T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells

Published OnlineFirst April 8, 2016; DOI: 10.1158/2326-6066.CIR-15-0231

Research Article Cancer Immunology Research T Cells Expressing CD19/CD20 Bispeciﬁc Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells Eugenia Zah1, Meng-Yin Lin1, Anne Silva-Benedict2,3, Michael C. Jensen2,4,5, and Yvonne Y. Chen1

Abstract

The adoptive transfer of T cells expressing anti-CD19 chimeric in vivo. To our knowledge, this is the first bispecific CAR capable of antigen receptors (CARs) has shown remarkable curative potential preventing antigen escape by performing true OR-gate signal against advanced B-cell malignancies, but multiple trials have also computation on a clinically relevant pair of tumor-associated reported patient relapses due to the emergence of CD19-negative antigens. The CD19-OR-CD20 CAR is fully compatible with leukemic cells. Here, we report the design and optimization of existing T-cell manufacturing procedures and implementable single-chain, bispecific CARs that trigger robust cytotoxicity against by current clinical protocols. These results present an effective target cells expressing either CD19 or CD20, two clinically vali- solution to the challenge of antigen escape in CD19 CAR T-cell dated targets for B-cell malignancies. We determined the structural therapy, and they highlight the utility of structure-based rational parameters required for efficient dual-antigen recognition, and we design in the development of receptors with higher-level complexity. demonstrate that optimized bispecific CARs can control both wild- Cancer Immunol Res; 4(6); 498–508. 2016 AACR. – type B-cell lymphoma and CD19 mutants with equal efficiency See related Spotlight by Sadelain, p. 473.

Introduction The probability of antigen escape by spontaneous mutation and selective expansion of antigen-negative tumor cells decreases Adoptive T-cell therapy has demonstrated clinical efficacy with each additional antigen that can be recognized by the CAR T against advanced cancers (1, 2). In particular, multiple clinical cells. Therefore, a potential prophylaxis against antigen escape is trials have shown that T cells programmed to express anti-CD19 to generate T cells capable of recognizing multiple antigens. Here, chimeric antigen receptors (CARs) have curative potential against we present the rational design and systematic optimization of relapsed B-cell malignancies (3–9). However, these trials have single-chain, bispecific CARs that efficiently trigger T-cell activa- also revealed critical vulnerabilities in current CAR technology, tion when either of two pan–B-cell markers, CD19 or CD20, is including susceptibility to antigen escape by tumor cells (7, 10). present on the target cell. These CARs can be efficiently integrated For example, a recent trial of CD19 CAR T-cell therapy revealed into primary human T cells and administered in the same manner that 90% of patients achieved a complete response, but 11% of as CAR T cells currently under evaluation in the clinic. those patients eventually relapsed with CD19-negative tumors A single-chain, bispecific CAR targeting CD19 and the human (10). Antigen escape has also been observed in the adoptive epidermal growth factor receptor 2 (HER2/neu) was previously transfer of T cells expressing NY-ESO1–specific T-cell receptors reported by Grada and colleagues (14). This bispecific receptor, (11) and in cancer vaccine therapy for melanoma (12, 13). termed TanCAR, efficiently triggered T-cell activation in response to either CD19 or HER2. However, because CD19 and HER2 are not typically expressed on the same cell, the TanCAR remains a proof-of-concept design. Importantly, Grada and colleagues 1Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles,California. 2Ben Towne Center for highlighted that the TanCAR is less strongly activated both in Childhood Cancer Research, Seattle Children's Research Institute, vitro and in vivo when challenged with target cells that express 3 Seattle, Washington. Department of Oncology and Hematology, HER2 alone, as compared with HER2/CD19 double-positive St. Luke's Regional Cancer Center, Duluth, Minnesota. 4Division of Pediatric Hematology-Oncology, University of Washington School of targets (14). This observation suggests that tumor cells can still Medicine, Seattle, Washington. 5Program in Immunology, Clinical escape TanCAR detection by eliminating CD19 expression. Research Division, Fred Hutchinson Cancer Research Center, Seattle, To effectively prevent antigen escape, the bispecific CAR must Washington. not only recognize two antigens, but also process both signals in a Note: Supplementary data for this article are available at Cancer Immunology true Boolean OR-gate fashion—i.e., either antigen input should Research Online (http://cancerimmunolres.aacrjournals.org/). be sufficient to trigger robust T-cell output. We thus refer to this Corresponding Author: Yvonne Y. Chen, University of California, Los Angeles, particular type of bispecific receptors as "OR-gate CARs." Here, we 420 Westwood Plaza, Boelter Hall 5531, Los Angeles, CA 90095. Phone: report on the development of CD19-OR-CD20 CARs, which 310-825-2816. Fax: 310-206-4107. E-mail: [email protected]. trigger robust T-cell–mediated cytokine production and cytotox- doi: 10.1158/2326-6066.CIR-15-0231 icity when either CD19 or CD20 is present on the target cell. We 2016 American Association for Cancer Research. demonstrate that the size and rigidity of CAR molecules can be

