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(CAR) T-celleterapi har vist betydeligt lovende for hæmatologiske maligniteter, hvilket har ført til, at US Food and Drug Administration har godkendt to CAR T-celle-baserede behandlinger til behandling af B-celle akut lymfatisk leukæmi og stor B-celle lymfom.

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Affiliations

¹ Department of Clinical Science, and.

² Department of Blood and Marrow Transplantation and Cellular Immunotherapy, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

³ Morsani College of Medicine, University of South Florida, Tampa, Florida, USA.

⁴ Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

Affiliations

¹ Department of Clinical Science, and.

² Department of Blood and Marrow Transplantation and Cellular Immunotherapy, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

³ Morsani College of Medicine, University of South Florida, Tampa, Florida, USA.

⁴ Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

Abstract

Chimeric antigen receptor (CAR) T cell therapy has shown considerable promise for hematologic malignancies, leading to the US Food and Drug Administration approval of two CAR T cell-based therapies for the treatment of B cell acute lymphoblastic leukemia and large B cell lymphoma. Despite success in hematologic malignancies, the treatment landscape of CAR T cell therapy for solid tumors has been limited. There are unique challenges in the development of novel CAR T cell therapies to improve both safety and efficacy. Improved understanding of the immunosuppressive tumor microenvironment and resistance mechanisms has led to encouraging approaches to mitigating these obstacles. This Review will characterize challenges with current CAR T designs for hematologic malignancies and solid tumors and emphasize preclinical and clinical strategies to overcome them with novel CAR T cell therapies.

Conflict of interest statement

Conflict of interest: MLD and RF received research funding from Novartis. MLD receives research funding from Precigen, Atara Biotherapeutics, and ELICIO Therapeutics. MLD has received licensing fees from Atara Biotherapeutics for CAR intellectual property. MLD has equity in Precision Biosciences and Adaptive Biotechnologies and unexercised options with Bellicum and CERtainty.

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precursor b-cell lymphoblastic leukemia-lymphoma / therapy*

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national center for biotechnology information

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Insight into next-generation CAR therapeutics: designing CAR T cells to improve clinical outcomes - PubMed

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Chimeric antigen receptor (CAR) T cell therapy has shown considerable promise for hematologic malignancies, leading to the US Food and Drug Administration approval of two CAR T cell-based therapies for the treatment of B cell acute lymphoblastic leukemia and large B cell lymphoma. Despite success in ...

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How close are we to CAR T-cell therapy for AML?

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How close are we to CAR T-cell therapy for AML?

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https://www.sciencedirect.com/science/article/pii/S1521692619300933?via%3Dihub

Artiklen omhandler hvor tæt man er på at kunne anvende CAR T-celle terapi imod akut myeloid leukæmi (AML). I artiklen bliver det beskrevet at det er svært at finde et AML-specifikt overfladeantigen, som kan være en begrænsning for metoden. Derfor forslår artiklen tre forskellige løsninger på dette, som vil kunne skabe en sikker og effektiv CAR T-celle terapi.

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STING agonist promotes CAR T cell trafficking and persistence in breast cancer PMID: 33382402

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STING agonist promotes CAR T cell trafficking and persistence in breast cancer PMID: 33382402

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CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from Th/Tc17 cells given with the STING agonists DMXAA or cGAMP greatly enhanced tumor control, which was associated with enhanced CAR T cell persistence in the TME. Using single-cell RNA sequencing, we demonstrate that DMXAA promoted CAR T cell trafficking and persistence, supported by the generation of a chemokine milieu that promoted CAR T cell recruitment and modulation of the immunosuppressive TME through alterations in the balance of immune-stimulatory and suppressive myeloid cells. However, sustained tumor regression was accomplished only with the addition of anti-PD-1 and anti-GR-1 mAb to Th/Tc17 CAR T cell therapy given with STING agonists. This study provides new approaches to enhance adoptive T cell therapy in solid tumors.

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https://pubmed.ncbi.nlm.nih.gov/31754328/

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Affiliations

¹ Shanghai Baize Medical Laboratory, Shanghai, China.

² Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.

³ Shanghai Cell Therapy Research Institute, Shanghai, China.

⁴ Shanghai Engineering Research Center for Cell Therapy, Shanghai, China.

Free PMC article

Free PMC article

Affiliations

¹ Shanghai Baize Medical Laboratory, Shanghai, China.

² Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.

³ Shanghai Cell Therapy Research Institute, Shanghai, China.

⁴ Shanghai Engineering Research Center for Cell Therapy, Shanghai, China.

Abstract

Cancer immunotherapy by chimeric antigen receptor-modified T (CAR-T) cells has shown exhilarative clinical efficacy for hematological malignancies. Recently two CAR-T cell based therapeutics, Kymriah (Tisagenlecleucel) and Yescarta (Axicabtagene ciloleucel) approved by US FDA (US Food and Drug Administration) are now used for treatment of B cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) respectively in the US. Despite the progresses made in treating hematological malignancies, challenges still remain for use of CAR-T cell therapy to treat solid tumors. In this landscape, most studies have primarily focused on improving CAR-T cells and overcoming the unfavorable effects of tumor microenvironment on solid tumors. To further understand the current status and trend for developing CAR-T cell based therapies for various solid tumors, this review emphasizes on CAR-T techniques, current obstacles, and strategies for application, as well as necessary companion diagnostics for treatment of solid tumors with CAR-T cells.

Keywords: CAR-T cells; CTC; chimeric antigen receptor; companion diagnostics; solid tumors.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Resistance to Immunotherapy: Mechanisms and Means for Overcoming.

Salkeni MA, Shin JY, Gulley JL. Salkeni MA, et al. Adv Exp Med Biol. 2021;1342:45-80. doi: 10.1007/978-3-030-79308-1_2. Adv Exp Med Biol. 2021. PMID: 34972962

CD8⁺ T Cells Expressing an HLA-DR1 Chimeric Antigen Receptor Target Autoimmune CD4⁺ T Cells in an Antigen-Specific Manner and Inhibit the Development of Autoimmune Arthritis.

Whittington KB, Prislovsky A, Beaty J, Albritton L, Radic M, Rosloniec EF. Whittington KB, et al. J Immunol. 2022 Jan 1;208(1):16-26. doi: 10.4049/jimmunol.2100643. Epub 2021 Nov 24. J Immunol. 2022. PMID: 34819392

References

Fournier C, Martin F, Zitvogel L, Kroemer G, Galluzzi L, Apetoh L. Trial watch: adoptively transferred cells for anticancer immunotherapy. Oncoimmunology. 2017;6:e1363139. - PMC - PubMed

Zhang H, Ye ZL, Yuan ZG, Luo ZQ, Jin HJ, Qian QJ. New strategies for the treatment of solid tumors with CAR-T cells. Int J Biol Sci. 2016;12:718-29. - PMC - PubMed

Heczey A, Louis CU, Savoldo B, Dakhova O, Durett A, Grilley B. et al. CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther. 2017;25:2214-24. - PMC - PubMed

Rupp LJ, Schumann K, Roybal KT, Gate RE, Ye CJ, Lim WA. et al. CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 2017;7:737. - PMC - PubMed

Hegde UP, Mukherji B. Current status of chimeric antigen receptor engineered T cell-based and immune checkpoint blockade-based cancer immunotherapies. Cancer Immunol Immunother. 2017;66:1113-21. - PMC - PubMed

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national center for biotechnology information

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Current Progress in CAR-T Cell Therapy for Solid Tumors - PubMed

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1 Shanghai Baize Medical Laboratory, Shanghai, China. 2 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA. 3 Shanghai Cell Therapy Research Institute, Shanghai, China. 4 Shanghai Engineering Research Center for Cell Therapy, Shanghai, China. Free PMC article Free PMC article 1 Shanghai Baize Medical Laboratory, Shanghai, China.

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- I modsætning til hæmatologiske maligniteter har den kliniske erfaring med CAR T-celleterapi til solide tumorer og hjernetumorer været mindre opmuntrende, hvor kun få patienter har opnået fuldstændige svar. Kliniske og prækliniske undersøgelser har identificeret flere "vejspærringer", herunder (1) en begrænset række af målrettede antigener og heterogen antigenekspression, (2) begrænset T-celle-fitness og overlevelse, før de når tumorsteder, (3) en manglende evne hos T-celler til effektivt at trafik til tumorsteder og trænge ind i fysiske barrierer, og (4) et immunsuppressivt tumormikromiljø.

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Affiliations

¹ Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

² Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Graduate School of Biomedical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

³ Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address: stephen.gottschalk@stjude.org.

Free PMC article

Free PMC article

Affiliations

¹ Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

² Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA; Graduate School of Biomedical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

³ Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address: stephen.gottschalk@stjude.org.

Abstract

Chimeric antigen receptor (CAR) T cell therapy has garnered significant excitement due to its success for hematological malignancies in clinical studies leading to the US Food and Drug Administration (FDA) approval of three CD19-targeted CAR T cell products. In contrast, the clinical experience with CAR T cell therapy for solid tumors and brain tumors has been less encouraging, with only a few patients achieving complete responses. Clinical and preclinical studies have identified multiple "roadblocks," including (1) a limited array of targetable antigens and heterogeneous antigen expression, (2) limited T cell fitness and survival before reaching tumor sites, (3) an inability of T cells to efficiently traffic to tumor sites and penetrate physical barriers, and (4) an immunosuppressive tumor microenvironment. Herein, we review these challenges and discuss strategies that investigators have taken to improve the effector function of CAR T cells for the adoptive immunotherapy of solid tumors.

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national center for biotechnology information

# data[].attributes.attachment_link.title
CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality? - PubMed

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Chimeric antigen receptor (CAR) T cell therapy has garnered significant excitement due to its success for hematological malignancies in clinical studies leading to the US Food and Drug Administration (FDA) approval of three CD19-targeted CAR T cell products. In contrast, the clinical experience with ...

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For at forbedre resultaterne for de maligne sygdomme kigges der på Immunsystemet, der virker via kræftimmunovervågning, der betragtes som et potentielt mål for at

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Biallelic loss of BCMA as a resistance mechanism to CAR T cell therapy in a patient with multiple myeloma

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Biallelic loss of BCMA as a resistance mechanism to CAR T cell therapy in a patient with multiple myeloma

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https://www.nature.com/articles/s41467-021-21177-5

CAR-terapi har vist en serie af relaps, ved multiple myeloma, dette er påvist at være sammenhængende med genet for BCMA der mister funktion ved deletion på det ene allel, hvilket hæmmer CAR proliferation og det derfor er vigtigt at overvåge dette gen ved behandling af multiple myeloma.

BCMA B-Cell Maturation antigen; spiller en vigtigt rolle i plasma celle funktion, hvilket så ikke ville kunne støtte behandleingen efter anden infusion af CAR.

Feb. 2021

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Introduction

Chimeric antigen receptor (CAR) T-cell therapy targeting B-cell maturation antigen (BCMA) has provided frequent, deep, and durable responses in relapsed, refractory multiple myeloma (MM), with initial Phase I/II studies reporting 73-100% overall response and 31-69% complete response1,2,3. However, progression-free survival in some studies have been <12 months, indicating myeloma recurrence despite the persistence of CAR T cells in a number of cases1,2. Importantly, among the small number of patients retreated with the same CAR T cell product at the time of progression, responses have been infrequent4,5. This highlights development of acquired resistance mechanisms6,7, which may preclude effectiveness of the second CAR T infusion, and may also explain relapse following the initial CAR T-cell therapy.

In this work, by performing single-cell transcriptome profiling on serially collected bone marrow (BM) samples, we show biallelic loss of BCMA as one of the resistance mechanisms to anti-BCMA CAR T-cell therapy with Idecabtagene Vicleucel in a patient with initial response but relapse with resistance to retreatment with the same CAR T-cell product. Furthermore, our results also highlight that MM cells may develop alternative paths to survive without BCMA.

Results

We evaluated samples from an individual patient who was diagnosed with IgG lambda MM with hypodiploidy and a complex karyotype with t(8;12) (q24;q14), clonal t(11;14) (q13;q32), and clonal deletion 13. The patient was treated with four lines of therapy including proteasome inhibitor, immunomodulatory agent, and anti-CD38 antibody before CAR T-cell therapy, with limited response. The patient was enrolled in a Phase I trial (CRB-401 ClinicalTrials.gov number, NCT02658929) of anti-BCMA CAR T-cell therapy with Idecabtagene Vicleucel (ide-cel) and received 150 × 10 ⁶ CAR+ T cells at day 0 following lymphodepletion with fludarabine (30 mg/m ² per day) and cyclophosphamide (300 mg/m ² per day) on days −5, −4, and −3, as reported in ref. 1. The patient developed grade 1 cytokine release syndrome and achieved partial response by 3 months. The patient relapsed 9 months after the first CAR T infusion and was treated a second time with identical lymphodepletion and using the same CAR T-cell product as the first infusion but at a higher dose of 450 × 10 ⁶ CAR+ T cells with no response (Fig. 1A, B).

A M spike and lambda free light chain evaluations for the patient. The y axis on left shows the M spike values (blue) and on the right it shows the lambda free light chain values (green). Time points ( x axis) marked with red labels also shows the longitudinal sample collection for single-cell RNA sequencing. B Expansion of CAR T cells ( y axis) measured with qPCR after first (blue) and second (red) infusions from day 0 to day 60 ( x axis). C Timeline of the eight samples collected for single-cell RNA sequencing. D Thirteen single-cell clusters from eight longitudinal bone marrow samples. Annotation of cell clusters are marked in the bottom part with color codes. Cell embedings are shown by using UMAP1 and UMAP2. E Ide-cel expression in single cells. Only limited number of cells are CAR+ at 2 weeks after the first infusion. None of the other time points show CAR+ cells. F Re-clustered T cells divided by time points from study screening to 1 month after second infusion and T-cell annotations for CD4+ and CD8+ cells are shown with color codes. G Percentage of particular T-cell types ( y axis) at each time point ( x axis) evaluated with single-cell RNA seqeuecning for T-cell clusters (top figure legend). Percentages are reflecting the % of particular cluster at given time point within all T cell populations. H Gene-set enrichment FDR values for differentially expressed genes for two samples collected two weeks after first (blue) and second (green) infusions. I Percentage of T cells ( y axis) expressing immune checkpoint inhibitors at each time point ( x axis).

Changes in BM microenvironment post-CAR T-cell therapy

To delineate changes in BM cellular components as a potential mechanism underlying lack of response to CAR T-cell reinfusion, we performed single-cell transcriptome profiling on serially collected BM samples (Fig. 1C). Clustering analysis from 37,658 cells from 8 time points, before the first CAR T cell infusion to 1 month after the second infusion, identified 13 clusters consisting of hematopoietic cells and MM cells (Fig. 1D and Supplementary Fig. 1). The BM sample before the first infusion was depleted of CD138+ cells by cell selection. A small number of MM cells were observed at 2 weeks after the first infusion of the CAR T-cell therapy. Thereafter, MM cells became undetectable and remained undetectable until eight months after the first infusion, when biochemical as well as cytological relapse occurred (Fig. 2A and Supplementary Fig. 1B). We observed a predicted suppression of B-cell count at study entry as an effect of the MM cell growth, with B-cell recovery at 1 month coinciding with anti-MM response (3% of all cells) and reaching 18% at 8 months after first infusion (Supplementary Fig. 1B), and again suppressed to 3% at relapse. We detected CAR+ T cells in the BM only at 2 weeks after first infusion, when maximal CAR+ T-cell expansion was observed in blood using reverse-transcription PCR (RT-PCR)-based detection (Fig. 1B, E). We did not detect infused CAR T cells in the BM with single-cell transcriptome profiling after the second CAR T infusion, but a limited expansion was confirmed in the blood using RT-PCR (Fig. 1E). The 6-log expansion of CAR+ T cells in the blood after the first infusion is consistent with observed expansion in the KarMMA study, where 5.5 log expansion was observed in the responding patients with a median peak CAR+ T-cell expansion at day 112. A lower expansion (2-log) with the second infusion may represent environmental influences or MM-intrinsic factors.

A Cell embedings for plasma/multiple myeloma (MM) cells (green colors) and B cells (cream colors) are shown from screening to 2 weeks after second infusion ( x axis). B cells are first detected at 1 month after first infusion and increased frequency until relapse. MM cells are detected at the 8 months after first infusion sample and remain same for further time points. B Soluble BCMA (sBCMA) level ( y axis) at the study screening, after first infusion, relapse, and after second infusion ( x axis) are shown. Time points makerd with * indicates the time points scRNAseq data also available. Screening refers to screening before first infusion (S1). Day 14 and Months 3 are refering to two weeks after first infusion (S2) and 3 months after first infusion (S4). Retreatment Screening is the relapse after first infusion (S6) and Retreatment Day 14 (S7) is 2 weeks after second infusion. C Expression levels of CD138, CCND1, XBP1, and RB1 in multiple myeloma (MM) cells and B cells. Normalized expression level scales are shown with legends. D Copy number predictions for each single cell (columns) from single-cell RNA sequencing data for chromosomal arms (rows). Deletions are shown with blue and gains are shown with red color for MM cells (left) and B cells (right). E Copy number estimates for CD138+ cells after second CAR T-cell infusion using whole exome sequencing. The top panel displays total copy number log-ratio where diploid state is shown with purple line and the second panel displays allele-specific log odds ratio data for allele-specific copy number calls with chromosomes alternating in blue and gray. Third panel shows the corresponding integer (black line for total copy number, red line for minor copy number) copy number calls. The bottom panel shows the predicted clonality of each events. Dark blue colors show regions with colonal copy number alterations and light blue color shows subclonal copy number events. F Percentage ( x axis) of single multiple myeloma/plasma cells with various copy number deletions (del) or gains ( y axis). Copy number events for each cell predicted using single-cell RNA sequencing. G Somatic mutaitons detected at relapse after second infusion with whole exome sequencing. Nonsense mutation which creates early stop codon in BCMA (top panel) and missense TP53 mutation (bottom panel) are shown in their amino acid locations. Protein domains are shown with color codes in each genes. H Clonal evolution of MM cells from diagnosis to relapse after second CAR T-cell infusion. I Co-occurrences of deletion 17p (del17p) and deletion 16p (del16p) on large-scale MM cohort. Clonal (red color), subclonal (yellow) deletions are shown for newly diagnosed MM patients (columns) and only patients with del16p and/or del17p are shown.

Re-clustering of the T-cell cluster showed an increased proportion of CD4+ helper and T-regulatory cells (Treg) 2 weeks after first infusion (Fig. 1F, G and Supplementary Fig. 2), and these two clusters had high expression of proliferation-related genes (Fig. 1H and Supplementary Data 1 and 2). However, Treg proportion remained similar at the second infusion, ruling out its impact on lack of expansion of CAR+ T cells. To detect any unusual endogenous T-cell activity that may potentially affect CAR T-cell function, we investigated inhibitory markers CD274 ( PD-L1), PDCD1 ( PD-1), LAG3, TIGIT at early and late time points including after second infusion. As can be seen (Fig. 1I and Supplementary Table 1), at no time point the proportion of the cells expressing these checkpoint inhibitors is higher then the base line. Moreover, the absence of change in these markers on endogenous T cells does not mean that the CAR+ T cells do not express these markers. However, in absence of detectable CAR T cells there is no direct way to look at expressed inhibitory markers on CAR T cells. Therefore, future studies will require investigation of resistance associated with presence of checkpoint inhibitors.

Role of tumor intrinsic factors in resistance

As we did not delineate a role of the BM milieu mediating suppression of CAR T-cell expansion and function following second infusion, we next explored tumor intrinsic factors. We evaluated soluble BCMA (sBCMA) level (produced predominantly by MM cells) in serum at different time points, and observed high levels before the first CAR T cell infusion, which dropped significantly to a very low level coincident with the clinical response; however, sBCMA remained low even at the time of relapse with increased burden of MM, indicating a lack of BCMA production by MM cells (Fig. 2B). We therefore investigated genomic changes in MM cells at the time of relapse. This patient had clonal t(11;14) translocation (96% of all cells) and clonal deletion 13 (94% of all cells) at the time of diagnosis. A similar clonal composition was observed by fluorescence in situ hybridization (FISH) analysis at study enrollment, when 4% of the cells also showed deletion 17p. Our single-cell transcriptomic analysis of BM samples identified three samples (at the time of relapse and post second CAR T-cell infusion) with significant numbers of MM cells, evidenced by expression of CD138 and XBP1 (markers of plasma cells), CCND1 (upregulated in this patient with t(11;14)), and lack of RB1 (downregulated in this patient with del13) (Fig. 2C). Imputation of copy number alterations from single-cell transcriptomic data showed that the majority of MM cells had a deletion of 16p, including the BCMA locus located on 16p13.13. (Fig. 2D). We further validated these findings using deep whole exome sequencing (WES) of purified CD138+ cells collected 2 weeks after the second CAR T infusion. Of note, copy number alterations detected by WES almost completely overlapped with CNAs predicted by single-cell RNA sequencing (scRNAseq), including deletion 16p (Fig. 2E). Before the first CAR T-cell infusion, 4% of BM MM cells showed deletion 17p, whereas after the second infusion both WES and scRNAseq prediction showed that del17p and del16p were clonal, and longitudinal scRNAseq analysis indicated that del17p and del16p co-occurred in the same clone (Fig. 2D, F). Interestingly, WES also identified a high subclonal (~70%) nonsense mutation (p.Q38*) in BCMA that creates an early stop codon in the BCMA gene (Fig. 2G and Supplementary Fig. 3). This biallelic BCMA loss, acquired with one copy deletion and a second copy loss-of-function mutation, provides the molecular basis for lack of BCMA expression in MM cells at the time of relapse.

Discussion

This case represents initial response followed by development of an acquired resistant phenotype as represented by both relapse and then lack of response to second CAR T infusion (Fig. 2H). BCMA represents an important component of plasma cell function, and thus its loss is not frequently observed. However, this case highlights a possibility that myeloma cells may be able to acuire alternative growth mechanisms to survive without BCMA expression and related signaling intermediates. Studies have shown that MM usually shows substantial inter and intra-tumor heterogeneity, which is closely related to progression, resistance to therapy, and recurrences8,9,10,11,12. Loss of several other targets for different treatments, such as CRBN with immunomodulatory agents or BCL-2 with venetoclax, has been associated with resistance to these treatments13,14. A single antigen targeting CAR T-cell treatment may also be affected by the loss of target as a result of tumor evolution and selection. Targeted antigen-negative relapse is one of the main reasons for resistance to CD19-directed CAR T-cell therapy and accounts for ~9-25% of cases of relapse in other hematological cancers3,4,6,7. In addition to antigen loss, immune-mediated rejection of the murine construct may play a role in resistance15. We did observe low level anti-drug antibodies (ADAs) at 6 months post first infusion, which persisted during the retreatment. ADA could have potential impact on CAR T cell expansion following second infusion. However, lack of BCMA expression was likely the predominant factor responsible for lack of response to second infusion. The extent of the role of ADA in this setting will need to be ascertained in a larger cohort of patients in the future. Previously, a large study evaluating CD19-targeted CAR T-cell therapy in B-cell malignincies showed that addition of fludarabine to cyclophosphamide-based lymphodepletion before the first infusion and an increased dose in the second infusion compared to the first infusion would increase the response rate16, both of which were followed in the currect study.

Here we describe emergence of a clone with loss of BCMA target leading to acquired resistance to retreatment. However, as it is equally important and previously shown in other hematological cancers, there are other possible factors such as microenvironmental changes and immune-mediated rejection of the murine construct that may contribute to resistance to CAR T-cell therapies. Here we only report one mechanism with limited power; however, future studies with larger sample size will be able to determine the dominant resistance factors and expected frequency of each mechanism in MM. We also observed a clonal TP53 missense mutation (p.P278T) (Fig. 2G) using WES, suggesting that both TP53 and BCMA had deletion in one allele and mutation in the second allele. We analyzed our data from 300 newly diagnosed MM patients and using a conservative estimate observed del16p in 6% patients (44% were subclonal deletions) and, interestingly, it co-occured with del17p in 77% of the del16p patients (sixfold encrichmnet, hypergeometric test p-value = 3.38e − 11, Fig. 2I). Importantly, we also observed that 36% of patients with del17p also carried del16p. These results support our previous observation regarding similar relative timing for both deletion events8,17, and may highlight the need to carefully examine for BCMA gene alterations in patients being retreated with subsequent BCMA targeting therapy at relapse from initial BCMA CAR T-cell treatment. The co-occurrence of 16p deletion in patients with del 17p also underscores the need to further evaluate the role of BCMA targted therapies in high-risk del17p MM. It would also be important to further investigate, with more sensitive methods, the presence and frequency of very low subclonality del16p. In general, WES data from 1300 newly diagnosed MM patients failed to detect any missense or nonsense mutations in BCMA18. This suggests that pressure of specific BCMA-targeted treatment can select for a very low level of biallelic deletion ( BCMA and TP53) in these patients (Fig. 2H). As BCMA has a functional role in MM, such BCMA-independent growth on one hand may indicate a more aggressive phenotype, but it may also suggest a new vulnerability that can be targeted by alternative therapies. Anecdotal instances of post-CAR T-cell sensitivity to various therapies have been reported and the index patient in this report has remained alive 3 years from CAR T-cell therapy.

This case represents molecular characteristics of MM. It identifies significant genomic evolution that may represent clonal selection and/or induction of new changes under the pressure of therapy. Our results suggest that BCMA-negative cell populations may get selected under strong treatments like CAR T-cell therapies. Although the platform we have used was not sensitive enough to detect the presence of low-level resistant cells at an early stage, our results still support a possible role for sensitive and deep sequencing of BCMA locus before CAR T-cell reinfusion or consideration of sequential BCMA-targeted therapies, to identify the outgrowth of a rare MM cell with BCMA loss. Recently, CAR T-cell therapy approach simultaneously targeting dual antigens BCMA and GPRC5D was shown as one approach to prevent BCMA escape-driven relapse19. The presence of subclonal changes may also provide clinically important information supporting dual antigen-targeted CAR T cell or other combination or maintenance therapies.

Methods

Patient samples

All eight samples for scRNAseq (CD138− sample before the first infusion (S1) and BM mononuclear cells from S2 to S8) and CD138+ sample for WES have been collected from an individual patients' posterior superior iliac spine area, who was enrolled in a phase I clinical study (CRB-401 ClinicalTrials.gov number, NCT02658929) of bb2121 involving patients with relapsed or refractory MM was initiated. The primary outcome results of this clinical trial have been published1. The study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation guidelines for Good Clinical Practice. The protocol was approved by Dana Farber/Harvard Cancer Center Institutional Review Board, and samples and data were obtained after a written informed consent was signed by the patient.

Single-cell RNA sequencing

For all eight samples, single-cell library constructions were performed using Chromium Single Cell 3′ Reagent kits v2. Each sample was processed individually according to 10× genomics protocols. Poly-A selected transcripts were reverse transcribed and full-length cDNA along with cell barcode identifiers were PCR amplified. The constructed libraries then sequenced with Illumina platform using paired-end sequencing. On average each sample sequenced with 118 M reads (range 87 M-141 M) (Suplementary Table 1). The Cell Ranger Suite (v3.1.0) from 10× genomics with GRCh38 reference genome was used to perform sample de-multiplexing, barcode processing and unique molecular identifier counting. Cellranger mkfastq and count funtions were used to quantify the expression values for captured single cells. The filtered gene-barcode matrix from Cellranger output then used for downstream analysis with Seurat (v3.1.5)20,21 to filter out Gel Bead-In Emulsions do not actually contain cells. Estimated number of cells per sample before additional filtering with Seurat20,21 was 5864 (range 4868-7801) and mean reads per cell was 20,682 (range 14,730-28,924). Additional quality control measurements can be found in Supplementary Tables 2 and 3.

Filtered counts then transferred to R and Seurat for downstream analysis. Only cells with at least 200 detected features and only feateres that are detected in 3 or more cells were kept for downstream analysis. After these additional filtering steps with Seurat, 4707 cells (range 3075-6818) per sample with 3695 reads per cell were used (Supplemantary Table 1 and Supplementary Fig. 4). Integration of multiple single-cell datasets was performed using anchored Analysis with SCTransform20,21 workflow and using 5000 integration features. First 20 dimensions for the Principle Componenet Analysis were used for clustering and Uniform Manifold Approximation and Projection analysis. Single-cell visualizations and downstream marker detections then performed as explained in Seurat website. Resolution was set to 0.3 for the clustering analysis. Known cell-type annotations were perfomed using SingleR(v1.4.0)22, as well as known gene surface markers for T, NK, B, plasma cells, monocytes, and erythyrocytes ( CD3D, CD3E, CD3G, CD4, CD8A, CD5, NCAM1, CCL5, KLRC1, KLRD1, KLRC2, CD79A, CD79B, CCND1, SLAMF7, XBP1, POU2AF1, CD38, IRF4, CD14, FCGR3A, CD68, PECAM1, HBB) (Supplementary Fig. 1). T-cell subgroups also identified using T-cell subgroup-specific markers ( CD4, CD8A, CCR4, CCR6, FOXP3, IL2RA, CCR7, IL7R, CD8A, CD8B, FASLG, IFNG, NKG7, GZMB, GZMH) (Supplemantary Fig. 2). Cytotoxic CD8+ T-cell makers were collected from Zavidij et al.23. Copy number analysis for the scRNAseq was done using CONICSmat(v0.1)24 and plasma cells were compared with B cells as reference set. Only the chromosomal arms that passed Bayesian information criteria > 0 and adjusted p-value < 1e−5 were considered significantly altered. Differentially expressed genes were detected using FindAllMarkers and FindMarkers function in the Seurat21 package. Gene-set enrichment analysis was done using molecular signature database (MSigDb) provided by Broad Institute25,26.

