T Cell Receptor Gene Transfer to Virus-Specific T Cells for Cellular Anti-Tumor Immunotherapy.

Abstract Patients with relapsed hematological malignancies after allogeneic stem cell transplantation (alloSCT) can be successfully treated by donor lymphocyte infusions (DLI). Since DLI consists of a variety of T cells with different specificities, the benificial anti-leukemic effect of DLI is often accompanied by Graft-versus-Host Disease (GvHD). Genetic modification of T cells to express T cell receptors (TCR) with defined anti-tumor specificity would be an attractive strategy to specifically eradicate the malignant cells without induction of GvHD. We previously demonstrated that transfer of the minor histocompatibility antigen HA-2 specific TCR to CMV specific T cells led to the generation of T cells with dual specificity for CMV as well as HA-2. CMV and EBV specific T cells are ideal target cells for TCR gene transfer, since the majority of human individuals have high frequencies of these T cells due to latent persistence of CMV and EBV. In addition, based on their virus specificity, these T cells do not induce GvHD in an alloSCT setting, and we hypothesize that due to frequent encounter with viral antigens, TCR transferred virus specific T cells will survive for a prolonged period of time in vivo. The aim of this study is to develop a clinical grade method for the generation of TCR transduced virus specific T cells for cellular immunotherapy. CMV and EBV specific T cells were isolated from healthy individuals using pentamers in combination with clinical grade available anti-biotin magnetic beads. Isolation by pentamer-coated beads induced stimulation, expansion and efficient transduction of virus specific T cells, leading to the generation of cell lines with high frequencies of virus specific (>80%) and transduced (20–40%) T cells. T cells were transduced with multi-cistronic retroviral vectors encoding the α and β chains of the HA-2 TCR linked by an IRES or 2A-like sequence. No differences in transduction efficiency and TCR surface expression were observed between the IRES and 2A-like vectors. The transduced virus specific T cells were shown to exhibit dual specificity and tetramer staining of the introduced TCR correlated with specific lysis of target cells endogenously-expressing HA-2. Furthermore, variation in surface expression of the introduced TCR was observed between T cells with different virus specificities. T cells directed against the HLA-A1 epitope of CMV-pp50, for example, efficiently expressed the HA-2 TCR, whereas T cells specific for the HLA-B8 epitope of EBV-EBNA-3A did not express the introduced TCR. Functional analyses demonstrated that TCR-transduced pp50 specific T cells were dual specific, recognizing HA-2 as well as pp50 positive target cells, whereas TCR-engineered EBNA-3A specific T cells were primarily EBNA-3A specific. The efficiency of surface expression of the transferred TCR was shown to be determined by intrinsic properties of the TCRs, illustrating that for TCR gene transfer purposes TCRs need to be selected that exhibit high competition potential, whereas recipient T cells need to express endogenous TCRs with low competition potential. For clinical application, TCRs will be transferred to virus specific T cells selected for their capacity to efficiently express the introduced TCR without loss of virus specificity. The safety, clinical and immunological efficacy of TCR gene transfer to virus specific T cells as cellular anti-tumor immunotherapy after alloSCT will be investigated in a clinical phase I/II study.

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Foamy Virus Based Vectors for CAR-T Cell Development

Blood ◽

10.1182/blood-2019-123022 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4646-4646

Author(s):

Emmanouil Simantirakis ◽

Vassilis Atsaves ◽

Ioannis Tsironis ◽

Margarita Gkyzi ◽

Kostas Konstantopoulos ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Peripheral Blood ◽

Transduction Efficiency ◽

Target Cells ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Ifn Γ

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

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Latent-Membrane 2a Specific CTL’s but Not Latent-Mebrane 1 Specific CTL’s Lysis Efficiently Hodgkin Lymphoma Cells.

Blood ◽

10.1182/blood.v104.11.4991.4991 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4991-4991

Author(s):

Max S. Topp ◽

Jan Diekamm ◽

Olaf Beck ◽

Georg Rauser ◽

Kai Hauschulz ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Adoptive Immunotherapy ◽

Cytokine Production ◽

Cd8 T Cell ◽

Rapid Expansion ◽

Target Cells ◽

Specific Lysis ◽

Healthy Donors

Abstract Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.

