Engineering of CD19-CAR T Cells from Non-Hodgkin Lymphoma Patients in a Closed System in Combination with Retronectin/OKT3 Stimulation

Abstract Background; Adoptive immunotherapy with chimeric antigen receptor (CAR) gene transduced CD19-CAR T cells, which are engineered to express extracellular single-chain immunoglobulin variable fragments to CD19, linked to cytoplasmic T cell activation domains including CD3-ζ, showed remarkable therapeutic benefits toward CD19+ B cell malignancies including acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma (B-NHL). For clinical setting, the phenotype of manufactured CAR T cells is an important factor; especially less differentiated T cells are anticipated to provide a long-lasting immune reconstitution. Furthermore, in order to avoid the risk of technical error and contamination during T cell manufacturing process, a closed system needs to be established. In this study, we addressed these issues and have established a novel CD19-CAR T cell manufacturing method from a small amount of blood in a closed system for clinical trial to treat patients with B-NHL. Methods; Peripheral blood was obtained from B-NHL patient volunteers and healthy donor volunteers who gave their written informed consents. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of blood using Ficoll-Paque PREMIUM density gradient centrifugation. PBMCs were stimulated in a plastic bag pre-coated with anti-CD3 monoclonal antibody (OKT3) and recombinant fibronectin fragment (RetroNectin®; RN). Following four days of stimulation, stimulated T cells were transferred into a 215 cm2 plastic bag pre-loaded with SFG-1928z retroviral vector (Brentjens et al., Clin Cancer Res. 2007) onto RN-coated substratum with low-temperature shaking (RBV-LTS method; Dodo et al., PLoS ONE, 2014). After one hour incubation, the bag was flipped over to facilitate more efficient utilization of the retroviral vector adsorbed on both top and bottom surfaces of the bag and further incubated. On Day 5, the transduction procedure was repeated, and the cells were transferred into 640 cm2 plastic bags until Day 10-14 for expansion. Closed system liquid handling was managed in all processes of manipulating T cells for stimulation, transduction, expansion and final product formulation. Results; We have previously reported that the fold expansion of T cells under stimulation with RN together with OKT3 enhanced cell proliferation while preserving the naïve phenotype of T cells in comparison to stimulation with OKT3 alone or OKT3 and anti-CD28 monoclonal antibody co-stimulation. Although B-NHL patients’ T cells showed much lower fold expansion compared to healthy donors’ T cells, 4/7 patients’ CAR T cells reached their target dose of 1 x 106 cells/kg from 30 ml of blood on Day 10. For the other 3 patients, 70-150 ml blood was estimated to be required to reach their target dose. The delay in proliferations was marked in B-NHL patients’ T cells compared to healthy donors’ T cells by Day 5, but B-NHL patients’ T cells represented significant catch-up growth, which was superior to healthy donor T cells during Day 7-14. Gene transfer efficiency of patients’ T cells (N = 7, 17.9 ± 4.7%) was equivalent to that of healthy donors’ T cells (N = 5, 22.2 ± 5.3%), and CAR T cells showed potent anti-tumor reactivity with cytokine productions against CD19 positive Raji cells in vitro. Comparing to CD3/CD28 beads stimulation method, RN/OKT3 stimulation method showed equivalent expansion. Furthermore, RN/OKT3 stimulated T cells preserved higher proportion of CD8+/CCR7+/CD45RA+/CD62L+ naïve phenotype T cells (43.6% in healthy donor and 30.9% in patient donor) compared to CD3/CD28 beads stimulated T cells (22.8% in healthy donor and 11.7% in patient donor). Conclusions; With our novel closed system manufacturing method utilizing RN/OKT3 stimulation combined with RBV-LTS transduction from a small amount of blood of B-NHL patients, we are able to manufacture a sufficient number of CAR T cells maintaining higher proportion of naïve phenotype, which is expected to improve the efficacy of adoptive immunotherapy. In our cell manufacturing, patients are not required to undergo leukapheresis, which is more invasive to patients. Based on our results, we employed our novel manufacturing method for the phase I/II clinical trial to treat patients with relapsed/refractory CD19+ B-NHL (clinicaltrials.gov, identifier; NCT02134262). Disclosures Chono: Takara Bio Inc.: Employment. Tahara:Takara Bio Inc.: Employment. Nukaya:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Membership on an entity's Board of Directors or advisory committees. Tsukahara:Takara Bio Inc.: Research Funding. Ohmine:Takara Bio Inc.: Research Funding. Ozawa:Takara Bio Inc.: Research Funding. Takesako:Takara Bio Inc.: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Successful Manufacturing of CAR T-Cells with Small Volume Peripheral Blood from Healthy Donors Using the Clinimacs Prodigy Device

