Subhash C. Sinha

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.

A general approach to γ-lactones via osmium-catalyzed asymmetric dihydroxylation. Synthesis of (−)- and (+)-muricatacin.

Abstract Both enantiomers of hydroxy γ-lactones have been prepared highly enantioselectively (92–99% ee) using either AD-mix-β or AD-mix-α with both β,γ- and γ,δ-unsaturated esters. The method is exemplified by the three-step synthesis of (−) and (+)-muricatacin in 74% yield and >99% ee.

Chemically programmed monoclonal antibodies for cancer therapy: Adaptor immunotherapy based on a covalent antibody catalyst

Proposing that a blend of the chemical diversity of small synthetic molecules with the immunological characteristics of the antibody molecule will lead to therapeutic agents with superior properties, we here present a device that equips small synthetic molecules with both effector function and long serum half-life of a generic antibody molecule. As a prototype, we developed a targeting device that is based on the formation of a covalent bond of defined stoichiometry between a 1,3-diketone derivative of an integrin αvβ3 and αvβ5 targeting Arg-Gly-Asp peptidomimetic and the reactive lysine of aldolase antibody 38C2. The resulting complex was shown to (i) spontaneously assemble in vitro and in vivo, (ii) selectively retarget antibody 38C2 to the surface of cells expressing integrins αvβ3 and αvβ5, (iii) dramatically increase the circulatory half-life of the Arg-Gly-Asp peptidomimetic, and (iv) effectively reduce tumor growth in animal models of human Kaposis sarcoma and colon cancer. This immunotherapeutic has the potential to target a variety of human cancers, acting on both the vasculature that supports tumor growth as well as the tumor cells themselves. Further, by use of a generic antibody molecule that forms a covalent bond with a 1,3-diketone functionality, essentially any compound can be turned into an immunotherapeutic agent thereby not only increasing the diversity space that can be accessed but also multiplying the therapeutic effect.

Genetic Incorporation of Multiple Unnatural Amino Acids into Proteins in Mammalian Cells

The ability to genetically incorporate unnatural amino acids (UAAs) at specific sites in the proteome of living cells provides a powerful tool to both investigate and engineer protein structure and function. A reassigned nonsense or frameshift codon is used to encode the UAA of interest, and an orthogonal aminoacyl-tRNA synthetase/tRNA (aaRS/ tRNA) pair specific for the UAA delivers the latter cotranslationally into the target protein. This technology has been used to genetically encode a large number of diverse amino acids, including chemically and photochemically reactive amino acids, biophysical probes, metal-ion chelators and redox-active amino acids in E. coli, S. cerevisiae, C. elegans, plant, and mammalian cells. Optimization of the various components of this system in E. coli has resulted in a significant enhancement in suppression efficiency, allowing the incorporation of multiple UAAs in the same polypeptide chain, as well as two different UAAs into a single protein. Unfortunately, suppression systems in mammalian cells are generally less efficient than in E. coli. Furthermore, the incorporation of multiple distinct UAAs into one protein in mammalian cells requires the development of mutually orthogonal aaRS/tRNA pairs capable of suppressing different nonsense/frameshift codons. Here we show that a previously developed enhanced suppression system can be used to incorporate O-methyltyrosine (OMeY, Figure 1b) in good yields at up to three different sites in an enhanced green fluorescent protein (EGFP) in HEK293T cells. We also show that the orthogonal PylRS/tRNAUUA Pyl pair can efficiently suppress the ochre (TAA) nonsense codon in mammalian cells, and in conjunction with the amber (TAG) suppressing EcTyrRS/tRNACUA Tyr pair can be used to incorporate two different UAAs into distinct sites of the same protein. The utility of this technology was demonstrated by generating fulllength anti-HER2 antibody conjugated to auristatin and the fluorophore Alexa Fluor 488 to obtain a defined antibody– drug–fluorophore conjugate. Recently, we reported a mammalian suppression system that offers a significant improvement in the efficiency of UAA incorporation, raising the possibility of its use for the expression of proteins containing multiple UAAs. This system encodes optimized expression cassettes for the aaRS/ tRNA pair and the nonsense mutant of the target gene within one plasmid, which can be used to deliver these genetic elements into mammalian cells either using a baculovirus expression vector, or directly by transient transfection. To evaluate the ability of this system to suppress multiple amber nonsense codons in the same polypeptide, we used pAcBac2 plasmids (Figure 1a) harboring an enhanced green fluorescent protein (EGFP) expression cassette encoding from one to three TAG codons at permissive sites: pAcBac2.tR4OMeYRS/GFP*-1 (Tyr39TAG), pAcBac2.tR4-OMeYRS/ GFP*-2 (Tyr39TAG, Tyr151TAG), and pAcBac2.tR4OMeYRS/GFP*-3 (Ser28TAG, Tyr39TAG, Tyr151TAG). This plasmid also encodes two copies each of the E. coli and Figure 1. Incorporation of one UAA into multiple sites of EGFP expressed in mammalian cells. a) pAcBac2.tR4-OMeYRS/GFP* encodes a CAG-promoter-driven mutant EGFP expression cassette, EcTyrRS gene driven by CMV promoter, and two 2X-tRNACUA Tyr cassettes in two different orientations. b) Structure of O-methyltyrosine (OMeY). c) Expression of the following EGFP mutants in HEK293T cells analyzed by fluorescence microscopy in the presence (+ UAA) or absence (-UAA) of 1 mm OMeY. Scale bar = 500 mm. d) Expression of the aforementioned EGFP mutants analyzed by SDS-PAGE in the presence (+) or absence ( ) of 1 mm OMeY.

