International Journal of Molecular Sciences

Article Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line

Chung Hyo Kang 1,2 , Yeongrin Kim 1,3, Heung Kyoung Lee 1, So Myoung Lee 1, Hye Gwang Jeong 2 , Sang Un Choi 1 and Chi Hoon Park 1,3,*

1 Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, P.O. Box 107, Daejeon 34114, Korea; [email protected] (C.H.K.); [email protected] (Y.K.); [email protected] (H.K.L.); [email protected] (S.M.L.); [email protected] (S.U.C.) 2 College of Pharmacy, Chungnam National University, Daejeon 34134, Korea; [email protected] 3 Medicinal Chemistry and Pharmacology, Korea University of Science and Technology, Daejeon 34113, Korea * Correspondence: [email protected]; Tel.: +82-42-860-7416; Fax: +82-42-861-4246

 Received: 18 August 2020; Accepted: 26 November 2020; Published: 1 December 2020 

Abstract: CD19 is the most promising target for developing chimeric-antigen (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.

Keywords: CD19; chimeric-antigen receptor; scFv; FMC63; 4G7; leukemia

1. Introduction T cells genetically modified to express chimeric-antigen receptor (CAR) specific to a tumor have shown dramatic results in preclinical and clinical tests [1]. Structurally, CAR is composed of an antigen-binding domain, which is typically a single-chain variable fragment (scFv) of monoclonal (mAb), a costimulatory domain, and a CD3ζ domain of T-cell receptor (TCR) [2]. Clinical trials of CAR -targeting CD19 have shown significant responses in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell (DLBCL), and (FL) etc. [3–11]. Two CD19 CAR T cell products are approved by the US FDA, and numerous clinical trials using CD19 CAR T cells are still ongoing by various institutions [12]. Each CD19 CAR T cell has a different CAR backbone, different gene transduction method, and different culture conditions [13,14]. For example, Kymriah, which is manufactured by Novartis, uses 4-1BB as the co-stimulatory domain, however, Yescarta, which is a Kite’s product, uses a CAR construct with CD28 as the co-stimulatory

Int. J. Mol. Sci. 2020, 21, 9163; doi:10.3390/ijms21239163 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 9163 2 of 12 domain [15]. In addition, the National Cancer Institute (NCI) adopted γ-retrovirus for gene transduction in T cells, while the University of Pennsylvania (UPenn) adopted lentivirus for this purpose [16]. Although they have quite different strategies to manufacture CD19 CAR T cells, there is one thing which is common in most institutions—CD19 scFv. Nearly 90% of the worldwide clinical anti-CD19 CAR T cell products use FMC63 as a CD19 scFv [17]. However, so far, numerous CD19 scFvs targeting the CD19 extracellular domain have been invented [18,19]. Therefore we postulated that it is meaningful to test these CD19 scFvs for developing potent CD19 CAR T cells. Here, we used the KHYG-1 NK/T cell line to test CAR construct with each CD19 scFv. It is not easy to make CAR T cells from peripheral blood mononuclear cells (PBMC) isolated from human blood. Because the KHYG-1 cell line has cytotoxic activity against target cells, KHYG-1 incorporated with CAR have the ability to lyse CAR-targeting cells [20–22]. Moreover, we proved that KHYG-1 is easy to culture it can easily transduce the CAR gene. Using the KHYG-1 cell line, we tested eight known CD19 scFvs for CAR T cells. Our in vitro and in vivo results revealed that 4G7 is as good as FMC63 for anti-CD19 CAR T cells.

2. Results

2.1. Usage of the KHYG-1 Cell Line to Screen CD19 scFvs for CD19 CAR T Cells We chose the KHYG-1 cell line to screen CD19 scFvs. Making CAR T cells from PBMC is relatively difficult, expensive, and time-consuming. Therefore we needed an easy system to test CAR constructs instead of CAR T cells from PBMC. Here, we used the KHYG-1 cell line, which is an NK/T-cell line. By gene transduction using lentiviral particle followed by puromycin selection, we obtained the KHYG-1 cell line stably expressing CAR . We tested several for culturing KHYG-1. Figure1A shows that IL2 or IL15, not IL-7, is indispensable for KHYG-1 growth. Although the NK-92 cell line is more popular than the KHYG-1 cell line among NK cell researchers, we found that KHYG-1 has several advantages over the NK-92 cell line. The NK-92 cell line needs a special medium, which is very expensive. However, we needed just RPMI1640 plus 10% FBS to culture KHYG-1. In addition, as shown in Figure1B, KHYG-1 has a high proliferation rate compared to NK-92. The doubling time of KHYG-1 is just 24 h, however, it is more than 72 h in NK-92. Therefore, we can get enough KHYG-1 cells very quickly compared to NK-92 cells. Moreover, KHYG-1 is very easily transduced by lentivirus compared to NK-92. To compare the transduction efficiency of these two cell lines, we measured the luminescence after luciferase gene transduction using lentivirus into KHYG-1 and NK-92. As we can see in Figure1C, the luminescence from KHYG-1 was much higher than NK-92. It means that we can easily transduce the CAR gene into KHYG-1 compared to NK-92. To optimize the best condition for lentiviral infection into KHYG-1, we tested the polybrene concentration (Figure1D). Polybrene is known to enhance the lentiviral infection in human cell lines. Data showed that polybrene increased the infection efficiency slightly. Although 10X polybrene concentration enhanced the transduction efficiency 3.5 fold, this high concentration of polybrene is very toxic to KHYG-1. The effect of polybrene on lentiviral infection into KHYG-1 seems to be very weak compared to other cancer cell lines. We checked whether transduction efficiency in KHYG-1 is enhanced by spinoculation. Spinoculation increased gene transduction greatly by more than 10-fold (Figure1E) and 1500 g seemed to be best for × KHYG-1 spinoculation. We tested which promoter is best for in the KHYG-1 cell line. We infected cytomegalovirus (CMV), elongation factor-1 alpha (EF1α), or phosphoglycerate kinase (PGK) promoter containing the luciferase gene into KHYG-1 using lentivirus. Figure1F shows that EF-1a promoter was superior to other promoters. Finally, we compared the lytic activity of CAR NK-92 and CAR KHYG-1 against tumor cells. Both FMC63 CAR NK-92 and FMC63 CAR KHYG-1 efficiently eliminated CD19-positive cell line, NALM-6 (Figure1G). Lytic activity of FMC63 CAR KHYG-1 seemed to be slightly better than FMC63 CAR NK-92. Neither mock NK-92 nor mock KHYG-1 lysed the CD19 target cells. These data means that both cell lines are able to perform target-specific lysis. Int. J. Mol. Sci. 2020, 21, 9163 3 of 12 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 12

