Current challenges and emerging opportunities of CAR-T cell therapies

Journal Pre-proof Current challenges and emerging opportunities of CAR-T cell therapies

Teresa Abreu, Nuno A. Fonseca, Nélio Gonçalves, João Nuno Moreira PII:

S0168-3659(19)30766-7

DOI:

https://doi.org/10.1016/j.jconrel.2019.12.047

Reference:

COREL 10090

To appear in:

Journal of Controlled Release

Received date:

4 October 2019

Revised date:

27 December 2019

Accepted date:

28 December 2019

Please cite this article as: T. Abreu, N.A. Fonseca, N. Gonçalves, et al., Current challenges and emerging opportunities of CAR-T cell therapies, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2019.12.047

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© 2019 Published by Elsevier.

Journal Pre-proof

Current challenges and emerging opportunities of CAR-T cell therapies Teresa Abreua,b, Nuno A. Fonsecaa,c , Nélio Gonçalves a, João Nuno Moreiraa,b*

a

CNC - Center for Neurosciences and Cell Biology, University of Coimbra, Faculty of Medicine (Polo 1), Rua Larga, 3004-504 Coimbra, Portugal. b

FFUC - Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal TREAT U, SA, Parque Industrial de Taveiro, Lote 44, 3045-508 Coimbra, Portugal

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c

E-mail addresses:

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[email protected] (T. Abreu); [email protected] (N.A. Fonseca); [email protected] (N. Gonçalves); [email protected] (J.N. Moreira).

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*Corresponding author:

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João Nuno Moreira, PharmD, MSc, PhD. Center for Neuroscience and Cell Biology University of Coimbra, Faculty of Medicine (Polo I), Rua Larga 3004-504 Coimbra, Portugal. Tel: +351 239820190 E-mail: [email protected]

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Abstract Infusion of chimeric antigen receptor (CAR)-genetically modified T cells (CAR-T cells) have led to remarkable clinical responses and cancer remission in patients suffering from relapsed or refractory B-cell malignancies. This is a new form of adoptive T cell therapy (ACT), whereby the artificial CAR enables the redirection of T cells endogenous antitumor activity towards a predefined tumor-associated antigen, leading to the elimination of a specific tumor. The early success in blood cancers has prompted the US Food and Drug Administration (FDA) to approve the first CAR-T cell therapies for the treatment of CD19-

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positive leukemias and lymphomas in 2017. Despite the emergence of CAR-T cells as one of

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the latest breakthroughs of cancer immunotherapies, their wider application has been hampered by specific life-threatening toxicities, and a substantial lack of efficacy in the

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treatment of solid tumors, owing to the strong immunosuppressive tumor microenvironment and the paucity of reliable tumor-specific targets.

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Herein, besides providing an overview of the emerging CAR-technologies and current clinical applications, the major hurdles of CAR-T cell therapies will be discussed, namely life-threatening

toxicities

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treatment-related

and

the

obstacles

posed

by

the

immunosupressive tumor-microenvironment of solid tumors, as well as the next-generation

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strategies currently designed to simultaneously improve safety and efficacy of CAR-T cell

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Keywords

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therapies in vivo.

CAR-T cells, B-cell malignancies, safety, cytokine release syndrome, neurotoxicity, solid tumors

Abbreviations aAPC ACT

artificial antigen-presenting cell Adoptive T cell therapy

ANG1

angiopoietin-1

ANG2 B-ALL

angiopoietin-2 acute lymphoblastic leukemia

BBB B-NHL CAR

Blood-brain barrier non-Hodgkin’s lymphoma chimeric antigen receptor 2

Journal Pre-proof CRP CRS

C-reactive protein cytokine-release syndrome

CSR CTLA-4 DLBCL

chimeric switch-receptor

EGFR

epidermal growth-factor receptor

GD2 MHC

disialoganglioside Major Histocompatibility Complex

MSLN

mesothelin

PD-1 scFv

programmed cell death protein 1 single-chain variable fragment

sCRS

severe cytokine-release syndrome

TAA TCR

tumor-associated antigen T cell receptor

TME

tumor-microenvironment

1.

Introduction

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cytotoxic T-lymphocyte-associated protein 4 diffuse large B-cell lymphoma

The establishment of immunotherapy (e.i. stimulation of the immune system to recognize

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and kill malignant cells) has shifted the paradigm of cancer treatment [1], which for decades, for some indications, has only relied on conventional methods like surgery, chemotherapy, and radiotherapy [2]. Among the different classes of cancer immunotherapies, including

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checkpoint inhibitor monoclonal antibodies (mAbs) [3], lymphocyte-activating cytokines [4],

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cancer vaccines [5], oncolytic viruses [6] and bispecific antibodies [7], adoptive T cell therapies (ACTs) are emerging as a revolutionary strategy to eliminate tumors [1].

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T cells are the preferred immune cell type for ACTs owing to their endogenous capacity to recognize and kill cancer cells, through the release of cytotoxic granules (e.g. perforin, granzyme) and cytokines (e.g. IL-2, IFNγ), and by triggering death-ligand/death receptormediated cell apoptosis [8]. In the early days, ACTs relied on the transfer of autologous tumor-infiltrating lymphocytes (TILs), isolated from patients resected tumors and further expanded ex-vivo to levels enabling a therapeutic effect [9]. The adoptive transfer of TILs has been

reported as the most effective immune-based treatment for patients with

metastatic melanoma [10], and recent data revealing their ability to target neoantigens in melanoma, further supports this long-recognized clinical benefit [11]. Unfortunately, not all tumors are infiltrated by sufficient numbers of TILs , limiting a wider application to other cancers [12]. This restriction has driven ACT towards the genetic manipulation of peripheral circulating T cells to express antitumor receptors. The first engineered T cells expressed cloned T cell receptors (TCRs), with affinity to a predetermined tumor antigen [13]. Despite being able to target both surface and cytoplasmatic tumor-associated proteins, possibly expanding the repertoire of targetable antigens, TCRs only become activated in the context 3

Journal Pre-proof of major histocompatibility complex (MHC) presentation. As many tumors downregulate the expression of MHC class I molecules to evade immune surveillance, cancer cells are rendered invisible to TCR-mediated recognition [1]. Aiming at overcoming this limitation, artificial chimeric antigen receptors (CARs) have been designed, combining the high-affinity of antibody-derived binding domains with the intracellular signaling domains of TCRs [14]. These novel artificial receptors redirect T cells cytotoxic activity and specificity towards predefined tumor-associated antigens (TAAs), independently from MHC expression [15]. In this context, T cells genetically modified to express CARs (CAR-T cells) specific for the Blymphocyte antigen CD19, have demonstrated unparalleled results in several clinical trials, with complete response rates ranging from 50% to 90% in the treatment of relapsed or refractory (r/r) hematologic B-cell malignancies [16–22]. This unprecedented clinical benefit

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has led the US Food and Drug Administration (FDA) to approve, in the second half of 2017, the first two CAR-T cell-based products, both harboring anti-CD19 CARs [23,24].

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However, despite the unchallenged clinical outcomes of CAR-T cells in the hematooncological field, their activity has been associated with severe side effects, such as the

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cytokine release syndrome and neurotoxicity, while the underlying mechanisms remain unknown [21,25–27]. Moreover, the translation of these therapies from liquid to solid tumors

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has been hampered by the physical barriers and the immunosupressive effects held by the tumor-microenvironment, which severely turns down CAR-T cells activity [28–30]. Therefore, substantial challenges regarding CAR-T cells safety and efficiency (particularly in solid

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tumors) still need to be overcome.

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Herein, a comprehensive overview of the emerging CAR-T cell technologies and their current clinical applications will be provided, as well as the most recently proposed

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mechanisms underlying CAR-T cell-related toxicities, and the next-generation strategies to finely tune CAR-T cells activation and improve safety in vivo. Moreover, the latest progress on CAR-T cells therapies for solid tumors, emphasizing the novel approaches to boost T cells efficacy and persistence at the tumor site will also be discussed.

2. Generations of CARs CARs are hybrid antigen receptors, which in a single molecule, redirect T cells specificity and cytolytic activity towards a predefined TAA , rapidly generating tumor-targeted T cells [15]. In fact, genetically manipulating autologous or allogeneic T cells to express CARs (CAR-T cells), endows T cells with supraphysiologic features, turning them into living therapeutic platforms [31]. In contrast to TCRs, which strictly depend on MHC for antigen presentation, being therefore, restricted to certain human leukocyte antigen (HLA) expression backgrounds, CARs do not require peptide processing for recognizing their target antigens [32]. Once the CAR binds the cognate tumor antigen, the T cell becomes activated, 4

Journal Pre-proof proliferates and kills the target cancer cells, independently from MHC or HLA expression. This makes CAR-T cells suitable for any patient, regardless of their HLA type [33]. The different CAR generations share a common backbone composed of a single polypeptide chain divided into two main moieties: an antibody (Ab)-derived antigen recognition motif, and TCR-derived signaling domains (Fig.1A.) [14]. The CAR extracellular binding domain is generally composed of

single-chain variable fragments (scFvs),

comprising the variable regions of both light and heavy chains from a target-reactive Ab, covalently linked by a flexible linker [14]. Nonetheless, there are alternative binding domains in preclinical evaluation including ligands [34], physiological receptors [35], peptides [36], nanobodies (single domain antibodies (VHHs)) [37] and DARPins (designed ankyrin repeat proteins) [38]. These recognition sequences dictate the CAR specificity and the binding

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affinity to the target antigen.

