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Building a better CAR: emerging high-throughput in vitro tools for CAR
selection and optimization
MEETING PRECLINICAL
DATA REQUIREMENTS FOR
CELL & GENE THERAPIES
EXPERT INSIGHT
Building a beter CAR: emerging
high-throughput in vitro tools for
CAR selecion and opimizaion
Darin Bloemberg, Scot McComb
& Risini Weeratna
Engineered cell-based therapies show great promise as potenial can-cer cures, paricularly regarding chimeric anigen receptor T cell (CAR-T) treatment of B-cell leukemia. While ani-CD19 CAR-T therapies demon-strate strong clinical responses in hematological malignancies, only a subset of paients show durable remission and trials targeing solid tu-mor anigens exhibit less encouraging results. The lack of wider CAR-T success likely results from the complexity of this therapeuic modality whereby many factors including the anigen binding domain character-isics (i.e. ainity, avidity), CAR-T phenotype, and micro-environment within individual tumors/tumor-types impact its eicacy. Hence, the ex-tensive toolbox generated and uilized to opimize CAR-T for leukemia may not be applicable to the development of CAR-T against other tumor types. Here, we outline the available in vitro techniques for designing, as-sessing, and opimizing CAR ectodomains in anicipaion that leveraging them will accelerate pre-clinical CAR-T development. These strategies exploit advancements in molecular biology, geneic engineering, biotech-nology and high-throughput analysis to manipulate and examine T-cell characterisics learned from decades of cellular immunology research.
Submited for peer review: May 1 2019 u Published: Jul 22 2019
Cell & Gene Therapy Insights 2019; 5(7), 681–692
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682
INTRODUCTION
Chimeric antigen receptors (CARs) were originally devised by recom-bining antibody variable regions with TCR constant regions to re-program T-cell speciicity [1–3]. Soon thereafter, several groups demonstrated that human CAR-T cells engineered to express an an-ti-CD19 CAR efectively eradi-cated B-cell malignancies in mice
[4–6] and showed clinical promise treating patients sufering from re-lapsed and/or refractory hemato-logical cancers [7–9]. While these results are encouraging, changes to CAR design, T-cell phenotypes, manufacturing processes and dos-ing/combination strategies will be required to improve response out-comes and validate CAR-T’s useful-ness in other tumor types [10–12]. In fact, in vitro analyses can provide relevant insight regarding ainity, tonic activation, signaling strength/ kinetics and CAR structure/expres-sion that can model or predict even-tual CAR-T function. As testing these myriad variables is not pos-sible using in vivo methods, their true optimization requires genuine high-throughput analyses and likely needs performed for every CAR-an-tigen combination.
TRADITIONAL
IMMUNOLOGY
Traditional immunological analyses are an important aspect of pre-clical CAR-T development. his in-cludes examining antigen-driven responses such as cytokine produc-tion (IL2, TNFα, IFNγ), cell prolif-eration and target cell killing using CAR-T cells generated from prima-ry human PBMCs [4–6]. Although
traditionally performed using ELISA [4], cell expansion [13,14]
and chromium release assays [5,6], multi-color low cytometry (FACS) has generally replaced these meth-ods [15,16]. Multi-parameter FACS allows simultaneous assessment of cytokine production, T cell pheno-type and diferentiation statues (i.e. efector, memory), all of which can eventually impact CAR-T function
[11,17]. Similarly, the availability of luorescent viability dyes has simpli-ied co-culture cytotoxicity testing and eliminated the need to handle chromium.
Although these techniques are highly relevant and remain a critical part of CAR development [15,16], there are drawbacks to applying them in screening experiments. Particularly, this includes expertise in culturing and manipulating pri-mary human T cells, the diversity of T-cell transduction and expansion protocols, and access to high-quality lentivirus/retrovirus. As a result, as-sessing CARs using these strategies is relatively low-throughput and re-quires large amounts of ‘hands-on’ time per CAR. Fortunately, sever-al high-throughput ansever-alysis tech-niques are adaptable to cell-based assays thereby allowing more rapid CAR assessment.
