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Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a (177)Lu-labeled radioimmunoconjugate based on (HER2)DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified (HER2)DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A″-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [(177)Lu]Lu(3+) to produce the completed radioimmunoconjugate — [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD — in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetallation over a 7 d incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD to that of a (177)Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD matched that of (177)Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8 respectively at the same timepoint. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs. 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.

Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11-Å deep pocket that permits site-specific conjugation of β-diketone-, β-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain (DVD) formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here we expand the scope of this ADC assembly strategy by mutating h38C2’s reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F (MMAF), h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.

Comparative analyses of four anthraquinone-fused enediyne biosynthetic gene clusters (BGCs) identified YpmL, a cytochrome P450 enzyme, unique to the yangpumicin (YPM) BGC. In vitro characterization of YpmL established it as a hydroxylase, catalyzing C-6 hydroxylation in YPM A biosynthesis. In vivo application of YpmL enabled engineered production of four new tiancimycin analogues (14-17). Evaluation of their cytotoxicity against selected human cancer cell lines shed new insights into enediyne structure-activity relationship.

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRβ) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRβ expressing cells. Multiple anti-mPDGFRβ antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

Peptide-based therapeutics have suffered from a short plasma half-life. On the other hand, antibodies suffer from poor penetration into solid tumors owing to their large size. Herein, we present a new molecular form, namely a hybrid complex between a hapten-labeled bispecific peptide and an anti-hapten antibody ("HyPEP-body"), that may be able to overcome the aforementioned limitation. The bispecific peptide containing a cotinine tag was synthesized by linking a peptide specific to fibronectin extra domain B (EDB) and a peptide able to bind and inhibit vascular endothelial growth factor (VEGF), yielding cot-biPEPEDB-VEGF . Simple mixing of cot-biPEPEDB-VEGF and anti-cotinine antibody (Abcot ) yielded the hybrid complex, HyPEPEDB-VEGF . HyPEPEDB-VEGF retained the characteristics of the included peptides, and showed improved pharmacokinetic behavior. Moreover, HyPEPEDB-VEGF showed tumor growth inhibition with excellent tumor accumulation and penetration. These findings suggest that the hybrid platform described here offers a solution for most peptide therapeutics that suffer from a short circulation half-life in blood.

A number of cancer-targeting peptide-drug conjugates (PDCs) have been explored as alternatives to antibody-drug conjugates (ADCs) for targeted cancer therapy. However, the much shorter circulation half-life of PDCs compared with ADCs in vivo has limited their therapeutic value and thus their translation into the clinic, highlighting the need to develop new approaches for extending the half-life of PDCs. Here, we report a new strategy for targeted cancer therapy of a PDC based on a molecular hybrid between an antihapten antibody and a hapten-labeled PDC. An anticotinine antibody (Abcot) was used as a model antihapten antibody. The anticancer drug SN38 was linked to a cotinine-labeled aptide specific to extra domain B of fibronectin (cot-APTEDB), yielding the model PDC, cot-APTEDB-SN38. The cotinine-labeled PDC showed specific binding to and cytotoxicity toward an EDB-overexpressing human glioblastoma cell line (U87MG) and also formed a hybrid complex (HC) with Abcot in situ, designated HC[cot-APTEDB-SN38/Abcot]. In glioblastoma-bearing mice, in situ HC[cot-APTEDB-SN38/Abcot] significantly extended the circulation half-life of cot-APTEDB-SN38 in blood, and it enhanced accumulation and penetration within the tumor and, ultimately, inhibition of tumor growth. These findings suggest that the present platform holds promise as a new, targeted delivery strategy for PDCs in anticancer therapy.

By exploiting a uniquely reactive lysine residue (Lys99) for site-specific attachment of small molecules, the humanized catalytic antibody h38C2 has been used as bioconjugation module in the assembly of chemically programmed antibodies and antibody-drug conjugates. Treatment of h38C2 with β-lactam-functionalized small molecules has been previously shown to result in covalent conjugation by selective formation of a stable amide bond with the e-amino group of the Lys99 residue. Here we report that heteroaryl methylsulfonyl (MS-PODA)-functionalized small molecules represent an alternative bioconjugation strategy through highly efficient, site-specific, and stable arylation of the Lys99 residue. A set of chemically programmed antibodies and antibody-drug conjugates assembled by Lys99 arylation provided proof-of-concept for the therapeutic utility of this alternative bioconjugation strategy. While being equally effective as β-lactam-functionalized ligands for bioconjugation with catalytic antibody h38C2, the MS-PODA moiety offers distinct synthetic advantages, making it highly attractive.

