Ibrutinib Treatment Reduces Myeloid Derived Suppressor Cell Numbers and Function in Chronic Lymphocytic Leukemia

Abstract In chronic lymphocytic leukemia (CLL), bidirectional interactions of leukemic B cells with components of a complex, yet incompletely defined tumor microenvironment (TME) are critical for leukemic cell survival and proliferation. Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, blocks signals that are crucial for survival of CLL cells which are delivered by the B cell receptor (BCR) and certain other receptors. However since BTK and its family members are expressed by other cell types, ibrutinib can also affect non-leukemic cells, thereby altering their function. Here, we focused on understanding how myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, and their main target, T cells, are affected by ibrutinib therapy. Using blood cells from a set of 20 previously untreated patients receiving ibrutinib, we analyzed circulating MDSCs and their subsets 15 days before and 1, 2 and 3 months after treatment initiation. As anticipated, at the first month time point the absolute CLL B-cell count increased significantly (P=0.024), followed by a progressive reduction at consecutive time points (P<0.001). In contrast, absolute MDSC numbers slowly and continuously decreased over time, achieving a significant reduction by the third month (P=0.009). When dividing MDSCs into their cellular subsets, the same pattern was observed for granulocyte-like MDSCs (gMDSCs) (P=0.005) but not for monocyte-like MDSCs (mMDSCs); for the latter an insignificant change was found. Of note, when comparing the changes of MDSC and gMDSC numbers to that of CLL B-cell counts, MDSC and gMDSC neared statistical significance at month one (P<0.1) and achieved significance at the second month (MDSCs: P<0.001; gMDSCs: P=0.033). We also observed differences in T-cell subpopulations shortly after ibrutinib treatment began. T cell counts increased significantly at the second month compared to pre-treatment (P=0.024); this was the case for both CD4+ and CD8+ cells (P = 0.022 and 0.010, respectively). In addition, CD8+ T cells maintained significance through the third month (P = 0.033). When exploring T cell subsets defined by cytokine production, we observed a spike at the second month of CD4+IL17F+ and of CD8+IL17F+, CD8+IL17A+ and CD8+FoxP3+ T cells. However, by the 3rd month, only the IFNγ-producing subset of CD4+ and CD8+ T cells were significantly higher (P = 0.049 and 0.042, respectively). Next we analyzed the function of gMDSCs and mMDSCs in the presence of ibrutinib in vitro, addressing the two main effects of these cells on T lymphocytes: suppression of T-cell proliferation and modulation of T-cell differentiation. Specifically, ibrutinib did not directly reduce T-cell expansion in the absence of MDSCs and did not alter the effect of CLL gMDSCs nor mMDSCs on T-cell proliferation, since the significant reduction induced by gMDSCs (P=0.047) and the insignificant, variable effect of mMDSCs were unchanged by the drug. However when we analyzed the influence of gMDSCs, mMDSCs and monocytes on naïve CD4+ T-cell differentiation in the presence or absence of ibrutinib, to our surprise the only T cell subpopulation directly compromised by ibrutinib was the Th1/IFNγ-producing subset. This was opposite that observed in co-cultures with gMDSCs, mMDSCs or normal monocytes in the presence of ibrutinib where Th1 cells expanded significantly (P= 0.021, 0.010, and 0.005, respectively). In the latter co-cultures not containing ibrutinib, more IL-4-producing (Th2) cells were found. Additionally, ibrutinib had a positive effect on IL-22+ (Th22) and FoxP3+ (Treg) cell numbers in the presence of MDSCs and monocytes. In summary, opposite to what we observed for CLL B and T cells, MDSC counts fell progressively after initiating ibrutinib therapy, possibly due to a direct effect of ibrutinib on BTK in MDSCs or an indirect effect mediated by diminishing signals from CLL B cells that would normally be delivered after BCR engagement. Ibrutinib did not directly alter the T cell suppressive ability of MDSCs, but it did skew T-cell differentiation to Th1 cells when MDSCs were present, in line with our finding higher Th1 cells in the blood after 3 months of treatment. Thus over time, ibrutinib shifts the CLL TME from an immunosuppressive to a more immune effective one. Disclosures Chen: Beigene: Research Funding; Verastem: Research Funding; Pharmacyclics: Research Funding. Barrientos:Pharmacyclics, an AbbVie Company: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy. Kolitz:Magellan Health: Consultancy, Honoraria. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

Download Full-text

CD19-Directed CAR T-Cell (CTL019) Product Viability and Clinical Outcomes in Non-Hodgkin Lymphomas and B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-197 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 197-197 ◽

Cited By ~ 4

Author(s):

Elise A. Chong ◽

Bruce L Levine ◽

Stephan A. Grupp ◽

Megan Davis ◽

Don L. Siegel ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Test Results ◽

