Stephen C. Alley

Antibody–drug conjugates: targeted drug delivery for cancer

The antibody-drug conjugate field has made significant progress recently owing to careful optimization of several parameters, including mAb specificity, drug potency, linker technology, and the stoichiometry and placement of conjugated drugs. The underlying reason for this has been obtained in pre-clinical biodistribution and pharmacokinetics studies showing that targeted delivery leads to high intratumoral free drug concentrations, while non-target tissues are largely spared from chemotherapeutic exposure. Recent developments in the field have led to an increase in the number of ADCs being tested clinically, with 3 in late stage clinical trials: brentuximab vedotin (also referred to as SGN-35) for Hodgkin lymphoma; Trastuzumab-DM1 for breast cancer; and Inotuzumab ozogamicin for non-Hodgkin lymphoma. This review highlights the recent pre-clinical and clinical advances that have been made.

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.

Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Potent antitumor activity of the anti-CD19 auristatin antibody-drug conjugate hBU12-vcMMAE against rituximab sensitive and resistant lymphomas

Despite major advances in the treatment of non-Hodgkin lymphoma (NHL), including the use of chemotherapeutic agents and the anti-CD20 antibody rituximab, the majority of patients eventually relapse, and salvage treatments with non-cross-resistant compounds are needed to further improve patient survival. Here, we evaluated the antitumor effects of the microtubule destabilizing agent monomethyl auristatin E (MMAE) conjugated to the humanized anti-CD19 antibody hBU12 via a protease-sensitive valine-citrulline (vc) dipeptide linker. hBU12-vcMMAE induced potent tumor cell killing against rituximab-sensitive and -resistant NHL cell lines. CD19 can form heterodimers with CD21, and high levels of CD21 were reported to interfere negatively with the activity of CD19-targeted therapeutics. However, we observed comparable internalization, intracellular trafficking, and drug release in CD21(low) and CD21(high), rituximab-sensitive and -refractory lymphomas treated with hBU12-vcMMAE. Furthermore, high rates of durable regressions in mice implanted with these tumors were observed, suggesting that both rituximab resistance and CD21 expression levels do not impact on the activity of hBU12-vcMMAE. Combined, our data suggest that hBU12-vcMMAE may represent a promising addition to the treatment options for rituximab refractory NHL and other hematologic malignancies, including acute lymphoblastic leukemia.

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]

Engineered anti-CD70 antibody-drug conjugate with increased therapeutic index

An anti-CD70 antibody conjugated to monomethylauristatin F (MMAF) via a valine-citrulline dipeptide containing linker has been shown previously to have potent antitumor activity in renal cell cancer xenograft studies. Here, we generated a panel of humanized anti-CD70 antibody IgG variants and conjugated them to MMAF to study the effect of isotype (IgG1, IgG2, and IgG4) and Fcγ receptor binding on antibody-drug conjugate properties. All IgG variants bound CD70+ 786-O cells with an apparent affinity of ∼1 nmol/L, and drug conjugation did not impair antigen binding. The parent anti-CD70 IgG1 bound to human FcγRI and FcγRIIIA V158 and mouse FcγRIV and this binding was not impaired by drug conjugation. In contrast, binding to these Fcγ receptors was greatly reduced or abolished in the variant, IgG1v1, containing the previously described mutations, E233P:L234V:L235A. All conjugates had potent cytotoxic activity against six different antigen-positive cancer cell lines in vitro with IC50 values of 30 to 540 pmol/L. The IgGv1 conjugate with MMAF displayed improved antitumor activity compared with other conjugates in 786-O and UMRC3 models of renal cell cancer and in the DBTRG05-MG glioblastoma model. All conjugates were tolerated to ≥40 mg/kg in mice. Thus, the IgG1v1 MMAF conjugate has an increased therapeutic index compared with the parent IgG1 conjugate. The improved antitumor activity of the IgG1v1 auristatin conjugates may relate to increased exposure as suggested by pharmacokinetic analysis. The strategy used here for enhancing the therapeutic index of antibody-drug conjugates is independent of the antigen-binding variable domains and potentially applicable to other antibodies. [Mol Cancer Ther 2008;7(9):2913–23]

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.

Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97

Identifying factors that determine the sensitivity or resistance of cancer cells to cytotoxicity by antibody-drug conjugates is essential in the development of such conjugates for therapy. Here the monoclonal antibody L49 is used to target melanotransferrin, a glycosylphosphatidylinositol-anchored glycoprotein first identified as p97, a cell-surface marker in melanomas. L49 was conjugated via a proteolytically cleavable valine-citrulline linker to the antimitotic drug, monomethylauristatin F (vcMMAF). Effective drug release from L49-vcMMAF likely requires cellular proteases most commonly located in endosomes and lysosomes. Melanoma cell lines with the highest surface p97 expression (80,000–280,000 sites per cell) were sensitive to L49-vcMMAF whereas most other cancer cell lines with lower p97 expression were resistant, as were normal cells with low copy numbers (≤20,000 sites per cell). Cell line sensitivity to L49-vcMMAF was found by immunofluorescence microscopy to correlate with intracellular fate of the conjugate. Specifically, L49-vcMMAF colocalized with the lysosomal marker CD107a within sensitive cell lines such as SK-MEL-5 and A2058. In contrast, in resistant cells expressing lower p97 levels (H3677; 72,000 sites per cell), L49-vcMMAF colocalized with caveolin-1, a protein prominent in caveolae, but not with CD107a. Thus, for antibody-drug conjugates targeting p97, antigen level and trafficking to the lysosomes are important factors for achieving robust in vitro cytotoxicity against cancer cells. Immunohistochemical analysis with L49 revealed that 62% of metastatic melanoma tumors had strong staining for p97. Overexpression of p97 in melanoma as compared with normal tissue, in conjunction with the greater sensitivity of tumor cells to L49-vcMMAF, supports further evaluation of antibody-drug conjugates for targeting p97-overexpressing tumors. [Mol Cancer Ther 2006;5(6):1474–82]

Flexibility of Duplex DNA on the Submicrosecond Timescale

Using a site-specific, Electron Paramagnetic Resonance (EPR)-active spin probe that is more rigidly locked to the DNA than any previously reported, the internal dynamics of duplex DNAs in solution were studied. EPR spectra of linear duplex DNAs containing 14-100 base pairs were acquired and simulated by the stochastic Liouville equation for anisotropic rotational diffusion using the diffusion tensor for a right circular cylinder. Internal motions have previously been assumed to be on a rapid enough time scale that they caused an averaging of the spin interactions. This assumption, however, was found to be inconsistent with the experimental data. The weakly bending rod model is modified to take into account the finite relaxation times of the internal modes and applied to analyze the EPR spectra. With this modification, the dependence of the oscillation amplitude of the probe on position along the DNA was in good agreement with the predictions of the weakly bending rod theory. From the length and position dependence of the internal flexibility of the DNA, a submicrosecond dynamic bending persistence length of around 1500 to 1700 A was found. Schellman and Harvey (Biophys. Chem. 55:95-114, 1995) have estimated that, out of the total persistence length of duplex DNA, believed to be about 500 A, approximately 1500 A is accounted for by static bends and 750 A by fluctuating bends. A measured dynamic persistence length of around 1500 A leads to the suggestion that there are additional conformations of the DNA that relax on a longer time scale than that accessible by linear CW-EPR. These measurements are the first direct determination of the dynamic flexibility of duplex DNA in 0.1 M salt.