Whole exome sequencing

WES data for tumor sample generated from CD138+ cells collected after the second infusion. Peripheral blood mononuclear cells were used as germline control. WES libraries generated using Twist Bioscience Human Core Exome Kit and sequenced as 75 bp paired-end reads with Illumina Novoseq platform. The average sequence coverage for targeted regions was 110× for tumor sample and 602× for germline DNA. We aligned paired-end reads using BWA-mem (v0.7.17-r1188)27 to GRCh38. We followed GATK (v4.0.11) best practice to mark duplicated reads with MarkDuplicates function and base quality score recalibration with ApplyBQSR28. Mutect229 was used to call mutations. Only mutation calls with at least 10× coverage for tumor and germline samples and passed FilterMutectCalls function were annoted using Variant Effect Predictor from Ensembl (v100). Allele-specific copy number calls as well as ploidy and purity of the sample were analyzed using FACETS (v0.6.1) (Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing)30.

FISH analysis

CD138+ sorted BM plasma cells were analyzed by FISH using commercially available probes specific for 8q24.1, del13, 17p13.1, gain11, gain1q22, t(4;14), t(11;14), t(14;16), t(14;20) by Mayo Clinic Laboratories. All probes were set up separately and for each probe, plasma cells (if possible) are scored and the result for each probe is reported.

Other statistical analysis

All other analyses were completed in the R programming language. Data preparation and processing were done using ggplot2, cowplot, and dplyr packages. R maftools was used for downstream analysis for the Single Nucleotide Variant (SNV) and small insertion deletion data. Protein domains were combined with SNV calls using ProteinPaint to generate lollipop plots.

Detecting CAR+ cells with qPCR

Copies of vector transgene per microgram genomic DNA was determined by quantitative PCR (qPCR) as previously described1. Briefly, CD3+ cells were isolated to high purity from whole blood. Genomic DNA from the purified CD3+ cell pellet was extracted and DNA concentration was determined. Purified CD3+ DNA (100 ng) was included in the qPCR reaction for specific quantification of the bb2121 transgene (Psi-gag) and a reference housekeeping gene ( RNaseP). Detection and quantification of the Psi-Gag sequence and RNaseP were achieved using target-specific oligonucleotide primers and dual-labeled oligonucleotide hydrolysis probes1. The amplified targets were detected in real time by Stratagene Mx3005P instrument using TaqMan® Universal PCR Master Mix, no UNG (Thermo Fisher Scientific), and quantified using a standard curve. Quantified copies of vector transgene per reaction is reported as copies per standardized input DNA (100 ng). Primer probe sequences are shown in Supplementary Table 4.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The single-cell RNA sequencing data generated in this study have been deposited in the GEO database under accession code GSE164551. In addition, the single-cell RNA sequencing dataset, BAM file for BCMA locus, SNP counts for allelic copy number, and meta data for single cells after clustering with Seurat are also available in the Harvard Dataverse database under accession code doi:10.7910/DVN/1RKYQ8 [ https://doi.org/10.7910/DVN/1RKYQ8]. The remaining data are available within the Article, Supplementary Information, or available from the authors upon request.

Acknowledgements

This study has been supported by NIH grants P01 CA155258 and P50 CA100707, Celgene Corporation and VA Healthcare System Grant 5I01BX001584, and Paula and Roger Riney Foundation grant.

Author information

Affiliations

1. Department of Data Science, Dana Farber Cancer Institute, Boston, MA, USA

Mehmet Kemal Samur & Anil Aktas Samur

2. Department of Biostatistics, Harvard T. H. Chan School of Public Health Boston, Boston, MA, USA

Mehmet Kemal Samur & Anil Aktas Samur

3. Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

Mehmet Kemal Samur, Mariateresa Fulciniti, Abdul Hamid Bazarbachi, Yu-Tzu Tai, Rao Prabhala, Alejandro Alonso, Adam S. Sperling, Kenneth C. Anderson & Nikhil C. Munshi

4. Department of Internal Medicine, Jacobi Medical Center, Albert Einstein College of Medicine, New York, NY, USA

Abdul Hamid Bazarbachi

5. VA Boston Healthcare System, Boston, MA, USA

Rao Prabhala & Nikhil C. Munshi

6. Bristol-Myers Squibb, San Francisco, CA, USA

Timothy Campbell & Kristen Hege

7. Bluebird Bio, Cambridge, MA, USA

Fabio Petrocca

8. Bristol-Myers Squibb, Seattle, WA, USA

Shari Kaiser

9. University Cancer Center of Toulouse Institut National de la Santé, Toulouse, France

Hervé Avet Loiseau

Contributions

M.K.S. and N.M. designed and conduct the study. M.K.S., M.F., A.A.S., A.H.B., Y.T., R.P., A.A., A.S., T.C., F.P., K.H., S.K., H.A.L., K.C.M., and N.M. collected the data. M.K.S., A.A.S., A.H.B., R.P., and N.M. analyzed the data. All authors discussed the data and wrote the manuscript.

Corresponding authors

Correspondence to Mehmet Kemal Samur or Nikhil C. Munshi.

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Competing interests

K.C.A. has received consulting fees from Bristol-Myers Squibb, Celgene, Gilead, Janssen, Precision Biosciences, Sanofi-Aventis, Takeda, and Tolero, and on the board of directors and stock options Oncopep. N.C.M. is a consultant for BMS, Janssen, Oncopep, Amgen, Karyopharm, Legened, Abbvie, Takeda, and GSK, and on the board of directors and stock options Oncopep. T.C., K.H., and S.K. are employed by Bristol-Myers Squibb. F.P. is employed by Bluebird Bio. Other authors declare no competing interests.

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Samur, M.K., Fulciniti, M., Aktas Samur, A. et al. Biallelic loss of BCMA as a resistance mechanism to CAR T cell therapy in a patient with multiple myeloma. Nat Commun 12, 868 (2021). https://doi.org/10.1038/s41467-021-21177-5
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# data[].attributes.attachment_link.title
Biallelic loss of BCMA as a resistance mechanism to CAR T cell therapy in a patient with multiple myeloma - Nature Communications

# data[].attributes.attachment_link.description
Relapse following BCMA targeted CAR T-cell therapy is frequently observed in patients with multiple myeloma (MM). Here, by single cell transcriptome profiling on serially collected bone marrow samples, the authors report biallelic loss of BCMA as the mechanism of resistance underlying both relapse and lack of response to a second CAR T infusion in a patient with MM.

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Artikel fra 2019:
En ny udvikling for kræft behandling.
- Chimeric antigen receptors er unikke receptor, der er målrettet efter tumor antigen.

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Introduction

Chimeric antigen receptors (CARs) are unique receptors that are designed to target a specific tumor antigen to functionally reprogram T lymphocytes. As T lymphocytes are genetically engineered to express these artificial receptors to target cancer cells, the type of therapy may be termed immunotherapy, gene therapy or cancer therapy ( 1). The human defense system can efficiently identify self and non-self molecules, including bacteria, viruses and abnormal cancer cells. The identification of tumor cells is based on their acquired antigenicity and immunogenicity via the expression of foreign antigens ( 2). However, cancer cells have the potential to subvert the immune system to their advantage, resulting in inadequate antitumor immunity, and tumor survival and progression ( 3). Immunotherapy is also termed biotherapy as the immune system in the body is naturally capable of detecting pathogens and cancerous cells. In recent years, immunotherapy has emerged as an important branch of treatment for similar types of disease; however, its protective mechanism may differ ( 4). Certain immunotherapies boost the immune system, whereas others directly target the cancer cells. Each treatment type has its advantages and disadvantages depending on the disease type ( 5). Tisagenlecleucel (Kymriah) is a medication used to treat patients with acute lymphoblastic leukemia (ALL) up to the age of 25 years. Similarly, axicabtagene ciloleucel (Yescarta) is approved for patients with large B-cell lymphoma, such as non-Hodgkin lymphoma (NHL), and those with cancer in a refractory and recurrent state or whose cancer is non-responsive to other treatments ( 6). With increasing awareness of the immune system, a number of innovative immunotherapies are being developed using methods which include inducing the immune system to function in an impertinent manner to target malignant cells. Another approach involves the administration of immune components, such as synthetic, modified immune proteins that are genetically engineered to target tumor antigens ( 7).

CAR T cell treatment has achieved success in treating hematopoietic malignancies; however, its effectiveness against solid tumors remains to be determined. In the following sections, the rapid progression in implementing the adoptive transfer of T cells and their mechanism of tumor cell eradication are discussed.

CAR T cell therapy

Architectural ideology of T cell engineering and CAR design

The uniqueness of chimeric receptors exists in their ability to fuse or split discrete vital functions, such as recognition, co-stimulation and activation, in different chains of a receptor molecule by imitating the complexity of the native T cell receptor (TCR) structure ( 11). T cells do not usually require costimulation for activation and to initiate proliferation, but in the process of establishing CAR T cells, the activation and proliferation of T cells require the presence of costimulatory molecules, which also assist in CAR T cell cytokine production. The strategy involves constructing an engineered chimeric receptor for T cells based on the integration of scFv fragments in the hinge area that separates scFv from the cell membrane. The exposure of scFv on the cell surface, in addition to other small functional molecules, enhances induction of the cytolytic function of the engineered T cell. Together, this coordination of a 'living drug' in the immune system fights against cancer ( 12). In addition, CAR T cells can remain stable for several years in the body as long-term memory cells. This feature allows them to recognize and kill cancer cells encountered in the circulation in the case of relapse. Another advantage of CAR T cells is that they specifically target only tumor cells and not auto-antigens. Therefore, it is safe and nonlethal to host cells ( 13). Once the synthetic immunoreceptor is expressed on the surface of an engineered T cell, its scFv specifically binds to target antigens expressed on a cancer cell. This binding subsequently results in the transduction of an activating signal into the genetically edited immune cell. The T cell then elicits its effector anticancer function ( 14). CAR T cell therapy is considered to be the first approach to reconfigure T lymphocytes with an antibody-specific scFv fragment obtained from monoclonal antibodies (mAbs) by replacing different parts of the TCRα and β chains. A recent report stated the potential of CAR γδ T cells in curing mucosal-derived malignant tumors as a novel strategy for CAR T cell therapy ( 15). The hybrid TCR functionally expresses and recognizes the analogous target antigen molecules in a non-MHC-restricted manner. Depending on the diverse biomarker selection and structural complexity, different generations of CAR model have been developed ( 16).

First-generation CARs

Second-generation CARs

Third-generation CARs

To extend the antitumor efficacy, third-generation CARs comprise two signaling domains and the CD3ζ chain, such as CD3ζ-CD28-OX40 and CD3ζ-CD28-4-1BB, to achieve improved activation signal, prolonged proliferation, elevated cytokine production and effective function ( 25). For example, a third-generation CAR consisting of α-CD19-CD3ζ-CD28-4-1BB reported complete remission rates by infiltrating and lysing cancer tissue in patients with chronic lymphocyte leukemia ( 26). In addition, certain CAR T cells function as memory cells, thus preventing tumor relapse. Despite the significant curative effect, the irrepressible activity of CARs and their increased antitumor efficacy is associated with life-threatening and unfavorable outcomes, with increased secretion of pro-inflammatory cytokines, multi-organ dysfunction, pulmonary failure and death ( 27).

Fourth-generation CARs

Mode of delivery

Gene therapy involves the delivery of DNA into cells and this can be accomplished using a number of methods summarized below. The most traditional method utilizes recombinant viruses (also known as viral vectors), biological nanoparticles and non-viral methods based on the direct delivery of naked DNA.

Viral vector

Viral vectors, such as γ-retrovirus, lentivirus and adenovirus vectors are generally used in gene therapy. Retrovirus transduction is one of the commonly used delivery methods in gene therapy. The method involves a reverse transcriptase that promotes the stable integration of artificial genes into the host genome ( 31). To create a vector, γ-retroviral coding sequences are substituted by a gene of interest. The retroviral vectors possess an innate capability to disturb the genomic section and results in neoplastic transformation. Thus, γ-retroviral vectors have been used in gene therapy applications ( 32). Lentiviral vectors are retroviruses derived from human immunodeficiency virus-1, which serve as a major tool for the delivery of transgenes into mammalian cells. The advantage of using lentivirus vectors is their efficient transduction and their stable integration and expression into non-dividing and dividing cells both in vitro and in vivo ( 33).

Non-viral delivery methods

Transposon transfection

The mechanism of transposon transfection is different from that of viral transfection. Delivery via transposons is a non-viral process that uses transposon DNA and a transposase enzyme for stable gene transfer. Two important vectors, namely piggyBac (PB) and sleeping beauty (SB), are frequently used ( 34). Transposons shift from one gene position to another position via a cut-and-paste mechanism. The mechanism of the transposon system using SB and PB involves four steps: i) The transposase enzyme helps in recognition and binding to the transposon; ii) a synaptic complex is produced by the coupling and binding of the repeat elements at both ends of the transposon; iii) the transposon excises the genetic element to be transposed, and iv) the excised element is reintegrated into the target location ( 35). The transposase for both vectors (SB and PB) consists of the DNA-binding domain, transposable catalytic domain and nuclear localization signal. The SB vector is a safer substitute for viral vectors owing to its innately low enhancer activity, non-pathogenic source and minimum epigenetic alterations at the incorporation site ( 36). By contrast, the PB vector has been shown to provide a large load capacity and additional competent transposition action in in vivo studies. PB vector-mediated CAR T therapy is considered safer than that based on the SB vector. Therefore, use of the PB vector results in greater CAR production, as evident from the production of CAR targeting CD19 using a PB vector ( 37).

Electroporation

Nanoparticles

The most critical step in CAR T cell therapy is T cell activation, for which cells need to be incubated with the viral vector including a CAR gene. However, stable insertional mutagenesis can be dangerous as its possible effects on humans remain to be fully elucidated ( 42). Retroviral or lentiviral vectors offer permanent gene integration into host cells, with the potential integration in close proximity with a proto-oncogene which can result in an oncogene. The various disadvantages associated with these transfer methods suggest that a safer alternative is needed ( 43).

The study by Smith et al elucidated the use of nanotechnology to resolve the tumor-targeting problem and make the therapy cost-effective. They reported that polymeric nanocarriers can efficiently deliver leukemia-specific CAR genes targeted to specific ligands on the host T cells in situ ( 44). A number of nanoparticles used for gene delivery have been investigated preclinically and reported. For example, magnetic nanoparticles, such as Fe3O4, integrate into the cell-penetrating peptide complexes. These oligonucleotides maintain stable cell transfection and plasmid transfection in addition to gene silencing and splice correction ( 45). A recently published study by Smith et al at the Fred Hutchinson Cancer Research Center, Seattle, confirmed that polymeric nanoparticles carrying DNA effectively introduced CAR genes into T cell nuclei and recognized leukemia cells distinctively ( 46). The use of nanoparticles to replace viral vectors for gene delivery has proved advantageous, as evident from the restriction of unspecific pro- or anti-inflammatory effects or pro- or antiproliferation effects ( 47).

Challenges of CAR T cell therapy

Advanced features of CAR T cell therapy

Although cancer cells have multiple lineages and heterogeneity, they possess common target antigens, such as CD19, CD20, CD22 and numerous others that allow CAR T cells to recognize tumor cells irrespective of cell lineage. Therefore, recent advances in this technique include more precise target antigens expressed by tumor cells ( 13, 52). This review places additional emphasis on new studies of CAR T cell therapy that distinguish diverse CAR T cells which can enhance tumor cell death. Certain models are already being implemented and others are in the clinical investigation status, including NK-CAR and clustered regularly interspaced short palindromic repeats (CRISPR)-CAR therapy ( Fig. 3A-E).

Bispecific CARs

A bispecific receptor is one that contains two distinct antigen recognition domains attached and placed with two distinct intracellular signaling domains that are expressed as two different CARs on a single cell surface. At present, bispecific CAR CD19/CD20 has been introduced as a novel synthetic molecule that can recognize and bind to more than one targeted tumor antigen on the cancer cell surface. Therefore, it can create a synergistic cascade of effector molecules when it encounters two tumor antigens ( 53). Additionally, the bispecific CAR conserves the cytolytic capacity of T cells, i.e., if one of the objective molecules is not accessible to CAR T cells due to a cellular hindrance such as mutation of a target antigen or loss of the target antigen commonly found in malignant cells, a bispecific CAR can counterbalance the tumor evasion ( 54). Investigated therapeutics include CD3 of T cells and tumor antigens, such as CD19, on malignant cells. The curative ability is evident from blinatumomab, an approved bispecific T cell-engager against relapsed/refractory B-ALL ( 55). Positive results have been reported in patients <15 years of age with relapsed/refractory ALL, with a heightened response in 26/36 (72%) patients in 9 months. Blinatumomab was discovered while studying a patient who was negative for minimal residual disease (MRD) and who was found to be MRD-positive following consolidated chemotherapy ( 56). Numerous other bispecific CARs have been investigated preclinically by several research organizations, including CD20/CD19 and CD20/CD3 ( 57, 58). Targeting T cells with bispecific CARs was shown to eliminate transplanted pediatric ALL in a study, whereas T cells targeted by CD20 CAR did not control the disease ( 59).

Tandem CARs (Tan-CARs)

Conventional CARs cannot meet higher expectations under certain circumstances, such as the downregulation or alteration of targeted antigens that can occur in cancerous cells. These conditions subsequently lead to antigenic loss or escape variations ( 60). To overcome this shortcoming, scientists are attempting to develop advanced technology, in which two particular antigen recognition sites are joined by a linker, placed in tandem on a single intracellular domain and expressed as a single CAR on a cell surface, termed a Tan-CAR. This model enables the synchronized targeting of both antigens on a single cancer cell or tumor microenvironment. In this manner, it enhances the activation and stimulation of T cells by increasing their avidity and expanding their therapeutic properties ( 61). Tan-CARs have several significant curative implications as they are as potent as standard disease models with single antigen-specific CARs. Additionally, they are more efficient and less noxious in a higher disease load setting ( 62). This may be attributed to optimized cytokine production and limited cell killing, as evident from preclinical studies on Tan-CAR T cells in a mouse tumor model, which confirmed its possibility for remedial implication in human disease consisting of B-cell antigen CD19 and human epidermal growth factor receptor 2 (HER2) ( 63). Recently, a trivalent CAR T cell was designed by a group of scientists by simultaneously co-targeting multiple antigens, such as HER2, IL-13 receptor α2 and ephrin A2, to overcome interpatient changeability with a propensity to target almost 100% of tumor cells ( 64).

Inhibitory CARs (I-CARs)

It has been reported that certain novel immunoinhibitory receptors are involved in T cell activation and the attenuation or termination of T cell responses, such as programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). These pathways are regarded as cancer immunotherapy breakthroughs ( 65). Recently, two immunologists won the Novel Prize in Physiology/Medicine, Dr James Allison and Dr Tasuku Honjo, for their profound discovery in the field of cancer immunotherapy. Dr Allison's work focused on the T cell surface protein CTLA-4; he found that the inhibition of immune cells and antibodies against CTL-4 eliminated malignant growth and prevented new tumor formation ( 66). This finding was tested on 14 patients with metastatic melanoma, with relapse observed in three patients. In 2011, the FDA approved an anti-CTLA-4 (ipilimumab) antibody as a treatment for high-grade melanoma ( 67).

Similarly, Dr Honjo's team investigated a novel T cell protein, PD-1, which was discovered in 1992 ( 68). They observed that PD-L1 presenting on healthy cells and malignant cells bind to PD-1. They reported another similar molecule, PD-L2, that also binds to PD-1. Based on these findings, they published a report that malignant cells produced PD-L1 and blocked PD-L1 using a counteracting antigen to inhibit tumor growth ( 69). The first clinical trials to target malignancy were launched in 2006, which indicated significant viability in a number of patients in 2012. The FDA approved the main PD-1 checkpoint inhibitors, nivolumab and pembrolizumab ( 70, 71), for treating melanoma in 2014 ( 72). However, a single treatment may not be sufficient, which is the reason that a consolidated treatment targeting CTLA-4 and PD-1 is currently being investigated. Both Dr Allison and Dr Honjo encouraged the merging of various strategies to generate more advanced forms of the drug for the immune system to inhibit tumor cells more effectively ( 73).

Physiological CARs

Initially, several CAR constructions contained scFv of murine origin. This is associated with the risk of an immune response to the modified cells, and the resulting anaphylaxis of CAR T cell transfer cannot be avoided. These disadvantages limit the persistence of the infused cells. Therefore, besides conventional CAR, a physiological CAR has been developed, also known as a receptor-ligand CAR, which can recognize and bind to tumor antigens, such as HER3 and HER4 ( 78). The physiological CAR consists of an antigen receptor and a CD3ζ intracellular signaling domain with or without a transmembrane region, which can also be engineered into immune cells to target the ligands expressed on tumor cells. This approach increases the capability of T lymphocytes to distinguish tumor-related targets and eliminate cancer cells ( 79). This physiological CAR is an emerging field of CAR T cell therapy with limited published reports. Experimental trials may have been initiated but the outcomes have not yet been published.

Universal CARs (uCARs)

Natural killer (NK)-CARs

NK cells are a type of cytotoxic T cell that are essential for natural immunity. The function of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. Immune cells generally identify MHC complexes expressed on infectious cell surfaces to elicit cytokine production, thus generating an immune response, culminating in the apoptosis or death of infectious cells ( 84). NK cells are the only immune cells that identify infected cells in the absence of MHC and antibodies, thus eliciting a rapid immune response. These are known as 'natural killers' as they do not require activation to destroy cells that are devoid of 'self' MHC class I molecule markers ( 85), making them important for destructive cells with missing MHC I markers.

Cancer cells that do not cause any inflammation are treated as self by the immune system and do not stimulate a T cell response. NK cells produce a number of cytokines, including tumor necrosis factor α, interferon γ and IL-10, which act as immune suppressors ( 86). The activation of NK cells leads to the gradual formation of cytolytic effectors cells, such as dendritic cells, macrophages and neutrophils, which consequently facilitate antigen-specific T and B cell responses. NK cell-mediated tumor cell lysis involves several receptors, including NKp44, NKp46, NKG2D, NKp30 and DNAM. Malignant cells usually express NKG2D in addition to ULBP and MICA ( 87, 88). The clinical effectiveness of CAR T cells has been shown for ALL; however, this therapeutic approach has not been confirmed for acute myeloid leukemia (AML), suggesting the need for other therapeutic options ( 89). Hypothetically, automotive NK cells have an additional favorable toxic effect in comparison to CAR T cells, particularly for avoiding unfavorable effects such as CRS. Additionally, in contrast to T cells, donor NK cells do not target non-hematopoietic cells, indicating that NK cell-mediated antitumor activity may be activated in the absence of graft-vs.-host disease ( 90) ( Fig. 4).

CRISPR CARs

Recently published reports suggest that CRISPR technology can deliver the CAR gene to the TRAC locus of T cells. In this regard, CRISPR-edited universal-CAR T cell therapy has been used in humans (NCT03166878 and NCT03229876). This technology is rapidly developing with the potential for gene correction ( 82). It is reported that anti-CD19 CAR to the TCR α constant locus (TRAC locus) not only results in increased T cell potency but also in the consistent expression of CAR in peripheral blood T cells ( 95). CRISPR-modified cells have been shown to perform well in generally constructed CAR T cells in a mouse model of ALL ( 96). It has also been demonstrated that targeting the TRAC locus turns on CAR signaling, thereby initiating successful internalization and stimulation of the CAR for single and repeated exposure to a tumor antigen. It also delays effector T cell differentiation and exhaustion ( 97). Multiplex genome editing is another attractive application of the CRISPR/Cas9 tool. Proficient genomic disturbance of multiple gene loci to create a universal donor cell and a potent effector T lymphocyte targeted to different inhibitory pathways, such as PD-1 and CTLA4, is established by incorporating several gRNAs into a CAR vector. Furthermore, two-fold knockout of the TCR gene and HLA class I can effectively permit the generation of an allogeneic uCAR T cell, in addition to CAR T cells that are universally Fas-resistant via three-fold gene disruption ( 98).

Advantages of CAR therapy over other therapies

The most notable advantage of CAR T cell therapy over other cancer therapies is the abrupt time intervention and single infusion of CAR T cells. Additionally, 2-3 weeks of proper care and observation is sufficient for the patient. CAR T cell therapy is regarded as a 'drug of the present day' and its efficacy may persist for decades as the cells can survive in the host body in the long term, with a constant ability to find and destroy cancer cells during relapse ( 2, 3). Currently, CAR T cell therapy is licensed for use in patients for whom transplantation has not been curative and who relapse following transplant. CAR T cell therapy is expected to be a substitute for different types of transplant ( 99). Clinical trials on blood cancer have shown that, even in patients with a refractory condition in which cancer reverted following several transplants, CAR T cell therapy was successful in completely eradicating the disease ( 100). Additionally, with CAR T cells, patients can live life without the risk of relapse and benefit from a sanatory treatment, such as stem cell transplantation. Therefore, CAR T cell therapy can be referred to as a 'living drug' ( 101).

CAR T cell therapy trials for solid tumors

Several clinical trials are currently examining the use of CAR T cell therapy against solid tumors and other diseases. Reports suggest that mesothelin-specific CAR mRNA-engineered T cells can induce antitumor activity in solid malignancies ( 102, 103). Furthermore, CAR technology has been used in organ transplantation using two novel HLA-A2-specific CARs, one representing a CD28-CD3d signaling domain (CAR) and the other missing an intracellular signaling domain (dCAR). The adoptive transfer of allospecific regulatory T cells (Tregs) provides a better safeguard from graft rejection compared with that of polyclonal Tregs ( 104). CAR comprising the ICOS signaling domain liaises with the effective antitumor effect on epidermal growth factor receptor variant III (EGFRvIII)-expressing glioma ( 105). The preclinical evaluation of CAR T cell therapy targeting the tumor antigen 5T4 in ovarian cancer has been associated with a successful outcome ( 106). An unexpected evolutionary finding was reported during the investigation of CAR targeting autoimmune diseases; therapy lacking autoimmune diseases that purposely target only the disease-causing cells, chimeric autoantibody receptor (CAAR), contain pemphigus vulgaris autoantigen, desmoglein (Dsg3), combined to CD137-CD3d signaling domains. Dsg3 CAAR-T cells exhibit accurate toxicity against anti-Dsg3-expressing cells ( 107). Certain solid tumor CAR targets are undergoing research with varied genetic products arising from gene mutations (EGFRvIII) ( 108) or modified glycosylation patterns (MUC1) ( 109), and cancer-testis antigen-derived peptides (MAGE), CAR specifically targets certain overexpressed antigens in breast cancer, lung cancer and pancreatic cancer, such as carcinoembryonic antigen ( 110) GD2, prostate-specific membrane antigen, HER2/ERBB2, MUC16 ( 111) and mesothelin or tumor-affiliated stoma (fibroblast activation protein and vascular endothelial growth factor receptor) ( 112).

Success rates of approved therapies

The global ELIANA trial reported a high success rate, with a 3-month complete remission rate of 83% with tisagenlecleucel, and a 6-month survival rate of 89% ( 117). Another trial, ZUMA-1, reported an equivalent results with an 82% overall response rate and 54% of complete remission rate following a single infusion of the therapeutic regime in 8 months. This indicates that the remission rate was more pronounced in pediatric B-ALL than that in adult relapsed/refractory DLBCL; however, reactivity was high in both diseases ( 118).

Furthermore, multiple clinical investigations have been implemented by Cellectis following UCART19. Cellectis is currently leading with two successful FDA Investigational New Drug (IND)-approved allogeneic CAR T approaches ( 119): UCART123 for patients with blastic plasmacytoid dendritic cell neoplasm and AML ( 120) and UCART22 IND for patients with B-ALL ( 121). Two other ongoing clinical trials are UCARTCS1 for suppressing CS1-expressing hematologic malignancies ( 122) and UCART38 for CD38-expressing hematologic malignancies. UCART38 is specifically developed to target T cell ALL, multiple myeloma, mantle cell lymphoma and NHL ( 123, 124). A list of CAR therapy clinical trials are listed in Table I.

Table I.
List of chimeric antigen receptor therapy clinical trials.

Future prospects

Although immunotherapy has achieved clinical success in treating blood cancer, the same success rates have not been observed for solid tumors. The various challenges associated with its safety, cost-effectiveness and quality require thorough investigation in order to implement this therapy for all cancer types. Another approach is to integrate CAR T cells with different types of immunotherapy to enhance its effectiveness. For example, CARs may be combined with certain checkpoint inhibitors, which limit tumor defense mechanisms against T cells. It is expected that future CAR T cell therapy regimens will target several diverse molecules for a particular type of tumor, such that CAR T cells can efficiently recognize cancerous cells even if these undergo mutations in their target molecules.

A number of CAR T therapies are already available to the market, but these are expensive, for example, $475,000 (€400,000) for Yescarta and $373,000 (€316,000) for Kymriah. According to experts, when hospitalization expenses and the costs of other drugs required for the treatment are also considered, the cost increases to almost $1,500,000 per cancer patient. Therefore, a possible solution is to reduce the cost of allogeneic CAR T treatment by supplying T lymphocytes from a healthy individual that can be readily utilized when a patient requires it, rather than genetically modifying each patient's T cells individually. Considerable scientific challenges also exist with regard to immunology, in addition to manufacturing, transportation and banking solutions to enhance the extensive treatment of patients. Therefore, scientists are investigating measures to overcome clinical challenges in terms of regulations. CAR T cells are available in several scientific frameworks, which may vary widely in different countries. These combined challenges and technology require standardization; however, CAR T cells offer patients hope of advanced treatment. As the first therapy is already available in the market, there is potential for a specific and improved alternative becoming available in upcoming decades.

Acknowledgements

We acknowledge the Science and Engineering Research Board for the generous funding.

Funding

Funding from the Science and Engineering Board, Department of Science and Technology, Government of India is duly acknowledged. ECR/2016/000584.

Availability of data and materials

Not applicable.

Authors' contributions

RM and NG conceptualized and co-wrote the manuscript. CRC, SA, PS and PG contributed to the literature review, organization and writing of various sections of the manuscript. NG is the PI and grant holder.