FigureFigure 1. Biological1. Biological characteristics characteristics of KHYG-1of KHYG-1 cells cells used us fored CD19for CD19 single-chain single-chain variable variable fragment fragment (scFv) screening.(scFv) screening. (A) The effect(A) The of effect cytokines, of cytokines, including including IL-2, IL-7, IL-2, and IL-7, IL-15, and on IL-15, the proliferationon the proliferation of KHYG-1 of cellsKHYG-1 was investigated. cells was investigated. After adding After IL-2, adding IL-7, IL-15,IL-2, IL-7, or IL-7 IL-15,/IL-15 or toIL-7/IL-15 the culture to the medium, culture proliferation medium, of KHYG-1proliferation was of analyzed KHYG-1 by was cell counting.analyzed by (B )cell Comparison counting. of() Comparison proliferation of in cell KHYG-1 proliferation and NK-92. in AfterKHYG-1 KHYG-1 and andNK-92. NK-92 After were KHYG-1 cultured and NK-92 in each were culture cultured medium in each containing culture medium IL-2, proliferation containing IL- was measured2, proliferation by a cell was titer-glo measured assay. by (aC cell) Comparison titer-glo assay. of transduction (C) Comparison efficiency of transduction in KHYG-1 efficiency and NK-92. in AfterKHYG-1 luciferase and transductionNK-92. After luciferase to KHYG-1 transduction and NK-92, to luminescenceKHYG-1 and NK-92, value was luminescence detected by value bright-glo was assaydetected for 6 days.by bright-glo (D) The assay effect for of 6 polybrene days. (D) onThe transduction effect of polybrene efficiency on in transduction KHYG-1. After efficiency luciferase in transductionKHYG-1. After with luciferase different concentrationstransduction with of polybrenedifferent concentrations into KHYG-1, of luminescence polybrene into was KHYG-1, measured usingluminescence a bright-glo was assay measured on the using 4th a day. bright-glo (E) KHYG-1 assay on cells the were4th day. infected (E) KHYG-1 with luciferase cells were containinginfected lentiviralwith luciferase particles containing with different lentiviral g-values. particles After with 5 days, different luminescence g-values. After was 5 measured. days, luminescence (F) Comparison was of genemeasured. expression (F) Comparison by promoter of typegene in expression KHYG-1. KHYG-1by promoter cells type were in transduced KHYG-1. withKHYG-1 pLVX-CMV-Luc, cells were pLVX-EF1transducedα-Luc, with or pLVX-hPGK-LucpLVX-CMV-Luc, lentivirus.pLVX-EF1α On-Luc, day or 4,pLVX-hPGK-Luc intensity of luminescence lentivirus. wasOn measuredday 4, throughintensity a bright-glo of luminescence assay. was (G) measur Antigen-specificed through cytotoxicity a bright-glo ofassay. KHYG-1 (G) Antigen-specific and NK-92. Mock cytotoxicity KHYG-1, FMC63of KHYG-1 CAR KHYG-1, and NK-92. mock Mock NK-92, KHYG-1, or FMC63 FMC63 CAR CAR NK-92 KHYG-1, cells mock (E:T ratio NK-92, 3:1 or 10:1)FMC63 were CAR co-cultured NK-92 4 withcells NALM-6 (E:T ratio (1.5 3:1 10or4 10:1)cells ),were a CD19 co-cultured positive with cell line NALM-6 expressing (1.5 × luciferase, 10 cells), fora CD19 5 h. Then, positive luminescence cell line × valuesexpressing were measured. luciferase, for 5 h. Then, luminescence values were measured.

