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Cd8 Tcells engineered to selectively attack surface antigen producing c...
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Cd8 Tcells engineered to selectively attack surface antigen producing cells.

This abstract from the APASL might be the most fundamentally promising of all. Epitope specific T cells can be engineered in large scale and without dangerous genetic manipulations that will attack surface antigen producing liver cells and also produce interferon gamma upon contact with these cells, which could clear neighboring cells from cccDNA.


Engineering T Cell Immunity Targeting HBV-expressing Hepatocellular Carcinoma for Clinical Use

Sarene Koh1,2, Noriko Shimasaki3, Rossarin Suwanarsuk4, Laurent Renia4, Adam Gehring1, Matti Sallberg2, Dario Campana3, Antonio Bertoletti1
1Singapore Institute for Clinical Sciences (A*STAR), Singapore, Singapore, 2Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, 3Department of Paediatrics, National University of Singapore, 4Singapore Immunology Network (A*STAR), Singapore, Singapore

Background/aims: The therapeutic efficacy of tumor-specific T-cell adoptive transfer for hepatocellular carcinoma (HCC) therapy needs to be evaluated before clinical translation. We previously showed that lymphocytes from patients with hepatitis B virus (HBV)-related HCC can be engineered to express HBV-specific T-cell receptors (TCR) through retroviral mediated gene transfer, and were capable of recognizing HCC cells expressing HBV-viral antigens from integrated HBV DNA. Due to potential safety issues that could limit the use of viral vectors in clinical application, we explored a safer and easier alternative strategy to redirect the specificity of T cells using mRNA electroporation.
Methods: Activated T cells were transfected with mRNA encoding HBV s183-191 TCR. HBV-specific TCR expression and function of electroporated cells were tested in vitro and in vivo in a xenograft tumor model produced by injecting HCC cell line constitutively expressing HBV surface antigen and luciferase into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. The kinetics of tumor development in vivo was measured by bioluminescence imaging.
Results: Approximately 90% of CD8 T cells expressed HBV TCR 24 h post-electroporation, 3 fold higher than what retroviral transduction routinely achieved. Electroporated T cells produced IFN-gamma, TNF-alpha and IL-2 after HBV-specific recognition, with a large contribution from transfected CD4 T cells. Cytokine production capacity, determined by mean fluorescence intensity, and cytotoxic activity was similar to that of retroviral transduced T cells. In vivo experiments showed electroporated T cells could suppress the growth of established tumors in mice.
Conclusions: Messenger RNA electroporation is a powerful tool to introduce TCR into human T lymphocytes with high efficiency. Electroporated cells are fully functional in vitro and in vivo. As stable integration of genes into host genome is avoided, this technology is particularly attractive for clinical translation since it can rapidly generate at low cost, large batches of antigen-specific T cells for immunotherapy.
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Avatar_m_tn
A very technical article to comprehend. So need to ask:
1. Would the technique only work for lmphocytes from patients with hepatitis B virus (HBV)-related HCC ?
2. "Activated T cells were transfected with mRNA encoding HBV s183-191 TCR. ". What is meant by "activated"? s183-191 is part of the HBV surface antigen?
3. How are these transfected T cells made to multiply in vitro? How long would it take to culture sufficient quantity to be used in therapeutic setting?

The paper seems to rely on the assumption that for HCC in HBV patients, the tumor cells consist of integrated hbvdna, thereby all tumor cells express hbv viral antigens. Is this a proven fact, especially that ALL tumor cells contain integrated hbvdna genome?

Finally why not simply test on HBV animal model instead of HCC (related to HBV) model? Is it because these engineered  T cells may wipe out all HBV infected cells?

Many thanks in advance.
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There is a study which shows HCC cells contained 80% of its normal DNA replaced by HBV DNA.    