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calibrated to match the specific antigens targeted, and the optimal every 48 hours. Dynabeads were removed 9 or 10 days after þ OR-gate CAR structure can be deduced from known structural isolation. CAR cells were enriched by magnetic bead–based requirements for single-input CARs. Finally, we show that the sorting (Miltenyi) and expanded by stimulation with irradiated – CD19-OR-CD20 CARs can control both wild-type and CD19 TM-LCLs at a T-cell:TM-LCL ratio of 1:7. Mock-transduced T cells mutant B-cell lymphomas with equal efficiency in vivo, thus were stimulated using the rapid expansion protocol as previously providing an effective safeguard against antigen escape in adop- described (20). Each in vitro experiment was repeated with T cells tive T-cell therapy for B-cell malignancies. from different donors (T cells were never pooled). See Supple- mentary Materials and Methods for additional details. Materials and Methods Cytotoxicity assay Plasmid construction Target cells (K562 cells) seeded at 1 104 cells/well in a 96-well fi Bispeci c CD19-CD20 CARs were constructed by isothermal plate were coincubated with effector cells at varying effector-to- assembly (15) of DNA fragments encoding the CD19 single-chain target (E:T) ratios in complete media without phenol red and with variable fragment (scFv) derived from the FMC63 mAb (16), the 5% HI-FBS for 4 hours. Supernatants were harvested and analyzed CD20 scFv derived from the Leu-16 mAb (17), an IgG4-based using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit extracellular spacer, the CD28 transmembrane domain, and the (Promega). cytoplasmic domains of 4-1BB and CD3 zeta. Sequences of extracellular spacers and linkers connecting scFv domains are Cytokine production quantification listed in Supplementary Table S1, and sequences of all OR-gate Target cells were seeded at 5 104 cells/well in a 96-well plate CARs are available in GenBank (accession numbers KX000905 to and coincubated with effector cells at an E:T ratio of 2:1 for 24 KX000911). All CARs were fused to a truncated epidermal growth hours. Cytokine concentrations in the culture supernatant were factor receptor (EGFRt) via a T2A peptide to facilitate antibody measured with the BD Cytometric Bead Array Human Th1/Th2 staining and sorting of CAR-expressing cells (18). Cytokine Kit II (BD Biosciences).

Cell line generation and maintenance / In vivo xenograft studies in NOD/SCID/gc (NSG) mice Parental Raji cells were obtained from the ATCC in 2003, and All in vivo experiments were approved by the UCLA Institutional parental K562 cells were a gift from Dr. Laurence Cooper in 2001. Animal Care and Use Committee. Six- to eight-week-old female fi Both cell lines were authenticated by short tandem repeat pro l- NSG mice were bred in house by the UCLA Department of þ ing at the University of Arizona Genetics Core in 2015. The Radiation and Oncology. EGFP , firefly luciferase (ffLuc)–expres- – Epstein Barr virus-transformed lymphoblastoid cell line (TM- sing Raji cells (5 105) were administered to NSG mice via tail- LCL) was made from peripheral blood mononuclear cells as þ þ þ þ vein injection. Seven days later, mice bearing engrafted tumors þ þ previously described (19). CD19 , CD20 , and CD19 /CD20 were treated with 10 106 mock-transduced or CAR /EGFRt K562 cells were generated by lentivirally transducing parental – cells via tail-vein injection. Tumor progression was monitored by K562 cells with CD19 and/or CD20 constructs. CD19 Raji cells bioluminescence imaging using an IVIS Lumina III LT Imaging were generated by CRISPR/Cas9-mediated gene editing. Raji cells System (PerkinElmer). Peripheral blood was obtained by retro- 6 (2 10 ) were transiently transfected with CD19-CRISPR-T2A- orbital bleeding 10 days and 20 days after tumor-cell injection, NM plasmid (2 mg) using the Amaxa Nucleofection Kit V (Lonza) and samples were analyzed by flow cytometry. and the Amaxa Nucleofector 2b Device (Lonza). After 24 hours, cells were coincubated with G418 sulfate (1.5 mg/mL; Enzo Life Statistical analysis Sciences) for 120 hours and further expanded in the absence of Statistical significance of in vitro results was analyzed using two- – fi antibiotics for 11 days. CD19 cells were rst enriched by staining tailed, unpaired, homoscedastic Student t test. Survival curves – cells with anti CD19-APC (allophycocyanin) followed by anti- were evaluated by the log-rank test, and statistical significance was APC microbeads (Miltenyi), and then removing labeled cells via determined by comparing log-rank test statistics against a c2 table – – magnetic bead based cell sorting. A pure CD19 population was with the degree of freedom equal to one. subsequently obtained by fluorescence-activated cell sorting using the FACSAria (II) at the UCLA Flow Cytometry Core Facility. All TM-LCL, Raji, and K562 cell lines were maintained in complete Results T-cell medium [RPMI-1640 (Lonza) with 10% heat-inactivated CD19 and CD20 CARs require distinct extracellular FBS (HI-FBS; Life Technologies)]. Human embryonic kidney 293T spacer lengths cells (ATCC) were cultured in DMEM (HyClone) supplemented Conventional CAR molecules comprise four main domains: with 10% HI-FBS. an scFv, an extracellular spacer, a transmembrane domain, and a cytoplasmic tail including costimulatory signals and the CD3z Generation of CAR-expressing primary human T cells chain (Fig. 1A). The CD19 CAR has superior activity when þ – þ CD8 /CD45RA /CD62L T cells were isolated from healthy constructed with a short extracellular spacer (21), but no sys- donor whole blood obtained from the UCLA Blood and Platelet tematic study has been reported for CD20 CARs. Therefore, we Center, stimulated with CD3/CD28 T-cell activation Dynabeads constructed and characterized a series of CD20 CARs incorpo- (Life Technologies) at a 1:1 bead:cell ratio, and lentivirally trans- rating long (IgG4 hinge-CH2-CH3; 229 aa), medium (IgG4 duced 72 hours later at a multiplicity of infection of 1.5. All T cells hinge-CH3; 119 aa), or short (IgG4 hinge; 12 aa) extracellular were expanded in complete T-cell medium supplemented with spacers connecting the Leu-16 scFv to the CD28 transmembrane penicillin–streptomycin (100 U/mL; Life Technologies) and fed domain (Supplementary Fig. S1A). All three CARs were efficient- þ IL2 (50 U/mL; Life Technologies) and IL15 (1 ng/mL; Miltenyi) ly expressed in primary CD8 human T cells (Supplementary

