Evaluation of Production Protocols for the Generation of NY-ESO-1-Speciﬁc T Cells

cells

Article Evaluation of Production Protocols for the Generation of NY-ESO-1-Speciﬁc T Cells

Wenjie Gong 1,2, Lei Wang 1, Sophia Stock 1 , Ming Ni 1,3, Maria-Luisa Schubert 1, Brigitte Neuber 1, Christian Kleist 4, Angela Hückelhoven-Krauss 1, Depei Wu 2, Carsten Müller-Tidow 1,5, Anita Schmitt 1, Hiroshi Shiku 6, Michael Schmitt 1,5 and Leopold Sellner 1,7,*

1 Department of Internal Medicine V, Heidelberg University Hospital, 69120 Heidelberg, Germany; [email protected] (W.G.); [email protected] (L.W.); [email protected] (S.S.); [email protected] (M.N.); [email protected] (M.-L.S.); [email protected] (B.N.); [email protected] (A.H.-K.); [email protected] (C.M.-T.); [email protected] (A.S.); [email protected] (M.S.) 2 Department of Hematology, First Afﬁliated Hospital of Soochow University, Suzhou 215006, China; [email protected] 3 Department of Hematology, The Afﬁliated Hospital of Guizhou Medical University, Guiyang 550025, China 4 Department of Nuclear Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany; [email protected] 5 National Center for Tumor Diseases (NCT), German Cancer Consortium (DKTK), 69120 Heidelberg, Germany 6 Department of Immuno-Gene Therapy, Mie University, Tsu 514-8507, Japan; [email protected] 7 Takeda Pharma Vertrieb GmbH & Co. KG, 10117 Berlin, Germany * Correspondence: [email protected]

Abstract: NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost- effectiveness considerations of cell production protocols are of importance for conducting clinical Citation: Gong, W.; Wang, L.; Stock, S.; studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Ni, M.; Schubert, M.-L.; Neuber, B.; Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin Kleist, C.; Hückelhoven-Krauss, A.; concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells gen- Wu, D.; Müller-Tidow, C.; et al. Evaluation of Production Protocols for erated according to the two protocols were investigated for differences in cell viability, transduction the Generation of NY-ESO-1-Specific T efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells Cells. Cells 2021, 10, 152. showed similar viability and transduction efficiency between both protocols. Protocol 1 generated https://doi.org/10.3390/cells10010152 higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for Received: 9 November 2020 efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 Accepted: 11 January 2021 displayed higher capacity of TNF-α generation, but lower cytotoxic capacities. Overall, both proto- Published: 14 January 2021 cols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently Publisher’s Note: MDPI stays neu- to achieve a cost-effective cellular product for further clinical evaluation. tral with regard to jurisdictional clai- ms in published maps and institutio- Keywords: NY-ESO-1-specific T cells; cell production protocols; adoptive cell therapy nal affiliations.

Copyright: © 2021 by the authors. Li- 1. Introduction censee MDPI, Basel, Switzerland. Adoptive cell therapy (ACT) with genetically engineered T cells expressing cancer- This article is an open access article speciﬁc T cell receptors (TCR) is a promising cancer immunotherapy approach. These distributed under the terms and con- recombinant TCRs can recognize surface as well as intracellular tumor antigens. The ditions of the Creative Commons At- cancer testis antigen New York esophageal squamous cell carcinoma-1 (NY-ESO-1) is tribution (CC BY) license (https:// an ideal immunotherapy target because NY-ESO-1 is widely expressed on a variety of creativecommons.org/licenses/by/ cancer entities including melanoma [1,2], multiple myeloma [3] and soft-tissue sarcoma 4.0/).

Cells 2021, 10, 152. https://doi.org/10.3390/cells10010152 https://www.mdpi.com/journal/cells Cells 2021, 10, 152 2 of 14

(STS) [2,4]. Importantly, NY-ESO-1 is only expressed on germinal tissue in adults [5,6]. Furthermore, NY-ESO-1 is an immunogenic protein as spontaneous humoral and cellular immune responses can be observed in cancer patients [7]. D’Angelo et al. demonstrated that engineered autologous T cells expressing NY-ESO-1c259 can mediate clinically meaningful antitumor effects in patients with metastatic synovial sarcoma [8]. Currently, numerous clinical trials using different NY-ESO-1-specific T cell products are initiated [9]. All of these studies are using different production protocols of genetically modified T cells. The manufacturing process can have an important impact on the efficacy of ACT [10]. Therefore, it is of great importance to evaluate different protocols for clinical application. Ideal tumor-specific T cell products should successfully expand in vivo, achieve long- term persistence and, importantly, efficiently eliminate cancer cells. For this purpose, genetically modified T cells with high viability as well as transduction efficiency are required. In addition, the composition of the final cell product can influence the therapeutic success of ACT: For example, less differentiated T cells such as naïve-like (TN) or stem cell memory-like (TSCM) T cells have been reported to promote engraftment, long-term persistence, and extended tumor control [11,12]. In this study, we evaluated two different “ready-to-use” manufacturing protocols using retroviral vectors to generate NY-ESO-1-specific T cells. Activation and transduction strategies as well as the interleukin-2 (IL-2) concentrations used for T cell culture were major differences in the protocols applied. Importantly, these factors not only resulted in different production efficiencies and cellular product compositions, but also strongly affected the overall costs of the T cell production process.

2. Materials and Methods 2.1. Primary Cells Peripheral blood (PB) samples from buffy coat of healthy donors (HDs) were obtained at the Heidelberg University Hospital. A ﬁcoll density gradient (Linaris, Dossenheim, Germany) was performed to purify mononuclear cells which were cryopreserved in liquid nitrogen until usage. The study was approved by the Ethics Committee of the University of Heidelberg (S-254/2016). Informed consent was obtained from all HDs at the Blood Bank Heidelberg.

2.2. Cell Lines The STS cell lines SW982 (NY-ESO-1+ HLA-A2+) and SYO-1 (NY-ESO-1- HLA-A2-) were kindly provided by Hiroshi Shiku (Mie University, Tsu, Japan). SW982 and SYO-1 cells were expanded in DMEM (Thermo Fisher Scientiﬁc, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientiﬁc, Waltham, MA, USA).

2.3. NY-ESO-1-Specific T Cells Generation Cryopreserved human PB mononuclear cells (PBMCs) from 6 different HDs were thawed and activated according to the two protocols, respectively. Protocol 1 was based on a NY-ESO-1 generation protocol employed in a clinical trial in Japan (NCT02869217). Protocol 2 was adapted from our clinical CD19 chimeric antigen receptor (CAR) trial for the treatment of CD19-positive hematological malignancies (NCT03676504) [13] using a third-generation retroviral vector system [14–17]. PBMCs from each HD were used for generation of NY-ESO-1-specific T cells applying the two protocols in parallel. This allows a direct comparison of the same donor samples between the two protocols. The main differences between the two protocols are illustrated in Figure1 and Table1. In both protocols, the first day of T cells activation was defined as day 0. Cells 2021, 10, 152 3 of 14 Cells 2021, 10, x FOR PEER REVIEW 3 of 14

FigureFigure 1.1. Time schedule until day 15 of of the the two two protocols protocols for for NY-ESO-1-specific NY-ESO-1-speciﬁc T T cell cell generation. generation. Abbreviations:Abbreviations: IL-2: interleukin-2, interleukin-2, FACS: FACS: fluorescence ﬂuorescence activated activated cell cell sorting, sorting, 51Cr:51Cr: Chromium-51. Chromium-51. ICS:ICS: intracellularintracellular cytokinecytokine staining.staining.

Table 1. Major differences in the protocols for the generation of NY-ESO-1 specific T cells. Table 1. Major differences in the protocols for the generation of NY-ESO-1 speciﬁc T cells.

