Gene-modified CD4 Research Articles

A novel costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine efficiently enhances anti-leukemia immunity.

Previous studies demonstrated that CD4+ T cells can uptake tumor antigen-pulsed dendritic cell-derived exosomes (DEXO), which harbor tumor antigen peptide/pMHC I complex and costimulatory molecules and show potent effects on inducing antitumor immunity. However, in preliminary study, CD4+ T cells targeted by leukemia cell-derived exosomes (LEXs) did not show the expected effects in inducing effective anti-leukemia immunity, indicating that LEX is poorly immunogenetic largely due to an inadequate costimulatory capacity. Therefore, LEX-based anti-leukemia vaccines need to be optimized. In this study, we constructed a novel LEX-based vaccine by combining CD4+ T cells with costimulatory molecules gene-modified LEXs, which harbor upregulated CD80 and CD86, and the anti-leukemia immunity of CD80 and CD86 gene-modified LEX-targeted CD4+ T cells was investigated. We used lentiviral vectors encoding CD80 and CD86 to successfully transduced the L1210 leukemia cells, and the expression of CD80 and CD86 was remarkably upregulated in leukemia cells. The LEXs highly expressing CD80 and CD86 were obtained from the supernatants of gene-transduced leukemia cells. Our data have shown that LEX-CD8086 could promote CD4+ T cell proliferation and Th1 cytokine secretion more efficiently than control LEXs. Moreover, CD4+ TLEX-CD8086 expressed the acquired exosomal costimulatory molecules. With acquired costimulatory molecules, CD4+ TLEX-CD8086 can act as APCs and are capable of directly stimulating the leukemia cell antigen-specific CD8+ CTL response. This response was higher in potency compared to that noted by the other formulations. Furthermore, the animal study revealed that the CD4+ TLEX-CD8086 significantly inhibited tumor growth and prolonged survival of tumor-bearing mice than other formulations did in both protective and therapeutic models. In conclusion, this study revealed that CD4+ TLEX-CD8086 could effectively induce more potential anti-leukemia immunity than LEX-CD8086 alone, suggesting that the utilization of a costimulatory molecule gene-modified leukemia cell-derived exosome-targeted CD4+ T cell vaccine may have promising potential for leukemia immunotherapy.

Control of HIV Infection InVivo Using Gene Therapy with a Secreted Entry Inhibitor.

HIV entry inhibitors are highly effective in controlling virus replication. We have developed a lentiviral vector that expresses a secreted entry inhibitor, soluble CD4 (sCD4), which binds to the HIV envelope glycoproteins and inactivates the virus. We have shown that sCD4 was secreted from gene-modified CD4+ T cells, as well as from human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs), and protected unmodified HIV target cells from infection in vitro. To investigate the in vivo application of our approach, we injected gene-modified HSPCs into NOD/SCID/γcnull (NSG) mice. NSG hosts supported multi-lineage differentiation of human gene-modified HSPCs. Upon challenge with HIV, humanized mice capable of secreting sCD4 demonstrated a reduction of viral load over time compared to control humanized mice. In contrast to gene therapy approaches that render only gene-modified HIV target cells resistant to infection, our approach also showed protection of unmodified CD4+ T cells in the peripheral blood and tissues. Our findings provide support for the continuous delivery of secreted entry inhibitors via gene therapy as an alternative to oral administration of antiretroviral drugs or injection of antiretroviral proteins, including antibodies.

Characterisation of gene-modified CD3-enhanced CD4+ T cells for cancer immunotherapy

Abstract TCR gene transfer is used to redirect the antigen specificity of T lymphocytes towards known tumour antigens. TCR gene therapies in murine studies have shown promising results. However, in the clinic they often generate sub-optimal responses, when compared to treatments with tumour infiltrating lymphocytes. Previous work to improve TCR gene therapy has demonstrated that transferring additional CD3 genes increases TCR expression of both endogenous and introduced TCR in CD4+ and CD8+ T cells. In vivo experiments demonstrated that CD8+ T cells, transduced with TCR and additional CD3 were more effective in tumour protection than T cells transduced with the TCR alone. Whilst, CD4+ T cells transduced with TCR and additional CD3 initially showed improved tumour protection, lethal toxicity, unrelated to tumour burden, was later observed. To investigate the effects of CD3 overexpression, CD3 genes only (no TCR genes) were transferred into purified CD4+ and CD8+ T cells. Following adoptive transfer, CD3-enhanced CD4+ T cells survived for longer and were recovered in higher percentages in spleen, lymph nodes, bone marrow and liver, compared to CD3-enhanced CD8+ T cells and mock transduced CD4+ T cells. The same trend was also seen in competition experiments where mice received a 1:1 ratio of CD3-enhanced CD4+ T cells and mock-transduced CD4+ T cells. Interestingly, this was observed despite a twofold downregulation of TCR levels in the CD3-enhanced CD4+ T cells, compared to their pre-transfer TCR levels. Current experiments are aimed at dissecting the mechanisms responsible for, and the physiological implications of the observed TCR downregulation.

