Juan F. Vera

Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety

T lymphocytes expressing a chimeric antigen receptor (CAR) targeting the CD19 antigen (CAR.19) may be of value for the therapy of B-cell malignancies. Because the in vivo survival, expansion and anti-lymphoma activity of CAR.19+ T cells remain suboptimal even when the CAR contains a CD28 costimulatory endodomain, we generated a novel construct that also incorporates the interleukin-15 (IL-15) gene and an inducible caspase-9-based suicide gene (iC9/CAR.19/IL-15). We found that compared with CAR.19+ T cells, iC9/CAR.19/IL-15+ T cells had: (1) greater numeric expansion upon antigen stimulation (10-fold greater expansion in vitro, and 3- to 15-fold greater expansion in vivo) and reduced cell death rate (Annexin-V+/7-AAD+ cells 10±6% for iC9/CAR.19/IL-15+ T cells and 32±19% for CAR.19+ T cells); (2) reduced expression of the programmed death 1 (PD-1) receptor upon antigen stimulation (PD-1+ cells <15% for iC9/CAR.19/IL-15+ T cells versus >40% for CAR.19+ T cells); and (3) improved antitumor effects in vivo (from 4.7- to 5.4-fold reduced tumor growth). In addition, iC9/CAR.19/IL-15+ T cells were efficiently eliminated upon pharmacologic activation of the suicide gene. In summary, this strategy safely increases the anti-lymphoma/leukemia effects of CAR.19-redirected T lymphocytes and may be a useful approach for treatment of patients with B-cell malignancies.

Activity of Broad-Spectrum T Cells as Treatment for AdV, EBV, CMV, BKV, and HHV6 Infections after HSCT

Rapidly generated broad-spectrum T cells can simultaneously treat multiple viral infections after hematopoietic stem cell transplant. Killing Multiple Viruses with One Stone Bone marrow or stem cell transplantation is becoming increasingly common for cancer as well as for other blood disorders and genetic diseases. Although patient outcomes are often good and are continuing to improve as technology evolves, the patients are still at risk for a variety of complications. One of the deadliest complications for newly transplanted patients is infection due to their severely compromised immune function. Viral infections are especially problematic, because many viruses have no specific treatments. In a small clinical trial, Papadopoulou et al. demonstrated a way to quickly generate antiviral T cells and give them to transplant patients, to help them safely clear up to four (and potentially five) simultaneous viral infections. It remains difficult to treat the multiplicity of distinct viral infections that afflict immunocompromised patients. Adoptive transfer of virus-specific T cells (VSTs) can be safe and effective, but such cells have been complex to prepare and limited in antiviral range. We now demonstrate the feasibility and clinical utility of rapidly generated single-culture VSTs that recognize 12 immunogenic antigens from five viruses (Epstein-Barr virus, adenovirus, cytomegalovirus, BK virus, and human herpesvirus 6) that frequently cause disease in immunocompromised patients. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term.

Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).

The clinical manufacture of antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy is limited by the complexity and time required to produce large numbers with the desired function and specificity. The culture conditions required are rigorous, and in some cases only achieved in 2-cm2 wells in which cell growth is limited by gas exchange, nutrients, and waste accumulation. Bioreactors developed to overcome these issues tend to be complex, expensive, and not always conducive to CTL growth. We observed that antigen-specific CTLs undergo 7 to 10 divisions poststimulation. However, the expected CTL numbers were achieved only in the first week of culture. By recreating the culture conditions present during this first week—low frequency of antigen-specific T cells and high frequency of feeder cells—we were able to increase CTL expansion to expected levels that could be sustained for several weeks without affecting phenotype or function. However, the number of 24-well plates needed was excessive and cultures required frequent media changes, increasing complexity and manufacturing costs. Therefore, we evaluated novel gas-permeable culture devices (G-Rex) with a silicone membrane at the base allowing gas exchange to occur uninhibited by the depth of the medium above. This system effectively supports the expansion of CTL and actually increases output by up to 20-fold while decreasing the required technician time. Importantly, this amplified cell expansion is not because of more cell divisions but because of reduced cell death. This bioprocess optimization increased T-cell output while decreasing the complexity and cost of CTL manufacture, making cell therapy more accessible.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

BACKGROUND AIMS Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. METHODS We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). RESULTS Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. CONCLUSIONS We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.

Kinetics of Tumor Destruction by Chimeric Antigen Receptor-modified T Cells

The use of chimeric antigen receptor (CAR)-modified T cells as a therapy for hematologic malignancies and solid tumors is becoming more widespread. However, the infusion of a T-cell product targeting a single tumor-associated antigen may lead to target antigen modulation under this selective pressure, with subsequent tumor immune escape. With the purpose of preventing this phenomenon, we have studied the impact of simultaneously targeting two distinct antigens present on tumor cells: namely mucin 1 and prostate stem cell antigen, both of which are expressed in a variety of solid tumors, including pancreatic and prostate cancer. When used individually, CAR T cells directed against either tumor antigen were able to kill target-expressing cancer cells, but tumor heterogeneity led to immune escape. As a combination therapy, we demonstrate superior antitumor effects using both CARs simultaneously, but this was nevertheless insufficient to achieve a complete response. To understand the mechanism of escape, we studied the kinetics of T-cell killing and found that the magnitude of tumor destruction depended not only on the presence of target antigens but also on the intensity of expression-a feature that could be altered by administering epigenetic modulators that upregulated target expression and enhanced CAR T-cell potency.

