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Cancer Res Commun

Cancer Res Commun

Cancer Research Communications

2767-9764

American Association for Cancer Research

CRC-22-0200

10.1158/2767-9764.CRC-22-0200

Version of Record

Research Article

Immunology

Immune Responses to Cancer

Immunomodulation

Hematological Cancers

Leukemias

Immunotherapy

Engineered/CAR T cells

Enhanced Costimulatory Signaling Improves CAR T-cell Effector Responses in CLL

CLL Costimulatory Phenotype Modulates CAR T-cell Responses

Collins McKensie A. Conceptualization Data curation Formal analysis Investigation Writing - original draft 123

Jung In-Young Investigation Writing - review and editing 24

https://orcid.org/0000-0002-8349-5322

Zhao Ziran Investigation Writing - review and editing 123

Apodaca Kimberly Investigation Writing - review and editing 23

Kong Weimin Investigation Methodology Writing - review and editing 24

Lundh Stefan Investigation Writing - review and editing 12

https://orcid.org/0000-0001-7900-8993

Fraietta Joseph A. Writing - review and editing 12345

https://orcid.org/0000-0003-3190-1891

Kater Arnon P. Conceptualization Resources Writing - review and editing 6

https://orcid.org/0000-0001-8498-4729

Sun Clare Conceptualization Resources Writing - review and editing 7

Wiestner Adrian Conceptualization Resources Writing - review and editing 7

https://orcid.org/0000-0001-7677-537X

Melenhorst J. Joseph Conceptualization Supervision Funding acquisition Methodology Writing - review and editing 1235

1 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

2 Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

3 Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

4 Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

5 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

6 Amsterdam UMC, University of Amsterdam, Department of Hematology, Cancer Center Amsterdam, Lymphoma and Myeloma Center Amsterdam, Amsterdam, the Netherlabds.

7 National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland.

Corresponding Author: J. Joseph Melenhorst, Center for ImmunoTherapy and Precision Immuno-Oncology, Cleveland Clinic Lerner Research Institute, 2111 East 96th Street, Cleveland, OH 44106. Phone: 216-215-5725; E-mail: melenhj@ccf.org

9 2022

30 9 2022

2 9 10891103

21 5 2022

17 8 2022

22 8 2022

© 2022 The Authors; Published by the American Association for Cancer Research

2022

Copyright held by the owner/author(s).

https://creativecommons.org/licenses/by/4.0/ This open access article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.

CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling.

Significance:

CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses.

http://dx.doi.org/10.13039/100014547 Parker Institute for Cancer Immunotherapy (Parker Institute) Collins McKensie A. Jung Inyoung Zhao Ziran Apodaca Kimberly Kong Weimin Lundh Stefan Fraietta Joseph A. Kater Arnon P. Sun Clare Wiestner Adrian Melenhorst J. Joseph http://dx.doi.org/10.13039/100000002 HHS | National Institutes of Health (NIH) Sun Clare Wiestner Adrian crossmarktrue

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pmcIntroduction

Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy that accounts for nearly one-third of adult leukemia diagnoses in the West (1). Standard-of-care chemoimmunotherapies and small molecules are initially efficacious but the majority of patients inevitably relapse with progressive disease (2). The only reliably curative therapy is allogeneic hematopoietic stem cell transplantation, but this comes with its own challenges, namely high morbidity and mortality, especially with a mainly elderly population and the difficulties associated with finding HLA-matched donors. CD19-directed cell therapies have shown promise, but with the exception of recent combination therapy trials where complete response rates can reach 40+% (3–5), only around one-fourth of patients with CLL treated with CD19-directed CAR T-cell therapy will achieve a complete remission (6–10). This was unexpected as the same therapy in pediatric acute lymphoblastic leukemia (ALL) has a complete response rate >80% (11–18). Further study to understand both tumor and T-cell biology in CLL is therefore warranted.

CLL is phenotypically, clinically, and genetically a highly heterogeneous disease. Patients stratify into fast and slow-progressing groups based on tumor immunoglobulin heavy chain variable region mutation status (19), driver and second-hit mutation status (20–23), and expression of ZAP70 (24–26) and CD38 (27–30). In addition, patients with CLL suffer from immune dysfunction mediated by maintenance of a protumor microenvironment (31–37) and endogenous T-cell dysfunctions including dysregulated cytokine production (38), reduced proliferation and cytotoxicity (39), and unstable immune synapse formation (40–42). The combined effects of CLL disease biology and impaired T-cell function influence the efficacy of autologous CAR T-cell therapies. Alterations in T-cell biology including terminal differentiation (43), exhaustion (39, 43–45), inverted CD4/CD8 ratios (43), impaired T-cell metabolic fitness (46, 47), and the advanced age of the patient population all impact clinical response rates (6, 48–50).

Significant work has been done to understand clinical progression in CLL. However, the field lacks a complete understanding of how tumor cells and engineered T cells interact and the subsequent impact on cell-based therapies. CLL cells residing in different biological niches have different phenotypes and activation profiles. The lymph node (LN), considered the “birth place” of CLL, is a highly organized, B-cell supportive environment (31–34), wherein CLL cells proliferate and maintain an activated profile (51–53) with higher expression of costimulatory and adhesion molecules (54–56). Peripheral blood (PB) CLL cells on the other hand, are generally unactivated and noncycling (50). Most CLL studies use PB-derived tumor cells, as these are the easiest to obtain. However, the differences in tumor phenotype between the LN and PB demonstrate that using only PB-derived CLL cells may inaccurately predict antitumor responses in other compartments.

