The Novel Anti-cMet Antibody seeMet 12 Potentiates Sorafenib Therapy and Radiotherapy in a Colorectal Cancer Model.

RATIONAL: cMet is abnormally regulated in gastrointestinal cancer, and is associated with increased invasiveness of the disease and poor overall survival. There are indications that targeted therapy against cMet, alone or in combination with additional cancer therapies, can help improve treatment outcome. Thus, in the present study we investigated the therapeutic efficacy of a novel cMet-targeting antibody therapy in gastrointestinal cancer models, and assessed potential augmenting effects in combination with tyrosine kinase inhibitor (TKI) targeted therapy or radiotherapy. METHODS: Three different cMet-targeting antibodies were first characterized with respect to antigen binding and effects on cell viability in vitro. The best performing candidate seeMet 12 was then further assessed for effects on colorectal cancer cell growth, proliferation and migration. Combinations with the TKI-inhibitor sorafenib or external beam radiotherapy were then evaluated for potential additive or synergistic effects in vitro using monolayer- and multicellular tumor spheroid assays. Finally, the combination of seeMet 12 and radiotherapy was evaluated in vivo in a proof-of-concept colorectal cancer xenograft study. RESULTS: Dose-dependent therapeutic effects were demonstrated for all three cMet-targeting antibodies. Monotherapy using seeMet 12 resulted in impaired cellular migration/proliferation and reduced tumor spheroid growth. Moreover, seeMet 12 was able to potentiate therapeutic effects in vitro for both sorafenib and radiotherapy treatments. Finally, the in vivo therapy study demonstrated promising results, where a combination of seeMet 12 and fractionated radiotherapy increased median survival by 79% compared to radiotherapy alone, and tripled maximum survival. CONCLUSION: The novel anti-cMet antibody seeMet 12 demonstrated therapeutic effects in cMet positive gastrointestinal cancer cells in vitro. Moreover, the addition of seeMet 12 augmented the effects of sorafenib and radiotherapy. An in vivo proof-of-concept study of seeMet 12 and radiotherapy further validated the results. Thus, cMet-targeted therapy should be further explored as a promising approach to increase therapeutic effects, circumvent treatment resistance, and reduce side effects.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

The beta domain is required for Vps4p oligomerization into a functionally active ATPase.

Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a beta domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the beta domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the beta domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.

Mutant p53 accumulates in cycling and proliferating cells in the normal tissues of p53 R172H mutant mice.

The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy.

Anti-c-Met antibodies recognising a temperature sensitive epitope, inhibit cell growth.

c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4oC when compared to 37oC. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met α-chain.