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Re-Engineering γ δ T Cells by α β T Cell Receptor Gene Transfer Creates Potent Effector Cells with Anti-Leukemic Reactivity.

Blood ◽

10.1182/blood.v106.11.1288.1288 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1288-1288

Author(s):

Lars T. van der Veken ◽

Renate S. Hagedoorn ◽

Marleen M. van Loenen ◽

Roel Willemze ◽

J.H. Frederik Falkenburg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Peripheral Blood ◽

Cytokine Production ◽

Target Cells ◽

Facs Analysis ◽

Effector Cells ◽

Tetramer Binding ◽

Potent Effector

Abstract Retroviral transfer of T cell receptors (TCRs) to peripheral blood derived T cells generates large numbers of T cells with the same antigen specificity, which can potentially be used for adoptive immunotherapy. One drawback of this procedure is the formation of mixed α β TCR dimers with unknown specificities due to pairing of endogenous and introduced TCR chains. To completely prevent the formation of mixed TCR dimers by TCR gene transfer to α β T cells we investigated whether γ δ T cells can serve as alternative host T cells for α β TCR transfer, since the γ δ TCR is not capable of forming dimers with the α β TCR. Peripheral blood derived γ δT cells were isolated by immunomagnetic bead isolation and subsequent FACS sorting, resulting in >99% pure populations of γ δT cells. The isolated γ δT cells were retrovirally transduced with three different TCRs specific for the hematopoietic minor histocompatibility antigen (mHag) HA-2 in the context of HLA-A2, for CMV-pp65 in the context of HLA-B7, or for the HLA class II restricted mHag DBY. The TCR-transduced γ δT cells expressed both the introduced TCRs and the endogenous γ δTCR at their cell surface as determined by FACS analysis. When γ δT cells transduced with the HLA class I restricted HA-2-TCR or CMV-TCR were stained with tetramers, only the CMV-TCR expressing γ δT cells but not the HA-2-TCR expressing γ δT cells were capable of strong antigen specific tetramer binding. In contrast, functional analysis indicated that all TCR-transduced γ δT cells specifically recognized peptide pulsed target cells leading to target cell lysis and IFNγ and IL-4 production, indicating that while the avidity of the HA-2-TCR engineered γ δT cells was insufficient for strong antigen specific tetramer binding, the avidity was high enough for the specific recognition of peptide pulsed target cells. However, the functional reactivity of the TCR-transduced γ δT cells against target cells presenting endogenously processed antigens was low. FACS analysis indicated that most γ δT cells lacked the expression of the coreceptors CD4 and CD8. Therefore, we investigated whether introduction of the relevant coreceptor could enhance the functionality of the redirected γ δT cells. Co-transfer of the CD8α β coreceptor to the HA-2-TCR and CMV-TCR transferred γ δT cells turned them into effective, antigen specific tetramer binders. Furthermore, expression of CD8α β by the HA-2-TCR and CMV-TCR transduced γ δT cells and CD4 by the DBY-TCR transduced γ δT cells generated powerful effector cells exerting high levels of antigen specific lysis of both peptide pulsed target cells and target cells presenting endogenously processed antigen. In addition, coreceptor expressing TCR-engineered γ δT cells produced high amounts of IFNγ and IL-4 when stimulated with peptide pulsed target cells or endogenously processed antigen. To investigate the anti-leukemic reactivity of TCR-transferred γ δT cells, we determined the antigen specific cytotoxicity and cytokine production against primary CML and AML cells by γ δT cells equipped with the HA-2-TCR and CD8α β . We observed both antigen specific cytolytic activity and cytokine production against both CML and AML cells expressing the hematopoiesis specific mHag HA-2, while HLA-A2+ leukemic cells lacking expression of the HA-2 mHag were not recognized. These data demonstrate that transfer of α β TCRs to γ δT cells generated potent effector cells for immunotherapy of leukemia, without the expression of potentially hazardous mixed TCR dimers.

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Generation of GMP-Grade CMV pp65-Specific CD8+ and CD4+ Donor T Cell Lines for Treatment of CMV Reactivation after Transplantation.