Advisory Committees ◽

Car T Cell ◽

Release Test ◽

Car T

Abstract Introduction: CTL019 is an anti-CD19 genetically modified autologous T-cell immunotherapy developed at the University of Pennsylvania (Penn) that was recently approved for treatment of relapsed/refractory pediatric and young adult B-cell acute lymphoblastic leukemia (ALL) and adult relapsed/refractory diffuse large B-cell lymphoma (DLBCL) as tisagenlecleucel (Novartis). For ALL, the FDA-approved dose is 0.2 to 5.0 x 106 CAR-positive viable T cells per kg of body weight for patients ≤ 50 kg or 0.1 to 2.5 x 108 CAR-positive viable T cells for pts > 50 kg; for DLBCL, the FDA-approved dose is 0.6 to 6.0 x 108 CAR-positive viable T cells. For CTL019 manufactured at Penn, the dose is determined by flow cytometric staining of CAR-positive T cells, which are cryopreserved in product bags along with replicate aliquots of the final formulation in vials, simultaneously cryopreserved for release testing. The CTL019 product release criteria include a post thaw viability assessment using a vial of replicate aliquot of the final formulation for Trypan blue exclusion or dual fluorescence automated cell counting (Luna-FL, Logos Biosystems). There are no published data examining the relationship between CTL019 viability release testing and clinical outcomes. Methods: We analyzed CTL019 post thaw viability release testing in patients treated on one prospective single institution clinical trial of CD19-expressing non-Hodgkin lymphomas (NHL) (NCT02030834) and two single-institution prospective pediatric ALL clinical trials (NCT01626495 and NCT02906371). Patients were assessed for response to therapy and CAR T-cell expansion. Receiver operating characteristic (ROC) curves were constructed for prediction of complete responses based on sensitivity and specificity of CAR T-cell product post thaw viability release test results. Results: 39 pts with relapsed/refractory NHL (24 diffuse large B-cell lymphoma and 15 follicular lymphoma) were enrolled and received the protocol-specified dose of CTL019. Best response rate was 56% (22/39) complete responses (CR). 123 pts with relapsed/refractory pediatric ALL were enrolled and received the protocol-specified dose of CTL019. Best response rate was 96% (118/123) CR/complete remission with incomplete blood count recovery (CRi). For patients with NHL infused with CTL019, product % viability had a median of 89.8% viability (range: 73.7%-97.7%); product % viability quintiles were as follows: 20%-tile=81.7%, 40%-tile=88.3%, 60%-tile=91.1%, 80%-tile=94.8%). ROC area for NHL patients was 0.47 (95%CI: 0.28-0.65). For patients with ALL infused with CTL019, product % viability had a median of 89.3% viability (range: 56.0%-98.4%); product % viability quintiles were as follows: 20%-tile=82.3%, 40%-tile=87.5%, 60%-tile=90.9%, 80%-tile=94.4%). ROC area for ALL patients was 0.52 (95%CI: 0.32-0.71). For patients with NHL, progression-free survival (PFS) was not significantly influenced by product viability release test results by Cox proportional hazards (HR: 1.0, 95%CI: 0.94-1.09, p=0.7). For patients with NHL, peak CAR T-cell expansion was not significantly correlated with product viability release test results (r2=0.12, p=0.5). Data collection for Cox analysis to investigate the effect of release test viability on PFS and correlation of release test viability with peak CTL019 expansion in ALL is ongoing and will be presented. Conclusions: Our data suggest that, within the ranges obtained in these trials, there is no clear dose-response relationship between CTL019 product viability release test results and clinical response rates in pediatric and young adult ALL or DLBCL. Figure Figure. Disclosures Chong: Novartis: Consultancy. Levine:Cure Genetics: Consultancy; Brammer Bio: Consultancy; CRC Oncology: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Siegel:Novartis: Research Funding. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Frey:Novartis: Consultancy; Servier Consultancy: Consultancy. Porter:Genentech: Other: Spouse employment; Novartis: Other: Advisory board, Patents & Royalties, Research Funding; Kite Pharma: Other: Advisory board. June:Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Schuster:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Efficacy and Toxicity of JCAR014 in Combination with Durvalumab for the Treatment of Patients with Relapsed/Refractory Aggressive B-Cell Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2018-99-116745 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1680-1680 ◽

Cited By ~ 8

Author(s):

Alexandre V. Hirayama ◽

Jordan Gauthier ◽

Kevin A. Hay ◽

Alyssa Sheih ◽

Sindhu Cherian ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Group 2

Abstract Introduction Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) have shown high overall response rates (ORR) in otherwise treatment-refractory CD19+ B-cell non-Hodgkin lymphoma (NHL); however, not all patients (pts) achieve complete remission (CR). PD-L1 expression on tumor cells and/or other tissues could impair the function of PD-1+ CAR-T cells and the efficacy of CD19 CAR-T cell immunotherapy. PD-1 pathway blockade may enhance the function and antitumor activity of CD19 CAR-T cells. Here we report preliminary data from a phase 1 dose-finding study (NCT02706405) of the safety and feasibility of combination therapy with JCAR014 CD19-specific 4-1BB-costimulated CAR-T cells and escalating doses of durvalumab, an anti-PD-L1 monoclonal antibody, in adults with relapsed/refractory aggressive B-cell NHL. Methods Pts are treated in one of two groups. All pts receive lymphodepletion chemotherapy with cyclophosphamide and fludarabine followed by infusion of JCAR014. Pts in group 1 receive the first infusion of durvalumab (225 mg, 750 mg, or 1500 mg) 21-28 days after treatment with JCAR014. Pts in group 2 receive the first dose of durvalumab (7.5 mg, 22.5 mg, 75 mg, 225 mg, 750 mg, or 1500 mg) 1 day prior to JCAR014 infusion. Up to 10 doses of durvalumab are administered after JCAR014 at the highest identified safe dose at 4-week intervals until toxicity or disease progression. We evaluated the safety, tolerability, and efficacy of the combination therapy and the pharmacokinetic profile of JCAR014 after infusion. Adverse events were graded using the Common Terminology Criteria for Adverse Events (CTCAE) v4.03, with the exception of cytokine release syndrome (CRS), which was graded according to consensus criteria (Lee, Blood 2014). Positron emission tomography/computed tomography was performed approximately 1, 2, 4, 6, 9, and 12 months after JCAR014 infusion and the best anti-tumor response was reported according to the Lugano criteria (Cheson, JCO 2014). Results Patient characteristics are shown in Table 1. Fifteen pts have been treated, including 6 in group 1 who received post-JCAR014 durvalumab doses of 225 mg (n = 3) and 750 mg (n = 3), and 9 in group 2 who received pre-JCAR014 durvalumab doses of 7.5 mg (n = 1), 22.5 mg (n = 1), 75 mg (n = 3), or 225 mg (n = 4). Durvalumab dose escalation is ongoing. JCAR014 manufacturing was successful for all pts. All pts received 2 x 106 JCAR014 CAR-T cells/kg, except the first 2 pts treated on the study who received 7 x 105 CAR-T cells/kg. Of the 13 pts who received JCAR014 at 2 x 106 CAR-T cells/kg, 5 pts (38%) developed CRS (2 grade 1, 2 grade 2, and 1 grade 4) and one (8%) developed grade 1 neurotoxicity. CRS and/or neurotoxicity occurred within 4 weeks of JCAR014 infusion, and were not observed when durvalumab was administered after JCAR014. With the exception of B cell aplasia, no autoimmune adverse events were observed. Twelve of 13 pts who received 2 x 106 CAR-T cells/kg were evaluable for response. One patient, who had grade 4 CRS and biopsy evidence of extensive CAR-T cell infiltration into persistent sites of disease, elected to receive hospice care and died on day 32 after JCAR014 infusion without full response evaluation. The overall response rate was 50% (5 CR, 42%; 1 PR, 8%). Of the 5 pts who achieved CR, 3 were in CR at the first restaging after JCAR014 and 2 subsequently converted to CR after the first post-JCAR014 durvalumab infusion. Only one patient who achieved CR has relapsed (median follow-up 10.6 months, range 3.7-11.8). Continued stable disease or evidence of regression was seen in 4 of 6 (67%) initially non-responding pts who continued durvalumab therapy (median 5 doses, range 1-6). CAR-T cell counts expanded in the peripheral blood within 14 days of JCAR014 infusion in all pts. Higher peak and day 28 CAR-T cell copy numbers in blood by qPCR were observed in responding pts. CAR-T cells were detected for a median of 5.1 months (range, 1.7 to 9.1 months) in responding pts. In vivo re-accumulation of CAR-T cells after the first post-JCAR014 durvalumab dose was observed in the blood of two patients in group 2. Conclusion The combination of JCAR014 with durvalumab for the treatment of adult pts with aggressive B-cell NHL appears safe; however, dose escalation is ongoing. Complete responses were observed both at initial restaging after JCAR014 infusion, and also subsequently in pts continuing durvalumab therapy after initially failing to achieve CR. Disclosures Hirayama: DAVA Oncology: Honoraria. Hay:DAVA Oncology: Honoraria. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy. Shadman:Verastem: Consultancy; Beigene: Research Funding; Mustang Biopharma: Research Funding; Gilead Sciences: Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy; Genentech: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy; Genentech: Consultancy; AstraZeneca: Consultancy; Acerta Pharma: Research Funding. Cassaday:Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Pfizer: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Kite Pharma: Research Funding; Incyte: Research Funding. Acharya:Juno Therapeutics: Research Funding; Teva: Honoraria. Riddell:Cell Medica: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; NOHLA: Consultancy. Maloney:Roche/Genentech: Honoraria; Juno Therapeutics: Research Funding; Janssen Scientific Affairs: Honoraria; GlaxoSmithKline: Research Funding; Seattle Genetics: Honoraria. Turtle:Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Bluebird Bio: Consultancy; Gilead: Consultancy; Nektar Therapeutics: Consultancy, Research Funding; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Caribou Biosciences: Consultancy; Aptevo: Consultancy.

