Identifying the Factors Modulating the Efficacy of CAR-T Cell Therapy

Abstract T cells modified to express tumour-directed chimeric antigen receptors (CARs) have shown anti-tumor efficacy in early phase clinical trials. There is no consensus yet about the optimal CAR T- cell dose needed for achieve optimal anti-leukemic activity. Early phase clinical trials have shown that the level of in vivo expansion of CAR T-cells rather than CAR T-cell dose determines the efficacy of CAR-T cell therapy. Here we identify factors that modify CAR-T cell expansion and anti-tumour activity in vitro and in vivo in a ‘humanised’ mouse model using CARs targeting CD19+ B-cell leukaemia (CAR19 T-cells). Human peripheral and cord blood T-cells were genetically modified using second generation of CAR19-T cells engineered to express CD3zeta and co-stimulatory CD28 domains cloned into PiggyBac-transposon vector. Antigen-specific stimulation using autologous mononuclear cells was used to enrich and expand CAR19 T-cells. CAR19 T-cells induced cytolysis of leukaemia cells in vitro and exhibited anti-tumour activity in vivo in xenograft mouse model of leukaemia. Antigen-specific stimulation induced rapid activation and proliferation of CAR19 T-cells, however, terminal effector differentiation and activated T cell death limited CAR19-T cell expansion. We have analysed CAR19 T-cell expansion using the panel of primary CD19+leukaemia cells expressing different levels of CD19 antigen. CAR19 T-cells expansion and cytolytic function correlated with the level of CD19 expression on target cells. In addition, increasing CD19 expression in leukaemia cells using hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA) promoted antigen-specific recognition of target cells by CAR19-T cells and increased their expansion. Treatment with 5-AZA also increased CAR expression on the CAR19 T-cells, however, cytotoxicity towards CAR19 T cells mediated by the agent delayed CAR19T-cell expansion. Interestingly, allogeneic stimulation of CAR19 T-cells using CD3/CD28 activation of TCR signalling or co-culture with allogeneic bone marrow stroma cells promoted CAR19 T-cell expansion, however, addition of antigen-specific target cells (CD19+leukaemia cells) attenuated allogeneic expansion indicating the antagonism between antigen-specific and allogeneic CAR19 T- cell activation. Effector to target (E:T) cell ratio appears to be a strong determinate of CAR19 T-cell expansion and cytolytic activity towards cancer cells. CAR19 T-cells used at high E:T ratios exhibit higher cytolytic activity compared to effector cells used at a low E:T. Cell division analysis demonstrats lower proliferation of CAR19 T-cells when used at a high E:T compared to those used at low E:T ratio. The repetitive antigen-specific activation of CAR19 T-cells occurring at a high density of target cells (low E:T ratio) promotes proliferation, however, also acts to induce terminal effector differentiation resulting in rapid T cell extinction due to activated T cell death. Thus the excessive proliferation and rapid extinction of CAR19 T-cells may account for the low efficacy of CAR19 T- cell therapy when given at low E:T ratios. Similar effects were observed in a stem cell reconstituted mouse model where stem cell-derived CD19+ B cells were targeted by CAR19 T-cells. A single infusion of CAR19-T cells infused at a high E:T ratio in mice with low B cell engraftment resulted in efficient B cell depletion while the same dose of CAR19 T-cells infused to mice with high B cell engraftment level(low E:T ratio) was less efficient. These findings show that rapid exhaustion of effector cells used at low E:T ratio prevents long lasting anti-tumor effects justifying the need of pre-conditioning in patients with advanced tumors. . Disclosures No relevant conflicts of interest to declare.

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Creation of the First Non-Human Primate Model That Faithfully Recapitulates Chimeric Antigen Receptor (CAR) T Cell-Mediated Cytokine Release Syndrome (CRS) and Neurologic Toxicity Following B Cell-Directed CAR-T Cell Therapy

Blood ◽

10.1182/blood.v128.22.651.651 ◽

2016 ◽

Vol 128 (22) ◽

pp. 651-651 ◽

Cited By ~ 9

Author(s):

