Determinants of response and resistance to CAR T cell therapy

Journal Pre-proof Determinants of response and resistance to CAR T cell therapy Stefanie Lesch, Mohamed-Reda Benmebarek, Bruno L. Cadilha, Stefan Stoiber, Marion Subklewe, Stefan Endres, Sebastian Kobold

PII:

S1044-579X(19)30219-6

DOI:

https://doi.org/10.1016/j.semcancer.2019.11.004

Reference:

YSCBI 1717

To appear in:

Seminars in Cancer Biology

Received Date:

30 September 2019

Revised Date:

28 October 2019

Accepted Date:

3 November 2019

Please cite this article as: Lesch S, Benmebarek M-Reda, Cadilha BL, Stoiber S, Subklewe M, Endres S, Kobold S, Determinants of response and resistance to CAR T cell therapy, Seminars in Cancer Biology (2019), doi: https://doi.org/10.1016/j.semcancer.2019.11.004

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Determinants of response and resistance to CAR T cell therapy Stefanie Lesch1*, Mohamed-Reda Benmebarek1*, Bruno L. Cadilha1*, Stefan Stoiber1*, Marion Subklewe2,3, Stefan Endres1,2 and Sebastian Kobold1,2,† 1

Center of Integrated Protein Science Munich (CIPS-M) and Division of Clinical

Pharmacology, Department of Medicine IV, Klinikum der Universität München, LMU Munich, Germany, Member of the German Center for Lung Research (DZL). 2

German Center for Translational Cancer Research (DKTK), partner site Munich,

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Munich Germany Department of Medicine III, Klinikum der Universität München, LMU Munich,

Germany

These authors contributed equally to this work.

†

Corresponding author

Sebastian Kobold, M.D.

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*

Division of Clinical Pharmacology, Klinikum der Universität München

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Lindwurmstraße 2a, 80337 München Phone: 0049-89-4400-57300 Fax: 0049-89-4400-57330

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[email protected]

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Abstract The remarkable success of chimeric antigen receptor (CAR)-engineered T cells in pre-B cell acute lymphoblastic leukemia (ALL) and B cell lymphoma led to the approval of anti-CD19 CAR T cells as the first ever CAR T cell therapy in 2017. However, with the number of CAR T cell-treated patients increasing, observations of tumor escape and resistance to CAR T cell therapy with disease relapse are demonstrating the current limitations of this therapeutic modality. Mechanisms hampering CAR T cell efficiency include limited T cell persistence, caused for example by T cell exhaustion and activation-induced cell death (AICD), as well as

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therapy-related toxicity. Furthermore, the physical properties, antigen heterogeneity and immunosuppressive capacities of solid tumors have prevented the success of CAR T cells in these entities. Herein we review current obstacles of CAR T cell

therapy and propose strategies in order to overcome these hurdles and expand CAR

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T cell therapy to a broader range of cancer patients.

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Introduction

Chimeric antigen receptors (CAR) are synthetic receptors that combine an antigenspecific extracellular domain, usually derived from the variable regions of an antibody, with the key functional intracellular domains of a T cell receptor (TCR). This architecture, upon antigen encounter triggers a potent TCR-independent T cell response. The design of CAR molecules compresses the information normally transmitted via the multiprotein TCR-complex (encoded in more than 10 kb DNA) into a single-chain molecule that is usually smaller than 2 kb. Nevertheless, the

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mechanisms by which CAR T cells exert their effector functions are quite akin to those mediated by intact TCR complexes. This topic has been extensively reviewed elsewhere [1]. Two CD19-targeted CAR T cell products are currently approved in Europe and the US: Yescarta (Axicabtagene ciloleucel) and Kymriah

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(Tisagenlecleucel).

The most notable structural difference between the two approved CAR T cell

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products is found in the costimulatory domain, 4-1BB for Kymriah and CD28 for Yescarta. Currently, Kymriah is approved for relapsed or refractory B cell

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malignancies, such as acute lymphoblastic leukemia (r/r ALL) for patients up to 25 years of age, and for relapsed or refractory diffuse large B cell lymphoma (DLBCL) after two or more lines of systemic therapy. Yescarta is approved for DLBCL and

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primary mediastinal large B cell lymphoma (PMBCL) after two or more lines of systemic therapy. These therapies were approved through accelerated programs from the EMA and FDA based on exceptional overall response rates in the single arm phase I/II trial ZUMA-1 [2, 3] and the single arm phase II trials ELIANA and

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JULIET [4-6], respectively.

However, at the time of approval, data on long-term progression-free and overall survival was not yet available. With the emergence of data regarding the long-term follow-up of these early cohorts, we now know that most patients will not benefit in the long run and will eventually succumb to their disease.

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Response rates to approved CAR T cell therapy

Adult B cell ALL patients have so far demonstrated the best rates of complete remission (CR), ranging from 83% to 93% [7-9]. Importantly, as anti-CD19-CAR T cell therapy is typically preceded by non-myeloablative chemotherapy, a fraction of responses is likely to be attributable to this pretreatment, rather than the CAR T cell therapy [10]. Children treated for B cell ALL have also shown remarkable results, with CR rates ranging between 67% and 90% [4, 11-13]. In contrast, DLBCL patients

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had CR rates between 43% [6] and 54% [3]. CR rates in chronic lymphocytic leukemia (CLL) patients ranged between 21% [14] and 29% in two studies. Thus,

there seems to be a correlation between disease biology (e.g. mutational load, intratumoral heterogeneity, tumor stroma) and response rates to CAR treatment (figure

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1).

