GRP78 expression in peripheral blood mononuclear cells is a new predictive marker for the benefit of taxanes in breast cancer neoadjuvant treatment.

BACKGROUND: Breast cancer treatment is tailored to the specific cancer subtype. Often, systemic treatment is given prior to surgery. Chemotherapy induces significant endoplasmic reticulum (ER) stress-mediated cell death and upregulation of 78-kDa glucose-regulated protein (GRP78). We hypothesized that chemotherapy induces ER stress not only in the tumor tissue but also in immune cells, which may affect the response to anti-cancer treatment. METHODS: We determined the surface expression of GRP78 on 15 different peripheral blood mononuclear cell (PBMC) subpopulations in 20 breast cancer patients at three time points of the neoadjuvant treatment, i.e., at baseline, after anthracycline treatment, and after taxanes treatment. For this purpose, we performed flow cytometric analyses and analyzed the data using ANOVA and the Tukey test. Serum cytokine levels were also evaluated, and their levels were correlated with response to treatment using the t-test after log transformation and Mann-Whitney U Wilcoxon W test. RESULTS: A significant increase in GRP78 expression in PBMCs was documented during the taxane phase, only in patients who achieved pathological complete response (pCR). GRP78-positive clones correlated with increased serum levels of interferon gamma (IFNγ). CONCLUSIONS: The presence of GRP78-positive clones in certain PBMC subpopulations in pCR patients suggests a dynamic interaction between ER stress and immune responsiveness. The correlation of GRP78-positive clones with increased levels of IFNγ supports the idea that GRP78 expression in PBMCs might serve as a new predictive marker to identify the possible benefits of taxanes in the neoadjuvant setting.

TNBC-derived Gal3BP/Gal3 complex induces immunosuppression through CD45 receptor.

A preliminary study investigating immunotherapy strategies for aggressive triple negative breast cancer (TNBC) revealed an overexpression of genes involved in the release of extracellular vesicles (EVs). Proteins expressed by EVs play a role in reprogramming the tumor microenvironment and impeding effective responses to immunotherapy. Galectin 3 (Gal3), found in the extracellular space of breast cancer cells, downregulates T-cell receptor expression. Gal3 binds to several receptors, including CD45, which is required for T-cell receptor activation. Previously, we reported a novel tumor escape mechanism, whereby TNBC cells suppress immune cells through CD45 intracellular signals. The objective of this study was to determine the potential association of Gal3 with TNBC-secreted EVs induction of immunosuppression via the CD45 signaling pathway. EVs were isolated from MDA-MB-231 cells and the plasma of patients with TNBC. Mass spectrometry revealed the presence of Gal3 binding protein (Gal3BP) in the isolated small EVs, which interacted with TNBC secreted Gal3. Gal3BP and Gal3 form a complex that induces a significant increase in T-regulatory cells in peripheral blood mononuclear cells (PBMCs). This increase correlates with a significant increase in suppressive interleukins 10 and 35. Blocking the CD45 receptor in PBMCs cultured with tumor-derived EVs impeded the immunosuppression exerted by the Gal3BP/Gal3 complex. This led to an increase in IFN-γ and the activation of CD4, CD8 and CD56 effector cells. This study suggests a tumor escape mechanism that may contribute to the development of a different immunotherapy strategy that complements current therapies used for TNBC.

Peptide-binding GRP78 protects neurons from hypoxia-induced apoptosis.