Abstract B106: Substantial enrichment of HIV- resistant CD4 T cells by combining the targeted integration of a resistance-cassette at the CCR5 locus with chemoselection

Abstract Engineered nucleases can be used to induce double stranded breaks at specific sites in the genome to facilitate therapeutic gene disruption or gene insertion. Gene insertion strategies may have a significant advantage over traditional lentiviral gene therapy since we have much greater control over transgene integration site, copy number, and expression. However, a major limitation in the clinical application of gene insertion technologies is the low efficiency of homology driven repair (HDR) achieved in hematopoietic cells. Here, we report the knock-in of a selection marker, which allows us to enrich for modified CD4 T cells, and to achieve clinically relevant levels of gene edited cell populations in the disease-context of HIV. Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4 T cells and represents an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining a high level of CCR5 gene disruption with preferential selection of gene-modified cells using a megaTAL nuclease. The CCR5-targeting megaTAL, combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site-specific DNA cleavage. This nuclease produces highly efficient CCR5 disruption in primary human CD4+ T cells in vitro (70-90%). To test the protective effects of gene disruption, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Next, to overcome the decline of HIV-resistant cells, observed in the clinical trials to date, we proposed to couple CCR5 gene-disruption with gene insertion of mutant human dihydrofolate reductase (mDHFR). The mDHFR chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We first tested our experimental approach by transducing cells with a lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.2uM. This approach resulted in a six-fold enrichment of gene modified primary CD4+ T cells ex vivo. For gene insertion, combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we merged the megaTAL nuclease and AAV gene-delivery treatments to integrate mDHFR into the CCR5 locus, thus producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.8kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in 0.1uM MTX compared to untreated controls ex vivo. Finally, we are assessing the therapeutic potential of our approach in NSG mice. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus. Eventually this system could be used to select for modified cells in patients using previously established protocols for MTX administration in the clinic. Citation Format: Biswajit Paul, Guillermo Romano Ibarra, David J. Rawlings, Hans-Peter Kiem. Substantial enrichment of HIV- resistant CD4 T cells by combining the targeted integration of a resistance-cassette at the CCR5 locus with chemoselection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B106.

44. Novel Combination of megaTAL Nuclease-Driven Genome Engineering with a Drug Selection Cassette Increases Efficiency of HIV Gene Therapy

Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4+ T cells and represent an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining high level of CCR5 gene disruption with preferential selection of gene modified cells. The CCR5-targeting megaTAL combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site specific DNA cleavage. This nuclease produces highly efficient CCR5 targeting in primary human CD4+ T cells in vitro (70-90% disruption). To test the protective effects of megaTAL treatment, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Based on the decline of CCR5 modified cells in the clinical trials to date, we hypothesized that we could improve maintenance of HIV resistant cells by expanding them either ex vivo or in vivo. We propose that coupling megaTAL nuclease treatment with drug selection will help us achieve therapeutically relevant levels of HIV-protected cells by enabling efficient selection only of CCR5-modified T-cells. The mutant human dihydrofolate reductase (mDHFR) chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We tested our experimental approach by transducing cells with lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.02uM. This approach resulted in a six fold enrichment of gene modified primary CD4+ T cells ex vivo. Previously we have shown that combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we combined megaTAL/AAV treatment to integrate the mutant DHFR into the CCR5 locus, producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.6kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in MTX compared to untreated controls ex vivo. Next, we have transplanted NSG mice with 1×106 gene modified cells/mouse to assess the therapeutic potential of our approach. Mice that engraft effectively will be treated with daily injections of 0, 0.5 and 2 mg/kg of MTX to monitor preferential selection and enrichment of our target cell population. Subsequent studies will assess the long term control of viremia in these mice following HIV challenge. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus.

Targeting Aurora Kinase with a Superior T-Cell Receptor Gene-Transfer Vector

[Background] Aurora Kinase A (AURKA) is a cancer-associated protein normally involved in the regulation of mitosis. It is over-expressed in a range of cancers, and has a 'cancer-testis' expression profile, making it a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor (TCR) sequences cognisant of HLA-A*0201-restricted AURKA antigen (aa207-215: YLILEYAPL) has previously been shown to transfer specific immunoreactivity against the target peptide in a HLA-restricted manner (Nagai K et al. Blood, 2012).While TCR gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumor-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous TCR chains. We have used a retroviral vector design bearing siRNAs that downregulates endogenous TCR chains, without affecting expression of the transgenic TCR sequences. The TCR expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the TCR α and β chains, further enhancing formation of the desired TCR. Via a simple, modular cloning method, we have cloned the α and β chains of the anti-AURKA -reactive TCR into this 'siTCR' vector. In this study, we then compared the activity of this vector against the original, 'conventional' vector without siRNAs across a panel of assays.[Methods] After codon-optimization to be resistant to built-in siRNAs for endogenous TCRs, full-length of an HLA-A*0201-restricted and AURKA207-215 nonamer -specific TCR α/β genes (Vα3 /J20/Cα, Vβ10.3(12)/J1.1/Cβ1, respectively) obtained from our established epitope-specific CD8+ T cell clone (AUR2) linked using T2A peptide sequence were cloned into the siTCR vector carrying those siRNAs using an Infusion cloning method (Takara Bio). Either conventional retroviral AURKA-specific TCR expression vector without those siRNAs or this new retroviral AURKA-siTCR vector was transduced into CD8+ T cells from healthy volunteers using anti-CD3 antibody and RetronectinTM (Takara Bio) as previously reported. The epitope-specific and leukemia specific cytotoxicity and IFN-γ production of these transfectants were examined using 51Cr-releasing assay, CD107a assay and ELISA. Introduced viral vector copy numbers of these two types of transfectants were measured using qPCR kit (Bio Line) according to manufacturer's instruction, on an ABI Prism 7500 (Applied Biosystems/Life Technologies). To examine the potential to target leukemia stem cells mediated by these AURKA-siTCR transfectants, we employed the side population of the AURKA mRNA highly expressing HLA-A*0201+ leukemia cell line, GANMO-1, using the dye exclusion method with Hoechst 33342 (Sigma). Concurrently the on-target/off-tumor adverse effect against hematopoietic progenitor cells mediated by these transfectants was examined using HLA-A*0201+ cord blood CD34+ cells as a target.[Results] TCRs expressed from the AURKA-siTCR vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed TCRs demonstrated by AURKA/HLA-A*0201 tetramer labeling was superior using the siTCR design, and this was achieved at lower vector copy numbers. AURKA mRNA tended to be overexpressed by the side population of GANMO-1, and AURKA-siTCR gene-modified CD8+ T cells successfully recognized and killed this population, but not normal cord blood CD34+ cells.[Conclusion] Maintaining TCR efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of TCR gene-transfer will be crucial for clinical applications of this technology. DisclosuresFujiwara:Celgene: Honoraria, Other: Travel, Acomodations, Expenses. Okamoto:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment.