Following CAR T cell therapy, ALL patients have a high risk of disease recurrence,

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with 41% of responders relapsing within 12 months in one study [4]. In patients with large B cell lymphoma, CR rates seem to be more durable, as the majority of

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patients who achieved CR maintained remission over a 12 month follow-up [3]. In the study by Porter and colleagues targeting CLL, none of the patients with CR (29%)

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have relapsed with a median duration of response of 40 months [15].

Pre-treatment considerations for CAR T cell therapy

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3.1. Patient selection

All patients receiving CAR T cell therapy have relapsed or refractory disease after chemotherapy or autologous stem cell transplantation [3, 4]. Patients are assessed for their capacity to cope with lymphodepletion chemotherapy or the potential toxicity of CAR T cells before being eligible for therapy [9]. Even if they are per se eligible for CAR T cell therapy, insufficient lymphocyte apheresis, poor lymphocyte viability, limited expansion ex vivo or disease progression during the manufacturing process are factors that will prevent patients from receiving CAR T cell therapy [3, 6]. Being 4

an indication for relapsed or refractory diseases, CAR T cell therapy outcome might be influenced by prior therapeutic regimens. Lastly, clinical studies are prone to introduce a certain bias due to rigorous patient selection, thus overestimating the potency of novel therapeutics. Translating any therapeutic modality into clinical routine is likely to reveal a lower overall therapeutic effectiveness.

3.2. Production platforms

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3.2.1. Choice of transgene vector

Currently, the majority of clinical and preclinical trials utilize gamma-retroviral and lentiviral vectors to genetically modify autologous T cells. In fact, Kymriah uses a lentiviral vector, whereas the Yescarta CAR is delivered with a gamma-retroviral

vector. While lentiviruses have the advantage of being able to infect non-dividing

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cells [16], both vector types harbor the risk of insertional oncogenesis [17, 18]. However, insertional oncogenesis has thus far not been reported after genetic

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manipulation of mature T cells [19, 20], in contrast to studies genetically modifying hematopoetic stem cells [21, 22]. An additional limitation is that the GMP-compatible

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production of viral vectors is cost-intensive and transgene size is limited [23].

An alternative platform uses transposon systems, such as Sleeping Beauty [24] and

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PiggyBac [25], which are less expensive, harbor fewer GMP restrictions and have been postulated to have a favorable safety profile in regards to transgene integration [24]. Furthermore, larger transgenes may be inserted into cells, this however comes at the cost of less efficient transposition [26].

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The above-mentioned approaches are particularly suited for long-term genetic modifications, as they result in stable integration of the transgene in the target cells. However, transient expression, which can be achieved via RNA-electroporation, also has certain advantages. With short-lived expression, this approach has been proposed to constitute a fitting way to screen for on-target off-tumor toxicities of novel CARs [27, 28]. In turn, this transient CAR expression is a double-edged sword as durable anti-tumoral efficacy would require repeated infusion of CAR T cells, potentially increasing the risk of an anti-CAR T cell immune response [29]. 5

3.2.2. Site-specific transgene integration

Increasing effort is being invested into directing the transgene towards specific genomic locations, with the proposed benefits of mitigating unforeseen genotoxicity and achieving optimal transgene function [30, 31]. Eyquem and colleagues demonstrated in vitro and in mouse models that targeting the CAR to the T cell receptor  constant (TRAC) locus reduced tonic signaling (antigen-independent CAR signaling, reviewed by Ajina et al. [32]), averted accelerated T cell differentiation and

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exhaustion, and increased the therapeutic potency of engineered T cells. This approach controlled both baseline CAR surface expression and the downregulation and recovery of CAR surface expression after antigen stimulation [33]. Achieving optimal CAR surface expression is of crucial importance to mitigate tonic CAR

signaling [33-35] and expression levels may furthermore be modulated to fine tune

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antigen sensitivity of CAR T cells [36].

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A remarkable success case, highlighting the importance of the site of transgene integration, was that of a 78-year-old patient with advanced relapsed and refractory

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CLL, treated with anti-CD19 CAR T cells (as part of the trial NCT01029366) [37]. Two years following CAR T cell treatment the patient was in CR and peak expansion of CAR T cells had been observed concurrently with CRS that resolved without anti-

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IL-6-receptor blockade. This was the second CAR T cell treatment this patient received, as the first treatment had failed to induce disease remission. As anti-IL-6receptor blockade was assumed to have hampered CAR T cell efficacy, the patient was eligible for repeated treatment.

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Deep sequencing of the pre-infusion product (for the second treatment) and sequential post-infusion analyses revealed that the initially polyclonal CAR T cell product was progressively dominated by the offspring of a single clone (94% of the CAR T cells at the peak of response). This CAR T cell clone demonstrated massive in vivo expansion (approximately 29 population doublings), followed by contraction upon disease clearance. This is in stark contrast with previous reports (with more than 40 patients treated with anti-CD19 CAR T cells) where the accumulation of CAR

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T cells in vivo was polyclonal or pauciclonal, at least [9]. Further analysis revealed that the lentiviral CAR vector integrated at intron 9 of TET2, disrupting this allele. In addition, this patient had a hypomorphic mutation (E1879Q, glutamic acid to glutamine) on his other TET2 allele (without CAR integration). This peculiar constellation led to loss of TET2 function in CAR T cells, reprogramming the epigenetic landscape of these T cells, thus boosting their anti-tumoral activity. The implications of such a finding go beyond this patient, as targeted transgene integration becomes feasible on a larger scale.

