Targeted in-vitro-stimulation reveals highly proliferative multi-virus-specific human central memory T cells as candidates for prophylactic T cell therapy

Abstract Background CD19-CAR T cells with costimulatory ligand of CD28 or 4-1BB have acquired well response in ALL and CLL, whereas it shows less effective in B-cell NHL. The microenvironment of lymphomas is much more complicated than that of leukemia, which containing physical barriers and higher immunosuppression levels preventing lymphoma cells from T cell attack. To overcome such T cell toleration, one can optimize T cell fitness by adding co-stimulatory domain or polarizing T cell differentiation. Some pre-clinical studies have reported the 3rd generation of CD19-CAR T cells with CD28 and 4-1BB domain in treating ALL, but the results were in controversy. Lenalidomide has been proved to have direct anti-tumor effects in killing DLBCL cell lines except its immunomodulatory functions. Therefore, we did preliminary investigation in vitro to seek whether the combination of lenalidomide and CD19 CAR-T cells with both CD28 and 4-1BB costimulatory domain could acquire better effects Method We first verified the proliferation inhibition of lenalidomide in treating both ABC-DLBCL cell lines (Su-DHL2 and OCI-Ly3) and GCB-DLBCL cell line OCI-Ly1. CY cell was primary cells isolated from GCB-DLBCL patients in Rui-jin Hospital. Under the maximum observed plasma concentration of lenalidmomide (2.2¦ÌM), the growth inhibition in both GCB-CY and OCI-Ly1 cell line was minimal, whereas the impact on ABC-DLBCL cell lines was more obvious. We further examined the efficiency of lenalidomide in vivo using a patient-derived mouse model. The primary lymphoma cells were obtained from a ABC-DLBCL patient and subcutaneously transplanted into NOD/SCID mouses. However, daily treated with lenalidomide could not delay the tumor growth (p>0.05) (Fig A, B, C). We next isolated CD3+ T cells from healthy donors, expanded with CD3/CD28 beads. The pLenti-EF1¦Á-CD19-28-BB-¦Æ-mcherry lentiviral vectors was generated and transduced in the expanded T cells to generate CD19 CAR-T cells. T cells transduced with pLenti-EFI¦Á-Actin-mcherry lentiviral vector were used as control. CD19-CAR T cells and T cells transdued with Actin-mcherry were pretreated with 2¦ÌM lenalidomide for 72 hours. LDH assay was then performed to identify the cytotoxicity of CD19-CAR T cells against CY in 7 hours. We found that lenalidomide substantially enhanced the anti-tumor function of CD19 CAR T cells and it also promoted the CD19-CAR T cells proliferation to some extent (Fig D, E). We therefore used three DLBCL patients CAR-T cells to identify the cytokine secretion. It was found that lenalidomide promoted Th1-biased cytokines secretion (IL-2, IFN-¦Ã, TNF-¦Á) and decreased Th2-biased cytokines (IL-6, IL-10). Interestingly, CAR-T cells secreted less IFN-¦Ã and TNF-¦Á but more IL-6 and IL-10 in killing OCI-Ly3 compared with OCI-Ly1 and CY (Fig F). The results leaded us to next determine the CD19-CAR T cell differentiation. A comparable increase of CD8+CD45RA-CD62L+ CD19 CAR T cells was observed as well as the CD4+CCR6-CCR4-CXCR3+ subset, indicating lenalidomide could induce CD19 CAR T cells differentiate to CD8+ central memory T cells and Th1 cells (Fig G). As the central memory T cells are more likely to home to the lymph nodes, we found that lenalidomide considerably increased the CD19-CAR T cell migration toward CCL21 and CCL19 in transwell system (Fig H). Conclusion In conclusion, our results indicate that lenalidomide could polarize CD19-CAR T cells to CD8 TCM and Th1 subset, which might contribute to the enhanced antitumor function of CD19 CAR-T cells. Meanwhile, by overexpressed CD62L, lenalidomide could promote the migrating capability of CD19 CAR-T cells. More in-vivo work shall be done to determine the combination therapy in the future. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Long‐term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges

International Journal of Cancer ◽

10.1002/ijc.33523 ◽

2021 ◽

Author(s):

Stefanie Herda ◽

Andreas Heimann ◽

Benedikt Obermayer ◽

Elisa Ciraolo ◽

Stefanie Althoff ◽

...

