Preparation Of Antibody-drug Conjugates Research Articles

Preparation of an Ultrahigh-DAR PDL1 monoclonal antibody-polymeric-SN38 conjugate for precise colon cancer therapy

Antibody-drug conjugates (ADCs) are the most potent active tumor-targeting agents used clinically. However, the preparation of ADCs with high drug-to-antibody ratios (DARs) remains a major challenge. Herein, a Fab-nondestructive SN38-loaded antibody-polymeric-drug conjugate (APDC), aPDL1-NPLG-SN38, was prepared that had a DAR as high as 72 for the first time, by increased numbers of payload binding sites via the carboxyl groups of poly (l-glutamic acid) (PLG). The bonding of Fc-III-4C peptide with PLG-graft-mPEG/SN38 (Fc-NPLG-SN38) was achieved using a click reaction between azide and DBCO groups. The aPDL1-NPLG-SN38 conjugate was then synthesized by the high-affinity interaction between the Fc-III-4C peptide in Fc-NPLG-SN38 and the crystallizable fragment (Fc) of PDL1 monoclonal antibody (aPDL1). This approach avoided the potential deleterious effects on the Fab structure of the monoclonal antibody. The aqueous environment used in its preparation helped maintain monoclonal antibody recognition capability. Through the specific recognition by aPDL1 of PDL1 that is highly expressed on MC38 tumors, the accumulation of aPDL1-NPLG-SN38 in the tumors was 2.8-fold greater than achieved with IgG-NPLG-SN38 that had no active tumor-targeting capability. aPDL1-NPLG-SN38 exhibited excellent therapeutic properties in both medium-sized and large MC38 tumor animal models. The present study provides the details of a novel preparation strategy for SN38-loaded ADCs having a high DAR.

Substrate Specificity of the Flavoenzyme BhaC1 That Converts a C-Terminal Trp to a Hydroxyquinone.

The preparation of protein-protein, protein-peptide, and protein-small molecule conjugates is important for a variety of applications, such as vaccine production, immunotherapies, preparation of antibody-drug conjugates, and targeted delivery of therapeutics. To achieve site-selective conjugation, selective chemical or enzymatic functionalization of proteins is required. We have recently reported biosynthetic pathways in which small, catalytic scaffold peptides are utilized for the generation of amino acid-derived natural products called pearlins. In these systems, peptide amino-acyl tRNA ligases (PEARLs) append amino acids to the C-terminus of a scaffold peptide, and tailoring enzymes encoded in the biosynthetic gene clusters modify the PEARL-appended amino acid to generate a variety of natural products. Herein, we investigate the substrate selectivity of one such tailoring enzyme, BhaC1, that participates in pyrroloiminoquinone biosynthesis. BhaC1 converts the indole of a C-terminal tryptophan into an o-hydroxy-p-quinone, a promising moiety for site-selective bioconjugation. Our studies demonstrate that BhaC1 requires a 20-amino acid peptide for substrate recognition. When this peptide was appended at the C-terminus of proteins, the C-terminal Trp was modified by BhaC1. The enzyme is sufficiently selective that only small changes to the sequence of the peptide are tolerated. An AlphaFold model for substrate recognition explains the selectivity of the enzyme, which may be used to install a reactive handle onto the C-terminus of proteins.

Bridged Cysteine Conjugations.

Preparation of antibody-drug conjugates (ADCs) with a highly homogeneous drug loading in general requires site-selective conjugation of a cytotoxic payload. Typically, functionality utilized for attachment of the payload is achieved through engineering of suitable chemical handles or by enzymatic modification of the antibody. Relatively few methods to produce ADCs with homogeneous drug loading via endogenous amino acid conjugation have been developed. Herein we describe a robust method for the conjugation of antibodies using a cysteine rebridging approach to produce ADCs with highly homogeneous drug-to-antibody ratios (DAR) at the native interchain disulfides, called ThioBridge®. The process described relies upon an elegant cascade of addition-elimination reactions carried out under mild aqueous conditions that can be readily applied to wild-type antibodies without the need for prior modification via recombinant or enzymatic means. Using this method, conversions to a conserved DAR ADC are typically in the range of 70-95% and overall process yields of >70% are readily achieved.

Structural Characterization of the Aggregates of Gemtuzumab Ozogamicin

The preparation of antibody drug conjugates (ADCs), particularly those containing hydrophobic payloads, may promote aggregate formation. The aggregate (high molecular mass species, HMMS) is an impurity that must be controlled in the final drug substance and drug product formulation to meet product safety and efficacy. Although there are numerous methods designed to mitigate protein aggregation, some of the reasons for the formation of aggregates in ADCs are unique and specific to the synthetic preparation and structure of each conjugate. Proper structural characterization and identification of causes of aggregation are key to improving the process for preparation of ADCs for minimizing aggregate formation. In this article, we have characterized aggregates generated during the preparation of a lysine conjugate, gemtuzumab ozogamicin, prepared from an IgG4 antibody (hP67.6) and a calicheamicin-based linker-payload. Using analytical and biophysical techniques, structural details of the aggregates formed in t...