Download Full-text

Adoptive T-Cell Therapy with TCL1-Specific TCR for B-Cell Lymphomas

Blood ◽

10.1182/blood-2018-99-115424 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3488-3488

Author(s):

Jinsheng Weng ◽

Kelsey Moriarty ◽

Yong Pan ◽

Man Chun John MA ◽

Rohit Mathur ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Malignancies ◽

Advisory Committees ◽

T Cell Therapy

Abstract Chimeric antigen receptor (CAR)-modified T-cell therapy targeting CD19 induces high response rates in patients with relapsed or refractory B-cell lymphomas. However, about 60% of patients experience primary or secondary resistance after CD19-targeted CAR T-cell therapy and a major of cause of failure appears to be due to loss of CD19 expression on the tumor. Therefore, novel targets for adoptive T-cell therapeutic approaches are needed to further improve clinical outcome in these patients. T-cell leukemia/lymphoma antigen1 (TCL1) is an oncoprotein that is overexpressed in multiple B-cell malignancies including follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). Importantly, it has restricted expression in only a subset of B cells among normal tissues. We previously identified a TCL1-derived HLA-A2-binding epitope (TCL170-79 SLLPIMWQLY) that can be used to generate TCL1-specific CD8+ T cells from peripheral blood mononuclear cells of both HLA-A2+ normal donors and lymphoma patients. More importantly, we showed that the TCL1-specific CD8+ T cells lysed autologous primary lymphoma cells but not normal B cells (Weng et al. Blood 2012). To translate the above discovery into clinic, we cloned the T-cell receptor (TCR) alpha and beta chains from a TCL1-specific CD8+ T-cell clone and showed that this TCL1-TCR could be transduced into polyclonal donor T cells using a lentiviral system with a transduction efficiency of >40% as determined by TCL170-79 tetramer positive T cells. Furthermore, we demonstrated that the TCL1-TCR-transduced T cells recognized T2 cells pulsed with TCL170-79 peptide producing IFN- γ >8 ng/ml and IL-2 >350 ng/ml but were not reactive to control HIV-Gag peptide (IFN- γ <0.1 ng/ml and IL-2 <0.2 ng/ml). The TCL1-TCR-transduced T cells recognized TCL170-79 peptide pulsed onto T2 cells at a concentration of 1-10 nM (IL-2 >10 ng/ml) suggesting it has moderate to high avidity. Importantly, TCL1-TCR-transduced T cells lysed HLA-A2+ (up to 43% lysis of Mino and 25% lysis of Jeko-1 at 40:1 Effector:Target ratio) but not HLA-A2- lymphoma cell lines (5.5% lysis of HLA A2- Raji and 2.3% lysis of Daudi at 40:1 Effector:Target ratio). TCL1-TCR-transduced T cells were also cytotoxic to HLA-A2+ primary lymphoma tumor cells (up to 48% lysis of CLL, 43% lysis of FL, 41% lysis of DLBCL, 46% lysis of splenic marginal zone lymphoma, and 11% lysis of MCL at 40:1 Effector:Target ratio) but not normal B cells derived from the same patients. Lastly, TCL1-TCR transduced T cells showed high efficacy in in vivo models. Adoptive transfer of the TCL1-TCR-tranduced T cells significantly reduced lymphoma tumor growth and extended survival in Mino mantle cell lymphoma cell line xenograft model (48% survival in TCL1-TCR-T treated group vs. 12.5% survival in control group at 10 weeks n=7-8 mice/group; P=0.02). Collectively, our data suggest that the high expression in B-cell tumors, restricted expression in normal tissues, and presence of an immunogenic CD8 T-cell epitope, make TCL1 a target for T cell-based therapeutic approaches in multiple B-cell malignancies. Our results also demonstrate that the TCL1-specific TCR-transduced T cells may serve as a novel adoptive immunotherapy approach for the treatment of patients with various B-cell malignancies (including FL, MCL, DLBCL, CLL). Acknowledgments: This study is supported by MD Anderson Moon Shot Program and CPRIT and the National Natural Science Foundation of China Grant (No. 81570189) Disclosures Neelapu: Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Poseida: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Karus: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153110 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 75-75