Keyword(s):

T Cells ◽

Memory T Cells ◽

Central Memory ◽

Central Memory T Cells ◽

In Vitro Expansion

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158 Inhibition of PI3Kδ improves tumor specific T cell immunity and metabolic fitness

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0158 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A172-A172

Author(s):

Guillermo Rangel Rivera ◽

Guillermo Rangel RIvera ◽

Connor Dwyer ◽

Dimitrios Arhontoulis ◽

Hannah Knochelmann ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Cell Therapy ◽

Tumor Immunity ◽

Mitochondrial Function ◽

T Cell Differentiation ◽

Tumor Control ◽

T Cell Therapy

BackgroundDurable responses have been observed with adoptive T cell therapy (ACT) in some patients. However, current protocols used to expand T cells often exhibit suboptimal tumor control. Failure in these therapies has been attributed to premature differentiation and impaired metabolism of the infused T cells. Previous work done in our lab showed that reduced PI3Kδ signaling improved ACT. Because PI3Kγ and PI3Kδ have critical regulatory roles in T cell differentiation and function, we tested whether inhibiting PI3Kγ could recapitulate or synergize PI3Kδ blockade.MethodsTo test this, we primed melanoma specific CD8+ pmel-1 T cells, which are specific to the glycoprotein 100 epitope, in the presence of PI3Kγ (IPI-459), PI3Kδ (CAL101 or TGR-1202) or PI3Kγ/δ (IPI-145) inhibitors following antigen stimulation with hgp100, and then infused them into 5Gy total body irradiated B16F10 tumor bearing mice. We characterized the phenotype of the transferred product by flow cytometry and then assessed their tumor control by measuring the tumor area every other day with clippers. For metabolic assays we utilized the 2-NBDG glucose uptake dye and the real time energy flux analysis by seahorse.ResultsSole inhibition of PI3Kδ or PI3Kγ in vitro promoted greater tumor immunity and survival compared to dual inhibition. To understand how PI3Kδ or PI3Kγ blockade improved T cell therapy, we assessed their phenotype. CAL101 treatment produced more CD62LhiCD44lo T cells compared to IPI-459, while TGR-1202 enriched mostly CD62LhiCD44hi T cells. Because decreased T cell differentiation is associated with mitochondrial metabolism, we focused on CAL101 treated T cells to study their metabolism. We found that CAL101 decreased glucose uptake and increased mitochondrial respiration in vitro, indicating augmented mitochondrial function.ConclusionsThese findings indicate that blocking PI3Kδ is sufficient to mediate lasting tumor immunity of adoptively transferred T cells by preventing premature differentiation and improving mitochondrial fitness. Our data suggest that addition of CAL101 to ACT expansion protocols could greatly improve T cell therapies for solid tumors by preventing T cell differentiation and improving mitochondrial function.

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124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.124 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A133-A133

Author(s):

Cheng-Fu Kuo ◽

Yi-Chiu Kuo ◽

Miso Park ◽

Zhen Tong ◽

Brenda Aguilar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

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CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽

10.1186/s12885-019-6131-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Lena Andersch ◽

Josefine Radke ◽

Anika Klaus ◽

Silke Schwiebert ◽

Annika Winkler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cell Lines ◽

T Cell Therapy ◽

Retinoblastoma Cell ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

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Circulating Memory T Cells Isolated from Hodgkin Lymphoma Patients Display Evidence of Exhaustion and Chronic Activation

Blood ◽

10.1182/blood.v124.21.4400.4400 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4400-4400

Author(s):

Catherine S. Diefenbach ◽

Bruce G. Raphael ◽

Kenneth B. Hymes ◽

Tibor Moskovits ◽

David Kaminetzky ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Memory T Cells ◽

