Antibody targeting of B-cell maturation antigen on malignant plasma cells.

B-cell maturation antigen (BCMA) is expressed on normal and malignant plasma cells and represents a potential target for therapeutic intervention. BCMA binds to two ligands that promote tumor cell survival, a proliferation inducing ligand (APRIL) and B-cell activating factor. To selectively target BCMA for plasma cell malignancies, we developed antibodies with ligand blocking activity that could promote cytotoxicity of multiple myeloma (MM) cell lines as naked antibodies or as antibody-drug conjugates. We show that SG1, an inhibitory BCMA antibody, blocks APRIL-dependent activation of nuclear factor-kappaB in a dose-dependent manner in vitro. Cytotoxicity of SG1 was assessed as a naked antibody after chimerization with and without Fc mutations that enhance FcgammaRIIIA binding. The Fc mutations increased the antibody-dependent cell-mediated cytotoxicity potency of BCMA antibodies against MM lines by approximately 100-fold with a > or = 2-fold increase in maximal lysis. As an alternative therapeutic strategy, anti-BCMA antibodies were endowed with direct cytotoxic activity by conjugation to the cytotoxic drug, monomethyl auristatin F. The most potent BCMA antibody-drug conjugate displayed IC(50) values of < or = 130 pmol/L for three different MM lines. Hence, BCMA antibodies show cytotoxic activity both as naked IgG and as drug conjugates and warrant further evaluation as therapeutic candidates for plasma cell malignancies.

Improved efficacy of alphavbeta3-targeted albumin conjugates by conjugation of a novel auristatin derivative.

Cellular handling of drug delivery preparations en route to the lysosomal compartment has been extensively studied, but little is known about cellular handling of drugs subsequent to their release from the delivery system. We studied a series of closely related drug targeting conjugates, consisting of albumins equipped with alpha vbeta 3-selective RGD-peptide homing ligands, PEG stealth domains, and either the antitubulin agent monomethyl auristatin E (MMAE) or a new F-variant (MMAF). Since MMAF has a C-terminal charge, this compound is potentially less prone to passive redistribution after its release from the carrier. We demonstrate that RGD-peptide-equipped albumin conjugates with MMAF were indeed more potent than MMAE conjugates, in killing both alpha vbeta 3-positive tumor cells and proliferating endothelial cells. Efficacy increased more in tumor cells than in endothelial cells, suggesting different drug redistribution behavior for the two cell types. Binding affinity and uptake of the conjugate and the cellular handling of released drug contributed to the final efficacy of drug-carrier conjugates, highlighting the importance of all aspects to be carefully considered in the design of targeted drug delivery preparations.

Evaluation of RGD-targeted albumin carriers for specific delivery of auristatin E to tumor blood vessels.

Induction of apoptosis in endothelial cells is considered an attractive strategy to therapeutically interfere with a solid tumor's blood supply. In the present paper, we constructed cytotoxic conjugates that specifically target angiogenic endothelial cells, thus preventing typical side effects of apoptosis-inducing drugs. For this purpose, we conjugated the potent antimitotic agent monomethyl-auristatin-E (MMAE) via a lysosomal cleavable linker to human serum albumin (HSA) and further equipped this drug-albumin conjugate with cyclic c(RGDfK) peptides for multivalent interaction with alphavbeta3-integrin. The RGD-peptides were conjugated via either an extended poly(ethylene glycol) linker or a short alkyl linker. The resulting drug-targeting conjugates RGDPEG-MMAE-HSA and RGD-MMAE-HSA demonstrated high binding affinity and specificity for alphavbeta3-integrin expressing human umbilical vein endothelial cells (HUVEC). Both types of conjugates were internalized by endothelial cells and killed the target cells at low nM concentrations. Furthermore, we observed RGD-dependent binding of the conjugates to C26 carcinoma. Upon i.v. administration to C26-tumor bearing mice, both drug-targeting conjugates displayed excellent tumor homing properties. Our results demonstrate that RGD-modified albumins are suitable carriers for cell selective intracellular delivery of cytotoxic compounds, and further studies will be conducted to assess the antivascular and tumor inhibitory potential of RGDPEG-MMAE-HSA and RGD-MMAE-HSA.

Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates.

The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valine-citrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.

Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity.

We have previously shown that antibody-drug conjugates (ADCs) consisting of cAC10 (anti-CD30) linked to the antimitotic agent monomethylauristatin E (MMAE) lead to potent in vitro and in vivo activities against antigen positive tumor models. MMAF is a new antimitotic auristatin derivative with a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its uncharged counterpart, MMAE, most likely due to impaired intracellular access. In vitro cytotoxicity studies indicated that mAb-maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (mAb-L1-MMAF) conjugates were >2200-fold more potent than free MMAF on a large panel of CD30 positive hematologic cell lines. As with cAC10-L1-MMAE, the corresponding MMAF ADC induced cures and regressions of established xenograft tumors at well tolerated doses. To further optimize the ADC, several new linkers were generated in which various components within the L1 linker were either altered or deleted. One of the most promising linkers contained a noncleavable maleimidocaproyl (L4) spacer between the drug and the mAb. cAC10-L4-MMAF was approximately as potent in vitro as cAC10-L1-MMAF against a large panel of cell lines and was equally potent in vivo. Importantly, cAC10-L4-MMAF was tolerated at >3 times the MTD of cAC10-L1-MMAF. LCMS studies indicated that drug released from cAC10-L4-MMAF was the cysteine-L4-MMAF adduct, which likely arises from mAb degradation within the lysosomes of target cells. This new linker technology appears to be ideally suited for drugs that are both relatively cell-impermeable and tolerant of substitution with amino acids. Thus, alterations of the linker have pronounced impacts on toxicity and lead to new ADCs with greatly improved therapeutic indices.

Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate.

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

Evaluation of antigen-based heteropolymer for treatment of systemic lupus erythematosus in a nonhuman primate model.

Autoantibodies that react with double-stranded DNA (dsDNA) are a hallmark for diagnosis of systemic lupus erythematosus (SLE) and are also considered the pathogenic subset that is most associated with lupus nephritis. As an agent to remove the pathogenic dsDNA antibodies from the circulation of SLE patients, we are developing an antigen-based heteropolymer (AHP). The AHP consists of a monoclonal antibody to the complement receptor (CR1) cross-linked to salmon testis dsDNA to effect clearance of anti-DNA antibodies by binding them to erythrocyte CR1. Utilizing a cynomolgus monkey model for SLE in which we infused plasma from SLE patients containing a high titer of high-avidity anti-dsDNA antibody, we have evaluated the safety and efficacy of AHP infusion. The results demonstrate that AHP rapidly (within 2 min of infusion) binds to monkey erythrocytes without causing any toxicological effects. We also demonstrate that human Ig (G+M) antibodies are rapidly bound to the AHP-erythrocyte complex. These events are mirrored in their kinetics by a substantial drop in the level of high-avidity dsDNA antibody in the plasma.