Brain ischemia has major consequences leading to the apoptosis of astrocytes and neurons. Glucose-regulated protein 78 (GRP78) known for its role in endoplasmic reticulum stress alleviation was discovered on several cell surfaces acting as a receptor for signaling pathways. We have previously described peptides that bind cell surface GRP78 on endothelial cells to induce angiogenesis. We have also reported that ADoPep1 binds cardiomyocytes to prevent apoptosis of ischemic heart cells. In this study we describe the effect of hypoxia on astrocytes and neurons cell surface GRP78. Under hypoxic conditions, there was an increase of more than fivefold in GRP78 on cell surface of neurons while astrocytes were not affected. The addition of the GRP78 binding peptide, ADoPep1, to neurons decreased the percentage of GRP78 positive cells and did not change the percent of astrocytes. However, a significant increase in early and late apoptosis of both astrocytes and neurons under hypoxia was attenuated in the presence of ADoPep1. Intravitreal administration of ADoPep1 to mice in a model of optic nerve crush significantly reduced retinal cell loss after 21 days compared to the crush-damaged eyes without treatment or by control saline vehicle injection. Histological staining demonstrated reduced GRP78 after ADoPep1 treatment. The mechanism of peptide neuroprotection was demonstrated by the inhibition of hypoxia induced caspase 3/7 activity, cytochrome c release and p38 phosphorylation. This study is the first report on hypoxic neuronal and astrocyte cell surface GRP78 and suggests a potential therapeutic target for neuroprotection.

BNT162b2 Booster Vaccination Induced Immunity against SARS-CoV-2 Variants among Hemodialysis Patients.

Background: The emergence of new SARS-CoV-2 variants, which evade immunity, has raised the urgent need for multiple vaccine booster doses for vulnerable populations. In this study, we aimed to estimate the BNT162b2 booster effectiveness against the spread of coronavirus variants in a hemodialysis population. Methods: We compared humoral and cell-mediated immunity in 100 dialysis patients and 66 age-matched volunteers, before and 2-3 weeks following the first booster vaccine dose. Participants were assessed for anti-spike (RBD) antibody titer, neutralizing antibodies against B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants, spike-specific T-cell responses by FACS and infection outbreak after the first and second booster. Results: Anti-spike antibody titer was significantly increased following the booster, with reduced humoral and cellular response in the dialysis patients. Neutralizing antibody levels increased significantly after the booster dose, with an inferior effect (≤2 fold) against Omicron compared with the Delta variant. Furthermore, CD4+ and CD8+ T-cell activation by Delta spike protein was preserved in 70% of PBMCs from the dialysis patients. A second booster dose tended to reduce breakthrough infections in the dialysis patients. Conclusions: Until the release of an updated vaccine, BNT162b2 booster doses will improve the humoral and cell-mediated immunity against variants. These findings support the importance of repetitive booster doses for hemodialysis patients.

Endothelial cell surface vimentin binding peptide induces angiogenesis under hypoxic/ischemic conditions.

We have previously identified several angiogenic peptides that bind cell surface proteins by screening a phage display peptide library on human umbilical endothelial cells exposed to hypoxic conditions. In this study we describe one of the selected peptides, SP. We found by protein precipitation of endothelial cell lysates that the 12 amino acid SP peptide binds cell surface vimentin. Surprisingly, vimentin was detected on the cell surface of about 30% of intact endothelial cells under both normoxic and hypoxic conditions, as was demonstrated by fluorocytometric analysis on viable cells. The assessment of SP in the induction of angiogenesis was established by a significant increase in endothelial cell proliferation and tube formation under hypoxic conditions and not under normoxic conditions. Cell proliferation and tube length increased two-fold in endothelial cells in the presence of 10 ng/ml SP peptide when compared to controls. The specificity of SP binding to vimentin was demonstrated by SP inhibition of anti-vimentin binding and by the inhibition of tube formation in cells transfected with siRNA against vimentin. Local intramuscular administrations of the peptide SP to ischemic hind limbs using the mouse hind limb ischemia model, demonstrated that SP inoculated at 1 and 10 µg, improved blood perfusion compared to inoculations with an irrelevant peptide or PBS. The recovery of blood perfusion correlated with the increase in the number of detectable capillaries in the ischemic limb. The development of novel peptides for the induction of pro-angiogenic activity may pave the way for new therapeutic strategies in the treatment of cardiovascular ischemic diseases.

A novel role for an old target: CD45 for breast cancer immunotherapy.

