The 4th World Antibody Drug Conjugate (WADC) Summit, organized by Hanson Wade was held on February 29‑March 1, 2012 in Frankfurt, Germany, which was also the location for the Antibody Drug Conjugate Summit Europe held in February 2011. During the one year between these meetings, antibody drug conjugates (ADCs) have confirmed their technological maturity and their clinical efficacy in oncology. Brentuximab vedotin (ADCETRIS (TM) ) gained approval by the US Food and Drug Administration in August 2011 and trastuzumab emtansine (T-DM1) confirmed impressive clinical efficacy responses in a large cohort of breast cancer patients. During the 4th WADC meeting, antibody-maytansinoid conjugates were showcased by representatives of ImmunoGen (T-DM1, SAR3419, lorvotuzumab mertansine/IMGN801, IMGN529 and IMG853) and Biotest (BT-062). Data on antibody-auristatin conjugates were presented by scientists and clinicians from Seattle Genetics and Takeda (brentuximab vedotin), Pfizer (5T4-MMAF), Agensys/Astella (AGS-16M8F), Progenics (PSMA-ADC) and Genmab (anti-TF ADCs). Alternative payloads such as calicheamicins and duocarmycin used for preparation of ADCs were discussed by Pfizer and Synthon representatives, respectively. In addition, emerging technologies, including site-directed conjugation (Ambrx), a protein toxin as payload (Viventia), hapten-binding bispecific antibodies (Roche), and use of light activated drugs (Photobiotics), were also presented. Last but not least, progresses in solving Chemistry Manufacturing and Control, and pharmacokinetic issues were addressed by scientists from Genentech, Pfizer, Novartis and Pierre Fabre.

Hodgkin/Reed-Sternberg (H-RS) and anaplastic large cell lymphoma (ALCL) cells express high levels of the cell surface receptor CD30, a member of the TNF receptor superfamily. To augment the antitumor activity of anti-CD30 based therapies, a novel synthetic anti-mitotic agent, monomethyl auristatin E (MMAE), has been conjugated to the anti-CD30 antibody cAC10. MMAE was synthetically modified to include a maleimide for conjugation to an antibody and a protease cleavable Val-Cit peptide linker to release the drug from the antibody. The in vitro and in vivo activity of SGN-35, the resulting antibody-drug conjugate, is outlined. SGN-35 has an average of four MMAE molecules per antibody. Cell surface binding, internalization and intracellular release of MMAE were monitored by immunofluoresence and isotope labeling methods. The H-RS cell line L540cy translocates SGN-35 to lysosomes within 16 hours. Analysis of the cellular contents demonstrated that MMAE is rapidly and efficiently liberated from the antibody, and MMAE accumulates inside the cells. After treating L540cy cells with 200 ng/mL of SGN-35 (6 nM MMAE), the intracellular concentration of MMAE reaches 800 nM after 24 hours, rising to nearly 1000 nM after 72 hours. From 24 to 72 hours, about 70% of the intracellular drug is liberated MMAE, while 30% is still bound to the antibody. In vitro potency of SGN-35 was measured by incubating various concentrations of SGN-35 with CD30+ tumor cells and measuring cell viability in culture. SGN-35 had potent, selective in vitro activity with IC50s ranging from 4 to 35 ng/mL for CD30+ cell lines, and an IC50 > 1000 ng/mL for the CD30− line WSU-NHL. This demonstrates that the conjugation of the novel cytotoxic agent MMAE to an antibody can be accomplished without compromising the antibody's specific binding and internalization. SCID mice were implanted with L540cy and treated with intravenous doses of SGN-35. In mice bearing L540cy xenografts, complete tumor regressions were achieved at doses as low as 2 mg/kg q4dx4. SGN-35 is also highly effective in treating mice with disseminated L540cy implants, at well tolerated doses. Based on the potent cytotoxicity, efficacy in xenograft models, and tolerability, SGN-35 is being advanced toward clinical development in CD30-positive hematologic malignancies.

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.

Effective antibody-drug conjugates (ADC) combine high drug-linker stability in circulation and efficient intratumoral release of drug. Conjugation of monomethyl auristatin E (MMAE) to the anti-CD30 monoclonal antibody (mAb), cAC10, produced a selective and potent ADC against CD30(+) anaplastic large cell lymphoma and Hodgkin's disease models. This ADC, cAC10-valine-citrulline-MMAE, uses a protease-sensitive dipeptide linker designed to release MMAE by lysosomal cathepsin B in target cells but maintain a stable linkage and attenuate drug potency in circulation. To evaluate ADC stability in vivo, we developed methods for measuring drug/mAb ratios at progressive times in plasma from ADC-treated mice and nonhuman primates. Anti-idiotype mAb permitted the capture and quantitation of mAb cAC10, whereas antidrug mAb and MMAE-conjugated horseradish peroxidase reporter provided quantitative detection of conjugated drug following its in vitro release by cathepsin B. These data were validated by an alternative ELISA using anti-idiotype and anti-MMAE mAbs for capture and detection, respectively. Both methods differentiated ADC with variable levels of drug loading and were subsequently applied to stability studies in severe combined immunodeficient mice and cynomolgus monkeys. Evaluation of ADC from mouse circulation showed the linker half-life to be approximately 144 hours (6.0 days), significantly greater than that reported for disulfide- or hydrazone-linked ADCs in mice or human trials. In cynomolgus monkey, the apparent linker half-life was approximately 230 hours (9.6 days), suggesting that the drug-linker will be highly stable in humans. These data represent the longest reported drug-linker half-life to date and provide the basis for the pronounced specificity and antitumor activity of cAC10-valine-citrulline-MMAE.

