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Promoter’s Choice Drive CAR-T Efficiency

The development and the functionality of Chimeric Antigen Receptor (CAR) therapy is highly linked to the stability and expression level of the receptor, for which the choice of the promoter is key. Although EF1α is a reference promoter to induce strong antigen binding and long-term CAR expression, we investigated the impact of this one and other constitutive promoters (MSCV, PGK, β2m and CMV) on a CAR123 construct in the Blastic Plasmocytoid Dendritic Cell Neoplasm (BPDCN). Our results indicate that lentiviral vector titer is affected by transgene nature and promoter activity. Interestingly, by moderating the expression level of CAR123 at the cell surface, its recognition capacity and selectivity were improved. We observed that in early stages after transduction, CAR123 expression level did J Biotechnol Biomed 2021; 4 (3): 63-83 DOI: 10.26502/jbb.2642-91280036 Journal of Biotechnology and Biomedicine 64 not affect lymphocyte’s phenotype and its cytotoxic capacity against BPDCN cell line. However, following long-term culture and decrease of cell-surface CAR expression, a hallmark of transition from activation to memory state of T-cells was observed, and only T-cells transduced with CAR-EF1α or -β2m promoters were still functional. We identified β2m as a promising promoter. Though it shows lower antigen-binding CAR-T cell and antigen recognition rates compared to EF1α, β2m promoter still present interesting CAR expression levels, stability, and binding capacity.


Introduction
Along the last two decades several immunotherapeutic strategies have been developed to generate an efficient treatment against cancers. Development of chimeric antigen receptors (CAR) [1] has been an important breakthrough since CARs allow the redirection of autologous T cells to target tumor cells. Several CARs targeting a broad range of tumor antigens are currently under development and are in clinical phases, especially for hematological malignancies [2][3][4][5].
We have developed a novel cellular immunotherapy against CD123 protein, which is an Interleukin-3 Receptor α chain expressed at the cell-surface, demonstrated to be involved in hematopoietic differentiation [6,7]. It was shown that CD123 is overexpressed in numerous hematologic malignancies, particularly in Blastic Plasmocytoid Dendritic Cell Neoplasm (BPDCN), acute myeloid leukemia [8] and acute lymphoblastic leukemia (ALL), whereas its expression is limited on normal hematopoietic stem cells, monocytes and endothelial cells [9,10].
The present study focuses on the CAR123 construct based on the B4D5 monoclonal antibody, which was recently demonstrated to eliminate BPDCN in vitro and in vivo [15]. This CAR123-chain was firstly vectorized by a Moloney murine leukemia virus vector. Since transgene introduction and regulation are crucial for CAR-T cell functionality and antigen recognition, this vector was replaced by the HIV-1 lentiviral vector, which is more adequate for this use in human T cells [15]. Also, in the context of gene therapy, Moloney derived vector presented safety issues [16][17][18], showing a propensity to integrate near gene transcription initiation sites inducing proto-oncogenes [19]. On the contrary, HIV genome was shown to integrate in distal regions of transcription initiation sites [20,21]. Thus, a lentiviral vector was selected for ongoing pre-clinical development because the risk of interference with flanking genes HIV-derived vectors is limited. More importantly, the type of promoter used to regulate the transgene expression is critical to ensure a long-term and stable expression of CAR-chain on human T cells [22]. Many constitutive promoters are described and can be used in lentiviral vectors, but few are able to sustain expression in human T cells.
In the present study, we investigated how different promoters influenced on our CAR123-chain expression, stability, and functionality, enabling to make a rational choice on the most appropriate one. Five different promoters were selected and assessed in primary human retroviral vector [23,26,27]; (iii) Human phosphoglycerate kinase (PGK), an endogenous housekeeping promoter showed to sustain a moderate and stable expression level [23,25]; (iv) Beta-2-Microglobulin (β2m) promoter, an ubiquitous and constitutive promoter especially strong in immune cells [28][29][30]; and (v) Human elongation factor 1 alpha (EF1α), a strong ubiquitous constitutive promoter classically used for stable gene transfer.
Here we report new CAR promoter optimizations to aim a stable and long-term cell surface expression on human T cells. We demonstrate that antigen binding is directly correlated to promoter intensity. Thus, by selecting a low intensity CAR123-T cell promoter we might attain a fine tuning anti-TAA engagement and reduce the "ontarget, off-tumor" effect.

Lentiviral vector production
Production and titration of lentiviral vector were formerly described [33].

CAR expression stability assay
To assess CAR cell surface expression, a truncated CD19 reporter (ΔCD19) was used as a selection marker [15]. Transduction efficiency was determined on the basis of ΔCD19 cell surface expression on transduced and analyzed with FlowJo software.

Evaluation of the binding capacity of the CAR in transduced cells
The expression of the CD123 CAR on cell surface of protein for 1h at room temperature, followed by PEstreptavidin (BD Biosciences, New Jersey, USA) incubation (dilution 1:1000).

In vitro Cytotoxicity assay
The CD123 + BPDCN cell line CAL-1 was kindly provided by Dr. Maeda, Nagasaki University, Japan. of target cells (CFSE + ). A cytotoxic assay was performed as described above.

