Natural Flt3Lg-Based Chimeric Antigen Receptor (Flt3-CAR) T Cells Successfully Target Flt3 on AML Cell Lines

Relapsed/refractory acute myeloid leukemia (AML) cannot be cured with chemotherapy alone, as the blasts survive the treatment. Chimeric antigen receptor (CAR) approaches for AML are being actively developed. CARs promote immune reactions through recognition of the target molecular epitopes at the surface of cancer cells. The recognition involves the extracellular portion of the CAR protein, which corresponds to either the antibody or the physiological binding partner of the targeted antigen. Here, we design a chimeric receptor with a full-length natural Flt3-ligand recognition module that targets Flt3 tyrosine kinase, known as an adverse marker in AML. We demonstrate specific killing of Flt3-positive THP-1 cells by Flt3-CAR T cells and the lack of cytotoxicity towards Flt3-negative U937 cells. We prove that the inherent cytolytic capacity of T cells is essential for the killing. Finally, we confirm the authenticity of targeting by its competitive dose-dependent inhibition with a soluble Flt3-ligand. The developed system can be viewed as a non-immunogenic functional equivalent of scFv-mediated targeting. The robust in vitro antitumor effects of Flt3-CAR T cells, combined with their low off-target cytotoxicity, hold promise for AML treatment.


Introduction
Acute myeloid leukemia (AML) is a hematopoietic neoplasm defined by misregulated clonal expansion of myeloid progenitor cells (blasts). First-line treatment for AML is based on intensive chemotherapy on the backbone of cytarabine and anthracycline. A significant proportion of patients require hematopoietic stem cell transplantation (HSCT) to secure long-term survival [1]. Both frontline chemotherapy and HSCT have been associated with multiple adverse effects, including long-term sequelae. Despite the intensity of the treatment approaches, leukemic blasts may develop resistance. The clear impossibility of the further intensification of the standard chemotherapy regimens necessitates the development of alternative and complementary treatment modalities, including, above all, immunotherapy.
Early steps in cancer immunotherapy involved the creation of vaccines, e.g., peptide vaccines, whole-cell vaccines, and dendritic cell-based vaccines, aimed at stimulating anticancer immune responses. For example, the primed dendritic cells were expected to promote the maturation of cytotoxic lymphocytes armed against tumor cells. This concept, on its own, failed to provide effective antitumor therapy [2]. Yet, the attempts to
The pmKate2 vector (Evrogen, [17]) was used as a template to introduce human c-Myc nuclear localization signal (NLS) at the C-terminal end of mKate2 by PCR. The product was subcloned into pLVX-EF-1α (Takara) vector.
The lentiviruses were packaged using HEK293T cell line. HEK293T cells were transfected with Lenti-X Packaging Single Shots (VSV-G) (Takara) according to optimized commercial protocol. The virus-containing supernatant was collected 48 h post-transfection, concentrated using Lenti-X Concentrator (Takara), aliquoted, and preserved at −80 • C until use.

Generation of CAR T Cells, CAR Jurkat Cells, and THP-1-mKate2 and U937-mKate2 Cell Lines
All cultures were maintained at 37 • C in a humidified atmosphere of 5% CO 2 . Primary donor T cells were isolated by apheresis on the CliniMACS Prodigy ® (Miltenyi Biotec). T cells were seeded in 24-well plates at 1 × 10 6 cells per well, activated with TransAct reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), transduced with CAR gene cargo viruses, and expanded for 7 days in TexMacs medium (Miltenyi Biotec). The culture medium was supplemented with IL-7 and IL-15 (25 ng/mL, Miltenyi Biotec). Jurkat cells were seeded in 24-well plates at 5 × 10 5 cells per well and transduced with CAR gene cargo viruses. Transduction efficiency was estimated by flow cytometry. EYFP-synthesizing cells were regarded as CAR-positive.
THP-1 and U937 cells were seeded at 5 × 10 5 cells per well and transduced with mKate2 cargo viruses. After transduction, the THP-1-mKate2 and U937-mKate2 cells were subjected to puromycin selection to eliminate non-transduced cells. Selection efficiency was confirmed by flow cytometry.

