A cell autonomous regulator of neuronal excitability modulates tau in Alzheimer's disease vulnerable neurons.

Neurons from layer II of the entorhinal cortex (ECII) are the first to accumulate tau protein aggregates and degenerate during prodromal Alzheimer's disease (AD). Gaining insight into the molecular mechanisms underlying this vulnerability will help reveal genes and pathways at play during incipient stages of the disease. Here, we use a data-driven functional genomics approach to model ECII neurons in silico and identify the proto-oncogene DEK as a regulator of tau pathology. We show that epigenetic changes caused by Dek silencing alter activity-induced transcription, with major effects on neuronal excitability. This is accompanied by gradual accumulation of tau in the somatodendritic compartment of mouse ECII neurons in vivo, reactivity of surrounding microglia, and microglia-mediated neuron loss. These features are all characteristic of early AD. The existence of a cell-autonomous mechanism linking AD pathogenic mechanisms in the precise neuron type where the disease starts provides unique evidence that synaptic homeostasis dysregulation is of central importance in the onset of tau pathology in AD.

TrkA expression directs the anti-neoplastic activity of MLK3 inhibitors in triple-negative breast cancer.

Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.

AD-linked R47H-TREM2 mutation induces disease-enhancing microglial states via AKT hyperactivation.

The hemizygous R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), a microglia-specific gene in the brain, increases risk for late-onset Alzheimer's disease (AD). Using transcriptomic analysis of single nuclei from brain tissues of patients with AD carrying the R47H mutation or the common variant (CV)­TREM2, we found that R47H-associated microglial subpopulations had enhanced inflammatory signatures reminiscent of previously identified disease-associated microglia (DAM) and hyperactivation of AKT, one of the signaling pathways downstream of TREM2. We established a tauopathy mouse model with heterozygous knock-in of the human TREM2 with the R47H mutation or CV and found that R47H induced and exacerbated TAU-mediated spatial memory deficits in female mice. Single-cell transcriptomic analysis of microglia from these mice also revealed transcriptomic changes induced by R47H that had substantial overlaps with R47H microglia in human AD brains, including robust increases in proinflammatory cytokines, activation of AKT signaling, and elevation of a subset of DAM signatures. Pharmacological AKT inhibition with MK-2206 largely reversed the enhanced inflammatory signatures in primary R47H microglia treated with TAU fibrils. In R47H heterozygous tauopathy mice, MK-2206 treatment abolished a tauopathy-dependent microglial subcluster and rescued tauopathy-induced synapse loss. By uncovering disease-enhancing mechanisms of the R47H mutation conserved in human and mouse, our study supports inhibitors of AKT signaling as a microglial modulating strategy to treat AD.

MAP4K4 promotes pancreatic tumorigenesis via phosphorylation and activation of mixed lineage kinase 3.

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.

Mixed Lineage Kinase 3 phosphorylates prolyl-isomerase PIN1 and potentiates GLI1 signaling in pancreatic cancer development.

The transcription factor Glioma-Associated Oncogene Homolog 1 (GLI1) is activated by sonic hedgehog (SHH) cascade and is an established driver of pancreatic ductal adenocarcinoma (PDAC). However, therapies targeting upstream hedgehog signaling have shown little to no efficacy in clinical trials. Here, we identify Mixed Lineage Kinase 3 (MLK3) as a druggable regulator of oncogenic GLI1. Earlier, we reported that MLK3 phosphorylated a peptidyl-prolyl isomerase PIN1 on the S138 site, and the PIN1-pS138 translocated to the nucleus. In this report, we identify GLI1 as one of the targets of PIN1-pS138 and demonstrate that PIN1-pS138 is upregulated in human PDAC and strongly associates with the upregulation of GLI1 and MLK3 expression. Moreover, we also identified two new phosphorylation sites on GLI1, T394, and S1089, which are directly phosphorylated by MLK3 to promote GLI1 nuclear translocation, transcriptional activity, and cell proliferation. Additionally, pharmacological inhibition of MLK3 by CEP-1347 promoted apoptosis in PDAC cell lines, reduced tumor burden, extended survival, and reduced GLI1 expression in the Pdx1-Cre x LSL-KRASG12D x LSL-TP53R172H (KPC) mouse model of PDAC. These findings collectively suggest that MLK3 is an important regulator of oncogenic GLI1 and that therapies targeting MLK3 warrant consideration in the management of PDAC patients.

Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8+ T cells.