The extracellular moiety is linked by a flexible hinge fragment to the transmembrane domain, which usually comprises the homodimer of the CD3 or CD8α human molecule

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[14,39]. It has been shown that the length of the CD8α transmembrane sequence, although

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lacking intrinsic signaling function, influences both cytokine production and proliferation of CAR-T cells [40]. In addition, the hinge domain can be conjugated with a spacer to further

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extend the scFv from the T cell’s surface [41]. The optimal spacer’s length depends on the distance between the binding epitope (in the target cell) and the T cell: longer spacers harboring the CH2CH3 domain of the immunoglobulin (Ig) G1 or IgG4 - are required for

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binding proximal epitopes, whereas shorter spacers - harboring solely the CH3 region - are

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required for distal epitopes [42–44]. The proximity between the CAR-T cell and the target cell might be relevant to exclude from the immunological synapse large phosphatases like CD45

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and CD148 capable of inhibiting the phosphorylation cascade triggered upon CAR engagement [45,46].

Currently, all CARs clinically tested, harbor the CD3 protein complex-ζ chain (CD3ζ), from the TCR signal-transduction moiety, as the intracellular signaling domain. The CD3ζ chain is then responsible for activating T cells lytic activity, upon antigen binding [14].

2.1. From the first- to the third-generation CARs of first-generation were dependent on the CD3ζ-mediated signaling for T cell activation, and although CD3ζ chain aggregation was sufficient to trigger T cells effector activity, these constructs have shown limited cytotoxicity owing to the low proliferation and persistence in vivo [47]. Functional augmentation was achieved with the intracellular incorporation of one or two co-stimulatory domains, derived from co-stimulatory receptor families (e.g., CD28, 4-1BB, ICOS or OX40) [48], generating second- and third-generation CARs (Fig.1B.), respectively, with enhanced antitumor efficacy and expansion in vivo [49,50]. 5

Journal Pre-proof The additional co-stimulatory signal enables T cells to elicit a robust cytokine response (i.e. secretion of IL-2 and IFNγ), which increases the cytolytic effect and improves T cell expansion upon repeated antigen exposure [32]. Among the available types of co-stimulatory domains, CD28 and 4-1BB are the most frequently tested. In a study conducted by Zhao et al. comparing both CD28 and 4-1BB tumor control kinetics, 4-1BB expressing CAR-T cells demonstrated longer persistence in vivo maintaining their proliferative capacity even after tumor eradication, making them suitable agents for preventing tumor recurrence [48]. On the other hand, CD28-based CARs have shown higher clonal expansion and IL-2 secretion in early phases of activation, leading to greater tumoricidal activity and faster tumor clearance, thus indicating their potential to induce remission in early stages of cancer [31,48].

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2.2. Fourth-generation CARs

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Fourth-generation CAR-T cells, also referred to as T cells redirected for universal cytokine-mediated killing (TRUCKs), are additionally engineered to constitutively or inducibly

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release transgenic cell products to a targeted-tumor tissue, upon CAR engagement (Fig.1B.) [51,52]. The local delivery of immune-modulating molecules, such as pro-inflammatory

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cytokines (e.g. IL-12, IL-15, IL-18) [52] and enzymes (e.g. heparanase) [53], allows T cells to convert the immunosupressive tumor-microenvironment into an immune-permissive one. One frequently overexpressed cytokine in CAR-T cells is IL-12, a potent molecule that

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enhances T cells secretion of IFNγ, granzyme B and perforin, and allows the recruitment of

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bystander NK cells to eliminate cancer cells not recognized by the CAR [54,55]. IL-12armored CAR-T cells have demonstrated enhanced antitumor efficacy and proliferation in

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several preclinical models, particularly in solid tumors, compared to conventional CARs [56– 58]. A phase I clinical trial using anti-MUC16ecto CAR-T cells secreting IL-12 has been conducted, and although the reported results had demonstrated little clinical benefit, no adverse events were observed [59]. Currently, other ongoing trials are testing the safety and efficacy of armored CAR-T cells secreting IL-12 [60]. An alternative cytokine is IL-18, which is known for inducing T cells to express IFNγ [61]. The transgenic expression of IL-18 has shown to increase T cells cytotoxic activity and expansion in preclinical models [62,63]. In this context, it has recently been reported that IL-18 can polarize CAR-T cells towards Tbethigh FoxO1low (transcription factors) effectors capable of inducing an acute inflammatory response against solid tumors [64,65].

2.3. The novel fifth-generation CAR

More recently, a novel CAR design has been developed, harboring a second-generation CAR backbone with an additional truncated cytoplasmic domain from the IL-2 receptor β6

Journal Pre-proof chain (IL-2Rβ), positioned between the CD3ζ and CD28 signaling domains, and a STAT3/5 (transcription factors)-binding tyrosine-X-X-glutamine motif (YXXQ) at the C terminus of CD3ζ (Fig.1B.) [66]. This fifth-generation CAR aims at triggering, at once, all three signals TCR (CD3ζ domain), co-stimulatory (CD28 domain) and cytokine (through the activation of JAK kinase and STAT3/5 transcription factors signaling pathways) - that are physiologically required for optimal T cell activation, in an antigen-dependent fashion. The authors have demonstrated, in both liquid and solid tumor models, that engineering CAR-T cells with

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cytokine-encoding genes enhanced their proliferation, survival and antitumor activity,

compared to conventional second-generation CAR-T cells [66]. Fig. 1. (double column figure) Schematic representation of the CAR structure and evolution of CAR generations. (A) CARs are hybrid receptors comprising an antibody-derived extracellular binding domain, generally in the form of a single-chain variable fragment, a transmembrane domain fused to a hinge fragment and an intracellular signaling domain responsible for T cells activation. The structure represents a second-generation CAR. (B) First-generation CARs only contained a CD3ζ chain for T cell activation in the intracellular domain. CARs from second- and third-generations harbor one and two additional co-stimulatory domains (CD), respectively, in the intracellular domain. Fourth-generation CARs are additionally engineered to constitutively or inducibly express transgenic products, as cytokines. The fifth-generation CAR is based on a second-generation CAR with an additional cytoplasmic domain derived from IL-2Rβ and a STAT3/5 binding motif, providing antigen-dependent cytokine signaling.

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3. Manufacturing The first step to generate CAR-T cells comprises the collection of unselected peripheral blood mononuclear cells (PBMC), either autologous or allogeneic, by leukapheresis [67]. The resulting product is then enriched in CD3-positive T cells, for example, through counterflow centrifugal elutriation, where cells are separated by size and density [68]. After purification, T cell activation and expansion can be accomplished by artificial antigen-presenting cell (aAPC) systems, typically consisting of magnetic beads coated with anti-CD3/CD28

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activating mAbs (CD3/CD28 Dynabeads), and further supplemented with exogenous IL-2 in

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the culture medium [69]. Other ex-vivo methods for T cell expansion include the use of aAPCs derived from engineering the K562 erythromyeloid cell line to express T cells’ co-

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stimulatory ligands [70]. Recently, an innovative expansion method has been described, in which a three-dimensional scaffold of mesoporous silica microrods, coated with supported

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lipid bilayers, was functionalized with T-cell activation cues, namely CD3 and CD28 Abderived agonists [71,72]. This APC-mimetic scaffold promoted higher polyclonal and antigen-

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specific expansion of T cells in comparison with conventional CD3/CD28 Dynabeads [71]. After expansion and activation, T cells are subsequently engineered to express the CAR

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transgene. Most clinical trials testing CAR-T cells, use ex-vivo genetic engineering techniques. T cells are transduced with retroviral vectors, mainly gamma-retroviral or

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lentiviral vectors, enabling a permanent genomic integration and thus long-term CAR expression [73]. Alternative methods of

non-viral

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electroporation

plasmids

to viral-mediated CAR transduction, include based

on

the

Sleeping

Beauty

(SB)

transposon/transposase system [74]. Briefly, the incorporation of the CAR transcript in the electrotransferred SB transposon into adenine/thymine dinucleotide base pairs is mediated by a SB transposase from a second DNA plasmid [75]. This strategy is currently under clinical investigation for the treatment of CD19-positive B cell malignancies (NCT00968760) [76]. Non-integrative gene transfer techniques based on mRNA transfection have also been applied for transient CAR expression. As the transgene is rapidly lost, these approaches require multiple CAR-T cell infusions, to achieve a clinical effect [77]. In situ strategies for targeting and genetically modifying T cells in vivo have been recently developed. For instance, the use of polymeric-based DNA nanocarriers, functionalized with anti-CD3 antibody fragments to target circulating T cells has been tested [78]. The intravenous administration of the CAR-encoding nanoparticles in a mouse model of B-cell acute lymphoblastic leukemia enabled tumor eradication in seven out of ten mice. It is noteworthy that the antitumor efficacy was comparable to that observed with conventional adoptive T-cell therapy, based on ex-vivo lentiviral transduction [78]. Agarwal et al. have 8

Journal Pre-proof generated CAR-T cells in vivo by specifically targeting lentiviral vectors to human CD8positive T cells [79]. A single intravenous administration of CD8-targeted lentivirus encoding anti-CD19 CARs in a humanized mouse model of acute lymphoblastic leukemia enabled the complete elimination of the tumor cells from bone marrow and spleen [79].

