Publications by authors named "Patrick J Hanley"

67 Publications

Identification of novel HLA-restricted preferentially expressed antigen in melanoma peptides to facilitate off-the-shelf tumor-associated antigen-specific T-cell therapies.

Cytotherapy 2021 Apr 5. Epub 2021 Apr 5.

Center for Cancer and Immunology Research, Children's National Hospital, Washington, DC, USA; GW Cancer Center, George Washington University, Washington, DC, USA; Division of Blood and Marrow Transplantation, Children's National Hospital, Washington, DC, USA. Electronic address:

Background Aims: Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized.

Methods: Peptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes.

Results: PRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes.

Conclusions: TAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.
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http://dx.doi.org/10.1016/j.jcyt.2021.03.001DOI Listing
April 2021

SARS-CoV-2-specific T cells are rapidly expanded for therapeutic use and target conserved regions of the membrane protein.

Blood 2020 12;136(25):2905-2917

Center for Cancer and Immunology Research and.

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.
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http://dx.doi.org/10.1182/blood.2020008488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746091PMC
December 2020

Virus-specific T-cell therapy to treat BK polyomavirus infection in bone marrow and solid organ transplant recipients.

Blood Adv 2020 11;4(22):5745-5754

Division of Bone Marrow Transplant and Immune Deficiency, Cincinnati Children's Hospital, Cincinnati, OH.

BK polyomavirus (BKPyV) infection is a major complication of hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT). Treatment options are limited, poorly effective, and have significant toxicities. Cellular therapy using T cells directed against BKPyV is an emerging therapy, and we report efficacy in controlling BKPyV-associated disease in highly immunocompromised patients. Virus-specific T cells (VSTs) against BKPyV were manufactured using either blood from the patient's stem cell donor (donor-derived VSTs) or from unrelated donors (third-party VSTs). VSTs were used to treat BKPyV in 38 HSCT recipients and 3 SOT recipients between June 2017 and December 2019. Overall response rate was 86% in patients treated for BK viremia, 100% in patients treated for hemorrhagic cystitis, and 87% in patients treated for both BK viremia and hemorrhagic cystitis. No infusional toxicity, de novo graft-versus-host disease, or rejection of the organ occurred attributable to the VST infusion. BKPyV-specific immune responses were demonstrated by interferon-γ production by peripheral blood mononuclear cells postinfusion in response to BKPyV antigens. VSTs are a safe and potentially effective strategy to treat BKPyV and associated symptoms in recipients of HSCT and SOT. Cellular therapy should be considered for all patients with BKPyV and underlying immune suppression at risk of complications. This trial was registered at www.clinicaltrials.gov as #NCT02532452.
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http://dx.doi.org/10.1182/bloodadvances.2020003073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686882PMC
November 2020

Processing laboratory considerations for multi-center cellular therapy clinical trials: a report from the Consortium for Pediatric Cellular Immunotherapy.

Cytotherapy 2021 Feb 12;23(2):157-164. Epub 2020 Nov 12.

Center for Cancer and Immunology Research, Center for Cancer and Blood Disorders, Children's National Hospital, Washington, DC, USA; The George Washington University, Washington, DC, USA. Electronic address:

``Cellular therapies first emerged as specialized therapies only available at a few "boutique" centers worldwide. To ensure broad access to these investigational therapies-regardless of geography, demographics and other factors-more and more academic clinical trials are becoming multi-center. Such trials are typically performed with a centralized manufacturing facility receiving the starting material and shipping the final product, either fresh or cryopreserved, to the patient's institution for infusion. As these academic multi-center trials increase in number, it is critical to have procedures and training programs in place to allow these sites that are remote from the production facility to successfully participate in these trials and satisfy regulatory compliance and patient safety best practices. Based on the collective experience of the Consortium for Pediatric Cellular Immunotherapy, the authors summarize the challenges encountered by institutions in shipping and receiving the starting material and final product as well as preparing the final product for infusion. The authors also discuss best practices implemented by each of the consortia institutions to overcome these challenges.
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http://dx.doi.org/10.1016/j.jcyt.2020.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855775PMC
February 2021

SARS-CoV-2 specific T-cells Are Rapidly Expanded for Therapeutic Use and Target Conserved Regions of Membrane Protein.

Blood 2020 Oct 26. Epub 2020 Oct 26.

The George Washington University, United States.