BACKGROUND: The mitogen-activated protein kinases (MAPKs) are important for T cell survival and their effector function. Mixed lineage kinase 3 (MLK3) (MAP3K11) is an upstream regulator of MAP kinases and emerging as a potential candidate for targeted cancer therapy; yet, its role in T cell survival and effector function is not known. METHODS: T cell phenotypes, apoptosis and intracellular cytokine expressions were analyzed by flow cytometry. The apoptosis-associated gene expressions in CD8+CD38+ T cells were measured using RT2 PCR array. In vivo effect of combined blockade of MLK3 and CD70 was analyzed in 4T1 tumor model in immunocompetent mice. The serum level of tumor necrosis factor-α (TNFα) was quantified by enzyme-linked immunosorbent assay. RESULTS: We report that genetic loss or pharmacological inhibition of MLK3 induces CD70-TNFα-TNFRSF1a axis-mediated apoptosis in CD8+ T cells. The genetic loss of MLK3 decreases CD8+ T cell population, whereas CD4+ T cells are partially increased under basal condition. Moreover, the loss of MLK3 induces CD70-mediated apoptosis in CD8+ T cells but not in CD4+ T cells. Among the activated CD8+ T cell phenotypes, CD8+CD38+ T cell population shows more than five fold increase in apoptosis due to loss of MLK3, and the expression of TNFRSF1a is significantly higher in CD8+CD38+ T cells. In addition, we observed that CD70 is an upstream regulator of TNFα-TNFRSF1a axis and necessary for induction of apoptosis in CD8+ T cells. Importantly, blockade of CD70 attenuates apoptosis and enhances effector function of CD8+ T cells from MLK3-/- mice. In immune-competent breast cancer mouse model, pharmacological inhibition of MLK3 along with CD70 increased tumor infiltration of cytotoxic CD8+ T cells, leading to reduction in tumor burden largely via mitochondrial apoptosis. CONCLUSION: Together, these results demonstrate that MLK3 plays an important role in CD8+ T cell survival and effector function and MLK3-CD70 axis could serve as a potential target in cancer.

Mixed lineage kinase 3 inhibition induces T cell activation and cytotoxicity.

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.

Development of Gleevec Analogues for Reducing Production of ß-Amyloid Peptides through Shifting ß-Cleavage of Amyloid Precursor Proteins.

Imatinib mesylate, 1a, inhibits production of ß-amyloid (Aß) peptides both in cells and in animal models. It reduces both the ß-secretase and γ-secretase cleavages of the amyloid precursor protein (APP) and mediates a synergistic effect, when combined with a ß-secretase inhibitor, BACE IV. Toward developing more potent brain-permeable leads, we have synthesized and evaluated over 75 1a-analogues. Several compounds, including 2a-b and 3a-c, inhibited production of Aß peptides with improved activity in cells. These compounds affected ß-secretase cleavage of APP similarly to 1a. Compound 2a significantly reduced production of the Aß42 peptide, when administered (100 mg/kg, twice daily by oral gavage) to 5 months old female mice for 5 days. A combination of compound 2a with BACE IV also reduced Aß levels in cells, more than the additive effect of the two compounds. These results open a new avenue for developing treatments for Alzheimer's disease using 1a-analogues.

Bidirectional regulation of Aß levels by Presenilin 1.

Alzheimer's disease (AD) is characterized by accumulation of the ß-amyloid peptide (Aß), which is generated through sequential proteolysis of the amyloid precursor protein (APP), first by the action of ß-secretase, generating the ß-C-terminal fragment (ßCTF), and then by the Presenilin 1 (PS1) enzyme in the γ-secretase complex, generating Aß. γ-Secretase is an intramembranous protein complex composed of Aph1, Pen2, Nicastrin, and Presenilin 1. Although it has a central role in the pathogenesis of AD, knowledge of the mechanisms that regulate PS1 function is limited. Here, we show that phosphorylation of PS1 at Ser367 does not affect γ-secretase activity, but has a dramatic effect on Aß levels in vivo. We identified CK1γ2 as the endogenous kinase responsible for the phosphorylation of PS1 at Ser367. Inhibition of CK1γ leads to a decrease in PS1 Ser367 phosphorylation and an increase in Aß levels in cultured cells. Transgenic mice in which Ser367 of PS1 was mutated to Ala, show dramatic increases in Aß peptide and in ßCTF levels in vivo. Finally, we show that this mutation impairs the autophagic degradation of ßCTF, resulting in its accumulation and increased levels of Aß peptide and plaque load in the brain. Our results demonstrate that PS1 regulates Aß levels by a unique bifunctional mechanism. In addition to its known role as the catalytic subunit of the γ-secretase complex, selective phosphorylation of PS1 on Ser367 also decreases Aß levels by increasing ßCTF degradation through autophagy. Elucidation of the mechanism by which PS1 regulates ßCTF degradation may aid in the development of potential therapies for Alzheimer's disease.

SMI-Ribosome inactivating protein conjugates selectively inhibit tumor cell growth.