Optimization of the piggybac transposon system for the sustained genetic modification of human T lymphocytes

Optimal implementation of adoptive T-cell therapy for cancer will likely require multiple and maintained genetic modifications of the infused T cells and their progeny so that they home to tumor sites and recognize tumor cells, overcome tumor immune evasion strategies, and remain safe. Retroviral vectors readily transduce T cells and integrate into the host cell genome, but have a limited capacity for multigene insertion and cotransduction and are prohibitively expensive to produce at clinical grade. Genetic modification of T cells using transposons as integrating plasmids is an attractive alternative because of the increased simplicity and cost of production. Of available transposons, piggyBac has the higher transposase activity and larger cargo capacity, and we now evaluate piggyBac for potential adoptive therapies with primary T cells. PiggyBac transposons mediated stable gene expression in approximately 20% of primary T cells without selection. Treatment and maintenance of T cells with interleukin-15 increased stable transgene expression up to approximately 40% and expression was sustained through multiple logs of expansion for over 9 weeks in culture. We demonstrate simultaneous integration of 2 independent transposons in 20% of T cells, a frequency that could be increased to over 85% by selection of a transgenic surface marker (truncated CD19). PiggyBac could also deliver transposons of up to 13 kb with 10,000-fold expansion of transduced T cells in culture and finally we demonstrate delivery of a functional suicide gene (iCasp9). PiggyBac transposons may thus be used to express the multiple integrated transgenes that will likely be necessary for the broader success of T-cell therapy.

Genetic Manipulation of Tumor-specific Cytotoxic T Lymphocytes to Restore Responsiveness to IL-7

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) can induce objective clinical responses in patients with malignant diseases. The option of providing a proliferative and survival advantage to adoptively transferred CTLs remains a challenge to improve their efficacy. Host lymphodepletion and administration of recombinant interleukin-2 (IL-2) are currently used to improve CTL survival and expansion after adoptive transfer, but these approaches are frequently associated with significant side effects and may increase proliferation of T regulatory cells. IL-7 is a crucial homeostatic cytokine that has been safely administered as a recombinant protein. However, while IL-7 induces robust expansion of naive and memory T lymphocytes, the lack of expression of the IL-7 receptor alpha chain (IL-7Ralpha) by CTLs precludes their response to this cytokine. We found that CTLs can be genetically modified to re-express IL-7Ralpha, and that this manipulation restores the response of these cells to IL-7 without apparent modification of their antigen specificity or dependency, and without changing their response to other common gamma (gammac) chain cytokines. This approach may allow selective expansion of CTLs without the unwanted effects associated with IL-2.

Nucleofection of DCs to generate multivirus-specific T cells for prevention or treatment of viral infections in the immunocompromised host

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. To prevent and treat these, we have produced and infused cytotoxic T lymphocytes (CTLs) with specificity for Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv), and shown that small numbers of infused cells proliferate in vivo and protect against all three viruses. Despite these encouraging results, broader implementation of this approach is limited by the need for infectious virus material (EBV), expensive production of clinical grade adenoviral vectors, and a prolonged (8-12 weeks) period of manufacture. There is also competition between virus-derived antigens within antigen-presenting cells (APCs), limiting extension to additional agents. We now describe an approach that uses DNA nucleofection of dendritic cells (DCs) with DNA plasmids that encode a range of immunodominant and subdominant viral antigens from CMV, EBV, BK, and Adv. Within 10 days, this methodology provides multivirus-reactive CTLs that lack alloreactivity. We further demonstrate that nucleofected DC stimulation can be combined with interferon-gamma (IFN-gamma) capture technology to produce even more rapid multivirus-CTL products for treatment of acute infection. These CTL generation procedures should increase the feasibility and applicability of T-cell therapy.

Reversal of Tumor Immune Inhibition Using a Chimeric Cytokine Receptor

The success of adoptively transferred tumor-directed T cells requires them to survive and expand in vivo. Most tumors, however, employ immune evasion mechanisms, including the production of inhibitory cytokines that limit in vivo T-cell persistence and effector function. To protect tumor-directed T cells from such negative influences, we generated a chimeric cytokine receptor in which the interleukin (IL) 4 receptor exodomain was fused to the IL7 receptor endodomain. We thereby inverted the effects of tumor-derived IL4 so that the proliferation and activation of tumor directed cytotoxic T cells was enhanced rather than inhibited in the tumor microenvironment, resulting in superior antitumor activity. These transgenic T cells were only activated in the tumor environment since triggering required exposure to both tumor antigen (signal 1) and tumor-derived IL4 (signal 2). This selectivity supports future clinical adaptation.

Cytotoxic T Lymphocytes Simultaneously Targeting Multiple Tumor-associated Antigens to Treat EBV Negative Lymphoma

Although immunotherapy with Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) can treat EBV-associated Hodgkin and non-Hodgkin lymphoma (HL/NHL), more than 50% of such tumors are EBV negative. We now describe an approach that allows us to consistently generate, in a single line, CTLs that recognize a wide spectrum of nonviral tumor-associated antigens (TAAs) expressed by human HL/NHL, including Survivin, MAGE-A4, Synovial sarcoma X (SSX2), preferentially expressed antigen in melanoma (PRAME) and NY-ESO-1. We could generate these CTLs from nine of nine healthy donors and five of eight lymphoma patients, irrespective of human leukocyte antigen (HLA) type. We reactivated TAA-directed T cells ex vivo, by stimulation with dendritic cells (DCs) pulsed with overlapping peptide libraries spanning the chosen antigens in the presence of an optimized Th1-polarizing, prosurvival/proliferative and Treg inhibitory cytokine combination. The resultant lines of CD4(+) and CD8(+), polycytokine-producing T cells are directed against a multiplicity of epitopes expressed on the selected TAAs, with cytolytic activity against autologous tumor cells. Infusion of such multispecific monocultures may extend the benefits of CTL therapy to treatment even of EBV negative HL and NHL.