4. Current clinical applications The earliest clinical trials of CAR-T cells in cancer, which involved patients suffering from late-stage solid tumors, namely advanced epithelial ovarian cancer [80] and metastatic renal cell carcinoma [30] have reported little therapeutic benefit and significant toxicity. In fact, the

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breakthrough of CAR-T cell-based immunotherapies was achieved with anti-CD19

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autologous CAR-T cells demonstrating significant antitumor activity against CD19-positive Bcell leukemias and lymphomas [17]. The CD19 antigen, an activating receptor required for

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normal B-cell development, was chosen as the initial target for B-cell malignancies due to its frequency, homogeneity and high-level expression in cancer cells [31]. It is noteworthy that,

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since CD19 expression is restricted to the B-cell lineage, healthy cells are also eliminated upon CAR activation, inducing profound B-cell aplasia in patients successfully treated with

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anti-CD19 CAR-T cell therapies [81]. Nonetheless, this on-target off-tumor effect is not lethal (elimination of non-vital B cells) and is clinically manageable with periodic infusions of pooled immunoglobulin [26], supporting CD19 as a nearly ideal target for CD19-positive blood

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cancers.

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Regarding the clinical evaluation of anti-CD19 CAR-T cells in the treatment of r/r B-cell leukemias, patients with acute lymphoblastic leukemia (r/r B-ALL) [19–22,24] and non-

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Hodgkin’s lymphomas (r/r B-NHL) [18], mainly diffuse large B-cell lymphoma (DLBCL) [16], appear to benefit the most from these therapies, reaching high remission rates ranging from 50% to 90%. Conversely, patients with chronic lymphocytic leukemia (r/r B-CLL) have shown mixed clinical responses to anti-CD19 CAR-T cells [82,83]. Additionally, anti-CD19 CAR-T cells, following autologous stem cell transplantation, are also being studied against multiple myeloma, as reported by a recently concluded phase I pilot trial (NCT02135406) [84,85], where the results revealed that anti-CD19 CAR-T cells may improve the duration of response to standard therapies [85]. In CD19-negative hematological cancers and in cases of CD19 loss or downregulation, due to: (1) induction of host immunity against the CAR transgene [31]; (2) generation of CD19-negative B-cell variants upon anti-CD19 CAR treatment pressure [86]; or (3) tumorinduced resistance mechanisms, such as lineage switch [87]; anti-CD19 CAR-T cells are no longer active and the disease relapses, followed by the loss of B-cell aplasia [21]. To overcome these challenges, alternative targets to CD19 for B-cell malignancies, such as CD22 [88], CD20 (NCT03576807, NCT03664635), Igκ (immunoglobulin kappa chain) [89] 9

Journal Pre-proof and ROR1 (tyrosine-protein kinase transmembrane receptor) (NCT02706392) are currently in preclinical and clinical evaluation . For example, CAR-T cells targeted towards CD22, a Bcell antigen whose expression remains after CD19 loss, have induced a remarkable complete remission rate (73%) in patients with r/r B-ALL, including patients previously treated with anti-CD19 immunotherapy, as reported in a phase I trial (NCT02315612) [88]. This was the first trial to reveal the significant clinical efficacy of anti-CD22 CARs in the treatment of r/r B-ALL, with comparable antileukemic activity and safety profile to that of anti-CD19 CARs [88]. For the treatment of other hematological cancers, such as acute myeloid leukemia (AML) and multiple myeloma, the most studied targets in clinical trials are CD123 [90], CD33 [91] and CD7 [92] for the first; BCMA (B-cell maturation antigen) [93] and CD138 [94] for the

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second.

Finally, one key component of ACTs, which has been widely applied in CAR-T cell trials is

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the administration of lymphodepletion regimens containing immunosuppressive cytotoxic agents, such as cyclophosphamide and fludarabine, before the adoptive T cell transfer

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[10,12,19,95,96]. It has been demonstrated that prior lymphodepletion enhances CAR-T cells effectiveness and persistence in vivo, through depletion of endogenous lymphocytes and

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immunosuppressive cells from the blood circulation and further promoting the homeostatic expansion of the transduced T cells [12,33].

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5. Translation of CAR-T cell technologies

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5.1. First-in-class CAR-T cell technologies

In August 2017, FDA approved the first CAR-T cell therapy, Tisagenlecleucel, marketed by Novartis as KYMRIAHTM, for the treatment of r/r B-ALL in children and young adults (up to 25 years of age) [97,98]. Tisagenlecleucel (CTL019), an anti-CD19 CAR-T cell product of single infusion, enables the lentiviral transduction of each patient autologous T cells with a gene encoding a second-generation anti-CD19 CAR harboring a 4-1BB co-stimulatory domain [24]. The approval was clinically supported by the multi-center, phase II ELIANA trial (NCT02435849), the first global study to evaluate anti-CD19 CAR-T cells efficacy and safety in children and young adults with r/r B-ALL. The results reported an impressive overall remission rate of 81% (75 patients infused) and a bloodstream persistence of 20 months [24]. This unprecedented success has prompted the investigation of CTL019 efficacy in the treatment of other B-cell malignancies, and during the last year, KYMRIAHTM was also approved by the FDA for the treatment of adult patients with r/r B-NHL, including DLBCL, high-grade B-cell lymphoma and DLBCL arising from follicular lymphoma [99,100]. The primary clinical evidence was provided by the international phase II JULIET trial 10

Journal Pre-proof (NCT02445248) [101]. The results reported an overall response rate of 52%, including 40% of complete responses, and no deaths were attributed to tisagenlecleucel, cytokine release syndrome, or neurologic events [101]. Nearly a year after the first approval in US, KYMRIAHTM was authorized for use in the European Union in its both therapeutic indications (r/r B-ALL and r/r DLBCL) [102]. Another first-in-class to receive FDA approval was Axicabtagene ciloleucel, the first antiCD19 CAR-T cell product to be approved for the treatment of adult patients with aggressive r/r B-NHL, including r/r DLBCL, transformed follicular lymphoma and primary mediastinal Bcell lymphoma [103]. Axicabtagene ciloleucel (AXI-CEL/KTE-C19) marketed by Kite, a Gilead Company as YESCARTATM, is a single dose CD19 CAR-T cell immunotherapy, whereby autologous T cells are retrovirally transduced to express a CD3ζ/CD28 second-generation

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CAR [23,103]. KTE-C19 approval was clinically supported by the multicenter, phase II ZUMA-1 trial (NCT02348216) which reported an objective response rate of 82%, with 54% of complete responses among the 111 patients enrolled [23]. More clinical trials testing

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(see detailed description in Table 1) [104].

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YESCARTATM’s safety and efficacy in other hematological cancers are currently ongoing

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Table 1. Examples of Axicabtagene ciloleucel’s clinical trials.

ZUMA-6

r/r DLBCL*

ZUMA-7

r/r DLBCL**

ZUMA-11

r/r DLBCL***

ZUMA-12

r/r DLBCL****

ZUMA-2

r/r MCL

ZUMA-3

adult r/r B-ALL

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Axicabtagene ciloleucel (AXI-CEL)

r/r Indolent NHL

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ZUMA-5

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Indication

KTE-X19 (formerly KTE-C19)

ZUMA-4

pediatric r/r B-ALL

ZUMA-8

r/r CLL

Status

ClinicalTrials.gov Referen ce number

Phase II, recruiting

NCT03105336

Phase II, active not recruiting

NCT02926833

Phase III, recruiting Phase I/II, recruiting Phase II, recruiting Phase II, active not recruiting

NCT03391466 NCT03704298 NCT03761056 NCT02601313 NCT02614066

Phase I/II, recruiting

NCT02625480 NCT03624036

* A

XI-CEL in combination with Atezolizumb (anti-PD-L1 mAb); ** AXI-CEL versus standard of care therapy; *** AXI-CEL in combination with Utomilumab (4-1BB/CD137 agonist); **** AXI-CEL as first-line therapy; DLBCL - diffuse large B cell lymphma; MCL - mantle cell lymphoma; ALL - acute lymphoblastic leukemia; NHL - non-Hodgkin lymphoma; CLL - chronic lymphocytic leukemia.

5.2. Emerging CAR-T cell products

The approval by the FDA of the first genetically modified autologous T cell immunotherapies using anti-CD19 CARs, has propelled research in this technology. 11

Journal Pre-proof Innovative CAR-T cell-based products are currently in preclinical and clinical stage for the treatment of both CD19-positive leukemias and other human cancers. One significant example is Cellectis leading candidate UCART19 (Universal Chimeric Antigen Receptor Tcells), an off-the-shelf product based on the genetic manipulation of allogeneic T cells, collected from non-HLA matched healthy donors [105]. In order to avoid potential complications of cellular rejection and graft-versus-host disease (GVHD) [106], CARtransduced allogeneic T cells are additionally disrupted from immunological key genes through the gene-editing technology TALEN® (activator-like effector nuclease), a restriction enzyme complex harboring a TALE (transcription activator-like effector)-derived binding domain [107]. UCART19 cells have already been applied in a clinical trial to treat r/r B-ALL, whereby allogeneic CAR-transduced T cells were gene-edited via TALEN to disrupt the

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expression of endogenous surface TCRs and CD52, in order to minimize the risk of GVHD and allow the administration of Alemtuzumab (anti-CD52) as pre-conditioning therapy [108].

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The results were promising with the only two infants treated achieving complete molecular remission. Currently, Cellectis UCART technology is in clinical investigation for CD19- and

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CD123-directed CARs (Table 2).

Despite the disappointing results reported from most clinical trials testing CAR-T cells

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against solid tumors, the emergence of new promising candidates is increasing. A significant example is Mustang Bio’s flagship product MB-101, a 4-1BB-based CAR-T cell targeting the IL-13 receptor alpha 2 (IL13Rα2), which is a glioma-specific antigenrelated with low survival

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rates [109]. In a clinical trial using MB-101 to treat a patient suffering from recurrent

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multifocal glioblastoma (NCT02208362), the results revealed a dramatic clinical response, with regression of all intracranial and spinal metastatic tumors sustained for more than 7

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months after the first infusion of anti-IL13Rα2 CAR-T cells [110]. However, despite the encouraging outcomes, the patient eventually experienced tumor recurrence, probably due to the downregulation of IL13Rα2 expression [110]. Besides MB-101, other Mustang Bio’s promising candidates are in clinical evaluation for the treatment of multiple solid cancers (Table 2).