Breast cancer subtypes have not shown significant response to current immunomodulatory therapies. Although most subtypes are treatable, triple negative breast cancer (TNBC), an aggressive highly metastatic cancer, comprising 10-20% of breast cancers, remains an unmet medical need. New strategies are needed in order to overcome flaws in the responsiveness to current TNBC therapies. Our aims were: first, to determine the efficacy of a novel immunomodulatory peptide, C24D, on TNBC and second, to elucidate the molecular mechanism by which C24D induces immune-modulating tumor killing. Using mass spectrometry analysis, we identified CD45 as the C24D binding receptor. In vitro and in vivo TNBC models were used to assess the efficacy of C24D in reversing TNBC-induced immunosuppression and in triggering immune-modulated tumor cell killing. The CD45 signal transduction pathway was evaluated by western blot and FACS analyses. We revealed that addition of PBMCs from healthy female donors to TNBC cells results in a cascade of suppressive CD45 intracellular signals. On binding to CD45's extra-cellular domain on TNBC-suppressed leukocytes, the C24D peptide re-activates the Src family of tyrosine kinases, resulting in specific tumor immune response. In vitro, immune reactivation by C24D results in an increase of CD69+ T and CD69+ NK cells, triggering specific killing of TNBC cells. In vivo, C24D induced CD8+ and activated CD56+ tumor infiltrated cells, resulting in tumor apoptosis. Our results should renew interest in molecules targeting CD45, such as the C24D peptide, as a novel strategy for TNBC immunotherapy.

Downregulation of CD45 Signaling in COVID-19 Patients Is Reversed by C24D, a Novel CD45 Targeting Peptide.

CD45, the predominant transmembrane tyrosine phosphatase in leukocytes, is required for the efficient induction of T cell receptor signaling and activation. We recently reported that the CD45-intracellular signals in peripheral blood mononuclear cells (PBMCs) of triple negative breast cancer (TNBC) patients are inhibited. We also reported that C24D, an immune modulating therapeutic peptide, binds to CD45 on immune-suppressed cells and resets the functionality of the immune system via the CD45 signaling pathway. Various studies have demonstrated that also viruses can interfere with the functions of CD45 and that patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are immune-suppressed. Given the similarity between the role of CD45 in viral immune suppression and our findings on TNBC, we hypothesized that the C24D peptide may have a similar "immune-resetting" effect on PBMCs from COVID-19 patients as it did on PBMCs from TNBC patients. We tested this hypothesis by comparing the CD45/TCR intracellular signaling in PBMCs from ten COVID-19 patients vs. PBMCs from ten healthy volunteers. Herein, we report our findings, demonstrating the immune reactivating effect of C24D via the phosphorylation of the tyrosine 505 and 394 in Lck, the tyrosine 493 in ZAP-70 and the tyrosine 172 in VAV-1 proteins in the CD45 signaling pathway. Despite the relatively small number of patients in this report, the results demonstrate that C24D rescued CD45 signaling. Given the central role played by CD45 in the immune system, we suggest CD45 as a potential therapeutic target.

Activation of GRP78 on endothelial cell membranes by an ADAM15-derived peptide induces angiogenesis.

Impaired angiogenesis is one of the features of ischemic diseases. We have previously identified, by screening a phage display peptide library, a peptide that induces angiogenesis in endothelial cells under hypoxic conditions by binding the cell's membrane heat shock protein GRP78. Protein data base search identified 4 amino acids (HWRR) of that synthetic peptide present on the ADAM15 metalloprotease domain, a protein considered to be involved in neovascularization. Three peptides were synthesized according to the ADAM15 sequence placing HWRR at different positions. Peptide ADoPep1 exhibited significant angiogenic properties under hypoxic conditions as determined by cell proliferation, migration and tube formation. In a mouse hind limb ischemia model, a single injection of the peptide restored blood perfusion. The identified peptide was found to activate GRP78 on endothelial cell membrane and siRNA directed against the GRP78 mRNA interfered with induction of angiogenesis by the peptide. The peptide binding induced a decrease in heat shock protein GRP78 that is overexpressed under hypoxic conditions. The mechanism of peptide-induced angiogenic activity involves inhibition of apoptosis as well as increased Akt phosphorylation and ERK 1/2 activation. The peptide did not induce VEGF receptor-2 protein synthesis and phosphorylation, suggesting a VEGF-independent mechanism of angiogenesis.