Agne Taraseviciute ◽

Leslie Kean ◽

Michael C Jensen

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

B Cell ◽

Cell Expansion ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

Abstract The advent ofadoptive T-cell therapy using CD19 Chimeric Antigen Receptor (CAR) T cells has revolutionized the treatment of relapsed and refractory acute lymphoblastic leukemia (ALL). CAR T cells have shown encouraging results in clinical trials, with complete remissions in 90% of patients with refractory B-cell ALL. However, CD19 CAR T cell therapy is associated with significant side effects, including cytokine release syndrome (CRS), encompassing fevers, myalgias, hypotension, respiratory distress, coagulopathy as well as neurologic toxicity, ranging from headaches to hallucinations, aphasia, seizures and fatal cerebral edema. Our understanding of CRS and neurologic toxicity has been significantly limited by the lack of animal models that faithfully recapitulate these symptoms. We chose the non-human primate (NHP), Macaca mulatta, given that it closely recapitulates the human immune system, to create an animal model of B-cell-directed CAR T cell therapy targeting CD20. Rhesus macaques (n=3) were treated with 30-40mg/kg cyclophosphamide followed 3-6 days later by an infusion of CAR T cells at a dose of 1x107 transduced cells/kg. Recipient animals were monitored for clinical signs and symptoms of CRS and neurotoxicity, and data were collected longitudinally to determine CAR T cell expansion and persistence, B cell aplasia, as well as clinical labs of CRS and cytokine levels. Prior to testing the CD20 CAR T cells, we performed a control experiment, in which 1x107/kg control T cells, transduced to express GFP only (without a CAR construct), were infused following cyclophosphamide conditioning. This infusion resulted in short-lived persistence of the adoptive cellular therapy, with disappearance of the cells from the peripheral blood by Day +14 (Figure 1, green traces) and no clinical signs of CRS (Figure 2) or neurologic toxicities. In contrast, recipients of 1x107 cells/kg CD20 CAR-expressing T cells (n = 3) demonstrated significant expansion of the CAR T cells, and persistence for as long as 43days post-infusion, which corresponded to concurrent B cell aplasia (Figure 1). These recipients also developed clinical signs and symptoms of CRS as well as neurologic toxicity which was manifested by behavioral abnormalities and extremity tremors, beginning between days 5 to 7 following CAR T cell infusion, with the onset of clinical symptoms coinciding with maximum CAR T cell expansion and activation. The neurologic symptoms were responsive to treatment with the anti-epileptic medicationlevetiracetam. The clinical syndrome was accompanied by elevations in CRP, Ferritin, LDH and serum cytokines, including IL-6, IL-8 and ITAC (Figure 2 A and B), recapitulating data from clinical trials using CD19 CAR T cells. An expansion of CD20 CAR T cells on day 7 following infusion was also observed in the CSF in the animals, and coincided with the onset of neurotoxicity. Strikingly, we also detected CD20 CAR T cells in multiple regions of the brain via flow cytometry, including the frontal, parietal, and occipital lobes, as well as the cerebellum, and demonstrated an increased number of infiltrating T cells by immunofluorescence in the brains of animals treated with CD20 CAR T cells when compared to healthy controls. These data demonstrate the successful establishment of a large animal model of B-cell directed CAR T cell therapy that recapitulates the most significant toxicities of CAR T cell therapy, including CRS and neurotoxicity. This model will permit a detailed interrogation of the mechanisms driving these toxicities as well as the pre-clinical evaluation of therapies designed to prevent or abort them after CAR T cell infusion. Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 1. Absolute numbers of GFP T cell (n=1) and CD20 CAR T cell (n=3) expansion and persistence in rhesus macaques (top graph). Maximum CD20 CAR T cell expansion occurred between day 7 and day 8 following CAR T cell infusion. Absolute numbers of B cells in rhesus macaques following GFP T cell (n=1) and CD20 CAR T cell (n=3) infusion (bottom graph). Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Figure 2. A. CRP, Ferritin and LDH levels were elevated following CD20 CAR T cell infusion, their peaks closely correlated with maximum CAR T cell expansion. No elevation of CRP, Ferritin or LDH was observed in Animal 1 which received GFP T cells. B. Elevations in IL-6, IL-8 and ITAC levels following CD20 CAR T cell infusion were highest surrounding the time of maximum CAR T cell expansion. Disclosures Kean: Juno Therapeutics, Inc: Research Funding. Jensen:Juno Therapeutics, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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First-in-Human Study of ET019003, a Next Generation Anti-CD19 T-Cell Therapy, in Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma (r/r DLBCL)

Blood ◽

10.1182/blood-2019-131414 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2870-2870 ◽

Cited By ~ 1

Author(s):