2.2.2.2. FMC63 FMC63 and and 4G7 4G7 scFvs scFvs Are Are Proved Proved to to BeBe ApplicableApplicable for Anti-CD19 CAR CAR Construct Construct OurOur lab lab has has the the CARCAR construct backbone. backbone. In this In this construct, construct, we can we change can change any scFvs any using scFvs XhoI using XhoIand and AvrII AvrII restriction restriction enzyme enzyme by cloning. by cloning. We made We eight made different eight di anti-CD19fferent anti-CD19 CAR plasmids, CAR plasmids, which whicheach eachhad a had CD19 a CD19 scFv, scFv,including including FMC63, FMC63, B43, 25C1, B43, BLY3, 25C1, 4G7, BLY3, HD37, 4G7, HB12a, HD37, and HB12a, HB12b. and VH HB12b. and VL sequences of each scFv are shown in Figure 2. We checked the availability of CD19 scFvs for CAR VH and VL sequences of each scFv are shown in Figure2. We checked the availability of CD19 scFvs forT CAR cells Tusing cells the using Jurkat the cell Jurkat line. cellEach line. anti-CD19 Each anti-CD19 CAR construct CAR was construct transiently was transfected transiently into transfected Jurkat intocells Jurkat (Figure cells 3A). (Figure CAR3A). Jurkat CAR cells Jurkat were cells incubated were incubated with NALM-6, with NALM-6, a CD19-positive a CD19-positive cell line. cell Only line. FMC63 and 4G7 CAR Jurkat cells secreted IL-2 (Figure 3B). To compare the lytic activities of each Only FMC63 and 4G7 CAR Jurkat cells secreted IL-2 (Figure3B). To compare the lytic activities of each CD19 CAR construct, we made lentivirus for CAR transduction into KHYG-1 cell by spinoculation. CD19 CAR construct, we made lentivirus for CAR transduction into KHYG-1 cell by spinoculation. After puromycin selection for 2 weeks, we confirmed the CAR expression in each KHYG-1. As shown After puromycin selection for 2 weeks, we confirmed the CAR expression in each KHYG-1. As shown in Figure 3C, each CAR protein was expressed in KHYG-1. Using these CD19 CAR KHYG-1 cells, we in Figure3C, each CAR protein was expressed in KHYG-1. Using these CD19 CAR KHYG-1 cells, performed lysis assay with NALM-6. FMC63 and 4G7 CAR KHYG-1 lysed NALM-6 very efficiently. weInterestingly, performed lysis anti-CD19 assay with KHYG-1 NALM-6. cell lines FMC63 equipped and 4G7 with CAR other KHYG-1 CD19 lysedscFvs, NALM-6 including very B43, effi 25C1,ciently. Interestingly,BLY3, HD37, anti-CD19 HB12a, and KHYG-1 HB12b, cell did lines not equippedlyse NALM-6 with (Figure other CD193D). Figure scFvs, 3E including shows that B43, the 25C1, in vitro BLY3, HD37,lytic HB12a,activity andof FMC63 HB12b, CAR did KHYG-1 not lyse was NALM-6 slightly (Figure higher3 thanD). Figure 4G7 CAR3E shows KHYG-1. that These the datain vitro meanlytic activitythat FMC63 of FMC63 and 4G7 CAR CAR KHYG-1 KHYG-1 was cells slightly are able higher to trigger than 4G7cell lysis CAR of KHYG-1. CD19 positive These cells. data mean that FMC63 and 4G7 CAR KHYG-1 cells are able to trigger cell lysis of CD19 positive cells. Int. J. Mol. Sci. 2020, 21, 9163 4 of 12 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 12

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 12

FigureFigure 2. CAR 2. CAR constructs constructs of of various various scFvsscFvs capable of of targeting targeting CD19. CD19. (A) Schematic (A) Schematic representation representation of Figureanti-CD19 2. CAR constructsCAR construct. of variousIt is a second-gener scFvs capableation of CAR targeting composed CD19. of anti-CD19(A) Schematic scFv, CD28representation of of anti-CD19 CAR construct. It is a second-generation CAR composed of anti-CD19 scFv, CD28 anti-CD19transmembrane CAR construct. region, CD28 It co-sis timulatorya second-gener domain,ation and CD3 CARζ signaling composed domain. of ( Banti-CD19) VH and VL scFv, CD28 transmembrane region, CD28 co-stimulatory domain, and CD3ζ signaling domain. (B)VH and VL transmembranesequences of eightregion, different CD28 anti-CD19 co-stimulatory scFvs. domain, and CD3ζ signaling domain. (B) VH and VL sequences of eight different anti-CD19 scFvs. sequences of eight different anti-CD19 scFvs.