Blood ◽

10.1182/blood-2020-136795 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Katie Palen ◽

Parameswaran Hari ◽

Nirav N. Shah ◽

Bryon Johnson

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Mononuclear Cells ◽

T Cell Therapy ◽

Cell Numbers ◽

Car T Cells ◽

Car T Cell ◽

Healthy Donors ◽

Car T

Introduction In recent years, CAR T-cell therapy has emerged as a potentially curative intent treatment for some patients with relapsed, refractory hematologic malignancies. Despite the exciting results, not all patients are able to receive CAR T-cells due to manufacturing failures. T-cells for CAR products are typically autologous and isolated from heavily pre-treated patients, which might account for some of the manufacturing failures and suboptimal clinical efficacy. T-cells collected either early into cancer diagnosis or prior to diagnosis may improve CAR T-cell expansion and limit manufacturing failure. We evaluated the feasibility of generating a CAR T-cell product manufactured from 50 ml of healthy donor blood. Methods Collaborators at Cell Vault collected 50 ml of whole blood from 3 healthy donors, isolated peripheral blood mononuclear cells (PBMCs), and cryopreserved the cells in cryovials at 5e6/vial (1.05-1.35e8 total cells). The vials were shipped to the Medical College of Wisconsin and stored frozen in liquid nitrogen until use. All PBMC vials for a given donor were thawed and pooled. Thawed PBMCs (0.93-1.17e8 cells) were loaded onto a CliniMACS Prodigy device, CD4 and CD8 T cells enriched by immunomagnetic sorting, and T cells placed in the culture chamber with IL-7, IL-15 and TransAct reagent to induce proliferation. On the second day of manufacturing, T cells were transduced with a lentiviral CAR vector encoding anti-CD19, 4-1BB and CD3z. Final CAR T-cell products for these pre-clinical studies were harvested on day 8 of manufacture. Results Starting enriched T-cell numbers from the 3 healthy donors ranged from 4.0-4.8e7 cells, the cells were 74-79% CD4/8+, and the average CD4/CD8 ratio was 1.4. On the day of CAR T harvest (day 8), total cells in the chamber had expanded to 3.6-4.6e9 cells (74-115 fold expansion), the cells were >99% CD3+, and the average CD4/CD8 ratio was 2.9 (Table 1). Final cell numbers were similar to what previously published CAR T manufacturing runs on the CliniMACS Prodigy (Zhu et al., Cytotherapy, 2018), that started with 1x108 enriched T-cells obtained from apheresed mononuclear cells. Cell surface CD19 CAR expression on the final cell products varied from 19.2-48.1%. While more than 50% of the starting T cells had a naïve (CD62L+ CD45RO-) phenotype, the final cell products contained greater than 80% central-memory (CD62L+ CD45RO+) T cells. Finally, the number of CD19 CAR T cells obtained from these pre-clinical manufacturing runs ranged from 7.82e8 to 2.21e9 cells. Conclusions 50 ml of cryopreserved PBMCs was adequate to manufacture clinically relevant CAR T-cell therapy doses from healthy donors not previously exposed to chemotherapy. Sufficient numbers of CAR T-cells were obtained to dose an 80 kg individual with at least 9e6 cells/kg which is greater than prescribed commercial doses of CD19 CAR T-cells. Further studies are indicated to determine if T-cells collected prior to disease modifying chemotherapies result in an improved product. These results demonstrate feasibility for generating CAR T cells from small volumes of whole blood collected at a time point before a cancer patient has been treated with multiple lines of therapy that could negatively impact starting T cell numbers and function. Disclosures Hari: GSK: Consultancy; Amgen: Consultancy; BMS: Consultancy; Takeda: Consultancy; Incyte Corporation: Consultancy; Janssen: Consultancy. Shah:TG Therapeutics: Consultancy; Celgene: Consultancy, Honoraria; Incyte: Consultancy; Kite Pharma: Consultancy, Honoraria; Cell Vault: Research Funding; Miltenyi Biotec: Honoraria, Research Funding; Lily: Consultancy, Honoraria; Verastim: Consultancy. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding.

