Improving Antibody-Tubulysin Conjugates through Linker Chemistry and Site-Specific Conjugation.

Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug-linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease-cleavable dipeptide linker, the ß-glucuronidase-cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity in vivo. In addition, site-specific conjugation can positively impact both acetate stability and in vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysin M.

Development of Novel Antibody-Camptothecin Conjugates.

We have developed a highly active and well-tolerated camptothecin (CPT) drug-linker designed for antibody-mediated drug delivery in which the lead molecule consists of a 7-aminomethyl-10,11-methylenedioxy CPT (CPT1) derivative payload attached to a novel hydrophilic protease-cleavable valine-lysine-glycine tripeptide linker. A defined polyethylene glycol stretcher was included to improve the properties of the drug-linker, facilitating high antibody-drug conjugate (ADC) drug loading, while reducing the propensity for aggregation. A CPT1 ADC with 8 drug-linkers/mAb displayed a pharmacokinetic profile coincident with parental unconjugated antibody and had high serum stability. The ADCs were broadly active against cancer cells in vitro and in mouse xenograft models, giving tumor regressions and complete responses at low (≤3 mg/kg, single administration) doses. Pronounced activities were obtained in both solid and hematologic tumor models and in models of bystander killing activity and multidrug resistance. Payload release studies demonstrated that two CPTs, CPT1 and the corresponding glycine analog (CPT2), were released from a cAC10 ADC by tumor cells. An ADC containing this drug-linker was well tolerated in rats at 60 mg/kg, given weekly four times. Thus, ADCs comprised of this valine-lysine-glycine linker with CPT drug payloads have promise in targeted drug delivery.

Structure-activity relationships of tubulysin analogues.

The tubulysins are an emerging antibody-drug conjugate (ADC) payload that maintain potent anti-proliferative activity against cells that exhibit the multi-drug resistant (MDR) phenotype. These drugs possess a C-11 acetate known to be hydrolytically unstable in plasma, and loss of the acetate significantly attenuates cytotoxicity. Structure-activity relationship studies were undertaken to identify stable C-11 tubulysin analogues that maintain affinity for tubulin and potent cytotoxicity. After identifying several C-11 alkoxy analogues that possess comparable biological activity to tubulysin M with significantly improved plasma stability, additional analogues of both the Ile residue and N-terminal position were synthesized. These studies revealed that minor changes within the tubulin binding site of tubulysin can profoundly alter the activity of this chemotype, particularly against MDR-positive cell types.

Glucuronide-Linked Antibody-Tubulysin Conjugates Display Activity in MDR+ and Heterogeneous Tumor Models.

Although antibody-drug conjugates (ADCs) find increasing applications in cancer treatment, de novo or treatment-emergent resistance mechanisms may impair clinical benefit. Two resistance mechanisms that emerge under prolonged exposure include upregulation of transporter proteins that confer multidrug resistance (MDR+) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next-generation ADCs. Recently, we developed the quaternary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amines and applied the linker to tubulysins, a highly potent class of tubulin binders that maintain activity in MDR+ cell lines. In this work, tubulysin M, which contains an unstable acetate susceptible to enzymatic hydrolysis, and two stabilized tubulysin analogues were prepared as quaternary ammonium-linked glucuronide-linkers and assessed as ADC payloads in preclinical models. The conjugates were potent across a panel of cancer cell lines and active in tumor xenografts, including those displaying the MDR+ phenotype. The ADCs also demonstrated potent bystander activity in a coculture model comprised of a mixture of antigen-positive and -negative cell lines, and in an antigen-heterogeneous tumor model. Thus, the glucuronide-tubulysin drug-linkers represent a promising ADC payload class, combining conjugate potency in the presence of the MDR+ phenotype and robust activity in models of tumor heterogeneity in a structure-dependent manner. Mol Cancer Ther; 17(8); 1752-60. ©2018 AACR.

Optimization of a PEGylated Glucuronide-Monomethylauristatin E Linker for Antibody-Drug Conjugates.

