A Panel of Artificial Antigen Presenting Cells (AAPC) Permits Immediate Sensitization and Rapid Generation of Virus-Specific T-Cells of Desired HLA Restriction for Adoptive Immunotherapy.

Abstract Adoptive transfer of donor-derived virus- specific T-cells can treat or prevent severe infections in allogeneic hematopoietic stem cell transplant recipients, but maybe ineffective in HLA-disparate recipients if the T-cells are restricted by unshared alleles. We previously described an AAPC consisting of mouse 3T3 fibroblasts transduced to express human B7.1, LFA-3, ICAM-1, β2-microglobulin and the alpha chain of HLA A*0201 (Papanicolaou et al. Blood2003;102:2498). To test the potential of AAPC to present CMV pp65 epitopes and elicit virus-specific T-cells restricted by other, less common alleles, we generated a panel of AAPC, each expressing one of a series of HLA class-I alleles, including HLA A*0201, A*0301, A*2402, B*0702, B*0801 and C*0401. We have subsequently tested the AAPC expressing HLA A*0201, A*2402 and B*0702 for their capacity to sensitize human T-cells when loaded with overlapping 15-mer peptides spanning the CMV pp65 sequence or transduced to express the CMV pp65 protein. T-cells from groups of up to 4 CMV seropositive donors, each expressing one of these HLA alleles, when sensitized with either peptide loaded or transduced AAPC expanded 15–38 fold in vitro over 21 days. The T-cells generated were predominantly CD8+ T-cells containing central memory and effector memory subsets. As shown in table.1, T-cells elicited by each AAPC exhibited a high level of HLA-restricted, CMV-specific cytotoxic activity despite the variability in the percentage of HLA-tetramer positive and IFNγ producing T-cells. Responses against unmodified autologous or allogeneic targets was <5%. Similarly, over 2% of the T-cells in the cultures generated IFNγ in response to secondary stimulation with autologous PBMC loaded with the peptide pool, versus 0.1–0.3% in controls. In comparative assays, the yields of specific CTL generated in response to AAPC transduced to express CMV pp65 were higher than those generated in response to pool-loaded AAPC in each of the 9 donors tested. Strikingly, staining by HLA-tetramers demonstrated that sensitization by mouse derived, CMV pp65 transduced AAPC generates high proportions of T cells (7–51%) recognizing epitopes known to be immunogenic when presented by that human HLA allele on CMV infected cells i.e. NLVPMAVTV by HLA A*0201, QYDPVAALF by A*2402 and RPHERNGFTV by B*0702. These data indicate that the human HLA expressing 3T3 cells process CMV pp65 protein and transport and present epitopes which are similar or identical to those presented on human, CMV-infected cells. Thus, a replenishable panel of 3T3 based AAPC, each expressing a single HLA allele, can be used for immediate sensitization and efficient expansion of human CMV-specific T-cells. Use of this panel permits rapid generation of virus-specific T-cells of desired HLA restriction for adoptive immunotherapy and maybe of particular value for treatment of patients receiving grafts from HLA-disparate donors. Table 1. Analysis of CTL Generated with AAPC (Transduced with CMV pp65 or Peptide Pulsed) Donors Fold T-cell Expansion % CD8 CD8 % Tetramer [+] CD8 % IFNγ 51-Cr Cytotoxicity All values represent average +/− SEM A*0201 Donors N=4 28 +/− 3 84% +/− 3 51% +/− 0.1 2.6% +/− 0.03 54% +/− 0.1 A*2402 Donors N=4 15 +/− 2 62% +/− 5 15% +/− 0.2 2.43% +/− 0.003 34% +/− 0.03 B*0702 Donors N=1 38 85% 7% 2% 53%

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Large-scale GMP-compliant CRISPR-Cas9–mediated deletion of the glucocorticoid receptor in multivirus-specific T cells

Blood Advances ◽

10.1182/bloodadvances.2020001977 ◽

2020 ◽

Vol 4 (14) ◽

pp. 3357-3367 ◽

Cited By ~ 4

Author(s):

Rafet Basar ◽

May Daher ◽

Nadima Uprety ◽

Elif Gokdemir ◽

Abdullah Alsuliman ◽

...