Bioconjugation through Mesitylene Thiol Alkylation.

The design and generation of complex multifunctional macromolecular structures by bioconjugation is a hot topic due to increasing interest in conjugates with therapeutic applications. In this regard, the development of efficient, selective, and safe conjugation methods is a major objective. In this report, we describe the use of the bis(bromomethyl)benzene scaffold as a linker for bioconjugation with special emphasis on antibody conjugation. We first performed the monothioalkylation of 1,3,5-tris(bromomethyl)benzene, which rendered the reactive dibromotrimethylbenzyl derivatives to be used in thiol bis-alkylation. Next, we introduced into the linker either a bis(Cys)-containing peptide or anti-CD4 and -CD13 monoclonal antibodies, previously subjected to partial reduction of disulfide bonds. Mass spectrometry, UV-vis spectra, and SDS-PAGE experiments revealed that this bis-alkylating agent for bioconjugation preserved both antibody integrity and antibody-antigen binding affinity, as assessed by flow cytometry. Taken together, our results show that the mesitylene scaffold is a suitable linker for thiol-based bioconjugation reactions. This linker could be applicable in the near future for the preparation of antibody drug conjugates.

A Hexa-rhodium Metallopeptide Catalyst for Site-Specific Functionalization of Natural Antibodies.

Preparation of antibody-drug conjugates (ADCs), an emerging novel class of highly targeted biological hybrid agents, necessitates precise control of conjugation reactivity. Antibodies have complex multistranded architectures, and specific modification of natural antibodies has proven quite challenging. Here, we demonstrate that cooperative activity of a multimetallic metallopeptide enables efficient site-specific antibody functionalization, based on molecular recognition of the constant Fc region. This interplay of multiple metal centers enables introduction of an orthogonal alkyne handle into monoclonal or polyclonal antibodies from different species in an Fc-specific fashion. Elaboration of this simple functionalization allows preparation of conjugates with fluorophore, affinity handle, and pharmacological agents. This method opens a new opportunity for quick and easy production of well-defined antibody conjugates from a variety of antibody sequence and species of origin.

Abstract 4334: A novel linker to enable alcohol-containing payloads for the preparation of antibody-drug conjugates

Abstract Antibody-drug conjugates (ADCs) have emerged as important therapeutics for treating cancer as evidenced by the recent FDA approval of brentuximab vedotin Adcetris™ and trastuzumab emtansineKadcyla™, and the large number of ADCs (>35) currently in clinical trials. ADCs are composed of two core units: the targeting antibody and the drug-linker. The drug-linker typically consists of a cytotoxic payload, a moiety for covalent attachment to the antibody, and a conditionally cleavable linker that is stable in circulation but releases the cytotoxic payload upon internalization into a target cell. The released payload will possess a residual chemical functional group that is used for attachment of the drug to the cleavable linker. Functional groups that have been exploited within the ADC field for this purpose include amines (primary and secondary), thiols, and carboxylic acids. Unfortunately, many potential ADC payloads do not possess any of these functional group ‘handles’ that allow for stable conjugation and release of the drug in an unmodified form. Use of these payloads often requires modification of the drug structure to introduce a handle for conjugation. To avoid this, we have developed new linker chemistry, the methylene-alkoxy-carbamate (MAC), which enables direct conjugation of drugs through alcohol functional groups. Such groups are present on a diverse array of synthetic drugs and cytotoxic natural products, thus, expanding the potential chemical space available for payload selection. As proof-of-concept, we have linked Auristatin E through the secondary alcohol of the norephedrine residue and characterized the stability and in vivo activity of the resulting ADCs. To further define the potential applicability of the MAC unit, we have examined its stability and release properties with a series of model systems including primary, secondary, and tertiary alcohols as well as a phenol. We have also explored the effect of substitution of the MAC nitrogen on these properties. This novel linker chemistry is currently being applied to new payloads that were inaccessible with previous technology. Citation Format: Robert V. Kolakowski, Karl Haelsig, Scott Jeffrey, Peter Senter. A novel linker to enable alcohol-containing payloads for the preparation of antibody-drug conjugates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4334.

Abstract 2486: Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar

Abstract Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. We previously developed a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle to conjugate a small molecule. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of trastuzumab (Herceptin®), but to a different epitope. The ADC m860 was generated by enzymatically adding a reactive keto-galactose to galactose-free m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F with a non-cleavable linker. It exhibited exceptional serum stability and potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers. This project was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and by federal funds from the National Cancer Institute, NIH, under contract N01-CO-12400, HHSN261200800001E as well as by the SU2C-St.Baldrick's Foundation. Citation Format: Zhongyu Zhu, Ramakrishnan Boopathy, Jinyu Li, Ponraj Prabakaran, Simona Colantonio, Yang Feng, Yanping Wang, Marzena A. Dyba, Dimiter S. Dimitrov. Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2486. doi:10.1158/1538-7445.AM2015-2486

Discovery of cytotoxic dolastatin 10 analogues with N-terminal modifications.

Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.