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References

# data[].attributes.attachment_link.title
CAR T cell therapy: A new era for cancer treatment (Review)

# data[].attributes.attachment_link.description
Cancer has recently been identified as the leading cause of mortality worldwide. Several conventional treatments and cytotoxic immunotherapies have been developed and made available to the market. Considering the complex behavior of tumors and the involvement of numerous genetic and cellular factors involved in tumorigenesis and metastasis, there is a need to develop a promising immunotherapy that targets tumors at both the cellular and genetic levels.

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CAR T cells, fra 2021.  (afsnit om Target selection)

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CAR T cells, fra 2021.  (afsnit om Target selection)

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* Beskriver at CAR-T celler blev udviklet på baggrund af manglen for T celler, der kunne opdage tumorceller, uden at skulle bruge specifikke antigen-processer og antistof-præsenterende celler.  Artiklen beskriver problematikker med specificitet, på grund af manglende reproducerbarhed af metoden.

* Der er også tale om at benytte andre ting der typisk findes i celler som  f.eks. overfladeproteiner, Karbohydrater, og glycolipid molekyler mm. til at give CAR-T cellerne egenskaberne der ønskes.  (cytotoksiske egenskaber)

* Dog er der også kritik af denne mulighed, da det potentielt kunne skabe CAR-T celler der er cytotoksiske for andre celler i kroppen da specificiteten ikke ville være så god igen hvis f.eks. glycolipider blev brugt, og det kunne f.eks. skade patientens organer. - Så det er en metode der stadig er under udvikling, for at det kan bruges til alle former for kræft.

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Laboratorio 7 Biotecnología Farmacéutica, Facultad de Farmacia, Universidad Autónoma Del Estado de Morelos, UAEM, Cuernavaca, Mexico

Coordinación de Investigación, Centro Médico Nacional "20 de Noviembre" ISSSTE, Mexico city, Mexico

Cancer is among the leading causes of death worldwide. Therefore, improving cancer therapeutic strategies using novel alternatives is a top priority on the contemporary scientific agenda. An example of such strategies is immunotherapy, which is based on teaching the immune system to recognize, attack, and kill malignant cancer cells. Several types of immunotherapies are currently used to treat cancer, including adoptive cell therapy (ACT). Chimeric Antigen Receptors therapy (CAR therapy) is a kind of ATC where autologous T cells are genetically engineered to express CARs (CAR-T cells) to specifically kill the tumor cells. CAR-T cell therapy is an opportunity to treat patients that have not responded to other first-line cancer treatments. Nowadays, this type of therapy still has many challenges to overcome to be considered as a first-line clinical treatment. This emerging technology is still classified as an advanced therapy from the pharmaceutical point of view, hence, for it to be applied it must firstly meet certain requirements demanded by the authority. For this reason, the aim of this review is to present a global vision of different immunotherapies and focus on CAR-T cell technology analyzing its elements, its history, and its challenges. Furthermore, analyzing the opportunity areas for CAR-T technology to become an affordable treatment modality taking the basic, clinical, and practical aspects into consideration.

Introduction

Cancer is a major health issue worldwide, causing a serious health problem that affects costs on public health, social development and, most importantly, reducing people's life expectancy ( Bray et al., 2018; Hassanpour and Dehghani 2017). In a broader context, cancer refers to more than 277 different types of cancer diseases ( Hassanpour and Dehghani 2017), which represent major challenges to find out specific diagnosis and treatment. Worldwide, the main incidence of cancer types in adults occurs in the prostate, lung, bronchus, colon, rectum, urinary bladder, breast, and thyroid ( Siegel et al., 2019), meanwhile, blood cancer is the main cancer type of concern in children´s health ( Kolonel and Wilkens). For years, the most effective cancer treatments have included surgery, chemotherapy, radiation therapy and bone marrow transplant ( Arruebo et al., 2011), these therapies are considered as the main treatment. Unfortunately, even considering the current technological improvements on such therapies, recurrence and metastasis are still the main causes of death ( Bray et al., 2018). In recent years, these common treatments have been challenged with the advent of immunotherapy.

Immunotherapies are designed to harness and manipulate part of the immune system to confer the ability to detect and specifically attack cancer cells ( Waldmann 2003). Immunotherapies introduce a different perspective into cancer treatment, not only in patients with refractory tumors, but also to ensure long-lasting clinical remission in patients that were historically considered incurable ( Dougan and Dranoff 2012; Guedan et al., 2019). Hence, in this paper we intend to expose the general panorama regarding immunotherapies, focusing the effort to the novel CAR-T cell therapy, its components, advantages, and limitations, summarizing the current challenges which need to be overcome and discussing the actual strategies to implement this therapy with a wider scope in benefit of a larger number of patients.

Immunotherapy, A New Approach for Cancer Treatment

Chemotherapy, radiation, and surgery are the most conventional cancer treatments, nevertheless, they have shown low efficacy and severe adverse effects. Therefore, during the last decade, researchers have addressed the need to improve the inconveniences with conventional cancer therapy, developing new strategies to achieve complete remission of the disease using molecular strategies. Under these circumstances, immunotherapy arises as a promise for revolutionary cancer treatment, because, compared with conventional therapeutics, it leads to lower side effects and can specifically target cancer cells by regulating the immune system´s machinery ( Papaioannou et al., 2016; Sang et al., 2019). Therefore, immunotherapy has become an important therapeutic alternative for patients whose immune systems are already compromised due to their advanced disease and/or failure of previous conventional therapies ( Ventola, 2017; Charmsaz et al., 2019). Table 1 covers the six major kinds of available cancer immunotherapies. The most recent immunotherapy that has shown promising results is the Chimeric Antigen Receptor (CAR)-T cell therapy, which is a type of Adoptive Cell Therapy (ACT). This therapeutic strategy entails genetically engineering a patient's T cells to express CARs that recognize, bind, attack and kill tumoral cells ( Mirzaei et al., 2017).

TABLE 1. Six major kinds of cancer immunotherapy.

A Brief History of Chimeric Antigen Receptor-T Cells Development

To start with the historical review of the discovery of these new technologies, it is necessary to date the first bone marrow transplant for leukemia patients that was reported in 1957 by Thomas and colleagues ( Thomas et al., 1957). A few years later, Miller J. and colleagues reported the immunological function of the timus, discovering the origin of T cells ( Miller, 1961).

However, it was not until 1986, when Steven Rosenberg and colleagues reported a study about Tumor-Infiltrating Lymphocytes (TILs), this therapy consists of cellular isolation of T lymphocytes from a tumor, that is subjected to an in vitro culture and expansion to infiltrate them back into the tumor to be treated, hence leading to elimination or clinical resection of the tumor ( Rosenberg et al., 1986; Lança and Silva-Santos, 2012; Garber 2019). This report allowed us to fix the gaze on the idea of "Patients' own immune cells can fight their own cancer".

Likewise, results of the successful T cell transfections of Sadelain and colleagues in 1992 and their methods publications for retrovirus-mediated gene transfer into primary T-lymphocytes in 1997, allowed genetic modifications providing the means of controlling immunity in an experimental or therapeutic setting ( Sadelain and Mulligan, 1992; Sadelain, 1997). Almost at the same time, Zelig Eshhar and colleagues designed a specific activation of cytotoxic lymphocytes through chimeric single chains, using an antibody-binding domain and the γ or ζ subunits of the immunoglobulin on the T-cell receptors, developing the First Generation of CAR-T cells ( Eshhar et al., 1993). Five years later, Dr. Sadelain's group demonstrated that the integration of a co-stimulatory signal like CD28 to a CAR-T enhanced survival, proliferation and remained active, leading to the development of the Second Generation of CARs ( Krause et al., 1998). The pioneering work of Dr. Carl June and his group (University of Pennsylvania) lead to the development of CAR therapy ( Kalos et al., 2011), with the approach of using modified T lymphocytes that carried a CAR to target CD19⁺ leukemic B cells ( Varadé et al., 2021). Such an approach was because the expression of the transmembrane glycoprotein CD19 is maintained during differentiation of B lineage at normal (14% in peripheral blood lymphocyte phenotype) ( Berron-Ruiz et al., 2016) and neoplastic B cells malignancies (80% of ALL, 88% of B cell lymphomas and 100% of B cell leukemias) ( Wang et al., 2012), as well as follicular dendritic cells. Nowadays, CD19 expression is widely used for the diagnosis of leukemias and lymphomas and is used as the main target for the redirection of T lymphocytes against these neoplastic associated molecules ( Scheuermann and Racila 1995; Tedder, 2009; Wang et al., 2012). In 2009, Isabelle Rivière group published the manufacturing validation of biologically functional T cells targeted to CD19 antigen basing their work on promising data in the eradication of systemic B cell malignancies. This publication launched phase I clinical trials in chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) ( Hollyman et al., 2009). CD19 expression is widely used for the diagnosis of leukemias and lymphomas and used as the main target for the redirection of T lymphocytes against these neoplastic associated molecules ( Scheuermann and Racila 1995; Tedder, 2009; Wang et al., 2012) even though there may be more specific receptors for tumor cells.

The publication of the phase I clinical trial results, proving that CAR-T therapy-induced molecular remissions in adults with chemotherapy-refractory ALL ( Brentjens et al., 2013; Grupp et al., 2013), was followed by a scale-up of bioprocess production. Finally, FDA approved the CD 19 CAR-T cell therapy (Tisagenlecleucel) for ALL in children and young adults ( U.S. Food and Drug Administrations, 2021a). In addition, in 2021 the FDA approved the use of CAR-T cells using the B-cell maturation antigen ("BCMA" target (Idecabtagene vicleucel) in multiple myeloma ( U.S. Food and Drug Administrations, 2021b).

During the first commercial lustrum of CAR immunotherapy, CAR-T therapy explored different applications in other malignancies associated with the initial biomolecules as large B-cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma and high-grade B-cell lymphoma (YESCARTA U.S. Food and Drug Administrations, 2021c). So far, most studies are carried out on the discovery of other targets such as CD20, CD22, CD30 and new targets in solid tumors to increase the scope of CAR-T therapy ( Elahi et al., 2018; Castella et al., 2020).

Profiting the clinical successes, the use of CAR-T cells has also been explored in a variety of cancers ( Petersen and Krenciute 2019) such as diffuse large B-cell lymphoma treatments in adults and multiple myeloma ( Mullard 2017; Iragavarapu and Hildebrandt 2021; Munshi, et al., 2021) in which the therapeutic products were recently approved by the Food and Drug Administration (FDA, United States). Meanwhile, the European Medicines Agency (EMA) has granted conditional approval, or their authorization is currently under review. CAR-T cells re-targeted against the ubiquitous B-cell antigen, CD19 are a remarkable innovation in the treatment of relapsed and/or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) ( Nie et al., 2020). The success of CAR T-cells therapy in cancer is exemplified by numerous clinical trials that have shown that 70-90% complete remission (CR) can be achieved in pediatric and adult patients treated with CD19-directed CAR T-cells ( Xu et al., 2019). Based on these growing CAR-T cell immunotherapy achievements, over 500 clinical trials worldwide analyzing CAR-T cells for the treatment of cancer are currently being under evaluation, and the majority are being performed in East Asia (269 trials), followed by the US (225 trials), and Europe (62 ongoing studies) (as registered at clinicaltrials.gov Q3 2020) ( Albinger et al., 2021). So far, five CAR-T cell therapy agents were approved by FDA for cancer treatment: Tisagenlecleucel (Kyrmriah®) for treatment of acute lymphoblastic leukemia (ALL) ( Mullard 2017), Axicabtagene ciloleucel (Yescarta™) for diffuse large B-cell lymphoma (DLBCL) and also FDA-approval for follicular lymphoma in april 2021 ( Bouchkouj et al., 2019), Brexucabtagene autoleucel (Tecartus™) for treatment of mantle cell lymphoma (MCL) ( Al-Juhaishi and Ahmed, 2021), Idecabtagene vicleucel (ABECMA®) for treatment of multiple myeloma ( Munshi et al., 2021) and Lisocabtagene maraleucel (Breyanzi®) for diffuse large B-cell lymphoma treatment ( Iragavarapu and Hildebrandt 2021) ( Table 2).

TABLE 2. Currently approved CAR-T cells products by FDA and EMA. These are the available products up to July 2021.

Chimeric Antigen Receptor T Cell Immunotherapy

CAR immunotherapy is a successful innovation in cancer therapies achieving impressive results in the treatment of resistant hematological malignancies highlighting its strong potential ( Neelapu et al., 2017). CAR-T cell immunotherapy was developed out of the need for T cells to be able to directly recognize tumor cells without the required antigen processing or presentation by professional antigen-presenting cells ( Varadé et al., 2021). This immunotherapy is based on T-cell engineering and the use of synthetic (recombinant) receptors, termed CARs, instead of the physiological (native) receptor for the antigen, the TCR ( Sadelain et al., 2009). These CARs are based on a specific antibody directed to a target surface molecule and were developed with the intent of combining the tumor recognition capabilities of antibodies with the powerful antitumor effector abilities of T cells. CAR expression is most often achieved using retroviral- or lentiviral-mediated gene transfer. The T cell's rapidly dividing nature facilitates viral integration, and its ability to establish memory serves to reinforce long-lasting transgene expression ( Grosser et al., 2019). Besides, CARs constructions include all the elements necessary for intracellular signaling and activation of helper and cytotoxic T lymphocytes ( Singh and McGuirk 2020).

Target Selection

CAR recognition of malignant cells depends on the designed chimeric recognition molecule, not on the traditional T-lymphocyte receptor (TCR) or human leukocyte antigen (HLA). This interaction between CAR and the target leads to the formation of immune synapses, where contact-dependent cytotoxicity occurs. Hence, choosing a target with high specificity and high coverage is the main objective for these therapies. Unfortunately, nowadays there is no guide with uniform criteria that shows how a target should be properly selected, however, it is important to consider that not only membrane proteins are useful, also carbohydrates and glycolipid molecules can be selected if they adapt to the clinical needs of the desired disease ( Wang et al., 2017; Wei et al., 2019).

It is recommended to identify and use a target that achieves adequate coverage of tumor cells with enough specificity to prevent CAR activation in other sites that could lead to serious damage in organs. Hence, this way, it is possible to avoid the main toxic side effects in CAR-T treatment, such as cytokine release syndrome (CRS), and the "off-tumor" effect caused by damage to non-tumor cells ( Liu et al., 2019).

Recently, different types of CAR have been designed to take into consideration the coverage of two selected targets and the specificity for different types of solid tumors that share proteins involved in cell malignancy. Developed strategies are known as, Pooled CAR: two CAR in the same vector; Dual CAR: cross-compliance where the first target has the signaling domain while the other one has the costimulatory domain, CAR in tandem: where two variable fraction chains are attached to the same antigen-binding domain and some others where activation or function would be inhibited by an inhibitory CAR. The previous designs will further expand CAR-T cell therapy in clinical trials ( Fedorov et al., 2013; Hartmann et al., 2017). Since novel targets are currently studied, in a further section, the specific antigens are presented as a strategy to improve the CAR-T cells application ( Table 3).

TABLE 3. Novel targets for hematologic malignancies and solid tumors.

The major advantage of cell-based therapies is the variety of surface proteins that can be used as targets, which allow the specific antigen recognition in an independent manner of the major histocompatibility complex (MHC), in contrast to TCR-mediated antigen recognition ( Li et al., 2018; Dwivedi et al., 2019), that deliver remarkable results in the clinic, specifically for the treatment of B cell malignancies ( Grupp et al., 2013; Porter et al., 2015).

Chimeric Antigen Receptor Designs

In detail, the CAR is a synthetic receptor that ligates to a surface antigen and transduces the protein target of recognition into a signaling cascade ( Brown and Mackall, 2019; Qin et al., 2021). The molecular architecture of CAR consists of four main components: 1) antigen-binding (recognition) domain, which provides the exclusive recognition of the target molecule based on monoclonal antibodies with variable regions that are linked into a single-stranded fragment (scFv) ( Shirasu and Kuroki, 2012); between the recognition and transmembrane domains lies a 2) hinge or spacer domain, which is a structure normally constructed by the flexible hinge sequences of CD8α, CD28 or the Fc region of immunoglobulins IgG1 or IgG4 ( Lee et al., 2019; Roselli et al., 2019), this is in charge of bringing an effective immunological synapse ( Fujiwara, et al., 2020). 3) A transmembrane domain, that is the anchor molecule to the cell membrane which impacts the regulation of the amount of CAR signaling into T cells via the control of the CAR surface expression level ( Dwivedi et al., 2019). 4) An intracellular signaling domain, that provides the intracellular portion of the TCR invariant chain CD3ζ, which triggers the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM´s) domains activates the signaling pathway of ZAP70 ( Milone et al., 2009). However, this signal is deficient to activate by itself the resting T cell. Hence, to improve activation, proliferation, and survival the incorporation of the costimulatory domain from other receptors, such as CD28, 4-1BB (CD137), should be included to the intracellular fragment ( Ramello et al., 2019) ( Figure 1A).

FIGURE 1. Development of CAR generations. (A) Archetype of a CAR. The four main domains that make up a CAR are distinguished: the recognition domain, the hinge domain, the transmembrane domain, and the intracellular domain. (B) CARs generations. First-generation CARs only own the CD3ζ in the intracellular domain; the second-generation CARs incorporate a costimulatory domain; the third-generation CARs include two costimulatory domains; and fourth-generation CARs are reinforced with genes that allow the expression of cytokines.

Simple CARs classification is given by the number of signaling groups that possess in the intracellular signaling domain, being the first-generation CARs those that in their signaling domain contain CD3ζ chain as the primary transmitter of endogenous TCR signals ( Ghosh et al., 2017). The first generation of chimeric receptors was primarily designed to mimic its T cell activation, a function that is sufficient to trigger cytolysis, but not enough to direct a sustained effective T cell response ( Sadelain et al., 2009). To broaden T cell function, a different kind of Intracellular signaling domain was designed, mimicking a normal activation of T lymphocytes, and ensuring the presence of a co-stimulatory signal. Diverse co-stimulatory signals have been proposed such as CD28, 4-1BB and OX40, as they normally activate cell survival and proliferation pathways (i.e., PI3K-Akt-mTORC1) ( Van Der Stegen et al., 2015). The second-generation of CARs have recently yielded impressive clinical results in patients with B cell malignancies, specifically in acute lymphoblastic leukemia ( Brentjens et al., 2011; Maude et al., 2014). The union of two costimulatory domains in the Intracellular signaling domain is called "third-generation" CARs; this strategy increases the differentiation towards T cell effectors (Teff) and improves persistence by prolonging T-cell survival ( Hombach et al., 2011). Further research has led to a novel generation of CARs named TRUCK cells (T-cells redirected for universal cytokine killing), also called "fourth generation" CARs that, in addition to costimulatory signal(s), is provided with a "nuclear factor of activated T cell-responsive expression" which can activate an innate immune response through the secretion of an element for an inducible transgenic product like IL-12 or another cytokine, ( Figure 1B). This CAR-T is expected to be applied in fields of virus infections, auto-immune diseases, or metabolic disorders ( Chmielewski et al., 2018; Date and Nair, 2021).

T-Cell Procurement

As the last element, the isolation of T cells is a crucial factor since these types of cells are needed to get a high-quality product. The requirements for CAR-T cell manufacturing process are T cells with CD3/CD4+ and CD3/CD8+ immunophenotype. For T cell harvesting the process starts from the mononuclear cell fraction notwithstanding that the final quantities of contaminating cells are dependent upon the type of blood cell isolation process, counterflow centrifugal elutriation or even the inherent properties of the patient's blood. As a strategy for obtaining viable and high purity of T cells (CD3/CD4+ and CD3/CD8+) additional purification steps are needed; At this point, immunomagnetic isolation has been introduced to shift the density gradient separation ( Fesnak and O'Doherty, 2017; Stroncek et al., 2017).

It has even been reported that the impact of CD4/CD8 T-cell selection on the starting apheresis product influences manufacturing feasibility, however, this strategy led to a direct increase in inflammatory toxicities ( Shah et al., 2020a; Shah et al., 2020b). Clinical recommended applications of CAR-T-cell therapy include infusion products at a 2:1 ratio being approximately CD4⁺ CAR-T (29 ± 18.1%) and CD8 + CAR-T (71 ± 18.1%) ( Itzhaki et al., 2020). Taking into consideration that on average one patient receives 3.1×10 ⁶ transduced viable T cells per kilogram of body weight (range, 0.2×10 ⁶ to 5.4×10 ⁶ cells per kilogram) for ALL treatment. Meanwhile, for lymphoma the recommended dose of infused CAR T-cells ranged from 0.66×10 ⁶/kg to 3.3×10 ⁹/m2 ( Maude et al., 2018; Cao et al., 2019), with a normal transduction rate around 20-60% ( Itzhaki et al., 2020).

Chimeric Antigen Receptor-T Cell Immunotherapy Challenges

CAR-T cell immunotherapy has found its success where conventional therapies fail, becoming a promising emerging technology that represents an important progress for the treatment of cancer malignancies. Furthermore, it also opens new potentials for the treatment of other diseases. However, this therapy is relatively new, and some significant challenges have been observed, mainly related to side effects, toxicity, T-cell exhaustion, and hostile tumor microenvironment (TME) ( Figure 2). In addition, the manufacturing process for offering clinical-grade cell therapies at a large-scale production and their distribution are currently time-consuming and costly. Therefore, making CAR-T cell immunotherapy available for as many patients as possible becomes a greater challenge. New processing techniques, quality control mechanisms and logistic developments are required to overcome these limitations and to develop robust technology. In the next paragraphs, an analysis of such challenges is presented.

FIGURE 2. The major challenges in CAR-T immunotherapy. Nowadays, the novel CAR-T cells therapy does face some significant challenges, such as side effects, a hostile tumor microenvironment (TME), and T-cell exhaustion. In addition, the challenge of scaling-up the manufacture of clinical-grade cell therapies needs to be addressed to make them available and affordable for more patients worldwide.

Side Effects and Toxicity

After CAR-T cell infusion, close patient monitoring is a crucial part of the treatment protocol due to the potential lethal toxicities of this immunotherapy. Fever, inflammation, abnormal liver enzyme elevation, breath difficulty, chills, confusion, dizziness, lightheadedness, severe nausea, vomiting, and diarrhea are some of reported side effects. All patients have developed long-term B cell aplasia, which can be alleviated by administration of gamma globulins ( Swiech et al., 2020; Zhao et al., 2020). There are two main categories of toxicity: Cytokine release Syndrome (CRS) and neurotoxicity (NTX) or CAR-T cell-related encephalopathy syndrome (CRES).

Cytokine Release Syndrome

CRS or "cytokine storm" is a systemic inflammatory response, caused by the release of inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-10, IL-15, interferon (IFN)-γ and tumor necrosis factor (TNF) by a large number of activated lymphocytes (B cells, T cells, and/or natural killer cells) and/or myeloid cells (macrophages, dendritic cells, and monocytes) which result in a wide range of clinical symptoms including fever, fatigue, headache, rash, joint pain and myalgia ( Chavez et al., 2019; Riegler et al., 2019). CRS is the most common adverse effect that occurs within a few days after the first infusion of CAR-T cells (the CRS of any grade occurred in 85% of patients). Severe CRS cases are characterized by tachycardia, hypotension, pulmonary edema, cardiac dysfunction, high fever, hypoxia, renal impairment, hepatic failure, coagulopathy, and irreversible organ damage ( Park et al., 2018). Fortunately, the effects of CRS can be attenuated by reducing the number of infused T cells and/or by the administration of anti-IL-6 receptor monoclonal antibody (tocilizumab) and steroids ( Swiech et al., 2020). Nowadays, the search for different biopharmaceuticals to avoid the CRS is increasing due to COVID-19 treatment, which exhibits a similar CRS profile. This means that the next findings could be useful to improve this type of treatment.

Neurotoxicity

NTX is another common complication of CAR-T cell immunotherapy that occurs in more than 40% of patients ( Belin et al., 2020). NTX described as CAR-T cell-related Encephalopathy (CRES) by Neelapu et al. (2018) and, more recently described under the name of ICANS (Immune effector Cell-Associated Neurotoxicity Syndrome) ( Lee et al., 2019), is less understood than CRS and usually appears within one to 3 weeks after CAR-T cell infusion, which is frequently correlated with CRS ( Siegler and Kenderian, 2020). Patients show a variety of symptoms such as confusion, obtundation, tremors, delirium, word-finding difficulty, and headaches; other symptoms such as aphasia, cranial nerve abnormalities and seizures have also been reported as part of NTX effect ( Neill et al., 2020). Nowadays, neurotoxicity management is treated by administering tocilizumab: an IL-6 receptor antibody; systemic corticosteroids; and dexamethasone ( Gust et al., 2018).

The prompt management of toxicities is essential to minimize the mortality associated with this immunotherapy, for that, researchers have developed different safety strategies for overcoming and prevent the CAR-T cells toxicities such as the design of new CARs generations ( Andrea et al., 2020; Rafiq et al., 2020). Toxicity management has become a critical step in the success of CAR-T cell immunotherapy. In fact, Tmunity Therapeutics developed a dual PSMA-specific, TGFβ-resistant CAR-modified autologous T cells (CAR-T-PSMA-TGFβRDN) for prostate cancer with the objective to expand the use of CAR-T from hematological malignancies to solid tumors. This clinical trial (NCT04227275) had a setback that led to its winding down after two patients died from a type of ICANS. Nevertheless, the study helped to identify the potential barriers of CAR-T therapies against solid tumors, which results in a better understanding of the adverse effects mechanisms that will aid to design safer candidates.

Chimeric Antigen Receptor-T Cell Exhaustion

CAR-T cells have raised enormous interest in cancer immunotherapy due to the high rates of complete remission. Nevertheless, a large fraction of patients that achieve remission have displayed disease relapse within a few years ( Maude et al., 2018; Park et al., 2018), the relapse rate varies from 21 to 45% in B-ALL and increases with the follow-up time ( Cheng et al., 2019). The treatment failure could be partially explained by CAR-T cells exhaustion induced by tumor microenvironment (TME) created by solid tumors. Similar phenomena appear to extend in hematological malignancies including chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and diffuse large B cell lymphoma (DLBCL), besides to an excessive antigen exposure ( Poorebrahim et al., 2020; Shen et al., 2020). The complex and heterogeneous TME affects the activation and function of the infiltrated effector T cells and thus impairs the persistence, proliferation, and potential of the T cells rendering them exhausted ( Tahmasebi et al., 2019). CAR-T cells exhaustion refers to a state of dysfunction characterized by physical deletion of antigen-specific T cells due to persisting antigen stimulation, the costimulatory domain of CAR structure and increased expression of inhibitory receptors, usually induced by chronic stimulation, such as cancer ( Wherry, 2011; Cheng et al., 2019). In vitro CAR-T cell studies have shown that during CAR-T cell exhausting process the loss of functionality against tumors is mainly triggered by the upregulated expression of inhibitory receptors such as PD-1, Lag3, Tim3 TIGIT, and the inhibition of PI3K/AKT pathway through CTLA-4. Nonetheless, cytokines play an important role in T cell activity or exhaustion, for instance, CAR-T cells decrease the ability of interleukin 2 (IL-2), Tumor necrosis factor α TNF- α, and interferon-γ (IFN-γ) among other cytokines ( Tang et al., 2021); Interleukin-5 (IL-5) increased proliferative capacity, decreased apoptotic state through IL-5 mediated reduction of mTORC1 activity ( Alizadeh et al., 2019).

Other factors such as transcriptional factors, metabolism, and epigenetic modification also play an important role in CAR-T cell exhaustion development ( Shen et al., 2020). This dysfunctional phenotype is associated with hallmarked loss of the CAR-T cell's capacity of expansion and persistence. That situation compromises the patient's clinical remission ( Fry et al., 2018). The reason why CAR-T cells lose in vivo persistence and potency remains unknown ( Balkhi, 2020), that is why the CAR-T cells exhaustion is a pivotal hurdle for successful CAR-T cell therapies. A possible approach to delay the exhaustion is the engineering of exhaustion-resistant CAR-T cells. Recent reports indicate that the discovery of certain transcription factors like TOX ( Seo et al., 2019) and NR4A ( Cheng et al., 2019), and the deletion or overexpression of AP-1 family transcription factor c-Jun ( Lynn et al., 2019) increases CAR-T cell resistance to exhaustion. Some studies have shown that CAR-T cells equipped with cytokines can enhance their own lifespan and expansion, promoting their own growth and proliferation ( Avanzi et al., 2018; Zhao et al., 2020). That is the reason for the TRUCKs development by genetically adding an inducible cytokine-producing cassette (e.g., IL- 12) ( Zhao et al., 2020). Lately, deleting PD-1 by CAR-T cells engineering (short hairpin RNAs or CRISPR/Cas9), blocking antibodies using PD-1 and blocking the IFN-γ signal have been used to increase the CAR-T therapy efficacy avoiding exhaustion ( Cheng et al., 2019; Rafiq et al., 2020). In this case, the discovery of the cellular mechanisms involved during cell proliferation and the maintenance of cell viability are a hindrance to the functioning of this therapy.

Tumor Microenvironment

The success of CAR T-cell immunotherapy has not yet been achieved in solid tumors. A possible reason is that the immunosuppressive nature of the tumor microenvironment (TME) affects the efficacy of adoptive immunotherapy. Solid tumors are highly infiltrated with stromal cells like cancer-associated fibroblasts (CAFs) and suppressive immune cells, including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), mast cells, and regulatory T cells (Tregs) which contribute to the establishment of a hostile and immunosuppressive TME capable of interfering the efficacy of CAR-T cell therapy ( Martinez and Moon, 2019; Rodriguez-Garcia et al., 2020). Strategies to overcome TME effect, include enabling T cells to resist tumor suppression in TME, such as transgene expression of dominant-negative receptors or signal converters, which can transform suppressive signals into stimulating signals ( Tang et al., 2021). Another opportunity to overcome the persistence and exhaustion of CAR-T cells is improving the trafficking delivery of the drug to the tumor site. For CAR-T cells, local injection is a popular investigation among trials. This could be done in an anatomical cavity (pleura, peritoneum), via a device placed surgically (for CNS tumors) or via intra-arterial delivery (such as hepatic artery catheterization), or by direct intratumoral injection ( Edeline et al., 2021).

