Construction and Molecular Characterization of a T-Cell Receptor-Like Antibody and CAR-T Cells Speciﬁc for Minor Histocompatibility Antigen HA-1H

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ORIGINAL ARTICLE Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells speciﬁc for minor histocompatibility antigen HA-1H

Y Inaguma1, Y Akahori2, Y Murayama1, K Shiraishi3, S Tsuzuki-Iba1, A Endoh1, J Tsujikawa1, A Demachi-Okamura3, K Hiramatsu3, H Saji4, Y Yamamoto1, N Yamamoto5, Y Nishimura6, T Takahashi3, K Kuzushima3, N Emi1 and Y Akatsuka1,3

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and β2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD = 14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

Gene Therapy (2014) 21, 575–584; doi:10.1038/gt.2014.30; published online 3 April 2014

INTRODUCTION CAR uses a single-chain variable region moiety (scFv) made of the A subset of patients with relapsed hematologic malignancies light chain and heavy-chain variable regions of a monoclonal following allogeneic stem cell transplantation (SCT) can be treated antibody (mAb), an extracellular hinge, a transmembrane – with donor lymphocyte infusions (DLI) where graft-versus-tumor and intracellular signaling domains such as CD3ζ chain.13 15 This (GVT) effect is mediated by donor T cells recognizing minor artificial structure results in precluding endogenous TCR from histocompatibility (H) antigens on tumor cells.1,2 Selective forming unwanted chimeric receptors. CARs enable T cells with GVT reactivity with minimal risk of graft-versus-host disease is MHC-independent, antibody-derived specificity and thus may thought to be induced by targeting minor H antigens expressed target any surface molecule on target cells. However, conventional only on patients’ hematopoietic cells. Among HLA-A*02:01- CAR strategy has the limitation of only targeting cell surface positive patients, minor H antigens such as HA-1 (ref. 3) and antigens on target cells. One possible way to attain intracellular HA-2 have been shown to be associated with antitumor responses 2 antigen targeting with a CAR is to use a TCR-like mAb as a source with minimal graft-versus-host disease. As preparation of T-cell of binding moiety for conferring antigen specificity to T cells.16–18 clones or lines for adoptive immunotherapy (ACT) is labor- While such TCR-like mAbs can also be administered alone,18 if intensive and time consuming, T-cell receptor (TCR) gene transfer armed as CAR-T cells, the necessity of frequent administration can to polyclonal T cells has become an attractive approach for the be avoided and long-lasting in vivo effect can be expected. rapid preparation of large numbers of T cells specific for target antigen of interest.4–6 However, problems such as induction of Generation of TCR-like mAbs in small animals has been technically unexpected specificities caused by chimeric TCR formation with challenging due to the high xenogenic immunogenicity of HLA molecules. Screening of non-immunized phage libraries,19,20 endogenous TCR or low expression of transduced TCR due to 21,22 competition with endogenous TCR have been reported.7–9 These peptide-loaded MHC (pMHC) immunization, and their 18,23,24 may be overcome by the use of γδT cells instead of αβT cells as combination also have been explored to produce effector cells,10 RNA interference specific for endogenous TCR5 or TCR-like mAbs targeting potentially therapeutic peptides finally TCR gene deletion with designer zinc finger nucleases.11 presented on various HLA molecules. Among these reports, a Another mode of adoptive T-cell therapy is to engineer T cells to promising scFv mAb against HLA-A2 complex incorporating express chimeric antigen receptors (CARs).12–15 The prototypical PR1, a proteinase 3-derived peptide that can induce efficient

1Department of Hematology, School of Medicine, Fujita Health University, Toyoake, Japan; 2Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan; 3Division of Immunology, Aichi Cancer Center Research Center, Nagoya, Japan; 4HLA Foundation Laboratory, Kyoto, Japan; 5Laboratory of Molecular Biology and Histochemistry, Fujita Health University Joint Research Laboratory, Toyoake, Japan and 6Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. Correspondence: Dr Y Akatsuka, Department of Hematology, School of Medicine, Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake 470-1192, Japan. E-mail: [email protected] Received 17 June 2013; revised 3 December 2013; accepted 6 January 2014; published online 3 April 2014 T-cell-like antibody specific for minor antigen Y Inaguma et al 576 complement-dependent cytolysis of myeloid leukemia progenitor In addition, antibody-blocking experiments demonstrated that cells but not normal hematopoietic cells, has been reported.18 #131 CD8 CAR-T cells were less sensitive to CD8 blockade while In this study, we describe the efficient isolation of #131, a novel they were completely blocked with HA-1 H/HLA-A2 tetramer. scFv mAb that binds with a conformational epitope of HA-1 CAR-T cells with lower KD (446 nM for clone #4 and 64.3 nM for H/HLA-A*02:01 by combining phage display screening of spleno- clone #9) did not exhibit cytotoxic activity superior to #131 CAR-T cytes from HLA-A2.1 (HHD) transgenic mice (Tgm) immunized cells. These data suggest that T cells with higher affinity antigen with tetramerized pMHC consisting of HA-1 H peptide, receptors than TCRs (average KD ranging between 1–100 μM) HLA-A*0201 and β2-microglobulin. We hypothesized that the are less able to recognize low-density peptide/MHC antigens as use of HLA-A2.1 (HHD) Tgm would be effective in offsetting reported in the case of affinity-matured TCR26 or CAR,12 and xenogenic immunogenicity of HLA-A*02:01 complexes to which that particularly CD8+ CAR-T cells may not be necessarily CD8- the murine T cells have been rendered tolerant during intrauterine dependent possibly due to failure to form complexes with CD3. development. We chose an HA-1 H minor H antigen epitope (VLHDDLLEA; the other allelic variant encodes VLRDDLLEA) derived from the HMHA1 gene product. The monomeric #131 RESULTS fi scFv mAb with high affinity (dissociation constant [KD] = 14.9 nM) Isolation of scFv speci c for HA-1 H/HLA-A2 complex successfully recognized HA-1 H/HLA-A2 but not HLA-A2 recon- A total of 5 × 108 phage clones were initially generated from stituted with other known HLA-A2-binding peptides including splenic B cells of three mice. After three rounds of panning, the HA-1 R. Next, we characterized primary human CD4 and CD8 T recovered phages were used to infect Escherichia coli, which cells modified with #131 and other scFVs with lower KD coupled to were spread on plates containing ampicillin without infection with the CD28 transmembrane and CD3ζ domains. Most of them helper phages (Figure 1a). A total of 144 clones randomly selected stained with HA-1 H/HLA-A2 tetramers as strongly as a cytotoxic T from 8.1 × 108 clones that had been recovered after the third lymphocyte (CTL) clone, EH6,25 specific for endogenously panning were tested by parallel ELISA screening for binding to HLA-A2- and HA-1 H-positive cells. Unexpectedly, however, both HA-1 H/HLA-A2 but not to MAGEA4/HLA-A2, as an irrelevant CD4 and CD8 #131 scFv CAR-T cells required ~ 100-fold control pMHC complex. Clones showing at least 10-fold higher greater antigen density to be pulsed exogenously in order to ELISA signal with HA-1H/HLA-A2 compared with MAGEA4/HLA-A2 exert cytotoxicity comparable to the cognate CTL clone, EH6. were further tested and categorized according to their VH CDR3