Western Blotting
The transduced CAR T cells and CAR Jurkat cells were collected, washed with Dulbecco's Phosphate Buffered Saline (DPBS, HyClone), and lysed in Laemmli sample buffer supplemented with β-mercaptoethanol (Bio-Rad, Hercules, CA, USA). The samples were boiled for 10 min at 100 • C before loading. The proteins were run on SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Chicago, IL, USA). The membrane was blocked overnight in 1% ECL Block (GE Healthcare). CARs were stained with anti-CD3ζ-chain antibody (Sigma) detected with anti-Rabbit-HRP (GE Healthcare). EYFP was stained with anti-GFP antibody (SCI-Store) detected with anti-Mouse-IgG-HRP (GE Healthcare).

Cell Culture
Human HEK293T, Jurkat, THP-1, and U937 cells were obtained from the Russian Cell Culture Collection of Vertebrate Cells (Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia).

CAR T and CAR Jurkat Cell Cytotoxicity Assay
Transduced T cells were rested under cytokine starvation for 3 days and transferred to RPMI complete medium. The 96-well plates for the assay were pre-coated with 10 µg/mL human plasma fibronectin (IMTEK) and washed with DPBS. CAR T cells were mixed with THP-1-mKate2 or U937-mKate2 cells at an effector-to-target ratio (E:T) of 1:1 or 5:1. The cells were incubated for 4 days (37 • C, 5% CO 2 ) in the IncuCyte ® S3 Live-Cell Imaging System (Sartorius, Göttingen, Germany). Each well was imaged at 9 locations; all experiments were repeated at least 3 times.
The competitive Flt3Lg binding experiments involved soluble Flt3Lg (SCI-Store) in final concentrations of 100, 20, 2, and 0.2 ng/mL. The ligand was added to the coculture medium before mixing of cell suspensions.

Cell Proliferation Assay
Proliferation of THP-1 cells in the presence of Flt3Lg was measured by optimized MTS assay. The cells were washed with serum-free RPMI medium and seeded in a 96-well plate, in low-serum RPMI medium. Soluble Flt3Lg (SCI-Store) was titrated from 30 ng/mL by 3-fold serial dilutions. Proliferation was assessed using CellTiter 96 ® AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer protocol.

Quantitative Data Handling
Statistical analysis of flow cytometry data was performed with Kaluza software. For the MTS assay, all measurements were made in triplicates and represented as the mean values. All experiments were repeated at least 3 times.

