809 time for TEA: t cells expanded on artificial antigen presenting cells?

809 Time for TEA T Cells Expanded on Artificial Antigen Presenting Cells? Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S312 CANCER IMMUNO[.]

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S312

CANCER - IMMUNOTHERAPY III

Jurkat clones, we demonstrate that this drug combination does not

signi cantly alter the expression of endogenous genes nearest to the

proviral integration site

806 Highly Ef cient Expression and

Puri cation of Hepatitis C Virus Core Protein in

E.coli

Yixin Bian,1 Guozhen Qiu,1 Tingting Li,1 Zixuan Chen,1 Wenjing

Wang,1 Chengyao Li.1

1 Southern Medical University, Guangzhou, Guangdong, China.

Hepatitis C virus (HCV) infection is major cause of chronic liver

diease and hepatocellular carcinoma, but currently there are no

prophylactic HCV vaccines available Therefore, the early diagnosis

and the early treatment are critical to hepatitis control Although

there are numerous commercial HCV diagnostic antigens available,

their speci city and sensitivity need to be improved HCV core

protein-encoding sequence is among the most conservative genes

in the HCV genome The core protein localizes to the endoplasmic

reticulum (ER) through a C-terminal hydrophobic region that is

cotranslationally inserted into the ER membrane In this study, a 360bp

cDNA encoding 120 amino acid of HCV type-1b core protein, which

lacked the C-terminal hydrophobic region, was ampli ed by PCR, and

then cloned into an expression plasmid pET-28a(+) The recombinant

plasmid pET-28a-core120 was transduced into E.coli BL21 The

expression of the recombinant core protein was induced by IPTG

Then the recombinant protein was puri ed by Ni+ -af nity column

and renatured by G-25 molecular sieve Finally, the expressed HCV

core protein, accounting for 30% of the total protein was identi ed

using SDS-PAGE, western blot and ELISA The results revealed its

speci c immunoreactivity with serum from patients with hepatitis

C In conclusion, high level of the truncated HCV core protein was

expressed in E.coli with high HCV speci city and good antigenicity

It may suggest the application on studying and developing of HCV

diagnostic antigens

807 Molecular Epidemiology of HCV Infection

among Recovery and Chronic Blood Donors in

Guangdong, China

Tingting Li,1 Jinfeng Zeng,2 Zixuan Chen,1 Lifang Shuai,1 Anqi

1 School of Biotechnology, Southern Medical University,

Guangzhou, Guangdong, China; 2 Shenzhen Blood Center,

Shenzhen, Guangdong, China.

Hepatitis C virus (HCV) infection is gaining importance in Asian

countries in recent years But epidemiological studies conducted on

recovery and chronic blood donors with HCV infection in China are

little known A study was carried out to determine the ratio between

HCV chronic and recovery infection in blood donors from Guangdong,

a province in southern China HCV genotyping and phylogenetic

analyses were also performed A total of 122 antibody positive

plasmas detected by both Lizhu and Ortho anti-HCV EIAs were tested

for viral load, con rmed by Nested-PCR, and then classi ed into

two statuses, chronic or recovery infection Approximately 37.5% of

con rmed anti-HCV carriers had no detectable viral RNA and were

considered to have cleared the virus and recovered from the infection

Chronic samples were ampli ed by 5’-NCR PCR and sequenced for

genotyping (215-218bp) The result showed genotype 1 (29.3%),

2(8.3%), 3 (29.3%), 6 (33.3%) was present in chronic blood donors

This initial study will help us to understand HCV infection among

recovery and chronic blood donors in China

808 Occult Hepatitis B Virus Infection in Shenzhen, China

Xin Zheng,1 Yixin Bian,2 Ling Zhang,1 Wenjing Wang,1 Lifang Shuai,3 Anqi Wang,1 Jean-Pierre Allain,4 Daniel Candotti,4

Chengyao Li.1

1 School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China; 2 Shenzhen Blood Centre, Shenzhen, China; 3 Guangzhou Centre of Disease Control of PLA, Guangzhou, China; 4 Department of Haematology, London, United Kingdom.