Author(s):

Jooeun Bae ◽

Shuichi Kitayama ◽

Laurence Daheron ◽

Zach Herbert ◽

Nikhil C. Munshi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

T Cell Differentiation ◽

Immune Checkpoints ◽

Publicly Traded ◽

Advisory Committees ◽

Induced Pluripotent

Abstract T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. Figure 1 Figure 1. Disclosures Munshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Preliminary Clinical Results of a Phase 1 Study Evaluating the Safety and Anti-Tumor Activity of ACTR707 in Combination with Rituximab in Subjects with Relapsed or Refractory CD20+ B-Cell Lymphoma

Blood ◽

10.1182/blood-2018-99-111622 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2966-2966 ◽

Cited By ~ 1

Author(s):

Ian W. Flinn ◽

Jonathon B. Cohen ◽

Luke P. Akard ◽

Samantha Jaglowski ◽

Michael Vasconcelles ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Dose Level ◽

Dose Escalation ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Advisory Committees

Abstract Background: Recent regulatory approvals of two CD19-targeted chimeric antigen receptor (CAR)-expressing autologous T lymphocyte therapies provide compelling evidence of the clinical potential of re-engineering T cells to specifically attack tumor cells, but the broader applicability of these therapies is constrained by safety considerations and target specificity. A universal approach to T cell therapy that enables flexibility in tumor target selection has been demonstrated by engineering autologous T cells to express an antibody-coupled T cell receptor (ACTR) composed of the ectodomain of the CD16 Fc receptor fused to costimulatory and CD3ζ signaling domains. Thus, the ACTR platform couples T cell anti-tumor effector functions, including cytotoxicity, cytokine production, and T cell proliferation, to target-specific therapeutic antibodies. Here we present the preliminary clinical findings of the ongoing, multicenter Phase 1 study, ATTCK-20-03 (NCT03189836), of ACTR707, a CD28-containing ACTR chimeric receptor, in combination with rituximab in subjects with relapsed or refractory CD20+ B cell lymphoma. Methods: The primary objectives of this first-in-human, dose escalation study are to evaluate the safety of the combination of ACTR707 and rituximab and to determine a maximum tolerated dose (MTD) and a proposed recommended phase 2 dose (RP2D). Other objectives include evaluation of antitumor activity, and assessment of ACTR T cell persistence, cytokine levels, and rituximab pharmacokinetics. Eligible subjects must have histologically confirmed relapsed or refractory CD20+ non-Hodgkin lymphoma and have received prior anti-CD20 mAb in combination with chemotherapy. Subjects received lymphodepleting chemotherapy (cyclophosphamide 400 mg/m2 and fludarabine 30 mg/m2) for 3 days, followed by rituximab (375 mg/m2) and ACTR707. Additional doses of rituximab were administered, one dose every 3 weeks in the absence of disease progression. The study is separated into 2 sequential phases, a dose escalation and a safety expansion phase. During the dose escalation phase, ACTR707 is being tested at increasing doses in combination with rituximab. Results: Six subjects were enrolled and received ACTR707 at the first dose level in combination with rituximab: 5 diagnosed with diffuse large B cell lymphoma (83%) and one with follicular lymphoma, Grade 3b (17%). Median age was 61 years (range: 57-76), 83% were male, 50% were treated with ≥3 lines of prior therapy, and 67% had no response to or relapse within 6 months from immediate prior therapy. ACTR707 was successfully manufactured for all subjects and demonstrated post-infusion expansion in the peripheral blood. ACTR+ T cells were detectable at Day 28 post-infusion for all subjects tested. No dose-limiting toxicities (DLTs) were observed at the first dose level in 4 DLT-evaluable subjects (2 subjects experienced disease progression during the DLT evaluation period). There were no cytokine release syndrome (CRS) or autoimmune adverse events (AEs), serious or severe (≥Gr3) neurotoxicity AEs, or deaths on treatment. AEs (all grades) reported in >1 subject included neutropenia (n=3), anemia, decreased appetite, febrile neutropenia, and thrombocytopenia (each in 2 subjects); the 2 events of febrile neutropenia were considered serious. Investigator-reported complete responses were observed in 3 of 6 subjects. These complete responses (duration of response range: 47+ to 81+ days) are ongoing as of the data cut-off. Enrollment into the second dose level is ongoing. Conclusions: ACTR707 in combination with rituximab induced complete responses in 3 of 6 subjects with relapsed or refractory aggressive CD20+ B cell lymphoma treated at the first dose level with ACTR707 in combination with rituximab, with no CRS, serious or severe (≥Gr3) neurotoxicity, or AEs leading to treatment discontinuation. ACTR+ T cells were detectable in all subjects and persisted. These results support the continued dose escalation of ACTR707 in combination with rituximab. Updated data, inclusive of preliminary dose level 2 and correlative biomarkers, will be presented. Disclosures Flinn: Verastem: Consultancy, Research Funding; Janssen: Research Funding; Pfizer: Research Funding; Kite: Research Funding; Forty Seven: Research Funding; BeiGene: Research Funding; ArQule: Research Funding; Takeda: Research Funding; TG Therapeutics: Research Funding; Incyte: Research Funding; Forma: Research Funding; Verastem: Research Funding; Novartis: Research Funding; Agios: Research Funding; Seattle Genetics: Research Funding; Trillium: Research Funding; Merck: Research Funding; Calithera: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Infinity: Research Funding; Portola: Research Funding; Pharmacyclics: Research Funding; Curis: Research Funding; Celgene: Research Funding. Cohen:BioInvent: Consultancy; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Research Funding; Infinity Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding. Akard:Gilead: Speakers Bureau; Celgene: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Jaglowski:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Consultancy. Vasconcelles:Unum Therapeutics: Employment. Ranger:Unum Therapeutics: Employment. Harris:Unum Therapeutics: Employment. Payumo:Unum Therapeutics: Employment. Motz:Unum Therapeutics: Employment. Bachanova:Gamida Cell: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Research Funding.

