Suppression of EBV-induced LCLs using CAR T cells redirected against HLA-DR.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 146-146
Author(s):  
Chungyong Han ◽  
Rohit Singh ◽  
Seon-Hee Kim ◽  
Beom K. Choi ◽  
Byoung S. Kwon
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Side Effect ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Hla Dr ◽  
Car T

146 Background: Recent studies demonstrated a therapeutic potential of T cells with chimeric antigen receptor (CAR) targeting CD19 in refractory B cell malignancies. However, CD19-CAR T cells frequently caused on-target off-tumor side effect, i.e. B cell aplasia, and led to the recurrence of CD19-negative leukemic cells. Alternative target antigen for B cell malignancies has to be excavated. Methods: We developed antibody clone, MVR, which specifically bound to HLA-DR that is highly expressed on malignant B cells. In particular, MVR recognized polymorphic region of HLA-DR, and indicated different binding affinity against various HLA-DR alleles. Based on MVR binding strength, PBMCs from high binder (MVRHigh) and low binder (MVRLow) were tested to generate MVR-CAR T cells. To evaluate the anti-tumor efficacy on B cell malignancies, MVR-CAR T cells were assessed for immune responses against Epstein-Barr virus (EBV)-induced lymphoblastoid cell line (LCL) in vitro and in vivo. Results: Final yield of MVR-CAR T cells generated from MVRHigh PBMCs was 10-fold lower than that of CD19-CAR T cells, presumably caused by "fratricide" among HLA-DR-upregulated MVR-CAR T cells. In contrast, fratricidal effect was ameliorated in MVR-CAR T cells generated from MVRLow PBMCs indicating that the interaction between MVR-CAR and MVRLow-HLA-DR was weak enough to achieve tolerance to fratricide. Of note, in spite of such low binding, MVRLow-LCLs were killed efficiently by the CAR T cells. Further quantitative analysis revealed that HLA-DR was far more upregulated on LCLs compared with normal T and B cells which did not undergo EBV-transformation. In accordance with this observation, MVR-CAR T cells successfully induced LCL-specific cytotoxicity without causing normal B cell damage in vitro and efficiently suppressed the outgrowth of metastasized tumors in LCL-xenografted immune-deficient mice. Conclusions: MVR-CAR T cells redirected against HLA-DR for B cell malignancies were evaluated for the cytotoxic efficacy in vitro and in vivo. Considering the alleviated on-target off-tumor side effect and the feasibility of targeting HLA-DR for CD19-deficient malignant B cells, MVR-CAR T cells can be an alternative option for B cell malignancies.

scholarly journals CD72 Nanobody-Based CAR-T Cells Have Potent Anti-Tumor Efficacy in B Cell Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1717-1717
Author(s):  
Matthew A Nix ◽  
William C Temple ◽  
William Karlon ◽  
Donghui Wang ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
In Vitro Cytotoxicity ◽  
B Cell Malignancies ◽  
Cytotoxicity Assays ◽  
Car T Cells ◽  
Car T