Pengcheng He ◽

Hong Liu ◽

Haibo Liu ◽

Mina Luo ◽

Hui Feng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cell Expansion ◽

T Cell Therapy ◽

Car T ◽

Equity Ownership ◽

T Cell Expansion ◽

The Absolute

Background : CD19-targeted CAR-T therapies have shown promising efficacy in treating B-cell malignancies. However, treatment-related toxicities, such as cytokine-release syndrome (CRS) and CAR T-cell-related encephalopathy syndrome (CRES), have been one of the major obstacles limiting the use of CAR-T therapies. How to minimize occurrence and severity of toxicity while maintaining efficacy is a major focus for T-cell therapies in development. ET019003 is a next generation CD19-targeted T-cell therapy developed by Eureka Therapeutics, built on the proprietary ARTEMISTM T-cell platform. The ET019003 construct is optimized with the co-expression of an ET190L1 Antibody-TCR (Xu et al, 2018) and novel co-stimulation molecule. We are conducting a First-in-human (FIH) study of ET019003 T cells in CD19+ r/r DLBCL patients. Methods: This FIH study aims to evaluate the safety and efficacy of ET019003 T-cell therapy in CD19+ patients with r/r DLBCL. As of July 2019, six subjects were administered ET019003 T cells. These subjects were pathologically confirmed with DLBCL that is CD19+ (by immunohistochemistry), whose disease have progressed or relapsed after 2-5 lines of prior therapies. All were high-risk patients with rapid tumor progression and heavy tumor burden. Each subject had a Ki67 proliferative index over 60%, 2/6 of the subjects had a Ki67 proliferative index over 90%. Moreover, 5/6 of the subjects had extra-nodal involvement. Following a 3-day preconditioning treatment with Fludarabine (25mg/m2/day)/ Cyclophosphamide (250mg/m2/day), patients received i.v. infusions of ET019003 T cells at an initial dose of 2-3×106 cells/kg. Additional doses at 3×106 cells/kg were administered at 14 to 30-day intervals. Adverse events were monitored and assessed based on CTCAE 5.0. Clinical responses were assessed based on Lugano 2014 criteria. Results: As of July 2019, six subjects have received at least one ET019003 T-cell infusion, and four subjects have received two or more ET019003 T-cell infusions. No Grade 2 or higher CRS was observed in the six subjects. One subject developed convulsions and cognitive disturbance. This subject had lymphoma invasion in the central nervous system before ET019003 T-cell therapy. The subject was treated with glucocorticoid and the symptoms resolved within 24 hours. Other adverse events included fever (6/6, 100%), fatigue (3/6, 50%), thrombocytopenia (3/6, 50%), diarrhea (2/6, 33%), and herpes zoster (1/6, 17%). ET019003 T-cell expansion in vivo (monitored by flow cytometry and qPCR) was observed in all six subjects after first infusion. The absolute peak value of detected ET019003 T cells ranged between 26,000 - 348,240 (median 235,500) per ml of peripheral blood. Tmax (time to reach the absolute peak value) was 6 - 14 days (median 7.5 days). For the four subjects who received multiple ET019003 T-cell infusions, the absolute peak values of detected ET019003 T cells after the second infusion were significantly lower than the absolute peak values achieved after the first infusion. For the two subjects who received three or more infusions of ET019003 T cells, no significant ET019003 T-cell expansion in vivo was observed after the third infusion. All six subjects completed the evaluation of clinical responses at 1 month after ET019003 T-cell therapy. All subjects responded to ET019003 T cells and achieved either a partial remission (PR) or complete response (CR). Conclusions: Preliminary results from six CD19+ r/r DLBCL patients in a FIH study show that ET019003 T-cell therapy is safe with robust in vivo T-cell expansion. The clinical study is on-going and we are monitoring safety as well as duration of response in longer follow-up. Reference: Xu et al. Nature Cell Discovery, 2018 Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership. Chang:Eureka Therapeutics: Equity Ownership. Liu:Eureka Therapeutics: Employment, Equity Ownership.

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CAR T cell therapy in B-cell acute lymphoblastic leukaemia: Insights from mathematical models

10.1101/2020.03.18.20038257 ◽

2020 ◽

Cited By ~ 1

Author(s):

Odelaisy León-Triana ◽

Soukaina Sabir ◽

Gabriel F. Calvo ◽

Juan Belmonte-Beitia ◽

Salvador Chulián ◽

...