Genetic Alterations

Several reports have highlighted a lack of efficacy and relapses in patients treated with CAR-T cells ( Sotillo et al., 2015; Fischer et al., 2017). Few reports have tackled the identification of genomic modifications post CAR-T cell therapy due to vector integration ( Nobles et al., 2020). Orlando et al. (2018) incorporated whole-exome DNA-seq and RNA-seq to investigate the extent to which CD19 mutations are the main mechanism causing relapse. They discovered de novo CD19 genetic alterations in exons 2-5 in 12 of 12 samples from patients, and loss-of-heterozygosity in 8 of 9 patients, concluding that homozygous mutations in CD19 are the main reason for acquired resistance to CAR-T cell therapy. Asnani et al. (2020) reported similar findings; they described skipping of exon 2 and exons 5-6 in patients with relapsed leukemia after CAR-Tc therapy. Exon 2 is essential for the integrity of CAR-T 19 epitope while exons 5-6 are responsible for the CD19 transmembrane domain ( Bagashev et al., 2018). CAR-T cells have been shown to improve the efficacy of immunotherapy; however, further research is needed to explore the impact of genome analysis to identify the prognosis once CAR-T cell transfusion is performed.

Manufacturing Process

The traditional technology to manufacture CAR-T cells consists in: 1) to isolate patient's T-cells (autologous) by apheresis; 2) to ship recovered cells to a central manufacturing site (bio-pharmaceutical companies) where 3) they are genetically engineered to express a CAR after which 4) they are expanded in the laboratory; then, 5) the CAR-T cells are returned to a hospital to be 6) infused into the patient for tumor eradication ( Figure 3) ( Roddie et al., 2019). The logistics involved in this traditional manufacturing and treatment with autologous CAR-T cells adds complexity for clinicians and patients because the period in between, referred to as "vein-to-vein time", ranges between three and 4 weeks in developed countries. This period can be daunting for the patients awaiting treatment and renders the CAR-T treatment complicated for patients with rapidly progressing diseases ( Aftab et al., 2020). Nowadays, this therapy presents these main manufacturing challenges and the picture in developed and underdeveloped countries is very different. Some of such challenges include:

FIGURE 3. A brief diagram of the CAR-T cells manufacturing process. After enough leukocytes have been harvested from a patient's blood via leukapheresis, T cells are shipped to a biopharmaceutical company where they are genetically engineered to express a CAR on the T cell surface. Then, the CAR-T cells are amplified in vitro; after that, the CAR-T cells are returned to the hospital for administration into the patient.

Packaging, Shipping, and Storage of Chimeric Antigen Receptor-T Cells

The clinical manufacture of CAR-T cells is currently a complex process that involves several steps across different geographic locations with multi-technologies and logistics. Any error in timing, transportation methods, cold chain, or storage, would result in cell damage directly impacting therapy efficiency, hence each one of the steps requires careful management, precise sample tracking and adequate preservation technologies to either freeze or cryopreserve patient samples ( Papathanasiou et al., 2020). Different shipments with dissimilar temperatures are required throughout the CAR-T cell manufacturing process, therefore, suitable cryopreservation during production is mandatory for quality control tests ( Stock et al., 2019).

Current Good Manufacturing Practices

CAR-T cells have a complex preparation process and the cGMP´s are a critical and a bottleneck in CAR-T cell production ( Wang and Rivière, 2016). The aim of cGMP´s is to provide a framework to ensure high-quality production in well-controlled facilities and equipment by well-trained and regularly trained staff. Likewise, it provides strict documentation processes that covers all operation aspects to demonstrate a continuous and adequate compliance ( Li et al., 2019). The CAR-T cells manufactured under GMP regulations should provide a system in which the cells will be prepared under controlled, auditable, reproducible conditions that result in providing adequate evidence of identity, safety, purity, and potency ( Gee, 2018). The manufacturing protocols must improve reproducibility, cost-effectiveness, and scalability that will enable a broad application of CAR-T cell therapies ( Fernández et al., 2019). According to International Organization for Standardization (ISO)5 CAR-T cell manufacturing requires GMP facilities as cell processing clean rooms, that must be equipped with 1) Facilities systems (e.g., air-handlers, 24/7 alarm monitoring systems); 2) Environmental monitoring equipment (e.g., viable and nonviable particle counters); 3) Manufacturing process equipment (e.g., cell washers, bioreactors); and 4) Analytical equipment (e.g., automatic cell counters, flow cytometers) ( Wang and Rivière, 2016). To optimize the GMP facilities the use of automatized manufacturing processes as CliniMACS or Cocoon is feasible to achieve reproducible processes in a closed system. Another key to maintaining a compliant GMP manufacturing environment is the highly skilled staff with extensive knowledge of GMP manufacturing, quality control, and quality assurance ( Wang and Rivière, 2016). One complete review of the requirements for CTL019 is presented by Levine et al. (2017), who explain the complete manufacturing process and the challenges involved in each stage.

Production of Lentiviral Vectors

The approval of LV gene therapy products ( e.g., Kymriah/Novartis) increased their demand and has created the need to improve their large-scale manufacture to clinical-grade LV ( Lester et al., 2018). Nevertheless, the LVs production has challenges, such as their inherent cytotoxicity, low stability, and the dependency on transient transfection impact, both upstream and downstream processes that are reflected in low yielding and cost-ineffective compared to another viral vector ( Ferreira et al., 2021). The level of GMP compliance required for the manufacture of LV is a combination of diverse factors including regulatory expertise, compliant facilities, validated and calibrated equipment, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and quality by design approach ( Levine et al., 2017; Dasgupta, et al., 2020). In addition, part of the commercialization success of this gene therapy product is the establishment, standardization, and implementation of stable cell lines to produce LVs facilitating GMP-compliant processes, providing an easier scale-up, reproducibility, biosafety, and cost-effectiveness ( Ferreira et al., 2021).

Staff and Training

Considering the complex nature of the therapy and its associated high-risk side effects, access to CAR-T cells is highly regulated, making them available only at certified centers and administered by trained staff. All staff involved in CAR-T cell manufacturing (from T cell collection to the manufacturer and back to the clinical unit) require a broad training with satisfactory levels of competency. Such abilities manage complications that could arise during the process hence being able to deliver the product ( Yakoub-Agha et al., 2020). Nowadays, there are only a few prepared professionals in this field, however, multidisciplinary collaboration is needed to create greater knowledge in this area. Academic participation is an important fact because the new personnel need to acquire different abilities to manufacture CAR-T cells.

Quality Control

As a living ''drug," a CAR-T cell has a complex preparation process and requires "whole-process quality control". During the production, well-controlled cold chain transport and storage play an important role in ensuring the cell products' quality and preventing the bacteria and mycoplasma contamination ( Li et al., 2019). Current requirements in the CAR-T cell´s quality control include the inspection of ex vivo transduced T-cell for the replicative virus and of the production materials. Furthermore, it should include a release test of the finished products to confirm identity, purity, safety, and potency, considering CAR-T cell´s characteristics as biological products, cell products, and gene therapy products ( Figure 4) ( Eyles et al., 2019; Li et al., 2019). In addition, the validation of the production process is essential for CAR-T cells to be considered a final product. Moreover, stability studies are needed to verify the storage conditions and their shelf life. Besides, the production of CAR-T cells needs more in-depth studies to evaluate T cells' quality in both relapsed and regression patients. These studies should provide data about the distribution of lymphocyte populations. This information can guarantee efficient cell transduction and propagation ensuring the CAR-T cells' quality, and subsequently it would be reflected in the decrease of CAR-T cells exhaustion in patients. To sum up, quality control is of ultimate importance for the success of CAR-T therapy ( Dai et al., 2019).

FIGURE 4. Quality control during CAR-T cell therapy. The median of total nucleated cells (TNC) is 98 × 10 ⁸ (range 9 - 341 × 10 ⁸) with a viability analysis of 98% for quality control (QC) in the leukapheresis products. However, the QC could be set in 10× 10 ⁸ CD3⁺ T cells with a viability analysis of 98% (99.6-100%) in a median volume of 237 ml (136-310 ml) for CD3⁺ T cell isolation (Data based in the report of Korell et al., 2020). The normal transduction rate is around 20-60% using lentiviral vectors. A 2:1 ratio immunophenotype of CD4+: CAR-T (29 ± 18.1%) and CD8+: CAR-T (71 ± 18.1%) is recommended for clonal CAR-T cell expansion under GMP conditions. Clonal expansion must reach 3.1×10 ⁶ transduced viable T cells per kilogram of body weight (range, 0.2×10 ⁶ to 5.4×10 ⁶ cells per kilogram); this CAR-T cells product is ready for cryopreservation. Cold chain must be controlled during transportation using liquid nitrogen. Prior infusion lymphodepleting chemotherapy with fludarabine and cyclophosphamide it is used. Finally, rate a CAR-T subclone identification followed by genome sequency should be evaluated for measure the molecular remission.

Scaling Up

CAR-T cells manufacturing should be scalable (that means, to have many single bioreactors for each patient) to have a broader scope to benefit patients without sacrificing the quality and reproducibility of the products ( Dai et al., 2019). Personalized therapies such as autologous cell-therapies require an "intensive scale-up", that is related to the fact of having many bioreactors to amplify the CAR-T cells for each patient, instead of increasing the volume as in common biopharmaceuticals. Besides, it depends upon the ability to accommodate multiple independent productions in parallel ( Wang and Rivière, 2016).

Limited Opportunity for Redosing

CAR-T cells manufacturing does not allow volumetric scale-up, consequently, cells must be prepared as a single batch limiting the quantity of available product. Under this scenario, patients may not have the opportunity to receive a new infusion of their CAR-T cells quickly and easily if needed ( Aftab et al., 2020; Papathanasiou et al., 2020).

Time Manufacturing

CAR-T cells can take up to 4 weeks to manufacture, during this window of time, the patients are extremely vulnerable with risk of disease progression and mortality while waiting for infusion ( Rafiq et al., 2020). Nevertheless, the use of novel strategies could help to overcome such a limitation, but it is necessary to consider the time required for transporting to prepare the adequate conditions for the patient. This takes around 6 weeks for the treatment to be applied at best.

Pricing and Patient Access

Pricing and patient access is the most important limitation to overcome to disclose the use of CAR-T cells all over the world. The current model of CAR-T cell manufacturing is highly centralized: firstly, the patient's T cells must be transported (often internationally) to the manufacturing facility, secondly, the CAR-T product is returned to the patient for infusion ( Hay and Cheung, 2019). The process is complex in each step, resulting in highly costly therapy that ranges between $373,000 and $475,000 USD per treatment (hospital expenses associated with the therapy are not considered in such average costs) that neither patients nor the health-care system can afford. This prohibitive cost and the reimbursement gap limit the patient's access and remain unsustainable in socio-economically underdeveloped countries, restricting even more the widespread use of CAR-T cell therapies. Until CAR-T cell therapy becomes affordable, its potential will remain untapped ( Yadav et al., 2020).

Regulatory Requirements

Another important bottleneck in cellular products is regulation. The CAR-T cells are globally considered within Advanced Therapy Medicinal Products (ATMP) and these products require licensure ( Marks 2019). Regulatory agencies are highly related to standard therapies, but cellular products have special requirements. US or EU regulatory authorities are working to define optimal guidelines that can globally harmonize the requirements for clinical manufacturing of ATMP ( Kaiser et al., 2015). Meanwhile underdeveloped countries face a greater challenge because CAR-T therapeutic use in clinics is hugely restricted, resulting in a poor understanding of the regulatory requirements by the authorities.

Strategies to Increase the Use of Chimeric Antigen Receptor-T Cells Technology. Discovery of New Biomarkers

Biomarkers are of main importance for cancer clinical management, as they can be used to define the criteria to identify the patients suitable for CAR-T therapy, prognosis, prediction of response to treatment and monitoring disease progression. Developing cancer immunotherapy has therapeutic implications and its success varies depending on the type of cancers ( Schumacher et al., 2015). On the other hand, CAR-T cell immunotherapy has achieved promising results mainly in patients with hematologic malignancies. On the other side, this type of therapy has more obstacles attacking solid tumors, including immunosuppressive tumor microenvironment (TME), lack of permeation of the CAR-T cells into tumors, limited transport, and specific tumor antigen ( Ling, 2020). Despite the significant advances, clinical improvement after CAR-T therapy has not been observed for all the patients who have undergone this immunotherapy. Such differences could rely on molecular response; hence it is necessary to fully understand remission and relapse in CAR-T therapy through biomarkers. Consequently, it is essential to identify the clinical features in the form of biomarkers that drive response, resistance, and adverse effects because of immunotherapies ( Townsend et al., 2018). Thus, for the respective CAR-T cell immunotherapy development and success it is essential to broaden the range of cancers that respond to such a kind of immunotherapy with the discovery of new biomarkers. The identification of new biomarkers improves clinical practices for early recognition and minimization of adverse effects while preserving the antitumor activity of the CAR-T cells ( Mirzaei et al., 2018), it opens the possibility to target other types of tumors and leads to the development of new strategies for numerous types of cancer treatments, positioning CAR-T cells as a therapeutic option in cancer medicine ( Ponterio et al., 2020). The first biomarker for CAR-T - therapy was CD19, a B cell surface protein expressed mainly on malignant B cells. Currently, scientific research is underway to find out biomarkers according to the stage of immunotherapy as follows: biomarkers to define patients baseline status; biomarkers for CAR-T-cell function, CAR-T cell exhaustion, CAR-T cell toxicity and biomarkers for cancer prognosis, response, and relapse ( Mirzaei et al., 2018). As for baseline biomarkers, cytokines of the immune system such as IL2, IL-5, IL-7, TNF-a, among others; lactate de Lactate dehydrogenase (LDH) and CD9 cells, are widely used. Meanwhile, for CAR-T cell function, the following biomarkers have been proposed: CD45RA, CD45RO, CD62L, CCR7, CD27, CD28 (differentiation markers), CD25, CD127, CD57, and CD137 (activation markers) ( Wang et al., 2019; Hong et al., 2021). Currently, there are no proven biomarkers that can be used to assess CAR-T cell exhaustion after infusion in patients. However, indirect parameters could be helpful for this purpose; for example, a sustained gain in B-cell recovery within 3 months after CART-therapy and the detection of LAG3+ cells could be considered due to CAR-T cell exhaustion. As well as high levels of expression of PD-1, LAG-3, TIM-3, and their receptors are associated with T cell exhaustion and poor response to CD19 CAR-T therapy ( Hong et al., 2021). Recently a model of T cell exhaustion assessment through flow cytometry has been proposed based upon PD-1 pathway blockade response, identifying three main stages: exhausted progenitors, intermediated exhausted, and terminally exhausted T cells ( Beltra et al., 2020). Relapse: IL-6, Protein C reactive, TNF-a, serum calcium, serum phosphate, uric acid ( Zhang et al., 2016). In Table 3, a summary of novel target antigens selection for CAR-T cells in hematologic malignancies and solid tumors under clinical trials is presented. Despite significant advances in CAR-T therapy, it is essential to continue exploring different cancer cell-type-specific biomarkers to develop more specific therapies.

Allogeneic Chimeric Antigen Receptor-T Cells

Currently, most CAR-T cell immunotherapy is generated using autologous T cells. This represents several disadvantages at different levels. For example, autologous cell therapy is performed for individual patients, the production can be long-time consuming and complicated, resulting in the increase of costs and it can limit the broader application of this therapy. In addition, the challenges due to the use of patient-derived T cells for CAR therapy include weaker proliferation, limited expansion, and low persistence of CAR-T cells. In some cases, CAR-T cell efficacy, most likely due to poor autologous immune cell strength in cancer patients caused by aggressive treatments ( Caldwell et al., 2021). One opportunity for improving some manufacturing-related issues is the use of allogeneic CAR-T cells for reducing time delays on autologous cell production. Generating universal CAR-T cells from allogeneic healthy donors could set the stage for overcoming high costs and high time production, making them easily available and with higher quality. Such a fact is very important for patients with aggressive cancers that require urgent therapy ( Caldwell et al., 2021). This strategy would broaden the number of patients that could receive this immunotherapy to make CAR-T cell therapy an off-the-shelf treatment at an affordable cost, that be readily available and would increase quality properties in T cells ( Cheng et al., 2019). Another advantage is that universal CAR-T cells could provide the possibility of manufacturing clinical-grade CAR-T products against various cancers "on-demand" ( Balkhi, 2020). However, it is important to also consider the up-coming challenges and disadvantages for using universal CAR-T cells. For example, immunologic mismatch between donor and recipient, if the administered allogeneic T cells attack healthy recipient tissues, may cause life-threatening graft-versus-host disease (GVHD), and if the recipient's immune system recognizes and reacts against the allogeneic T cells, these may be rapidly eliminated by the host immune system limiting the expected therapeutic effect ( Depil et al., 2020). A possible solution is a genetic elimination or disruption of the TCR gene and/or HLA class I loci on the donor to eliminate GVHD ( Cheng et al., 2019), also, the use of allogeneic anti-CD19 CAR with deleted α-TCR chain and CD52 gene loci has demonstrated effectiveness to eliminate GVHD ( Cheng et al., 2019; Balkhi, 2020).

Different allogeneic strategies have appeared to improve immunotherapy challenges such as the use of induced pluripotent stem cells (iPSC). iPSCs offer the opportunity to be unlimitedly cultured in vitro and be successfully differentiated towards the lymphoid lineage as to NK lymphocytes that are innate lymphocytes that kill malignant cells in an HLA-independent manner ( Kennedy et al., 2012). The use of iPSCs provides unlimited ''off-the-shelf'' NK cells offering doses without limitations, standardized and cost-effective products ( Zeng et al., 2017). iPSCs can generate immune effectors with the capacity of easily being genetically modified to augment their applicability, potency, and persistence ( Nianias and Themeli, 2019). The possibility of effortlessly generating NK cells from iPSCs (iPSC-derived NK cells) opens the great option to use the CAR constructions as an alternative to T cells, to produce CAR-iPSCNK cells with rapid responses against malignant cells without causing GVHDs ( Euchner et al., 2021). Compared to CAR-T cells, CAR-NK cells offer some more advantages, such as a lack or a decrease in cytokine release syndrome and neurotoxicity in an autologous setting, another point in its favor is the multiple mechanisms for activating cytotoxic activity (NKG2D, KIR´s, CD16, NKp30, NKp44, NKp46), still maintaining its infiltration capacity into solid tumors and into the resistant tumor microenvironment ( Xie et al., 2020). CAR-NK preclinical studies showed effectiveness against hematologic malignancy targets (CD19 and CD20), as well as solid tumor targets demonstrating their potential to be an allogeneic therapeutic ( Caldwell et al., 2021). Besides CAR-iPSCNK strategy, virus-specific T cells, memory T cells, genetically modified αβ T cells, and γδ T cells are other effective ways to tackle allogeneic CAR immunotherapy. All these strategies have in common their effectiveness to attack malignant cells, and the possibility to use CAR construction to improve toxicity effects, costs and efficiency of CAR-T manufacturing, and the persistence of reducing GVHD risk ( Martinez and Moon, 2019; Yazdanifar et al., 2020).

Other Strategies to Improve Chimeric Antigen Receptor-T Cells Outcomes

Dual or tandem CARs, consist of the coexpression of two separate CARs in each T cell, which recognize two different antigens rather than one. Some dual CARs have entered clinical trials in hematological malignancies targeting CD19/CD20, and CD19/CD22 ( Zah et al., 2016; NCT03241940) and solid tumors. CAR specific for HER2/MUC1 had promising in vitro results in a breast cancer model. It is important to mention that dual-target CAR specific HER2/IL13Ra2 showed greater success than single-target CARs in a xenograft glioma model ( Martinez and Moon, 2019). Dual CAR is a promising method to address antigen heterogeneity and to prevent the relapse caused by antigen escape ( Huang et al., 2020). Synthetic Notch (synNotch) receptors have been applied in CAR-T cells to improve safety. SynNotch receptors recognize one specific tumor antigen and then transcriptional activation domains are released, promoting the local expression of a CAR ( Han et al., 2019). Moreover, synNotch-regulated CAR expression prevents constitutive signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state ( Choe et al., 2021). Synthetic Notch (synNotch) receptors have been applied in CAR-T cells to improve safety. SynNotch receptors recognized one specific tumor antigen and then transcriptional activation domains were released, promoting the local expression of a CAR ( Han et al., 2021). Moreover, synNotch-regulated CAR expression prevents constitutive signaling and exhaustion, maintaining a higher fraction of the T cells in a naïve/stem cell memory state ( Choe et al., 2021). Inhibitory chimeric antigen receptor (iCAR), which incorporates inhibitor receptors, such as PD-1 and CTLA-4, play a crucial role in attenuating or terminating T cells response, therefore, they are considered a safety strategy ( Yu et al., 2019). iCAR shows antigen-specific suppression of T cell cytokine secretion, cytotoxicity, and proliferation of rapid and selective kills of target cells that only express one antigen, whereas off-target cells, that co-expresses another inhibitory ligand recognized by iCAR, are protected from attack. This allows T cells to distinguish target cells from the off-target cells ( Han et al., 2019). With this said, it is important to highlight the following aspects for a successful clinical application: the iCARs' affinity and expression level, the appropriate selection of target antigen and the optimization of the CAR/iCAR ratio ( Gargett and Brown, 2014). Currently, Jan and cols., have developed ON and OFF switches for CAR-T cells using lenalidomide, a clinically approved drug, which facilitates the proteasome degradation of several target proteins ( Jan et al., 2021). In addition, to efficiently treat solid tumor, innovative combinations strategies such as vaccines, biomaterials, and oncolytic virus are good prospects, because they allow either to directly enhance T cells' function or to recruit endogenous immune cells as well as remodeling the TME ( Huang et al., 2020).

Fully Automated Manufacturing Process

Worldwide, the number of patients who require CAR-T cell immunotherapy quickly increases. That is the reason why the standardization of the manufacturing process (following GMP's), the quality control and expanding the production to hospitals making the treatment accessible for a larger number of patients, are highly needed. The industry has developed automated and closed cell manufacturing platforms to adapt to this scenario. Examples of this effort are the automatized platforms Cocoon® (Lonza) and CliniMACS Prodigy (Miltenyi Biotec), both allow reproducible and rapid production of cells, and every step is strictly documented. Cocoon® Platform streamlines patient-scale cell therapy manufacturing making them more efficient, reliable, and flexible. These features speed up CAR-T manufacturing, cut costs and decrease manufacturing failure rates that may be associated with more hands-on methods. In 2020 Lonza and Sheba Medical Center announced the first patient has been treated at Sheba Medical Center with CD19 CAR-T cell immunotherapy manufactured using Lonza's Cocoon Platform. Another example for automated and supervised manufacturing is CliniMACS, which allows T cell activation, transduction, amplification, and final harvesting of CAR-T cells, with high transduction frequencies and high cell numbers in the same device. This method fully controls the multi-step process through a flexible programming suite ( Lock et al., 2017; Köhl et al., 2018).

Network Collaboration

Unfortunately, most of the patients cannot afford CAR-T therapy due to the high costs and the lack of facilities around the world to produce them. The efforts focused on quality production following the GMP's had been successful to a certain extent, but the road is still long to bring this technology to a massive application.

To achieve this, it is necessary to generate a collaborative network between various stakeholders such as the academia, the industry, and the hospitals, for the establishment of adequate and robust legislation for this type of product in each country where such a technology is applied. Thus, universities should provide knowledge and innovation to future professionals for them to do research on new biomarkers discoveries, on the development of stable cell lines, and on developing and validating orthogonal analysis methodologies for the product quality and for them to learn about different production systems. The industry applying GMP's, quality control and automation processes can support the establishment of more standardized and reliable products that have a high quality, and it can also develop on-site production units that allow lower transport and storage costs. Hospitals must have adequate facilities for the management of this technology, with trained personnel in the management of production units in situ (if applicable), as well as medical personnel and health professionals for the effective treatment of patients. Encompassing all these activities there must be robust and specific legislation in this area that guarantees the quality of this type of advanced therapy. Additionally, regulation is an essential part of such a process. Nowadays, there is sparse information about regulatory guidelines in different countries, the EMA and FDA guidelines ( Iglesias-López et al., 2019) have established the baseline of such requirements for these technologies but the behavior and results on different phases of clinical trials will address novel considerations. One challenge but at the same time one opportunity will be to establish local regulation of these technologies in every country that needs to be approved by competent authorities for smooth implementation of CAR-T cell clinical programs. All this collaborative effort is the key to allow worldwide access to CAR-T cell therapy to a larger number of individuals with cancer.

Finally, the idea of promoting the use of CAR-T cells technology in a greater number of countries rests on its potential application in various diseases in both adults and children. To our knowledge, the application of CAR-T cells technology has been mainly used in developed countries ( Elahi et al., 2018), revealing a large gap in the rest of the world. In fact, the only clinical study conducted in Latin America was made in Brazil (Cunha Oct 16, 2019) in a 63-year-old man that was affected by BLBCL. Therefore, it is important to consider that for this technology to be enforceable in developing countries various policies must be created and implemented. On the one hand, technology transfer agreements can be established among centers with experience in the subject and treatment centers in developing countries, however, this continues to foster scientific-technological dependence. On the other hand, developing countries should work on integrating their own elements (academy-industry-hospital and government), generating treatments of this type but geared to the specific needs of its population and adapting them to the available resources. However, it is important to highlight that in this case, government support is essential to start setting up treatment centers for advanced therapies in hospitals and health institutions. In addition, international support from non-profit institutions in terms of financial resources and support for the training of professionals is also necessary.

Discussion

Current and widely used methods for cancer treatment consist of surgery, chemotherapy, radiation therapy and immunotherapy (mainly monoclonal antibodies). Despite the health care system efforts, with the increasing incidence of cancer, its clinical management continues to be a major challenge. Recently, remarkable progress has been achieved towards adoptive immunotherapies through clinical application and consistent evolution of ACT strategies. Such immunotherapies, in combination with conventional methods have proven major efficiency for cancer treatment. In the last few years, CAR-T cell therapy, a type of ACT, has demonstrated potential in clinical trials, leading to complete and durable responses in patients with late-stage and treatment-refractory disease. In fact, there are currently 5 FDA-approved products on the market, which have been tested in a few hundred patients during clinical trials in developed countries around the world. Furthermore, the first one was approved for commercial use in 2017 but the cost of such a treatment is substantially high (from $ 375,000.00 to $475,000.00 without considering operation and indirect costs) so, considering its potential application in the treatment of different types of diseases there is a need to improve the technology to make it more accessible to the world's population.

From a pharmaceutical point of view, CAR-T cells are considered as advanced therapy products or biopharmaceuticals (depending on the country) whose quality must be demonstrated through their identity, safety, and efficacy. This novel technology is the set of synergy between three main elements: the targets (antigens against the CAR will be directed and that, ideally, are required to be only expressed in malignant cells); chimeric receptors (whose sophistication allow to bind to said antigen and that are designed to provide better selectivity towards tumor cells, and properly, T cells (containing CD3 +/CD4 + and CD3 +/CD8 +, in an adequate proportion) that are genetically modified to express the CAR and guarantee the success of such therapy.

Each of the previous three elements presents significant challenges. As discussed in this work, the search for biomarkers is an exhaustive task aimed at identifying target molecules that are specific to a certain type of disease, which will help improve efficacy and reduce adverse effects. Likewise, the improvement in the development of CARs to promote an effective union with the receptor and the generation of an effective immune response against the disease and improve activation, proliferation, and survival is the subject of study. Furthermore, many efforts are directed at the selection of the T cells used in this technology, such cells must comply with adequate phenotypic characteristics; being genetically stable; being able to transduce within a high percentage (also putting on the table the need of vector optimization for this purpose); being expanded to reach the required levels for the therapy; being enough stable to avoid the cell exhaustion; and, accomplishing all the requirements of a cell therapy product at the level of identity, purity and efficacy. Furthermore, it is necessary to focus on the reduction of adverse effects related to CRS and NTX and in the application of this technology in solid tumors with the idea of increasing the safety and efficacy of this therapy, among other points of improvement.

Alternatively, the need to generate lower-cost production systems, with robust production in each batch (patient-specific), but that meet the necessary quality requirements to be applied to the patient is also a constant challenge. Currently, the use of automated systems applicable in situ seems a promising alternative. These processes must be in complete harmony with the requirements established by international and regional health authorities.

Finally, considering all the aspects of this therapy, the multidisciplinary participation of health care professionals in various stages is required. Through intensive collaborations between academia, industry, hospitals, and government, both at the international and regional level, it will be possible to achieve a broader accessibility to this technology to a larger number of patients. This situation should be able to overlap in the case of developing countries, who must seek different strategies by applying their own generated knowledge, making an efficient use of the existing infrastructure, and seeking collaborations at various levels to obtain the necessary information and resources. The implementation of these strategies is likely to increase the number of treatments all over the world.

Author Contributions

All authors conceptualized, designed, and assisted with the draft/approval of this manuscript.

Funding

The financial support for this work was by I0031-2019-03MACROPRONAII-SALUD-2019 Programa Nacional Estratégico para la Leucemia Infantil, CONACYT postdoctoral fellowship to AH-L (CVU: 253979), CONACYT INFR-2014 226271 and CONACYT doctoral fellowship to MA T-G (02986).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We gratefully acknowledge Facultad de Farmacia, Universidad Autónoma del Estado de Morelos. AH-L (CVU: 253979) is the recipient of a postdoctoral fellowship from CONACYT during development of this work.

References

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Chimeric Antigen Receptor-T Cells: A Pharmaceutical Scope

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Cancer is among the leading causes of death worldwide. Therefore, improving cancer therapeutic strategies using novel alternatives is a top priority on the contemporary scientific agenda. An example of such strategies is immunotherapy, which is based on teaching the immune system to recognize, attack, and kill malignant cancer cells.