Combinatorial hematopoietic stem cell transplantation and vaccination reduces viral pathogenesis following SHIV89.6P-challenge.

Development of curative approaches for HIV-1 infected patients requires novel approaches aimed at eliminating viral reservoirs and replacing potential target cells with infection-resistant immune cell populations. We have previously shown that autologous transplantation of genetically modified hematopoietic stem cells (HSCs) with lentiviral vectors encoding the mC46-fusion inhibitor results in a significant reduction in viral pathogenesis following challenge with the highly pathogenic dual tropic, SHIV89.6P strain. In this study, we used a combinatorial approach in which following engraftment of genetically modified HSCs, pigtailed macaques were vaccinated with a previously developed vaccinia-based vaccine expressing SIV-Gag, Pol. Using this dual therapy approach, lower viremia was detected in both the acute and chronic phase of disease with levels reaching near the lower limits of detection. In comparison with macaques receiving HSCT only, the combination approach resulted in a further log decrease in plasma viremia. Similar to our previous studies, positive selection of all CD4(+) T-cell subsets was observed; however, higher gene-modified CD4(+) T-cell levels were observed during the chronic phase when vaccination was included suggesting that combining vaccination with HSCT may lower the necessary threshold for achieving viremic control.

Concomitant Administration of Gene-Modified T Cells Expressing a Chimeric CD16-CD3ζ Receptor with Mogamulizmab Synergistically Suppresses Adult T Cell Leukemia Cells in Vivo

[Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab.[Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014).[Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface. DisclosuresOkamoto:CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.

A quantitative comparison of anti-HIV gene therapy delivered to hematopoietic stem cells versus CD4+ T cells.

Gene therapy represents an alternative and promising anti-HIV modality to highly active antiretroviral therapy. It involves the introduction of a protective gene into a cell, thereby conferring protection against HIV. While clinical trials to date have delivered gene therapy to CD4+T cells or to CD34+ hematopoietic stem cells (HSC), the relative benefits of each of these two cellular targets have not been conclusively determined. In the present analysis, we investigated the relative merits of delivering a dual construct (CCR5 entry inhibitor + C46 fusion inhibitor) to either CD4+T cells or to CD34+ HSC. Using mathematical modelling, we determined the impact of each scenario in terms of total CD4+T cell counts over a 10 year period, and also in terms of inhibition of CCR5 and CXCR4 tropic virus. Our modelling determined that therapy delivery to CD34+ HSC generally resulted in better outcomes than delivery to CD4+T cells. An early one-off therapy delivery to CD34+ HSC, assuming that 20% of CD34+ HSC in the bone marrow were gene-modified (G+), resulted in total CD4+T cell counts ≥180 cells/ µL in peripheral blood after 10 years. If the uninfected G+ CD4+T cells (in addition to exhibiting lower likelihood of becoming productively infected) also exhibited reduced levels of bystander apoptosis (92.5% reduction) over non gene-modified (G-) CD4+T cells, then total CD4+T cell counts of ≥350 cells/ µL were observed after 10 years, even if initially only 10% of CD34+ HSC in the bone marrow received the protective gene. Taken together our results indicate that: 1.) therapy delivery to CD34+ HSC will result in better outcomes than delivery to CD4+T cells, and 2.) a greater impact of gene therapy will be observed if G+ CD4+T cells exhibit reduced levels of bystander apoptosis over G- CD4+T cells.