Chemotherapy Controls Metastasis Through Stimulatory Effects on GRP78 and Its Transcription Factor CREB3L1.

To achieve a cure for metastatic breast cancer, further understanding of molecular drivers of the metastatic cascade is essential. Currently, chemotherapy regimens include doxorubicin and paclitaxel which act in part by inducing the unfolded protein response (UPR). The master regulator of the UPR, glucose regulated protein 78 (GRP78), localizes on the surface of tumor cells and is associated with metastatic disease. Cyclic AMP responsive element binding protein 3-like 1 (CREB3L1), a member of the UPR, is a breast cancer metastasis suppressor that acts on cyclic AMP to promote the expression of target genes including GRP78. The aim of the present study was to evaluate the effects of chemotherapy on CREB3L1 and cell-surface GRP78 expression and its association with the development of breast cancer metastasis. For this purpose, we use breast cancer cells migration in vitro assays and an in vivo metastatic mouse model. The results showed that chemotherapy activated CREB3L1 and enhanced cell-surface GRP78 expression specifically in triple-negative breast cancer cells (TNBC), reducing their migration and metastatic potential. CREB3L1 knockout (KO) in the triple negative MDAMB231 cell line using CRISPR/Cas9 technology led to inhibition of GRP78 expression and abrogation of the CREB3L1 metastatic suppression function. Inoculation of CREB3L1-KO MDAMB231 cells into a mouse metastatic model induced a massive metastatic profile which chemotherapy failed to prevent. These findings elucidate a potential pathway to the development of a novel treatment strategy for metastatic TNBC based on modulating CREB3L1 and cell-surface GRP78 expression by chemotherapy and GRP78-targeted drugs.

Angiogenesis induced by novel peptides selected from a phage display library by screening human vascular endothelial cells under different physiological conditions.

Angiogenesis is a process modulated by several endogenous vascular growth factors as well as by oxygen conditions. For example VEGF failed to induce useful therapeutic angiogenesis in clinical trials. We used a combinatory phage display peptide library screening on human umbilical endothelial cells under normoxia and hypoxia conditions in order to identify novel peptides that bind endothelial cells. The identified peptides induced angiogenesis as demonstrated by endothelial cell proliferation, migration and tube formation. Injection of peptides into the ears of mice resulted in increased numbers of blood vessels. Peptides did not induce VEGF receptor gene expression indicating a possible VEGF unrelated mechanism.

Prevention of melanoma metastases in lungs of BAT treated and peptide immunized mice.

BAT is an immune-modulatory monoclonal antibody that exhibits strong lymphocyte-mediated anti-tumor activity against a variety of murine and human tumors. Peptide A is a vaccine we have developed by screening a phage display peptide library on BAT monoclonal antibody. Anti-tumor activity was obtained in mice inoculated with B16 melanoma by either a single injection with BAT or immunization with peptide A. The aim of this study was to follow and compare histopathologically the process of prevention of melanoma metastases in lungs of treated and immunized mice. Mice were sacrificed on different days after tumor inoculation, their lungs were weighed and the number of metastases was counted. The lungs were then fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin. Histological examination of tumor inoculated mice on day 10 revealed the existence of microscopic melanoma lesions (0.01-0.012 mm) that increased gradually in number and size and on day 21, most of the metastases were large and spanned entire lobes, from the pleura to the hilum, measuring up to 3.5 mm and coexisting with scattered, small metastases showing the same morphology and pattern of lung involvement. On day 24, the lungs of untreated mice were massively infiltrated by coalescing metastases replacing up to 50% of the lung tissue and measuring up to 7.0 mm. The number of lung metastases and weight was dramatically decreased by a single injection of BAT monoclonal antibody ten days post tumor inoculation. The treated mice clearly had fewer and smaller metastases in different mice at the different days post tumor inoculation. On day 21, there were few small metastases measuring up to 1.6 mm and on day 24 no lung metastases were detected in this group that appeared with a completely normal lung structure. Immunization with peptide A started one day post tumor inoculation and was compared to immunization with control peptide N. Fourteen days post tumor inoculation, mice immunized with peptide A had only 1-2 metastases (0.012-0.076 mm) and on day 24 ranged up to 2 mm compared to control immunized mice where the tumor developed up to 5-7 mm. Foci of lung inflammation in both the untreated, treated or immunized mice were rare, small, and not preferentially associated with the lung metastases. They were composed mainly of small lymphocytes and a few macrophages. This study is the basis of histopathological understanding of metastases prevention in lungs of mice immunized or treated by BAT monoclonal antibody.