Blood ◽

10.1182/blood.v108.11.2931.2931 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2931-2931

Author(s):

Maarten L. Zandvliet ◽

J.H. Frederik Falkenburg ◽

Inge Jedema ◽

Roelof Willemze ◽

Henk-Jan Guchelaar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Cd4 T Cells ◽

Target Cells ◽

Specific Cell ◽

Specific Response ◽

Specific Lysis ◽

T Cell Lines

Abstract Reactivation of CMV remains a major cause of morbidity and mortality in immunocompromised recipients of allogeneic stem cell transplantation. Antiviral pharmacotherapy may not be sufficient due to significant toxicity and moderate efficacy. It has been shown that adoptive transfer of donor-derived CMV-specific T cells may be an effective strategy to control established CMV infection. For a persistent function in vivo the presence of both virus-specific CD8+ and CD4+ T cells is essential. Therefore, we developed an optimized protocol for the generation of CMV pp65-specific CD8+ and CD4+ T cell lines which is fully compliable with Good Manufacturing Practice (GMP) conditions. Enrichment for CMV-specific T cells followed by only a short culture period is likely to retain maximal in vivo potential. PBMCs from 7 CMV seropositive donors were stimulated with recombinant pp65 protein (7–70 μg/ml) and/or HLA-A*0201/HLA-B*0702 restricted immunodominant pp65 peptides (NLV/TPR). Peptides used were clinical grade, and recombinant protein was gamma-irradiated (50 kGy, −80 C°) to eliminate possible microbiological contamination. High dose gamma-irradiation of pp65 protein resulted in partial degradation, but antigenic presentation was maintained. IFNγ producing cells were enriched using the IFNγ secretion assay (Miltenyi Biotec) at day 1 after stimulation, and cultured with autologous feeders (10x) and IL-2 (10 or 50 IU IL-2/ml) with or without CD3/28 expansion beads. Addition of high concentrations of protein during initial stimulation had a negative effect on enrichment probably due to non-specific stimulation of cells. Addition of immunodominant pp65 peptides promoted isolation efficiency and proliferation of epitope-specific CD8+ T cells in some donors. Cell lines were analyzed at different time points (day 4–15) using peptide-MHC tetramer and phenotypic markers. In addition, pp65-specificity was evaluated by intracellular IFNγ staining after restimulation with a pp65 protein-spanning pool of 15-mer peptides. CMV-specific lysis was tested in a 51-Cr release assay on pp65-transduced target cells. Enrichment of IFNγ producing cells after pp65 protein stimulation resulted in pp65-specific cell lines consisting of both CD8+ and CD4+ T cells. The T cell subset distribution directly after enrichment did not change during culture and was reproducible for each donor. Moreover, the composition of T cell lines reflected the pp65-specific response in donor PBMC starting material. The CD8+ compartment contained the known immunodominant tetramer staining cells (range 5–100%). The majority of both CD8+ and CD4+ T cells produced IFNγ upon restimulation with the pp65 peptide-pool, and showed CMV-specific lysis of target cells. The phenotype of pp65-specific T cells was predominant CD28+/CD45RO+ and CD45RA−/CCR7−/CD62L−, although CCR7 and CD62L were transiently expressed at day 4 and 7 after stimulation. Cryopreservation did not affect the composition or functionality of T cell lines. In conclusion, this procedure yields GMP-grade T cell lines comprising both CD8+ and CD4+ CMV-specific T cells. Processing and presentation of CMV protein by donor antigen-presenting cells enables selection of the full pp65-specific donor repertoire, without restrictions related to HLA or known epitopes. The choice for a moderate or more vigorous expansion after enrichment remains arbitrary and needs to be evaluated in clinical trials.

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Development of Novel Non-Immunoglobulin Centyrin-Based Cars (CARTyrins) Targeting Human Bcma

Blood ◽

10.1182/blood.v128.22.4557.4557 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4557-4557 ◽

Cited By ~ 3

Author(s):