Co-Administration Of Gene-Modified CD4+ T Cells Targeting HLA Class I-Restricted WT1 Epitope Diversely Enhances The Antitumor Effect Mediated By Redirected T-Cell Based Adoptive Immunotherapy Against Human Leukemia

PurposeIn the context of redirected T-cell based antitumor adoptive immunotherapy, the therapeutic roles played by co-infused CD4+ T cells genetically redirected to the predefined HLA class I-restricted epitope which had been originally recognized by effector CD8+ T cells has not yet been fully discussed. In this study, using an HLA class I-restricted WT1 -specific T-cell receptor (TCR) gene transfer, we in detail examined antileukemia functionality mediated by these gene-modified CD4+ T cells co-infused with similarly gene-modified effector CD8+ T cells as the redirected T cell-based adoptive immunotherapy. MethodsUsing our unique retroviral vector expressing HLA-A*2402-restricted and WT1235-243-specific TCR a/b genes and shRNAs for endogenous TCRs (WT1-siTCR vector), we genetically modified both CD4+ and CD8+ T cells from the same healthy donor or leukemia patients (termed WT1-siTCR/CD4 and WT1-siTCR/CD8, respectively). First, target-responsive cellular outputs mediated by WT1-siTCR/CD4 was thoroughly examined using flowcytometry, ELISA, 51Cr-release assay, CFSE dilution assay and bioluminescence assay. Next we similarly assessed impacts of WT1-siTCR/CD4 on the antileukemia functionality mediated by concurrentWT1-siTCR/CD8 both in vitro and in vivo. Eventually, we assessed the in vivo therapeutic efficacy of combined administration of WT1-siTCR/CD8 with WT1-siTCR/CD4 using a xenografted mouse model. ResultsThe transcription factor profile demonstrated that WT1-siTCR/CD4 turned a terminal effector, but not regulatory phenotype. Activated WT1-siTCR/CD4 expressed cell-surface CD40L. Target-responsive cytokine production profile of WT1-siTCR/CD4 represented the Th1 helper function in the context of HLA-A*2402. HLA class II molecules expressed by leukemia cells facilitated the recognition of leukemia cells by WT1-siTCR/CD4 in the context of HLA-A*2402. WT1-siTCR/CD4 displayed the delayed cytocidal activity determined by 51Cr release assay. WT1-siTCR/CD4 could produce IFN-g in response to freshly isolated leukemia cells. WT1-siTCR/CD4 displayed the leukemia trafficking activity in vivo. WT1-siTCR/CD4 represented the potential to migrate into bone marrow via CXCR4/CXCL12 axis both in vitro and in vivo. Concurrent WT1-siTCR/CD4 augmented IFN-g production and cytotoxic degranulation mediated by WT1-siTCR/CD8 in response to the cognate epitope via humoral factors. Consequently, the cytocidal activity against autologous leukemia cells mediated by WT1-siTCR/CD8 was augmented in the presence of WT1-siTCR/CD4, both of them generated from normal lymphocytes of the same patient with leukemia in a complete remission. Upon the target recognition, activated WT1-siTCR/CD4 recruited WT1-siTCR/CD8 via CCL3/4-CCR5 axis. Proliferative response and differentiation into central memory T-cell subset mediated by WT1-siTCR/CD8 in response to the cognate epitope and leukemia cells were enhanced in the presence of autologousWT1-siTCR/CD4, but not gene-modified CD4+ T cells (NGM-CD4). CD127 expression on activated WT1-siTCR/CD8 also increased in parallel to this differentiation. Co-infused WT1-siTCR/CD4 augmented the tumor trafficking and persistence of WT1-siTCR/CD8 in vivo, resulting in the greater suppression of leukemia cells in a xenografted mouse model. Finally, in the therapeutic mouse model, co-infusion of WT1-siTCR/CD8 with of WT1-siTCR/CD4 significantly suppressed the growth of inoculated leukemia cells compared to that in mice received co-infusion of WT1-siTCR/CD8 with NGM-CD4 (Fig.1). Correlation between the therapeutic efficacy and survival of infused gene-modified T cells was also observed. ConclusionIn results, the combined infusion of WT1-siTCR/CD8 with WT1-siTCR/CD4, but not NGM-CD4 obviously demonstrates the enhanced antileukemia efficacy via diverse mechanisms. Now we have just started a clinical trial using gene-modified T cells with WT1-siTCR vector for the treatment of patients with refractory acute myeloid leukemia and myeloid dysplastic syndrome. Because redirected T cells employed in this trial encompassed both WT1-siTCR/CD4 and WT1-siTCR/CD8, we are planning to clinically verify the significance of WT1-siTCR/CD4 in the redirected T cell-based antileukemia adoptive immunotherapy.(Fig.1) [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CD4 reconstitution and reduction of reservoir in HIV-positive patients following a single infusion of CCR5 modified autologous CD4 T cells (SB-728-T)

