SLT-VEGF Reduces Lung Metastases, Decreases Tumor Recurrence, and Improves Survival in an Orthotopic Melanoma Model

SLT-VEGF is a recombinant cytotoxin comprised of Shiga-like toxin (SLT) subunit A fused to human vascular endothelial growth factor (VEGF). It is highly cytotoxic to tumor endothelial cells overexpressing VEGF receptor-2 (VEGFR-2/KDR/Flk1) and inhibits the growth of primary tumors in subcutaneous models of breast and prostate cancer and inhibits metastatic dissemination in orthotopic models of pancreatic cancer. We examined the efficacy of SLT-VEGF in limiting tumor growth and metastasis in an orthotopic melanoma model, using NCR athymic nude mice inoculated with highly metastatic Line IV Cl 1 cultured human melanoma cells. Twice weekly injections of SLT-VEGF were started when tumors became palpable at one week after intradermal injection of 1 × 106 cells/mouse. Despite selective depletion of VEGFR-2 overexpressing endothelial cells from the tumor vasculature, SLT-VEGF treatment did not affect tumor growth. However, after primary tumors were removed, continued SLT-VEGF treatment led to fewer tumor recurrences (p = 0.007), reduced the incidence of lung metastasis (p = 0.038), and improved survival (p = 0.002). These results suggest that SLT-VEGF is effective at the very early stages of tumor development, when selective killing of VEGFR-2 overexpressing endothelial cells can still prevent further progression. We hypothesize that SLT-VEGF could be a promising adjuvant therapy to inhibit or prevent outgrowth of metastatic foci after excision of aggressive primary melanoma lesions

Prognostic Biomarkers in Melanoma: Tailoring Treatments to the Patient

BACKGROUND: It is often difficult to accurately predict how a melanoma will progress because melanomas can be so diverse in their genetic and histological makeup. OBJECTIVE: We sought to characterize the current state and progression of biomedical markers towards their utilization as prognostic indicators for patients with melanoma. METHODS: A literature search of the research repository databases PubMed and GoogleScholar was conducted using the following inclusion criteria: (1) published within the last 10 years, and (2) use of overall survival, disease progression, or clinical outcome as primary endpoints. Search terms included various permutations of biomarkers, prognostic, immunologic, serologic, visual, and melanoma. Results were evaluated for statistical power, results significance, and experimental design integrity. RESULTS: The prognostic capabilities of clinical tests for malignant melanoma have made great strides in the last few years, with several serologic and immunohistochemical biomarkers being preliminarily linked to various measures of clinical prognosis. While clinical feasibility of a single sensitive and specific biomarker remains unfeasible, use of select combinations of tested biomarkers remain viable. CONCLUSION: Diagnostic and prognostic genetic assays have begun to cross over from research to commercial application, giving physicians additional tools during the early stages of diagnosis to optimize and individualize treatments

Use of Peptide Library Screening To Detect a Previously Unknown Linear Diagnostic Epitope: Proof of Principle by Use of Lyme Disease Sera

Diagnostic peptides previously isolated from phage-displayed libraries by affinity selection with serum antibodies from patients with Lyme disease were found to give reproducible serum reactivity patterns when tested in two different enzyme-linked immunosorbent assay formats. In addition, the hypothetical possibility that peptides selected by this type of “epitope discovery” technique might identify the original antigens eliciting antibody responses was tested by searching for sequence similarities in bacterial protein databases. In support of this hypothesis, our search uncovered similarities between peptides representing two different sequence motifs and sequences in the VlsE and BBA61 antigens of Borrelia burgdorferi. Utilizing synthetic peptides, we verified that the sequence KAASKETPPALNK, located at the C terminus of the VlsE antigen, had the same reactivity pattern to sera from patients with extracutaneous Lyme disease as the diagnostic peptide SKEKPPSLNWPA, with which it shared a 7-amino-acid-residue match (consensus residues are underlined). A peptide with conservative mutations of five of the consensus residues was nonreactive, strongly suggesting that the VlsE sequence represents the epitope that originally elicited antibody responses in these patients. The diagnostic sensitivity of this new VlsE epitope was relatively low (30%) compared to that (100%) of the well-documented C(6) diagnostic peptide of VlsE when tested in our small cohort of 10 patients with Lyme disease. Nonetheless, the identification of this previously unknown epitope serves as a proof of the principle of the hypothetical ability of “epitope discovery” techniques to detect specific microbial antigens with diagnostic relevance in infectious diseases