Burton Earle Barnett ◽

Xinxin Wang ◽

David L. Hermanson ◽

Yening Tan ◽

Eric M. Osertag ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

B Cell ◽

Tumor Cells ◽

Surface Expression ◽

Domain Swapping ◽

Target Cells ◽

High Quality ◽

Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapy is a promising type of cancer therapy and substantial progress has been made in developing adoptive T cell approaches for B cell malignancies. B cell maturation antigen (BCMA) is an attractive target for patients with multiple myeloma (MM) due to its high level of expression on tumor cells and restricted expression on normal tissues. Traditionally, the antigen-binding domain of a CAR is a single chain variable fragment (scFv) comprised of heavy chain (HC) and light chain (LC) variable fragments joined by a flexible linker that has been derived from a non-human monoclonal Ab (mAb). However, there are a number of disadvantages to scFv-based CARs including the limited availability of scFv, their potential to elicit antibody responses, and their association with tonic signaling due, in part, to inherent instability and flexibility of the structure and the potential for both HC/LC domain swapping and multimer formation through framework region interactions. Thus, replacement with alternative binding technologies may improve CAR-T efficacy in the clinic. Centyrins are alternative scaffold molecules that bind protein targets with high affinity and specificity, similar to scFv molecules. However, unlike scFv, Centyrins are smaller, derived from human consensus tenascin FN3 domains and are predicted to have decreased immunogenicity. Additionally, a monomeric Centryin in CAR format (i.e. CARTyrin molecule) is less likely to engage in domain swapping or interact with other Centyrins at the cell surface, thereby limiting the potential for the tonic signaling that drives the functional exhaustion of CAR T cells. Centyrins can be isolated against virtually any antigen through ex vivo panning of an extensive Centyrin library, yielding many distinct binders with a range of affinities and target epitopes. Panning with soluble BCMA protein yielded a large pool of BCMA-specific Centyrins, from which 11 distinct monomeric binders and 1 non-monomeric binder were selected for further study in CAR format. In addition, we tested numerous signal peptides, linkers, transmembrane domains and signaling domains to determine optimal configuration. We then created all CARTyrins by fusing each Centyrin with a CD8a leader peptide, spacer and transmembrane domain, as well as an intracellular signaling domain derived from both 4-1BB and CD3ζ. High quality mRNA of each CARTyrin construct was produced in order to rapidly screen CARTyrin cell surface expression and functionality in human pan T cells against BCMA+ targets. We also constructed scFv-based CARs against CD19 and BCMA for comparison. Previously CD3/CD28-stimulated T cells were electroporated (EP) with mRNA encoding each of the 12 anti-BCMA CARTyrins and, the following day, analyzed for surface expression of CARTyrin and their ability to degranulate against BCMA+ tumor cells. All 12 CARTyrins were detected on the cell surface and the 11 monomeric CARTyrins imparted BCMA-specific killing capacity to T cells. Notably, in these assays, CARTyrins were functionally comparable to scFv-based CARs against BCMA or to CD19-specific scFv-based CARs in a parallel assay with CD19+ tumor cells. The 11 functional anti-BCMA CARTyrins were further characterized for functional avidity by determining their activity against a panel of target cells with titrated levels of surface BCMA expression. To create this panel, various amounts of high quality BCMA mRNA were electroporated into BCMA- K562 tumor cells. After 4 hours of co-culture with the panel of BCMA expressing cells, CARTyrin+ T cell activity was measured as a function of CD107a expression. We observed a range of activities by each CARTyrin and show that this assay can be utilized to determine the minimal effective dose of BCMA needed to induce killing by CARTyrin+ cells. Furthermore, we establish that certain BCMA-specific CARTyrins are responsive to target cells with extremely low levels of surface BCMA expression. These results confirm that Centyrins are viable replacements for scFv in the construction of functional CARs and establish their potential utility in generating novel BCMA-specific CAR molecules, as well as other novel targetable tumor antigens. Disclosures Barnett: Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

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IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes

Blood ◽

10.1182/blood-2002-07-2224 ◽

2003 ◽

Vol 101 (6) ◽

pp. 2167-2174 ◽

Cited By ~ 83

Author(s):

Els Verhoeyen ◽

Valerie Dardalhon ◽

Odile Ducrey-Rundquist ◽

Didier Trono ◽

Naomi Taylor ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

Gene Transfer ◽

Lentiviral Vectors ◽

Target Cells ◽

Interleukin 7 ◽

Efficient Gene ◽

Cognate Antigen ◽

Therapy Target

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell–based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3–displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)–displaying HIV-1–derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7–displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7–displaying vectors could constitute interesting tools for T-cell–targeted gene therapy.