Abstract Background: Approximately 50% of pediatric B-ALL patients treated with clinically approved CD19-targeting CAR-T cells do not remain in remission one year after therapy. CD22-targeting CAR-T cells appear to be curative in only a small fraction of CD19-refractory patients and this therapeutic strategy is primarily used as a bridge to stem cell transplant. Additional immunotherapeutic targets thus remain urgently needed. Our laboratory recently used cell surface proteomics to identify CD72 as a B-cell specific marker especially upregulated on poor prognosis, KMT2A/MLL-rearranged B-ALL (Nix et al., Cancer Discovery (2021)). In this published work, we used a best-in-class nanobody library displayed on yeast to develop binders to CD72. We demonstrated for the first time that fully synthetic nanobodies can generate CAR-T cells that are highly potent in vitro and in vivo. While we previously focused on these "nanoCARs" in KMT2A/MLLr B-ALL, in this follow-up study we aimed to 1) further expand our nanoCAR indications to other CD72-expressing B-cell malignancies; 2) biophysically characterize our synthetic nanobodies; 3) evaluate the potential for further humanization of the nanobody binder amino acid sequence while retaining anti-tumor efficacy; and 4) characterize the potency and T-cell immunophenotypes in the context of our lead nanobody binder ("NbD4") placed on different CAR backbones. Methods: Flow cytometry of primary patient samples for CD72 was performed in a CLIA-certified laboratory. NbD4 nanobody was recombinantly expressed in E. coli and biolayer interferometry was used to determine the binding affinity to recombinantly-expressed CD72 extracellular domain. CAR-T cells were generated from peripheral blood donor CD4+ and CD8+ cells (1:1) ratio via lentiviral transduction. In vitro cytotoxicity assays were performed at a range of effector:tumor ratios. In vivo studies were performed in human cell line orthotopic xenografts in NSG mice. 1e6 luciferase-labeled Jeko cells were implanted at Day 0 followed by administration of 4e6 CAR-T cells at Day 6. Tumor burden was assessed by bioluminescence. Results: Flow cytometry on primary non-Hodgkin B-cell lymphoma obtained from fine needle aspiration biopsy (n = 5) confirmed CD72 surface expression (not shown), consistent with RNA-seq across larger cohorts. Biolayer interferometry demonstrated that NbD4 bound with surprisingly low affinity to recombinant CD72 (K D ~800 nM) (Fig. 1A), with both slow on rate (k on 8.38e4 M -1s -1) and rapid off rate (k off 6.82e-2 s -1). This affinity stands in contrast to that reported for FMC63 single chain variable fragment (scFv) used in clinically approved CD19-targeting CAR-T cells (K D 0.3-5 nM), despite similar in vitro and in vivo efficacy of both products. Our NbD4 framework region shows ~82% homology to a human IgG variable heavy domain, significantly higher than FMC63 (~59% homology). We made additional substitutions in the framework domain to increase human homology up to 89%. In vitro cytotoxicity assays with SEM B-ALL cells showed several humanized variants with similar efficacy to NbD4 (Fig. 1B). We further evaluated the impact of placing NbD4 on different CAR backbones, including combinations of CD28 or 4-1BB costimulatory domains and CD8 or IgG4-based transmembrane and hinge regions (Fig. 1C). In vivo, CD72 nanoCARs with Backbone 3 showed significantly increased potency (Fig. 1D). Indeed, tumors treated with Backbone 3 CAR-Ts showed complete tumor clearance and did not develop new tumors even after re-challenge with 1e6 Jeko cells at Day 52 (Fig. 1D). Preliminary characterization of effector and memory CAR-T cell phenotypes before exposure to tumor suggested that Backbone 3 had an increased number of naïve T cells compared to empty CAR and CD19 CAR-T cells (data not shown). Conclusions: Our results demonstrate that our fully synthetic CD72 nanoCARs can effectively eliminate CD72-expressing B-cell malignancy models despite low nanobody binding affinity. Humanized NbD4 variants may serve as clinical candidates with even further reduction in possible immunogenicity of the llama amino acid framework. Alterations to the CAR backbone have a major impact on anti-tumor efficacy and phenotypes of our synthetic nanobodies. CD72-targeting therapies may be effective therapeutics not only KMT2A/MLLr B-ALL but also across a broader spectrum of refractory B-cell malignancies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ligand Based CAR T-Cell Targeting BAFF Receptors Asa Novel Therapy for B Cell Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Reshmi Parameswaran ◽  
Derek Wong ◽  
Keman Zhang ◽  
Abhishek Asthana ◽  
Marcos de Lima ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Advisory Board ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Car T