BackgroundSB-728-T is a zinc finger nuclease-mediated, CCR5-modified, autologous CD4 T-cell product. We investigated whether CCR5 knockout might render a survival advantage to the gene-modified CD4 T cells in HIV-infected patients and potentially improve antiviral immune function. MethodsNine aviraemic and chronically HIV-infected patients with suboptimum CD4 counts (200–500 cells per μL) were infused with 10–30 billion SB-728-T cells. The treated patients were male (seven white, two Hispanic) with an average age of 49 years (SD 6·5), and 20·8 years (SD 6·5) of diagnosed HIV infection. All participants were on antiretroviral therapy before the study and for the duration of the study. FindingsWe observed that a single infusion of SB-728-T was well tolerated and led to short-term and long-term CD4 reconstitution, with a median increase of 223 cells (95% CI 79–688) at day 14 and 103 cells (14–368) at month 12. This was primarily driven by an increase in central memory CD4 T cells (TCM) (r=0·9, p=0·083). CD4 reconstitution also correlated with reduced inflammation in the innate immune system, whereby patients who showed comparatively less improvement in CD4 cell counts had overexpression of activation markers, as well as a gene expression signature of upregulated genes downstream of the type II interferon pathway. CCR5-modified CD4 T cells peaked near day 14 (median 8·5% CCR5-modified allele of peripheral CD4 T cells; 95% CI 5·2–12·0) and were sustained for longer than 12 months (median 4·8% [3·1–8·0] at 6 months; and 5·6% [3·3–8·8] at 12 months). By contrast, CCR5 modification was maintained specifically in the central memory T-cell compartment (median 6%, 7%, and 8% at approximately 15 days, 6 months, and 12 months, respectively). At month 12, we observed reductions in total cell-associated HIV DNA concentrations using digital droplet PCR and longitudinal linear regression analysis (median −0·6 log HIV copies per 106 peripheral blood mononuclear cells [95% CI −0·03 to −0·9]) which were related to both CD4 reconstitution (r=−0·86, p=0·024) and exposure to CCR5-modified cells (r=-0·75, p=0·002). InterpretationSB-728-T infusion is safe and is associated with sustained improvements in CD4 cell counts. Decreased inflammation might provide a survival advantage after the infusion of central memory CD4 T cells. SB-728-T could have a role in the observed decrease in latent reservoir size. Clinically robust techniques such as digital droplet PCR for quantification of HIV in peripheral blood mononuclear cells are useful to guide clinical assessment of the antiviral effect of interventions in aviraemic HIV patients who are well controlled on antiretroviral therapy. FundingThis research was supported by funding to R-PS from the National Institutes of Health(U19 AI096109-03) and amfAR.

Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model

AbstractDespite continued progress in the development of novel antiretroviral therapies, it has become increasingly evident that drug-based treatments will not lead to a functional or sterilizing cure for HIV+ patients. In 2009, an HIV+ patient was effectively cured of HIV following allogeneic transplantation of hematopoietic stem cells (HSCs) from a CCR5−/− donor. The utility of this approach, however, is severely limited because of the difficulty in finding matched donors. Hence, we studied the potential of HIV-resistant stem cells in the autologous setting in a nonhuman primate AIDS model and incorporated a fusion inhibitor (mC46) as the means for developing infection-resistant cells. Pigtail macaques underwent identical transplants and Simian-Human Immunodeficiency Virus (SHIV) challenge procedures with the only variation between control and mC46 macaques being the inclusion of a fusion-inhibitor expression cassette. Following SHIV challenge, mC46 macaques, but not control macaques, showed a positive selection of gene-modified CD4+ T cells in peripheral blood, gastrointestinal tract, and lymph nodes, accounting for >90% of the total CD4+ T-cell population. mC46 macaques also maintained high frequencies of SHIV-specific, gene-modified CD4+ T cells, an increase in nonmodified CD4+ T cells, enhanced cytotoxic T lymphocyte function, and antibody responses. These data suggest that HSC protection may be a potential alternative to conventional antiretroviral therapy in patients with HIV/AIDS.

Development of a novel redirected T-cell–based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia

AbstractAlthough adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell–based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) –restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/β genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8+ T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell–based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Therapeutically Infused Redirected T Cells Targeting WT1 Successfully Inhibited Leukemia Stem Cells in Vivo

