Tag Archives: T-cell

From clinic to mouse to clinic: New HIV gene therapy shows promise!

Yesterday a team of University of Pennsylvania researchers – led by Dr Pablo Tebas, Professor Carl June, and Dr Bruce Levine – announced the successful conclusion of a clinical trial to evaluate the safety of a new gene therapy technique for treating HIV. It is a result that may eventually allow millions of HIV positive people to control the infection without having to take daily medication.

Two technicians in Penn Medicine's Clinical Cell and Vaccine Production Facility hold up a bag of modified T cells. Image: Penn Medicine

Two technicians in Penn Medicine’s Clinical Cell and Vaccine Production Facility hold up a bag of modified T cells. Image: Penn Medicine

Their study, published in the New England Journal of Medicine, involved taking a sample of T-cells from 12 patients and then using an adenoviral vector to introduce into these cells an enzyme known as a zinc-finger nuclease (ZFN) that has been targeted to the CCR5 receptor gene so that it introduces a mutation called CCR5-delta-32.  They then expanded the number of T-cells in vitro until they had billions of the transformed T-cells ready for transplant back into the patients.

Most HIV strains need to bind to CCR5 to infect T-cells, and the CCR5-delta-32 mutation prevents this binding and subsequent infection, as was dramatically demonstrated in the case of the “Berlin patient”, so the Pennsylvania team are hoping that their method will enable long-term control of HIV infection in patients, so that they may no longer need to take anti-retroviral medication.

An important part of the development of this therapy was its evaluation in vivo in an animal model of HIV infection. To do this they turned to mice rather than the more usual SIV/macaque model, as the sequence of the CCR5 gene at the site targeted by ZFN in macaques is not conserved with humans and would require the design and assembly of a distinct ZFN binding set for testing in SIV infection. Mice don’t normally become infected with HIV, but by using NOG mice that have been genetically modified so that their own immune system do not develop and then transplanting human immune cells into the mice they were able to produce mice with “humanized” immune systems that could be used to evaluate the ability of their ZFN modified T-cells to block HIV infection. In a paper published in the journal Nature Biotechnology in 2008, the team led by Carl June reported that the transformed human T-cells could successfully engraft and proliferate when transplanted into the NOG mice, and protect against subsequent HIV infection.

To our knowledge, genome editing that is sufficiently robust to support therapy in an animal model has not been shown previously. The ZFN-guided genomic editing was highly specific and well tolerated, as revealed by examination of the stability, growth and engraftment characteristics of the genome-modified sub-population even in the absence of selection…We also observed a threefold enrichment of the ZFN-modified primary human CD4+ T cells and protection from viremia in a NOG mouse model of active HIV-1 infection. As predicted for a genetically determined trait, the ZFN-modified cells demonstrated stable and heritable resistance in progeny cells to HIV-1 infection both in vitro and in vivo. These results demonstrate that ZFN-mediated genome editing can be used to reproduce a CCR5 null genotype in primary human cells.”

Following this they also undertook more extensive regulatory studies in mice to demonstrate that there were no toxicities associated with the ZFIN transformation of the T-cells.

While the clinical trial announced yesterday focused on the safety of the technique, the authors also reported that HIV RNA became undetectable in one of four patients who could be evaluated, and that the blood level of HIV DNA decreased in most patients, which bodes well for future trials when larger quantities of ZFN-modified cells will be transplanted.

This is not the first time that the pioneering work of Bruce Levine and Carl June has caught our attention, they are the same researchers who have hit the headlines with an innovative “Chimeric Antibody Receptor” gene therapy for leukemia that is part of the cancer immunotherapy revolution now underway. Their latest breakthrough is another indication of how gene therapy is becoming an important part of 21st century medicine.

Paul Browne

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Mice and macaques pave the way for effective HIV vaccines

There is encouraging news this week on the prospects for an effective vaccine against HIV. A  research team led by Professor Mariano Esteban at the Spanish Superior Scientific Research Council (CSIC) have announced that the vaccine MVA-B elicited a persistent immune response against HIV in  85% of volunteers in a phase 1 clinical trial. MVA-B is a therapeutic vaccine, it is not intended to block infection but rather to keep HIV levels in the body at levels well below those at which the virus can cause illness.