Keyword(s):

T Cells ◽

Glucocorticoid Receptor ◽

Large Scale ◽

Genetically Engineered ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Management Of Complications ◽

Rapid Generation

Abstract Virus-specific T cells have proven highly effective for the treatment of severe and drug-refractory infections after hematopoietic stem cell transplant (HSCT). However, the efficacy of these cells is hindered by the use of glucocorticoids, often given to patients for the management of complications such as graft-versus-host disease. To address this limitation, we have developed a novel strategy for the rapid generation of good manufacturing practice (GMP)–grade glucocorticoid-resistant multivirus-specific T cells (VSTs) using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) gene-editing technology. We have shown that deleting the nuclear receptor subfamily 3 group C member 1 (NR3C1; the gene encoding for the glucocorticoid receptor) renders VSTs resistant to the lymphocytotoxic effect of glucocorticoids. NR3C1-knockout (KO) VSTs kill their targets and proliferate successfully in the presence of high doses of dexamethasone both in vitro and in vivo. Moreover, we developed a protocol for the rapid generation of GMP-grade NR3C1 KO VSTs with high on-target activity and minimal off-target editing. These genetically engineered VSTs promise to be a novel approach for the treatment of patients with life-threatening viral infections post-HSCT on glucocorticoid therapy.

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Adoptive Immunotherapy for CMV Infection Complicating Haploidentical Transplants: Challenges Posed by the HLA Restriction of Immuno- Dominant Donor T Cells

Blood ◽

10.1182/blood.v112.11.2918.2918 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2918-2918

Author(s):

Aisha Hasan ◽

Ekatarina Doubrovina ◽

Deepa Trivedi ◽

Nancy A. Kernan ◽

Susan Prockop ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Immunotherapy ◽

Antiviral Treatment ◽

Host Cells ◽

Unrelated Donor ◽

Hematopoietic Stem ◽

Hla Alleles ◽

Hla Restriction

Abstract T-cell depleted hematopoietic stem cell transplants (HSCT) from HLA haplotypedisparate related donors are being increasingly used for the treatment of patients lacking an HLA-compatible related or unrelated donor. Acute and chronic graft versus host disease can be prevented without added prophylaxis if techniques achieving extensive T-cell depletion are employed. Improved conditioning regimens and transplants providing high doses of CD34+ cells have also insured consistent engraftment. However, these patients have a higher and more prolonged risk of serious viral infections than recipients of matched unmodified or T-cell depleted grafts. Adoptive immunotherapy with donor-derived pathogen-specific T-cells can restore effective resistance in these patients. However, T-cells sensitized and propagated in vitro usually respond to only 1–2 immunodominant epitopes from any antigeneic protein presented by different donor HLA alleles. We questioned how often T-cells generated from an HLA-haplotype disparate donor would be restricted by donor-unique, rather than shared HLA alleles since such T-cells would be inactive against virus-infected cells of a haplotype disparate host. Accordingly, we tested 8 cytomegalovirus (CMV) seropositive HLA haplotype disparate parent or sibling donors, as well as 4 patients, each transplanted from one of these donors 1 or more years previously. In each case, T-cells were sensitized with autologous DCs loaded with a pool of overlapping 15-mers spanning the sequence of CMVpp65, an immunodominant protein of cytomegalovirus, for 21–28 days. Using a matrix of peptide subpools, we then mapped the epitopes eliciting IFNγ+ T-cell responses and then determined their HLA-restriction by assaying their specific cytotoxicity against a panel of EBV B-cell lines loaded with the immunogenic epitope each sharing a single HLA allele with the donor. Of the 8 donors tested, 6 responded to CMV pp65 epitopes presented by HLA alleles shared with the haplotype matched recipient, while 2 donors responded exclusively to epitopes presented by donor unique HLA alleles. Recipients of transplants from the latter two donors experienced prolonged CMV viremia, one with CMV chorioretinitis, requiring months of antiviral treatment. CMV-specific T-cells isolated from each of the four transplanted patients over 1 year post transplant were specific for epitopes presented by HLA alleles shared by their donors. In the patients transplanted from donors whose CMV-pp65 specific T-cells were restricted by donor-unique HLA alleles, the epitope targeted and HLA restriction of the engrafted T-cell differed from that of the transplant donors own T-cells, suggesting that these T-cells may have developed from donor precursors differentiating in the host thymus. Thus, in a significant proportion of cases (2/8 in this study), virus-specific T-cells generated in vitro from HLA haplotype disparate donors may be restricted by donor-unique HLA alleles and, as a consequence, inactive against virus-infected host cells. For such patients, techniques insuring selective generation of T-cells specific for epitopes presented by shared alleles should be employed. Our findings may also in part explain the prolonged susceptibility of these patients to viral infection. Since immunodominant T-cells reactive against the same epitopes are also those detected at highest frequency in the donor’s blood, they would likely be the only virus-reactive T-cells transferred in a T-cell depleted graft. As a consequence, effective CMV-specific responses might be delayed until new T cells are generated from precursors developing in the host thymus.