ANTIBODY OPTIMIZATION
FOR CAR DESIGN
Monoclonal antibody (Mab)-de-rived single chain variable fragments (scFvs) constitute the large majority of CAR ectodomains (ECDs) and selecting an appropriate antibody is key to CAR design. High-speci-icity scFvs are generally desired for CAR applications to limit of-target toxicity; therefore, implementing
robust and comprehensive proto-cols to identify antibody candidates is paramount to downstream CAR development.
Mabs typically undergo signii-cant characterization (i.e., speciic-ity, ainspeciic-ity, avidspeciic-ity, internalization) and optimization (i.e., ainity mod-ulation, humanization) during their development. However, apart from knowledge that the scFv derived from FMC63 used in many CD19 CAR-T products possesses relatively high ainity Ka values (0.4 nM and 0.2 nM for the antibody and scFv, respectively [18]), it is currently un-clear which antibody properties cor-respond to their eventual function as CARs [19]. Furthermore, opti-mal Mab characteristics likely vary across antigens and/or tumor types. For example, lower-ainity Mabs improve CAR selectivity for cancer antigens which are overexpressed on tumor cells but also present in nor-mal tissues [14], while higher-ain-ity Mabs may be appropriate with tumor-restricted antigens such as EGFRvIII or when clinical efects of of-tumor killing are reasonably manageable, as with CD19 [20]. Importantly, our understanding of optimal antibody ainity for bal-ancing T-cell cytolytic responses with persistence and exhaustion re-mains a complex problem [21].
Relevantly, the steric interactions between puriied/recombinant an-tibody/scFv and antigen do not recapitulate a CAR and its antigen due to a number of factors: 1) vari-ation in CAR surface expression, pre-clustering and association with additional molecules; 2) antigen density and presentation on target cells; 3) epitope availability; and 4) post-transcriptional modiications. hese issues are further compound-ed in the context of multi-targetcompound-ed
CAR-T cells and optimizing other structural regions (i.e., hinge and signaling), which cannot be evalu-ated or predicted using these meth-ods. Furthermore, as novel antigen binding domains (ABD) such as single-domain antibodies (sdAb) derived from camelidae species are introduced into CARs [22], such legacy Mab knowledge and char-acterization techniques may be-come less useful. Similarly, cellular factors, such as the numerous gene editing strategies being investigated, necessitate cell-based screening as-says performed in high-throughput formats.
IN VITRO
TUMOR
MODELLING
High-throughput imaging
A number of relatively high-through-put detection techniques are amena-ble to CAR-T cytotoxic evaluation to supplement or replace FACS-based methods. In practice, these assays mirror common drug screen-ing protocols. hey are enabled by genetically engineering target cells to express reportable markers (i.e., luciferase or β-galactosidase [23]) thereby negating the need for via-bility dyes or markers (i.e., LDH, GAPDH). While irely luciferase is infamous for its short half-life
[24], engineered luciferase mutants demonstrate robust function in this context [25]. In fact, generating tar-get cell lines with stable expression of signal peptide-deicient luciferase enables spectroscopic CAR-T cyto-toxicity testing in 384 well plates without additional washing steps
[25].
High-throughput luores-cence-based microscopic analyses are
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also increasingly available as many life sciences suppliers provide systems capable of live-cell imaging single- to 384-well plate formats. By engineer-ing CAR-T and target cells with dif-ferent luorescent proteins, both can be tracked and imaged over time for parallel examination of target inter-action, CAR/target proliferation and cytotoxicity thereby representing a powerful and informative in vitro as-say [26]. Notably, spectroscopy and microscopy may be preferential to FACS for CAR-T analyses as they are more compatible with adherent target cells and can assess time-based efects. In our own hands, imaging GFP-expressing CAR-T cells co-cul-tured with mKate-labelled tumor cells provides consistent data regard-ing target cell killregard-ing and CAR-T proliferation (Figure 1).