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Are you sure? Replaced is very different to integrated.
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Researchers Discover How HBV Integrates to Cause Cancer
The Asian Cancer Research Group, a nonprofit research organization
funded by an international consortium of pharmaceutical companies, has discovered exactly how the HBV integrates into liver cells' genetic blueprint to cause abnormal cell growth or cancer.In this study, researchers
sequenced the entire genome (which contains all the DNA and information
needed to build and maintain an organism) of cancerous liver cells from
people with hepatitis B to find out how the virus integrates into liver cells and causes cancer.
Researchers found HBV had integrated into 86.4% of liver tumors' cancer cells. Meanwhile, the integration rate was only 30.7% in adjacent, cancerfree liver cells. Researchers also identified the genes—including
three new ones—that appear to help HBV integrate into liver cells and spur
tumor growth.  The discovery should help researchers better understand the molecular mechanisms and clinical impact of HBV integration, which could lead to more effective liver cancer treatments. In 2008, liver cancer killed 695,000 people worldwide.
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1. Would the technique only work for lmphocytes from patients with hepatitis B virus (HBV)-related HCC ?
Each electropretically introduced messenger RNA will produce the specific Tcell receptor for the specific class I epitope it was made for. In this case they engineered them to recognize a particular class i epitope from the HBv surface protein. This cd8 cell will recognize any cell that will present this particular epitope on its surfaced in the MHC class I context (embedded in the presenting groove of the MHC I surface protein complex).

The reason they are using HBV HCC as current therapy target is simply, that it is easier to start clinical trials on such patients due to the pressing need and the threat of upcoming death.

They have previously introduced the specific TCRs using a transfer vector  virus,  virus mediated gene transfer-, that holds many safety risks and is unlikely to translate into a treatment for chronic hepatitis.

This new technique uses the a method where the cells are suspended in a solution of the TCR messenger RNa, then stronf electric field pulses are applied, that introduce short temporary rifts in the cell membrane, allowing outside material - the messenger RNA-  to slip in. The rifts close rapidly, due to the self closing feature of membrane blebs.

Since the proteins produced are made from the messenger RNA, not from gene expression, these transfected lymphocytes will not be able to multiply, since the mRNA will be diluted out with each cell division.
Thus enough needs to be made ex vivo for the therapy session, and the cd8 recipient lymphocytes need to be expanded ahead of the electroporation to the high numbers needed for the in vivo application.

2. "Activated T cells were transfected with mRNA encoding HBV s183-191 TCR. ". What is meant by "activated"? s183-191 is part of the HBV surface antigen?
The cells are activated by a cytokine cocktail prior to electroporation.
The hbv s183-191 is indeed one of the well known class i surface antigen epitopes, a peptide processed for presentation on the MHCs on the hepatocyte surfaces.

3. How are these transfected T cells made to multiply in vitro? How long would it take to culture sufficient quantity to be used in therapeutic setting?

See above and regarding the efficiency of the process we have to rely on their statement that high numbers of these Tcells can be easily made for clinical applications.

why not simply test on HBV animal model instead of HCC (related to HBV) model? Is it because these engineered  T cells may wipe out all HBV infected cells?

they will probably soon come out with hbv infected chimeric mice, treated with these human engineered Tcells.

Yes, these Tcells will wipe out all surface antigen carrying cells, that means mostly all infected cells, if supplied in high enough numbers.

Please note, that these Tcells are fundamentally different from the engineered Tcells of Ulrike Protzer in Munich, which uses an antibody against surface antigen Bcell epitopes fused to the TCR. This is unlikely to have any realistic future application in vivo.

The Bertoletti approach reported here however is fascinating, because it mimics the natural clearance of HBV, in an externally controlled fashion and it should have very very low risk. These Tcells are almost like the ones that clear the infection in case of acute HBV. Hopefully they will overcome all the practical hurdles to produce sufficient numbers and the needed variety to mimic this clearance in chronic patients.







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Avatar_m_tn
"There is a study which shows HCC cells contained 80% of its normal DNA replaced by HBV DNA. "

No, it means that 80 percent of the HCC samples had some  integration of HBV into its genome.
In a similar study there was almost no difference found in the integration frequency between tumor and surrounding liver tissue. But the integration sites were more targeted to oncogenic places in the genome.
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Avatar_m_tn
What clinics do this procedure?
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Avatar_m_tn
It sounds like a real breakthrough and very much looking forward to HBV studies.  Two things that stand out other than the explanation that you gave, are low cost and rapid production.  Are we to expect a lot more studies on this approach in the near future since they can engineer these cells with minimal effort?    

Can they not use this therapy for a lot of tumors/cancers?  I understand that they are targeting HBV expressing HCC, just wondering if can be used on others.  
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Avatar_m_tn
I am sure the Bertoletti group works with maximum effort to pursue this technology within the limits of its staff and budgets.