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Rapid Generation of Multivirus-Specific T Lymphocytes for the Prevention and Treatment of Respiratory Viral Infections

Blood ◽

10.1182/blood-2018-99-113168 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3332-3332

Author(s):

Spyridoula Vasileiou ◽

Annie Turney ◽

Manik Kuvalekar ◽

Shivani Mukhi ◽

Ayumi Watanabe ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Viral Antigen ◽

T Cell Subsets ◽

Effector Memory ◽

Cell Transplant ◽

Activation Markers ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Tract Infections

Abstract Acute upper and lower respiratory tract infections (RTIs) due to community-acquired respiratory viruses (CARVs) including respiratory syncytial virus (RSV), influenza, parainfluenza virus (PIV) and human metapneumovirus (hMPV) are a leading cause of morbidity and mortality worldwide, with individuals whose immune systems are naïve (e.g. children) or compromised being most vulnerable. In allogeneic hematopoietic stem cell transplant (HSCT) recipients, the incidence of CARV-related respiratory viral infection reaches 29%. Most patients initially present with mild symptoms of upper RTI and in 50% of cases the infection progresses to a lower RTI with severe symptoms including bronchiolitis and pneumonia and mortality rates as high as 50%. Currently there are no approved vaccines nor antiviral drugs for hMPV and PIV, while the preventative vaccine for Influenza is not indicated earlier than 6 months post-HSCT. Aerosolized ribavirin is FDA-approved for the treatment of RSV infections, but it is logistically difficult to administer and comes at a considerable cost. Thus, the lack of approved antiviral agents combined with the high cost of antiviral therapy emphasize the need for alternative treatment strategies for CARVs. Our group has previously demonstrated the safety and clinical efficacy of using adoptive T-cell transfer for the treatment of both latent [Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), human herpesvirus 6 (HHV6)] and lytic [adenovirus (AdV)] viruses in recipients of allo-HSCT by generation of multivirus-specific T cell (VST) lines. Given that susceptibility to CARVs is highly associated with underlying immune deficiency, we wanted to explore the potential for extending this approach to Influenza, RSV, hMPV and PIV3 infections. In order to do so, we exposed PBMCs from healthy donors to a cocktail of pepmixes (overlapping peptide libraries) spanning immunogenic antigens derived from our target viruses [Influenza - NP1 and MP1; RSV - N and F; hMPV - F, N, M2-1 and M; PIV3 - M, HN, N and F] followed by expansion in the presence of activating cytokines in a G-Rex device. Over 10-13 days we achieved an average 8.5 fold expansion [increase from 0.25x107 PBMCs/cm2 to mean 1.9±0.2x107 cells/cm2; n=12). Cells were comprised almost exclusively of CD3+ T cells (96.2±0.6%; mean±SEM), with a mixture of cytotoxic (CD8+) and helper (CD4+) T cells and a phenotype consistent with immediate effector function and long term memory, as evidenced by upregulation of the activation markers CD25, CD69, and CD28 as well as expression of central (CD45RO+/CD62L+) and effector memory markers (CD45RO+/CD62L−), with minimal PD1 or Tim3 expression. Anti-viral specificity of multi-R-VSTs was tested in an IFNγ Elispot assay using each of the individual stimulating antigens as an immunogen and all 12 lines screened proved to be reactive against all 4 of the target viruses [Influenza: mean 735±75.6 SFC/2x105, RSV: 758±69.8, hMPV: 526±100.8, PIV3: 391±93.7]. As demonstrated by intracellular cytokine staining, the immune response was mediated by both CD4+ and CD8+ T cell subsets, and the majority of IFNγ-producing cells also produced TNFα. In addition, the cells secreted GM-CSF as measured by Luminex array, with baseline levels of Th2/suppressive cytokines. Furthermore, upon antigenic stimulation our VSTs produced the effector molecule Granzyme B suggesting the cytolytic potential of these expanded cells, which was confirmed in a standard Cr51-release assay against viral pepmix-loaded autologous PHA blasts. Viral antigen-loaded targets were specifically recognized and lysed by our VSTs, while there was no evidence of activity against non-infected autologous or allogeneic targets. In conclusion, we have shown that it is feasible to rapidly generate a single preparation of polyclonal multi-respiratory (multi-R)-VSTs with specificities directed to Influenza, RSV, hMPV and PIV3 and a total of 12 encoded antigens using GMP-compliant manufacturing methodologies. The expanded cells are Th1-polarized, polyfunctional and selectively able to react to and kill viral antigen-expressing targets with no auto- or alloreactivity, attesting to both their selectivity and their safety for clinical use in HSCT recipients. We anticipate such multi-R-VSTs will provide clinical benefit in preventing or treating CARV infections in the immunocompromised. Disclosures Vera: Viracyte: Equity Ownership. Tzannou:Viracyte: Consultancy, Equity Ownership. Leen:Viracyte: Equity Ownership.