Download Full-text

Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽

10.1182/blood-2019-129640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 776-776

Author(s):

Claire Roddie ◽

Maeve A O'Reilly ◽

Maria A V Marzolini ◽

Leigh Wood ◽

Juliana Dias Alves Pinto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Peak Car ◽

Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

Download Full-text

Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Blood ◽

10.1182/blood-2019-124031 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 196-196

Author(s):

Bishwas Shrestha ◽

Kelly Walton ◽

Jordan Reff ◽

Elizabeth M. Sagatys ◽

Nhan Tu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Graft Versus Host ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Fund Research

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

Download Full-text

B-CLL Mediated Resistance to CAR T Cell Therapy Via Insufficient Activation Is CAR-Independent

Blood ◽

10.1182/blood-2020-137638 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 44-44

Author(s):

McKensie Collins ◽

Weimin Kong ◽

Inyoung Jung ◽

Stefan M Lundh ◽

J. Joseph Melenhorst

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Cytokine Production ◽

Cell Function ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T

Chronic Lymphocytic Leukemia (CLL) is a B cell malignancy that accounts for nearly 1/3rd of adult leukemia diagnoses in the Western world. Conventional chemo-immunotherapies initially control progression, but in the absence of curative options patients ultimately succumb to their disease. Chimeric Antigen Receptor (CAR) T cell therapy is potentially curative, but only 26% of CLL patients have a complete response. CLL-stimulated T cells have reduced effector functions and B-CLL cells themselves are believed to be immunosuppressive. Our work demonstrates that insufficient activation of CAR T cells by CLL cells mediates some of these effects and that the results are conserved between ROR1- and CD19-targeting CARs. Results: In this study we used an in vitro system to model the in vivo anti-tumor response in which CAR T cells serially engage with CLL cells. Multiple stimulations of CD19 or ROR1-targeting CAR T cells with primary CLL cells recapitulated many aspects of known T cell dysfunction including reduced proliferation, cytokine production, and activation. While the initial stimulation induced low level proliferation, subsequent stimulations failed to elicit additional effector functions. We further found that these functional defects were not permanent, and that CAR T cell function could be restored by switching to a stimulus with an aAPC (artificial Antigen Presenting Cell) control cell line. The aAPCs are well-characterized as potent stimulators of CAR T cell effector responses. Flow cytometry revealed that CLL-stimulated CAR T cells retained a non-activated, baseline differentiation profile, suggesting that CLL cells fail to stimulate CAR T cells rather than rendering them non-functional. One mechanism that could dampen activation is immune suppression. We assessed this at a high level by stimulating CAR T cells with CLL cells and aAPCs mixed at known ratios. However, even cultures containing 75% CLL cells stimulated proliferation and cytokine production. Extensive immune-phenotyping revealed high level expression of the IL-2 Receptor on 90% (18/20) of the B-CLL cells tested. Since cytokine sinking via IL-2 receptor expression is a well-known mechanism of regulatory T cell suppression, we hypothesized that CLL cells similarly sink IL-2, blunting T cell activation. To test this, we supplemented IL-2 into CLL/CAR T cell co-cultures and showed that this rescued proliferation but only partially restored cytokine production. In contrast to our hypothesis, analysis of cytokine production by flow cytometry showed that CLL-stimulated CAR T cells did not produce IL-2 following a 6- or 12-hour stimulus, but TNFα was expressed after 12-hours. Similarly, CAR T cell degranulation, a prerequisite for target cell lysis was triggered after CLL recognition. These data again suggested that CLL cells insufficiently stimulate CAR T cell cytokine production, but also showed that cytolytic activity against CLL cells is intact. We further proposed that CLL cells express insufficient levels of co-stimulatory and adhesion molecules to activate CAR T cells. Flow cytometry showed that most CLL cells expressed co-stimulatory and adhesion molecules at low levels; we hypothesized that up-regulating these molecules would enhance CAR T cell targeting of CLL cells. CLL cells were activated with CD40L and IL-4, which increased expression of CD54, CD58, CD80, and CD86. Stimulating CAR T cells with activated CLL cells enhanced CAR T cell proliferation and induced cell conjugate formation, indicating cell activation. Therefore, improving CLL stimulatory capacity can rescue T cell dysfunctions. To assess whether IL-2 addition and CD40 ligation were synergistic, we combined the two assays; however, we saw no additional improvement over IL-2 addition alone, suggesting that the two interventions may act upon the same pathway. Importantly, we also showed that rescue of CAR T cell function via IL-2 addition or CD40 ligation was not CAR-specific, as we observed the functional defects and subsequent rescue with both a ROR1-targeting CAR and the gold standard CD19-targeting CAR. Conclusions: Together, these data show that CAR T cell "defects" in CLL are actually insufficient activation, and improving the stimulatory capacity of CLL cells may enable better clinical responses. Further, this effect is not CAR-specific and these results may therefore be broadly applicable to multiple therapies for this disease. Disclosures Melenhorst: IASO Biotherapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Novartis: Other: Speaker, Research Funding; Johnson & Johnson: Consultancy, Other: Speaker; Simcere of America: Consultancy; Poseida Therapeutics: Consultancy.