Central Memory ◽

Effector Memory ◽

T Cell Exhaustion ◽

Differentiation Markers ◽

Cell Markers ◽

Cell Exhaustion ◽

Central Memory T Cells

Abstract Background: In Hodgkin lymphoma (HL) the malignant Hodgkin Reed-Sternberg (HRS) cells comprise only a small fraction of the total cellular tumor population. These HRS cells orchestrate an inflammatory microenvironment of reactive cells that propagate a permissive milieu for HL growth, contributing to an ineffective local anti-tumor immune response. The peritumoral CD4 and CD8 T cells in HL patients show high expression of the receptor programmed death-1 (PD-1), involved in the functional impairment and “exhaustion” of T cells. Growing data suggests that this HL-mediated immune suppression may have effects that extend beyond the tumor microenvironment. High systemic levels of inflammatory cytokines and chemokines in HL patients has been reported. We characterized the systemic immune profile of HL patients with both newly diagnosed (ND) and relapsed (R) disease. Methods: Informed consent for correlative blood testing was obtained from patients with ND (n=8) or R (n=5) HL treated at the NYU Perlmutter Cancer Center or NY Presbyterian/Weil Cornell since January of 2013. Blood samples were drawn pre-treatment, and at sequential timepoints during and after therapy. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll separation method and cells were frozen for subsequent analysis. The frozen PBMC were then stained with fluorescent-conjugated antibodies against T cell surface molecules in 10-color FACS analysis. The analyses were performed after gating live cells for CD4, CD8 and memory and effector T cell markers. Patient samples were compared to normal controls matched for age and sex (n=18). Results: The median HL patient age was 32 (22-72), and 8 subjects were male. All ND HL patients were treated with ABVD (range 4-6 cycles) +/- consolidative radiation; R patients had median of 3 prior therapies. One patient out of 5 had prior autologous stem cell transplant (SCT), and 1 had prior allogeneic SCT, but was not on immunosuppression. Eight patients (6ND, 2R) responded to therapy (8 CR); 5 patients (1ND, 4R) progressed on therapy or had stable disease. HL patients displayed a high frequency of the exhaustion marker PD-1 on CD4 central memory T cells (CD4+CD45RO+CD27+) compared to normal matched controls (NC): mean 41, standard error (SE) 4.8 for HL patients vs. mean 22.2, SE 1.3 for NC (p = 0.0002) (Figure 1A). PD-1 expression was similarly elevated on CD8 central memory T cells (CD8+CD45RO+CD27+) of HL patients: mean 55, SE 3.3 vs. NC: mean 40, SE 3.3 (p = 0.003) (Figure 1B). HL patients also displayed an increased frequency of PD-1 expression on CD27 negative CD4 effector T cells: mean 43, SE 4, vs. NC: mean 28.5, SE 2.4 (p = 0.003) (Figure 2). In 4 of the HL patients who responded to therapy, PD-1 expression on central memory CD4+ cells declined after therapy: mean 30.1 vs. mean increase of +2.67 in 3 patients who progressed on therapy (p< 0.009). A higher number of subjects in prospective analysis is underway, to confirm whether a response to therapy may be correlated with a reversal of the suppressed phenotype of T cells in these patients. Conclusion: HL patients have evidence of chronic activation/exhaustion in their central memory and effector T cells, suggesting that ineffective immune clearance of the HRS cells may be a systemic rather than local phenomenon. In patients with progressive disease for whom this phenotype persists it is worthy of investigation whether this immune dysfunction is a cause or consequence of resistance to therapy. This may be rationale for immune targeted therapy in patients with relapsed or resistant disease. Figure 1. Evidence for increased levels of T cell exhaustion in central memory T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4, CD8) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells were determined in: A) CD4+CD45RO+CD27+ and B) CD8+CD45RO+CD27+ T cells. Figure 1. Evidence for increased levels of T cell exhaustion in central memory T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4, CD8) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells were determined in: A) CD4+CD45RO+CD27+ and B) CD8+CD45RO+CD27+ T cells. Figure 2. Evidence for increased levels of T cell exhaustion in effector memory CD 4+ T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells was determined in CD4+RO+CD27- T cells Figure 2. Evidence for increased levels of T cell exhaustion in effector memory CD 4+ T cells of HL patients. PBMC were stained with specific fluorescent conjugated antibodies against T cell markers (CD3, CD4) together with differentiation markers (CD45RO, CD27) and PD1 and analyzed using FACS (LSR-II). The proportion of PD1+ T cells was determined in CD4+RO+CD27- T cells Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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Mobilization of CD8+ Central Memory T-Cells with Enhanced Reconstitution Potential in Mice By a Combination of G-CSF and GMI-1271-Mediated E-Selectin Blockade

Blood ◽

10.1182/blood.v126.23.512.512 ◽

2015 ◽

Vol 126 (23) ◽

pp. 512-512 ◽

Cited By ~ 1

Author(s):

Ingrid G Winkler ◽

Valerie Barbier ◽

Kristen J Radford ◽

Julie M Davies ◽

Jean-Pierre Levesque ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Memory T Cells ◽

