You are here

Osada, Takuya

Positions:

Associate Professor of Surgery

Surgery, Surgical Sciences
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1986

M.D. — University of Tokyo (Japan)

Ph.D. 1997

Ph.D. — University of Tokyo (Japan)

Grants:

Developing a HER3 Vaccine to Prevent Resistance to Endocrine Therapy

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
September 30, 2012
End Date
September 29, 2018

Oncogenic Signaling Networks

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
September 30, 2012
End Date
September 29, 2018

Targeting the WNT/beta-catenin Pathway in Triple Negative Breast Cancer

Administered By
Medicine, Medical Oncology
AwardedBy
Department of Defense
Role
Investigator
Start Date
September 30, 2013
End Date
March 29, 2018

Clinical Oncology Research Career Development Program

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Research Candidate
Start Date
September 29, 2009
End Date
July 31, 2015

Targeting hCG-beta for Breast Cancer Immunotherapy

Administered By
Duke Cancer Institute
AwardedBy
National Institutes of Health
Role
Research Analyst
Start Date
January 01, 2004
End Date
December 31, 2009

Redirecting Specificity of Viral Specific T Cells

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Research Scientist
Start Date
June 01, 2003
End Date
May 31, 2008

Dendritic Cell Mobilization and Active Immunotherapy

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Investigator
Start Date
February 16, 2001
End Date
January 31, 2007
Show More

Publications:

In Vivo Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior.

Purpose: Hsp90, a chaperone to numerous molecular pathways in malignant cells, is elevated in aggressive breast cancers. We hypothesized that identifying breast cells with elevated Hsp90 activity in situ could result in early detection of aggressive breast cancers.Experimental Design: We exploited the uptake of an Hsp90 inhibitor by malignant cells to create an imaging probe (HS131) of Hsp90 activity by linking it to a near-infrared (nIR) dye. HS131 uptake into cells correlated with cell membrane expression of Hsp90 and was used to image molecular subtypes of murine and human breast cancers in vitro and in murine models.Results: HS131 imaging was both sensitive and specific in detecting the murine 4T1 breast cancer cell line, as well as subclones with differing metastatic potential. Highly metastatic subclones (4T07) had high HS131 uptake, but subclones with lower metastatic potential (67NR, 168FARN) had low HS131 uptake. We generated isogenic cell lines to demonstrate that overexpression of a variety of specific oncogenes resulted in high HS131 uptake and retention. Finally, we demonstrated that HS131 could be used to detect spontaneous tumors in MMTV-neu mice, as well as primary and metastatic human breast cancer xenografts. HS131 could image invasive lobular breast cancer, a histologic subtype of breast cancer which is often undetectable by mammography.Conclusions: An HSP90-targeting nIR probe is sensitive and specific in imaging all molecular subtypes of murine and human breast cancer, with higher uptake in aggressive and highly metastatic clones. Clinical studies with Hsp90-targeting nIR probes will be initiated shortly. Clin Cancer Res; 1-12. ©2017 AACR.

Authors
Osada, T; Kaneko, K; Gwin, WR; Morse, MA; Hobeika, A; Pogue, BW; Hartman, ZC; Hughes, PF; Haystead, T; Lyerly, HK
MLA Citation
Osada, T, Kaneko, K, Gwin, WR, Morse, MA, Hobeika, A, Pogue, BW, Hartman, ZC, Hughes, PF, Haystead, T, and Lyerly, HK. "In Vivo Detection of HSP90 Identifies Breast Cancers with Aggressive Behavior." Clinical cancer research : an official journal of the American Association for Cancer Research (October 9, 2017).
PMID
28993342
Source
epmc
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Publish Date
2017
DOI
10.1158/1078-0432.ccr-17-1453

A Fluorescent Hsp90 Probe Demonstrates the Unique Association between Extracellular Hsp90 and Malignancy in Vivo.

Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by overexpressing p110HER2. Whole body cryoslicing, imaging, and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an "Achilles heel" for the early diagnosis of metastatic disease and targeted drug delivery.

Authors
Crowe, LB; Hughes, PF; Alcorta, DA; Osada, T; Smith, AP; Totzke, J; Loiselle, DR; Lutz, ID; Gargesha, M; Roy, D; Roques, J; Darr, D; Lyerly, HK; Spector, NL; Haystead, TAJ
MLA Citation
Crowe, LB, Hughes, PF, Alcorta, DA, Osada, T, Smith, AP, Totzke, J, Loiselle, DR, Lutz, ID, Gargesha, M, Roy, D, Roques, J, Darr, D, Lyerly, HK, Spector, NL, and Haystead, TAJ. "A Fluorescent Hsp90 Probe Demonstrates the Unique Association between Extracellular Hsp90 and Malignancy in Vivo." ACS chemical biology 12.4 (April 2017): 1047-1055.
PMID
28103010
Source
epmc
Published In
ACS Chemical Biology
Volume
12
Issue
4
Publish Date
2017
Start Page
1047
End Page
1055
DOI
10.1021/acschembio.7b00006

Vaccination targeting human HER3 alters the phenotype of infiltrating T cells and responses to immune checkpoint inhibition.

Expression of human epidermal growth factor family member 3 (HER3), a critical heterodimerization partner with EGFR and HER2, promotes more aggressive biology in breast and other epithelial malignancies. As such, inhibiting HER3 could have broad applicability to the treatment of EGFR- and HER2-driven tumors. Although lack of a functional kinase domain limits the use of receptor tyrosine kinase inhibitors, HER3 contains antigenic targets for T cells and antibodies. Using novel human HER3 transgenic mouse models of breast cancer, we demonstrate that immunization with recombinant adenoviral vectors encoding full length human HER3 (Ad-HER3-FL) induces HER3-specific T cells and antibodies, alters the T cell infiltrate in tumors, and influences responses to immune checkpoint inhibitions. Both preventative and therapeutic Ad-HER3-FL immunization delayed tumor growth but were associated with both intratumoral PD-1 expressing CD8+ T cells and regulatory CD4+ T cell infiltrates. Immune checkpoint inhibition with either anti-PD-1 or anti-PD-L1 antibodies increased intratumoral CD8+ T cell infiltration and eliminated tumor following preventive vaccination with Ad-HER3-FL vaccine. The combination of dual PD-1/PD-L1 and CTLA4 blockade slowed the growth of tumor in response to Ad-HER3-FL in the therapeutic model. We conclude that HER3-targeting vaccines activate HER3-specific T cells and induce anti-HER3 specific antibodies, which alters the intratumoral T cell infiltrate and responses to immune checkpoint inhibition.

Authors
Osada, T; Morse, MA; Hobeika, A; Diniz, MA; Gwin, WR; Hartman, Z; Wei, J; Guo, H; Yang, X-Y; Liu, C-X; Kaneko, K; Broadwater, G; Lyerly, HK
MLA Citation
Osada, T, Morse, MA, Hobeika, A, Diniz, MA, Gwin, WR, Hartman, Z, Wei, J, Guo, H, Yang, X-Y, Liu, C-X, Kaneko, K, Broadwater, G, and Lyerly, HK. "Vaccination targeting human HER3 alters the phenotype of infiltrating T cells and responses to immune checkpoint inhibition." Oncoimmunology 6.6 (January 2017): e1315495-.
PMID
28680745
Source
epmc
Published In
OncoImmunology
Volume
6
Issue
6
Publish Date
2017
Start Page
e1315495
DOI
10.1080/2162402x.2017.1315495

Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET.

The human growth factor receptor type 2 (HER2) is overexpressed in breast as well as other types of cancer. Immuno-PET, a noninvasive imaging procedure that could assess HER2 status in both primary and metastatic lesions simultaneously, could be a valuable tool for optimizing application of HER2-targeted therapies in individual patients. Herein, we have evaluated the tumor-targeting potential of the 5F7 anti-HER2 Nanobody (single-domain antibody fragment; ∼13 kDa) after (18)F labeling by 2 methods.The 5F7 Nanobody was labeled with (18)F using the novel residualizing label N-succinimidyl 3-((4-(4-(18)F-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ((18)F-SFBTMGMB; (18)F-RL-I) and also via the most commonly used (18)F protein-labeling prosthetic agent N-succinimidyl 3-(18)F-fluorobenzoate ((18)F-SFB). For comparison, 5F7 Nanobody was also labeled using the residualizing radioiodination agent N-succinimidyl 4-guanidinomethyl-3-(125)I-iodobenzoate ((125)I-SGMIB). Paired-label ((18)F/(125)I) internalization assays and biodistribution studies were performed on HER2-expressing BT474M1 breast carcinoma cells and in mice with BT474M1 subcutaneous xenografts, respectively. Small-animal PET/CT imaging of 5F7 Nanobody labeled using (18)F-RL-I also was performed.Internalization assays indicated that intracellularly retained radioactivity for (18)F-RL-I-5F7 was similar to that for coincubated (125)I-SGMIB-5F7, whereas that for (18)F-SFB-5F7 was lower than coincubated (125)I-SGMIB-5F7 and decreased with time. BT474M1 tumor uptake of (18)F-RL-I-5F7 was 28.97 ± 3.88 percentage injected dose per gram of tissue (%ID/g) at 1 h and 36.28 ± 14.10 %ID/g at 2 h, reduced by more than 90% on blocking with trastuzumab, indicating HER2 specificity of uptake, and was also 26%-28% higher (P < 0.05) than that of (18)F-SFB-5F7. At 2 h, the tumor-to-blood ratio for (18)F-RL-I-5F7 (47.4 ± 13.1) was significantly higher (P < 0.05) than for (18)F-SFB-5F7 (25.4 ± 10.3); however, kidney uptake was 28-36-fold higher for (18)F-RL-I-5F7.(18)F-RL-I-5F7 is a promising tracer for evaluating HER2 status by immuno-PET; however, in settings in which renal background is problematic, strategies for reducing its kidney uptake may be needed.

Authors
Vaidyanathan, G; McDougald, D; Choi, J; Koumarianou, E; Weitzel, D; Osada, T; Lyerly, HK; Zalutsky, MR
MLA Citation
Vaidyanathan, G, McDougald, D, Choi, J, Koumarianou, E, Weitzel, D, Osada, T, Lyerly, HK, and Zalutsky, MR. "Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET." Journal of nuclear medicine : official publication, Society of Nuclear Medicine 57.6 (June 2016): 967-973.
PMID
26912425
Source
epmc
Published In
Journal of nuclear medicine : official publication, Society of Nuclear Medicine
Volume
57
Issue
6
Publish Date
2016
Start Page
967
End Page
973
DOI
10.2967/jnumed.115.171306

WE-FG-BRA-01: Cancer Treatment Utilizing Photo-Activation of Psoralen with KV X-Rays.

This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with immunogenic anti-cancer potential. Psoralen therapies have been limited due to the requirement for psoralen activation by UVA light. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and reradiate (phosphoresce) at UV wavelengths.The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry to investigate treatment induced apoptosis. Methylene blue staining, and WST assays were also used. X-PACT was then evaluated in an in-vivo pilot study on BALBc mice with syngeneic 4T1 tumors, including control arms for X-PACT components. Analysis focused on tumor growth delay.A multivariable regression analysis of 36 independent in-vitro irradiation experiments demonstrated that X-PACT induces significant tumor cell apoptosis and cytotoxicity on all three tumor cell lines in-vitro (p<0.0001). Neither psoralen nor phosphor alone had a strongly significant effect. The in-vivo studies show a pronounced tumor growth delay when compared to controls (42% reduction at 25 days, p=0.0002).These studies demonstrate for the first time a therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation generate UVA light in-situ (including deep seated lesions) which in-turn photo-activates powerful anticancer therapeutics which may lead to short and long term therapeutic effect. This work was supported by Immunolight Llc.

Authors
Oldham, M; Yoon, S; Meng, B; Fathi, Z; Beyer, W; Adamson, J; Alcorta, D; Osada, T; Lyerly, K; Dewhirst, M; Fecci, P; Walder, H; Spector, N
MLA Citation
Oldham, M, Yoon, S, Meng, B, Fathi, Z, Beyer, W, Adamson, J, Alcorta, D, Osada, T, Lyerly, K, Dewhirst, M, Fecci, P, Walder, H, and Spector, N. "WE-FG-BRA-01: Cancer Treatment Utilizing Photo-Activation of Psoralen with KV X-Rays." Medical physics 43.6 (June 2016): 3823-.
PMID
28047541
Source
epmc
Published In
Medical physics
Volume
43
Issue
6
Publish Date
2016
Start Page
3823
DOI
10.1118/1.4957901

X-Ray Psoralen Activated Cancer Therapy (X-PACT).

This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of solid cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with current application to proliferative disease and extracorporeal photopheresis (ECP) of cutaneous T Cell Lymphoma. An immunogenic role for light-activated psoralen has been reported, contributing to long-term clinical responses. Psoralen therapies have to-date been limited to superficial or extracorporeal scenarios due to the requirement for psoralen activation by UVA light, which has limited penetration in tissue. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and re-radiate (phosphoresce) at UV wavelengths. The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry in combination with complimentary assays, and the in-vivo mouse study. In an in-vitro study, we show that X-PACT induces significant tumor cell apoptosis and cytotoxicity, unlike psoralen or phosphor alone (p<0.0001). We also show that apoptosis increases as doses of phosphor, psoralen, or radiation increase. Finally, in an in-vivo pilot study of BALBc mice with syngeneic 4T1 tumors, we show that the rate of tumor growth is slower with X-PACT than with saline or AMT + X-ray (p<0.0001). Overall these studies demonstrate a potential therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. In summary, X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation are delivered to a specific tumor site to generate UVA light which in-turn unleashes both short- and potentially long-term antitumor activity of photo-active therapeutics like psoralen.

Authors
Oldham, M; Yoon, P; Fathi, Z; Beyer, WF; Adamson, J; Liu, L; Alcorta, D; Xia, W; Osada, T; Liu, C; Yang, XY; Dodd, RD; Herndon, JE; Meng, B; Kirsch, DG; Lyerly, HK; Dewhirst, MW; Fecci, P; Walder, H; Spector, NL
MLA Citation
Oldham, M, Yoon, P, Fathi, Z, Beyer, WF, Adamson, J, Liu, L, Alcorta, D, Xia, W, Osada, T, Liu, C, Yang, XY, Dodd, RD, Herndon, JE, Meng, B, Kirsch, DG, Lyerly, HK, Dewhirst, MW, Fecci, P, Walder, H, and Spector, NL. "X-Ray Psoralen Activated Cancer Therapy (X-PACT)." PloS one 11.9 (January 2016): e0162078-.
Website
http://hdl.handle.net/10161/13034
PMID
27583569
Source
epmc
Published In
PloS one
Volume
11
Issue
9
Publish Date
2016
Start Page
e0162078
DOI
10.1371/journal.pone.0162078

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

INTRODUCTION: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment. METHODS: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform. RESULTS: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. CONCLUSIONS: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Authors
Ren, XR; Wang, J; Osada, T; Mook, RA; Morse, MA; Barak, LS; Lyerly, HK; Chen, W
MLA Citation
Ren, XR, Wang, J, Osada, T, Mook, RA, Morse, MA, Barak, LS, Lyerly, HK, and Chen, W. "Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth." Breast cancer research : BCR 17.1 (December 2015): 528-.
PMID
25778364
Source
epmc
Published In
Breast Cancer Research
Volume
17
Issue
1
Publish Date
2015
Start Page
528

An Anti-HER2 Nanobody Labeled with 18F Using a Residualizing Label for Assessing HER2 Status

Authors
Vaidyanathan, G; McDougald, D; Choi, J; Koumarianou, E; Pruszynski, M; Osada, T; Lyerly, H; Lahoutte, T; Zalutsky, MR
MLA Citation
Vaidyanathan, G, McDougald, D, Choi, J, Koumarianou, E, Pruszynski, M, Osada, T, Lyerly, H, Lahoutte, T, and Zalutsky, MR. "An Anti-HER2 Nanobody Labeled with 18F Using a Residualizing Label for Assessing HER2 Status." October 2015.
Source
wos-lite
Published In
European Journal of Nuclear Medicine and Molecular Imaging
Volume
42
Publish Date
2015
Start Page
S102
End Page
S102

CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.

Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

Authors
Osada, T; Patel, SP; Hammond, SA; Osada, K; Morse, MA; Lyerly, HK
MLA Citation
Osada, T, Patel, SP, Hammond, SA, Osada, K, Morse, MA, and Lyerly, HK. "CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1." Cancer immunology, immunotherapy : CII 64.6 (June 2015): 677-688.
PMID
25742933
Source
epmc
Published In
Cancer Immunology, Immunotherapy
Volume
64
Issue
6
Publish Date
2015
Start Page
677
End Page
688
DOI
10.1007/s00262-015-1671-y

Effect of alphavirus vaccine encoding HER2 during concurrent anti-HER2 therapies on induction of oligoclonal T cell and antibody responses against HER2.

Authors
Gwin, WR; Hobeika, A; Osada, T; Hartman, Z; Cheng, Q; Broadwater, G; Kimmick, GG; Blackwell, KL; Morse, M; Lyerly, K
MLA Citation
Gwin, WR, Hobeika, A, Osada, T, Hartman, Z, Cheng, Q, Broadwater, G, Kimmick, GG, Blackwell, KL, Morse, M, and Lyerly, K. "Effect of alphavirus vaccine encoding HER2 during concurrent anti-HER2 therapies on induction of oligoclonal T cell and antibody responses against HER2." May 20, 2015.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
33
Issue
15
Publish Date
2015

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Authors
Osada, T; Nagaoka, K; Takahara, M; Yang, XY; Liu, C-X; Guo, H; Roy Choudhury, K; Hobeika, A; Hartman, Z; Morse, MA; Lyerly, HK
MLA Citation
Osada, T, Nagaoka, K, Takahara, M, Yang, XY, Liu, C-X, Guo, H, Roy Choudhury, K, Hobeika, A, Hartman, Z, Morse, MA, and Lyerly, HK. "Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors." Journal of immunotherapy (Hagerstown, Md. : 1997) 38.4 (May 2015): 155-164.
PMID
25839441
Source
epmc
Published In
Journal of Immunotherapy
Volume
38
Issue
4
Publish Date
2015
Start Page
155
End Page
164
DOI
10.1097/cji.0000000000000075

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment.We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform.We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo.This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Authors
Ren, X-R; Wang, J; Osada, T; Mook, RA; Morse, MA; Barak, LS; Lyerly, HK; Chen, W
MLA Citation
Ren, X-R, Wang, J, Osada, T, Mook, RA, Morse, MA, Barak, LS, Lyerly, HK, and Chen, W. "Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth." Breast cancer research : BCR 17 (February 15, 2015): 20-.
PMID
25849870
Source
epmc
Published In
Breast Cancer Research
Volume
17
Publish Date
2015
Start Page
20
DOI
10.1186/s13058-015-0528-9

Extracellular Hsp90 is actively trafficked and internalized in aggressive forms of breast cancer.

Authors
Burianek, LE; Hughes, PF; Osada, T; Alcorta, DA; Smith, AP; Spector, NL; Lyerly, HK; Haystead, TA
MLA Citation
Burianek, LE, Hughes, PF, Osada, T, Alcorta, DA, Smith, AP, Spector, NL, Lyerly, HK, and Haystead, TA. "Extracellular Hsp90 is actively trafficked and internalized in aggressive forms of breast cancer." 2015.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
26
Publish Date
2015

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization.

N-succinimidyl 4-guanidinomethyl-3-[(*)I]iodobenzoate ([(*)I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [(131)I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[(131)I]iodobenzoate (iso-[(131)I]SGMIB) wherein this bulky group was moved from ortho to meta position.Boc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors - trastuzumab (Tras) and a nanobody 5F7 (Nb) - were labeled using iso-[(*)I]SGMIB and [(*)I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed.When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[(131)I]SGMIB were significantly higher than those for Boc2-[(131)I]SGMIB (70.7±2.0% vs 56.5±5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[(131)I]SGMIB than with [(131)I]SGMIB (Nb, 33.1±7.1% vs 28.9±13.0%; Tras, 45.1±4.5% vs 34.8±10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5F7 Nb indicated similar residualizing capacity over 6h; however, at 24h, radioactivity retained intracellularly for iso-[(131)I]SGMIB-Nb was lower than for [(125)I]SGMIB-Nb (46.4±1.3% vs 56.5±2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[(125)I]SGMIB and [(131)I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [(131)I]SGMIB-Tras.Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.

Authors
Choi, J; Vaidyanathan, G; Koumarianou, E; McDougald, D; Pruszynski, M; Osada, T; Lahoutte, T; Lyerly, HK; Zalutsky, MR
MLA Citation
Choi, J, Vaidyanathan, G, Koumarianou, E, McDougald, D, Pruszynski, M, Osada, T, Lahoutte, T, Lyerly, HK, and Zalutsky, MR. "N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization." Nuclear medicine and biology 41.10 (November 2014): 802-812.
PMID
25156548
Source
epmc
Published In
Nuclear Medicine and Biology
Volume
41
Issue
10
Publish Date
2014
Start Page
802
End Page
812
DOI
10.1016/j.nucmedbio.2014.07.005

Functional genomic screens and identification of signaling pathways in oxaliplatin-resistance in colorectal cancer.

Authors
Mettu, NB; Uronis, JM; Osada, T; Lu, M; Osada, K; Mook, R; Chen, W; Morse, M; Lyerly, HK; Wood, K; Hsu, SD
MLA Citation
Mettu, NB, Uronis, JM, Osada, T, Lu, M, Osada, K, Mook, R, Chen, W, Morse, M, Lyerly, HK, Wood, K, and Hsu, SD. "Functional genomic screens and identification of signaling pathways in oxaliplatin-resistance in colorectal cancer." May 20, 2014.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
32
Issue
15
Publish Date
2014

Designing effective vaccines for colorectal cancer.

Achieving long-term control of colorectal cancers with therapeutic vaccines that generate potent anti-tumor T cell and antibody responses has been a goal for more than two decades. To date, clinical trials of these vaccines have demonstrated induction of immune responses, but clinical benefit has been limited. Improved vector delivery systems with enhanced immunostimulatory properties, decreased immunogenicity against vector and improved antigen presentation are some of the key features of modern tumor vaccines. Furthermore, an improved understanding of the various immunosuppressive factors in the tumor microenvironment and regional lymph nodes, coupled with a burgeoning ability to impair inhibitory immune synapses, highlights a growing opportunity to induce beneficial antigen-specific responses against tumor. The combination of improved antigenic delivery systems, coupled with therapeutic immune activation, represents state-of-the-art colorectal vaccine design concepts with the goal of augmenting immune responses against tumor and improving clinical outcomes.

Authors
Patel, SP; Osada, T; Lyerly, HK; Morse, MA
MLA Citation
Patel, SP, Osada, T, Lyerly, HK, and Morse, MA. "Designing effective vaccines for colorectal cancer." Immunotherapy 6.8 (January 2014): 913-926. (Review)
PMID
25313570
Source
epmc
Published In
Immunotherapy
Volume
6
Issue
8
Publish Date
2014
Start Page
913
End Page
926
DOI
10.2217/imt.14.61

Modulation of immune system inhibitory checkpoints in colorectal cancer

T cell infiltration of colorectal cancer is associated with improved clinical outcome, underlining the importance of the immune system in cancer control; however, immune checkpoints, including the inhibitory T cell molecules CTLA-4 and PD-1 that temper the native immune response, mitigating autoimmunity, are coopted by tumors to facilitate escape from immune surveillance. Blockade of CTLA-4 and PD-1 and its ligand PD-L1, expressed by many tumors, have shown impressive activity in melanoma, renal cell carcinoma, and lung cancer. Immune checkpoint inhibition has been less well studied in colorectal cancer, but preclinical and clinical investigations are in progress. © Springer Science+Business Media New York 2013.

Authors
Patel, SP; Osada, T; Osada, K; Hurwitz, H; Lyerly, HK; Morse, MA
MLA Citation
Patel, SP, Osada, T, Osada, K, Hurwitz, H, Lyerly, HK, and Morse, MA. "Modulation of immune system inhibitory checkpoints in colorectal cancer." Current Colorectal Cancer Reports 9.4 (December 1, 2013): 391-397.
Source
scopus
Published In
Current Colorectal Cancer Reports
Volume
9
Issue
4
Publish Date
2013
Start Page
391
End Page
397
DOI
10.1007/s11888-013-0184-3

A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer.

OBJECTIVE: To determine whether 1 of 2 vaccines based on dendritic cells (DCs) and poxvectors encoding CEA (carcinoembryonic antigen) and MUC1 (PANVAC) would lengthen survival in patients with resected metastases of colorectal cancer (CRC). BACKGROUND: Recurrences after complete resections of metastatic CRC remain frequent. Immune responses to CRC are associated with fewer recurrences, suggesting a role for cancer vaccines as adjuvant therapy. Both DCs and poxvectors are potent stimulators of immune responses against cancer antigens. METHODS: Patients, disease-free after CRC metastasectomy and perioperative chemotherapy (n = 74), were randomized to injections of autologous DCs modified with PANVAC (DC/PANVAC) or PANVAC with per injection GM-CSF (granulocyte-macrophage colony-stimulating factor). Endpoints were recurrence-free survival overall survival, and rate of CEA-specific immune responses. Clinical outcome was compared with that of an unvaccinated, contemporary group of patients who had undergone CRC metastasectomy, received similar perioperative therapy, and would have otherwise been eligible for the study. RESULTS: Recurrence-free survival at 2 years was similar (47% and 55% for DC/PANVAC and PANVAC/GM-CSF, respectively) (χ P = 0.48). At a median follow-up of 35.7 months, there were 2 of 37 deaths in the DC/PANVAC arm and 5 of 37 deaths in the PANVAC/GM-CSF arm. The rate and magnitude of T-cell responses against CEA was statistically similar between study arms. As a group, vaccinated patients had superior survival compared with the contemporary unvaccinated group. CONCLUSIONS: Both DC and poxvector vaccines have similar activity. Survival was longer for vaccinated patients than for a contemporary unvaccinated group, suggesting that a randomized trial of poxvector vaccinations compared with standard follow-up after metastasectomy is warranted. (NCT00103142).