Catalytic enantioselective retro-aldol reactions: Kinetic resolution of beta-hydroxyketones with aldolase antibodies

High enantiomeric enrichment after 50% conversion: Racemates of aldols can be resolved by the title reaction [Eq.(1)] by use of the aldolase antibody 38C2 or 33F12; the ee values of the unconverted aldols are greater than 95% in most cases. Since the antibodies also catalyze the aldol reaction-that is, the reverse reaction-it is possible to prepare both enantiomers using the same antibody catalysts.

The Membrane Subunit NuoL(ND5) Is Involved in the Indirect Proton Pumping Mechanism of Escherichia coli Complex I

Complex I pumps protons across the membrane by using downhill redox energy. Here, to investigate the proton pumping mechanism by complex I, we focused on the largest transmembrane subunit NuoL (Escherichia coli ND5 homolog). NuoL/ND5 is believed to have H+ translocation site(s), because of a high sequence similarity to multi-subunit Na+/H+ antiporters. We mutated thirteen highly conserved residues between NuoL/ND5 and MrpA of Na+/H+ antiporters in the chromosomal nuoL gene. The dNADH oxidase activities in mutant membranes were mostly at the control level or modestly reduced, except mutants of Glu-144, Lys-229, and Lys-399. In contrast, the peripheral dNADH-K3Fe(CN)6 reductase activities basically remained unchanged in all the NuoL mutants, suggesting that the peripheral arm of complex I was not affected by point mutations in NuoL. The proton pumping efficiency (the ratio of H+/e−), however, was decreased in most NuoL mutants by 30–50%, while the IC50 values for asimicin (a potent complex I inhibitor) remained unchanged. This suggests that the H+/e− stoichiometry has changed from 4H+/2e− to 3H+ or 2H+/2e− without affecting the direct coupling site. Furthermore, 50 μm of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), a specific inhibitor for Na+/H+ antiporters, caused a 38 ± 5% decrease in the initial H+ pump activity in the wild type, while no change was observed in D178N, D303A, and D400A mutants where the H+ pumping efficiency had already been significantly decreased. The electron transfer activities were basically unaffected by EIPA in both control and mutants. Taken together, our data strongly indicate that the NuoL subunit is involved in the indirect coupling mechanism.