3D culture
3D culture techniques are quick-ly becoming an alternative model to traditional 2D/monolayer cell culture for testing cancer therapeu-tics. hese systems better mimic the transition in oxygen and nutrient levels across physical space typical of solid tumors, resulting in cancer stem cell and necrotic core develop-ment, and can additionally include multiple cell types [27]. Although creating tumor spheroids has typi-cally been time consuming and re-quired relatively complex labware, ultra-low attachment multi-well culture plates are easing the tech-nical skill needed for 3D spheroid generation from numerous cancer cell lines [28]. Combining such approaches with the imaging tech-niques described above may result in a much more relevant context in which to examine solid tumor
killing in vitro; a strategy CAR-T researchers are actively using [29].
Zebraish xenograph
Moving one step closer to in vivo models, zebraish are increasingly used for cancer drug discovery due to their relative fecundity, rapid de-velopment and ease of manipulation compared to immunocompromised mice [30]. Importantly, many human cancers have been demonstrated to xenograft well in zebraish, thereby incorporating 3D tumor properties as well as mechanisms of invasive-ness, metastasis, and angiogenesis into this cancer model [31]. Although the function of CAR-T cells in ze-braish tumor models has not to our knowledge yet been demonstrated, they may represent a useful pre-clini-cal tool for eiciently evaluating mul-tiple CAR constructs.
METABOLIC ASSESSMENT
In addition to canonical immune responses, T cells dramatically alter their metabolism upon activation, characterized by a spike in glycolytic activity [32,33]. Furthermore, these responses may denote or predict speciic diferentiation phenotypes, where TCM cells possess increased capacity for increasing mitochon-drial oxygen usage [34]. Although measuring metabolic changes has traditionally been relatively la-borious, new technologies allow high-throughput metabolic assess-ment using small numbers of live cells [35]. In fact, activation through CARs with diferent co-stimulatory domains causes development of T cells with diverse metabolic proiles
metabolic analysis may help validate the particular diferentiation pheno-types of manufactured CAR-T cells.
COMPUTATIONAL
MOD-ELING OF
RECEPTOR-AN-TIGEN INTERACTIONS
Computer modelling techniques are used extensively in targeted small molecule [37] and antibody [38]
development. his type of predic-tive 3D modeling is often used to humanize scFv derived from mouse Mabs [39]. While 3D structural modeling and binding prediction of entire CARs with their antigens has not yet been reported, researchers have begun to simulate CAR sig-naling elements [40] in order to un-derstand and predict their function. Accurately modeling CAR-antigen
binding in silico would represent a major advancement, enabling the screening of CAR constructs de-rived from rational design and ma-chine learning strategies.
ASSESSING CARS USING
IMMORTALIZED CELLS
Assessing CAR function in cell lines has the obvious advantage of not re-quiring manipulation of donor-de-rived primary human cultures. Many studies utilize Jurkat cells (an immortalized human T cell line
[41]) for in vitro CAR testing as they increase IL2 output in response to CD3 stimulation as well as through CAR [42,43] and recombinant TCR
[44] signaling. Relevantly, a Jurkat line containing retrovirally-deliv-ered open reading frames for EGFP,
f
FIGURE 1
High-content live imaging of CAR-T:target cell co-cultures.
Human T cells derived from donor PBMCs were transduced with disinct CAR leniviruses (CAR 1 or CAR 2) that co-express GFP and cultured with target cells stably expressing mKate2. The igure illustrates the increase in CAR-T proliferaion (as indicated by increased green count) and corresponding reducion in tumor cells (red count) as depicted in middle panel with CAR1.