Authors
Morse, MA; Niedzwiecki, D; Marshall, JL; Garrett, C; Chang, DZ; Aklilu, M; Crocenzi, TS; Cole, DJ; Dessureault, S; Hobeika, AC; Osada, T; Onaitis, M; Clary, BM; Hsu, D; Devi, GR; Bulusu, A; Annechiarico, RP; Chadaram, V; Clay, TM; Lyerly, HK
MLA Citation
Morse, MA, Niedzwiecki, D, Marshall, JL, Garrett, C, Chang, DZ, Aklilu, M, Crocenzi, TS, Cole, DJ, Dessureault, S, Hobeika, AC, Osada, T, Onaitis, M, Clary, BM, Hsu, D, Devi, GR, Bulusu, A, Annechiarico, RP, Chadaram, V, Clay, TM, and Lyerly, HK. "A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer." Ann Surg 258.6 (December 2013): 879-886.
PMID
23657083
Source
pubmed
Published In
Annals of Surgery
Volume
258
Issue
6
Publish Date
2013
Start Page
879
End Page
886
DOI
10.1097/SLA.0b013e318292919e

Abstract C86: Tethered Hsp90 inhibitors carrying optical or radioiodinated probes reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells.

Authors
Barrott, JJ; Smith, AP; Osada, T; Ramanujam, N; Zalutsky, MR; Lyerly, K; Haystead, TAJ
MLA Citation
Barrott, JJ, Smith, AP, Osada, T, Ramanujam, N, Zalutsky, MR, Lyerly, K, and Haystead, TAJ. "Abstract C86: Tethered Hsp90 inhibitors carrying optical or radioiodinated probes reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells." November 1, 2013.
Source
crossref
Published In
Molecular cancer therapeutics
Volume
12
Issue
11_Supplement
Publish Date
2013
Start Page
C86
End Page
C86
DOI
10.1158/1535-7163.TARG-13-C86

Optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells.

Inhibitors of heat-shock protein 90 (Hsp90) have demonstrated an unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained, but could be influenced by ectopically expressed Hsp90 in tumors. In this work, we synthesized Hsp90 inhibitors that can carry optical or radioiodinated probes via a polyethyleneglycol tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein occurs at the plasma membrane. In mice, we observed exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors that target ectopic Hsp90 can be used to detect breast cancer malignancies through noninvasive imaging.

Authors
Barrott, JJ; Hughes, PF; Osada, T; Yang, X-Y; Hartman, ZC; Loiselle, DR; Spector, NL; Neckers, L; Rajaram, N; Hu, F; Ramanujam, N; Vaidyanathan, G; Zalutsky, MR; Lyerly, HK; Haystead, TA
MLA Citation
Barrott, JJ, Hughes, PF, Osada, T, Yang, X-Y, Hartman, ZC, Loiselle, DR, Spector, NL, Neckers, L, Rajaram, N, Hu, F, Ramanujam, N, Vaidyanathan, G, Zalutsky, MR, Lyerly, HK, and Haystead, TA. "Optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells." Chem Biol 20.9 (September 19, 2013): 1187-1197.
PMID
24035283
Source
pubmed
Published In
Chemistry and Biology
Volume
20
Issue
9
Publish Date
2013
Start Page
1187
End Page
1197
DOI
10.1016/j.chembiol.2013.08.004

Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment.

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.

Authors
Hanks, BA; Holtzhausen, A; Evans, KS; Jamieson, R; Gimpel, P; Campbell, OM; Hector-Greene, M; Sun, L; Tewari, A; George, A; Starr, M; Nixon, A; Augustine, C; Beasley, G; Tyler, DS; Osada, T; Morse, MA; Ling, L; Lyerly, HK; Blobe, GC
MLA Citation
Hanks, BA, Holtzhausen, A, Evans, KS, Jamieson, R, Gimpel, P, Campbell, OM, Hector-Greene, M, Sun, L, Tewari, A, George, A, Starr, M, Nixon, A, Augustine, C, Beasley, G, Tyler, DS, Osada, T, Morse, MA, Ling, L, Lyerly, HK, and Blobe, GC. "Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment." J Clin Invest 123.9 (September 2013): 3925-3940.
Website
http://hdl.handle.net/10161/13297
PMID
23925295
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
123
Issue
9
Publish Date
2013
Start Page
3925
End Page
3940
DOI
10.1172/JCI65745

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients.

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.

Authors
Morse, MA; Chaudhry, A; Gabitzsch, ES; Hobeika, AC; Osada, T; Clay, TM; Amalfitano, A; Burnett, BK; Devi, GR; Hsu, DS; Xu, Y; Balcaitis, S; Dua, R; Nguyen, S; Balint, JP; Jones, FR; Lyerly, HK
MLA Citation
Morse, MA, Chaudhry, A, Gabitzsch, ES, Hobeika, AC, Osada, T, Clay, TM, Amalfitano, A, Burnett, BK, Devi, GR, Hsu, DS, Xu, Y, Balcaitis, S, Dua, R, Nguyen, S, Balint, JP, Jones, FR, and Lyerly, HK. "Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients." Cancer Immunol Immunother 62.8 (August 2013): 1293-1301.
PMID
23624851
Source
pubmed
Published In
Cancer Immunology, Immunotherapy
Volume
62
Issue
8
Publish Date
2013
Start Page
1293
End Page
1301
DOI
10.1007/s00262-013-1400-3

Correction: phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion.

Authors
Hamilton, E; Blackwell, K; Hobeika, AC; Clay, TM; Broadwater, G; Ren, XR; Chen, W; Castro, H; Lehmann, F; Spector, N; Wei, J; Osada, T; Lyerly, HK; Morse, MA
MLA Citation
Hamilton, E, Blackwell, K, Hobeika, AC, Clay, TM, Broadwater, G, Ren, XR, Chen, W, Castro, H, Lehmann, F, Spector, N, Wei, J, Osada, T, Lyerly, HK, and Morse, MA. "Correction: phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion." J Transl Med 11 (2013): 82-.
PMID
23536971
Source
pubmed
Published In
Journal of Translational Medicine
Volume
11
Publish Date
2013
Start Page
82
DOI
10.1186/1479-5876-11-82

Biomarkers and correlative endpoints for immunotherapy trials.

Immunotherapies for lung cancer are reaching phase III clinical trial, but the ultimate success likely will depend on developing biomarkers to guide development and choosing patient populations most likely to benefit. Because the immune response to cancer involves multiple cell types and cytokines, some spatially and temporally separated, it is likely that multiple biomarkers will be required to fully characterize efficacy of the vaccine and predict eventual benefit. Peripheral blood markers of response, such as the ELISPOT assay and cytokine flow cytometry analyses of peripheral blood mononuclear cells following immunotherapy, remain the standard approach, but it is increasingly important to obtain tissue to study the immune response at the site of the tumor. Earlier clinical endpoints such as response rate and progression-free survival do not correlate with overall survival demonstrated for some immunotherapies, suggesting the need to develop other intermediary clinical endpoints. Insofar as all these biomarkers and surrogate endpoints are relevant in multiple malignancies, it may be possible to extrapolate findings to immunotherapy of lung cancer.

Authors
Morse, MA; Osada, T; Hobeika, A; Patel, S; Lyerly, HK
MLA Citation
Morse, MA, Osada, T, Hobeika, A, Patel, S, and Lyerly, HK. "Biomarkers and correlative endpoints for immunotherapy trials." Am Soc Clin Oncol Educ Book (2013). (Review)
PMID
23714525
Source
pubmed
Published In
American Society of Clinical Oncology educational book / ASCO. American Society of Clinical Oncology. Meeting
Publish Date
2013
DOI
10.1200/EdBook_AM.2013.33.e287

Modulation of Immune System Inhibitory Checkpoints in Colorectal Cancer

T cell infiltration of colorectal cancer is associated with improved clinical outcome, underlining the importance of the immune system in cancer control; however, immune checkpoints, including the inhibitory T cell molecules CTLA-4 and PD-1 that temper the native immune response, mitigating autoimmunity, are coopted by tumors to facilitate escape from immune surveillance. Blockade of CTLA-4 and PD-1 and its ligand PD-L1, expressed by many tumors, have shown impressive activity in melanoma, renal cell carcinoma, and lung cancer. Immune checkpoint inhibition has been less well studied in colorectal cancer, but preclinical and clinical investigations are in progress. © 2013 Springer Science+Business Media New York.

Authors
Patel, SP; Osada, T; Osada, K; Hurwitz, H; Lyerly, HK; Morse, MA
MLA Citation
Patel, SP, Osada, T, Osada, K, Hurwitz, H, Lyerly, HK, and Morse, MA. "Modulation of Immune System Inhibitory Checkpoints in Colorectal Cancer." Current Colorectal Cancer Reports (2013): 1-7.
Source
scival
Published In
Current Colorectal Cancer Reports
Publish Date
2013
Start Page
1
End Page
7
DOI
10.1007/s11888-013-0184-3

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models.

INTRODUCTION: The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance. METHODS: Using a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs. RESULTS: The signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings. CONCLUSIONS: Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.

Authors
Xia, W; Petricoin, EF; Zhao, S; Liu, L; Osada, T; Cheng, Q; Wulfkuhle, JD; Gwin, WR; Yang, X; Gallagher, RI; Bacus, S; Lyerly, HK; Spector, NL
MLA Citation
Xia, W, Petricoin, EF, Zhao, S, Liu, L, Osada, T, Cheng, Q, Wulfkuhle, JD, Gwin, WR, Yang, X, Gallagher, RI, Bacus, S, Lyerly, HK, and Spector, NL. "An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models." Breast Cancer Res 15.5 (2013): R85-.
PMID
24044505
Source
pubmed
Published In
Breast Cancer Research
Volume
15
Issue
5
Publish Date
2013
Start Page
R85
DOI
10.1186/bcr3480

Abstract P4-08-07: Novel insight into the tumor “flare” phenomenon and lapatinib resistance

Authors
Piede, JA; Zhao, S; Liu, L; Lyerly, HK; Osada, T; Wang, T; Xia, W; Spector, N
MLA Citation
Piede, JA, Zhao, S, Liu, L, Lyerly, HK, Osada, T, Wang, T, Xia, W, and Spector, N. "Abstract P4-08-07: Novel insight into the tumor “flare” phenomenon and lapatinib resistance." Cancer Research 72.24 Supplement (December 15, 2012): P4-08-07-P4-08-07.
Source
crossref
Published In
Cancer Research
Volume
72
Issue
24 Supplement
Publish Date
2012
Start Page
P4-08-07
End Page
P4-08-07
DOI
10.1158/0008-5472.SABCS12-P4-08-07

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

Authors
Osada, T; Berglund, P; Morse, MA; Hubby, B; Lewis, W; Niedzwiecki, D; Yang, XY; Hobeika, A; Burnett, B; Devi, GR; Clay, TM; Smith, J; Kim Lyerly, H
MLA Citation
Osada, T, Berglund, P, Morse, MA, Hubby, B, Lewis, W, Niedzwiecki, D, Yang, XY, Hobeika, A, Burnett, B, Devi, GR, Clay, TM, Smith, J, and Kim Lyerly, H. "Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects." Cancer Immunol Immunother 61.11 (November 2012): 1941-1951.
PMID
22488274
Source
pubmed
Published In
Cancer Immunology, Immunotherapy
Volume
61
Issue
11
Publish Date
2012
Start Page
1941
End Page
1951
DOI
10.1007/s00262-012-1248-y

Characterization of an oxaliplatin sensitivity predictor in a preclinical murine model of colorectal cancer.

Despite advances in contemporary chemotherapeutic strategies, long-term survival still remains elusive for patients with metastatic colorectal cancer. A better understanding of the molecular markers of drug sensitivity to match therapy with patient is needed to improve clinical outcomes. In this study, we used in vitro drug sensitivity data from the NCI-60 cell lines together with their Affymetrix microarray data to develop a gene expression signature to predict sensitivity to oxaliplatin. To validate our oxaliplatin sensitivity signature, patient-derived colorectal cancer explants (PDCCE) were developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice from resected human colorectal tumors. Analysis of gene expression profiles found similarities between the PDCCEs and their parental human tumors, suggesting their utility to study drug sensitivity in vivo. The oxaliplatin sensitivity signature was then validated in vivo with response data from 14 PDCCEs treated with oxaliplatin and was found to have an accuracy of 92.9% (sensitivity = 87.5%; specificity = 100%). Our findings suggest that PDCCEs can be a novel source to study drug sensitivity in colorectal cancer. Furthermore, genomic-based analysis has the potential to be incorporated into future strategies to optimize individual therapy for patients with metastatic colorectal cancer.

Authors
Kim, MK; Osada, T; Barry, WT; Yang, XY; Freedman, JA; Tsamis, KA; Datto, M; Clary, BM; Clay, T; Morse, MA; Febbo, PG; Lyerly, HK; Hsu, DS
MLA Citation
Kim, MK, Osada, T, Barry, WT, Yang, XY, Freedman, JA, Tsamis, KA, Datto, M, Clary, BM, Clay, T, Morse, MA, Febbo, PG, Lyerly, HK, and Hsu, DS. "Characterization of an oxaliplatin sensitivity predictor in a preclinical murine model of colorectal cancer." Mol Cancer Ther 11.7 (July 2012): 1500-1509.
PMID
22351745
Source
pubmed
Published In
Molecular cancer therapeutics
Volume
11
Issue
7
Publish Date
2012
Start Page
1500
End Page
1509
DOI
10.1158/1535-7163.MCT-11-0937

Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells.

INTRODUCTION: Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy. METHODS: We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine. RESULTS: HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation. CONCLUSION: These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.

Authors
Ren, X-R; Wei, J; Lei, G; Wang, J; Lu, J; Xia, W; Spector, N; Barak, LS; Clay, TM; Osada, T; Hamilton, E; Blackwell, K; Hobeika, AC; Morse, MA; Lyerly, HK; Chen, W
MLA Citation
Ren, X-R, Wei, J, Lei, G, Wang, J, Lu, J, Xia, W, Spector, N, Barak, LS, Clay, TM, Osada, T, Hamilton, E, Blackwell, K, Hobeika, AC, Morse, MA, Lyerly, HK, and Chen, W. "Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells. (Published online)" Breast Cancer Res 14.3 (June 7, 2012): R89-.
PMID
22676470
Source
pubmed
Published In
Breast Cancer Research
Volume
14
Issue
3
Publish Date
2012
Start Page
R89
DOI
10.1186/bcr3204

Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy.

Although cellular immunotherapy based on autolgous dendritic cells (DCs) targeting antigens expressed by metastatic cancer has demonstrated clinical efficacy, the logistical challenges in generating an individualized cell product create an imperative to develop alternatives to DC-based cancer vaccines. Particularly attractive alternatives include in situ delivery of antigen and activation signals to resident antigen-presenting cells (APCs), which can be achieved by novel fusion molecules targeting the mannose receptor and by recombinant viral vectors expressing the antigen of interest and capable of infecting DCs. A particular challenge in the use of viral vectors is the well-appreciated clinical obstacles to their efficacy, specifically vector-specific neutralizing immune responses. Because heterologous prime and boost strategies have been demonstrated to be particularly potent, we developed two novel recombinant vectors based on alphaviral replicon particles and a next-generation adenovirus encoding an antigen commonly overexpressed in many human cancers, carcinoembryonic antigen (CEA). The rationale for developing these vectors, their unique characteristics, the preclinical studies and early clinical experience with each, and opportunities to enhance their effectiveness will be reviewed. The potential of each of these potent recombinant vectors to efficiently generate clinically active anti-tumor immune response alone, or in combination, will be discussed.

Authors
Osada, T; Morse, MA; Hobeika, A; Lyerly, HK
MLA Citation
Osada, T, Morse, MA, Hobeika, A, and Lyerly, HK. "Novel recombinant alphaviral and adenoviral vectors for cancer immunotherapy." Semin Oncol 39.3 (June 2012): 305-310. (Review)
PMID
22595053
Source
pubmed
Published In
Seminars in Oncology
Volume
39
Issue
3
Publish Date
2012
Start Page
305
End Page
310
DOI
10.1053/j.seminoncol.2012.02.013

Effect of the loss of the type III TGF beta receptor during tumor progression on tumor microenvironment: Preclinical development of TGF beta inhibition and TGF beta-related biomarkers to enhance immunotherapy efficacy.

Authors
Hanks, BA; Holtzhausen, A; Gimpel, P; Jamieson, R; Campbell, OM; Sun, L; Augustine, CK; Tyler, DS; Osada, T; Morse, M; Ling, LE; Lyerly, HK; Blobe, GC
MLA Citation
Hanks, BA, Holtzhausen, A, Gimpel, P, Jamieson, R, Campbell, OM, Sun, L, Augustine, CK, Tyler, DS, Osada, T, Morse, M, Ling, LE, Lyerly, HK, and Blobe, GC. "Effect of the loss of the type III TGF beta receptor during tumor progression on tumor microenvironment: Preclinical development of TGF beta inhibition and TGF beta-related biomarkers to enhance immunotherapy efficacy." May 20, 2012.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
30
Issue
15
Publish Date
2012

Effect of the vaccine Ad5 [E1-, E2b-]-CEA(6D) on CEA-directed CMI responses in patients with advanced CEA-expressing malignancies in a phase I/II clinical trial

Authors
Morse, M; Hobeika, A; Chaudhry, A; Amalfitano, A; Niedzwiecki, D; Clay, TM; Osada, T; Devi, G; Burnett, BK; Weinhold, K; Hsu, SD; Blobe, GC; Xu, Y; Nguyen, S; Dua, R; Balcaitis, S; Gabitzsch, E; Balint, J; Jones, F; Lyerly, HK
MLA Citation
Morse, M, Hobeika, A, Chaudhry, A, Amalfitano, A, Niedzwiecki, D, Clay, TM, Osada, T, Devi, G, Burnett, BK, Weinhold, K, Hsu, SD, Blobe, GC, Xu, Y, Nguyen, S, Dua, R, Balcaitis, S, Gabitzsch, E, Balint, J, Jones, F, and Lyerly, HK. "Effect of the vaccine Ad5 [E1-, E2b-]-CEA(6D) on CEA-directed CMI responses in patients with advanced CEA-expressing malignancies in a phase I/II clinical trial." May 20, 2012.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
30
Issue
15
Publish Date
2012

Immunodominant liver-specific expression suppresses transgene-directed immune responses in murine pompe disease.

Pompe disease can be treated effectively, if immune tolerance to enzyme replacement therapy (ERT) with acid α-glucosidase (GAA) is present. An adeno-associated viral (AAV) vector carrying a liver-specific regulatory cassette to drive GAA expression (AAV-LSPhGAA) established immune tolerance in GAA knockout (KO) mice, whereas ubiquitous expression with AAV-CBhGAA provoked immune responses. Therefore, we investigated the hypothesis that immune tolerance induced by hepatic-restricted expression was dominant. AAV-LSPhGAA and AAV-CBhGAA were administered singly or in combination to groups of adult GAA-KO mice, and AAV-LSPhGAA induced immune tolerance even in combination with AAV-CBhGAA. The dual vector approach to GAA expression improved biochemical correction of GAA deficiency and glycogen accumulations at 18 weeks, and improved motor function testing including wire-hang and grip-strength testing. The greatest efficacy was demonstrated by dual vector administration, when both vectors were pseudotyped as AAV8. T cells from mice injected with AAV-LSPhGAA failed to proliferate at all after an immune challenge with GAA and adjuvant, whereas mock-treated GAA-KO mice mounted vigorous T cell proliferation. Unlike AAV-LSPhGAA, AAV-CBhGAA induced selective cytokine and chemokine expression in liver and spleen after the immune challenge. AAV-CBhGAA transduced dendritic cells and expressed high-level GAA, whereas AAV-LSPhGAA failed to express GAA in dendritic cells. The level of transduction in liver was much higher after dual AAV8 vector administration at 18 weeks, in comparison with either vector alone. Dual vector administration failed to provoke antibody formation in response to GAA expression with AAV-CBhGAA; however, hepatic-restricted expression from dual vector expression did not prevent antibody formation after a strong immune challenge with GAA and adjuvant. The relevance of immune tolerance to gene therapy in Pompe disease indicates that hepatic expression might best be combined with nonhepatic expression, achieving the benefits of ubiquitous expression in addition to evading deleterious immune responses.

Authors
Zhang, P; Sun, B; Osada, T; Rodriguiz, R; Yang, XY; Luo, X; Kemper, AR; Clay, TM; Koeberl, DD
MLA Citation
Zhang, P, Sun, B, Osada, T, Rodriguiz, R, Yang, XY, Luo, X, Kemper, AR, Clay, TM, and Koeberl, DD. "Immunodominant liver-specific expression suppresses transgene-directed immune responses in murine pompe disease." Hum Gene Ther 23.5 (May 2012): 460-472.
Website
http://hdl.handle.net/10161/15089
PMID
22260439
Source
pubmed
Published In
Human Gene Therapy
Volume
23
Issue
5
Publish Date
2012
Start Page
460
End Page
472
DOI
10.1089/hum.2011.063

Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition [corrected].

BACKGROUND: Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. METHODS: We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. RESULTS: This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). CONCLUSIONS: dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. TRIAL REGISTRY: ClinicalTrials.gov NCT00952692.

Authors
Hamilton, E; Blackwell, K; Hobeika, AC; Clay, TM; Broadwater, G; Ren, X-R; Chen, W; Castro, H; Lehmann, F; Spector, N; Wei, J; Osada, T; Lyerly, HK; Morse, MA
MLA Citation
Hamilton, E, Blackwell, K, Hobeika, AC, Clay, TM, Broadwater, G, Ren, X-R, Chen, W, Castro, H, Lehmann, F, Spector, N, Wei, J, Osada, T, Lyerly, HK, and Morse, MA. "Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition [corrected]. (Published online)" J Transl Med 10 (February 10, 2012): 28-.
PMID
22325452
Source
pubmed
Published In
Journal of Translational Medicine
Volume
10
Publish Date
2012
Start Page
28
DOI
10.1186/1479-5876-10-28

Histological and molecular evaluation of patient-derived colorectal cancer explants.

Mouse models have been developed to investigate colorectal cancer etiology and evaluate new anti-cancer therapies. While genetically engineered and carcinogen-induced mouse models have provided important information with regard to the mechanisms underlying the oncogenic process, tumor xenograft models remain the standard for the evaluation of new chemotherapy and targeted drug treatments for clinical use. However, it remains unclear to what extent explanted colorectal tumor tissues retain inherent pathological features over time. In this study, we have generated a panel of 27 patient-derived colorectal cancer explants (PDCCEs) by direct transplantation of human colorectal cancer tissues into NOD-SCID mice. Using this panel, we performed a comparison of histology, gene expression and mutation status between PDCCEs and the original human tissues from which they were derived. Our findings demonstrate that PDCCEs maintain key histological features, basic gene expression patterns and KRAS/BRAF mutation status through multiple passages. Altogether, these findings suggest that PDCCEs maintain similarity to the patient tumor from which they are derived and may have the potential to serve as a reliable preclinical model that can be incorporated into future strategies to optimize individual therapy for patients with colorectal cancer.

Authors
Uronis, JM; Osada, T; McCall, S; Yang, XY; Mantyh, C; Morse, MA; Lyerly, HK; Clary, BM; Hsu, DS
MLA Citation
Uronis, JM, Osada, T, McCall, S, Yang, XY, Mantyh, C, Morse, MA, Lyerly, HK, Clary, BM, and Hsu, DS. "Histological and molecular evaluation of patient-derived colorectal cancer explants." PLoS One 7.6 (2012): e38422-.
PMID
22675560
Source
pubmed
Published In
PloS one
Volume
7
Issue
6
Publish Date
2012
Start Page
e38422
DOI
10.1371/journal.pone.0038422

Evidence for phenotypic plasticity in aggressive triple-negative breast cancer: human biology is recapitulated by a novel model system.

Breast cancers with a basal-like gene signature are primarily triple-negative, frequently metastatic, and carry a poor prognosis. Basal-like breast cancers are enriched for markers of breast cancer stem cells as well as markers of epithelial-mesenchymal transition (EMT). While EMT is generally thought to be important in the process of metastasis, in vivo evidence of EMT in human disease remains rare. Here we report a novel model of human triple-negative breast cancer, the DKAT cell line, which was isolated from an aggressive, treatment-resistant triple-negative breast cancer that demonstrated morphological and biochemical evidence suggestive of phenotypic plasticity in the patient. The DKAT cell line displays a basal-like phenotype in vitro when cultured in serum-free media, and undergoes phenotypic changes consistent with EMT/MET in response to serum-containing media, a unique property among the breast cancer cell lines we tested. This EMT is marked by increased expression of the transcription factor Zeb1, and Zeb1 is required for the enhanced migratory ability of DKAT cells in the mesenchymal state. DKAT cells also express progenitor-cell markers, and single DKAT cells are able to generate tumorspheres containing both epithelial and mesenchymal cell types. In vivo, as few as ten DKAT cells are capable of forming xenograft tumors which display a range of epithelial and mesenchymal phenotypes. The DKAT model provides a novel model to study the molecular mechanisms regulating phenotypic plasticity and the aggressive biology of triple-negative breast cancers.

