Purpose: An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 monoclonal antibody (mAb) cAC10, with eight drug moieties per mAb, was previously shown to have potent cytotoxic activity against CD30 ؉ malignant cells. To determine the effect of drug loading on antibody-drug conjugate therapeutic potential, we assessed cAC10 antibody-drug conjugates containing different drug-mAb ratios in vitro and in vivo.Experimental Design: Coupling MMAE to the cysteines that comprise the interchain disulfides of cAC10 created an antibody-drug conjugate population, which was purified using hydrophobic interaction chromatography to yield antibody-drug conjugates with two, four, and eight drugs per antibody (E2, E4, and E8, respectively). Antibody-drug conjugate potency was tested in vitro against CD30؉ lines followed by in vivo xenograft models. The maximum-tolerated dose and pharmacokinetic profiles of the antibody-drug conjugates were investigated in mice.Results: Although antibody-drug conjugate potency in vitro was directly dependent on drug loading (IC 50 values E8

We describe the in vitro and in vivo properties of monoclonal antibody (mAb)-drug conjugates consisting of the potent synthetic dolastatin 10 analogs auristatin E (AE) and monomethylauristatin E (MMAE), linked to the chimeric mAbs cBR96 (specific to Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological malignancies). The linkers used for conjugate formation included an acid-labile hydrazone and protease-sensitive dipeptides, leading to uniformly substituted conjugates that efficiently released active drug in the lysosomes of antigen-positive (Ag+) tumor cells. The peptide-linked mAb-valine-citrulline-MMAE and mAb-phenylalanine-lysine-MMAE conjugates were much more stable in buffers and plasma than the conjugates of mAb and the hydrazone of 5-benzoylvaleric acid-AE ester (AEVB). As a result, the mAb-Val-Cit-MMAE conjugates exhibited greater in vitro specificity and lower in vivo toxicity than corresponding hydrazone conjugates. In vivo studies demonstrated that the peptide-linked conjugates induced regressions and cures of established tumor xenografts with therapeutic indices as high as 60-fold. These conjugates illustrate the importance of linker technology, drug potency and conjugation methodology in developing safe and efficacious mAb-drug conjugates for cancer therapy.

The chimeric monoclonal antibody cAC10, directed against CD30, induces growth arrest of CD30 ؉ cell lines in vitro and has pronounced antitumor activity in severe combined immunodeficiency (SCID) mouse xenograft models of Hodgkin disease. We have significantly enhanced these activities by conjugating to cAC10 the cytotoxic agent monomethyl auristatin E (MMAE) to create the antibody-drug conjugate cAC10-vcMMAE. MMAE, a derivative of the cytotoxic tubulin modifier auristatin E, was covalently coupled to cAC10 through a valine-citrulline peptide linker. The drug was stably attached to the antibody, showing only a 2% release of MMAE following 10-day incubation in human plasma, but it was readily cleaved by lysosomal proteases after receptormediated internalization. Release of MMAE into the cytosol induced G 2 /Mphase growth arrest and cell death through the induction of apoptosis. In vitro, cAC10-vcMMAE was highly potent and selective against CD30 ؉ tumor lines (IC 50 less than 10 ng/mL) but was more than 300-fold less active on antigennegative cells. In SCID mouse xenograft models of anaplastic large cell lymphoma or Hodgkin disease, cAC10-vcMMAE was efficacious at doses as low as 1 mg/kg. Mice treated at 30 mg/kg cAC10-vcMMAE showed no signs of toxicity. These data indicate that cAC10-vcMMAE may be a highly effective and selective therapy for the treatment of CD30 ؉ neoplasias.