Background: Autologous T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have shown rapid and durable responses in B cell malignancies. Although CD19 CAR-T cells have demonstrated remarkable success, CD19-negative relapses occur in 30-45% of patients, highlighting the need for adoptive immunotherapies with alternative targeting approaches. B-cell activating factor (BAFF) is a critical B cell survival factor. Receptors of BAFF (BAFF-R, TACI and BCMA) are expressed by a wide range of B cell neoplasms, including ALL, CLL, NHL and MM, making them attractive therapeutic targets. We developed a novel ligand-based CAR that when expressed in T cells, targets and eliminates malignant B cells expressing BAFF receptors (BAFF CAR-T). This approach has several potential advantages over CD19 targeting CAR-T therapy: CD19 is expressed on all B cells, but BAFF receptors are expressed only on mature B cells, making it a more specific antigen for targeting and potentially narrowing down the side effect profile. BAFF CAR-T cells are a potential therapeutic strategy to treat CD19 CAR-T relapsed patients as well as chemotherapy resistant patients. Methods: BAFF ligand was fused to a second generation CAR backbone containing 4-1BB costimulatory and CD3ζ intracellular signaling domains. T cells were isolated from human blood, activated and transduced with BAFF-CAR lentiviral particles. In vitro tumor cell killing was analyzed using calcein-AM cytotoxicity assay. For in vivo testing of BAFF CAR-T cytotoxicity, we used mantle cell lymphoma (MCL) Jeko-1 xenograft model. Immunocompromised NSG mice were subcutaneously injected with human MCL cell line Jeko-1 (10.106 cells at day 0). Once these mice developed measurable tumors, we injected T cells transduced with empty vector (control T cells) or BAFF-CAR T cells (10 x 106 cells) or PBS intra-tumorally as a one-time injection. Tumor volumes were measured every other day using calipers. Results: BAFF CAR-T cells showed significant cytotoxicity in vitro (not shown) and in vivo against human MCL cell line Jeko-1. Mice treated with BAFF-CAR-T showed significant reduction in tumor volume compared to mice treated with control T cells and PBS (Figure 1A, B). Tumor progression was observed after control T cell and PBS treatment, whereas the cohort treated with BAFF CAR-T did not show any tumor progression, and with complete or near-complete tumor eradication. Survival analysis showed the BAFF CAR-T treated cohort had significantly longer survival compared to control-T cell and PBS treated cohorts (Figure 1C). Mice were sacrificed when tumor volume reached 2 cm3. Conclusion: Our data suggest that targeting BAFF receptors with a novel, ligand-based BAFF-CAR-T is a feasible and effective immunotherapeutic strategy to eliminate malignant B cells, warranting further development. BAFF-CAR-T cells have therapeutic potential against a wide spectrum of B cell malignancies, including CD19 negative relapsed disease. Clinical grade expansion and clinical trials are in development for BAFF CAR-T therapy non Hodgkin lymphoma patients. Disclosures Parameswaran: Luminary Therapeutics: Consultancy; Luminary therapeutics: Research Funding. de Lima:Kadmon: Other: Personal Fees, Advisory board; BMS: Other: Personal Fees, advisory board; Incyte: Other: Personal Fees, advisory board; Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding. Caimi:Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board; ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau.

scholarly journals CD19-CAR-T Cells Bearing a KIR/PD-1-Based Inhibitory CAR Eradicate CD19+HLA-C1− Malignant B Cells While Sparing CD19+HLA-C1+ Healthy B Cells

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2612
Author(s):  
Lei Tao ◽  
Muhammad Asad Farooq ◽  
Yaoxin Gao ◽  
Li Zhang ◽  
Congyi Niu ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Killer Cell ◽  
Xenograft Model ◽  
Sustained Remission ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

B cell aplasia caused by “on-target off-tumor” toxicity is one of the clinical side effects during CD19-targeted chimeric antigen receptor (CAR) T (CD19-CAR-T) cells treatment for B cell malignancies. Persistent B cell aplasia was observed in all patients with sustained remission, which increased the patients’ risk of infection. Some patients even died due to infection. To overcome this challenge, the concept of incorporating an inhibitory CAR (iCAR) into CAR-T cells was introduced to constrain the T cells response once an “on-target off-tumor” event occurred. In this study, we engineered a novel KIR/PD-1-based inhibitory CAR (iKP CAR) by fusing the extracellular domain of killer cell immunoglobulin-like receptors (KIR) 2DL2 (KIR2DL2) and the intracellular domain of PD-1. We also confirmed that iKP CAR could inhibit the CD19 CAR activation signal via the PD-1 domain and CD19-CAR-T cells bearing an iKP CAR (iKP-19-CAR-T) exerted robust cytotoxicity in vitro and antitumor activity in the xenograft model of CD19+HLA-C1− Burkitt’s lymphoma parallel to CD19-CAR-T cells, whilst sparing CD19+HLA-C1+ healthy human B cells both in vitro and in the xenograft model. Meanwhile, iKP-19-CAR-T cells exhibited more naïve, less exhausted phenotypes and preserved a higher proportion of central memory T cells (TCM). Our data demonstrates that the KIR/PD-1-based inhibitory CAR can be a promising strategy for preventing B cell aplasia induced by CD19-CAR-T cell therapy.