Authors
D'Amato, NC; Ostrander, JH; Bowie, ML; Sistrunk, C; Borowsky, A; Cardiff, RD; Bell, K; Young, LJT; Simin, K; Bachelder, RE; Delrow, J; Dawson, A; Yee, LD; Mrózek, K; Clay, TM; Osada, T; Seewaldt, VL
MLA Citation
D'Amato, NC, Ostrander, JH, Bowie, ML, Sistrunk, C, Borowsky, A, Cardiff, RD, Bell, K, Young, LJT, Simin, K, Bachelder, RE, Delrow, J, Dawson, A, Yee, LD, Mrózek, K, Clay, TM, Osada, T, and Seewaldt, VL. "Evidence for phenotypic plasticity in aggressive triple-negative breast cancer: human biology is recapitulated by a novel model system." PLoS One 7.9 (2012): e45684-.
PMID
23049838
Source
pubmed
Published In
PloS one
Volume
7
Issue
9
Publish Date
2012
Start Page
e45684
DOI
10.1371/journal.pone.0045684

Increasing vaccine potency through exosome antigen targeting.

While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA "cancer vaccines" using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations.

Authors
Hartman, ZC; Wei, J; Glass, OK; Guo, H; Lei, G; Yang, X-Y; Osada, T; Hobeika, A; Delcayre, A; Le Pecq, J-B; Morse, MA; Clay, TM; Lyerly, HK
MLA Citation
Hartman, ZC, Wei, J, Glass, OK, Guo, H, Lei, G, Yang, X-Y, Osada, T, Hobeika, A, Delcayre, A, Le Pecq, J-B, Morse, MA, Clay, TM, and Lyerly, HK. "Increasing vaccine potency through exosome antigen targeting." Vaccine 29.50 (November 21, 2011): 9361-9367.
PMID
22001882
Source
pubmed
Published In
Vaccine
Volume
29
Issue
50
Publish Date
2011
Start Page
9361
End Page
9367
DOI
10.1016/j.vaccine.2011.09.133

Truncated ErbB2 expressed in tumor cell nuclei contributes to acquired therapeutic resistance to ErbB2 kinase inhibitors.

ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185(ErbB2)). In addition to p185(ErbB2), truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2(+) breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2(+) breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2(+) breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs.

Authors
Xia, W; Liu, Z; Zong, R; Liu, L; Zhao, S; Bacus, SS; Mao, Y; He, J; Wulfkuhle, JD; Petricoin, EF; Osada, T; Yang, X-Y; Hartman, ZC; Clay, TM; Blackwell, KL; Lyerly, HK; Spector, NL
MLA Citation
Xia, W, Liu, Z, Zong, R, Liu, L, Zhao, S, Bacus, SS, Mao, Y, He, J, Wulfkuhle, JD, Petricoin, EF, Osada, T, Yang, X-Y, Hartman, ZC, Clay, TM, Blackwell, KL, Lyerly, HK, and Spector, NL. "Truncated ErbB2 expressed in tumor cell nuclei contributes to acquired therapeutic resistance to ErbB2 kinase inhibitors." Mol Cancer Ther 10.8 (August 2011): 1367-1374.
PMID
21673090
Source
pubmed
Published In
Molecular cancer therapeutics
Volume
10
Issue
8
Publish Date
2011
Start Page
1367
End Page
1374
DOI
10.1158/1535-7163.MCT-10-0991

Phase I study utilizing a novel antigen-presenting cell-targeted vaccine with Toll-like receptor stimulation to induce immunity to self-antigens in cancer patients.

PURPOSE: The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self-antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity. EXPERIMENTAL DESIGN: Two phase I studies were conducted with CDX-1307, a vaccine composed of human chorionic gonadotropin beta-chain (hCG-β) fused to an MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose escalation of single-agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) and the Toll-like receptor (TLR) 3 agonist polyinosinic-polycytidylic acid (poly-ICLC) and TLR7/8 agonist resiquimod to activate the APC. RESULTS: CDX-1307 induced consistent humoral and T-cell responses to hCG-β when coadministered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and nonelevated levels of serum hCG-β. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions. CONCLUSIONS: APC targeting and activation induce adaptive immunity against poorly immunogenic self-antigens which has implications for enhancing the efficacy of cancer immunotherapy.

Authors
Morse, MA; Chapman, R; Powderly, J; Blackwell, K; Keler, T; Green, J; Riggs, R; He, L-Z; Ramakrishna, V; Vitale, L; Zhao, B; Butler, SA; Hobeika, A; Osada, T; Davis, T; Clay, T; Lyerly, HK
MLA Citation
Morse, MA, Chapman, R, Powderly, J, Blackwell, K, Keler, T, Green, J, Riggs, R, He, L-Z, Ramakrishna, V, Vitale, L, Zhao, B, Butler, SA, Hobeika, A, Osada, T, Davis, T, Clay, T, and Lyerly, HK. "Phase I study utilizing a novel antigen-presenting cell-targeted vaccine with Toll-like receptor stimulation to induce immunity to self-antigens in cancer patients." Clin Cancer Res 17.14 (July 15, 2011): 4844-4853.
PMID
21632857
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
17
Issue
14
Publish Date
2011
Start Page
4844
End Page
4853
DOI
10.1158/1078-0432.CCR-11-0891

HER2 overexpression elicits a proinflammatory IL-6 autocrine signaling loop that is critical for tumorigenesis.

HER2 overexpression occurs in approximately 25% of breast cancers, where it correlates with poor prognosis. Likewise, systemic inflammation in breast cancer correlates with poor prognosis, although the process is not understood. In this study, we explored the relationship between HER2 and inflammation, comparing the effects of overexpressing wild-type or mutated inactive forms of HER2 in primary human breast cells. Wild-type HER2 elicited a profound transcriptional inflammatory profile, including marked elevation of interleukin-6 (IL-6) expression, which we established to be a critical determinant of HER2 oncogenesis. Mechanistic investigations revealed that IL-6 secretion induced by HER2 overexpression activated Stat3 and altered gene expression, enforcing an autocrine loop of IL-6/Stat3 expression. Both mouse and human in vivo models of HER2-amplified breast carcinoma relied critically on this HER2-IL-6-Stat3 signaling pathway. Our studies offer the first direct evidence linking HER2 to a systemic inflammatory mechanism that orchestrates HER2-mediated tumor growth. We suggest that the HER2-IL-6-STAT3 signaling axis we have defined in breast cancer could prompt new therapeutic or prevention strategies for treatment of HER2-amplified cancers.

Authors
Hartman, ZC; Yang, X-Y; Glass, O; Lei, G; Osada, T; Dave, SS; Morse, MA; Clay, TM; Lyerly, HK
MLA Citation
Hartman, ZC, Yang, X-Y, Glass, O, Lei, G, Osada, T, Dave, SS, Morse, MA, Clay, TM, and Lyerly, HK. "HER2 overexpression elicits a proinflammatory IL-6 autocrine signaling loop that is critical for tumorigenesis." Cancer Res 71.13 (July 1, 2011): 4380-4391.
PMID
21518778
Source
pubmed
Published In
Cancer Research
Volume
71
Issue
13
Publish Date
2011
Start Page
4380
End Page
4391
DOI
10.1158/0008-5472.CAN-11-0308

Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations.

Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

Authors
Osada, T; Chen, M; Yang, XY; Spasojevic, I; Vandeusen, JB; Hsu, D; Clary, BM; Clay, TM; Chen, W; Morse, MA; Lyerly, HK
MLA Citation
Osada, T, Chen, M, Yang, XY, Spasojevic, I, Vandeusen, JB, Hsu, D, Clary, BM, Clay, TM, Chen, W, Morse, MA, and Lyerly, HK. "Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations." Cancer Res 71.12 (June 15, 2011): 4172-4182.
PMID
21531761
Source
pubmed
Published In
Cancer Research
Volume
71
Issue
12
Publish Date
2011
Start Page
4172
End Page
4182
DOI
10.1158/0008-5472.CAN-10-3978

Survival rates among patients vaccinated following resection of colorectal cancer metastases in a phase II randomized study compared with contemporary controls

Authors
Morse, M; Niedzwiecki, D; Marshall, J; Garrett, CR; Chang, DZ; Aklilu, M; Crocenzi, TS; Cole, DJ; Dessureault, S; Hobeika, A; Osada, T; Clary, BM; Hsu, SD; Devi, G; Bulusu, A; Annechiarico, R; Chadaram, V; Clay, TM; Lyerly, HK
MLA Citation
Morse, M, Niedzwiecki, D, Marshall, J, Garrett, CR, Chang, DZ, Aklilu, M, Crocenzi, TS, Cole, DJ, Dessureault, S, Hobeika, A, Osada, T, Clary, BM, Hsu, SD, Devi, G, Bulusu, A, Annechiarico, R, Chadaram, V, Clay, TM, and Lyerly, HK. "Survival rates among patients vaccinated following resection of colorectal cancer metastases in a phase II randomized study compared with contemporary controls." May 20, 2011.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
29
Issue
15
Publish Date
2011

A dendritic cell-based vaccine effects on T-cell responses compared with a viral vector vaccine when administered to patients following resection of colorectal metastases in a randomized phase II study.

Authors
Lyerly, HK; Hobeika, A; Niedzwiecki, D; Osada, T; Marshall, J; Garrett, CR; Chang, DZ; Aklilu, M; Crocenzi, TS; Cole, DJ; Dessureault, S; Hsu, SD; Bulusu, A; Clary, BM; Annechiarico, R; Devi, G; Chadaram, V; Clay, TM; Morse, M
MLA Citation
Lyerly, HK, Hobeika, A, Niedzwiecki, D, Osada, T, Marshall, J, Garrett, CR, Chang, DZ, Aklilu, M, Crocenzi, TS, Cole, DJ, Dessureault, S, Hsu, SD, Bulusu, A, Clary, BM, Annechiarico, R, Devi, G, Chadaram, V, Clay, TM, and Morse, M. "A dendritic cell-based vaccine effects on T-cell responses compared with a viral vector vaccine when administered to patients following resection of colorectal metastases in a randomized phase II study." May 20, 2011.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
29
Issue
15
Publish Date
2011

MHC class I-presented tumor antigens identified in ovarian cancer by immunoproteomic analysis are targets for T-cell responses against breast and ovarian cancer.

PURPOSE: The purpose of this study is to test whether peptide epitopes chosen from among those naturally processed and overpresented within MHC molecules by malignant, but not normal cells, when formulated into cancer vaccines, could activate antitumor T-cell responses in humans. EXPERIMENTAL DESIGN: Mixtures of human leukocyte antigen A2 (HLA-A2)-binding ovarian cancer-associated peptides were used to activate naive T cells to generate antigen-specific T cells that could recognize ovarian and breast cancers in vitro. Combinations of these peptides (0.3 mg of each peptide or 1 mg of each peptide) were formulated into vaccines in conjunction with Montanide ISA-51 and granulocyte monocyte colony stimulating factor which were used to vaccinate patients with ovarian and breast cancer without evidence of clinical disease in parallel pilot clinical trials. RESULTS: T cells specific for individual peptides could be generated in vitro by using mixtures of peptides, and these T cells recognized ovarian and breast cancers but not nonmalignant cells. Patient vaccinations were well tolerated with the exception of local erythema and induration at the injection site. Nine of the 14 vaccinated patients responded immunologically to their vaccine by inducing peptide-specific T-cell responses that were capable of recognizing HLA-matched breast and ovarian cancer cells. CONCLUSION: Mixtures of specific peptides identified as naturally presented on cancer cells and capable of activating tumor-specific T cells in vitro also initiate or augment immune responses toward solid tumors in cancer patients.

Authors
Morse, MA; Secord, AA; Blackwell, K; Hobeika, AC; Sinnathamby, G; Osada, T; Hafner, J; Philip, M; Clay, TM; Lyerly, HK; Philip, R
MLA Citation
Morse, MA, Secord, AA, Blackwell, K, Hobeika, AC, Sinnathamby, G, Osada, T, Hafner, J, Philip, M, Clay, TM, Lyerly, HK, and Philip, R. "MHC class I-presented tumor antigens identified in ovarian cancer by immunoproteomic analysis are targets for T-cell responses against breast and ovarian cancer." Clin Cancer Res 17.10 (May 15, 2011): 3408-3419.
PMID
21300761
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
17
Issue
10
Publish Date
2011
Start Page
3408
End Page
3419
DOI
10.1158/1078-0432.CCR-10-2614

A dendritic cell-based vaccine effects on T-cell responses compared with a viral vector vaccine when administered to patients following resection of colorectal metastases in a randomized phase II study.

2533 Background: CD8+ T cell responses to colorectal cancer are associated with longer survival. This has led to the hypothesis that cancer vaccines, capable of activating T cell responses, may improve clinical outcome. Vaccines based on antigen-presenting cells/dendritic cells (DC) and viral vectors, potent stimulators of adaptive immunity, have been associated with enhanced survival in prostate cancer patients. We compared rates of tumor antigen-specific T cell and antibody responses between a DC and a poxvector vaccine. The clinical outcome data is reported elsewhere.74 patients with no evidence of disease after colorectal cancer metastasectomy and completion of peri-operative chemotherapy were randomized 1:1 to receive injections of one of either: DC mixed with PANVAC-VF (poxvectors encoding CEA, MUC1, CD54, CD58, CD80) or PANVAC-VF along with local injections of GM-CSF. Peripheral blood was drawn before and after completing the immunizations for analysis of CEA and MUC-1 immune (T cell and antibody) responses by ELISPOT and ELISA.T cell responses against CEA were significantly more frequent in the DC arm (69 versus 41%, p=0.02) although the magnitude of the T cell response among responders was similar. There was a trend for improved relapse-free survival among patients with CEA-specific T cell responses (log rank p = 0.10). The antibody response to CEA was more frequent with the PANVAC alone (100% versus 67%, (p= 0.018)) and the antibodies in serum from vaccinated patients could bind to CEA-expressing tumor cells and mediated ADCC. No antibody response was induced against MUC-1. The antibody response against CEA did not correlate with clinical benefit. Few deaths were observed limiting comparison of survival by immune response.A dendritic cell vaccine leads to a greater frequency of CEA-specific T cell responses which is associated with enhanced RFS. Ongoing studies are evaluating the role of additional immunostimulatory cytokines and modulation of regulatory cell populations and molecules in enhancing the adaptive immune response to the DC-based vaccine.

Authors
Lyerly, HK; Hobeika, A; Niedzwiecki, D; Osada, T; Marshall, J; Garrett, CR; Chang, DZ; Aklilu, M; Crocenzi, TS; Cole, DJ; Dessureault, S; Hsu, SD; Bulusu, A; Clary, BM; Annechiarico, R; Devi, G; Chadaram, V; Clay, TM; Morse, M
MLA Citation
Lyerly, HK, Hobeika, A, Niedzwiecki, D, Osada, T, Marshall, J, Garrett, CR, Chang, DZ, Aklilu, M, Crocenzi, TS, Cole, DJ, Dessureault, S, Hsu, SD, Bulusu, A, Clary, BM, Annechiarico, R, Devi, G, Chadaram, V, Clay, TM, and Morse, M. "A dendritic cell-based vaccine effects on T-cell responses compared with a viral vector vaccine when administered to patients following resection of colorectal metastases in a randomized phase II study." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29.15_suppl (May 2011): 2533-.
PMID
28022328
Source
epmc
Published In
Journal of Clinical Oncology
Volume
29
Issue
15_suppl
Publish Date
2011
Start Page
2533

Survival rates among patients vaccinated following resection of colorectal cancer metastases in a phase II randomized study compared with contemporary controls.

3557 Background: Patients with completely resected metastases from colorectal cancer (CRC) remain at high risk of recurrence and death despite adjuvant chemotherapy. Recently, survival of prostate cancer patients was enhanced by antigen-presenting cell therapy. We investigated whether administration of an antigen-presenting cell vaccine based on dendritic cells (DC) after metastasectomy would reduce the risk of recurrence and increase survival.Patients (n=74) with no evidence of disease after resection of CRC metastases and completion of their physician-determined peri-operative chemotherapy were randomized 1:1 to four immunizations with: DC modified with the PANVAC-VF poxvectors encoding CEA, MUC1, CD54, CD58, and CD80 or the PANVAC-VF poxvectors along with GM-CSF at the injection site. We report recurrence-free survival (RFS) at 2 years and overall survival (OS). CEA specific T cell responses were measured by ELISPOT. Data from a prospectively registered, comparable, contemporary control group of patients who had undergone metastasectomy for CRC were also available.The arms of the study and contemporary controls were well balanced. The majority of the toxicities for the DC and PANVAC arms respectively were grade 1, 2 injection site reactions (63% versus 64%), low grade fevers (17% vs 31%), myalgia (11% vs 11%), and fatigue (26% vs 34%). The two year RFS was similar in all groups (50, 56 and 55% for the DC arm, the PANVAC arm and the contemporary control group, respectively). However, there was a trend for improved RFS among patients with CEA-specific T cell responses (log rank p = 0.10). At a median follow-up of 40 months, 2 of 37 patients treated with DC and 5 of 37 treated with PANVAC alone have died, with a combined survival rate exceeding that of the unvaccinated control patients.Patients vaccinated after metastasectomy experienced a longer survival relative to contemporary controls. A phase III study of OS comparing patients vaccinated after resection with the DC vaccine and observation is warranted.

Authors
Morse, M; Niedzwiecki, D; Marshall, J; Garrett, CR; Chang, DZ; Aklilu, M; Crocenzi, TS; Cole, DJ; Dessureault, S; Hobeika, A; Osada, T; Clary, BM; Hsu, SD; Devi, G; Bulusu, A; Annechiarico, R; Chadaram, V; Clay, TM; Lyerly, HK
MLA Citation
Morse, M, Niedzwiecki, D, Marshall, J, Garrett, CR, Chang, DZ, Aklilu, M, Crocenzi, TS, Cole, DJ, Dessureault, S, Hobeika, A, Osada, T, Clary, BM, Hsu, SD, Devi, G, Bulusu, A, Annechiarico, R, Chadaram, V, Clay, TM, and Lyerly, HK. "Survival rates among patients vaccinated following resection of colorectal cancer metastases in a phase II randomized study compared with contemporary controls." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29.15_suppl (May 2011): 3557-.
PMID
28020295
Source
epmc
Published In
Journal of Clinical Oncology
Volume
29
Issue
15_suppl
Publish Date
2011
Start Page
3557

Polyclonal immune responses to antigens associated with cancer signaling pathways and new strategies to enhance cancer vaccines.

Aberrant signaling pathways are a hallmark of cancer. A variety of strategies for inhibiting signaling pathways have been developed, but monoclonal antibodies against receptor tyrosine kinases have been among the most successful. A challenge for these therapies is therapeutic unresponsiveness and acquired resistance due to mutations in the receptors, upregulation of alternate growth and survival pathways, or inadequate function of the monoclonal antibodies. Vaccines are able to induce polyclonal responses that can have a multitude of affects against the target molecule. We began to explore therapeutic vaccine development to antigens associated with these signaling pathways. We provide an illustrative example in developing therapeutic cancer vaccines inducing polyclonal adaptive immune responses targeting the ErbB family member HER2. Further, we will discuss new strategies to augment the clinical efficacy of cancer vaccines by enhancing vaccine immunogenicity and reversing the immunosuppressive tumor microenvironment.

Authors
Clay, TM; Osada, T; Hartman, ZC; Hobeika, A; Devi, G; Morse, MA; Lyerly, HK
MLA Citation
Clay, TM, Osada, T, Hartman, ZC, Hobeika, A, Devi, G, Morse, MA, and Lyerly, HK. "Polyclonal immune responses to antigens associated with cancer signaling pathways and new strategies to enhance cancer vaccines." Immunol Res 49.1-3 (April 2011): 235-247.
PMID
21136201
Source
pubmed
Published In
Immunologic Research
Volume
49
Issue
1-3
Publish Date
2011
Start Page
235
End Page
247
DOI
10.1007/s12026-010-8186-6

Development of an assay to predict oxaliplatin sensitivity from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues

Authors
Freedman, JA; Osada, T; Tsamis, KA; Morse, M; Clary, BM; Lyerly, HK; Nevins, JR; Clay, TM; Hsu, SD
MLA Citation
Freedman, JA, Osada, T, Tsamis, KA, Morse, M, Clary, BM, Lyerly, HK, Nevins, JR, Clay, TM, and Hsu, SD. "Development of an assay to predict oxaliplatin sensitivity from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues." February 1, 2011.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
29
Issue
4
Publish Date
2011

Development of an assay to predict oxaliplatin sensitivity from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues.

429 Background: Genomic profiling has improved our understanding of the underlying biology of tumors, accuracy of diagnosing disease, predictions of the courses of disease, and ability to determine the therapeutic agents that will be most effective in the treatment of particular tumors. However, in order for assays involving microarray data to be useful in the clinical setting, the ability to generate reliable and consistent data from FFPE tissues is essential.Cancer cell lines from the NCI-60 collection exhibiting greatest sensitivity or resistance to oxaliplatin were identified. These cells were grown in culture, fixed for 24 hours in formalin, and paraffin-embedded. RNA from the FFPE cells was isolated, amplified, and hybridized to Affymetrix arrays. A Bayesian binary regression analysis was used to generate a predictor of oxaliplatin sensitivity from the gene expression data. Metastatic derived xenografts (MDXs) from resected colorectal tumors were established and treated with oxaliplatin. Samples from tumors prior to treatment were paraffin-embedded and used for RNA extraction, amplification, and hybridization to Affymetrix arrays. The gene expression signature predicting sensitivity to oxaliplatin was then validated with response data from MDXs treated with oxaliplatin.A predictor consisting of 300 genes that could predict sensitivity to oxaliplatin was generated using FFPE samples. Significant correlation was observed between the predicted probability of oxaliplatin sensitivity and the tumor growth inhibition measurement for a given MDX (p=0.0012).Reliable and consistent predictions of oxaliplatin sensitivity can be obtained from gene expression data from FFPE tissues. This method has potential utility in the clinical setting. The ability to predict response to a therapeutic in a FFPE sample has the potential to guide the choice of therapeutics, resulting in an option that could be most effective in treating an individual with metastatic colorectal cancer. No significant financial relationships to disclose.

Authors
Freedman, JA; Osada, T; Tsamis, KA; Morse, M; Clary, BM; Lyerly, HK; Nevins, JR; Clay, TM; Hsu, SD
MLA Citation
Freedman, JA, Osada, T, Tsamis, KA, Morse, M, Clary, BM, Lyerly, HK, Nevins, JR, Clay, TM, and Hsu, SD. "Development of an assay to predict oxaliplatin sensitivity from formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29.4_suppl (February 2011): 429-.
PMID
27985824
Source
epmc
Published In
Journal of Clinical Oncology
Volume
29
Issue
4_suppl
Publish Date
2011
Start Page
429

Depletion of human regulatory T cells.

Regulatory T cells (Treg) have become increasingly relevant in the study of human disease including cancer. Treg cells have been shown to inhibit anti-tumor immune responses, and elevated Treg levels have been associated with certain types of cancer. Similarly, depletion of Tregs by various methods can also enhance anti-tumor immune responses. We have found a prevalence of Treg in cancer patients when compared to normal volunteers. In addition, we have shown that the depletion of Treg using the IL-2 fusion protein denileukin diftitox decreased Treg function and increased antigen-specific T cell response to a cancer vaccine. These results indicate the potential for combining Treg depletion with anti-cancer vaccines to enhance tumor antigen-specific immune responses and the need to explore the dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

Authors
Hobeika, AC; Morse, MA; Osada, T; Peplinski, S; Lyerly, HK; Clay, TM
MLA Citation
Hobeika, AC, Morse, MA, Osada, T, Peplinski, S, Lyerly, HK, and Clay, TM. "Depletion of human regulatory T cells." Methods Mol Biol 707 (2011): 219-231.
PMID
21287338
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
707
Publish Date
2011
Start Page
219
End Page
231
DOI
10.1007/978-1-61737-979-6_14

CSPG4 protein as a new target for the antibody-based immunotherapy of triple-negative breast cancer.

BACKGROUND: The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)-based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb. METHODS: CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided. RESULTS: CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell-derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm(2); difference of mean = 121360.0 μm(2), 95% confidence interval = 91010.7 to 151709.4 μm(2); P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis. CONCLUSIONS: This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.

Authors
Wang, X; Osada, T; Wang, Y; Yu, L; Sakakura, K; Katayama, A; McCarthy, JB; Brufsky, A; Chivukula, M; Khoury, T; Hsu, DS; Barry, WT; Lyerly, HK; Clay, TM; Ferrone, S
MLA Citation
Wang, X, Osada, T, Wang, Y, Yu, L, Sakakura, K, Katayama, A, McCarthy, JB, Brufsky, A, Chivukula, M, Khoury, T, Hsu, DS, Barry, WT, Lyerly, HK, Clay, TM, and Ferrone, S. "CSPG4 protein as a new target for the antibody-based immunotherapy of triple-negative breast cancer." J Natl Cancer Inst 102.19 (October 6, 2010): 1496-1512.
PMID
20852124
Source
pubmed
Published In
Journal of the National Cancer Institute
Volume
102
Issue
19
Publish Date
2010
Start Page
1496
End Page
1512
DOI
10.1093/jnci/djq343

Ligand-independent toll-like receptor signals generated by ectopic overexpression of MyD88 generate local and systemic antitumor immunity.

