Exhausted CLL T Cells Mediated By PD1 Expression an Important Mechanism for CD19 CAR Efficacy in CLL in the Adoptive Transfer TCL1 Mouse Model

Abstract Background: The first two autologous CD19 chimeric antigen receptor T (CAR T) cells targeting CD19 have now been approved for the treatment of ALL and refractory lymphomas. Despite impressive responses in these diseases, results remain inconsistent in chronic lymphocytic leukaemia (CLL). It is unknown if this reflects CAR design or an effect of the underlying function of CLL T cells. These 2nd generation CAR T cells require CD28 or 41BB co-stimulatory signalling domains, but these have not been compared directly in humans. Pre-clinical models afford the opportunity to do this, however, modelling of CAR T cells has mostly been performed in vitro or using immunodeficient mice, limiting the ability to study more complex immune biology. CLL is associated with a tumour supportive microenvironment and T cells exhibit multiple functional defects and features of exhaustion. These T cell defects in CLL are closely recapitulated in Eμ-TCL1 (TCL1) mice, and induced in healthy mice by adoptive transfer (AT) of murine CLL cells. We aimed to demonstrate the effect of CLL T cell dysfunction on CAR T cell efficacy and compare CD28 and 41BB directly. Methods: Immunocompetent C57BL/6 mice (WT) received AT of pooled 20 x106 TCL1 cells from fully leukemic TCL1 mice from the same background. Syngeneic donor CAR T cells were either pooled spleens from WT mice or WT mice given AT CLL with CLL load >80%. Both groups were aged matched (approx. 3 months). Splenoctyes were enriched for CD3+ with magnetic beads then activated with anti CD3/CD28 Dynabeads (Thermofisher) and rIL-2 (Roche). They were transduced with retroviral supernatant from either SFG-m19BBmZ-GFP (CD19-41BB) or MSGV-1D3-28Z-1.3mut (CD19-CD28) and cultured for 4 days when they were injected into 48 mice in total. Mice were given 100mg/kg intraperitoneal cyclophosphamide on D-1 followed by 6-8 x106 CAR T cells (or untransduced T cells). Mice were bled weekly to assess CLL load and T cell subsets and were culled when they appeared sick or peripheral blood (PB) CLL>70%. Results: CAR T cells derived from WT and AT T cells exhibit different phenotypes. WT CAR T cells proliferate more readily in culture and exhibit significantly higher transduction efficiencies in the CD8 subset although CD4 transduction is preserved. Following activation and transduction WT CAR T cells have a CD4: CD8 ratio of 1:1 whilst those from AT are heavily skewed to CD8. In both groups >90% T cells are CD44+. PD1+ expression in both CD4 and CD8 subsets is significantly higher in AT compared to WT CAR T cells. Mice treated with the CD19 -41BB CAR derived from WT and AT T cells or untransduced T cells did not respond, whereas 100% of mice treated with CD19-CD28 CAR derived from WT T cells had a complete response with loss of normal B cells 1 week post CAR T cells injection compared to 50% of mice treated with CD19-CD28 from AT T cells. All non-responding mice were culled at week 8 due to progressive leukaemia as were control mice treated with untransduced T cells. All mice with an established response had a continued complete response for 5 weeks following CAR T cell injection. Half of these mice were culled for phenotypic comparison and the other half observed for survival analysis. Those mice that responded and culled at week 8 had equal spleen size (0.1g) to age matched WT mice controls whilst non-responding mice had significantly larger spleens (0.5-3.3g). CAR T cells were only detectable in the PB +1 week post injection. In the PB there was restoration of CD4: CD8 ratios in responding mice compared to leukemic mice. PD1 expression in the spleen and bone marrow in CD3+CD8+ and CD4+ T cells normalised in responding mice compared to non-responding mice. Conclusion: AT of TCL1 CLL into immunocompetent mice is a viable model to study in vivo CAR T cell function and the host immune response. CAR T cells derived from WT T cells lead to a complete response in all of the mice but this response is significantly reduced if T cells exposed to CLL are used. Time to relapse for these responding mice has not been reached. We postulate that failure of the CD19 -41BB CAR in vivo relates to rejection of the GFP construct. There are significant differences in PD1 expression between WT and AT derived CAR T cells, which suggest strategies to repair exhausted T cells may improve the clinical response to CAR T cells in CLL. This provides the rationale for our on going studies of PD1/PDL1 blocking drugs in combination with CAR T cells in this immunocompetent pre-clinical model. Disclosures Gribben: Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Acerta Pharma: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria; Pharmacyclics: Honoraria; NIH: Research Funding; Kite: Honoraria; TG Therapeutics: Honoraria; Wellcome Trust: Research Funding; Cancer Research UK: Research Funding; Unum: Equity Ownership; Roche: Honoraria; Abbvie: Honoraria.

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Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Blood ◽

10.1182/blood-2021-149642 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2776-2776

Author(s):

Salvatore Fiorenza ◽

George S. Laszlo ◽

Tinh-Doan Phi ◽

Margaret C. Lunn ◽

Delaney R. Kirchmeier ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Set Domain ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Privately Held

Abstract Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

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Early Clinical Experience of CD19 x CD22 Dual Specific CAR T Cells for Enhanced Anti-Leukemic Targeting of Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-113126 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 278-278 ◽

Cited By ~ 25

Author(s):

