants, Ctx-VY and Ctx-Y104D, exhibit a restored binding ability with EGFRS492R, which harbors the most common resistance substitution, S492R. Ctx-W52D exhibits restored binding with EGFR harboring another common cetuximab resistance substitution, G465R (EGFRG465R). All the evolved cetuximab variants effectively inhibit EGFR activation and downstream signaling and induce the internalization and degradation of EGFRS492R and EGFRG465R as well as EGFRWT. The evolved cetuximab variants (Ctx-VY, Ctx-Y104D and Ctx-W52D) with one or two amino acid substitutions in the complementarity-determining region inherit the optimized physical and chemical properties of cetuximab to a great extent, thus ensuring their druggability. Our data collectively show that structure-guided and phage-assisted evolution is an efficient and general approach for reversing receptor mutation-mediated resistance to therapeutic antibody drugs.

Structure of the Streptococcus pyogenes NAD+ Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes.

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a ß-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

An amphipathic Bax core dimer forms part of the apoptotic pore wall in the mitochondrial␣membrane.

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

A Trimeric Hydrophobic Zipper Mediates the Intramembrane Assembly of SARS-CoV-2 Spike.

The S protein of SARS-CoV-2 is a type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the NMR structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in a membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeats. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchors of viral fusion proteins can form highly specific oligomers, but the exact function of these oligomers remains unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

Inhibitor Development against p7 Channel in Hepatitis C Virus.

Hepatitis C Virus (HCV) is the key cause of chronic and severe liver diseases. The recent direct-acting antiviral agents have shown the clinical success on HCV-related diseases, but the rapid HCV mutations of the virus highlight the sustaining necessity to develop new drugs. p7, the viroporin protein from HCV, has been sought after as a potential anti-HCV drug target. Several classes of compounds, such as amantadine and rimantadine have been testified for p7 inhibition. However, the efficacies of these compounds are not high. Here, we screened some novel p7 inhibitors with amantadine scaffold for the inhibitor development. The dissociation constant (Kd) of 42 ARD-series compounds were determined by nuclear magnetic resonance (NMR) titrations. The efficacies of the two best inhibitors, ARD87 and ARD112, were further confirmed using viral production assay. The binding mode analysis and binding stability for the strongest inhibitor were deciphered by molecular dynamics (MD) simulation. These ARD-series compounds together with 49 previously published compounds were further analyzed by molecular docking. Key pharmacophores were identified among the structure-similar compounds. Our studies suggest that different functional groups are highly correlated with the efficacy for inhibiting p7 of HCV, in which hydrophobic interactions are the dominant forces for the inhibition potency. Our findings provide guiding principles for designing higher affinity inhibitors of p7 as potential anti-HCV drug candidates.

NMR Model of the Entire Membrane-Interacting Region of the HIV-1 Fusion Protein and Its Perturbation of Membrane Morphology.

HIV-1 envelope glycoprotein (Env) is a transmembrane protein that mediates membrane fusion and viral entry. The membrane-interacting regions of the Env, including the membrane-proximal external region (MPER), the transmembrane domain (TMD), and the cytoplasmic tail (CT), not only are essential for fusion and Env incorporation but also can strongly influence the antigenicity of the Env. Previous studies have incrementally revealed the structures of the MPER, the TMD, and the KS-LLP2 regions of the CT. Here, we determined the NMR structure of the full-length CT using a protein fragment comprising the TMD and the CT in bicelles that mimic a lipid bilayer, and by integrating the new NMR data and those acquired previously on other gp41 fragments, we derived a model of the entire membrane-interacting region of the Env. The structure shows that the CT forms a large trimeric baseplate around the TMD trimer, and by residing in the headgroup region of the lipid bilayer, the baseplate causes severe exclusion of lipid in the cytoleaflet of the bilayer. All-atom molecular dynamics simulations showed that the overall structure of the MPER-TMD-CT can be stable in a viral membrane and that a concerted movement of the KS-LLP2 region compensates for the lipid exclusion in order to maintain both structure and membrane integrity. Our structural and simulation results provide a framework for future research to manipulate the membrane structure to modulate the antigenicity of the Env for vaccine development and for mutagenesis studies for investigating membrane fusion and Env interaction with the matrix proteins.

A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike.

The S protein of the SARS-CoV-2 is a Type I membrane protein that mediates membrane fusion and viral entry. A vast amount of structural information is available for the ectodomain of S, a primary target by the host immune system, but much less is known regarding its transmembrane domain (TMD) and its membrane-proximal regions. Here, we determined the nuclear magnetic resonance (NMR) structure of the S protein TMD in bicelles that closely mimic a lipid bilayer. The TMD structure is a transmembrane α-helix (TMH) trimer that assembles spontaneously in membrane. The trimer structure shows an extensive hydrophobic core along the 3-fold axis that resembles that of a trimeric leucine/isoleucine zipper, but with tetrad, not heptad, repeat. The trimeric core is strong in bicelles, resisting hydrogen-deuterium exchange for weeks. Although highly stable, structural guided mutagenesis identified single mutations that can completely dissociate the TMD trimer. Multiple studies have shown that the membrane anchor of viral fusion protein can form highly specific oligomers, but the exact function of these oligomers remain unclear. Our findings should guide future experiments to address the above question for SARS coronaviruses.