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Targeted transgene integration may be achieved with the use of site-specific endonuclease platforms such as CRISPR-Cas9 [33], TALEN [33] and ZFN [38]

which introduce a DNA-double strand break, enabling specific alterations at the

desired location [39]. Thereafter, a repair matrix (with the desired transgene) can be introduced into the cell and is copied into the location of the double strand break.

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Viral vectors and double-stranded DNA have been successfully used as repair

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3.2.3. In vivo T cell transduction

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matrices in this context [33, 40].

CAR T cell manufacturing requires ex vivo activation, engineering and expansion of patient T cells. To circumvent this time-consuming process, new approaches have

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been devised, to enable the in vivo delivery of CAR-encoding DNA selectively into T cells. In a preclinical mouse model, using synthetic nanoparticles, Smith and colleagues reported that in vivo engineered CD19-specific CAR T cells were as efficient as ex vivo manufactured CAR T cells in controlling tumor growth [41]. More recently, Pfeiffer et al. described an approach for in vivo reprogramming of

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human CD8+ T cells to express an anti-CD19 CAR, which could successfully eradicate CD19+ lymphoma cells [42]. Standard CAR T cell manufacturing protocols have been studied extensively and long-term follow up studies of patients treated with ex vivo engineered T cells revealed no evidence of retroviral gene-transfermediated oncogenesis [20]. In vivo genetic engineering, however, bears the risk of gene transfer into off-target cells, raising serious safety concerns. A careful examination of unintentional cell transformation must be carried out before this strategy can be translated into clinical application. 7

3.2.4. Off-the-shelf CAR T cells

The mainstay of trials has focused on modifying autologous T cells for cancer therapy. Increasing effort is being invested to make adoptive T cell therapy amenable to patients with, for example, insufficient T cell numbers by creating “offthe-shelf” CAR T cells from healthy donors. Multiple distinct approaches rely on the ability to knock-out genes encoding for certain surface proteins, such as TCR- and HLA-molecules [43, 44], thereby reducing the risk for graft-versus-host disease and

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potentially allowing for HLA-incompatible T cell infusions. This strategy has already been successfully applied to a small number of patients [44]. However, for certain approaches, extensive lymphodepletion is required prior to allogeneic CAR T cell

transfer, to prevent the rejection of the transferred cells by the recipient’s immune system [44, 45]. An alternative approach would be to utilize well-characterized

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human cell lines with a short in-patient half-life. One example of this is the

immortalized natural killer cell line NK92 [46]. However, the safety, large scale

Post-transfer determinants of efficacy of current CAR T cell therapies

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CAR T cells remain to be determined.

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feasibility and efficacy of off-the-shelf CAR T cell products compared to autologous

4.1 Toxicity

CAR T cells will induce toxicity as a result of antigen-recognition and consequent triggering of potent T cell effector functions. This may result in the lysis of antigen-

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expressing healthy cells (direct toxicity), whereas cytokine release syndrome (CRS) constitutes a form of systemic off-target CAR T cell toxicity. In contrast to naturally occurring TCR-activated T cells, CAR T cells may require less external costimulation to trigger a full-blown T cell response and may therefore be more likely to cause severe toxicity. Moreover, CAR T cell-related toxicity may exceed the severity of adverse events observed for monoclonal antibodies directed against the same target, as CAR T cell avidity is higher [47] and the triggered effector functions are extremely potent [48]. 8

Previous trials with the CD28 superagonist (TGN1412) taught valuable lessons on CRS, the severity of this adverse event frequently requiring targeted therapy in addition to corticosteroids. Tocilizumab is an anti-IL6-receptor antibody which can help resolve severe CRS [8, 49]. Furthermore, tocilizumab is being tested as a preemptive mitigation strategy for severe CRS (NCT02906371). Fever, hypotension, hypoxia and neurological dysfunction coupled with increased serum levels of cytokines such as IL-6, IFN-, CXCL10, MIP-1, MCP-1, CXCL9, IL-8, IL-10, MIP-1 and of soluble receptors IL-1R and IL-2Rhave been described to follow CAR T

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cell therapy and are now comprised under the definition of CRS [50]. Cytokine levels peak concurrently with levels of the administered CAR T cells, and their effects may trigger life-threatening capillary leakage with hypoxia and hypotension. In some cases, severity of CRS can be predicted from pre-treatment tumor burden

(measurement of blasts in the bone marrow) [11]. Nonetheless, there are several

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other factors that may influence CRS severity such as the type of lymphodepletion

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[51] or the costimulatory domain employed in the CAR [6, 52]

Like blinatumomab, a bispecific antibody for CD3 and CD19, anti-CD19 CAR T cells

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have been shown to cause neurotoxicity, however, the mechanism is still unclear [8, 9, 11]. A preclinical study conducted by Sterner and colleagues showed that the inhibition of GM-CSF with the monoclonal antibody lenzilumab reduced CRS and

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neuroinflammation associated with CAR T cell therapy. To test if this approach constitutes a novel strategy to prevent toxicity following CAR T cell therapy, a phase II clinical trial evaluating the combination of lenzilumab and axicabtagene ciloleucel in patients with relapsed or refractory diffuse large B-cell lymphoma will be launched

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by the end of 2019 [53].