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 (IC50 values E8<E4<E2), the in vivo antitumor activity of E4 was comparable with E8 at equal mAb doses, although the E4 contained half the amount of MMAE per mAb. E2 was also an active antitumor agent but required higher doses. The maximum-tolerated dose of E2 in mice was at least double that of E4, which in turn was twice that of E8. MMAE loading affected plasma clearance, as E8 cleared 3-fold faster than E4 and 5-fold faster than E2. Conclusions: By decreasing drug loading per antibody, the therapeutic index was increased demonstrating that drug loading is a key design parameter for antibody-drug conjugates.

Proc Amer Assoc Cancer Res, Volume 45, 2004 624 Antibodies that target tumor-associated antigens are currently used in the clinic to treat cancer, in some cases as front-line therapy. Even with these successes there is a need to improve the therapeutic activity of antibody-based therapies. We have recently shown that an antibody-drug conjugate (ADC) targeted to CD30 has potent and selective activity against CD30+ malignancies such as Hodgkin’s disease (HD) and anaplastic large cell lymphoma (ALCL) (Blood 102: 1458-1465, 2003). This ADC, cAC10-vcMMAE, consists of a chimeric anti-CD30 antibody, cAC10, conjugated with 8 molecules of the cytotoxic drug monomethyl auristatin E (MMAE). The drug moieties were attached via a Val-Cit peptide linker to the 8 cysteine residues that comprise the 4 inter-chain disulfide bonds of the cAC10 antibody. cAC10-vcMMAE first binds to CD30 expressed on the surface of CD30+ cells and, upon internalization, drug moieties are enzymatically released and inhibit tubulin polymerization. In the present work, we sought to optimize the therapeutic potential of cAC10-vcMMAE conjugates by assessing the impact of drug loading. ADCs with 2 and 4 drugs per mAb, cAC10-vcMMAE2 and cAC10-vcMMAE4, respectively, were constructed and compared to cAC10-vcMMAE8. Cytotoxicity of the ADCs against CD30+ cell lines in vitro was found to be dependent on drug loading; as drug loading increased ADC cytotoxicity was enhanced. Karpas-299, an ALCL line, and L540cy, a HD cell line, were implanted into SCID mice to compare the efficacy of the ADCs. Unlike the in vitro findings, decreasing drug loading did not necessarily reduce potency in vivo . Complete regressions were achieved with cAC10-vc-MMAE8 and cAC10-vcMMAE4 at the same dose of antibody even though the latter contained half the amount of cytotoxic drug. The difference between cAC10-vcMMAE2 and cAC10-vcMMAE4 was dependent on drug load in that twice the dose was required to achieve complete regressions with cAC10-vcMMAE2. The clearance of the cAC10-ADCs was directly related to the drug loading as cAC10-vcMMAE8 cleared faster than cAC10-vcMMAE4. Similarly, cAC10-vcMMAE2 cleared the slowest of any ADC tested, and was comparable in clearance with the naked cAC10 antibody. In contrast to the in vivo efficacy, there was evidence of a correlation between drug loading and the maximum tolerated dose (MTD). The MTDs of cAC10-vc-MMAE2 and cAC10-vc-MMAE4 in mice were at least four-fold and two-fold higher, respectively, than cAC10-vc-MMAE8. Our data indicates that decreasing the drug loading from eight to four drugs per antibody doubles the therapeutic window of the cAC10-vcMMAE. These results demonstrate that drug loading is a key design parameter in optimizing the therapeutic window of ADCs.

626 PSMA, a 100kDa type II membrane glycoprotein possesses a number of characteristics that make it a suitable candidate for prostate cancer specific therapy. PSMA expression is highly restricted to prostate tissue with strongest expression in both primary and metastatic prostate cancers and PSMA expression is upregulated upon androgen withdrawal. The extracellular domain of PSMA is accessible to agents in the extracellular peri-tumoral fluid thus making it possible to target prodrugs for enzymatic activation. Two discrete enzymatic functions for PSMA have been described. PSMA harbors the hydrolytic properties of N-acetylated alpha-linked acidic dipeptidase ( NAALADase) and is able to hydrolyze the neuropeptide N-acetyl-aspartyl-l-glutamate (NAAG). In addition, PSMA also functions as a pteroyl poly-gamma-glutamyl carboxypeptidase (folate hydrolase) and is able to progressively hydrolyze gamma glutamyl linkages of both poly gamma glutamated folates and methotrexate analogs with varying length glutamate chains. In order to develop prodrugs that can be activated within prostate cancers by PSMA’s enzymatic activity, a PSMA specific peptide carrier is required. In the present study a number of peptide substrates in which the amino acid portion consisted primarily of alpha or gamma carboxyl linked glutamic and or aspartic acids of various chain lengths were coupled to the amino terminus of 8-O-(12Aminododecanoyl)-8-O-debutanoylthapsigargin (12ADT), a potent thaspigargin (Tg) analog. Tg is a highly potent natural plant product that induces proliferation independent apoptosis in all cell types. To selectively and efficiently deliver this anti-tumoral effect to sites of prostatic cancer, prodrugs that were activated within prostate cancer by PSMA’s enzymatic activity were developed. These substrates were characterized on the basis of rates of PSMA hydrolysis and stability in human plasma. All substrates were hydrolyzed by PSMA to varying extents and stable in human plasma. Some of the synthesized 12ADT prodrugs were selectively toxic to PSMA expressing human prostate cancer cell lines. Efforts are underway to test lead therapeutic prodrugs in animal models against PSMA-producing prostate cancer xenografts. The aforementioned prodrug candidates that are especially activated by PSMA represent a novel therapy that could be given to men with prostate cancer while avoiding significant systemic toxicity.