Keyword(s):

Mathematical Model ◽

T Cells ◽

T Cell ◽

Side Effects ◽

B Cell ◽

Target Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T

AbstractImmunotherapies use components of the patient immune system to selectively target cancer cells. The use of CAR T cells to treat B-cell malignancies – leukaemias and lymphomas– is one of the most successful examples, with many patients experiencing long-lasting complete responses to this therapy. This treatment works by extracting the patient’s T cells and adding them the CAR group, which enables them to recognize and target cells carrying the antigen CD19+, that is expressed in these haematological tumors.Here we put forward a mathematical model describing the time response of leukaemias to the injection of CAR T-cells. The model accounts for mature and progenitor B-cells, tumor cells, CAR T cells and side effects by incorporating the main biological processes involved. The model explains the early post-injection dynamics of the different compartments and the fact that the number of CAR T cells injected does not critically affect the treatment outcome. An explicit formula is found that provides the maximum CAR T cell expansion in-vivo and the severity of side effects. Our mathematical model captures other known features of the response to this immunotherapy. It also predicts that CD19+ tumor relapses could be the result of the competition between tumor and CAR T cells analogous to predator-prey dynamics. We discuss this fact on the light of available evidences and the possibility of controlling relapses by early re-challenging of the tumor with stored CAR T cells.

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Tisagenlecleucel and Axicabtagene Ciloleucel Expansion Kinetics and CAR T Cell Attributes in the Infusion Products Are Early Predictors of Clinical Efficacy

Blood ◽

10.1182/blood-2021-152309 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3877-3877

Author(s):

Cristiana Carniti ◽

Chiara Monfrini ◽

Vanessa Aragona ◽

Martina Magni ◽

Cristina Vella ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Expansion ◽

B Cell Lymphoma ◽

Advisory Board ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

Abstract Background: CD19-directed CAR-T cell therapy has shown promising efficacy in relapsed/refractory (R/R) B-cell malignancies in clinical trials resulting in the approval and commercialization of two products (tisagenlecleucel/Tisa-cel and axicabtagene ciloleucel/Axi-cel) for R/R diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). However, relapses occur in 60-65% of patients (pts) and thus a better understanding of the early determinants of response is critical to improve long-term survival in the real-world scenario. Aims of the study: To assess whether CAR-T cell expansion after infusion represents a crucial determinant to sustain effective anti-tumor responses to both Tisa-cel and Axi-celTo evaluate differences in CAR-T cell kinetics due to the use of CD28 or 4-1BB costimulatory moleculesTo identify immune phenotypic features of infusion products accounting for CAR-T cell expansion and survival probability Methods: We analyzed samples from 43 pts [29 DLBCL, 8 high grade B-cell lymphoma (HGBCL) and 6 PMBCL] treated with Axi-cel (n=22) and Tisa-cel (n=21) at the Fondazione IRCCS Istituto Nazionale Tumori prospectively collected between November 2019 and April 2021. CAR-T cells were monitored in the peripheral blood (PB) on days 0, 4, 7, 10, 14, 21, 28 and monthly post infusion by flow cytometry (FCM). Cells were stained with CD19 CAR Detection Reagent (Miltenyi), CD3, CD4, CD8, CD45, CD14, CD45RO, CD62L, CD197, CD279, CD223 and CD366. Residual cells obtained from washings of 32 infused commercial CAR-T bags (10 Tisa-cel and 22 Axi-cel) were also analyzed by FCM. Data were acquired on a BD FACSCanto II (BD Biosciences) and a MACSQuant® Analyzer MQ10 (Miltenyi) and analyzed using FlowJo software, version 10. Results: The median time to maximal expansion of CAR-T cells was at day 10 post infusion with no differences between Axi-cel and Tisa-cel [median concentration at day 10 (C 10) 25 for Axi-cel vs 26 CAR-T cells/µl for Tisa-cel; p, ns], nor among the different histologies (median C 10 33 for DLBCL vs 19 for HGBCL vs 18 CAR-T cells/µl for PMBCL; p, ns). On the contrary, CAR-T peak concentration (C max) was higher in responders at 3 months post infusion (RE, n=28) (defined as pts achieving complete or partial response by PET/CT) than in non responders (NR, n=13) (median C max 87 in RE vs 26 in NR CAR-T cells/µl; p<0.01; Fig 1A). Consistently, the magnitude of CAR-T cell expansion in the first 30 days was higher in RE than in NR [median area under the curve (AUC 0-30) 189 vs 50; p<0.005; Fig 1B]. Circulating CAR-T cells were enriched in subpopulations representing naïve T cells (CD8+ T N; CD45RO−/CD62L+) in RE (median 0.4% in RE vs 0.04% in NR, p<0.05) while NR had significantly higher levels of effector memory T cells (CD8+ T EM; CD45RO+/CD62L+) (median 26.5% in RE vs 66.2% in NR, p<0.05). Additionally, the extent of CAR-T cell expansion predicted the progression free survival (PFS), but not the overall survival (OS), irrespective of the product used (Fig 2, p<0.05) and the overall survival was improved by salvage treatment with bispecifc antibodies. Finally, we evaluated whether CAR-T cell expansion was influenced by the immune phenotypic attributes of the infused products. A significant enrichment of central memory populations (CD8+ T CM; CD45RO−/CCR7+/CD62L+) among CAR-T cells within the infusion products of pts with longer PFS was documented, as compared with those with shorter PFS (CD8+ T CM; median 15.2% vs 3.1%; p<0.005). Conclusion: To the best of our knowledge, this is the first study assessing the clinical utility of early CAR-T cell monitoring in lymphoma pts receiving both commercial anti-CD19 CAR-T cell therapies. We provide evidence that in pts treated with Axi-cel and Tisa-cel: i) the in vivo kinetics of the CAR-T cell products are similar, consistent with the fact that no differences in the outcome of Axi-cel and Tisa-cel treated pts were detected; ii) CAR-T cell expansion is critical for efficacy and predicts the PFS; iii) circulating CAR-T cells in responders have a naïve phenotype; iv) a memory signature in the CAR-T cell product before infusion is associated with in vivo expansion and survival. Figure 1 Figure 1. Disclosures Chiappella: Celgene Bristol Myers Squibb: Other: lecture fee, advisory board; Incyte: Other: lecture fee; Novartis: Other: lecture fee; Astrazeneca: Other: lecture fee; Servier: Other: lecture fee; Takeda: Other: advisory board; Gilead Sciences: Other: lecture fee, advisory board; Clinigen: Other: lecture fee, advisory board; Roche: Other: lecture fee, advisory board; Janssen: Other: lecture fee, advisory board. Corradini: AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Consultancy; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Honoraria; KiowaKirin; Incyte; Daiichi Sankyo; Janssen; F. Hoffman-La Roche; Kite; Servier: Consultancy; Novartis; Gilead; Celgene: Consultancy, Other: Travel and accommodations; BMS: Other: Travel and accommodation; Sanofi: Consultancy, Honoraria; Amgen; Takeda; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations; Incyte: Consultancy; Novartis, Janssen, Celgene, BMS, Takeda, Gilead/Kite, Amgen, AbbVie: Other: travel and accomodations.