HA-1/A2-Db in adjuvant

Immunization

Isolation of VH and VL from splenic B cells

Primary phage clones (5x108)

Panning (3 times) against HA-1H/A2

Screening of resultant phage clones (8.1x108) for specificity by ELISA

Non- HLA-A2 HA-1H/A2 Total reactive reactive specific

144 7 137 18

Figure 1. Schematic representation of the generation and selection of HA-1 H/HLA-A2-specific scFv mAb. (a) HLA-A2.1 (HHD)-transgenic mice were subcutaneously immunized with suspension composed of 50 μg HA-1 H/HLA-A2-Db tetramers emulsified with Montanide ISA51 adjuvant at 7-day intervals. One week after the third immunization, splenocytes were harvested and B-lymphocyte-rich fractions were used to construct a library by a phage-display system. The phage library was then concentrated by three-round panning against coated HA-1 H/ HLA-A2 in the presence of irrelevant MAGEA4/HLA-A2 monomers. (b) The table shows the total number of screened scFv cp3 clones after the panning and their specificities. (c) The binding activities of the representative cp3 clones assessed by ELISA. Binding of the seven clones to bottom-fixed HA-1 H/HLA-A2 antigens was assessed by twofold serial dilutions down to 128-fold. (d) BIAcore surface plasmon resonance of selected clones. BIAcore measurements were performed to measure binding affinity of candidate-tetramerized scFv mAbs as described in the Materials and Methods. HA-1 H, HMHA1 histocompatibility (minor) HA-1-derivied peptide (amino-acid position 153–161), VLHDDLLEA. Note that strong spikes were recorded at buffer exchange due to the high sensitivity of BIAcore 3000 instrument.

Gene Therapy (2014) 575 – 584 © 2014 Macmillan Publishers Limited T-cell-like antibody specific for minor antigen Y Inaguma et al 577 amino-acid sequences. Among 144 clones screened, 18 (12.5%) were not stained (data not shown). The stabilized expression level showed preferential binding to HA-1/HLA-A2, 137 showed similar of HLA-A2 on T2 cells loaded with the above-mentioned peptides binding to both pMHC complexes, and 7 showed reactivity to was verified as increased MFI with anti-HLA-A2 mAb (Figure 2b). neither of them (Figure 1b). As shown in Figure 1b, among Interestingly, HA-1R peptide was almost incapable of stabilizing 18 specific binders to HA-1H/HLA-A2 we identified seven different HLA-A2 molecule on T2 cells, confirming the previous report that groups containing 1–8 identical clones per group based on their HA-1R is a ~ 2 log inferior binder to HLA-A2 compared with CDR3 amino-acid sequences. The binding activities of the HA-1H.3 These data suggested that under exogenously pulsed representative cp3 clones from each group were assessed by conditions, monomeric #131 binds to efficiently HA-1H peptide ELISA. As shown in Figure 1c, binding of #131 to bottom-fixed and weakly to HA-1R peptide presented by HLA-A2 molecules. HA-1H/HLA-A2 antigens was detected until 64-fold dilution when Furthermore, monomeric #131 binding was concentration- the cutoff OD value was set to 0.5, while that of #4 and #9 was dependent against T2 cells labeled at fixed concentration detected until 16-fold dilution. We also used surface plasmon (10 μM) of HA-1H peptide (Figure 2c) and also on the concentra- resonance to measure binding affinity of candidate scFv tion of HA-1H peptide added to T2 cells (Figure 2d). Use of a antibodies. Among three representative clones (#4, #9 and #131) fluorescence amplification increased the detection limit of tested, clone #131 bound with best affinity to soluble HA-1H/HLA- HA-1H presented on T2 from 1 μM to 0.1 μM, suggesting that A2 monomer, mainly because of its balanced association and the monomeric #131 scFv did bind to HA-1H peptide/HLA-A2 dissociation rate, with a dissociation constant (KD) of 14.9 nM complexes at concentrations as low as 0.1 μM (see Supplementary (Figure 1d), compared with KD of 446 nM for clone #4 and 64.3 nM Figure 1). for clone #9, respectively. We also tested binding affinity to To further evaluate the affinity of the monomeric #131 scFv, we MAGE3A/HLA-A2, but we were unable to detect any resonance quantified HA-1H/HLA-A2 molecules on T2 cells as antibody- (data not shown). Collectively, we chose clone #131 for binding capacity (ABC) as previously reported.27 By interpolation further study. on the calibration curve (Figure 3a) and the obtained mean fluorescence intensity (MFI) for individual T2 cells pulsed with HA-1H peptide diluted serially by 10-fold from 10 μM to 0.01 μM Characterization of #131 scFv specificity and affinity (Figure 3b), we could determine specific antibody-binding To further confirm #131 specificity, we examined whether capacity (SABC) as number of cell surface antigens. For example, monomeric #131 scFv could distinguish peptides presented by T2 cells pulsed with 10 μM HA-1H peptide possessed 29 815 HA-1H cell surface HLA-A2 molecules on T2 cells at a peptide peptide/HLA-A2 complexes per cell with MFI shift from 24.67 concentration of 10 μM by using flow cytometry. As shown in (control T2 cells) to 176.81 (Figure 3b). On the other hand, using Figure 2a, #131 scFv monomer detected T2 cells pulsed with an MHC stabilization assay, the MFI shift assessed by anti-HLA-A2 HA-1H (bold solid line) but not other peptides that bound HLA-A2, mAb binding was from 75.5 to 157 (Figure 2b). These data indicate such as Flu-A M1, MAGEA4, HBV Env and HER2/neu. On the other that the monomeric #131 scFv did not fail to bind to HA-1H- hand, T2 cells pulsed with HA-1R (human HA-1H counterpart) were bound, stabilized HLA-A2 complexes. The peptide concentration weakly stained (dashed line) while those pulsed with 1 μM HA-1R detection limit of 0.1 μM corresponding to 1963 antigens per cell is

