Xinqun Zhang

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 14C-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. Clin Cancer Res; 16(3); 888–97

The Pharmacologic Basis for Antibody-Auristatin Conjugate Activity

Antibody-drug conjugates (ADCs) made with auristatin antimitotic agents have shown significant preclinical and clinical oncology activity. SGN-75 is composed of the anti-CD70 antibody h1F6 conjugated to monomethylauristatin F through a noncleavable maleimidocaproyl linkage. To understand the pharmacologic basis of the activity of this ADC, its pharmacokinetics and biodistribution were evaluated in a mouse xenograft model with use of a dual-radiolabeled ADC. The concentrations of antibody, total auristatin (conjugated plus unconjugated), and unconjugated auristatin were measured simultaneously in serum, tumor, and 16 normal tissues. Serum pharmacokinetic parameters for antibody and total auristatin were similar with very little unconjugated auristatin observed, demonstrating a high degree of stability. The kinetic values in normal tissues generally tracked with serum: the first time point (1 h) had the highest antibody and total auristatin concentrations with low unconjugated auristatin concentrations, with the exception of organs expected to be involved in hepatobiliary clearance of the ADC, where total and unconjugated auristatin concentrations peaked at 4 h and then rapidly decreased. In tumors, antibody concentrations were maximal at 1 day, with total auristatin increasing until 2 days. Intratumoral unconjugated auristatin was a substantial fraction of the total auristatin and reached concentrations much higher than in normal tissues. The exposure of the tumor to total and unconjugated auristatin was tens to hundreds times higher than normal tissue exposure. The data establish the pharmacologic basis of activity of the ADC through specific tumor targeting, intratumoral auristatin retention, and ADC stability in the systemic circulation.

Anti-CD30 diabody-drug conjugates with potent antitumor activity

Anti-CD30 diabodies were engineered with two cysteine mutations for site-specific drug conjugation in each chain of these homodimeric antibody fragments. Diabodies were conjugated with ∼4 equivalents of the anti-tubulin drugs, monomethyl auristatin E or F, via a protease-cleavable dipeptide linker, to create the conjugates, diabody-vcE4 and diabody-vcF4, respectively. Diabody conjugation had only minor (<3-fold) effects on antigen binding. Diabody-vcF4 was potently cytotoxic against the antigen-positive cell lines, Karpas-299 (34 pmol/L IC50) and L540cy (22 pmol/L IC50), and was 8- and 21-fold more active than diabody-vcE4 against these cell lines, respectively. Clearance of diabody-vcF4 (99-134 mL/d/kg) was 5-fold slower than for the nonconjugated diabody in naive severe combined immunodeficient mice. Diabody-vcF4 had potent and dose-dependent antitumor activity against established Karpas-299 xenografts and gave durable complete responses at well-tolerated doses. Biodistribution experiments with diabody-[3H]-vcF4 (0.72-7.2 mg/kg) in tumor-bearing mice showed a dose-dependent increase in total auristatin accumulation in tumors (≤520 nmol/L) and decrease in relative auristatin accumulation (≤8.1 %ID/g), with peak localization at 4 to 24 h after dosing. Diabody-vcF4 had ∼4-fold lower cytotoxic activity than the corresponding IgG1-vcF4 conjugate in vitro. A similar potency difference was observed in vivo despite 25- to 34-fold faster clearance of diabody-vcF4 than IgG1-vcF4. This may reflect that dose-escalated diabody-vcF4 can surpass IgG1-vcF4 in auristatin delivery to tumors, albeit with higher auristatin exposure to some organs including kidney and liver. Diabody-drug conjugates can have potent antitumor activity at well-tolerated doses and warrant further optimization for cancer therapy. [Mol Cancer Ther 2008;7(8):2486–97]

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.

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.

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.

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

Abstract 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.

Abstract 3694: Relationship between in vivo antitumor activity of ADC and payload release in preclinical models

Antibody-drug conjugates (ADC) deliver cytotoxic payloads to target cells via receptor-mediated internalization. We have demonstrated that SGN-35 (cAC10-vcMMAE) binds to CD30 on target cells and releases intracellular monomethyl auristatin E (MMAE) in vitro, contributing to potent killing of CD30+ cells. Using a set of auristatin-based ADCs, we evaluated whether the intratumoral concentration of the released payload is a predictive indicator of antitumor activity in preclinical models. We first treated CD30+, anaplastic large cell lymphoma (ALCL) Karpas 299 tumors with SGN-35 or a non-binding control ADC (IgG-vcMMAE). The plasma ADC and MMAE PK measurements were similar in SGN-35 and IgG-vcMMAE treated tumor-bearing animals. In contrast, SGN-35 generated 5-fold greater intratumoral MMAE exposure than IgG-vcMMAE, which correlated with tumor regression observed in the SGN-35- treated group. We then examined the relationship between released MMAE and in vitro cytotoxicity by targeting different receptors expressed on the same cells. The lymphoma cell line L82 expresses CD30, CD70, and CD71 receptors. Although auristatin-based ADCs targeting each of these targets has a different IC50, the intracellular MMAE was similar when 50% growth inhibition was achieved (approximately 200 nM). In vivo, CD30, CD70, or CD71-directed ADCs demonstrated similar antitumor activity for a given dose level (0.5, 1, or 3 mg/kg). Concordantly, equal intratumoral MMAE concentrations were observed for a given dose level, regardless of the antigen target. The cell permeable MMAE has been shown to mediate bystander killing in vitro. To evaluate whether intratumoral MMAE mediates bystander killing in vivo, an admixed tumor model consisting CD30+ and CD30- Karpas 299 cells was treated with SGN-35. As predicted, both CD30+ and CD30- tumor cells were killed, presumably due to intratumoral diffusion of MMAE across cell membranes. In contrast, a conjugate with a highly polar form of auristatin (MMAF) was unable to kill the CD30-negative cell population of the admixed tumors. These preclinical results provide evidence that the concentration of released MMAE within the tumor correlates with the ADC antitumor activity in vitro and in vivo. Moreover, the in vivo bystander activity of MMAE may improve utility of this chemotype in tumors with heterogeneous antigen expression. Citation Format: Fu Li, Xinqun Zhang, Kim Emmerton, Mechthild Jonas, Jocelyn Setter, Bill Arthur, Nicole Okeley, Robert Lyon, Dennis Benjamin, Che-Leung Law. Relationship between in vivo antitumor activity of ADC and payload release in preclinical models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2014-3694