Another innovative example is Celyad’s leading technologies, CYAD-01 and CYAD-101, based on the genetic manipulation of autologous and allogeneic T cells, respectively, to express non-conventional CARs harboring a natural killer group 2D (NKG2D) activating receptor (expressed on NK cells), as the antigen-binding domain [111]. These NKG2D-based CARs have the advantage to: (1) bind various stress-inducible ligands (NKG2D ligands), which are known to be overexpressed in a broad panel of liquid and solid tumors, but not on normal cells; and to (2) physiologically provide co-stimulatory signals by association with the native co-stimulatory molecule DAP10 (adaptor molecule DNAX-activating protein of 10 kDa), thus recapitulating the function of a second-generation CAR [112,113]. Both CYAD-01 and CYAD-101 products are currently being evaluated in phase I trials for the treatment of 12

Journal Pre-proof several hematological and solid tumors, including metastatic colorectal cancer (Table 2) [111,114].

Table 2. Emerging CAR-T cell-based candidate products. Company

CARtechnology

Indication

Target

Status

NCT02808442 (PALL)

adult r/r B-ALL

UCART22

adult B-ALL

UCARTCS1 UCARTCLL1

IND

MM

CS1

IND

AML

CLL1

Preclinical

r/r B-NHL

CD19

Phase I/II clinical trial

MM

BCMA

IND

FLT3

Preclinical

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ALLO-715*** ALLO-819***

AML

r/r malignant glioblastoma r/r AML and BPDCN glioblastoma multiforme MM prostate, pancreatic, gastric and bladder cancers

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MB-101

CD22

e-

ALLO-501**

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MB-102 MB-103 MB-104 MB-105 MB-106

r/r B-NHL

Hematological and solid tumors

NCT02735083 NCT02746952 (CALM) NCT03190278 (AML123)

----

NCT03939026 (ALPHA) ----

IL-13Rα2

NCT02208362

CD123

NCT02159495

HER2

Phase I clinical trial

NCT03696030

CS1

NCT03710421

PSCA

NCT03873805

CD20

r/r AML/MDS

Mustang Bio

Phase I Clinical trial

CD123

pr

AML

Pr

Cellectis

UCART123

CD19

oo

Advanced lymphoide malignancies*

f

pediatric r/r B-ALL UCART19

ClinicalTrials.gov Reference number

Phase I/II clinical trial Phase I/II clinical trial

CYAD-101

mCRC

CYAD-211

MM

BCMA

CYAD-221

B cell malignancies

CD19

CYAD-231

undisclosed

NKG2D

NCT03466320 (DEPLETHINK) NCT03018405

(THINK)

NKG2D

mCRC

NCT03277729

Phase I clinical trial

NCT03310008 (SHRINK) NCT03692429 (AlloSHRINK)

Preclinical

----

13

Journal Pre-proof *Previously exposed to UCART19/ALLO-501; **collaboration between Servier and Allogene; ***licensed to Allogene; IND - Investigational new drug by FDA; AML - Acute myeloid leukemia; MM - Multiple myeloma; BPDCN - Blastic plasmacytoid dendritic cell neoplasm; MDS - Myelodysplastic syndromes; mCRC - Metastatic colorectal cancer; CLL1 C-type lectin-like molecule-1; BCMA - B-cell maturation antigen; FLT3 - FMS-like tyrosine kinase 3 ; HER2 - human epidermal growth factor receptor 2; PSCA - prostate stem cell antigen; NKG2D - natural killer group 2D activating receptor.

6. The underlying mechanisms of CAR-T cells toxicity

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Despite the outstanding responses reported in phase I/II clinical trials using CAR-T cells

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for r/r CD19-positive B-cell cancers, these therapies have been associated with unique toxicities, which can be severe or even lethal [81,115]. Several clinical studies, including the

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aforementioned ELIANA, JULIET and ZUMA-1 trials, have reported grade 3 and grade 4 adverse events, namely cytokine release syndrome, febrile neutropenia, infection and

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neurologic events [23,24,100,101]. For instance, in the ELIANA trial two patients succumbed to grade 4 neurotoxicity, one to encephalopathy and the other to delirium [24]. In fact,

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neurotoxicity and cytokine-release syndrome are reported as the two most-common lifethreatening toxicities related to CAR-T cells, mainly with anti-CD19 therapies [27].

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Conversely, the occurrence of on-target, off-tumor effects in the form of B-cell aplasia on hematological malignancies is clinically manageable, having no lethal effect on patient’s

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health. Nevertheless, off-tumor toxicities have a significant impact on CAR-T cell therapies for solid tumors, as it will be further discussed. Therefore, the following topics will only refer

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to cytokine-release syndrome and neurotoxicity, addressing the most recently proposed mechanisms for both toxicities.

6.1. Cytokine-release syndrome 6.1.1. Clinical features The most frequently reported toxicity associated with CAR-T cell therapies is the cytokinerelease syndrome (CRS) [27,81,115,116]. The CRS, occurring early after the infusion, is related with CAR-T cells supraphysiologic activation and expansion in vivo, upon target recognition, and subsequent release of high levels of inflammatory cytokines from both activated T cells and myeloid-derived cells (e.g., macrophages, dendritic cells and monocytes), resulting in a systemic inflammatory response [116,117]. It is noteworthy that this syndrome has also been observed upon treatment with other targeted-T cell therapies,

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Journal Pre-proof such as bispecific T-cell-engaging antibodies (BiTEs), namely the anti-CD19/CD3 BiTE, blinatumomab [7]. CAR-T cell-mediated CRS has been associated with peak levels of several serum cytokines, including IFNγ, TNFα (tumor necrosis factor-α), IL-2, IL-6, IL-10, ferritin, and CRP (C-reactive

protein),

which

is

released

by

hepatocytes

in

response

to

IL-6

[19,24,81,115,118,119]. In a clinical study, the cytokines and clinical biomarkers from 51 CTL019-treated patients were measured, and serum elevations of specific cytokines, namely IFNγ, IL-8, sIL-2Rα (soluble IL-2 receptor-α), IL-6, sIL-6R (soluble IL-6 receptor), sgp130 (serum

glycoprotein

130),

MCP1

(monocyte

chemoattractant

protein 1), MIP-1α

(macrophage inflammatory protein 1α), MIP-1β (macrophage inflammatory protein 1β) and GM-CSF (granulocyte-macrophage colony-stimulating factor), in the first month of treatment

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were associated with severe CRS (sCRS) [119]. In fact, early peaks of IFNγ, IL6, sIL-6R, sgp130 and sIL-1RA were considered as predictive markers for the development of sCRS

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[119,120]. CRP has also been considered a reliable indicator of CRS’s onset and severity in other clinical studies [115,121].

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Beyond the cytokine signature, CAR-T cells peak expansion further promoted by prior lymphodepletion regimen, and higher tumor burden at the time of the infusion are other two

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recognized factors associated with high-grade CRS [19,115,121,122]. The symptoms can range from mild to severe, including high fevers, hypotension, tachycardia, hypoxia, fatigue, and life-threatening events such as disseminated intravascular coagulation and multi-organ

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insufficiency [26,27,118].

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dysfunction, namely hepatoxicity, renal failure and respiratory and cardiovascular

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6.1.2. CRS’s pathophysiology: the key role of IL-6 and IL-1 Despite the unknown pathogenesis of CRS, emerging evidence highlights the central role of IL-6 [119], whose physiological function is to bind either sIL-6R (soluble) or IL-6 receptor (IL-6R) at the cell-membrane and subsequently induce the intracellular activation of the JAK/STAT signaling pathway [123]. In fact, it has been consistently reported in several trials that peak serum concentrations of IL-6 upon infusion of anti-CD19 CAR-T cells correlates with the occurrence of sCRS [18,21,115]. The role of IL-6 is further supported by the clinical efficacy of the anti-IL-6R mAb tocilizumab, that blocks IL-6 from binding both IL-6R and sIL6R [118]. Tocilizumab has been established as first line therapy for the treatment of CRS, since it readily reverses the symptomatology without preventing CAR-T cells activity, in contrast to systemic corticosteroids (second-line therapy), which protracted use has shown to abrogate CAR-T cells antitumor efficacy and proliferation [20,27,118]. It remains unclear, though, whether the main sources of IL-6 during sCRS are activated CAR-T cells or other immune bystander cells instead [120]. 15

Journal Pre-proof Recently, preclinical studies using humanized murine models of sCRS have provided new mechanistic insights into the pathophysiology of CRS, highlighting the contribution of hostderived myeloid cells to this syndrome. According to some authors, monocytes are the major source of IL-6 during CRS, rather than CAR-T cells [120,122]. In a study conducted by Norelli et al., the depletion of circulating monocytes from mice before anti-CD19 CAR-T cell treatment enabled the complete suppression of CRS incidence and mortality [122]. Surprisingly, pre-depleting monocytes also led to a substantial decrease in CAR-T cells expansion in vivo, suggesting that besides being primarily responsible for CRS, these myeloid-derived cells might have an important role in CAR-T cells antitumor efficacy. Moreover, a time-course analysis revealed that IL-1, a monocyte-derived cytokine, was released 24 hours before IL-6. Since IL-1 is known to induce IL-6 secretion, the release of IL-

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1 from circulating monocytes, already activated by CAR-T cells, might be the first responsible for CRS’s onset [122]. The hypothesis was further supported by CRS responsiveness to the

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IL-1 receptor antagonist, anakinra, which was as effective as tocilizumab in preventing CRS mortality. Unexpectedly, anakinra also provided protection against post-CRS lethal

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neurotoxicity, suggesting that IL-1 might be a key-factor in both toxicities and a possible target for alternative pharmacological approaches [122].