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CAR-T (KTE-X19) Celle terapi i cancerpatienter med tilbagefaldene cancer (Mantle-Cell Lymphoma) 2020

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CAR-T (KTE-X19) Celle terapi i cancerpatienter med tilbagefaldene cancer (Mantle-Cell Lymphoma) 2020

# data[].attributes.body
Clinical trial - Forsøget inkluderede 74 patienter, hvor der blev produceret KTE-x19 til 71 af dem og administreret til 68.

84-98% viste et delvist reaktion, 67% viste en complet reaktion.
Patienterne der blev administreret KTE-X19 viste en seriøs og livstruende reaktion ud fra kroppens reaktion på KTE-X19, men efter 12 måneder viste 61-84% en fuldstændig overlevelses rate hvor canceren ikke voksede.

# data[].attributes.attachment_link.title
KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma - PubMed

# data[].attributes.attachment_link.description
KTE-X19 induced durable remissions in a majority of patients with relapsed or refractory mantle-cell lymphoma. The therapy led to serious and life-threatening toxic effects that were consistent with those reported with other CAR T-cell therapies. (Funded by Kite, a Gilead company; ZUMA-2 ClinicalTri…

# data[].attributes.headline
Chimeric antigen receptor T cells targeting CD147 for non-small cell lung cancer therapy PMID: 34896852

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Chimeric antigen receptor T cells targeting CD147 for non-small cell lung cancer therapy PMID: 34896852

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CD147-CART cells have strong anti-tumor activity against NSCLC cells in vivo in both CDX and PDX models and no adverse side effects. Our findings show that CD147-CART immunotherapy for NSCLC is safe and effective, which is an ideal and promising medical patch for treating NSCLC.

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https://pubmed.ncbi.nlm.nih.gov/33025047/

"Chimeric antigen receptor (CAR)-T-cell therapy in non-small-cell lung cancer (NSCLC): current status and future perspectives"

Status: Der er stadig mange forhindringer ved brug af metoden, og man regner med at identificere patienter, der har en høj risiko for at være "resistent" overfor CAR T-cell terapi. Ellers virker metoden lovende for at virke for patienter med NSCLC.

Der er 85% af patienter med lungecancer der har typen NSCLC, og selvom der forvejen er mange andre behandlingsmetoder, har prognosen for patienterne ikke blevet væsentlig bedre

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Chimeric antigen receptor (CAR)-T-cell therapy in non-small-cell lung cancer (NSCLC): current status and future perspectives - PubMed

# data[].attributes.attachment_link.description
There has been a rapid progress in developing genetically engineered T cells in recent years both in basic and clinical cancer studies. Chimeric antigen receptor (CAR)-T cells exert an immune response against various cancers, including the non-small-cell lung cancer (NSCLC). As novel agents of immun…

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Et review af de forventede muligheder i fremtiden med CAR T-celle terapi

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Et review af de forventede muligheder i fremtiden med CAR T-celle terapi

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Artiklen er fra d. 22 januar 2021

Der undersøges muligheden for at udvikle CAR T-celle terapi til behandling af hjernetumor af typen glioblastoma. Der har tidligere været forsøg med udviklingen af en terapi type som er rettet imod EGFRvIII, IL-13 alpha2 receptorer og HER2, men uden større succes, med den forventede grund at der har været mangel på de ønskede target antigener.
For at overkomme udfordringen er der forsøgt at designe en terapi baseret på chlorotoxin peptid. Ved patologiske undersøgelser af glioblastoma celler er der fundet en ukendt ligand. Ved udnersøgelse af dette er der fundet at denne ligand virker afhængig af metalloproteinase-2, som kan identificeres af den nye type af CAR T-celle. Umiddelbart af det de har fundet, er den ikke skadelig overfor andet væv og blodkar.
Der er ikke umiddelbart en afklaring om det vil fungere endnu, men det forventes at undersøges nærmere.

http://europepmc.org/article/MED/33483715

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Recent advances and discoveries in the mechanisms and functions of CAR T cells.

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This Review discusses the major advances and changes made over the last three years to our understanding of chimeric antigen receptor (CAR) T cell efficacy and safety. Recently, the field has gained insight into how various molecular modules of the CAR influence signaling and function.

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Behandlingsmuligheder ved relaps af hæmatologisk cancer og primær behandlingsresistent sygdom er begrænsede.

Chimeric antigen receptor (CAR)-T-celler er et lovende immunologisk behandlingsprincip.

Behandling med CD19 CAR-T-celler fra kommercielle og nonkommercielle produkter etableres p.t. på Rigshospitalet

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Overlevelsen ved en række hæmatologiske cancerformer er øget markant i de seneste år. Et eksempel er akut lymfoblastær leukæmi (ALL), hvor bedre risikostratificering kombineret med intensivering af kemoterapien har medført overlevelsesrater på op til 90% hos børn og yngre voksne [1, 2]. Prisen for den mere intensive kemoterapi har dog været alvorlige akutte bivirkninger og fysiske eller psykosociale senfølger hos en stor del af patienterne [3]. Hos patienter med højrisiko-ALL og hos patienter med recidiv efter standardbehandling anvendes allogen stamcelletransplantation, som kombinerer intensiv kemoterapi med vedvarende immunologisk kontrol af leukæmicellerne via effekten af donorderiverede lymfocytter. Ved denne tilgang helbredes op til 70%, men med risiko for alvorlige bivirkninger som følge af helkropsbestråling, kemoterapi og graft-versus-host-sygdom og med en nonrelapsmortalitet på omkring 10% [4, 5]. For en række andre kræftformer hos børn, inkl. solide tumorer og cancer i centralnervesystemet, har udviklingen været mere beskeden, og hos børn med recidiv af ALL ses der fortsat høje mortalitetsrater [6, 7]. Et tilsvarende billede ses hos voksne, hvor langtidsoverlevelsen f.eks. efter recidiv af ALL er på 10-30% og ca. 20% ved behandlingsrefraktært eller recidiverende diffust storcellet B-cellelymfom (DLBCL) [8-10]. Der er således behov for udvikling af nye behandlingsprincipper, der kombinerer effektiv sygdomskontrol med en mere acceptabel bivirkningsprofil.

CANCERIMMUNTERAPI

Cancerimmunterapi udgør et gennembrud i behandlingen af cancer og udnytter immunsystemets evne til at genkende og dræbe cancerceller. P.t. bliver nye former for immunterapi godkendt i hastigt tempo til stadig flere kræftformer, og de nye behandlingsmuligheder tyder på, at langt flere patienter, selv med fremskreden sygdom, kan blive langtidsoverlevere. Cancerimmunterapi omfatter forskellige behandlingsprincipper, hvoraf de såkaldte checkpointinhibitorer (CPI) er inde i en betydelig udvikling. CPI er antistoffer, der binder til receptorer på immunceller og/eller cancerceller og blokerer for inhibitoriske immunologiske signaler. Klinisk effekt af CPI er påvist over en bred række af hæmatologiske og solide cancerformer og skyldes aktivering/styrkelse af et T-cellebaseret immunrespons mod muterede tumorproteiner [11]. For flere cancerformer med lave mutationsrater, herunder akut leukæmi, er cancercellernes immunaktiverende effekt imidlertid så ringe, at der ikke induceres et naturligt immunrespons, og ved disse cancerformer har CPI begrænset effekt.

CHIMERIC ANTIGEN RECEPTOR-T-CELLER

En ny lovende strategi til generering af et effektivt anticancerimmunrespons ved de kræftformer, der ikke fremkalder et sufficient naturligt immunrespons, er anvendelse af chimeric antigen receptor (CAR)-T-celler. Udviklingen af CAR-T-celler er et lysende eksempel på, hvordan detaljeret grundvidenskabelig immunologisk indsigt kan føre til et muligt paradigmeskifte i cancerbehandlingen. Ved behandling med CAR-T-celler modificeres patientens egne T-celler ex vivo til på celleoverfladen at udtrykke en CAR, der er en kimærisk antigenreceptor bestående af den antigenbindende del af et immunglobulinmolekyle koblet til T-cellereceptorens intracellulære domæne (Figur 1). For at opnå tilstrækkelig proliferation og aktivering efter infusion i patienten modificeres constructet i de nyere CARs til at indeholde et eller flere kostimulerende domæner (oftest CD28 eller 4-1BB). Uden disse ekstra signaler bliver både naturlige T-celler og CAR-T-celler anerge og dermed klinisk ineffektive [12-14].

Patientbehandlingen med CAR-T-celler er kompleks og kræver et tværfagligt setup med henblik på timing, leukaferese, genmodificering, opbevaring og administrering af genmodificerede celler samt mulighed for hurtig adgang til en intensivafdeling pga. risiko for alvorlige akutte toksiciteter. Behandlingen indledes med høst af autologe lymfocytter fra patienten ved leukaferese. De høstede T-celler transduceres in vitro under GMP-vilkår med anvendelse af en viral vektor (retro- eller lentivirus), der bærer CAR- constructet, og cellepopulationen ekspanderes med anvendelse af cytokiner. Efter denne procedure, der som oftest tager 10-14 dage, skal CAR-T-celleproduktet kontrolleres for bl.a. virus og kan herefter reinfunderes intravenøst, som oftest forudgået af en kort forbehandling af patienten med lymfocytreducerende kemoterapi (Figur 2).

KLINISKE UNDERSØGELSER

Behandling med CAR-T-celler rettet imod maligne B-cellesygdomme er på nuværende tidspunkt den mest veletablerede, og de kliniske resultater har været bemærkelsesværdige, hvad angår både effekt og bivirkningsprofil. CAR-T-celler rettet mod CD19-receptoren på maligne celler har således i flere kliniske 1-2-studier vist høj antitumoreffekt hos børn og voksne med fremskreden ALL, kronisk lymfatisk leukæmi og B-cellelymfom [13]. De anvendte CAR-T-celleprodukter i disse studier adskiller sig i CAR-design, T-celleaktiverings- og transduktionsmetoder samt celledoser. Et af de bedst undersøgte CAR-T-celleprodukter er udviklet på University of Pennsylvania og Children's Hospital of Philadelphia og er efterfølgende i samarbejde med Novartis godkendt til klinisk anvendelse til CD19-positiv hæmatologisk cancer. Dette produkt, tisagenlecleucel, er undersøgt i et internationalt fase 2-studie af recidiverende/behandlingsrefraktær CD19+-B-celle-ALL hos 75 børn og yngre voksne i 25 centre. Resultaterne viste en recidivfri overlevelse og en samlet overlevelse på henholdsvis 73% (95% konfidens-interval (KI): 60-82) og 90% (95% KI: 81-95) ved seks måneder og på henholdsvis 50% (95% KI: 35-64) og 76% (95% KI: 63-86) ved 12 måneder [15]. Samme produkt blev undersøgt i et fase 2-studie med voksne med behandlingsrefraktært eller recidiverende DLBCL. Af 93 behandlede patienter responderede 52% (95% KI:41-62), 40% med komplet respons og 12% med partielt respons. Blandt de responderende var 79% i live ved 12-måneders opfølgning [16]. I et studie af et lignende CAR-T-celleprodukt fra Kite/Gilead (axicabtagenciloleucel) hos patienter med progredierende B-cellelymfom fandt man responsrater på mere end 80% og over 70% progressionsfri overlevelse efter 24 måneder hos de patienter, der var i remission ved tremånedersopfølgning. For alle behandlede var den progressionsfri og den samlede overlevelse på henholdsvis 39% og ca. 50% ved 24-månedersopfølgning [17]. Dette har medført godkendelse i både USA og Europa af disse to nu kommercielt tilgængelige CAR-T-celleprodukter til behandling af patienter, som er op til 25 år og har recidiverende eller refraktær B-ALL, samt til patienter med behandlingsrefraktært eller recidiverende DLBCL efter flere systemiske behandlinger. Behandling med CAR-T-celler er meget dyr, og i Danmark har Medicinrådet i starten af 2019 anbefalet brugen af tisagenlecleucel til B-ALL i ovenfor anførte situationer. Derimod anbefaler Medicinrådet hverken tisagenlecleucel eller axicabtagenciloleucel som standardbehandling hos patienter med relaps/refraktært DLBCL, idet det vurderes, at der mangler evidens for den kliniske merværdi i forhold til den bedst tilgængelige behandling, samt at der ikke er et rimeligt forhold mellem lægemidlets kliniske merværdi og meromkostningerne ved denne behandling.

VIDEREUDVIKLING AF CAR-T-CELLEBEHANDLING

På trods af høje primære responsrater er recidiv efter CAR-T-behandling en betydelig risiko. Forskelle i de observerede respons- og recidivrater afspejler dels forskelle i CAR- constructet, dels forskelle i celledosering og sammensætningen af de transducerede celler og forskelle i anvendelsen af den indledende lymfocytreducerende kemoterapi. Recidiver kan skyldes tab af CAR-T-cellerne, tab af deres cytotoksiske aktivitet eller udvikling af CD19-negativ leukæmi. Nye strategier for at imødegå dette er sikring af bedre CAR-T-celleekspansion bl.a. via indsættelse af flere kostimulatoriske domæner i CAR- constructet eller supplerende transduktion med andre aktivatorer som f.eks. cytokinreceptorer og cytokiner. Kombinationsbehandling med CPI er en anden lovende strategi til modvirkning af tab af CAR-T-celleaktivitet. En anden mulighed, der p.t. afprøves, er anvendelse af bispecifikke CAR-T-celler rettet mod en kombination af antigene epitoper. F.eks. har en kombination af CD22- og CD19-specificitet vist lovende resultater ved tilbagefald af CD19-negativ B-ALL efter primær behandling med CD19-CAR-T-celler [18, 19].

Udvidelse af anvendelsen af CAR-T-celler til behandling af solide tumorer og andre hæmatologiske cancere er et vigtigt mål, men resultaterne har hidtil været skuffende. Det drejer sig bl.a. om kræftformer som glioblastom, gliom, mesoteliom og neuroblastom samt akut myeloid leukæmi. Den foreløbig begrænsede succes på dette område skyldes bl.a. det immunhæmmende tumormikromiljø, der gør CAR-T-cellerne anerge, og udfordringer med at identificere egnede tumorspecifikke target-molekyler uden risiko for krydsreaktivitet over for raske væv [20, 21]. En mere fuldstændig gennemgang af dette mangeartede forskningsområde ligger uden for målet med denne artikel, men er resumeret i en aktuel oversigtsartikel [22].

Behovet for adgang til CAR-T-celler rettet mod et større spektrum af cancertyper og ønsket om at øge effekten via supplerende aktiveringsmekanismer forklarer dynamikken på dette forskningsområde. Feltet er i en hastig udvikling med mere end 300 igangværende kliniske undersøgelser primært i USA og Asien, rettet mod mere end 20 forskellige cellulære overflademarkører [23, 24]. Flere af CAR-T-celleprodukterne er udviklet i universitetsmiljøer, og omkring en tredjedel af de kliniske studier udgår fra nonindustrielle forskningsinstitutioner, bl.a. pga. udfordringer med pris og tilgængelighed af de kommercielle produkter. Herhjemme er klinisk anvendelse af det kommercielle tisagenlecleucel tilgængelig på Rigshospitalet, ligesom behandling med egenproducerede CAR-T-celler er undervejs gennem et samarbejde mellem Herlev Hospital og Rigshospitalet.

BIVIRKNINGER OG RISICI

Toksicitet ved CAR-T-cellebehandling forårsages af inflammatoriske reaktioner som følge af forstyrrelser i den normale immunologiske homøostase. Hos hovedparten af patienterne ses der varierende grader af cytokin-release-syndrom (CRS) i dagene umiddelbart efter infusionen af CAR-T-celler med høj feber, myalgier, generel utilpashed og evt. capillary leak-syndrom, der kan medføre hypoksi, hypotension, nyresvigt og koagulopati [25]. Syndromet skyldes stærkt øgede niveauer af cirkulerende inflammatoriske cytokiner bl.a. interleukin (IL)-6 og ses særligt hos patienter med stor tumorbyrde pga. øget initial aktivering af CAR-T-cellerne [12, 15, 26]. Forløbet er i de fleste tilfælde mildt, og i sværere tilfælde kan tilstanden behandles med en kombination af kortikosteroid og cytokininhibitorer primært rettet mod IL-6 (f.eks. tocilizumab). Hos nogle få patienter er der dog rapporteret om dødelig udgang [13, 27]. En innovativ og lovende strategi til kontrol af svære tilfælde af CRS er neutralisering af aggressive CAR-T-celler via kotransduktion af inducerbare "selvmordsgener" (f.eks. caspase 9), der selektivt inducerer apoptose i CAR-T-cellerne efter aktivering ved indgivelse af en kemisk inducer hos patienter, der udvikler ukontrollerede immunreaktioner [28].

Neurotoksicitet er en anden relativt hyppig komplikation, som indtræder 1-3 uger efter CAR-T-celleinfusion. Symptomerne er generelt forbigående med en varighed på 5-10 dage og omfatter tremor, afasi, konfusion, hallucinationer og undertiden kramper [29]. Symptomerne tilskrives til dels inflammatoriske mekanismer og ledsages i svære tilfælde af EEG-forandringer [24, 30].

Anvendelsen af CAR-T-celler rettet mod CD19-receptoren ledsages generelt af et reduceret niveau af normale, raske B-celler ( on-target off-tumour toxicity) med resulterende reduktion i plasmaceller og hypogammaglobulinæmi, hvilket nødvendiggør substitutionsbehandling med immunglobulin [14, 16]. Hos hovedparten af patienterne ses der efter CAR-T-cellebehandling tillige korterevarende anæmi, trombocytopeni og neutropeni, som bl.a. tilskrives den indledende lymfocytreducerende kemoterapi. Hos nogle patienter varer cytopenien dog i flere måneder efter CAR-T-cellebehandling, men dette har kun i få tilfælde medført alvorlige infektioner eller blødning [30].

KONKLUSION

CAR-T-cellebehandling er et helt nyt behandlingsprincip inden for cellulær immunterapi og har vist meget lovende kliniske resultater. Hos patienter med behandlingsrefraktær hæmatologisk cancer ses der responsrater på op til 90% med bivirkninger, der oftest er forbigående. CAR-T-celler og andre former for cellulær immunterapi kan potentielt ændre de terapeutiske strategier inden for en række kræftformer i de kommende år. Perspektivet er, at flere kan blive kræftoverlevere og med væsentligt færre senfølger, da behovet for toksisk kemoterapi og bestråling reduceres.

Korrespondance: Klaus Müller. E-mail: kgmuller@gmail.com

Antaget: 20. august 2019

Publiceret på Ugeskriftet.dk: 14. oktober 2019

Interessekonflikter: ingen. Forfatternes ICMJE-formularer er tilgængelige sammen med artiklen på Ugeskriftet.dk

Taksigelser: Børnecancerfonden takkes for støtte til udvikling og implementering af CAR-T-cellebehandling i Danmark.

Litteratur: Findes i artiklen publiceret på Ugeskriftet.dk

# data[].attributes.attachment_link.description
Overlevelsen ved en række hæmatologiske cancerformer er øget markant i de seneste år. Et eksempel er akut lymfoblastær leukæmi (ALL), hvor bedre risikostratificering kombineret med intensivering af kemoterapien har medført overlevelsesrater på op til 90% hos børn og yngre voksne [1, 2].

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https://linkinghub.elsevier.com/retrieve/pii/S0753-3322(19)35307-7
- Genredigerede T-celler ser ud til at være gavnlige til at kontrollere inflammation ved allergisk astma.

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STING agonist hjælper CAR T celler med at fordele sig og være udholdende overfor brystkræft.
Der laves stadig kliniske forsøg med dem.

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. 2021 Feb 1;218(2):e20200844.

doi: 10.1084/jem.20200844.

Affiliations

¹ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

³ Center for Cancer Research, National Cancer Institute, Bethesda, MD.

⁴ Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC.

⁵ Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

⁶ Computational Medicine Program, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Free PMC article

Nuo Xu et al. J Exp Med.

Free PMC article

. 2021 Feb 1;218(2):e20200844.

doi: 10.1084/jem.20200844.

Affiliations

¹ Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

³ Center for Cancer Research, National Cancer Institute, Bethesda, MD.

⁴ Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC.

⁵ Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC.

⁶ Computational Medicine Program, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Abstract

CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from Th/Tc17 cells given with the STING agonists DMXAA or cGAMP greatly enhanced tumor control, which was associated with enhanced CAR T cell persistence in the TME. Using single-cell RNA sequencing, we demonstrate that DMXAA promoted CAR T cell trafficking and persistence, supported by the generation of a chemokine milieu that promoted CAR T cell recruitment and modulation of the immunosuppressive TME through alterations in the balance of immune-stimulatory and suppressive myeloid cells. However, sustained tumor regression was accomplished only with the addition of anti-PD-1 and anti-GR-1 mAb to Th/Tc17 CAR T cell therapy given with STING agonists. This study provides new approaches to enhance adoptive T cell therapy in solid tumors.

Conflict of interest statement

Disclosures: N. Xu reported a patent to STING on CAR T therapy against solid tumor pending. A.C. Robeson reported personal fees from Precision BioSciences, Inc. outside the submitted work. B.G. Vincent reported "other" from GeneCentric Therapeutics outside the submitted work. N.P. Restifo reported a patent to UNC/NCI pending. J.S. Serody reported grants from Merck Inc. and Glaxo Smith Kline outside the submitted work; in addition, J.S. had a patent for the use of STING agonist to enhance CAR T cell function pending. No other disclosures were reported.

Figures

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References

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national center for biotechnology information

# data[].attributes.attachment_link.title
STING agonist promotes CAR T cell trafficking and persistence in breast cancer - PubMed

# data[].attributes.attachment_link.description
CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from ...

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Chimeric antigen receptor -T cell therapy: Applications and challenges in treatment of allergy and asthma - PubMed (nih.gov)
Der er flere tilgange til at bruge CAR-T-cellen i allergisk astma. Eksempelvis fungal astma er meget farlig sygdom. Af denne grund er behandling af denne sygdom meget vigtig. Svampedræbende CAR-T-celler med dectin-1 som antigenbindende domæne for CAR kan bruges til behandling af fungal astma. Her konstrueres CAR-Tregs celler mod allergisk luftvejsreaktion og hermed undertrykke IgE overproducing, eosinofil betændelse og hypersecretion. IgE receptorer kan bruges til at konstruere CARs med ScFvs til behandling af allergisk astma

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doi: 10.1016/j.biopha.2019.109685.

Affiliations

¹ Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran; Cancer Gene Therapy Research Center (CGRC), Zanjan University of Medical Sciences, Zanjan, Iran.

² Department of Immunology, Health Faculty, Tehran University of Medical Sciences, Tehran, Iran.

³ Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran. Electronic address: SS.Athari@gmail.com.

Free article

Free article

doi: 10.1016/j.biopha.2019.109685.

Affiliations

¹ Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran; Cancer Gene Therapy Research Center (CGRC), Zanjan University of Medical Sciences, Zanjan, Iran.

² Department of Immunology, Health Faculty, Tehran University of Medical Sciences, Tehran, Iran.

³ Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran. Electronic address: SS.Athari@gmail.com.

Abstract

Despite the current advancements, cancer treatment approaches have limitations restricting their cure rate. Immunotherapy techniques are among novel and promising cancer therapeutic approaches. Therapeutic antibodies and adoptive cell therapy (ACT) are the main branches of immunotherapy. T lymphocytes and genetically engineered cells are among important cells which can be used in ACT. This review has focused on recent advances in engineered cell-based immunotherapy based on T lymphocytes with chimeric antigen receptors (CARs). CARs are recombinant receptors expressing T cell signaling domains with or without co-stimulatory molecules. CAR-T cells are expanded ex vivo and re-infused to patients in order to improve their therapeutic efficacy. Nowadays, the beneficial function of CAR-T cell therapy has been indicated in various diseases including hematological malignancies, solid tumors, autoimmune diseases, and allergic diseases such as asthma. Furthermore, antigen-specific T regulatory cells (T regs) and gene-edited T cells seem to be beneficial in controlling inflammation in allergic asthma. In fact, dysregulated function of T regs is responsible for dominance of T helper 2 immune response and progression of allergic asthma. CAR-T reg cells can also be designed and reproduced using iT reg population to manage asthma. In addition, universal CAR-T cells can be modified to selectively target multiple antigens. The fourth generation CAR-T cells (i.e. TRUCK cells) represent novel strategies to cure asthma and allergic diseases as well. Despite the advantages of CAR-T cells, their applications can be associated with some unwanted reactions such as cytokine storm, anaphylaxis, neurotoxicity, etc. For clinical application, there is a need to prevent and manage these complications by optimizing ACT protocols.

Keywords: Adoptive cell therapy; Allergy; Asthma; CAR-T cell; Immunotherapy; T regulatory cells.

Conflict of interest statement

Declaration of Competing Interest The authors declare that the current research was conducted in the absence of any commercial or financial relationships which could be construed as a potential conflict of interest.

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# data[].attributes.attachment_link.title
Chimeric antigen receptor -T cell therapy: Applications and challenges in treatment of allergy and asthma - PubMed

# data[].attributes.attachment_link.description
doi: 10.1016/j.biopha.2019.109685. 1 Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran; Cancer Gene Therapy Research Center (CGRC), Zanjan University of Medical Sciences, Zanjan, Iran. 2 Department of Immunology, Health Faculty, Tehran University of Medical Sciences, Tehran, Iran. 3 Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.

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CAR T-celler til faste tumorer: Nye strategier til at finde, infiltrere og overleve i tumormikromiljøet.

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Finding the Tumor: Tumor Associated Antigens, Expression Level, and Susceptibility to CAR T Cells

The first major difference between solid tumors and hematological tumors is that it is more difficult to find an ideal target antigen. Unlike cancers such as ALL or CLL in which the tumor cells universally express the B-cell marker CD19, solid tumors rarely express one tumor specific antigen. For most solid tumors, it is more common to find a tumor associated antigen (TAA) where the antigen is enriched on tumors but also expressed at low levels on normal tissues (see ). This is the case for many frequently targeted TAAs for solid tumors, including CEA, ERBB2, EGFR, GD2, mesothelin, MUC1, and PSMA ( 1, 14, 18).

Table 1

Lack of tumor antigen specificity increases the potential risk of significant on-target off-tumor toxicity. This was the case for a patient with metastatic colon cancer who received an infusion of CAR T cells targeted to the antigen HER2 (ERBB2) and died 5 days later ( 58). The cause of death was attributed to low levels of HER2 on the epithelial cells of the lung, which were attacked by the CARs. Another example of on-target, off-tumor toxicity was found with a high affinity anti-GD2 CAR for neuroblastoma, in which low levels of GD2 in the brain resulted in fatal encephalitis ( 59). These catastrophic events underscore the importance of finding a safe TAA, given the possibility that even low levels of the target antigen on normal tissues can result in significant toxicity. These acute responses also highlight that the binding affinity of a CAR is related to both safety and efficacy, and that higher affinity is not necessarily better. An in vivo study found that CAR T cells targeting ICAM-1, a marker associated with many solid tumors including thyroid cancer (but also expressed on many normal tissues as an adhesion marker), was safer and more effective when bearing CARs with micromolar affinity than with those with higher, nanomolar affinity ( 39, 40). Additionally, the authors found that the CAR with lower affinity showed less exhaustion and enhanced proliferation in vivo. In another approach to limiting CAR toxicity, one group interested in treating colorectal cancer created a CAR targeting GUCY2C, a receptor that is conserved in at least 95% of metastatic tumor at tenfold greater levels, but is not targeted by T cells when expressed in normal epithelial tissues because it is restricted to luminal membranes ( 33). The CAR was shown to be safe and effective in both immunocompetent mice with metastatic tumors and human xenograft models. Antigens that are aberrantly or overexpressed on tumors but are also expressed on normal tissues can thus be cautiously explored to serve as targets for solid tumors and their metastases.

Suicide genes [reviewed by ( 60)] are genes coexpressed with the CAR construct that can induce cell death when activated by an agent such as a drug or antibody. Suicide genes have been integral to improving the safety of CAR T cells, particularly as they move into clinical trials. These genes include inducible caspase 9 (iC9) and truncated EGFR (tEGFR or EGFRt) (), which can trigger antibody-mediated cell death, and herpes simplex virus thymidine kinase (HSV-TK), which disrupts DNA replication and also induces apoptosis via Fas-mediated cell death ( 68).

Many groups have used immunoproteomics to discover TAAs using autoantibodies against immunogenic antigens expressed by tumor cells (either on the surface or in the cytosol) ( 69). These antigens may be entirely unidentified proteins (neoantigens) or peptides that are mutated from the wild type (neoepitopes) ( 70). A few examples of TAAs identified using proteomics include the markers PSMA1, LAP3, ANXA3, and maspin, which were identified by one group as biomarkers for colon cancer ( 71). Other novel potentially targetable TAAs include olfactomedin 4, CD11b, and integrin alpha-2, which were found to be overexpressed in colorectal cancer with liver metastases ( 72). Neoantigens can also be found using DNA or RNA sequencing or whole exome screening to identify somatic mutations in tumors ( 73- 75). A study using whole exome sequencing of melanoma samples found multiple mutated epitopes in 5 of 8 patients, as well as the presence of T cell clones reactive to 8 of the 9 neoepitopes ( 73). Another study used whole genome sequencing to identify somatic mutations in glioblastoma multiforme (GBM) samples, and found neoepitope-specific tumor infiltrating lymphocytes (TILs) in all five studied patients ( 70). Whole exome sequencing for neoantigen prediction was recently employed in a long term study of PDA patients, and the authors found that greater numbers of neoantigens, combined with greater numbers of CD8⁺ TILs, correlated with increased survival ( 76). Many of the neoantigens with lasting immunogenicity in long term survivors were contained within the tumor-associated MUC16 antigen; with metastatic progression, loss of MUC16 clones was seen, indicating a role for the loss of those neoantigens in tumor progression and metastasis.