Generating Flt3-CAR
To target Flt3, we engineered a chimeric antigen receptor Flt3-CAR comprising fulllength polypeptide sequence of Flt3Lg (soluble isoform) known to bind Flt3 with high affinity (2.5 × 10 10 M −1 [18]). The Flt3Lg moiety in the extracellular part of Flt3-CAR is supposed to be non-immunogenic, as it corresponds to a real human cytokine.
Our Flt3-CAR contains a combined ICOS and 4-1BB co-stimulatory domain and a classical CD3ζ activating domain. It is a natural ligand-based third generation CAR intended for targeting Flt3 receptors on blast cells. The EYFP tag, attached to the C terminus via a self-cleaving P2A peptide linker, facilitates the evaluation of transduction efficiency. We also produced gene constructs encoding Flt3-CAR without EYFP (Flt3-CAR EYFP-) and CAR19 (targeting CD19 antigen; Figure 1A).
Primary donor T cells were activated, transduced with CAR gene cargo lentiviruses, and analyzed by Western blotting. High bands corresponding to CD3ζ domain of CAR were observed for all studied constructs ( Figure 1B). CAR19 T cells were used as a positive control. For both Flt3-CAR and Flt3-CAR EYFP-, bands of the same weight were successfully detected. The double bands observed for Flt3-CARs apparently reflect partial glycosylation of the Flt3Lg moiety. The same cell lysates were used for anti-EYFP immunoblotting, demonstrating the proper cleavage of EYFP and the absence of residual fusions ( Figure 1C).
Based on these findings, EYFP-synthesizing cells were regarded as CAR-positive. Relative counts of EYFP-positive cells indicated the average transduction efficiency of 49% ( Figure 1D). Primary donor T cells were activated, transduced with CAR gene cargo lentiviruses, and analyzed by Western blotting. High bands corresponding to CD3ζ domain of CAR were observed for all studied constructs ( Figure 1B). CAR19 T cells were used as a positive control. For both Flt3-CAR and Flt3-CAR EYFP-, bands of the same weight were successfully detected. The double bands observed for Flt3-CARs apparently reflect partial glycosylation of the Flt3Lg moiety. The same cell lysates were used for anti-EYFP immunoblotting, demonstrating the proper cleavage of EYFP and the absence of residual fusions ( Figure 1C).
Based on these findings, EYFP-synthesizing cells were regarded as CAR-positive. Relative counts of EYFP-positive cells indicated the average transduction efficiency of 49% ( Figure 1D).

Flt3-CAR T Cells Specifically Kill Flt3-Positive Cells
Human cell lines THP-1 (Flt3+) and U937 (Flt3-) were used as AML model. The immunophenotypes [19] were confirmed by staining with anti-Flt3-PE antibodies ( Figure  2A,B). Both target cell lines were transduced with mKate2 cargo lentivirus to make them fluorescent ( Figure 2C,D) and thereby distinguishable from the CAR T cells during coincubation experiments.

Flt3-CAR T Cells Specifically Kill Flt3-Positive Cells
Human cell lines THP-1 (Flt3+) and U937 (Flt3-) were used as AML model. The immunophenotypes [19] were confirmed by staining with anti-Flt3-PE antibodies (Figure 2A,B). Both target cell lines were transduced with mKate2 cargo lentivirus to make them fluorescent ( Figure 2C,D) and thereby distinguishable from the CAR T cells during co-incubation experiments.

Specific Cytolytic Capacity of T Cells Is Necessary for Flt3-CAR-Mediated Killing
To assess the significance of T cell functional identity for Flt3-CAR-induced killing, we developed a Flt3-CAR T cell model with impaired cytolytic activity. We used Jurkat cells as a non-cytotoxic T cell equivalent and transduced them with Flt3-CAR, Flt3-CAR EYFPand CAR19 gene cargo lentiviruses ( Figure 1A). Upon transduction, Jurkat cells produced Flt3-CAR, Flt3-CAR EYFP-, and CAR19 identically with the donor-derived T cells, as confirmed by Western blotting (Figure 4A). Similar bands of appropriate size were detected with anti-CD3ζ staining. Transduced with Flt3-CAR, the Jurkat population was 43.1% EYFP-positive ( Figure 4B). Eventually, we developed Jurkat cell lines expressing Flt3-CAR, Flt3-CAR EYFPand CAR19 proteins.

Specific Cytolytic Capacity of T Cells Is Necessary for Flt3-CAR-Mediated Killing
To assess the significance of T cell functional identity for Flt3-CAR-induced killing, we developed a Flt3-CAR T cell model with impaired cytolytic activity. We used Jurkat cells as a non-cytotoxic T cell equivalent and transduced them with Flt3-CAR, Flt3-CAR-EYFP-and CAR19 gene cargo lentiviruses ( Figure 1A). Upon transduction, Jurkat cells produced Flt3-CAR, Flt3-CAR EYFP-, and CAR19 identically with the donor-derived T cells, as confirmed by Western blotting (Figure 4A). Similar bands of appropriate size were detected with anti-CD3ζ staining. Transduced with Flt3-CAR, the Jurkat population was 43.1% EYFP-positive ( Figure 4B). Eventually, we developed Jurkat cell lines expressing Flt3-CAR, Flt3-CAR EYFP-and CAR19 proteins. .