Rebecca Gardner ◽

Colleen Annesley ◽

Olivia Finney ◽

Corinne Summers ◽

Adam J. Lamble ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dose Level ◽

T Cell Subsets ◽

Preclinical Testing ◽

Car T Cells ◽

Car T Cell ◽

Dual Specificity ◽

Car T

Abstract Introduction: Advances in chimeric antigen receptor (CAR) T cell therapy have yielded complete remission (CR) rates in relapsed/refractory B-ALL (rrB-ALL) of 70-95%. However, disease recurrence after CD19 or CD22 CAR therapy is greater than 50% at 1 year, and approximately half of recurrences are due to antigen escape. To reduce antigen escape and optimize the durability of remission, we sought to design a CAR T cell product with dual specificity that is capable of simultaneously targeting both CD19 and CD22. Preclinical testing of our bi-specific CAR showed a preference for signaling through CD22 over the CD19 CAR. In contrast, dual transduced T cells signaled through both the CD19 and CD22 CAR with lytic activity and cytokine production similar to single transduced CAR T cells of the same specificity. Therefore, we opted to move forward with dual transduced T cells for clinical use. We are currently testing SCRI-CAR19x22v1 in PLAT-05 (NCT03330691), a phase 1 clinical trial for pediatric and young adult patients with CD19+CD22+ rrB-ALL, with the primary objectives to determine the feasibility of manufacturing products with dual specificity, to assess the safety of the cryopreserved product infusion, and to describe the full toxicity profile. Methods: Subjects undergo apheresis, after which the CD4 and CD8 T cell subsets are immunomagnetically selected and seeded at a prescribed ratio for co-culture in a closed-system G-Rex bioreactor. Following anti-CD3xCD28 bead stimulation, T cells are transduced with two separate SIN lentiviral vectors that direct the expression of a CD19-specific FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR with an Her2tG tag and expression of a CD22-specific m971scFv:IgG4hinge:CH2(L235D)-CH3:CD28tm:4-1BB:ζ CAR with an EGFRt tag, creating three distinct populations of CAR T cells (anti-CD19, anti-CD22, and anti-CD19x 22). Transduced cells are expanded in serum free media formulation with IL-7, IL-15, and IL-21. Following lymphodepleting chemotherapy, cryopreserved products are thawed and infused at the protocol-prescribed dose level. Cytokine release syndrome (CRS) is graded according to Lee et al. (Blood 2014) and is treated according to our early intervention strategy of tocilizumab and dexamethasone for persistent, mild CRS. Results: Seven subjects (ages 1-26 yr) with rrB-ALL have been enrolled with 4 treated at dose level 1 (1 x 106 CAR T cells/kg) and 3 treated at dose level 2 (3 x 106 CAR T cells/kg). The mean culture time was 7.9 days (range 7-11) and subjects received infusions with a mean CD8:CD4 ratio of 1.7 (range 0.2 - 3.1). CD8 CAR composition, on average, consisted of 21.6 % CD19 CAR, 37.8 % CD22 CAR, and 40.6 % CD22xCD19 CAR T cells. CD4 CAR composition, on average, consisted of 25.8 % CD19 CAR, 30.6 % CD22 CAR, and 43.6 % CD22xCD19 CAR T cells (Figure). Peak engraftment occurred between days 7 and 14 for all patients and was predominantly composed of the CD19 CAR population with median peak values for CD19 CAR, CD22 CAR, and CD19xCD22 CAR T cell populations of 9.1%, 1.2%, and 2.4%, respectively. A CR was achieved in 5/7 (71%) subjects by day 21, 4 of which were minimal residual disease negative. The two subjects without a CR did not exhibit evidence of CAR T cell engraftment; one had previously received CD19 CAR T cells, and the other had progressive disease and pursued alternative therapy at day 10. Therapy was well tolerated with no dose limiting toxicities. CRS occurred in 5 subjects (Grade 1) with 2 of these subjects experiencing mild neurotoxicity (Grade 1). Four subjects received tocilizumab +/- dexamethasone, and two of these received multiple doses of dexamethasone. Conclusions: Preclinical testing showed superior efficacy against both CD19 and CD22 when using two separate CARs and dual transduction, compared to a single bi-specific CAR. Preliminary analysis of PLAT-05 supports feasibility of product manufacturing, and toxicity and response rates that are consistent with the reported CD19 CAR T cell experience. While the infused SCRI-CAR19x22v1 products consist of a near-uniform distribution of the 3 distinct populations, we observed selective in vivo expansion of the CD19 CAR T cell population. Further investigation is required to understand the mechanism of CD19 CAR dominance in vivo. Continued accrual of subjects is ongoing to further assess the impact of dual antigen targeting on the prevention of antigen escape and the potential to provide a more durable remission. Figure. Figure. Disclosures Park: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

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Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors

Blood ◽

10.1182/blood-2021-146995 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1727-1727

Author(s):

Sherly Mardiana ◽

Olga Shestova ◽

Stephan A. Grupp ◽

Marco Ruella ◽

David M. Barrett ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Tumor Cells ◽

Therapeutic Efficacy ◽

Research Funding ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19 or CD22 in leukemia and lymphoma, BCMA in multiple myeloma, and EGFRvIII in glioblastoma. This observation forms the basis of current efforts to develop multi-targeting CAR T cells to prevent antigen-negative escape. Antigen density is an important factor modulating CAR T cell response, since antigen expression below a certain threshold fails to trigger the full range of T cell functions. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two dimly-expressed antigens will result in enhanced CAR T cell signaling and anti-tumor function, approaching that seen in response to one highly-expressed antigen. This is important given the heterogeneity of antigen expression in various cancers. Therefore, the bi-specific CAR T cells currently being developed to prevent antigen-negative escape could also be used to enhance efficacy against low antigen density (LAD) tumors. Results from this study will provide a novel rationale for using multi-specific CAR T cells and illuminate the mechanisms of successful CAR T cell therapy. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 or CD22, or both in cis, herein referred to as CAR19, CAR22, or CAR19/22, respectively (Figure 1A). For in vitro functional characterization, we performed co-culture assay of T cells and B cell leukemia cell line NALM6, which is known to express high levels of both CD19 and CD22. To assess T cell function against LAD tumor cells, primary patients' B-ALL samples expressing low antigen density in comparison to the NALM6 cell line were used (Figure 1B). CAR T cell anti-tumor potency was determined by assessing CAR T cell cytotoxicity and cytokine production. For in vivo therapeutic study, primary patients' B-ALL samples with dimly expressed CD19 and CD22 were used to evaluate and compare the therapeutic efficacy of mono- versus bi-specific CAR T cells. Additionally, we generated a LAD tumor model by deleting the highly expressed CD19 and CD22 from the ALL cell line NALM6 using CRISPR/Cas9, transducing the now antigen-negative cell line with CD19 and CD22, followed by single cell cloning to generate a cell line expressing low antigen density for both the CD19 and CD22. We engrafted tumor cells in NSG mice, followed by administration of CAR19, CAR22, CAR19/22 or untransduced T cells. Therapeutic efficacy was assessed by measuring tumor burden using either flow cytometry or bioluminescent imaging. RESULTS Cytotoxicity assay revealed that the bi-specific CAR19/22 T cells killed tumor cells more rapidly than CAR19 or CAR22 T cells. Further, compared to mono-specific CAR T cells, the bi-specific CAR19/22 T cells produced significantly more pro-inflammatory cytokines including IL-2 and IFNg, in response to stimulation with LAD primary samples or NALM6 cells. This increased cytokine-producing capacity compared to mono-specific CAR T cells was maintained following repeated antigen stimulation when in vitro exhaustion assay was performed. In vivo, enhanced tumor elimination was observed in mice receiving bi-specific CAR19/22 T cells compared to either of the mono-specific CAR T cells, in both low antigen density primary ALL and NALM6 tumor models. This translated to increased survival rates seen in mice treated with the bi-specific CAR19/22 T cells (Figure 1C-D). CONCLUSIONS Here we showed that bi-specific CAR19/22 T cells are superior to mono-specific CAR19 or CAR22 T cells, not only against LAD tumors but also tumor cells expressing high antigen density, NALM6. This was demonstrated by their enhanced cytokine-producing function, cytotoxic capacity, and therapeutic efficacy in vivo. Results from this study provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing antigen-negative escape. Figure 1 Figure 1. Disclosures Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Ruella: viTToria biotherapeutics: Research Funding; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

