A novel mouse monoclonal antibody targeting ErbB2 suppresses breast cancer growth.

Overexpression of ErbB2 in breast cancer is associated with increased recurrence and worse prognosis. Accumulating evidences suggest that molecular targeted therapy is a promising anticancer strategy. In this study, we produced a novel anti-ErbB2 monoclonal antibody, 6G10, that recognized an epitope distinct from the trastuzumab binding site. 6G10 induced aggregation of BT474 breast cancer cells and inhibited proliferation of various breast cancer cell lines including BT474. A growth inhibition assay showed that 6G10 had EC(50) values comparable to trastuzumab, indicating that the drugs have a similar level of potency. Furthermore, intraperitoneal administration of 6G10 completely inhibited the growth of xenografted tumors derived from BT474 and SK-BR-3 cells. These data suggested that 6G10 has great therapeutic potential and could be administered to patients alternatively, or synergistically, with trastuzumab.

New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

The improvement of an anti-CD22 immunotoxin: conversion to single-chain and disulfide stabilized form and affinity maturation by alanine scan.

HA22-LR is a recombinant immunotoxin for the treatment of B-cell malignancies that contains the Fv portion of an anti-CD22 antibody fused to a functional portion of Pseudomonas exotoxin A. In the present study, we attempted to improve this molecule. First, we produced a single-chain version of HA22-LR (scdsFv-HA22-LR) in which a peptide linker was introduced between the disulfide-linked light and heavy chains to enable production via single fermentation. No difference in cytotoxic activity was observed between scdsFv-HA22-LR and prototype HA22-LR. Next, we attempted to increase the affinity of scdsFv-HA22-LR by using alanine scanning mutagenesis of complementarity determining regions (CDRs) to assess the specific contribution of each CDR residue to the antigen binding. We found that mutation of asparagine 34 in VLCDR1, which is located at the VL/VH interface, to alanine (N34A) caused a substantial increase in affinity and activity. Estimated KD values measured by fluorescence-activated cell sorting were lowered by 10-fold: 0.056 nM in the N34A mutant compared to 0.58 nM in wild type (WT). Cell viability assays of CD22-positive B-cell lymphoma and leukemia cell lines showed that the N34A mutant had increased cytotoxicity ranging from ~2 (HAL-1, IC 50(WT): 2.37 ± 0.62 ng/ml, IC 50(N34A): 1.32 ± 0.41 ng/ml) to 10 (SUDHL-6, IC 50(WT): 0.47 ± 0.090 ng/ml, IC 50(N34A): 0.048 ± 0.018 ng/ml)-fold compared to WT immunotoxin. The present study suggests that the N34A mutant of scdsFv-HA22-LR could have important consequences in a clinical setting.

A flow cytometry method to quantitate internalized immunotoxins shows that taxol synergistically increases cellular immunotoxins uptake.

Tumor microenvironments present significant barriers to penetration by antibodies, immunoconjugates, and other immunotoxins. In this report, we illustrate a novel strategy to increase tumor cell uptake of immunotoxin by combination with Taxol. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing phase II testing in mesothelioma. Using novel flow cytometry and gel filtration methods, we quantified SS1P uptake in individual tumor cells along with levels of shed mesothelin (sMSLN), a barrier of SS1P therapy. The validity of our flow cytometric method was confirmed by the ability to similarly quantitate tumor cell uptake of Herceptin and an immunotoxin targeting HER2/neu. SS1P uptake peaked several hours after SS1P was cleared from the blood, reflecting an intratumor distribution process of SS1P that is independent of blood supply. Using the methods developed, we demonstrated that Taxol could improve SS1P penetration into tumors in parallel with an associated reduction of sMSLN in tumor extracellular fluid. Our findings offer a mechanistic rationale to combine SS1P with Taxol or another cytotoxic drug as a strategy to increase immunotoxin uptake by tumor cells. Further, we suggest one basis to understand why chemotherapy and antibody-based therapies cooperate when combined in cancer treatment.

Azoospermia in mice with targeted disruption of the Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) gene.

Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) is a member of the Aatyk family of kinases that comprises Aatyk1, Brek/Lmtk2/Aatyk2, and Aatyk3. Although several potential roles have been proposed for Brek and other Aatyk family members, the physiological functions of these kinases remain unclear. Here, we report that Brek(-/-) male mice are infertile, with azoospermia. Detailed histological analysis revealed that Brek(-/-) germ cells differentiated normally until the round-spermatid stage, but failed to undergo the normal change in morphology to become elongated spermatids. Testicular somatic cells appeared normal in these mice. Expression of Brek in testis was restricted to the germ cells, suggesting that the maturations of germ cells in Brek(-/-) mice are affected in a cell-autonomous manner. On the basis of these findings, we concluded that Brek is essential for a late stage of spermatogenesis. Further clarification of the mechanism by which Brek regulates spermatogenesis may help identify new targets for reproductive contraceptives and treatments against infertility.

Involvement of BREK, a serine/threonine kinase enriched in brain, in NGF signalling.

We identified AATYK2 (Apoptosis-Associated Tyrosine Kinase 2) through a database search as a kinase specifically expressed in the brain. After characterization, we renamed it BREK (Brain-Enriched Kinase). Mouse BREK mRNA is expressed predominantly in brain, especially in olfactory bulb, olfactory tubercle, hippocampus, striatum, cerebellum, and cerebral cortex. Levels of expression and phosphorylation of BREK were high at 0-2 weeks after birth, suggesting that BREK is involved in neural development and functions during the early postnatal period. Phosphoamino acid analysis following in vitro kinase reaction revealed that BREK is a catalytically active, serine/threonine kinase. In PC12 cells, BREK was phosphorylated rapidly upon stimulation with nerve growth factor (NGF) in a protein kinase C-dependent pathway. In differentiated PC12 cells, BREK was enriched in cell bodies and growth cones, and also present along neurites. Introduction of a kinase-defective mutant of BREK into PC12 cells enhanced both ERK phosphorylation and neurite outgrowth in response to NGF, suggesting that BREK is a negative regulator of NGF-induced neuronal differentiation. Thus, we conclude that BREK is a new member of the family of protein serine/threonine kinases and that it plays important roles in NGF-TrkA signalling.