FigureFigure 3. FMC63 3. FMC63 and and 4G7 4G7 CAR CAR constructs constructs eff effectivelyectively recognize recognize antigenantigen CD19. (A (A) )After After transfection transfection of CD19of CAR CD19 genes CAR intogenes Jurkat into Jurkat cells, CARcells, expressionCAR expression was was confirmed confirmed through through the the Western Western blot. blot. (B ()B CD19) CARCD19 Jurkat CAR (2 Jurkat105 cells) (2 × 10 co-cultured5 cells) co-cultured with 2 with10 42 NALM-6× 104 NALM-6 cells cells for 21for h. 21 IL-2 h. IL-2 level level secreted secreted by by CD19 × × CARCD19 Jurkat CAR was Jurkat measured was measured through through ELISA ELISA assay. assay. (C) After (C) After transduction transduction of eightof eight di ffdifferenterent CD19 CD19 CAR CAR genes into KHYG-1 cells, CAR expression was confirmed through Western blot. (D) Tumor- genes into KHYG-1 cells, CAR expression was confirmed through Western blot. (D) Tumor-killing abilitykilling of eight ability diff erentof eight CD19 different CAR CD19 KHYG-1 CAR cells KHYG-1 against cells luciferase-expressing against luciferase-expressing NALM-6. NALM-6. CD19 CAR FigureKHYG-1CD19 3. cellsFMC63 CAR (E:T KHYG-1 ratioand 3:14G7 cells or CAR(E:T 10:1) ratio wereconstructs 3:1 co-cultured or 10:1) effectively were with co-cultured luciferase-expressing recognize with luciferase-expressing antigen NALM-6 CD19. ((1.5A NALM-6) After104 cells transfection) (1.5 × 104 cells) for 7 h. Then, luminescence values were measured. (E) FMC63 CAR KHYG-1 or× 4G7 offor CD19 7 h. Then, CAR luminescencegenes into Jurkat values cells, were CAR measured. expression (E) FMC63 was CARconfirmed KHYG-1 through or 4G7 CARthe Western KHYG-1 blot. (B) CAR KHYG-1 cells were incubated with NALM-6 at different E:T ratios (1:1, 3:1, and 10:1) and CD19cells were CAR incubated Jurkat (2 with × 10 NALM-65 cells) co-cultured at different with E:T ratios 2 × 10 (1:1,4 NALM-6 3:1, and 10:1)cells andfor 21 diff h.erent IL-2 incubation level secreted by different incubation times (6 h and 23 h) to see their lytic activity. CD19times CAR (6 h and Jurkat 23 h) was to see measured their lytic through activity. ELISA assay. (C) After transduction of eight different CD19 CAR genes into KHYG-1 cells, CAR expression was confirmed through Western blot. (D) Tumor- killing ability of eight different CD19 CAR KHYG-1 cells against luciferase-expressing NALM-6. CD19 CAR KHYG-1 cells (E:T ratio 3:1 or 10:1) were co-cultured with luciferase-expressing NALM-6 (1.5 × 104 cells) for 7 h. Then, luminescence values were measured. (E) FMC63 CAR KHYG-1 or 4G7 CAR KHYG-1 cells were incubated with NALM-6 at different E:T ratios (1:1, 3:1, and 10:1) and different incubation times (6 h and 23 h) to see their lytic activity.

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2.3. 4G7 CAR T cells are as Effective as FMC63 CAR T Cells in Eradicating Leukemic Tumor Cells 2.3. 4G7 CAR T Cells Are as Effective as FMC63 CAR T Cells in Eradicating Leukemic Tumor Cells As 4G7 had proven to be as good as FMC63 in lysing NALM-6 in KHYG-1 cell model, we determinedAs 4G7 had to make proven anti-CD19 to be as good CAR as T FMC63 cells with in lysing 4G7 NALM-6 scFv to compare in KHYG-1 with cell FMC63. model, we We determined made 4G7 toand make FMC63 anti-CD19 CAR T CAR cells Twith cells PBMC with 4G7 isolated scFv from to compare healthy with donor’s FMC63. blood. We As made shown 4G7 in and Figure FMC63 4A, CARCAR T expressions cells with PBMC were isolatedconfirmed from in healthyboth CAR donor’s T cells. blood. Both As CAR shown T cells in Figureselectively4A, CAR lysed expressions the CD19- werepositive confirmed cell line in (Figure both CAR 4B). TU937, cells. a BothCD19 CAR negative T cells cell selectively line, was lysed not lysed the CD19-positive by CD19 CAR cell T cells. line (FigureAlso, IFN-4B).γ U937, and IL-2 a CD19 were negative secreted cellwhen line, FMC63 wasnot and lysed 4G7 CAR by CD19 T cells CAR were T cells.incubated Also, with IFN- NALM-γ and IL-26, not were with secreted U937 (Figure when FMC634C). We and performed 4G7 CAR in T vivo cells assay were with incubated an NSG with mice NALM-6, model. notNALM-6 with U937 cells (Figureexpressing4C). Weluciferase performed werein injected vivo assay into withNSG anmice NSG by micei.v. route. model. After NALM-6 1 day, cellsCAR expressing T cells were luciferase injected wereby i.v. injected route. intoIn the NSG in micevivo bymodel, i.v. route. both AfterFMC63 1 day,and CAR4G7 TCAR cells T were cells injectederadicated by i.v.NALM-6, route. Inwhile the incontrol vivo model, CAR T both cells FMC63(FITC CAR and 4G7T) did CAR not T (Figure cells eradicated 5A,B). The NALM-6, survival while curve control also shows CAR Tsimilar cells (FITCefficacy CAR between T) did 4G7 not (FigureCAR T5 andA,B). FMC63 The survival CAR T curve cells also(Figure shows 5C). similar These edatafficacy suggest between that 4G74G7 CARCAR TT andcells FMC63 are as effective CAR T cellsas FMC63 (Figure CAR5C). T These cells against data suggest B-cell thatleukemia. 4G7 CAR T cells are as e ffective as FMC63 CAR T cells against B-cell leukemia.