Obtaining and producing the messenger RNA that codes for a very particular Tcell receptor for a particular class I epitope is not at all trivial. It has to be done by cloning and transcribing  from a cd8 Tcell that carries that adapted TCR gene for that epitope naturally.

The use of this technology for the HBV HCC takes advantage of the fact that the surface antigen is expressed in the majority of these cancer cells and literally in all of them in a particular cancer of a given patient, since cancers are normally clones from single cells.

As such other cancers that express unique marker proteins with known class I epitopes, that are not present in normal tissues, can be approached by this methodology.
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Avatar_m_tn
This is still research work in its early stages in the Bertoletti lab in Singapore.
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Avatar_m_tn
Thanks for explanation.  Can you expound a little on you statement about needed variety?  Do you mean different epitopes?  
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Avatar_m_tn
Needed variety means different epitopes. Also, epitopes are of varying capacity to elicit a response in terms of TCR response strength, density of presentation on the MHCs, availability from cytosolic processing of proteins  ( proteasome cutting efficacy, relative abundance of soluble protein units)  into the class I pathway.

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Avatar_m_tn
I stand corrected.  Sounds like i should be saying intergrated.  I am most impress by studyforhope knowledge and explanation.  
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Avatar_m_tn
It does seem the concept had been tested in the mouse model by Protzer et al using a virus to deliver the gene.


T Cells Expressing a Chimeric Antigen Receptor That Binds Hepatitis B Virus Envelop Proteins Control Virus Replication in Mice.
Authors
Krebs K, Böttinger N, Huang LR, Chmielewski M, Arzberger S, Gasteiger G, Jäger C, Schmitt E, Bohne F, Aichler M, Uckert W, Abken H, Heikenwalder M, Knolle P, Protzer U.
Journal

Gastroenterology. 2013 Apr 29. pii: S0016-5085(13)00684-7. doi: 10.1053/j.gastro.2013.04.047. [Epub ahead of print]
Affiliation

Institute of Virology, Technische Universität München / Helmholtz Zentrum München, Trogerstr. 30, 81675 München, Germany.
Abstract

BACKGROUND & AIMS: Antiviral agents suppress hepatitis B virus (HBV) replication but do not clear the infection. A strong effector T-cell response is required to eradicate HBV, but this does not occur in patients with chronic infection. T cells might be directed toward virus-infected cells by expressing HBV-specific receptors, and thereby clear HBV and help to prevent development of liver cancer. In mice, we studied whether redirected T cells can engraft following adoptive transfer, without prior T-cell depletion, and whether the large amounts of circulating viral antigens inactivate the transferred T cells or lead to uncontrolled, immune-mediated damage.

METHODS: CD8(+) T cells were isolated from mice and stimulated using an optimized protocol. Chimeric antigen receptors (CARs) that bind HBV envelope proteins (S-CAR) and activate T cells were expressed on the surface of cells using retroviral vectors. S-CAR-expressing CD8(+) T cells, which carried the marker CD45.1, were injected into CD45.2(+) HBV transgenic mice. We compared these mice with mice that received CD8(+) T cells induced by vaccination, cells that express a CAR without a proper signaling domain, or cells that express a CAR that does not bind HBV proteins (controls).

RESULTS: CD8(+) T cells that expressed HBV-specific CARs recognized different HBV subtypes and were able to engraft and expand in immune-competent HBV transgenic mice. Following adoptive transfer, the S-CAR-expressing T cells localized to and functioned in the liver; they rapidly and efficiently controlled HBV replication, compared with controls, causing only transient liver damage. The large amount of circulating viral antigen did not impair or over-activate the S-CAR grafted T cells.

CONCLUSION: T cells with a CAR specific for HBV envelop proteins localize to the livers of mice to reduce HBV replication, causing only transient liver damage. This immune-cell therapy might be developed for patients with chronic hepatitis B, regardless of their HLA-type.
Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.
PMID
23639914 [PubMed - as supplied by publisher]
Full text: Elsevier Science
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Please note that Protzners Tcells have an unnatural chimeric construct using a fused antibody instead of the regular MHC-peptide recognizing TCR. They are strangely not activated by MHC presentation, but by binding to full size surface proteins on their Bcell epitopes. It relies on the fact, that some of the surface antigen subunits do not get processed into particles , but float lonely on the cell membrane of infected liver cells.
This is certainly a very interesting idea, it has no equivalent in nature. the mouse studies seem promising, but i have heard, that there are substantial safety problems with this approach.
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