scholarly journals Automated Generation and Phenotypic Characterization of Clinical Grade CD19 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1675-1675
Author(s):  
Ashish Sharma ◽  
Anne Roe ◽  
Filipa Blasco Lopes ◽  
Ruifu Liu ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Central Memory ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown enormous promise in the treatment of certain B cell malignancies. Access to treatment is still limited due to a variety of issues, including pricing and centralized manufacturing models. Generation of CAR-T cells using an automated platform, followed by rigorous functional phenotyping, may contribute to the development of a robust long-lasting therapy. METHODS: Here, we used the Miltenyi Prodigy (Miltenyi Biotech, Bergisch Gladbach, Germany) to automate the process of manufacturing genetically manipulated T cells in a closed system. The system obviates the need for clean room infrastructure. We tested the feasibility of utilizing the Miltenyi Prodigy to manufacture CAR-T cells using a CD19 scFV vector with the 4-1BB co-stimulatory domain. (Lentigen Technology, Inc, Gaithersburg, MD). The purity, differentiation capacity and effector function of the enriched CAR-T cells was studied using high-dimensional flow cytometry. Finally, the functional potential of these cells was tested in vitro and by treating NOD-SCID-gamma (NSG) mice infused with B cell lymphoma cells (Raji B cell), with the CAR-T cells. RESULTS: Starting with 1 x 108 CD4 and CD8 cells from donor apheresis products, CAR-T cells were expanded for 12 days in culture media containing with TransAct (Miltenyi Biotech), IL7 and IL15. The mean fold-expansion at day 12 was 44 ± 5.6, range 39-50 (n=3). The mean transduction efficiency of CAR-T vector was 20%, range 10-25% (n=3), which is similar to other reported methods. The CD19 CAR-T product was enriched in both the CD4 and CD8 T cells subsets, and showed high-level of cytotoxicity against CD19+ cell lines in vitro and in vivo (Figure 1: Mice treated with the CD19-CAR T demonstrated a marked reduction in disease burden as compared to T cell control as measured by bioluminescence imaging and flow cytometric analysis). The CAR-T product was enriched in cell subsets with both effector (CD27-CCR7-; ~20% of total cells) and central memory phenotypes (CD27+CCR7+; ~30% of total T cells). The effector CD4 and CD8 T cells showed increased expression of major functional T cell differentiation transcription factors (i.e. T-bet and GATA3) critical for the development of anti-tumor responses. Whereas, the central CD4 and CD8 T cells were enriched for the expression of TCF7 (a stemness related member of the WNT signaling known to increase longevity of these cells). The frequencies and phenotypes of these cells were maintained in peripheral blood of NSG mice infused with B cell lymphoma cells (Raji B cells), 1 week after treatment. A significant expansion of CD8+ effector T cells and a dramatic reduction in tumor burden was observed over the next 4 weeks in all major organs. Interestingly, we observed that smaller proportion of central-memory like cells (with higher TCF7 levels) continued to persist 6 weeks post-treatment, potentially contributing to a long-lived recallable response. Based on these data we have initiated a phase 1 clinical trial to test the therapeutic potential of the CAR-T product in patients with advanced/refractory B cell lymphoma. The first clinical grade manufacturing run resulted in a CD19 + cell yield of 1.4 x109. CONCLUSION: Our data highlight that the automated CAR-T generation platform (i.e. Miltenyi Prodigy) is effective at the generating purified functionally competent CAR-T cells. Further, findings from our phenotyping analyses show that the CAR-T product is enriched in both effector and central memory subsets and is effective at tumor clearance in vivo. Thus far, we have treated one patient with CD19 CAR-T manufactured on this platform and 2 more have been enrolled. Though this initial study is based on CD19 CAR-T cells, the approach described here could easily be utilized to genetically engineer T cells with gene constructs that are more relevant for specific cancers and infectious diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Derek P. Wong ◽  
Nand K. Roy ◽  
Keman Zhang ◽  
Anusha Anukanth ◽  
Abhishek Asthana ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Viral Gene ◽  
Car T Cells ◽  
Car T ◽  
Almost All ◽  
Viral Gene Delivery

AbstractB cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.