As a CSIC press release published online on EureakAlert! notes the MVA-B vaccine, created by inserting four HIV genes from the B subtype of HIV – the subtype accounting for most HIV infections in Europe and North America – into a vector derived from the Modified Ankara Vaccinia virus (a smallpox vaccine and shown to be safe in both animal studies and extensive human use), notes that:

In 2008, MVA-B already showed very high efficiency in mice as well as macaque monkeys against Simian’s immunodeficiency virus (SIV). Due to it’s high immunological response in humans, Phase I clinic trials will be conducted with HIV infected volunteers, to test its efficiency as a therapeutic vaccine.”

This is indeed true, a 2007 study in mice revealed that the MVA-B vaccine induced a strong immune response , while a paper published in 2008 by the same group demonstrated that a very similar MVA vaccine was able to induce a robust response involving both the HIV-1-specific CD4+ helper T-cells  and CD8+ cytotoxic T cells in Rhesus macaques, and was able to control virus levels in macaques infected with the SHIV 89.6P hybrid virus whereas in unvaccinated monkeys the levels of virus rose and most developed an AIDS-like illness.

There is a question over whether the immune response generated by the MVA-B vaccine will be able to restrict HIV in humans, after all the MRK-Ad5 vaccine which failed to restrict the HIV virus in human trials and the pathogenic SIV MAC239 – considered a better model for HIV infection than SHIV 89.6p – in macaque monkeys had successfully controlled SHIV 89.6P in earlier studies.

Some reassurance on this issue comes from a study at Oregon Health and Science University (OHSU) that was announced earlier this year, where a group led by Dr. Louis Picker used a different vaccine vector – one based on Cytomegalovirus – to elicit a very similar broad immune response , with strong memory T-cell involvement, to that induced by MVA-B, and found that it induced long-term control the highly pathogenic SIV MAC239 strain. This was the highest degree of control demonstrated to date against this SIV strain, and indeed the cytomegalovirus vaccine is one of the first to demonstrate any ability to control SIV MAC239 levels.

Professor Esteban and his colleagues are certainly not resting on their laurels either, further clinical trials of the MVA-B vaccine are planned, to determine whether it can protect against HIV.  In the meantime they are also seeking to improve on this vaccine.  Earlier this year they published a paper in the open-access journal PloS One where they deleted a gene in the MVA vector to yield a new MVA-B  vaccine that showed in mice a substantial increase in the magnitude and breath of the immune response compared with their original MVA-B vaccine, and an even better  memory T-cell response. They now plan to evaluate this improved vaccine in a non-human primate model of HIV infection, and it will be interesting to see if they choose to use a more stringent model of infection such as SIV MAC239 rather than SHIV-89.6P.

Despite the setbacks and disappointments over the past two decades, it is clear from the work being done at the CSIC and OHSU that real progress is being made towards the development of both prophylactic and therapeutic  vaccines against HIV, and it is just as clear that animal research continues to play a vital role in that progress.

Paul Browne

A breakthrough against Chronic Lymphocytic Leukemia…thank the mice!

A challenge that science communicators frequently face when discussing the process whereby a scientific discovery eventually leads to a medical breakthrough is the time that this often takes, indeed by the time that the reports of exciting clinical trial outcomes start to appear in the press the role of the scientists who made the initial discoveries is often relegated to a passing comment…if it is mentioned at all. An example of this comes from the Weizmann Wave blog, produced by the Weizmann Institute of Science.

You may remember reports last month on the very promising results of a small clinical trial where a new immunotherapy technique was used to eradicate cancer cells in patients with Chronic Lymphocytic Leukemia (CLL), a blood cancer for which currently available treatments are often inadequate.  That trial, conducted by scientists at the University of Pennsylvania led by Professor Carl June, involved removing T-cells from the patient, treating the cells with a lentiviral vector that encodes for a Chimeric Antigen Receptor which recognises a protein named CD19 that is found on B-cells, including the cancer cells responsible for CLL, and then infusing the transformed T-cells back into the patients.  As the reported in the Los Angeles Times the results were dramatic, within a few weeks of the infusion the modified T-cells expanded rapidly and targeted the cancer cells in all three paients, so that a year later two of the three patients were still in complete remission.