Download Full-text

Treatment with Acalibrutinib, Ibrutinib and CD19 CAR T Cells Restore the Number of Granulocytic Myeloid Derived Supressor Cells in CLL-Bearing Mice

Blood ◽

10.1182/blood-2019-126697 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3032-3032

Author(s):

Arantxa Romero-Toledo ◽

Robin Sanderson ◽

John G. Gribben

Keyword(s):

T Cells ◽

T Cell ◽

Cell Population ◽

Research Funding ◽

Cell Function ◽

Car T Cells ◽

Car T Cell ◽

T Cell Function ◽

Car T ◽

Secondary Lymphoid Organs

The complex crosstalk between malignant chronic lymphocytic leukemia (CLL) cells and the tumor microenvironment (TME) is not fully understood. CLL is associated with an inflammatory TME and T cells exhibit exhaustion and multiple functional defects, fully recapitulated in Eµ-TCL1 (TCL1) mice and induced in healthy mice by adoptive transfer (AT) of murine CLL cells, making it an ideal model to test novel immunotherapies for this disease. Myeloid-derived suppressor cells (MDSCs), a non-leukemic cell type within the TME, are immature myeloid cells with the ability to suppress T cell function and promote Treg expansion. In humans, CLL cells can induce conversion of monocytes to MDSCs provoking their accumulation in peripheral blood (PB). MDSCs include two major subsets granulocytic (Gr) and monocytic (M)-MDSC. In mice, Gr-MDSCs are defined as CD11b+Ly6G+Ly6Clo and M-MDSC as CD11b+Ly6G-Ly6Chi. Both murine and human MDSCs express BTK. We observed that in CLL-bearing mice, MDSCs cells are lost in PB as disease progresses. Treatment with both BTK inhibitors (BTKi), ibrutinib (Ibr) and acalabrutinib (Acala), result in shift of T cell function from Th2 towards Th1 polarity and increase MDSC populations in vivo. We aimed to determine whether combination treatment with BTKi and chimeric antigen receptor (CAR) T cells renders recovery of the MDSC population in CLL-bearing mice. To address this question we designed a two-part experiment, aiming to mimic the clinically relevant scenario of pre-treatment of CLL with BTKi to improve CAR T cell function. Part 1 of our experiment consisted of 4 groups (n=12) of 2.5 month old C57/Bl6 mice. Three groups had AT with 30x106 TCL1 splenocytes. A fourth group of WT mice remained CLL-free as a positive control and donors for WT T cells. When PB CLL load reached >10% (day 14) animals were randomized to either Ibr or Acala at 0.15 mg/l in 2% HPBC or no treatment for 21 days. All animals from part 1 were culled at day 35 post-AT and splenic cells were isolated, analyzed and used to manufacture CAR T cells. WT, CLL, Ibr and Acala treated T cells were activated and transduced with a CD19-CD28 CAR to treat mice in part 2. Here, 50 WT mice were given AT with 20x106 TCL1 splenocytes for CLL engraftment. All mice were injected with lymphodepleting cyclophosphamide (100mg/kg IP) one day prior to IV CAR injection. At day 21 post-AT, mice were treated with WT CAR, CLL CAR, IbrCAR, AcalaCAR or untransduced T cells. MDSC sub-populations were monitored weekly in PB and SP were analysed by flow cytometry. As malignant CD19+CD5+ cells expands in PB, the overall myeloid (CD19-CD11b+) cell population was not affected, but MDSCs significantly decreased (p<0.0001). Treatment with Acala, but not Ibr restores total MDSCs. However, MDSC impairment occurs in the Gr- but not M- MDSC population and both Acala and Ibr restores this population (Figure 1a). When we examined the spleen, treatment with both Ibr (p<0.001) and Acala (p<0.001) reduced CD5+CD19+ cells, whereas neither BTKi affected the overall myeloid (CD19-CD11b+) cell population. Gr-MDSCs were restored by both treatments whilst M-MDSCs were only restored after Ibr treatment (p<0.001 in each case). In part 2 of this experiment we observed that treatment with all CAR-T cell groups provokes the clearance of all CD19+CD5+ cells. The overall CD19-CD11b+ population stays the same across all mice groups 35 days after treatment in PB with any group of CAR and untransduced T cells. Overall MDSC population is maintained following all CAR T cells compared to CLL-bearing mice (p<0.0001) and it is the Gr- but not the M- MDSC population which is recovered in PB (Figure 1b). These parts of the experiments can of course be influenced by treatment with cyclophosphamide. We conclude that novel therapies for CLL treatment have an effect not only in CLL cells but also in non-malignant cell components of the TME. In this animal model of CLL, the rapid expansion of CLL cells in PB and secondary lymphoid organs provokes loss of MDSC, particularly the Gr-MDSC subpopulation is affected. Treatment with BTKi and CAR T cells provokes clearance of CLL cells in PB and spleen allowing MDSC recovery; suggesting this may be BTK and ITK independent. We continue to explore secondary lymphoid organs to further characterize the shift of the CLL microenvironment from an immunosuppressive to an immune effective one and its impact on immune function in this model. Disclosures Sanderson: Kite/Gilead: Honoraria. Gribben:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