CAR T cells Targeting Human Immunoglobulin Light Chains Eradicate Mature B Cell Malignancies While Sparing a Subset of Normal B Cells

2021 ◽  
pp. clincanres.2754.2020
Author(s):  
Raghuveer Ranganathan ◽  
Peishun Shou ◽  
Sarah Ahn ◽  
Chuang Sun ◽  
John West ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Human Immunoglobulin ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Car T

Donor-Derived Anti-CD19 Chimeric-Antigen-Receptor-Expressing T Cells Cause Regression Of Malignancy Persisting After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 151-151 ◽  
Author(s):  
James N Kochenderfer ◽  
Mark E. Dudley ◽  
Robert O. Carpenter ◽  
Sadik H Kassim ◽  
Jeremy J. Rose ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Cell Malignancies ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Grade 3

Abstract Progressive malignancy is a leading cause of death in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). To improve treatment of B-cell malignancies that persist despite alloHSCT, we conducted a clinical trial of allogeneic T cells genetically modified to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Ten patients were treated on this trial. Four patients were recipients of human-leukocyte-antigen (HLA)-matched unrelated donor (URD) transplants and 6 patients were recipients of HLA-matched sibling transplants. T cells for genetic modification were obtained from each patient’s healthy alloHSCT donor. Patients received a single infusion of anti-CD19-CAR T cells. Cell doses ranged from 1x106 to 10x106 T cells/kg. A mean of 58% of the infused cells expressed the CAR. Patients did not receive chemotherapy or other anti-malignancy therapy with the CAR-T-cell infusions, so the responses observed in these patients are not confounded by the effects of chemotherapy. In contrast to other reports of successful treatment of B-cell malignancies with anti-CD19-CAR T cells, the patients on this study were not lymphocyte-depleted at the time of the CAR-T-cell infusions. Two patients with chronic lymphocytic leukemia (CLL) refractory to standard unmanipulated allogeneic donor lymphocyte infusions (DLIs) had regressions of large malignant lymph node masses after infusion of allogeneic anti-CD19-CAR T cells. One of these CLL patients obtained a complete remission that is ongoing 9 months after treatment with allogeneic anti-CD19-CAR T cells. This patient also had complete eradication of blood B cells within 9 days after her CAR-T-cell infusion. Another patient had tumor lysis syndrome requiring rasburicase treatment as his CLL dramatically regressed in lymph nodes, bone marrow, and blood within 2 weeks of his anti-CD19-CAR-T-cell infusion. A patient with mantle cell lymphoma obtained a partial remission that is ongoing 3 months after infusion of anti-CD19-CAR T cells. A fourth patient with diffuse large B-cell lymphoma has ongoing stable disease 11 months after infusion of anti-CD19-CAR T cells. The other 6 treated patients all had short periods of stable malignancy or progressive disease after their CAR-T-cell infusions. Specific eradication of blood B cells occurred after infusion of CAR T cells in 3 of 4 patients with measurable blood B cells pretreatment. None of the patients treated on this study developed GVHD after their anti-CD19-CAR-T-cell infusions, despite the fact that 6 of 10 treated patients had experienced GVHD at earlier time-points after their most recent alloHSCT. One patient, who had a history of cardiac dysfunction with prior acute illnesses, had temporary cardiac dysfunction after infusion of anti-CD19-CAR T cells. The most prominent toxicities experienced by patients were fever and hypotension; these peaked 5 to 12 days after CAR-T-cell infusions and resolved within 14 days after the T-cell infusions. Two patients had Grade 3 fever, and 2 patients had Grade 3 hypotension. No patients experienced Grade 4 toxicities that were attributable to the CAR-T-cell infusions. Elevated levels of serum interferon gamma were detected in 3 patients at the time that they were experiencing toxicities. We detected cells containing the anti-CD19-CAR gene in the blood of 8 of 10 patients. The peak blood levels of CAR T cells varied from undetec to 2.8% of peripheral blood mononuclear cells. The persistence of the CAR T cells in the blood of patients was limited to one month or less. When we assessed T cells from the blood of patients ex vivo, we found elevated levels of the T-cell inhibitory molecule programmed cell death protein-1 (PD-1) on CAR+ T cells compared to CAR-negative T cells. These results show for the first time that small numbers of donor-derived allogeneic anti-CD19-CAR T cells can cause regression of highly treatment-resistant B-cell malignancies after alloHSCT without causing GVHD. Malignancies that were resistant to standard DLIs regressed after anti-CD19-CAR-T-cell infusions. Future goals for improving this approach include enhancing the persistence of anti-CD19-CAR T cells and reducing toxicities. Infusion of allogeneic T cells genetically modified to recognize malignancy-associated antigens is a promising approach for treating residual malignancy after alloHSCT. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5812-5812
Author(s):  
Alla Dolnikov ◽  
Swapna Rossi ◽  
Ning Xu ◽  
Guy Klamer ◽  
Sylvie Shen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Target Cells ◽  
Car T Cells ◽  
Anti Tumour Activity ◽  
Car T ◽  
Pre Treatment ◽  
Tumour Activity