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Clonal Kinetics and Single Cell Transcriptional Profiling of Adoptively Transferred CD19 CAR-T Cells

Blood ◽

10.1182/blood-2018-99-111350 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 702-702

Author(s):

Alyssa Sheih ◽

Laila-Aicha Hanafi ◽

Valentin Voillet ◽

Hannah A. DeBerg ◽

Reed M. Hawkins ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Adoptive Transfer ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Cell Clones ◽

Car T

Abstract INTRODUCTION: When introduced into polyclonal T cells, chimeric antigen receptors (CAR) redirect specificity of the engineered T cells to an antigen recognized by the CAR. We conducted a phase I/II clinical trial of treatment of relapsed and refractory CD19-positive B cell malignancies using a defined formulation of CD4+ and CD8+ CD19-specific CAR-T cells (NCT01865617). Little is known about the transcriptional heterogeneity of CAR-T cells in the infused product and their clonal kinetics after adoptive transfer. METHODS: To understand the factors that impact clonal CAR-T cell behavior in vivo, we performed TCRBV sequencing and single cell transcriptional profiling (10X Genomics) on CD8+ CAR-T cells isolated from infused products and the blood of treated patients. TCRBV sequencing was performed on 0.8 to 1.5 million cells from the infused product and 700-65,000 CAR-T cells from blood after CAR-T infusion. For single-cell RNA sequencing (scRNAseq), we obtained paired 5' gene expression and V(D)J data from individual CAR-T cells isolated from infused products and from the blood at the peak of in vivo expansion, after contraction, and at a late time point. RESULTS: High-throughput sequencing of the TCRBV genes revealed that CAR-T cells were polyclonal in the infused products, and during in vivo expansion and contraction, and at late times (≥ 3 months) after adoptive transfer. We evaluated the diversity of the TCRBV repertoire using the Shannon entropy index, and found that clonal diversity was highest in the infused product and declined at later time points after adoptive transfer. Loss of diversity after adoptive transfer was due to both expansion of higher frequency CAR-T cell clones and loss of low-frequency clones. We identified distinct CAR-T cell clones in the infused product and in blood at multiple time points after infusion that exhibited different kinetics of expansion and contraction. To examine the transcriptional programs that regulate the fate of CAR-T cells after infusion, we performed scRNAseq on CD8+ CAR-T cells, and found transcriptional heterogeneity in the infused products, which declined in CD8+ CAR-T cells isolated from patient blood after adoptive transfer. Gene set enrichment analysis showed that the infused products expressed higher levels of genes associated with hypoxia, glycolysis, and proliferation, and lower levels of genes associated with cytotoxicity compared to CAR-T cells isolated after adoptive transfer. In the infused product, genes associated with cytotoxicity were expressed at higher levels in CAR-T cells harboring clonotypes that were subsequently represented at relatively higher levels in vivo after adoptive transfer. CONCLUSIONS: There is transcriptional heterogeneity in the infused product and distinct CAR-T cell clones exhibit different kinetics of expansion and contraction after infusion. A better understanding of the kinetics of clonal expansion of CAR-T cells after adoptive transfer may provide insight into strategies to improve CAR-T cell immunotherapy. Disclosures Turtle: Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Membership on an entity's Board of Directors or advisory committees; Juno/Celgene: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Ibrutinib before Apheresis May Improve Tisagenlecleucel Manufacturing in Relapsed/Refractory Adult Diffuse Large B-Cell Lymphoma: Initial Results from a Phase 1b Study

Blood ◽

10.1182/blood-2020-134270 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 3-4

Author(s):

Julio C. Chavez ◽

Frederick L. Locke ◽

Ellen Napier ◽

Carl Simon ◽

Andrew Lewandowski ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Biomedical Research ◽