Pharmacokinetics of Roxadustat: A Population Analysis of 2855 Dialysis- and Non-Dialysis-Dependent Patients with Chronic Kidney Disease.

BACKGROUND: Roxadustat is a novel, small-molecule, first-in-class therapeutic that stimulates erythropoiesis by inhibiting hypoxia-inducible factor prolyl hydroxylase enzymes. This agent (roxadustat) is in clinical development for the treatment of anemia in patients with non-dialysis-dependent (NDD) and dialysis-dependent (DD) chronic kidney disease. A population pharmacokinetic analysis was undertaken to evaluate the effect of intrinsic and extrinsic factors on roxadustat pharmacokinetics. METHODS: Non-linear mixed-effects models implemented in NONMEM software were fitted to 8209 pharmacokinetic samples from 2855 DD and NDD subjects enrolled in four phase III studies with roxadustat dose concentrations of 20-400 mg as orally administered tablets. Effects of intrinsic and extrinsic factors were evaluated using a stepwise covariate modeling procedure in combination with the full covariate approach, and defined no-effect boundaries for exposure were based on the difference in exposure between 70 and 100 mg of roxadustat (i.e., - 30%, + 43%). RESULTS: A two-compartment model with first-order absorption adequately described roxadustat pharmacokinetics, with parameter estimates (relative standard error) for apparent clearance of 1.1 (0.0223) L/h in NDD subjects, and apparent central and peripheral volumes of distribution of 14.9 (0.0278) L and 9.5 (0.0872) L, respectively. Stepwise covariate modeling identified bodyweight, dialysis status, race, and dose as statistically significant covariates on apparent clearance, and bodyweight, sex, and albumin as statistically significant covariates on apparent central volume of distribution. However, the effects of these covariates did not result in roxadustat area under the curve or maximum plasma concentration changes outside of the defined no-effect boundaries. The effects of concomitant oral iron, clopidogrel, and staggered sevelamer, calcium carbonate, or calcium acetate were investigated using a full covariate approach but did not result in roxadustat area under the curve or maximum plasma concentration changes outside of the defined no-effect boundaries. CONCLUSIONS: A population pharmacokinetic model was developed for the pharmacokinetics of roxadustat in the target population. None of the investigated intrinsic or extrinsic factors resulted in a significant change in roxadustat exposure outside of the defined no-effect boundaries.

The Diversity and Similarity of Transmembrane Trimerization of TNF Receptors.

Receptors in the tumor necrosis factor receptor superfamily (TNFRSF) regulate proliferation of immune cells or induce programmed cell death, and many of them are candidates for antibody-based immunotherapy. Previous studies on several death receptors in the TNFRSF including Fas, p75NTR, and DR5 showed that the transmembrane helix (TMH) of these receptors can specifically oligomerize and their oligomeric states have direct consequences on receptor activation, suggesting a much more active role of TMH in receptor signaling than previously appreciated. Here, we report the structure of the TMH of TNFR1, another well studied member of the TNFRSF, in neutral bicelles that mimic a lipid bilayer. We find that TNFR1 TMH forms a defined trimeric complex in bicelles, and no evidences of higher-order clustering of trimers have been detected. Unexpectedly, a conserved proline, which is critical for Fas TMH trimerization, does not appear to play an important role in TNFR1 TMH trimerization, which is instead mediated by a glycine near the middle of the TMH. Further, TNFR1 TMH trimer shows a larger hydrophobic core than that of Fas or DR5, with four layers of hydrophobic interaction along the threefold axis. Comparison of the TNFR1 TMH structure with that of Fas and DR5 reveals reassuring similarities that have functional implications but also significant structural diversity that warrants systematic investigation of TMH oligomerization property for other members of the TNFRSF.

Evolutionarily conserved sequence motif analysis guides development of chemically defined hydrogels for therapeutic vascularization.

Biologically active ligands (e.g., RGDS from fibronectin) play critical roles in the development of chemically defined biomaterials. However, recent decades have shown only limited progress in discovering novel extracellular matrix-protein-derived ligands for translational applications. Through motif analysis of evolutionarily conserved RGD-containing regions in laminin (LM) and peptide-functionalized hydrogel microarray screening, we identified a peptide (a1) that showed superior supports for endothelial cell (EC) functions. Mechanistic studies attributed the results to the capacity of a1 engaging both LM- and Fn-binding integrins. RNA sequencing of ECs in a1-functionalized hydrogels showed ~60% similarities with Matrigel in "vasculature development" gene ontology terms. Vasculogenesis assays revealed the capacity of a1-formulated hydrogels to improve EC network formation. Injectable alginates functionalized with a1 and MMPQK (a vascular endothelial growth factor-mimetic peptide with a matrix metalloproteinase-degradable linker) increased blood perfusion and functional recovery over decellularized extracellular matrix and (RGDS + MMPQK)-functionalized hydrogels in an ischemic hindlimb model, illustrating the power of this approach.

Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat (FG-4592) for Treatment of Anemia in Chronic Kidney Disease: A Placebo-Controlled Study of Pharmacokinetic and Pharmacodynamic Profiles in Hemodialysis Patients.

Roxadustat (FG-4592), an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that stimulates erythropoiesis, was evaluated in a phase 1b study in patients with end-stage renal disease with anemia on hemodialysis. Seventeen patients, on epoetin-alfa maintenance therapy with stable hemoglobin levels ≥10 g/dL, had epoetin-alfa discontinued on day 3 and were enrolled in this double-blind placebo-controlled study. Two cohorts were randomized 3:1 (roxadustat: placebo). Patients received single doses of roxadustat (1 or 2 mg/kg) or placebo 1 hour after hemodialysis on day 1 and 2 hours before dialysis on day 8. Maximum plasma concentration and area under the plasma concentration-time curve for patients receiving roxadustat were slightly more than dose proportional and elimination half-life ranged from 14.7 to 19.4 hours. Roxadustat was highly protein bound (99%) in plasma, and dialysis contributed a small fraction of the total clearance: only 4.56% and 3.04% of roxadustat recovered from the 1 and 2 mg/kg dose groups, respectively. Roxadustat induced transient elevations of endogenous erythropoietin that peaked between 7 and 14 hours after dosing and returned to baseline by 48 hours after dosing. Peak median endogenous erythropoietin levels were 96 mIU/mL and 268 mIU/mL for the 1- and 2-mg/kg doses, respectively, within physiologic range of endogenous erythropoietin responses to hypoxia at high altitude or after blood loss. No serious adverse events were reported, and there were no treatment- or dose-related trends in adverse event incidence.

Structural basis of transmembrane coupling of the HIV-1 envelope glycoprotein.

The prefusion conformation of HIV-1 envelope protein (Env) is recognized by most broadly neutralizing antibodies (bnAbs). Studies showed that alterations of its membrane-related components, including the transmembrane domain (TMD) and cytoplasmic tail (CT), can reshape the antigenic structure of the Env ectodomain. Using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of an Env segment encompassing the TMD and a large portion of the CT in bicelles. The structure reveals that the CT folds into amphipathic helices that wrap around the C-terminal end of the TMD, thereby forming a support baseplate for the rest of Env. NMR dynamics measurements provide evidences of dynamic coupling across the TMD between the ectodomain and CT. Pseudovirus-based neutralization assays suggest that CT-TMD interaction preferentially affects antigenic structure near the apex of the Env trimer. These results explain why the CT can modulate the Env antigenic properties and may facilitate HIV-1 Env-based vaccine design.

Structural Characterization of the N-Terminal Domain of the Dictyostelium discoideum Mitochondrial Calcium Uniporter.

The mitochondrial calcium uniporter (MCU) plays a critical role in mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multicomponent system, including the pore-forming subunit MCU and several regulators (MICU1, MICU2, and Essential MCU REgulator, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as high-order oligomers. Mutagenesis showed that the acidic residues Asp60, Glu72, and Glu74, which appeared to mediate the interface II, as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex was disrupted in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

HIV-1 fusion inhibitors targeting the membrane-proximal external region of Env spikes.

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.

DNA-Mediated Assembly of Multispecific Antibodies for T Cell Engaging and Tumor Killing.

Targeting T-cells against cancer cells is a direct means of treating cancer, and has already shown great responses in clinical treatment of B-cell malignancies. A simple way to redirect T-cells to cancer cells is by using multispecific antibody (MsAb) that contains different arms for specifically "grabbing" the T-cells and cancer cells; as such, the T-cells are activated upon target engagement and the killing begins. Here, a nucleic acid mediated protein-protein assembly (NAPPA) approach is implemented to construct a MsAb for T-cell engaging and tumor killing. Anti -CD19 and -CD3 single-chain variable fragments (scFvs) are conjugated to different l-DNAs with sequences that form the Holliday junction, thus allowing spontaneous assembly of homogeneous protein-DNA oligomers containing two anti-CD19 and one anti-CD3 scFvs. The new MsAb shows strong efficacy in inducing Raji tumor cell cytotoxicity in the presence of T-cells with EC50 ≈ 0.2 × 10-9 m; it also suppresses tumor growth in a Raji xenograft mouse model. The data indicates that MsAbs assembled from protein-DNA conjugates are effective macromolecules for directing T-cells for tumor killing. The modular nature of the NAPPA platform allows rapid generation of complex MsAbs from simple antibody fragments, while offering a general solution for preparing antibodies with high-order specificity.