Figure 4. FMC63 and 4G7 CAR T cells effectively responded to CD19-positive NALM-6 cells. (A) After

transduction of anti-CD19 CAR lentiviruses into primary human T cells, CAR expression was confirmed throughFigure 4. the FMC63 Western and blot. 4G7 Anti-human CAR T cells CD3effectively antibody resp wasonded used to to CD19-positive detect CD3ζ of NALM-6 CAR. (B) cells. Tumor-killing (A) After abilitytransduction of FMC63, of anti-CD19 4G7, and CAR FITC lentiviruses CAR T cells in againstto primary luciferase-expressing human T cells, CAR NALM-6 expression and U937. was FMC63,confirmed 4G7, through and FITCthe Western CAR T blot. cells Anti-human (2 105 cells) CD3 wereantibody co-cultured was used with to detect luciferase-expressing CD3ζ of CAR. (B) × NALM-6Tumor-killing (2 10 ability4 cells of) or FMC63, U937 (24G7,10 and4 cells) FITC for CAR indicated T cells incubationagainst luciferase-expressing times at the E:T ratio NALM-6 10:1. × × 5 Then,and U937. luminescence FMC63, values 4G7, wereand FITC measured. CAR (TC )cells FMC63, (2 × 4G7, 10 orcells) FITC were CAR co-cultured T cells (1.5 with105 cells)luciferase- were 4 4 × co-culturedexpressing NALM-6 with NALM-6 (2 × 10 cells cells) (1.5 or U937104 cells) (2 × 10 or cells) U937 for cells indicated (1.5 10 incubation4 cells) for times 21 h at at the the E:T E:T ratio ratio × × 5 10:1.10:1. TheThen, amount luminescence of IFN-γ valuesand IL-2 were secreted measured. by CAR (C) FMC63, T cells was 4G7, measured or FITC CAR through T cells the (1.5 ELISA × 10 assay. cells) were co-cultured with NALM-6 cells (1.5 × 104 cells) or U937 cells (1.5 × 104 cells) for 21 h at the E:T ratio 10:1. The amount of IFN-γ and IL-2 secreted by CAR T cells was measured through the ELISA assay.