Research Funding ◽

B Cell Lymphoma ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background: Tisagenlecleucel (tisa-cel), an autologous anti-CD19 chimeric antigen receptor (CAR)-T cell therapy, has demonstrated durable responses and a manageable safety profile in adult patients (pts) with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). It has previously been suggested that prior therapy with ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, may improve tisa-cel manufacturing, in vivo cellular kinetics, and antitumor efficacy (Fraietta et al. Blood. 2016). Moreover, since BTK signaling is involved in direct pro-inflammatory polarization of macrophages, as well as indirectly by T cells, it is hypothesized that ibrutinib may mitigate CAR-T cell-related toxicities such as cytokine release syndrome (CRS) and neurological events (NE). We report the initial results from a Phase Ib, multicenter, open-label trial evaluating the safety and tolerability of tisa-cel in combination with ibrutinib in adult pts with r/r DLBCL. Methods: Adult pts with r/r DLBCL who received >2 prior lines of systemic therapy, including pts who progressed after or were ineligible for autologous stem cell transplant, were enrolled. The study design has 2 nonrandomized arms. In Arm 1, pts received ibrutinib 560 mg/d for ~4 weeks prior to leukapheresis; in Arm 2, pts were exposed to ibrutinib after leukapheresis. In both arms, ibrutinib was continued throughout lymphodepleting chemotherapy, tisa-cel infusion, and post infusion for up to 24 months. Lymphodepleting chemotherapy, ending at least 2 days before tisa-cel infusion, was either fludarabine (25 mg/m2) and cyclophosphamide (250 mg/m2) daily for 3 days or bendamustine (90 mg/m2) daily for 2 days. Pts received a single infusion of tisa-cel (target dose: 0.6-6.0×108 viable CAR+ T cells). Primary endpoints are incidence and severity of adverse events and ibrutinib dose interruptions/modifications. Secondary endpoints include best overall response (BOR) by Lugano criteria and cellular kinetics of tisa-cel. Results: As of June 9, 2020, 10 pts have been treated and observed through at least the Day 28 assessment: 4 in Arm 1 and 6 in Arm 2. Median age was 59 (range, 32-67) in Arm 1 and 64 (range, 58-76) in Arm 2. Median number of prior therapies was 3.5 (range, 2-5) in Arm 1 and 2 (range, 2-3) in Arm 2. Three of 10 pts (Arm 1, n=1; Arm 2, n=2) had an activated B-cell-like subtype of DLBCL. Six of 10 pts (Arm 1, n=1; Arm 2, n=5) had grade 1 CRS (by Lee scale) and 1 pt had NE (Arm 2, grade 1 by ASTCT criteria; Table). One pt in Arm 2 had grade 3 neutropenia lasting >28 days post tisa-cel infusion. No other pts had grade 3 or 4 neutropenia or thrombocytopenia lasting >28 days. No major bleeding events were observed. Ibrutinib-related bradycardia and atrial fibrillation (both grade 2) were each observed in 1 pt in Arm 1; supraventricular tachycardia (grade 1) related to tisa-cel was observed in 1 pt in Arm 2. No pt required tocilizumab or ICU admission. As of data cutoff, BOR in Arm 1 was complete response (CR) in 2 pts and partial response (PR) in 2 pts, with no relapses. BOR in Arm 2 was CR in 2 pts, PR in 1 pt, and progressive disease in 3 pts (Table). CAR-T cell expansion in vivo by qPCR was in line with data from the pivotal JULIET trial, except for 1 pt in Arm 2 whose transgene levels were below the limit of quantification at all points in time and who progressed at Day 28. Median viability of the leukapheresis material was 96.80% (range, 88.8-97.3) in Arm 1 and 90.95% (range, 88.1-94.7) in Arm 2. A naïve/stem cell-like central memory phenotype (CD45RA+/CCR7+) was observed in 24.05% (median; range, 15.9-37.0) of CD8+ T cells in the leukapheresis material for Arm 1 and in 8.12% (median; range, 1.3-20.4) for Arm 2 (Fig.1A). Fig.1B shows total CAR+ manufactured cells in each arm. The median dose of the final product was 3.9×108 CAR+ T cells in Arm 1 (range, 3.4-4.6×108 CAR+ T cells; median viability 92.25%) and 1.7×108 CAR+ T cells in Arm 2 (range, 1.2-3.0×108 CAR+ T cells; median viability 85.8%; Fig.1C). IFNγ secretion of tisa-cel in vitro in response to CD19+ target cells was similar between the 2 arms, whereas median normalized IL-2 responses were 23.1 fg/CAR+ cell in Arm 1 (range, 16.7-43.8) and 1.1 fg/CAR+ cell in Arm 2 (range, 0-17.3). Conclusions: These results support the feasibility of administering ibrutinib to pts with DLBCL throughout tisa-cel therapy. When given before apheresis, ibrutinib may improve CAR-T cell manufacturing, although further studies are needed to confirm this finding. Disclosures Chavez: AstraZeneca: Speakers Bureau; Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding; Bayer: Consultancy; BeiGene: Speakers Bureau; Karyopharm: Consultancy; Genentech: Speakers Bureau; AbbVie: Consultancy; Epizyme: Speakers Bureau; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Speakers Bureau; Verastem: Consultancy; Pfizer: Consultancy. Locke:Kite, a Gilead Company: Consultancy, Research Funding; Calibr: Consultancy; Celgene/Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; GammaDelta Therapeutics: Consultancy; Cellular Biomedicine Group: Other: Consultancy with grant options; Allogene: Consultancy; Wugen: Consultancy. Simon:Novartis: Current Employment. Lewandowski:Novartis Institutes for BioMedical Research: Current Employment. Awasthi:Novartis Institutes for BioMedical Research: Current Employment. Engels:Novartis Institutes for BioMedical Research: Current Employment. Georgala:Novartis Pharmaceuticals Corporation: Current Employment. Bondanza:Novartis Institutes for BioMedical Research: Current Employment. Schuster:AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria; Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding.

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Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after CD19 CAR-T Cell Immunotherapy

Blood ◽

10.1182/blood.v130.suppl_1.805.805 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 805-805

Author(s):