BAT monoclonal antibody immunotherapy of human metastatic colorectal carcinoma in mice.

BAT monoclonal antibody exhibited anti-tumor activity mediated by T and NK cells. We have evaluated the efficacy of murine and humanized BAT for the treatment of human colorectal carcinoma liver metastases in nude mice. HM7, a human colorectal carcinoma was injected into the spleen to colonize the liver. A single intravenous administration of both BAT antibodies significantly reduced the number of metastases and liver weights. Histological examinations demonstrated lymphocyte accumulation near remnant tumors and in tumor-free tissues of BAT treated mice. The efficacy of humanized BAT in the regression of hepatic metastases in human colorectal carcinoma has potential clinical use.

Cancer disease predictive diagnosis: BAT/CD3-positive lymphocytes in cancer patients.

BAT is an immune-activating monoclonal antibody produced against Daudi cell membranes and selected for stimulating lymphocyte proliferation. The anti-tumor activity of BAT is related to its immunostimulatory properties. Both T and NK cells mediate the anti-tumor activity of BAT. CD4-positive T cells respond to BAT activation by proliferation and INF-gamma production. The aim of the study was to assess the probability that the BAT monoclonal antibody binding capacity to T cells is a marker for different cancers. Human peripheral blood T cells from colon, breast and prostate cancer patients, as well as healthy volunteer donors, were tested for the percentage of binding to BAT mAb (BAT/CD3 cells) by FACS analysis. All patients were tested before undergoing surgery or treatment, and their diagnosis was confirmed by histology. The results showed that the percentage of BAT monoclonal antibody binding to CD3-positive T cells in the peripheral blood was different in cancer patients with diverse tumor types. We found that lymphocytes from the blood of healthy donors contained 25% BAT/CD3 cells. In colon and breast cancer patients, a significant decrease to 13 and 11% of BAT/CD3 cells was found. In contrast, these cells increased ><50% in patients with prostate cancer. These findings may have a potential diagnostic significance and also assist in the evaluation of strategies for the therapeutic use of BAT for different cancer patients.

Cell surface GRP78: A potential marker of good prognosis and response to chemotherapy in breast cancer.

The 78-kDa glucose-regulated protein (GRP78) is a stress induced heat shock protein which, under limiting conditions, functions as a cell surface signaling receptor. Tumor cells are considered to be subjected to a physiologically stressful microenvironment due to their excessive growth. The role of GRP78 in tumor survival has been of notable interest. The present study aimed to assess the potential prognostic and predictive value of cell surface GRP78 expression in breast cancer tumor cells. Cell surface and cytoplasmic expression of GRP78 was examined by immunohistochemical staining of GRP78 in breast cancer archival paraffin-embedded tumor specimens. The cohort studied included breast cancer patients with operable T1,2, estrogen receptor-positive, node-negative cancer who were assessed using the Oncotype DX gene profile, as well as patients with locally advanced disease prior to and following neoadjuvant systemic treatment. GRP78 values were compared between the 2 groups, and prior to and following systemic treatment. Association analyses between GRP78 expression and prognostic markers were also performed. Cox regression analysis was used to examine the impact of these variables on disease-free survival (DFS). No differences in cytoplasmic GRP78 expression were observed. By contrast, the rates of cell surface GRP78 expression were 74.1% in the early stage operable patients, 36% in neoadjuvant systemic treatment patients prior to treatment and 62.5% in patients following systemic treatment (P<0.039). Positive cell surface GRP78 expression was associated with increased expression of the progesterone receptor (P=0.024), p53 expression (P=0.022) and improved DFS (P=0.047). In the case of GRP78 positivity, a trend for a superior response to chemotherapy was observed (P=0.19). The results of the present study indicated that cell surface GRP78 may be used as a marker for good prognosis in breast cancer and a potential marker for response to chemotherapy.

