Recently there have been two widely publicized updates relating to SB728-T, a gene therapy for HIV infection developed by Sangamo BioSciences. On Wednesday March 5th the New England Journal of Medicine (NEJM) published results from a phase I study conducted by Pablo Tebas and colleagues at the University of Pennsylvania, and on the following day at CROI 2014 Gary Blick* presented new data derived from subsequent trials. The therapy is technologically complex: it involves extracting CD4 T cells from HIV-positive individuals and then modifying them with a method that aims to disable the gene that encodes the CCR5 receptor (which most HIV variants use as a foothold to gain entry into cells). The CD4 T cells are then expanded in number and ultimately infused back into the individual, typically with around 10% successfully modified so that they no longer express CCR5. The method for disabling the CCR5 gene involves an enzyme called a zinc finger nuclease (ZFN), which targets and breaks the DNA containing the gene; cellular repair enzymes then stitch the DNA back together in a way that prevents the gene from making a functional CCR5 receptor. The ZFN is delivered into the CD4 T cells by an adenovirus vector during the laboratory modification procedure. The goal of the therapy is to create a population of CD4 T cells that are resistant to infection by HIV.
The results described in NEJM relate to a clinical trial begun in 2009. There were 12 participants, all receiving antiretroviral therapy (ART): six classified as immunological responders with CD4 T cell counts above 450 and a nadir of no lower than 300, and six immunological non-responders with relatively low CD4 T cell counts despite HIV suppression (between 200 and 500 after a minimum of two years of ART). The primary purpose of the study was to evaluate the safety of a single infusion of gene-modified CD4 T cells, with secondary analyses including CD4 T cell count changes, persistence (and trafficking to gut mucosa) of modified cells, and viral load measurements.
CD4 T cell counts increased in all participants, although the paper notes there was variation between individuals, with seven experiencing large increases and the remainder more modest changes. After 36 weeks of follow up, the median increase was 615 cells. CD4/CD8 ratios, which are typically slow to improve on ART alone, rose significantly from a median of 0.99 at baseline to 2.62 after one week, before declining to 1.14 at week 36. Tracking the gene-modified CD4 T cells is complex, because only approximately 25% show a consistent, quantifiable genetic signature associated with the CCR5 gene disruption; the number measured by this technique is therefore multiplied by four to estimate the total. The median number of gene-modified CD4 T cells in blood was 250 cells per cubic millimeter one week after infusion, representing 13.9% of CD4 T cells, but declined thereafter with an average half-life of 48 weeks. Modified CD4 T cells remain detectable in the blood of all participants, and in the individual followed for the longest period (3.5 years), the level is now 13 cells per cubic millimeter, representing 1.7% of circulating CD4 T cells. Among 11 participants who agreed to repeat rectal biopsies, the median frequency of gene-modified cells was 0.8% of rectal mononuclear cells at day 21 after infusion with subsequent measurements ranging from 0.2-0.4%.
To assess effects on viral load, ART was interrupted in the six immune responder participants four weeks after the CD4 T cell infusion. The duration of the interruption was set at 12 weeks, but in two cases ART was restarted early (due to concern about rapidly rising viral load and three consecutive viral loads over 100,000 copies, respectively). In three of the four remaining individuals, viral load rebounded but subsequently declined to levels similar to their historical pre-ART setpoints at the end of the interruption. In the final participant, a surprising result was observed: viral load rebounded but declined to undetectable levels just before ART was resumed (the viral load graph in the paper appears to indicate there may have been a slight, temporary blip after treatment was begun again, but this is not clarified in the text). Subsequent analyses revealed that this individual is heterozygous for the CCR5-Δ32 mutation, meaning that in his CD4 T cells one copy of the CCR5 gene is already disabled (each cell contains two CCR5 genes, one on each set of chromosomes). As a result, in CCR5-Δ32 heterozygotes there is less work for the gene therapy to do: it only has to disable one CCR5 gene in each CD4 T cell in order to completely abrogate expression of the CCR5 receptor.
The need to disable two CCR5 genes per cell in most recipients adds an additional wrinkle to attempts to measure the number of CD4 T cells modified by the approach. Because laboratory studies indicate that approximately 33% of modified CD4 T cells have both CCR5 genes disabled, the estimated number of modified cells has to be multiplied by 1/3 to reach an approximate number of CD4 T cells that completely lack CCR5 receptor expression. In CCR5-Δ32 heterozygotes, the multiplier is 2/3. Using these formulas, the researchers were able to demonstrate a statistically significant inverse correlation (rho = -0.90, P=0.037) between the estimated numbers of CD4 T cells lacking CCR5 and the viral load decline during the ART interruption, although this is not reported in the paper itself but rather in figure S7 of the supplementary appendix.
Side effects from the administration of the CD4 T cells were generally mild to moderate infusion reactions comprising transient fever, chills, myalgia, arthralgia, and headache. In one case these symptoms were severe enough to lead to a visit to the emergency room within 24 hours after the infusion. A temporary garlic-like body odor was common after infusion due to the metabolism of the substance DMSO, which is used as a cryopreservative during the storage of the extracted CD4 T cells.
The media coverage of the NEJM paper has generally failed to note that the data are not new; Carl June first presented the trial at CROI in 2011. The results have informed the design of several subsequent studies being conducted by Sangamo BioSciences, including those covered in the presentation by Gary Blick at CROI 2014. Specifically, the apparent correlation between numbers of gene-modified cells and viral load reductions spurred an evaluation of whether the immunosuppressive drug cyclophosphamide (Cytoxan) could be used to deplete CD4 T cells prior to the infusion and thereby enhance the uptake of the modified CD4 T cells (essentially create immunological “space” for the new cells to expand into).