Cameron J. Turtle ◽

Kevin A. Hay ◽

Laila-Aicha Hanafi ◽

Juliane Gust ◽

W. Conrad Liles ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Endothelial Activation ◽

Advisory Board ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

High Concentrations

Abstract CD19 chimeric antigen receptor (CAR)-modified T cell therapy has produced impressive results in patients (pts) with CD19+B cell malignancies; however, treatment can be complicated by neurologic adverse events (AEs). The presentation and pathogenesis of neurotoxicity (NT) are incompletely understood. We report a clinical and pathologic study evaluating NT in 133 B-ALL, NHL and CLL pts treated with lymphodepletion chemotherapy and CD19 CAR-T cells (NCT01865617). Neurologic AEs were observed in 53 of 133 pts (40%; 19% grade [gr] 1-2; 16% gr 3; 2% gr 4; 3% gr 5). Most neurologic AEs were reversible. Delirium, headache, and language disturbances were the most frequently observed neurologic AEs, presenting in 66%, 55%, and 34% of pts with NT, respectively. Multivariable analysis showed that higher burden of malignant B cells in marrow, lymphodepletion with cyclophosphamide and fludarabine, and higher infused CAR-T cell dose were associated with increased risk of NT and cytokine release syndrome (CRS). The severity of NT correlated with higher peak concentrations of cytokines that may activate endothelial cells (EC), such as IL-6, IFN-γ, and TNF-α. Pts who developed gr ≥3 NT had more severe CRS with higher CAR-T cell counts in blood and evidence of vascular dysfunction. In severe NT, hypofibrinogenemia with elevated PT, aPTT, and d-dimer were consistent with EC activation. We confirmed in vivo EC activation during NT by demonstrating high concentrations of serum angiopoietin-2 (Ang-2) and von Willebrand Factor (VWF), which are released from Weibel-Palade bodies on EC activation. In vitro, serum from pts with NT induced activation and VWF release from human umbilical vein ECs (HUVECs), which suggests that the finding of a reduced fraction of high molecular weight VWF multimers in vivo during gr ≥4 NT results from consumption on platelets and sequestration on activated EC. Consistent with this explanation, we found reduced activity of the VWF-cleaving protease ADAMTS13 relative to VWF levels and more severe thrombocytopenia in pts with gr ≥4 NT. We considered that cytokine-induced EC activation might alter the integrity of the blood-brain barrier (BBB) during NT. During severe NT, CSF analyses demonstrated increased permeability of the BBB to protein and leukocytes, including CAR-T cells. The gradient between serum and CSF cytokines observed before lymphodepletion was lost during acute NT, consistent with inability of the BBB to shield the CNS from high concentrations of plasma cytokines. Brain vascular pericytes (BVP) play a critical role in vascular and BBB support, and when exposed to high TNF-α or IFN-γ concentrations BVP exhibited stress (increased cleaved caspase-3 and reduced PDGFRβ) and secreted IL-6 and VEGF, further promoting EC activation and BBB permeability. Neuropathologic examination of the brain of a patient with fatal NT demonstrated disrupted endothelium by CD31 immunohistochemistry and EC activation was confirmed by intravascular VWF binding and CD61+ platelet microthrombi. Further evidence of breach of the BBB included red blood cell extravasation from multiple vessels, vascular lesions with karyorrhexis, perivascular CD8+ T cell infiltration, and fibrinoid vessel wall necrosis. CAR-T cells were detected in the CNS. We investigated strategies to reduce the risks of severe NT. Logistic probability curves demonstrated that reduction in CAR-T cell dose to reduce the peak in vivo CAR-T cell blood count was associated with reduced risk of NT, but that the narrow therapeutic index of this approach would lead to loss of anti-tumor efficacy. To maintain CAR-T cell peak counts in blood, we developed a strategy to identify pts early after CAR-T cell infusion who might be at risk of subsequent severe NT and could be candidates for early intervention. Classification tree modeling demonstrated that in the first 36 hours after CAR-T cell infusion pts with fever ≥38.9°C, serum IL-6 ≥16 pg/mL, and MCP-1 ≥1343.5 pg/mL were at high risk of subsequent gr ≥4 NT (sensitivity 100%; specificity 94%). We also investigated whether pts with pre-existing endothelial activation were at higher risk of NT. Before lymphodepletion, pts who developed gr ≥4 NT had higher Ang-2:Ang-1 ratios than those with gr ≤3 NT, indicating that endothelial activation before lymphodepletion or CAR-T cell infusion may be a risk factor for NT that identifies pts who would benefit from a modified treatment regimen. Disclosures Turtle: Adaptive Biotechnologies: Other: Advisory board; Bluebird Bio: Other: Advisory board; Gilead Sciences: Other: Advisory board; Precision Biosciences: Other: Advisory board; Celgene: Other: Advisory board; Juno Therapeutics: Other: Advisory board, Patents & Royalties, Research Funding. Liles: Juno Therapeutics: Consultancy, Other: Advisory board. Li: Juno Therapeutics: Employment, Equity Ownership. Yeung: Gilead: Research Funding. Riddell: Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney: Celgene: Other: Advisory board; Kite Pharmaceuticals: Other: Advisory board; Juno Theraapeutics: Other: Advisory board, Patents & Royalties, Research Funding; Roche/Genetech: Other: Advisory board.

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Optimal Dual-Targeted CAR Construct Simultaneously Targeting Bcma and GPRC5D Prevents Bcma-Escape Driven Relapse in Multiple Myeloma

Blood ◽

10.1182/blood-2019-126145 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 136-136 ◽

Cited By ~ 8

Author(s):

Carlos Fernandez de Larrea ◽

Mette Staehr ◽

Andrea Lopez ◽

Yunxin Chen ◽

Terence J Purdon ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Car T Cells ◽