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Engineered Cytokine Signaling to Improve CAR T Cell Effector Function

Frontiers in Immunology ◽

10.3389/fimmu.2021.684642 ◽

2021 ◽

Vol 12 ◽

Author(s):

Matthew Bell ◽

Stephen Gottschalk

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Expansion ◽

Cytokine Signaling ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

Adoptive immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) is a promising approach to improve outcomes for cancer patients. While CAR T cell therapy is effective for hematological malignancies, there is a need to improve the efficacy of this therapeutic approach for patients with solid tumors and brain tumors. At present, several approaches are being pursued to improve the antitumor activity of CAR T cells including i) targeting multiple antigens, ii) improving T cell expansion/persistence, iii) enhancing homing to tumor sites, and iv) rendering CAR T cells resistant to the immunosuppressive tumor microenvironment (TME). Augmenting signal 3 of T cell activation by transgenic expression of cytokines or engineered cytokine receptors has emerged as a promising strategy since it not only improves CAR T cell expansion/persistence but also their ability to function in the immunosuppressive TME. In this review, we will provide an overview of cytokine biology and highlight genetic approaches that are actively being pursued to augment cytokine signaling in CAR T cells.

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Myeloid Cells in Peripheral Blood Mononuclear Cell (PMBC) Concentrates Inhibit the Expansion of Chimeric Antigen Receptor (CAR) T Cells

Blood ◽

10.1182/blood.v126.23.383.383 ◽

2015 ◽

Vol 126 (23) ◽

pp. 383-383

Author(s):

David F. Stroncek ◽

Daniel W. Lee ◽

Jiaqiang Ren ◽

Marianna Sabatino ◽

Hanh Khuu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Myeloid Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T ◽

