Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10-11-10-8 M) against a variety of HER2-expressing cancer cell lines. Several peptide-linked variants of the lead compound were prepared and conjugated to trastuzumab to afford ADCs with drug-to-antibody (DAR) ratios ranging from 3 to 5. The ADCs exhibited antigen-dependent cytotoxicity in vitro and potently suppressed tumor xenograft growth in vivo in a target-dependent manner. Moreover, the ADCs were well-tolerated in both mouse and rat. This work demonstrates for the first time that PBD polyamide hybrids can serve as effective ADC payloads.

Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

BACKGROUND: We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. METHODS: In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. RESULTS: TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. CONCLUSIONS: Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection.

Development of a Novel DNA Mono-alkylator Platform for Antibody-Drug Conjugates.

Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing. A phosphoramidate prodrug of the payload enables low ADC aggregation even at drug-to-antibody ratios of 5:1 while still delivering a bystander-capable payload that is effective in multidrug resistant (MDR)-overexpressing cell lines. The platform was comparable in xenograft studies to the clinical benchmark DNA mono-alkylator ADC platform DGN459 but with a significantly better tolerability profile in rats. Thus, the activity and tolerability profile of this new platform make it a viable option for the development of ADCs.

Chemically programmed bispecific antibodies that recruit and activate T cells.

Bispecific antibodies (biAbs) that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next-generation antibody therapeutics. Of particular interest are biAbs of relatively small size (∼50 kDa) that can redirect cytotoxic T cells through simultaneous binding of tumor cells. Here we describe a conceptually unique class of biAbs in which the tumor cell specificity of a humanized antibody fragment that recognizes CD3 on T cells is chemically programmed through a C-terminal selenocysteine (Sec) residue. We demonstrate that through chemically programmed specificity for integrin α(4)ß(1) or folate receptor 1 (FOLR1), and common specificity for CD3, these hybrid molecules exert potent and specific in vitro and ex vivo cytotoxicity toward tumor cell lines and primary tumor cells in the presence of primary T cells. Importantly, the generic nature of chemical programming allows one to apply our approach to virtually any specificity, promising a broad utility of chemically programmed biAbs in cancer therapy.

Discovery and Optimization of a STING Agonist Platform for Application in Antibody Drug Conjugates.

While STING agonists have proven to be effective preclinically as anti-tumor agents, these promising results have yet to be translated in the clinic. A STING agonist antibody-drug conjugate (ADC) could overcome current limitations by improving tumor accessibility, allowing for systemic administration as well as tumor-localized activation of STING for greater anti-tumor activity and better tolerability. In line with this effort, a STING agonist ADC platform was identified through systematic optimization of the payload, linker, and scaffold based on multiple factors including potency and specificity in both in vitro and in vivo evaluations. The platform employs a potent non-cyclic dinucleotide STING agonist, a cleavable ester-based linker, and a hydrophilic PEG8-bisglucamine scaffold. A tumor-targeted ADC built with the resulting STING agonist platform induced robust and durable anti-tumor activity and demonstrated high stability and favorable pharmacokinetics in nonclinical species.

Application of strain-promoted azide-alkyne cycloaddition and tetrazine ligation to targeted Fc-drug conjugates.

We have previously described an approach whereby antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In the present work, we describe methodology for improving the efficacy of these Fc-Sec conjugates by incorporating cytotoxic drugs. The Fc-Sec protein is first programmed to target specific tumor cell types by attachment of a bifunctional linker that contains a "clickable" handle (e.g., cyclobutane or cyclooctyne) in addition to a tumor cell-binding peptide or small molecule. Following Fc-Sec conjugation, a cytotoxic warhead is then attached by cycloaddition reactions of tetrazine or azide-containing linker. To validate this approach, we used a model system in which folic acid (FA) is the targeting moiety and a disulfide-linked biotin moiety serves as a cytotoxic drug surrogate. We demonstrated successful targeting of Fc-Sec proteins to folate-receptor expressing tumor cells. Tetrazine ligation was found to be an efficient method for biotin "arming" of the folate-targeted Fc-Sec proteins. We also report novel bioconjugation methodologies that use [4 + 2] cycloaddition reactions between tetrazines and cyclooctynes.