Car T Cell ◽

Dual Targeting ◽

Car T ◽

Previously Treated

Multiple myeloma (MM) remains generally incurable, calling for the development of novel treatment strategies such as chimeric antigen receptor (CAR) T cell therapy. Most clinically tested CAR T cell therapies for MM target B cell maturation antigen (BCMA), but despite high response rates, many patients relapse (Raje N. NEJM 2019). BCMA negative-low MM cells are implicated as a reservoir preceding relapse (Brudno J. JCO 2018; Cohen A. JCI 2019). Our aims are to (1) evaluate whether upfront simultaneous targeting of an additional antigen such as G protein-coupled receptor class C group 5 member D (GPRC5D; Smith EL. Sci Trans Med 2019) can mitigate BCMA escape-mediated relapse in MM, and (2) compare dual targeting strategies to identify an optimal approach. Dual targeting for CD19/CD22 malignancies has been investigated, and multiple approaches are feasible; however, approaches have yet to be comprehensively compared head to head. Here, we compare 2 parallel production and 3 single-vector dual targeting strategies (Fig. 1A). To enhance clinical translatability, all strategies are built on the BCMA(125)/4-1BBζ CAR (BCMA scFv 125; Smith EL. Mol Ther 2018), which is currently under multi-center clinical investigation (NCT03430011; Mailankody S. ASH 2018). We confirmed that all dual targeted approaches lyse, proliferate, and secrete polyfunctional cytokines specifically in response to BCMA and GPRC5D mono- and dual-positive cell lines and/or primary patient MM aspirate samples. Activity in vivo was confirmed using the bone marrow-tropic OPM2 MM model (endogenously BCMA+GPRC5D+). In all experiments MM cells (2 x 106) were injected IV into NSG mice and engrafted/expanded for 14 days before treatment. A high dose of all dual targeted CAR T cell approaches (3 x 106 CAR+) induced long-term disease control (median overall survival (mOS) BCMA(125) non-signaling del control 32d vs other groups mOS not reached; p < 0.05). Prevention of latent BCMA escape-mediated relapse was evaluated by re-challenge of previously treated long-surviving mice with 2 x 106 OPM2 BCMA CRISPR KO (OPM2BCMA KO) cells at day 100 without re-treatment. While mice previously treated with BCMA(125)/41BBζ CAR T cells succumbed to OPM2BCMA KO disease, dual targeted approaches prevented OPM2BCMA KO growth (mOS BCMA mono-targeted arm 37d post re-challenge vs other groups mOS not reached; p < 0.05). To better recapitulate human MM and distinguish among dual targeting approaches, we modeled established BCMA heterogeneous disease by spiking 5-10% OPM2BCMA KO into bulk OPM2WT cells for injection. Each OPM2 population was modified to express distinct luciferases for simultaneous in vivo monitoring by bioluminescent imaging (BLI). Treatment with a moderate (5 x 105) dose of CAR T cells eradicated OPM2WT cells in all groups, but anti-GPRC5D CARs with CD28 co-stimulation, whether included within a mixed T cell population or in a bicistronic construct (Fig. 1A ii, iv), failed to control OPM2BCMA KO cells (Fig. 1B). Correspondingly, 4-1BB-only containing CAR T cells had increased in vivo expansion (2.1-4.1-fold increase CAR T cell BLI at day 7 over CD28 containing groups; p < 0.05). As this result is likely from greater activation-induced cell death in the CD28-containing approaches that was not rescued by 4-1BB, we later compared 4-1BB-only containing approaches (Fig. 1A i, iii, v). These 3 dual targeting approaches effectively controlled OPM2WT disease at moderate (1 x 106 CAR+) and low (2.5 x 105 CAR+) doses. However, when using a sub-therapeutic dose (2.5 x 105 CAR+) in the OPM2BCMA KO-spiked model, the tandem scFv-single stalk design was least effective in controlling OPM2BCMA KO disease (Fig 1C). At a dose that is sub-therapeutic to control OPM2WT disease (1 x 105 CAR+), the bicistronic dual 4-1BB design (Fig. 1A iii) was more effective in eradicating tumor compared with the parallel production approach (6-fold difference tumor BLI at day 28; p < 0.05). These results indicate that upfront dual targeting of BCMA/GPRC5D with CAR T cells can mitigate BCMA escape-mediated relapse in a model of MM. While parallel infusion of separate BCMA- and GPRC5D-targeted CAR T cells is effective, a single bicistronic vector encoding two 4-1BB-containing CARs avoids the practical challenges of parallel manufacturing, and uniquely may provide superior anti-MM efficacy. Figure Disclosures Fernandez de Larrea: Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy.

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First Results from the Dose Escalation Segment of the Phase I Clinical Study Evaluating Cyad-02, an Optimized Non Gene-Edited Engineered NKG2D CAR T-Cell Product, in Relapsed or Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

Blood ◽

10.1182/blood-2020-139667 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36 ◽

Cited By ~ 1

Author(s):

Dries Deeren ◽

Johan A. Maertens ◽

Tara Lin ◽

Yves Beguin ◽

Benjamin Demoulin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase I ◽

Research Funding ◽

Next Generation ◽

Cell Product ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background T-cells engineered to express a chimeric antigen receptor (CAR) based on the NKG2D receptor (NKG2D CAR) targeting the 8 NKG2D ligands (MICA/B, ULBP1-6) over-expressed by a large variety of malignancies have been developped to treat patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Previously, CYAD-01, the first generation of NKG2D CAR T-cell products, was evaluated in several Phase I clinical trials and showed initial signals of objective clinical responses in patients with r/r AML and MDS, albeit with short durability. Preclinical data have shown that NKG2D ligands MICA and MICB are transiently upregulated on activated CAR T-cells, and target-dependent killing of CAR T-cells post-infusion can potentially occur, leading to short in vivo persistence. In an effort to increase the persistence and potency of the NKG2D CAR T-cells, CYAD-02 was developed as a next-generation product using a non-gene editing approach to silence the expression of MICA and MICB. Aim MICA and MICB were down-regulated by inserting a single optimized short hairpin RNA (shRNA) targeting both MICA and MICB within the NKG2D CAR construct. This next-generation NKG2D CAR T-cell product is manufactured with the OptimAb process, resulting in CAR T-cells with a higher frequency of early memory T-cells secreting high levels of cytokines upon activation, and is referred to as CYAD-02. Results As compared to CYAD-01, CYAD-02 cell expansion in vitro was 3-fold increased. In an in vivo AML model, CYAD-02 showed 10-fold higher engraftment 1 week after injection and improved anti-tumor activity as compared to CYAD-01 manufactured with the initial mAb process. This led to a 2.6-fold increase of mouse survival as compared to CYAD-01 in a stress-test aggressive AML model where the dose of CYAD-01 was titrated down for minimal activity (figure). The first-in-human study evaluating CYAD-02, the CYCLE-1 study (NCT04167696), has been initiated in early 2020 in patients with r/r AML/MDS. The study evaluates three dose-levels of CYAD-02 (1x108, 3x108 and 1x109 cells/infusion), administered as a single infusion after non-myeloablative preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days, CyFlu) according to a classical Fibonacci 3+3 design. As of August 2020, 6 patients have been treated with CYAD-02 at the dose of 1x108 or 3x108 cells/infusion. To date, the results demonstrate the safety and tolerability for CYAD-02 in patients with r/r AML and MDS with no dose-limiting toxicity observed. The study is currently enrolling at 1x109 cells/infusion. The CYAD-02 safety profile and preliminary clinical activity data together with the pharmacokinetics evaluation from the complete dose escalation segment will be provided at the time of presentation. Conclusion/summary The CYAD-02 is the first autologous CAR T-cell product based on the non-gene edited shRNA technology used to treat patients. This next generation NKG2D CAR T-cell product is currently investigated in the CYCLE-1 Phase I study in r/r AML/MDS patient population, a difficult to target disease due in part to the absence of truly AML-specific surface antigens, its rapid clinical progression and the absence of disease control by the CyFlu preconditioning. Both the anti-MICA and MICB shRNA hairpin and the OptimAb manufacturing process for CYAD-02 aim to improve CAR T-cell persistence and clinical responses. Figure Disclosures Lin: Mateon Therapeutics: Research Funding; Aptevo: Research Funding; Abbvie: Research Funding; Ono Pharmaceutical: Research Funding; Incyte: Research Funding; Gilead Sciences: Research Funding; Jazz: Research Funding; Astellas Pharma: Research Funding; Bio-Path Holdings: Research Funding; Celgene: Research Funding; Celyad: Research Funding; Genetech-Roche: Research Funding; Seattle Genetics: Research Funding; Tolero Pharmaceuticals: Research Funding; Trovagene: Research Funding; Prescient Therapeutics: Research Funding; Pfizer: Research Funding. Demoulin:Celyad Oncology: Current Employment. Fontaine:Celyad Oncology: Current Employment. Sotiropoulou:Celyad Oncology: Current Employment. Alcantar-Orozco:Celyad Oncology: Current Employment. Breman:Celyad Oncology: Current Employment. Dheur:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