Severe side effects could be limited by the introduction of suicide genes into CAR T cells (reviewed in [54]), which permit the selective ablation of transferred cells. This can be achieved via antibody-mediated targeting of truncated surface molecules [55], inducible Caspase 9 [56-58] or HSV-TK in conjunction with the antiviral agent ganciclovir [59]. However, as a downside, systemic depletion of the therapy will simultaneously abrogate anti-tumor efficacy. Currently, clinical testing in patients is

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under way for multiple tumor entities as fourth generation specific CAR (incorporating three costimulatory domains as well as the iCas9 suicide gene) in ten different clinical trials listed in clinicaltrials.gov. These therapeutic strategies are still in their early stages of validation. Thus, comprehensive management guidelines should be set in place to ensure a rapid and standardized response to the wide gamut of patient characteristics and tumor entities.

An alternative strategy may be to engineer CAR T cells with Boolean logic-circuits, most commonly referred to as AND- or OR-gated CAR T cells. Different levels of

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control over CAR T cell stimulation have been achieved by separating the CD3 chain and the costimulus into two distinct receptors [60, 61], by the use of synthetic Notch receptor CARs [62] or by combining chimeric receptors with small molecules

inducing costimulation [63, 64]. Such modifications allow for a modular response of CAR T cells, varying according to the presence or in other cases the absence of

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antigens [65]. Another approach may be to generate “universal” CAR T cells in

combination with a tumor-specific adaptor [66]. CAR T cell specificity and activity

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may then be controlled by application or withdrawal of the adaptor molecule [66]. The downside is the dependence of the CAR T cell response on the

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pharmacokinetics of the adaptor molecule.

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4.2 CAR T cell failure mechanisms and predictors of response

4.2.1. Exhaustion and AICD

T cell exhaustion is a complex phenomenon, characterized by sequential loss of effector functions, such as their ability to produce cytokines, proliferate and mediate

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lysis of target cells [67-69]. It is accompanied by a distinct transcriptional profile [70, 71] and upregulation of inhibitory receptors, such as PD-1, TIM-3 and LAG-3 [69]. Multiple environmental and T cell intrinsic mechanisms contribute towards the acquisition of an exhausted phenotype. These include, but are not limited to chronic antigen exposure [68, 72], presence of immunosuppressive ligands and immunosuppressive cell types [73-76] or tonic CAR signaling [69]. An attractive, but difficult to implement strategy aims at mitigating immunosuppression at the tumor

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site. A more straightforward approach may be to abate CAR T cell dysfunction by optimizing CAR design [77]. There may be a variable expression of inhibitory receptors and cytokine production in different CAR T cells. Zolov and colleagues described that CD28 costimulation increased the percentage of TIM-3 positive cells, as opposed to when the costimulatory domain was 4-1BB, with more PD-1 and LAG3 being expressed on CAR T cells [78]. In contrast, Long and colleagues observed that 4-1BB-containing CAR T cells directed against GD2 had a lower expression of PD-1, TIM-3 and LAG-3 compared to CD28-CAR T cells [69]. While exhaustion of tumor-specific T cells may severely hinder their ability to combat disease, strategies

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to reverse exhaustion and restore T cell function are being investigated and are met with success in some studies [79-81]. For example, the ex vivo expansion of tumorinfiltrating lymphocytes from pancreatic cancer patients could be improved by employing PD-1-blocking or 4-1BB-stimulatory antibodies [82].

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AICD is another limiting factor of CAR T cell persistence and efficacy [35, 83, 84]. In contrast to exhaustion, AICD is irreversible, therefore, preventive measures must be

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employed. Several groups have successfully reduced the frequency of CAR T cell AICD in preclinical studies by optimizing CAR structure [85], transgene vector design

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[35] and the choice of costimulatory molecules [84]. Gomes da Silva and colleagues showed reduced tonic 4-1BB costimulation and consequently less AICD when utilizing a lentiviral vector instead of a gamma-retroviral vector. Similar results could

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be achieved by reducing CAR surface expression via an IRES element [35]. On the other hand, Künkele and colleagues reported, that third-generation CAR T cells were more susceptible to AICD in comparison to second-generation CAR T cells, indicating that excessive stimulation of T cells may have deleterious consequences [84]. Data from Hudecek and colleagues highlighted the role of the extracellular

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spacer domain in CAR T cell functionality, as IgG-based spacers led to the sequestration, activation and deletion of CAR T cells in murine models. The clinical significance of this finding is still unclear, as the observed phenomenon relied on the binding of the spacer domain to murine FcɣR [85].

4.2.2. Quest for the optimal T cell phenotype

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Preclinical data indicated that central memory CAR T cells directed towards CD19 improved anti-tumor efficacy in a xenograft mouse model compared to effector memory CAR T cells [86]. This may be due to increased persistence in vivo [86]. It has also been reported that stem cell memory T cells, a subset of memory T cells, display an even superior capacity for self-renewal [87]. Likewise, in the case of TET2 deficient CAR T cells mentioned above, these highly potent CAR T cells predominantly displayed a central memory phenotype at peak response [37]. In contrast, CAR T cells without a TET-2 deficiency were composed mainly of effector memory and effector CD8+ cells in the same study [37]. This however, did not hinder

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patients’ response to CAR T cell therapy, suggesting that the differentiation state of CAR T cells may not be the sole driver of disease regression following CAR T cell

therapy [37]. Nevertheless, strategies to skew CAR T cell differentiation towards a memory phenotype are being successfully undertaken [88, 89]. In this context, 4-

1BB and CD28 costimulation were shown to promote differential metabolic programs

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in CAR T cells, favoring memory differentiation or the development of an effector phenotype, respectively [90]. Therefore, the optimized choice of costimulatory

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domain may help pave the way for increased CAR T cell persistence and

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effectiveness [90, 91].