Protocol Protocol 1 (P1) 1 (P1) Protocol Protocol 2 (P2)2 (P2) GT-T551GT-T551 45%45% RPMI RPMI 1640 1640 2% human serum albumin 45% Click’s45% Click’s Medium Medium (EHAA) Culture Medium 2% human serum albumin Culture Medium 0.6% human serum 10% heat-inactivated(EHAA) FBS 2 mM L-glutamine 0.6% human serum 10% heat-inactivated FBS Anti-CD3 (5 µg/mL) + Anti-CD3 (1 µg/mL) + Activation RetroNectin (25 µg/mL) Anti-CD282 mM L-glutamine (1 µg/mL) Cytokines 600Anti-CD3 U/mL IL-2 (5 μg/mL) + Anti-CD3100 U/mL (1 IL-2μg/mL) + RetronectinActivation used for RetroNectin20 µg/mL (25 μg/mL) Anti-CD28 7 µg/mL (1 μg/mL) transduction Cytokines 600 U/mL IL-2 100 U/mL IL-2 1 mL retrovirus 2000 g 32 ◦C 2 h 1 mL retrovirus 37 ◦C 1 h RetronectinTransduction used for transduc- (spinoculation)20 μg/mL (static)7 μg/mL tion 1 mL retrovirus 2000 g 32 °C Protocol 1: GT-T551 (Takara Bio, Shiga, Japan) supplemented1 mL with retrovirus 0.6% human 37 °C serum 1 h (ZenBio, Durham,Transduction NC, USA) and 2% human serum2 h albumin (US biological, Swampscott, MA, USA) was used for cell culturing.(spinoculation) On day −1, the plates were(static) pre-coated with 5 µg/mL anti-CD3 (Biozol, Eching, Germany) plus 25 µg/mL retronectin (Takara Bio). On day 0,Protocol PBMCs 1: from GT-T551 HDs were (Takara added Bio, to Shiga, the pre-coated Japan) supplemented plates and the with culture 0.6% medium human was se- supplementedrum (ZenBio, Durham, with 600 NC, U/mL USA) IL-2 and (Novartis 2% human Pharma, serum Basel, albumin Switzerland). (US biological, Transduction Swamp- wasscott, performed MA, USA) on was day used 4 with for retronectin cell culturing. (20 µ Ong/mL, day Takara−1, the Bio)plates paired were with pre-coated spinoculation with 5 (centrifugationμg/mL anti-CD3 at (Biozol, 2000× g ,Eching, 32 ◦C for Germany) 2 h). plus 25 μg/mL retronectin (Takara Bio). On day 0,Protocol PBMCs 2:from 45% HDs RPMI were 1640 added (Thermo to the pr Fishere-coated Scientific) plates and and the 45% culture Click’s medium Medium was (EHAA)supplemented (Irvine with Scientific, 600 U/mL Santa IL-2 Ana, (Novartis CA, USA), Pharma, supplemented Basel, Switzerland). with 10% heat-inactivated Transduction FBSwas (Thermoperformed Fisher on day Scientific) 4 with andretronectin 2 mM L-glutamine (20 μg/mL, (ThermoTakara Bio) Fisher paired Scientific) with spinocula- was used fortion cell (centrifugation culturing. On at day2000×− 1,g, 32 the °C plates for 2 wereh). pre-coated with 1 µg/mL anti-CD3 plus 1 µg/mLProtocol anti-CD28 2: 45% (Biozol). RPMI 1640 On day (Thermo 0, PBMCs Fisher from Scientific) HDs were and added 45% toClick’s the pre-coated Medium plates.(EHAA) The (Irvine culture Scientific, medium Santa was Ana, supplemented CA, USA), supplemented with 100 U/mL with IL-2 10% on heat-inactivated day 2 of T cell culturing.FBS (Thermo Transduction Fisher Scientific) was performed and 2 mM onL-gl dayutamine 3 with (Thermo retronectin Fisher (7 Scientific)µg/m, Takara was used Bio) pairedfor cell with culturing. a static On condition day −1, (37 the◦ Cplates for 1 were h without pre-coated centrifugation). with 1 μg/mL anti-CD3 plus 1 μg/mLFor anti-CD28 both protocols, (Biozol). culture On day medium 0, PBMC wass changedfrom HDs routinely were added on days to the 7, pre-coated 10 and 14 withplates. addition The culture of fresh medium IL-2. Transduction was supplemented was performed with 100 with U/mL the MS3II-NY-ESO-1-siTCRIL-2 on day 2 of T cell retroviralculturing. vectorTransduction based on was the perfor Moloneymed murine on day leukemia 3 with retronectin virus. Detailed (7 μg/m, information Takara Bio) on thepaired two with protocols a static is condition provided in(37 Supplementary °C for 1 h without Figure centrifugation). S1. For both protocols, culture medium was changed routinely on days 7, 10 and 14 with 2.4.addition Flow Cytometryof fresh IL-2. for SurfaceTransduction Markers was performed with the MS3II-NY-ESO-1-siTCR ret- roviralCells vector were based evaluated on the by Moloney multiparametric murine leukemia flow cytometry. virus. Detailed Near-IR information Dead Cell on Stain the Kittwo (Thermo protocols Fisher is provided Scientific) in Supplementary was used to exclude Figure deadS1. cells. HLA-peptide monomers were kindly provided by Prof. H. Shiku (Mie University, Tsu, Japan). PE-conjugated HLA NY-ESO-1-tetramer were produced according to the protocol of the NIH Tetramer

Cells 2021, 10, 152 4 of 14

Core Facility (Emory University, Atlanta, GA, USA) and were used to detect NY-ESO-1- speciﬁc TCR expression. The following ﬂuorochrome-conjugated antibodies were used for immunophenotyping of following surface markers: anti-CD3-V510 (AmCyan), anti-CD8- PerCP, anti-CD45RA-APC, anti- programmed cell death protein 1 (PD-1)-Alexa Fluor 488, anti-T cell immunoglobulin mucin-3 (TIM-3)-Brilliant Violet 421 and anti-CXCR3-Alexa Fluor 488 (all from Biolegend, San Diego, CA, USA), anti-CD4-Alexa Fluor 700, anti-CCR7- PE-Cy7, anti-CD62L-eFluor 450 and anti-CD3-eFlour 610 (all from eBioscience, San Diego, CA, USA). Data were acquired on an LSR II device (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA). The gating strategies are illustrated in Supplementary Figure S2.

2.5. Intracellular Cytokine Staining Intracellular cytokine staining was performed as reported previously [18]. In brief, T cells were co-cultured with SW982 cells (NY-ESO-1+ HLA-A2+) or SYO-1 cells (NY-ESO- 1- HLA-A2-) for 6 h in 96-well U-bottom microplates (Greiner BioOne, Frickenhausen, Germany). The cytokine secretion inhibitor Brefeldin A (BFA) was added for intracellular cytokine retention. Cells were then stained with NEAR-IR and surface marker antibodies followed by ﬁxation and permeabilization using the Foxp3 ﬁx/perm buffer set (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, cells were stained with anti-interferon gamma (IFN-γ)-Alexa Fluor 488 (Biolegend) and anti-tumor necrosis factor (TNF)-BV421 (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 30 min. The gating strategies are illustrated in Supplementary Figure S2.

2.6. Cytotoxicity Assay The specific anti-tumor efficacy of NY-ESO-1-specific T cells was measured using a 12-h Chromium-51 (51Cr; Hartmann Analytic, Braunschweig, Germany) release assay. SW982 cells (NY-ESO-1+ HLA-A2+) served as target cells, whereas SYO-1 cells (NY-ESO-1- HLA-A2-) were used as negative control. Cell lines were labeled with 51Cr for 2 h and co-incubated with NY-ESO-1 specific T cells or non-transduced T cells (effector cells) for 12 h using effector to target cell (E:T) ratios of 10:1, 5:1, 2.5:1 and 1:1 (5:1 only for non- transduced T cells) in 96-well U-bottom microplates (Greiner Bio-One) at 37 ◦C and 5% CO2. Maximum release and spontaneous release were determined by incubating the target cells with 1% Triton X-100 (Merck KGaA, Darmstadt, Germany) and medium alone, respectively. In addition, 75 µL of culture supernatant was diluted in Ultima Gold liquid scintillation cocktail (PerkinElmer, Waltham, MA, USA) and measured on a γ-counter (PerkinElmer). All experiments were performed in triplicates. Specific lysis was calculated according to the following formula: % specific lysis = (51Cr release in the test well—Spontaneous 51Cr release)/(maximum 51Cr release—Spontaneous 51Cr release) × 100.

2.7. Statistical Analysis Statistical analysis was performed using Excel (Microsoft, Redmond, WA, USA). p-values were calculated using the paired t-test. p-values < 0.05 were considered statisti- cally signiﬁcant. Graphs and tables were designed using Excel, PowerPoint (Microsoft), Origin 2020 (OriginLab, Northampton, MA, USA) and Prism 6 (GraphPad Software Inc., San Diego, CA, USA). If not otherwise mentioned, results are presented as mean ± standard deviation (SD).