Abstract 982: Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities

A common theme in treating cancer is the use of combination chemotherapy, where multiple drugs with different mechanisms of action are combined to elicit synergistic activity or overcome differential drug sensitivities. Antibody-drug conjugates (ADCs) have emerged as a powerful approach for treating cancer, combining the tumor targeting specificity of monoclonal antibodies with the potent cell-killing activity of cytotoxic drugs. Like other therapies, these agents are increasingly being tested in combination with unconjugated, clinically approved anticancer agents. In addition, emerging data demonstrates that insensitivity to a particular ADC can be overcome through delivery of a different payload using the same antibody. For these reasons, the development of ADCs that can deliver two complementary payloads to a tumor would likely be a significant advancement in ADC technology. To enable dual-drug conjugation, we utilized a multiplexing drug carrier that contains cysteine residues with orthogonal protecting groups and identified novel conditions for utilization of these protecting groups on a folded protein. Sequential cysteine unmasking enables discrimination between conjugation sites to allow for site-specific drug conjugation. This strategy provides homogeneous ADCs bearing 16 total drugs per antibody, split evenly between the two drug linkers. Importantly, this strategy is flexible, as it does not require engineered antibodies or custom enzymes for drug-linker conjugation. To demonstrate the potential benefits of ADC dual drug delivery, this strategy was applied to the construction of ADCs bearing two classes of auristatin drug linkers that have different physiochemical properties and complementary anti-cancer activities. Dual-auristatin ADCs were tested in cell line and xenograft models that have differential sensitivities to the individual auristatin components, including those with heterogeneous antigen expression or high levels of drug efflux transporters. The data from these studies demonstrate that the dual-auristatin ADCs were active on cells and tumors that are refractory to treatment with either of the individual component drugs. This work highlights the potential for delivering two synergistic or complementary payloads on a single ADC and presents a flexible method for constructing dual-drug ADCs with site-specific and homogeneous drug loading. Citation Format: Matthew R. Levengood, Xinqun Zhang, Kim K. Emmerton, Joshua H. Hunter, Peter D. Senter. Development of homogeneous dual-drug ADCs: Application to the co-delivery of auristatin payloads with complementary antitumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 982. doi:10.1158/1538-7445.AM2017-982

Abstract 4465: Differential MMAE delivery from ADCs utilizing the valine-citrulline-PAB and β-glucuronide cleavable linker systems

The previously described β-glucuronide linker system (Jeffrey S.C. et al, Bioconj. Chem. 17, 831-840, 2006) was designed to release the toxic payload from an antibody-drug conjugate via the activity of β-glucuronidase, a known lysosomal enzyme. This linker system was shown to be suitable for the generation of potent ADCs utilizing the microtubule disrupting agent monomethylauristatin E (MMAE) as the delivered payload, and is thus an alternative to the protease-cleavable linker valine-citrulline-p-amino benzyl alcohol (vc-PAB). We have recently observed that ADCs utilizing the β-glucuronide linker system to deliver an MMAE payload exhibit potent cytotoxic activity in some cell lines that are resistant to conjugates utilizing the vc-PAB linker to deliver the same drug. To better understand this phenomenon, we have used mass spectrometry to compare the concentration and distribution of MMAE released from both glucuronide and vc-PAB linked ADCs in cell culture. Our data demonstrate that cellular accumulation of released MMAE is considerably greater when delivered by a glucuronide ADC relative to a vc-PAB ADC. We have also employed live-cell fluorescence microscopy to monitor the process of enzymatic cleavage of glucuronide and vc-PAB conjugates. The results of these studies suggest that the two linker systems deliver drug via distinct cellular mechanisms, possibly due to unique intracellular locations of drug release. We propose a model for how these unique linker systems can lead to differences in cellular accumulation of free drug and ultimately increased cytotoxicity. Citation Format: Nicole M. Okeley, Heather A. VanEpps, Xinqun Zhang, Jocelyn R. Setter, Patrick J. Burke, Joseph Z. Hamilton, Robert P. Lyon. Differential MMAE delivery from ADCs utilizing the valine-citrulline-PAB and β-glucuronide cleavable linker systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2014-4465