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AB G4S Linker VL VH V ScFv H ScFv #1 V H V VL VLL ScFv #2 Extracellular spacer Transmembrane domain IgG4 Spacer CD28 tm Cell membrane Cell membrane

ζ ζ 4-1BB Costimulatory domain CD3ζ Signaling domain CD3ζ Signaling domain

Figure 1. Schematics of single-input and bispeciﬁc CARs. A, a second-generation, single-input CAR. B, a bispeciﬁc, OR-gate CAR. C, schematic of four OR-gate CARs containing variations in extracellular spacer length and ordering of scFv domains. Hinge, CH2, and CH3 are domains within human IgG4. CD28tm is the CD28 transmembrane domain. EGFRt was fused to all CAR constructs via a T2A cleavage peptide to facilitate staining for CAR expression and sorting.

þ Fig. S1B). When challenged with CD20 target cells, T cells CD19, consistent with previous reports of single-input CD19 expressing the long-spacer CAR consistently showed greater CARs (21). target-cell lysis, cytokine production, and T-cell proliferation Although the long-spacer CARs did not target CD19, they could (Supplementary Fig. S1C–S1E), indicating functional superior- respond to CD20 (Fig. 2A). The 20-19 short CAR, which contained ity of the long-spacer CAR for CD20 detection. a short spacer and the CD20 scFv in the membrane-distal position, also performed well in cytokine production and was the most þ Bispecific and single-input CARs share antigen-specific efficient at lysing CD20 targets (Fig. 2). Given the scFv orienta- structural requirements tion in this construct, the CD19 scFv and the G4S linker between Second-generation, CD19-OR-CD20 CARs were constructed by the two scFv domains could serve as a proxy spacer that projects taking the standard four-domain CAR architecture and incorpo- the CD20 scFv away from the T-cell membrane, to a position rating two scFvs connected in tandem via a G4S flexible linker conducive for CD20 binding. We thus hypothesized that modi- (Fig. 1B). In light of the different preferences in extracellular spacer fying the G4S linker while keeping the 20-19 short CAR config- length for CD19 and CD20 single-input CARs, a panel of CD19- uration might further enhance CD20 response without compromis- þ OR-CD20 CARs was constructed to systematically evaluate the ing the receptor's sensitivity toward CD19 target cells. effects of spacer length and the ordering of the two scFv domains (Fig. 1C). Each CAR was constructed in the scFv #1 (VL-VH) – scFv Sequence modifications on scFv linkers enable effective #2 (VH-VL) orientation to minimize potential mispairing of VL and targeting of disparate antigens VH domains between the two scFvs. All four OR-gate CARs were A new panel of 20-19 short CARs was constructed with various produced at full length and expressed on the surface of primary linker sequences inserted between the two scFv domains (Fig. 3A). þ human CD8 T cells (Supplementary Fig. S2A and S2B). The original, short (G4S)1 flexible linker was compared against a þ When challenged with CD19 target cells, the OR-gate CARs long (G4S)4 flexible linker, a short (EAAAK)1 rigid linker, and a showed superior cytokine production and target-cell lysis when a long (EAAAK)3 rigid linker (22, 23). CARs with modified linker short extracellular spacer was used, regardless of scFv domain sequences were expressed at full length and localized to the cell order (Fig. 2). Both long-spacer CARs failed to produce IL2 surface (Supplementary Fig. S2C and S2D). þ (Fig. 2A) and had minimal lysis activity against CD19 targets To evaluate the utility of OR-gate CARs in preventing antigen – (Fig. 2B). Thus, short-spacer CARs were more effective in targeting escape, a mutant CD19 lymphoma cell line was generated by