Characterization of occult HBV infection(OBI) in blood donors in China remains far unknown This study is to explore the molecular characterization of occult hepatitis B virus (HBV) infection in blood donors in Shenzhen, China The HBsAg negative donors plasmas collected for HBV/HCV/HIV screening from 2003 by the ELISA were detected for HBV DNA by Nucleic Acid Testing (NAT) in blood donors from Shenzhen blood center All samples of China origin OBIs were quanti ed for HBV DNA loads by real-time PCR(QPCR) The ampli ed basic core promoter/precore, pre-S/S, and whole genome were cloned and sequenced, phylogenetic analysis were performed based on one to three sequences of HBV clones Twenty-nine samples

of OBI screened from 165371 donors were con rmed and genotyped, according to sequences of Pre-S/S region Genotype C was more frequent (8 strains) than genotype B (5 strains) OBI donors were obtained for 8 females (27.6%) and 21males(72.4%) Viral load ranged between unquanti able and 7321IU/ml (median 17.4IU/ml)

Sequence analyses of full length genome showed occult isolates were variants from clones The amino acid substitutions of core and pre-s/s open reading frame in OBI were more frequent than HBsAg+

sample in genotype B(P< 0.03), but not in the core region of genotype C(P>0.05) Frequency of diversity of core regular element in OBIs was signi cantly higher than that in wildtype HBV(P<0.01) Amino acid substitutions were concentrated in the immunoreactive regions

of the Pre-S/S and core proteins.Twelve OBI samples were mutated

in B cell epitope and CD8+ T cell epitope of pre-S/S while 7 OBI samples were mutated in B cell epitope and CD4+ Th cell epitope and CTL of core region There were deletions in pre-s/s and insertion

in CURS region.The results suggested that mutations happened in regular element and immunoreactive region on the HBV pre-S/S and core proteincould affect HBV replication

Cancer – Immunotherapy III

809 Time for TEA: T Cells Expanded on Arti cial Antigen Presenting Cells?

Hillary N Gibbons,1 Carrie Yuen,1 Sergio Giralt,2 Carl H June,3,4

Laurence J N Cooper.1

1 Pediatrics, M.D Anderson Cancer Center, Houston, TX; 2 Stem Cell Transplantation, M.D Anderson Cancer Center, Houston, TX;

3 Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA; 4 Abramson Cancer Research Institute, University of Pennsylvania Medical Center, Philadelphia, PA.

Patients who rapidly recover their absolute lymphocyte count (ALC) after ablative therapy have improved disease-free and event-free survival Therefore, we developed a method using arti cial antigen presenting cells (aAPC) to numerically expand T cells from peripheral blood mononuclear cells (PBMC) and umbilical cord blood (UCB) for adoptive transfer A clinically-appealing approach

to rapidly expand T cells was based upon using banked clinical-grade aAPC derived from irradiated K562 that had been modi ed to express the T-cell costimulatory molecules CD86, and 41BBL and membrane bound version of IL-15 By co-expressing the high af nity Fc receptor, CD64, we were able to load desired monoclonal antibodies, such as

clinical grade anti-CD3 (OKT3) to provide a proliferative signal The T-cell to aAPC ratio was systematically varied (from 10:1 to 1:2) and

T cells were numerically expanded up to 105 fold

Phenotype was examined as a measure of persistence and function

Surprisingly, varying the ratios of aAPC changed the relative outgrowth of CD4+ versus CD8+ T cells The cultures containing the fewest aAPC (10:1) yielded more CD8+ T cells, while the culture with the most aAPC (1:2) yielded more CD4+ T cells (p value = 05)

The aAPC supported the expansion of T-cell populations containing central memory T cells (CD95+CD28+CD62L+) and effector memory