100 140 Flu-A M1, MAGEA4, HA-1R 120 80 HBV Env, HER2/neu 100 Flu-A M1 60 HA-1R HA-1H 80 60 HA-1H Counts Counts 40 40 MAGEA4, HBV Env, 20 HER2/neu 20 0 0 100 101 102 103 104 0 200 400 600 800 1000 #131 scFv monomer-PE Fluorescence Intensity (HLA-A2) MFI MFI

#131 scFv monomer concentration (g/ml) Peptide concentration (M)

Figure 2. Characterization of #131 scFv specificity and affinity. (a) Specificity was evaluated by incubating T2 cells with 10 μM of the indicated peptides for 1 h, and cells were stained with PE-conjugated #131 scFv monomer and analyzed by flow cytometry. HA-1R, H151R polymorphic version of HA-1 H; Flu-A M1, influenza A matrix protein58-66; HBV Env, Hepatitis B virus envelope183-191. Non-pulsed T2 cells is shown as a shaded cytogram. Representative data from two independent experiments are shown. (b) MHC stabilization assay. T2 cells were pulsed with each of the indicated peptides (10 μM) at 27 °C overnight, followed by incubation at 37 °C for 3 h. Surface HLA-A2 molecules were then stained with Alexa 647-conjugated HLA-A2-specific mAb and analyzed by flow cytometry. Non-pulsed T2 cells is shown as a shaded cytogram. Representative data from two independent experiments are shown. (c) Binding characterization of #131 scFv monomer. Titrated concentrations of the PE-labeled #131 scFv monomer were tested with T2 cells pulsed with 10 μM HA-1 H peptide at 37 °C for 2 h. Representative data from two independent experiments are shown. (d) Sensitivity of #131 scFv monomer. T2 cells were incubated with the indicated concentrations of the HA-1 H or HA-1R peptides for 2 h and used for the binding assay with #131 scFv monomer. Representative data from one of at least two independent experiments are shown.

© 2014 Macmillan Publishers Limited Gene Therapy (2014) 575 – 584 T-cell-like antibody specific for minor antigen Y Inaguma et al 578 construct (Figure 4a) on days 3 and 4 in parallel. We compared whether the transduced T cells with different constructs would exert any growth advantage or disadvantage. As shown in Figure 4b, not only non-transduced cells but also scFv-28z CAR-transduced T cells (designated to as #4–28z, #9–28z and #131–28z, respectively) demonstrated an average of ~ 1000-fold calibration beads expansion after a single cycle of bead stimulation over the course of transduction and growth in 21 days. Addition of IL-7 or IL-15 to blank beads the IL-2-supplemented cultures did not change the growth capacity when CD3/CD28 beads29 were used for initial stimulation (data not shown). The percentages and MFI of T cells expressing Antigen binding capacity (ABC) the individual CAR with anti-CD8 mAb and HA-1H/HLA-A2 tetramers after magnetic bead-based separation for CD4 or CD8 MFI are shown in Figure 4c, while they were not stained with irrelevant fi 300 HIV/HLA-A2 tetramers (data not shown). Interestingly, the speci c 100 nM MFI value of CD4- and CD8-sorted T cells expressing individual 240 10 nM CARs (designated to as #4–28z (CD4), #4–28z (CD8), #9–28z (CD4), 1 M #9–28z (CD8), #131–28z (CD4) and #131–28z (CD8), respectively) 180 1 nM 10 M was slightly stronger than that of the cognate HA-1H/HLA-A2- fi 25 Count 120 speci c EH6-CTL (Figure 4c) when a minimally saturating concentration of HA-1H/HLA-A2 tetramers was used. In addition, 60 of note is that a subset of CD4 T cells tend to be resistant 0 to retroviral transduction (Figure 4c, lower panel), but we used 100 101 102 103 104 these lines throughout the experiments for this revision. Anti-mouse IgG-FITC Cytotoxicity of CAR-T cells

HA-1H concentrationon We evaluated the cytolytic abilities of six CAR-T cell lines, two MFI ABC SABC 2 cells (M) mock-transduced T cell lines and EH6-CTL using a 4-h CRA. We conducted peptide reconstitution assays using 51chromium- Irrelevant scFv 24.67 4104 0 labeled T2 cells (Figure 5) or K562/A2 cells (Supplementary 1 nM 25.28 4213 109 Figure 2) by pulsing the peptides (HA-1H and HA-1R) for 2 h at room temperature, and then using as targets. The #131–28z (CD8) 10 nM 26.26 4388 284 cells could lyse T2 cells pulsed with as low as 10 nM cognate HA-1H 100 nM 35.52 6067 1963 peptide (corresponding to the concentration at the half maximal 1 µM 84.46 15359 11255 lysis) while T2 cells pulsed with HA-1H allelic counterpart HA-1R µ were not recognized. Similar cytotoxic activities were observed for 10 M 176.81 33919 29815 #4–28z (CD4), #4–28z (CD8) and #131–28z (CD4) cells, but the – – Figure 3. Quantification of copy number of HA-1 H/HLA-A2 same trend was observed only in #4 28z (CD8) and #131 28z complexes per cell. (a) Calibration curve using QIFI kit (DAKO). (CD8) cells when target cells were changed to K562/A2 Beads coated with well-determined quantities of mouse mAb were (Supplementary Figure 2). Both #9–28z (CD4) and #9–28z (CD8) detected with F(ab)2 fragment of FITC-conjugated goat anti-mouse cells demonstrated cytotoxicity to not only HA-1H but also HA-1R IgG. The MFI and indicated mouse IgG molecules per bead (as peptide-pulsed T2 cells, suggesting its lower specificity. antigen-binding capacity, ABC) were plotted. (b) Detection of The cognate EH6-CTL25 lysed T2 cells pulsed with HA-1H, at HA-1 H/HLA-A2 complexes on T2 cells pulsed with 10-fold serially concentrations as low as 10pM (its half-maximal lysis was obtained diluted HA-1 H peptide. After 2-h incubation with indicated at the concentration of 100 pM), ~ 2 log lower than that required concentration of HA-1H peptide, T2 cells were first incubated with for the #131–28z (CD8) cells (Figure 4b). saturating concentration of #131 scFv mAb and then stained – with FITC-conjugated anti-mouse IgG mAb. These procedures were Although the expression levels of #131 28z (CD8) detected by performed in parallel with bead preparation as in (a). (c) Estimation HA-1H/HLA-A2 tetramer was stronger than that of EH6-CTL of specific antigen-binding capacity of T2 cells pulsed with HA-1H. (Figure 4c), the minimal HA-1H peptide concentration necessary The obtained MFI was interpreted to ABC using the calibration for T-cell recognition was nearly 2 logs different. curve, and then SABC was calculated by subtraction of background antigen equivalent (MFI of stained with irrelevant scFv) from observed ABC. Reactivity to endogenously HA-1H-expressing target cells by selected CAR-T cells We next studied whether CAR-T cells selected for positive lytic not necessarily low when considering a report from Wang et al.28 activity specific for HA-1H peptide-pulsed target cells were able to demonstrating that the mean ABC value for CD20 on normal B kill target cells that express HA-1H epitope endogenously. To cells is ~ 140 000 and that on B-cells from chronic lymphocytic prepare versatile artificial target cells, K562 cells that possessed leukemia is 22 000. only HA-1R alleles homozygously were stably transduced with HLA-A*02:01 followed by minigenes encoding either HA-1H or HA-1R 9-mer peptide (designated as to K562/A2/HA-1H, K562/A2/ Generation of CAR-T cells from primary human T cells HA-1R, respectively). For this experiment, T2 cells were excluded As retroviral vectors can transfer genes into rapidly dividing cells because they cannot transport cytosolic peptides to endoplasmic with high efficiency, we used anti-CD3/anti-CD28 coated magnetic reticulum. As shown in Figure 6a, EH6-CTL lysed not only K562/A2 29 30 beads instead of the rapid expansion protocol with anti-CD3 cells pulsed with 10 nM HA-1H but also K562/A2/HA-1H. None of (clone OKT3) for activation and expansion of human primary T the CAR-T cells (that is, #4–28z (CD8), #4–28z (CD4), #131–28z cells. Activated T cells (2 × 105) purified from a normal volunteer (CD8) and #131–28z (CD4)) however were able to lyse K562/A2/ were infected with a retroviral vector containing #4, #9 or #131 HA-1H efficiently. In contrast, both #131–28z (CD8) and #131–28z