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On the other hand, Giavridis et al. have demonstrated that macrophages, rather than monocytes, are the main source of IL-6 during CRS [124]. According to the study, the colocalization of both CAR-T cells and myeloid-derived cells at the tumor site was required for

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the secretion of IL-6 and other proinflammatory cytokines, including IL-1, that were ultimately

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responsible for inducing CRS [124]. This suggests that the occurrence of CRS in CAR-T cell therapies might be related tothe extension of myeloid infiltration at the tumor, rather than

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strictly depending on CAR-T cells supra-activation. Another interesting finding was the direct involvement of macrophage-derived iNOS (inducible nitric oxide synthase) in CRS pathophysiology. When activated by IL-6 and IL-1, macrophages produced high levels of iNOS, known to induce abnormal NO secretion, which in turn led to vasodilation and hypotension, both clinical features of CRS [124]. The administration of anakinra enabled the downregulation of iNOS levels and subsequent prevention of CRS mortality, once again highlighting IL-1 as a suitable target to tackle CRS without compromising CAR-T cells activity [124].

6.2. Neurotoxicity 6.2.1. Clinical features Neurotoxicity, also named CAR‑ T cell‑ related encephalopathy syndrome, is the second most frequent life-threatening event associated with CAR-T cell therapies, generally occurring during or following sCRS [27]. The most frequently reported symptoms associated 16

Journal Pre-proof with neurotoxicity are confusion, aphasia, delirium, disorientation, somnolence, tremors, language disturbance, ataxia, and in severe cases, patients might develop seizures and cerebral edema [16,18,23,24,101,125]. Despite the recognized correlation between highgrade CRS and neurotoxicity, since patients with sCRS onset shortly after CAR-T cells infusion are at higher risk to subsequently develop severe neurotoxicity [21,115], the mechanisms underlying this lethal effect remain poorly understood. Recently, a study was conducted aimed at better characterizing neurotoxicity pathophysiology and identifying risk factors with predictive value for neurotoxicity [25]. The neurologic events of 133 patients treated with anti-CD19 CAR-T cells revealed that 53 patients had at least one grade 1 neurologic event within the first month of treatment. The symptoms reported were in agreement with other trials [16,18,24], and although these were

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reversible for the majority of patients, 4 patients died due to cerebral edema (2), brainstem hemorrhage (1) and cortical laminar necrosis (1) [25]. According to a multivariable analysis,

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baseline characteristics as higher disease burden, higher infused dose of CAR-T cells, preexistence of neurologic comorbidities and a cyclophosphamide/fludarabine-based pre-

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lymphodepletion regimen, which increases CAR-T cells expansion in vivo, were considered risk factors for developing neurotoxicity. Not surprisingly, patients who experienced sCRS

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with an early high fever onset after CAR-T cells infusion, subsequently developed severe neurotoxicity [25].

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6.2.2. Neurotoxicity’s pathophysiology: the emerging role of endothelial cells Similarly to sCRS, high serum levels of CRP, ferritin, INFϒ, TNFα and peak

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concentrations of IL-6 within the first days of treatment were associated with higher risk of severe neurotoxicity [25]. The results indicated that CAR-T cells activation and further release of high levels of serum cytokines, including those that activate endothelial cells (e.g. IL-6, INFϒ and TNFα), induced a systemic state of endothelial activation and vascular dysfunction that extended to the CNS. Besides the clinical evidence of vascular leak and enhanced endothelial permeability in patients with severe neurotoxicity, the widespread endothelial activation was further confirmed by alterations in the serum levels of endothelial cell (EC) regulators, such as increased levels of ANG2 (angiopoietin-2), low ratios of ANG1 (angiopoietin-1) to ANG2, and elevated concentrations of the VWF (von Willebrand factor) [25,126]. The increased blood-brain barrier (BBB) permeability enabled the transit into the cerebrospinal fluid (CSF) of CAR-T cells and inflammatory cytokines, such as INFϒ and TNFα, that activated brain vascular pericytes to secrete high amounts of IL-6, further enhancing ECs activation and BBB permeability [25]. Once in the CNS, CAR-T cells maintain this loop of continued EC and pericyte activation that ultimately results in lethal BBB 17

Journal Pre-proof disruption, as observed in the autopsies of patients who succumbed to severe neurotoxicity [126]. In this plausible mechanism, early EC activation is neurotoxicity’s main triggering factor. Accordingly, patients with (1) preexisting neurologic comorbidities, (2) elevated ANG2:ANG1 ratios prior to the treatment and (3) early rises in ANG2 levels right after CAR-T cell infusion, might be at higher risk of developing severe CAR-related neurotoxicity, and should be given a lower dose of CAR-T cells [25]. Since the occurrence of sCRS is associated with increased risk of subsequent severe neurotoxicity, CRS management with tocilizumab and corticosteroids has been the first strategy to prevent this lethal effect [25,122]. However, although highly effective in reverting CRS, tocilizumab fails to prevent subsequent neurotoxicity. In fact, as tocilizumab is not able to penetrate the BBB, the peripheral blockade of IL-6R might increase CSF-IL-6 levels and

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transiently aggravate neurotoxicity [118]. For this reason and given the central role of the vascular endothelium in this model, strategies to stabilize ECs and prevent BBB disruption

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might be more relevant to protect patients from neurotoxicity. For instance, normalizing the ANG1/ANG2 ratio with BowANG1 (recombinant human ANG1 protein) [127], platelets

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hypertransfusion to augment ANG1 levels, or exchanging plasma to deplete inflammatory cytokines could potentially be tested in a clinical context [25]. Another noteworthy point is the

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apparent higher frequency of severe neurotoxicity in patients treated with immunotherapies targeting CD19 compared to other antigens, as also reported in clinical trials using blinatumomab [7]. This indicates the ability of anti-CD19 immunotherapies to elicit robust T

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cell activation and expansion in vivo.

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7. Strategies to improve CAR-T cells safety and efficacy As discussed above, managing CAR-related life-threatening toxicities is one of the key hurdels of CAR-T cell therapies. In fact, improving safety and controlling CAR-T cells supraphysiologic activity is of utmost importance, owing to the CAR-associated deaths reported in clinical trials. As living therapeutic platforms, CAR-T cells are capable of intelligent sensing and response behaviors, which make them difficult to control once infused into patients [31]. The need for better safety outcomes has led to the development of innovative control strategies, such as, switch-based control systems, combinatorial antigen recognition approaches and CARs sensitive to microenvironmental stimuli [41].

7.1. Switch-based control In the switch-based control group, CAR-T cells activity is controlled upon the addition of an exogenous switch molecule that can either induce T cells suppression, by elimination 18

Journal Pre-proof switches, or activate T cells cytotoxicity, through the design of ON-switch CAR-systems or bifunctional intermediate switches.

7.1.1. Elimination switches The incorporation of a suicide switch (“OFF-switch”) into the CAR-encoding vector as long been recognized as a reliable strategy to rapidly eliminate the transfused T cells by apoptosis, upon the occurrence of undesired toxicities [31]. Currently, the most frequently used suicide switch is the inducible caspase-9 (iCasp9) system, where the CASP9 gene encodes for a modified split form of the human caspase-9 apoptotic protein, that is activated by a small dimerizing drug (AP1903), added upon a toxic event specifically related to CAR-T

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cells activity [128]. CAR-T cells expressing the iCasp9 suicide system are already in clinical

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evaluation for the treatment of neuroblastoma (NCT01822652) and sarcoma (NCT01953900) with anti-GD2 (disialoganglioside) CARs [129,130].

Alternative switch-based elimination

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strategies include CAR-T cells expressing epitope tags that are recognized by clinically approved mAbs [41]. For instance, CAR-T cells expressing the truncated human epidermal

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growth-factor receptor (huEGFRt) devoid of both N-terminal extracellular binding- and intracellular signaling domains, can be targeted by cetuximab, an anti-EGFR mAb, which

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induces T cell death by endogenous antibody-dependent cellular cytotoxicity (ADCC) [131]. Although specifically eliminating transduced T cells with minimal immunogenic potential,

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cancer recurrence.

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these suicide systems might lead to irreversible elimination of therapeutic CAR-T cells and

7.1.2. ON-switch CARs

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In contrast to suicide switches that induce irreversible suppression of CAR-T cells functions, strategies based on ON-switch systems provide positive control of CAR-T cells activity, enabling the precise regulation of CAR-T cells activation. A clear example of an ONswitch CAR is the splitCAR in which the antigen-binding domain is dissociated from the signaling motif, or otherwise, the CD3ζ chain is segregated from the CD28/4-1BB costimulatory domain in the intracellular region [132]. T cells transduced with splitCARs only become fully activated (e.i. lytic activity and proliferative potential) upon: (1) engagement with the target antigen and (2) addition of a dimerizing agent capable of assembling the splited domains [132]. The application of this technology would ultimately allow physicians to remotely control the timing and the strength of CAR-T cells cytolytic response by titrating the concentration of the exogenous chemical inducer [31].