T-cell responses to SARS-CoV-2 have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T-cells can be expanded from convalescent donors, and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a GMP-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited IFN-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T cell responses, which may be critical for the development of effective vaccine and T cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve anti-viral control while mitigating uncontrolled inflammation.
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http://dx.doi.org/10.1182/blood.2020008488DOI Listing
October 2020

Engineered Antigen-Specific T Cells Secreting Broadly Neutralizing Antibodies: Combining Innate and Adaptive Immune Response against HIV.

Mol Ther Methods Clin Dev 2020 Dec 21;19:78-88. Epub 2020 Aug 21.

George Washington University Cancer Center, George Washington University, Washington, DC, USA.

While antiretroviral therapy (ART) can completely suppress viremia, it is not a cure for HIV. HIV persists as a latent reservoir of infected cells, able to evade host immunity and re-seed infection following cessation of ART. Two promising immunotherapeutic strategies to eliminate both productively infected cells and reactivated cells of the reservoir are the adoptive transfer of potent HIV-specific T cells and the passive administration of HIV-specific broadly neutralizing antibodies also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). The simultaneous use of both as the basis of a single therapeutic has never been explored. We therefore sought to modify HIV-specific T cells from HIV-naive donors (to allow their use in the context of allotransplant, a promising platform for sterilizing cures) so they are able to secrete a broadly neutralizing antibody (bNAb) directed against the HIV envelope to elicit ADCC. We designed an antibody construct comprising bNAb 10-1074 heavy and light chains, fused to IgG3 Fc to elicit ADCC, with truncated cluster of differentiation 19 (CD19) as a selectable marker. HIV-specific T cells were expanded from HIV-naive donors by priming with antigen-presenting cells expressing overlapping HIV antigens in the presence of cytokines. T cells retained specificity against Gag, Nef, and Pol peptides (218.55 ± 300.14 interferon γ [IFNγ] spot-forming cells [SFC]/1 × 10) following transduction (38.92 ± 25.30) with the 10-1074 antibody constructs. These cells secreted 10-1074 antibodies (139.04 ± 114.42 ng/mL). The HIV-specific T cells maintained T cell function following transduction, and the secreted 10-1074 antibody bound HIV envelope (28.13% ± 19.42%) and displayed ADCC activity (10.47% ± 4.11%). Most critically, the 10-1074 antibody-secreting HIV-specific T cells displayed superior suppression of HIV replication. In summary, HIV-specific T cells can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadens the target range of the immune therapy, and provides further insight into what defines an effective anti-HIV response.
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http://dx.doi.org/10.1016/j.omtm.2020.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508916PMC
December 2020

Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay.

Cytotherapy 2021 Jan 11;23(1):65-76. Epub 2020 Sep 11.

Center for Cancer and Immunology Research, Children's National Hospital, Washington, DC, USA; Division of Blood and Marrow Transplantation, Children's National Hospital, Washington, DC, USA; The George Washington University Cancer Center, Washington, DC, USA. Electronic address:

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) expression. Combinations of IL-15/IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7- effector memory (CD45RO+ CCR7- CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO- CCR7- CD3+), and naive (CD45RO- CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 10 PBMCs.
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http://dx.doi.org/10.1016/j.jcyt.2020.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785615PMC
January 2021

Virus-Specific T Cell Therapies for HIV: Lessons Learned From Hematopoietic Stem Cell Transplantation.

Front Cell Infect Microbiol 2020 7;10:298. Epub 2020 Jul 7.

Center for Cancer and Immunology Research, Children's National Hospital, Washington, DC, United States.

Human immunodeficiency virus (HIV) has caused millions of deaths and continues to threaten the health of millions of people worldwide. Despite anti-retroviral therapy (ART) substantially alleviating severity and limiting transmission, HIV has not been eradicated and its persistence can lead to other health concerns such as cancer. The only two cases of HIV cure to date are HIV cancer patients receiving an allogeneic hematopoietic stem cell transplantation (allo-HSCT) from a donor with the CCR5 Δ32 mutation. While this approach has not led to such success in other patients and is not applicable to HIV individuals without cancer, the encouraging results may point toward a breakthrough in developing a cure strategy for HIV. Adoptive transfer of virus-specific T cells (VSTs) post HSCT has been effectively used to treat and prevent reactivation of latent viral infections such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), making VSTs an attractive therapeutic to control HIV rebound. Here we will discuss the potential of using adoptive T cell therapies in combination with other treatments such as HSCT and latency reversing agents (LRAs) to achieve a functional cure for HIV.
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http://dx.doi.org/10.3389/fcimb.2020.00298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381350PMC
July 2020

Emerging trends in COVID-19 treatment: learning from inflammatory conditions associated with cellular therapies.