Infection No. of cells (x10

Days after stimulation

EH6-CTL Mock (CD8) #4-28z (CD8) #9-28z (CD8) #131-28z (CD8) (174) (3.8) (6.5) (765) (5.3) (322) (6.4) (538)

1.3 95.5 99.0 0.5 19.1 80.2 5.3 94.3 6.8 92.9 1.3 1.9 0.5 0 0.2 0.5 0.3 0.1 0.1 0.1

HA-1H/HLA-A2

CD8 Mock (CD4) #4-28z (CD4) #9-28z (CD4) #131-28z (CD4) (2.6) 1.1 0 0.4 0.7 0.4 1.7 0.8 1.7 98.9 0 38.4 60.5 34.8 63.1 38.5 59.0 97.3 0.2 (3.3) (4.2)(986) (5.8) (229) (5.2) (446) 2.5 0

HIV/HLA-A2 HA-1H/HLA-A2

Figure 4. Retroviral transduction of primary human T cells with various scFv CAR. (a) A diagram of the recombinant retroviral LZRSpBMN- based vector encoding scFv-28z is shown (LTR, long terminal repeat; CD28, part of the extracellular region and all of the transmembrane and intracellular regions of CD28; CD3-ζ, the entire cytoplasmic region of the CD3-ζ molecule). (b) In vitro expansion of T cells following activation with anti-CD3/anti-CD28-coated magnetic beads and transduction with or without scFv CAR vector on day 2 and 3. Data are representative of two independent experiments. (c) Expression levels of individual scFv on sorted CD8 and CD4 T cells were compared with that of TCR on EH6-CTL, which is speciﬁc for cognate HA-1H epitope in the context of HLA-A*02:01 by staining with PE-conjugated HA-1H/HLA-A2 tetramer. Mock means that T cells were infected with supernatant from packaging cell line. Data are representative of two independent experiments.

(CD4) cells were able to produce interferon-γ (IFN-γ), tissue DISCUSSION necrosis-α (TNF-α) and IL-2 against K562/A2/HA-1H (Figures 6b–d). In this study we describe the first scFv that binds specifically to a Of note is that #4–28z (CD4) cells were able to produce large complex formed by a minor H antigen peptide, HA-1H, and the amounts of TNF-α and IL-2, but #4–28z (CD8) cells showed no HLA-A*02:01 molecule. It has been suggested that the majority of significant trend. Collectively, although the selected CAR-T cells clones isolated from conventional phage mAb libraries may not be failed to exert evaluable cytotoxic activity by conventional 4-h of high affinity because they should be naive to the antigens.31 CRA, it was found that at least #4–28z (CD4), #131–28z (CD8) and Thus, we used a phage mAb library that was constructed from #131–28z (CD4) CAR-T cells were potent to produce inflammatory HLA-A2.1-Tgm B cells that had been immunized with HA-1H/HLA- cytokines in response to endogenously presented HA-1H/A2 A2 tetramer. We hypothesized that use of HLA-A2.1-Tgm would complexes. facilitate efficient induction of high-affinity mAbs because B cells were tolerant to xenogeneic HLA-A2 complexes presenting murine endogenous peptides. We also incorporated a panning procedure in the presence of irrelevant MAGEA4/HLA-A2 mono- Blocking studies for CD8 #131–28z CAR-T cells mers to block binders to immunogenic streptavidin and peptide/ To this end, we examined the mode of recognition of these two HLA-A2 molecules other than HA-1H/HLA-A2 in addition to effector cells by blocking experiments (Figure 7). Lytic activity of depletion of streptavidin binders originally used by Cohen CD8/#131–28z CAR-T cells (independently generated from posi- et al.32 In the end we found that 12.5% (18 out of 144) of the tively sorted CD8 T cells at the beginning) against HA-1H-pulsed resultant phage mAbs showed preferential binding to HA-1H/HLA- T2 cells was slightly blocked by CD8 mAb (clone Hit8a) while that A2 and one of these, clone #131, showed a high binding affinity 18 of EH6-CTL was profoundly blocked. Anti-HLA-A2 mAb (clone (KD = 14.9 nM) to HA-1H/HLA-A2. Sergeeva et al. reported that BB7.2) showed partial inhibition to CD8/#131–28z CAR-T cells but 78% of hybridoma clones generated were HLA-A2-specific and not E6-CTL, suggesting that #131 scFv contacts HLA-A2 residues only 1 out of 2850 clones recognized PR1/HLA-A2, indicating our that are close to BB7.2 recognition. HA-1H/HLA-A2 tetramer screening approach was capable of concentrating targeted clones. profoundly inhibited lytic activity of CD8/#131–28z CAR-T cells and As expected, monomeric #131 scFv did not bind to HA-1R (due to EH6-CTL, but its effect was significantly stronger in inhibition of its low affinity to HLA-A2) or irrelevant HLA-A2-restricted peptides #131 scFv binding (Figure 7). Considering that #131–28z (CD4) on T2 cells, but unexpectedly, did bind to T2 cells pulsed CAR-T cells also lysed T2 cells pulsed with 10 nM HA-1H peptide exogenously with HA-1Q, a nonameric peptide corresponding to (Figure 5), CD8 coreceptor is unlikely involved in the facilitated murine Hmha1 (Supplementary Figure 3a). The binding to HA-1Q/ recognition. HLA-A2 was reflected as cytotoxicity against T2 cells pulsed with

