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Targeting CARVs using ex-vivo-expanded VSTs
dual/multiple co-existing infections are common, with fre- quencies that may exceed 40% among children under 5- years of age and are associated with increased risk of mor- bidity and hospitalization.22-26 Among immunocompro- mised allogeneic HSCT recipients up to 40% experience CARV infections that can range from mild (associated symptoms including rhinorrhea, cough and fever) to severe (bronchiolitis and pneumonia) with associated mortality rates as high as 50% in those with LRTI.5-9 The therapeutic options are limited. For hMPV and PIV-3, there are currently no approved preventative vaccines nor ther- apeutic antiviral drugs, while the off-label use of the nucleoside analog RBV and the investigational use of DAS-181 (a recombinant sialidase fusion protein) have had limited clinical impact.10,11,27,28 The preventative annual Influenza vaccine is not recommended for allogeneic HSCT recipients until at least six months post transplant (and excluded in recipients of intensive chemotherapy or anti-B-cell antibodies), while neuraminidase inhibitors are not always effective for the treatment of active infec- tions.12 For RSV, aerosolized RBV is FDA-approved for the treatment of severe bronchiolitis in infants and children, and it is also used off-label for the prevention of upper or lower RTI and treatment of RSV pneumonia in HSCT recipients.13,15,16 However, its widespread use is limited by
A
B
Figure 4. Multi-respiratory virus-targeted cells (multi-R-VST) are exclusively reactive against virus-infected targets. (A) Cytolytic potential of multi-R-VST evaluated by standard 4-hour Cr51 release assay using autologous pepmix- pulsed PHA blasts as targets (E:T 40:1; n=8) with unloaded phytohemagglutinin (PHA) blasts as a control. Results are presented as percentage of specific lysis (mean±SEM). (B) Demonstration that multi-R-VST show no activity against either non-infected autologous or allogeneic PHA blasts, as assessed by Cr51 release assay.
the cumbersome nebulization device and ventilation sys- tem required for drug delivery, as well as the considerable associated cost. For example, in 2015, aerosolized RBV cost $29,953 per day, with five days representing a typical treatment course.14 Thus, the lack of approved treatments combined with the high cost of antiviral agents led us to explore the potential for using adoptively-transferred T cells to prevent and/or treat CARV infections in this patient population.
The pivotal role of functional T-cell immunity in medi- ating viral control of CARV has only recently attracted attention. For example, a retrospective study of 181 HSCT patients with RSV URTI, reported lymphopenia (defined as ALC ≤100/mm3) as a key determinant in identifying patients whose infections would progress to LRTI, while RSV neutralizing antibody levels were not significantly associated with disease progression.29 Furthermore, in a recent retrospective analysis of 154 adult patients with hematologic malignancies with or without HSCT treated for RSV LRTI, lymphopenia was significantly associated with higher mortality rates.30 Both of these studies are sug- gestive of the importance of cellular immunity in mediat- ing protective immunity in vivo.
Our group has previously demonstrated the feasibility and clinical utility of ex vivo-expanded VST to treat a range of clinically problematic viruses including the latent virus- es CMV, EBV, BKV, HHV-6 and AdV.17,31-33 Our initial stud- ies (and those of others)34-37 explored the safety and activi- ty of donor-derived T-cell lines, but more recently we have developed an “off the shelf” universal T-cell platform whereby VST specific for all five viruses (CMV, EBV, BKV, HHV-6, AdV) were prospectively generated and banked, thus ensuring their immediate availability for administra- tion to immunocompromised patients with uncontrolled infections. Indeed, in our recent phase II clinical trial, we administered these partially HLA-matched VST to 38 patients with a total of 45 infections that had proven refractory to conventional antiviral agents and achieved an overall response rate of 92%, with no significant toxicity.18 This precedent of clinical success using adoptively trans- ferred T cells, as well as the absence of effective therapies for a range of CARV, prompted us to explore the potential for extending the therapeutic scope of VST therapy to Influenza, RSV, hMPV and PIV-3 infections post HSCT. In this context, one could consider the option of prophylactic VST administration seasonally to high-risk patients [e.g. young (<5 years) and elderly adults, patients with impaired immune systems]. Alternatively, these cells could be used therapeutically in patients with URTI who have failed conventional antiviral medications in order to prevent LRT progression.
Thus, using our established, GMP-compliant VST man- ufacturing methodology, we demonstrated the feasibility of generating VST reactive against a spectrum of CARV- derived antigens chosen on the basis of both their immunogenicity to T cells and their sequence conserva- tion [Influenza – NP1 and MP1;20,38,39 RSV – N and F;15,16,20 hMPV–F,N,M2-1andM;21 PIV3–M,HN,NandF19] from 12 donors with diverse haplotypes. The expanded cells were polyclonal (CD4+ and CD8+), Th1-polarized and polyfunctional, and were able to lyse viral antigen- expressing targets while sparing non-infected autologous or allogeneic targets, attesting to both their virus specifici- ty and their safety for clinical use. Finally, to assess the clinical significance of these findings we examined the
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