Download Full-text

B Cell Maturation Antigen-Specific CAR T Cells for Relapsed or Refractory Multiple Myeloma: A Meta-Analysis

Blood ◽

10.1182/blood-2019-121967 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3113-3113 ◽

Cited By ~ 3

Author(s):

Nico Gagelmann ◽

Francis Ayuketang Ayuk ◽

Djordje Atanackovic ◽

Nicolaus Kroeger

Keyword(s):

T Cells ◽

T Cell ◽

High Risk ◽

Research Funding ◽

Response Rates ◽

Overall Response ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background Cellular immunotherapies represent an enormously promising strategy for relapsed/refractory multiple myeloma (RRMM). Chimeric antigen receptor (CAR) T cells targeting B cell maturation antigen (BCMA) have shown impressive results in early-phase clinical studies. Here, we summarize the current body of evidence on the role of anti-BCMA CAR T cell therapy for RRMM. Methods We performed a systematic literature review to identify all publicly available prospective studies. We searched Medline, Cochrane trials registry, and www.clinicaltrials.gov. To include the most recent evidence, meeting abstracts from international hematology congresses were added. A conventional meta-analysis was conducted using meta and metafor packages in R statistical software. Pooled event rates and 95% confidence intervals (CIs) were calculated using the inverse variance method within a random-effects framework. Main efficacy outcomes were overall response, complete response (CR), and minimal residual disease (MRD). Furthermore, relapse rates, progression-free survival, and overall survival were evaluated. In terms of safety, outcomes were cytokine release syndrome (CRS), neurotoxicity, and hematologic toxic effects. Results Fifteen studies comprising a total of 285 patients with heavily pretreated RRMM were included in quantitative analyses. Patients received a median of seven prior treatment lines (such as proteasome inhibitors, immunomodulatory drugs, monoclonal antibodies, stem cell transplantation) which included autologous stem cell transplantation in 90% of patients. The median age of patients was 59 years and median follow-up duration ranged from 1.1 to 11.3 months. Most studies used 4-1BB (or CD137), a member of the TNF receptor superfamily, as an activation-induced T-cell costimulatory molecule. Most studies used fludarabine and cyclophosphamide for lymphodepletion while one study used busulfan and cyclophosphamide and one study used cyclophosphamide only. Most studies used the former Lee criteria for CRS grading. Anti-BCMA CAR T cells resulted in a pooled overall response of 82% (95% CI, 74-88%). The pooled proportion of CR in all evaluable patients was 36% (95% CI, 24-50%). Within responders, the pooled proportion of MRD negativity was 77% (95% CI, 67-85%). Higher dose levels of infused CAR+ cells were associated with higher overall response rates resulting in a pooled proportion of 88% (95% CI, 78-94%). In addition, peak CAR T cell expansion appeared to be associated with responses.The presence of high-risk cytogenetics appeared to be associated with lower overall response rates resulting in a pooled proportion of 68% (95% CI, 47-83%). The presence of extramedullary disease at time of infusion did not influence outcome and was associated with similar response rates compared with RRMM patients who did not have extramedullary disease, resulting in a pooled proportion of 78% (95% CI, 47-93%). The pooled relapse rate of all responders was 45% (95% CI, 27-64%) and the median progression-free survival was 10 months. In terms of overall survival, pooled survival rates were 84% (95% CI, 60-95%) at last follow-up (median, 11 months). In terms of safety, the pooled proportion of CRS of any grade was 69% (95% CI, 51-83%). Notably, the pooled proportions of CRS grades 3-4 and neurotoxicity were 15% (95% CI, 10-23%) and 18% (95% CI, 10-31%). Peak CAR T cell expansion appeared to be more likely in the setting of more severe CRS in three studies. Most hematologic toxic effects of grade 3 or higher were neutropenia (85%), thrombocytopenia (70%), and leukopenia (60%). Conclusion Anti-BCMA CAR T cells showed high response rates, even in high-risk features such as high-risk cytogenetics and extramedullary disease at time of CAR T cell infusion. Toxicity was manageable across all early-phase studies. However, almost half of the patients who achieved a response eventually relapsed. Larger studies with longer follow-up evaluating the association of response and survival are needed. Disclosures Ayuk: Novartis: Honoraria, Other: Advisory Board, Research Funding. Kroeger:Medac: Honoraria; Sanofi-Aventis: Honoraria; Neovii: Honoraria, Research Funding; Riemser: Research Funding; JAZZ: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; DKMS: Research Funding.