Although critical for initiating and regulating immune responses, the therapeutic use of individual cytokines as anticancer immunotherapeutic agents has achieved only modest clinical success. Consequently, many current strategies have focused on the use of specific immunotherapeutic agonists that engage individual receptors of innate immune networks, such as the Toll-like receptor (TLR) system, each resulting in specific patterns of gene expression, cytokine production, and inflammatory outcome. However, these immunotherapeutics are constrained by variable cellular TLR expression and responsiveness to particular TLR agonists, as well as the specific cellular context of different tumors. We hypothesized that overexpression of MyD88, a pivotal regulator of multiple TLR signaling pathways, could circumvent these constraints and mimic coordinated TLR signaling across all cell types in a ligand-independent fashion. To explore this hypothesis, we generated an adenoviral vector expressing MyD88 and show that Ad-MyD88 infection elicits extensive Th1-specific transcriptional and secreted cytokine signatures in all murine and human cell types tested in vitro and in vivo. Importantly, in vivo intratumoral injection of Ad-MyD88 into established tumor masses enhanced adaptive immune responses and inhibited local tumor immunosuppression, resulting in significantly inhibited local and systemic growth of multiple tumor types. Finally, Ad-MyD88 infection of primary human dendritic cells, tumor-associated fibroblasts, and colorectal carcinoma cells elicited significant Th1-type cytokine responses, resulting in enhanced tumor cell lysis and expansion of human tumor antigen-specific T cells. Thus, Ad-MyD88 initiated robust antitumor activity in established murine tumor microenvironments and in human contexts, suggesting its potential effectiveness as a clinical immunotherapeutic strategy.

Authors
Hartman, ZC; Osada, T; Glass, O; Yang, XY; Lei, G-J; Lyerly, HK; Clay, TM
MLA Citation
Hartman, ZC, Osada, T, Glass, O, Yang, XY, Lei, G-J, Lyerly, HK, and Clay, TM. "Ligand-independent toll-like receptor signals generated by ectopic overexpression of MyD88 generate local and systemic antitumor immunity." Cancer Res 70.18 (September 15, 2010): 7209-7220.
PMID
20823152
Source
pubmed
Published In
Cancer Research
Volume
70
Issue
18
Publish Date
2010
Start Page
7209
End Page
7220
DOI
10.1158/0008-5472.CAN-10-0905

An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer.

Therapeutic anticancer vaccines are designed to boost patients' immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression.

Authors
Morse, MA; Hobeika, AC; Osada, T; Berglund, P; Hubby, B; Negri, S; Niedzwiecki, D; Devi, GR; Burnett, BK; Clay, TM; Smith, J; Lyerly, HK
MLA Citation
Morse, MA, Hobeika, AC, Osada, T, Berglund, P, Hubby, B, Negri, S, Niedzwiecki, D, Devi, GR, Burnett, BK, Clay, TM, Smith, J, and Lyerly, HK. "An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer." J Clin Invest 120.9 (September 2010): 3234-3241.
Website
http://hdl.handle.net/10161/4330
PMID
20679728
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
120
Issue
9
Publish Date
2010
Start Page
3234
End Page
3241
DOI
10.1172/JCI42672

Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo.

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.

Authors
Morse, MA; Wei, J; Hartman, Z; Xia, W; Ren, X-R; Lei, G; Barry, WT; Osada, T; Hobeika, AC; Peplinski, S; Jiang, H; Devi, GR; Chen, W; Spector, N; Amalfitano, A; Lyerly, HK; Clay, TM
MLA Citation
Morse, MA, Wei, J, Hartman, Z, Xia, W, Ren, X-R, Lei, G, Barry, WT, Osada, T, Hobeika, AC, Peplinski, S, Jiang, H, Devi, GR, Chen, W, Spector, N, Amalfitano, A, Lyerly, HK, and Clay, TM. "Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo." Int J Cancer 126.12 (June 15, 2010): 2893-2903.
PMID
19856307
Source
pubmed
Published In
International Journal of Cancer
Volume
126
Issue
12
Publish Date
2010
Start Page
2893
End Page
2903
DOI
10.1002/ijc.24995

Adenovirus vaccine immunotherapy targeting WT1-expressing tumors.

IMPORTANCE OF THE FIELD: Tumor associated antigens (TAAs) offer specific targets for developing cancer immunotherapies. In particular, viral vectors encoding transgenic TAAs have been used in recent vaccination strategies. Wilm's Tumor gene (WT1) is a robust TAA which is overexpressed in many malignancies and has been recently used to develop a novel recombinant adenovirus (Ad-WT1) for antitumor immunotherapy. AREAS COVERED IN THIS REVIEW: The lines of evidence over the past two decades leading to the development of Ad-WT1 immunotherapy are reviewed, including preclinical studies and clinical trials using WT1-based vaccines and TAA-expressing adenoviral vectors for antitumor therapy. WHAT THE READER WILL GAIN: The fundamental immunogenic properties of WT1-based vaccines are detailed, as well as the recent progress in using adenoviral vectors for eliciting a TAA-specific immune response. The reader will also gain an understanding of the evidence supporting Ad-WT1 antitumor therapy in vivo. TAKE HOME MESSAGE: Ad-WT1 elicits a potent CD4(+) and CD8(+) T cell immune response and can effectively inhibit tumor growth in vivo, thus making it an important potential cancer therapy worthy of future investigation.

Authors
Clarke, JM; Morse, MA; Lyerly, HK; Clay, T; Osada, T
MLA Citation
Clarke, JM, Morse, MA, Lyerly, HK, Clay, T, and Osada, T. "Adenovirus vaccine immunotherapy targeting WT1-expressing tumors." Expert Opin Biol Ther 10.6 (June 2010): 875-883. (Review)
PMID
20380487
Source
pubmed
Published In
Expert Opinion on Biological Therapy
Volume
10
Issue
6
Publish Date
2010
Start Page
875
End Page
883
DOI
10.1517/14712591003798278

Use of gene expression signatures to predict in vivo sensitivity of human metastatic colorectal cancer to chemotherapy and to identify novel drug combinations.

Authors
VanDeusen, JB; Osada, T; Morse, M; Clary, BM; Lyerly, HK; Nevins, JR; Clay, TM; Febbo, PG; Hsu, SD
MLA Citation
VanDeusen, JB, Osada, T, Morse, M, Clary, BM, Lyerly, HK, Nevins, JR, Clay, TM, Febbo, PG, and Hsu, SD. "Use of gene expression signatures to predict in vivo sensitivity of human metastatic colorectal cancer to chemotherapy and to identify novel drug combinations." May 20, 2010.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
28
Issue
15
Publish Date
2010

Effect of a novel recombinant alphaviral vector on tolerance to self-antigen in the setting of elevated regulatory T cells.

Authors
Morse, M; Hobeika, A; Osada, T; Berglund, P; Negri, S; Niedzwiecki, D; Hubby, B; Burnett, B; Clay, TM; Lyerly, HK
MLA Citation
Morse, M, Hobeika, A, Osada, T, Berglund, P, Negri, S, Niedzwiecki, D, Hubby, B, Burnett, B, Clay, TM, and Lyerly, HK. "Effect of a novel recombinant alphaviral vector on tolerance to self-antigen in the setting of elevated regulatory T cells." May 20, 2010.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
28
Issue
15
Publish Date
2010

Abstract 5338: Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T cell killing mediated by CEA/CD3-bispecific T cell-engaging BiTE antibody

Authors
Osada, T; Hsu, D; Hammond, S; Hobeika, A; Devi, G; Clay, TM; Lyerly, HK; Morse, MA
MLA Citation
Osada, T, Hsu, D, Hammond, S, Hobeika, A, Devi, G, Clay, TM, Lyerly, HK, and Morse, MA. "Abstract 5338: Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T cell killing mediated by CEA/CD3-bispecific T cell-engaging BiTE antibody." Cancer Research 70.8 Supplement (April 15, 2010): 5338-5338.
Source
crossref
Published In
Cancer Research
Volume
70
Issue
8 Supplement
Publish Date
2010
Start Page
5338
End Page
5338
DOI
10.1158/1538-7445.AM10-5338

An adenoviral vaccine encoding full-length inactivated human Her2 exhibits potent immunogenicty and enhanced therapeutic efficacy without oncogenicity.

PURPOSE: Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in immunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. EXPERIMENTAL DESIGN: We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. RESULTS: Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. CONCLUSIONS: Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials.

Authors
Hartman, ZC; Wei, J; Osada, T; Glass, O; Lei, G; Yang, X-Y; Peplinski, S; Kim, D-W; Xia, W; Spector, N; Marks, J; Barry, W; Hobeika, A; Devi, G; Amalfitano, A; Morse, MA; Lyerly, HK; Clay, TM
MLA Citation
Hartman, ZC, Wei, J, Osada, T, Glass, O, Lei, G, Yang, X-Y, Peplinski, S, Kim, D-W, Xia, W, Spector, N, Marks, J, Barry, W, Hobeika, A, Devi, G, Amalfitano, A, Morse, MA, Lyerly, HK, and Clay, TM. "An adenoviral vaccine encoding full-length inactivated human Her2 exhibits potent immunogenicty and enhanced therapeutic efficacy without oncogenicity." Clin Cancer Res 16.5 (March 1, 2010): 1466-1477.
PMID
20179231
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
16
Issue
5
Publish Date
2010
Start Page
1466
End Page
1477
DOI
10.1158/1078-0432.CCR-09-2549

Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T-cell killing mediated by CEA/CD3-bispecific T-cell-engaging BiTE antibody.

BACKGROUND: Novel technologies to redirect T-cell killing against cancer cells are emerging. We hypothesised that metastatic human colorectal cancer (CRC) previously treated with conventional chemotherapy would be sensitive to T-cell killing mediated by carcinoembryonic antigen (CEA)/CD3-bispecific T-cell-engaging BiTE antibody (MEDI-565). METHODS: We analysed proliferation and lysis of CEA-positive (CEA+) CRC specimens that had survived previous systemic chemotherapy and biologic therapy to determine whether they could be killed by patient T cells engaged by MEDI-565 in vitro. RESULTS: At low concentrations (0.1-1 ng ml(-1)), MEDI-565+ T cells caused reduced proliferation and enhanced apoptosis of CEA+ human CRC specimens. High levels of soluble CEA did not impair killing by redirected T cells and there was no increase in resistance to T-cell killing despite multiple rounds of exposure. CONCLUSIONS: This study shows for the first time that metastatic CRC specimens derived from patients previously treated with conventional chemotherapy can be lysed by patient T cells. Clinical testing of cancer immunotherapies, such as MEDI-565 that result in exposure of tumours to large numbers of T cells, is warranted.

Authors
Osada, T; Hsu, D; Hammond, S; Hobeika, A; Devi, G; Clay, TM; Lyerly, HK; Morse, MA
MLA Citation
Osada, T, Hsu, D, Hammond, S, Hobeika, A, Devi, G, Clay, TM, Lyerly, HK, and Morse, MA. "Metastatic colorectal cancer cells from patients previously treated with chemotherapy are sensitive to T-cell killing mediated by CEA/CD3-bispecific T-cell-engaging BiTE antibody." Br J Cancer 102.1 (January 5, 2010): 124-133.
PMID
19953093
Source
pubmed
Published In
British Journal of Cancer
Volume
102
Issue
1
Publish Date
2010
Start Page
124
End Page
133
DOI
10.1038/sj.bjc.6605364

Molecular characterization of putative chordoma cell lines.

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Authors
Brüderlein, S; Sommer, JB; Meltzer, PS; Li, S; Osada, T; Ng, D; Möller, P; Alcorta, DA; Kelley, MJ
MLA Citation
Brüderlein, S, Sommer, JB, Meltzer, PS, Li, S, Osada, T, Ng, D, Möller, P, Alcorta, DA, and Kelley, MJ. "Molecular characterization of putative chordoma cell lines." Sarcoma 2010 (2010): 630129-.
PMID
21253487
Source
pubmed
Published In
Sarcoma
Volume
2010
Publish Date
2010
Start Page
630129
DOI
10.1155/2010/630129

Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity.

Recombinant serotype 5 adenovirus (Ad5) vectors lacking E1 expression induce robust immune responses against encoded transgenes in pre-clinical models, but have muted responses in human trials because of widespread pre-existing anti-adenovirus immunity. Attempts to circumvent Ad5-specific immunity by using alternative serotypes or modifying capsid components have not yielded profound clinical improvement. To address this issue, we explored a novel alternative strategy, specifically reducing the expression of structural Ad5 genes by creating E1 and E2b deleted recombinant Ad5 vectors. Our data show that [E1-, E2b-]vectors retaining the Ad5 serotype are potent immunogens in pre-clinical models despite the presence of significant Ad5-specific immunity, in contrast to [E1-] vectors. These pre-clinical studies with E1 and E2b-deleted recombinant Ad5 vectors suggest that anti-Ad immunity will no longer be a limiting factor, and that clinical trials to evaluate their performance are warranted.

Authors
Osada, T; Yang, XY; Hartman, ZC; Glass, O; Hodges, BL; Niedzwiecki, D; Morse, MA; Lyerly, HK; Amalfitano, A; Clay, TM
MLA Citation
Osada, T, Yang, XY, Hartman, ZC, Glass, O, Hodges, BL, Niedzwiecki, D, Morse, MA, Lyerly, HK, Amalfitano, A, and Clay, TM. "Optimization of vaccine responses with an E1, E2b and E3-deleted Ad5 vector circumvents pre-existing anti-vector immunity." Cancer Gene Ther 16.9 (September 2009): 673-682.
PMID
19229288
Source
pubmed
Published In
Cancer Gene Therapy
Volume
16
Issue
9
Publish Date
2009
Start Page
673
End Page
682
DOI
10.1038/cgt.2009.17

Physiology and therapeutics of vascular endothelial growth factor in tumor immunosuppression.

Vascular endothelial growth factor (VEGF), known as a primary mediator of tumor-induced angiogenesis, is now understood to have a role in tumor-associated immunosuppression. Initially, VEGF was identified to alter the growth and maturation of the immature granulocyte-macrophage progenitors, and more recently it has been noted that it prevents dendritic cell (DC) precursors from developing into mature, antigen-presenting DC. VEGF is associated with recruitment of macrophages to the tumor stroma and VEGF inhibition of myeloid progenitor maturation is associated with the development tumor associated macrophages (TAM) which possess immunosuppressive capacity as well. Therapies intended to inhibit VEGF or VEGF receptors have demonstrated improved anti-tumor immunity and enhanced responses to cancer vaccines.

Authors
Johnson, B; Osada, T; Clay, T; Lyerly, H; Morse, M
MLA Citation
Johnson, B, Osada, T, Clay, T, Lyerly, H, and Morse, M. "Physiology and therapeutics of vascular endothelial growth factor in tumor immunosuppression." Curr Mol Med 9.6 (August 2009): 702-707. (Review)
PMID
19689297
Source
pubmed
Published In
Current molecular medicine
Volume
9
Issue
6
Publish Date
2009
Start Page
702
End Page
707

Induction of Wilms' tumor protein (WT1)-specific antitumor immunity using a truncated WT1-expressing adenovirus vaccine.

PURPOSE: Wilms' tumor protein (WT1) is overexpressed in most leukemias and many solid tumors and is a promising target for tumor immunotherapy. WT1 peptide-based cancer vaccines have been reported but have limited application due to HLA restriction of the peptides. We sought to vaccinate using adenoviral (Ad) vectors encoding tumor-associated antigens such as WT1 that can stimulate tumor-associated antigen-specific immunity across a broad array of HLA types and multiple class I and class II epitopes. EXPERIMENTAL DESIGN: We developed a novel Ad vector encoding a truncated version of WT1 (Ad-tWT1) lacking the highly conserved COOH terminus zinc finger domains and tested its ability to stimulate WT1-specific immune responses and antitumor immunity in two murine models of WT1-expressing tumors. RESULTS: Despite encoding a transcription factor, we found that Ad-tWT1-transduced murine and human dendritic cells showed cytoplasmic expression of the truncated WT1 protein. In addition, vaccination of C57BL/6 mice with Ad-tWT1 generated WT1-specific cell-mediated and humoral immune responses and conferred protection against challenge with the leukemia cell line, mWT1-C1498. Moreover, in a tumor therapy model, Ad-tWT1 vaccination of TRAMP-C2 tumor-bearing mice significantly suppressed tumor growth. CONCLUSIONS: This is the first report of a WT1-encoding Ad vector that is capable of inducing effective immunity against WT1-expressing malignancies. Based on these findings, Ad-tWT1 warrants investigation in human clinical trials to evaluate its applications as a vaccine for patients with WT1-expressing cancers.

Authors
Osada, T; Woo, CY; McKinney, M; Yang, XY; Lei, G; Labreche, HG; Hartman, ZC; Niedzwiecki, D; Chao, N; Amalfitano, A; Morse, MA; Lyerly, HK; Clay, TM
MLA Citation
Osada, T, Woo, CY, McKinney, M, Yang, XY, Lei, G, Labreche, HG, Hartman, ZC, Niedzwiecki, D, Chao, N, Amalfitano, A, Morse, MA, Lyerly, HK, and Clay, TM. "Induction of Wilms' tumor protein (WT1)-specific antitumor immunity using a truncated WT1-expressing adenovirus vaccine." Clin Cancer Res 15.8 (April 15, 2009): 2789-2796.
PMID
19351755
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
15
Issue
8
Publish Date
2009
Start Page
2789
End Page
2796
DOI
10.1158/1078-0432.CCR-08-2589

Antibody-based immunotherapy: targeting CSPG4 on human basal breast cancer stem cells

Authors
Wang, X; Osada, T; Wang, Y; Miyazato, P; Thorne, S; DeLeo, A; Lyerly, HK; Clay, T; Ferrone, S
MLA Citation
Wang, X, Osada, T, Wang, Y, Miyazato, P, Thorne, S, DeLeo, A, Lyerly, HK, Clay, T, and Ferrone, S. "Antibody-based immunotherapy: targeting CSPG4 on human basal breast cancer stem cells." January 15, 2009.
Source
wos-lite
Published In
Cancer Research
Volume
69
Issue
2
Publish Date
2009
Start Page
314S
End Page
315S

Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines.

CD4(+)CD25(high)FoxP3(+) regulatory T (Treg) cells limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. In preclinical and clinical studies, we assessed the immune consequences of FoxP3(+) Treg-cell depletion in patients with advanced malignancies. We demonstrated that a CD25(high) targeting immunotoxin (denileukin diftitox) depleted FoxP3(+) Treg cells, decreased Treg-cell function, and enhanced antigen-specific T-cell responses in vitro. We then attempted to enhance antitumor immune responses in patients with carcinoembryonic antigen (CEA)-expressing malignancies by Treg-cell depletion. In a pilot study (n = 15), denileukin diftitox, given as a single dose or repeated dosing, was followed by immunizations with dendritic cells modified with the fowlpox vector rF-CEA(6D)-TRICOM. By flow cytometric analysis, we report the first direct evidence that circulating CD4(+)CD25(high)FoxP3(+) Treg cells are depleted after multiple doses of denileukin diftitox. Earlier induction of, and overall greater exposure to, the T-cell response to CEA was observed in the multiple-dose group, but not the single-dose group. These results indicate the potential for combining Treg-cell depletion with anticancer vaccines to enhance tumor antigen-specific immune responses and the need to explore dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

Authors
Morse, MA; Hobeika, AC; Osada, T; Serra, D; Niedzwiecki, D; Lyerly, HK; Clay, TM
MLA Citation
Morse, MA, Hobeika, AC, Osada, T, Serra, D, Niedzwiecki, D, Lyerly, HK, and Clay, TM. "Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines." Blood 112.3 (August 1, 2008): 610-618.
PMID
18519811
Source
pubmed
Published In
Blood
Volume
112
Issue
3
Publish Date
2008
Start Page
610
End Page
618
DOI
10.1182/blood-2008-01-135319

The effect of anti-VEGF therapy on immature myeloid cell and dendritic cells in cancer patients.

Impairment of dendritic cells (DC), the most effective activators of anticancer immune responses, is one mechanism for defective antitumor immunity, but the causes of DC impairment are incompletely understood. We evaluated the association of impaired DC differentiation with angiogenesis-associated molecules D-dimer, vascular endothelial growth factor (VEGF), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor (PAI-1) in peripheral blood from 41 patients with lung, breast, and colorectal carcinoma. Subsequently, we studied the effect of administration of the anti-VEGF antibody (bevacizumab) on DC maturation and function in vivo. Compared with healthy volunteers, cancer patients had a bias toward the immunoregulatory DC2, had deficits in DC maturation after overnight in vitro culture, and had a significant increase in immature myeloid cell progenitors of DC (0.50 +/- 0.31% vs. 0.32 +/- 0.16% of peripheral blood mononuclear cells, respectively, P = 0.011). A positive correlation was found between the percentage of DC2 and PAI-1 (R = 0.50) and between immature myeloid cells and VEGF (R = 0.52). Bevacizumab administration to cancer patients was associated with a decrease in the accumulation of immature progenitor cells (0.39 +/- 0.30% vs. 0.27 +/- 0.24%, P = 0.012) and induced a modest increase in the DC population in peripheral blood (0.47 +/- 0.23% vs. 0.53 +/- 0.30%). Moreover, anti-VEGF antibody treatment enhanced allo-stimulatory capacity of DC and T cell proliferation against recall antigens. These data suggest that DC differentiation is negatively associated with VEGF levels and may be one explanation for impaired anticancer immunity, especially in patients with advanced malignancies.

Authors
Osada, T; Chong, G; Tansik, R; Hong, T; Spector, N; Kumar, R; Hurwitz, HI; Dev, I; Nixon, AB; Lyerly, HK; Clay, T; Morse, MA
MLA Citation
Osada, T, Chong, G, Tansik, R, Hong, T, Spector, N, Kumar, R, Hurwitz, HI, Dev, I, Nixon, AB, Lyerly, HK, Clay, T, and Morse, MA. "The effect of anti-VEGF therapy on immature myeloid cell and dendritic cells in cancer patients." Cancer Immunol Immunother 57.8 (August 2008): 1115-1124.
PMID
18193223
Source
pubmed
Published In
Cancer Immunology, Immunotherapy
Volume
57
Issue
8
Publish Date
2008
Start Page
1115
End Page
1124
DOI
10.1007/s00262-007-0441-x

Depletion of human regulatory T cells (Treg) and antigen-specific immune responses to cancer vaccines

Authors
Clay, TM; Hobeika, A; Osada, T; Serra, D; Niedzwiecki, D; Lyerly, HK; Morse, MA
MLA Citation
Clay, TM, Hobeika, A, Osada, T, Serra, D, Niedzwiecki, D, Lyerly, HK, and Morse, MA. "Depletion of human regulatory T cells (Treg) and antigen-specific immune responses to cancer vaccines." May 20, 2008.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
26
Issue
15
Publish Date
2008

Depletion of human regulatory T cells (Treg) and antigen-specific immune responses to cancer vaccines.

3010 Background: CD4+CD25+FoxP3+ regulatory T cells (Treg) limit antigen-specific immune responses and are a cause of suppressed anticancer immunity. Conversely, depletion of Treg leads to immune enhancement. The immunotoxin denileukin diftitox which selectively targets lymphocytes expressing CD25 may deplete FoxP3+ Treg.We evaluated the proliferative potential of PBMC to various antigens in vitro following exposure to denileukin diftitox. We then performed a pilot study in which patients with advanced CEA expressing malignancies, being immunized with autologous dendritic cells modified with a fowlpox vector encoding CEA (rF-CEA(6D)-TRICOM), received denileukin diftitox 18 mcg/kg, once, 4 days before the immunizations began, or 9 mcg/kg prior to each of the 4 immunizations. ELISPOT, cytokine flow cytometry, and ELISA were used to measure the T cell and antibody response.In vitro, escalating doses of denileukin diftitox depleted FoxP3+ Treg, decreased Treg function in vitro, and enhanced antigen-specific T cell responses. In the pilot study (n=15), denileukin diftitox was associated with a 74 ± 6% decrease in Treg in those receiving multiple doses, but not in those receiving a single dose. An earlier peak in the vaccine-induced CEA-specific T cell responses, and significant levels (>0.5%) of circulating CD8+ and CD4+ CEA-specific T cells were also seen in the multiple dose group. Conversely, a single dose of denileukin diftitox enhanced anti-CEA, but not antifowlpox vector, antibody responses. Multiple doses abolished the anti-CEA antibody response.These results indicate the potential for combining Treg depletion with anticancer vaccines to enhance tumor antigen specific immune responses. No significant financial relationships to disclose.

Authors
Clay, TM; Hobeika, A; Osada, T; Serra, D; Niedzwiecki, D; Lyerly, HK; Morse, MA
MLA Citation
Clay, TM, Hobeika, A, Osada, T, Serra, D, Niedzwiecki, D, Lyerly, HK, and Morse, MA. "Depletion of human regulatory T cells (Treg) and antigen-specific immune responses to cancer vaccines." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 26.15_suppl (May 2008): 3010-.
PMID
27947631
Source
epmc
Published In
Journal of Clinical Oncology
Volume
26
Issue
15_suppl
Publish Date
2008
Start Page
3010

Apoptosis induction by p38 MAPK inhibitor in human colon cancer cells.

BACKGROUND/AIMS: The p38 mitogen-activated protein kinases (p38 MAPKs) function in a wide variety of signaling pathways. However, the role of p38s is cell type- and stimulus-dependent. The present study aimed to evaluate the effects of p38 MAPK inhibitor on human colon cancer cells. METHODOLOGY: The effect of p38 MAPK inhibitor, FR167653, on DLD-1 and SW480 was investigated related to cell proliferation, apoptosis induction and caspase activity. Additionally, the effect of FR167653 on colon cancer cell migration, MMPs production and ability to adhere to extracellular matrix was investigated. RESULTS: Inhibitor of p38 MAPK dose-dependently suppressed the proliferative activity of both cell lines, and increased the induction of cell apoptosis. The caspase-3, 8, and 9 activities were accompanied in the pathway. Neither cell migration, MMPs production, nor the ability to adhere extracellular matrix were affected by FR167653. CONCLUSIONS: Inhibitor of p38 MAPK suppressed the proliferation of colon cancer cells by induction of cell apoptosis through the caspase activation. The present results suggest the pro-oncogenic role ofp38 in colon cancer, and its inhibition would be a novel strategy for the prevention and treatment of colon cancer.