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Anti-CD19 Chimeric Antigen Receptor-Modified T Cell Therapy for B Cell Non-Hodgkin Lymphoma and Chronic Lymphocytic Leukemia: Fludarabine and Cyclophosphamide Lymphodepletion Improves In Vivo Expansion and Persistence of CAR-T Cells and Clinical Outcomes

Blood ◽

10.1182/blood.v126.23.184.184 ◽

2015 ◽

Vol 126 (23) ◽

pp. 184-184 ◽

Cited By ~ 29

Author(s):

Cameron J Turtle ◽

Carolina Berger ◽

Daniel Sommermeyer ◽

Laila-Aicha Hanafi ◽

Barbara Pender ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Research Funding ◽

T Cell Subsets ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Clinical Responses

Abstract BACKGROUND: Autologous T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) have demonstrated activity in patients with relapsed or refractory B cell NHL and CLL. The functional heterogeneity that is inherent in CAR-T cell products that are manufactured from undefined T cell subsets has hindered definition of dose-response relationships and identification of factors that may impact efficacy and toxicity, such as the lymphodepletion regimen and infused cell dose. We manufactured anti-CD19 CAR-T cells from a defined composition of CD4+ and CD8+ T cell subsets to treat adults with relapsed or refractory B cell NHL or CLL. T cell subsets were enriched from each patient, transduced with a CD19 CAR lentivirus and separately expanded in vitro before formulation for infusion in a 1:1 ratio of CD8+:CD4+ CAR+ T cells at one of three dose levels (2x105, 2x106 or 2x107 CAR-T cells/kg). CAR-T cells were administered 48-96 hours after lymphodepletion with either cyclophosphamide (Cy, 60 mg/kg)+/- etoposide or Cy (60 mg/kg) and fludarabine (25 mg/m2 daily for 3-5 days (Cy/Flu). RESULTS: Adult patients with relapsed/refractory CD19 expressing B cell NHL (n=28, median age 59 years, range 36-70) or CLL (n=6, median age 60 years, range 54-64) were treated with at least one CAR-T cell infusion. NHL histologies include diffuse large B cell or transformed NHL (DLBCL, n=18), follicular NHL (FL, n= 6) or mantle cell lymphoma (MCL, n=4). 15 patients had failed prior autologous (n=13) or allogeneic (n=3) transplants. Twelve of the 28 NHL patients received lymphodepletion with Cy-based regimens without fludarabine. Expansion of CAR-T cells and clinical responses were observed in 50% (CR=1 (DLBCL), PR=5 (2 FL, 2 DLBCL, 1 MCL), no response=6). Patients were treated at all three dose levels without dose limiting toxicity or severe cytokine release syndrome (sCRS). With this regimen, we observed short CAR-T cell persistence in most patients and demonstrated a CD8-mediated immune response to the murine scFv component of the CAR transgene that correlated with loss of CAR-T cells. Retreatment with CAR-T cells with or without chemotherapy in 5 patients led to no significant T cell expansion or clinical responses. To minimize transgene rejection fludarabine was added to the lymphodepletion regimen administered to the subsequent 16 NHL patients. Clinical responses were evaluated in 12 of 16 patients (2 not yet evaluable, 2 early deaths). Addition of Flu to the lymphodepletion regimen increased the CR rate to 42%, compared to 8% with Cy alone. Clinical responses were identified in 6 of 8 patients with DLBCL (3 CR, 3 PR) and 2 of 3 patients with FL (2 CR). The overall response rate was 67%. We noted higher peak CAR-T cell levels in blood in the Cy/Flu group (n=13) compared with the Cy only group (n=11) (CD8+ CAR-T cells, median 31.9 cells/ml vs 0.55 cells/ml, p = 0.009; CD4+ CAR-T cells, median 16.5 cells/ml vs 0.31 cells/ml, p= 0.007), and CAR-T cell persistence was longer in Flu-treated patients (see Figure 1 for patients treated at 2 x 107/kg). Surprisingly, 2 of 7 patients who received 2 x 107 CAR-T cells/kg experienced dose-limiting toxicity necessitating dose de-escalation. Markedly elevated IL-6 levels were observed within the first day after CAR-T cell infusion in patients who subsequently developed severe toxicity, which may provide an opportunity to test early interventional approaches to minimize toxicity. Six patients with relapsed and refractory CLL received CAR-T cells. Five of 6 restaged patients had complete clearance of blood and/or marrow disease by high-resolution flow cytometry 4 weeks following treatment. Overall clinical responses included 3 CR, 1 PR and 2 no response. One patient with a PR died from refractory pulmonary aspergillus infection. Patients with CR remain in remission at 1-10 months after therapy. CONCLUSION: Immunotherapy with CD19 CAR-T cells of defined subset composition is feasible in patients with NHL and CLL and has potent anti-tumor activity. Toxicity is related to cell dose. The addition of Flu to a Cy-based lymphodepletion regimen results in greater CAR-T cell expansion and persistence, and improves the CR rate after CD19 CAR-T cell therapy. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy. Maloney:Juno Therapeutics: Research Funding; Janssen Scientific Affairs: Honoraria; Seattle Genetics: Honoraria; Roche/Genentech: Honoraria.