Chaperone-Targeting Cytotoxin and Endoplasmic Reticulum Stress-Inducing Drug Synergize to Kill Cancer Cells12

Diverse physiological and therapeutic insults that increase the amount of unfolded or misfolded proteins in the endoplasmic reticulum (ER) induce the unfolded protein response, an evolutionarily conserved protective mechanism that manages ER stress. Glucose-regulated protein 78/immunoglobulin heavy-chain binding protein (GRP78/BiP) is an ER-resident protein that plays a central role in the ER stress response and is the only known substrate of the proteolytic A subunit (SubA) of a novel bacterial AB5 toxin. Here, we report that an engineered fusion protein, epidermal growth factor (EGF)-SubA, combining EGF and SubA, is highly toxic to growing and confluent epidermal growth factor receptor-expressing cancer cells, and its cytotoxicity is mediated by a remarkably rapid cleavage of GRP78/BiP. Systemic delivery of EGF-SubA results in a significant inhibition of human breast and prostate tumor xenografts in mouse models. Furthermore, EGF-SubA dramatically increases the sensitivity of cancer cells to the ER stress-inducing drug thapsigargin, and vice versa, demonstrating the first example of mechanism-based synergism in the action of a cytotoxin and an ER-targeting drug

<i>In vivo</i> anti-sarcoma response by IGF1R and ROR1 CAR T cells in a localized tumor mouse model.

<p>(A) The experimental schedule of tumor cell injection, CAR T cell infusion and BLI monitoring. Prior to testing, all mice displayed normal healthy status. (B) Bioluminescent imaging (BLI) of tumor growth in NOD/SCID mice (four groups, n = 12–13 each) treated with a sarcoma patient derived T cells expressing IGF1R CAR (IGZ), ROR1 CAR (RGZ) or mock T cells. One group mice were untreated. Two mice in the untreated group died of tumor progression on day 13 and 20 and the other two mice died of unknown causes on day 12 and 13. Three mice in the mock group died of tumor progression on day 7, 12 and 19 andone died of unknown causes on day 13. Three mice in the IGZ group died of unknown causes on day 10 and two died of unknown courses on day 17 and 18. Two mice in the RZG group died of tumor progression on day 12 and 50 andone died of unknown causes on day 13. (C) Bioluminescent intensity of the mice treated with the T cells. All <i>p</i> values were shown in the right panel table and were verified independently. (D) Animal survival after T-cell therapy. All <i>p</i> values were determined by the Mantel-Haenszel logrank test and are shown in the right panel table.</p

<i>In vitro</i> anti-sarcoma cytotoxicity of IGF1R and ROR1 CAR T cells.