AbstractAbstract 4221 Background & Purpose:Recent findings regarding leukemia stem cell (LSC) have emphasized the importance of suppression of LSC for the achievement of durable remission, the first requisite to establish cure of leukemia. For this subject, successful graft-vs.-leukemia (GvL) effect in allogeneic hematopoietic stem cell transplantation (allo-HSCT) against human leukemias has strongly illustrated the importance of anti-leukemia immunity. Additionally, WT1, one of well-known leukemia-associated antigens, has been obviously demonstrated to be expressed by LSCs in bone marrow niche (Saito Y et al, Sci Transl Med.2010). On the other hand, cell-cycle quiescence of LSCs in bone marrow niche is importantly implicated in their chemoresistance. Taking all above, in this study, we set out to answer questions whether therapeutically adopted T-cell immunity towards WT1 enabled to suppress LSC in vivo, and whether cell-cycle status of leukemia cells affected the sensitivity to cyotocidal activity mediated by WT1-specific cytotoxic T cells (CTLs). Methods:Approval for this study was obtained from the Institutional Review Board of Ehime University Hospital. Written informed consent was given by all patients, healthy volunteers in accordance with the Declaration of Helsinki. Peripheral CD8+ T cells obtained from AML or ALL patients in complete remission (CR) or healthy individuals were gene-modified to express HLA-A*2402-restricted and WT1235–243 nonamer -specific T-cell receptor (TCR) using our unique TCR-a/b gene expression vector carrying silencers for endogenous TCRs (WT1-siTCR vector) were generated as effector cells. Bone marrow CD34+ leukemia (L-BMCD34+) cells isolated using immunomagnetic beads from HLA-A*2402 positive or negative patients with AML or ALL were serially transplanted into NOD/scid/γcnull (NOG) mice as previously reported (Ochi T et al. Blood, 2011). 12 weeks later, engrafted leukemia cells in murine bone marrow were examined using a flowcytometry. In some experiments, after engrafted, first transplanted mice were treated with intraperitoneal administration of high dose (150mg/kg) of cytosine arabinoside (Ara-C). A week later, those mice received intravenous administration of gene-modified autologous CD8+ T cells to express WT1-specific TCR or non-gene-modified (NGM) ones in combination with intraperioneal administration of 500u of IL-2 every 2 days. A week after therapeutic T-cell infusion, bone marrow cells were harvested, and transplantation into second mice. 12 weeks later, engrafted human leukemia cells in murine bone marrow were assessed. Next, using a time-lapse photo assay and fluorescent ubiqutination-based cell-cycle indicator (Fucci)-labeled K562-A24 cells which are known to produce high amounts of WT1 mRNA and are positive for HLA-A*2402, we directly assessed the impact of cell-cycle status of leukemia cells on their sensitivity to redirected CTL towards WT1 in vitro. Results:Using isolated L-BMCD34+ cells, LSCs were detectable as leukemia initiating cell in serially transplanted NOG mice. High dose of Ara-C treatment alone was unable to eradicate LSCs. An experiment using samples from a patient with HLA-A*2402+ ALL revealed that intravenously infused gene-modified autologous peripheral CD8+ T cells in CR successfully reduced leukemia burden in bone marrow which were refractory to high dose of Ara-C. In serial transplantation experiments using samples from AML patients, therapeutic infusion of redirected CD8+ T cells to express WT1-specific TCR, but not NGM ones in nadir state successfully eradicated LSCs out of murine bone marrow. In vitro time-lapse photo assay directly illustrated that retargeted CD8+T cells towards WT1 killed fucci-labeled K562-A24 cells irrelevantly to cell-cycle status of target leukemia cells. Summary:In this study, when leukemia mass burden was reduced, therapeutically infused gene-modified CD8+ T cells targeting WT1 successfully enabled to inhibit LSCs in vivo. Furthermore cell-cycle status of leukemia cells which is importantly implicated in their chemoresistance in bone marrow niche, did not affect WT1-specific cytocidal activity mediated by genetically redirected CTLs at all. Although it is preliminary, our observation encourages us to actively introduce redirected T cell-based antileukemia adoptive immunotherapy, aiming at a cure of leukemias. Disclosures:No relevant conflicts of interest to declare.

Redirected CD4+ T Cells towards HLA Class I-Restricted WT1 Epitope Successfully Display Multifactorial Helper Function for the Enhancement of Antileukemia Efficacy Mediated by Redirected T-Cell Based Immunocelltherapy.

AbstractAbstract 3153 Purpose:In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods:HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results:WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion:Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures:No relevant conflicts of interest to declare.

In Vivo Safety and Persistence of Endoribonuclease Gene-Transduced CD4+ T Cells in Cynomolgus Macaques for HIV-1 Gene Therapy Model

BackgroundMazF is an endoribonuclease encoded by Escherichia coli that specifically cleaves the ACA sequence of mRNA. In our previous report, conditional expression of MazF in the HIV-1 LTR rendered CD4+ T lymphocytes resistant to HIV-1 replication. In this study, we examined the in vivo safety and persistence of MazF-transduced cynomolgus macaque CD4+ T cells infused into autologous monkeys.Methodology/Principal FindingsThe in vivo persistence of the gene-modified CD4+ T cells in the peripheral blood was monitored for more than half a year using quantitative real-time PCR and flow cytometry, followed by experimental autopsy in order to examine the safety and distribution pattern of the infused cells in several organs. Although the levels of the MazF-transduced CD4+ T cells gradually decreased in the peripheral blood, they were clearly detected throughout the experimental period. Moreover, the infused cells were detected in the distal lymphoid tissues, such as several lymph nodes and the spleen. Histopathological analyses of tissues revealed that there were no lesions related to the infused gene modified cells. Antibodies against MazF were not detected. These data suggest the safety and the low immunogenicity of MazF-transduced CD4+ T cells. Finally, gene modified cells harvested from the monkey more than half a year post-infusion suppressed the replication of SHIV 89.6P.Conclusions/SignificanceThe long-term persistence, safety and continuous HIV replication resistance of the mazF gene-modified CD4+ T cells in the non-human primate model suggests that autologous transplantation of mazF gene-modified cells is an attractive strategy for HIV gene therapy.

