Current strategies to produce homogeneous antibody-drug conjugates (ADCs) rely on mutations or inefficient conjugation chemistries. Here we present a strategy to produce site-specific ADCs using a ...highly reactive natural buried lysine embedded in a dual variable domain (DVD) format. This approach is mutation free and drug conjugation proceeds rapidly at neutral pH in a single step without removing any charges. The conjugation chemistry is highly robust, enabling the use of crude DVD for ADC preparation. In addition, this strategy affords the ability to precisely monitor the efficiency of drug conjugation with a catalytic assay. ADCs targeting HER2 were prepared and demonstrated to be highly potent and specific in vitro and in vivo. Furthermore, the modular DVD platform was used to prepare potent and specific ADCs targeting CD138 and CD79B, two clinically established targets overexpressed in multiple myeloma and non-Hodgkin lymphoma, respectively.
Site-specific antibody-drug conjugate (ADC) technologies are highly desirable for the production of therapeutics with well-defined biochemical and pharmacological characteristics. We have developed a ...strategy to produce site-specific ADCs using a highly reactive lysine residue embedded in a dual-variable-domain (DVD) format. Here we provide protocols for the engineering, expression, and purification of the DVDs used for this strategy. We also provide a protocol for DVD-drug conjugation and describe methods for their biochemical characterization, including a catalytic assay to monitor conjugation efficiency.
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.
Abstract
Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasms which tend to have poor outcomes, and the development of new targets and strategies to treat these ...cancers is sorely needed. Antibody–drug conjugate (ADC) therapy has been shown to be a promising targeted therapy for treating many cancers, but has only rarely been tried in patients with TNBC. A major reason the efficacy of ADC therapy in the setting of TNBC has not been more fully investigated is the lack of appropriate target molecules. In this work we were able to identify an effective TNBC target for use in immunotherapy. We were guided by our previous observation that in some breast cancer patients the protein tropomyosin receptor kinase B cell surface protein (TrkB) had become immunogenic, suggesting that it was somehow sufficiently chemically different enough (presumably by mutation) to escaped immune tolerance. We postulated that this difference might well offer a means for selective targeting by antibodies. We engineered site-specific ADCs using a dual variable domain (DVD) format which combines anti-TrkB antibody with the h38C2 catalytic antibody. This format enables rapid, one-step, and homogeneous conjugation of β-lactam-derivatized drugs. Following conjugation to β-lactam-derivatized monomethyl auristatin F, the TrkB-targeting DVD-ADCs showed potency against multiple breast cancer cell lines, including TNBC cell lines. In addition, our isolation of antibody that specifically recognized the breast cancer-associated mutant form of TrkB, but not the wild type TrkB, indicates the possibility of further refining the selectivity of anti-TrkB DVD-ADCs, which should enhance their therapeutic index. These results confirmed our supposition that TrkB is a potential target for immunotherapy for TNBC, as well as for other cancers with mutated cell surface proteins.
Homogeneous antibody-drug conjugates (ADCs) that use a highly reactive buried lysine (Lys) residue embedded in a dual variable domain (DVD)-IgG1 format can be assembled with high precision and ...efficiency under mild conditions. Here we show that replacing the Lys with an arginine (Arg) residue affords an orthogonal ADC assembly that is site-selective and stable. X-ray crystallography confirmed the location of the reactive Arg residue at the bottom of a deep pocket. As the Lys-to-Arg mutation is confined to a single residue in the heavy chain of the DVD-IgG1, heterodimeric assemblies that combine a buried Lys in one arm, a buried Arg in the other arm, and identical light chains are readily assembled. Furthermore, the orthogonal conjugation chemistry enables the loading of heterodimeric DVD-IgG1s with two different cargos in a one-pot reaction and thus affords a convenient platform for dual-warhead ADCs and other multifaceted antibody conjugates.
Hwang, Nilchan et al. convert a reactive lysine in a catalytic antibody to a reactive arginine, reveal its location by X-ray crystallography, and utilize the engineered antibody for site-selective antibody functionalization including its pairing with the original antibody for orthogonal dual cargo conjugation.
Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and ...function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody-siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.
Stable and Potent Selenomab-Drug Conjugates Li, Xiuling; Nelson, Christopher G.; Nair, Rajesh R. ...
Cell chemical biology,
04/2017, Letnik:
24, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Selenomabs are engineered monoclonal antibodies with one or more translationally incorporated selenocysteine residues. The unique reactivity of the selenol group of selenocysteine permits ...site-specific conjugation of drugs. Compared with other natural and unnatural amino acid and carbohydrate residues that have been used for the generation of site-specific antibody-drug conjugates, selenocysteine is particularly reactive, permitting fast, single-step, and efficient reactions under near physiological conditions. Using a tailored conjugation chemistry, we generated highly stable selenomab-drug conjugates and demonstrated their potency and selectivity in vitro and in vivo. These site-specific antibody-drug conjugates built on a selenocysteine interface revealed broad therapeutic utility in liquid and solid malignancy models.