T Cell Expansion

Abstract Introduction: While autologous anti-CD19 and anti-GD2 CAR T cell therapy has shown promise results in children with B cell acute lymphocytic leukemia (ALL) and sarcoma respectively, T cells from some patients fail to expand in culture. The anti-CD19 and anti-GD2 CAR T cell products manufactured by our center and the factors affecting CAR T cell expansion were reviewed. Methods: The manufacturing methods for both types of CAR T cells were similar; autologous PBMC concentrates collected by apheresis were used as starting material, anti-CD3/CD28 beads were used for T cell enrichment, and IL-2 plus CD3/CD28 beads were used for T cell activation and expansion. Retroviral vector transduction of CD19 anti-GD2 CAR T cells was performed on days 2 and 3. Anti-CD19 CAR T cells from 28 patients were expanded over 7 to 11 days and anti-GD2 CAR T cells from 9 patients expanded over 10 to 11 days. The target dose of transduced cells was 1 or 3x106/Kg for both clinical protocols. Results: Following transduction and expansion, the 28 anti-CD19 CAR T cell products contained a mean of 1,130±916 x106 transduced T cells. Four of the 28 had poor expansion defined as not yielding enough transduced cells for one dose (1x106 CAR cells/kg). When the characteristics of the PBMC concentrates collected from the 4 patients whose T cells expanded poorly was compared with that of the other 24, those collected from the poor expanders contained greater quantities of monocytes (39.8±12.9% vs 15.3±10.8%, p=0.0014) and less lymphocytes (42.3±8.4% vs 75.3±14.1%; p<0.001). The poorly expanding apheresis products also contained less CD3+ cells (27.4±10.6% vs 59.7±19.5%; p=0.0045) and more CD56+ cells (26.9±14.5% vs 6.1±5.5%; p<0.001), but there was no difference in the quantity of CD19+ cells. The sum of the proportion of granulocytes and monocytes in each PBMC concentrate was inversely associated with T cell expansion (r= -0.59). A comparison of the quantity of anti-GD2 CAR T cells produced from the 9 sarcoma patients with the quantity of anti-CD19 CAR T cells produced from the 22 ALL patients whose cells were also expanded over 10 to 11 days revealed that the anti-GD2 CAR T cell products contained less transduced cells (197±205x106 vs 1,313±917x106; p=0.0083). The PMBC concentrates collected from sarcoma patients contained more monocytes (31.4±12.4% vs 18.5±13.7%; p=0.019) than those collected from the ALL patients. Of the 9 anti-GD2 CAR T cell products, one failed to meet dose (prescribed doses ranged from 1 x 105/kg-3 x 106/kg); the apheresis product from this patient contained large quantities of myeloid cells: 37% monocytes and 55% granulocytes. However, for sarcoma patients there was no relationship between the sum of granulocytes and monocytes in the PBMC concentrate and the yield of transduced anti-GD2 CAR T cells (r=-0.11). Among the 5 products that expanded poorly, manufacturing was repeated for 2 ALL and 1 sarcoma patient using a cryopreserved aliquot of their PBMC concentrate but incorporating an upfront step to deplete myeloid cells by plastic adherence, with or without density gradient separation. All three products expanded well following myeloid cell depletion. For the ALL patients 0 and 2.4x106 transduced anti-CD19 CAR T cells were produced initially compared to 147x106 and 160x106 transduced cells using the second manufacturing procedure. For the sarcoma patient 4.8x106 transduced anti-GD2 CAR T cells were produced by the first procedure and 5,200x106 by the second procedure. Conclusions: These results show that the expansion of autologous T cells for CAR T cell therapy is highly variable and the variability is due at least in part to the contamination of the starting apheresis concentrate with myeloid cells. More rigorous removal of myeloid cells prior to initiating cell culture was associated with improved T cell expansion. Suppression of expansion by myeloid cells appears to more problematic in sarcoma patients than in leukemia patients. Disclosures Sabatino: Kite Pharma: Employment. Mackall:Juno: Patents & Royalties: CD22-CAR.

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P04.01 Immunomonitoring of CD19. CAR T-cells in Large B-Cell Lymphoma- a two-center experience

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-itoc8.30 ◽

2021 ◽

Vol 9 (Suppl 1) ◽

pp. A16-A16

Author(s):