Cell-targeting conjugates of Saporin 6, a ribosome inactivating protein (RIP), were prepared using the Saporin Ala 157 Cys mutant, a small molecule inhibitor (SMI) of integrins αvß3/αvß5, and a potent cytotoxin, auristatin F (AF). The conjugates selectively and potently inhibited proliferation of tumor cells expressing the target integrins. We anticipate that the small molecule-RIP bioconjugate approach can be broadly applied using other small molecule drugs.

Gleevec shifts APP processing from a ß-cleavage to a nonamyloidogenic cleavage.

Neurotoxic amyloid-ß peptides (Aß) are major drivers of Alzheimer's disease (AD) and are formed by sequential cleavage of the amyloid precursor protein (APP) by ß-secretase (BACE) and γ-secretase. Our previous study showed that the anticancer drug Gleevec lowers Aß levels through indirect inhibition of γ-secretase activity. Here we report that Gleevec also achieves its Aß-lowering effects through an additional cellular mechanism. It renders APP less susceptible to proteolysis by BACE without inhibiting BACE enzymatic activity or the processing of other BACE substrates. This effect closely mimics the phenotype of APP A673T, a recently discovered mutation that protects carriers against AD and age-related cognitive decline. In addition, Gleevec induces formation of a specific set of APP C-terminal fragments, also observed in cells expressing the APP protective mutation and in cells exposed to a conventional BACE inhibitor. These Gleevec phenotypes require an intracellular acidic pH and are independent of tyrosine kinase inhibition, given that a related compound lacking tyrosine kinase inhibitory activity, DV2-103, exerts similar effects on APP metabolism. In addition, DV2-103 accumulates at high concentrations in the rodent brain, where it rapidly lowers Aß levels. This study suggests that long-term treatment with drugs that indirectly modulate BACE processing of APP but spare other BACE substrates and achieve therapeutic concentrations in the brain might be effective in preventing or delaying the onset of AD and could be safer than nonselective BACE inhibitor drugs.

Chemically Programmed Bispecific Antibodies in Diabody Format.

Chemically programmed bispecific antibodies (biAbs) endow target cell-binding small molecules with the ability to recruit and activate effector cells of the immune system. Here we report a platform of chemically programmed biAbs aimed at redirecting cytotoxic T cells to eliminate cancer cells. Two different antibody technologies were merged together to make a novel chemically programmed biAb. This was achieved by combining the humanized anti-hapten monoclonal antibody (mAb) h38C2 with the humanized anti-human CD3 mAb v9 in a clinically investigated diabody format known as Dual-Affinity Re-Targeting (DART). We show that h38C2 × v9 DARTs can readily be equipped with tumor-targeting hapten-derivatized small molecules without causing a systemic response harming healthy tissues. As a proof of concept, we chemically programmed h38C2 × v9 with hapten-folate and demonstrated its selectivity and potency against folate receptor 1 (FOLR1)-expressing ovarian cancer cells in vitro and in vivo Unlike conventional biAbs, chemically programmed biAbs in DART format are highly modular with broad utility in terms of both target and effector cell engagement. Most importantly, they provide tumor-targeting compounds access to the power of cancer immunotherapy.

Chemically Programmed Bispecific Antibody Targeting Legumain Protease and αvß3 Integrin Mediates Strong Antitumor Effects.

A chemically programmed bispecific antibody (cp-bsAb) that targeted cysteine protease legumain and αvß3 integrin has been prepared using the aldolase antibody chemical programming (AACP) strategy. In vitro evaluation of the anti-legumain, anti-integrin cp-bsAb and its comparison with cpAbs targeting either integrin or legumain have shown that the former possesses superior functions, including receptor binding and inhibitory effects on cell proliferation as well as capillary tube formation, among all three cpAbs. The anti-legumain, anti-integrin cp-bsAb also inhibited growth of primary tumor more effectively than either anti-legumain or anti-integrin cpAb as observed in the MDA-MB-231 human breast cancer mouse model. The AACP-based cp-bsAb, which contains a generic aldolase antibody, can also serve as a suitable platform for combination therapy, where two equally potent compounds are used to target extracellular receptors.

Synthesis and evaluation of the aldolase antibody-derived chemical-antibodies targeting α5ß1 integrin.

Integrin α5ß1 is an important therapeutic target that can be inhibited using an aldolase antibody (Ab)-derived chemical-Ab (chem-Ab) for the treatment of multiple human diseases, including cancers. A fairly optimized anti-integrin α5ß1 chem-Ab 38C2-4e was obtained using an in situ convergent chemical programming (CP) approach, which minimized the time and effort needed to develop a chem-Ab. Multiple Ab-programming agents (PAs) 4a-e could be prepared rapidly using the Cu-catalyzed alkyne-azide coupling (Cu-AAC) reaction of an α5ß1 inhibitor 2 with multiple linkers 3a-e, either before or after conjugating the linkers into Ab 38C2 binding sites. In these two-steps processes, the products after step 1 can be used in the next step without performing an extensive purification or analysis of the Ab-PAs or Ab-linker conjugates affording chem-Abs 38C2-(4a-e). Flow cytometry assay was used to determine the binding of the chem-Abs to U87 human glioblastoma cells expressing α5ß1 integrin and identify 38C2-3e as the strongest binder. Further studies revealed that 38C2-3e strongly inhibited proliferation of U87 cells and tube formation of HUVEC in the matrigel assay, as well as tumor growth and metastasis of 4T1 cells in vivo.