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Safety and Preliminary Efficacy of "Ready to Administer" Cytomegalovirus (CMV)-Specific T Cells for the Treatment of Patients with Refractory CMV Infection

Blood ◽

10.1182/blood.v128.22.388.388 ◽

2016 ◽

Vol 128 (22) ◽

pp. 388-388 ◽

Cited By ~ 1

Author(s):

Ifigeneia Tzannou ◽

Kathryn S. Leung ◽

Caridad Martinez ◽

Swati Naik ◽

Stephen Gottschalk ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplant ◽

Fixed Dose ◽

Effector Memory ◽

Cell Transplant ◽

Decision Tool ◽

Widespread Application ◽

Hematopoietic Stem

Abstract Despite advances in antiviral drugs, Cytomegalovirus (CMV) infections remain a significant cause of morbidity and mortality in immunocompromised individuals. We have recently demonstrated in hematopoietic stem cell transplant (HSCT) recipients that adoptively-transferred virus-specific T cells, generated from healthy 3rd party donors and administered as an "ready to administer" product, can be curative, even in patients with drug-refractory CMV infections. However, broader implementation has been hindered by the postulated need for extensive panels of T cell lines representing a diverse HLA profile, as well as the complexities of large scale manufacturing for widespread clinical application. To address these potential issues, we have developed a decision tool that identified a short list of donors who provide HLA coverage for >90% of the stem cell transplant population. Furthermore, to generate banks of CMV-specific T cells from these donors, we have created a simple, robust, and linearly scalable manufacturing process. To determine whether these advances would enable the widespread application of "ready to administer" T cells, we generated CMV cell banks (Viralym-C™) from 9 healthy donors selected by our decision tool, and initiated a fixed-dose (2x107 cells/m2) Phase I clinical trial for the treatment of drug-refractory CMV infections in pediatric and adult HSCT recipients. To generate the Viralym-C™ banks, we stimulated donor peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries spanning the immunodominant CMV antigens pp65 and IE1. Cells were subsequently expanded in a G-Rex device, resulting in a mean fold expansion of 103±12. The lines were polyclonal, comprising both CD4+ (21.3±6.7%) and CD8+ (74.8±6.9%) T cells, and expressed central CD45RO+/CD62L+ (58.5±4.2%) and effector memory markers CD45RO+/CD62L- (35.3±12.2%). Furthermore, the lines generated were specific for the target antigens (IE1: 419±100; pp65 1070±31 SFC/2x105, n=9). To date, we have screened 12 patients for study participation, and from our bank of just 9 lines we have successfully identified a suitable line for all patients within 24 hours. Of these, 6 patients have been infused; 5 received a single infusion and 1 patient required 2 infusions for sustained benefit. There were no immediate infusion-related toxicities; and despite the HLA disparity between the Viralym-C lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (GvHD). One patient developed a transient fever a few hours post-infusion, which spontaneously resolved. Based on viral load, measured by quantitative PCR, or symptom resolution (in patients with disease), Viralym-C™ cells controlled active infections in all 5 evaluable patients; 4 patients had complete responses, and 1 patient had a partial response within 4 weeks of cell infusion. One patient with CMV retinitis had complete resolution of symptoms following Viralym-C™ infusion. In conclusion, our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" Viralym-C™ cells that have been generated from a small panel of healthy, eligible CMV seropositive donors identified by our decision support tool. These data suggest that cost-effective, broadly applicable T cell anti-viral therapy may be feasible for patients following HSCT and potentially other conditions. Disclosures Tzannou: ViraCyte LLC: Consultancy. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties. Kakarla:ViraCyte LLC: Employment.