Authors
Tsuchiya, T; Tsuno, NH; Asakage, M; Yamada, J; Yoneyama, S; Okaji, Y; Sasaki, S; Kitayama, J; Osada, T; Takahashi, K; Nagawa, H
MLA Citation
Tsuchiya, T, Tsuno, NH, Asakage, M, Yamada, J, Yoneyama, S, Okaji, Y, Sasaki, S, Kitayama, J, Osada, T, Takahashi, K, and Nagawa, H. "Apoptosis induction by p38 MAPK inhibitor in human colon cancer cells." Hepatogastroenterology 55.84 (May 2008): 930-935.
PMID
18705300
Source
pubmed
Published In
Hepato-gastroenterology
Volume
55
Issue
84
Publish Date
2008
Start Page
930
End Page
935

Epigallocatechin gallate affects human dendritic cell differentiation and maturation.

BACKGROUND: Epigallocatechin gallate (EGCG), a component of green tea catechin with the strongest biological activity, has been focused in recent years because of its anti-inflammatory and immunomodulatory activities. Dendritic cells (DCs) are professional antigen-presenting cells, capable of priming naive T cells, and play the key roles in the activation of T-cell-mediated immune responses. OBJECTIVE: We aimed to investigate the effect of EGCG on human monocyte-derived DCs (MODCs) and, consequently, on the T-cell-mediated immune response. METHODS: The induction of apoptosis, and the detailed phenotypic and functional changes of MODCs, generated by culture of peripheral blood monocytes in the presence of GM-CSF and IL-4, induced by EGCG was investigated and compared with the effects of dexamethasone. RESULTS: Epigallocatechin gallate induced apoptosis and affected the phenotype of the developing DCs. The expressions of CD83, CD80, CD11c, and MHC class II, which are molecules essential for antigen presentation by DCs, were downregulated by EGCG. EGCG also suppressed the endocytotic ability of immature DCs, whereas dexamethasone-treated DCs had higher endocytotic ability than control DCs. Most importantly, mature DCs treated with EGCG inhibited stimulatory activity toward allogeneic T cells while secreting high amounts of IL-10. CONCLUSION: Epigallocatechin gallate induces immunosuppressive alterations on human MODCs, both by induction of apoptosis and suppression of cell surface molecules and antigen presentation.

Authors
Yoneyama, S; Kawai, K; Tsuno, NH; Okaji, Y; Asakage, M; Tsuchiya, T; Yamada, J; Sunami, E; Osada, T; Kitayama, J; Takahashi, K; Nagawa, H
MLA Citation
Yoneyama, S, Kawai, K, Tsuno, NH, Okaji, Y, Asakage, M, Tsuchiya, T, Yamada, J, Sunami, E, Osada, T, Kitayama, J, Takahashi, K, and Nagawa, H. "Epigallocatechin gallate affects human dendritic cell differentiation and maturation." J Allergy Clin Immunol 121.1 (January 2008): 209-214.
PMID
17935769
Source
pubmed
Published In
Journal of Allergy and Clinical Immunology
Volume
121
Issue
1
Publish Date
2008
Start Page
209
End Page
214
DOI
10.1016/j.jaci.2007.08.026

Detailed analysis of cytomegalovirus (CMV)-specific T cells expanded for adoptive immunotherapy of CMV infection following allogeneic stem cell transplantation for malignant disease.

BACKGROUND: Cytomegalovirus (CMV) infection and its treatment causes significant morbidity following allogeneic stem cell transplantation (SCT) for malignancies. We studied the phenotype, function and growth kinetics of CMV pp65 antigen (Ag)-specific T cells expanded in a short-term culture for adoptive therapy. METHODS: Peripheral blood mononuclear cells (PBMC) from CMV-seropositive donors were cultured in various conditions with CMV pp65((495-503)) peptide to determine the most effective method for generating CMV-specific T cells. CMV-expanded cultures were tested for frequency, phenotype and functionality using peptide-MHC tetramer analysis, cytokine flow cytometry and cytolytic assays. A patient undergoing allogeneic SCT was administered CMV pp65-specific T cells generated from the donor based on these data, and recipient PBMC were analyzed following T-cell infusion. RESULTS: CMV pp65-specific T cells were consistently generated from CMV-seropositive donors at high frequencies (20-40% of CD8+ T cells), secreted interferon-gamma (IFN-gamma) in response to CMV peptide and had lytic activity against CMV peptide-expressing targets. Cultured CMV-specific T cells were infused into a SCT recipient without toxicity. DISCUSSION: Stimulating donor PBMC to generate functional, Ag-specific T cells for infusion into SCT recipients was accomplished consistently using readily available technology. We observed no toxicity in one patient receiving T cells and were able to monitor infused cells. These findings support further study of this approach as a prophylaxis against the risk of infection in patients receiving allogeneic transplantation from CMV-seropositive donors.

Authors
Hobeika, A; Osada, T; Serra, D; Peplinski, S; Hanson, K; Tanaka, Y; Niedzwiecki, D; Chao, N; Rizzieri, D; Lyerly, H; Clay, T; Morse, M
MLA Citation
Hobeika, A, Osada, T, Serra, D, Peplinski, S, Hanson, K, Tanaka, Y, Niedzwiecki, D, Chao, N, Rizzieri, D, Lyerly, H, Clay, T, and Morse, M. "Detailed analysis of cytomegalovirus (CMV)-specific T cells expanded for adoptive immunotherapy of CMV infection following allogeneic stem cell transplantation for malignant disease." Cytotherapy 10.3 (2008): 289-302.
PMID
18418774
Source
pubmed
Published In
Cytotherapy (Informa)
Volume
10
Issue
3
Publish Date
2008
Start Page
289
End Page
302
DOI
10.1080/14653240801927040

A study of dendritic and endothelial cell interactions in colon cancer in a cell line and small mammal model.

AIM: Historically, cancer therapy directly targeting tumor cells have yielded suboptimal clinical results, and therefore anti-angiogenic therapy that targets tumor cells indirectly through impairing tumor vasculature is now considered to be one of the novel approaches potentially effective against various types of cancer. In this study, we evaluated whether lysates of endothelium could be effectively pulsed in dendritic cells (DCs), to enhance their anti-tumor effects. METHODS: For this purpose, we prepared DCs of BALB/c mouse, incubated them with lysates of autologous or xenogeneic endothelium, and tested their anti-tumor effects in two syngeneic models of colon cancer. RESULTS: DCs pulsed with the respective endothelium lysates significantly inhibited the growth of subcutaneous tumors as well as pulmonary metastases in mice, and their anti-tumor effect was superior to that of unpulsed DCs. Immunohistopathological analysis showed significant decrease in the mean vascular density of tumors, correlating well with the extent of tumor inhibition. In vitro analysis of splenocytes isolated from immunized mice revealed an induction of cytotoxic T lymphocytes and activation of natural killer cells, with a lytic activity against activated endothelium but not tumor cells. In addition, antibodies reacting with activated endothelium, but not tumor cells, were detected in murine sera by ELISA, and their function was confirmed by complement-dependent cytotoxicity assay. CONCLUSIONS: Our present results suggest that lysates of endothelium can be effectively pulsed in DCs and enhance their anti-tumor effects through induction of anti-angiogenesis, and therefore should have important clinical implications for adjuvant cancer therapy.

Authors
Yoneyama, S; Okaji, Y; Tsuno, NH; Kawai, K; Yamashita, H; Tsuchiya, T; Yamada, J; Sunami, E; Osada, T; Kitayama, J; Takahashi, K; Nagawa, H
MLA Citation
Yoneyama, S, Okaji, Y, Tsuno, NH, Kawai, K, Yamashita, H, Tsuchiya, T, Yamada, J, Sunami, E, Osada, T, Kitayama, J, Takahashi, K, and Nagawa, H. "A study of dendritic and endothelial cell interactions in colon cancer in a cell line and small mammal model." Eur J Surg Oncol 33.10 (December 2007): 1191-1198.
PMID
17314028
Source
pubmed
Published In
European Journal of Surgical Oncology
Volume
33
Issue
10
Publish Date
2007
Start Page
1191
End Page
1198
DOI
10.1016/j.ejso.2007.01.013

Long term disease-free survival and T cell and antibody responses in women with high-risk Her2+ breast cancer following vaccination against Her2.

BACKGROUND: The HER2-inhibiting antibody trastuzumab, in combination with chemotherapy, significantly improves survival of women with resected, HER2-overexpressing breast cancers, but is associated with toxicities including a risk of cardiomyopathy. Additionally, the beneficial effect of trastuzumab is expected to decrease once the drug is discontinued. We proposed to address these concerns by using cancer vaccines to stimulate HER2 intracellular domain (ICD)-specific T cell and antibody responses. METHODS: Subjects with stage II (> or = 6 +LN), III, or stage IV breast cancer with > 50% HER2 overexpressing tumor cells who were disease-free after surgery and adjuvant therapy were eligible. Vaccines consisted of immature, cultured DC (n = 3), mature cultured DC (n = 3), or mature Flt3-ligand mobilized peripheral blood DC (n = 1) loaded with ICD, or tetanus toxoid, keyhole limpet hemocyanin or CMV peptide as controls, and were administered intradermally/subcutaneously four times at 3 week intervals. ICD-specific T cell and antibody responses were measured. Cardiac function was determined by MUGA or ECHO; long term disease status was obtained from patient contact. RESULTS: All seven patients successfully underwent DC generation and five received all 4 immunizations. There were no toxicities greater than grade 1 or ejection fraction decrements below normal. Delayed-type hypersensitivity (DTH) reactions at the injection site occurred in 6/7 patients and HER2 specificity was detected by cytokine flow cytometry or ELISPOT in 5 patients. At more than 5 years of follow-up, 6/7 had detectable anti-ICD antibodies. One patient experienced a pulmonary recurrence at 4 years from their study immunizations. This recurrence was resected and they are without evidence of disease. All patients are alive and disease-free at 4.6-6.7 years of follow-up. CONCLUSION: Although this was a small pilot study, the well-tolerated nature of the vaccines, the lack of cardiac toxicity, significant immunogenicity, and a 100% 4.5-year survival rate suggest that vaccination with HER2 ICD protein-containing DC is appropriate for further study in this population. TRIAL REGISTRATION: ClinicalTrials.gov NCT00005956.

Authors
Morse, MA; Hobeika, A; Osada, T; Niedzwiecki, D; Marcom, PK; Blackwell, KL; Anders, C; Devi, GR; Lyerly, HK; Clay, TM
MLA Citation
Morse, MA, Hobeika, A, Osada, T, Niedzwiecki, D, Marcom, PK, Blackwell, KL, Anders, C, Devi, GR, Lyerly, HK, and Clay, TM. "Long term disease-free survival and T cell and antibody responses in women with high-risk Her2+ breast cancer following vaccination against Her2. (Published online)" J Transl Med 5 (September 6, 2007): 42-.
PMID
17822557
Source
pubmed
Published In
Journal of Translational Medicine
Volume
5
Publish Date
2007
Start Page
42
DOI
10.1186/1479-5876-5-42

Adenovirus-human HER2 vaccine inhibits breast cancer growth and vaccine induced antibodies (VIA) are efficacious against Herceptin-refractory human breast cancer

Authors
Wei, J; Hartman, Z; Osada, T; Yang, XY; Lei, G; Jiang, H; Chong, G; Amalfitano, A; Lyerly, HK; Morse, M; Clay, T
MLA Citation
Wei, J, Hartman, Z, Osada, T, Yang, XY, Lei, G, Jiang, H, Chong, G, Amalfitano, A, Lyerly, HK, Morse, M, and Clay, T. "Adenovirus-human HER2 vaccine inhibits breast cancer growth and vaccine induced antibodies (VIA) are efficacious against Herceptin-refractory human breast cancer." JOURNAL OF IMMUNOLOGY 178 (April 1, 2007).
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
178
Publish Date
2007

Immunotherapeutic targeting of Wilms' tumor protein.

The expression of Wilms' tumor protein (WT1)-derived peptides on malignant cell surfaces and recognition of those peptides by cellular and humoral immune responses suggest that WT1 may be a promising potential target antigen in immunotherapeutic trials. With a high frequency of expression in hematopoietic as well as solid tumors, WT1 is a broadly applicable target. Both in vivo mouse model and in vitro human studies have demonstrated the ability of WT1-specific cytotoxic T-lymphocytes to lyse WT1-expressing malignancies without harming normal tissue. WT1-peptide vaccination, in combination with adjuvants, has demonstrated the ability to activate WT1-specific immune responses and evidence of clinical activity. Because peptide-based vaccines are human leukocyte antigen-restricted, other more broadly applicable strategies are now being developed to activate WT1-specific immune responses, including the use of WT1-specific viral vectors.

Authors
Hutchings, Y; Osada, T; Woo, CY; Clay, TM; Lyerly, HK; Morse, MA
MLA Citation
Hutchings, Y, Osada, T, Woo, CY, Clay, TM, Lyerly, HK, and Morse, MA. "Immunotherapeutic targeting of Wilms' tumor protein." Curr Opin Mol Ther 9.1 (February 2007): 62-69. (Review)
PMID
17330403
Source
pubmed
Published In
Current Opinion in Molecular Therapeutics
Volume
9
Issue
1
Publish Date
2007
Start Page
62
End Page
69

Natural killer cell activation and dendritic cell-based vaccines

Natural killer (NK) cells are the key players of the innate immune system, which can immediately limit or eliminate dangerous challenges by pathogens or tumor cells to the host. Recent studies have demonstrated the reciprocal activation of NK cell and dendritic cell (DC) through NK-DC interactions, elucidating the functional links between these two cell lineages. More details in NK-DC interactions, such as subsets of cells, molecular pathways involved and the possible anatomical sites, have been investigated and reported. Murine experiments have demonstrated that injection of mature DCs induces rapid recruitment of NK cells to lymph nodes, and that these NK cells provide interferon-γ(IFN-γ) for type 1 (Th1) priming of T cells. Thus, an increasing body of in vivo evidence is indicating that NK-DC interactions during the early phase of innate immunity can impact the quality and the magnitude of the subsequent adaptive immune response. Importantly, these studies imply that NK cells might not serve merely as cytotoxic effector cells combating virally-infected cells and malignant tumors, but might also play an important role as immunoregulatory cells with a significant influence on adaptive immunity. However, there is a relative paucity of information from the clinical side regarding NK cell function in adaptive immunity, as few DC vaccine studies have attempted to evaluate the antigen-nonspecific, yet potentially clinically-relevant, NK response to immunization. In this article, we will review studies focusing on NK-DC interactions and highlight the most recent clinical findings relating to the potential role of NK cells in DC-based vaccine therapy.

Authors
Osada, T; Clay, TM; Woo, CY; Morse, MA; Lyerly, HK
MLA Citation
Osada, T, Clay, TM, Woo, CY, Morse, MA, and Lyerly, HK. "Natural killer cell activation and dendritic cell-based vaccines." Minerva Biotecnologica 19.3 (2007): 91-104.
Source
scival
Published In
Minerva Biotecnologica
Volume
19
Issue
3
Publish Date
2007
Start Page
91
End Page
104

Recent clinical progress in virus-based therapies for cancer.

As our knowledge of the molecular basis of cancer expands, viral vectors have been increasingly studied as potential antitumour therapeutic agents. With their ability to invade and replicate within target cells, viruses have been utilised as oncolytic agents to directly lyse tumour cells. Viruses can also deliver their genetic payload into infected cells, allowing for the repair of defective tumour suppressor genes, disruption of oncogenic pathways, and production of cytokines that activate the immune system. Finally, viruses encoding tumour-associated antigens can infect dendritic cells, triggering the development of a tumour-specific immune response. The ability to engineer viruses with high levels of tumour specificity and efficient rates of infection has enhanced the safety profile of these agents, allowing for the development of viable therapeutic options that have been examined in the clinic, either alone or in conjunction with more conventional therapies. This review highlights the principles underlying virus-based therapies for cancer, with an emphasis on recent developments from the clinic.

Authors
Woo, CY; Osada, T; Clay, TM; Lyerly, HK; Morse, MA
MLA Citation
Woo, CY, Osada, T, Clay, TM, Lyerly, HK, and Morse, MA. "Recent clinical progress in virus-based therapies for cancer." Expert Opin Biol Ther 6.11 (November 2006): 1123-1134. (Review)
PMID
17049011
Source
pubmed
Published In
Expert Opinion on Biological Therapy
Volume
6
Issue
11
Publish Date
2006
Start Page
1123
End Page
1134
DOI
10.1517/14712598.6.11.1123

Dendritic cell-based immunotherapy.

Dendritic cells (DCs) play a crucial role in the induction of antigen-specific T-cell responses, and therefore their use for the active immunotherapy of malignancies has been studied with considerable interest. More than a decade has passed since the publication of the first clinical data of DC-based vaccines, and through this and subsequent studies, a number of important developmental insights have been gleaned. These include the ideal source and type of DCs, the discovery of novel antigens and methods of loading DCs, the role of DC maturation, and the most efficient route of immunization. The generation of immune responses against tumor antigens after DC immunization has been demonstrated, and favorable clinical responses have been reported in some patients; however, it is difficult to pool the results as a whole, and thus the body of data remains inconclusive, in part because of varying DC preparation and vaccination protocols, the use of different forms of antigens, and, most importantly, a lack of rigorous criteria for defining clinical responses. As such, the standardization of clinical and immunologic criteria utilized, as well as DC preparations employed, will allow for the comparison of results across multiple clinical studies and is required in order for future trials to measure the true value and role of this treatment modality. In addition, issues regarding the optimal dose and clinical setting for the application of DC vaccines remain to be resolved, and recent clinical studies have been designed to begin to address these questions.

Authors
Osada, T; Clay, TM; Woo, CY; Morse, MA; Lyerly, HK
MLA Citation
Osada, T, Clay, TM, Woo, CY, Morse, MA, and Lyerly, HK. "Dendritic cell-based immunotherapy." Int Rev Immunol 25.5-6 (September 2006): 377-413. (Review)
PMID
17169781
Source
pubmed
Published In
International Reviews of Immunology (Informa)
Volume
25
Issue
5-6
Publish Date
2006
Start Page
377
End Page
413
DOI
10.1080/08830180600992456

NK cell activation by dendritic cell vaccine: a mechanism of action for clinical activity.

Recent reports revealed that dendritic cell (DC)-natural killer (NK) cell interaction plays an important role in tumor immunity, but few DC vaccine studies have attempted to evaluate the non-specific, yet potentially clinically relevant, NK response to immunization. In this study, we first analyzed in vitro activation of NK cells by DCs similar to those used in clinical trials. Subsequently, NK cell responses were analyzed in a phase I clinical trial of a vaccine consisting of autologous DCs loaded with a fowlpox vector encoding CEA. The data were compared with the clinical outcome of the patients. DC enhances NK activity in vitro, partly by sustaining NK cell survival and by enhancing the expression of NK-activating receptors, including NKp46 and NKG2D. Among nine patients in our clinical trial, NK cytolytic activity increased in four (range 2.5-5 times greater lytic activity) including three who had increased NK cell frequency, was stable in two and decreased in three. NKp46 and NKG2D expression showed a good correlation with the patients' NK activity. When patients were grouped by clinical activity (stable disease/no evidence of disease (stable/NE, n=5) vs progressive disease (N=4) at 3 months), the majority in the stable/NE group had increases in NK activity (P=0.016). Anti-CEA T cell response was enhanced in all the nine patients analyzed, but was not significantly different between the two groups (P=0.14). Thus, NK responses following DC vaccination may correlate more closely with clinical outcome than do T cell responses. Monitoring of NK response during vaccine studies should be routinely performed.

Authors
Osada, T; Clay, T; Hobeika, A; Lyerly, HK; Morse, MA
MLA Citation
Osada, T, Clay, T, Hobeika, A, Lyerly, HK, and Morse, MA. "NK cell activation by dendritic cell vaccine: a mechanism of action for clinical activity." Cancer Immunol Immunother 55.9 (September 2006): 1122-1131.
PMID
16273350
Source
pubmed
Published In
Cancer Immunology, Immunotherapy
Volume
55
Issue
9
Publish Date
2006
Start Page
1122
End Page
1131
DOI
10.1007/s00262-005-0089-3

Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells.

Endothelial progenitor cells (EPCs) have been recently found to exist circulating in peripheral blood of adults, and home to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells (PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Few reports exist in the literature concerning the characteristics of EPCs, especially related to their surface antigen expressions, except for endothelial markers. Here, we aimed to investigate the surface expression of differentiation markers, and the functional activities of early-outgrowth of EPCs (EO-EPCs), especially focusing on their antigen-presenting ability. EO-EPCs were generated from PBMNCs, by culture in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and, additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a mixed-lymphocyte reaction, and could generate cytotoxic lymphocytes, with the ability to lyze tumor cells in an antigen-specific manner. The antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity against angiogenic vessels can be expected. In conclusion, EO-EPCs should be an interesting alternative for the development of new therapeutic strategies to combat cancer, either as the effectors or as the targets of cancer immunotherapy.

Authors
Asakage, M; Tsuno, NH; Kitayama, J; Kawai, K; Okaji, Y; Yazawa, K; Kaisaki, S; Osada, T; Watanabe, T; Takahashi, K; Nagawa, H
MLA Citation
Asakage, M, Tsuno, NH, Kitayama, J, Kawai, K, Okaji, Y, Yazawa, K, Kaisaki, S, Osada, T, Watanabe, T, Takahashi, K, and Nagawa, H. "Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells." Cancer Immunol Immunother 55.6 (June 2006): 708-716.
PMID
16133110
Source
pubmed
Published In
Cancer Immunology, Immunotherapy
Volume
55
Issue
6
Publish Date
2006
Start Page
708
End Page
716
DOI
10.1007/s00262-005-0057-y

Role of natural killer cell function in dendritic cell-based vaccines.

Recent studies have elucidated the functional links between natural killer (NK) cells and, demonstrating the reciprocal activation of these cell types through NK-DC interactions. The subsets of cells and molecular pathways involved in such interactions have been defined, and the possible anatomical sites of these interactions have also been reported. Murine experiments have demonstrated that injection of mature DCs induces rapid recruitment of NK cells to lymph nodes and that these NK cells provide interferon-gamma for Type 1 priming. Thus, there is an increasing body of in vivo evidence indicating that NK-DC interactions during the early phase of innate immunity can impact the quality and magnitude of the subsequent adaptive immune response. Importantly, these studies imply that NK cells might not serve merely as cytotoxic lymphocytes combating viral pathogens and malignant tumors, but must also be considered as important immunoregulatory cells with a significant influence on adaptive immunity. In contrast to the large volume of knowledge obtained through basic research, there is a relative paucity of information regarding NK cell function in adaptive immunity from clinical trials, as few DC vaccine studies have attempted to evaluate the nonspecific, yet potentially clinically relevant, NK response to immunization. In this article, the authors will review studies focusing on NK-DC interactions and highlight the most recent clinical findings relating to the potential role of NK cells in DC-based vaccine therapy.

Authors
Woo, CY; Clay, TM; Lyerly, HK; Morse, MA; Osada, T
MLA Citation
Woo, CY, Clay, TM, Lyerly, HK, Morse, MA, and Osada, T. "Role of natural killer cell function in dendritic cell-based vaccines." Expert Rev Vaccines 5.1 (February 2006): 55-65. (Review)
PMID
16451108
Source
pubmed
Published In
Expert Review of Vaccines
Volume
5
Issue
1
Publish Date
2006
Start Page
55
End Page
65
DOI
10.1586/14760584.5.1.55

Immunization with fowlpox vector-modified dendritic cell in patients with carcinoembryonic antigen-expressing cancer: Phase I clinical study

A dendritic cell (DC), which can induce a primary immune response, is known to be the most potent antigen-presenting cell in the immune system. Many studies have been performed to evaluate the safety and efficacy of DC-based immunotherapy. At Duke University Medical Center, various methods of tumor antigen-loading to DC (i.e., peptide, protein, mRNA, virus vectors) were tried in patients with carcinoembryonic antigen (CEA)-expressing colorectal/lung cancers, HER2-expressing breast cancers, or prostate specific antigen (PSA)-expressing prostate cancers. In this article, we report the results of our latest study, a phase I clinical study of immunization with DCs modified with fowlpox vector encoding carcinoembryonic antigen and costimulatory molecules. We administered one or two cycles of four triweekly subcutaneous/intradermal injections of CEA-expressing DCs. Among the 14 patients enrolled (11 with colorectal cancer, 3 with non-small cell lung cancer), 12 patients completed at least one cycle of immunization. There were no immunization-related grade 3/4 toxicities. One patient showed a decrease in the CEA level and regression of the lymphadenopathy that occurred several months after completion of immunization. Five other patients were stable through at least one cycle of immunization. Immune monitoring assays showed induction of CEA-specific immune response in all immune responders. Thus, this vaccine strategy is safe, induces potent CEA-specific immune responses, and warrants further study.

Authors
Osada, T; Morse, MA
MLA Citation
Osada, T, and Morse, MA. "Immunization with fowlpox vector-modified dendritic cell in patients with carcinoembryonic antigen-expressing cancer: Phase I clinical study." Biotherapy 20.6 (2006): 541-548.
Source
scival
Published In
Biotherapy
Volume
20
Issue
6
Publish Date
2006
Start Page
541
End Page
548

Sulforaphane induces inhibition of human umbilical vein endothelial cells proliferation by apoptosis.