Cytotherapy 2020 09 7;22(9):474-481. Epub 2020 May 7.

Center for Cancer and Immunology Research, Center for Cancer and Blood Disorders, Children's National Hospital and the George Washington University Cancer Center, George Washington University, Washington, DC, USA.

Coronavirus disease 2019 (SARS-CoV2) is an active global health threat for which treatments are desperately being sought. Even though most people infected experience mild to moderate respiratory symptoms and recover with supportive care, certain vulnerable hosts develop severe clinical deterioration. While several drugs are currently being investigated in clinical trials, there are currently no approved treatments or vaccines for COVID-19 and hence there is an unmet need to explore additional therapeutic options. At least three inflammatory disorders or syndromes associated with immune dysfunction have been described in the context of cellular therapy. Specifically, Cytokine Release Syndrome (CRS), Immune Reconstitution Inflammatory Syndrome (IRIS), and Secondary Hemophagocytic Lymphohistiocytosis (sHLH) all have clinical and laboratory characteristics in common with COVID19 and associated therapies that could be worth testing in the context of clinical trials. Here we discuss these diseases, their management, and potential applications of these treatment in the context of COVID-19. We also discuss current cellular therapies that are being evaluated for the treatment of COVID-19 and/or its associated symptoms.
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http://dx.doi.org/10.1016/j.jcyt.2020.04.100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252029PMC
September 2020

EBV-directed viral-specific T-lymphocyte therapy for the treatment of EBV-driven lymphoma in two patients with primary immunodeficiency and DNA repair defects.

Pediatr Blood Cancer 2020 03 18;67(3):e28126. Epub 2019 Dec 18.

Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio.

Children with ataxia telangiectasia (AT), a primary immunodeficiency caused by mutations in ATM, which is critical for repairing DNA defects, are at risk for the development of hematologic malignancy, frequently driven by infection with Epstein-Barr virus (EBV). Conventional chemotherapy is poorly tolerated by patients with AT, with excessive toxicity even when doses are reduced. Here, we report on two patients with AT and EBV-positive neoplasms who were treated with EBV-targeted viral-specific T cells (VST). One patient had a prolonged complete response to VSTs while the other had a partial response. Therapy was well tolerated without infusion toxicity or graft-versus-host disease.
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http://dx.doi.org/10.1002/pbc.28126DOI Listing
March 2020

HIV-Specific T Cells Can Be Generated against Non-escaped T Cell Epitopes with a GMP-Compliant Manufacturing Platform.

Mol Ther Methods Clin Dev 2020 Mar 11;16:11-20. Epub 2019 Oct 11.

Center for Cancer and Immunology Research, Children's National Health System, Washington, DC 20010, USA.

Although anti-retroviral therapy (ART) is successful in suppressing HIV-1 replication, HIV latently infected reservoirs are not eliminated, representing a major hurdle in efforts to eradicate the virus. Current strategies to eradicate HIV involve two steps: (1) the reactivation of latently infected cells with latency reversing agents (LRAs) to expose persisting HIV, and (2) the elimination of these cells with immune effectors while continuing ART to prevent reinfection. HIV-specific T cells (HSTs) can kill reactivated HIV-infected cells and are currently being evaluated in early-stage immunotherapy trials. HIV can mutate sequences in T cell epitopes and evade T cell-mediated killing of HIV-infected cells. However, by directing T cells to target multiple conserved, non-escaped HIV epitopes, the opportunity for viral escape can be reduced. Using a good manufacturing practice (GMP)-compliant platform, we manufactured HSTs against non-escape epitope targets (HST-NEETs) from HIV and HIV-seronegative donors. HST-NEETs expanded to clinically relevant numbers, lysed autologous antigen-pulsed targets, and showed a polyfunctional pro-inflammatory cytokine response. Notably, HST-NEETs recognized multiple conserved, non-escaped HIV epitopes and their common variants. We propose that HST-NEETs could be used to eliminate reactivated virus from latently infected cells in HIV individuals following LRA treatment. Additionally, HST-NEETs derived from HIV-negative individuals could be used post-transplant for HIV individuals with hematologic malignancies to augment anti-viral immunity and destroy residual infected cells.
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http://dx.doi.org/10.1016/j.omtm.2019.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838524PMC
March 2020

Driving the CAR to the Bone Marrow Transplant Program.

Curr Hematol Malig Rep 2019 12;14(6):561-569

Center for Cancer and Immunology Research, Children's National Health System, Washington, DC, USA.