Download Full-text

Axl-RTK Inhibition Modulates T Cell Functions and Synergizes with Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies

Blood ◽

10.1182/blood-2018-99-112209 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 728-728 ◽

Cited By ~ 1

Author(s):

Reona Sakemura ◽

Nan Yang ◽

Michelle J. Cox ◽

Sutapa Sinha ◽

Mehrdad Hefazi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Phenotype ◽

Inhibitory Receptors ◽

B Cell Malignancies ◽

Advisory Committees ◽

Cell Functions

Abstract Despite the remarkable outcomes and recent FDA approval of CD19 directed chimeric antigen receptor T (CART19) cell therapy in B cell malignancies, the durable responses in diffuse large B cell lymphoma are less than 40% and CART activity in chronic lymphocytic leukemia (CLL) is further limited. This is thought to be related to loss of CART persistence, poor trafficking to lymph nodes and inhibition by the leukemic microenvironment. Therefore, strategies to enhance CART cell function to overcome these limitations are needed. Recent studies have shown that abnormal expression of the receptor tyrosine kinase (RTK) AXL is associated with poor prognosis in human cancers. AXL signaling is associated with tumor proliferation, survival, metastasis, and drug resistance. Inhibition of AXL RTK with TP-0903, a high affinity AXL inhibitor has been found to induce robust apoptosis of CLL B cells. Based on the significant modulation of T cell functions observed with BTK inhibitor, we examined the role of AXL RTK inhibition with TP-0903 on T cell function in CLL and other B cell malignancies. First, we investigated the effect of AXL inhibition on T cell phenotype in normal donors. When naïve T cells were stimulated with PMA/Ionomycin and cultured with low dose TP0903, cytokine production was favorably altered through the promotion of Th1 and reduction of Th2 cytokines. This was associated with a significant reduction of inhibitory receptors (Fig 1a). Western blot of T cell lysates suggests low dose TP-0903 results in inhibition of LCK. When effector T cells and regulatory T cells (Treg) were treated with TP-0903 for 3 days, there was a preferential reduction of Treg (Fig 1b). Next, we investigated the influence of TP-0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. Similar to our findings on naïve T cells, TP-0903 treatment led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP-0903 treatment also significantly downregulated inhibitory receptors on activated CART cells, including a reduction of canonical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1c). The combination of CART19 cells and TP-0903 yielded a synergistic antitumor activity against JeKo in vitro, at low E:T ratios (Fig 1d). Western blot of T cell lysates revealed phosphorylation of LCK was remarkably reduced in the presence of TP-0903, suggesting a mechanism for the observed Th1 polarization. We compared the transcriptome of activated CART cells treated with TP-0903 and more than 100 genes were differentially expressed compared to non-treated cells. Among these genes, immune synapse related genes such as cell junction and cell migration related genes were significantly increased in activated CART cells treated with TP-0903. To investigate the effect of AXL RTK inhibition of CART cells with TP-0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of JeKo into NSG mice. A week after the injection of JeKo, mice were treated with either vehicle alone, TP-0903 (20mg/kg/day) alone, 0.5x106 of CART19 alone, or TP-0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of JeKo. Mice treated with CART19 and TP-0903 rejected the JeKo tumor challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1e). Finally, we validated our preclinical findings in a correlative analyses of Phase I clinical trial of TP-0903 for patients with solid tumors (NCT02729298). Blood T cells from 3 patients were isolated and analyzed before and a week after treatment with TP-0903. Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned Phase I clinical trial of TP-0903 in relapsed/refractory CLL (NCT03572634). In summary, we demonstrated for the first time that AXL inhibitior is capable of polarizing T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes with CART cells in B cell malignancies. These findings encourage further study of TP-0903 as an enhancer of T cell immunotherapies. Disclosures Mouritsen: Tolero Pharmaceuticals: Employment. Foulks:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria. Ding:Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Download Full-text

CLL B Cells Develop Resistance to Ibrutinib By Reinvigorating the IL-4R - IL-4 Axis Blocked By Bruton's Tyrosine Kinase Inhibitors Including Acalabrutinib and Zanubrutinib

Blood ◽

10.1182/blood-2019-127255 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 477-477

Author(s):

Shih-Shih Chen ◽

Constantine S. Tam ◽

Alan G. Ramsay ◽

Priyadarshini Ravichandran ◽

Natalia C. Couto-Francisco ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Cell Subset ◽