Some studies have explored the use of CD40 agonists to boost T cell immunity to solid tumors (CD40 is expressed on dendritic cells and other antigen-presenting cells (APCs) and binds CD40 ligand on T cells to stimulate immune response) ( 77). Using CD40 agonists to augment T cell response to weakly immunogenic tumor antigens or cold tumors is particularly useful in cancers such as PDA that tend to lack mutational burden and often have no baseline immunogenicity. In a murine model of spontaneous PDA (KPC mice), combining chemotherapy with CD40 agonists showed T cell infiltration and neoantigen-specific response and tumor regression ( 78). The data was consistent with the hypothesis that CD40 activated pre-existing tumor-reactive TILs, showing that priming can overcome suboptimal T cell reactivity to antigen and induce an immune response with subsequent tumor control. These studies using neoepitopes show that tumors can induce secondary immune responses against previously unrecognized antigens, and that endogenous immunity to neoantigens may control tumor spread. This bears significance for adoptive T cell therapy including that which is CAR-based. Many of these methods to screen for neoepitopes rely on identifying pre-existing TCR reactivity and, thus, rely on the inherent immunogenicity of neopeptides; however, identifying neoepitopes and using CAR T cells to target them could theoretically bypass this issue because the scFv of a CAR does not rely on MHC presentation.

Solid tumors tend to display a large degree of antigen heterogeneity. Many tumors have only a subset of cells that express the target antigen. Even in the setting of a uniformly expressed TAA, there is the possibility of antigen loss or antigen escape, where the target antigen disappears from the surviving tumor ( 79). This has already been observed with CD19 negative relapses in leukemia post CAR19 T cell transfer, and the mechanisms are not well characterized ( 80). One study discovered a splice mutation that resulted in a form of CD19 that was missing the specific epitope targeted by the CD19 CAR ( 81, 82). In a phase I study using an EGFRvIII-specific CAR to treat GBM, a single dose of the CAR T cells resulted in downregulation of the EGFR/EGFRvIII receptor and appeared to promote T cell resistance, although administration was shown to be safe and potentially effective ( 83). In a glioma model, an IL13Rα2 specific CAR T cell that also had transgenic expression of IL-15 successfully killed tumor, proliferated, and produced cytokine in vivo; however, recurrent tumors demonstrated IL13Rα2 downregulation ( 84). Dual or tandem CARs, which recognize two antigens rather than one, have been created to address both antigen heterogeneity and the threat of antigen loss. Such dual CARs have entered clinical trials in hematological malignancies targeting CD19/CD20 and CD19/CD22 [( 85); NCT03241940]. For solid tumors, a CAR specific for both HER2 and MUC1 had promising in vitro results in a breast cancer model, and a dual-target CAR specific for HER2 and IL13Rα2 showed greater success than single-target CARs in a xenograft glioma model ( 86, 87).

Also relevant to antigen heterogeneity is the concept of epitope spreading [reviewed by ( 88)], a phenomenon in which a different epitope of a previously tolerated antigen becomes targeted by T cells. In the context of CAR T cell therapy, this means that even if a tumor does not uniformly express the originally targeted antigen, lysis of some cells by CARs might release tumor-specific neoantigens or epitopes that would be processed and presented by APCs to TILs to induce a secondary immune response against the tumor. Evidence for epitope spreading has been shown in melanoma, where TILs reactive to tumor neoantigens were discovered after vaccination with melanoma antigens (MAGE) ( 89). Another study using a viral-based vaccine for MUC1 and IL-2 induced epitope spreading and correlated with improved survival of patients with NSCLC ( 90), and a case study using mRNA electroporated mesothelin CARs displayed an immune response that suggested epitope spreading in two patients with MPM and metastatic pancreatic cancer ( 91). In a mouse pancreatic cancer model with tumors of low mutational burden and no predicted neoepitopes, introduction of the neoantigen ovalbumin (OVA) spurred a memory immune response leading to tumor clearance and no evidence of antigen escape, while the same tumors provoked no T cell response in immune competent mice without ovalbumin ( 92). Further understanding and inducing epitope spreading has significant potential to bolster the effectiveness of CAR T cells, especially in tumors with high heterogeneity, low mutational burden, and evidence of antigen escape.

For traditional CAR T cells, the target antigen must be expressed on the cell surface in order to engage with a T cell. However, only about 1% of total cellular proteins are actually expressed on the cell surface, meaning that a huge number of potential tumor target antigens are not available to a CAR T cell ( 62). Recently, to open the doors to targeting intracellular antigens with CAR T cells, Patel et al. ( 93) showed success in an in vivo myeloma study with a CAR/TCR hybrid that recognized the antigen NY-ESO-1 in the context of HLA-A2. These TCR-CARs were shown to effectively bind an HLA-A2⁺ T cell artificially engineered to express NY-ESO-1. TCR-CARs that recognize antigen in combination with MHC can thus recognize both extra- and intra-cellular antigens in the way that wild-type or modified TCRs can. Walseng et al. ( 62) also created a TCR-CAR composed of a soluble TCR directed against either the melanoma-associated antigen MART1 or TGFβR2 (a neoantigen peptide) joined to a CAR signaling component. The result was a versatile receptor that bound antigen in an MHC-I restricted manner, but with signaling and killing similar to that of a CAR. They demonstrated that this construct could be transduced not only into T cells but also into a NK cell, with successful in vitro killing.

Tumor Infiltration

Even when a target antigen for a solid tumor is identified, a CAR T cell must be able to reach the tumor site. In hematological cancers, circulating CAR T cells in the bloodstream have already reached their destination. In solid tumors, there are multiple barriers that a CAR T cell must surmount in order to reach the tumor site [for full reviews of the tumor microenvironment, see ( 94, 95)]. Chemokine-receptor mismatch can prevent migrating lymphocytes from following a chemotactic gradient. Surface markers like selectins on endothelial cells that bind circulating lymphocytes and induce signaling cascades for subsequent extravasation into sites of inflammation are necessary, as are the corresponding receptors on T cells. Additionally, physical barriers such as cancer associated fibroblasts (CAFs) and abnormal vasculature at the tumor site can block T cell entry ( 95).

The presence of blood vessels known as high endothelial venules (HEVs) are hypothesized to be critical for T cell infiltration and have been associated with tumor regression in cancers such as melanoma. However, these blood vessels are distorted and immature in many solid tumors, particularly at the core of the tumor where the fewest TILs are found ( 96). Anti-angiogenic therapy targeting VEGF, CD276, or endothelin B receptor has been shown to normalize tumor vasculature and could be used in combination with targeted therapy like CAR T cells to increase tumor infiltration ( 97, 98). Notably, one study that performed qPCR on melanoma lesions observed that high HEV density positively correlated with the number of genes encoding for chemokines known to recruit TILs, including CCL2, CCL5, CXCL9-13, CCL19, and CCL21 ( 99). In colorectal cancer, expression of CXCL9, 10 and 11 were positively correlated with the presence of CD8⁺ and CD4⁺ TILs and with post-operative survival ( 100).

Given the importance of chemokines in lymphocyte migration and homing, varying methods have been used to deliver chemokines intratumorally to attract TILs. One study employed a vaccinia virus to deliver the chemokine CXCL11 intratumorally in a subcutaneous mouse model of MPM and observed significantly increased levels of T cell infiltration and anti-tumor efficacy after intravenous mesothelin-directed CAR T cell injection ( 101). The same group also developed a CXCL11/mesothelin CAR that increased intratumoral levels of CXCL11 but did not improve anti-tumor activity. The investigators hypothesized that this was due to chronic chemokine secretion inducing hypofunction in the T cells, and/or the anti-angiogenic effects that CXCL11 can exert on its surroundings. However, another study that engineered "armored" mesothelin CAR T cells that constitutively expressed both the cytokine IL-7 and the chemokine CCL19 showed complete tumor regression and prolonged survival in a solid tumor mouse model () ( 66). The study also showed that lymphodepletion before CAR T cell injection decreased efficacy, suggesting that IL-7 and CCL19 recruited endogenous anti-tumor TILs as well. CAR T cells have also been transduced to express chemokine receptors with beneficial results, as in the case of lentivirally engineered mesothelin CAR/CCR2 T cells that displayed greater than 12-fold increased homing and tumor regression in subcutaneous human MPM tumors and a GD2/CCR2b CAR T cells that showed greater than 10-fold increased homing in neuroblastoma tumors in vivo () ( 64, 65).

Another promising approach to augment CAR T cell infiltration into tumor sites is the development of a CAR targeting FAP (fibroblast activation protein), which is expressed on multiple types of stromal cells that are associated with nearly all epithelial tumors ( 102). FAP has been shown to play a role in epithelial-to-mesenchymal transition (EMT) in pancreatic ductal adenocarcinomas (PDA) among other tumor types ( 103). In one study, where human MPM tumor samples and fibroblast samples were shown to be positive for FAP by immunohistochemistry, FAP CAR T cells efficiently killed MPM cells in vitro. The same CAR T cells inhibited tumor growth and lengthened the survival of immunodeficient mice with intraperitoneal (IP) tumor xenografts ( 104). However, another study showed little efficacy of a FAP CAR in a syngeneic mouse model using multiple tumor types and observed lethal toxicity, which was attributed to FAP expression on bone marrow-derived stem cells (BMSCs) ( 105). The authors reported that this may have been due to the use of mouse tumor lines with limited FAP expression, while robust FAP staining by IHC was observed in multiple human tumor samples, indicating that human tumor cell lines may have been better targets for the study.

One method of entirely circumventing the hurdle of suboptimal T cell homing (and also potentially avoiding on-target off-tumor toxicity) is regional/local CAR T cell administration, which has already been tested in patients with solid tumors with varying degrees of success. One phase 0 study that enrolled patients with metastatic breast cancer demonstrated that intratumoral administration of mRNA c-Met CAR T cells was safe and resulted in tumor cell death, and showed other signs of anti-tumor inflammation including macrophage recruitment ( 20). Recently, in a study using a xenograft mouse model of human breast cancer metastatic to the brain, intracranial and intratumoral administration of HER2-specific CAR T cells showed improved antitumor activity compared with intravenous delivery, with complete tumor eradication and 100% survival even after tumor rechallenge ( 106). Another study showed that regional delivery of a HER2-BBζ CAR T cell cleared medulloblastomas in NSG mice and required a significantly lower dose than intravenous delivery ( 107). The same CAR in nonhuman primates with HER2 positive medulloblastomas showed no toxicity after intraventricular delivery. A mouse study using a CEA CAR for peritoneal carcinomatosis (colorectal cancer metastasized to the peritoneal cavity) showed that regional intraperitoneal (IP) delivery resulted in better antitumor response than intravenous delivery, even after tumor rechallenge and at distal tumor sites ( 108). Finally, a study of intracavitary administration of pan-ErbB/IL-4 CAR T cells targeting patient derived MPM xenografts in SCID mice showed tumor regression or cure in all mice ( 23).

TIL Survival in the Tumor Microenvironment

Once a CAR T cell finds its way into the tumor, the battle is far from over. The tumor microenvironment (TME) has been extensively characterized as hostile for T cells [see ( 95, 109), and ( 110) for reviews of the tumor microenvironment and the different cell types it comprises]. The glycolytic metabolism of tumor cells renders the environment hypoxic, acidic, low in nutrients, and prone to oxidative stress ( 1, 109). In an inflammatory environment, tumors cells often upregulate ligands such as programmed cell death ligand 1 (PD-L1) and Galectin-9 that bind to inhibitory receptors on T cells (see ). The tumor microenvironment also relies on stromal cells like cancer associated fibroblasts (CAFs) and suppressive immune cells, including myeloid-derived suppressor cells (MDSCs), tumor associated macrophages (TAMs), tumor associated neutrophils (TANs), mast cells, and regulatory T cells (Tregs) () ( 95). These cells and tumor cells secrete soluble factors like vascular endothelial growth factor (VEGF) and transforming growth factor β (TGFβ), which contribute to abnormal tumor vasculature, promote anti-inflammatory polarization of TAMs and other immune cells, and are implicated in EMT ( 116). They also produce reactive oxygen species (ROS) and molecules like lactate, indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), soluble Fas, and adenosine, which contribute to the suppression of the T cell immune response () ( 117, 121).

Table 2

A2AR

Adenosine 2A receptor

Adenosine

CTLA-4

Cytotoxic T lymphocyte antigen-4

CD80, CD86

CD160

Cluster of differentiation 160

MHC Class I, herpesvirus entry mediator (HVEM) ( 112)

LAG-3

Lymphocyte activation gene 3

MHC Class II

PD-1

Programmed cell death 1

Programmed cell death ligand 1 (PD-L1), PD-L2

TIM-3

T cell immunoglobulin-3

Galectin-9 (Gal9), phosphatidylserine (PtdSer), high mobility group protein B1 (HMGB1), Ceacam-1 ( 113)

TIGIT

T cell immunoglobulin and ITIM domain

PVR (CD155) >> PVRL2 (CD112), PVRL3 ( 113)

When they are activated, effector T cells generally switch from oxidative phosphorylation (oxphos) to glycolysis to facilitate faster proliferation, while memory T cells and Tregs rely on oxphos and fatty acid oxidation (FAO) when activated [for a full review on T cell activation and metabolism see ( 122)]. However, both of these metabolic resources are limited in the TME because glucose is depleted by tumor cells, leaving glycolytic T cells nutrient-deprived. The lack of glucose results in lowered AKT/mTOR signaling, downregulation of the glucose receptor Glut1, and reduced capacity for glycolysis ( 120). Low oxygen concentrations in the TME limit oxphos as well. Overall, this results in significant depletion of both major sources of T cell nutrients. In a study of resected tumor tissue from 54 patients with clear cell renal carcinoma (ccRCC), CD8⁺ TILs showed very low levels of activation and proliferation, and although T cell Glut1 remained expressed, TILs did not uptake glucose ( 123). The study also observed that mitochondria (crucial for T cell activation), undergo remodeling during glycolysis, lose function and release detrimentally high levels of ROS. This comprehensive study is one of many to show the effects of TME hypoglycemia on suppressing T cell activation via glycolytic and mitochondrial pathways.

The hypoxic conditions in the TME provide particular challenges for memory T cells, the metabolisms of which rely heavily on oxygen. Some studies have recently begun to identify and test modifications to CAR T cells to improve their function in low-oxygen conditions. Kawalekar et al. ( 6) found that BBζ CAR T cells had increased mitochondrial spare respiratory capacity (SRC) compared with 28ζ CAR T cells, resulting in greater metabolic efficiency even in nutrient-poor, oxygen depleted conditions such as the TME. Because the BBζ costimulatory has been implicated in promoting memory-associated metabolic pathways such as fatty acid oxidation as well as increased persistence (further discussed under "Intracellular signaling pathways of the CAR" below), the increased SRC observed in these T cells was hypothesized to aid their survival in low-oxygen conditions.

One approach designed to protect T cells from the oxidative stress inflicted by ROS in the TME was the design of a CAR T cell coexpressing catalase, an enzyme that reduces hydrogen peroxide to water and oxygen ( 124). The authors tested both CEA and HER2 CAR T cells in vitro and found that CAR-CAT displayed a reduced oxidative state and improved proliferation and cytotoxicity compared with CAR alone. Another study harnessed the hypoxia associated with the TME to develop a CAR coexpressing the oxygen-sensitive domains of HIF1α, a transcription factor that is stabilized in response to hypoxia ( 125). In vitro, the strategy enabled very low CAR expression at normal oxygen levels, but highly increased levels of CAR expression together with HIF1α in hypoxic conditions. While this approach does not address the detrimental effects of low oxygen or ROS in the TME, it does provide proof of concept for a novel type of safety switch that uses the hypoxic TME to a therapeutic advantage.

T cell exhaustion [reviewed by ( 111)] is characterized by chronic antigen exposure that spurs loss of effector and memory phenotypes, inability to produce cytokines like IFNγ, TNFα, and IL-2, and upregulates expression of inhibitory receptors (IRs) that further shut down effector functions upon binding to inhibitory ligands or soluble factors in the TME () ( 109).

Checkpoint Blockade

One of the most popular and successful strategies to combat T cell exhaustion is the use of checkpoint inhibitors, in which either an IR or its ligand is blocked with an antibody. Drugs targeting PD-L1 (atezolizumab), PD-1 (pembrolizumab, nivolumab), and CTLA-4 (ipilimumab) have been used independently and in combination with CAR T cell therapy with success in many patients [reviewed in ( 126)]. Currently, atezolizumab and pembrolizumab are used to treat metastatic NSCLC and are being actively studied for use in other solid tumors as well. Pembrolizumab was recently approved by the FDA for first line use in combination with chemotherapy in lung cancer ( 127, 128). Nivolumab has shown significant responses in a phase I/II trial with HCC, among others ( 17). Ipilimumab was shown to lengthen the survival of metastatic melanoma patients in a phase III study from 2010, and it has shown promising results in mouse mesothelioma models as well as in many other preclinical studies ( 129, 130). Ipilimumab has also been used in combination with VEGF inhibitors to treat metastatic melanoma in phase I trials. In one study, anti-CTLA-4 therapy combined with anti-VEGF antibodies resulted in an increase in anti-tumor response resistant to the immunosuppressive effects of the ligand galectin-1 ( 131). Another study by the same authors showed ipilimumab and anti-CTLA-4 therapy resulted in humoral immunity to galectin-3, which is also a suppressive tumor ligand ( 132).

Preclinically, combining CARs with checkpoint blockade antibodies has shown promising results. CAR T cells have also been engineered to secrete checkpoint inhibitor antibodies themselves. Anti CAIX CAR T cells engineered to secrete anti-PD-L1 antibodies showed significantly improved activity compared to standard CAR T cells, with increased cytokine production and immune cell recruitment as well as significantly reduced tumor size in a human ccRCC mouse model ( 14). In another study, a CAR19 T cell designed to constitutively secrete anti-PD-1 also showed enhanced anti-tumor activity in a CD19⁺ lung cancer xenograft model, with increased T cell proliferation and cytotoxicity, and prolonged survival ( 133). A similar study also used MUC16-ecto targeting CARs secreting anti-PD-1 scFvs in syngeneic and xenograft mouse models of PD-L1⁺metastatic ovarian cancers, and showed superiority over CAR T cells plus PD-1 checkpoint inhibitors ( 134). Along a similar line of thinking, an anti-PD-L1 CAR has shown in vitro cytotoxicity ( 135); it has yet to be seen whether these CARs might be successful in vivo, either alone or as adjuvant therapy. CAR T cells engineered to secrete PD-1, CTLA-4, or PD-L1 antibodies have gone to clinical trials for MUC1, EGFR, EGFRvIII, and mesothelin expressing cancers ( 136).

Dominant negative genes for IRs have also been successfully introduced to CAR T cells in many preclinical studies, as in the case of a mesothelin CAR T cell (with either a CD28 or 4-1BB costimulatory domain) overexpressing dominant negative PD-1 ( 137). The authors observed tumor clearance with the dominant negative PD-1 CARs, while repeated doses of PD-1 blocking antibody in combination with either the mesothelin-28ζ or mesothelin-BBζ CAR was able to prevent growth but not eradicate the tumor. These results show that a genetic built-in resistance to checkpoint inhibition has advantages over a blocking antibody that must be repeatedly administered and may cause resistance. Recently, CRISPR/Cas9 technology has been used to knock out the gene for the IR itself, which has been done for both PD-1 and LAG-3 in CD19-BBζ CAR T cells. In both cases, tumors were eradicated in mouse xenograft models using the IR knockout CAR T cells ( 138, 139). This approach has recently been translated to solid tumors as well: CRISPR/Cas9 was used to knock out PD-1 in T cells while simultaneously transducing them with a CD133-specific CAR, and the resulting PD-1 KO CARs showed improved tumor control in a mouse glioma model compared with control CD133 CARs ( 140).

With the optimization of gene editing methods, CRISPR/Cas9 edited CARs are already moving into clinical trials: a PD-1 knockout CD19 CAR has is being studied in a phase I clinical trial ( NCT03298828).

Switch receptors are designed to mitigate the effects of inhibitory ligands on T cell function while simultaneously enhancing T cell activity. In a switch receptor, the ligand-binding external IR domain is fused to the cytoplasmic signaling domain of an activating molecule. For example, a PD-1/CD28 switch receptor was engineered into mesothelin-BBζ or PSCA-BBζ CAR T cells, and both switch-receptor CARs performed significantly better than wild type CARs at eradicating tumor in xenograft NSG mouse models ( 141). In a breast cancer model, the investigators engineered a MUC1 CAR that coexpressed a cytokine switch receptor (4/7ICR) with an IL-4 receptor extracellular domain fused to an IL-7 intracellular signaling domain ( 142). The 4/7ICR MUC1 CARs proliferated, suppressed tumor growth in vivo and did not show markers of exhaustion, while MUC1 CARs without the switch receptor did. PSCA CAR T cells that also contained a 4/7ICR switch receptor proliferated and killed better in the presence of IL-4 and showed significantly improved tumor reduction compared to T cells with the CAR alone in NSG mice with subcutaneous pancreatic cancer ( 57).

Similar to switch receptors are bispecific T cell engagers (BiTEs), which also subvert suppressive signals from the TME by binding both a tumor ligand and a T cell marker (i.e., CD3). Recently, a humanized EGFRvIII-specific scFv linked to an anti-CD3 scFv showed significant control of glioma xenografts and prolonged survival of mice ( 143). Potentially, the use of bispecific antibodies in conjunction with CAR T cells could play a role in recruiting TILs and in deterring immunosuppressive signals. Another strategy is to have a fusion protein bind not to the T cell but to the tumor itself. Recently, a PD-L1/TGFβR2 fusion protein was developed for use with TGFβ expressing urothelial carcinoma ( 144). In this study, a PD-L1 antibody fused to a TGFβ receptor was able to accomplish both blockade of PD-L1 on the tumor and binding of TGFβ to attenuate its presence in the TME. Excitingly, the authors also observed a greater presence of chemokines like CXCL11 in the tumor as well as antigen-specific killing by T cells. With the right target tumor ligands, BiTEs could be a promising strategy to augment the function of CAR T cells in solid tumors.

Understanding the metabolism and transcriptional profiles of exhausted or exhausting TILs has significant impacts on the success of therapies like checkpoint blockade and could lead to the production of more functional CAR T cells via metabolic reprogramming. In a mouse melanoma model, one group showed that promoting fatty acid catabolism in vaccine-induced CD8⁺ TILs using a PPARα agonist combined with anti PD-1 therapy significantly improved anti-tumor activity ( 145). Another group showed that CTLA-4, PD-1, and PD-L1 blockade increased glucose concentrations in the TME to favor glycolysis in T cells, improving their function in a mouse sarcoma model ( 120). These studies provide clues into the roles of ligands like PD-L1 on tumor metabolism, in addition to their known inhibitory effects on T cell IRs.

Despite the success of PD-1 therapy in treating NSCLC and melanoma, as well as its use in multiple other clinical trials, it is inadequate to characterize markers like PD-1 as solely inhibitory. Much of what we know about PD-1 in the context of exhaustion comes from chronic viral infection models, and it has become clear that PD-1 can in fact be a marker of activation and positive prognosis if expressed on certain subsets of T cells in cancer, while some subsets of exhausted T cells may have low PD-1 expression ( 111, 141). In the same study by Zhang et al. ( 145) that found a synergistic effect of PD-1 blockade with fatty acid catabolism in a melanoma model, hypoxia-driven hypofunction in CD8⁺ TILs was accompanied by lower PD-1 expression (but increased LAG-3 expression), and increased PD-1 signaling was hypothesized to be associated with metabolic reprogramming from glycolysis to FAO in low glucose environments like the TME. Another study suggested that PD-1 was in fact not linked to exhaustion, and that prolonged antigen exposure alone could cause T cells to become exhausted ( 146). In a recent analysis of varied NSCLC patient samples, a population of CD8⁺ TILs with high PD-1 expression did not appear exhausted, and genes involved in cycling and proliferation such as Ki-67, as well as genes involved in trafficking and metabolism, were upregulated ( 147). The PD-1 high TILs also showed greater glucose, lipid and fatty acid uptake than patient TILs with lower PD-1 expression. This data challenges the understanding of PD-1 being solely an inhibitory receptor and sheds new light on Zhang et al.'s observation that an increase in PD-1 expression results in lower capacity for glycolysis. Further showing a role for PD-1 outside of exhaustion, a study comparing CD4⁺ TILs in 34 patients with metastatic melanoma, grouped into young vs. old, showed that the younger patients had a greater percentage of memory T cells that expressed PD-1, Ki-67, and HLA-DR (another activation marker), compared with age matched controls; these memory and activation phenotypes were less distinct in older patients ( 148). However, supporting the hypothesis of PD-1 as a marker of hypofunction, but not necessarily of terminal exhaustion, one study demonstrated that mesothelin/BBζ T cells that had high PD-1 expression and a hypofunctional phenotype in an vivo human mesothelioma model recovered the ability to produce cytokines and had lower PD-1 expression after 24 h out of the tumor ( 126). These data show that PD-1 can have highly variable functions which likely depend on T cell phenotype, metabolism, tumor type, and other factors in the TME, and also helps explain why only a fraction of patients respond to PD-1 blockade even when their tumors have high PD-L1 expression.

Transcription Factors

Transcription factors such as T-box transcription factor TBX21 (T-bet) and Eomesodermin (Eomes) are involved in determining T cell fate and their discovery has led to further insight into the mechanisms of T cell exhaustion. T cells high in Eomes and PD-1 have been shown to be terminally exhausted, while those with high T-bet and medium PD-1 levels appear to retain proliferative potential despite displaying other classically defined features of exhaustion ( 111). Hypoglycemia and hypoxia in the TME have been shown to decrease T-bet expression in TILs that also lose effector functions ( 145). As an example of Eomes' role in CAR T cell exhaustion, a case study with a patient with refractory diffuse large B cell lymphoma (DLBCL) who received CAR19 T cell therapy combined with PD-1 blockade showed decreased Eomes as well as decreased PD-1 levels in peripheral blood CAR T cells ( 149). The patient had a clinically significant response to the treatment, indicating that PD-1 blockade improved the efficacy of the CAR T cells.

Other transcription factors and signaling cascades have been investigated as biomarkers to predict T cell function and patient prognosis after adoptive transfer of CAR T cells. A study using IL-18/CEA CAR T cells to treat immunocompetent mice with advanced pancreatic carcinoma showed that constitutive secretion of the proinflammatory cytokine IL-18 resulted in CARs with high T-bet in conjunction with low levels of the transcription factor FOXO1 and showed improved antitumor efficacy ( 150). In an analysis of CAR19 T cells derived from patients with CLL, the circulating CARs from complete responders had upregulated genes associated with memory differentiation status, including IL-6 and STAT3, and were observed to lose function upon IL-6/STAT3 blockade ( 119). Nonresponders, on the other hand, had upregulated genes associated with more of an effector phenotype as well as glycolysis, exhaustion and cell death by apoptosis. Finally, the paper showed that CD8⁺ cells that were CD27 positive and PD-1 negative were positively predictive of response to CAR19 T cell therapy. Investigating the role of these biomarkers in CAR T cell response for solid tumors and whether they have an impact on patient survival may elucidate the transcriptional profiles of functional CARs.

Differentiation and Memory

Naive, central memory and effector memory, and terminally differentiated effector T cells all have distinct markers of differentiation. Differential expression of CCR7, CD62L, CD25, CD45RA, CD45R0, CD95, and IL-7Rα, among others, can identify subsets of T cells ( 151, 152). Tregs, CD4⁺ T cells that express CD25, CTLA-4, and FOXP3, are another distinct subset that inhibits T cell effector function; studies have shown that with CAR T cell therapy, Treg presence lowers CAR antitumor activity. Checkpoint blockade targeting CTLA-4 may be one way to address this problem. Other surface markers like IL-2Ra and KLRG1 have been shown to be associated with effector-like phenotypes, while IL-7Rα and the chemokine receptor CXCR3 are associated with memory-like cells ( 153). Cytokines such as IL-2, IL-12, IL-27, and IFNγ are also traced to effector-differentiated T cells, while IL-10, IL-21, IL-7, IL-15, and TGFβ are associated with a memory phenotype. Genes such as T-bet, Id2, Blimp-1, Batf and Stat4 have been associated with effector phenotypes, while Id3, Bcl-6, Tcf-7, Stat3, Foxo1, and Eomes are all proposed to be upregulated in memory-like T cells ( 150, 153).

Gattinoni et al. ( 154) describe a human stem cell memory T cell (TSCM) population that expresses both the classical markers of naive cells as well as certain memory cell markers including CD95 and IL-2Rβ, which the authors determined to be crucial identifiers of TSCM. These cells were found in 2-3% of circulating blood lymphocytes of healthy donors, and could also be induced from naive T cells by culturing them in the presence of a glycogen synthase kinase 3β (GSK3β) inhibitor. (Inhibition of GSK3β has been described to stabilize β-catenin and halt differentiation to effector T cells while promoting memory characteristics). These TSCM cells demonstrated increased proliferation in response to the cytokines IL-7 and IL-15 compared with effector memory T cells, while still retaining their phenotype.

The memory phenotype has consistently been shown to favor T cell survival, proliferation, and prolonged presence of TILs at tumor sites. However, only a few studies focusing on T cell differentiation and memory up to this point have been done in solid tumor models. Still, valuable lessons can be learned from models of ACT, GVHD, or hematological malignancies. For example, a study of ACT in both NSG mice and nonhuman primates showed that purified TCM cells persist better than standard T cells, and form stable memory pools ( 155). Cieri et al. ( 156) published an in vitro culture protocol for inducing a stem cell memory-like phenotype in T cells using IL-7 and IL-15 during CD3/CD28 bead activation of naive precursor cells. When transduced with a transgenic TCR, these T cells displayed memory-associated qualities including proliferation, cytokine production, and expression of CD45R0 upon exposure to target antigen, while retaining markers indicative of naive T cells including CD45RA and CD62L. These T cells were very similar to those described by Gattinoni et al. ( 154) above, except for the expression of CD45R0. The authors found enhanced proliferation in these cells compared with cells expressing a central memory or effector memory phenotype, and this translated to increased expansion and cytotoxicity compared with other subsets of T cells in a mouse GVHD model. The results of these experiments suggest that, as stated by the authors, using naive cells for ACT may significantly improve clinical outcomes. Similarly, using IL-7/IL-15 with naive T cells during transduction and culture conditions to produce more TSCM CAR T cells is a promising strategy for the creation of CAR T cells that both kill and proliferate better in the body. In one such study for solid tumors, T cells bearing a third-generation GD2 CAR (signaling domains for CD28 and OX40), as well as an inducible caspase 9 suicide gene, were activated with CD3/CD28 stimulation along with a variety of cytokines, with the hope of identifying conditions to promote a memory phenotype ( 157). Addition of IL-7 and IL-15 led to the greatest antigen-specific cytotoxicity in vitro along with the highest percentage of stem cell memory and central memory subsets as identified by CD45RA, CCR7, and CD95. The authors predicted better proliferation, survival, and antitumor activity of GD2-CD28-OX40 CAR T cells cultured in IL-7/IL-15.