Sulforaphane (SUL), one of the isothiocyanates (ITCs), has recently been focused due to its inhibitory effects on tumor cell growth in vitro and in vivo, which is dependent on the direct effect on cancer cells. In the present study, we aimed to investigate the potential anti-angiogenic effect of SUL and its mechanism of action. Using the human umbilical vein endothelial cells (HUVECs) as a model of angiogenesis, we investigated the effect of SUL on the various steps of angiogenesis, including the proliferation of endothelial cells, tubular formation, and matrix metalloproteinase (MMP) production. Sulforaphane induced a dose-dependent decrease in the proliferative activity of endothelial cells, which was dependent on cell apoptosis. Also SUL inhibited tube formation on matrigel, but did not affect MMP production. The present results demonstrate the anti-angiogenic activity of SUL and its potential use as an anti-cancer drug is suggested.

Authors
Asakage, M; Tsuno, NH; Kitayama, J; Tsuchiya, T; Yoneyama, S; Yamada, J; Okaji, Y; Kaisaki, S; Osada, T; Takahashi, K; Nagawa, H
MLA Citation
Asakage, M, Tsuno, NH, Kitayama, J, Tsuchiya, T, Yoneyama, S, Yamada, J, Okaji, Y, Kaisaki, S, Osada, T, Takahashi, K, and Nagawa, H. "Sulforaphane induces inhibition of human umbilical vein endothelial cells proliferation by apoptosis." Angiogenesis 9.2 (2006): 83-91.
PMID
16821112
Source
pubmed
Published In
Angiogenesis
Volume
9
Issue
2
Publish Date
2006
Start Page
83
End Page
91
DOI
10.1007/s10456-006-9034-0

Targeting Id1 and Id3 inhibits peritoneal metastasis of gastric cancer.

Inhibitor of DNA binding (Id) proteins are essential for cell differentiation, proliferation, migration, invasion and angiogenesis. Recently, they have been shown to correlate with less differentiated phenotypes, high malignant potential and poor clinical outcome in various kinds of tumors. In an attempt to develop new strategies for the treatment of peritoneal metastasis of gastric cancer, we prepared an Id1, 3 double-knockdown gastric cancer cell line, MKN45, by RNA interference and investigated its effects on the development of metastatic nodules in the peritoneal cavity. Both cell proliferation and migration capabilities were decreased in Id1, 3 double-knockdown cells, as was their ability to bind to laminin, which could be explained by the decreased expression of integrin alpha6. These are important steps in the metastatic process. In a mouse model, the number of peritoneal metastatic nodules formed by Id1, 3 double-knockdown cells was reduced compared to mock-transfected control cells, as was the size of individual tumors. In this study, we clearly demonstrated that Id1, 3 double-knockdown significantly impaired the ability of gastric cancer cells to form peritoneal metastasis. Id should be considered an ideal target for the treatment and prevention of gastric cancer, and RNA interference is an attractive and promising strategy to achieve it.

Authors
Tsuchiya, T; Okaji, Y; Tsuno, NH; Sakurai, D; Tsuchiya, N; Kawai, K; Yazawa, K; Asakage, M; Yamada, J; Yoneyama, S; Kitayama, J; Osada, T; Watanabe, T; Tokunaga, K; Takahashi, K; Nagawa, H
MLA Citation
Tsuchiya, T, Okaji, Y, Tsuno, NH, Sakurai, D, Tsuchiya, N, Kawai, K, Yazawa, K, Asakage, M, Yamada, J, Yoneyama, S, Kitayama, J, Osada, T, Watanabe, T, Tokunaga, K, Takahashi, K, and Nagawa, H. "Targeting Id1 and Id3 inhibits peritoneal metastasis of gastric cancer." Cancer Sci 96.11 (November 2005): 784-790.
PMID
16271072
Source
pubmed
Published In
Cancer Science
Volume
96
Issue
11
Publish Date
2005
Start Page
784
End Page
790
DOI
10.1111/j.1349-7006.2005.00113.x

Ex vivo expanded human CD4+ regulatory NKT cells suppress expansion of tumor antigen-specific CTLs.

NKT cells can produce large amounts of both Th1- and Th2-type cytokines and are an important regulatory cell type. To elucidate their role in acquired immunity, we examined the effect of human Valpha24+Vbeta11+ NKT cells or CD1d-specific ligand alpha-galactosylceramide (alphaGalCer) on the in vitro generation of antigen-specific CTLs from PBMCs using autologous MART-1(26-35) peptide-pulsed dendritic cells as stimulators. Flow cytometry using tetramer for MART-1(26-35) peptide revealed that NKT cells have inhibitory effects on CTL generation. Cytokine analysis using cytometric bead array assay and ELISA showed higher IL-4 and IL-10 secretion in the alphaGalCer(+) and/or NKT cell(+) culture setting, whereas IL-13 secretion in the culture was not affected by the presence of alphaGalCer. The CD4+ NKT cell subset seemed to play a major role in this inhibitory effect by secreting large amounts of Th2-type cytokines. Interestingly however, unlike recent reports utilizing mouse models, IL-13 was not a main effector molecule in our human system. Culture with alphaGalCer in the presence of cytokine-neutralizing antibodies for the Th2 cytokines, IL-4, IL-5 and IL-10, resulted in enhanced CTL generation, suggesting the dominant role of Th2 cytokines over Th1 cytokines. Thus, CD4+ NKT cells can work as immunoregulatory T cells that suppress anti-tumor immune response and, therefore, NKT cells or alphaGalCer could be used as therapeutic modalities to modulate systemic immune responses, such as autoimmune diseases. Conversely, the use of NKT cells along with anti-Th2 cytokine-neutralizing antibodies or CD4-negative NKT cell subset could enhance the generation of antigen-specific CTLs for adoptive immunotherapy.

Authors
Osada, T; Morse, MA; Lyerly, HK; Clay, TM
MLA Citation
Osada, T, Morse, MA, Lyerly, HK, and Clay, TM. "Ex vivo expanded human CD4+ regulatory NKT cells suppress expansion of tumor antigen-specific CTLs." Int Immunol 17.9 (September 2005): 1143-1155.
PMID
16027139
Source
pubmed
Published In
International Immunology
Volume
17
Issue
9
Publish Date
2005
Start Page
1143
End Page
1155
DOI
10.1093/intimm/dxh292

CD11b-mediated migratory property of peripheral blood B cells.

BACKGROUND: CD11b belongs to the integrin family and is expressed on neutrophils, monocytes, natural killer cells, and a subset of lymphocytes. Although CD11b expressed on neutrophils and monocytes has been extensively investigated and has been reported to play an important role in the migration of these subsets of leukocytes, the function of CD11b expressed on a subset of B cells has not yet been clarified. OBJECTIVE: To elucidate the functional activity of CD11b expressed on B cells, we characterized the CD11b-expressing cells among the B-cell population and investigated their migratory ability. METHODS: Isolated peripheral blood CD19 + B cells were analyzed by flow cytometry. The migratory ability of B cells was evaluated by the transwell assay, and the contribution of CD11b to this ability was investigated by using an anti-CD11b blocking mAb. RESULTS: The majority of CD27 - IgD + naive B cells were CD11b - , whereas most CD27 + memory cells were CD11b +. Among the CD27 + memory cells, expression of CD11b was stronger on the IgD - cells than on the IgD + cells. In the transwell assay, the migrating cells were predominantly CD27 + IgD - cells, most of which expressed CD11b. The addition of an anti-CD11b blocking mAb resulted in the significant reduction of the number of migrating B cells. CONCLUSION: Memory B cells express CD11b and, in contrast with naive B cells, have high migratory ability. CD11b plays an essential role in the homing process of memory cells.

Authors
Kawai, K; Tsuno, NH; Matsuhashi, M; Kitayama, J; Osada, T; Yamada, J; Tsuchiya, T; Yoneyama, S; Watanabe, T; Takahashi, K; Nagawa, H
MLA Citation
Kawai, K, Tsuno, NH, Matsuhashi, M, Kitayama, J, Osada, T, Yamada, J, Tsuchiya, T, Yoneyama, S, Watanabe, T, Takahashi, K, and Nagawa, H. "CD11b-mediated migratory property of peripheral blood B cells." J Allergy Clin Immunol 116.1 (July 2005): 192-197.
PMID
15990794
Source
pubmed
Published In
Journal of Allergy and Clinical Immunology
Volume
116
Issue
1
Publish Date
2005
Start Page
192
End Page
197
DOI
10.1016/j.jaci.2005.03.021

Correlation of clinical outcome with natural killer (NK) response to an anti-cancer, dendritic cell-based vaccine.

Authors
Morse, M; Osada, T; Hobeika, A; Chui, S; Clay, T; Lyerly, HK
MLA Citation
Morse, M, Osada, T, Hobeika, A, Chui, S, Clay, T, and Lyerly, HK. "Correlation of clinical outcome with natural killer (NK) response to an anti-cancer, dendritic cell-based vaccine." June 1, 2005.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
23
Issue
16
Publish Date
2005
Start Page
187S
End Page
187S

Correlation of clinical outcome with natural killer (NK) response to an anti-cancer, dendritic cell-based vaccine.

2585 Background: Cancer vaccines have generally been developed to activate antigen-specific T cell responses, but few studies have attempted to evaluate the non-specific, yet potentially clinically-relevant, NK response to immunization. We hypothesized that the NK response would predict clinical benefit in immunized patients.In a phase I clinical trial of a vaccine consisting of autologous dendritic cell (DC) loaded with a fowlpox vector encoding CEA (rF-CEA(6D)-TRICOM, we measured CEA-specific immune responses by ELISPOT assay and reported increases in 12/14 patients (Proc ASCO2004, abst 2508). Using archived peripheral blood specimens (n=9) from before and after all immunizations, we measured CD3-CD56+ NK% and NK cytolytic activity against the NK target K562. These data were compared with the clinical outcome of the patients.There were no differences in the percentage of NK cells or NK markers NKG2A, NKG2C, NKG2D before or after immunization. In contrast, NK cytolytic activity increased in 4 patients (range 2.5 to 5 times greater lytic activity), was stable in 2 and decreased in 3. When patients were grouped by clinical activity (stable disease/no evidence of disease (n=5) versus progressive disease (N=4) at three months), we observed that the majority of those with stable disease/no evidence of disease had increases in their NK activity (Chi Square, P= 0.0163). The NK results predicted more closely the clinical outcome than did T cell responses (Chi Square, P=NS).NK responses following immunization with a dendritic cell vaccine are associated with clinical benefit as indicated by a lack of progressive disease. Monitoring of NK response during vaccine studies should be routinely performed. No significant financial relationships to disclose.

Authors
Morse, M; Osada, T; Hobeika, A; Chui, S; Clay, T; Lyerly, HK
MLA Citation
Morse, M, Osada, T, Hobeika, A, Chui, S, Clay, T, and Lyerly, HK. "Correlation of clinical outcome with natural killer (NK) response to an anti-cancer, dendritic cell-based vaccine." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 23.16_suppl (June 2005): 2585-.
PMID
27945884
Source
epmc
Published In
Journal of Clinical Oncology
Volume
23
Issue
16_suppl
Publish Date
2005
Start Page
2585

Phase I study of immunization with dendritic cells modified with fowlpox encoding carcinoembryonic antigen and costimulatory molecules.

PURPOSE: To determine the safety and immunologic and clinical efficacy of a dendritic cell vaccine modified to hyperexpress costimulatory molecules and tumor antigen. EXPERIMENTAL DESIGN: In this phase I study, we administered one or two cycles of four triweekly s.c./intradermal injections of ex vivo generated dendritic cells modified with a recombinant fowlpox vector encoding carcinoembryonic antigen (CEA) and a triad of costimulatory molecules [rF-CEA(6D)-TRICOM]. Controls consisted of immature dendritic cells loaded with tetanus toxoid and a HLA A2-restricted peptide derived from cytomegalovirus pp65 protein. RESULTS: Fourteen patients (11 with colorectal cancer and 3 with non-small cell lung cancer) were enrolled and 12 completed at least one cycle of immunization. There were no grade 3/4 toxicities directly referable to the immunizations. One patient had a decrease in the CEA level from 46 to 6.8 and a minor regression in adenopathy that occurred several months after completion of the immunizations. Five other patients were stable through at least one cycle of immunization (3 months). Direct analysis of peripheral blood mononuclear cells using the ELISpot assay showed an increase in the frequency of CEA-specific T cells in 10 patients (range, 10-541 CEA-specific cells/10(5) peripheral blood mononuclear cells). There was a trend for a greater peak frequency of CEA-specific T cells among those with either a minor response or a stable disease following at least one cycle of therapy. A second cycle was not associated with higher T-cell frequencies. Cytokine flow cytometry showed CEA-specific immune response among both CD4(+) and CD8(+) T cells in all immune responders. CONCLUSION: This immunization strategy is safe and activates potent CEA-specific immune responses.

Authors
Morse, MA; Clay, TM; Hobeika, AC; Osada, T; Khan, S; Chui, S; Niedzwiecki, D; Panicali, D; Schlom, J; Lyerly, HK
MLA Citation
Morse, MA, Clay, TM, Hobeika, AC, Osada, T, Khan, S, Chui, S, Niedzwiecki, D, Panicali, D, Schlom, J, and Lyerly, HK. "Phase I study of immunization with dendritic cells modified with fowlpox encoding carcinoembryonic antigen and costimulatory molecules." Clin Cancer Res 11.8 (April 15, 2005): 3017-3024.
PMID
15837756
Source
pubmed
Published In
Clinical cancer research : an official journal of the American Association for Cancer Research
Volume
11
Issue
8
Publish Date
2005
Start Page
3017
End Page
3024
DOI
10.1158/1078-0432.CCR-04-2172

A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer.

BACKGROUND: There is a continued need to develop more effective cancer immunotherapy strategies. Exosomes, cell-derived lipid vesicles that express high levels of a narrow spectrum of cell proteins represent a novel platform for delivering high levels of antigen in conjunction with costimulatory molecules. We performed this study to test the safety, feasibility and efficacy of autologous dendritic cell (DC)-derived exosomes (DEX) loaded with the MAGE tumor antigens in patients with non-small cell lung cancer (NSCLC). METHODS: This Phase I study enrolled HLA A2+ patients with pre-treated Stage IIIb (N = 4) and IV (N = 9) NSCLC with tumor expression of MAGE-A3 or A4. Patients underwent leukapheresis to generate DC from which DEX were produced and loaded with MAGE-A3, -A4, -A10, and MAGE-3DPO4 peptides. Patients received 4 doses of DEX at weekly intervals. RESULTS: Thirteen patients were enrolled and 9 completed therapy. Three formulations of DEX were evaluated; all were well tolerated with only grade 1-2 adverse events related to the use of DEX (injection site reactions (N = 8), flu like illness (N = 1), and peripheral arm pain (N = 1)). The time from the first dose of DEX until disease progression was 30 to 429+ days. Three patients had disease progression before the first DEX dose. Survival of patients after the first DEX dose was 52-665+ days. DTH reactivity against MAGE peptides was detected in 3/9 patients. Immune responses were detected in patients as follows: MAGE-specific T cell responses in 1/3, increased NK lytic activity in 2/4. CONCLUSION: Production of the DEX vaccine was feasible and DEX therapy was well tolerated in patients with advanced NSCLC. Some patients experienced long term stability of disease and activation of immune effectors.

Authors
Morse, MA; Garst, J; Osada, T; Khan, S; Hobeika, A; Clay, TM; Valente, N; Shreeniwas, R; Sutton, MA; Delcayre, A; Hsu, D-H; Le Pecq, J-B; Lyerly, HK
MLA Citation
Morse, MA, Garst, J, Osada, T, Khan, S, Hobeika, A, Clay, TM, Valente, N, Shreeniwas, R, Sutton, MA, Delcayre, A, Hsu, D-H, Le Pecq, J-B, and Lyerly, HK. "A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. (Published online)" J Transl Med 3.1 (February 21, 2005): 9-.
PMID
15723705
Source
pubmed
Published In
Journal of Translational Medicine
Volume
3
Issue
1
Publish Date
2005
Start Page
9
DOI
10.1186/1479-5876-3-9

Enumerating antigen-specific T-cell responses in peripheral blood: a comparison of peptide MHC Tetramer, ELISpot, and intracellular cytokine analysis.

Detection of the circulating antigen-specific T-cell response to immunization is an important biologic end point in clinical trials of cancer vaccines. Typically employed assays are peptide MHC tetramer, ELISpot, and intracellular cytokine analysis. Although there is no agreement on the definition of a positive response in these assays, many groups have chosen a number of T cells greater than 2 standard deviations above the mean of the negative controls. The authors wished to determine how well this cutoff performed for each of these assays in detecting positive and negative T-cell responses to a model antigen, the immunodominant HLA-A*0201-restricted epitope of cytomegalovirus (CMV) pp65. For each assay, the mean + 2 standard deviations of the response for CMV seronegatives was the point that best separated the two groups. Using this value, each assay had a sensitivity of 87.5% and specificity of 95% to 100% and exhibited a high degree of concordance (kappa 0.76-0.9) with the other two. The authors conclude that currently available immunologic assays perform well in detecting biologically relevant levels of antigen-specific T cells. These assays will better define the quantity and quality of protective immune responses to viral disease and offer insight into the requirements for protective anti-cancer immunity.

Authors
Hobeika, AC; Morse, MA; Osada, T; Ghanayem, M; Niedzwiecki, D; Barrier, R; Lyerly, HK; Clay, TM
MLA Citation
Hobeika, AC, Morse, MA, Osada, T, Ghanayem, M, Niedzwiecki, D, Barrier, R, Lyerly, HK, and Clay, TM. "Enumerating antigen-specific T-cell responses in peripheral blood: a comparison of peptide MHC Tetramer, ELISpot, and intracellular cytokine analysis." J Immunother 28.1 (January 2005): 63-72.
PMID
15614046
Source
pubmed
Published In
Journal of Immunotherapy
Volume
28
Issue
1
Publish Date
2005
Start Page
63
End Page
72

Intracellular Cytokine Assays

This chapter provides an overview of intracellular cytokine assays. Intracellular cytokine assays are a relatively new method of identifying cytokine production by individual T cells and have the ability to correlate cytokine expression with cell surface phenotype without cell separation. In addition, this highly sensitive flow cytometric method allows for the rapid detection of low frequency T cells expressing cytokine in response to specific antigen stimulation. The unique capabilities of this method make it a model assay for clinical and research applications. The overall premise of intracellular cytokine assays is direct detection of intracellular cytokine expression in response to antigen stimulation. Intracellular cytokine assays can be performed using various sources of cells and antigen depending on the target(s) of interest. T cell stimulation can be performed directly on whole blood, peripheral blood mononuclear cells, in vitro manipulated lymphocytes, isolated cells, and lymph nodes; although using whole blood for these assays provides a more physiological environment and may have an effect on the T cell response to stimulation. Since these assays are most often used to detect very low frequency events, appropriate control antigens are particularly important to ensure clear antigen specific response. © 2005 Elsevier Ltd. All rights reserved.

Authors
Hobeika, AC; Morse, MA; Clay, TM; Osada, T; Mosca, PJ; Lyerly, HK
MLA Citation
Hobeika, AC, Morse, MA, Clay, TM, Osada, T, Mosca, PJ, and Lyerly, HK. "Intracellular Cytokine Assays." 2005. 336-340.
Source
scival
Publish Date
2005
Start Page
336
End Page
340
DOI
10.1016/B978-012455900-4/50290-7

Regulatory and effector T cell subsets and dendritic cells in breast cancer.

Authors
Chui, SY; Morse, MA; Doldo, T; Osada, T; Clay, TM; Lyerly, HK; Khan, S; Gattis, A; Hobeika, AC
MLA Citation
Chui, SY, Morse, MA, Doldo, T, Osada, T, Clay, TM, Lyerly, HK, Khan, S, Gattis, A, and Hobeika, AC. "Regulatory and effector T cell subsets and dendritic cells in breast cancer." July 15, 2004.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
22
Issue
14
Publish Date
2004
Start Page
884S
End Page
884S

Phase I study of immunization with dendritic cells (DC) modified with recombinant fowlpox encoding carcinoembryonic antigen (CEA) and the triad of costimulatory molecules CD54, CD58, and CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies.

Authors
Morse, M; Clay, T; Hobeika, A; Osada, T; Panicali, D; Lyerly, HK
MLA Citation
Morse, M, Clay, T, Hobeika, A, Osada, T, Panicali, D, and Lyerly, HK. "Phase I study of immunization with dendritic cells (DC) modified with recombinant fowlpox encoding carcinoembryonic antigen (CEA) and the triad of costimulatory molecules CD54, CD58, and CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies." July 15, 2004.
Source
wos-lite
Published In
Journal of Clinical Oncology
Volume
22
Issue
14
Publish Date
2004
Start Page
165S
End Page
165S

Tumor-infiltrating effector cells of alpha-galactosylceramide-induced antitumor immunity in metastatic liver tumor.

BACKGROUND: alpha-Galactosylceramide (alpha-GalCer) can be presented by CD1d molecules of antigen-presenting cells, and is known to induce a potent NKT cell-dependent cytotoxic response against tumor cells. However, the main effector cells in alpha-GalCer-induced antitumor immunity are still controversial. METHODS: In order to elucidate the cell phenotype that plays the most important role in alpha-GalCer-induced antitumor immunity, we purified and analyzed tumor-infiltrating leukocytes (TILs) from liver metastatic nodules of a colon cancer cell line (Colon26), comparing alpha-GalCer- and control vehicle-treated mice. Flow cytometry was performed to analyze cell phenotype in TILs and IFN-gamma ELISA was performed to detect antigen-specific immune response. RESULTS: Flow cytometry analysis showed a significantly higher infiltration of NK cells (DX5+, T cell receptor alphabeta (TCR)-) into tumors in alpha-GalCer-treated mice compared to vehicle-treated mice. The DX5+TCR+ cell population was not significantly different between these two groups, indicating that these cells were not the main effector cells. Interestingly, the CD8+ T cell population was increased in TILs of alpha-GalCer-treated mice, and the activation level of these cells based on CD69 expression was higher than that in vehicle-treated mice. Moreover, the number of tumor-infiltrating dendritic cells (DCs) was increased in alpha-GalCer-treated mice. IFN-gamma ELISA showed stronger antigen-specific response in TILs from alpha-GalCer-treated mice compared to those from vehicle-treated mice, although the difference between these two groups was not significant. CONCLUSIONS: In alpha-GalCer-induced antitumor immunity, NK cells seem to be some of the main effector cells and both CD8+ T cells and DCs, which are related to acquired immunity, might also play important roles in this antitumor immune response. These results suggest that alpha-GalCer has a multifunctional role in modulation of the immune response.

Authors
Osada, T; Nagawa, H; Shibata, Y
MLA Citation
Osada, T, Nagawa, H, and Shibata, Y. "Tumor-infiltrating effector cells of alpha-galactosylceramide-induced antitumor immunity in metastatic liver tumor. (Published online)" J Immune Based Ther Vaccines 2.1 (July 13, 2004): 7-.
PMID
15251043
Source
pubmed
Published In
Journal of Immune Based Therapies and Vaccines
Volume
2
Issue
1
Publish Date
2004
Start Page
7
DOI
10.1186/1476-8518-2-7

Regulatory and effector T cell subsets and dendritic cells in breast cancer.

9697 Background: Successful immune responses against breast cancer may depend on the balance between immune stimulation mediated through dendritic cells (DC) & cytolytic T cells, and immune inhibition mediated in part by CD4+CD25+ regulatory T cells (Treg) and CTLA4 expression on T cells. We hypothesized that low anti-tumor immunity in breast cancer patients might be partially explained by aberrations in the frequencies of these different components of the immune response compared with healthy individuals.We analyzed whole blood from 26 breast cancer patients (9 stage I, 8 stage II, 3 stage III, 6 stage IV) for Treg, DC subsets, CD8+CD45RA+CCR7- cytolytic effectors, and CTLA4 expressing T cells. Controls were 41 healthy volunteers.Breast cancer patients overall had higher levels of Treg (37+/-14% of CD4+ T cells) than controls (26.3 +/- 9%; p=0.002). Treg were higher in patients with stage II-IV disease, positive lymph nodes, and age < 65. Patients <65 also tended to have higher levels of CTLA4 on both CD4 and CD8+ T cells. Across all patients, there was a high positive correlation (0.82) of CTLA4 expression on Treg and CD8+ T cells, suggesting that the presence of this suppressive molecule may be a more generalized phenomenon in some individuals. The only groups that differed in CD8+CD45RA+CCR7- cytolytic effectors were premenopausal patients who had lower levels than post-menopausal women (37 vs 54%, p=0.04). Across all the patients, there was a modest negative correlation (-0.44) between Treg and CD8+ CD45RA+CCR7- suggesting that Treg may inhibit development of cytotoxic effector T cells. Finally, patients receiving chemotherapy and those with Stage IV disease had lower levels of immunostimulatory CD11c+ DC and higher levels of immunomodulatory CD123+ DC than other patients suggesting a possible negative impact of chemotherapy and advanced disease on stimulatory DC.This data highlights the inhibitory environment of the immune system in breast cancer patients, particularly those with advanced disease and younger age. Modification of this inhibitory environment will be needed to increase the activity of immunotherapy interventions in these patients. No significant financial relationships to disclose.