7.1.3. Bifunctional switches

19

Journal Pre-proof A different approach relies on the use of bispecific molecules as intermediate bifunctional switches capable of recruiting CAR-T cells to the tumor site by simultaneously targeting specific markers on T cells and TAAs [133]. An established clinical application of this technology is blinatumomab, capable of inducing immune antitumor responses by directly engaging CD3-positive, non-engineered T cells with CD19-expressing tumor cells [7]. A different form of the bifunctional molecule is the folate-fluorescein isothiocyanate conjugate (folate-FITC) capable of targeting anti-FITC CAR-T cells towards FR (folate receptor)positive cancer cells [134]. Recently, Lu et al. (2019) have demonstrated that the administration of folate-targeted FITC conjugate (referred to as EC17 CAM) was critical to trigger anti-FITC CAR-T cells activation, proliferation, and persistence against FR-positive hematologic and solid tumors [135]. The promising preclinical results are paving the way for

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clinical investigation, as exemplified by Endocyte’s anti-FITC CAR technology, which is progressing towards clinical evaluation [136].

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A more innovative approach uses peptide-specific switchable CAR-T cells (sCAR-T) directed to a peptide neo-epitope (PNE) molecule coupled with a TAA-specific Fab. Instead

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of recognizing an antigen, sCAR-T cells specifically bind the PNE/Ab-based switch (e.g. PNE-CD19, PNE-CD20), allowing to temporally control the interactions between T cells and

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target cells, in a dose-titratable manner, lowering the risk of excessive CAR activation [137]. For instance, in a preclinical study, sCAR-T cells specific for a switch targeted to HER2 (human epidermal growth factor receptor 2), induced complete remission in patient-derived

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pancreatic tumor models, demonstrating the efficacy of this switchable CAR system to treat

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aggressive and disseminated tumors [138]. In a different perspective, both anti-FITC and sCARs can be classified as universal

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immune receptors (UIRs), more specifically as tag-specific UIRs, in which the extracellular domain binds an intermediate adaptor molecule instead of a TAA [139]. In fact, the development of universal CAR designs is a promising strategy to simultaneously improve safety and efficacy of CAR-T cell therapies. One significant example is the highly innovative tag-specific UIR named SUPRA (split, universal, and programmable) CAR recently developed by Cho et al. [140]. The SUPRA CAR is a slipt receptor system harboring both an universal receptor (zipCAR) expressed on T cells and an independent tumor-targeting scFv molecule (zipFv). Briefly, the zipCAR comprises the CAR intracellular signaling domains fused with an extracellular leucin zipper adaptor, which binds the cognate leucine zipper on the scFv molecule, within the zipFv [140]. After zipFv-mediated recognition of the target antigen, the leucine zippers pair and activate the zipCAR, triggering T cells antitumor activity. Impressive advantages arise from this technology, such as: (1) the ability to target multiple tumor-antigens without genetically re-engineering T cells; (2) the controllability of SUPRA CAR activity levels by adjusting the zipFvs dose and the affinity between both leucine zippers by varying their configurations; and (3) the possibility to express multiple orthogonal zipCARs 20

Journal Pre-proof in the same T cell, each one containing a distinct intracellular domain, enabling the tuning of different signaling pathways through combinatorial antigen recognition [140,141]. Therefore, the SUPRA CAR arises as a programmable split system able to finely tune, at once, multiple parameters of CAR-T cells activity simultaneously increasing their efficacy, specificity and safety.

7.2. Combinatorial antigen recognition To avoid potential on-target, off-tumor toxicities, combinatorial strategies, based on NOTand AND-gate circuits, have been developed to increase T cells specificity and subsequent

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ability to discriminate target cells from bystander cells [31].

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7.2.1. NOT-gate circuits

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In NOT-gate circuits CAR-T cells are able to override self-activation upon binding to a ‘negative antigen’ present on non-cancer cells [31]. For instance, transducing CAR-T cells

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with an additional inhibitory CAR (iCAR), comprising an extracellular binding domain specific for a healthy tissue antigen fused to an intracellular domain derived from co-inhibitory

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receptors, such as PD-1 (programmed cell death protein 1) and CTLA-4 (cytotoxic Tlymphocyte-associated protein 4), enables suppression of their activity whenever they encounter a non-targeted cell [142]. Briefly, once the PD-1-/CTLA-4-based iCAR binds to a

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non-malignant cell, it initiates an inhibitory signaling cascade that blocks CAR-T cells

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activation and further cytotoxic activity [142]. These NOT-gated CAR-T cells are thus able to distinguish cancer cells from normal cells, and since the iCAR effect is temporary (i.e.

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reversible restriction), transduced T cells become fully functional once the activator CAR engages with the target TAA [142].

7.2.2. AND-gate circuits Conversely, AND-gate dual-receptor systems provide complementary signals that drive towards CAR-T cells activation, instead of inhibiting their activity. For instance, CAR-T cells can be engineered to express synthetic Notch receptors (synNotch) that release an intracellular transcription factor upon binding a first target antigen, which in turn induces the expression of a CAR specific for a second target antigen [41,143]. In this system, CAR-T cells only activate and proliferate in the presence of both target antigens, further increasing T cells-specificity. Conditional CAR-T cells are another example of an AND-gate circuit, whereby the CD3ζ signaling chain is separated from the co-stimulatory domain in two different CARs that recognize distinct antigens. In principle, the activation of either CD3ζ or co-stimulatory CARs alone would not be sufficient to activate T cells cytolytic activity, and only cells expressing both target antigens would be eliminated [144]. However, it has been 21

Journal Pre-proof demonstrated that single activation of the CD3ζ-only CAR could mediate tumor cell killing, making these CAR-T cells unable to discriminate between single- and double-positive target cells [46].

7.3. Stimuli-responsive CAR A different approach to improve safety relies on CARs that are only activated upon contacting with certain physical features at the tumor-site, allowing for localized tumor recognition and site-specific CAR-T cell activation. One example is masked CARs (mCARs), harboring a masking peptide with a protease-cleavable linker that blocks the CAR extracellular domain [28]. This construction prevents T cells from off-target activation until

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they reach the tumor-microenvironment and encounter locally secreted proteases that cleave the linker and disengage the inhibitory peptide, allowing the unmasked CARs to bind their target antigens [41]. In a preclinical study, a third generation mCAR targeted to EGFR

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associated with a better safety profile [145].

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demonstrated antitumor efficacy in vivo comparable to that of conventional CARs, but Another microenvironmental feature that has been exploited to locally activate CAR-T

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cells is the hypoxic milieu generated upon abnormal tumor-vasculature [146]. These unconventional CARs comprise an oxygen-sensitive subdomain which is stable under hypoxic conditions and degraded upon normal oxygen concentrations [147]. As reported by

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Juillerat et al., despite the low expression levels of oxygen-sensitive CARs on transduced T

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cells, designing CAR-T cells responsive to oxygen variations might be another potential

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strategy to regulate their cytolytic properties and improve safety [147].

8. The solid tumor challenge Despite the remarkable results in the treatment of CD19-positive blood cancers, the extension of CAR-T therapies to solid cancers has been hampered by numerous obstacles. The cause for reduced antitumor efficacy in tackling solid tumors is likely multifactorial, yet, the existence of a surrounding immunosuppressive tumor-microenvironment (TME) is pointed as the main barrier to clinical success [148]. The following topics will address the TME’s intrinsic hostile features and the current strategies developed to counteract them.

8.1. The effects of the tumor-microenvironment on CAR-T cells activity In contrast to the accessibility of peripheral hematological tumors, solid cancers are difficult to reach, forcing CAR-T cells to successfully: (1) traffic to the tumor site, which requires the expression of adhesion molecules on both T cells and tumor vasculature, as well 22

Journal Pre-proof as a match between tumor-derived chemokines and the respective chemokine receptors on T cells surface; (2) infiltrate the stromal region; (3) bind the target antigen to become activated and (4) rapidly expand to relevant therapeutic densities in order to face the tumor burden [148,149]. Accordingly, unlikely to blood cancers, solid tumors are physically supported by a cohesive extracellular matrix produced by stromal cells. Stromal cells are responsible for tumor growth and angiogenesis, providing nutrients, growth factors, chemokines and matrix, as well as for blocking anticancer therapies, by contributing to the generation of an immunosuppressive environment that knockdown T cells effector functions [149]. In fact, stroma-associated antigens have been recognized as potential targets for CAR-T cell therapies against solid tumors [150]. For instance, Wang et al. have developed a CAR specific for murine FAP (fibroblast activation protein), which is overexpressed in tumor

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stroma, and the results revealed an effective reduction in tumor growth followed by a decrease in FAP+ stromal cells [151].

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Additional modification of CAR-T cells to co-express the appropriate chemokine receptors to pair with the cognate tumor-derived chemokines, as CCR2b in anti-GD2 CARs [152],

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CCR2 in anti-mesothelin (MSLN) CARs [153] and CCR4 in anti CD30-CARs [154], has demonstrated to improve intratumoral trafficking and antitumor activity. However, it’s

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noteworthy that the surface expression of additional chemokine receptors can lead to anomalous chemokine signaling in T cells and subsequent off-target side effects [155]. A more recent strategy to promote CAR-T cells infiltration is based on the integrin trans-

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regulation phenomenon. It consists on inhibiting the phosphorylation of the α4 integrin by the

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protein kinase A (PKA) to further enhance integrin αLβ2-mediated T cell migration [156]. In this respect, it was demonstrated that B16 melanoma tumors in α4 (S988A) mutant mice (e.i.

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inhibition of α4 PKA-mediated phosphorylation) presented higher concentrations of T cells, than wild type mice, which supports integrin trans-regulation as a plausible strategy for homing solid tumors [157].