Specific Cytolytic Capacity of T Cells Is Necessary for Flt3-CAR-Mediated Killing
To assess the significance of T cell functional identity for Flt3-CAR-induced killing, we developed a Flt3-CAR T cell model with impaired cytolytic activity. We used Jurkat cells as a non-cytotoxic T cell equivalent and transduced them with Flt3-CAR, Flt3-CAR-EYFP-and CAR19 gene cargo lentiviruses ( Figure 1A). Upon transduction, Jurkat cells produced Flt3-CAR, Flt3-CAR EYFP-, and CAR19 identically with the donor-derived T cells, as confirmed by Western blotting ( Figure 4A). Similar bands of appropriate size were detected with anti-CD3ζ staining. Transduced with Flt3-CAR, the Jurkat population was 43.1% EYFP-positive ( Figure 4B). Eventually, we developed Jurkat cell lines expressing Flt3-CAR, Flt3-CAR EYFP-and CAR19 proteins. .

Soluble Flt3Lg Promotes Proliferation of THP-1 Cells
Flt3 is known to mediate the abnormal proliferation of AML blasts [20]. The acute monocytic leukemia-derived THP-1 cells express wild-type Flt3. Using an optimized MTS assay, we showed that soluble Flt3Lg stimulates THP-1 proliferation (Figure 6). With the addition of recombinant human Flt3Lg, the proliferation of THP-1 cells increased in a dose-dependent manner (ED 50 = 0.9 ng/mL) reaching saturation at 10 ng/mL. Vaccines 2021, 9, x. 8 of 12

Soluble Flt3Lg Promotes Proliferation of THP-1 Cells
Flt3 is known to mediate the abnormal proliferation of AML blasts [20]. The acute monocytic leukemia-derived THP-1 cells express wild-type Flt3. Using an optimized MTS assay, we showed that soluble Flt3Lg stimulates THP-1 proliferation (Figure 6). With the addition of recombinant human Flt3Lg, the proliferation of THP-1 cells increased in a dose-dependent manner (ED50 = 0.9 ng/mL) reaching saturation at 10 ng/mL.

Flt3-CAR T Cells Recognize the Flt3Lg-Binding Site of Flt3 on the Surface of THP-1 Cells
The recognizing part of Flt3-CAR comprises wild-type Flt3Lg, which is presumed to interact with Flt3 via the Flt3Lg-binding surface. To test the authenticity of such recognition, we performed a series of functional tests in the presence of soluble Flt3Lg. The soluble human Flt3Lg was added to culture medium upon co-incubation of THP-1-mKate2 cells with Flt3-CAR T cells, non-transduced T cells, or no effector cells for 20 h. The addition of soluble Flt3Lg led to the dose-dependent inhibition of Flt3-CAR T-mediated killing of THP-1 cells (Figure 7). An excess of soluble Flt3Lg (100 ng/mL, which exceeded the saturating concentration 10-fold), markedly interfered with the cytotoxic effects, while lower concentrations of Flt3Lg (2 ng/mL and 0.2 ng/mL, around ED50 doses) did not prevent the Flt3-CAR T cell activity. These experiments confirm that the Flt3-CAR receptor recognizes its target molecule, Flt3, via the Flt3Lg-binding site of the receptor. The recognizing part of Flt3-CAR comprises wild-type Flt3Lg, which is presumed to interact with Flt3 via the Flt3Lg-binding surface. To test the authenticity of such recognition, we performed a series of functional tests in the presence of soluble Flt3Lg. The soluble human Flt3Lg was added to culture medium upon co-incubation of THP-1-mKate2 cells with Flt3-CAR T cells, non-transduced T cells, or no effector cells for 20 h. The addition of soluble Flt3Lg led to the dose-dependent inhibition of Flt3-CAR T-mediated killing of THP-1 cells (Figure 7). An excess of soluble Flt3Lg (100 ng/mL, which exceeded the saturating concentration 10-fold), markedly interfered with the cytotoxic effects, while lower concentrations of Flt3Lg (2 ng/mL and 0.2 ng/mL, around ED 50 doses) did not prevent the Flt3-CAR T cell activity. These experiments confirm that the Flt3-CAR receptor recognizes its target molecule, Flt3, via the Flt3Lg-binding site of the receptor.