Authors
Chui, SY; Morse, MA; Doldo, T; Osada, T; Clay, TM; Lyerly, HK; Khan, S; Gattis, A; Hobeika, AC
MLA Citation
Chui, SY, Morse, MA, Doldo, T, Osada, T, Clay, TM, Lyerly, HK, Khan, S, Gattis, A, and Hobeika, AC. "Regulatory and effector T cell subsets and dendritic cells in breast cancer." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 22.14_suppl (July 2004): 9697-.
PMID
28016077
Source
epmc
Published In
Journal of Clinical Oncology
Volume
22
Issue
14_suppl
Publish Date
2004
Start Page
9697

Phase I study of immunization with dendritic cells (DC) modified with recombinant fowlpox encoding carcinoembryonic antigen (CEA) and the triad of costimulatory molecules CD54, CD58, and CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies.

2508 Background: We hypothesize that the activity of vaccines based on DC loaded with tumor antigens will be enhanced by modifications that increase antigen expression and costimulatory activity of the DC.In an ongoing phase I study, we are administering 1, 2, or 3 cycles of 4 triweekly subcutaneous / intradermal injections of ex vivo generated DC (5 x 10E6 cells) modified with the recombinant fowlpox vector rF-CEA(6D)-TRICOM (2.5 x 10E7 pfu/5 x 10E7 DC).Thus far, 14 patients have been enrolled into the first 2 cohorts. There were no grade 3/4 toxicities directly referable to the immunizations. One patient had a decrease in the CEA level from 46 to 6.8 and a minor regression in supraclavicular adenopathy that occurred several months after completion of the immunizations. Three other patients were stable through at least 1 cycle of immunization (3 months). Direct analysis of T cells in peripheral blood using the ELISpot assay demonstrated an increase in the frequency of T cells specific for the CEA-expressing vector in 10 of the 12 patients completing participation (range 10 to 541 CEA-specific cells/100,000 peripheral blood mononuclear cells (PBMC).) A greater CEA-specific response was observed for patients with either a minor response or stable disease following at least 1 cycle of therapy compared with those who progressed (mean of 241 vs 50 CEA-specific cells/100,000 PBMC, P=0.03). Cytokine flow cytometry demonstrated that the CEA-specific immune response occurred amongst both CD4 and CD8+ T cells in all responders. Changes in the frequency of CD4+CD25+ regulatory T cells were observed and, in some patients, were inversely correlated with the frequency of CEA-specific CD4+ T cells suggesting that regulatory mechanisms might impact the greatest magnitude of CEA-specific T cells that can be achieved.This immunization strategy is safe and feasible and activates potent CEA-specific immune responses. This study will continue into the third cohort. [Table: see text].

Authors
Morse, M; Clay, T; Hobeika, A; Osada, T; Panicali, D; Lyerly, HK
MLA Citation
Morse, M, Clay, T, Hobeika, A, Osada, T, Panicali, D, and Lyerly, HK. "Phase I study of immunization with dendritic cells (DC) modified with recombinant fowlpox encoding carcinoembryonic antigen (CEA) and the triad of costimulatory molecules CD54, CD58, and CD80 (rF-CEA(6D)-TRICOM) in patients with advanced malignancies." Journal of clinical oncology : official journal of the American Society of Clinical Oncology 22.14_suppl (July 2004): 2508-.
PMID
28014999
Source
epmc
Published In
Journal of Clinical Oncology
Volume
22
Issue
14_suppl
Publish Date
2004
Start Page
2508

Flt3-ligand as a vaccine adjuvant: Results in a study of Flt3-ligand plus tetanus toxoid immunization

Dendritic cells (DC) efficiently process and present antigens to the effector arm of the immune system, thereby stimulating immunity against antigens of both foreign and self origin. Administration of Flt3-ligand (FL) has been reported to increase dendritic cell (DC) numbers in mice and humans. As a result, FL has attracted interest as an adjuvant for vaccine immunotherapy. To investigate whether FL might increase the immune response to a model recall antigen, we administered FL 25 μg/kg/d subcutaneously to six healthy volunteers followed by a standard injection of intramuscular tetanus toxoid (TT). A control cohort of six healthy volunteers received tetanus toxoid alone. Compared to subjects who received only TT, subjects who received Flt3L and TT had greater TT-specific DTH reactivity. In contrast, FL did not augment peripheral blood mononuclear cell proliferative responses or antibody responses to TT. FL resulted in inconsistent TT-specific T cell responses as measured by interferon-gamma ELISPOT and cytokine flow cytometry. We conclude that while FL mobilization of DC may enhance in vivo immune responses to a known immunogenic recall antigen, there are inconsistent effects on immune response detected by in vitro assays. Further study will be required to determine which individuals might experience augmentation of the immune response with FL.

Authors
Chui, S; Clay, TM; Mosca, PJ; Hobeika, AC; Osada, T; Galibert, L; Caron, D; Lyerly, HK; Morse, MA
MLA Citation
Chui, S, Clay, TM, Mosca, PJ, Hobeika, AC, Osada, T, Galibert, L, Caron, D, Lyerly, HK, and Morse, MA. "Flt3-ligand as a vaccine adjuvant: Results in a study of Flt3-ligand plus tetanus toxoid immunization." Journal of Applied Research 4.4 (2004): 536-549.
Source
scival
Published In
The journal of applied research
Volume
4
Issue
4
Publish Date
2004
Start Page
536
End Page
549

Comparison of antigen-specific T cell responses induced by transduction of human dendritic cells with E1- and E1-, E2b- adenoviral vectors: Development of adenovirus vectors for DC-based anti-tumor immunotherapy

Authors
Venturi, CB; Osada, T; Serraz, D; Hartman, Z; Evelyn, C; Morse, MA; Clay, TM; Amalfitano, A; Lyerly, HK
MLA Citation
Venturi, CB, Osada, T, Serraz, D, Hartman, Z, Evelyn, C, Morse, MA, Clay, TM, Amalfitano, A, and Lyerly, HK. "Comparison of antigen-specific T cell responses induced by transduction of human dendritic cells with E1- and E1-, E2b- adenoviral vectors: Development of adenovirus vectors for DC-based anti-tumor immunotherapy." April 14, 2003.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
17
Issue
7
Publish Date
2003
Start Page
C331
End Page
C331

Suppression of antigen-specific CTL generation by human NKT cell secretion of TH2 cytokines

Authors
Osada, T; Morse, MA; Clay, TM; Shimosaka, A; Lyerly, HK
MLA Citation
Osada, T, Morse, MA, Clay, TM, Shimosaka, A, and Lyerly, HK. "Suppression of antigen-specific CTL generation by human NKT cell secretion of TH2 cytokines." April 14, 2003.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
17
Issue
7
Publish Date
2003
Start Page
C77
End Page
C78

Dendritic cells cultured in anti-CD40 antibody-immobilized plates elicit a highly efficient peptide-specific T-cell response.

The function of dendritic cells (DCs), antigen-presenting cells that can initiate and regulate cellular and humoral responses, is highly influenced by their level of maturation. Immature DCs may be harmful in anti-tumor immunotherapy, because they can induce immunotolerance rather than immunostimulation. In this study, the authors sought to determine the optimal culture conditions for obtaining fully mature DCs. When DCs were cultured in agonistic anti-CD40 monoclonal antibody-immobilized plates, they showed a higher expression of the maturation marker CD83 than DCs cultured without CD40 ligation or those cultured in medium supplemented with anti-CD40 monoclonal antibody. In addition, when interferon-gamma (IFN-gamma) was added to the medium, additive up-regulation of CD83 expression was observed. These DCs treated with both maturation signals showed a higher secretion of interleukin-12. To evaluate the capacity of antigen presentation, specific cytotoxic T lymphocytes were generated using autologous DC pulsed with a human lymphocyte antigen-A24-restricted peptide epitope derived from carcinoembryonic antigen. Interferon-gamma-secreting CD8+ T cells were analyzed by flow cytometry using the cellular affinity matrix technology. Dendritic cells, matured with CD40 ligation and IFN-gamma, were more efficient at eliciting an antigen-specific T-cell response in vitro than DCs stimulated with anti-CD40 monoclonal antibody or IFN-gamma alone. A cytotoxicity assay using carcinoembryonic antigen-expressing tumor cell lines also showed that DCs matured with both signals were more efficient at inducing cytotoxic T lymphocytes. These results demonstrate that DC culture in an anti-CD40 monoclonal antibody-immobilized plate in medium supplemented with IFN-gamma has a positive impact on DC maturation and may be optimal for eliciting an antigen-specific T-cell response without the need for CD4+ T-helper epitopes.

Authors
Osada, T; Nagawa, H; Takahashi, T; Tsuno, NH; Kitayama, J; Shibata, Y
MLA Citation
Osada, T, Nagawa, H, Takahashi, T, Tsuno, NH, Kitayama, J, and Shibata, Y. "Dendritic cells cultured in anti-CD40 antibody-immobilized plates elicit a highly efficient peptide-specific T-cell response." J Immunother 25.2 (March 2002): 176-184.
PMID
12074047
Source
pubmed
Published In
Journal of Immunotherapy
Volume
25
Issue
2
Publish Date
2002
Start Page
176
End Page
184

Cholesterol granuloma of the breast mimicking carcinoma: report of a case.

Cholesterol granuloma of the breast is a rare benign condition which is often clinically and radiologically indistinguishable from breast carcinoma. We herein report the case of a 62-year-old asymptomatic woman who was found on a routine breast examination to have an elastic hard mass, measuring 0.9 cm in diameter, in the upper outer quadrant of the left breast. Physical examination and ultrasonography strongly suggested a carcinomatous lesion. A cytological examination of a fine-needle aspiration biopsy specimen was inconclusive because of the paucity of epithelial cells. A histological examination of excisional biopsy materials showed scattered cholesterol crystals arranged in irregular, parallel arrays, surrounded by histiocytes and giant cells, which were consistent with a diagnosis of cholesterol granuloma. This case report indicates the importance of performing a histological examination to establish the final diagnosis of cholesterol granuloma. We believe that a better awareness of this breast disease might help to prevent both a misdiagnosis and unnecessary surgery.

Authors
Osada, T; Kitayama, J; Nagawa, H
MLA Citation
Osada, T, Kitayama, J, and Nagawa, H. "Cholesterol granuloma of the breast mimicking carcinoma: report of a case." Surg Today 32.11 (2002): 981-984.
PMID
12444435
Source
pubmed
Published In
Surgery Today
Volume
32
Issue
11
Publish Date
2002
Start Page
981
End Page
984
DOI
10.1007/s005950200196

Decreased synthesis of matrix metalloproteinase-7 and adhesion to the extracellular matrix proteins of human colon cancer cells treated with troglitazone.

PURPOSE: In the present study, we investigated the effect of troglitazone, a selective ligand and agonist of PPAR-gamma, on the metastatic potential of human colon cancer cells. METHODS: High- and low-PPAR-gamma expression clones of the colon cancer cell line, HT29, namely clones 21 and 3 respectively, were used. We investigated the effect of troglitazone on the proliferation, on the adhesion to extracellular matrix proteins and on the synthesis of matrix metalloproteinases (MMPs) of colon cancer cells. RESULTS: Troglitazone inhibited the proliferation of both subclones, in a dose-dependent manner, and the inhibitory effect correlated with the level of PPAR-gamma expression. Troglitazone strongly inhibited the production of MMP-7, an enzyme associated with invasiveness of cancer cells, by both subclones. In addition, troglitazone caused a strong decrease in the adhesion of clone 21 to extracellular matrix (ECM) proteins, laminin and type IV collagen. This effect was independent of beta1-integrins expression CONCLUSION: In addition to inhibition of cancer cell growth, troglitazone had an inhibitory effect on two important events associated with the metastatic potential of cancer cells, production of MMPs and adhesion to ECM proteins. Consequently, troglitazone is a promising agent for the treatment and prevention of colon cancer metastasis.

Authors
Sunami, E; Tsuno, NH; Kitayama, J; Saito, S; Osada, T; Yamaguchi, H; Tomozawa, S; Tsuruo, T; Shibata, Y; Nagawa, H
MLA Citation
Sunami, E, Tsuno, NH, Kitayama, J, Saito, S, Osada, T, Yamaguchi, H, Tomozawa, S, Tsuruo, T, Shibata, Y, and Nagawa, H. "Decreased synthesis of matrix metalloproteinase-7 and adhesion to the extracellular matrix proteins of human colon cancer cells treated with troglitazone." Surg Today 32.4 (2002): 343-350.
PMID
12027200
Source
pubmed
Published In
Surgery Today
Volume
32
Issue
4
Publish Date
2002
Start Page
343
End Page
350

Peripheral blood dendritic cells, but not monocyte-derived dendritic cells, can augment human NK cell function.

Dendritic cells (DCs) are essential antigen-presenting cells with a wide variety of functions relating to both adaptive and innate immunity. Recently, interactions of DCs with natural killer (NK) cells and NK1.1-positive T cells have been reported in mice. However, in humans, this interaction is not well understood. Here we report the use of a coculture method to analyze the modulation of NK cell function in antitumor immunity by DCs. We found that peripheral blood DCs (PDCs) enhanced NK cell activity in cytotoxicity assay, even without direct contact between DC and NK cells. In contrast, neither monocyte-derived DCs (MoDCs), nor TNF-alpha-treated MoDCs, stimulated NK lytic activity. Secretion of IL-12 and TNF-alpha into the PDC-NK coculture supernatant was increased. However, blocking antibodies against these cytokines could not completely abolish the upregulation of NK activity, suggesting the presence of other soluble factor(s) that affect DC-NK cell interaction. To summarize, this study demonstrates for the first time the direct activation of human NK cells by DC-NK cell interaction in vitro, suggesting that DCs may have a central role linking the innate and adaptive immune responses. Moreover, in stimulating NK cell function, PDCs appear to have a different potential from MoDCs.

Authors
Osada, T; Nagawa, H; Kitayama, J; Tsuno, NH; Ishihara, S; Takamizawa, M; Shibata, Y
MLA Citation
Osada, T, Nagawa, H, Kitayama, J, Tsuno, NH, Ishihara, S, Takamizawa, M, and Shibata, Y. "Peripheral blood dendritic cells, but not monocyte-derived dendritic cells, can augment human NK cell function." Cell Immunol 213.1 (October 10, 2001): 14-23.
PMID
11747352
Source
pubmed
Published In
Cellular Immunology
Volume
213
Issue
1
Publish Date
2001
Start Page
14
End Page
23
DOI
10.1006/cimm.2001.1858

Clustered cancer cells show a distinct adhesion behavior from single cell form under physiological shear conditions.

It remains a question whether hematogeneous metastasis arises from a single cancer cell attached to the local endothelium or from a cluster of cancer cells trapped in the vascular bed in the target organ. Adhesive interaction of the single cell form and the clustered form of cancer cells was examined under flow conditions, using two subclones of mouse colon adenocarcinoma Colon 26. A subclone NL17, but not NL14, formed many clusters composed of tumor cells and platelets just after the addition of platelet rich plasma (PRP). Under the shear of 1.0 dyn/cm3, the clustered form of NL17 tethered on laminin or mouse endothelial cell line in hepatic sinusoids (HSE) more frequently than the single cell form of NL17 and NL14. However, all of the clusters showed only transient attachment and never underwent stable arrest on coated laminin, while the single cell form of NL14 and NL17 underwent immediate arrest under shear conditions. On HSE stimulated with TNF-alpha, a small number of NL17 clusters made stable adhesion, although all the clusters detached if the shear stress was increased above 4.0 dyn/cm2. In contrast, the single form of arrested NL17 as well as NL14 remained adherent even at shear of 8.0 dyn/cm2. Compared with single cell, binding of cancer cell clusters to laminin and HSE showed lower resistance to shear stress, although they had adhesive interactions more frequently in flow condition. Since NL17 cells form significantly more metastases by intravenous injection in vivo, our data suggest that "stable adhesion" observed in our flow assay system is not always a prerequisite for clustered cancer cells to develop into metastatic lesions.

Authors
Yano HKitayama, J; Hatano, K; Tsuno, N; Osada, T; Watanabe, T; Tsuruo, T; Muto, T; Nagawa, H
MLA Citation
Yano HKitayama, J, Hatano, K, Tsuno, N, Osada, T, Watanabe, T, Tsuruo, T, Muto, T, and Nagawa, H. "Clustered cancer cells show a distinct adhesion behavior from single cell form under physiological shear conditions." J Exp Clin Cancer Res 20.3 (September 2001): 407-412.
PMID
11718222
Source
pubmed
Published In
Journal of Experimental and Clinical Cancer Research
Volume
20
Issue
3
Publish Date
2001
Start Page
407
End Page
412

Dendritic cells activate antitumor immunity for malignant intracranial germ cell tumor: a case report.

We report a 22-year-old male patient with a history of intracranial malignant germ cell tumor (GCT) who had undergone tumor resection twice, followed by radiation and chemotherapy. The tumor had rapidly recurred along the entire ventricular wall with extensive invasion into the brain parenchyma. The serum level of human beta-chorionic gonadotropin (beta-hCG) was 232.3 ng/ml on admission. Although tissue samples of the recurrent tumor could not be obtained, the previous histological diagnosis of germinoma and elevated serum beta-hCG levels suggested recurrence of malignant GCT. The patient declined chemotherapy but accepted dendritic cell (DC)-based immunotherapy. DC inoculation five times resulted in rapid tumor shrinkage and a significant decrease in the serum level of beta-hCG. Here we discuss the effectiveness of immunotherapy using DCs for recurrent intracranial malignant GCTs.

Authors
Osada, T; Fujimaki, T; Takamizawa, M; Tsuno, NH; Kirino, T; Shibata, Y
MLA Citation
Osada, T, Fujimaki, T, Takamizawa, M, Tsuno, NH, Kirino, T, and Shibata, Y. "Dendritic cells activate antitumor immunity for malignant intracranial germ cell tumor: a case report." Jpn J Clin Oncol 31.8 (August 2001): 403-406.
PMID
11574635
Source
pubmed
Published In
Japanese Journal of Clinical Oncology
Volume
31
Issue
8
Publish Date
2001
Start Page
403
End Page
406

Thoracic empyema associated with recurrent colon cancer: report of a case and review of the literature.

Many types of infections associated with colorectal cancer have been reported. Here, we describe a rare case of thoracic empyema that was observed during immunotherapy for recurrent colon cancer. Culture of the pleural fluid yielded Streptococcus bovis, which is known to be associated with gastrointestinal lesions, especially colorectal malignancies. The possible correlation between these two clinical entities-empyema and colon cancer-is discussed.

Authors
Osada, T; Nagawa, H; Masaki, T; Tsuno, NH; Sunami, E; Watanabe, T; Muto, T; Shibata, Y
MLA Citation
Osada, T, Nagawa, H, Masaki, T, Tsuno, NH, Sunami, E, Watanabe, T, Muto, T, and Shibata, Y. "Thoracic empyema associated with recurrent colon cancer: report of a case and review of the literature." Dis Colon Rectum 44.2 (February 2001): 291-294. (Review)
PMID
11227950
Source
pubmed
Published In
Diseases of the Colon and Rectum
Volume
44
Issue
2
Publish Date
2001
Start Page
291
End Page
294

Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma.

BACKGROUND/AIMS: Glutamine synthetase (GS) catalyzes the synthesis of glutamine, a major energy source of cells, and is upregulated in a subset of human hepatocellular carcinomas (HCCs). GS expression may be related to tumor recurrence, since GS-expressing tumors have a growth advantage in that they are independent of the extracellular glutamine supply. However, there are no studies concerning the prognostic value of GS expression in patients with HCC. METHODS: Seventy-three patients with a single advanced HCC nodule who underwent curative hepatectomy were included in the study. GS expression in the HCC nodules was analyzed immunohistochemically and was compared with clinicopathologic features and the behavior of the tumors. Survival curves were assessed according to the Kaplan-Meier product-limit method and multivariate analysis based on the Cox regression model was performed. RESULTS: GS expression was strong in 26 cases (35.6%, high-GS group) and weak or absent in 47 cases (64.4%, low-GS group). Univariate analysis showed that the high-GS group had a significantly shorter disease-free survival time than the low-GS group (p=0.042). Multivariate analysis revealed that GS expression (p=0.021), as well as Child's classification (p=0.005) and portal invasion (p=0.039), was a significant and independent prognostic parameter that affected tumor recurrence. CONCLUSION: The results of this study indicate that GS expression may enhance the metastatic potential in HCC, and GS immunostaining may be helpful in identifying HCC patients at high risk for disease recurrence.

Authors
Osada, T; Nagashima, I; Tsuno, NH; Kitayama, J; Nagawa, H
MLA Citation
Osada, T, Nagashima, I, Tsuno, NH, Kitayama, J, and Nagawa, H. "Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma." J Hepatol 33.2 (August 2000): 247-253.
PMID
10952242
Source
pubmed
Published In
Journal of Hepatology
Volume
33
Issue
2
Publish Date
2000
Start Page
247
End Page
253

Cyclooxygenase-2 overexpression correlates with tumour recurrence, especially haematogenous metastasis, of colorectal cancer.

Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs), known to inhibit cyclooxygenase (COX), reduce the risk of colorectal cancer. COX is a key enzyme in prostaglandin biosynthesis, and two isoforms of COX, COX-1 and COX-2, have been identified. Recently COX-2 has been reported to frequently overexpress in colorectal neoplasms and to play a role in colorectal tumorigenesis and tumour progression. In this study, using immunohistochemistry, we examined COX-2 expression in advanced human colorectal cancer and its correlation with clinicopathological features. COX-2 expression was observed mainly in the cytoplasm of cancer cells in all the specimens examined, but some stromal cells and endothelial cells were also stained. According to the grade of COX-2 expression of the cancer cells, patients were divided into high- and low-COX-2 expression groups. High-COX-2 expression significantly correlated with tumour recurrence, especially haematogenous metastasis. These results suggest that a selective COX-2 inhibitor can be a novel class of therapeutic agents not only for tumorigenesis but also for haematogenous metastasis of colorectal cancer. To our knowledge, this is the first report on the correlation between COX-2 overexpression and recurrence of colorectal cancer.

Authors
Tomozawa, S; Tsuno, NH; Sunami, E; Hatano, K; Kitayama, J; Osada, T; Saito, S; Tsuruo, T; Shibata, Y; Nagawa, H
MLA Citation
Tomozawa, S, Tsuno, NH, Sunami, E, Hatano, K, Kitayama, J, Osada, T, Saito, S, Tsuruo, T, Shibata, Y, and Nagawa, H. "Cyclooxygenase-2 overexpression correlates with tumour recurrence, especially haematogenous metastasis, of colorectal cancer." Br J Cancer 83.3 (August 2000): 324-328.
PMID
10917546
Source
pubmed
Published In
British Journal of Cancer
Volume
83
Issue
3
Publish Date
2000
Start Page
324
End Page
328
DOI
10.1054/bjoc.2000.1270

Expression of platelet-derived endothelial cell growth factor correlates with good prognosis in patients with colorectal carcinoma.

BACKGROUND: Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor that has potent chemotactic activity for endothelial cells. Although it is expressed in the majority of colorectal tumors, and some reports suggest that its high expression is related to poor prognosis, to the authors' knowledge there is yet no consensus regarding whether PD-ECGF expression is a prognostic factor. To investigate the prognostic value of PD-ECGF and its role in tumor angiogenesis, an immunohistochemical study of PD-ECGF expression and tumor vasculature was performed and their relation with the clinicopathologic factors in patients with advanced colorectal carcinoma was evaluated. METHODS: Formalin fixed, paraffin embedded specimens from 86 colorectal carcinoma patients (40 cases in the muscularis propria and 46 cases in the subserosa) were immunostained for PD-ECGF and CD31 as a marker for vascular endothelial cells and expression of PD-ECGF was evaluated using an image analysis system. Patients were divided into high expression and low expression groups based on PD-ECGF expression, and were divided into high vascular grade and low vascular grade groups based on the microvessel density. Correlations between PD-ECGF expression and vascular grade and between PD-ECGF expression,vascular grade, and the clinicopathologic features of the patients were evaluated statistically. RESULTS: PD-ECGF expression was observed predominantly in the tumor stroma and not in tumor cells. The cells that stained strongly for PD-ECGF were confirmed to be macrophages infiltrating the interstitial tissue of the tumor. High PD-ECGF expression was found in 56 cases (65.1%) and low expression was detected in 30 cases (34.9%). Thirty-one of 86 tumors (36.0%) showed high vascular grade and 55 (64.0%) showed low vascular grade. No correlation between PD-ECGF expression and vascular grade was found, but there was an inverse correlation between PD-ECGF expression and the rate of incidence of lymph node and hematogenous metastasis. These correlations were statistically significant. Vascular grade was not found to correlate with the clinicopathologic features. CONCLUSIONS: Patients with high PD-ECGF expression had a lower rate of incidence of lymphatic and hematogenous metastasis, with a consequently better prognosis than patients with low PD-ECGF expression. PD-ECGF expression did not correlate with vascular grade, suggesting that PD-ECGF plays little role in tumor angiogenesis of colorectal carcinoma. Based on these data, the authors conclude that macrophages infiltrating the tumor stroma produce PD-ECGF and play important roles in the immune reaction against the tumor rather than in tumor angiogenesis.

Authors
Saito, S; Tsuno, N; Nagawa, H; Sunami, E; Zhengxi, J; Osada, T; Kitayama, J; Shibata, Y; Tsuruo, T; Muto, T
MLA Citation
Saito, S, Tsuno, N, Nagawa, H, Sunami, E, Zhengxi, J, Osada, T, Kitayama, J, Shibata, Y, Tsuruo, T, and Muto, T. "Expression of platelet-derived endothelial cell growth factor correlates with good prognosis in patients with colorectal carcinoma." Cancer 88.1 (January 1, 2000): 42-49.
PMID
10618604
Source
pubmed
Published In
Cancer
Volume
88
Issue
1
Publish Date
2000
Start Page
42
End Page
49

E-selectin can mediate the arrest type of adhesion of colon cancer cells under physiological shear flow.