Purpose Of Review: The US Food and Drug Administration (FDA) approved two commercially available chimeric antigen receptor (CAR) T cell therapies for the treatment of relapsed B cell acute lymphoblastic leukemia (B-ALL) children and young adults less than 25 years of age and non-Hodgkin lymphoma in adults after promising results from early-phase single and multi-institutional clinical trials. In this review, we provide an overview of the practical aspects of a chimeric antigen T cell receptor (CAR-T) program development and the steps necessary for its successful implementation.

Recent Findings: CAR-T therapy is a complex process and poses significant challenges as institutions prepare to deliver this therapy as a standard of care for the eligible patients. It requires a rigorous infrastructure with specific clinical, administrative, and regulatory demands. Institutions that led the clinical trials for CAR-T have adopted various approaches to integrate commercial CAR-T products into their program. Delivering commercial CAR-T cells outside the scope of clinical trials requires careful planning, allocation of resources, and utilization of existing infrastructure. Institutions may need to adapt the existing recommendations and guidelines and tailor them to meet the needs of their program and ensure appropriate financial reimbursement for this expensive but promising immunotherapy.
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http://dx.doi.org/10.1007/s11899-019-00544-6DOI Listing
December 2019

Impact of Mesenchymal Stromal Cell Delivery Through Cardiopulmonary Bypass on Postnatal Neurogenesis.

Ann Thorac Surg 2020 04 26;109(4):1274-1281. Epub 2019 Sep 26.

Children's National Heart Institute, Children's National Hospital, Washington, DC; Center for Neuroscience Research, Children's National Hospital, Washington, DC; George Washington University School of Medicine and Health Science, Washington, DC. Electronic address:

Background: Neurodevelopmental impairment is an important challenge for survivors after neonatal surgery with cardiopulmonary bypass (CPB). The subventricular zone, where most neural stem/progenitors originate, plays a critical role in cortical maturation of the frontal lobe. Promoting neurogenesis in the subventricular zone is therefore a potential therapeutic target for preserving cortical growth. Mesenchymal stromal cells (MSCs) promote endogenous regeneration in the rodent brain. We investigated the impact of MSC delivery through CPB on neural stem/progenitor cells and neuroblasts (ie, young neurons) in the piglet subventricular zone.

Methods: Two-week-old piglets (n = 12) were randomly assigned to one of three groups: (1) control, (2) deep hypothermic circulatory arrest, and (3) circulatory arrest, followed by MSC administration. MSCs (10 × 10 per kg) were delivered through CPB during the rewarming period. Neural stem/progenitors, proliferating cells, and neuroblasts were identified with immunohistochemistry at 3 hours after CPB.

Results: CPB-induced insults caused an increased proliferation of neural stem/progenitors (P < .05). MSC delivery reduced the acute proliferation. MSC treatment increased the number of neuroblasts in the outer region of the subventricular zone (P < .05) where they form migrating chains toward the frontal lobe. Conversely, the thickness of the neuroblast-dense band along the lateral ventricle was reduced after treatment (P < .05). These findings suggest that MSC treatment changes neuroblast distribution within the subventricular zone.

Conclusions: MSC delivery through CPB has the potential to mitigate effects of CPB on neural stem/progenitor cells and to promote migration of neuroblasts. Further investigation is necessary to determine the long-term effect of MSC treatment during CPB on postnatal neurogenesis.
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http://dx.doi.org/10.1016/j.athoracsur.2019.08.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093227PMC
April 2020

Generation of Norovirus-Specific T Cells From Human Donors With Extensive Cross-Reactivity to Variant Sequences: Implications for Immunotherapy.

J Infect Dis 2020 02;221(4):578-588

Center for Cancer and Immunology Research, Children's National Health System, Washington, District of Columbia, USA.

Background: Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences.

Methods: Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome.

Results: We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates.

Conclusions: Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.
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http://dx.doi.org/10.1093/infdis/jiz491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325618PMC
February 2020

Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization.

J Transl Med 2019 09 23;17(1):321. Epub 2019 Sep 23.

George Washington University Cancer Center, George Washington University, Washington, DC, USA.

Background: Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB.

Methods: CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed.

Results: We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008).

Conclusions: CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.
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http://dx.doi.org/10.1186/s12967-019-2055-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6757414PMC
September 2019

The pipeline of antiviral T-cell therapy: what's in the clinic and undergoing development.

Transfusion 2020 01 30;60(1):7-10. Epub 2019 Aug 30.

