A phase I clinical trial using armored GPC3 CAR T cells for children with relapsed/refractory liver tumors.

TPS2647 Background: CAR T therapies have been successful against hematologic malignancies, but have benefited only a handful of patients with solid cancers. Glypican 3 (GPC3) is an attractive immunotherapeutic target due to its preferential expression on multiple pediatric and adult solid cancers and lack of expression on non-malignant tissues. GPC3-CAR T cells were tested preclinically and inclusion of the 4-1BB costimulatory endodomain with IL-15 and IL-21 co-expression enabled CAR T cells to expand and persist the most in vitro and in vivo and led to robust antitumor activity in vivo. We are now testing GPC3-CAR T cells with IL15 and IL-21 for the first time in children with relapsed/refractory liver tumors. Methods: In this Phase 1 trial (GAP, NCT02932956), we are evaluating patients in 3 cohorts: 1) GPC3-CAR alone; 2) GPC3-CAR and IL-15; 3) GPC3-CAR with IL-15 and IL-21. We will 1) define the safety and establish the Recommended Phase 2 Dose (RP2D) of GPC3-CAR T cells co-expressing IL-15 and IL-21; 2) determine persistence and anti-tumor activity of GPC3-CAR T cells; 3) examine changes in gene and protein expression in the tumor microenvironment associated with potential immune escape mechanisms. Inclusion criteria are the following: age ≤18; histology proven, GPC3-positive tumor; life expectancy>12 weeks; Child-Pugh-Turcotte score<7; serum AST<5 times ULN; total bilirubin<3 times ULN for age; INR ≤1.7; absolute neutrophil count>500/μl; platelet count>20,000/μl; Hgb≥9.0 g/dl. Toxicity will be monitored using the Common Terminology Criteria of Adverse Events v4. The RP2D will be determined by the standard 3+3 dose escalation method using 5 dose levels. Persistence will be quantified using RT-PCR and flow cytometry. Antitumor activity will be defined by 3D imaging using RECIST 1.1 criteria and the immune-related response criteria. Immune-escape will be examined using single cell RNA sequencing and imaging of paraffin-embedded tissues using codetection by indexing to evaluate candidate proteins. Data will be analyzed via descriptive statistics. Cohort 1 of this study is now open for enrollment. Clinical trial information: NCT02932956.

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CD22 CAR Optimization for Improved in-Human Activity Following Inadequate CD22 CAR Activity in Phase 1 Clinical Trial PLAT-04

Blood ◽

10.1182/blood-2021-147928 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 403-403

Author(s):

Corinne Summers ◽

Blake Baxter ◽

Colleen Annesley ◽

Jason Yokoyama ◽

Stephanie Rhea ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

T Cell ◽

Phase 1 ◽

Chromium Release Assay ◽

Phase 1 Clinical Trial ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Background: CD19 targeting chimeric antigen receptor (CAR) T cells have induced unprecedented remission rates in high-risk precursor B Acute Lymphoblastic Leukemia (ALL); however recurrent disease with CD19 antigen escape variants is not uncommon. Therefore, we developed a novel CD22 targeting CAR, and following preclinical validation, tested it in a first-in-human pediatric and young adult phase 1 clinical trial, PLAT-04 (NCT03244306). Four subjects were treated at 2 dose levels (DL) (1x10 6/kg (DL1) and 3x10 6/kg (DL2)). The CD22 CAR T cell product (SCRI-CAR22v1) was successfully manufactured (n=4) and no dose limiting toxicity (DLTs), cytokine release syndrome (CRS) or neurotoxicity was observed. However, all subjects had minimal CAR T cell expansion, with 3 of 4 subjects demonstrating persistent or progressive disease at day 21 evaluation despite continued CD22 expression on leukemic blasts. Based on the poor in vivo expansion and lack of activity, enrollment was voluntarily halted to interrogate and optimize the CAR construct for enhanced performance. Methods: Human T cells were transduced to express one of two CD22 CAR constructs. We designed SCRI-CAR22v2, a CD22 CAR that utilizes the same scFv as SCRI-CAR22v1 but with a shorter linker between M971 VH and VL and a shorter hinge with differing transmembrane region, and both using CD8 alpha (Figure A). This construct maintained the truncated EGFR extracellular tag (EGFRt) for tracking and potential in vivo suicide mechanism. The two transduced CAR T cell products were compared preclinically by flow cytometry, chromium release assay and in an in vivo murine model to understand differing T cell activity between the CAR constructs. Additionally, SCRI-CAR22v2 is currently under investigation in a dose finding phase 1 clinical trial, PLAT-07 (NCT04571138). Results: Following use of cetuximab-APC and biotinylated anti-human Fab antibody for surface EGFRt and CAR detection, the SCRI-CAR22v1 expresses lower levels of EGFRt but similar CAR levels on the cell surface demonstrated by MFI (Figure B). Biotinylated, soluble CD22 antigen was also used to evaluate CD22 CAR receptor activity and, as measured by MFI, a higher affinity is suggested via SCRI-CAR22v2 as compared to SCRI-CAR22v1 (Figure B). K562 cells expressing low, medium or high CD22 were used to evaluate the impact of surface antigen expression on the CAR activity level. SCRI-CAR22v2 demonstrates improved targeted cell lysis at all 3 antigen quantity levels by chromium release assay (Figure C). In NSG mice inoculated with Raji tumor cells expressing ffluc, SCRI-CAR22v2 demonstrated improved survival compared to SCRI-CAR22v1 (Figure D) and clearance of Raji tumor cells (Figure E). Based on this promising preclinical data, we initiated enrollment onto PLAT-07, a phase 1 dose finding trial (2x10 5cells/kg (DL1), 5x10 5cells/kg (DL2) and 1x10 6cells/kg (DL3)) of SCRI-CAR22v2. To date, 3 subjects have been enrolled and successfully infused at DL1. All had prior CD19-CAR therapy and 2 lacked CD19 leukemic expression at the time of SCRI-CAR22v2 infusion. At the time of cell infusion, one subject had only extramedullary disease, one had MRD of <1% and one subject had a larger disease burden of 30% ALL. None experienced a DLT and all were MRD negative in the bone marrow at day 28 and the subject with EMD demonstrated a complete metabolic response by PET scan. Figure F exhibits the improved expansion and engraftment of the SCRI-CAR22v2 cells as compared to SCRI-CAR22v1 DL1 (n=3) and DL2 (n=1), and higher peak levels of CD22 CAR T cells as compared to SCRI-CAR22v1 DL1 and DL2 (Figure G). Conclusions: Despite encouraging preclinical data, SCRI-CAR22v1 demonstrated poor expansion and engraftment in a Phase 1 trial. Notably, minor CAR alterations lead to encouraging in-human activity in early clinical findings. Our experience suggests a shorter linker and hinge as well as incorporation of an CD8 alpha transmembrane region improves the clinical activity of CD22 targeted CAR T cells in subjects with recurrent disease following CD19 CAR T cells. Further evaluation is needed to elucidate the critical CAR components and/or assays at the preclinical level that can best predict which CAR should be brought to the clinic for further evaluation. Figure 1 Figure 1. Disclosures Orentas: Lentigen: Patents & Royalties. Jensen: BMS: Patents & Royalties; Umoja Biopharma: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bluebird Bio: Research Funding. Gardner: Novartis: Consultancy; BMS: Patents & Royalties.