Abstract T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals In Vitro-Selected Nanobody-Based Cellular Therapy Targeting CD72 for Treatment of Refractory B-Cell Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1337-1337
Author(s):  
Matthew Nix ◽  
Yu-Hsiu T. Lin ◽  
Huimin Geng ◽  
Makeba Marcoulis ◽  
Paul Phojanakong ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
B Cell ◽  
Board Of Directors ◽  
B Cell Malignancies ◽  
Advisory Committees ◽  
Car T ◽  
Equity Ownership

Introduction: B-cell acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr; also known as KMT2Ar) have particularly dismal clinical outcomes. Although CAR T immunotherapies targeting CD19 have shown impressive responses treating MLLr B-ALL and other B cell malignancies, relapse, often with loss of relevant CD19 epitope, remains a major clinical concern. The mixed results of CD19 CAR T as a monotherapy underscores the need to pursue additional immunotherapy targets and novel therapeutic modalities for high-risk patients. Results and Methods: Data with existing CAR-T's suggest that increased target antigen density frequently correlates with increased tumor elimination. Therefore, we aimed to define the cell surface proteomic landscape of B-ALL to identify novel, MLLr-enriched candidates for targeted immunotherapy of this poor-prognosis subtype. As an initial screen, using N-glycoprotein capture and mass spectrometry, we quantified differentially abundant cell surface proteins in MLLr (n= 4) versus non-MLLr (n= 5) B-ALL cell lines (Figure 1). Label-free proteomics (n= 3 replicates) quantified >900 high-confidence membrane proteins (FDR=0.05). Principal component analysis identified unique cell surfaceome signatures between B-ALL subtypes, implying different surface landscapes associated with specific genetic alterations. The MLLr B-ALL "surfaceome" is notably characterized by increased expression of adhesion molecules not identified by RNA-sequencing alone. We focused on CD72 as a novel immunotherapy target given significant enrichment on MLLr B-ALL vs. other B-ALL subtypes, near equivalent antigen density to CD19, undetectable expression on HSPCs, T-cells, and other normal tissues, and reported widespread expression on other mature B-cell malignancies. Analysis of transcriptome and ChIP-seq data suggested increased CD72 expression in MLLr B-ALL is not regulated directly by the MLL-AF4 oncoprotein but instead a function of increased CD72 expression at pro-B-cell stage. Flow cytometry and immunohistochemistry on primary samples confirmed high expression of CD72 both in MLLr B-ALL as well as DLBCL. Recombinant CD72 ECD was panned against a fully in vitro nanobody yeast display library (McMahon et al., Nat Struct Mol Biol(2018)) resulting in isolation of multiple unique, highly-specific CD72 nanobody binders with KD's < 5nM. Nanobodies were incorporated into 2nd generation CAR constructs and transduced into normal donor CD8+ T-cells and assessed in vitro for tumor cell lysis, cytokine release, and exhaustion marker expression. Nanobody clone Nb.D4 outperformed others in lysis of B-ALL and DLBCL cells lines displaying a broad range of CD72 expression, had no activity versus CD72 negative cells, and showed similar efficacy to that found with a clinically-used CD19 CAR. To assess in vivo activity, CD72(Nb.D4) CAR-T's at 1:1 CD4:CD8 ratio were injected at an effector:tumor ratio of 5:1 into tumor-bearing NSG mice (luciferase-labeled SEM or MLLr PDX). In vivo results confirmed strong anti-tumor effect of CD72 nanobody CAR-T's, equivalent to clinical CD19 CAR, and significantly increased survival in mice (Figure 2). A CRISPR interference-generated antigen escape model of CD19 was also effectively eliminated by CD72 CAR-T's. We also introduce "antigen escape profiling", where cell surface proteomics of a CRISPRi CD72-knockdown model demonstrated extensive surfaceome rewiring with potential implications for leukemia cell trafficking and adhesion in the setting of acquired resistance. Given CD72's role as a BCR signaling inhibitory receptor, we are currently examining its influence on proximal B-cell receptor signaling and relationship to combination therapies affecting this pathway. Conclusions:By characterizing the surface proteomic landscape of B-ALL, we develop a resource for the research community and identify CD72 as a promising therapeutic target. We demonstrate that a novel, fully recombinant nanobody library can generate potent cellular therapies, which may be extended to other targets in the future. We anticipate that antigen escape profiling will prove broadly useful for anticipating mechanisms of resistance to novel immunotherapies. CD72 CAR-T's are a promising strategy across a range of B-cell malignancies, particularly those refractory to CD19 therapy. Disclosures Nix: UCSF: Patents & Royalties. Wiita:UCSF: Patents & Royalties; Indapta Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Protocol Intelligence: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Application of Novel T Cell Immunotherapeutics to Drive Antigen-Specific Activation, Expansion, and Differentiation of CD19 Chimeric Antigen Receptor T Cells (CAR T-cells)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Moriah Rabin ◽  
Mengyan Li ◽  
Scott Garforth ◽  
Jacqueline Marino ◽  
Jian Hua Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Effector Memory ◽  
Mhc Molecules ◽  
Car T Cells ◽  
Car T