Center for Cancer and Immunology Research, Children's National Medical Center, Washington, District of Columbia.

Virus-specific T cells allow targeting of multiple pathogens in patients after hematopoietic stem cell transplantation and have demonstrated potential efficacy for cytomegalovirus, Epstein-Barr Virus, and adenovirus. Novel targets may include BK virus, JC virus, varicella zoster virus, human herpesvirus 6, Aspergillus, human parainfluenza virus-3, herpes simplex virus Type I, Zika virus, and mycobacteria. Generation of patient-specific products and third-party products may expand feasibility of therapy.
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http://dx.doi.org/10.1111/trf.15501DOI Listing
January 2020

Immunotherapy of Relapsed and Refractory Solid Tumors With Ex Vivo Expanded Multi-Tumor Associated Antigen Specific Cytotoxic T Lymphocytes: A Phase I Study.

J Clin Oncol 2019 09 29;37(26):2349-2359. Epub 2019 Jul 29.

Children's National Health System, Washington, DC.

Purpose: Tumor-associated antigen cytotoxic T cells (TAA-Ts) represent a new, potentially effective and nontoxic therapeutic approach for patients with relapsed or refractory solid tumors. In this first-in-human trial, we investigated the safety of administering TAA-Ts that target Wilms tumor gene 1, preferentially expressed antigen of melanoma, and survivin to patients with relapsed/refractory solid tumors.

Materials And Methods: TAA-T products were generated from autologous peripheral blood and infused over three dose levels: 1, 2, and 4 × 10 cells/m. Patients were eligible for up to eight infusions administered 4 to 7 weeks apart. We assessed dose limiting toxicity during the first 45 days after infusion. Disease response was determined within the context of a phase I trial.

Results: There were no dose-limiting toxicities. Of 15 evaluable patients, 11 (73%) with stable disease or better at day 45 postinfusion were defined as responders. Six responders remain without progression at a median of 13.9 months (range, 4.1 to 19.9 months) after initial TAA-Ts. Patients who were treated at the highest dose level showed the best clinical outcomes, with a 6-month progression-free survival of 73% after TAA-T infusion compared with a 38% 6-month progression-free survival with prior therapy. Antigen spreading and a reduction in circulating tumor-associated antigens using digital droplet polymerase chain reaction was observed in patients after TAA-T infusion.

Conclusion: TAA-Ts safely induced disease stabilization, prolonged time to progression, and were associated with antigen spreading and a reduction in circulating tumor-associated antigen DNA levels in patients with relapsed/refractory solid tumors without lymphodepleting chemotherapy before infusion. TAA-Ts are a promising new treatment approach for patients with solid tumors.
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http://dx.doi.org/10.1200/JCO.19.00177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804838PMC
September 2019

Safety and feasibility of virus-specific T cells derived from umbilical cord blood in cord blood transplant recipients.

Blood Adv 2019 07;3(14):2057-2068

Center for Cancer and Immunology Research, Children's National Health System and Department of Pediatrics, The George Washington University, Washington, DC.

Adoptive transfer of virus-specific T cells (VSTs) has been shown to be safe and effective in stem cell transplant recipients. However, the lack of virus-experienced T cells in donor cord blood (CB) has prevented the development of ex vivo expanded donor-derived VSTs for recipients of this stem cell source. Here we evaluated the feasibility and safety of ex vivo expansion of CB T cells from the 20% fraction of the CB unit in pediatric patients receiving a single CB transplant (CBT). In 2 clinical trials conducted at 2 separate sites, we manufactured CB-derived multivirus-specific T cells (CB-VSTs) targeting Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV) for 18 (86%) of 21 patients demonstrating feasibility. Manufacturing for 2 CB-VSTs failed to meet lot release because of insufficient cell recovery, and there was 1 sterility breach during separation of the frozen 20% fraction. Delayed engraftment was not observed in patients who received the remaining 80% fraction for the primary CBT. There was no grade 3 to 4 acute graft-versus-host disease (GVHD) associated with the infusion of CB-VSTs. None of the 7 patients who received CB-VSTs as prophylaxis developed end-organ disease from CMV, EBV, or adenovirus. In 7 patients receiving CB-VSTs for viral reactivation or infection, only 1 patient developed end-organ viral disease, which was in an immune privileged site (CMV retinitis) and occurred after steroid therapy for GVHD. Finally, we demonstrated the long-term persistence of adoptively transferred CB-VSTs using T-cell receptor-Vβ clonotype tracking, suggesting that CB-VSTs are a feasible addition to antiviral pharmacotherapy.
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http://dx.doi.org/10.1182/bloodadvances.2019000201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650740PMC
July 2019

Generation of Zika virus-specific T cells from seropositive and virus-naïve donors for potential use as an autologous or "off-the-shelf" immunotherapeutic.