Blick reported results involving three different Cytoxan doses: 200mg, 500mg and 1000mg per meter squared. There were three, six and three participants in each dose group respectively. Two individuals receiving 500mg withdrew consent due to nausea and vomiting, and the protocol was subsequently amended to allow prophylactic antiemetic treatment prior to Cytoxan administration. Six weeks after receiving the CD4 T cell infusions (ranging from around 8-35 billion cells), participants underwent a 16-week ART interruption, with the exception of an individual in the 200mg group whose viral tropism (CCR5 vs. CXCR4) could not successfully be evaluated. Blick reported that the highest numbers of gene-modified CD4 T cells were seen in recipients of the 1000mg Cytoxan dose. Initial CD4 T cell count increases also appeared to be somewhat greater in this group. However both of these measures seemed to decline to levels that overlapped with the other groups by the end of the ART interruption. In terms of viral load, one individual in the 1000mg Cytoxan dose group experienced a decline of close to 2 logs from the peak during ART interruption, while another saw a drop to around 1 log below their pre-ART baseline; in other cases the drops were in the 0.3-1 log range. Two participants in the highest dose group remain off ART, based on protocol criteria that allowed the interruption to be continued if viral load stayed beneath 10,000 copies and CD4 T cell counts were maintained above 500 (the wisdom of this is could be questionable given recent evidence that cumulative exposure to detectable viral load is associated with an increased risk of mortality; levels of inflammatory biomarkers in these individuals were not reported). No statistical comparisons of the viral load outcomes between the different dose groups were provided in the presentation.
Based on these very limited numbers, Blick speculated that perhaps the highest Cytoxan dose is allowing the number of gene-modified CD4 T cells to get close to a level where a functional cure might be achieved. An additional arm administering 1,500mg of Cytoxan (preceded by prophylactic antiemetics) has now been added to the study. Blick also mentioned an individual under his care who is a participant in another Sangamo BioSciences clinical trial, which enrolled only CCR5-Δ32 heterozygotes (a design based on the promising data described in the NEJM paper). This person has now been off ART for over 31 weeks and continues to maintain viral load at or below the limit of detection (Sangamo BioSciences has previously provided updates on this individual via press releases at 7, 14 and 20 weeks of follow up). The complete data from this trial has not yet been presented or published.
Overall the results are encouraging, but preliminary. Many media stories have emphasized the exciting possibility of achieving ART-free containment of HIV, but it’s important to note that, as of now, there is only one CCR5-Δ32 heterozygous study participant who has maintained viral load levels beneath detection for an extended period. Because the CCR5-Δ32 heterozygote described in the NEJM paper restarted ART, it is not known if viral load would have remained controlled in that case. For the two individuals in the Cytoxan study who remain off ART, further follow up and additional studies are needed to assess if the relatively low but detectable viral load is leading to elevated inflammation, as is typical in untreated HIV infection, or if the gene therapy is ameliorating those effects.
The mechanism of viral load control also needs to be better understood; Dale Ando from Sangamo BioSciences has presented evidence that improved HIV-specific CD8 T cell responses may be playing a role, possibly suggesting that, in some cases, gene-modified HIV-specific CD4 T cells have been created that are protected from HIV infection and therefore able to provide appropriate help to CD8 T cells. As yet however, no data on HIV-specific CD4 T cell responses has been reported (at least to my knowledge). Because the play of chance would influence how many HIV-specific CD4 T cells are extracted and successfully modified by SB728-T for a given individual, this could be a potential contributor to the heterogeneous outcomes seen in the trials, along with other immunological factors such as variation in HLA genes that affect the quality and targeting of virus-specific CD4 and CD8 T cells responses. A better understanding of these issues might help further refine the approach in order to improve success (e.g. perhaps combining with therapeutic vaccination to try and boost HIV-specific T cell immunity prior to ART interruption). Although it’s only given for a short period, Gary Blick’s report indicates Cytoxan may have some drawbacks as an adjunctive modality.
Researchers are also pursuing the possibility of introducing CCR5 modification at the level of stem cells (based on the cure achieved in Timothy Brown, who received a stem cell transplant from a CCR5-Δ32 homozygote), although there are significant barriers to making this type of delivery possible in people who do not have cancer diagnoses requiring stem cell transplantation.
* Some community members with long memories were surprised to see Blick presenting these data. In the past Blick has been involved in studies of a controversial purported therapy called extracorporeal hyperthemia (a procedure for heating the blood outside the body that was associated with some recipient deaths), which briefly drew publicity, and harsh criticism, in the early 1990s. Blick is still listed as holding patents on hyperthermia, both as a treatment for HIV and hepatitis C.
N Engl J Med. 2014 Mar 6;370(10):901-10. doi: 10.1056/NEJMoa1300662.
Tebas P1, Stein D, Tang WW, Frank I, Wang SQ, Lee G, Spratt SK, Surosky RT, Giedlin MA, Nichol G, Holmes MC, Gregory PD, Ando DG, Kalos M, Collman RG, Binder-Scholl G, Plesa G, Hwang WT, Levine BL, June CH.
BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.
METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance.
RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients.
CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).
N Engl J Med. 2014 Mar 6;370(10):968-9. doi: 10.1056/NEJMe1400593.
Kay MA1, Walker BD.