Engineering of Human T-Cells with a Novel Aurora-A Kinase-Specific T-Cell Receptor Gene Transfer Confers Anti-Leukemia Reactivity

AbstractAbstract 4290 Background & Purpose:Recently we have identified a novel HLA-A*0201-restricted antigenic 9mer epitope (aa207-215: YLILEYAPL) derived from Aurora-A kinase (Aurora-A) which is capable of generating anti-leukemia cytotoxic T-lymphocytes (CTL).(Blood, 2009) To improve the feasibility of Aurora-A targeting cellular immunotherapy against leukemia, we have established a Aurora A207-215-specific CTL clone, and have obtained the full-length T-cell receptor (TCR) α/β genes for TCR gene transfer. In this study, using human leukemia cell lines and patients' leukemia cells, we examined the anti-leukemia reactivity of engineered T-cells with Aurora-A-specific TCR gene transfer. Methods:Full-length of an HLA-A*0201-restricted and Aurora-A207-215-specific TCR α/β genes (Vα3/J20/Cα, Vβ10.3(12)/J1.1/Cβ1, respectively) were cloned into bicistronic GaLV-pseudotyped retroviral vector. Using Retronectin (Takara Bio. Japan)-coated plates, Aurora-A-specific TCR α/β genes were inserted into lymphocytes. Whether this vector was capable of generating a functional Aurora-A207-215-specific TCR heterodimer was examined using Jurkat/MA cells (kindly gifted from Prof. Erik Hooijberg, Netherlands). The epitope-specific and leukemia specific cytotoxicity and IFN-γ production of gene-modified normal CD8+ and CD4+ T-cells were examined by 51Cr-releasing assay and ELISA. HLA-A*0201-restriction of engineered T-cell responses was examined by inhibition assay with antibodies, and HLA-A*0201 transduced human leukemia cell line: MEG01 which abundantly expresses Aurora-A. In vivo anti-leukemia effect of gene-modified CD8+T-cells was examined using NOD/SCID/γcnull (NSG) mice. Eventually, the on-target adverse effect of these Aurora-A-specific TCR-gene transferred CD8+T-cells against autologous hematopoietic progenitor cells was examined using cord blood CD34+ cells. Results:The Aurora-A specific-TCR expressing retroviral vector was capable of generating a functional TCR in Jurkat/MA cells which could produce luciferase in response to Aurora-A peptide on C1R-A2 cells in a dose dependent manner. Aurora-A-specific TCR-transduced CD8+ T-cells produced IFN-γ and exerted cytotoxicity against Aurora-A peptide-loaded C1R-A2 cells in an HLA-A*0201 restricted fashion. These engineered CD8+ T-cells also killed HLA-A*0201+ leukemia cell line and patient leukemia cells, but not HLA-A*0201+ normal PBMC and normal mitotic PHA-stimulated lymphoblasts. The anti-leukemia effect of These engineered CD8+ T cells was significantly abrogated by the anti-HLA-class I monoclonal antibody (MoAb), but not by anti-HLA-DR-MoAb. These engineered CD8+ T-cells killed HLA-A*0201-transduced MEG01 cells which were abundantly expressing Aurora-A, but not parent HLA-A*0201-negative MEG01 cells. Aurora-A-specific TCR gene transduced CD4+ T-cells produced IFN-γ in response to the epitope recognition, which was also in an HLA-A*0201-restricted fashion. Furthermore, Aurora-A-specific TCR-transduced CD8+ T cells did not damage the viability of autologous cord blood CD34+ cells in vitro. Finally, These engineered CD8+ T-cells successfully inhibited the engraftment and growth of inoculated leukemia cell line cells in the NOD/SCIDγ/cnull mice. Background:In this study, Aurora-A kinase-specific TCR gene transferred T-cells successfully recognized the target epitope and exerted the target-specific cytotoxicity. Additionally these engineered CD8+ T-cells exerted anti-leukemia effect both in vitro and in vivo. While those these transfectants did not damage autologous hematopoietic progenitor cells in vitro. Collectively, the novel anti-leukemia adoptive therapy using Aurora-A-specific TCR-gene transferred T-cells appears promising, and further investigations are warranted for the clinical application. Disclosures:No relevant conflicts of interest to declare.

Eradication of Established B-cell Lymphoma by CD19-specific Murine T Cells is Dependent on Host Lymphopenic Environment and Can be Mediated by CD4+ and CD8+ T Cells

B-cell malignancies seem to be particularly amenable to immunotherapy and as such make particularly attractive targets for adoptive T-cell therapy. Murine T cells gene-modified to express a chimeric immune receptor specific for CD19+ (aCD19z) efficiently kill CD19 B-cell lymphoma cells in vitro. aCD19z T cells also secrete high levels of interleukin-2 during culture with target cells in a CD86 independent manner. aCD19z T cells proved effective at eradicating established B-cell lymphoma in a syngeneic model system when combined with a lymphodepleting preconditioning regimen. In mice deficient of T, B, and natural killer cells (severe combined immunodeficient/Beige), aCD19z T cells efficiently eradicated long-term (13 d) established tumors with 100% of treated animals remaining tumor free for greater than 77 days. Although gene-modified CD4+ and CD8+ were both active in this setting, poor engraftment by CD8+ T cells coupled with the rigorous expansion of CD4+ cells in the Balb/c background suggests that CD4+ T cells may be playing a predominant role in lymphoma rejection in this model. Taken together, the therapeutic effectiveness of aCD19z T cells in this model supports a recently opened phase 1 trial of this receptor in non-Hodgkin lymphoma.