Figure 5. Epitope reconstitution assays. T2 cells were labeled with 51Cr and distributed in 96-well round-bottomed plates, pulsed with serial dilutions of the indicated peptides for 2 h at room temperature, and then used as targets for CD4- or CD8-sorted human primary T cells that had been gene-modiﬁed with indicated scFvs and EH6-CTL as cognate CTL in a standard 51Cr release assay (E:T ratio 10:1). Data are representative of two independent experiments. Half-maximal lysis for EH6-CTL and #131-28z (CD8) were shown as dashed line with arrow head.

HA-1Q by CD8/#131–28z CAR-T cells (Supplementary Figure 3b); staining was slightly stronger than that of HA-1H-specific CTL however, EH6-CTL did not lyse T2 cells even pulsed with 1 μM clone, EH6, the #131–28z (CD8) cells were unable to lyse K562/A2/ HA-1Q (Supplementary Figure 3c). In addition, murine thymoma HA-1H efficiently while they could produce pro-inflammatory cell line, EL4 (H-2Db background), expressing HLA-A2 and human cytokines. The failure in cytotoxicity with #131–28z (CD8) cells may β2-microglobulin was not lysed at all by CD8/#131–28z CAR-T cells be explained by several findings. First, the #131–28z (CD8) cells (Supplementary Figure 3d). These findings suggest that HA-1Q required 100-fold higher peptide concentration compared with peptide can be presented in association with HLA-A*02:01 at a EH6-CTL irrespective of slightly superior HA-1H/ HLA-A2 tetramer level comparable to HA-1H, but because of its very low cell surface binding. Second, CD8 co-receptor was not employed effectively by copy number (discussed later) even though endogenously CD8/#131–28z cells, unlike EH6-CTL as shown by blocking studies. presented, the murine B cells were likely not tolerant and able The coreceptor CD8 is thought to contribute to the antigen- to mount immune responses when they were challenged recognition process by binding to α3 domain of the MHC class I by HA-1H/HLA-A2 tetramers. As a result, the generation of molecule and by promoting intracellular signaling, serving to high-affinity #131 scFv was eventually possible. Elucidation of enhance TCR stimuli triggered by cognate ligands.33 It is possible the mechanism of cross-reactivity between HA-1H and HA-1Q that the #131–28z molecule failed to recruit CD8 resulting in could require three-dimensional analysis and is beyond the scope insufficient activation of #131–28z (CD8) cells. However, this may of current studies. Nevertheless, it is of note that both use of not be critical because #131–28z (CD4) cells showed similar HLA-A2.1 Tgm for immunization and depletion of potential HLA- cytotoxic activities against HA-1H-pulsed T2 cells. Third, A2 binders other than HA-1H/HLA-A2 using irrelevant HLA-A2 cell surface HA-1H/HLA-A2 complexes have been reported to be monomers resulted in the efficient generation of scFv specific for ~ 10 per cell by comparing the signal intensity of the naturally targeted peptide/HLA complexes. As the degree of polymorphism occurring peptide with a known amount of synthetic HA-1H among HLA-A alleles is low compared with amino acid differences peptide.3 In the case of PR1/HLA-A2, it has been shown that T2 between HLA and H-2 molecules, we surmise that the use of a cells pulsed with 1μM PR1 present 10 000 copies per cell, which phage library prepared from HLA-A2.1 Tgm can facilitate the corresponds to 100 copies per T2 cell pulsed with 10 nM efficient generation of TCR-like mAb specific for peptides peptides.34 Fourth, in our quantification analysis based on presented at least on other HLA-A alleles rather than using non antibody-binding capacity, the detection limit of 10 nM HA-1H Tgm. This should be also the case when phage libraries from on T2 cells corresponded to ~ 300 copies per cell. It appears that human B cells are to be constructed. ~ 100 copies per cell may be the threshold for detection by scFv or Next we questioned whether endogenously HLA-A*02:01 and probably CAR-T cells when evaluated by cytotoxicity. Thus, it is HA-1H-expressing cells were susceptible to CAR-T cells when #131 possible that #131–28z (CD8) cells could not lyse target cells that scFv monomer failed to detect them by flow cytometry even using were expressing HA-1H peptides endogenously at ~ 10 copies per fluorescence amplification. We transduced T cells with #131 cell. In terms of cytokine-producing capacity, however, this range scFv with transmembrane and intracellular signaling domain may be sufficient even with #131–28z (CD8) cells. If this is the case, consisting of CD28 and CD3-ζ. Although expression of #131 scFv it is intriguing to know why EH6-CTL lysed such target cells on #131–28z (CD8) cells assessed by HA-1H/HLA-A2 tetramer efficiently. A serial triggering model of T-cell activation has been

Figure 6. Recognition of endogenously HA-1H expressing target cells assessed by cytotoxicity and cytokine production. (a) The human primary CD4 or CD8 T cells gene-modiﬁed with either #4–28z or #131–28z vectors and cognate EH6-CTL were co-cultured with 51Cr-labeled, HLA-A*02:01-transduced K562 cells (both HA-1 alleles are HA-1 R) stably expressing HA-1 H or HA-1R peptides from corresponding minigene vectors in a 4-h standard chromium release assays at an E:T ratio of 10:1. Positive controls are HLA-A*02:01-transduced K562 cells pulsed with 10 nM HA-1 H peptide. (b–d) Cytokine production assessed by ELISA for (b) interferon-γ,(c) tissue necrosis factor-α and (d) interleukin-2. Number of effector T cells and stimulators was 10 000 and 5000 per well, respectively. Positive controls are HLA-A*02:01-transduced K562 cells pulsed with 1 nM HA-1 H peptide. *Po0.01; **Po0.05; NS, not signiﬁcant.