Clinically, work has been done to determine biomarkers associated with memory that are predictive of response as well as to actually manufacture CAR T cells with optimized differentiation status for infusion into patients. In a study examining T cell memory in ovarian carcinoma patients, it was found that increased presence of CD8⁺ effector memory cells, as well as the chemokine CXCL9, was significantly associated with long-term survival ( 158). The authors also implicated the signaling proteins STAT5B, PLCγ1, and NFATc2 as being relevant to survival, with lower levels of these signals correlating with hypofunctional T cells and shorter survival times in patients.

In the study by Fraietta et al. ( 119) discussed above, the authors looked at both the original unmodified T cells from CLL patients and the corresponding CAR transduced infusion products. All sets of T cells from responding patients showed markers associated with early memory, non-exhausted T cells. As glycolysis is a hallmark of effector/exhausted T cell metabolism, and the T cells of nonresponders displayed upregulated genes for exhaustion and glycolysis, the authors used a glycolysis inhibitor while manufacturing CAR T cells and observed increased numbers of memory CAR T cells along with enhanced proliferation upon exposure to antigen. Blocking glycolysis is another approach that could be used in solid tumor-targeting CAR T cells to push formation of memory T cells during activation and transduction, particularly given ( 119) evidence that the initial differentiation status of a patient's apheresed T cells may significantly affect the efficacy and persistence of the infusion product. Memory CAR T cells have been manufactured for clinical use already in hematopoetic malignancies such as leukemia; Wang et al. ( 155) used a protocol using magnetic separation to select CD8⁺ CD45RA⁺ CD62L⁺ TCM cells to transduce with a CD19 CAR while culturing with IL-2 and IL-15, and several variations of these CARs (which have CD28 costimulatory domains) are being tested in a phase I study. Also for leukemia, a GMP protocol for manufacturing CAR T cells highly enriched for TCM and TSCM phenotypes has been recently developed ( 159) in which, on average, 50% of the T cells were TCM and 46% TSCM. The authors reported that the results were consistently achieved even with very few T cells available to start. The use of CAR T cells enriched for TCM and TSCM has reached the clinic even in solid tumors: the GD2-CD28-OX40 CAR manufactured in IL-7/IL-15 ( 157) is currently in a phase I trial to treat patients with sarcoma, osteosarcoma, neuroblastoma, and melanoma ( NCT02107963).

Tissue Resident Memory Cells

Another memory T cell subset that may be of special importance, especially in treating solid tumors, are tissue resident memory cells (Trms), reviewed in ( 160). Trms have been shown to permanently reside at sites of prior infection or inflammation and quickly respond to pre-recognized antigen, recruiting other immune cells, and increasing the local anti-tumor immune response at a very early stage ( 161). CD8⁺ Trms are characterized by high surface levels of CD103 and the activation marker CD69 and low CD62L and CCR7, and it is believed that TGFβ and IL-15 are both important soluble factors that promote T cell differentiation to a Trm phenotype. Despite having many memory markers, Trms secrete high levels of cytokines such as granzyme B and perforin. Interestingly, Chang et al. ( 153) and Wakim et al. ( 162) found that Trms do not tend to have high T-bet as other memory T cells do. Mackay et al. ( 163) describe downregulation of T-bet, but with necessary residual activity, as one of the factors driving skin Trms, in addition to downregulation of Eomes.

While a significant amount of this research has been done on Trms in skin, some data shows that analysis of Trms across various tissues obtained from humans retain similar phenotypes, particularly in CD8⁺ Trms ( 164). One examination of the transcriptional profiles of Trms in multiple tissue types showed that the gene Hobit ("homolog of Blimp1 in T cells") was upregulated, and together with Blimp1 was a driver of a Trm phenotype ( 165). CD103 was not expressed on all Trms described, which has also been shown in other tissues including the brain ( 162). Interestingly, the genes Hobit and Blimp1 were also upregulated in activated NK cells, suggesting a similar signaling pathway during activation of both Trms and NK cells. Other studies offer support for this parallel, including one by Lotem et al. ( 166) that reported regulation of activation and proliferation in both mature CD8⁺ T cells and NK cells by the transcription factor Runx3. High levels of Runx3 have been shown by several studies to decrease CD4⁺ and increase CD103 expression on T cells, biasing them toward a cytotoxic CD8⁺ Trm phenotype. In a study by Cruz-Guilloty et al. ( 167), Runx3 also was reported to drive an increase in Eomes expression and granzyme B and perforin secretion in differentiating CD8⁺ T cells, while T-bet expression peaked early, at around 2 days, and then decreased over a week of differentiation. The authors also reported Runx3 to regulate CD103 in resting NK cells. While ongoing research is required to parse out more information on Trms, induction of a Trm phenotype using TGFβ and IL-15, or via a genetic engineering, could be a powerful way to improve upon ACT or CAR T cell efficacy. In a study of TILs in lung cancer patients, transcriptome analysis showed that CD103 and other Trm-linked markers were significantly increased in the patients with high numbers of TILs; moreover, having a higher percentage of Trms was reported to predict better survival ( 168). In a study using an orthotopic head and neck cancer model in mice, a cancer vaccine was more successful after induction of Trms, and Trms were still detectable at the tumor site 90 days later ( 169). Additionally, Trms protected against tumor re-grafting even when recruitment of additional effector T cells was blocked, showing that Trms alone can mount a successful antitumor response and tumor rejection upon rechallenge. These data show that Trms may be critical to successful tumor infiltration and protection against tumor relapse, and the induction of a Trm phenotype is likely to increase therapeutic outcomes.

Overcoming Other Immunosuppressive Factors in the TME

Administration of cytokines to polarize the tumor mileu to be more hospitable to T cells and improve CAR T cell recruitment and functionality has been tested in both preclinical and clinical trials. Local delivery of IL-12, which induces inflammatory immune cell recruitment, augmented the anti-tumor activity of adoptively transferred anti-VEGFR-2 CAR T cells and led to prolonged survival of mice bearing five different subcutaneous tumor types ( 170). In the study, treatment of IL-12 plus VEGFR2 CAR T cells, but neither alone, reduced VEGFR2-positive intratumoral MDSCs, providing strong support for the combination of IL-12 with CAR T cells. Due to positive responses like these, CAR T cells that constitutively secrete cytokines, termed "armored" CARs [reviewed by Yeku et al. ( 171)] have been created to enhance T cell infiltration and function in solid tumors (). Particularly, the cytokine IL-12 has been an attractive tool for this. In a mouse xenograft model of ovarian cancer, MUC16 CAR/IL-12 T cells lengthened survival and showed increased persistence and tumor cytotoxicity ( 53). More recently, in a syngeneic mouse model of peritoneal carcinomatosis (metastasized from ovarian cancer), IP-delivery of MUC16 CAR/IL-12 T cells was found to confer longer survival, even when administered to mice with significant disease progression ( 67).

Some other strategies to boost CAR T cell function in the TME include inhibiting suppressive soluble factors, like adenosine, IDO1, and VEGF, and protecting against the immune suppression of non-tumor cells in the TME like MDSCs, TAMs, and stromal cells. In a study using HER2 CAR T cells in a syngeneic tumor model, blockade of the adenosine 2A receptor significantly improved the efficacy of the CAR T cells by enhancing activation and cytokine production ( 118). Additionally, the authors reported that PD-1 blockade further augmented the T cell immune response. Another study demonstrated significant slowing of tumor growth in a xenograft colon cancer model by combining blockade of IDO1 (negatively correlated with patient survival in colon cancer) with EGFRvIII CAR T cell transfer ( 172). VEGF blockade has been successful in solid tumors such as melanoma, and VEGF-targeted CARs have shown efficacy in multiple preclinical solid tumor models ( 131, 132, 170).

Increasing antitumor response can also involve either depleting anti-inflammatory cells in the TME or inducing more inflammatory phenotypes in other immune cells. Research in mouse breast cancer models has suggested that targeting TAMs may be effective for treating progressive cancer, as TAMs were associated with more anti-inflammatory activity and tumor immune evasion ( 173). Another study demonstrated that in murine ovarian cancer models, macrophages were associated with resistance to VEGF blockade. When macrophages were depleted, survival was prolonged, and in macrophage deficient mice, resistance was not observed unless macrophages were reintroduced into the tumors ( 174). TAMs are, therefore, a highly active subset of immune cells that seem to promote tumor survival and immune evasion. In a subcutaneous mouse model of ovarian cancer, tumor rejection by HER2 CAR T cells was shown to require the presence of M1 (inflammatory) macrophages and IFNγ receptors on stromal cells, demonstrating that tumor-specific attack by T cells, even functional ones, may not be enough to clear tumors; stromal cell targeting (for example, with FAP CARs) and recruitment of other types of inflammatory immune cells may be necessary ( 175). Depleting MDSCs can also improve T cell responses, as shown in a study with a GD2 CAR in which the CAR T cell alone had no anti-tumor activity in a xenograft sarcoma model, but in combination with MDSC reduction using all-trans retinoic acid, led to significant antitumor activity ( 176). Noman et al. ( 177) demonstrated in vivo that hypoxia in the TME plays a significant role in upregulation of PDL-1 on MDSCs and on their subsequent suppression of TILs. PDL-1 upregulation was determined to be dependent on HIF1α, and PDL-1 blockade prevented T cell suppression by MDSCs. In another study (described in section Tumor Infiltration) using a CEA CAR, blockade of PD-L1 positive MDSCs and Tregs in the TME augmented CAR T cell anti-tumor function ( 108).

Intracellular Signaling Pathways of the CAR

It is also important to study the signaling pathway of the CAR itself, particularly how different costimulatory domains may affect T cell activation, metabolic needs, differentiation pathways, and the propensity to exhaust. Adding a costimulatory molecule to the original CD3ζ cytoplasmic domain revolutionized the functionality of the CAR T cell; now, there is a broad array of signaling molecules that can be used. The most common costimulatory molecules are 4-1BB and CD28, and depending on the CAR and tumor type, many studies focus on one or the other. Some have studied adding a third costimulatory domain, like ICOS or OX40. Some studies have demonstrated little significant advantage of one design over another. In a study comparing 4-1BB vs. CD28 in an EphA2 CAR, both CARs displayed equally potent antitumor activity in a xenograft mouse glioma model, and creating a third-generation CAR with both domains did not improve T cell performance over the second-generation CARs ( 27). In the study described in section Tumor Infiltration that used mesothelin/IL-7/CCL19 CARs to treat murine mesothelin-expressing PDA, there was also no difference between 4-1BBζ and CD28ζ CARs ( 66).

Many studies implicate 4-1BB as promoting superior differentiation phenotype and persistence. A recent study used phosphoproteomics to report on the kinetics of the 4-1BB vs. CD28 domains in CAR T cells. The authors found that 4-1BBζ CARs and CD28ζ CARs signaled through the same intermediates, but CD28ζ CARs had more and faster changes in protein phosphorylation, which seemed to drive them toward an effector phenotype. On the other hand, 4-1BBζ CARs were shown to express more memory-related genes and performed better in vivo than their CD28ζ CAR counterparts ( 178). Another study comparing 4-1BB and CD28 signaling in a PSCA CAR to treat patient derived prostate cancer xenografts found 4-1BB to be superior to CD28, with 4-1BBζ CARs leading to less exhaustion and better antigen selectivity (however, in vitro killing was equal between the two CARs) ( 179). In the aforementioned study of a regionally delivered HER2 CAR in xenograft models of brain-metastasized breast cancer, 4-1BBζ CARs also showed superior proliferation and less exhaustion than CD28ζ CARs ( 106). The evidence for 4-1BB preferentially expressing memory markers so far has been borne out clinically: an ex vivo study showed that in both CD19 and mesothelin CARs across multiple donors' T cells, 4-1BB promoted better proliferation, central memory differentiation, and greater levels of fatty acid oxidation and mitochondria generation than CD28, while CD28 was linked to increased glycolysis and an effector phenotype ( 6). Other 4-1BB based CARs from in vivo studies described in this review include HER2 CARs in models of medulloblastoma and gastric cancer (92,30); GD2 CARs in models of neuroblastoma and patients with melanoma ( 29, 30); mesothelin CARs in preclinical models of mesothelioma ( 64); and FAP CARs used in models of tumor associated stroma ( 102). Clinically, data is rare so far for solid tumors, but a case study described by Brown et al. ( 42) showed tumor regression induced by T cells expressing a 4-1BBζ IL13Rα2 CAR in a patient with glioblastoma.

Numerous studies have engineered effective CARs that signal through the CD28ζ domain, many of which target the same antigens and are used in similar disease models as 4-1BBζ-signaling CARs. These include humanized HER2 CARs that were shown to have a central memory phenotype in the context of treating breast cancer xenografts ( 35); IL13Rα2 CARs that showed proliferation and cytotoxicity in a mouse model of glioblastoma ( 41); and FAP CARs in IP mouse models of MPM ( 104). Other CARs mentioned in this review that use the CD28ζ costimulatory domain include L1CAM CARs for ovarian cancer in mice ( 45), MET CARs for MPM ( 48), MUC16 CARs in mouse models of ovarian cancer and peritoneal carcinomatosis ( 53, 67), and NKG2D CARs in Ewing's sarcoma models ( 54). Studies have also used CD28 with dual CARs, such as HER2/MUC1 bispecific CARs in in-vitro breast cancer models and HER2/IL13Rα2 CARs in xenograft glioma models ( 86, 87). Clinically, a HER2/CD28ζ CAR was used to treat progressive glioblastoma in a phase I trial that showed efficacy in some patients ( 38).

Finally, third generation CARs have also been studied in preclinical and clinical settings. A recent study comparing third generation GD2 CARs to treat in vivo models of neuroblastoma found 4-1BB/CD28 CARs to be superior to CD28/OX40ζ CARs in terms of activation, exhaustion, and in vivo antitumor efficacy ( 180). Successful in vivo studies using 4-1BB/CD28 third generation CARs include an ICAM-1 CAR for a mouse model of thyroid cancer ( 40), a GPC3 CAR in a patient derived xenograft of model of HCC ( 32), and a VEGFR2 CAR against multiple tumor types in vivo ( 161). However, the study that observed fatal toxicity with the use of a FAP CAR in syngeneic mice used a 4-1BB/CD28ζ third generation CAR, and the case report (mentioned in section 1) of a patient death was after administration of HER2 4-1BB/CD28ζ CAR T cells ( 58). A third generation mesothelin CAR using ICOS/4-1BBζ showed significantly better tumor control and better T cell persistence than ICOSζ or 4-1BBζ CARs alone in a mesothelin-expressing pancreatic xenograft NSG mouse model ( 181). This study also provided significant insight into the signaling pathways that may be required for optimal CAR T cell activation and differentiation. Lower surface CAR expression corresponded to less tonic signaling (signaling in the absence of antigen), which is linked to exhaustion and has been observed in both CD28ζ and 4-1BBζ CARs. Additionally, the authors found that ICOS/4-1BBζ CARs only performed better than second generation CARs when ICOS was proximal to the transmembrane domain. In the clinic, GD2 CARs with CD28/OX40 costimulatory domains are currently in phase I trials for neuroblastoma ( 31).

The site of gene integration has also been shown to have significant impact on CAR function. A study with a CD19/28ζ CAR used CRISPR to insert the CAR under the control of the TCR promoter (at the TRAC locus), while simultaneously knocking out the TCR via insertion of the CAR gene ( 182). The results indicated enhanced proliferation, more memory cells, and much less exhaustion. The TRAC locus CAR was also hypothesized to have reduced tonic signaling that would push T cells toward terminal differentiation and exhaustion.

Future Directions and Conclusions

CAR T cell therapy remains extremely expensive, and patients with severely depleted immune systems may not have viable T cells for autologous CAR T cell generation; additionally, concerns about immunogenicity of certain CAR designs may render therapy less effective in patients that develop an immune response to the CAR. New approaches are needed to make CAR T cells not only functional, but also more efficient and accessible.

Technology such as CRISPR/Cas9 as a highly specific and efficient method of genome editing has become translatable to patients in the past few years. Beyond its use in generating IR knockout CARs, CRISPR/Cas9 has been used to knock out or replace the native TCR in CAR and TCR engineered T cells, which has been shown to provide higher antigen sensitivity and specificity ( 183). In addition to its implications for T cell function, the use of CRISPR is extremely promising in the field of CAR T cell therapy because it can be used to knock out HLA as well as the endogenous TCR, meaning CARs can be made from allogeneic cells without the threat of cross reactivity and GVHD or rejection. This could dramatically reduce the cost, time and resources required to generate CAR T cells for every patient ( 184). This has recently been done successfully with a TCR and HLA class I double knockout CAR19 ( 185). Investigators also developed a CAR that knocked out Fas as well as the TCR and HLA-1 genes, which showed enhanced antitumor activity in vivo against a leukemic cell line, with longer survival than unmodified CAR T cells. Thus, CRISPR can be used not only to knock out inhibitory receptors, but also to knock out the TCR and HLA to generate universal or "off the shelf" CARs ( 186). Moreover, these modifications can be accomplished simultaneously with high precision and efficiency.

Universal CARs have also been developed using other systems of genome editing, including transcription activator-like effector nucleases (TALENs), which create double-stranded breaks in DNA for efficient gene knockouts. Recently, TALEN-mediated editing was used to knock out the TCR-α chain in CAR19 T cells. The subsequent universal CAR T cells, which were from allogeneic donors, induced remission in two infants with B cell ALL ( 187). Before the advent of CRISPR/Cas9, zinc finger nucleases (ZFNs), proteins that recognize three base pairs at a time to bind to DNA, were also used to remove surface expression of molecules like HLA from allogeneic T cells ( 185, 188). ( 189) used ZFNs to disrupt both the TCR β- and α-chain genes while also lentivirally transducing the T cells to express a WT-1 recognizing TCR. This led to superior in vivo antitumor activity and eliminated off-target reactivity.

In some cases where the scFv is murine-derived, there is the potential for the development of anti-mouse antibodies that could reject the CAR T cells. Many studies have adopted the use of a humanized scFv, and these humanized CARs have also gone to clinical trials ( 8). However, humanizing the scFv is a long and onerous process and few fully humanized sequences are currently known. Thus, some recent studies have proposed alternatives to the scFv antigen-recognition domain. One of these alternatives is an affinity molecule from the type III domain of human fibronectin (Fn3), which is similar to the scFv of an antibody but is smaller and has a less complex structure without disulfide bonds, enabling easier in vitro generation of specific binding domains ( 190). Additionally, its smaller size may enable the Fn3 to recognize epitopes that scFvs cannot. Fn3 domains specific for CD20, CEA, EGFR, IGF-1R, and VEGFR2 have been developed. A VEGFR2-specific Fn3 CAR with a CD28ζ costimulatory domain showed in vitro antigen-specific T cell activation and cytotoxicity, and another Fn3 CAR engineered to target EGFR with both CD28 and 4-1BBζ costimulatory domains showed efficacy that was comparable to a traditional CAR against a xenograft lung cancer model ( 191, 192). Another alternative to the scFv is the use of antibody mimetic proteins, such as designed ankyrin repeat proteins (DARPins), synthetic proteins mimicking naturally occurring ankyrin membrane proteins that can be generated with antigen-binding specificity and are smaller and more stable than scFvs. Recently, a HER2-specific DARPin CAR was shown to perform as well as a traditional HER2 CAR in vivo against a human ovarian cancer cell line ( 193).

Significant research has been done with CAR T cells in terms of identifying target antigens, avoiding toxicity, improving CAR T cell trafficking and entry into the tumor site, and promoting better signaling, less exhaustion, and memory phenotypes in solid tumors. Additionally, combination therapy with checkpoint inhibitors, armored CARs, and suppression of other inhibitory factors in the TME has been shown to aid in CAR T cell efficacy in solid tumors, with some of these approaches already being used in clinical trials. Solid tumors pose a wide array of challenges that hematological malignancies do not, hence the need for multi-pronged strategies in addressing them. However, it is clear that our understanding of the TME is increasing at a rapid rate. As the signaling pathways between T cells and other TME cellular components, as well as the intracellular signaling cascades specific to CAR T cell activation and exhaustion, become further understood, CARs hold the promise for greater success in treating solid tumors.

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CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment

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The first major difference between solid tumors and hematological tumors is that it is more difficult to find an ideal target antigen. Unlike cancers such as ALL or CLL in which the tumor cells universally express the B-cell marker CD19, solid tumors rarely express one tumor specific antigen.

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Ved hjælp af terapeutisk brug af overførte T-celler kan cancerceller blive dræbt.

T-celler medierer MHC-ubegrænset tumorcelledrab

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https://link.springer.com/article/10.1186/s13287-021-02420-8
Ny CAR T-terapi er en stråle af håb i behandlingen af ​​alvorligt syge AML-patienter. Mere effektive og mindre skadelige immunterapi-baserede tilgange såsom adoptiv-overførende T-celleterapi under udvikling til behandling af AML.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854376/
Aktuelle fremskridt i CAR-T-celleterapi for solide tumorer. Primært fokuseret på at forbedre CAR-T-celler og overvinde de ugunstige virkninger af tumormikromiljø på solide tumorer.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407347/
Innovative strategier til at fremme CAR T-celleterapi for solide tumorer. Forskellige tilgange til at øge effektiviteten af ​​CAR T-celler gennem direkte optimering af CAR-konstruktioner eller gennem innovative kombinationsstrategier såsom vacciner, biomaterialer og onkolytisk virus er opstået. Ud over at beskrive de vigtigste forhindringer, der begrænser promoveringen af ​​CAR T-celler, fokuserer artiklen på at gennemgå nye igangværende strategier for at omgå disse begrænsninger.

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Abstract

Acute myeloid leukemia (AML) is a serious, life-threatening, and hardly curable hematological malignancy that affects the myeloid cell progenies and challenges patients of all ages but mostly occurs in adults. Although several therapies are available including chemotherapy, allogeneic hematopoietic stem cell transplantation (alloHSCT), and receptor-antagonist drugs, the 5-year survival of patients is quietly disappointing, less than 30%. alloHSCT is the major curative approach for AML with promising results but the treatment has severe adverse effects such as graft-versus-host disease (GVHD). Therefore, as an alternative, more efficient and less harmful immunotherapy-based approaches such as the adoptive transferring T cell therapy are in development for the treatment of AML. As such, chimeric antigen receptor (CAR) T cells are engineered T cells which have been developed in recent years as a breakthrough in cancer therapy. Interestingly, CAR T cells are effective against both solid tumors and hematological cancers such as AML. Gradually, CAR T cell therapy found its way into cancer therapy and was widely used for the treatment of hematologic malignancies with successful results particularly with somewhat better results in hematological cancer in comparison to solid tumors. The AML is generally fatal, therapy-resistant, and sometimes refractory disease with a disappointing low survival rate and weak prognosis. The 5-year survival rate for AML is only about 30%. However, the survival rate seems to be age-dependent. Novel CAR T cell therapy is a light at the end of the tunnel. The CD19 is an important target antigen in AML and lymphoma and the CAR T cells are engineered to target the CD19. In addition, a lot of research goes on the discovery of novel target antigens with therapeutic efficacy and utilizable for generating CAR T cells against various types of cancers. In recent years, many pieces of research on screening and identification of novel AML antigen targets with the goal of generation of effective anti-cancer CAR T cells have led to new therapies with strong cytotoxicity against cancerous cells and impressive clinical outcomes. Also, more recently, an improved version of CAR T cells which were called modified or smartly reprogrammed CAR T cells has been designed with less unwelcome effects, less toxicity against normal cells, more safety, more specificity, longer persistence, and proliferation capability. The purpose of this review is to discuss and explain the most recent advances in CAR T cell-based therapies targeting AML antigens and review the results of preclinical and clinical trials. Moreover, we will criticize the clinical challenges, side effects, and the different strategies for CAR T cell therapy.

Introduction

Some immunotherapy approaches have emerged with effective applications and interestingly they achieved more successful results when administered in hematological malignancies and solid tumors [ 6]. Over the last decade, it was illustrated that immunotherapy-based strategies are attractive for AML patients resistant to chemotherapy. These novel therapies can specifically target the antigens on leukemic stem cells and leukemic blasts so less toxicity is achieved by using the therapy [ 7].

Vaccine therapy, monoclonal antibodies, checkpoint inhibitors, stem cell transplantation, and CAR T cell therapy are some of the immunotherapy techniques with promising outcomes in the treatment of AML [ 7].

Several new therapeutic agents based on antibodies have been developed targeting key molecules of AML. For instance, gemtuzumab ozogamicin (Mylotarg®; Pfizer) is a humanized mAb linked to an anti-tumor antibiotic, calicheamicin, and so is considered as an antibiotic-antibody conjugate. It was approved by the United States Food and Drug Administration (FDA) for treatment of relapsed AML in adults and children with confident therapeutic results [ 8]. The ADC treatment alongside other antibody-based therapies such as bispecific T cell engager (BiTE) antibodies achieved successful outcomes.

In this review, we will discuss the current therapeutic approaches for AML with a special focus on the CAR T cell therapy, structure, generation, and function of CARs, trials on the CAR T cells for the treatment of AML, possible challenges and toxicity of CARs, as well as the suggestions for improving the CAR T cells' safety and efficacy and for decreasing the toxicity.

Therapeutic approaches for AML

Chemotherapy

Antigen targeted therapy

Hedgehog (Hh) signaling pathway

Fms-like tyrosine kinase 3 (FLT3)

FLT3 is a transmembrane tyrosine kinase and its mutant forms are mostly overexpressed in AML [ 21]. Midostaurin is one of the important preventing agents against FLT3, which has shown beneficial effects on repressing the AML and increasing the survival rate especially when it is used along with chemotherapy agents [ 22].

Isocitrate dehydrogenase 1,2 (IDH1/2)

Ivosidenib and Enasidenib are other chemical agents used to restrain the functionality of IDH1 and IDH2 mutant forms (in cancerous cells), respectively. The mutant forms cause a cascade of harmful intracellular changes such as enhanced 2-hydroxyglutarate production, an oncometabolite that inhibits the enzymes with an important role in histone methylation. The subsequent changes in methylation processes cause transcriptional dysregulation and therefore resulted in the promotion of proliferation and blockade of differentiation of myeloid cells. The drugs targeting IDH1 and IDH2 reverse these harmful changes and therefore cause therapeutic effects in AML patients [ 23].

B cell leukemia/lymphoma-2 (BCL2) and P53

E-selectin

E-selectin is an overexpressed cell adhesion molecule on leukemic blasts. Uproleselan (GMI-1271) is an E-selectin blocker that can increase the anti-cancer chemotherapy response against AML [ 27].

Polo-like kinase-1 (PLK1)

PLK1 is a key mitosis regulator and helps the cell for the right passage through mitosis processes. It is thought that PLK1 has a role in DNA modification and replication procedures. Volasertib and Rigosertib are important suppressors of PLK1 and developed for the treatment of AML [ 28, 29].

Cyclin-dependent kinases (CDKs)

CDKs, the members of the protein kinases family, are also important target molecules in AML. CDKs have critical roles in cell cycle regulation, cell differentiation, mRNA preparation, and transcription regulation. CHK1 protein kinase overexpression in AML is correlated with limited responses to chemotherapy and a shorter duration of survival. Prexasertib is a CHK1 inhibitory agent that can restrict the replication processes [ 30]. Moreover, FDA granted palbociclib as the CDK4/6 inhibitory agent for the treatment of breast cancer. It has a good potential to be used as a therapy in other cancers such as AML [ 31].

Vaccine therapy

Monoclonal antibody therapy

Bispecific antibody therapy

Bispecific antibodies (BAB) composed of two distinct variable domains, one specific for a tumor antigen and the other specific for a CD3 receptor on the T cell, have been developed in recent years with some superiority in comparison to monospecific antibodies. BABs can detect the tumor cell and simultaneously activate the killing mechanisms in T cells by engaging them towards the detected tumor cell. An anti-CD3/CD33 bispecific antibody (AMG330) and an anti-CD3/CD123 antibody named flotetuzumab are two good examples of bispecific antibodies, both evaluated for the treatment of AML. Compared to old-fashioned mAbs, BABs have some advantages such as increased safety, more efficacy in tumor cell killing, and reduced exhaustion of the immune cells which is particularly characterized by deletion or inhibited functions of T cells [ 43, 44].

Checkpoint inhibitors

Allogeneic hematopoietic stem cell transplantation (alloHSCT)

CAR T cell therapy

CAR T cell structure

Isolation, engineering, proliferation, and administration of CAR T cells. In the first stage, T cells are isolated from patients through leukapheresis and then harvested and activated. After that, the engineering and construction of the CARs are done in T cells. After the proliferation of the produced CAR T cells, the cells are re-injected into patients

The process of CAR T cell development and its various generations

Different generations of CAR T cells. CAR signaling and applications are updated with each new generation. The first generation consists of an intracellular domain (CD3ζ chain), the second generation of CAR T cells has been composed of the addition of CD28 to CD3 as co-stimulatory molecules. In the third generation of CAR T cells, OX-40, a second co-stimulatory molecule, has been added. In the fourth generation, NFAT has been added to the induction of cassette containing the IL-12 gene promoter. NFAT, nuclear factor of activated T cells

CAR T cell function

In contrast to the classical T cells with recognizing the antigens by T cell receptor (TCR), chimeric antigen receptor (CAR) in engineered CAR T cells has a role in antigen recognition. As well, CAR T cells can identify broader types of antigens in the independence pathway of MHC. Antigen recognition through ScFv followed by intracellular signaling activates the CAR T cell against target cells. After activation, the CAR T cell specifically exerts its functionality through the secretion of anti-tumor cytokines, perforin, and granzymes into the tumor microenvironment (TME) [ 69, 70]. Therewith, immune system surveillance, recruitment of other immune cells, tumor cell elimination, and inhibition of tumor relapse can be increased by using CAR T cell as a living drug.