V Blumenberg ◽

S Völkl ◽

G Busch ◽

S Baumann ◽

M Winkelmann ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

B Cell Lymphoma ◽

Car T Cells ◽

Car T Cell ◽

Large B Cell Lymphoma ◽

Car T ◽

T Cell Expansion

BackgroundCD19. CAR T-cells for the treatment of relapsed and refractory (r/r) Diffuse Large B-Cell Lymphoma (DLBCL) demonstrated complete responses in 40%-58% of the patients. Recently, others could associate high tumor volume and low CAR T-cell expansion in vivo with poor outcome. We hypothesize, that the expansion and immunphenotype of (CAR) T cells in vivo determine treatment response and depend on patient- and disease associated factors.Materials and MethodsPatients with r/r DLBCL (n=34) were treated with either Axi-cel or Tisa-cel at the University Hospitals of Erlangen and Munich (LMU). The CAR T-cell product and peripheral blood were collected on day 0, 4, 7, 14, 30, 60 and 90 post transfusion. CAR T-cells were detected through flow cytometry utilizing a two step-staining with a biotinylated CD19 protein. Effector:Target (E:T) Ratios were estimated as absolute peak expansion of CAR T-cells (/ul) per tumorvolume (cm3). Responder (R, complete or partial remission) were compared to Non-Responder (NR, stable or progressive disease) according to response assessment with PET-CT three months after transfusion.ResultsCAR T-cell expansion peaked between day 7 and day 14 after transfusion with a greater expansion of CD8+compared to CD8- CAR T-cells on day 14 (59.27% vs 37.42%, p=0.021). The ratio of CD8+ and CD8- CAR T-cells did not differ between R and NR, however R exhibited higher E:T ratios of CD3+ CAR T-cells compared to NR (20.94 vs 12.81, p=0.015) and an increased E:T ratio of CD8+ CAR T-cells correlated with better progression-free survival (p=0.033). Interestingly, high CRP and ferritin levels at baseline were inversely associated with the E:T ratio (p=0.048 and p=0.017). CD3+ CAR T-cells of R showed earlier peak expression of PD-1 than NR (day 7 vs day 21). Further, peak expansion of CD3+ CAR T-cells correlated with higher PD-1 expression in R but not in NR (p=0.003 vs p=0.12). In addition, R revealed an increased relative frequency of effector memory differentiated CD3+ CAR T-cells (CCR7-CD45RA-, p=0.02), whereas CAR T-cells in NR showed an increased relative frequency of a naïve phenotype (CCR7+CD45RA+, p=0.001) on day 7 post infusion.ConclusionsFlow-based immunomonitoring with longitudinal characterization of CAR T-cells demonstrated a correlation of the E:T ratio with treatment response and survival. Increased inflammatory conditions at baseline correlated with diminished E:T ratios. Notably, in R CAR peak expansion was positively associated with higher PD-1 expression suggestive for superior CAR T-cell activation. In addition, greater memory differentiation was associated with efficacy during the time of peak expansion. Multiparameter analysis with other clinical covariates will show, whether CAR T-cell expansion and immunphenotypes can predict patient outcome.Disclosure InformationV. Blumenberg: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Novartis, Gilead Sciences, Janssen, BMS/Celgene. S. Völkl: None. G. Busch: None. S. Baumann: None. M. Winkelmann: None. B. Tast: None. D. Nixdorf: None. G. Hänel: None. L. Frölich: None. C. Schmidt: None. R. Jitschin: None. F. Vettermann: None. W. Kunz: None. D. Mougiakakos: None. M. von Bergwelt: None. V. Bücklein: None. A. Mackensen: None. M. Subklewe: None.

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Distribution Measurements of Chimeric Antigen Receptor-Modified T Cells Against CD19

10.21203/rs.3.rs-63462/v1 ◽

2020 ◽

Author(s):

Zhitao Ying ◽

Ting He ◽

Xiaopei Wang ◽

Wen Zheng ◽

Ningjing Lin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Target Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Backgroud: The unprecedented efficacy of chimeric antigen receptor (CAR) T-cell immunotherapy of CD19+ B-cell malignancies has opened a new and useful way for the treatment of malignant tumor. Nonetheless, there are still formidable challenges in the field of CAR-T cell therapy, such as the biodistribution of CAR-T cells in vivo.Methods: We demonstrated the distribution of CAR-T cells in the absence of target cells or with target cells in the mice and the dynamic changes in the patient blood over time after infusion were deteced by qPCR and FACS. Results: CAR-T cells still proliferated in the mice without target cells and peaked at 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at 6 weeks. In the clinical trial, we found that CAR-T cells peaked at 7-21days after infusion and can last for as long as 510 days in the peripheral blood of patients. Simultaneously, mild side-effects were noted which can be effectively controlled within two months in these patients.Conclusions: CAR-T cells can expand themselves with or without target cells in mice. CAR-T cells can persistence for a long time in patients.

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CD19/BAFF-R Dual-Targeted CAR T Cells for the Treatment of Mixed Antigen-Negative Variants of Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2021-151293 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2783-2783

Author(s):

Xiuli Wang ◽

Zhenyuan Dong ◽

Wen-Chung Chang ◽

Wesley Cheng ◽

Vibhuti Vyas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Dual Targeting ◽