CD40L-expressing, CD28ζ-signaling CAR T cells show enhanced CAR T-cell activation and improved the antigen-presenting cell (APC) phenotype of CLL cells (57). We therefore sought to use CD40L-mediated CLL activation to mimic the phenotype of LN-derived cells and directly test the impact of enhanced costimulation on 4-1BB–signaling CAR T-cell function. We address the gap in knowledge in understanding how CLL cells negatively impact allogeneic CAR T-cell products and how tumor cells from different compartments interact with these therapies. To study this, we developed an in vitro system wherein we show that CLL-mediated T-cell defects are also seen in a healthy donor–derived CAR T-cell setting; these dysfunctions are therefore not dependent on the endogenous T-cell defects seen in a patient setting, as detailed above. This is of interest as allogeneic therapies become more developed, particularly in a disease such as CLL where patients may benefit from a more-potent allogeneic CAR T-cell product. We show that these defects are rescuable, and attributable to poor costimulation by CLL cells, rather than permanent dysfunction or immune suppression. Furthermore, we show that LN-resident CLL cells are better targets for CAR T-cell lytic activity than circulating tumor cells, and this costimulatory phenotype can be modeled in vitro. Herein we describe the consequences of CLL/CAR T-cell interactions and show that the activated CLL compartment drives antitumor CAR T-cell responses. This represents a rational point of departure to design the next generation of cell-based therapeutics for this disease.

Materials and Methods

Antibodies and Flow Cytometry

The following antibodies and fluorescent reagents were used in this study: Live/Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, #L34957), Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG F(abʹ)2 Fragment Specific (Jackson ImmunoResearch, #115-065-072, RRID:AB_2338565), PE-streptavidin (BioLegend, #405204, RRID:AB_2921282), and PE-anti-CAR19 (Novartis, custom antibody). Additional antibodies can be found in Supplementary Table S1. Cell sorts were performed on a FACS Aria II machine managed by the University of Pennsylvania Flow Core.

Cell Lines

The artificial APC (aAPC) cell lines were created and validated in-house by transducing K562 cells (ATCC, CCL-243, RRID: CVCL_0004) to stably express CD64, CD86, 4-1BBL, and either ROR1 or CD19. The CD40 ligand–expressing Ltk cell line (Ltk-CD40L; refs. 58, 59) was obtained from Dr. Cees van Kooten (Leiden University Medical Center, Leiden, the Netherlands). Cells were tested for Mycoplasma using the Cambrex MycoAlert kit (Promega, #LT07-118). The most recent test was in February, 2022. Furthermore, the cell lines have been regularly authenticated by the University of Arizona Genetics Core using short tandem repeat profiling.

Paired LN and PB CLL Samples

Patients were enrolled in the Institutional Review Board–approved protocol: Natural History Study of monoclonal B-cell lymphocytosis, CLL/small lymphocytic lymphoma, lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia, and splenic marginal zone lymphoma (ClinicalTrials.gov number NCT00923507). Samples were obtained after written informed consent in accordance with the Declaration of Helsinki, and applicable federal regulations. PB mononuclear cells (PBMC) were isolated by density gradient centrifugation and cryopreserved. LN biopsies were mechanically disaggregated into single-cell suspensions and cryopreserved.

CAR T-cell Generation

Normal donor apheresis products were obtained from the University of Pennsylvania Human Immunology Core. PBMCs were isolated via Ficoll density gradient centrifugation and cryopreserved in 70% OpTmizer 5 (OpT5) medium, 20% human AB serum, and 10% DMSO. T cells were isolated using the Miltenyi Pan T cell Isolation Kit (Miltenyi Biotec, #130-096-535) and cultured in OpT5 medium with T-cell expansion supplement (Thermo Fisher Scientific, #A1048501), 2 mmol/L Glutamax, and 5% human AB serum supplemented with 100 U/mL hIL2 unless otherwise stated. Cells were activated using anti-CD3/anti-CD28 Dynabeads at a ratio of 3 beads:1 T cell. T cells were transduced with CAR-expressing lentivirus on day 1. Cells were counted and resuspended at 5.0 × 105 cells/mL on days 3, 5, and 7. On day 9, cells were counted and cryopreserved. Absolute cell counts were obtained using the Luna fluorescence-based automatic cell counter (Logos Biosystems).

Lentivirus Production

VSVg-pseudotyped anti-CD19 or anti-ROR1 CAR (CAR19 and CAR-ROR1, respectively) lentivirus was produced using HEK293T cells (ATCC, CRL-11268). Cells were seeded on day −1 in R10 medium (RPMI + 10% FBS + 1% Penicillin/Streptomycin). On day 0, the cells were transfected with VSVg, RSV/Rev, Gag/Pol, and CAR plasmids using lipofectamine 2000 (Thermo Fisher Scientific, #11668019). Supernatant was collected at 24 and 48 hours and concentrated using an ultracentrifuge overnight at 4°C and 8,861 Relative Centrifugational Force (RCF), followed by 2.5 hours at 4°C at 76,800 RCF. Virus was aliquoted and stored at −80°C.

B-CLL Isolation

Primary CLL PBMCs were obtained from the Stem Cell and Xenograft Core at the University of Pennsylvania (Philadelphia, PA). B-CLL cells were isolated using the Miltenyi B-CLL Isolation Kit (Miltenyi Biotec, #130-103-466). A full list of CLL donors used can be found in Supplementary Table S2.