Cytotherapy 2019 08 4;21(8):840-855. Epub 2019 Jul 4.

Center for Cancer and Immunology Research, Children's National Health System, Washington, DC, USA; The George Washington University, Washington, DC, USA; Division of Blood and Marrow Transplantation, Children's National Health System, Washington, DC, USA. Electronic address:

Background: Zika virus (ZIKV) infection can cause severe birth defects in newborns with no effective currently available treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for patients with ZIKV infection. However, extending this strategy to the ZIKV setting has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T cells and characterized the cellular immune responses against ZIKV antigens.

Methods: T-cell products were generated from peripheral blood of ZIKV-exposed donors, ZIKV-naive adult donors and umbilical cord blood by stimulation with pentadecamer (15mer) overlapping peptide libraries spanning four ZIKV polyproteins (C, M, E and NS1) using a Good Manufacturing Practice-compliant protocol.

Results: We successfully generated T cells targeting ZIKV antigens with clinically relevant numbers. The ex vivo-expanded T cells comprised both CD4 and CD8 T cells that were able to produce Th1-polarized effector cytokines and kill ZIKV-infected HLA-matched monocytes, confirming functionality of this unique T-cell product as a potential "off-the-shelf" therapeutic. Epitope mapping using peptide arrays identified several novel HLA class I and class II-restricted epitopes within NS1 antigen, which is essential for viral replication and immune evasion.

Discussion: Our findings demonstrate that it is feasible to generate potent ZIKV-specific T cells from a variety of cell sources including virus naïve donors for future clinical use in an "off-the-shelf" setting.
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http://dx.doi.org/10.1016/j.jcyt.2019.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744970PMC
August 2019

T-cell receptor sequencing demonstrates persistence of virus-specific T cells after antiviral immunotherapy.

Br J Haematol 2019 10 20;187(2):206-218. Epub 2019 Jun 20.

Center for Cancer and Immunology Research, Children's National Health System, Washington, DC, USA.

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (P = 0·0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT.
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http://dx.doi.org/10.1111/bjh.16053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786907PMC
October 2019

Fresh versus Frozen: Effects of Cryopreservation on CAR T Cells.

Authors:
Patrick J Hanley

Mol Ther 2019 07 13;27(7):1213-1214. Epub 2019 Jun 13.

Program for Cell Enhancement and Technology for Immunotherapy, Center for Cancer and Immunology Research, Center for Cancer and Blood Disorders, Children's National Health System, and The George Washington University, Washington, DC 20010, USA. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2019.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612752PMC
July 2019

Beyond CAR T Cells: Other Cell-Based Immunotherapeutic Strategies Against Cancer.

Front Oncol 2019 10;9:196. Epub 2019 Apr 10.

GW Cancer Center, The George Washington University, Washington, DC, United States.

Chimeric antigen receptor (CAR)-modified T cells have successfully harnessed T cell immunity against malignancies, but they are by no means the only cell therapies in development for cancer. Systemic immunity is thought to play a key role in combatting neoplastic disease; in this vein, genetic modifications meant to explore other components of T cell immunity are being evaluated. In addition, other immune cells-from both the innate and adaptive compartments-are in various stages of clinical application. In this review, we focus on these non-CAR T cell immunotherapeutic approaches for malignancy. The first section describes engineering T cells to express non-CAR constructs, and the second section describes other gene-modified cells used to target malignancy. CAR T cell therapies have demonstrated the clinical benefits of harnessing our body's own defenses to combat tumor cells. Similar research is being conducted on lesser known modifications and gene-modified immune cells, which we highlight in this review.
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http://dx.doi.org/10.3389/fonc.2019.00196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467966PMC
April 2019

Mycobacteria-Specific T Cells May Be Expanded From Healthy Donors and Are Near Absent in Primary Immunodeficiency Disorders.

Front Immunol 2019 29;10:621. Epub 2019 Mar 29.

Center for Cancer and Immunology Research, Children's National Health System, Washington, DC, United States.