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids.

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

Targeting cell surface alpha(v)beta(3) integrin increases therapeutic efficacies of a legumain protease-activated auristatin prodrug.

Novel monomethylauristatin E (MMAE) prodrug 8 was designed and prepared that bound cell surface glycoprotein integrin αvß3, and was activated using legumain protease as a catalyst. Upon activation, prodrug 8 strongly induced the death of MDA-MB-435 cells that express integrin αvß3 on cell surface. Efficacies of prodrug 8 were also determined in vivo using animal models of 4T1 murine breast cancer, D121 Lewis lung carcinoma, and MDA-MB-435 human breast cancer. The results demonstrated that prodrug 8 decreased tumor growth and metastasis effectively. In comparison to the parent cytotoxin, MMAE, and prodrug 3, prodrug 8 was less toxic to mouse white blood cells. The latter caused no loss in weight gain of mice at a dose 3 mg/kg, which is over 30 times in excess to MMAE (0.1 mg/kg). We hypothesize that overexpression and colocalization of integrin αvß3 and legumain protease on tumor cells, tumor vasculature, and/or tumor microenvironments can be exploited to enhance the efficacy and selectivity of potent cytotoxins, such as MMAE, which is otherwise too toxic to use for therapy.

Tissue factor-activated coagulation cascade in the tumor microenvironment is critical for tumor progression and an effective target for therapy.

Tissue factor (TF), a rate-limiting enzyme cofactor in activating coagulation, is highly expressed in a wide spectrum of human tumor and tumor stromal cells. Using TF-deficient cancer cells and a conditional TF-knockout mouse model, we show that TF expressed by cancer cells, but not by the host stromal cells, plays a critical role in tumor growth. In the tumor microenvironment, serum coagulation factors are readily extravasated and therefore lead to continuous TF-mediated activation of coagulation proteases. To target this highly specific cascade of serine proteases, we used both a TF:VIIa inhibitor and doxorubicin-based prodrugs that are selectively activated by TF:FVIIa, FXa, and thrombin. Treatment with the TF:FVIIa inhibitor led to growth retardation in breast tumor models. In contrast, treatment with the prodrug eliminated primary tumor cells and lung metastases without apparent toxicity. Our findings offer preclinical proof of principle that targeting the coagulation cascade that is activated in the tumor microenvironment can be a highly effective approach for cancer therapy.

Chemically programmed antibodies targeting multiple alpha(v) integrins and their effects on tumor-related functions in vitro.

Integrins αvß3 and αvß6 are highly expressed on tumor cells and/or by the tumor vasculature of many human cancers, and represent promising targets for anticancer therapy. Novel chemically programmed antibodies (cpAbs) targeting these integrins were prepared using the catalytic aldolase Antibody (Ab) programming strategy. The effects of the cpAbs on cellular functions related to tumor progression were examined in vitro using tumor cell lines and their cognate integrin ligands, fibronectin and osteopontin. The inhibitory functions of the conjugates and their specificity were examined based on interference with cell-cell and cell-ligand interactions related to tumor progression. Cell binding analyses of the anti-integrin cpAbs revealed high affinity for tumor cells that overexpressed αvß3 and αvß6 integrins, and weak interactions with αvß1 and αvß8 integrins, in vitro. Functional analyses demonstrated that the cpAbs strongly inhibited cell-cell interactions through osteopontin binding, and they had little or no immediate effects on cell viability and proliferation. On the basis of these characteristics, the cpAbs are likely to have a broad range of activities in vivo, as they can target and antagonize one or multiple αv integrins expressed on tumors and tumor vasculatures. Presumably, these conjugates may inhibit the establishment of metastastatic tumors in distant organs through interfering with cell adhesion more effectively than antibodies or compounds targeting one integrin only. These anti-integrin cpAbs may also provide useful reagents to study combined effect of multiple αv integrins on cellular functions in vitro, on pathologies, including tumor angiogenesis, fibrosis, and epithelial cancers, in vivo.

An efficient chemical approach to bispecific antibodies and antibodies of high valency.

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with beta-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (D-Lys(6))-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (D-Lys(6))LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (D-Lys(6))-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors alpha(v)beta(3) and alpha(v)beta(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.