Download Full-text

Combination of NKTR-255, a Polymer Conjugated Human IL-15, with CD19 CAR T Cell Immunotherapy in a Preclinical Lymphoma Model

Blood ◽

10.1182/blood-2019-125960 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2866-2866 ◽

Cited By ~ 2

Author(s):

Cassie Chou ◽

Simon Fraessle ◽

Rachel Steinmetz ◽

Reed M. Hawkins ◽

Tinh-Doan Phi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Board Member ◽

Ad Hoc ◽

Advisory Board ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Background CD19 CAR T immunotherapy has been successful in achieving durable remissions in some patients with relapsed/refractory B cell lymphomas, but disease progression and loss of CAR T cell persistence remains problematic. Interleukin 15 (IL-15) is known to support T cell proliferation and survival, and therefore may enhance CAR T cell efficacy, however, utilizing native IL-15 is challenging due to its short half-life and poor tolerability in the clinical setting. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL15Rα and exhibits reduced clearance, providing sustained pharmacodynamic responses. We investigated the effects of NKTR-255 on human CD19 CAR T cells both in vitro and in an in vivo xenogeneic B cell lymphoma model and found improved survival of lymphoma bearing mice receiving NKTR-255 and CAR T cells compared to CAR T cells alone. Here, we extend upon these findings to further characterize CAR T cells in vivo and examine potential mechanisms underlying improved anti-tumor efficacy. Methods CD19 CAR T cells incorporating 4-1BB co-stimulation were generated from CD8 and CD4 T cells isolated from healthy donors. For in vitro studies, CAR T cells were incubated with NKTR-255 or native IL-15 with and without CD19 antigen. STAT5 phosphorylation, CAR T cell phenotype and CFSE dilution were assessed by flow cytometry and cytokine production by Luminex. For in vivo studies, NSG mice received 5x105 Raji lymphoma cells IV on day (D)-7 and a subtherapeutic dose (0.8x106) of CAR T cells (1:1 CD4:CD8) on D0. To determine optimal start date of NKTR-255, mice were treated weekly starting on D-1, 7, or 14 post CAR T cell infusion. Tumors were assessed by bioluminescence imaging. Tumor-free mice were re-challenged with Raji cells. For necropsy studies mice received NKTR-255 every 7 days following CAR T cell infusion and were euthanized at various timepoints post CAR T cell infusion. Results Treatment of CD8 and CD4 CAR T cells in vitro with NKTR-255 resulted in dose dependent STAT5 phosphorylation and antigen independent proliferation. Co-culture of CD8 CAR T cells with CD19 positive targets and NKTR-255 led to enhanced proliferation, expansion and TNFα and IFNγ production, particularly at lower effector to target ratios. Further studies showed that treatment of CD8 CAR T cells with NKTR-255 led to decreased expression of activated caspase 3 and increased expression of bcl-2. In Raji lymphoma bearing NSG mice, administration of NKTR-255 in combination with CAR T cells increased peak CAR T cell numbers, Ki-67 expression and persistence in the bone marrow compared to mice receiving CAR T cells alone. There was a higher percentage of EMRA like (CD45RA+CCR7-) CD4 and CD8 CAR T cells in NKTR-255 treated mice compared to mice treated with CAR T cells alone and persistent CAR T cells in mice treated with NKTR-255 were able to reject re-challenge of Raji tumor cells. Additionally, starting NKTR-255 on D7 post T cell infusion resulted in superior tumor control and survival compared to starting NKTR-255 on D-1 or D14. Conclusion Administration of NKTR-255 in combination with CD19 CAR T cells leads to improved anti-tumor efficacy making NKTR-255 an attractive candidate for enhancing CAR T cell therapy in the clinic. Disclosures Chou: Nektar Therapeutics: Other: Travel grant. Fraessle:Technical University of Munich: Patents & Royalties. Busch:Juno Therapeutics/Celgene: Consultancy, Equity Ownership, Research Funding; Kite Pharma: Equity Ownership; Technical University of Munich: Patents & Royalties. Miyazaki:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Turtle:Allogene: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, 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; Kite/Gilead: Other: Ad hoc advisory board member.