high-affinity receptor expressed on #131–28z (CD8) might preclude the T cells from attaining sequential engagement with the limited number of HA-1H/HLA-A2 complexes. The findings that #4–28z (CD4) CAR-T cells possessing scFv whose KD was 446 nM, 30-fold higher than that of #131, produced large amount of TNF-α and IL-2 may support this hypothesis. In contrast, when target cells expressing a sufficient number of target antigens where a single engagement of individual CAR is enough for T-cell activation, high affinity at 'antibody level' would not limit CAR-T cells to activate and lyse targets. Our data suggest that problems in efficient T-cell activation with high-affinity receptors could occur not only in TCR but also CAR as previously reported by Chmielewski et al.12 More extensive binding studies using other scFv CARs obtained throughout the experiments other than #4 and #9 will address the appropriate range of affinity to low-density Figure 7. Inhibition of cytotoxicity by mAbs and tetramers. cell surface HA-1H/HLA-A2 complexes. Chromium-labeled T2 cells were incubated with indicated mAbs fi + In summary, we have demonstrated the generation of the rst or tetramers, and were tested by human primary CD8 cytotoxic T fi fi CAR-T cells speci c for minor H antigen, HA-1H, presented on HLA- lymphocytes gene-modi ed with #131-28z vector at an E:T ratio of fi fi 1:1. Pre-determined concentrations of mAbs (typically 10 or 20 A*02:01. Our data con rm that T cells with too-high-af nity CAR μgml− 1) or tetramers (10 μgml− 1) were used. The mAbs used for paradoxically may fail to get appropriately activated and kill target this purpose were against the following antigens: HLA-A2 (BB7.2), cells expressing a very low number of target antigens as CD4 (RPA-T4), CD8 (HIT8a). *Po0.05. previously reported12 and that CD8 coreceptor may be dispen- sable. Further studies with T cells expressing scFv with lower affinity similar to TCR level will be warranted. postulated where one MHC–peptide complex can sequentially engage several TCR molecules to achieve a critical threshold of ~ 200 triggered TCRs that is required for sufficient T-cell activation MATERIALS AND METHODS (reviewed by Valitutti et al.35) As the affinity of TCR for peptide/ Mice b− / − − / − MHC complexes is relatively low, with dissociation constants KD HLA-A2.1 (HHD) Tgm; H-2D β2m double knockout mice introduced 36 − b ranging from 1–100 μM, while that of #131 scFv is 14.9 nM; such a with a human β2m HLA-A2.1 (α1, α2)-H-2D (α3 transmembrane

© 2014 Macmillan Publishers Limited Gene Therapy (2014) 575 – 584 T-cell-like antibody specific for minor antigen Y Inaguma et al 582 cytoplasmic; HHD) monochain construct gene were generated in the Immunization of mice with HA-1H/HLA-A2-Db tetramers Département SIDA-Retrovirus, Unite d’ Immunite Cellulaire Antivirale, HLA-A2.1 (HHD) Tgm mice at 6–8 weeks of age were subcutaneously 37,38 Institut Pasteur and kindly provided by Dr FA Lemonnier. immunized with a 300-μl suspension composed of 50 μg HA-1 H/HLA- The experimental design was approved by the Animal Care Committee A2-Db tetramers emulsified with Montanide ISA51 adjuvant (SEPPIC, Paris, of the Aichi Cancer Center Research Institute, and the animals were cared France) at 7-day intervals. One week after the third immunization, for in accordance with institutional guidelines as well as the Guidelines for splenocytes were harvested and frozen until use. Proper Conduct of Animal Experiments. Phage library preparation and isolation of mAbs against Blood samples and cell lines HA-1 H/HLA-A2 The study design and purpose, after prior approval by the Institutional A phage library was constructed as previously reported with Review Board of the Aichi Cancer Center, were fully explained, and written modifications.43 In brief, frozen spleen cells (~107 cells) from the consent was obtained from healthy blood donors. Peripheral blood immunized HLA-A2.1 (HHD) Tgm were thawed and used as gene sources mononuclear cells (PBMCs) were isolated and subjected to B-lymphoid of mAbs. Following isolation of total RNA by the guanidine thiocyanate cell line (B-LCL) induction and isolation of CD8+ T cells with a CD8+ T method, cDNA synthesis was performed using random hexamer and Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). B-LCLs, T2 SuperScript III RT First-Strand Synthesis System (Life Technologies, (T-cell/B-cell hybridoma deficient for transporter associated with antigen Carlsbad, CA, USA). cDNA was amplified with murine JH primer mix for processing), K562 (homozygous for the Arg allele of the HA-1 genotype), VH, and random hexamer (Life Technologies) for Vκ and Vλ, respectively, EL6 (murine lymphoma, H-2b), EL4S3-Rob-A2-HHD (EL4 transduced with digested with appropriate restriction enzymes (SfiI and XhoI for VH, human β2-microglobulin-HLA-A2-Db), 293T, Phoenix-Galv cells and their NcoI and AscI for Vκ and Vλ, respectively), and finally cloned into pSCCA5 transfectants were cultured as previously described.39 The HA-1H/HLA-A2- vector. Using a phage-display system, the scFv form of a mAb fused to a specific cytotoxic T cell clone, EH6-CTL,25 was used as a positive control. truncated cp3 (Fab-cp3) was expressed on the phage surface.43