Discussion
The main obstacle in developing CAR T cell therapy for AML is the lack of a suitable antigen for specific blast cell recognition. Most of the candidate target molecules for AML are also expressed on hematopoietic progenitor cells [21]. Among the characterized tumor-associated antigens, growth factor receptors are of particular interest. The dysregulation of normal paracrine signaling by aberrant activity of growth factor receptors is typical in cancers [22,23]. Often, pathological cells (blasts) can be discriminated from nonleukemic cells by elevated expression of non-mutated growth factor receptors.
Two major strategies for the alleviation of the abnormal growth factor receptor signaling are inhibition of the excessive signaling [24] and the targeted elimination of the transformed cells [21]. Several molecular targets for CAR T cell-mediated elimination of AML blasts have been considered. However, the killing of tumor cells is often accompanied by the elimination of normal hematopoietic cells, which also carry target antigens on their surfaces.
A number of projects have focused on the design and selection of single-chain antibody fragments to be used in chimeric receptors [25][26][27]. The efficiency of CD123-CAR T cell-mediated killing was shown to correlate with the density of the antigen molecules on the target cells [26]. This finding inspired a massive screening for optimal affinity among slightly different scFv, ultimately yielding CAR T cells capable of distinguishing AML blasts from normal cells. Another group [27] introduced point mutations into anti-CD123 scFv to modulate its affinity, thereby reducing the cytotoxicity towards normal cells. In this study, we used a full-length natural ligand (Flt3Lg) of the target molecule (Flt3) as a recognition module in chimeric receptor Flt3-CAR. The estimated affinity of binding between Flt3 and Flt3Lg (2.5 × 10 10 M −1 [18]) is high enough to ensure effective targeting.
A similar approach was reported by Wang et al. [15], who used the isolated receptorbinding domain of Flt3Lg as a recognition module in chimeric receptors. The developed