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MDSC Suppression of CAR T Cells Can be Reduced By Targeted Signaling Disruption

Blood ◽

10.1182/blood-2019-122752 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4438-4438 ◽

Cited By ~ 1

Author(s):

Estelle V Cervantes ◽

Justin C. Boucher ◽

Sae Bom Lee ◽

Kristen Spitler ◽

Kayla Reid ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Research Funding ◽

Target Cells ◽

Car T Cells ◽

Car T Cell ◽

Cell Counts ◽

Car T

CAR T cells are genetically modified with an extracellular scFv, transmembrane domain, and intracellular costimulatory and CD3ζ domains. Two treatments received the approval from the FDA for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma. However, CAR T cell persistence remains a problem. A reason for this may be that myeloid cells such as myeloid derived suppressor cells (MDSCs) may be contributing to the reduced persistence of CAR T cells. MDSCs originate from myeloid cells and have been implicated in the suppression of the immune system in the tumor microenvironment. To determine what effect MDSCs might have during CAR T cell production we co-cultured MDSCs during CAR transduction. We found gene transfer was lower for m19z (38.7% vs 46.8%), m1928z (24.1% vs 39.1%), and m19hBBz (35.8% vs 46.2%) CAR T cells co-cultured with MDSCs compared to those that were not (Fig 1A). There was also a reduction in total T cell counts for m19z (58%), m1928z (88%), and m19hBBz (65%) after MDSC co-culture. This data suggests MDSCs present during CAR T cell production can alter gene transfer and total T cell counts. We also investigated the effect MDSCs can potentially have on CAR T cells when present during CAR T cell antigen stimulation. CAR T cells co-cultured with MDSCs in vitro had significant reductions in m19z, m1928z, and m19hBBz CAR T cell mediated killing against target cells (Fig 1B). We also found significantly lower production of IFNγ in m19z, m1928z, and m19hBBz CAR T cells cultured with MDSCs compared to cells cultured with total BM (Fig 1C). After 24hr stimulation with MDSCs and target cells, there was lower expression of activation markers PD1 and LAG3 by CAR T cells compared to culture without MDSCs. This suggests that MDSCs reduce CAR T cell activation, killing, and cytokine production. We also looked at how CAR proliferation after antigen stimulation is affected by MDSC co-culture and found MDSCs significantly reduced CAR proliferation in vitro. To evaluate if we could create a more resistant CAR T cell to MDSC suppression null mutations were incorporated into a CD28 CAR. We mutated the YMNM and PRRP subdomains of CD28 which signal through PI3K and ITK respectively leaving only PYAP active (mut06). When MDSCs were co-cultured with mut06 T cells during production mut06 had a smaller reduction in gene transfer (21% vs 38%) and T cell counts (80% vs 88%) compared to m1928z. Mut06 also had a significantly higher expression of PD1 and TIM3 compared to m1928z after production with MDSCs. In vitro when mut06 was co-cultured with MDSCs it had the same killing ability as m1928z without MDSCs and was significantly better at killing compared to m1928z co-cultured with MDSCs (Fig 1B). To examine the effect of MDSCs on CAR T cells in vivo we injected C57BL6 mice with CAR T cells followed by MDSCs a week later. In these ongoing experiments we found mut06 had significantly higher numbers of CAR T cells in the blood compared to m1928z (Fig1D). Overall our data shows that MDSCs can suppress CAR T cell function when present during production as well as CAR stimulation. It also suggests that by optimizing CD28 CAR signaling using mut06 that we were able to generate a CAR T cell that is more resistant to MDSCs. Furthermore, we may be able to recapitulate the effect of mut06 with targeted inhibitors. Figure 1 Disclosures Davila: GlaxoSmithKline: Consultancy; Novartis: Research Funding; Adaptive: Consultancy; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; Anixa: Consultancy; Atara: Research Funding.