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A IFNγ TNFα IL2 5,000 12,000 14,000 8,000 4,000 10,000 8,000 2,000 3,000

pg/mL 6,000 1,500 2,000 4,000 1,000 1,000 Figure 2. 2,000 500 Functional CD19-OR-CD20 CARs can be 0 0 0 constructed by linking scFv domains in Parental CD19 CD20 Parental CD19 CD20 Parental CD19 CD20 tandem. A, IFNg, TNFa, and IL2 production K562 K562 K562 K562 K562 K562 K562 K562 K562 by T cells expressing single-input or OR- gate CARs. Cytokine levels in media were Mock CD19 Short CD20 Long measured after a 24-hour coincubation 19-20 Long 20-19 Long 19-20 Short 20-19 Short with K562 target cells. B, target-cell lysis activity of CD19-OR-CD20 CARs following a 4-hour coincubation with K562 target cells. Reported values are the mean of B Parental K562 CD19 K562 CD20 K562 triplicates, with error bars indicating one 70 70 70 SD. Results are representative of two independent experiments performed with 50 50 50 CAR T cells derived from two different donors. 30 30 30

% Lysis 10 10 10

–10 10:1 3:1 1:1 –10 10:1 3:1 1:1 –10 10:1 3:1 1:1 E:T Ratio E:T Ratio E:T Ratio

19–20 Long 20–19 Long 19–20 Short 20–19 Short

CRISPR/Cas9-mediated genome editing of Raji lymphoma The original OR-gate CAR with a (G4S)1 linker had lower – þ cells (Supplementary Fig. S3). As expected, the single-input CD19 toxicity against mutant (CD19 /CD20 ) Raji compared with – CAR-T cells showed no response to CD19 target cells (Fig. 3B–D). WT Raji, indicating suboptimal CD20 targeting. Increasing In contrast, T cells expressing OR-gate CARs efﬁciently lysed both the length and/or rigidity of the linker sequence improved þ þ – wild-type (WT; CD19 /CD20 ) and CD19 target cells (Fig. 3D). the OR-gate CARs' ability to recognize CD20, resulting in

A 20–19 Short CD20 scFv Linker CD19 scFv Hinge CD28 tm 4-1BB CD3ζT2A EGFRt Linker = i. (G4S)1 ii. (G4S)4 iii. (EAAAK)1 iv. (EAAAK)3 Mock CD19 Short

B CD69 CD137 CD107a C IFNγ TNFα IL2 CD20 Long

6,000 ** 2,000 3,000 ** 2,000 ** 400 (G4S)1 ** ** ** ** ** ** ** ** ** ** 3,000 ** 2,500 1,500 ** 1,500 300 * (G4S)4 4,000 2,000 **

MFI ** 2,000 1,000 1,500 1,000 200 (EAAAK)1 2,000 pg/mL 1,000 1,000 500 100 500 500 (EAAAK)3 0 0 0 0 0 0

D CD19 Short CD20 Long (G4S)1 (G4S)4 (EAAAK)1 (EAAAK)3 100 100 100 100 100 100 WT Raji 80 80 80 80 80 80 CD19- Raji 60 60 60 60 60 60 40 40 40 40 40 40 20 % Lysis 20 20 20 20 20 0 0 0 0 0 0 10:1 3:1 1:1 10:1 3:1 1:1 10:1 3:1 1:1 –20 10:1 3:1 1:1 –20 –20 10:1 3:1 1:1 –20 –20 –20 10:1 3:1 1:1 E:T Ratio E:T Ratio E:T Ratio E:T Ratio E:T Ratio E:T Ratio

Figure 3. Rational structural modiﬁcations improve OR-gate CAR activity against mutant tumor cells. A, design of 20-19 short CARs incorporating linkers with increased – length and/or rigidity. B, CD69, CD137, and CD107a surface expression (MFI) by CAR T cells after a 24-hour coincubation with CD19 Raji cells. C, IFNg, TNFa, and IL2 production by the CAR T cells in B. D, cell lysis by single-input and OR-gate CAR T cells after 4-hour coincubation with WT and CD19– Raji cells. Reported values are the mean of triplicates, with error bars indicating one SD. P values were calculated by two-tailed Student t test; , P < 0.05; , P < 0.01. Data in B–E are representative of two independent experiments performed with CAR T cells derived from two different donors.