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548 Phase 2 study of FLX475 in combination with ipilimumab in advanced melanoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.548 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A578-A578

Author(s):

Rakesh Goyal ◽

Nicole Nasrah ◽

Dan Johnson ◽

William Ho

Keyword(s):

Clinical Trial ◽

T Cells ◽

Antitumor Activity ◽

Regulatory T Cells ◽

Response Rate ◽

Advanced Melanoma ◽

Phase 2 ◽

Car T Cells ◽

Phase 2 Study ◽

Car T

BackgroundRegulatory T cells (Treg) can dampen antitumor immune responses in the tumor microenvironment (TME) and have been shown to correlate with poor clinical outcome. Translational studies have demonstrated an accumulation of Treg in tumors after treatment with immunotherapies including CAR-T cells and anti-CTLA-4, which could potentially reflect a mechanism of adaptive immune resistance.1–2 CCR4, the receptor for the chemokines CCL17 and CCL22, is the predominant chemokine receptor on human Treg and is responsible for the migration and accumulation of Treg in the TME. Preclinical studies with orally available CCR4 antagonists have demonstrated potent inhibition of Treg migration into tumors, an increase in the intratumoral Teff/Treg ratio, and antitumor efficacy as a single agent and in combination with checkpoint inhibitors, including anti-CTLA-4.3 In a first-in-human trial conducted in healthy volunteers, the oral CCR4 antagonist FLX475 was demonstrated to be well tolerated with outstanding pharmacokinetic and pharmacodynamic properties.4 An ongoing Phase 1/2 clinical trial of FLX475 is examining the safety and preliminary antitumor activity of FLX475 as monotherapy and in combination with pembrolizumab in subjects with several types of advanced cancer.5 Given the preclinical data demonstrating a significant enhancement of the antitumor activity of anti-CTLA-4 when combined with FLX475, a Phase 2 study investigating the combination of FLX475 and ipilimumab is now being conducted in subjects with advanced melanoma.MethodsThis clinical trial is a Phase 2, multicenter, open-label, single-arm study to determine the antitumor activity of FLX475 in combination with ipilimumab in subjects with advanced melanoma previously treated with an anti-PD-1 or anti-PD-L1 agent. The primary objectives of the study are to evaluate objective response rate, and the safety and tolerability of this combination. The study will first examine the safety of the combination of the 100 mg PO QD recommended Phase 2 dose of FLX475 and the approved 3 mg/kg IV Q3W dose of ipilimumab as part of a safety run-in phase, prior to examining the degree of antitumor activity in approximately 20 subjects. Evidence of an overall response rate (ORR) notably greater than the expected ORR of ipilimumab monotherapy alone in such subjects, which has been shown to be approximately 14%,6 would provide preliminary clinical evidence in support of the clinical hypothesis that CCR4 blockade by FLX475 can significantly enhance the antitumor activity of an anti-CTLA-4 checkpoint inhibitor.Trial RegistrationClinicalTrials.gov Identifier: NCT04894994ReferencesO’Rourke D, Nasrallah M, Desai A, Melenhorst J, Mansfield K, Morrissette J, Martinez-Lage M, Brem S, Maloney E, Shen A, Isaacs R, Mohan S, Plesa G, Lacey S, Navenot J, Zheng Z, Levine B, Okada H, June C, Brogdon J, Maus M. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med 2017;9:eaaa0984. doi: 10.1126/scitranslmed.aaa0984.Sharma A, Subudhi S, Blando J, Vence L, Wargo J, Allison JP, Ribas A, Sharma P. Anti-CTLA-4 immunotherapy does not deplete FOXP3+ regulatory T cells (Tregs) in human cancers-Response. Clin Cancer Res 2019;25:1233–1238.Marshall L, Marubayashi S, Jorapur A, Jacobson S, Zibinsky M, Robles O, Hu D, Jackson J, Pookot D, Sanchez J, Brovarney M, Wadsworth A, Chian D, Wustrow D, Kassner P, Cutler G, Wong B, Brockstedt D, Talay O. Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4. J Immunother Cancer 2020;8:e000764.van Marle S, van Hoogdalem E, Johnson D, Okal A, Kassner P, Wustrow D, Ho W, Smith S. Pharmacokinetics, pharmacodynamics, and safety of FLX475, an orally-available, potent, and selective small-molecule antagonist of CCR4, in healthy volunteers. J Immunother Cancer 2018; 6(Suppl 1):P484(SITC 2018).Powderly J, Chmielowski B, Brahmer J, Piha-Paul S, Bowyer S, LoRusso P, Catenacci D, Wu C, Barve M, Chisamore M, Nasrah N, Johnson D, Ho W. Phase I/II dose-escalation and expansion study of FLX475 alone and in combination with pembrolizumab in advanced cancer. Journal of Clinical Oncology 2020;38(15_suppl): TPS3163 (ASCO 2020).Long G, Mortier L, Schachter J, Middleton M, Neyns B, Sznol M, Zhou H, Ebbinghaus S, Ibrahim N, Arance A, Ribas A, Blank C and Robert C. Society for Melanoma Research 2016 Congress. Pigment Cell & Melanoma Research 2017;30:76–156.Ethics ApprovalThis study has been approved by the Institutional Review Board at each investigational site.