It’s exciting stuff but as the Weizmann Wave reports the Press Release issued by Penn Medicine noted that this was a “cancer treatment breakthrough 20 years in the making” but “didn’t, however, explain those “20 years in the making.””. The Weizmann Wave goes on to discuss the pioneering basic scientific research undertaken by Professor Zelig Eshhar at theWeizman Institute of Science in the late 1980’s, which you can read about here.

Of course between the basic research undertaken by Prof. Eshhar and his colleagues in the 1980’s and the clinical trial whose outcome was announced last month there was a lot of work to be done. It would be impractical to describe all the different discoveries that made this immunotherapy possible, but one discovery in particular highlights the importance of animal research to this breakthrough.

There have been previous attempts to use Chimeric Antigen Receptors to target T-cells to attack cancer, but these had disappointing results in clinical trials.  A major improvement made by the University of Pennsylvania team was to include an additional motif – named the CD137 co-stimulatory molecule- which greatly enhances the cancer killing ability of the infused T-cells.  In a recent paper published in the Journal of Cancer the University of Pennsylvania team point out that the decision to include CD137 (called 4-1BB in mice) in their Chimeric Antigen Receptor construct was based on promising results in studies undertaken in mice:

 Our group has tested a CAR directed against CD19 linked to the CD137 (4-1BB) co-stimulatory molecule signaling domain to enhance activation and signaling after recognition of CD19. By inclusion of the 4-1BB signaling domain, in vitro tumor cell killing, and in-vivo anti-tumor activity and persistence of CART-19 cells in a murine xenograft model of human ALL (acute lymphocytic leukemia) is greatly enhanced”

Indeed, in a paper published by Professor June and colleagues in the journal Molecular Therapy in 2009 they describe this work in much more detail, highlighting just how groundbreaking the results were:

Previous in vitro studies have characterized the incorporation of CD137 domains into CARs.10,11,29 Our results represent the first in vivo characterization of these CARs and uncover several important advantages of CARs that express CD137 that were not revealed by the previous in vitro studies. We demonstrated that CARs expressing the CD137 signaling domain could survive for at least 6 months in mice bearing tumor xenografts. This may have significant implications for immunosurveillance, as well as for tumor eradication. For example, in a mouse prostate cancer xenograft model, survival of CAR+ T cells for at least a week was required for tumor eradication.30

Long-term survival of the CARs did not require administration of exogenous cytokines, and these results significantly extend the duration of survival of human T cells expressing CARs shown in previous studies.17,31 To our knowledge, this is the first report demonstrating elimination of primary leukemia xenografts in a preclinical model using CAR+ T cells. Furthermore, complete eradication was achieved in some animals in the absence of further in vivo therapy, including prior chemotherapy or subsequent cytokine support.

The long-term control of well-established tumors by immunotherapy has rarely been reported. Most preclinical models in a therapeutic setting have tested tumors that have been implanted for a week or less before initiation of therapy.32 After establishing leukemia 2–3 weeks before T cell transfer, we found that many animals had long-term control of leukemia for at least 6 months. The efficacy of targeted, adoptive immunotherapy in this xenograft model of primary human ALL compares favorably to our prior experience testing the antileukemic efficacy of single cytotoxic (ref. 27 and data not shown) or targeted agents,26 where we have observed extension of survival but not cure of disease. Additionally, we have not previously observed the ability to control xenografted ALL for a period of as long as 6 months.”

These results led directly to the clinical trial reported last month.

So there you have it, behind the headlines are years of graft by hard-working and innovative scientists, who utilised a wide range of experimental approaches – among which animal studies figure prominently – to develop a novel therapy for CLL. As Professor Bruce Levine points out in the video above, the key to success is often keeping one hand in the basic research lab and the other in the clinic.

Paul Browne

Addendum: Scienceblogger Erv has written an excellent commentary on this study