3. Results 3.1. Viability, Transduction Efﬁciency, and Expansion Viability, transduction efficiency and expansion of T cells were determined longitudinally during the NY-ESO-1-specific T cell generation with PBMCs from six HDs. Cells were evaluated on days 10 and 14 of the T cell production process as harvesting for clinical application is usually performed in this time frame. Protocol 1 yielded significantly higher T cells on day 10 (P1 vs. P2: 28.21 ± 12.30 vs. 15.60 ± 3.73 × 106 cells, p = 0.02, day 10; Cells 2021, 10, x FOR PEER REVIEW 5 of 14

3. Results 3.1. Viability, Transduction Efficiency, and Expansion Viability, transduction efficiency and expansion of T cells were determined longitudinally during the NY-ESO-1-specific T cell generation with PBMCs from six HDs. Cells Cells 2021, 10, 152 were evaluated on days 10 and 14 of the T cell production process as harvesting5 of 14 for clinical application is usually performed in this time frame. Protocol 1 yielded significantly higher T cells on day 10 (P1 vs. P2: 28.21 ± 12.30 vs. 15.60 ± 3.73 × 106 cells, p = 0.02, day 10; 45.99 ± 22.81 vs. 28.47 ± 6.65 × 106 cells, p = 0.06, day 14; Figure 2A). The relative fold expansion 6 45.99 ± 22.81 wasvs. 28.47significantly± 6.65 ×higher10 cells, on dayp = 10 0.06, and day day 14; 14 Figureapplying2A). protocol The relative 1 (P1 fold vs. P2: 88 ± 37 vs. expansion was37 significantly ± 9, p = 0.007, higher day 10; on 148 day ± 1067 andvs. 74 day ± 21, 14 applyingp = 0.02, day protocol 14; Figure 1 (P1 2B). vs. The P2: viability (P1 ± ± ± ± 88 37 vs. 37vs. 9,P2:p =91 0.007, ± 5% dayvs. 89 10; ± 148 3%, p67 = 0.50, vs. 74 day21, 10;p 88= 0.02,± 6% dayvs. 87 14; ± Figure 4%, p2 =B). 0.47, The day 14; Figure ± ± p ± ± p viability (P1 vs.2C) P2: and 91 transduction5% vs. 89 efficiency3%, = 0.50, (P1 day vs. 10;P2: 8834 ± 14%6% vs. vs. 87 23 ± 4%,5%, p= = 0.47,0.08, day day 10; 30 ± 13% 14; Figure2C) and transduction efficiency (P1 vs. P2: 34 ± 14% vs. 23 ± 5%, p = 0.08, day 10; vs. 21 ± 5%, p = 0.11, day 14; Figure 2D) of NY-ESO-1-specific T cells generated employing 30 ± 13% vs. 21 ± 5%, p = 0.11, day 14; Figure2D) of NY-ESO-1-specific T cells generated the two protocols were similar on day 10 and day 14. Protocol 1 generated more NY-ESO- employing the two protocols were similar on day 10 and day 14. Protocol 1 generated more 1-specific T cells on day 10 (P1 vs. P2: 8.00 ± 5.13 vs. 2.93 ± 0.92 × 106 cells, p = 0.04; Figure NY-ESO-1-specific T cells on day 10 (P1 vs. P2: 8.00 ± 5.13 vs. 2.93 ± 0.92 × 106 cells, p = 0.04; 2D). However, the differences of absolute numbers of NY-ESO-1-specific T cells did not Figure2D). However, the differences of absolute numbers of NY-ESO-1-specific T cells did reach statistical significance on day 14 (P1 vs. P2: 11.83 ± 9.57 vs. 4.79 ± 2.07 × 106 cells, p = not reach statistical significance on day 14 (P1 vs. P2: 11.83 ± 9.57 vs. 4.79 ± 2.07 × 106 cells, 0.08; Figure 2E). p = 0.08; Figure2E).

Figure 2. Viability,Figure transduction 2. Viability, transduction efficiency and efficiency cell expansion. and cell (A expansion.,B) Expansion (A,B of) Expansionall T cells, ( ofC) allviability, T cells, ( (DC)) transduction vi- efficiency andability, (E) NY-ESO-1 (D) transduction specific T efficiency cell expansion and ( Ewere) NY-ESO-1 assessed specific on day T10 cell and expansion day 14 of the were production assessed on(n = 6) using protocol 1 (P1)day and 10 andprotocol day 142 (P2). of the Mean production values (weren = 6) calculat using protocoled for each 1 (P1) group; and error protocol bars 2 indicate (P2). Mean standard values deviation. Statistical significancewere calculated was calculated for each group;with a errorpaired bars two-way indicate student standard t-test. deviation. Significance Statistical is represented significance as * wasfor p-values < 0.05 and ** forcalculated p-values with< 0.01. a paired two-way student t-test. Significance is represented as * for p-values < 0.05 and ** for p-values < 0.01. 3.2. Distribution of Different NY-ESO-1-Specific T Cells Subsets 3.2. Distribution of Different NY-ESO-1-Specific T Cells Subsets The two protocols exerted a similar influence on the distribution of CD8+ and CD4+ + + The twoNY-ESO-1-specific protocols exerted a T similar cells in influence the cell product: on the distribution On day 7, more of CD8 CD4and+ than CD4 CD8+ NY-ESO-1- + + NY-ESO-1-specificspecific T cells T cells in were the cell the product: main subsets On day detectable 7, more CD4withinthan the T CD8 cell cultureNY-ESO-1- in both protocols. specific T cellsCD8 were+ NY-ESO-1-specific the main subsets detectable T cells progressively within the T increased cell culture while in both CD4 protocols.+ NY-ESO-1-specific T + + CD8 NY-ESO-1-specificcells gradually T cells decreased progressively in the course increased of production while CD4 andNY-ESO-1-specific the ratio between CD8+ and + T cells graduallyCD4 decreased+ NY-ESO-1-specific in the course T cells of production was reversed and along the ratio with between the culture CD8 durationand regardless + CD4 NY-ESO-1-specificof the protocol T cells used was (Supplemen reversed alongtary Figure with the S3). culture At the duration end of the regardless production, of comparable the protocol usedproportions (Supplementary of CD8+ NY-ESO-1-specific Figure S3). At the endT cells of theas well production, as CD4+ comparableNY-ESO-1-specific T cells + + proportions offor CD8 bothNY-ESO-1-specific protocols were observed T cells as(Figure well as3A). CD4 NY-ESO-1-specific T cells for both protocolsThe were NY-ESO-1-specific observed (Figure3 A).T cells were divided into four subsets according to CD45RA The NY-ESO-1-specific T cells were divided into four subsets according to CD45RA and CCR7 expression: naïve-like T (TN, CD45RA+CCR7+), central memory-like T (TCM, + + and CCR7 expression: naïve-like T (TN, CD45RA CCR7 ), central memory-like T (TCM, − + − − CD45RA CCR7 ), effector memory-like T (TEM, CD45RA CCR7 ), and terminally dif- + − ferentiated effector-like T (TE, CD45RA CCR7 ) cells. After in vitro activation, almost all T cells express CD95 [15]. Thus, T cells defined as T cells can be regarded as stem N cell memory-like T (TSCM-like) cells in our study [19]. Protocol 1 rendered significantly + higher proportions of TE cells among all CD3 NY-ESO-1-specific T cells on day 10 and day 14 (P1 vs. P2: 66 ± 13% vs. 36 ± 17%, p = 0.01, day 10; 57 ± 12% vs. 37 ± 11%, p = 0.03, day 14; Figure3B,C), whereas significantly higher proportions of T CM (P1 vs. P2: 2 ± 1% vs. 8 ± 6%, p = 0.04, day 10; 2 ± 1% vs. 5 ± 2%, p = 0.02, day 14; Figure3B,C) and T EM cells Cells 2021, 10, x FOR PEER REVIEW 6 of 14

CD45RA-CCR7+), effector memory-like T (TEM, CD45RA-CCR7-), and terminally differentiated effector-like T (TE, CD45RA+CCR7-) cells. After in vitro activation, almost all T cells express CD95 [15]. Thus, T cells defined as TN cells can be regarded as stem cell memory- like T (TSCM-like) cells in our study [19]. Protocol 1 rendered significantly higher proportions of TE cells among all CD3+ NY-ESO-1-specific T cells on day 10 and day 14 (P1 vs. P2: Cells 2021, 10, 152 6 of 14 66 ± 13% vs. 36 ± 17%, p = 0.01, day 10; 57 ± 12% vs. 37 ± 11%, p = 0.03, day 14; Figure 3B,C), whereas significantly higher proportions of TCM (P1 vs. P2: 2 ± 1% vs. 8 ± 6%, p = 0.04, day 10; 2 ± 1% vs. 5 ± 2%, p = 0.02, day 14; Figure 3B,C) and TEM cells (P1 vs. P2: 9 ± 10% vs. 20 (P1 vs. P2: 9 ±± 7%,10% p vs. = 0.03, 20 ± day7%, 10;p = 12 0.03, ± 13% day vs. 10; 23 12 ±± 7%,13% p = vs. 0.048, 23 ± day7%, 14;p =Figure 0.048, 3B,C) day 14; were observed Figure3B,C) wereon day observed 10 and onday day 14 10applying and day protocol 14 applying 2. protocol 2.