The anti-CD20 antibody rituximab is useful in the treatment of certain B-cell malignancies, most notably nonHodgkin's lymphoma. Its efficacy has been increased when used in combination with chemotherapy, yet anti-CD20 monoclonal antibodies (mAbs) directly conjugated with drugs such as doxorubicin (Dox) have failed to deliver drug or to demonstrate antitumor activity. We have produced anti-CD20 antibody-drug conjugates that possess potent antitumor activity by using the anti-mitotic agent, monomethyl auristatin E (MMAE), linked via the lysosomally cleavable dipeptide, valine-citrulline (vc). Two anti-CD20 conjugates, rituximab-vcMMAE and 1F5-vcMMAE, were selectively cytotoxic against CD20؉ B-lymphoma cell lines, with IC 50 values ranging from 50 ng/mL to 1 g/mL. Unlike rituximab, which showed diffuse surface localization, rituximabvcMMAE capped and was internalized within 4 hours after binding to CD20 ؉ B cells. Internalization of rituximabvcMMAE was followed by rapid G 2 -M phase arrest and onset of apoptosis. Anti-CD20 antibody-drug conjugates prepared with Dox were internalized and localized as with rituximab-vcMMAE, yet these were not effective for drug delivery (IC 50 > 50 g/mL). Consistent with in vitro activity, rituximab-vcMMAE showed antitumor efficacy in xenograft models of CD20-positive lymphoma at doses where rituximab or rituximab-Dox conjugates were ineffective. These data indicate that anti-CD20 -based antibody-drug conjugates are effective antitumor agents when prepared with a stable, enzyme-cleavable peptide linkage to highly potent cytotoxic agents such as MMAE.

Immunotoxins are hybrid molecules composed of a cell-surface bin domain and a protein toxin moiety that together target sifc cell popaons for elimination. These Vascular leak syndrome (VLS) is the dose-limiting toxicity found in many clinical trials utilizing immunotoxins, including those prepared with blocked ricin, ricin A chain (in native and deglycosylated forms), and saporin (1-10). VLS is characterized in humans by hypoalbuminemia, weight gain, and edema, resulting from the extravasation offluids and proteins from the vascular system into the periphery. VLS restricts the use of immunotoxins in humans and in many cases necessitates either a significant reduction in dose or a complete cessation of therapy. While peripheral edema is clinically manageable, pulmonary edema can be life threatening. Recently, VLS was found to have contributed to the death of two B-cell lymphoma patients who were treated with anti-CD22-deglycosylated ricin A chain (9). Other toxic effects in patients treated with immunotoxins may be a result of VLS, including tachycardia, nausea, aphasias as a result ofcerebral edema, and myocardial damage (4).Other proteins also have been found to induce VLS in humans. Systemic administration of the cytokine interleukin 2 (IL-2) results in the development ofVLS when approaching doses that may provide antitumor efficacy in patients with metastatic cancer (11,12). VLS has also been observed in patients following treatment with granulocyte/macrophage colony-stimulating factor or the anti-GD3 antibody R24 (13,14).BR96 sFv-PE40 is a single-chain immunotoxin fusion protein that has been shown to cure established human tumor xenografts in both athymic rats and mice (15,16). In this study, a rat model for VLS was established following administration of BR96 sFv-PE40 at doses beyond those required for cures of tumor xenografts in rodent models. The identification of an animal model for VLS that closely approximates the human VLS response to immunotoxins provides an opportunity to evaluate specific drugs for their ability to block immunotoxin-induced VLS and to determine whether they affect the antitumor activity of BR96 sFv-PE40. MATERIALS AND METHODSReagents. The single-chain immunotoxin fusion protein BR96 sFv-PE40 was expressed in Escherichia coli and purified as described (16). Diphenhydramine hydrochloride was purchased from Elkins-Sinn (Cherry Hill, NJ). Cyclosporine A (CsA) was purchased from Sandoz Pharmaceutical. 15-Deoxyspergualin (DSG) was purchased from Nippon Kayaku (Tokyo). Dexamethasone (Dex) was purchased from Anpro Pharmaceuticals (Arcadia, CA).Toxicity Studies. Six-to 8-week-old female Wistar Furth and Rowett, nu/nu (athymic) rats (Harlan-Sprague-Dawley) were i.v. injected with various amounts of BR96 sFv-PE40 (0.25-4 mg/kg). After 24 hr, they were euthanized by exposure to C02, and the tissues were analyzed using gross and microscopic techniques. Cardiac blood was collected from comatose animals and placed either in serum collection tubes for blood chemistry analysis or in EDT...