Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of cytokines that mediate leukocyte chemotaxis.

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. Cancer Res; 70(4); 1544-54. ©2010 AACR.

The human P-chemokine receptor CCR5 is an important cofactor for entry of human immunodeficiency virus-type 1 (HIV-I). The murineform of CCR5, despite its 82 percent identity to the human form, was not functional as an HIV-1 coreceptor. HIV-1 entry function could be reconstituted by fusion of various individual elements derived from the extracellular region of human CCR5 onto murine CCR5. Analysis of chimeras containing elements from human CCR5 and human CCR2B suggested that a complex structure rather than single contact sites is responsible for facilitation of viral entry. Further, certain chimeras lacking the domains necessary to signal in response to their natural chemokine ligands retained vigorous HIV-1 coreceptor activity. T h e discovery of che~iokines as modulators of the replication cycle of HIV-1 (1) and the subseauent identification of human chemokine receptors as essential cofactors in cell entry by HIV-1 (2) have provided a new perspective on the biology of this ~a t h o g e n i c human virus. Human CCR5 u ( 3 ) , a seven-transmembrane receptor for the chemokines MIP-la, MIP-1P, and RANTES, confers susceptibility to infection by certain macrophage-tropic strains of HIV-1 in the presence of human CD4 (4, 5). T o investigate structural features of CCR5 that contribute to this function. we developed a transient transfectiol~-infection system in which human CD4 is expressed in COS-7 cells in conjunction with a natural chemokine receDtor or receutor variants. A synthet~c epitopc i.ngineered Into the NH,-termlnus of each ~h e m o k~n e receotor Der-L . mitted rapid verification (7f surface expression bv conventional il~~munoassav inethods. ~i a n s f e c t e d cells were exposkd to a macrophage-tropic strain of HIV-1, such as Ba-L ( 6 ) , and cell entry was quantitated by measurement of intracellular exmession of the viral capsid protein p24 by means of fluorescence-activated cell sorting (FACS).Expression of CD4 alone in COS-7 cells was insufficient to confer susceptibility to infection by Ba-L, because fewer than 0.7% of the CD4-positive cells exhibited detect-