Synthetic peptides Selection of phage mAbs on biotinylated complexes Peptides VLHDDLLEA (HA-1H),3 VLRDDLLEA (HA-1R), VLQDDLLEA (murine Selection of phages exhibiting HA-1 H/HLA-A2 binding activity was HA-1 counterpart, HA-1Q), FLLTRILTI (HVB Env – ), GILGFVFTL (Influenza performed by a panning method that was essentially the same as 183 191 44–46 AMP , Flu-A M1), GVYDGREHTV (MAGEA4 ), ALCRWGLLL (HER2/ that described previously with some modifications. Phages were first 58-66 230-239 − 1 neu ) and SLYNTVATL (HIV Gag ) were purchased from Bio-Synthesis, preincubated with 1% Triton X-100/PBS solution with 100 μgml 5-13 77-85 − 1 Inc. (Lewisville, TX, USA). They were reconstituted in an appropriate solvent streptavidin and 100 μgml non-biotinylated MAGEA4/HLA-A2 mono- and frozen until use. mers for 1 h to block the streptavidin and HLA-A2 binders, followed by incubation in a Maxisorp Loose plate (Nunc, Roskilde, Denmark) that had been coated with biotinylated HA-1H/HLA-A2 via NeutrAvidin (Life Generation of pMHC monomers and tetramers Technologies) for 1 h. Then, the well was washed 10 times with 0.1% fi fi The human β2m-HLA-A2.1 (α1, α2)-H-2Db (α3) single-chain gene construct Tween-20/PBS, ve times with PBS and nally once with high-purity Milli-Q was PCR-amplified from genomic DNA isolated from HLA-A2.1 (HHD) Tgm water (Millipore, Billerica, MA, USA). Finally, bound phages were eluted spleen cells. For monomer production, the leader sequence of the original with 50 mM glycine (pH 3.0), and the eluent was immediately neutralized construct was removed and substituted with a prokaryote ribosome with 1.5 M Tris-HCl, and then infected to E. coli, DH12S cells. The phage binding site for expression in E. coli and BamHI site was introduced at the clones obtained through this process were used for the next round of 3′ end of Db exon 4 corresponding to the C terminus of the α3 domain panning. After the third round of panning, DH12S cells infected with with the primers as follows (EcoRI and BamHI sites for cloning are the selected phages were spread on appropriate agar plate and incubated underlined, respectively): sense 5′-ATGAATTCTAAGGAGGATATTAAAATG at 30 °C overnight. ATCCAGCGTACTCCAAAG-3′ and antisense 5′-ATGGATCCCCATCTCAGG GTGAGGGGCTTGGGCAAACC-3′. The PCR product was cloned into a Enzyme-linked immunosorbent assay (ELISA) pHN1+ vector (kindly provided by the late Dr Don C Wiley, Howard To determine the specificity of the mAbs presented on cp3 against Hughes Medical Institute, Harvard University, Cambridge, MA, USA) that HA-1H/HLA-A2, immunoplates (96-well; Thermo Fisher Scientific, had been modified to join a BamHI site followed by the sequence for the Cambridge, MA, USA) were in parallel coated either with HA-1H/HLA-A2 biotinylating enzyme BirA, expressed as inclusion bodies in XA-90 E. coli, or MAGEA3/HLA-A2 tetramers as previously reported.43 For the reactivity purified and refolded with HA-1H peptide essentially as described study, the plates were then incubated with 50 ng (or twofold serial dilution previously.40,41 In addition, for screening purposes, HA-1H and MAGEA3 for titration ranging from 1 × to 128 × ) of purified Fab-cp3 in PBS(+) at 4 °C peptides were refolded with HLA-A2 heavy chain and β2-microglobulin in overnight. After the plates had been washed with PBS(+), a custom-made a conventional manner.41 Part of the resultant pMHC complexes and pMHC rabbit anti-cp3 polyclonal antibody (MBL) was added and incubated at incorporating HIV peptide (as negative control) were biotinylated and then room temperature for 1 h. After washing with PBS(+), peroxidase- converted into tetramers with PE-labeled streptavidin. conjugated goat anti-mouse IgG (H+L chain; MBL) was added. Finally, bound scFv-cp3 was detected by incubation with OPD substrate Flow cytometry and MHC stabilization assay (Sigma-Aldrich, St Louis, MO, USA) reading absorbance at 492 nm. The scFv gene-transduced CAR-T cells were incubated with optimized concentration (1–2 μgml− 1) of tetramer at room temperature for 15 min Surface plasmon resonance followed by FITC-conjugated anti-CD3 (BD Biosciences, San Diego, CA, Kinetics and affinities of the monomeric HA-1H/HLA-A2 binding to USA) and Tricolor anti-CD8 mAb (Caltag, Burlingame, CA, USA) on ice for three scFv antibodies (#4, #9 and #131) were measured by surface 15 min. Peptide-pulsed T2, K562/A2 cells or K562 transfectants were plasmon resonance using a BIAcore 3000 at Institute for Antibodies Co., stained with PE-labeled #131 monomer (see below) on ice for 15 min. If Ltd. (Nagoya, Japan). Binding studies were performed at 25 °C using a HBS- fi necessary, the signal of #131 monomer-PE was ampli ed by FASER EP running buffer (GE Healthcare, Uppsala Sweden) containing 10 mM fi Kit PE Ampli cation (Miltenyi Biotec) according to the manufacturer's HEPES, pH7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20 surfactant. The scFvpp instructions. Copy number of HA-1H/HLA-A2 complexes per cell was antibodies were captured on CM5 surfaces at densities of 230–1140 RU measured as ABC using the QIFI kit (DAKO, Glostrup, Denmark) as (resonance units) by amine coupling procedure. The HA-1H/HLA-A2 27 fl described previously. Finally, cells were analyzed with a FACSCalibur ow analyte was diluted to 1,000 nM and tested at 25 nM,50nM, 100 nM, cytometer and CellQuest software (BD Biosciences). 150 nM and 200 nM. MHC stabilization assays were performed as described earlier.42 Briefly, T2 cells were pulsed with each of the peptides (10 μM) at 27 °C overnight, followed by incubation at 37 °C for 3 h. Surface HLA-A2 molecules were Preparation of monomeric scFv mAbs then stained with Alexa 647-conjugated HLA-A2-specific mAb (clone BB7.2, The scFv-cp3 molecules were purified with anti-cp3 mAb-conjugated BD Biosciences). Expression was measured by FACSCalibur (BD Biosciences) Sepharose beads. After isolation of phage particles, the gene encoding an and MFI was recorded. scFv cp3 molecule was converted into another expression vector(pYA208 )