DOI: 10.18609/cgi.2019.078
686
ECFP, and mCherry driven by re-sponse elements for NFAT, NFκB and AP1, respectively, has been generated, thereby allowing FACS-based detection of their transcrip-tional activity [45]. A similar cell line (NFAT and NFkB reporting) was also used to validate CAR func-tion and screen scFv libraries [46]. Although a novel scFv was not identiied in this experiment, such an approach represents an extreme-ly powerful tool for CAR develop-ment. We have similarly generated a Jurkat line where tdTomato expres-sion reports CD69 transcriptional activity, providing a useful tool for detecting functional CAR-induced activation signaling [Unpublished data].
Although Jurkat cells adequately report T-cell activation, they display weak cytotoxicity. If interested, im-mortalized natural killer (NK) cells can be used to examine such efects. he best known of these, NK92, was derived from a fast-growing non-Hodgkin lymphoma and con-stitutes somewhat-activated NK cells with cytotoxic efects against numerous cells (notably, it is CD16-negative) [47,48]. NK92 can be engineered using retroviral trans-duction or electroporation, though the latter is more diicult [25,48]. A CD3ζ-containing CAR delivered via mRNA electroporation enabled NK92-directed killing of various CLL cell lines [49], demonstrating their utility in this context.
TOWARDS
HIGH-THROUGHPUT
CAR SCREENING &
OPTIMIZATION
Evidently, there are numerous as-pects of CAR-T to examine and
optimize and an equal number of ways to measure their activity. As each possesses ‘pros’ and ‘cons’, no single technique adequately and eiciently detects and/or predicts CAR-T function. Despite this, be-cause CAR ECDs perform the rel-atively simple function of antigen binding (much like antibody vari-able regions), their optimization is amenable to genuine high-through-put assessment.
CAR ECTODOMAINS:
HINGE, SCFV & LINKER
As mentioned, a current unan-swered question contributing to the bottleneck in CAR develop-ment is selecting antibodies from which to derive ABDs. Mabs are the most common source of CAR ECDs and the process of creating antibodies has been expanded and simpliied by 40 years of scientiic advancement, where hundreds are assessed during antibody generation projects. Given the cost/complexity of testing scFv, transferring them to CAR molecules, and screening for CAR activity, shortlisting candi-dates from large hybridoma librar-ies is a fundamental and paramount decision currently made with little insight. To remedy this, we suggest that CAR binding and simple recep-tor functions should irst be assessed in cost-eicient and high through-put cell-based assays (Figure 2).
An example strategy involves detecting canonical T-cell activa-tion using CAR-expressing Jurkat cells. Here, various scFv sequenc-es are cloned into a modular CAR backbone plasmid to minimize gene synthesis. Jurkat cells are then electroporated with CAR-express-ing plasmids, co-cultured with
speciic target cells, and assessed for activation markers (i.e., IL2 production, CD69, CD25). In our hands, such an approach involving high-throughput FACS assessment of CD69 reliably reports speciic CAR-mediated activation in re-sponse to numerous cancer anti-gens [Bloemberg et al.; Manuscript in Preparaion] and allows speciicity and signaling magnitude to be ad-justed by altering hinge/linker for-mats. hus, this strategy can iden-tify ABDs with desired speciicity and optimize CAR constructs as ac-tual antigen receptors thereby solv-ing the Mab-related shortcomsolv-ings mentioned above. Furthermore, due to the rapid decrease in DNA fragment synthesis and robustness of novel molecular cloning strate-gies [50,51], appropriately-designed CAR-expressing plasmids can be created in arrayed and library for-mats, enabling the assessment of large numbers of CAR constructs.
In fact, such cell-based strategies are compatible with large-format and micro-luidic screening devices, opening the opportunity for fully automated analyses. Taking this one step further, this establishes the pos-sibility of bypassing antibody gener-ation and/or screening during CAR development. Here, libraries or ar-rays of DNA fragments encoding ECDs derived from rational design, machine learning, mutational mat-uration, immunized animal cells or un-screened hybridomas can be cloned into a modular CAR plas-mid, delivered to Jurkat cells, and CAR-mediated activation of these cells can be determined after short-term co-cultures. In combination with properly-designed reporter cell lines [45,46], such an approach could greatly accelerate the CAR development cycle.