T cells (CD95+CD28negCD62Lneg), suggesting that the culture conditions support a range of T-cell differentiation There were more central memory T cells (CD4+CD62L+CCR7+) when cultured with the fewest aAPC (p value = 0.03) Since programmed death 1 (PD-1) expression is correlated with an inferior anti-tumor response, we demonstrated that T cells cultured with the fewest aAPC showed less PD-1 expression To evaluate function, the T cells were activated and found to produce IL-2 and IFN-g with minimal production of IL-10 and TGF-b These data demonstrate that OKT3-loaded aAPC can

be used to ef ciently propagate T cells containing desired subsets

in a clinically-amenable approach, given that the aAPCs have been prepared as a master cell bank The observation that different ratios

of CD4 and CD8 T cells results in culture based on the aAPC ratio provides a platform to generate desired mixes of these T cells to enhance a therapeutic effect

810 Oxaliplatin Increases the Antitumor Effect

of Vector-Delivered Interleukin-12 Expression

in a Murine Model of Hepatic Colorectal Cancer Metastases

Manuela Gonzalez-Aparicio,1 Itsaso Mauleon,1 Pilar Alzuguren,1

Sandra Hervas-Stubbs,1 Uxua Manchengo,1 Julien Crettaz,1

Gloria González-Aseguinolaza,1 Jose Medina-Echeverz,1 Pedro Berraondo,1 Jesus Prieto,1,2 Ruben Hernandez-Alcoceba.1

1 Gene Therapy and Hepatology, FIMA, Pamplona, Navarra, Spain; 2 CIBERehd, University Clinic of Navarra, Pamplona, Navarra, Spain.

Objectives: The appearance of liver metastases is one of the leading causes of death in colorectal cancer patients New therapeutic options are needed for the management and prevention of this disease Interleukin-12 (IL-12) is an immunostimulatory cytokine with proven antitumor effect in different animal models of this disease Despite evidences indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant bene t in cancer patients The dif culties in obtaining a suitable expression pattern and the lack of co-stimulatory factors contribute to this poor performance We propose a new approach to overcome these limitations

Methods: A High-Capacity (“gutless”) Adenoviral vector carrying a liver-speci c, Mifepristone-inducible system for the expression of

IL-12 (HC-Ad/RUmIL-IL-12) was used in combination with chemotherapy (oxaliplatin or gemcitabine) Tumors were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells Results: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimes allowed the maintenance of safe and ef cient levels

of IL-12 in vivo Two cycles of 10 daily inductions were needed to eradicate hepatic lesions A robust stimulation of speci c CD8+ T cell responses was observed, but complete protection against tumor re-challenge was only observed in less than 25% of the animals The incorporation of oxaliplatin (5 mg/kg) three days before starting the induction regime achieved (i) increased stimulation of the immune response against cancer cells (ii) efficient elimination of liver metastases with a single cycle of IL-12 induction and (iii) improved protection or response against an experimental tumor re-challenge

In contrast, gemcitabine (60 mg/kg) cooperated with a single cycle of IL-12 expression, but was not bene cial in the long-term management

of tumor-bearing mice, in part due to the toxicity of the combined protocol

Conclusions: Long-term controlled expression of IL-12 using a

HC-Ad vector in combination with oxaliplatin is effective and clinically applicable against hepatic colon cancer metastases

811 Optimizing Dendritic Cell Vaccination

by Combination with Oncolytic Adenovirus Coexpressing Interleukin-12 and Granulocyte-Macrophage Colony Stimulating Factor

Song-Nan Zhang,1 Jing-Hua Huang,2 Ji Young Yoo,3 Kyung-Ju Choi,4 Chae-Ok Yun.5

1 Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea; 2 Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea; 3 Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea; 4 Brain Korea

21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea; 5 Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea.

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy However, clinical trials have indicated that DC vaccination alone is insuf cient to effectively treat cancer patients Immunosuppressive microenvironments induced by tumors

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