Dynamic Profiling of Antitumor Activity of CAR T Cells Using Micropatterned Tumor Arrays

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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SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models

Science Translational Medicine ◽

10.1126/scitranslmed.abd8836 ◽

2021 ◽

Vol 13 (591) ◽

pp. eabd8836

Author(s):

Axel Hyrenius-Wittsten ◽

Yang Su ◽

Minhee Park ◽

Julie M. Garcia ◽

Josef Alavi ◽

...

Keyword(s):

Ovarian Cancer ◽

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Solid Tumors ◽

Mouse Models ◽

Solid Tumor ◽

Car T Cells ◽

Therapeutic Benefits ◽

Car T

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening “on-target/off-tumor” toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.

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Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells

Advanced Materials ◽

10.1002/adma.201900192 ◽

2019 ◽

Vol 31 (23) ◽

pp. 1900192 ◽

Cited By ~ 71

Author(s):

Qian Chen ◽

Quanyin Hu ◽

Elena Dukhovlinova ◽

Guojun Chen ◽

Sarah Ahn ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Photothermal Therapy ◽

Tumor Infiltration ◽

Car T Cells ◽

Car T

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Photothermal Therapy: Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells (Adv. Mater. 23/2019)

Advanced Materials ◽

10.1002/adma.201970166 ◽

2019 ◽

Vol 31 (23) ◽

pp. 1970166 ◽

Cited By ~ 6

Author(s):

Qian Chen ◽

Quanyin Hu ◽

Elena Dukhovlinova ◽

Guojun Chen ◽

Sarah Ahn ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Photothermal Therapy ◽

Tumor Infiltration ◽

Car T Cells ◽

Car T

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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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Inducible Activation of MyD88 and CD40 in CAR T Cells Results in Controllable and Potent Antitumor Activity in Preclinical Solid Tumor Models

Cancer Discovery ◽

10.1158/2159-8290.cd-17-0263 ◽

2017 ◽

Vol 7 (11) ◽

pp. 1306-1319 ◽

Cited By ~ 43

Author(s):

Melinda Mata ◽

Claudia Gerken ◽

Phuong Nguyen ◽

Giedre Krenciute ◽

David M. Spencer ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Solid Tumor ◽

Tumor Models ◽

Car T Cells ◽

Car T ◽

Inducible Activation

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

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.tps2647 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. TPS2647-TPS2647 ◽

Cited By ~ 1

Author(s):

David Henry Michael Steffin ◽

Sai A Batra ◽

Purva Rathi ◽

Linjie Guo ◽

Wenpeng Li ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

Antitumor Activity ◽

Immune Escape ◽

Liver Tumors ◽

Phase 1 ◽

Car T Cells ◽

Gene And Protein Expression ◽

Car T

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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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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Antitumor activity of CAR-T cells targeting the intracellular oncoprotein WT1 can be enhanced by vaccination

Blood ◽

10.1182/blood-2017-08-802926 ◽

2018 ◽

Vol 132 (11) ◽

pp. 1134-1145 ◽

Cited By ~ 14

Author(s):

Yasushi Akahori ◽

Linan Wang ◽

Motohiro Yoneyama ◽

Naohiro Seo ◽

Satoshi Okumura ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Tumor Cell ◽

Xenograft Model ◽

Tumor Cell Lines ◽

Car T Cells ◽

Dc Vaccine ◽

Murine Xenograft Model ◽

Key Points ◽

Car T

Key Points T cells expressing a CAR consisting of scFv #213 targeting WT1 peptide/HLA-A*2402 complex killed HLA-A*2402+ WT1+ tumor cell lines. The therapeutic efficacy of #213 scFv CAR-T cells was shown to be enhanced by DC vaccine in a murine xenograft model.

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Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models

Cancers ◽

10.3390/cancers13174301 ◽

2021 ◽

Vol 13 (17) ◽

pp. 4301

Author(s):

Marianna Csaplár ◽

János Szöllősi ◽

Stephen Gottschalk ◽

György Vereb ◽

Árpád Szöőr

Keyword(s):

T Cells ◽

T Cell ◽

Antitumor Activity ◽

Cytolytic Activity ◽

Effector Memory ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Differentiation Status

Correlative studies of clinical studies for hematological malignancies have implicated that less differentiated, CD8+-dominant CAR T cell products have greater antitumor activity. Here, we have investigated whether the differentiation status of CAR T cell products affects their antitumor activity in preclinical models of solid tumors. We explored if different activation/expansion protocols, as well as different co-stimulatory domains in the CAR construct, influence the short- and long-term efficacy of CAR T cells against HER2-positive tumors. We generated T cell products that range from the most differentiated (CD28.z; OKT3-antiCD28/RPMI expansion) to the least differentiated (41BB.z; OKT3-RetroNectin/LymphoONE expansion), as judged by cell surface expression of the differentiation markers CCR7 and CD45RA. While the effect of differentiation status was variable with regard to antigen-specific cytokine production, the most differentiated CD28.z CAR T cell products, which were enriched in effector memory T cells, had the greatest target-specific cytolytic activity in vitro. These products also had a greater proliferative capacity and maintained CD4+ T cells upon repeated stimulation in vitro. In vivo, differentiated CD28.z CAR T cells also had the greatest antitumor activity, resulting in complete response. Our results highlight that it is critical to optimize CAR T cell production and that optimal product characteristics might depend on the targeted antigen and/or cancer.

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