Characterization of Genetically Modified T-Cell Receptors that Recognize the CEA:691-699 Peptide in the Context of HLA-A2.1 on Human Colorectal Cancer Cells

Carcinoembryonic antigen (CEA) is a tumor-associated protein expressed on a variety of adenocarcinomas. To develop an immunotherapy for patients with cancers that overexpress CEA, we isolated and genetically modified a T-cell receptors (TCRs) that specifically bound a CEA peptide on human cancer cells. HLA-A2.1 transgenic mice were immunized with CEA:691-699. A CEA-reactive TCR was isolated from splenocytes of these mice and was genetically introduced into human peripheral blood lymphocytes via RNA electroporation or retroviral transduction. Amino acid substitutions were introduced throughout the complementarity determining regions (CDR1, CDR2, and CDR3) of both TCR alpha and beta chains to improve recognition of CEA. Murine lymphocytes bearing the CEA-reactive TCR specifically recognized peptide-loaded T2 cells and HLA-A2.1(+) CEA(+) human colon cancer cells. Both CD8(+) and CD4(+) human lymphocytes expressing the murine TCR specifically recognized peptide-loaded T2 cells. However, only gene-modified CD8(+) lymphocytes specifically recognized HLA-A2.1(+) CEA(+) colon cancer cell lines, and tumor cell recognition was weak and variable. We identified two substitutions in the CDR3 of the alpha chain that significantly influenced tumor cell recognition by human peripheral blood lymphocytes. One substitution, T for S at position 112 (S112T), enhanced tumor cell recognition by CD8(+) lymphocytes, and a second dually substituted receptor (S112T L110F) enhanced tumor cell recognition by CD4(+) T cells. The modified CEA-reactive TCRs are good candidates for future gene therapy clinical trials and show the power of selected amino acid substitutions in the antigen-binding regions of the TCR to enhance desired reactivities.

Gene-Modified CD8+ T Cells Undergo Functional Polarization to Effector and Central Memory Cells in Response to Antigen Exposure.

Adoptive transfer (AT) of autologous T cells genetically-redirected against tumor antigens has considerable potential as cancer immunotherapy [Kershaw, Nat Rev Immunol. 2005]. However, the in vivo persistence of AT T cells is critical for tumor control and requires the development (in vitro or in vivo) of a memory T cell subset. We investigated the generation of memory T cell subsets in a novel chimeric T cell receptor-expressing T cell product prior to, and after exposure to cognate antigen. Gene-modified T cells (LeY-T) express a chimeric receptor comprising a single chain variable fragment (scFv) specific for Lewis Y (LeY) antigen coupled to the intracellular signaling domains of CD3 zeta and CD28, capable of inducing T cell effector granule release and target killing [Westwood PNAS 2005]. To produce LeY-T cells, PBMC from healthy donors (n=20) or multiple myeloma patients (n=2) were cultured with anti-OKT3 (30ng/ml) and IL-2 (600IU/ml) for three days, followed by two rounds of transduction with retroviral supernatant. Subsequently, T cells were expanded in high dose IL-2 (600IU/ml) from day 5 onwards. T cells were harvested for this study on culture days 10–12, CD8+ and CD4+ T cells expressed the chimeric protein (50–60)%. LeY CD8+ T cell subsets were assessed as naïve (N), central memory (CM), effector memory (EM) or effector (E) based on three features:-phenotype (CD45RA, CCR7, CD28, CD27 and perforin);homeostatic cytokine (IL-15/IL-7) proliferation;response to Lewis antigen contact including cell proliferation and cytokine secretion.We repeatedly observed that CD8+ LeY-T cells analyzed directly from the initial expansion culture demonstrate an effector memory (EM) phenotype (CD45RA−/CCR7−/CD28+/perforinhi and variable CD27 expression) (Figure 1A). Furthermore in vitro expanded LeY CD8 T cells express IL- 15R beta (CD122) and the common gamma chain (CD132), they proliferate in response to IL-15 (86% cell division, division index 1.82), but less with IL-7 (30% cell division, division index 0.56). Baseline expanded CD8+ LeY-T cells respond to the presence of LeY antigen by proliferating and secreting IFN-gamma (4–8% of CD8 T cells) but not IL-2. Importantly, no IFN-gamma secretion was seen in control T cells transduced with empty vector (Figure 1B, OVCAR cells). Furthermore, no IFN-gamma was secreted by the control or the CD8+ LeY-T cells in response to the Lewis antigen negative cell line (Figure 1C, HCT116 cells). To explore the memory component further, we examined the functional status of the CD8+ LeY-T cells seven and 30 days following a 48-hour exposure to LeY antigen (OVCAR cells), and compared this to CD8+ LeY-T cell functional status at baseline. Thus, direct from transduction, expansion culture LeY CD8+ T cells were largely EM phenotype (95%) a small population of cells (1–5)% had a CM phenotype (CD45RA−/CCR7+/CD28+/perforinlo). In contrast, seven days after Lewis antigen contact the EM cells had decreased to (76–88)% and CM increased to (10–21)%; this distribution was retained up to day 30 post-antigen exposure. In addition, seven days after Lewis antigen exposure, CD8+ LeY-T cells retain the capacity to proliferate in response to Lewis antigen and to secrete IFN-gamma, at no stage do these cells secrete IL-2. In conclusion, the CD8+ LeY-T cells produced by in vitro transduction and expansion culture have an EM functional status direct from in vitro culture indicating that they are an appropriate starting population for in vivo adoptive transfer. After exposure to LeY expressed on tumor cell lines in vitro, CD8+ LeY T cells show further polarization to either EM or CM cells. These results suggest that the LeY-chimeric T cells have the potential to form long-term memory populations in vivo after adoptive transfer. [Display omitted]