4.2.3. CD4 vs. CD8 CAR T cells

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Besides identifying the optimal differentiation state of CAR T cells for maximal efficacy, the choice of T cell subset is under intensive investigation. Turtle and colleagues have reported a clinical trial utilizing an anti-CD19-CAR T cell product of defined CD4 and CD8 composition to treat B cell ALL [9] and 93% of patients achieved remission as determined by high-resolution flow cytometry. In contrast,

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Wang et al. showed that a mix of CD4+ and CD8+ glioblastoma-targeting CAR T cells was inferior to CD4+ CAR T cells (anti-IL13Ra2) alone in controlling tumors in vivo [92]. CD4+ CAR T cells were less susceptible to tumor-challenge-induced exhaustion, even after repetitive challenges, as opposed to CD8+ CAR T cells. Activation-induced exhaustion, mainly occurred in CD8+ T cells (surface expression of LAG-3, 2B4, PD-1, TIM-3 and CD57 was higher in CD8+ T cells). Similarly, higher expression of terminal differentiation-associated transcription factors TBX, GATA3 and EOMES was observed in CD8+ T cells, whereas higher expression of memory 12

associated transcription factors (LEF1, FOXP1, and KLF7) was seen in CD4+ T cells [92]. Intriguingly, the optimal composition of CD4+ and CD8+ T cells may depend on the choice of costimulation, as distinct subpopulations of T cells may require different costimulatory signals. In this context, it was shown that CD4+ CAR T cells with ICOS costimulation improved the persistence of co-transferred CD8+ CAR T cells in an in vivo mouse model [91]. Additionally, 4-1BB-costimulation was most effective in prolonging persistence of CD8+ CAR T cells [91]. The impact of other variables, such as the targeted disease entity on the relevance of the CD4+ vs. CD8+ ratio has not

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yet been determined.

4.3. Tumor escape mechanisms

4.3.1. Antigen loss

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Antigen loss is not unique to CAR T cell therapy, but rather a limitation of targeted

therapy. Clinical relapse due to CD19 immune escape following immunotherapy was

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first reported in 2011 by Topp and colleagues in a phase II trial investigating the efficacy of the CD19-targeted T cell engaging antibody blinatumomab in MRD-

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positive B-ALL patients [93]. A global phase II study of the anti-CD19 CAR T tisagenlecleucel in young patients with B-ALL (NCT02435849) reported an overall remission rate of 81% at day 28 after T cell infusion [94], but 59% of the patients

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relapsed within 12 months. In this study the CD19 status of 16 relapsed patients was analyzed and it was shown that in 94% of cases the relapses resulted from antigen loss and only one patient had CD19-positive recurrence. If only the specific epitope is lost from the targeted molecule, employing CAR T cells which recognize a different epitope within the same molecule may be a feasible strategy. Antigen loss has

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furthermore been observed for anti-CD22 CAR T therapy in B-ALL patients (some of which already received CD19-directed immunotherapy) by Fry and colleagues in a phase I trial [95]. Following anti-leukemic activity that was comparable to tisagenlecleucel, seven out of eight patients relapsed with lymphoblasts that showed decreased CD22 density or complete CD22 loss.

Interestingly, most ALL patients with disease relapse following 4-1BB-CAR treatment, show either antigen negative relapse or failure of CAR T cell persistence 13

[9, 11]. Patients treated with a CD28-CAR were seen to more frequently relapse with antigen positive disease [7]. Intriguingly, this study also found that long-term response and overall survival seemed independent from CD28-CAR T cellpersistence (beyond induction of CR), suggesting that therapy responsiveness and disease resistance are closely linked to the costimulation-driven CAR T cell phenotype [7].

4.3.2. Mechanisms of antigen loss

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A group of researchers analyzed antigen loss following anti-CD19 CAR T cell infusion in pediatric patients with B-ALL and found several CD19 splice variants

expressed by relapsing lymphoblasts, including one variant with a deletion of exon-2, which contains the extracellular epitope of CD19 that is recognized by CD19 CAR T (FMC63). Furthermore, variants were found, that showed significantly reduced

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incorporation in the plasma membrane and up to 90% of the CD19 isoform remained cytosolic [5]. One unique escape mechanism was described by Ruella et al., where

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accidental transduction of one malignant blast led to coexpression of CD19 and antiCD19-CAR on the cancer cell, presumably masking the CD19 epitope and rendering

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the offspring of this transduced clone resistant to CD19-targeted CAR T cell therapy [96].

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4.3.3. Strategies to overcome antigen loss

One approach to prevent antigen loss following CAR T cell therapy is to simultaneously target more than one tumor-associated antigen with multi-specific CAR T cells. For example, CD123 was found to be highly expressed in patients with

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relapsing CD19-negative disease after targeted therapy. Therefore, targeting CD123 in combination with CD19 could enhance the likelihood to eradicate leukemic clones that would otherwise have a selective advantage following CD19 CAR T cell therapy. Ruella and colleagues used a xenograft model of primary blasts isolated from patients relapsing with CD19-negative disease to demonstrate the efficient antileukemia activity of the dual-targeted approach. In addition, the group showed that the dual expression of anti-CD19 and anti-CD123 CAR is more effective in a B-ALL model than either CAR alone or the pooled CAR T cell product [97]. Combining 14

several dual CAR and the simultaneous or sequential infusion of CAR T cells targeting different antigens are areas of active clinical investigation [95, 98]. Fousek and colleagues took this approach one step further and equipped T cells with a trivalent CAR targeting CD19, CD20 and CD22. In a preclinical model they demonstrated that trivalent CAR T cells effectively mitigated CD19-negative relapse of B-ALL [99]. Another approach is based on a “universal” CAR that uses an adaptor molecule to confer T cells with antigen specificity. This strategy may therefore be employed to

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simultaneously target several tumor associated antigens by application of several distinct adaptor molecules. In addition, novel antigens can be targeted without re-

engineering CAR T cells, making the system adjustable to evolving disease [100].