Download Full-text

A Failure to Start: Aborted Activation of CAR T Cells in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-122063 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 681-681

Author(s):

McKensie Collins ◽

Weimin Kong ◽

Inyoung Jung ◽

Meng Wang ◽

Stefan M Lundh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Research Funding ◽

Cell Dysfunction ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Dysfunction ◽

Low Levels

Introduction: Chronic Lymphocytic Leukemia (CLL) is a CD19+ B-cell malignancy that accounts for approximately 25% of adult leukemia diagnoses in the developed world. While conventional therapies have some efficacy, there are few curative therapeutic options and many patients ultimately progress to relapsed or refractory disease. CD19-targeting chimeric antigen receptor (CAR) T cell therapy has provided some hope, but induces complete remission in only 26% of patients. This suboptimal response rate is believed to be due to T cell dysfunction and immune-suppression by CLL cells, the mechanisms of which are poorly understood. Results: To understand the causes of CAR T cell dysfunction in CLL we investigated the defects that CLL cells induced in normal donor CD19-targeting CAR T cells. CAR T cells were repeatedly stimulated at 5-day intervals with either primary CLL cells from patients or a CD19-expressing control cell line (aAPC). Repeat stimulation of CAR T cells with aAPCs resulted in 5.36 ± .94 population doublings after three stimulations, whereas CLL cells only evoked 2.39 ± .92 population doublings. We performed phenotyping, proliferation analysis, and cytokine analysis of stimulated CAR T cells. CLL-stimulated T cells appeared un-activated, with low levels of PD-1, LAG3, and TIM3, low levels of cytokine production, and a high proportion of non-cycling cells as measured by Ki67 staining. We first hypothesized that CLL cells induce an altered epigenetic program that prevents effector function and is stabilized by successive stimulations. To test this, we stimulated CAR T cells with CLL cells or aAPCs as indicated in Fig. 1A. CLL-stimulated CAR T cells failed to proliferate or produce cytokines, but subsequent stimulation with aAPCs rescued these functions (Fig. 1B). Further, CLL-stimulated CAR T cells did not differentiate, suggesting that CLL cells do not induce stable defects but rather insufficiently activate CAR T cells (Fig. 1C). These cells also appeared un-activated as indicated by low levels of PD-1 and Ki67. We then used flow cytometry to assess expression of costimulatory and inhibitory molecules on the primary CLL samples. We found that the levels of co-stimulatory and adhesion molecules, namely CD80/CD86 and CD54/CD58 respectively were found at low frequencies, and where present were expressed at low levels. This suggested that one mechanism behind the lack of CAR T cell effector responses may be that a lack of co-stimulation prevents proper CAR T cell targeting of these cells. Towards this, we incubated CLL cells with a murine fibroblast line expressing CD40 ligand for 24 hours with IL-4 to activate the CLL cells. We found that this activation significantly increased expression of CD80, CD86, CD54, and CD58 on the CLL cells. We then used these cells to stimulate CAR T cells in our re-stimulation assay and found that CAR T cells were able to proliferate in response to these activated CLLs (Fig. 1D). In addition, CAR T cells stimulated with activated CLL cells formed more cell-to-cell conjugates than those stimulated with un-activated CLL cells. These data suggest not only that insufficient activation of CAR T cells may be responsible for the poor response rates to CAR T cell therapy in CLL, but also implicate a need for additional co-stimulation in this CAR T cell setting. Another contributing factor may be immune suppression by CLL cells. To determine if CLL cells are immune-suppressive, we used a co-culture assay to stimulate CAR T cells with aAPCs and CLL cells mixed at known ratios. Interestingly, all mixed cultures proliferated similarly, suggesting that CLL cells did not prevent T cell activation in the presence of a strong activation signal. We also found that CLL cells are responsive to IL-2, as addition of this cytokine to culture media prolongs survival of CLL cells out to 10 days depending on the dose. This suggests that CLL cells express a functional IL-2 receptor and may be taking up IL-2 from the culture media, further impairing T cell activation. In support of this, supplementing IL-2 into our CLL/CAR T cell co-cultures rescued T cell proliferative capacity. Taken together, these data suggest that T cell dysfunction in CLL is the result of insufficient activation rather than true functional defects. Disclosures June: Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; Incyte: Research Funding.