Advisory Committees

Bruton's tyrosine kinases inhibitors (BTKis) represent major advances in CLL therapy. However resistance to this form of therapy is emerging, and such patients often progress more rapidly. Hence there is an important need for therapies that address resistance. Microenvironmental input like IL-4 is critical for CLL disease progression. Compared with normal B cells, CLL cells exhibit significantly higher levels of surface membrane (sm) IL-4 receptor (IL4-R) and contain increased amounts of pSTAT6, a downstream mediator of IL-4R signaling. IL-4 stimulation of CLL B cells suppresses smCXCR4 and increases smIgM, thus promotes CLL cell retention and expansion. In this study, we aimed to examine if smIL-4R expression, IL4R signaling, and IL-4-producing cells are altered in patients sensitive or resistant to BTKis. To do so, T and B cell subset changes were studied overtime in 12 acalabrutinib-treated CLL patients, 6 zanubrutinib-treated CLL patients, 30 ibrutinib-sensitive and 5 ibrutinib-resistant CLL patients, 4 of which exhibited BTK mutations. Consistent with only ibrutinib inhibiting T-cell kinase (ITK), T-cell subset analyses revealed no changes in Th1, Th2, Th17, Th9, and Th22 cells after zanubrutinib or acalabrutinib treatment. In contrast, a Th1-biased T-cell immunity was observed in patients responsive to ibrutinib. In patients progressing on ibrutinib, significantly reduced Th2 T cells were found during the resistant as well as sensitive periods. In an in vitro T-cell function assay using T cells collected before and after the treatment with each BTKi, only ibrutinib treated patients exhibited a reduced ability of T cells to support CLL B cell survival. We next studied changes in CLL B cells, including numbers of IL-4, -10 and -13 producing B cells after BTKi treatment. IL-13 producing CLL B cells were not changed. IL-10 producing CLL B cells were reduced in both ibrutinib sensitive and resistant patients, but not in zanubrutinib or acalabrutinib treated patients. Importantly, IL-4 producing CLL B cells were significantly decreased in patients treated with all 3 BTKi. Significantly reduced smIL-4R levels, impaired IL-4R signaling, decreased smIgM and increased smCXCR4 were also seen in patients treated with each BTKi. To understand the mechanism responsible for inhibition of IL-4 production in CLL cells treated with BTKis, we stimulated CLL cells through IgM, Toll-like receptor and CD40L, finding that only anti-IgM stimulation significantly increased IL-4 production and p-STAT6 induction. We then explored the function of IL-4. IL-4 enhanced CLL B cell survival in vitro and this action was blocked by all 3 BTKis. Moreover, adhesion of CLL B cells to smIL-4R expressing stromal cells was decreased by IL-4 and IL-4R neutralizing antibodies, especially in M-CLL cases. In in vivo studies transferring autologous T cells and CLL PBMCs into alymphoid mice, we found less CLL B cells in mouse spleens post ibrutinib than zanubrutinib or acalabrutinib treatment. This might be due to the suppressed Th2 cells found only in ibrutinib, while IL-4 producing B cells were reduced in all 3 BTKi treated mice. These results support the idea that IL-4 promotes CLL B cell adhesion and growth in tissues. Finally, we investigated the IL-4/IL-4R axis in ibrutinib-resistant patients. Although IL-4 producing T cells remain reduced during the sensitive and resistant phases, CLL B cell production of IL-4 and expression of and signaling through smIL-4R returned when patients developed ibrutinib-resistance. When comparing paired ibrutinib-sensitive and -resistant CLL B cells collected from 3 patients in a xenograft model that requires T cell help, we found ibrutinib-resistant CLL B cells grew in vivo with only minimal (~15%) numbers of autologous T cells compared to B cells collected from ibrutinib-sensitive phase; this suggested a reduced requirement for T-cell help for growth of ibrutinib-resistant CLL cells. In summary, we found IL-4 is a key survival factor in the CLL microenvironment that also improves leukemia cell adhesion to stromal cells expressing smIL-4R. IL-4 production and signaling can be stimulated in CLL B cells through the B-cell receptor, and are consistently blocked by BTKis. Moreover, the recovered ability of ibrutinib-resistant CLL B cells to produce and respond to IL-4 leads to disease progression, suggesting blocking the IL-4/IL-4R axis is a potential treatment for ibrutinib-resistant CLL patients. Disclosures Chen: Pharmacyclics: Research Funding; Beigene: Research Funding; Verastem: Research Funding; ArgenX: Research Funding. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Kolitz:Boeringer-Ingelheim: Research Funding; Roche: Research Funding; Astellas: Research Funding. Zhou:BeiGene: Employment. Barrientos:Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Rai:Pharmacyctics: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Targeting Lymphomas Using Non-Engineered, Multi-Antigen Specific T Cells

Blood ◽

10.1182/blood-2018-99-113140 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1685-1685 ◽

Cited By ~ 1

Author(s):

George Carrum ◽

Premal Lulla ◽

Ifigeneia Tzannou ◽

Ayumi Watanabe ◽

Manik Kuvalekar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Post Infusion ◽

Equity Ownership ◽

T Cell Lines ◽

Large B Cell

Abstract Immunotherapy is emerging as a potent therapy for a range of hematologic malignancies including lymphomas. Indeed adoptive transfer of T cells genetically engineered to express the CD19 chimeric antigen receptor (CAR) has now received FDA approval for the treatment of patients with refractory diffuse large B cell lymphomas (DLBCL). We have developed a non-engineered T cell-based therapy to treat patients with all types of lymphomas: Hodgkin's (HL) and non-Hodgkin's lymphoma (NHL). The approach uses single T cell lines that simultaneously target a range of tumor-associated antigens (TAAs) that are frequently expressed by these tumors, including PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin. We can consistently prepare these lines by culturing PBMCs in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail, and adding autologous DCs as APCs that are loaded with pepmixes (15mer peptides overlapping by 11 amino-acids) spanning all 5 target antigens. The use of whole antigen should remove the HLA restriction imposed by the use of transgenic TCRs specific for single peptides, while targeting multiple antigens simultaneously would reduce the risk of tumor immune evasion. We have generated 42 clinical-grade multiTAA-specific T cell lines, comprising CD3+ T cells (mean 98±1.1%) with a mixture of CD4+ (mean 48±4.3%) and CD8+ (mean 37±4%) T cells, which expressed central and effector memory markers (CD45RO+/CD62L+/CCR7+ -- mean 14±3%; CD45RO+/CD62L+/CCR7- -- 10±2.2%; CD45RO+/CD62L-/CCR7- -- 28.3±3.6%) (n=42). The expanded lines recognized the targeted antigens PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin (range 0-463, 0-496, 0-330, 0-379 and 0-304 spot forming units (SFU)/2x105 input cells, respectively in IFNg ELIspot, n=34). None of the lines reacted against non-malignant autologous recipient cells (3±3.8% specific lysis; E:T 20:1). We have treated 33 patients: 13 with HL, 17 with aggressive NHL (diffuse large B-cell, mantle cell, or T cell lymphomas) and 3 with indolent NHLs (FL and marginal zone lymphoma). Patients received 0.5-2x107 multiTAA-T cells/m2. Of 18 patients who were infused as adjuvant therapy all but 2 remain in remission (range 3-42 months post-infusion). Fifteen patients have received multiTAA-specific T cells to treat active disease, all of whom had failed a median of 4 lines of prior therapy. Of these, 5 had transient disease stabilization followed by disease progression, 4 have ongoing stable disease, 3-18 months post-multiTAA-specific T cells while the remaining 6 (3 with HL and 3 with DLBCL) have all had complete and durable responses ( 4 to 41 months), as assessed by PET imaging. These clinical responses correlated with the detection of tumor-reactive T cells in patient peripheral blood post-infusion directed against both targeted antigens as well as non-targeted TAAs including MAGEA2B and MAGE C1, indicating induction of antigen/epitope spreading. Notably, no patient, including the complete responders, had infusion-related systemic- or neuro-toxicity. Thus, infusion of autologous multiTAA-targeted T cells directed to PRAME, SSX2, MAGEA4, NY-ESO-1 and Survivin has been safe and provided durable clinical benefit to patients with lymphomas. Disclosures Brenner: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Cell Medica: Research Funding; Tessa Therapeutics: Research Funding; Viracyte: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Cytosen: Membership on an entity's Board of Directors or advisory committees. Rooney:Marker: Equity Ownership. Vera:Marker: Equity Ownership. Leen:Marker: Equity Ownership.