Even upon CAR-T cells successfully tumor infiltration, the surrounding microenvironment is endowed with harsh living conditions, including hypoxia, abnormal vascularization, compact extracellular matrix, nutritional depletion, acidic pH, oxidative stress, and inhibitory cytokines and soluble factors (e.g. prostaglandin E2, adenosine, TGFβ, IL-4 and IL-10), that hinder T cells expansion and persistence [148,149]. Applying fourth-generation CAR-T cells (TRUCK cells), capable of secreting activating cytokines (e.g. IL-12, IL-15, IL-18), or fifthgeneration CARs, endowed with the ability to trigger endogenous cytokine signaling, might help to enhance T cells proliferative capacity and survival [155]. In addition, the presence of high numbers of immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAM) or neutrophils (TAN), in the TME severely suppresses CAR-T cells cytotoxic functions and prevents the recruitment of other effector immune cells to the tumor 23

Journal Pre-proof tissue [149]. In a clinical trial testing the treatment of neuroblastoma with anti-GD2 thirdgeneration CARs (NCT01822652), a robust increase in circulating immunosupressive myeloid cells, mainly M2 (inhibitory phenotype)-polarized macrophage-like cells, was observed [96]. These findings suggest that the TME recruited immunosuppressive cells in response to anti-GD2 CAR-T cells infusion, to counteract the treatment. Similarly, O’Rourke et al. reported high numbers

of non-transduced immunosuppressive Tregs and

overexpression of inhibitory molecules (IDO1, PD-L1 and IL-10) after the infusion of CAR-T cells targeting the variant III of the EGFR (EGFRvIII), in a clinical trial to treat recurrent glioblastoma (NCT02209376) [158]. Once more, the results suggest the occurrence of a compensatory tumor-mediated immunosuppressive response, upon CAR-T cells activation. In addition, T cells intrinsic regulatory mechanisms, such as the up-regulation of co-inhibitory

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receptors (e.g. CTLA4 and PD-1), whose ligands are overexpressed in tumor cells, and the activation of pro-apoptotic intracellular pathways (e.g. Fas receptor-Fas ligand) further

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amplify the immunosuppressive milieu and contribute for T cells anergy and exhaustion

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[159].

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8.2. In search of alternative solid tumor antigens Beyond the physical, chemical, metabolic and immunosuppressive barriers of the TME, the difficulty in identifying unique tumor-associated targets, owing to the inherent

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heterogeneity of solid tumors, is another challenge to overcome [160]. Most of the antigens

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expressed are neither tumor-specific, nor restricted to cancer cells, particularly enhancing the risk of on-target, off-tumor toxicities in healthy tissues, as reported in the first clinical trials

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using CAR-T cells for the treatment of solid tumors [161]. For example, the application of multiple-targeting CAR designs based on OR-gate circuits, such as multi [162], pooled [163] and tandem [164] CAR-T cells (Fig.2.), instead of conventional one-target-based constructions, might be a reliable strategy to enhance the repertoire of targetable TAAs [41].

Fig. 2. (1.5 column figure) Schematic representation of multiple-signal CAR-T cells. In multi-CAR-T cells a single T cell expresses several CAR constructs with distinct antigenic specificities. In tandem

24

Journal Pre-proof CAR-T cells, T cells express CARs designed with two extracellular binding domains that recognize different antigens. Pooled CAR-T cells comprise T cell products harboring two or more single-targeting CAR T-cell types with distinct antigen specificities.

Currently, alternative targets for CAR recognition are being investigated, including: (1) neoantigens, (i.e. tumor-associated peptides resulting from the expression of tumor-specific mutations) [148], such as EGFRvIII [158]; (2) antigens arising from abnormal glycosylation patterns, as the glycoproteins MUC1 and MUC16 [59,60]; (3) tumor stroma/tumor vasculature-associated antigens, as FAP [151] and VEGF receptor-2 [165]; (4) tumor-specific antigens expressed at low levels on healthy cells, as MSLN [166], IL-13Rα2 [110], GD2 [167], and PSMA (prostate specific membrane antigen) [168]; and (5) developmental or

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oncofetal antigens, as CEA (carcinoembrionic antigen) [169]. From those, MSLN, PSMA, GD2, IL-13Rα2, HER2, MUC1, MUC16 and EGFRvIII are currently the most studied antigens

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for targeting solid tumors in clinical trials [170,171].

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8.3. Overcoming the immune suppression

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Novel approaches are being developed to prevent tumor-mediated T cells suppression, mainly by impairing the PD-1/CTLA4 inhibitory axis, which is known to induce T cell exhaustion (e.i. diminished cytokine secretion, cytolytic activity and expansion in vivo) and

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apoptosis [159]. A corresponding example is the disruption of genes encoding PD-1/CTLA-4 inhibitory receptors, using the CRISPR/Cas9 (clustered regularly interspaced short

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palindromic repeats-associated 9) technology to gene editing CAR-transduced T cells.

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Recent results from a preclinical study reported a greater antitumor activity of PD-1 deficient anti-CD19 CAR-T cells in tumor xenograft models engrafting PD-L1+ tumors [172]. Currently, CRISPR/Cas9 gene edited CAR-T cells are in clinical investigation for the treatment of MSLN-positive solid tumors (NCT03545815 and NCT03747965). Following the line of gene silencing, Cherkassky et al. successfully co-transduced antiMSLN CAR-T cells with vectors expressing shRNAs targeted against PD-1 genes [173]. The cotransduction with PD-1 shRNAs and subsequent downregulation of PD-1 intrinsic levels enabled CAR-T cells to further expand and lyse MSLN/PD-L1-positive cancer cells [173]. The same study also reports the development of a PD-1 dominant negative receptor (DNR) capable of competing with the endogenous receptor for binding its ligands (PD-Ls) and restoring CAR-T cells activity, by preventing PD-1:PD-Ls interactions (Fig.3.) [173]. Of note, these truncated suppressive receptors have also been applied in CAR-T cell therapies to counteract the negative effects of the tumor-derived immunosuppressive cytokine TGFβ using a dominant-negative form of the TGFβ receptor II [168].

25

Journal Pre-proof Another promising approach consists in converting PD-1/CTLA4 inhibitory signals into activating ones through a chimeric switch-receptor (CSR), harboring a truncated form of the PD-1 receptor as the extracellular domain fused with the cytoplasmic signaling domains of the CD28 co-stimulatory molecule (Fig.3.) [174]. In the earliest report of this technology, CD8-positive cytotoxic T cells genetically engineered to express the PD1:CD28 chimera were capable of engaging PD-L1-positive tumor cells and provide a PD-L1-mediated costimulatory signal, via CD28, resulting in increased cytotoxic activity and proliferation of the transduced T cells [174]. More recently, Liu et al. managed to co-transduce anti-MSLN and anti-PSCA

(prostate

stem

cell

antigen)

second-generation

CAR-T

cells

with a

PD1:CD28CSR, thus converting a second-generation co-stimulatory signal into a thirdgeneration one, upon PD-L1 binding [175]. In the study, T cells transduced with both CAR

oo

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and PD1:CD28 CSR had shown increased survival, proliferation and antitumor activity in xenograft models for pleural mesothelioma and prostate cancer, further supporting the

pr

potential of CSR technology to circumvent tumor-induced T cell hypofunction and exhaustion

Jo u

rn

al

Pr

e-

[175].

Fig. 3. (1,5 column figure) Schematic representation of a PD-1 dominant negative receptor (DNR) and a PD1:CD28 chimeric switch receptor (CSR) in comparison with a second-generation CAR. The PD-1 DNR is a truncated suppressive receptor, only harboring the extracellular and transmembrane portions of the PD-1 receptor, capable of competing with the endogenous receptor for binding its ligands, mainly PD-L1, and prevent PD-L1-mediated T cells inhibition. The CSR combines the same truncated form of the PD1 receptor fused with the cytoplasmatic signaling domains of the CD28 co-stimulatory molecule (PD1:CD28 chimera), allowing to convert a PD-L1-mediated inhibitory signal into an activating one, via CD28, further enhancing T cells cytotoxic activity.

A feasible alternative is to combine CAR-T cell therapy with the systemic administration of mAbs capable of blocking the interactions between PD-1/CTLA4 inhibitory receptors and their ligands [3]. In fact, combining CAR-T cells with checkpoint blockade therapy have shown to enhance CAR-T cells effector activity and to decrease T cell exhaustion in preclinical [176,177] and clinical studies [178]. Nonetheless, the clinical benefits from combining CAR-T cell therapies with immune checkpoint inhibitors will have to be further investigated, since the improvements reported in CAR-T cells expansion and persistence in 26

Journal Pre-proof vivo are still controversial [96]. Moreover, for a synergistic effect it is required high and frequent Ab doses, since lower doses have shown to be insufficient to inhibit tumor growth or to improve CAR-T cells functional capacity against solid tumors [173,179]. Currently, newer combinatorial approaches are emerging, whereby Ab-derived checkpoint inhibitors are produced and secreted by T cells themselves, taking advantage of CAR-T cells as factories [155]. Accordingly, anti-CAIX (carbonic anhydrase IX) CAR-T cells secreting anti-PD-L1 mAbs have been developed by transducing T cells with lentiviral vectors encoding both anti-CAIX CAR and anti-PD-L1 IgGs [180]. The block of PD-1:PD-L1 interaction enhanced anti-CAIX CAR-T cells survival and antitumor activity, by diminishing PD-L1-mediated T cell exhaustion. Moreover, the local release of anti-PD-L1 mAbs enabled the recruitment of NK cells to the tumor site, that enhanced cancer cell killing by ADCC,

oo

f

leading to a five-fold reduction in tumor growth in a mouse model of renal carcinoma [180]. Similarly, Li et al. reported increased tumor cell killing in a lung carcinoma mouse model,

pr

mediated by anti-CD19 CAR-T cells genetically engineered to constitutively secrete anti-PD1 scFvs derived from anti-PD-1 mAbs [179]. These innovative CAR-T cell systems are

e-

currently in clinical investigation for MUC1- (NCT03179007), EGFR- (NCT03182816) and