Discussion
The main obstacle in developing CAR T cell therapy for AML is the lack of a suitable antigen for specific blast cell recognition. Most of the candidate target molecules for AML are also expressed on hematopoietic progenitor cells [21]. Among the characterized tumorassociated antigens, growth factor receptors are of particular interest. The dysregulation of normal paracrine signaling by aberrant activity of growth factor receptors is typical in cancers [22,23]. Often, pathological cells (blasts) can be discriminated from non-leukemic cells by elevated expression of non-mutated growth factor receptors.
Two major strategies for the alleviation of the abnormal growth factor receptor signaling are inhibition of the excessive signaling [24] and the targeted elimination of the transformed cells [21]. Several molecular targets for CAR T cell-mediated elimination of AML blasts have been considered. However, the killing of tumor cells is often accompanied by the elimination of normal hematopoietic cells, which also carry target antigens on their surfaces.
A number of projects have focused on the design and selection of single-chain antibody fragments to be used in chimeric receptors [25][26][27]. The efficiency of CD123-CAR T cellmediated killing was shown to correlate with the density of the antigen molecules on the target cells [26]. This finding inspired a massive screening for optimal affinity among slightly different scFv, ultimately yielding CAR T cells capable of distinguishing AML blasts from normal cells. Another group [27] introduced point mutations into anti-CD123 scFv to modulate its affinity, thereby reducing the cytotoxicity towards normal cells. In this study, we used a full-length natural ligand (Flt3Lg) of the target molecule (Flt3) as a recognition module in chimeric receptor Flt3-CAR. The estimated affinity of binding between Flt3 and Flt3Lg (2.5 × 10 10 M −1 [18]) is high enough to ensure effective targeting.
A similar approach was reported by Wang et al. [15], who used the isolated receptorbinding domain of Flt3Lg as a recognition module in chimeric receptors. The developed FLT3L CAR T cells showed specific activity towards Flt3-positive THP-1, MV4-11, and MOLM13 cell lines, albeit that they also showed a weak cytotoxic effect towards the Flt3negative cell line U937 as soon as effector : target ratio (E:T) = 1:1. The full-length sequence of soluble human Flt3Lg cytokine used by us in this study is about 50 amino acids longer than the fragment used by Wang et al. [15]. We demonstrate that Flt3-CAR with the fulllength soluble Flt3Lg polypeptide does not affect the proliferation of the Flt3-negative U937 cells, even at E:T = 5:1, indicating negligible off-target activity and higher specificity of activation compared with the use of an isolated binding domain [15]. Moreover, we show that an excess of soluble Flt3Lg effectively inhibits the interaction of Flt3-CAR with Flt3. Based on this result, we conclude that Flt3-CAR recognizes its target directly through the Flt3Lg binding site.
The non-specific activation of chimeric receptors can be pronounced. One study [28] dealt with CD44v6-CAR T cells activated towards target-negative cell line HL-60. The authors managed to reduce the tonic signaling by supplementing the extracellular hinge of CAR with a fragment of low-affinity nerve-growth-factor receptor, and thereby suppress the non-specific killing. Thus, the specificity of CAR T cell-mediated killing can be tremendously enhanced by deliberate combination of original human sequences in chimeric receptor constructs. As our Flt3-CAR construct comprises the non-mutated full-length Flt3Lg sequence, the chimeric receptor is supposed to interact with Flt3 as a fully identical equivalent of Flt3Lg [28].
Leukemic blasts may adapt to immunotherapy through mutations in the target epitope. Considering the outgrowth of AML clones resistant to Flt3-CAR T cell therapy, it is reasonable to expect that such clones will have a defective ligand binding site in the Flt3 molecule. Since Flt3Lg stimulates proliferation in AML (as we demonstrate by using an in vitro model), disruption of the Flt3Lg binding site in Flt3 may deprive blast cells of the proliferative advantage.
Apart from that, we should mention the prognostic significance of the endogenous Flt3Lg levels for AML patients undergoing induction chemotherapy [29]. According to the study, a sustained increase in the concentration of soluble Flt3Lg in the blood plasma (up to 16 ng/mL on day 22 of chemotherapy) correlates with better prognosis. By contrast, unsteady kinetics or consistently low plasma levels of Flt3Lg (within the total range of 0.2-9.5 ng/mL) indicate increased likelihood of refractoriness or relapse. Within the specified range of soluble Flt3Lg concentrations, the Flt3-CAR T cells can efficiently recognize and kill their AML targets.
In summary, our results provide a proof-of-concept that a full-length cytokine sequence within chimeric antigen receptor allows targeting corresponding growth factor receptors with high specificity and affinity. In particular, Flt3-CAR T cells can be used to eliminate AML blast cells with prominent surface expression of Flt3. Indications for such therapy may involve endogenous blood levels of soluble Flt3Lg. AML patients with low blood levels of soluble Flt3Lg (incidentally associated with high risks of relapsed/refractory disease) are more likely to respond. Further research, and notably in vivo testing, will be required to define the clinical prospects of the developed approach.

Informed Consent Statement:
The study involving human material was reviewed and approved by the Independent Ethics Committee at Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. All participants provided their written informed consent to participate in the study.

Data Availability Statement:
The raw data supporting the conclusions of this article are available on request, without undue reservation.