N-terminally LRMK-linked HER-2 peptides, AE-37 [p776(774-788)] and AE-47 [Ava-F7(776-788)], aid differentiation of E75-TCR+CD8+ cells to perforin-positive cells.

The objective of this study was to discover whether the peptides LRMK and LRMK-Ava linked to the N-terminus of peptides HER-2 (774-788) and HER-2 (776-788), respectively, help differentiation of E75-TCR(+)CD8(+) cells. Activation was quantified in terms of proliferation of E75-TCR(+)CD8(+) cells expressing high, medium and low density amounts of the specific TCR. Differentiation to functional CD8(+) cells was quantified as induction of Perforin (Perf), the lytic-enzyme which mediates the effector function of CD8(+) cells, in E75-TCR(+)CD8(+) cells. Peripheral blood mononuclear cells (PBMCs) of 3 patients activated with E75(+)AE-37 and E75(+)AE-47 more greatly increased the number of E75-TCR(Hi) CD8(+)Perf(+) cells than PBMCs activated by AE-47 alone or AE-47(+) E75. E75 plus cytokines and cytokines alone activated more E75-TCR(Low) cells than did AE-37 and AE-47. E75(+) AE-37 and AE-37 also induced differentiation of small- and medium-size activated CD8(+) cells from BRC ascites, in allogeneic activation, to Perf(+) cells. Preferential differentiation of E75-TCR(+)CD8(+)Perf(+) cells in distinct patients by AE-37 and AE-47 indicates that cancer vaccines will benefit from such correct individual and disease-associated help. Additional studies using the natural peptides p776 and F7 are needed to understand whether the LRMK-(Ava) tetra-, or pentamer augments or inhibits differentiation of CD8(+) cells, compared with native, natural HER-2 peptides and/or protects CD8(+) cells activated by E75 and by other HLA-I bound peptides from death. Our findings also develop a model for uniform quantification of differentiated CD8(+) effectors.

Distinct patient responses to activation of T-cells by free HER-2, G89 (777-789) and protected LRMK-linked HER-2, {AE-39 [p776 (Ava-774-788)], AE-47 [(Ava-776-788)] and AE-37[p776 (774-788)]} peptides could lead to development of personalized cancer vaccines.

The objective of this study was to find whether the peptide LRMK linked to the N-terminus of HER-2, 774-788 and shorter peptides create a T helper antigen, which can replace all functional HER-2 peptides in a cancer vaccine. Of the 6 LRMK-HER-2 peptides tested in the presence of IL-12, AE-37, AE-38 and AE-39 induced higher IFN-gamma in PBMCs from 4 healthy donors than the other peptides. AE-37 and AE-39 contained the immunogenic HER-2 peptide, p776 (774-788), while AE-38 contained the truncated HER-2 peptide G89 (777-788). The free, unprotected HER-2 peptide G89 (777-789) activated IFN-gamma production in PBMCs from another BRC patient. Responses to free p776 and F7 could not be tested. Three out of 11 patients responded strongly only to AE-37, while 3 of 10 patients responded strongly only to G89. One responded only to AE-39. All 3 patients diagnosed with DICS of mixed HER(hi), ER+ PR+ type responded to AE-37. Three out of 4 patients diagnosed with luminal cancer and one HERhi, ER- PR- BRC patient responded only to G89. AE-37, at low concentration, helped to expand more E75-TCR(Hi+Med) cells than the negative control AE-47, for IFN-gamma induction AE-47, but was a weaker helper than AE-47 at higher concentrations, and eliminated E75-TCR(Hi) cells. The strongest and most consistent effect of AE-37 was the elimination of CD4(Hi) CD25(Hi) cells. When LRMK is linked to AE-37, the side-chains of L(P-10) and R(P-9) are positioned away from MHC; LRMK forms a bi-strophynx (rotating-double-hook-like) structure when attached to AE-37 and the minimum HER-2 peptide in the peptide-binding groove (PBG). K(P-8) anchors the bi-strophynx to HLA-DR outside the PBG in a novel site DRalpha, 49-52. The HER-2 amino acids, Y(P-1) and R(P3) point towards TCR; R(P-9) and M(P-8) can contact the TCRValpha1. The positions of K(P-8) in AE-37 and Ava (epsilonV)(P-8) in AE-39 modulated immunogenicity of p776. It is unknown whether LRMK adds TCR contact points to the minimal HLA-DR-bound HER-2 peptide (780-788) or activates T-cells of other specificities to produce cytokines and die. Preferential activation of Th1 cells in distinct individuals by AE-37, G89 and AE-39 indicates that cancer vaccines will benefit from correct individual and disease-associated help. Emergence of distinct daughters of luminal and myoepithelial BRC stem cells during metastasis through "de-differentiation" and "reverse differentiation" of drug-resistant cancer requires personalized vaccines, which use an optimal-helper antigen.

BRAK/CXCL14 is a potent inhibitor of angiogenesis and a chemotactic factor for immature dendritic cells.

BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., K(d), approximately 2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.