The aim of this study was to determine whether colon cancer cells flowing in blood exhibit the same adhesion pattern to the vascular bed as leucocytes using a flow adhesion system. In shear flow conditions, five colon cancer cell lines showed less tethering to E-selectin substrates than polymorphonuclear cells (PMN). However, some of the Colo201 cells formed complete arrest on E-selectin in continuous shear flow which was never observed in PMN cells. Colo201 cells expressed both sialyl Le-x and sialyl Le-a at similar levels in flow cytometry. However, the staining pattern showed marked contrast under the fluorescein microscope. The cell membrane of Colo201 cells was uniformly stained with anti-sialyl Le-a MAb, whereas anti-sialyl Le-x MAb only stained in the patchy areas. Pretreatment of Colo201 cells with anti-sLe-a decreased tethering, while anti-sLe-x significantly inhibited the arrest formation. Our data suggest that E-selectin alone can mediate colon cancer cell lodgement and subsequent metastasis without the contribution of integrin molecules and that the different distribution of E-selectin ligands may affect the adhesion behaviour of colon cancer cells in flow conditions.

Authors
Kitayama, J; Tsuno, N; Sunami, E; Osada, T; Muto, T; Nagawa, H
MLA Citation
Kitayama, J, Tsuno, N, Sunami, E, Osada, T, Muto, T, and Nagawa, H. "E-selectin can mediate the arrest type of adhesion of colon cancer cells under physiological shear flow." Eur J Cancer 36.1 (January 2000): 121-127.
PMID
10741305
Source
pubmed
Published In
European Journal of Cancer
Volume
36
Issue
1
Publish Date
2000
Start Page
121
End Page
127

α-Glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro

α-Glycosylceramides, such as α-galactosylceramide and α- glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, Vα14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by α-glycosylceramides. We recently reported that Vα24 TCR+ NKT cells, the human homologues of murine Vα14 TCR+NK1.1+ cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether α- glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of α-glycosylceramides in cancer immunotherapy in humans. Here, we show that α-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3-CD56+ NK cells. Even though Vα24 TCR-NKT cells proliferated remarkably in response to α-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of α-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+NK cells in the liver.

Authors
Ishihara, S; Nieda, M; Kitayama, J; Osada, T; Yabe, T; Kikuchi, A; Koezuka, Y; Porcelli, SA; Tadokoro, K; Nagawa, H; Juji, T
MLA Citation
Ishihara, S, Nieda, M, Kitayama, J, Osada, T, Yabe, T, Kikuchi, A, Koezuka, Y, Porcelli, SA, Tadokoro, K, Nagawa, H, and Juji, T. "α-Glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro." Journal of Immunology 165.3 (2000): 1659-1664.
PMID
10903777
Source
scival
Published In
Journal of Immunology
Volume
165
Issue
3
Publish Date
2000
Start Page
1659
End Page
1664

MMP-1 is a prognostic marker for hematogenous metastasis of colorectal cancer.

BACKGROUND: Degradation of basement membrane and extracellular matrix by matrix metalloproteinases (MMPs) is believed to be an essential step in the complicated process of hematogenous metastasis. MMP-1 is a member of collagenases, a family of MMPs that degrades collagens type I, II, and III, main components of the interstitial stroma. The purpose of this study was to investigate the expression of MMP-1 in colorectal cancer and its correlation with hematogenous metastasis. Patients and Methods. We examined 133 cases of colorectal cancer (Dukes A: 72; Dukes B: 26; Dukes C: 23; Dukes D: 12). Sections were cut from formalin-fixed, paraffin-embedded samples containing the deepest site of cancer invasion and stained immunohistochemically with a monoclonal antibody to MMP-1. According to the area of the tumor that was stained, patients were divided into high- and low-MMP-1 expression groups. RESULTS: MMP-1 expression was observed in the cytoplasm of cancer cells, some stromal cells, and a few normal epithelial cells of colonic mucosa. High MMP-1 expression was found in 47 (35.3%) cases and low in 86 (64.7%). Hematogenous metastasis was identified in 14 (29.8%) of high-MMP-1 groups and 12 (13.9%) of low-MMP-1 groups. MMP-1 expression significantly correlated with hematogenous metastasis of colorectal cancer, but no correlation was found between MMP-1 expression and the other clinicopathological features investigated. CONCLUSIONS: MMP-1 expression may be a novel marker for hematogenous metastasis of colorectal cancer, and its inhibition may be a strategy for prevention of metastasis.

Authors
Sunami, E; Tsuno, N; Osada, T; Saito, S; Kitayama, J; Tomozawa, S; Tsuruo, T; Shibata, Y; Muto, T; Nagawa, H
MLA Citation
Sunami, E, Tsuno, N, Osada, T, Saito, S, Kitayama, J, Tomozawa, S, Tsuruo, T, Shibata, Y, Muto, T, and Nagawa, H. "MMP-1 is a prognostic marker for hematogenous metastasis of colorectal cancer." Oncologist 5.2 (2000): 108-114.
PMID
10794801
Source
pubmed
Published In
The oncologist
Volume
5
Issue
2
Publish Date
2000
Start Page
108
End Page
114

Inhibition of haematogenous metastasis of colon cancer in mice by a selective COX-2 inhibitor, JTE-522.

It is proposed that non-steroidal anti-inflammatory drugs (NSAIDs) reduce colorectal tumorigenesis by inhibition of cyclooxygenase (COX). COX is a key enzyme in the conversion of arachidonic acid to prostaglandins and two isoforms of COX have been characterized, COX-1 and COX-2. Multiple studies have shown that COX-2 is expressed at high levels in colorectal tumours and play a role in colorectal tumorigenesis. Recently it has been reported that selective inhibition of COX-2 inhibits colon cancer cell growth. In this study we investigated the effect of a selective COX-2 inhibitor (JTE-522) on haematogenous metastasis of colon cancer. For this purpose, we selected a murine colon cancer cell line, colon-26, that constitutively expresses the COX-2 protein. The subclone P expressed a high level of COX-2 and the subclone 5 expressed a low level. The colon-26 subclones were injected into the tail vein of BALB/c mice. JTE-522 was given intraperitoneally every day from the day prior to cancer cell injection, and the mice were sacrificed 16 days after cell injection. Lung metastases were compared between groups with and without JTE-522. In the mice injected with subclone P, the number of lung metastatic nodules was significantly reduced in the treated group. However, in the mice injected with subclone 5, there was little difference between the control and the treated groups. These results indicate that there may be a direct link between inhibition of haematogenous metastasis of colon cancer and selective inhibition of COX-2, and that selective COX-2 inhibitors may be a novel class of therapeutic agents not only for colorectal tumorigenesis but also for haematogenous metastasis of colon cancer.

Authors
Tomozawa, S; Nagawa, H; Tsuno, N; Hatano, K; Osada, T; Kitayama, J; Sunami, E; Nita, ME; Ishihara, S; Yano, H; Tsuruo, T; Shibata, Y; Muto, T
MLA Citation
Tomozawa, S, Nagawa, H, Tsuno, N, Hatano, K, Osada, T, Kitayama, J, Sunami, E, Nita, ME, Ishihara, S, Yano, H, Tsuruo, T, Shibata, Y, and Muto, T. "Inhibition of haematogenous metastasis of colon cancer in mice by a selective COX-2 inhibitor, JTE-522." Br J Cancer 81.8 (December 1999): 1274-1279.
PMID
10604722
Source
pubmed
Published In
British Journal of Cancer
Volume
81
Issue
8
Publish Date
1999
Start Page
1274
End Page
1279
DOI
10.1038/sj.bjc.6694262

Laminin mediates tethering and spreading of colon cancer cells in physiological shear flow.

Under the physiological shear condition, cultured colon cancer cells bound to laminin (LM), but not to fibronectin or vitronectin. Most of the tethered cells did not roll, but arrested immediately and spread within 10-30 min on LM under the continuous presence of shear flow. The tethering of Colo201 was partially inhibited by monoclonal antibodies (mAbs) to alpha6 integrin and a combination of mAbs to beta1 and beta4 integrins, but not by mAb to 67KD laminin receptor. Some Colo201 cells still tethered at 4 degrees C. This suggests that alpha6beta1 and alpha6beta4 integrins participate in Colo201 tethering on LM, although other non-integrin molecules play roles. In contrast, the spread of Colo201 was effectively inhibited by the mAbs to integrin alpha2, alpha6 and beta1 chains. The effect of anti-alpha2 plus anti-alpha6 mAbs was almost equal to anti-beta1, suggesting that Colo201 cells mainly use alpha2beta1 and alpha6beta1 integrins for spreading on LM. When the cells were perfused on subconfluent endothelial cells (HUVEC) cultured on LM, they did not tether on HUVEC but did on coated LM exposed at intercellular gap area. Immunohistochemistry revealed that LM abundantly existed in the cytosol of human portal and hepatic vein endothelial cells. These data suggest that LM can mediate from tethering to spreading of colon cancer cells under the blood flow and plays an essential role in haematogeneous metastasis.

Authors
Kitayama, J; Nagawa, H; Tsuno, N; Osada, T; Hatano, K; Sunami, E; Saito, H; Muto, T
MLA Citation
Kitayama, J, Nagawa, H, Tsuno, N, Osada, T, Hatano, K, Sunami, E, Saito, H, and Muto, T. "Laminin mediates tethering and spreading of colon cancer cells in physiological shear flow." Br J Cancer 80.12 (August 1999): 1927-1934.
PMID
10471041
Source
pubmed
Published In
British Journal of Cancer
Volume
80
Issue
12
Publish Date
1999
Start Page
1927
End Page
1934
DOI
10.1038/sj.bjc.6690622

CD8(+)NKR-P1A (+)T cells preferentially accumulate in human liver.

A unique subset of T cells that co-express NKR-P1, which is a lectin type of NK receptor and is thought to have a major role in triggering NK activity, has been identified. In mice, NK1.1 (mouse NKR-P1C)(+) T cells, called NKT cells, preferentially accumulate in the liver and bone marrow. They predominantly use invariant Valpha14 chain TCR and phenotypically are CD4(+)CD8(-) or CD4(-)CD8(-) T cells. In this study, we analyzed, phenotypically and functionally, the NKR-P1A (analogue of murine NKR-P1C)(+) T cells resident in the human liver. Here, we show that in complete contrast to the NKT cells in the mouse liver, the majority of NKR-P1A(+) T cells in the human liver are CD8(+) and their TCR repertoire is not skewed to Valpha24 TCR, the homologue of murine Valpha14 TCR. Almost all of the NKR-P1A(+) T cells in the human liver expressed CD69, suggesting that they were activated. Furthermore, the NKR-P1A(+) T cells in the human liver exhibited strong cytotoxicity against a variety of tumor cell lines including K562, Molt4 and some colonic adenocarcinoma cell lines.

Authors
Ishihara, S; Nieda, M; Kitayama, J; Osada, T; Yabe, T; Ishikawa, Y; Nagawa, H; Muto, T; Juji, T
MLA Citation
Ishihara, S, Nieda, M, Kitayama, J, Osada, T, Yabe, T, Ishikawa, Y, Nagawa, H, Muto, T, and Juji, T. "CD8(+)NKR-P1A (+)T cells preferentially accumulate in human liver." Eur J Immunol 29.8 (August 1999): 2406-2413.
PMID
10458753
Source
pubmed
Published In
European Journal of Immunology
Volume
29
Issue
8
Publish Date
1999
Start Page
2406
End Page
2413
DOI
10.1002/(SICI)1521-4141(199908)29:08<2406::AID-IMMU2406>3.0.CO;2-F

Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis.

BACKGROUND: The authors previously reported increased ubiquitin (Ub) immunoreactivity in hepatocellular carcinomas (HCCs) and suggested a possible correlation between changes in ubiquitinated protein levels and multistep hepatocarcinogenesis. The current study was performed to identify one of these ubiquitinated proteins (42 kDa) and to analyze the clinical significance of its accumulation. METHODS: The protein was purified using two-dimensional gel electrophoresis and identified by amino acid sequence analysis. The authors studied the expression of this protein in 101 HCCs and 23 precancerous lesions by immunohistochemical methods and in 26 HCCs by immunoblot analysis. A survival analysis was performed on patients with advanced HCC using the Kaplan-Meier method with approximate chi-square statistics for the log rank test. RESULTS: The target protein for ubiquitination was identified as glutamine synthetase (GS). Accumulation of GS was found in 19 of 49 advanced HCCs (38.8%) by immunohistochemical methods and in 9 of 16 (56.3%) by immunoblot analysis, whereas the frequency was much lower in early HCCs (12.9% and 33.3%, respectively) and precancerous lesions (4.3% by immunostaining). In the Ub immunoblot analysis of strongly GS positive specimens, an intense 42-kDa ubiquitinated band was observed. Nine of 21 (42.9%) nodule-in-nodule type HCCs showed a GS positive, high-grade component within a GS negative, low-grade component, indicating the acquisition of GS expression during progression. Among 23 patients with a single advanced HCC nodule, the relapse free survival time was significantly shorter in the GS positive group than in the GS negative group. CONCLUSIONS: The results of this study demonstrate the acquisition of GS expression during hepatocarcinogenesis and the possible regulation of GS enzyme activity by a Ub-dependent proteolytic system. Moreover, GS might play a significant role in promoting the metastatic potential of HCC.

Authors
Osada, T; Sakamoto, M; Nagawa, H; Yamamoto, J; Matsuno, Y; Iwamatsu, A; Muto, T; Hirohashi, S
MLA Citation
Osada, T, Sakamoto, M, Nagawa, H, Yamamoto, J, Matsuno, Y, Iwamatsu, A, Muto, T, and Hirohashi, S. "Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis." Cancer 85.4 (February 15, 1999): 819-831.
PMID
10091759
Source
pubmed
Published In
Cancer
Volume
85
Issue
4
Publish Date
1999
Start Page
819
End Page
831

Histopathological prognostic factors influencing long-term prognosis after surgical resection for hepatic metastases from colorectal cancer

OBJECTIVE: We aimed to present new histopathological features of metastatic liver nodules as more reliable prognostic factors after surgical resection for colorectal metastatic cancer. METHODS: Clinicopathological features, including newly proposed histopathological ones, of 63 consecutive patients were reviewed retrospectively to determine which most strongly correlated with long-term prognosis after hepatectomy for metastatic tumors from colorectal cancers, using univariate and multivariate analysis. RESULTS: The 1-, 3-, and 5-year cancer-related survival rates after hepatectomy were 87.8%, 55.2%, and 47.3%, respectively. New histopathological features we proposed, which are expansive growth, marginal fibrosis, and peritumorous lymphocytic infiltration of hepatic tumor, were significant prognostic factors for cancer-related survival after hepatectomy in an univariate analysis. Also in a multivariate analysis, i.e., a stepwise Cox regression analysis, infiltrative, i.e., not expansive, growth of hepatic tumor was one of significant and independent poor prognostic factors for survival after hepatectomy, with moderate to severe lymphatic vessel involvement of the primary colorectal lesion, microscopic cancer invasion at the surgical margin of hepatectomy, and extrahepatic distant metastases. CONCLUSIONS: Our results suggest that our proposed new histopathological features of hepatic metastases were good predictors of prognosis after surgical resection for hepatic metastases from colorectal cancer. Especially, infiltrative growth of hepatic tumor is strongly correlated with a poor prognosis after hepatectomy.

Authors
Nagashima, I; Oka, T; Hamada, C; Naruse, K; Osada, T; Muto, T
MLA Citation
Nagashima, I, Oka, T, Hamada, C, Naruse, K, Osada, T, and Muto, T. "Histopathological prognostic factors influencing long-term prognosis after surgical resection for hepatic metastases from colorectal cancer." American Journal of Gastroenterology 94.3 (1999): 739-743.
PMID
10086660
Source
scival
Published In
The American Journal of Gastroenterology (Elsevier)
Volume
94
Issue
3
Publish Date
1999
Start Page
739
End Page
743
DOI
10.1016/S0002-9270(98)00826-0

Increased ubiquitin immunoreactivity in hepatocellular carcinomas and precancerous lesions of the liver.

BACKGROUND/AIMS: Ubiquitin covalently attaches to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Increased ubiquitin immunoreactivity was recently observed immunohistochemically in human malignant tumors. To clarify the change in protein metabolism during hepatocarcinogenesis, we studied ubiquitin immunoreactivity in hepatocellular carcinomas (HCCs) and precancerous lesions using immunohistochemistry and immunoblot analysis. METHODS: A total of 72 HCCs (37 advanced, 19 early, 16 early-advanced (advanced HCC component in early HCC nodule) type HCCs) and 18 precancerous lesions (8 atypical adenomatous hyperplasias (AAHs), 10 adenomatous hyperplasias (AHs)) were studied immunohistochemically. Immunoblot analysis was also performed in advanced HCC and early HCC cases. RESULTS: Non-tumorous hepatocytes were either immunonegative or weakly stained in their nuclei. Advanced HCCs showed strong immunoreactivity in most cases, while early HCCs showed relatively weaker immunoreactivity. In 14 of 16 early-advanced type tumors, the inner portion of the nodules, which corresponds to advanced HCC, showed stronger immunoreactivity than the outer low-grade portion. In 8 of 8 AAHs and 7 of 10 AHs, positive but weak staining was found. Immunoblot analysis showed an increase in 42 kDa ubiquitinated protein(s) in 8 of 16 advanced HCC cases (50%) and in 1 of 6 early HCC cases (16.7%), as well as an increase in several other bands in tumor tissues. CONCLUSIONS: The intensity of ubiquitin staining appeared to increase in a stepwise manner from AH to advanced HCC, and the results suggest a possible correlation between changes in the ubiquitinated proteins and multistep hepatocarcinogenesis.

Authors
Osada, T; Sakamoto, M; Nishibori, H; Iwaya, K; Matsuno, Y; Muto, T; Hirohashi, S
MLA Citation
Osada, T, Sakamoto, M, Nishibori, H, Iwaya, K, Matsuno, Y, Muto, T, and Hirohashi, S. "Increased ubiquitin immunoreactivity in hepatocellular carcinomas and precancerous lesions of the liver." J Hepatol 26.6 (June 1997): 1266-1273.
PMID
9210613
Source
pubmed
Published In
Journal of Hepatology
Volume
26
Issue
6
Publish Date
1997
Start Page
1266
End Page
1273

Immunoreaction at 43 kDa with anti-ubiquitin antibody in breast neoplasms.

Protein ubiquitination has been implicated in ATP-dependent protein turnover and normal cell proliferation. To investigate whether the ubiquitin-mediated system is functionally involved in the cancerous state, we examined changes in protein ubiquitination in 52 surgically resected primary breast tumors. Immunohistochemically, ubiquitin (Ub) was identified in the cytoplasm of cancer cells, which were stained more strongly than adjacent normal ductal epithelium. Corresponding immunoblot analysis of normal and neoplastic regions of human breast showed that the immunoreaction for Ub at about 43 kDa was increased in all of the tumors (100%), regardless of the clinical stage or histologic grade. This protein, which gave a single spot on two-dimensional gel electrophoresis, had partial amino acid sequences which were identical to those of actin family members. Our results suggest that ubiquitination of this 43-kDa protein may be involved in the carcinogenesis or biological characteristics of human breast neoplasms.

Authors
Iwaya, K; Nishibori, H; Osada, T; Matsuno, Y; Tsuda, H; Sato, S; Kono, H; Fukutomi, T; Suzuki, M; Torikata, C; Iwamatsu, A; Hirohashi, S
MLA Citation
Iwaya, K, Nishibori, H, Osada, T, Matsuno, Y, Tsuda, H, Sato, S, Kono, H, Fukutomi, T, Suzuki, M, Torikata, C, Iwamatsu, A, and Hirohashi, S. "Immunoreaction at 43 kDa with anti-ubiquitin antibody in breast neoplasms." Jpn J Cancer Res 88.3 (March 1997): 273-280.
PMID
9140112
Source
pubmed
Published In
Japanese journal of cancer research : Gann
Volume
88
Issue
3
Publish Date
1997
Start Page
273
End Page
280

E-cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma.

In human hepatocellular carcinoma (HCC), the liver is the major target organ of metastasis, which is known as intrahepatic metastasis. To analyze the mechanism of this metastasis, we established two sublines from the human HCC cell line Li7. Subline Li7HM produced multiple liver metastasis, whereas subline Li7NM never did so after intrasplenic injection into nude mice. Two-dimensional gel electrophoresis and immunoblot analysis showed that only Li7NM expressed vimentin and lacked E-cadherin expression, indicating that this clone had undergone epithelial-mesenchymal transition. We then transfected mouse E-cadherin complementary DNA into Li7NM cells and found that the transfectant cells (EM16.21B.3) formed liver metastasis (8/16 mice) after intrasplenic injection and liver tumors (11/13 mice) after intrahepatic injection, whereas the control cell line formed no tumors. These results suggest that E-cadherin plays an important role in the process of intrahepatic metastasis of HCC.

Authors
Osada, T; Sakamoto, M; Ino, Y; Iwamatsu, A; Matsuno, Y; Muto, T; Hirohashi, S
MLA Citation
Osada, T, Sakamoto, M, Ino, Y, Iwamatsu, A, Matsuno, Y, Muto, T, and Hirohashi, S. "E-cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma." Hepatology 24.6 (December 1996): 1460-1467.
PMID
8938181
Source
pubmed
Published In
Hepatology
Volume
24
Issue
6
Publish Date
1996
Start Page
1460
End Page
1467
DOI
10.1053/jhep.1996.v24.pm0008938181

Human colorectal carcinomas specifically accumulate Mr 42,000 ubiquitin-conjugated cytokeratin 8 fragments.

Recent studies have shown that various tumor cells accumulate ubiquitin (Ub)-conjugated proteins, the profiles of which differ from those of normal cells. To identify the Ub-conjugated proteins accumulated specifically by human carcinoma cells, a two-dimensional immunoblot analysis of 31 surgically resected human primary colorectal carcinoma tissues was performed using an anti-Ub monoclonal antibody, KM691. Two distinct Mr 42,000 and 45,000 proteins in the Triton X-insoluble fractions of carcinoma tissues reacted with this antibody, whereas only one Mr 45,000 protein reacted in normal tissues. The Mr 42,000 Ub-conjugated proteins were specific to carcinoma tissues from 25 patients (80.6%). One of the purified Mr 42,000 proteins was digested with Achromobacter protease I. This protein was identified as a cytokeratin 8 (CK 8) fragment based on both molecular mass determination and molecular mass searching of Achromobacter protease I-digested fragments of proteins registered in a protein sequence data base. Two-dimensional immunoblot analysis with an anti-CK 8 antibody confirmed that all of the Mr 42,000 proteins were CK 8 degradation products. These results demonstrate that human colorectal carcinomas specifically accumulate Mr 42,000 Ub-conjugated CK 8 fragments. This accumulation was observed frequently not only in advanced (18/22, 81.8%), but also in early stage cases (7/9, 77.8%), suggesting that it occurs even in the early stages of colorectal carcinoma progression.

Authors
Nishibori, H; Matsuno, Y; Iwaya, M; Osada, T; Kubomura, N; Iwamatsu, A; Kohno, H; Sato, S; Kitajima, M; Hirohashi, S
MLA Citation
Nishibori, H, Matsuno, Y, Iwaya, M, Osada, T, Kubomura, N, Iwamatsu, A, Kohno, H, Sato, S, Kitajima, M, and Hirohashi, S. "Human colorectal carcinomas specifically accumulate Mr 42,000 ubiquitin-conjugated cytokeratin 8 fragments." Cancer Res 56.12 (June 15, 1996): 2752-2757.
PMID
8665509
Source
pubmed
Published In
Cancer Research
Volume
56
Issue
12
Publish Date
1996
Start Page
2752
End Page
2757

Surgical resection for small hepatocellular carcinoma.

BACKGROUND: Surgical resection for hepatocellular carcinoma (HCC) can be curative in selected patients, particularly in those with a solitary small HCC (s-sHCC; 2 cm or less in diameter). However, even these patients often have a risk of tumor recurrence or death from underlying liver dysfunction. Therefore it is important to determine which clinicopathologic features are related to the long-term prognosis after resection of s-sHCC. METHODS: Fifty patients with s-sHCC underwent partial hepatectomy at our department between 1977 and 1992. Six (12%) died of liver failure in hospital after operation. Eight clinicopathologic features were examined in the remaining 44 patients with regard to their long-term prognosis by use of univariate and multivariate analyses. RESULTS: The 1-, 3-, and 5-year survival rates were 90%, 75%, and 53%, respectively. The corresponding disease-free survival rates were 80%, 53%, and 30%, respectively. None of the following parameters was significantly related to survival rate or disease-free survival rate: presence of vascular invasion or capsular formation, the distance of free surgical margin (1 cm or more or not), serum alpha-fetoprotein level, positive hepatitis B surface antigen, and preoperative transarterial embolization. Complicated liver function was the only significant factor related to survival rate and disease-free survival rate. CONCLUSIONS: A good hepatic reserve is an important factor in treating patients with s-sHCC by surgical resection, even for a long-term prognosis. Liver transplantation should be considered for patients with severe cirrhosis and s-sHCC, even though a curative resection might be possible.

Authors
Nagashima, I; Hamada, C; Naruse, K; Osada, T; Nagao, T; Kawano, N; Muto, T
MLA Citation
Nagashima, I, Hamada, C, Naruse, K, Osada, T, Nagao, T, Kawano, N, and Muto, T. "Surgical resection for small hepatocellular carcinoma." Surgery 119.1 (January 1996): 40-45.
PMID
8560384
Source
pubmed
Published In
Surgery
Volume
119
Issue
1
Publish Date
1996
Start Page
40
End Page
45
Show More