Figure 3. DistributionFigure 3. Distributionof different ofNY-ESO-1-specific different NY-ESO-1-specific T cell subsets. T cell (A subsets.) The evolution (A) The evolutionof NY-ESO-1-specific of NY-ESO-1- CD4+ and CD8+ T cells (specificn = 6) was CD4 assessed+ and CD8 on +dayT cells 10 and (n = day 6) was14 of assessed the production on day 10using and protocol day 14 of 1 the(P1) production and protocol using 2 (P2); (B,C) + comparison ofprotocol the proportion 1 (P1) and of protocol TN, TCM 2, T (P2);EM, and (B,C T)E comparison cells among of CD3 the proportion NY-ESO-1-specific of TN,TCM T,T cellsEM, ( andn = 6) TE oncells day 10 and day 14 betweenamong protocols CD3+ NY-ESO-1-specific1 and 2. NY-ESO-1-specific T cells (n =T 6)cells on subsets day 10 andwere day divided 14 between according protocols to CD45RA 1 and 2. and NY- CCR7 expression. TN cells were defined as CD45RA+CCR7+, TCM cells as CD45RA-CCR7+, TEM cells as CD45RA-CCR7- and TE cells ESO-1-specific T cells subsets were divided according to CD45RA and CCR7 expression. TN cells were as CD45RA+CCR7- T cells. Mean values were calculated for each group; error bars indicate standard deviation. Statistical defined as CD45RA+CCR7+,T cells as CD45RA−CCR7+,T cells as CD45RA−CCR7− and T cells significance was calculated with a paired two-wayCM student t-test. SignificanceEM is represented as * for p-valuesE < 0.05. as CD45RA+CCR7− T cells. Mean values were calculated for each group; error bars indicate standard deviation. Statistical3.3. Absolute significance Numbers was calculated of Different with Su absets paired among two-way NY-ESO-1-Specific student t-test. Significance T Cells is represented as * for p-values < 0.05. Due to slower expansion (Figure 2D), protocol 2 resulted in lower absolute numbers of CD8+ TN cells NY-ESO-1-specific T cells on day 14 compared to protocol 1, but this was 3.3. Absolute Numbers of Different Subsets among NY-ESO-1-Speciﬁc T Cells without statistical significance (Figure 4A). No differences were observed in absolute Due to slower expansion (Figure2D), protocol 2 resulted in lower absolute numbers numbers of CD4+ TN, CD8+ TCM, and CD4+ TCM cells (Figure 4B–D). Protocol 2 increased the of CD8+ T cells NY-ESO-1-specific T cells on day 14 compared to protocol 1, but this was N numbers of CD8+ TEM cells on day 14 (P1 vs. P2: 0.06 ± 0.07 vs. 0.21 ± 0.11 × 106 cells, p = without statistical significance (Figure4A). No differences were observed in absolute numbers + + + of CD4 TN, CD8 TCM, and CD4 TCM cells (Figure4B–D). Protocol 2 increased the numbers + 6 of CD8 TEM cells on day 14 (P1 vs. P2: 0.06 ± 0.07 vs. 0.21 ± 0.11 × 10 cells, p = 0.02, + Figure4E ) rather than CD4 TEM cells (Figure4F), while protocol 1 increased the amounts of + 6 CD8 TE cells (P1 vs. P2: 2.70 ± 2.00 vs. 0.58 ± 0.32 × 10 cells, p = 0.046, day 10; 3.41 ± 2.64 6 + vs. 0.98 ± 0.56 × 10 cells, p = 0.043, day 14, Figure4G) rather than CD4 TE cells (Figure4H). Cells 2021, 10, x FOR PEER REVIEW 7 of 14

0.02, Figure 4E) rather than CD4+ TEM cells (Figure 4F), while protocol 1 increased the amounts of CD8+ TE cells (P1 vs. P2: 2.70 ± 2.00 vs. 0.58 ± 0.32 × 106 cells, p = 0.046, day 10; Cells 2021, 10, 152 3.41 ± 2.64 vs. 0.98 ± 0.56 × 106 cells, p = 0.043, day 14, Figure 4G) rather than7 of CD4 14 + TE cells (Figure 4H).

Figure 4. AbsoluteFigure number 4. Absolute of different number NY-ESO-1-specific of different NY-ESO-1-speciﬁc T cell subsets. ( TA cell–H)subsets. Differences (A– Hin) Differencesthe absolute in amounts the of

NY-ESO-1-specificabsolute TN (A amounts,B), TCM (C of,D NY-ESO-1-specific), TEM (E,F) and TE T(G,HN (A) ,cellsB), T (CMn = (6)C ,onD), day TEM 10(E and,F) andday T14E were(G,H )compared cells (n = 6between) protocol (P1) andon protocol day 10 and 2 (P2). day Mean 14 were values compared were calculat betweened protocolfor each group; (P1) and error protocol bars indicate 2 (P2). Meanstandard values deviation. Statistical significancewere calculated was calculated for each with group; a paired error two-way bars indicate student standard t-test. Significance deviation. Statistical is represented significance as * for wasp-values < 0.05. calculated with a paired two-way student t-test. Significance is represented as * for p-values < 0.05.

3.4. Exhaustion and Homing Markers The exhaustion markers T cell immunoglobulin mucin-3 (TIM-3) and programmed cell death protein 1 (PD-1), as well as the homing markers CD62L and CXCR3, were evaluated applying both protocols. Compared to protocol 1, protocol 2 significantly decreased Cells 2021, 10, 152 8 of 14 the expression of TIM-3 (P1 vs. P2: 81 ± 20% vs. 51 ± 28%, p = 0.004, day 10; 80 ± 8% vs. 51 ± 14%, p = 0.005, day 14; Figure 5A). PD-1 levels were similar between the two protocols (Figure 5B). No significant differences were observed in the co-expression TIM-3+ PD-1+ subsets between the two protocols (Figure 5 5CC).). Furthermore,Furthermore, thethe proportionsproportions of ofCXCR3 CXCR3++ (Figure5 5D)D) andand CD62LCD62L+ cellscells (Figure (Figure 55E)E) amongamong NY-ESO-1-specific TT cellscells werewere similarsimilar between the twotwo protocols.protocols.

+ Figure 5. ExhaustionExhaustion and homing markersmarkers ofof NY-ESO-1-specificNY-ESO-1-specific T T cells. cells. (A (–AC–)C Comparison) Comparison of of TIM-3 TIM-3(A), PD-1+ (A+),( BPD-1), and+ (B TIM-3), and+ TIM-3PD-1++(PD-1C) cells+ (C among) cells among NY-ESO-1-specific NY-ESO-1-specific T cells (Tn cells= 5) between(n = 5) between protocol protocol1 (P1) and 1 (P1) protocol and 2protocol (P2); (D 2,E (P2);) comparison (D,E) comparison of CXCR3 of+ (CXCR3D) and+ CD62L (D) and+ (CD62LE) cells+ among(E) cells NY-ESO-1- among NY-ESO-1-specificspecific T cells (n = T 5) cells between (n = 5) protocol between 1 (P1)protocol and protocol1 (P1) and 2 (P2).protocol Mean 2 (P2). values Mean were values calculated were for calculatedeach group; for error each bars group; indicate error standard bars indicate deviation. standard Statistical deviation. significance Statistical was calculatedsignificance with was a pairedcalcu- latedtwo-way with student a pairedt-test. two-way Significance student is t represented-test. Significance as ** for is prepresented-values < 0.01. as ** for p-values < 0.01.