The emergence of antibody-drug conjugates (ADC), such as brentuximab vedotin and ado-trastuzumab emtansine, has led to increased efforts to identify new payloads and develop improved drug-linker technologies. Most antibody payloads impart significant hydrophobicity to the ADC, resulting in accelerated plasma clearance and suboptimal in vivo activity, particularly for conjugates with high drug-to-antibody ratios (DAR). We recently reported on the incorporation of a discrete PEG24 polymer as a side chain in a ß-glucuronidase-cleavable monomethylauristatin E (MMAE) linker to provide homogeneous DAR 8 conjugates with decreased plasma clearance and increased antitumor activity in xenograft models relative to a non-PEGylated control. In this work, we optimized the drug-linker by minimizing the size of the PEG side chain and incorporating a self-stabilizing maleimide to prevent payload de-conjugation in vivo Multiple PEG-glucuronide-MMAE linkers were prepared with PEG size up to 24 ethylene oxide units, and homogeneous DAR 8 ADCs were evaluated. A clear relationship was observed between PEG length and conjugate pharmacology when tested in vivo Longer PEG chains resulted in slower clearance, with a threshold length of PEG8 beyond which clearance was not impacted. Conjugates bearing PEG of sufficient length to minimize plasma clearance provided a wider therapeutic window relative to faster clearing conjugates bearing shorter PEGs. A lead PEGylated glucuronide-MMAE linker was identified incorporating a self-stabilizing maleimide and a PEG12 side chain emerged from these efforts, enabling highly potent, homogeneous DAR 8 conjugates and is under consideration for future ADC programs. Mol Cancer Ther; 16(1); 116-23. ©2016 AACR.

The Methylene Alkoxy Carbamate Self-Immolative Unit: Utilization for the Targeted Delivery of Alcohol-Containing Payloads with Antibody-Drug Conjugates.

A strategy for the conjugation of alcohol-containing payloads to antibodies has been developed and involves the methylene alkoxy carbamate (MAC) self-immolative unit. A series of MAC ß-glucuronide model constructs were prepared to evaluate stability and enzymatic release, and the results demonstrated high stability at physiological pH in a substitution-dependent manner. All the MAC model compounds efficiently released alcohol drug surrogates under the action of ß-glucuronidase. To assess the MAC technology for ADCs, the potent microtubule-disrupting agent auristatin E (AE) was incorporated through the norephedrine alcohol. Conjugation of the MAC ß-glucuronide AE drug linker to the anti-CD30 antibody cAC10, and an IgG control antibody, gave potent and immunologically specific activities in vitro and in vivo. These studies validate the MAC self-immolative unit for alcohol-containing payloads within ADCs, a class that has not been widely exploited.

Development of Novel Quaternary Ammonium Linkers for Antibody-Drug Conjugates.

A quaternary ammonium-based drug-linker has been developed to expand the scope of antibody-drug conjugate (ADC) payloads to include tertiary amines, a functional group commonly present in biologically active compounds. The linker strategy was exemplified with a ß-glucuronidase-cleavable auristatin E construct. The drug-linker was found to efficiently release free auristatin E (AE) in the presence of ß-glucuronidase and provide ADCs that were highly stable in plasma. Anti-CD30 conjugates comprised of the glucuronide-AE linker were potent and immunologically specific in vitro and in vivo, displaying pharmacologic properties comparable with a carbamate-linked glucuronide-monomethylauristatin E control. The quaternary ammonium linker was then applied to a tubulysin antimitotic drug that contained an N-terminal tertiary amine that was important for activity. A glucuronide-tubulysin quaternary ammonium linker was synthesized and evaluated as an ADC payload, in which the resulting conjugates were found to be potent and immunologically specific in vitro, and displayed a high level of activity in a Hodgkin lymphoma xenograft. Furthermore, the results were superior to those obtained with a related tubulysin derivative containing a secondary amine N-terminus for conjugation using previously known linker technology. The quaternary ammonium linker represents a significant advance in linker technology, enabling stable conjugation of payloads with tertiary amine residues. Mol Cancer Ther; 15(5); 938-45. ©2016 AACR.

Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation.

The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology.

A highly cytotoxic DNA cross-linking pyrrolobenzodiazepine (PBD) dimer with a valine-alanine dipeptide linker was conjugated to the anti-CD70 h1F6 mAb either through endogenous interchain cysteines or, site-specifically, through engineered cysteines at position 239 of the heavy chains. The h1F6239C-PBD conjugation strategy proved to be superior to interchain cysteine conjugation, affording an antibody-drug conjugate (ADC) with high uniformity in drug-loading and low levels of aggregation. In vitro cytotoxicity experiments demonstrated that the h1F6239C-PBD was potent and immunologically specific on CD70-positive renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines. The conjugate was resistant to drug loss in plasma and in circulation, and had a pharmacokinetic profile closely matching that of the parental h1F6239C antibody capped with N-ethylmaleimide (NEM). Evaluation in CD70-positive RCC and NHL mouse xenograft models showed pronounced antitumor activities at single or weekly doses as low as 0.1 mg/kg of ADC. The ADC was tolerated at 2.5 mg/kg. These results demonstrate that PBDs can be effectively used for antibody-targeted therapy.

SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.

Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

Development of orally active inhibitors of protein and cellular fucosylation.