CAR T cell therapy translation to solid tumors

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Whether this approach is feasible on a clinical scale remains to be determined.

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While CAR T cell therapy has shown unparalleled clinical efficacy in hematologic malignancies, solid tumors pose a much greater challenge to CAR T cell therapy [101-103]. This is due to numerous hindrances, most notably limited immune cell

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trafficking to the tumor site [104, 105], tumor heterogeneity in regard to antigen expression [106-108] and a highly immunosuppressive environment [109, 110].

Across cancer cells, most tumor-associated antigens are heterogeneously

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expressed both within individuals and between patient cohorts [111-114]. This heterogeneity further encompasses the mutational load, tumor-educated immune cells, as well as the non-cellular hindrance of the dense extracellular matrix, which poses an added challenge for CAR T cells in solid tumors [115]. This was underscored by a study from Beatty and colleagues, who employed anti-mesothelin CAR T cells for the treatment of metastatic adenocarcinoma. In one patient, metastatic liver lesions were seen to undergo a complete reduction in FDG uptake after one month, whereas the primary tumor experienced no reduction at all [116]. 15

This heterogeneity may very well explain the observed disparity in clinical responses for different diseases and disease subtypes.

It is striking that among hematological malignancies, the disease entities which display more “solid” tumor characteristics (e.g. immunosuppressive microenvironment, extracellular matrix barrier, slower tumor onset and latency), show poorer clinical responses to CAR T cell therapy, despite targeting the same antigen [15]. Characteristic of CLL and multiple myeloma (MM), for example, is their capacity to shape lymphoid and bone marrow niches, respectively. Within said

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niches leukemic cells interact with different types of stromal cells, such as mesenchymal stromal cells, monocyte-derived nurse-like cells, as well as T cells to promote tumor cell escape and hinder T cell anti-tumoral responses. The tumor

microenvironment of CLL for example was shown to induce T cell immune synapse dysfunction [117]. Therefore, CLL and MM display many characteristics which are

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frequently found in solid tumors and the success of immunomodulatory drugs, such as lenalidomide, in the treatment of CLL and MM [118, 119] was the result of their

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capacity to mitigate some of the effects of this immunosuppressive environment

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[120].

5.1. Postulated mechanisms of resistance in solid tumors

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5.1.1. Tumor infiltration

Strategies to improve T cell recruitment have recently garnered interest in light of the fact that higher numbers of tumor-infiltrating lymphocytes (TILs) correlate with a better clinical outcome [121]. This was shown for several solid malignancies, such as gliomas

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[122], colorectal [123], breast [124] and cervical cancers [125]. Anarchic tumor vasculature [126] or integrin and chemokine modulation within the tumor microenvironment [127-131] are known mechanisms by which a solid tumor becomes less permeable to the immune system. To exit the blood flow and access the tissue, leukocytes interact with endothelial cells, modulating the expression of selectins, chemokine receptors and integrins [132].

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However, depending on the tumor entity, the expression of chemokines may vary considerably, influencing the recruitment of immune cells. For instance ovarian and breast cancers can up-regulate CCL22 and CCL28, central chemokine ligands for the recruitment of T regulatory (Treg) cells [133, 134]. Colorectal tumors upregulate CCL2 and thus recruit tumor-associated macrophages (TAMs) [135]. Melanomas have been shown to modulate the expression of a variety of chemokine ligands such as CCL2, CCL3, CCL4, CCL5, CXCL9 and CXCL10 [129, 136] thereby decreasing the amount of pro-inflammatory immune cells in the tumor environment.

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Limited CAR T cell access to the tumor may be overcome by direct intratumoral or intracompartmental delivery of the T cell product [137]. Even peripheral expansion of tumor-specific T cells can increase anti-tumor efficacy, as showed by Ma et al. in a

study that used vaccination to enhance proliferation, activation and polyfunctionality [138]. However, not all tumor entities are easily accessible. Alternatively, CAR T cells

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may be modified to express matrix-degrading enzymes [139], or chemokine receptors [140] to potentiate their migratory and infiltrative capacity. These

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approaches have shown promising results in numerous preclinical studies. Kershaw and colleagues have equipped T cells with CXCR2 [141]. Di Stasi and colleagues

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incorporated CCR4 into CAR T cells in a murine model of Hodgkin lymphoma [142] whereas Rapp and colleagues have used the same chemokine receptor to improve T cell therapy in a murine pancreatic tumor model [143]. In a human neuroblastoma

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tumor model CCR2b was proven to be of benefit to the infiltration of CAR T cells by Craddock and colleagues [140]. In addition to using CX3CR1 to improve the trafficking of T cells to colon cancer, Siddiqui and colleagues could also demonstrate that the chemokine gradient between tumor and systemic circulation played a crucial

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role [144].