Background: While chimeric antigen receptor T cells (CAR T-cells) induce dramatic remissions of refractory or recurrent B cell malignancies, the durability of these remissions is frequently limited by subsequent reduction in circulating CAR T-cells and/or by diminution of their effector function. We hypothesized that we could overcome this therapeutic limitation and increase the functional activity and longevity of CAR T-cells by selectively deriving them from virus-specific effector memory T cells. We have developed biologics we termed synTacs (artificial immunological synapse for T-cell activation), which selectively activate and expand antigen-specific CD8+ T cells in vitro and in vivo by recapitulating signals delivered at the immunological synapse. The synTacs consist of dimeric Fc domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to virus-derived peptides. Treatment of PBMCs from CMV-exposed donors with synTacs presenting a CMV-derived peptide (pp65-NLVPMVATV) induce vigorous and selective ex vivo and in vivo expansion of highly functional CMV-specific CD8+ T cells, with potent antiviral activity. We used these synTacs to selectively generate CAR T-cells from CMV-specific effector memory CD8+ T cells, which could be further expanded by restimulation with the CMV-specific synTacs. Methods: We treated PBMCs from CMV-exposed donors in media supplemented with either IL-2 or IL-7/12/15 with a synTac containing the CMV-derived pp65 peptide presented by HLA-A2 MHC molecules linked to ligands capable of stimulating CD28- or 4-1BB-dependent costimulatory pathways. PBMCs activated either with anti-CD3/CD28 or the CMV-specific synTacs were transduced with lentivirus expressing an anti-CD19 CAR and a GFP reporter gene. CMV-specific CD8+ T cells were quantified by tetramer staining and CAR T-cells were detected by GFP expression determined by flow cytometric analysis. The functional activity of the CD19 CAR T-cells was determined by a B cell-specific cytotoxic assay. Results: After 7 days, treatment of PBMCs with CMV-specific synTacs rapidly induced robust activation and >50-fold expansion of CMV-specific CD8+ T cells expressing effector memory markers. Treatment of the PBMCs with CMV-specific synTacs selectively activated CMV-specific T cells and enabled them to be specifically transduced with a CD19-specific CAR lentivirus and converted into CD19 CAR T-cells. These CMV-specific CD19 CAR T-cells displayed potent dose-responsive cytotoxic activity targeting purified primary B cells. Furthermore, these CMV-specific CD19 CAR T-cells could be selectively expanded by in vitro treatment with CMV-specific synTacs. Conclusions: SynTacs are versatile immunotherapeutics capable of selective in vitro and in vivo activation and expansion of virus-specific CD8+ T cells with potent antiviral cytotoxic activity. After selective lentiviral transduction and conversion into CD19 CAR T-cells, their co-expression of the CMV-specific T cell receptor enabled them to be potently stimulated and activated by in vitro treatment with CMV synTacs. The modular design of synTacs facilitates efficient coupling of other costimulatory ligands - such as OX40 or GITRL - or cytokines, such as IL-2, IL-7, or IL-15, to enable the selective in vivo delivery of defined costimulatory signals or cytokines to the CAR T-cells expressing CMV-specific TCR. This strategy has the potential to boost the in vivo activity of tumor-specific CAR T-cells after infusion and enable more durable and potent treatment of refractory/recurrent B cell malignancies. Disclosures Almo: Cue Biopharma: Current equity holder in publicly-traded company, Patents & Royalties: Patent number: 62/013,715, Research Funding. Goldstein:Cue Biopharma: Research Funding.

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