3.5. Functional Evaluation of NY-ESO-1-Specific T Cells 3.5. Functional Evaluation of NY-ESO-1-Specific T Cells 51Cr release assay was performed on day 14 of T cells production. The STS cell line 51Cr release assay was performed on day 14 of T cells production. The STS cell line SW982 (NY-ESO-1+ HLA-A2+) was used as target cells. NY-ESO-1-specific T cells showed SW982 (NY-ESO-1+ HLA-A2+) was used as target cells. NY-ESO-1-specific T cells showed - - specificspecific lysis ofof SW982SW982 cellscells comparedcompared to to NY-ESO-1 NY-ESO-1− HLA-A2HLA-A2−SYO-1SYO-1 cells cells (Supplementary (Supplementary Figure S4A). The lysis of SW82 cells by by NY-ESO-1-specific NY-ESO-1-specific T cells was significantly significantly higher compared to non-transduced T cells with an E:T ratio ofof 5:1.5:1. SignificantlySignificantly more SW982 cells were were lysed lysed by by NY-ESO-1-specific NY-ESO-1-specific T T cells cells generated generated with with protocol protocol 2 in 2 incomparison comparison to protocolto protocol 1, especially 1, especially at lower at lower E:T ratios E:T ratios (P1 vs. (P1 P2: vs. 18 P2:± 6% 18 vs.± 346% ± 3%, vs. 34p = ±0.01,3%, E:Tp == 2.5:1 0.01, ;E:T 5 ± =3% 2.5:1; vs. 22 5 ± ± 3%6%, vs. p =22 0.02,± 6%, E:T p= =1:1; 0.02, Figure E:T =6A). 1:1; Figure6A). Intracellular cytokine staining of IFN-γ and TNF-α was performed on day 15 of T cell generation. NY-ESO-1-specific T cells generated with both production protocols were incubated for 6 h with the STS cell line SW982 cells (NY-ESO-1+ HLA-A2+). There was no difference in IFN-γ levels in NY-ESO-1-specific T cells generated with the two production protocols (Figure6B). In contrast, protocol 1 significantly upregulated TNF- α levels of all CD3+ (P1 vs. P2: 54 ± 10% vs. 35 ± 13%, p = 0.03) as well as CD8+ (P1 vs. P2: 65 ± 7% vs. 38 ± 13%, p = 0.02) but not of CD4+ NY-ESO-1-specific T cells (Figure6C). Furthermore, the distribution of multifunctional NY-ESO-1-specific T cells capable of both IFN-γ and TNF-α production were assessed. There were no significant differences in the proportion of multifunctional NY-ESO-1-specific T cells generated with the two different production protocols (Figure6D). No relevant baseline cytokine production of the NY-ESO-1-specific T cells was detectable with stimulation using NY-ESO-1− HLA-A2− SYO-1 cells or without stimulation (Supplementary Figure S4B,C).

Cells 2021, 10, x FOR PEER REVIEW 9 of 14 Cells 2021, 10, 152 9 of 14

Figure 6. Functional evaluation of NY-ESO-1-specific T cells. (A) Cytotoxicity of NY-ESO-1-specific T Figure 6. Functional evaluation of NY-ESO-1-specific T cells. (A) Cytotoxicity of NY-ESO-1-spe- 51 cificcells T cultivated cells cultivated with protocolwith protocol 1 (P1) 1 or (P protocol1) or protocol 2 (P2) 2 was(P2) determinedwas determined by byCr 51 releaseCr release assay assay after + + afterco-culture co-culture with with NY-ESO-1 NY-ESO-1HLA-A2+HLA-A2SW982+ SW982 target target cells cells for for 12 12 h. h. Different effector (NY-ESO- (NY-ESO-1- 1-specificspecific T T cells) cells) to to target target (SW982 (SW982 cells) cells) ratios ratios (10:1, (10:1, 5:1, 5:1, 2.5:1, 2.5:1, 1:1) 1:1) were were used used in in technical technical triplicates tripli- to catesassess to average assess average lysis; (B –lysis;D) Intracellular (B–D) Intracellular production production of IFN-γ ofand IFN- TNF-γ andα in TNF- NY-ESO-1-specificα in NY-ESO-1- T cells specificcultivated T cells with cultivated protocol 1with (P1) protocol or protocol 1 (P1) 2 (P2) or protocol was determined 2 (P2) was after determined stimulation after with stimulation SW982 cells γ withfor 6 SW982 h (n = 3).cells Overall, for 6 h IFN-(n = γ3).( BOverall,) and TNF- IFN-α ((CB)) productionand TNF-α as(C) well production as multifunctional as well as multifunc- NY-ESO-1- tional NY-ESO-1-specific T cells producing both TNF-α and IFN-γ (D) were determined in CD3+, specific T cells producing both TNF-α and IFN-γ (D) were determined in CD3+, CD4+, and CD8+ T CD4+, and CD8+ T cells. Mean values were calculated for each group; error bars indicate standard cells. Mean values were calculated for each group; error bars indicate standard deviation. Statistical deviation. Statistical significance was calculated with a paired two-way student t-test. Significance significance was calculated with a paired two-way student t-test. Significance is represented as * for is represented as * for p-values < 0.05. non-T: non-transduced T cells generated under the same culturep-values conditions. < 0.05. non-T: non-transduced T cells generated under the same culture conditions.

4. DiscussionIntracellular cytokine staining of IFN-γ and TNF-α was performed on day 15 of T cell generation.ACT with NY-ESO-1-specific T cells recognizing T cells the generat cancered testiswith both antigen production NY-ESO-1 protocols is a promising were in- cubatedtreatment for option 6 h with for solidthe STS tumors cell line such SW982 as STS. cells The (NY-ESO-1 ex vivo expansion+ HLA-A2 and+). There manipulation was no differenceof NY-ESO-1-specific in IFN-γ levels T cells in NY-ESO-1-specific is a crucial step for T cell therapeutics generated success. with the However, two production various protocolsprotocols (Figure differing 6B). in cultureIn contrast, conditions protocol including 1 significantly activation, upregulated transduction TNF- asα welllevels as of sup- all CD3plemented+ (P1 vs. cytokines P2: 54 ± 10% are being vs. 35 used ± 13%, to generatep = 0.03) theseas well cells. as CD8 By comparing+ (P1 vs. P2: two 65 ± completely 7% vs. 38 ±different 13%, p = manufacturing 0.02) but not of protocols, CD4+ NY-ESO-1-specific instead of elaborating T cells a single(Figure aspect 6C). Furthermore, of a protocol, thisthe distributionstudy aims of to multifunctional provide the community NY-ESO-1-specific “ready-to-use” T cells manufacturing capable of both protocolsIFN-γ and for TNF- the αgeneration production of NY-ESO-1-specificwere assessed. There T cells were (and no potentially significant ACTs differences with other in the targets). proportion Both pro- of multifunctionaltocols can provide NY-ESO-1-specific highly functional T tumor-specific cells generated T cells with and the have two individual different production advantages protocolsand disadvantages. (Figure 6D). No relevant baseline cytokine production of the NY-ESO-1-specific T cellsBoth was protocols detectable generated with stimulation NY-ESO-1-specific using NY-ESO-1 T cells- withHLA-A2 a high- SYO-1 viability cells that or without reached stimulationapproximately (Supplementary 90% during the Figure production S4B,C). process. The T cell growth factor IL-2 is widely used for cultivation of T cells to maintain T cell proliferation and survival and serves as 4.the Discussion basis of most adoptive T cell therapy protocols. IL-2 was supplemented to the culture mediumACT inwith both T cells production recognizing strategies, the cancer although testis the antigen concentration NY-ESO-1 differed is a promising dramatically: treat- menta higher option concentration for solid tumors of IL-2 such was as used STS. inTh protocole ex vivo 1expansion (600 U/mL) and compared manipulation to protocol of NY- 2 (100 U/mL) and might be a key parameter for the higher proliferation of T cell using ESO-1-specific T cells is a crucial step for therapeutic success. However, various protocols protocol 1. Retrovirus-mediated gene transfer requires proliferating cells [20]. The higher differing in culture conditions including activation, transduction as well as supplemented