Central Memory ◽

Donor Blood ◽

Central Memory T Cells

Abstract T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations. Central memory cells (TCM CD62L+ CCR7+) and their precursors, stem central memory T-cells (TSCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy. The challenge remains to harvest good quality TCM-cells for these immunotherapy approaches. The bone marrow (BM) is the major reservoir of CD8+ TCM and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8+ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset. First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period. Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8+ TCM cells, and their apparent migration (increase in numbers) to the blood, while CD8+ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8+ but not CD4+ TCM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8+ but not CD4+ central memory T-cells. Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8+ TCM/SCM (CD62Lhi, CCR7+) cells over treatment with G-CSF alone (p<0.05). To determine the functional consequences of this skewed mobilization following GMI-1271 co-administration, 25 uL of mobilized blood was transplanted into irradiated congenic B6.SJL recipients together with 2x105 congenic BM cells to analyze long-term donor T-cell engraftment in the recipient mice. We found G-CSF mobilized donor blood did not contribute CD8+ TCM cells that can persist post-transplant (<0.5% at 20 weeks post-transplant). In contrast when donor mice were mobilized with G-CSF together with E-selectin blockade (GMI-1271), we found elevated levels of donor blood derived CD8+ T-cells demonstrating robust long-term CD8+ T-cell persistence / regeneration (5.3 ±3.2% of total recipient T-cells, p=0.04). This dramatic boost in donor CD8+ T-cell reconstitution in mobilized blood following GMI-1271 co-administration is likely to be due to the long-term persistence and in vivo amplification of CD8+ TCM cells from donor mobilized blood. Similar in vivo enhancing effects of GMI-1271 were also observed with other mobilizing agents such as combined CXCR4 and VLA-4 blockade and GM-CSF resulting in a significant 4.9-fold boost in donor CD8+ reconstitution with GMI-1271. Importantly, only 12 hours of E-selectin blockade was sufficient to achieve this boost in CD8+ TCM numbers in the blood following G-CSF. In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo. Thus, it is possible that E-selectin blockade boosts mobilization of CD8+ TCM/SCM with stem-like properties into the blood by loosening factors retaining CD8+ TCM/SCM in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset. In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8+ TCM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8+ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering. Disclosures Winkler: GlycoMimetics Inc: Research Funding. Barbier:GlycoMimetics Inc: Research Funding. Davies:GlycoMimetics Inc: Research Funding. Smith:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000896 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000896

Author(s):

Talia Velasco-Hernandez ◽

Samanta Romina Zanetti ◽

Heleia Roca-Ho ◽

Francisco Gutierrez-Aguera ◽

Paolo Petazzi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

High Affinity ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundThere are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19– either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19– B-ALL relapses and CD19– preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.MethodsHere, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.ResultsConformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.ConclusionsWe report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22–CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

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Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma

Cancers ◽

10.3390/cancers12010139 ◽

2020 ◽

Vol 12 (1) ◽

pp. 139 ◽

Cited By ~ 5

Author(s):

Jing Cui ◽

Herui Wang ◽

Rogelio Medina ◽

Qi Zhang ◽

Chen Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Therapeutic Potential ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

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Naive CD4+ T Cells Exert Superior Alloreactive Capacity Than Central Memory CD4+ T Cells in Fully HLA-Matched Setting

Blood ◽

10.1182/blood.v118.21.4702.4702 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4702-4702

Author(s):

Bachra Choufi ◽

Mathilde Cherel ◽

Stephanie Thiant ◽

Damien Despicht ◽

jean-Paul Dessaint ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Acute Gvhd ◽

Memory T Cells ◽

Central Memory ◽

Ifn Gamma ◽

Sibling Pairs ◽

Central Memory T Cells ◽

Female Sibling

Abstract Abstract 4702 Background: Allogeneic stem cell transplantation provides donor-derived mature T cells which are involved in post-transplant immune reactions including engraftment, graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) activity, and thus play a major role in determining clinical outcome. In a prior clinical study, we showed that receiving a high percentage of CD4+CCR7+ T cells that includes naive and central memory (CM) subsets, was correlated with increased incidence and severity of acute GVHD. In a recent update of our results on 126 patients, we confirmed our previous results with a particular impact of CD4+ naive subset on acute GVHD development. The aim of the present study was to investigate in vitro the alloreactive response of CD4+CCR7+CD45RA+ naïve T cells and CD4+CCR7+CD45RAneg central memory T cells in HLA-A, -B, -C, -DRB1 and -DQB1 allele matched setting (so called 10/10 match) Materials and methods: The alloreactivity was investigated by mixed lymphocyte dendritic cell reaction using five HLA-identical healthy male and female sibling pairs to include at least a H-Y mismatch. Stimulators were mature dendritic cells (derived from monocytes of the male sibling) co-cultured with each one of the three highly purified CD4+ T cell subsets (naive, central memory and effector memory) from the female sibling. Alloreactive response was assessed by 3H thymidine incorporation at day 5, and functionally at day 4, 6 and 10 by IFN-gamma ELISpot and measurement of Th1/Th2/Th17 cytokines in co-culture supernatants. Results: Four out of five sibling pairs developed an alloreactive response to mHAs. Maximal proliferation was supported by the CCR7+CD45RA+ naive CD4+ T cells with proliferation indices from 1.75 to 3.85. This proliferation was accompanied by a functional differentiation: only naive CD4+ T cells were able to produce significant amounts of IL-6, TNF-alpha and IFN-gamma at day 6 and day 10 (120 to 174 IFN-gamma spots at day 10). Conclusion: This study demonstrates the superior capacity of naive CD4+ T cells to mount a primary alloreactive response as compared to central memory T cells. Their proliferative response associated with a pro-inflammatory differentiation makes naive CD4+ T cells potential acute GVHD inducers. These in vitro results confirm what we have observed in clinical studies and may also lend support to approaches of selective T cell depletion for GVHD prevention. Disclosures: No relevant conflicts of interest to declare.

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