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– equally efficient elimination of both WT and CD19 Raji target results demonstrate that OR-gate CARs can efficiently detect and – cells (Fig. 3D). lyse CD19 escape mutants in vitro, and that rational modifica- In addition to enhanced cytotoxicity, modified OR-gate CARs tions to the CAR structure successfully improved CD20 targeting expressed more activation and degranulation markers, and they while maintaining high CD19 sensitivity. produced significantly more IFNg, TNFa, and IL2 compared with the original CAR with a (G4S)1 linker (Fig. 3B and C). The OR-gate Bispecificity does not affect CAR T-cell growth, differentiation, CAR with a (G4S)4 linker showed similar levels of effector output exhaustion profile, or lytic capability in vitro compared with the single-input CD20 CAR (Fig. 3B–D). Thus, Despite comparable activation marker expression and lytic linker modifications successfully compensated for impairments capabilities, CD19 and OR-gate CAR T cells showed disparate in CD20 targeting imposed by the short extracellular spacer, IL2 production upon antigen stimulation (Supplementary Fig. which was necessary for efficient CD19 targeting. S4B). To investigate potential effects of this difference in cytokine The increase in CD20 targeting efficiency obtained by linker production, we examined the cell differentiation pattern and modifications did not compromise CD19 targeting capability. All proliferation rate of the various CAR T-cell lines during extended 20-19 short CARs, regardless of linker type, showed robust CD69, coincubation with Raji target cells in the absence of exogenous CD137, and CD107a expression when challenged with WT Raji or cytokines. þ – CD19 /CD20 K562 target cells (Supplementary Fig. S4A). Com- Across multiple donors, we consistently observed a slight but pared with the single-input CD19 CAR, OR-gate CARs triggered statistically significant elevation in the central memory (TCM) comparable expression of activation and degranulation markers, population among cultured CD19 CAR T cells compared with as well as IFNg production in response to CD19 stimulation OR-gate CAR T cells prior to antigen stimulation (Fig. 4A). (Supplementary Fig. S4A and S4B). The OR-gate CAR T cells However, this statistical difference disappeared after 6 days of þ could lyse CD19 target cells as efficiently as single-input CD19 coincubation with WT Raji targets, with no major fluctuation CAR T cells (Supplementary Fig. S4C). Taken together, these in cell-type distribution (Fig. 4A), indicating that the level of

A Pre-antigen stimulation 6 days post-antigen stimulation 140 ** 140 n.s. 120 * * 120 n.s. n.s. 100 100 80 80 60 60 40 40 % T-cell Subtype % T-cell Figure 4. 20 20 CD19 and OR-gate CAR T cells exhibit similar 0 0 differentiation and cell proliferation following k t 4 3 k t 4 antigen stimulation. A, distribution of T-cell c r ) c ) o S K)3 subtypes prior to coincubation with WT Raji Mo h 4 AK) Mo (G A (G4S cell targets and 6 days after target-cell EA ( D19 Shor (EAAA CD19 S C addition. Cells were surface-stained for CD45RA and CCR7 expression. B, T-cell − + + − TCM (CD45RA , CCR7 ) TEMRA (CD45RA , CCR7 ) proliferation after coincubation with WT Raji cells. CD19 and OR-gate CAR T cells show − − + + similar proliferation, whereas CD20 CAR T TE/EM (CD45RA , CCR7 ) T (CD45RA , CCR7 ) naïve cells show signiﬁcantly less proliferation compared with CD19 CAR T cells (P < 0.01 for all time points except for day 6). C, expansion B C of mixed CD19 and CD20 CAR T cells stimulated with WT Raji cells over 8 days. 2.5 5 ** Values shown are the mean of triplicates, with error bars indicating one SD. P values were 2 4 calculated by two-tailed Student t test; n.s., not statistically signiﬁcant (P > 0.1); , P < 0.05; 1.5 3 ** , P < 0.01. Results are representative of three 1 2 ** independent experiments using CAR T cells * derived from three different donors.

Fold expansion 0.5 1 Fold expansion 0 0 0 2 4 6 8 0 2 4 6 8 Days post–target cell addition Days post–target cell addition

Mock CD19 Short CD20 Long

(G4S)4 (EAAAK)3

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A B 1.5 1.5

1 1

0.5 0.5 in survival Fold change in survival Fold change 0 0 0 2 4 6 0 2 4 6 Days post–target cell addition Days post–target cell addition

Mock CD19 Short CD20 Long

Figure 5. (G4S)4 (EAAAK)3 OR-gate CAR T cells maintain robust lysis capability through repeated antigen stimulation. Mock- Day 8: transduced and CAR T cells were coincubated with C 48 h post-stimulation WT Raji targets for 6 days, and then coincubated Pre-antigen stimulation with CD19– mutant Raji targets for another 6 days. with CD19-Raji A, survival of WT Raji cells coincubated with effector 120 120 – T cells. B, survival of CD19 Raji cells coincubated 100 100 with T cells that were previously challenged with WT Raji cells. (Note: Mock-transduced T cells had been 80 80 overwhelmed by WT Raji by Day 6 and were not 60 60 rechallenged with mutant Raji.) C, exhaustion 40 40 marker staining of CAR T cells before antigen % Positive stimulation, 48 hours after coincubation with WT 20 20 Raji (Day 2), and 48 hours or 6 days after subsequent – 0 0 coincubation with CD19 Raji cells (Days 8 and 12, k rt g 3 4 3 respectively). At each time point, cells were c o n ) ort ng o h S)4 K h S) M S Lo 4 A Lo 4 AK) surface-stained for Lag-3, Tim-3, and PD-1 and then 9 0 (G 0 (G A 1 2 19 S 2 analyzed for the simultaneous expression of one, D (EAA D (EA C CD C two, or three markers. Values shown are the mean of CD triplicates, with error bars indicating one SD. Day 2: Day 12: Results are representative of three independent 48 h post-stimulation 6 days post-stimulation experiments using CAR T cells derived from three with WT Raji with CD19-Raji different donors. 120 120 100 100 80 80 60 60 40 40 % Positive 20 20 0 0

ck g )4 )3 rt )4 )3 o n K ong S hort L M S Lo 0 (G4S (G4 9 2 19 Sho 20 1 D D (EAAAK (EAAA D C CD C C No marker One marker Two markers Three markers

antigen-stimulated IL2 production does not have a major impact in the CD20 CAR T-cell population (Fig. 4C). Thus, simultaneous on cell-type differentiation in vitro. Furthermore, CD19 and administration of a mixture of two CAR T-cell lines can result in OR-gate CAR T cells had similar proliferation rates upon antigen the selective expansion of one at the expense of the other. This stimulation (Fig. 4B). Thus, the amount of IL2 produced by behavior highlights an important benefit of using a single, bis- OR-gate CAR T cells is sufficient to support robust T-cell prolif- pecific CAR T-cell product that can ensure the maintenance of eration, an observation that was later confirmed in vivo. both CD19 and CD20 recognition capabilities while avoiding Despite producing similar IL2 concentrations as OR-gate CAR T growth competition between multiple T-cell products. cells, CD20 CAR T cells showed significantly weaker proliferation We next investigated whether the tumor-lysis capability and compared with CD19 CAR T cells (Fig. 4B). When single-input exhaustion profile of the OR-gate CARs differ from those of the CD19 and CD20 CAR T-cell lines were mixed and cocultured with single-input CARs in response to initial antigen stimulation and WT Raji target cells, the CD19 CAR T cells proliferated significantly subsequent emergence of antigen-escape mutants. At a 2:1 E:T more than CD20 CAR T cells, ultimately leading to a net decrease ratio, all CAR T-cell lines eliminated WT Raji cells after 6 days

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A CD19 Short + (G4S)4 + (EAAAK)3 + (EAAAK)3 + Mixed Raji Mixed Raji Mixed Raji WT Raji

d6 Min = 1.5e5 Max = 1.0e8

d18 Min = 2.0e7 Max = 2.0e8

d21 Min = 2.0e7 Max = 2.0e8

B n.s.

** n.s. ** %Survival

Days after tumor inoculation

Figure 6. OR-gate CARs abrogate the effects of antigen escape in vivo. A, tumor progression in NSG mice bearing WT or mixed (75% WT, 25% CD19–) Raji xenografts. Bioluminescence imaging was performed on days 6, 18, and 21 after tumor injection (T cells were injected on day 7). B, survival of mice bearing mixed Raji tumor xenografts and treated with T cells expressing no CAR, the single-input CD19 CAR, or OR-gate CARs. n ¼ 5 in all test groups. P values were calculated by log-rank test analysis; n.s., not statistically signiﬁcant (P > 0.1); , P < 0.1; , P < 0.05. Results represent one independent trial.

(Fig. 5A), at which point each sample was rechallenged with cells but failed to control the mixed Raji population (Fig. 6; – CD19 mutant Raji cells at a 2:1 E:T ratio. Target-cell count Supplementary Fig. S6A). Postmortem analysis revealed the out- – indicated that both OR-gate and CD20 CAR T cells efficiently growth of CD19 mutants in the mixed Raji xenograft, confirming – eliminated CD19 targets, whereas CD19 CAR T cells failed to antigen escape from single-input CAR T-cell therapy (Supplemen- curb the outgrowth of escape mutants (Fig. 5B). Surface antibody tary Fig. S6B). staining revealed consistent upregulation of PD-1, Tim-3, and In contrast with the single-input CD19 CAR, OR-gate CARs Lag-3 upon antigen stimulation across all CAR constructs (Fig. 5C; could target WT and mixed Raji tumors with equal efficiency – Supplementary Fig. S5). Upon subsequent challenge with CD19 (Fig. 6; Supplementary Fig. S6C), demonstrating that OR-gate target cells, exhaustion marker expression was sustained in OR- CAR T cells were unaffected by the loss of CD19 expression on gate and CD20 CAR T cells but declined in CD19 CAR T cells (Fig. tumor cells and only require a single antigen to trigger robust 5C; Supplementary Fig. S5). Taken together, these results indicate antitumor functions. The OR-gate CARs were as effective as the that OR-gate CAR T cells retain robust effector function upon single-input CD19 CAR in controlling the growth of WT Raji cells repeated antigen presentation, and they exhibit equivalent lysis (Supplementary Fig. S6D), confirming that the broadening of capabilities and exhaustion marker expression patterns as CD20 antigen specificity did not compromise in vivo antitumor efficacy – þ CAR T cells in response to CD19 escape mutants. against CD19 targets. Taken together, these results demonstrate that OR-gate CARs can efficiently target malignant B cells and OR-gate CARs prevent tumor antigen escape in vivo abrogate the effects of tumor antigen loss in vivo. To determine the in vivo functionality of OR-gate CARs, NOD/ Although OR-gate CARs were unaffected by the loss of CD19 / — SCID/gc (NSG) mice were injected with either a pure popu- antigen, none of the CAR T-cell lines including second-gener- – lation of WT Raji cells or a 3:1 mixture of WT and CD19 Raji cells. ation CD19 CAR T cells challenged with WT Raji cells—could þ Mice bearing established xenografts were then treated with CD8 completely eradicate engrafted tumors under the conditions CAR T cells. As expected, single-input CD19 CAR T cells signif- tested. Analysis of retro-orbital blood samples obtained 3 days icantly extended the survival of animals engrafted with WT Raji after T-cell injection confirmed the presence of CAR T cells

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WT Raji Mixed Raji Preinjection Postmortem ACB * * WT Raji Mixed Raji ** * * 0.5 ** 0.5 30 30 100 100 + + * 25 25 0.4 * 0.4 + 80 80 20 20 0.3 60 60

0.3 EGFRt EGFRt * + + 15 15

0.2 0.2 % PD-1 40 40 10 10 * ** 0.1 0.1 5 5 20 20 % CD8 % CD8 0 0 0 0 0 0 t 4 3 t 4 3 4 3 rt 4 3 rt 4 3 ) r ) rt ) ) ) rt )4 )3 ) ) S o S K) K ho S K o o S hor 4 h 4 ho A A K h S S S G4 4S A S G4 AAK G (G AA (G4S) A 9 ( Sh G 9 ( ( A A 1 AA ( 1 A 19 S EAAAK) 19 E 19 E E 19 E D ( ( ( ( (EAA ( C CD CD CD CD CD

D Postmortem Preinjection WT Raji Mixed Raji 120 120 120 − + T (CD45RA , CCR7 ) 100 100 100 CM 80 80 − − 80 TE/EM (CD45RA , CCR7 )

60 60 60 + − TEMRA (CD45RA , CCR7 ) 40 40 40 + + 20 20 20 Tnaïve (CD45RA , CCR7 ) % T-cell Subtype % T-cell 0 0 0 t 4 3 rt 3 rt 4 3 ) o )4 ) o or S h S K h S) K) 4 AK) S 4 A 4 A Sh G A 9 (G 9S (G A 9 ( A 1 AA A 1 E E E D ( D ( ( C C CD1

Figure 7. CD19 and OR-gate CAR T cells persist, upregulate PD-1, and exhibit similar differentiation subtypes in vivo. A, presence of CAR T cells in peripheral blood 3 days after T-cell injection. Cells from retro-orbital blood were stained for the expression of human CD8 and EGFRt, which was cotranslated with each CAR as a T2A fusion. B, percentage of CAR T cells present in the bone marrow collected at the time of sacriﬁce. C, PD-1 expression of CAR T cells in the bone marrow of animals bearing WT Raji tumors. D, preinjection and postmortem T-cell subtype distributions. "WT Raji" and "Mixed Raji" labels indicate the type of tumor engrafted in the animal from which the T cells were harvested. P values were calculated by two-tailed Student t test; n.s., not signiﬁcant (P > 0.1); , P < 0.05; , P < 0.01.

– (Fig. 7A). Both OR-gate CAR T-cell lines showed expansion in vivo resulting from the emergence of CD19 tumor cells (7, 10). To (Supplementary Fig. S6E). Finally, CAR-expressing T cells could arm CD19 CAR T cells against antigen escape, we constructed be found in the bone marrow of all animals at the time of sacrifice novel CARs capable of OR-gate signal processing—i.e., receptors (Fig. 7B), and all Raji cells retained either CD19 expression or that can trigger robust T-cell responses as long as the target cells CD19/CD20 dual expression (Supplementary Fig. S7). Therefore, express either CD19 or CD20. Results of our in vitro and in vivo inability to clear the tumor burden was not due to absence of CAR characterization experiments indicate that the CD19-OR-CD20 T-cell proliferation or the loss of both targeted antigens. CARs can safeguard against the effects of antigen escape by We next investigated the cell-type differentiation and exhaus- targeting malignant B cells through CD20 when CD19 expres- tion marker expression patterns of the CAR T cells. Results indicate sion has been lost. a significant increase in PD-1 expression but little change in cell- The choice of CD19 and CD20 as the target-antigen pair type distribution after in vivo antigen exposure (Fig. 7C and D). provides several important advantages. First, CD19 and CD20 PD-1 expression was significantly higher for CD19 CAR T cells are both clinically validated B-cell antigens expressed on the vast compared with OR-gate CAR T cells (Fig. 7C). However, PD-1 is majority of malignant B cells (10, 25, 26), making the CD19-OR- known to be upregulated upon T-cell activation, and prolonged CD20 CAR a clinically applicable construct that addresses a real PD-1 does not necessarily indicate lack of effector function in challenge facing adoptive T-cell therapy. Second, efforts to broad- CAR T cells (24). Given the proven clinical efficacy of the second- en the recognition capability of CAR T cells are often met with the generation CD19 CAR, the inability to achieve tumor clearance undesirable side effect of increased on-target, off-tumor toxicity. here is likely a result of the aggressive tumor line and specific However, this trade-off does not apply to the case of CD19 and tumor and T-cell dosages evaluated in this animal study. CD20, because both are exclusively expressed on B cells and have the same off-tumor toxicity profile. Finally, the ubiquitous expression of CD19 and CD20 on B cells and their known and Discussion predicted roles in promoting B-cell survival (27, 28) suggest that CD19 CAR T-cell therapy has yielded remarkable clinical simultaneous loss of both antigens would be a very low-prob- outcomes in the treatment of acute and chronic B-cell malig- ability event. Therefore, targeting CD19 and CD20 is expected nancies (3–9). However, this treatment strategy's vulnerability to to provide an effective safeguard against antigen escape by antigen escape has been highlighted by multiple cases of relapse malignant B cells.