Gene Therapy (2014) 575 – 584 © 2014 Macmillan Publishers Limited T-cell-like antibody specific for minor antigen Y Inaguma et al 583 encoding His-tag and a C-terminal Avi-tag fused form and biotin ligase, Statistical analysis transformed into DH12S cells, cultured with 2x YTAIB (ampicillin 200 The unpaired two-tailed Student's t-test was used for data analysis − 1 μgml , IPTG 0.5 mM, biotin 2 μM) medium. As scFv-His-Avi-form (http://www.physics.csbsju.edu/stats/t-test.html), and a P-value o0.05 was antibodies were secreted in the culture supernatant, they were collected considered to be statistically significant. by centrifugation, followed by 50% ammonium sulfate precipitation. The pellet was suspended with PBS, applied on Ni-NTA-agarose column, washed with 0.1% Tween-20/PBS, PBS and 10mM imidazole/PBS, and CONFLICT OF INTEREST finally eluted with 500mM imidazole/PBS. After several times of dialysis fl against PBS, they were concentrated by Amicon Ultra (Millipore). Protein The authors declare no con ict of interest. concentration was estimated by SDS-PAGE and Coomassie blue staining and western blot analysis was performed with NeutrAvidin-HRP (Thermo Fisher Scientific). Finally, R-phycoerythrin-labeled monomeric scFv ACKNOWLEDGEMENTS Abs were generated with direct conjugation (R-Phycoerythrin Labeling We thank Dr W Ho for critically reading the manuscript; Ms Sayoko Ogata and Kit—NH2; DOJINDO, Kumamoto, Japan). Ms Hiromi Tamaki for their technical expertise. This study was supported in part by Grant-in-Aid for Scientific Research (C)(24591435), from the Ministry of Education, Culture, Science, Sports and Technology, Japan; Grants for Research on the Human Genome, Tissue Engineering Food Biotechnology and the Second and Third Team Expression vectors Comprehensive 10-year Strategy for Cancer Control, from the Ministry of Health, For expression of scFv on mammalian cells, the variable heavy-chain Labour and Welfare, Japan; and a grant from the Japan Leukemia Research Fund region (VH) and the variable and constant light-chain region (VL CL) of an (2013). This study was supported in part by Grant-in-Aid for Scientific Research (C) Fab fragment were amplified by PCR. After digestion with SfiI and AscI for (24591435), from the Ministry of Education, Culture, Science, Sports and Technology, the VL CL region, the PCR products were subcloned into an original Japan; Grants for Research on the Human Genome, Tissue Engineering Food pYA128 vector. For transduction of T cells, an expression cassette was Biotechnology and the Second and Third Team Comprehensive 10-year Strategy for constructed (referred to as #clone-28z, Figure 4a) was generated by Cancer Control, from the Ministry of Health, Labour and Welfare, Japan; and a grant combining leader sequence of the murine immunoglobulin kappa light from the Japan Leukemia Research Fund (2013). chain, scFv, the transmembrane and truncated intracellular signaling domains of CD28 and CD3-ζ as previously reported by Kochenderfer et al.30 with minor modifications. The cassette was subcloned into LZRSpBMN-Z REFERENCES retroviral vector (a kind gift from Dr G Nolan, Stanford University, Stanford, 1 Kolb HJ, Mittermuller J, Clemm C, Holler E, Ledderose G, Brehm G et al. Donor CA, USA) with modification of packaging signal sequence according to 47 leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia Lee et al. in marrow transplant patients. Blood 1990; 76 (12): 2462–2465. 2 Marijt WA, Heemskerk MH, Kloosterboer FM, Goulmy E, Kester MG, van der Hoorn MA et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or fi Preparation of CAR-T cell lines HA-2-speci c T cells can induce complete remissions of relapsed leukemia. Proc Natl Acad Sci USA 2003; 100: 2742–2747. The retrovirus producer was prepared by transfecting the construct 3 den Haan JM, Meadows LM, Wang W, Pool J, Blokland E, Bishop TL et al. into a retrovirus packaging cell line, Phoenix-GALV using X-tremeGENE 9 The minor histocompatibility antigen HA-1: a diallelic gene with a single amino (Roche Applied Science, Mannheim, Germany). CD8 T cells, CD4 T cells or acid polymorphism. Science 1998; 279: 1054–1057. both were positively selected by means of anti-CD8, anti-CD4 magnetic 4 Heemskerk MH, Hoogeboom M, de Paus RA, Kester MG, van der Hoorn MA, fi beads (MACS system; Miltenyi Biotec, Auburn, CA, USA). Puri ed Goulmy E et al. Redirection of antileukemic reactivity of peripheral T lymphocytes T cells were activated with CD3/CD28 T-cell expander Dynabeads using gene transfer of minor histocompatibility antigen HA-2-specific T-cell (Life Technologies) at 1:1 ratio for 2 days in Advanced RPMI 1640 receptor complexes expressing a conserved alpha joining region. Blood 2003; (Life Technologies) supplemented with 4% pooled human serum, 2 mM 102: 3530–3540. − 1 L-glutamine in the presence of 30 U ml Interleukin-2 (IL-2) and 10 5 Ochi T, Fujiwara H, Okamoto S, An J, Nagai K, Shirakata T et al. Novel adoptive − 1 ng ml IL-7 (all from R&D Systems, Minneapolis, MN, USA). For the T-cell immunotherapy using a WT1-specific TCR vector encoding silencers for transductions, retroviral supernatant was centrifuged onto 24- or 48-well endogenous TCRs shows marked antileukemia reactivity and safety. Blood 2011; coated with Retronectin (Takara Bio, Shiga, Japan) plates for 2 h at 2000 g 118: 1495–1503. ’ at 32 °C as per manufacturer s instruction. T cells that had been activated 6 Udyavar A, Geiger TL. Rebalancing immune specificity and function in cancer by for 2 days as above were then plated, which was repeated on the following T-cell receptor gene therapy. Arch Immunol Ther Exp 2010; 58:335–346. − 1 day. During transduction, T cells were cultured with 30 U ml IL-2 and 7 Bendle GM, Linnemann C, Hooijkaas AI, Bies L, de Witte MA, Jorritsma A et al. − 1 − 1 10 ng ml IL-7; after that T cells were expanded with 30 U ml IL-2 with Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. − 1 − 1 – or without 10 ng ml IL-7 or 5 ng ml IL-15 for a total of 10 21 days Nat Med 2010; 16: 565–570. – before assays (that is, 8 19 days after completion of the transduction). For 8 HeemskerkMH,HagedoornRS,vanderHoornMA,vanderVekenLT,HoogeboomM, fi CD4/CD8 subset analyses, gene modi ed bulk T cells were positively Kester MG et al. Efficiency of T-cell receptor expression in dual-specific T cells is sorted by means of anti-CD8 or anti-CD4 magnetic beads (Miltenyi controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 Biotec). complex. Blood 2007; 109:235–243. 9 van Loenen MM, de Boer R, Amir AL, Hagedoorn RS, Volbeda GL, Willemze R et al. Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc Natl Acad Sci USA 2010; 107: 10972–10977. Chromium release assay 10 van der Veken LT, Hagedoorn RS, van Loenen MM, Willemze R, Falkenburg JH, 51 3 Target cells were labeled with 0.1 mCi of Cr for 2 h, and 1 × 10 target Heemskerk MH. Alphabeta T-cell receptor engineered gammadelta T cells cells per well were mixed with CTL at the E:T ratio indicated. After mediate effective antileukemic reactivity. 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