Statistical analysis
Independent experiments were originally performed 3 times and then repeated until a significant variation was reported for demonstration key points. The data were analyzed using GraphPad Prism.

CAR expression level affects lentiviral vector production
CARs immunotherapy is highly dependent on lentivirus To assess the impact of the promoters' expression level on the titer of the CAR vector, we compared and measured the functional titer of the CAR123 vector production by qPCR ( Figure 2B). between CMV and β2m ( Figure 2C). Yet, for the membrane protein, the titer was higher with β2m promoter than with CMV promoter (18.2-fold increase for CAR-chain and 16.8-fold increase for hCD123).
Taken together, these results evidenced that cell-surface protein expression, particularly membrane CAR-chain expression, was clearly deleterious for vector production. As a result, CAR vectors with weak promoters in HEK-293T like β2m and PGK ensure better production than strong promoters like EF1α and CMV promoters. This observation is crucial for CAR development and manufacturing, but improving CAR vector production should not be done at the expense of CAR functionality in primary human T-cells.

Expression stability of CAR123-chain is related to the promoter
Expression level and stability are two critical parameters to generate functional CAR-T cells and we sought to study whether promoters could affect them.
Since no direct immunolabeling of CAR protein could be performed, we used the 3 rd generation CAR123 construct encompassing a CD19 truncated protein (ΔCD19) as a reporter protein to determine the promoter's activity and stability. CD3 + T cells were These results confirm that EF1α promoter presents better yields for CAR expression. Nevertheless, β2m promoter also ensures high and stable CAR expression in primary human T cells in comparison to CMV, MSCV and PGK promoters.

CAR expression level is associated to target binding and cytotoxic activity
We determined that the expression level of ΔCD19  Figure 4B). Though, none of the "low-binding" promoters were able to equal EF1α promoter yields with this approach.
These results confirmed that CAR binding capacity is tightly related to the CAR expression level. And only EF1α promoter allows the generation of CAR-T cells with a "high-binding" capacity due to a high expression level. Among "low-binding" group, β2m promoter is the most interesting promoter with a slightly superior binding capacity compared to the others.
To study the incidence of expression stability of CAR- This decrease in cytotoxicity for PGK, MSCV and CMV promoters was correlated with the loss of CAR expression observed between day 6 and day 37 ( Figure   3A). However, β2m promoter showed a singular profile.
Though it shared the "low-binding" profile with PGK, MSCV and CMV promoters, it demonstrated to maintain a stable protein expression, with higher expression level, and was able to sustain a cytotoxic activity comparable to EF1α at day 40.
Thereby, among all promoters tested, this observation points out the advantage of using either EF1α to generate CAR with a "high-binding" profile, or β2m promoters for a "low-binding" profile. Both promoters ensure an efficient level of expression of CAR123-chain in human T-cells to preserve a prolonged efficiency.  hinge of human IgG4 and 28BBz backbone [55]. Reporter ΔCD19 was a truncated version of CD19 protein (1-313aa)

Differentiation of CAR123-T cells is not promoter-dependent after CAL-1 co-culture
co-expressed through a P2A cleavage site. Differentiation phenotype was determined through CD62L and CD45RA cell surface expression, 6 days after transduction and 12 days after being exposed to CAL-1 at a ratio E:T of 10:1. (C) PD-1 expression in CD8 + ΔCD19 + population before and after 12 days of stimulation. Columns represent mean ± SEM of 3 different donors. Significance was determined using a Mann-Whitney test (ns: non-significant).

Discussion
In this work, we investigated whether promoters with different activity could modulate CAR-chain production, expression, antigen recognition and subsequent cytotoxic function against the targeted antigen. Several constitutive promoters were tested to determine their effect on CAR-T cells reactivity against CD123 antigen using our CD123 CAR construct.
Firstly, we evidenced that lentiviral vector titer is affected by transgene nature and promoter activity: type I membrane-bound proteins, such as the CAR construct, CD123 presents a differential level of expression between most of BPDCN blasts and normal hematopoietic cells [11,41]. In this context, "on-target, off-tumor" toxicity can be deleterious for endothelial  Also, it has been demonstrated that introduction of a CAR in a TRAC locus, that mimics TCR regulation, prevents tonic CAR signaling with a basal low level of expression and allows its modulation after stimulation [48]. Thus, β2m promoter, regulated by inflammation signals like Interferons α/β and γ through ISRE motifs, could ensure a CAR expression profile closer to a natural TCR regulation [28,29].
Our work demonstrates that not all promoters are suited for CAR-T cells development. EF1α remains a robust choice for further development in the context of CD123 B4D5 CAR-T cells. Nevertheless, regarding production, expression level and binding capacity, β2m promoter should be considered as a fine-tuned approach to address the "on-target off-tumor" issue, using low affinity paratopes [52]. Although this study focuses on the CD123 targeting, our results could also be profitable to other CAR sharing the same targeting problem, such as CD33 on hepatic Kupffer cells [53] or CD44 on keratinocytes [54].