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Figure 5. In vivo, both FMC63 and 4G7 CAR T cells eliminated acute-lymphoblastic-leukemia-cell FigureNALM-6. 5. In (A vivo,–C) NOD.Cg-PrkdcscidIl2rgtm1Wjl both FMC63 and 4G7 CAR T/SzJ cells (NSG) eliminated mice were acute-lymphoblastic-leukemia-cell injected intravenously (i.v.) with NALM-65 106 Luc-expressing. (A–C) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ NALM-6 cells. Next day, mice (NSG) were mice administered were injected with 1intravenously107 FMC63, (i.v.) 4G7, × × withand FITC5 × 10 CAR6 Luc-expressing T cells or phosphate NALM-6 bu cells.ffered Next saline day, (PBS) mice control were (i.v.). administered From day with 5, tumor 1 × 10 progression7 FMC63, 4G7,was observedand FITC via CAR bioluminescence T cells or ph imaging.osphate buffered (A) Represents saline the(PBS) luminescence control (i.v.). image, From and day (B )5, indicates tumor progressionthe luminescence was observed value of via each bioluminescence group. (C) Percentage imaging. survival (A) Represents was calculated. the luminescence image, and (B) indicates the luminescence value of each group. (C) Percentage survival was calculated. 3. Discussion 3. DiscussionBecause of the high response rate of anti-CD19 CAR T cells in leukemia patients, numerous anti-CD19Because CAR of the T high cell clinicalresponse trials rate areof anti-CD19 in process CAR worldwide T cells in [ 23leukemia–27]. Although patients, numerous there are many anti- CD19qualified CAR CD19 T cell , clinical trials most are of in clinical process trials worldwide of CD19 [23–27]. CAR T cellsAlthough use FMC63 there are exclusively many qualified [13,28]. CD19Recently, antibodies, several institutesmost of clinical have been trials trying of CD19 to use CAR novel T cells CD19 use scFvs, FMC63 which exclusively they developed, [13,28]. forRecently, clinical severaltrial of CARinstitutes T cell have therapy been [trying29–31]. to We use searched novel CD19 for CD19 scFvs, scFvs which in they literature developed, including for clinical patents trial and ofscientific CAR T papers. cell therapy We found [29–31]. that moreWe searched CD19 scFvs for thatCD19 bind scFvs CD19 in eliteratureffectively exist.including We triedpatents to make and scientificCAR constructs papers. withWe found these that CD19 more scFvs, CD19 and scFvs finally th weat bind got eightCD19 CD19effectively CAR exist. constructs We tried as shown to make in CARFigure constructs2. Here, we with tested these eight CD19 known scFvs, CD19 and antibodies,finally we FMC63,got eight 4G7, CD19 25C1, CAR B43, constructs HD37, BLY3, as shown HB12a, in Figureand HB12b, 2. Here, for we CD19 tested CAR eight T cells.known These CD19 antibodies antibodies, are FMC63, old, because, 4G7, 25C1, except B43, for HD37, HB12a BLY3, and HB12a, HB12b andwhich HB12b, were developedfor CD19 CAR in 2011, T cells. these These CD19 antibodies scFvs were are developed old, because, in the 1980sexcept or for 1990s. HB12a They and were HB12b also whichproduced were in developed different ways. in 2011, FMC63 these is producedCD19 scFvs by we immunizingre developed BALB in /thec mice 1980s with or JVM3, 1990s. a They B-CLL were cell alsoline [produced19]. 4G7 was in different derived byways. immunizing FMC63 is mice produced with B-CLL by immunizing cells. HD37 BALB/c was produced mice with by immunizing JVM3, a B- CLLBALB cell/c miceline [19]. with 4G7 hairy was cell derived leukemia by cellsimmunizing [32]. B43 mice was derivedwith B-CLL by immunizing cells. HD37 BALBwas produced/c mice with by immunizingBurkitt’s lymphoma BALB/c mice cells with [33]. hairy These cell CD19 leukemia scFvs cells were [32]. cloned B43 was in our derived CAR by construct immunizing comprised BALB/c of miceCD28 with and CD3Burkitt’sζ cytoplasmic lymphoma domains. cells [33]. We These used CD19 KHYG-1 scFvs cell were to screen cloned CD19 in our scFvs. CAR Because construct the comprisedKHYG-1 cell of lineCD28 is veryand easyCD3 toζ cytoplasmic culture, we selecteddomains. KHYG-1 We used for KHYG-1 our study cell instead to screen of NK-92, CD19 which scFvs. is Becausea very popular the KHYG-1 cell line cell in line CAR is very T cell easy research to culture, as it canwe growselected on KHYG-1 very simple for our medium study (RPMI1640 instead of NK-92,supplemented which is with a very 10% popular FBS), and cell it line grows in CAR very T fast cell (Figure research1B). as On it can the grow contrary, on very NK-92 simple needs medium special (RPMI1640medium, which supplemented is very expensive, with 10% and FBS), it grows and it very grows slowly. very Infast addition, (Figure we1B). can On more the contrary, easily transduce NK-92 needsCAR genespecial into medium, KHYG-1 which cell line is very than expensive, the NK-92 celland lineit grows (Figure very1C). slowly. Most researchers In addition, use we the can NK-92 more easilycell line transduce for CAR CAR T-cell gene study into [34 –KHYG-139]. However, cell line several than research the NK-92 groups cell useline KHYG-1 (Figure cell1C). line Most for researchersCAR T-cell studyuse the [20 NK-92–22]. Usingcell line this for system, CAR T-cell we made study eight [34–39]. different However, anti-CD19 several CAR research KHYG-1 groups cells, usewhich KHYG-1 had di ffcellerent line CD19 for CAR scFvs T-cell to each study other. [20–22]. CAR KHYG-1 Using this data system, show thatwe made only FMC63 eight different and 4G7 anti- CAR CD19KHYG-1 CAR lyse KHYG-1 NALM-6 cells, cells which very ha effidciently differentin vitroCD19. ThescFvs Jurkat to each system other. also CAR shows KHYG-1 that FMC63 data show and that4G7 only CAR FMC63 Jurkat and cells 4G7 secrete CAR IL-2 KHYG-1 when incubatedlyse NALM-6 with cells CD19-positive very efficiently cell in lines. vitro. Based The Jurkat on these system data, alsowe made shows CD19 that CAR FMC63 T cells and with 4G7 FMC63 CAR Jurkat or 4G7 cells scFvs secrete from peripheral IL-2 when blood incubated mononuclear with CD19-positive cells (PBMC). cellThese lines. two Based CAR on T cellsthese lysed data, CD19-positivewe made CD19 cell CAR lines, T cells and with secreted FMC63 IFN- orγ 4G7and scFvs IL-2 whenfrom peripheral incubated bloodwith CD19-positivemononuclear cells cell lines.(PBMC). As These shown two in CAR Figure T5 cells, the lysed anti-tumor CD19-positive e fficacy cell of 4G7lines, CAR and Tsecreted cells is IFN-as goodγ and as IL-2 FMC63 when CAR incubated T cells. with Although CD19-positive our data cell strongly lines.suggest As shown that in 4G7 Figure CAR 5, Tthe cells anti-tumor are very efficacy of 4G7 CAR T cells is as good as FMC63 CAR T cells. Although our data strongly suggest