Cells 2021, 10, 152 10 of 14

proliferation rate observed applying protocol 1 may be, at least in part, responsible for the trend of the higher transduction efficiency and overall faster and superior yield of NY-ESO-1-specific T cells when compared to protocol 2. However, even the 6x lower concentration of IL-2 in protocol 2 was enough to provide highly functional, genetically modified T cells. Importantly, the high variability between different cell donor batches, especially using the higher IL-2 concentration, might have limited a clearer picture here. Retronectin, the fragment of human fibronectin, enhances transduction efficiency by co-localizing viral vectors and the cells of interest, such as hematopoietic progenitor cells [21] and T lymphocytes [22]. Retronectin was used for transduction in both protocols. However, the concentration of retronectin and the binding strategy of retronectin with the virus differed: applying protocol 1, the retrovirus was pre-loaded on a higher concentration of retronectin (20 µg/mL) by 2 h centrifugation (spinoculation), before a second 10-min centrifugation of activated T cells was performed. In protocol 2, a lower concentration of retronectin (7µg/mL) was used to adsorb the retrovirus at 37 ◦C for 1 h (static), followed by a 5-min centrifugation of activated T cells. It was shown previously that, compared to static transduction, spinoculation could not only lead to a more than eightfold adsorption of retroviral vectors, but also increase the transduction efficiency by more than five fold [22]. Moreover, Chono et al. demonstrated that the gene transfer efficiency of lentiviral vectors was doubled after spinoculation when compared to a static pre-load [23]. Apart from retronectin and transduction strategy, efficient retroviral vector transduction depends on T cell division given that retroviral vectors can only integrate into the genome of proliferating cells. Protocol 1 led to a higher expansion of NY-ESO-1-specific T cells reaching significance on day 10. By making full use of high concentration of both retronectin and IL-2 applying protocol 1, a higher transduction efficiency was expected. However, in contrast to the above- mentioned studies, our data only showed a trend for increased transduction efficiency without reaching significance applying protocol 1. Nevertheless, the more labor- and cost-intensive protocol 1 could generate a significantly higher absolute number of NY-ESO- 1-specific T cells. A comparable proportion of CD8+ and CD4+ NY-ESO-1-specific T cells was generated with both protocols. Our observation that the duration of T cell culture can alter the balance between CD8+ cells and CD4+ cells towards CD8+ cells is in line with previous studies [15,24]. It was previously reported that anti-CD3/retronectin-based activation enriched for CD8+ T cells while anti-CD3/anti-CD28-based activation enriched for CD4+ T cells [25,26]. However, this effect was not observed in the current study at the relevant time points at day 10 and day 14 of the T cell production. In general, the role of CD4+ T cells in TCR-engineered T cells with MHC class 1 restrictions is not fully defined yet. When addressing the subsets of NY-ESO-1-specific T cells in detail, we observe that protocol 1 augmented the TE cell subpopulation among NY-ESO-1-specific T cells. In contrast, protocol 2 enriched for TCM and TEM cells. No significant differences in the relative and absolute numbers of TN cells between the two production protocols were observed. Accumulating evidence indicates that less-differentiated T cells such as TN cells are important for longer persistence of engineered cells and sustained anti-tumor control in ACT [12,27–29]. The combination of anti-CD3/retronectin to activate T cells has been suggested to be superior for generating CD8+ T cells with a naïve-like phenotype compared to anti-CD3/anti-CD28-based activation [25]. This stands in contrast to the observation in our study. One of the explanations may be that the much higher IL-2 concentration used in protocol 1 compromised the effect of anti-CD3/retronectin-based activation. Kaartinen et al. evaluated various concentration of IL-2 ranging from 0 IU/mL to 300 IU/mL to generate CD19 CAR-T cells and reported that high IL-2 concentrations can decrease TCM cells and augment TE cells [24]. It is of note that, in contrast to previous findings in other ACT trials, no correlation of treatment response with the T cell phenotype in the applied cell product could be identified in a clinical trial evaluating NY-ESO-1-specific T cells in patients with synovial sarcoma [8]. However, the limited patient numbers in this study Cells 2021, 10, 152 11 of 14

(n = 12) may limit clear conclusions and further analysis is necessary to evaluate the impact of less-differentiated T cells in NY-ESO-1-specific T cell products. T cells of cancer patients undergo “exhaustion” as a deterioration of T cell function due to permanent exposure to tumor antigens [30]. Exhausted T cells display lower proliferation rates, less cytokine production, and lower cytotoxicity. The exhaustion is characterized by the overexpression of multiple inhibitory receptors such as TIM-3 and PD-1 [31]. TIM-3 expression on T cells is induced and maintained following chronic stimulation [32]. In our study, TIM-3 levels of NY-ESO-1-specific T cells were significantly higher under the conditions of protocol 1 when compared to protocol 2. This is in line with a previous study showing that TIM-3 expression can be increased by IL-2 in a dose-dependent manner [33]. In contrast, PD-1 levels of NY-ESO-1 specific T cells between the two protocols were similar. In particular, TIM-3+PD-1+ CD8+ T cells have been described to be in a more exhausted state compared to T cells expressing PD-1 alone [34,35]. In the current study, no significant differences in the proportion of TIM-3+PD-1+ cells were identified between the two protocols. Although exhausted T cells can be, in general, less effective in anti-tumor activity, it is unclear whether the sole higher expression of TIM-3 on these cells may have a negative effect on in vivo efficacy. Further evaluation is required to define the impact of TIM-3 and PD-1 expression on clinical outcome using NY-ESO-1-specific T cells. The efficient homing of T cells and infiltration of the tumor tissue is critical in restrain- ing tumor progression. CXCR3 is the predominant chemokine receptor mediating TE cells recruitment to tumor tissue via its ligands CXCL9 and CXCL10, and has been demonstrated to be crucial for tumor control and survival [36,37]. Melanoma with low expression of ligands for CXCR3 is poorly infiltrated by T cells [38]. Moreover, CD62L has been reported to be a critical component for homing of T cells to the lymph node during initial homeostatic proliferation [39]. In the current study, we observed similar proportions of CXCR3+ cells and CD62L+ cells among the NY-ESO-1-specific T cells using the two protocols. Both homing markers were expressed on more than half of the NY-ESO-1-specific cells, and efficient homing into tumor tissue and lymph nodes can be expected using both protocols. NY-ESO-1-specific T cells generated by the two protocols displayed specific lysis of NY-ESO-1+ HLA-A2+ SW982 cells. However, the lytic capacity of NY-ESO-1 specific T cells generated by applying protocol 2 was superior compared to T cells employing protocol 1. The reasons for this difference in lytic capacity is unclear and is unexpected when taking into consideration the higher enrichment of TE cells generated using protocol 1. However, the higher expression of TIM-3 in T cells generated according to this protocol may be involved in this finding. CD8+ NY-ESO-1-specific T generated using protocol 1 displayed a significantly higher TNF-α level. This might partially be related to the higher percentage of TE cells among NY- ESO-1-specific T cells using protocol 1. Similar percentages of IFN-γ+ NY-ESO-1-specific T cells and multifunctional NY-ESO-1-specific T cells producing both IFN-γ and TNF-α between both protocols were observed. This raises the question of why NY-ESO-1-specific T cells produced by protocol 1 secreted more TNF-α but had lower lytic activity when compared to cells generated using protocol 2. According to Chao Ma et al., polyfunctional T cells are highly functional T cells that dominate the anti-tumor immune response. These cells can secrete 100 times more copies of a given protein than non-polyfunctional T cells in spite of their relatively low proportion among all T cells [40]. In addition, a previous study in mouse models has demonstrated that the overall quality of an immune response is best reflected by the effector function of polyfunctional T cells [41]. Therefore, the secretion of a single cytokine is probably not sufficient to assess anti-tumor efficacy. The discrepancy between lysis and TNF-α secretion in our study implied the role of multifunction T cells. Furthermore, other factors, e.g., the higher expression of exhaustion markers such as TIM-3 in NY-ESO-1-specific T cells generated with protocol 1 may be accountable for the differences in cytotoxicity. Importantly, ex vivo lysis and cytokine production assays are crucial to determine general functionality of genetically modified T cells, but the results cannot directly reflect their efficacy in clinical application. Cells 2021, 10, 152 12 of 14

