A Novel Antibody-Toxin Congujate to Treat Mantle Cell Lymphoma

Introduction

Mantle cell lymphoma (MCL), while sensitive to several chemotherapeutic regimens including rituximab, cure rates remain low and new treatments are needed.

We have identified a membrane bound "activated" matriptase, as an attractive antigen target for the highly selective delivery of toxins to epithelial tumors and MCL. Matriptase, a member of type II transmembrane serine proteases, is a glycoprotein (80-90 kDa) synthesized as a latent single-chain structure. Activation generates a disulfide-linked-two-chain fully active enzyme through an auto-activation step. Reactive oxygen species (ROS), as well as acidic environments present in tumors can activate matriptase. In addition to roles in initiating carcinogenesis, the enzyme also plays important roles in tumor progression, including invasiveness and metastasis.

Importantly, while matriptase is present in a latent form on epithelial cells and B-cells, activated matriptase expression is restricted to the membranes of epithelial tumors and some B-cell malignancies .

Methods

Materials: The M-69 antibody was generated by CY. Bortezomib was from the CINJ pharmacy.

Cell Culture: The MCL cells (JeKo-1, Mino, Maver and Z138) were cultured in 1X RPMI Media 1640 (Life Technologies) containing 10% fetal bovine serum (FBS) at 37ºC.

Western blotting: The M-69 antibody was used for western analysis and Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) purchased from Millipore was used as a control.

Cytotoxicity assay: Cell viability was measured by the trypan blue dye exclusion method using a Vi-Cell XR© cell viability analyzer (Beckman Coulter). The cytotoxicity data were analyzed using GraphPad Prism 4 software (GraphPad Software Inc., CA).

Animal studies: NOD/SCID/IL2 receptor gamma chain null (NOD/SCID/IL2gnull, NSG) mice were obtained from the Jackson Laboratory. JeKo-1 MCL cells were used for the xenograft studies. Once tumors were palpable, the mice were treated i.p. with anti-matriptase antibody (M-69) conjugated with MMAE (ADC). Controls used were M-69 antibody alone and saline. In a second experiment mice were treated with the ADC alone, bortezomib alone or the combination. Tumor size and body weights were measured twice a week.

Results

The linkage of monomethyl auristatin-E (MMAE), a potent tubulin-inhibitor, to M69 used the releasable linker technology which is based on Seattle Genetics' valine-citrulline-PABA linker that is cleavable by cathepsin B in the lysosome, while showing stability in circulation. MALDI-TOF mass spectrometry of the ADC showed a 7000 Da increase of the average M.W. of the antibody that corresponds to an average of 3.5 drug molecules linked to each mAb molecule.

MCL (JeKo-1, Mino, Maver and Z138) cell lines were found to overexpress activated matriptase using the M-69 antibody. These cell lines were sensitive to the ADC with IC50s at single digit µg/ml of the conjugate.

We elected to use the JeKo-1 cell line for the initial xenograft studies. We found that the 5 mg/kg dose of the ADC weekly produced a better anti-tumor effect (90% growth inhibition) than a 1 mg/kg dose, without any body weight loss. In combination with bortezomib, treatment with the ADC led to some complete responses.

Conclusions

The mouse antibody to human matriptase, M-69, conjugated to MMAE potently inhibited growth of four MCL cell lines and inhibited xenografts of the JeKo-1 MCL in NSG mice alone and produced enhanced effects in combination with bortezomib without significant side effects.