Mycobacterial Infections can be severe in patients with T-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. Restoration of T-cell immunity via stem cell transplantation facilitates control of mycobacterial infections, but presence of active infections during transplantation is associated with a higher risk of mortality. Adoptive T cell immunotherapy has been successful in targeting viruses, but has not been attempted to treat mycobacterial infections. We sought to expand and characterize mycobacterial-specific T-cells derived from healthy donors in order to determine suitability for adoptive immunotherapy. Mycobacteria-specific T-cells (MSTs) were generated from 10 healthy donors using a rapid expansion protocol targeting five known mycobacterial target proteins (AG85B, PPE68, ESXA, ESXB, and ADK). MSTs were compared to T-cells expanded from the same donors using lysate from or purified protein derivative from (sensitin). MST expansion from seven patients with primary immunodeficiency disorders (PID) and two patients with IFN-γ autoantibodies and invasive infections. MSTs expanded from healthy donors recognized a median of 3 of 5 antigens, with production of IFN-γ, TNF, and GM-CSF in CD4 T cells. Comparison of donors who received BCG vaccine ( = 6) to those who did not ( = 4) showed differential responses to PPE68 ( = 0.028) and ADK ( = 0.015) by IFN-γ ELISpot. MSTs expanded from lysate or sensitin also recognized multiple mycobacterial antigens, with a statistically significant differences noted only in the response to PPE68 ( = 0.016). MSTs expanded from patients with primary immunodeficiency (PID) and invasive mycobacterial infections showed activity against mycobacterial antigens in only two of seven subjects, whereas both patients with IFN-γ autoantibodies recognized mycobacterial antigens. Thus, MSTs can be generated from donors using a rapid expansion protocol regardless of history of BCG immunization. Most tested PID patients had no detectable T-cell immunity to mycobacteria despite history of infection. MSTs may have clinical utility for adoptive immunotherapy in T-cell deficient patients with invasive mycobacterial infections.
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http://dx.doi.org/10.3389/fimmu.2019.00621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450173PMC
August 2020

Critical testing and parameters for consideration when manufacturing and evaluating tumor-associated antigen-specific T cells.

Cytotherapy 2019 03 28;21(3):278-288. Epub 2019 Mar 28.

Program for Cell Enhancement and Technologies for Immunotherapy, Center for Cancer and Immunology Research; Center for Cancer and Blood Disorders, and the Division of Blood and Marrow Transplantation; Children's National Health System and The George Washington University, Washington, DC, USA. Electronic address:

The past year has seen remarkable translation of cellular and gene therapies, with U.S. Food and Drug Administration (FDA) approval of three chimeric antigen receptor (CAR) T-cell products, multiple gene therapy products, and the initiation of countless other pivotal clinical trials. What makes these new drugs most remarkable is their path to commercialization: they have unique requirements compared with traditional pharmaceutical drugs and require different potency assays, critical quality attributes and parameters, pharmacological and toxicological data, and in vivo efficacy testing. What's more, each biologic requires its own unique set of tests and parameters. Here we describe the unique tests associated with ex vivo-expanded tumor-associated antigen T cells (TAA-T). These tests include functional assays to determine potency, specificity, and identity; tests for pathogenic contaminants, such as bacteria and fungus as well as other contaminants such as Mycoplasma and endotoxin; tests for product characterization, tests to evaluate T-cell persistence and product efficacy; and finally, recommendations for critical quality attributes and parameters associated with the expansion of TAA-Ts.
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http://dx.doi.org/10.1016/j.jcyt.2019.02.004DOI Listing
March 2019

Proposal for the International Society for Cell & Gene Therapy position statement on assays for the quality control and potency assessment of adoptive cellular immunotherapies.

Cytotherapy 2019 03 16;21(3):367-375. Epub 2019 Mar 16.

Centre for Cell, Gene & Tissue Therapeutics, Royal Free Hospital, London, UK; Department of Haematology, Cancer Institute, University College London, London, UK. Electronic address:

Translation of cell and gene therapies from pre-clinical experiments to clinical trials and final drug licensing brings requires the development, verification and even validation of the assays essential for the definition of the drug product. The technical and scientific challenges in doing this are far greater than they seem at first and are compounded by a lack of approved standards for assays used to support (c)GMP manufacture. This paper highlights some of those challenges and proposes solutions based on the experience of our colleagues using similar assay platforms in regulated pathology laboratories.
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http://dx.doi.org/10.1016/j.jcyt.2019.02.001DOI Listing
March 2019

Advancing cellular therapies towards standard of care: a focus on testing of cellular therapy products.

Cytotherapy 2019 03 7;21(3):275-277. Epub 2019 Mar 7.

