Alan F. Wahl

Development of potent monoclonal antibody auristatin conjugates for cancer therapy

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.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.

Lymphocyte Activation Antigen CD70 Expressed by Renal Cell Carcinoma Is a Potential Therapeutic Target for Anti-CD70 Antibody-Drug Conjugates

Metastatic renal cell carcinoma (RCC) is an aggressive disease refractory to most existing therapeutic modalities. Identifying new markers for disease progression and drug targets for RCC will benefit this unmet medical need. We report a subset of clear cell and papillary cell RCC aberrantly expressing the lymphocyte activation marker CD70, a member of the tumor necrosis factor superfamily. Importantly, CD70 expression was found to be maintained at the metastatic sites of RCC. Anti-CD70 antibody-drug conjugates (ADC) consisting of auristatin phenylalanine phenylenediamine (AFP) or monomethyl auristatin phenylalanine (MMAF), two novel derivatives of the anti-tubulin agent auristatin, mediated potent antigen-dependent cytotoxicity in CD70-expressing RCC cells. Cytotoxic activity of these anti-CD70 ADCs was associated with their internalization and subcellular trafficking through the endosomal-lysosomal pathway, disruption of cellular microtubule network, and G2-M phase cell cycle arrest. The efficiency of drug delivery using anti-CD70 as vehicle was illustrated by the much enhanced cytotoxicity of antibody-conjugated MMAF compared with free MMAF. Hence, ADCs targeted to CD70 can selectively recognize RCC, internalize, and reach the appropriate subcellular compartment(s) for drug release and tumor cell killing. In vitro cytotoxicity of these ADCs was confirmed in xenograft models using RCC cell lines. Our findings provide evidence that CD70 is an attractive target for antibody-based therapeutics against metastatic RCC and suggest that anti-CD70 ADCs can provide a new treatment approach for advanced RCC patients who currently have no chemotherapeutic options.

Antibody–drug conjugates: an emerging modality for the treatment of cancer

Antibody–drug conjugates (ADCs) offer promise as a therapeutic modality that can potentially reduce the toxicities and poor therapeutic indices caused by the lack of specificity of conventional anticancer therapies. ADCs combine the potency of cytotoxic agents with the target selectivity of antibodies by chemically linking a cytotoxic payload to an antibody, potentially creating a synthetic molecule that will deliver targeted antitumor therapy that is both safe and efficacious. The ADC repertoire contains a range of payload molecules, antibodies, and linkers. Two ADC molecules, Kadcyla® and Adcetris®, have been approved by the FDA, and many more are currently in clinical development.

Selective tumor sensitization to taxanes with the MAB-drug conjugate CBR96-doxorubicin

The chimeric monoclonal antibody cBR96 conjugated to doxorubicin (cBR96‐Dox) is selectively internalized by a wide variety of human carcinomas expressing an extended form of Lewis Y antigen (Ley). Endocytosis is followed by cleavage and release of free doxorubicin from the endocytic vesicles and subsequent cytotoxicity. Combination studies with standard anti‐cancer agents, undertaken to further increase the potency of this targeted therapy, identified significant synergistic anti‐tumor activity of cBR96‐Dox and either of the taxanes paclitaxel or docetaxel. Treatment with cBR96‐Dox 24 hr prior to paclitaxel resulted in a steady increase in the percentage of G2 tumor cells and corresponding increase in sensitivity to taxanes. Cell cycle analysis indicated the cBR96‐delivered doxorubicin was most effective against S‐phase cells, yet cells exposed to even subtoxic levels progressed to and arrested in G2, at a point of high sensitivity to the anti‐tubulin agent paclitaxel. The synergy obtained by staged combination of cBR96‐Dox and paclitaxel in vitro was reflected in significant anti‐tumor efficacy in vivo against xenograft models of human lung and breast tumors that could not be achieved by either agent alone. The staged combination elicited significant or complete regressions of established human Ley‐positive tumor xenografts using significantly reduced drug levels. Taken together, these data demonstrate a mechanistic approach to the selective elimination of Ley‐positive tumors by using targeted doxorubicin followed by taxane treatment.

Synthesis and Activity of Fluorinated Derivatives of Sulindac Sulphide and Sulindac Sulphone

The synthesis of fluorinated derivatives of the sulphide and sulphone metabolites of sulindac, a non-steroidal anti-inflammatory agent with chemopreventative activity, is reported. n n n nThe key step in the synthesis is a Pummerer-rearrangement of the parent sulindac sulphoxide using (diethylamino)sulphur trifluoride as an activating agent and as a source of the fluoride nucleophile. The reaction leads to the formation of the 4-fluoromethylthio and 4-fluoromethylsulphonyl derivatives of sulindac (4a and 4b, respectively). Sulindac sulphide is 2.5 times more potent as a COX-1 inhibitor compared with its fluorinated counterpart 4a. The sulphones were inactive as COX-1 inhibitors, and none of the compounds inhibited COX-2 concentrations up to 0.1 mM. Cytotoxicity assays showed that 4a and 4b were as cytotoxic as sulindac sulphide and sulindac sulphone on 3719 colorectal carcinoma cell lines. Compound 4b was the most potent compound on RCA cells with an IC50 of 95 μM (sulindac sulphide 140μM, sulindac sulphone 175 μM). n n n nFluorinated sulindac derivatives warrant further investigation since we have shown that cytotoxic activity can be retained or even increased independently from COX-inhibitory properties. This could help minimize the undesired side-effects associated with chronic sulindac administration.

Centrifugal elutriation to obtain synchronous populations of cells.

Counterflow centrifugal elutriation is a noninvasive method for separating large numbers of cells on the basis of their size and mass. For mammalian cells, this method is useful for separating mixed populations of cells, in particular cells at different stages of the cell division cycle without perturbing cell metabolism or using synchronizing agents. This unit describes a method for separating 2 x 108 cells using the standard JE‐6B rotor or larger numbers of cells in the JE‐5.0 rotor. To verify the purity and to characterize the cell cycle positions of cells in the elutriated populations, the unit includes protocols for measuring nascent DNA synthesis by [3H]thymidine incorporation and for detecting DNA synthesis and content by propidium iodide flow cytometry alone or in combination with bromodeoxyuridine incorporation.