The key role played by fucose in glycoprotein and cellular function has prompted significant research toward identifying recombinant and biochemical strategies for blocking its incorporation into proteins and membrane structures. Technologies surrounding engineered cell lines have evolved for the inhibition of in vitro fucosylation, but they are not applicable for in vivo use and drug development. To address this, we screened a panel of fucose analogues and identified 2-fluorofucose and 5-alkynylfucose derivatives that depleted cells of GDP-fucose, the substrate used by fucosyltransferases to incorporate fucose into protein and cellular glycans. The inhibitors were used in vitro to generate fucose-deficient antibodies with enhanced antibody-dependent cellular cytotoxicity activities. When given orally to mice, 2-fluorofucose inhibited fucosylation of endogenously produced antibodies, tumor xenograft membranes, and neutrophil adhesion glycans. We show that oral 2-fluorofucose treatment afforded complete protection from tumor engraftment in a syngeneic tumor vaccine model, inhibited neutrophil extravasation, and delayed the outgrowth of tumor xenografts in immune-deficient mice. The results point to several potential therapeutic applications for molecules that selectively block the endogenous generation of fucosylated glycan structures.

Expanded Utility of the ß-Glucuronide Linker: ADCs That Deliver Phenolic Cytotoxic Agents.

The ß-glucuronide linker has been used for antibody-drug conjugates (ADCs) to deliver amine-containing cytotoxic agents. The linker is stable in circulation, hydrophilic and provides ADCs that are highly active in vitro and in vivo. To extend the utility of the ß-glucuronide linker toward phenol-containing drugs, an N,N'-dimethylethylene diamine self-immolative spacer was incorporated with the linker for release of the potent cytotoxic phenol psymberin A. Exposure of the drug-linker to ß-glucuronidase resulted in facile drug release. The corresponding ADCs were active and immunologically selective against CD30-positive L540cy and CD70-positive Caki-1 cell lines.

Design, synthesis, and biological evaluation of antibody-drug conjugates comprised of potent camptothecin analogues.

Antibody-drug conjugates (ADCs) were prepared with potent camptothecin analogues attached to monoclonal antibodies (mAbs) via dipeptide or glucuronide-based linkers. Aniline-containing camptothecin analogues were employed to provide a site of linker attachment via carbamate bonds that would be stable in circulation. The camptothecin analogues, 7-butyl-10-amino-camptothecin and 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, are generally 10-1000 times more potent than camptothecin. Dipeptide and glucuronide drug linkers were employed containing self-immolative spacers that release drug following lysosomal degradation upon ADC internalization into antigen-positive cell lines. The camptothecin drug linkers were conjugated to three antibodies: chimeric BR96, chimeric AC10, and humanized 1F6, which bind to the Lewis-Y antigen on carcinomas, CD30 on hematologic malignancies, and CD70 present on hematologic malignancies and renal cell carcinoma, respectively. ADCs bearing the potent camptothecin analogue, 7-butyl-9-amino-10,11-methylenedioxy-camptothecin, were highly potent and immunologically specific on a panel of cancer cell lines in vitro, and efficacious at well-tolerated doses in a renal cell carcinoma xenograft model.

Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system.

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody-drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.

Synthesis of the northern sector (C8-C19) of rapamycin via Chan rearrangement and oxidation of an alpha-acyloxyacetate.

Two routes to the masked tricarbonyl segment of the immunosuppressant rapamycin comprising C8-C19 were explored beginning from D-xylose. The first approach employed a protected form of 2,4,5-trihydroxypentanol to obtain dithiane 43, which failed to react with dimethyl oxalate to give a 1,2,3-tricarbonyl unit corresponding to the northern sector of rapamycin. A second approach employing carboxylic acid 61 derived from 43 utilized base-mediated (Chan) rearrangement of alpha-acyloxyacetate 62 with trapping of the resultant enediolate as bis silyl ether 63. Epoxidation of this diene afforded masked tri-keto ester 65 which underwent acid-catalyzed methanolysis to produce cyclic ketal 67.

Contribution of linker stability to the activities of anticancer immunoconjugates.

The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.

Minor groove binder antibody conjugates employing a water soluble beta-glucuronide linker.

The minor groove binder beta-glucuronide drug-linker 3 was constructed from amino CBI 1 and determined to be a substrate for Escherichia coli beta-glucuronidase (EC 3.2.1.31), resulting in facile drug release. Compound 3 was conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) to give antibody-drug conjugates (ADCs) with potencies comparable to that of free drug 1. The ADCs were largely monomeric at intermediate loading levels (4-5drug/mAb), in contrast to highly aggregated p-aminobenzylcarbamate dipeptide-based ADCs of 1 previously reported. Significant levels of immunologic specificity were observed with cAC10-3 by comparing antigen positive versus negative cell lines and binding versus non-binding control ADCs. The water soluble beta-glucuronide linker is stable in plasma and effectively delivers drugs to target cells leading to potent cytotoxic activities.