Pharmacological induction of cell surface GRP78 contributes to apoptosis in triple negative breast cancer cells.

Breast cancer tumor with triple-negative receptors (estrogen, progesterone and Her 2, receptors) is the most aggressive and deadly subtype, with high rates of disease recurrence and poor survival. Here, we show that induction in cell surface GRP78 by doxorubicin and tunicamycin was associated with CHOP/GADD153 upregulation and increase in apoptosis in triple negative breast cancer tumor cells. GRP78 is a major regulator of the stress induced unfolded protein response pathway and CHOP/GADD153 is a pro-apoptotic transcription factor associated exclusively with stress induced apoptosis. The blocking of cell surface GRP78 by anti-GRP78 antibody prevented apoptosis, suggesting that induction of cell surface GRP78 by doxorubicin and tunicamycin is required for apoptosis. A better understanding of stress induction of apoptotic signaling in triple negative breast cancer cells may help to define new therapeutic strategies.

The presence of anti-GRP78 antibodies in the serum of patients with colorectal carcinoma: a potential biomarker for early cancer detection.

BACKGROUND: The identification of new biomarkers is required for early diagnosis of colorectal carcinoma patients (CRC), since about 20% of these patients are initially diagnosed with a distant metastatic disease. GRP78, a heat shock protein, functions also as a cell surface signaling receptor of cells under physiological stress. GRP78 was found to be expressed on the cell surface of various tumor cells. The presence of autoantibodies to GRP78 in cancer patient's serum was found to be correlated with a poor prognosis. In this study we aimed to identify anti-GRP78 antibodies in the serum of 85 patients diagnosed by colonoscopy, as an early detection biomarker. METHODS: We developed an ELISA assay with recombinant GRP78 immobilized on 96-well culture plates and used an anti-IgG antibody to measure the sole anti-GRP78 IgGs. RESULTS: Testing for anti-GRP78 showed a significant increase in antibody titer in patients with a polyp and in CRC patients (p<0.001) compared to healthy subjects. CONCLUSIONS: This is the first study showing the presence of anti-GRP78 at the very early stages of CRC.

Colon cancer cells expressing cell surface GRP78 as a marker for reduced tumorigenicity.

BACKGROUND: The glucose regulated heat shock protein 78 (GRP78) is a central regulator of ER (endoplasmic reticulum) stress due to its pro-survival property. Up regulated GRP78 expression in tumor cells has been correlated with aggressive malignancies whereas some reports have predicted an improved prognosis. Over-expression of GRP78 in the ER promotes its localization to the cell surface on several cell types including tumor cells. METHODS: In order to elucidate whether GRP78 receptor positive and negative tumor cells manifest different properties in colorectal cancer, we first artificially separated GRP78 positive and negative sub-populations from HM7 and HCT116 cell lines using anti GRP78 antibody coated magnetic beads. RESULTS: Only GRP78 negative cells were highly proliferative, induced significant growth in tumor size in nude mice and metastasized to the liver in a human metastatic colorectal carcinoma model in mice. In contrast, GRP78 positive cells manifested reduced proliferation, colony formation, tumor growth and liver metastases. The reduced tumorigenicity of GRP78 positive subpopulation was abrogated by silencing GRP78 expression using siRNA oligomers. In our efforts to induce cell surface GRP78, we subjected the cells to doxorubicin and taxol that increased significantly the percent of GRP78 positive population. Cells pre-incubated with doxorubicin exhibited reduced proliferation and tumor growth in mice. CONCLUSION: This study demonstrates the significance of cell surface GRP78 in colon cancer, which may be used as a marker for reduced tumorigenicity.

Peptide-binding heat shock protein GRP78 protects cardiomyocytes from hypoxia-induced apoptosis.