The current landscape of clinical trials co-engineering T cells is rather limited, with two clinical trials currently listed. One (NCT01740557) aims at incorporating CXCR2 into TILs, while the other (NCT03602157) combines CCR4 with a CD30-specific CAR. Moreover, these trials have not yet reported results. 5.1.2. Tumor microenvironment

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Upon entry into the tumor bed, T cells will be faced with mechanisms that will hamper their cytotoxic and proliferative capacity. This can be mediated in a contactdependent manner or via soluble factors secreted by tumor cells or immunosuppressive cells. Potent mediators of local immunosuppression include but are not limited to factors such as TGF-β, IDO, checkpoint molecules, as well as immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs) or Treg cells [127, 145, 146]. The extent to which a solid tumor is able to shape the immune system is crucial to the transition from the state of equilibrium to tumor escape [147]. Several components of the adaptive and innate immune system (e.g. NK cells, CD8+

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cytotoxic T cells and CD4+ T helper 1 T cells) help prevent tumorigenesis in healthy individuals [147]. These cell subtypes are the main effectors of anti-tumor immunity, releasing proinflammatory cytokines and ultimately mediating tumor cell lysis [147].

Dendritic cells are key regulators of the adaptive T cell response. If T cells encounter antigen-presenting cells in a proinflammatory milieu, T cells will be effectively primed

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and are consequently able to recognize and mount a potent response against their target cells. However, if this interaction occurs in an environment deprived of

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inflammatory mediators, T cells may be rendered anergic [148]. The presence of immunosuppressive ligands (IL-10, VEGF) secreted by the tumor cells may

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exacerbate this further [149, 150]. Besides impairing the anti-tumoral function of dendritic cells, IL-10 also prevents proper antigen-processing and presentation via the downregulation of TAP1 and TAP2 in tumor cells, depriving CAR T cells from the

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backup of the endogenous immune system [151]. TGF-β is another extensively characterized cytokine produced by both tumor cells and Treg cells and inhibits T cell activation, proliferation and differentiation [152-154]. The immunosuppressive function of inhibitory molecules such as CTLA-4 [155] and PD-1 [156] are mediated by cell-to-cell interactions driving T cells into an anergic state. Understanding these

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mechanisms has led to the successful implementation of combinatorial treatment strategies aimed at potentiating adoptive T cell therapy for several tumor entities.

Besides an abundance of immunosuppressive cells and ligands, physical factors such as hypoxia, lack of nutrients or low pH also contribute to the dysfunction of infiltrating immune cells [157, 158]. One elegant strategy exploits the lack of oxygen within the tumor environment by fusing the CAR with the oxygen sensitive

18

subdomain of HIF-1a [159]. CAR expression was therefore responsive to oxygen availability and would be upregulated within a hypoxic tumor microenvironment. The complex nature of the tumor microenvironment requires comprehensive approaches that integrate multiple diagnostic parameters to better stratify patients and assess both therapeutic options and patient prognosis. To this end Galon and colleagues have demonstrated the predictive power of an immunoscore that incorporates information about the type, quantity and location of immune cells within human colorectal tumors [123, 160].

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Besides the combination of CAR T cells with checkpoint blockade, the knockout of PD-1 in CAR T cells has shown promising preclinical results. Rupp and colleagues used the CRISPR-Cas9 system to disrupt PD-1 in anti-CD19 CAR T cells and reported enhanced in vitro cytotoxicity and significantly improved anti-tumor

response in a subcutaneous xenograft model [81]. This approach is translatable to

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other immune checkpoint molecules and multiplexed knockout may pave the way for

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improved CAR T cells therapy in highly immunosuppressive tumors [161].

Another preclinical study addressed this issue by combining ibrutinib, a tyrosine

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kinase inhibitor, with anti-CD19-specific CAR T cell product. It was demonstrated that the improved anti-tumor response of the CAR T cells by concurrent ibrutinib therapy was accompanied by a diminished PD-1 expression on CAR T cells

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providing a new tool to reduce anti-CD19-CAR T cell exhaustion [162].

Alternatively, the development of chimeric switch receptors, which convert inhibitory signals into T cell stimulatory ones [163, 164] may be an attractive strategy. One example of these receptors would be the PD-1-CD28 fusion receptor. Upon binding

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of tumor-derived PD-L1 transmits a CD28 costimulus to the modified T cell. Similarly, the incorporation of a dominant negative TGF-β receptor II on CAR T cells has proven effective, mitigating the effects of this immunosuppressive cytokine. Based on encouraging preclinical results [165], a phase I clinical trial is currently examining the therapeutic use of said dominant negative TGF-β receptor II CAR T cells for castration-resistant prostate cancer (NCT03089203).

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The combination of CAR T cells with oncolytic viruses has emerged as a promising strategy to overcome the limited success of CAR T cells in solid tumors, acting as vehicles for the delivery of therapeutic transgenes into the tumor microenvironment. Oncolytic adenoviruses engineered to express IL-15 and RANTES [166], or IL-2 and TNF have been reported to enhance accumulation and persistence of CAR T cells in the tumor tissue [167]. To better address the antigen heterogeneity of solid tumors, Wing and colleagues combined CAR T cells with an oncolytic adenovirus expressing a bispecific T cell engager (BiTE) targeting a second tumor antigen. One group successfully combined an oncolytic adenovirus expressing a PD-L1 blocking mini-

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antibody with CAR T cells, enhancing their therapeutic efficiency in solid tumors [168].

An alternative mechanism by which solid tumors could be made more susceptible to CAR T cell therapy is the combination with low-dose irradiation. DeSelm and

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colleagues found that activated CAR T cells secrete the death-inducing ligand

TRAIL. They found that TRAIL has a strong apoptotic effect on antigen-negative

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tumor cells after sensitization to TRAIL-mediated cell death by low-dose irradiation. They describe that irradiation enhanced tumor cell killing through a pathway

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independent of target expression [169].