Application of a water-soluble pyridyl disulfide amine linker for use in Cu-free click bioconjugation.

Described herein is the design and synthesis of a discrete heterobifunctional PEG-based pyridyl disulfide/amine-containing linker that can be used in the Cu-free click preparation of bioconjugates. The title PEG-based pyridyl disulfide amine linker is a potentially useful reagent for preparing water-soluble disulfide-linked cargos. It may be particularly valuable in expanding the field of Cu-free click-based bioconjugations to include reductively labile antibody, polymer, or nanoparticle-based drug conjugates.

An engineered selenocysteine defines a unique class of antibody derivatives.

Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin alpha(4)beta(1). Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin alpha(4)beta(1) and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

Application of a trifunctional reactive linker for the construction of antibody-drug hybrid conjugates.

A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin alpha(4)beta(1)-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin alpha(4)beta(1). The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.

In vitro evaluation of alkylcarbonyloxymethyl (ACOM) ethers as novel prodrugs of phenols for topical delivery: ACOM prodrugs of acetaminophen.

The fluxes (J(IPM)) of a series of alkylcarbonyloxymethyl (ACOM) ethers of acetaminophen (APAP) were measured through hairless mouse skin from suspensions of each prodrug in isopropyl myristate (IPM). Solubilities in IPM, estimated solubilities in pH 4.0 buffer (S(4.0)) and flux data for the 4-ACOM-APAP prodrugs were incorporated into the Roberts-Sloan (RS) database to give new estimates for the independent variables of the RS equation: logJ(IPM)=x+ylogS(IPM)+(1-y)logS(4.0)-zM(W). All but one of the 4-ACOM-APAP derivatives hydrolyzed completely on permeation through mouse skin. Three out of the five prodrugs permeated the skin better than APAP, with a maximum fourfold increase in flux. Biphasic solubility - not solubility in a single solvent - was shown to have the greatest impact on flux. A fit of the new n=66 database to the RS equation gave the following values for x, y, z, and r(2): x=-0.545, y=0.511, z=0.00253, r(2)=0.915. These results demonstrate that the topical delivery of a model phenol, acetaminophen, can be improved by transiently masking the phenolic hydroxyl group as an ACOM ether.

A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy.

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development.

Harnessing the fcµ receptor for potent and selective cytotoxic therapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcµR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcµ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcµ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcµ-drug conjugate was selectively toxic for FcµR-expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcµ-drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcµ-drug conjugate in immunodeficient NOD/SCID/IL-2Rγ(null) (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcµ-drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcµR. FcµR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcµR as a valuable therapeutic target in CLL and for IgM-based antibody-drug conjugates as a new targeting platform.

Evaluation of alkyloxycarbonyloxymethyl (AOCOM) ethers as novel prodrugs of phenols for topical delivery: AOCOM prodrugs of acetaminophen.

The maximum fluxes of a series of alkyloxycarbonyloxymethyl (AOCOM) ethers of acetaminophen (APAP) through hairless mouse skin from isopropyl myristate, IPM (J(MMIPM)) were measured. The J(MMIPM), solubilities in IPM (S(IPM)), water (S(AQ)) and pH 4.0 buffer (S4.0) and molecular weights MW were then fitted to the Roberts-Sloan (RS) equation: log JM = x + y log S(LIPID) + (1-y) log S(AQ)-zMW. Only one of the prodrugs gave an improvement in the flux obtained by APAP itself. The general lack of improvement in flux seems to be due to the fact that there was no improvement in the S(AQ) values of the AOCOM derivatives compared to APAP. When the n = 5 members of the AOCOM series were added to the n = 66 database of J(MMIPM) to give n = 71 and fitted to the RS equation where S(LIPID) was S(IPM), the following coefficients were obtained: x = -0.562, y = 0.501, z = 0.00248, r2 = 0.923. These results demonstrate the importance of improving S(AQ) for prodrugs to improve their solubilities in the skin and hence the flux of the parent drug. The RS equation, which is derived directly from Fick's law, explains this dependence of flux on S(AQ).