Finally, it is important to highlight that, in contrast to protocol 1, protocol 2 is relying on FBS for T cell cultivation. We are using FBS for the generation of GMP-grade CAR-T cells in our clinical trial (NCT03676504) but extensive washing steps at the end of the production process are necessary to reduce the amount of FBS in the final cell product. Furthermore, the use of FBS for clinical cell therapy application may be restricted in some countries. In these terms, the FBS-free protocol 1 may have advantages over protocol 2 in specific occasions where FBS has to be avoided for GMP-grade T cell cultivation. In conclusion, rather than evaluating a single aspect of an ACT production protocol, we compared two “ready-to-use” protocols that can be directly adapted by the community. Both protocols can efficiently manufacture functional NY-ESO-1-specific T cells. However, both protocols endow advantages and disadvantages: Protocol 1 can achieve a faster T cell + expansion with enrichment of CD8 TE NY-ESO-1-specific T cells. In contrast, protocol 2 can promote a higher number of less-differentiated T cells with a lower expression of exhaustion markers. This is particularly important, as the crucial problem in current therapies with genetically modified T cells is longevity of the T cell population and function. Here, transduced TN cells are particularly helpful as they can later differentiate into effector T cells, thus ensuring a long-lasting action of genetically altered T cells. Protocol 1 as the more expensive and more labor-intensive protocol may be preferred if there is need to quickly generate the cellular product. Protocol 2 seems to be more cost-effective and may generate a lower number of NY-ESO-1-specific T cells that are potentially of higher quality and functionality. A higher proportion of NY-ESO-1-specific T cells with a more favorable phenotype may need a lower absolute number of cells transfused for efficient engraftment and tumor control. The transfer of a lower number of T cells may also be accompanied by a more favorable safety profile. In vivo studies will be necessary to confirm the comparability or even superiority of one of these protocols.

Supplementary Materials: The following are available online at http://www.mdpi.com/xxx/s1. Figure S1: Details of protocol 1 and protocol 2, Figure S2: Gating strategy from a representative production, Figure S3: The evolution of NY-ESO-1-specific CD4+ and CD8+ T cells on days 7 and 10 of the production using protocol 1 and 2, Figure S4: Specificity of NY-ESO-1-specific T cells for NY-ESO-1+HLA-A2+ cells. Author Contributions: Conceptualization, W.G., L.S.; Methodology, W.G., L.S., H.S.; experimental design discussion L.W., S.S., M.N., M.-L.S., B.N., C.K., A.H.-K., D.W., C.M.-T., A.S., H.S. and M.S.; Resources: H.S.; Data analysis, W.G., L.S.; Writing—original draft preparation, W.G.; writing—review and editing, all authors; supervision, L.S., M.S.; project administration, L.S.; funding acquisition, L.S., M.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by the DKTK (Deutsches Konsortium für Translationale Krebsforschung) and the NCT-HD-CAR-1 grant from the German Cancer Research Center (DKFZ). Leopold Sellner was supported by the “Physician Scientist Program” of the Medical Faculty of Heidelberg, the NCT Heidelberg School of Oncology (HSO), and the “Clinician Scientist Program” of the German Society of Internal Medicine (DGIM). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the University of Heidelberg (S-254/2016). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Conflicts of Interest: L.S.: Employment, Takeda. L.W.: Employment, TolerogenixX Ltd. A.S.: travel grants, Hexal and Jazz Pharmaceuticals, research grant, Therakos/Mallinckrodt, co-founder and part-time employee, TolerogenixX Ltd.

References 1. Barrow, C.; Browning, J.; MacGregor, D.; Davis, I.D.; Sturrock, S.; Jungbluth, A.A.; Cebon, J. Tumor antigen expression in melanoma varies according to antigen and stage. Clin. Cancer Res. 2006, 12, 764–771. [CrossRef][PubMed] 2. Park, T.S.; Groh, E.M.; Patel, K.; Kerkar, S.P.; Lee, C.C.; Rosenberg, S.A. Expression of MAGE-A and NY-ESO-1 in Primary and Metastatic Cancers. J. Immunother. 2016, 39, 1–7. [CrossRef][PubMed] Cells 2021, 10, 152 13 of 14

3. van Rhee, F.; Szmania, S.M.; Zhan, F.; Gupta, S.K.; Pomtree, M.; Lin, P.; Batchu, R.B.; Moreno, A.; Spagnoli, G.; Shaughnessy, J.; et al. NY-ESO-1 is highly expressed in poor-prognosis multiple myeloma and induces spontaneous humoral and cellular immune responses. Blood 2005, 105, 3939–3944. [CrossRef][PubMed] 4. Endo, M.; de Graaff, M.A.; Ingram, D.R.; Lim, S.; Lev, D.C.; Briaire-de, B.I.; Somaiah, N.; Bovée, J.V.; Lazar, A.J.; Nielsen, T.O. NY-ESO-1 (CTAG1B) expression in mesenchymal tumors. Mod. Pathol. 2015, 28, 587–595. [CrossRef] 5. Wang, R.F.; Wang, H.Y. Immune targets and neoantigens for cancer immunotherapy and precision medicine. Cell Res. 2017, 27, 11–37. [CrossRef][PubMed] 6. Wei, R.; Dean, D.C.; Thanindratarn, P.; Hornicek, F.J.; Guo, W.; Duan, Z. Cancer testis antigens in sarcoma: Expression, function and immunotherapeutic application. Cancer Lett. 2020, 479, 54–60. [CrossRef] 7. Fratta, E.; Coral, S.; Covre, A.; Parisi, G.; Colizzi, F.; Danielli, R.; Nicolay, H.J.; Sigalotti, L.; Maio, M. The biology of cancer testis antigens: Putative function, regulation and therapeutic potential. Mol. Oncol. 2011, 5, 164–182. [CrossRef] 8. D’Angelo, S.P.; Melchiori, L.; Merchant, M.S.; Bernstein, D.; Glod, J.; Kaplan, R.; Grupp, S.; Tap, W.D.; Chagin, K.; Binder, G.K.; et al. Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1c259 T Cells in Synovial Sarcoma. Cancer Discov. 2018, 8, 944–957. [CrossRef] 9. Thomas, R.; Al-Khadairi, G.; Roelands, J.; Hendrickx, W.; Dermime, S.; Bedognetti, D.; Decock, J. NY-ESO-1 Based Immunotherapy of Cancer: Current Perspectives. Front. Immunol. 2018, 9, 947. [CrossRef] 10. Dwarshuis, N.J.; Parratt, K.; Santiago-Miranda, A.; Roy, K. Cells as advanced therapeutics: State-of-the-art, challenges, and opportunities in large scale biomanufacturing of high-quality cells for adoptive immunotherapies. Adv. Drug Deliv. Rev. 2017, 114, 222–239. [CrossRef] 11. Hinrichs, C.S.; Borman, Z.A.; Gattinoni, L.; Yu, Z.; Burns, W.R.; Huang, J.; Klebanoff, C.A.; Johnson, L.A.; Kerkar, S.P.; Yang, S.; et al. Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy. Blood 2011, 117, 808–814. [CrossRef][PubMed] 12. Xu, Y.; Zhang, M.; Ramos, C.A.; Durett, A.; Liu, E.; Dakhova, O.; Liu, H.; Creighton, C.J.; Gee, A.P.; Heslop, H.E.; et al. Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15. Blood 2014, 123, 3750–3759. [CrossRef][PubMed] 13. Schubert, M.L.; Schmitt, A.; Sellner, L.; Neuber, B.; Kunz, J.; Wuchter, P.; Kunz, A.; Gern, U.; Michels, B.; Hofmann, S.; et al. Treat- ment of patients with relapsed or refractory CD19+ lymphoid disease with T lymphocytes transduced by RV-SFG.CD19.CD28.4- 1BBzeta retroviral vector: A unicentre phase I/II clinical trial protocol. BMJ Open 2019, 9, e026644. [CrossRef][PubMed] 14. Hoffmann, J.M.; Schubert, M.L.; Wang, L.; Hückelhoven, A.; Sellner, L.; Stock, S.; Schmitt, A.; Kleist, C.; Gern, U.; Loskog, A.; et al. Differences in Expansion Potential of Naive Chimeric Antigen Receptor T Cells from Healthy Donors and Untreated Chronic Lymphocytic Leukemia Patients. Front. Immunol. 2017, 8, 1956. [CrossRef][PubMed] 15. Stock, S.; Hoffmann, J.M.; Schubert, M.L.; Wang, L.; Wang, S.; Gong, W.; Neuber, B.; Gern, U.; Schmitt, A.; Müller-Tidow, C.; et al. Influence of Retronectin-Mediated T-Cell Activation on Expansion and Phenotype of CD19-Specific Chimeric Antigen Receptor T Cells. Hum. Gene Ther. 2018, 29, 1167–1182. [CrossRef][PubMed] 16. Stock, S.; Übelhart, R.; Schubert, M.L.; Fan, F.; He, B.; Hoffmann, J.M.; Wang, L.; Wang, S.; Gong, W.; Neuber, B.; et al. Idelalisib for optimized CD19-specific chimeric antigen receptor T cells in chronic lymphocytic leukemia patients. Int. J. Cancer 2019, 145, 1312–1324. [CrossRef] 17. Yoo, H.J.; Liu, Y.; Wang, L.; Schubert, M.L.; Hoffmann, J.M.; Wang, S.; Neuber, B.; Hückelhoven-Krauss, A.; Gern, U.; Schmitt, A.; et al. Tumor-Specific Reactive Oxygen Species Accelerators Improve Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies. Int. J. Mol. Sci. 2019, 20, 2469. [CrossRef] 18. Wang, L.; Gong, W.; Wang, S.; Neuber, B.; Sellner, L.; Schubert, M.; Hückelhoven-Krauss, A.; Kunz, A.; Gern, U.; Michels, B.; et al. Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells. Cytotherapy 2019, 21, 566–578. [CrossRef] 19. Gattinoni, L.; Klebanoff, C.A.; Restifo, N.P. Paths to stemness: Building the ultimate antitumour T cell. Nat. Rev. Cancer 2012, 12, 671–684. [CrossRef] 20. Lesbats, P.; Engelman, A.N.; Cherepanov, P. Retroviral DNA Integration. Chem. Rev. 2016, 116, 12730–12757. [CrossRef] 21. Lee, H.J.; Lee, Y.S.; Kim, H.S.; Kim, Y.K.; Kim, J.H.; Jeon, S.H.; Lee, H.W.; Kim, S.; Miyoshi, H.; Chung, H.M.; et al. Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells. Biologicals 2009, 37, 203–209. [CrossRef][PubMed] 22. Dodo, K.; Chono, H.; Saito, N.; Tanaka, Y.; Tahara, K.; Nukaya, I.; Mineno, J. An efficient large-scale retroviral transduction method involving preloading the vector into a RetroNectin-coated bag with low-temperature shaking. PLoS ONE 2014, 9, e86275. [CrossRef][PubMed] 23. Chono, H.; Goto, Y.; Yamakawa, S.; Tanaka, S.; Tosaka, Y.; Nukaya, I.; Mineno, J. Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation. J. Biochem. 2011, 149, 285–292. [CrossRef][PubMed] 24. Kaartinen, T.; Luostarinen, A.; Maliniemi, P.; Keto, J.; Arvas, M.; Belt, H.; Koponen, J.; Loskog, A.; Mustjoki, S.; Porkka, K.; et al. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion. Cytotherapy 2017, 19, 689–702. [CrossRef][PubMed] Cells 2021, 10, 152 14 of 14