Download Full-text

Allogeneic Anti-CD19 CAR T Cells Manufactured from Healthy Donors Provide a Unique Cellular Product with Distinct Phenotypic Characteristics Compared to CAR T Cells Generated from Patients with Mature B Cell Malignancies

Blood ◽

10.1182/blood-2019-123018 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3228-3228 ◽

Cited By ~ 3

Author(s):

Charlotte Graham ◽

Agnieszka Jozwik ◽

Ruby Quartey-Papafio ◽

Nikolaos Ioannou ◽

Ana M Metelo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

B Cell Malignancies ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

Despite the success of autologous anti-CD19 CAR T cell therapy in B-Acute lymphoblastic leukaemia (B-ALL) and Diffuse Large B Cell Lymphoma (DLBCL), treatment failures occur. One contributing factor may be the intrinsic T cell fitness of the CAR T cell product that is influenced by the underlying malignancy and prior treatments. With the advent of gene editing, 'off the shelf' non-HLA matched healthy donor (HD) CAR T cells are under investigation for the treatment of patients (pts) in clinical trials. UCART19 (S68587) is a first-in-class allogeneic CAR T cell product expressing a second generation anti-CD19 CAR with TALEN®-mediated gene knockouts of T cell receptor alpha chain (TRAC) and CD52 to prevent graft versus host disease and to render them resistant to anti-CD52 antibody used for lymphodepletion. Preliminary clinical trial data on the use of UCART19 in B-ALL was previously reported at ASH (Benjamin et al, 2018). The phenotypic and functional characteristics of CAR T cell products manufactured from B-ALL, Chronic Lymphocytic Leukaemia (CLL) and DLBCL pts were compared to young adult healthy donor (HD) CAR T cell products. In addition, potential effects related to knocking out TRAC in HD TCR-CAR T cells were examined. Thawed PBMCs from B-ALL, CLL, DLBCL pts and HDs underwent T cell enrichment, activation with anti-CD3/CD28 beads and IL-2, followed by transduction with anti-CD19 4-1BB CD3ζ lentiviral CAR construct and expansion. HD TCR- CAR T cells were manufactured by electroporation of HD CAR T cells with mRNA coding for TRAC TALEN® and residual TCRαβ+cells were removed by magnetic bead selection. CAR expression levels, T cell subsets, and exhaustion markers were examined by flow cytometry. Expression of activation markers CD25 and CD69 was measured in response to co-culture with the CD19+cell line NALM-6. Cytotoxicity against NALM-6 and Raji was assessed and antigen-mediated proliferation measured over 14 days. HD CAR T cells (n=11) expanded significantly more during manufacture than CAR T cells derived from B-ALL (n=9), CLL (n=8) or DLBCL (n=8) pts. As expected, the electroporation step resulted in a transient decrease in viability which recovered over time in culture (n=10). Median CAR expression level was higher on CLL CAR T cell products compared to those from B-ALL pts and HDs, thought to be due to a higher CD4:CD8 ratio in some CLL products. As a consequence of TCR knockout, CD3 expression was lost on HD TCR- CAR T cells (n=10), apart from a small population of γδ CAR T cells. CLL and DLBCL CD8+CAR+cells expressed higher levels of PD1 than HD CD8+CAR+cells. DLBCL CD4+CAR+cells also expressed significantly higher levels of PD1 than HD or HD TCR-CD4+CAR+T cells. CAR+CD8+CD27+PD1- T cells have been previously described as a functionally important population that correlated with clinical outcome in pts who received CLL CAR T cells (Fraietta et al, 2018). We found HD (n=13) and HD TCR- (n=10) CAR T cells had significantly more CD8+CD27+PD1- CAR T cells compared to those derived from CLL (n=8) and DLBCL (n=6) pts, but similar levels to B-ALL pts (n=10). In the absence of CD19 antigen, DLBCL CAR+CD8+ T cells (n=6) had greater expression of CD25 and CD69. However, in response to stimulation with CD19+ NALM-6 cells, HD (n=12), HD TCR- (n=10) and B-ALL (n=10) CAR T cells had higher fold increase in CD69+ cells compared to DLBCL (n=6) CAR T cells. On paired analysis (n=6), no difference was seen in activation in response to CD19 antigen on HD compared to HD TCR- CAR T cells. All CAR T cell products demonstrated comparable cytotoxicity against NALM-6 and Raji cell lines in short term in vitro assays. However, long-term cytotoxicity will be evaluated in a murine model. We performed a detailed comparison of the phenotypic and functional characteristics of CAR T cells derived from pts with B-cell malignancies and HDs. DLBCL CAR T cells showed lower antigen specific activation but higher baseline activation which could lead to more differentiated exhausted T cells. CAR T cells derived from HDs show a higher proportion of the therapeutically relevant CAR+CD8+CD27+PD1- cells compared to patients with mature B cell malignancies (CLL and DLBCL), which is maintained after TRAC knockout. This suggests allogeneic CAR T cells, such as UCART19, may provide a more effective product for pts with T cell dysfunction. Disclosures Graham: Gillead: Other: Funding to attend educational meeting; Servier: Research Funding. Jozwik:Servier: Research Funding. Metelo:Pfizer: Research Funding; Allogene: Research Funding. Almena-Carrasco:Servier: Employment. Peranzoni:Servier: Employment. Ramsay:Celgene Corporation: Research Funding; Roche Glycart AG: Research Funding. Dupouy:Servier: Employment. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Patten:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. Benjamin:Amgen: Honoraria; Allogene: Research Funding; Gilead: Honoraria; Servier: Research Funding; Eusapharm: Consultancy; Pfizer: Research Funding; Takeda: Honoraria; Novartis: Honoraria.