A Humanized Aldolase Antibody for Selective Chemotherapy and Adaptor Immunotherapy

Mouse monoclonal antibody 38C2 is the prototype of a new class of catalytic antibodies that were generated by reactive immunization. Through a reactive lysine, 38C2 catalyzes aldol and retro-aldol reactions using the enamine mechanism of natural aldolases. In addition to its remarkable versatility and efficacy in synthetic organic chemistry, 38C2 has been used for the selective activation of prodrugs in vitro and in vivo and thereby emerged as a promising tool for selective chemotherapy. Adding another application with relevance for cancer therapy, designated adaptor immunotherapy, we have recently shown that 38C2 can be chemically programmed to target tumors by formation of a covalent bond of defined stoichiometry with a beta-diketone derivative of an integrin alpha(v)beta(3) targeting RGD peptidomimetic. However, a major limitation for the transition from preclinical to clinical evaluation is the human anti-mouse antibody immune response that mouse 38C2 is likely to elicit in a majority of patients after single administration. Here, we report the humanization of mouse 38C2 based on rational design guided by molecular modeling. In essence, the catalytic center of mouse 38C2, which encompasses a deep hydrophobic pocket with a reactive lysine residue at the bottom, was grafted into a human antibody framework. Humanized 38C2 IgG1 was found to bind to beta-diketone haptens with conserved affinities and revealed strong catalytic activity with identical k(cat) and slightly higher K(M) values compared to the parental mouse antibody. Furthermore, humanized 38C2 IgG1 revealed efficiency in prodrug activation and chemical programming comparable to the parental mouse antibody.

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.

Synthesis of All Four Isomers of Disparlure Using Osmium-Catalyzed Asymmetric Dihydroxylation.

Disparlure, the sex attractant emitted by the female gypsy moth, Porthetiu dispar (L.), and its (-)-enantiomer were synthesized in 43% yield and >99% ee, starting from undecanal and using the asymmetric dihydroxylation (AD) reaction. Similarly, the,two trans- isomers of disparlum were synthesized in 51% yield and 95% ee.

Small molecule drug activity in melanoma models may be dramatically enhanced with an antibody effector

Monoclonal antibody (mAb) 38C2 belongs to a group of catalytic antibodies that were generated by reactive immunization and contains a reactive lysine. 38C2 catalyzes aldol and retro‐aldol reactions, using an enamine mechanism, and mechanistically mimics natural aldolase enzymes. In addition, mAb 38C2 can be redirected to target integrins αvβ3 and αvβ5 through the formation of a covalent bond between a β‐diketone derivative of an arginine–glycine–aspartic acid (RGD) peptidomimetic and the reactive lysine residue in the antibody combining site to provide the chemically programmed mAb cp38C2. In this study, we investigated the potential of enhancing the activity of receptor‐binding small molecule drug (SCS‐873) through antibody conjugation. Using a M21 human melanoma xenograft model in nude mice, cp38C2 inhibited the growth of the tumor by ˜81%. The chemically programmed antibody was shown to be highly active at a low concentration while SCS‐873 alone was ineffective even at dosages ˜1,000‐fold higher than those used for the chemically programmed antibody. In vitro programming of the catalytic antibody was shown to be as effective as in vivo programming. In an experimental metastasis assay, treatment with mAb cp38C2 significantly prolonged overall survival of tumor‐bearing severe combined immuno‐deficient (SCID) mice when compared to treatment with unprogrammed mAb 38C2, SCS‐873 alone or the integrin‐specific monoclonal antibody LM609. In vitro, cp38C2 inhibited human and mouse endothelial and human melanoma cell adhesion, migration and invasion. Additionally, cp38C2 inhibited human and mouse endothelial cell proliferation and was active in complement‐dependent cytotoxicity assays. These studies establish the potential of chemically programmed monoclonal antibodies as a novel and effective class of immunotherapeutics that combine the merits of traditional small molecule drug design with immunotherapy.