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Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

Nature Communications ◽

10.1038/s41467-020-19672-2 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Lintao Liu ◽

Enguang Bi ◽

Xingzhe Ma ◽

Wei Xiong ◽

Jianfei Qian ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Effector Memory ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

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Baseline and early post-treatment clinical and laboratory factors associated with severe neurotoxicity following 19-28z CAR T cells in adult patients with relapsed B-ALL.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.7024 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 7024-7024 ◽

Cited By ~ 14

Author(s):

Jae Hong Park ◽

Bianca Santomasso ◽

Isabelle Riviere ◽

Brigitte Senechal ◽

Xiuyan Wang ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

T Cell ◽

Blood Parameters ◽

Serum Biomarkers ◽

Post Treatment ◽

Factors Associated ◽

Car T Cells ◽

Car T

7024 Background: CD19-specific chimeric antigen receptor (CAR) modified T cells produce high anti-tumor activity in relapsed or refractory (R/R) ALL, but can be associated with cytokine release syndrome (CRS) and neurotoxicity (NTX). Herein, we report baseline and post-treatment clinical and laboratory factors associated with severe NTX (≥Grade 3) in our phase I clinical trial of CD19-specific 19-28z CAR T cells for adult patients (pts) with R/R B-ALL (NCT01044069). Methods: 51 adult pts with R/R B-ALL were treated with 19-28z CAR T cells following conditioning chemotherapy at MSKCC. In order to identify clinical and serum biomarkers associated with severe NTX (sNTX), we examined demographic, treatment, and clinical blood parameters as well as in vivo CAR T expansion and serum cytokines, and performed univariate and multivariate analysis. Results: In this cohort of ALL pts, 20, 8, 2, 18 and 3 pts experienced Gr 0, 1, 2, 3, and 4 NTX, respectively. No pt developed grade 5 NTX. Disease burden (≥50% blasts) at the time of T cell infusion (p = 0.0045) and post-treatment ≥Gr3 CRS (p = 0.0010) were significantly associated with sNTX, but we found no association with age, weight, T cell dose, choice of conditioning chemotherapy (Flu/Cy s. Cy), and prior lines of treatment. Among the clinical and blood parameters, fever, low PLT, high ferritin and MCHC as well as elevated GM-CSF, IFNγ, IL-15, IL-5, IL-10, IL-2 at day 3 of T cell infusion at day 3 of T cell infusion were significantly associated with sNTX (all p < 0.01). While some of these cytokines were also elevated in severe CRS cases, IL-5 and IL-2 at day 3 were unique to sNTX. Furthermore, in vivo peak CAR T expansion at day 7 (p = 0.0001) significantly correlated with sNTX (p < 0.01). Lastly, multivariate analysis revealed baseline PLT < 60 or MCHC > 33.2% and morphologic disease ( > 5% blasts) has 95% sensitivity and 70% specificity of identifying sNTX pts. Conclusions: These data provide a characterization of early clinical and serum biomarkers of sNTX in adult pts receiving 19-28z CAR T cells and should help identify appropriate pts for early intervention strategy to mitigate NTX. Clinical trial information: NCT01044069.