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Fresh Versus Cryopreserved/Thawed Bispecific Anti-CD19/CD20 CAR-T Cells for Relapsed, Refractory Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2019-125328 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4465-4465 ◽

Cited By ~ 2

Author(s):

Nirav N. Shah ◽

Fenlu Zhu ◽

Dina Schneider ◽

Winfried Krueger ◽

Andrew Worden ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Viability ◽

Research Funding ◽

Phase 1 ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Introduction Chimeric Antigen Receptor modified T (CAR-T) cell therapies have revolutionized the relapsed, refractory B cell malignancy landscape. Due to the complex steps involved with cell production, some third-party companies require T cells to be cryopreserved prior to shipping, while most manufacturers deliver modified CAR-T cells to the treating center in a cryopreserved state. This is vastly different to the approach taken with traditional cell based therapies, specifically allogeneic transplant (allo-HCT), an immunological treatment that relies on a graft-versus-tumor (GVT) effect to prevent disease relapse. Historically, "fresh" stem cells were felt to be superior to cryopreserved products due to concerns that cryopreservation may damage T cells and other mononuclear cells delaying engraftment and limiting GVT reactivity. As a result, in clinical practice most allo-HCT products are still given as fresh infusions without cryopreservation. In a Phase 1 clinical trial evaluating the safety of a bispecific anti-CD19, anti-CD20 CAR (LV20.19CAR), CAR-T cells were produced in a point-of-care fashion utilizing the CliniMACS Prodigy device. Local manufacturing allowed flexibility to administer either fresh LV20.19CAR-T cells without cryopreservation, or if indicated, thawed CAR-T cells post-cryopreservation. Methods Patients (pts) were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of 41BB/CD3z LV20.19CAR-modified T cells for adults with relapsed, refractory B cell NHL including DLBCL, MCL, FL, and CLL. The starting dose was 2.5x10^5 cells/kg with a target dose of 2.5x10^6 cells/kg. All pts received low dose fludarabine (30 mg/m2) x 3 days +cyclophosphamide (500 mg/m2) x 1 day for lymphodepletion. In the Phase 1 dose-escalation cohorts, pts received fractionated CAR-T cells over two days (30% on Day 0 and 70% on Day+1), while expansion cohort pts received CAR-T cells as a single infusion. The goal for all pts was to infuse fresh CAR-T cell prior to cryopreservation, however, CAR-T cell could be cryopreserved and infused at a later date for clinical / logistical reasons. Results A total of 20 pts received LV20.19CAR T cell therapy (Table 1). Fourteen pts received fresh CAR-T cells immediately post-harvest, 5 pts received post-thaw CAR-T cells, and 1 patient received a mixed fresh/cryopreserved product and was not included in this analysis. Reasons for cryopreserved administration was delay due to active infection (N=3), patient preference (N=1), and unexplained neutropenia (N=1). Among 19 evaluable pts, the CR rate (79% vs 40%), mean ferritin, mean CRP, and incidence of CRS and neurotoxicity were all higher in the fresh infusion group (Table 1), but not statistically significant. In terms of LV20.19 CAR-T product characteristics, mean cell viability at infusion was 93% for the fresh infusion group versus 63% for cryopreserved pts (p<0.01). Point-of-care administration allowed final cell doses to be adjusted for diminished viability among pts receiving cryopreserved product. Figure 1 demonstrates the in-vivo expansion and persistence of LV20.19CART cells among fresh versus post-thaw pts. The peak percentage of CAR-T cells within the CD3 compartment was higher in pts given fresh cell infusions (Figure 2), but was not statistically significant (p=0.08). Conclusions Cryopreservation is known to diminish cell viability and increase clinical costs associated with freezing and storage. To date, there is limited clinical data evaluating outcomes of pts receiving fresh CAR-T cells compared to thawed CAR-T cells post-cryopreservation. Although it is presumed that in-vivo CAR-T cell activity is comparable in both scenarios, among our pts, both cell viability and in-vivo expansion favored pts who received a fresh infusion. Unlike third-party CAR-T cell products where viability is unknown at the time of infusion, we adjusted the final dose to accommodate decreased cell viability. CR rates and incidence of CRS and NTX were higher among fresh infused pts suggesting greater in-vivo activity, although findings were not statistically significant, partially a result of the small sample size. While our findings are limited by small numbers in each cohort and variability in cell dose and diagnosis, these data suggest that cryopreservation of CAR-T cells may impact clinical responses and is a logistical step that needs further investigation. Disclosures Shah: Cell Vault: Consultancy, Equity Ownership; Oncosec: Equity Ownership; Lentigen: Honoraria, Research Funding; Exelexis: Equity Ownership; Geron: Equity Ownership; Celgene: Other: Advisory Board; Incyte: Consultancy; Oncosec: Equity Ownership; Kite Pharma: Other: Advisory Board. Zhu:Miltenyi Biotec: Research Funding. Schneider:Lentigen Technology, A Miltenyi Biotec Company: Employment. Krueger:Lentigen Technology, A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hamadani:Sanofi Genzyme: Research Funding, Speakers Bureau; Otsuka: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Takeda: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Merck: Research Funding; Medimmune: Consultancy, Research Funding. Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hari:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; Cell Vault: Equity Ownership; AbbVie: Consultancy, Honoraria. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Equity Ownership.

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