<p>(A) Cytotoxicity against IGF1R⁺ target cells including sarcoma cell lines by SB modified IGF1R CAR T cells derived from two healthy donors. PBL1-IGZ, PBL1-IG, PBL2-IGZ and PBL2-mock T cells were generated by transfection of PBMCs derived from two healthy donors (PBL1 and PBL2) using pKT2-CaIG:Z or pKT2-CaIG plus pCMV-SB100X plasmids or without DNA. PBL-CD19CAR T cells were used as control. PBL1-IGZ: Rh1 vs K562 and OS17 vs K562, <i>p</i> < 0.001 at all 3 E/T ratios. PBL1-IG: Rh1 vs K562 and OS17 vs K562, <i>p</i> < 0.01. PBL2-IGZ: Rh1 vs K562, <i>p</i> < 0.001, OS17 vs K562, <i>p</i> < 0.01. Killing of Rh1 and OS17 in each CAR T cell group was also significant compared to the corresponding tumor cells in mock T cell group (<i>p</i> < 0.05). (B) Cytotoxicity of IGF1R CAR T cells against a panel of sarcoma cell lines. Comparisons in PBL1-IGZ and PBL2-IGZ were conducted between each sarcoma line and K562, <i>P</i> = 0.0001 at all E/T ratios. (C) Cytotoxicity against sarcoma cells by IGF1R CAR T cells derived from four more healthy donors but not by mock T cells. Comparisons in PBL3-IGZ, PBL4-IGZ, PBL5-IGZ and PBL6-IGZ were conducted between Rh30 vs K562 and SaOS2 vs K562, <i>P</i> = 0.0001 at all E/T ratios.(D) Expression of human IGF1R in R- transfected cell line confirmed by flow cytometry. (E) Specific cytotoxicity against human IGF1R transfected cell line by IGF1R CAR T cells. Comparisons in PBL3-IGZ, PBL4-IGZ, PBL5-IGZ and PBL6-IGZ were conducted between R- and R-/IGF1R, <i>P</i> = 0.0001 at all E/T ratios. (F) Expression of ROR1 in DB and RBV-LCL cell lines. ROR1^- K562 and ROR1⁺ RPMI8226 were used as control (data not shown). (G) Cytotoxicity against ROR1⁺ target cells including a sarcoma cell line by ROR1 CAR T cells derived from two healthy donors. Comparisons in PBL7-RGZ, PBL7-GZ, PBL8-RGZ and PBL8-GZ were conducted between each target cell and K562 at all E/T ratios. PBL7-RGZ: SaOS2 vs K562, <i>p</i> = 0.0001, 0.0008, 0.0001 (E/T of 60:1, 20:1 and 6:1). PBL7-GZ: SaOS2 vs K562, <i>p</i> = 0.0221, 0.0762, 0.1121. PBL8-RGZ: SaOS2 vs K562, <i>p</i> = 0.0001, 0.0009, 0.0041. PBL8-GZ: SAOS2 vs K562, <i>p</i> = 0.2585, 0.8439, 0.9298. <i>P</i> values for DB vs K562 and EBV-LCL vs K562 were not shown. All cytotoxicity data shown are mean ± S.E. of triplicates.</p

<i>In vivo</i> anti-sarcoma activity of IGF1R and ROR1 CAR T cells in a disseminated mouse model.

<p>(A) The experimental schedule of tumor cell injection, CAR T cell infusion and BLI monitoring. Prior to testing, all mice displayed normal healthy status. B) Bioluminescent imaging (BLI) of tumor growth in NSG mice (three groups, n = 6–8 each) treated with a sarcoma patient derived T cells expressing IGF1R CAR (IGZ), ROR1 CAR (RGZ) or mock T cells. SaOS2-fflucN cells were transduced with a lentiviral vector expressing humanized firefly luciferase and truncated nerve growth factor receptor (NGFR). Two mice in the mock group died of tumor progression on day 8. Four mice in IGZ group died of unknown causes on day 8, 9, 16 and 24, probably due to cytokine storms. (C) Bioluminescent intensity of the mice treated with the T cells. All <i>p</i> values were shown in the right panel table and were verified independently. (D) Animal survival after T-cell therapy. All <i>p</i> values were determined using Mantel-Haenszel logrank test and shown in the right panel table. The <i>p</i> values were independently confirmed. Note that <i>p</i> > 0.05 between mock vs IGZ was likely due to a small sample size.</p

Cytokine profiling of IGF1R and ROR1 CAR T cells in response to sarcoma stimulation.

<p>(A) Significant production of IFN-γ, TNF-α, and IL-13 by IGF1R and ROR1 CAR T cells derived from a healthy donor. Data shown are mean ± S.E. of duplicates. One out of two representative data is shown. (B) Antigen-specific production of IFN-γ by IGF1R and ROR1 CAR T cells derived from three sarcoma patients. Data shown are mean ± S.E. of duplicates.</p