Targeting Treg cells in combination with CAR T cell therapy may be another

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promising avenue to improve patient response. One example of this would be the combination of CAR T cells with mogamulizumab. This drug depletes CCR4+ cells (thus depleting Treg cells) and is currently under investigation in several clinical trials for cancer therapy, one of which combining mogamulizumab and nivolumab (NCT02705105). However, the depletion of Treg cells may come at the cost of

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triggering severe autoimmune reactions. In this case the tumor-specific modulation of Treg cell activity would be superior to their systemic depletion. Di Pilato and colleagues have reported that genetic disruption of the CARD11-BCL10-MALT1 (CBM) complex in Treg cells triggers genetic instability, resulting in an altered transcriptional profile, with more IFN- and TNF being secreted by those cells. The authors recapitulated these findings through systemic pharmacological inhibition, thereby transforming murine syngeneic “cold” melanoma tumors into “hot” tumors.

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This consequently primed them for effective anti-PD-1-therapy [170]. Such a therapy may similarly render the tumor microenvironment permissive to CAR T cell therapy.

With CAR T cell therapy only recently gaining the status of a clinically approved therapy, and despite their undeniable success in hematological malignancies, we are bound to see more improvements tackling the current solid tumor bottlenecks for successful CAR T cell therapy.

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5.1.3 Lack of tumor-specific target antigens

A major limiting factor preventing the successful use of CAR T cells in numerous

disease subtypes is the lack of truly tumor-specific antigens, as virtually all targets

for CAR T cell therapy are also expressed on healthy tissue. Despite CD19 not being specific to cancerous cells, the off-target killing of healthy B cells is a rather benign

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side effect, with the intravenous substitution of immunoglobulins being a readily

available treatment strategy [171]. This may however not be the case with antigens

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expressed on vital tissue, with several clinical reports underscoring the potentially severe consequences of off-target CAR T cell activation [48, 172, 173]. Therefore,

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the identification of truly tumor-specific targets for CAR T cell therapy is of crucial importance. In an attempt to broaden the range of suitable targets for CAR T cell therapy, it was shown that a single-chain variable fragment (scFv) against peptide-

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MHC complexes can redirect CAR T cells towards intracellular antigens [47, 174]. If this approach is used, scFv affinity may have to be tuned down to a TCR-like receptor affinity to avoid nonspecific recognition of HLA-molecules which are lacking the target peptide [47].

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Furthermore, unlike hematological tumors such as ALL or CLL which homogeneously express CD19, there currently is no universally expressed antigen within the heterogeneous solid tumor landscape, rendering the single target approach an overly simplistic one. Besides targeting multiple antigens, CAR T cells which target non-cancerous cells in the tumor environment, such as cancerassociated fibroblasts [175, 176], or the tumor vasculature [177] may help to overcome antigen heterogeneity on cancer cells.

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6.

Conclusion

The emergence of CAR T cell therapy has been as transformative as it has been effective in the treatment of relapsed or refractory hematological malignancies. Despite this, many patients show no response or relapse with therapy-resistant disease. Further, treatment-related toxicities are a caveat of this promising new therapy. Especially in the context of solid tumors, CAR T cells have been largely ineffective. This is due to numerous obstacles, which are characteristic of solid tumor

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entities, and successful translation of CAR T cell therapy to these malignancies will require a more comprehensive understanding of both tumor- and CAR T cell biology. Multiple genetic modifications may be required to help CAR T cells overcome the

translational bottlenecks and make them an effective and safe therapeutic strategy in solid tumors. Ultimately, this will require CAR T cells to be able to access the tumor

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site, to resist the tumor-driven immune suppression, persist and directly or indirectly recognize and mediate tumor cell lysis. The success of CAR T cell therapy thus far

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solid tumors to CAR T cell therapy.

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has rightly been heralded, though much work still remains to break the resistance of

Funding Source

SK receives funding from TCR2 Inc, Boston Conflict of Interest

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SK is an inventor of several patents in the field of cellular therapy. SK received speaker honoraria from GSK and Novartis.

7.

Acknowledgements

This study was supported by grants from the international doctoral program “i-Target: Immunotargeting of cancer” funded by the Elite Network of Bavaria (to SK and SE), the Melanoma Research Alliance (grant number 409510 to SK), the Marie-

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Sklodowska-Curie “Training Network for the Immunotherapy of Cancer (IMMUTRAIN)” funded by the H2020 program of the European Union (to SE and SK), the Else Kröner-Fresenius-Stiftung (to SK and SFK), the German Cancer Aid (to SK), the Ernst-Jung-Stiftung (to SK) by LMU Munich‘s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative (to SE and SK), by the Bundesministerium für Bildung und Forschung (to SE and SK), by the European Research Council Starting Grant (grant number 756017 to SK), by the DFG, the Hector foundation, the Bayerisches Staatsministerium für Wirtschaft (M4 award) and the José-Carreras Cancer Foundation to SK.

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The authors have no conflict of interest to declare. References

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Figure captions

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Figure 1: Hematological malignancies complete response rates to CAR T cell therapy

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Figure 2: Current limitations to CAR T cell therapy.

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Figure 3: Resistance mechanisms to CAR T cell therapy.

Figure 4: Solid tumors response rate to CAR T cell therapy.

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Figure 5: CAR T cell therapy translation bottlenecks to solid tumors.

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Figure 6: CAR T cell therapy – an overview.

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