25. Yu, S.S.; Nukaya, I.; Enoki, T.; Chatani, E.; Kato, A.; Goto, Y.; Dan, K.; Sasaki, M.; Tomita, K.; Tanabe, M.; et al. In vivo persistence of genetically modified T cells generated ex vivo using the fibronectin CH296 stimulation method. Cancer Gene Ther. 2008, 15, 508–516. [CrossRef][PubMed] 26. Gargett, T.; Brown, M.P. Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2. Cytotherapy 2015, 17, 487–495. [CrossRef] [PubMed] 27. Hinrichs, C.S.; Borman, Z.A.; Cassard, L.; Gattinoni, L.; Spolski, R.; Yu, Z.; Sanchez-Perez, L.; Muranski, P.; Kern, S.J.; Logun, C.; et al. Adoptively transferred effector cells derived from naive rather than central memory CD8+ T cells mediate superior antitumor immunity. Proc. Natl. Acad. Sci. USA 2009, 106, 17469–17474. [CrossRef] 28. Klebanoff, C.A.; Scott, C.D.; Leonardi, A.J.; Yamamoto, T.N.; Cruz, A.C.; Ouyang, C.; Ramaswamy, M.; Roychoudhuri, R.; Ji, Y.; Eil, R.L.; et al. Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy. J. Clin. Investig. 2016, 126, 318–334. [CrossRef] 29. Kondo, T.; Imura, Y.; Chikuma, S.; Hibino, S.; Omata-Mise, S.; Ando, M.; Akanuma, T.; Iizuka, M.; Sakai, R.; Morita, R.; et al. Generation and application of human induced-stem cell memory T cells for adoptive immunotherapy. Cancer Sci. 2018, 109, 2130–2140. [CrossRef] 30. Wherry, E.J.; Kurachi, M. Molecular and cellular insights into T cell exhaustion. Nat. Rev. Immunol. 2015, 15, 486–499. [CrossRef] 31. Pauken, K.E.; Wherry, E.J. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015, 36, 265–276. [CrossRef] [PubMed] 32. Das, M.; Zhu, C.; Kuchroo, V.K. Tim-3 and its role in regulating anti-tumor immunity. Immunol. Rev. 2017, 276, 97–111. [CrossRef] [PubMed] 33. Mujib, S.; Jones, R.B.; Lo, C.; Aidarus, N.; Clayton, K.; Sakhdari, A.; Benko, E.; Kovacs, C.; Ostrowski, M.A. Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway. J. Immunol. 2012, 188, 3745–3756. [CrossRef][PubMed] 34. Sakuishi, K.; Apetoh, L.; Sullivan, J.M.; Blazar, B.R.; Kuchroo, V.K.; Anderson, A.C. Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J. Exp. Med. 2010, 207, 2187–2194. [CrossRef][PubMed] 35. Jin, H.T.; Anderson, A.C.; Tan, W.G.; West, E.E.; Ha, S.J.; Araki, K.; Freeman, G.J.; Kuchroo, V.K.; Ahmed, R. Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection. Proc. Natl. Acad. Sci. USA 2010, 107, 14733–14738. [CrossRef] [PubMed] 36. Mullins, I.M.; Slingluff, C.L.; Lee, J.K.; Garbee, C.F.; Shu, J.; Anderson, S.G.; Mayer, M.E.; Knaus, W.A.; Mullins, D.W. CXC chemokine receptor 3 expression by activated CD8+ T cells is associated with survival in melanoma patients with stage III disease. Cancer Res. 2004, 64, 7697–7701. [CrossRef] 37. Mikucki, M.E.; Fisher, D.T.; Matsuzaki, J.; Skitzki, J.J.; Gaulin, N.B.; Muhitch, J.B.; Ku, A.W.; Frelinger, J.G.; Odunsi, K.; Gajewski, T.F.; et al. Non-redundant requirement for CXCR3 signalling during tumoricidal T-cell trafficking across tumour vascular checkpoints. Nat. Commun. 2015, 6, 7458. [CrossRef] 38. Harlin, H.; Meng, Y.; Peterson, A.C.; Zha, Y.; Tretiakova, M.; Slingluff, C.; McKee, M.; Gajewski, T.F. Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res. 2009, 69, 3077–3085. [CrossRef] 39. Schuster, K.; Gadiot, J.; Andreesen, R.; Mackensen, A.; Gajewski, T.F.; Blank, C. Homeostatic proliferation of naïve CD8+ T cells depends on CD62L/L-selectin-mediated homing to peripheral LN. Eur. J. Immunol. 2009, 39, 2981–2990. [CrossRef] 40. Ma, C.; Cheung, A.F.; Chodon, T.; Koya, R.C.; Wu, Z.; Ng, C.; Avramis, E.; Cochran, A.J.; Witte, O.N.; Baltimore, D.; et al. Multifunctional T-cell analyses to study response and progression in adoptive cell transfer immunotherapy. Cancer Discov. 2013, 3, 418–429. [CrossRef] 41. Darrah, P.A.; Patel, D.T.; De Luca, P.M.; Lindsay, R.W.; Davey, D.F.; Flynn, B.J.; Hoff, S.T.; Andersen, P.; Reed, S.G.; Morris, S.L.; et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major. Nat. Med. 2007, 13, 843–850. [CrossRef][PubMed]