CAR T cell therapy in hematological malignancies

CAR T cell therapy with hopeful success results has been developed in various hematological malignancies without significant efficacy in solid tumors. Immunosuppressive conditions of the solid tumor microenvironment, antigen heterogeneity, and other related hurdles reduce the CAR T cell efficacy in the context of solid tumor tissue [ 58]. CAR T cell-based therapies with manageable cytotoxicity and high efficacy are widely utilized in hematological cancers such as acute and chronic forms of leukemia, lymphoma, and multiple myeloma [ 13].

CD22 another overexpressed antigen on B cell cancers can be targeted by the CD22-CAR T cell. The impressive anti-tumor function of CD22-CAR T cell was reported in relapsed cancer after CD19-CAR T cell therapy or in eradicating the ALL tumor cells [ 77].

CAR T cell therapy and potential targets in AML

In AML, due to the difficult identification of target antigens, CAR T cell therapy is encountered various challenges and no approved study exists related to CAR T cell therapy for AML yet. Therefore, increasing the understanding of the AML microenvironment can be contributed to discovering the enthusiastic and proper target antigens (Table 1). Based on this, several clinical studies have been developed to investigate the CAR T cell therapeutic function against predisposed antigens such as CD7, CD33, CD38, CD44, CD70, CD123, CLL, FLT3l, FRβ, Le-Y, LILRB2, NKG2D, PR1, and WT1. In this review, various types of CAR T cells against candidate antigens have been described (Table 2).

Table 1 AML target molecules

Table 2 CAR T cell against AML target antigens

CD7-CAR T cell

CD7 is a transmembrane glycoprotein expressed by T cells, NK cells, and cord blood myeloid progenitors with a co-stimulatory role in B and T cell lymphoid development interactions. CD7 is also expressed by leukemic cells like AML (30%) but not by healthy myeloid cells. So, it can be a potential candidate for eradicating tumor cells selectively with no toxicity on normal cells [ 107]. For engineering CAR T cells, CD7 removal requires limiting the T cell fratricide due to the CD7 expression by T cells [ 108].

In two studies by Gomes-Silva et al. [ 85, 108], CD7-CAR T cell was engineered against CD7⁺ tumor cells in a xenograft model of AML. Before CAR T cell generation, the CD7 gene of primary activated T cells was removed by CRISPR/Cas9 strategy. Then, the second generation of CD7-knockout (CD7 KO) CD28- CD3ζ-CD7-CAR T cell was designed by using the ScFv derived from the anti-CD7 antibody. Astonishingly, findings manifested the high cytolytic effects against AML include notable elimination of primary AML blasts and leukemia colony-forming cells, no toxicity on healthy and erythroid cells, and a high concentration of IFN-γ. Moreover, reduction of tumor burden indicated that CD7-CAR T cell prevents systemic leukemia progression. In consequence, CD7-CAR T cell can be a potent treatment for refractory or relapsed AML.

CD33-CAR T cell

CD33 as another potential target antigen is a transmembrane protein of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family with regulatory effect on leukocytes in immune responses. CD33 is expressed on normal progenitor cells, myeloid cells, and more than 90% of AML cells possessing diagnostic and therapeutic capabilities [ 109].

In a phase I trial study conducted by Wang et al. [ 113], the safety and efficacy of CD33-CAR T cells were assessed in relapsed and refractory AML patients. They administered a total of 1.12 × 10 ⁹ autologous T cells with 38% anti-CD33 CAR expression. As a consequence, CD33-CAR T cells displayed notable cytolytic functions against CD33+ blasts such as remarkable tumor degradation in the early stage as well as high maintenance of CAR T cell number and cytotoxicity. However, disease progression, pancytopenia, high fever, and CRS toxicity were observed as adverse effects due to the production of a high level of proinflammatory cytokines. Based upon this, safety measures should be considered to reducing the CD33-CAR T cell.

In a preclinical study, to increase the CAR T cell safety, Kenderian et al. [ 86] generated the transiently expressed mRNA-modified second-generation CD33-CAR T cell by using the ScFv derived from GO. The study findings disclosed that CD33-CAR T cells can strongly eliminate the human AML cells and myelodysplastic syndrome blasts in mouse xenografts. The reported in vivo and in vitro results of assessing the CD33-CAR T cells were the significant proliferation of CAR T cell, prominent anti-tumor activity, increased level of cytokine production, degranulation, and leukemia burden reduction. Additionally, they suggested that CD33 gene eradication through the CRISPR/Cas9 gene-editing strategy as a CD33 knockout (KO) HSPC contributes to increasing the safety of CAR T cells. In this regard, the results of a study demonstrated that the combined administration of CD33-CAR T cell with CD33 KO HSPC led to targeting the AML cells specifically and reducing the myelotoxicity [ 114].

CD38-CAR T cell

In a study, Yoshida and colleges engineered CD38-CAR T cells to target leukemia cells. They showed that ATRA increased the CD38 expression on AML cells, and subsequently, remarkable cytotoxicity of CD38-CAR T cell combined with ATRA was observed in eliminating the tumor cells [ 88].

CD44v6-CAR T cell

CD70-CAR T cell

CD70 is a ligand for CD27 identified as a type II transmembrane glycoprotein, a member of the TNF family. CD70 expression is upregulated on APCs and can be expressed on T and B cells which are triggered by stimulatory factors. CD70 expression has been evidenced on AML bulk cells and leukemic stem cells (LSC) but not on normal hematopoietic stem cells (HSCs) [ 91, 92].

In a preclinical study conducted in the AML xenograft model, a second-generation CD27-CD3ζ-CD70-CAR T cell was generated and validated. Strong capability of proliferation, robust potential cytolytic function in eliminating the tumor cells, increased level of TNF-α and IFN-γ production, and no toxicity on healthy HSCs are the reported applications of the CD70-CAR T cell [ 92]. These promising outcomes suggest the applicability of CD70 antigen as a proper choice for developing CAR T cell against AML.

CD123-CAR T cell

Another proper target antigen for AML cell therapy is the IL-3 receptor α subunit (IL3Rα) named CD123. CD123 overexpression has been evidenced on leukemic stem cells (LSCs) and AML blasts and no significant expression on normal hematopoietic stem cells [ 122]. In a clinical study, anti-CD123 neutralizing monoclonal antibody demonstrated insufficient efficacy against AML [ 123].

Gill and colleagues engineered a second-generation CD123-CAR T cell to target CD123⁺ AML cells in the xenograft model. Interestingly, strong anti-tumor cytotoxicity, establishing memory T cells, proliferation, persistence, degranulation, and effector cytokine production were reported as results of using CD123-CAR T cell [ 94].

In a study, Thokala et al. engineered CD123-CAR T cells by using different chains of VL and VH from various CD123 specific monoclonal antibodies (mAbs) instead of only one specific antibody. Findings revealed the CD123-CAR T cell cytotoxicity against CD123⁺ AML cells with no effect on CD123 − B cell lymphoma cells and tumor burden reduction. Concerning myelotoxicity of CD123 like CD33, it remains a problem with targeting this antigen for AML therapy. Surprisingly, CAR T cells with ScFv composed of VL and VH from various mAbs presented the low off-tumor toxicity and lysis effect on healthy hematopoietic stem cells compared to CAR T cells with VL and VH chains of only one mAb [ 124].

FLT3-CAR T cell

In a preclinical study [ 95], a second-generation FLT3-4-1BB-CD3-CAR T cell was engineered by using the anti-human FLT3 antibody-derived ScFv to targeting the FLT3+ tumor cells. According to in vitro investigations, IFN-γ and IL-2 were produced by the FLT3-CAR T cell encountering AML cell lines. Withal, inhibition of leukemia cell proliferation was observed by in vivo evaluation of FLT3-CAR T cell function. Toxicity assessment of FLT3-CAR T cell in xenograft model indicated no significant toxicity to multipotent myeloid progenitors (MMPs) and hematopoietic stem cells (HSCs) along with equivalent toxicity to common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) describing the less hematologic toxicity of FLT3-CAR T cell.

In another preclinical study, Wang et al. [ 130] engineered FLT3L-4-1BB-CD3ζ-CAR T cell to assessing its functionality against FLT3L+ leukemia cells. As a result, prominent anti-tumor cytotoxicity of CAR T cell against FLT3+ leukemia cells as well as the influential killing of the ITD type FLT3 cells compared to the wild type FLT3 cells was observed by using the CAR T cell. Therewith, AML xenograft model survival was lengthened by the effect of FLT3L CAR T cell. FLT3L-CAR T cell indicated less off-tumor toxicity on healthy progenitor and hematopoietic stem cells.

CLL1-CAR T cell

C-type lectin-like molecule-1 (CLL1) as an inhibitory receptor is a type II transmembrane glycoprotein that can be a potential candidate for AML CAR T cell therapy. CLL1 expression accounts for 92% in AML cases, which is overexpressed on differentiated myeloid cells and AML blasts. As well, CLL overexpression has been evidenced on leukemic stem cells (LSCs) but not on normal HSCs which proposes the less off-tumor toxicity of CLL1-CAR T cell. Currently, targeting CLL1 by specific monoclonal antibody has revealed an effective therapeutic function against AML along with reducing tumor burden [ 131, 132].

In a previous study, the third generation of CLL1-CAR T cell composed of anti-CCL1 ScFv fused to CD28, 4-1BB, and CD3 signaling domains was generated. This anti-CCL1 CAR T cell showed the in vitro and in vivo strong functionality against CLL1⁺ tumor cells with a wide production of effector cytokines and chemokines including GM-CSF, TNF-α, IFN-γ, and IL-13 [ 97].

Moreover, two other related studies reported the results of using second-generation CAR T cells to targeting CLL1⁺ AML cells with sparing normal myeloid precursor cells. Increased persistence and cytotoxicity, efficient tumor elimination, anti-inflammatory cytokine production, and managing tumor relapse were reported as results of CLL1-CAR T cell activity [ 98, 133]. Importantly, it has been reported that using the inducible caspase 9 (iC9) strategy transduced into T cell along with the CAR gene increased the safety of CLL1-CAR T cells by controlling its function. This approach contributed to inhibiting the CAR T cell overactivity by triggering apoptosis [ 134].

LeY-CAR T cell

Lewis Y (LeY) is a carbohydrate tumor-associated antigen related to blood-group members. Its expression has been reported by various epithelial-derived cancers and less expression by healthy tissues [ 134]. Several studies have noticed the relationship between LeY antigen and progression of solid tumors and hematological malignancies. Due to LeY expression on early myeloid progenitor cells, it can be a proper targeting choice for AML treatment [ 99]. Hence, to target tumors, T cells like engineered CAR T cells have been developed to redirect against LeY tumor cells.

In a preclinical study, Peinert et al. [ 99] generated the anti-LeY CAR T cell and then investigated its functionality against AML and MM (multiple myeloma) cells. According to the reported results, engineered LeY-CAR T cells specifically targeted the LeY⁺ cells by producing the IFN-γ.

To conduct clinical trial studies for AML and MM adoptive cell therapy by the CAR T cell, Neeson et al. [ 135] engineered the CD28-CD3z-LeY CAR T cell by using the autologous T cells based on LeY-T ex vivo transduction and expansion protocol. CD8+ modified T cells showed a cytolytic response against LeY⁺ tumor cells and high-level production of IFN-γ.

FRβ-CAR T cell

In a preclinical study [ 101], the first anti-FRβ-CAR T cell (m909) was engineered to targeting AML cells in vitro and in vivo. The effective cytolytic function of m909 CAR T cell was observed against FRβ + cells in vitro and regression of AML tumor cells in vivo with no toxicity on HPSCs. Moreover, they presented the enhancement functionality of CAR T cells due to the elevated expression of FRβ in the presence of ATRA. In another study [ 101], a high-affinity (HA) anti-FRβ ScFv-CAR T cell was generated for AML treatment. The strong anti-tumor function and potential lyse myeloid-lineage target cells without toxicity on HSCs were observed through using HA-FRβ-CAR T cells in vitro and in vivo.

Consequently, FRβ can be an encouraging and proper target antigen for AML CAR T cell therapy powerful activity in combination with ATRA and without toxicity on normal cells.

LILRB4-CAR T cell

Monocytic AML (M5) is a common type of AML in children including 20% of AML cases along with poor outcomes. LILRB4 as a potential target antigen in AML belongs to the family of the leukocyte immunoglobulin-like receptor-B family. LILRB4 expression was reported on healthy monocytic cells and monocytic AML cells in all stages [ 139].

In a preclinical study, 41BB-CD3ζ-anti-LILRB4-CAR T cell was engineered by using the humanized ScFv to target LILRB4 AML cells specifically. Interestingly, applying powerful cytotoxicity to AML cells in vitro, decrement of xenograft model tumor burden in vivo, and reduction of off-tumor toxicity by sparing normal progenitors and HSPCs were found as results of using LILRB4-CAR T cell [ 102]. These results have illustrated the potential capacity of LILRB4-CAR T cell therapy in targeting the AML selectively and specifically.

NKG2D-CAR T cell

In a phase I clinical trial study conducted by Baumeiste et al. [ 145], autologous first-generation NKG2D-CD3ζ-CAR T cell was engineered and validated in AML and myelodysplastic syndrome (MDS) patients. For safety and efficacy evaluation, single intravenous administration of NKG2D-CAR T cell was performed by a dose of 1 × 10 ⁶ up to 3 × 10 ⁷ T cells. As a result, in one AML patient, an objective clinical response with a high level of IFN-γ production was reported without any high-grade toxicities. Interestingly, it has been reported there are no long-term toxicities like myeloablation and B cell aplasia, infusion toxicity, autoimmunity, and CRS.

PR1-CAR T cells

In the intracellular process, leukemia-associated-antigens and neoantigens are presented to T cells by HLA-II molecules. TCR-mimic (TCRm) CARs have been developed to targeting the HLA-presented antigens [ 10].

One of the important leukemia-associated antigens that arose from Proteinase 3 and neutrophil elastase is Proteinase 1 (PR1), HLA A2-restricted nonameric peptide. These proteinases are normally expressed in neutrophils azurophilic granules and are overexpressed on myeloid leukemia blasts [ 146]. To targeting PR1+ AML cells, Ma et al. developed the anti-PR1/HLA-A2-second-generation CAR T cell by using the ScFv derived from anti-PR1/HLA-A2 TCR-like antibody so-called h8F4. As a consequence, the PR1-CAR T cell exhibited the effective avidity to the PR1+ target cell and preferentially targeted the human AML cell lines and primary AML blasts with high cytotoxicity in vitro. Also, outcomes of evaluating the CAR T cell off-target toxicity have illustrated that leukemia progenitor cells were preferentially suppressed by anti-PR1/HLA2-2-CAR T cell rather than normal hematopoietic progenitors [ 105]. The successfully reported findings of the h8F4-CAR suggesting the potential of endogenous self-antigens for targeting by CAR T cell in AML.

WT1-CAR T cells

In a study conducted by Rafiq et al. [ 106], WT1-CAR T cell as another TCRm CAR was developed and validated by using the HLA-A*02:01-a peptide that arose from WT1 antigen. In practice, WT1⁺/HLA-A*02:01⁺ primary tumor cells or cell lines were distinguished and lysed by WT1-CAR T cell. Based on this, prominent cytotoxicity and significant secretion of IL-2 and IFN-γ were observed against primary AML cells or cell lines.

CAR T cell improvement strategies, side effects, and challenges

Despite the more beneficial effects of CAR T cells, their proliferation, persistence, and anti-tumor functions may decrease encountering some challenges in hematological malignancies or solid tumors (Fig. 3).

Implications of the association between the CAR T cell and cancer cells in AML. The production of a CAR T cell in patients with AML leads to its identification and binding to TAAs or TSAs on the surface of the cancer cell and has a variety of effects, including activation of macrophages to produce ROS and NOS, activation of NK cells to secrete perforin and granzyme, and secretion of cytokines that strengthen the body's immune system, which eventually kills tumor cells

Improvement strategies

CAR T cell delivery

Intravenous infusion (Systemic delivery) and intracranial infusion (local delivery) are both CAR T cell delivery strategies that have a significant role in increasing the safety and efficacy of CAR T cell therapy.

Encouragingly, successful results of CAR T cell local delivery have been evidenced in solid tumors. For an instance, efficient tumor remission was observed by intracranial infusion of IL13Rα-CAR T cells in glioblastoma [ 154].

CAR T cell production

Appropriate concentration (10 ⁸-10 ⁹ cells) of CAR T cell requires impressive efficacy. However, some challenges led to insufficient CAR T cell production. Ex vivo increasing number of CAR T cells is an important phase of CAR T cell manufacturing process after engineering can be improved the function quality of produced cells. For this reason, non-significant expansion of engineered T cells may lead to the insufficient response of CAR T cells.

To expand the CAR T cells, synthetic APCs and stimulatory cytokines like IL-2, IL-7, and IL-15 along with anti-CD3 and anti-CD28 antibodies can be used as expansion trigger factors [ 155, 156].

Further, patients' lymphopenia arose from chemotherapy as another major reason for CAR T cell failure production. Importantly, different types of infused T cells impressed the anti-tumor efficacy of CAR T cell therapy. As an instance, central memory T CD8⁺ cells have remarkably indicated the effective cytotoxicity against tumor cells [ 157, 158, 159].

CAR T cell persistence and efficacy

Cancer relapse after CAR T cell therapy

Autologous and allogeneic CAR T cells

Using the autologous or allogeneic T cells as another principal challenge of CAR T cell therapy has remained unclear. The engineer of autologous CAR T cell is identified as a safe cell therapy with restricted autoimmune responses and GVHD. However, using these types of cells encounters some disadvantages which include high costs and time, elaborate types of equipment, and specialized experts. Disappointingly, the insufficient number of T cells in some patients with lymphopenia and unsuccessful choice for serious disorders are the other challenges of using autologous T cells [ 168]. Contrarily, allogeneic T cells have more advantages including low-time and low-cost engineering procedures, producing high-quality and high-quantity CAR T cells, and noticeable treatment. Encouragingly, the GVHD effect of using allogeneic T cells can be inhibited through the inactivation of related genes, and also, they can be safely acted after allogeneic HSCT [ 169].

Tumor immune-escape strategies

Side effects

Cytokine release syndrome (CRS)

Same as other therapeutic approaches, some side effects also have been reported in CAR T cell therapy. One of the major disadvantages of CAR T cells is related to cytokine production phenomena such as macrophage activation syndrome, hemophagocytic lymphohistiocytosis, and cytokine release syndrome (CRS). CRS may occur in patients with great tumor burden and due to the robust activity of CAR T cells or administration of a high dose of CAR T cell [ 173]. Strong production of cytokines such as Il-6, IFN-y, IL-1β, GM-CSF, and TNF-α during the CAR T cell function induces the CRS response followed by fevers, hypoxia, myalgia, hypotension, vascular leakage, and neurological disorders [ 174].

On-target/off-tumor toxicity

Anaphylaxis

The major population of engineered CAR T cells is composed of the murine ScFv. Based on this, these types of CAR T cells encountering cellular and humoral rejection caused by murine protein immunogenicity which led to the anaphylaxis outbreak. Acute anaphylaxis as an immediate interaction occurs because of the host immune system reaction after recognizing the foreign component has entered [ 181].

Neurotoxicity

Neurotoxicity is nervous system toxicity accompanied by expressive aphasia, delirium, seizure, and confusion symptoms. The incidence etiology of neurotoxicity is unclear; however, it may happen by inflammation derived from cytokine overproduction which has been mostly reported in patients treated by the CD19-CAR T cell [ 182].

Insertional oncogenesis

Besides, insertional oncogenesis is another side effect that comes about through the transferring of CAR transgene into T cells by lentiviral or retroviral vectors which can increase the induced malignant transformation risk. Fortunately, the incidence rate of insertional oncogenesis is less; however, considering accurate and safe measures is required in the CAR T cell engineering procedure [ 183].

Hematological toxicity

Hematological toxicity as another adverse effect of CAR T cell therapy is caused after allogeneic hematopoietic stem cell transplantation (alloHSCT) and CAR T cell administration. Neutropenia, anemia, and thrombocytopenia are the reported examples of hematopoietic system toxicities after CAR T cell therapy. Normal HSPC destruction by CAR T cell due to the presence of common antigen on malignant cells and HSPCs like CD33 is identified as hematological toxicity in AML patients [ 184, 185].

CAR T cell therapy challenges regarding their toxicity

CAR T cell toxicity management

To mitigate the toxicity and improve the clinical application and efficacy of CAR T cell therapy, various approaches have been developed. Some strategies are used to control the CAR T cell activity with switch-off mechanisms to avoid the CAR T cell overactivity, and some of them have been developed to decrease mentioned side effects derived from CAR T cells. In addition, using the high-affinity ScFv, humanized or human-derived ScFv rather than murine, utilization of local delivery instead of intravenously, and optimizing the CAR T cell manufacturing and infusion process can be improved the clinical therapeutic applications of CAR T cell [ 182, 186].

Inhibition of CRS and Neurotoxicity

Inhibition of anaphylaxis

Intending to overcome anaphylaxis toxicity, efforts for using the humanized or human ScFv are undergoing, to improve the survival and function of CAR T cells [ 192].

Inhibition of hematological toxicity

In hematological malignancies, the incidence of hematological toxicity is possible due to the CAR T cell activity. To overcome this toxicity, common antigens between leukemic cells and HSPCs should be removed from HSPCs by gene-editing methods like CRISPR/Cas9 system and then used for alloHSCT. As an example, in a CD33⁺ AML patient, alloHSCT with CD33 negative HSPCs along with anti-CD33-CAR T cell therapy can eradicate the CD33⁺ blasts without effect on CD33 neagative HSPCs which results in reducing the hematopoietic toxicity [ 114, 193]. Collectively, combinational therapy of alloHSCT and CAR T cell therapy as a novel therapeutic strategy would be improved cancer treatment like AML.

Inhibition of "on-target/off-tumor" toxicity and CAR T cell overactivity

Various controllable strategies have been developed to switch off the CAR T cell, optimize the CAR T cell function, and prevent the CAR T cell overactivation which mitigated the CAR T cell-derived side effects like on-target/off-tumor toxicity. Accordingly, development strategies include the mRNA electroporation delivery system, suicide genes, CRISPR/Cas9 gene-editing strategy, and smart or multi-targeted CAR T cells ("Advanced generations of CAR T cell" section).

mRNA electroporation

CAR T cell transient delivery systems like mRNA electroporation strategy as well as transposon/transposase are used to limit CAR T cell expression and activation. A biodegradable CAR like RNA-CAR123 is an example of using an mRNA electroporation mechanism that gradually decays the CAR T cell expression and activation in AML cases [ 193].

Suicide genes

Eliminating genes

Eliminating genes are known as another mechanism of CAR T cell-selective exhaustion. To induce the CAR T cell death, CD20 or EGFR identified as cell surface antigens can be used in CAR structure. Then, the infusion of CD20 (rituximab) or EGFR (cetuximab) mAbs trigger the CAR T cell death through their interaction with mentioned eliminating genes [ 197, 198].

CRISPR/Cas9 genome editing system

An important gene-modifying strategy with a high potential operation is Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/CRISPR-Associated 9 (Cas9). This strategy is used to developing the next-generation CAR T cells with high efficacy and safety by genome manipulation of antigens, enzymes, checkpoints, and cytokines [ 61, 64].

Advanced generations of CAR T cell

In a study by Cartellieri et al. [ 201], a flexible CAR platform so-called universal CAR T cell was engineered to target CD33 and/or CD123 AML blasts in vitro and in vivo. Universal CAR regulating the CAR T cell function by on/off switching mechanism. As a result, universal CAR T cell lyses the AML blasts and AML cell lines potentially by dual-targeting the CD33 and CD123 antigens. Interestingly, no on-target/off-tumor toxicity and xenogeneic graft-versus-host disease were reported in using the universal CAR. In practice, two or multiple antigens targeted simultaneously can be reduced the risk of tumor escape, on-target/off-tumor toxicity, and increased the specificity of antigen targeting.

Considering the lack of proper specific targets of AML, new potential targets can be discovered to improve the AML treatment. Hence, in a study by Perna et al. [ 200], the discovery and analysis of AML new target antigens were conducted by combining the proteomic and genomic resulted from data from AML patients and healthy volunteers to finding the ideal targets of AML for CAR T cell therapy. As a consequence, four hopeful and potential target antigens were found to include ADGRE2, CCR1, CD70, and LILRB2 which could be multiple targeted by engineering appropriate smart CARs. More than that, they predicted identified antigen overexpression on the majority of AML blasts along with limited expression on normal cells and activated T cells, expression in a majority of AML patients, and less off-tumor toxicity on healthy cells. Consequently, recognizing the new target antigens in AML and establishing the powerful smart CAR T cells can hopefully increase the success rate of CAR T cell therapy in AML patients.

Concluding remarks

In conclusion, since there is no approved CAR T cell therapy approach for AML, discovering the predisposed and potential AML target antigens and performing the various strategies to improving the CAR T cells' safety and efficacy can lead to a breakthrough in AML CAR T cell therapy.

Availability of data and materials

Abbreviations

CAR:

Chimeric antigen receptor

GvHD:

Graft-versus-host disease

CAR-NK:

CAR-transduced NK cells

NK cells:

Natural killer cells

ICIs:

Immune checkpoint inhibitors

ACTs:

Adaptive cell therapies

NHL:

Non-Hodgkin lymphomas

DLBCL:

Diffused large B cell lymphoma

FL:

Follicular lymphoma

ALL:

Lymphoblastic leukemia

MM:

Multiple myeloma

AML:

Acute myeloid leukemia

CRS:

Cytokine release syndrome

CTLs:

Cytotoxic T lymphocytes

NCRs:

Cytotoxicity receptors

SHP-1:

SH-2 containing protein tyrosine phosphatase

ITIMs:

Immunoreceptor tyrosine-based inhibitory motifs

hESCs:

Human embryonic stem cells

SCGM:

Stem Cell Growth Medium

bNHL:

B cell non-Hodgkin's lymphoma

RCC:

Renal cell carcinoma

CAIX:

Carbonic anhydrase IX

EpCAM:

Epithelial cell adhesion molecule

iCAS9:

Inducible caspase 9

Acknowledgements

We would like to thank German Cancer Research Center (DKFZ) for accepting to pay the article publication charge.

Author information

Affiliations

1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Faroogh Marofi, Navid Shomali, Ali Hassanzadeh, Behzad Baradaran & Hossein Saeedi

2. College of Medicine, University of Sulaimani, Sulaimaniyah, Iraq

Heshu Sulaiman Rahman

3. Department of Medical Laboratory Sciences, Komar University of Science and Technology, Chaq-Chaq Qularaise, Sulaimaniyah, Iraq

Heshu Sulaiman Rahman

4. Department of Pharmaceutical Chemistry, College of Pharmacy, University of Alkafeel, Najaf, 54001, Iraq

Zaid Mahdi Jaber Al-Obaidi

5. Department of Chemistry and Biochemistry, College of Medicine, University of Kerbala, Karbala, 56001, Iraq

Zaid Mahdi Jaber Al-Obaidi

6. Faculty of Biology and Ecology, Yanka Kupala State University of Grodno, Grodno, Belarus

Abduladheem Turki Jalil

7. Department of Health and Rehabilitation Sciences, College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, Al Kharj, Saudi Arabia

Walid Kamal Abdelbasset

8. Department of Physical Therapy, Kasr Al-Aini Hospital, Cairo University, Giza, Egypt

Walid Kamal Abdelbasset

9. Faculty of Nursing, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy, Bangkok, 10210, Thailand

Wanich Suksatan

10. Sechenov First Moscow State Medical University, Moscow, Russia

Aleksei Evgenievich Dorofeev

11. DigiCare Behavioral Research, Casa Grande, AZ, USA

Max Stanley Chartrand

12. Taneja College of Pharmacy, University of South Florida, Tampa, FL, USA

Yashwant Pathak

13. Department of Pharmaceutics, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia

Yashwant Pathak

14. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Majid Ahmadi

15. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Safa Tahmasebi

16. German Cancer Research Center, Toxicology and Chemotherapy, No. 2, Floor 4 Unit (G401), 69120, Heidelberg, Germany

Mostafa Jarahian

Contributions

All authors contributed to the conception and the main idea of the work. F.M, H.S.R, N.SH, A.H, S.T, A.E.D, H.S, and M.A drafted the main text, figures, and tables. M.J and B.B supervised the work and provided the comments and additional scientific information. Z.M.J.A, A.T.J, W.K.A, W.S, M.S.C, and Y.P also reviewed and revised the text. All authors read and approved the final version of the work to be published.

Corresponding authors

Correspondence to Safa Tahmasebi or Mostafa Jarahian.

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Marofi, F., Rahman, H.S., Al-Obaidi, Z.M.J. et al. Novel CAR T therapy is a ray of hope in the treatment of seriously ill AML patients. Stem Cell Res Ther 12, 465 (2021). https://doi.org/10.1186/s13287-021-02420-8
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Novel CAR T therapy is a ray of hope in the treatment of seriously ill AML patients - Stem Cell Research & Therapy

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Acute myeloid leukemia (AML) is a serious, life-threatening, and hardly curable hematological malignancy that affects the myeloid cell progenies and challenges patients of all ages but mostly occurs in adults. Although several therapies are available including chemotherapy, allogeneic hematopoietic stem cell transplantation (alloHSCT), and receptor-antagonist drugs, the 5-year survival of patients is quietly disappointing, less than 30%.