Download Full-text

Expression of the Tigit/CD226/CD155 Receptors/Ligand System in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128308 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5454-5454 ◽

Cited By ~ 2

Author(s):

Francesca Arruga ◽

Giulia Guerra ◽

Denis Baev ◽

Catherine Hoofd ◽

Marta Coscia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

B Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees

Introduction: T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a surface receptor mainly expressed by CD8+, regulatory T lymphocytes and natural killer (NK) cells, but not by normal B cells. It performs as an inhibitory immune checkpoint, activated through binding of CD155. TIGIT competes with CD226 for CD155 binding, resulting in opposite outcomes: while CD226 enhances cytotoxicity of T lymphocytes and NK cells, TIGIT exerts immunosuppressive effects. Whether TIGIT engagement triggers an alternative signaling cascade, or whether it simply prevents CD226 activation, remains an open point. Tumor-infiltrating T lymphocytes generally express high levels of the molecule, together with the other checkpoint inhibitor PD-1. On this basis, antagonist antibodies targeting TIGIT are under evaluation to restore immunity and treat cancer patients, alone or in various combinations. Chronic lymphocytic leukemia (CLL), the most common adult leukemia, is characterized by a highly heterogeneous clinical outcome. Several molecular markers can help in stratifying patients, including the presence or absence of somatic mutations in B cell receptor, cytogenetic aberrations and single gene mutations. Interestingly, CLL cells express several T cell specific antigens, including CD5. A previous report indicates that, in CLL, TIGIT is expressed by circulating CD4+T cells, increasing during disease progression, while nothing is known about its expression on CLL cells. Aim:This work was undertaken with the aim of studying expression of the TIGIT/CD226/CD155 axis in CLL. Methods:We assembled a cohort of 101 primary CLL samples (40% females, mean age of 61). All patients were either untreated or had not received treatment in the 6 months prior to analysis. PBMC samples were tested for expression of TIGIT, CD155 and CD226 in both T and B subsets. A multiparametric flow cytometry strategy was designed, combining anti-TIGIT, anti-CD155 and anti-CD226 antibodies with a panel of B- (anti-CD19, anti-CD5, anti-CD38, anti-CD49d and anti-CD73) and T-mono/NK specific (anti-CD3, anti-CD8, anti-CD4, anti-CD14 and anti-CD56) markers. The number of TIGIT molecules on leukemic cells was estimated by interpolating values of mean fluorescence intensity (MFI) of each sample with that of PE-Quantibrite beads. Results:CLL cells heterogeneously express surface TIGIT, ranging from 0.2 to 81% (mean value 20%, median 10%, SEM ±2.145). The estimated number of molecules per cell was in the range of 32.5-3571 (mean 1140, median 841.1, SEM ±83.6). Expression of TIGIT was independent of gender or age at diagnosis and there was no correlation between TIGIT levels and lymphocyte counts in peripheral blood. In contrast, in this cohort of untreated patients, we observed a significantly lower TIGIT expression in samples with advanced disease (RAI III-IV) compared to early stages (RAI 0-I). Accordingly, low TIGIT associated with unmutated (UM) IGHVgenes and with an unfavorable FISH profile (trisomy 12, deletion 17 and deletion 11 vs. deletion 13 or normal karyotype). Lower, although not significant, TIGIT levels were observed in NOTCH1-mutated CLL samples (n=11) compared to counterpart (n=89). Looking at the T cell population, we observed overall higher TIGIT levels in the CD8+vs CD4+subset (mean %TIGIT+cells in CD8+56.7±1.8 vs 27.2±1.3 in CD4+). In line with reported observations, we found a modest but significant increase of TIGIT+T cells in advanced stage CLLs, at variance with what observed on the leukemic B cell side. Accordingly, we observed higher percentages of TIGIT+/CD4+cells in CLL samples carrying UM IGHVgenes. CD226 and CD155 were more homogeneously expressed in all subsets without significant differences, both in CLL and T cell components. Conclusions: This work shows that CLL cells express the immunomodulatory molecule TIGIT, particularly in the early stages of the disease in untreated patients. While further studies are needed to characterize its functional implications as well as treatment effect on TIGIT expression, it is tempting to speculate that TIGIT expression by CLL cells may serve to trigger an immunosuppressive behavior in these cells, which is no longer needed when the disease becomes advanced. This observation represents a starting point for future studies investigating the role of TIGIT in CLL and hints to a possible use of anti-TIGIT antibodies to target different cellular components of the disease. Disclosures Hoofd: iTeos Therapeutics: Employment. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Research Funding. Gaidano:Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Furman:Acerta Pharma: Consultancy; Beigene: Consultancy; Incyte: Consultancy; Janssen: Consultancy; Oncotracker: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Deaglio:VelosBio Inc.: Research Funding; Verastem Inc: Research Funding; iTeos Therapeutics: Research Funding.

Download Full-text