Myocardial ischemia is a severe stress condition that causes extensive biochemical changes triggering cardiac cell death. The 78-kDa glucose-regulated protein (GRP78), a heat shock protein present in all cells and a widely used marker of endoplasmic reticulum stress, functions in controlling the structural maturation of nascent glycoproteins. However, GRP78 was also found to be expressed on the cell surface of several cells such as endothelial cells, macrophages, and tumor cells where it functions as a receptor for a variety of ligands in signaling pathways. Recently, we have identified peptides from two different sources that specifically bind GRP78 protein. We have shown that binding of these peptides to endothelial cell surface GRP78 resulted in angiogenesis. In this study, we first established the presence of cell surface GRP78 on cardiac myocytes. Analysis of cardiomyocytes under hypoxia determined the significant increase in cell surface GRP78 in addition to gene expression and total protein. Apoptosis that was significantly increased in cardiomyocytes under hypoxic conditions was inhibited by the presence of the peptide-binding GRP78 during hypoxia. Inhibition of apoptosis was mediated by the binding of the peptide to cardiomyocytes cell surface GRP78 resulting in blocking caspase-3/7 activation. Silencing GRP78 RNA that reduced GRP78 receptor abrogated the peptide activity. Apoptosis of cardiac cells induced by myocardial infarction in a mouse model was also significantly inhibited by the administration of the peptide to mouse hearts. Our findings may make ADoPep1 a useful therapeutic tool for relieving of ischemia.

Therapeutic angiogenesis of mouse hind limb ischemia by novel peptide activating GRP78 receptor on endothelial cells.

Therapeutic angiogenesis emerged as a non-invasive mean of promoting neovascularization in ischemic tissues. We have searched for new molecules that induce angiogenesis by screening a phage display combinatory peptide library on endothelial cells. One of the selected peptides identified by binding to endothelial cells under hypoxic conditions was further studied. The aim of this study was to assess the therapeutic value of this peptide, RoY, in a mouse hind limb ischemia model and to identify it's receptor on endothelial cells. RoY, a 12 amino-acid synthetic peptide, induced in vitro angiogeneic activity under hypoxic conditions by increasing endothelial cell proliferation, migration and tube formation. In order to assess its therapeutic properties in ischemic tissues, a hind limb ischemia model was induced in C57BL mice by a femoral artery excision. A single local intramuscular injection of RoY peptide to the operated limb, significantly restored blood perfusion and alleviated hind limb ischemia as determined by a laser Doppler imager. Increased capillary density in histological sections corroborated these findings. Protein precipitation and mass spectroscopy studies identified GRP78, a heat shock protein, as the peptide-binding membrane receptor that was increased on endothelial cell membranes under hypoxic conditions. This study demonstrates the efficacy of RoY peptide in alleviation of hind limb ischemia. In addition, it provides evidence that GRP78 is an angiogenic receptor on hypoxic endothelial cells.

A mimotope peptide-based anti-cancer vaccine selected by BAT monoclonal antibody.

Combinatorial phage display peptide libraries are employed to identify small molecules which bind with high affinity to receptor molecules and which mimic the interaction with natural ligands. We used a synthetic combinatory phage display peptide library to screen for peptides that bind BAT monoclonal antibody, an immune modulatory and anti-tumor antibody, to serve as the basis for an anti-cancer vaccine. Two distinct mimotopes, peptides A and B, were isolated, with repeated Proline, Arginine, and Isoleucine amino acids. Mimotope binding was determined by direct binding and by inhibition of BAT binding to the peptide bound phages and to Daudi cells. Immunization of mice with the peptides induced cellular and humoral responses. Cellular response was manifested by significant increase in cytolitic activity. Humoral response was manifested by production of specific antibodies. Serum purified IgG fraction contained anti-peptide antibodies that identified BAT binding mimotopes and competed with BAT binding on Daudi cells. These "BAT like" antibodies exhibited similar immune stimulatory properties to BAT. Immunization of mice with the peptides prevented tumor growth. These finding are the basis for the development of an anti-cancer vaccine.