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (Cκ ) fusion protein. N-terminal amino acid sequencing using Edman degradation demonstrated that over 95% of the recombinant protein produced is [-2]proPSA, thereby showing for the first time that recombinant [-2]proPSA can be produced as a major fraction. We also generated a recombinant chicken antibody specific to [-2]proPSA but not cross-reactive to recombinant [-7]proPSA-Cκ , [-5]proPSA-Cκ , and PSA purified from human seminal fluid in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Also, the recombinant chicken antibody reacted to recombinant [-2]proPSA protein bound to an anti-PSA antibody coated on the micrometer plate in a sandwich ELISA. All of these results suggest that the N-terminus of the [-2]proPSA-Cκ fusion protein resides on the exterior of the protein, thus allowing exposure to the antibody.

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRβ) x cotinine single-chain variable fragment (scFv)-kappa constant region (C

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.

Antibodies have been used for cancer therapy for two decades, but they suffer from poor localization and penetration into solid tumors because of their large size. Peptides can be an alternative for treatment of solid tumors due to their small size. However, therapeutic use of peptides is limited because of their short plasma half-life. We present a new molecular platform based on a hybrid complex between bispecific peptide and antibody, designated as HyPEP-body, composed of bispecific peptides labeled with a hapten as a pharmacophore and an anti-hapten antibody. Two different peptides are joined together with linker and then, labeled with the hapten. The bispecific pharmacophore are bound to the anti-hapten antibody through antigen-antibody interaction. The pharmacophores which can be chemically optimized or replaced with other peptides are in charge of therapeutic function, while the antibody imparts long plasma half-life. As a model study, we constructed HyPEP-body that simultaneously binds extra domain B (EDB) and vascular endothelial growth factor (VEGF) with EDB binding peptide, VEGF binding peptide, cotinine and anti-cotinine antibody. Complexation with the antibody extended the plasma half-life of the bispecific pharmacophores. Furthermore, bispecific pharmacophores released from the antibodies distributed more widely in the tumor tissue than the antibodies. Finally, treatment of the HyPEP-body inhibited tumor growth in U87MG, human glioblastoma, xenograft mouse model. This versatile hybrid platform will enhance the value of peptide therapeutics and suggest an innovative solution to the development of bispecific antibodies that has suffered from productivity issues. Citation Format: Byeongjun Yu, Dobeen Hwang, Sangyong Jon, Junho Chung. Hybrid complex between bispecific peptide and antibody for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2999.

A truncated precursor form of prostate-specific antigen (PSA), [-2]proPSA, is a well-known biomarker for prostate cancer. To develop a biomarker assay, highly purified [-2]proPSA is required as a standard reference and for generation of a specific antibody. In this study, we generated an efficient mammalian expression system for producing a recombinant [-2]proPSA-human kappa constant domain (C

Nanomaterial bioconjugates have gained unabated interest in the field of sensing, imaging and therapy. As a conjugation process significantly affects the biological functions of proteins, it is crucial to attach them to nanomaterials with control over their orientation and the nanomaterial-to-protein ratio in order to amplify the binding efficiency of nanomaterial bioconjugates to targets. Here, we describe a targeting nanomaterial platform utilizing carbon nanotubes functionalized with a cotinine-modified dextran polymer and a bispecific anti-HER2 × cotinine tandem antibody. This new approach provides an effective control over antibody orientation and density on the surface of carbon nanotubes through site-specific binding between the anti-cotinine domain of the bispecific tandem antibody and the cotinine group of the functionalized carbon nanotubes. The developed synthetic carbon nanotube/bispecific tandem antibody conjugates (denoted as SNAs) show an effective binding affinity against HER2 that is three orders of magnitude higher than that of the carbon nanotubes bearing a randomly conjugated tandem antibody prepared by carbodiimide chemistry. As the density of a tandem antibody on SNAs increases, their effective binding affinity to HER2 increases as well. SNAs exhibit strong resonance Raman signals for signal transduction, and are successfully applied to the selective detection of HER2-overexpressing cancer cells.

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type. These results suggest that the HU protein functions as a negative modulator of seqA expression.