Development and properties of beta-glucuronide linkers for monoclonal antibody-drug conjugates.

A beta-glucuronide-based linker for attaching cytotoxic agents to monoclonal antibodies (mAbs) was designed and evaluated. We employed the cytotoxic auristatin derivatives MMAE (1a) and MMAF (1b) and doxorubicin propyloxazoline (DPO, 2) to give the beta-glucuronide drug-linkers 9a, 9b, and 17, respectively. Cysteine-quenched derivatives of 9b and 17 were determined to be substrates for E. coli beta-glucuronidase, resulting in facile drug release. The beta-glucuronide MMAF drug-linker 9b was highly stable in rat plasma with an extrapolated half-life of 81 days. Each drug-linker when conjugated to mAbs c1F6 (anti-CD70) and cAC10 (anti-CD30) gave monomeric antibody-drug conjugates (ADCs) with as many as eight drugs per mAb and had high levels of immunologically specific cytotoxic activity on cancer cell lines. cAC10-9a displayed pronounced antitumor activity in a subcutaneous Karpas 299 lymphoma tumor model. A single dose treatment led to cures in all animals at the 0.5 mg/kg dose level and above, and the conjugate was well tolerated at 100 mg/kg. In mice with subcutaneous renal cell carcinoma xenografts, the MMAF conjugate c1F6-9b was tolerated at 25 mg/kg and efficacious at 0.75 mg/kg. These results demonstrate that the beta-glucuronide linker system is an effective strategy for targeting cytotoxic agents providing ADCs with high degrees of efficacy at well-tolerated doses.

Dipeptide-based highly potent doxorubicin antibody conjugates.

Highly potent and novel derivatives of doxorubicin were linked to monoclonal antibodies (mAbs) for site-specific drug delivery. Drug linker 5 consisted of a dipeptide linker attached directly to the daunosamine nitrogen of the n-butyldiacetate doxorubicin derivative 2a. Upon hydrolysis of the peptide linker and acetate groups, the free daunosamine nitrogen is able to form the highly potent 2-pyrrolinodoxorubicin (3a). The second approach involved the use of an oxazolidine carbamate (13) to mask an activating aldehyde group until proteolytic hydrolysis releases 3a. Both drug linkers were shown to be substrates for the lysosomal enzyme cathepsin B. Each molecule was conjugated to the mAbs c1F6 (anti-CD70) and cAC10 (anti-CD30) to give potent drug conjugates against renal cell carcinoma and anaplastic large cell lymphoma cell lines, respectively. The activities were immunologically selective, since antigen negative cell lines were much less sensitive to treatment with the drug conjugates. The approaches described here for attaching highly potent doxorubicin derivatives to mAbs are novel and allow for control of drug stability while covalently bound to the delivery agent.

Design, synthesis, and in vitro evaluation of dipeptide-based antibody minor groove binder conjugates.

Antibody-drug conjugates (ADCs) were prepared consisting of DNA minor groove binder drugs (MGBs) attached to monoclonal antibodies (mAbs) through peptide linkers designed to release drugs inside the lysosomes of target cells. The site of linker attachment on the MGB was at the 5-position on the B-ring, since model studies showed that attachment of an electron-withdrawing group (i.e., acyl, carbamoyl) at this position increased the stability of the molecule. Because of the hydrophobic nature of the MGBs, several measures were required to overcome their tendencies to induce mAb aggregation upon conjugation. This is exemplified in the series of ADCs containing the amino-CBI drug 1. Initial adducts were prepared using the peptide sequence valine-citrulline, attached to a self-immolative para-aminobenzyl carbamate spacer. The resulting ADCs were completely aggregated. Removal of the self-immolative spacer, introduction of a more hydrophilic valine-lysine sequence, and incorporation of a tetraethyleneglycol unit between the mAb and the peptide resulted in conjugates that were nonaggregated, even with as many as eight drugs per mAb. These results were extended to include the hydroxy aza-CBI drug 2, which was linked to the valine-lysine sequence through a para-aminobenzyl ether self-immolative spacer. The resulting mAb conjugates were monomeric and released the hydroxy aza-CBI drug upon treatment with human cathepsin B. In vitro cytotoxicity assays established that the mAb-MGB drug conjugates were highly cytotoxic and effected immunologically specific cell kill at subsaturating doses. The results provide a general strategy for MGB prodrug design and illustrate the importance of linker hydrophilicity in making nonaggregated, active mAb-MGB conjugates.