Download Full-text

Clinical Responses In Patients Infused With T Lymphocytes Redirected To Target κ-Light Immunoglobulin Chain

Blood ◽

10.1182/blood.v122.21.506.506 ◽

2013 ◽

Vol 122 (21) ◽

pp. 506-506 ◽

Cited By ~ 6

Author(s):

Carlos A. Ramos ◽

Barbara Savoldo ◽

Enli Liu ◽

Adrian P. Gee ◽

Zhuyong Mei ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

B Cell ◽

Light Chain ◽

Stable Disease ◽

Research Funding ◽

B Cell Malignancies ◽

Car T Cells ◽

Car T

Abstract Adoptive transfer of T cells with a CD19-specific chimeric antigen receptor (CAR) to treat B-cell malignancies shows remarkable clinical efficacy. However, long-term persistence of T cells targeting CD19, a pan-B cell marker, causes sustained depletion of normal B cells and consequent severe hypogammaglobulinemia. In order to target B-cell malignancies more selectively, we exploited the clonal restriction of mature B-cell malignancies, which express either a κ or a λ-light immunoglobulin (Ig) chain. We generated a CAR specific for κ-light chain (CAR.κ) to selectively target κ+ lymphoma/leukemia cells, while sparing the normal B cells expressing the reciprocal λ-light chain, thus minimizing the impairment of humoral immunity. After preclinical validation, we designed a phase I clinical trial in which patients with refractory/relapsed κ+ non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL) are infused with autologous T cells expressing a CAR.κ that includes a CD28 costimulatory domain. The protocol also included patients with multiple myeloma with the aim of targeting putative myeloma initiating cells. Three dose levels (DL) are being assessed, with escalation determined by a continual reassessment method: 0.2 (DL1), 1 (DL2) and 2 (DL3) ×108 T cells/m2. Repeat infusions are allowed if there is at least stable disease after treatment. End points being evaluated include safety, persistence of CAR+T cells and antitumor activity. T cells were generated for 13 patients by activating autologous PBMC with immobilized OKT3 (n=5) or CD3/CD28 monoclonal antibodies (n=8). In 2 patients with >95% circulating leukemic cells, CD3 positive selection was performed using CliniMACS. After transduction, T cells (1.2×107±0.5×107) were expanded ex vivo for 18±4 days in the presence of interleukin (IL)-2 to reach sufficient numbers for dose escalation. CAR expression was 81%±13% by flow cytometry (74,112±23,000 transgene copy numbers/mg DNA). Products were composed predominantly of CD8+ cells (78%±10%), with a small proportion of naïve (5±4%) and memory T cells (17%±12%). CAR+ T cells specifically targeted κ+ tumors as assessed by 51Cr release assays (specific lysis 79%±10%, 20:1 E:T ratio) but not κ–tumors (11%±7%) or the NK-sensitive cell line K562 (26%±13%). Ten patients have been treated: 2 on DL1, 3 on DL2 and 5 on DL3. Any other treatments were discontinued at least 4 weeks prior to T-cell infusion. Patients with an absolute leukocyte count >500/µL received 12.5 mg/kg cyclophosphamide 4 days before T-cell infusion to induce mild lymphopenia. Infusions were well tolerated, without side effects. Persistence of infused T cells was assessed in blood by CAR.κ-specific Q-PCR assay and peaked 1 to 2 weeks post infusion, remaining detectable for 6 weeks to 9 months. Although the CAR contained a murine single-chain variable fragment (scFv), we did not detect human anti-mouse antibodies following treatment and CAR.κ+T cell expansion continued to be observed even after repeated infusions. We detected modest (<20 fold) elevation of proinflammatory cytokines, including IL-6, at the time of peak expansion of T cells, but systemic inflammatory response syndrome (cytokine storm) was absent. No new-onset hypogammaglobulinemia was observed. All 10 patients are currently evaluable for clinical response. Of the patients with relapsed NHL, 2/5 entered complete remission (after 2 and 3 infusions at dose level 1 and 3, respectively), 1/5 had a partial response and 2 progressed; 3/3 patients with multiple myeloma have had stable disease for 2, 8 and 11 months, associated with up to 38% reduction in their paraprotein; and 2/2 patients with CLL progressed before or shortly after the 6-week evaluation. In conclusion, our data indicate that infusion of CAR.κ+ T cells is safe at every DL and can be effective in patients with κ+ lymphoproliferative disorders. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Rooney:Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Download Full-text