Immuno-Gene Therapy Scientific Committee, International Society for Cell and Gene Therapy, Vancouver, Canada; Department of Haematology, Cancer Institute, University College London, London, UK; Centre for Cell, Gene & Tissue Therapeutics, Royal Free Hospital, London, UK.

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http://dx.doi.org/10.1016/j.jcyt.2019.02.003DOI Listing
March 2019

Build a Bank: Off-the-Shelf Virus-Specific T Cells.

Authors:
Patrick J Hanley

Biol Blood Marrow Transplant 2018 12 18;24(12):e9-e10. Epub 2018 Oct 18.

Center for Cancer and Immunology Research, Center for Cancer and Blood Disorders, and Program for Cell Enhancement and Technologies for Immunotherapy, Children's National Health System, The George Washington University, Washington, DC. Electronic address:

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http://dx.doi.org/10.1016/j.bbmt.2018.10.010DOI Listing
December 2018

EBV/LMP-specific T cells maintain remissions of T- and B-cell EBV lymphomas after allogeneic bone marrow transplantation.

Blood 2018 11 27;132(22):2351-2361. Epub 2018 Sep 27.

Center for Cancer and Immunology Research, Children's National Health System and George Washington University, Washington, DC.

Autologous T cells targeting Epstein-Barr virus (EBV) latent membrane proteins (LMPs) have shown safety and efficacy in the treatment of patients with type 2 latency EBV-associated lymphomas for whom standard therapies have failed, including high-dose chemotherapy followed by autologous stem-cell rescue. However, the safety and efficacy of allogeneic donor-derived LMP-specific T cells (LMP-Ts) have not been established for patients who have undergone allogeneic hematopoietic stem-cell transplantation (HSCT). Therefore, we evaluated the safety and efficacy of donor-derived LMP-Ts in 26 patients who had undergone allogeneic HSCT for EBV-associated natural killer/T-cell or B-cell lymphomas. Seven patients received LMP-Ts as therapy for active disease, and 19 were treated with adjuvant therapy for high-risk disease. There were no immediate infusion-related toxicities, and only 1 dose-limiting toxicity potentially related to T-cell infusion was seen. The 2-year overall survival (OS) was 68%. Additionally, patients who received T-cell therapy while in complete remission after allogeneic HSCT had a 78% OS at 2 years. Patients treated for B-cell disease (n = 10) had a 2-year OS of 80%. Patients with T-cell disease had a 2-year OS of 60%, which suggests an improvement compared with published posttransplantation 2-year OS rates of 30% to 50%. Hence, this study shows that donor-derived LMP-Ts are a safe and effective therapy to prevent relapse after transplantation in patients with B cell- or T cell-derived EBV-associated lymphoma or lymphoproliferative disorder and supports the infusion of LMP-Ts as adjuvant therapy to improve outcomes in the posttransplantation setting. These trials were registered at www.clinicaltrials.gov as #NCT00062868 and #NCT01956084.
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http://dx.doi.org/10.1182/blood-2018-07-863654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6265652PMC
November 2018

HIV-Specific, Ex Vivo Expanded T Cell Therapy: Feasibility, Safety, and Efficacy in ART-Suppressed HIV-Infected Individuals.

Mol Ther 2018 10 21;26(10):2496-2506. Epub 2018 Sep 21.

UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Adoptive T cell therapy has had dramatic successes in the treatment of virus-related malignancies and infections following hematopoietic stem cell transplantation. We adapted this method to produce ex vivo expanded HIV-specific T cells (HXTCs), with the long-term goal of using HXTCs as part of strategies to clear persistent HIV infection. In this phase 1 proof-of-concept study (NCT02208167), we administered HXTCs to antiretroviral therapy (ART)-suppressed, HIV-infected participants. Participants received two infusions of 2 × 10 cells/m HXTCs at a 2-week interval. Leukapheresis was performed at baseline and 12 weeks post-infusion to measure the frequency of resting cell infection by the quantitative viral outgrowth assay (QVOA). Overall, participants tolerated HXTCs, with only grade 1 adverse events (AEs) related to HXTCs. Two of six participants exhibited a detectable increase in CD8 T cell-mediated antiviral activity following the two infusions in some, but not all, assays. As expected, however, in the absence of a latency reversing agent, no meaningful decline in the frequency of resting CD4 T cell infection was detected. HXTC therapy in ART-suppressed, HIV-infected individuals appears safe and well tolerated, without any clinical signs of immune activation, likely due to the low residual HIV antigen burden present during ART.
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http://dx.doi.org/10.1016/j.ymthe.2018.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171327PMC
October 2018