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HIV-specific T Cell Cytotoxicity Mediated by RANTES Via the Chemokine Receptor CCR3

Journal of Experimental Medicine ◽

10.1084/jem.188.3.609 ◽

1998 ◽

Vol 188 (3) ◽

pp. 609-614 ◽

Cited By ~ 47

Author(s):

Fabienne Hadida ◽

Vincent Vieillard ◽

Brigitte Autran ◽

Ian Clark-Lewis ◽

Marco Baggiolini ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chemokine Receptor ◽

Mononuclear Cells ◽

Cytolytic Activity ◽

Target Cells ◽

Peripheral Blood Mononuclear ◽

Specific Lysis ◽

Stromal Cell Derived Factor ◽

Hiv 1

CC chemokines produced by CD8+ T cells are known to act as HIV-suppressive factors. We studied the possible role of these chemokines in HIV-1–specific killing of target cells. We found that the activity of cytotoxic T lymphocytes (CTLs) in CTL lines or freshly isolated peripheral blood mononuclear cells from HIV-1–infected individuals is markedly enhanced by RANTES (regulated on activation, normal T cell expressed and secreted) and virtually abolished by an antibody neutralizing RANTES or the RANTES receptor antagonist RANTES(9-68). Lysis was mediated by CD8+ major histocompatibility complex class I–restricted T cells and was obtained with target cells expressing epitopes of the HIV-1LAI proteins Gag, Pol, Env, and Nef. The cytolytic activity observed in the presence or absence of added RANTES could be abolished by pretreatment of the CTLs with pertussis toxin, indicating that the effect is mediated by a G protein–coupled receptor. The chemokines monocyte chemotactic protein (MCP)-3, MCP-4, and eotaxin acted like RANTES, whereas macrophage inflammatory protein (MIP)-1α, MIP-1β, MCP-1, and stromal cell–derived factor 1 were inactive, suggesting a role for the eotaxin receptor, CCR3, and ruling out the involvement of CCR1, CCR2, CCR5, and CXCR4. CTL activity was abrogated by an antibody that blocks CCR3, further indicating that specific lysis is triggered via this chemokine receptor. These observations reveal a novel mechanism for the induction of HIV-1–specific cytotoxicity that depends on RANTES acting via CCR3.

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The Functional Activity of Genetically Engineered T Cell Receptor Transferred T Cells Is Highly Dependent on Pairing Properties of the Transferred TCR α and β Chains.

Blood ◽

10.1182/blood.v104.11.1753.1753 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1753-1753

Author(s):

Mirjam H.M. Heemskerk ◽

Manja Hoogeboom ◽

Renate Hagedoorn ◽

Michel G.D. Kester ◽

Roel Willemze ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Target Cells ◽

Antigen Specificity ◽

Tetramer Staining ◽

Cell Immunity ◽

The Individual

Abstract The genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T cell immunity towards viral infections and malignancies. Transfer of virus- or tumor-specific TCRs has demonstrated to endow T cells with redirected antigen specificity. We demonstrated redirected anti-leukemic reactivity of CMV specific T cells using gene transfer of minor histocompatibility antigen HA-2 specific TCRs. The HA-2-TCR-modified T cells exerted high cytolytic activity against HA-2 expressing target cells, including leukemic cells, and not against target cells negative for the HA-2 mHag. After cloning of the TCR-transferred T cells, we demonstrated that the HA-2-TCR cell surface expression, measured by HA-2-tetramer staining, was variable on the transduced T cell clones, and that the cytolytic capacity of the T cells correlated with the level of HA-2-TCR expression. Since we could demonstrate that this variation in HA-2-TCR expression was not due to differences in transgene expression, we investigated whether the endogenous TCRs influenced the expression of the introduced TCR. CMV-A2 specific T cells were isolated from peripheral blood and transduced with the HA-2-TCR. In control transduced CMV specific T cells we observed 5 different high affinity CMV specific TCRs. CMV specific T cells transduced with the HA-2-TCR that expressed predominantly the HA-2-TCR, expressed only one of these types of CMV-TCR, and in CMV specific T cells with low HA-2-TCR expression two different types of CMV-TCRs were found. These data indicated that the level of expression of the introduced TCR is strongly influenced by the endogenous TCR. To investigate whether this was due to differences in promotor activity of the endogenous and retrovirally introduced TCR, the three CMV-TCRs were characterized and transferred into unselected peripheral T cells. T cells transferred with the weak competitior CMV-TCR that was strongly downregulated in CMV specific T cells by introduction of the HA-2-TCR, showed low CMV specific cytotoxicity and no tetramer staining. In contrast, T cells transferred with the strong competitor CMV-TCR that was modestly downregulated in CMV specific T cells by introduction of the HA-2-TCR, revealed strong CMV specific cytotoxic activity and tetramer staining. These data demonstrate that the introduced and endogenous TCRs compete for cell surface expression, and that this competition is dependent on characteristics of the different TCRs and independent of whether the TCR is retrovirally introduced or naturally expressed. To investigate whether the cell surface expression of the different TCRs was determined by preferential pairing properties of the individual TCR chains, TCR α and β deficient Jurkat 76 cells were transduced with the three CMV-specific TCRs or with chimeric TCRs consisting of the TCR α chain of one TCR with the TCR β chain of another TCR. TCRαβ membrane expression revealed that TCRs with a strong competitor phenotype expressed higher levels of TCRαβ than the TCR that was a weak competitor. TCRαβ expression of Jurkat cells transduced with chimeric TCRs indicated that the expression level of the different TCRs was determined by the pairing properties of the individual TCR α and β chains and not by differences in protein expression. In conclusion these data demonstrated that introduced and endogenous TCRs compete for cell surface expression in favor of the TCR that has the highest intrinsic pairing properties.