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BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved antitumor activity

10.1101/2021.11.17.469041 ◽

2021 ◽

Author(s):

Taylor L Hickman ◽

Eugene Choi ◽

Kathleen R Whiteman ◽

Sujatha Muralidharan ◽

Tapasya Pai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Solid Tumor ◽

Cell Function ◽

Cell Metabolism ◽

Car T Cells ◽

Car T

Purpose: The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. Experimental Design: We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. Results: Expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. Conclusions: Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).

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The S enantiomer of 2-hydroxyglutarate increases central memory CD8 populations and improves CAR-T therapy outcome

Blood Advances ◽

10.1182/bloodadvances.2020002309 ◽

2020 ◽

Vol 4 (18) ◽

pp. 4483-4493

Author(s):

Iosifina P. Foskolou ◽

Laura Barbieri ◽

Aude Vernet ◽

David Bargiela ◽

Pedro P. Cunha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Cd8 T Cells ◽

Ex Vivo ◽

Phase 1 ◽

Therapeutic Success ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

Abstract Cancer immunotherapy is advancing rapidly and gene-modified T cells expressing chimeric antigen receptors (CARs) show particular promise. A challenge of CAR-T cell therapy is that the ex vivo–generated CAR-T cells become exhausted during expansion in culture, and do not persist when transferred back to patients. It has become clear that naive and memory CD8 T cells perform better than the total CD8 T-cell populations in CAR-T immunotherapy because of better expansion, antitumor activity, and persistence, which are necessary features for therapeutic success and prevention of disease relapse. However, memory CAR-T cells are rarely used in the clinic due to generation challenges. We previously reported that mouse CD8 T cells cultured with the S enantiomer of the immunometabolite 2-hydroxyglutarate (S-2HG) exhibit enhanced antitumor activity. Here, we show that clinical-grade human donor CAR-T cells can be generated from naive precursors after culture with S-2HG. S-2HG–treated CAR-T cells establish long-term memory cells in vivo and show superior antitumor responses when compared with CAR-T cells generated with standard clinical protocols. This study provides the basis for a phase 1 clinical trial evaluating the activity of S-2HG–treated CD19-CAR-T cells in patients with B-cell malignancies.

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CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo

Blood ◽

10.1182/blood-2011-03-344275 ◽

2012 ◽

Vol 119 (3) ◽

pp. 696-706 ◽

Cited By ~ 180

Author(s):

De-Gang Song ◽

Qunrui Ye ◽

Mathilde Poussin ◽

Gretchen M. Harms ◽

Mariangela Figini ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Car T Cells ◽

Human T Cell ◽

Human T Cells ◽

Car T ◽

Cd27 Costimulation

AbstractThe costimulatory effects of CD27 on T lymphocyte effector function and memory formation has been confined to evaluations in mouse models, in vitro human cell culture systems, and clinical observations. Here, we tested whether CD27 costimulation actively enhances human T-cell function, expansion, and survival in vitro and in vivo. Human T cells transduced to express an antigen-specific chimeric antigen receptor (CAR-T) containing an intracellular CD3 zeta (CD3ζ) chain signaling module with the CD27 costimulatory motif in tandem exerted increased antigen-stimulated effector functions in vitro, including cytokine secretion and cytotoxicity, compared with CAR-T with CD3ζ alone. After antigen stimulation in vitro, CD27-bearing CAR-T cells also proliferated, up-regulated Bcl-XL protein expression, resisted apoptosis, and underwent increased numerical expansion. The greatest impact of CD27 was noted in vivo, where transferred CAR-T cells with CD27 demonstrated heightened persistence after infusion, facilitating improved regression of human cancer in a xenogeneic allograft model. This tumor regression was similar to that achieved with CD28- or 4-1BB–costimulated CARs, and heightened persistence was similar to 4-1BB but greater than CD28. Thus, CD27 costimulation enhances expansion, effector function, and survival of human CAR-T cells in vitro and augments human T-cell persistence and antitumor activity in vivo.

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