Int. J. Mol. Sci. 2020, 21, 9163 7 of 12 powerful against CD19-positive tumors, there is no clinical trial of CD19 CAR T cells using 4G7 scFv. Based upon our data, we postulate that the therapy with 4G7 CAR T cells against B-cell lymphoma would be as successful as therapy with FMC63 CAR T cells. Our data show that only FMC63 and 4G7 are effective for CD19 CAR T cells. Currently, we do not know exactly why only these two scFvs are effective in CD19 CAR T cells. One reason could be that spacer and transmembrane domain might cause changes in 3-dimensional structure in scFvs to the binding affinity to the epitope. In further study, we can try to make a CD19 CAR construct that has a different spacer or transmembrane domain to increase the activity of CAR T cells. Here we suggest that KHYG-1 is very useful cell line for CAR T-cell study. Also, we suggest the 4G7 scFv can be a potent CD19 scFv for making CD19 CAR T cells. In vitro and in vivo data demonstrate that 4G7 CAR T cells perform anti-cancer activity as effective as FMC63 CAR T cells. Since the current FMC63 CAR T cells show excellent efficacy in patients, the development of new CD19 CAR T cells should be focused on FMC63 CAR-T-cell-resistant patients. Although FMC63 and 4G7 recognize a similar conformational epitope centered on residue R144 in CD19, deep mutational scanning experiments demonstrated that the residues of CD19 which affect the binding to CD19 are slightly different between FMC63 and 4G7 [40–43]. Therefore, it is plausible that the 4G7 CAR T cell can be used for FMC63 CAR-T-cell resistant patients. In addition, recent papers demonstrate that the transmembrane or spacer domain of CAR influences the function of CAR T cell [44]. Therefore, in further study, we can try making 4G7 CAR T cells with optimized transmembrane or spacer domains to increase the anti-tumor effect.

4. Materials and Methods

4.1. Cell Culture NALM-6 and Jurkat cells were cultured in RPMI1640 medium (SH30027; HyClone Laboratory Tools, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (FBS) (16000-044; Gibco Life Technologies, Carlsbad, CA, USA). KHYG-1 and T cells were maintained in RPMI1640 supplemented with 10% FBS and 200 IU/mL rhIL-2 (202-IL-500; R&D Systems, Minneapolis, MN, USA). NK-92 cells were cultured with MyelocultTM H5100 (05150; STEMCELL Technologies, Vancouver, Canada) with 500 IU/mL rhIL-2. Cells were incubated in a humidified incubator at 37 ◦C with 5% CO2.

4.2. Construct of CARs Eight CD19 scFvs (FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b) and FITC and scFvs were synthesized by Macrogene (Daejeon, Korea). scFvs recognize the fluorescein derivative called fluorescein isothiocyanate (FITC). Since FITC did not exist in the cells, CAR-targeting FITC was used as a negative control. Eight CD19 CAR and FITC CAR genes were inserted into the pLVX-CMV-puro (632164; Clontech, Mountain View, CA, USA) vector or into pLVX-EF1α vector (631982; Clontech) in which IRES-ZsGreen was deleted.

4.3. Lentivirus Production and Generation of CAR KHYG-1 On day 0, 1.5 107 293T cells were seeded in a 150-mm dish. Next day, eight different CD19 × CAR, FITC CAR, and luciferase vectors were co-transfected with lentiviral packaging plasmids, pRSV-Rev (12253; Addgene, Watertown, MA, USA), pMDLg/pRRE (12251; Addgene) and pMD2.G (12259; Addgene) to 293T cells. On day 2, the medium of transfected 293T cells was replaced with fresh medium. On day 3–4, lentivirus supernatants were collected from the transfected 293T cells and were filtered with 0.45 µm a polyethersulfone (PES) membrane filter (SLHP033RB; Merck Millipore, Burlington, MA, USA). If necessary, lentivirus supernatants were concentrated using Lenti-X concentrator (631232; Clontech). Int. J. Mol. Sci. 2020, 21, 9163 8 of 12

4.4. Human T Cell Isolation and Generation of CAR T Cells Whole blood was obtained from the Korean Red Cross Blood Services and peripheral blood mononuclear cells (PBMCs) were separated using density-gradient centrifugation. Whole blood was diluted with an equal volume of PBS containing 2% FBS. Fifteen mL of Lymphoprep (07851; STEMCELL Technologies) was added to the conical centrifuge tube, then 30 mL of diluted whole blood was added. Samples were centrifuged at 800 g for 20 min without a break and the yellow middle layer × (mononuclear cells, MNCs) was transferred to a new conical centrifuge tube. After adding the wash buffer (PBS with 2% FBS) to MNCs, tubes were centrifuged at 300 g for 8 min. This was repeated twice. × On day 0, PBMC were cultured in culture medium containing Dynabeads Human T-Activator CD3/CD28 (11132D; Thermo Fisher Scientific, Waltham, MA, USA) and 200 IU/mL rhIL-2 to activate only T cells. On the day 2, the Dynabeads were removed with an EasySep Magnet (18001; STEMCELL Technologies) and then mixed with lentivirus supernatants. Eight µg/mL of polybrene was added to the T cell-lentivirus mixture and spinoculation was done for 90 min at 1000 g. After centrifugation, × transduced T cells were incubated in a medium containing rhIL-2. On day 7, to evaluate the function of CAR T cells, cytotoxicity assay and release assay were performed, as well as Western blot to confirm CAR expression.

4.5. Proliferation Assay To compare the effect of interleukins on proliferation of KHYG-1 cells, 1 105 KHYG-1 cells were × cultured in medium containing IL-2 (202-IL-500; R&D Systems), IL-7 (200-07; PeproTech, Cranbury, NJ, USA), IL-15 (200-15; PeproTech), or IL-7/IL-15, respectively. On days 1, 2, and 4, the number of KHYG-1 cells cultured in each medium were counted using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Hercules, CA, USA). To compare the proliferation of KHYG-1 and NK-92 cells, 2 103 NK-92 and KHYG-1 cells were × seeded in a 96-well plate and incubated at 37 ◦C. On days 1, 3, and 4, equal volumes of CellTiter-Glo assay reagent (G7572; Promega, Madison, WI, USA) was added to each well and shaken for 2 min. To stabilize the luminescence signal, the plate was left at room temperature for 10 min, and then the luminescence was measured via an EnVision reader (PerkinElmer, Waltham, MA, USA).