Car T Cell Therapy ◽

Car T

Abstract The results of clinical trials evaluating CD19-targeting chimeric antigen receptor (CAR) T cells are impressive, with overall response rates of up to 90% in B cell acute lymphoblastic leukemia (ALL) and 50-80% in lymphoma. Despite initial responses, antigen-negative relapse is common following treatment with CD19-targeted therapies and is estimated to occur in up to 39% of patients. One approach of addressing this problem is to utilize dual-targeting CAR T cells, a strategy that has recently been applied to CD19/CD22 for ALL, CS1/BCMA, and BCMA/GPRC5D for multiple myeloma. Dual-targeting CARs can simultaneously target two tumor antigens and, therefore, potentially eradicate heterogeneous tumors. The initial response to dual-targeted CAR T cells is expected to provide greater tumor coverage compared to single-targeted therapy, and can potentially circumvent antigen escape. Moreover, if one tumor antigen becomes downregulated during treatment, the second targeting domain will continue to be reactive to tumors. Therefore, it is critical to identify novel targets that can be combined with CD19 in a dual-targeted immunotherapeutic platform. To expand the potential for dual targeting of ALL, we developed a CAR T cell therapy against a novel target, B-cell activating factor receptor (BAFF-R), based on the remarkable specificity of anti-BAFF-R antibodies that we previously generated. BAFF-R is expressed almost exclusively on B cells, including in patients with CD19-negative relapse, making it an ideal immunotherapeutic target. Studies demonstrate that the role of BAFF-R in B cell function and survival is conclusive, an important feature that may mitigate the tumor's ability to escape therapy through antigen loss, particularly if a non-redundant role for BAFF-R is confirmed. BAFF-R-CAR T cells demonstrate in vitro effector function and in vivo therapeutic efficacy in CD19-negative models, including patient-derived xenograft models, and are currently being evaluated clinically for the treatment of ALL (NCT04690595). We hypothesized that simultaneous targeting of CD19 and BAFF-R in a bispecific CAR platform could confer a therapeutic advantage and avoid the challenges of sequential administration of CD19 and BAFF-R monospecific CAR T cells. We leveraged our experience with CD19- and BAFF-R-CAR T cells to develop a dual-targeting, bispecific CAR with a 41BB costimulatory domain (CD19/BAFF-R dual CAR). Here, we identified the optimal orientation of the single-chain variable fragment (loop scFv) domains within the dual construct and tested the CD19/BAFF-R dual CAR T cells for their in vitro effector function and in vivo anti-leukemia activity. To evaluate the specific targeting to CD19 and BAFFR, we developed Nalm-6-BAFF-R-knockout (KO) and Nalm-6-CD19-KO cell lines. CD19/BAFF-R dual CAR T cells specifically released IFN-g following incubation with Nalm-6 CD19-/- or BAFF-R-/- cells (P<0.001) compared with un-transduced mock T cells. Both CD4+ and CD8+ CAR T cell populations exhibited effector function. To evaluate the antigen-dependent targeting of the CD19/BAFF-R dual CAR T cells in vivo, we utilized a mixed B-cell leukemia model that simulates clinical tumor heterogeneity. NOD-scid IL2Rgammanull (NSG) mice were inoculated with 2-3x10 5 of mixed Nalm-6 BAFF-R-/- and CD19-/- cells at a 1:1 ratio with a single injection. 1x10 6 CD19/BAFF-R dual CAR, CD19, or BAFF-R single CAR-T cells were administered intravenously 9-10 days later. Tumor growth was monitored by bioluminescent imaging weekly. We observed superior tumor eradication (P<0.01) and survival (P<0.01) (Figure 1) by CD19/BAFF-R dual CAR T cells compared to either single-targeting CAR and mock T cells. The adoptively transferred CD19/BAFF-R dual CAR T cells were able to persist in vivo. Our unique CD19/BAFF-R dual-targeting CAR T cells will be the first to target this combination of tumor-associated antigens. Our study demonstrated the reliability of bispecific CD19/BAFF-R dual CAR T cell therapy in inducing remission in ALL consisting of CD19-/- and BAFF-R-/- tumors. We hypothesize that simultaneous immunotherapy targeting of heterogeneous leukemic cell populations may diminish the likelihood of antigen escape and may have a significant impact on leukemia treatment by improving the therapeutic benefits of CAR T cell therapy. Figure 1 Figure 1. Disclosures Wang: Pepromene Bio, Inc.: Consultancy. Forman: Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Allogene: Consultancy; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company. Kwak: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company. Qin: Pepromene Bio, Inc.: Consultancy, Current equity holder in publicly-traded company.

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Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-129025 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3921-3921 ◽

Cited By ~ 1

Author(s):

Cesar Sommer ◽

Hsin-Yuan Cheng ◽

Yik Andy Yeung ◽

Duy Nguyen ◽

Janette Sutton ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Target Cells ◽

Employment Equity ◽

T Cell Therapy ◽

Cell Therapies ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

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