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IL-7 and IL-15 allow the generation of suicide gene–modified alloreactive self-renewing central memory human T lymphocytes

Blood ◽

10.1182/blood-2008-05-156059 ◽

2009 ◽

Vol 113 (5) ◽

pp. 1006-1015 ◽

Cited By ~ 113

Author(s):

Shin Kaneko ◽

Sara Mastaglio ◽

Attilio Bondanza ◽

Maurilio Ponzoni ◽

Francesca Sanvito ◽

...

Keyword(s):

T Cells ◽

Suicide Gene ◽

Central Memory ◽

Effector Memory ◽

Humanized Mouse Model ◽

Hematopoietic Stem ◽

Gene Therapy Approach ◽

Activation Induced Cell Death ◽

Cells Cultured

Abstract Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (TEM) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (TCM) phenotype. To this, we generated suicide gene–modified TCM lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene–modified TCM cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene–modified T cells cultured with IL-7 and IL-15 persisted, differentiated in TEM cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene–modified TCM cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.

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Treatment of Adenovirus Infection by Adoptive Immunotherapy: Adenovirus Capsid Hexon Is the Main Target Protein of Adenovirus-Specific Cellular Immunity.

Blood ◽

10.1182/blood.v108.11.2930.2930 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2930-2930

Author(s):

Olga Arbach ◽

Marco Frentsch ◽

Barbara Chmielewicz ◽

Marco Kaiser ◽

Heinz Ellerbrock ◽

...

Keyword(s):

T Cells ◽

Adoptive Immunotherapy ◽

Cd4 T Cells ◽

Ex Vivo ◽

Cytokine Profile ◽

Target Antigen ◽

Hematopoietic Stem ◽

Hexon Protein ◽

Antigenic Targets

Abstract Introduction: Adenovirus (AdV) infection is a severe complication after hematopoietic stem cell transplantation, particularly in paediatric patients. Control of AdV infection particularly seems to be dependent on CD4+ T-cells but their main AdV antigenic targets have remained unknown so far. In order to design protocols for targeted adoptive immunotherapy the identification of the main AdV antigenic targets is a fundamental prerequisite. Methods: We here adapted a novel technique to directly assess the entire repertoire of CD4+ T-cells specific AdV antigens according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. The cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells were assessed by intracellular analysis of AdV-induced CD154+ CD4+ T-cells. Results: AdV-lysate-specific CD4+ T-cells reacted predominantly with AdV-hexon capsid protein. Furthermore, AdV-hexon (serogroup B) specific CD4+ T-cells crossreacted with recombinant hexon protein derived from various other AdV-serotypes and were characterized by a Th1-like cytokine profile. Conclusion: Our results prove the effectiveness of antigen-induced CD154-expression for assessment of the entire repertoire of CD4+ T-cells specific for pathogens, for the identification of immunodominant target antigen from pathogens. We demonstrate that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive CD4+ T-cells with a Th1-like cytokine profile for adoptive T-cell therapies.

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Rapidly expanded partially HLA DRB1–matched fungus-specific T cells mediate in vitro and in vivo antifungal activity

Blood Advances ◽

10.1182/bloodadvances.2020001565 ◽

2020 ◽

Vol 4 (14) ◽

pp. 3443-3456

Author(s):

Gloria Castellano-González ◽

Helen M. McGuire ◽

Fabio Luciani ◽

Leighton E. Clancy ◽

Ziduo Li ◽

...

Keyword(s):

T Cells ◽

Fungal Infection ◽

Fungal Infections ◽

Interferon Γ ◽

Third Party ◽

Interleukin 17 ◽

Cell Transplant ◽

Hematopoietic Stem

Abstract Invasive fungal infections are a major cause of disease and death in immunocompromised hosts, including patients undergoing allogeneic hematopoietic stem cell transplant (HSCT). Recovery of adaptive immunity after HSCT correlates strongly with recovery from fungal infection. Using initial selection of lymphocytes expressing the activation marker CD137 after fungal stimulation, we rapidly expanded a population of mainly CD4+ T cells with potent antifungal characteristics, including production of tumor necrosis factor α, interferon γ, interleukin-17, and granulocyte-macrophage colony stimulating factor. Cells were manufactured using a fully good manufacturing practice–compliant process. In vitro, the T cells responded to fungal antigens presented on fully and partially HLA-DRB1 antigen–matched presenting cells, including when the single common DRB1 antigen was allelically mismatched. Administration of antifungal T cells lead to reduction in the severity of pulmonary and cerebral infection in an experimental mouse model of Aspergillus. These data support the establishment of a bank of cryopreserved fungus-specific T cells using normal donors with common HLA DRB1 molecules and testing of partially HLA-matched third-party donor fungus-specific T cells as a potential therapeutic in patients with invasive fungal infection after HSCT.