9. Future Perspectives

Pr

MSLN-positive (NCT03182803) advanced solid tumors.

al

The consistently high response rates observed in blood cancer patients treated with anti-

rn

CD19 CAR-engineered T cells, have emerged CAR-T cells as one of the major breakthroughs of cancer immunotherapies in the last years. Moreover, the FDA approval of

Jo u

the two first anti-CD19 CAR-T cell therapies as reliable options to treat refractory B-cell malignancies, have prompted several biopharmaceutical companies to develop novel CAR-T cell-based products, which are currently in preclinical and clinical evaluation for the treatment of both liquid and solid tumors, as detailed in Table 2. However, the clinical success in hematological cancers is stained by life-threatening side effects that have led to some CAR-associated deaths in clinical trials. Besides, the extension of this ACT to solid malignancies has reported little clinical effect owing to the strong immunosuppressive environment and the lack of specific-tumor antigens. Fortunately, new strategies are arising with the potential to overcome these challenges. The employment of highly innovative safety technologies based on (1) switch-based control systems, including universal CAR designs, (2) combinatorial antigen recognition strategies, (3) stimuliresponsive constructions, and (4) multiple-targeting CARs, will hopefully allow to finely tune and redirect CAR-T cells activity in order to avoid undesired toxicities. In the field of solid tumors, the recognition of the PD-1/CTLA4 inhibitory axis as a key factor

on

TME-mediated immunosuppression have propelled the investigation of 27

Journal Pre-proof unconventional strategies, including gene silencing technologies, suppressive truncatedreceptors, and combinatorial therapies using Ab-derived checkpoint inhibitors to impair these inhibitory mechanisms. In addition, equipping CAR-T cells to secrete immune-modulating molecules, like cytokines and Ab-derived checkpoint inhibitors, or to express fifth-generation CARs,

further

enhances

the

possibility

to

modulate

the

TME,

converting

the

immunosuppressive milieu into an immune permissive one. The future generation of CAR-technology is driving towards the creation of multifunctional smart T cells, with the potential to generate complex synthetic immune responses and to adapt their activity to each cancer type, by remodeling the TME in their favor.

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f

Acknowledgements This work was supported by the European Regional Development Fund (ERDF), through

pr

the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P.,

e-

under projects Cancel Stem (reference POCI-01-0145-FEDER-016390), CENTRO-01-0145FEDER-000012 (HealthyAging2020), Euronanomed 2 (FCT reference ENMed/0005/2015)

rn

Conflict of interests

al

Pr

and CNC.IBILI (FCT reference UID/NEU/04539/2019).

Jo u

The authors declare no conflict of interest.

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[174] M.E. Prosser, C.E. Brown, A.F. Shami, S.J. Forman, M.C. Jensen, Tumor PD-L1 costimulates primary human CD8+ cytotoxic T cells modified to express a PD1: CD28 chimeric receptor, Mol. Immunol. 51 (2012) 263–272. doi:10.1016/j.molimm.2012.03.023. [175] X. Liu, R. Ranganathan, S. Jiang, C. Fang, J. Sun, S. Kim, K. Newick, A. Lo, C.H. June, Y. Zhao, E.K. Moon, A chimeric switch-receptor targeting PD1 augments the efficacy of second-generation CAR T cells in advanced solid tumors, Cancer Res. 76 (2016) 1578–1590. doi:10.1158/0008-5472.CAN-15-2524. [176] T. Gargett, W. Yu, G. Dotti, E.S. Yvon, S.N. Christo, J.D. Hayball, I.D. Lewis, M.K. Brenner, M.P. Brown, GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected from Activation-induced Cell Death by PD-1 Blockade, Mol. Ther. 24 (2016) 1135–1149. doi:10.1038/mt.2016.63. [177] L.B. John, C. Devaud, C.P.M. Duong, C.S. Yong, P.A. Beavis, N.M. Haynes, M.T. Chow, M.J. Smyth, M.H. Kershaw, P.K. Darcy, Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells, Clin. Cancer Res. 19 (2013) 5636–5646. doi:10.1158/1078-0432.CCR-13-0458. [178] E.A. Chong, J.J. Melenhorst, S.F. Lacey, D.E. Ambrose, B. Levine, C.H. June, S.J. Schuster, PD-1 Blockade Modulates Chimeric Antigen Receptor ( CAR ) Modified T Cells and Induces Tumor Regression : Refueling the CAR, Blood. (2017). doi:10.1182/blood-2016-09-738245. [179] S. Li, N. Siriwon, X. Zhang, S. Yang, T. Jin, F. He, Y.J. Kim, J. Mac, Z. Lu, S. Wang, X. Han, P. Wang, Enhanced cancer immunotherapy by chimeric antigen receptor– modified T cells engineered to secrete checkpoint inhibitors, Clin. Cancer Res. 23 (2017) 6982–6992. doi:10.1158/1078-0432.CCR-17-0867. [180] E.R. Suarez, D.-K. Chang, J. Sun, J. Sui, G.J. Freeman, S. Signoretti, Q. Zhu, W.A. Marasco, Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model, Oncotarget. 7 (2016) 34341–34355. doi:10.18632/oncotarget.9114.

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Journal Pre-proof Table 1. Examples of Axicabtagene ciloleucel’s clinical trials. ClinicalTrials.gov Reference number

ZUMA-5

r/r Indolent NHL

Phase II, recruiting

NCT03105336

ZUMA-6

r/r DLBCL*

Phase II, active not recruiting

NCT02926833

ZUMA-7

r/r DLBCL**

ZUMA-11

r/r DLBCL***

ZUMA-12

r/r DLBCL****

ZUMA-2

r/r MCL

Phase III, recruiting Phase I/II, recruiting Phase II, recruiting Phase II, active not recruiting

ZUMA-3

adult r/r B-ALL

ZUMA-4

pediatric r/r B-ALL

ZUMA-8

r/r CLL

* A X I C E L

NCT03391466

i n

NCT03704298

c o m b i n a t i

NCT03761056 NCT02601313 NCT02614066

Phase I/II, recruiting

f

KTE-X19 (formerly KTE-C19)

Status

oo

Axicabtagene ciloleucel (AXI-CEL)

Indication

NCT02625480 NCT03624036

e-

pr

on with Atezolizumb (anti-PD-L1 mAb); ** AXI-CEL versus standard of care therapy; *** AXI-CEL in combination with Utomilumab (4-1BB/CD137 agonist); **** AXI-CEL as first-line therapy; DLBCL - diffuse large B cell lymphma; MCL - mantle cell lymphoma; ALL - acute lymphoblastic leukemia; NHL - nonHodgkin lymphoma; CLL - chronic lymphocytic leukemia.

Company

Pr

Table 2. Emerging CAR-T cell-based candidate products. CARtechnology

Indication

Target

Status

NCT02808442 (PALL)

al

pediatric r/r B-ALL Advanced lymphoide malignancies*

rn

UCART19

adult r/r B-ALL

Phase I Clinical trial

NCT02735083 NCT02746952 (CALM) NCT03190278 (AML123)

AML

CD123

UCART22

adult B-ALL

CD22

IND

UCARTCS1

MM

CS1

IND

UCARTCLL1

AML

CLL1

Preclinical

ALLO-501**

r/r B-NHL

CD19

Phase I/II clinical trial

ALLO-715***

MM

BCMA

IND

ALLO-819***

AML

FLT3

Preclinical

MB-101

r/r malignant glioblastoma r/r AML and BPDCN glioblastoma multiforme MM prostate, pancreatic, gastric and bladder cancers

IL-13Rα2

NCT02208362

CD123

NCT02159495

r/r B-NHL

CD20

MB-102 MB-103 Mustang Bio

CD19

UCART123

Jo u

Cellectis

ClinicalTrials.gov Reference number

MB-104 MB-105 MB-106

HER2

Phase I clinical trial

----

NCT03939026 (ALPHA) ----

NCT03696030

CS1

NCT03710421

PSCA

NCT03873805 Phase I/II clinical trial

NCT03277729

42

Journal Pre-proof r/r AML/MDS

Phase I/II clinical trial

Hematological and solid tumors

NCT03466320 (DEPLETHINK) NCT03018405 (THINK)

NKG2D mCRC

Phase I clinical trial

CYAD-101

mCRC

CYAD-211

MM

BCMA

CYAD-221

B cell malignancies

CD19

CYAD-231

undisclosed

NKG2D

Preclinical

NCT03310008 (SHRINK) NCT03692429 (AlloSHRINK)

----

Jo u

rn

al

Pr

e-

pr

oo

f

*Previously exposed to UCART19/ALLO-501; **collaboration between Servier and Allogene; ***licensed to Allogene; IND - Investigational new drug by FDA; AML - Acute myeloid leukemia; MM - Multiple myeloma; BPDCN - Blastic plasmacytoid dendritic cell neoplasm ; MDS - Myelodysplastic syndromes; mCRC - Metastatic colorectal cancer; CLL1 C-type lectin-like molecule-1; BCMA - B-cell maturation antigen; FLT3 - FMS-like tyrosine kinase 3 ; HER2 - human epidermal growth factor receptor 2; PSCA - prostate stem cell antigen; NKG2D - natural killer group 2D activating receptor.

43

Journal Pre-proof

f oo pr ePr al rn

  

CRS and neurotoxicity are the most-common life-threatening CAR-mediated toxicities; Novel safety devices are emerging to tune CAR-T cells activity and reduce toxicity; CAR-T cells efficacy in solid tumors is hindered by the immunosuppressive TME; Strategies to inhibit the PD-1/CTLA4 axis allow CAR-T cells to modulate the TME.

Jo u



44