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Playing the Game Together: Coexpression of a Single Chain T Cell Receptor and a T Cell Receptor Constant-Alpha Domain Triggers Tumor Reactivity.

Blood ◽

10.1182/blood.v108.11.3716.3716 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3716-3716

Author(s):

Simone Thomas ◽

Ralf H. Voss ◽

Ratna Intan ◽

Renate Engel ◽

Juergen Kuball ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Receptor ◽

Cell Receptor ◽

Surface Expression ◽

Target Cells ◽

Single Chain ◽

Double Chain ◽

Human T Cells

Abstract Grafting T cells by tumor-antigen specific T cell receptors (TCR) could trigger the initiation of effector function and redirect T cell cytotoxicity towards tumors. We utilized various HLA-A2.1 transgenic mice to bypass human MDM2- and p53-specific self-tolerance. In contrast to the use of HuCD8×A2Kb transgenic mice to generate an MDM2-specific CD8-dependent TCR, we generated a high-affinity, CD8-independent p53-specific TCR in single human A2.1 transgenic mice. The efficiency of double chain (dc) TCR modified T cells could be affected by the incorrect TCR α/β chain pairing between endogenous and transgenic TCR constructs to form hybrid TCR potentially leading to autoimmunity. To address this concern, chimeric A2.1-restricted peptide-specific murine single chain (sc) TCRs were constructed (Vα-Li-VβCβ) and retrovirally transduced into human T cells. Despite detectable surface expression, these chimeric receptors were not able to convey any MDM2- or p53-specific cytolytic activity. Therefore we developed a truncated TCR-alpha domain (Cα) comprising solely the TCRα signal peptide, the ecto-domain, the transmembrane region as well as the cytoplasmic tail and cotransduced these construct with the scTCRs. We anticipated that Cα would stabilize scTCR expression by interacting with the single chain beta chain. Indeed, this approach not only led to increased expression levels of the chimeric scTCRs, but also induced specific lysis of A2.1 positive MDM2 or p53 peptide-pulsed target cells as well as solid tumor cell lines. Recognition of malignant targets by p53 specific scTCR transduced CD4 and CD8-positive T cells was equivalent to that observed with double-chain p53 TCR gene modified effector cells. To test whether this concept is applicable to human TCRs as well, we constructed a human gp100-specific scTCR and a human Cα domain. In contrast to the gp100-specific double chain TCR, only a marginal expression pattern was observed for the human scTCR / Cα constructs. Introduction of an additional disulfide bond within the constant domains in order to stabilize TCR surface expression showed no effect. Since murine TCR are expressed on human T cells to a much higher extent, the human constant β-domain of the scTCR was replaced by murine Cβ. Comparable to the murine scTCR concept, the chimerized scTCR coexpressed with murine Cα demonstrated high cell surface expression and triggered cytotoxicity of malignant A2.1/gp100-positive targets. In summary, our results lay a commonly applicable conceptual basis for the construction of therapeutic scTCR to prevent recombination of natural and transgenic dcTCR alpha and beta chains.

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