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CAR T Cells Targeted with a High Affinity Scfv Against the HCMV Glycoprotein Gb As Adoptive T Cell Therapy after Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v128.22.5721.5721 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5721-5721 ◽

Cited By ~ 2

Author(s):

Renata Stripecke ◽

Laura Gerasch ◽

Sebastian Theobald ◽

Bala Sai Sundarasetty ◽

Maksim Mamonkin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Target Cells ◽

Hematopoietic Stem ◽

High Affinity ◽

Car T Cells ◽

Car T ◽

Infected Cells

Abstract Introduction: Reactivation of human cytomegalovirus (HCMV) in immune compromised patients after hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality, particularly after cord blood transplantation (CBT). Adoptive transfer of T cells expanded in vitro is currently used as therapy for drug-refractory HCMV disease. A major limitation of this approach is the requirement of HLA-restricted HCMV-specific memory T cells. An alternative approach exploring HLA-independent T cell recognition was sought. Because the HCMV envelope glycoprotein B (gB) is highly expressed during lytic infection and in latently infected cells, we hypothesized that T cells can be redirected to recognize and kill HCMV-specific cells by means of a gB-specific chimeric antigen receptor (CAR). We have synthesized and tested a gB-specific CAR derived from the SM5-1 monoclonal antibody which binds with high affinity (KD 5.7x1011) to a conserved antigenic and non-glycosylated domain of gB. Methods: We generated two codon-optimized SM5-derived scFvs (VH->VL and VL->VH) and fused with an existing CAR backbone containing an IgG Fc spacer and intracellular signaling domains. CARs containing either CD28.zeta or 4-1BB.zeta were synthesized and expressed in T cells following a standard retroviral transduction protocol yielding 80-90% transduction rate. Expression of the CARs on T cells was confirmed by flow cytometry using goat anti-human immunoglobulin reactive against the IgG Fc region. 293T cells co-expressing gB and dTomato were used for in vitro cytotoxicity assays. Results: T cells expressing gB-CAR/CD28.zeta were cytotoxic against gB+ target cells producing 90% killing of 293T/gB-dTom cells compared with control CD19 CAR/CD28.zeta cells at an effector-to-target ratio 3:1 for 48 h (parental 293T cells were not killed). The cytolytic activity correlated with expansion of CAR T cells and concomitant loss of gB-dTom expression in the remaining viable 293T cells. Sequential co-culture of these gB-CAR T cells with freshly seeded 293T/gB-dTom resulted into further elimination of target cells. We are currently evaluating the effects of different gB-CAR T cell designs in the killing of HCMV-infected cell lines and primary cells using HCMV laboratory strains expressing the GFP and Gaussia Luciferase reporter genes. Pilot experiments indicated that gB-CAR/CD28.zeta cells with the scFv in the VL->VH orientation resulted into more clustering and killing of HepG2 cells infected with HCMV-GFP after 24h of co-culture than a control CD19 CAR/CD28.zeta. Humanized mice transplanted with cord blood CD34+ stem cells and challenged with these HCMV laboratory strains will be used to evaluate the in vivo effectivity of cord blood-derived donor-matched gB-CAR-T cells to eliminate acute and latent HCMV infections. Conclusion: These studies explore a novel approach in preventing HCMV reactivation in immunosuppressed patients by redirecting T cells expressing a high-affinity gB-CAR to eliminate HCMV-infected cells in a TCR/MHC-independent manner. Disclosures No relevant conflicts of interest to declare.

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CrkL is required for donor T cell migration to GvHD Target organs

10.1101/854042 ◽

2019 ◽

Author(s):

Nathan H. Roy ◽

Mahinbanu Mammadli ◽

Janis K. Burkhardt ◽

Mobin Karimi

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Adaptor Protein ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Peripheral Tissues ◽

T Cell Migration ◽

Related Family

ABSTRACTThe success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.

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