4.6. Cytotoxicity Assay To generate target cells expressing luciferase, lentiviral luciferase was transduced into NALM-6. In 96-well plates, CAR NK-92, CAR KHYG-1, or CAR T cells were co-cultured with luciferase-expressing NALM-6 at indicated E:T ratio and incubation time. At the end of incubation time, the Bright-Glo Luciferase assay reagent (E2650; Promega) was added to each well containing effector/target cell mixture. After shaking the plate for 5 min at room temperature, luminescence was detected using an EnVision reader (PerkinElmer).

4.7. Cytokine Release Assay Eight CD19 CAR (FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b) and FITC CAR genes were transduced into Jurkat or T cells by electroporation or lentiviral infection, respectively. CAR Jurkat and CAR T cells were co-cultured with NALM-6 for the indicated E:T ratio and indicated incubation time in 96-well plates. At the end of incubation, effector–target cell mixtures were harvested in 1.5 mL microtubes and centrifuged at 4 ◦C, 13,000 rpm for 10 min. Only supernatants were collected and cytokine level was measured using human interleukin-2 (IL-2, 431801; BioLegend, San Diego, CA, USA) and human interferon gamma (IFN-γ, 430104; BioLegend) ELISA assay .

4.8. Western Blotting CAR transduced Jurkat, KHYG-1, and T cells were harvested and cell lysates were made by 1 sample × buffer (10% glycerol, 2% SDS, 50 mM Tris-HCl (pH 6.8), 3% β-mercaptoethanol). Lysates were boiled Int. J. Mol. Sci. 2020, 21, 9163 9 of 12

10 min, at 95 ◦C. Samples were loaded on 4–15% gradient Mini-PROTEAN TGX gels (456-1086; Bio-Rad Laboratories) and electrophoresis was performed at 100 V for 75 min. The transfer step was conducted at 250 mA for 60 min. Using the blocking buffer (5% skimmed milk in 1 TBS-T), membrane was × blocked for 60 min. After washing with 1X TBS-T, the membrane was incubated with primary antibody (CD3ζ (551034; BD Biosciences, San Jose, CA, USA), GAPDH (5174; Technology, Danvers, MA, USA)) overnight at 4 C. Next day, the membrane was washed with 1 TBS-T and incubated with a ◦ × horseradish peroxidase (HRP)-conjugated secondary antibody (31430 and 31460; Thermo Fisher Scientific). Signal intensity was detected using SuperSignal West Pico solution (1863096 and 1863097; Thermo Fisher Scientific) and Sensi-Q2000 Chemidoc (LugenSci, Bucheon, Korea) instruments.

4.9. Mouse Xenograft Model The NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and the experimental procedure was conducted according to guidelines approved by the Laboratory Animal Care and Use Committee of the Korea Research Institute of Chemical Technology (project code: 2020-6C-01-01, approval date: 20 January 2020). The NSG mice were injected intravenously (i.v.) with 4 105 luciferase-expressing NALM-6 cells. On day 1, control (PBS), FMC63 CAR T, 4G7 CAR × T, or FITC CAR T (1 107) cells were administered by i.v. to NSG mice. To measure tumor progression, × d-luciferin (122799; PerkinElmer) was administered intraperitoneally to mice, and luminescence was monitored using the IVIS Spectrum In vivo Imaging System (PerkinElmer).

Author Contributions: Conceptualization, C.H.K. and C.H.P.; formal analysis, C.H.K., Y.K., and C.H.P.; funding acquisition, C.H.P.; investigation, C.H.K., Y.K., and C.H.P.; methodology, C.H.K., Y.K., H.K.L., and S.M.L.; supervision, H.G.J., S.U.C., and C.H.P.; validation, C.H.P.; writing—original draft, C.H.K.; writing—review and editing, C.H.P. All authors have read and agreed to the published version of the manuscript. Funding: This Research was supported by the Korea Research Institute of Chemical Technology (BSK20-402, SI1931-40). Acknowledgments: We gratefully acknowledge the financial support of the Korea Research Institute of Chemical Technology. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

ALL Acute lymphoblastic leukemia CAR Chimeric antigen receptor CLL Chronic lymphocytic leukemia CMV Cytomegalovirus DLBCL Diffuse large B-cell lymphoma EF1α Elongation factor-1 alpha FL Follicular lymphoma HRP Horseradish peroxidase IFN-γ Interferon-gamma IL-2 Interleukin-2 mAb Monoclonal antibody MNC Mononuclear cells NCI National Cancer Institute NK Natural killer NSG NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PES Polyethersulfone PGK Phosphoglycerate kinase scFv Single-chain variable fragment TCR T-cell receptor UPenn University of Pennsylvania Int. J. Mol. Sci. 2020, 21, 9163 10 of 12

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