CAR ENDODOMAINS:
CO-STIMULATION &
ACTIVATION SIGNALING
In our experience, Jurkat cells dis-play limited use for examining CAR endodomain activity, as they do not suiciently replicate import-ant physiological T cell attributes such as speciic downstream efects of diferent activation signals (i.e., CD3ζ-only versus CD28-41BB-CD3ζ versus other/novel signaling domains) or their associated long-term stimulation efects (i.e., dif-ferentiation phenotype and exhaus-tion). For these reasons, screening CAR ECD to demonstrate recep-tor functionality is a useful step in shortlisting scFv sequences before transducing primary human T cells to examine other aspects of CAR-T function.
TRANSLATION INSIGHT
CAR-T is the most complicated human therapeutic. Unsurprisingly, its development is complex and per-forming CAR-T research requires signiicant technical and academic expertise alongside a dedicated re-search staf and supportive institu-tion. he lack of success in non-he-matological malignancies is a major stumbling point in the long-term adoption of CAR-T, but may po-tentially be explained by over-op-timization of current therapies for leukemia [52]. Of course, many anatomical, structural, and cellular/ humoral factors associated with the micro-environment of solid tumors present roadblocks that impair the simple translation of CAR-T: after all, diferent cancers never respond similarly to the same therapeutic. his suggests that modern CAR-T
688
688
DOI: DOI: 10.18609/cgi.2019.078 Cell & Gene Therapy Insights - ISSN: 2059-7800f
FIGURE 2
Overview of typical CAR development pipeline.
development technologies will not only enhance our understanding of CAR design and immuno-oncol-ogy, but will also lead to efective solid tumor therapies.
he high-throughput in vitro techniques discussed here will ex-pand the toolbox of CARs available for potential therapeutic applica-tion and provide key data to answer looming technical and academic questions regarding CAR biology. For example, how does ABD af-inity relate to on- versus of-target CAR speciicity and to on-target, of-tumor responses? Potentially more importantly, understand-ing what antibody properties pre-dict auto-activating (tonic) signals within CAR-T cells remains a key mystery. Improved ABD discovery and design will ultimately be key to accelerating CAR-T therapeutic development.
A related question is how best to optimize CAR signaling for a strong antigen-speciic response and a long-term T-cell persistence. Although the desired function of CAR-T cells is antigen-speciic activation and target cell killing, the in vitro responsiveness T cells is inversely correlated to their in
vivo anti-cancer function [53–55]. his adds to long established dog-ma in T-cell biology, that strong and chronic antigenic signaling
causes T-cell exhaustion and aner-gy [56]. hese properties of CAR constructs could be assessed by combining the high throughput FACS-based screening platforms and live real-time microscopy systems. Overall, we believe that high-throughput cell-based in
vi-tro methods for CAR assessment
will quicken their development by customizing CAR-T characteristics for particular malignancies.
FINANCIAL & COMPETING INTERESTS DISCLOSURE
Darin Bloemberg, Scott McComb, and Risini Weeratna wrote the manuscript as employees of the National Research Council Canada and received no outside assistance or inluence regarding its content. Darin Bloemberg and Scott McComb have no personal or inancial conlicts-of-interests to disclose; Risini Weeratna owns stock in Pizer Inc. Darin Bloemberg, Scott McComb, and Risini Weeratna are co-inventors of a provisional patent application regarding novel CAR targeting domains.
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AFFILIATIONS
Darin Bloemberg, Scot McComb, Risini Weeratna†
Naional Research Council Canada, Otawa, ON, Canada
†Author for correspondence:
