Orthogonal Cysteine Protection Enables Homogeneous Multi-Drug Antibody-Drug Conjugates.

A strategy for the preparation of homogeneous antibody-drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site-specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug-linkers that have differing physiochemical properties and exert complementary anti-cancer activities. Dual-auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual-drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site-specific protein modification.

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.

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.

Novel Auristatins with High Bystander and Cytotoxic Activities in Drug Efflux-positive Tumor Models.

Auristatins, a class of clinically validated anti-tubulin agents utilized as payloads in antibody-drug conjugates, are generally classified by their membrane permeability and the extent of cytotoxic bystander activity on neighboring cells after targeted delivery. The drugs typically fall within two categories: membrane permeable monomethyl auristatin E-type molecules with high bystander activities and susceptibility to efflux pumps, or charged and less permeable monomethyl auristatin F (MMAF) analogs with low bystander activities and resistance to efflux pumps. Herein, we report the development of novel auristatins that combine the attributes of each class by having both bystander activity and cytotoxicity on multidrug-resistant (MDR+) cell lines. Structure-based design focused on the hydrophobic functionalization of the N-terminal N-methylvaline of the MMAF scaffold to increase cell permeability. The resulting structure-activity relationships of the new auristatins demonstrate that optimization of hydrophobicity and structure can lead to highly active free drugs and antibody-drug conjugates with in vivo bystander activities.

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.

Novel peptide linkers for highly potent antibody-auristatin conjugate.

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.

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.

Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models.

Antibody-drug conjugates (ADC) comprise targeting antibodies armed with potent small-molecule payloads. ADCs demonstrate specific cell killing in clinic, but the basis of their antitumor activity is not fully understood. In this study, we investigated the degree to which payload release predicts ADC activity in vitro and in vivo ADCs were generated to target different receptors on the anaplastic large cell lymphoma line L-82, but delivered the same cytotoxic payload (monomethyl auristatin E, MMAE), and we found that the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity independent of target expression or drug:antibody ratios. Intratumoral MMAE concentrations consistently correlated with the extent of tumor growth inhibition in tumor xenograft models. In addition, we developed a robust admixed tumor model consisting of CD30(+) and CD30(-) cancer cells to study how heterogeneity of target antigen expression, a phenomenon often observed in cancer specimens, affects the treatment response. CD30-targeting ADC delivering membrane permeable MMAE or pyrrolobenzodiazepine dimers demonstrated potent bystander killing of neighboring CD30(-) cells. In contrast, a less membrane permeable payload, MMAF, failed to mediate bystander killing in vivo, suggesting local diffusion and distribution of released payloads represents a potential mechanism of ADC-mediated bystander killing. Collectively, our findings establish that the biophysical properties and amount of released payloads are chief factors determining the overall ADC potency and bystander killing. Cancer Res; 76(9); 2710-9. ©2016 AACR.

SGN-LIV1A: a novel antibody-drug conjugate targeting LIV-1 for the treatment of metastatic breast cancer.

In this article, we describe a novel antibody-drug conjugate (ADC; SGN-LIV1A), targeting the zinc transporter LIV-1 (SLC39A6) for the treatment of metastatic breast cancer. LIV-1 was previously known to be expressed by estrogen receptor-positive breast cancers. In this study, we show that LIV-1 expression is maintained after hormonal therapy in primary and metastatic sites and is also upregulated in triple-negative breast cancers. In addition to breast cancer, other indications showing LIV-1 expression include melanoma, prostate, ovarian, and uterine cancer. SGN-LIV1A consists of a humanized antibody conjugated through a proteolytically cleavable linker to monomethyl auristatin E, a potent microtubule-disrupting agent. When bound to surface-expressed LIV-1 on immortalized cell lines, this ADC is internalized and traffics to the lysozome. SGN-LIV1A displays specific in vitro cytotoxic activity against LIV-1-expressing cancer cells. In vitro results are recapitulated in vivo where antitumor activity is demonstrated in tumor models of breast and cervical cancer lineages. These results support the clinical evaluation of SGN-LIV1A as a novel therapeutic agent for patients with LIV-1-expressing cancer.

Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate.

PURPOSE: SGN-35 is an antibody-drug conjugate (ADC) containing the potent antimitotic drug, monomethylauristatin E (MMAE), linked to the anti-CD30 monoclonal antibody, cAC10. As previously shown, SGN-35 treatment regresses and cures established Hodgkin lymphoma and anaplastic large cell lymphoma xenografts. Recently, the ADC has been shown to possess pronounced activity in clinical trials. Here, we investigate the molecular basis for the activities of SGN-35 by determining the extent of targeted intracellular drug release and retention, and bystander activities. EXPERIMENTAL DESIGN: SGN-35 was prepared with (14)C-labeled MMAE. Intracellular ADC activation on CD30(+) and negative cell lines was determined using a combination of radiometric and liquid chromatograhpy/mass spectrometry-based assays. The bystander activity of SGN-35 was determined using mixed tumor cell cultures consisting of CD30(+) and CD30(-) lines. RESULTS: SGN-35 treatment of CD30(+) cells leads to efficient intracellular release of chemically unmodified MMAE, with intracellular concentrations of MMAE in the range of 500 nmol/L. This was due to specific ADC binding, uptake, MMAE retention, and receptor recycling or resynthesis. MMAE accounts for the total detectable released drug from CD30(+) cells, and has a half-life of retention of 15 to 20 h. Cytotoxicity studies with mixtures of CD30(+) and CD30(-) cell lines indicated that diffusible released MMAE from CD30(+) cells was able to kill cocultivated CD30(-) cells. CONCLUSIONS: MMAE is efficiently released from SGN-35 within CD30(+) cancer cells and, due to its membrane permeability, is able to exert cytotoxic activity on bystander cells. This provides mechanistic insight into the pronounced preclinical and clinical antitumor activities observed with SGN-35.

Anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia.

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR(+), HEL9217 and TF1-alpha, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNFalpha-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR(-) and MDR(+) AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.

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.