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•Selenomab-drug conjugates are antibody-drug conjugates based on selenocysteine•Iodoacetamide-based selenomab-drug conjugates have excellent stability•Selenomab-drug conjugates are potent and specific cancer therapeutics•The location of selenocysteine in selenomab-drug conjugates impacts their potency
Li et al. harness the high reactivity of the 21st natural amino acid selenocysteine to rapidly and efficiently assemble antibody-drug conjugates that reveal excellent stability, potency, and selectivity in diverse in vitro and in vivo models of human cancers.
While all clinically translated antibody-drug conjugates (ADCs) contain a single-drug payload, most systemic cancer chemotherapies involve use of a combination of drugs. These regimens improve ...treatment outcomes and slow development of drug resistance. We here report the generation of an ADC with a dual-drug payload that combines two distinct mechanisms of action.
Virtual DNA crosslinking agent PNU-159682 and tubulin polymerization inhibitor monomethyl auristatin F (MMAF) were conjugated to a HER2-targeting antibody
site-specific conjugation at engineered selenocysteine and cysteine residues (thio-selenomab).
The dual-drug ADC showed selective and potent cytotoxicity against HER2-expressing cell lines and exhibited dual mechanisms of action consistent with the attached drugs. While PNU-159682 caused S-phase cell cycle arrest due to its DNA-damaging activity, MMAF simultaneously inhibited tubulin polymerization and caused G2/M-phase cell cycle arrest.
The thio-selenomab platform enables the assembly of dual-drug ADCs with two distinct mechanisms of action.
Abstract
We have established a set of technologies called Tunable Drug Conjugates (TDCs) that will differentiate from other DCs via a rapid payload release/rapid systemic clearance approach intended ...to quickly drive high toxic “payload” concentrations within tumor cells while minimizing toxicities to patients. TDCs use proprietary Silicon-based linker chemistries (SiLinkers) in novel Payload Cassettes (PCs) that enable the rapid and uniform release of multiple and/or mixed payloads both in the endosome after internalization and within the necrotic microenvironment of tumors. PCs can incorporate multiple copies of the same payload or optimal ratios of different therapeutic agents that work together synergistically to kill cancer cells. A second technology - a Dual variable Domain-Fab (DVD-Fab) targeting capability provides a proprietary means of specifically targeting a broad array of cell surface proteins. DVD-Fab containing TDCs should be cleared from the body within hours, enabling better management of side-effects. Our Silinker system is differentiated by the exquisite and tunable sensitivity of the linkers to cleavage under the modestly acidic conditions encountered in the endosomal and lysosomal compartments of a cell, and in the microenvironment of solid tumors. Good stability at physiological pH (and in human plasma) compares to rapid cleavage at relevant lower pHs as demonstrated using model systems. SiLinkers exhibit good stability at 37°C in pH7.4 buffer (and plasma) and show rapid hydrolysis at pH ~5.5 to 6.5 (t1/2 = 15 - 120 min). Confocal microscopy studies with imaging constructs demonstrate the translation of the in vitro data to cellular systems. We assembled SiLinker based prototype TDCs to target the folate receptor α (FAR) with folic acid as the targeting ligand and vinblastine analogs as the payload(s). We used single and PC drug conjugates to show cellular and in vivo proof-of-concept. We tested our TDCs in cell lines overexpressing FAR and showed that cytotoxicity was blocked by excess folic acid. Subsequent in vivo studies, using a nude mouse xenograft model, showed good antitumor activity for these prototype TDCs. BB-01 (a single payload construct) showed a clear dose response and had cures in 3 out of 5 mice after 24 days on a 3 μmol/kg TIW schedule. BB-03 (a triple PC construct) shows essentially complete tumor regression compared to modest inhibition by BB-01 at doses that deliver equivalent amounts of payload, demonstrating the synergistic potential of the PC concept to enhance the activity. We have also synthesized DVD-Fab TDCs. DVD-Fabs contain a reactive chemical group for seamless connection with our SiLinkers and PCs. We will describe conjugation chemistries and present cellular and in vivo Xenograft data for prototype DVD-Fab TDCs.
Citation Format: Sara C. Hickey, Alex R. Nanna, Hanh N. Nguyen, Leslie Ofori, Christoph Rader, Michael K. Rood, Jutta Wanner, Doug S. Werner. Tunable Drug Conjugates: a differentiated drug conjugate (DC) platform abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5153. doi:10.1158/1538-7445.AM2017-5153
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