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In principle, multiple receptor configurations can be adopted with our observations, quantitative imaging studies would be to achieve bispecific signal computation, such as coexpressing required to confirm its accuracy. two different CARs in one T cell (29) or mixing two CAR T-cell Based on our understanding of the structural requirements lines, each targeting a different antigen (30). Instead, we chose for productive CAR/antigen interactions, we arrived at the to engineer dual-antigen recognition capability into a single optimal structure for a bispecificCARthroughsystematic, CAR molecule due to a number of advantages. First, compared rational design. By adjusting the extracellular spacer length with expressing two separate single-input CARs in one T cell, and linker sequence between the two scFv domains, we were the bispecificCARhasasignificantly smaller DNA footprint able to independently optimize CD19 and CD20 targeting (reduced by 40% in DNA length), and previous studies have without compromising the CAR's ability to recognize either shown that construct size significantly affects viral vector pack- antigen. In vitro and in vivo results demonstrate that the OR-gate aging and transduction efficiency (31, 32). Most clinical T-cell CARs are as efficient in CD19 targeting as the clinically suc- products are administered as a polyclonal population without cessful single-input CD19 CAR, and the OR-gate CARs succeed þ – prior sorting for CAR cells (3, 5, 6), thus the ability to achieve in controlling CD19 mutant lymphoma cells where the single- high transduction efficiency has a direct impact on clinical input CAR fails. efficacy. Genetic compactness becomes particularly critical A potential concern with the design of bispecificCARsisthe when features such as suicide genes (33) and additional sig- increased number of peptide fusion points in the receptor nal-processing units (e.g., inhibitory receptors; ref. 34) also protein. However, almost all of the components used in the need to be integrated into the CAR T cells to ensure safety or OR-gate CARs presented in this study have been tested in the enhance efficacy. Second, compared with mixing two different clinic, including the two scFv domains, the long and short single-input CAR T-cell lines, the ability to produce and admin- extracellular spacers, as well as the transmembrane and cyto- ister a single, bispecific CAR T-cell product significantly reduces plasmic signaling domains (3–5, 7, 8, 39). Long, flexible linker treatment costs and increases the probability of successful T-cell peptides consisting of glycine and serine residues have been production within a short clinical timeframe. Third, the CD19 used in fusion peptides such as rVIIa-FP, which is now being CAR has outperformed all other CARs evaluated in the clinic to evaluated in a clinical trial (40, 41). It remains possible that date, including the CD20 CAR (35, 36). Our data demonstrate rigid peptide linkers or new combinations of previously tested that in a coculture, CD19 CAR T cells have a significant growth peptide components could result in immunogenicity. However, advantage over CD20 CAR T cells, resulting in a net decline in available evidence suggests that the probability is low and will CD20 CAR T-cell count despite the presence of CD20 antigen have to be verified through extensive testing in preclinical and (Fig. 4C). Therefore, it is probable that coadministering two clinical studies. single-input CAR T-cell populations would result in the dis- Several studies have explored the contribution of individual proportionate expansion of CD19 CAR T cells at the expense of CAR components to T-cell functionality, with results suggesting CD20 CAR T cells, thereby compromising this strategy's ability that the scFv framework regions, extracellular spacer length, and – to safeguard against CD19 mutants when they emerge later in costimulatory signals all play important and nonobvious roles in the treatment period. By making each T cell capable of bispe- enabling robust T-cell–mediated response to tumor antigens cific antigen recognition, the OR-gate CAR design maximizes (21, 36, 42–45). It remains possible that additional modifications thenumberofTcellsthatcanrecognizeanescapemutantwhen to each of these domains could further increase the OR-gate CAR's it appears. For these reasons, we chose to engineer a single CAR functionality. Increasingly detailed mechanistic understanding of molecule capable of dual-antigen recognition by attaching two CAR signaling and the systematic incorporation of such knowl- tandem scFv domains to the standard CAR chassis. Our data edge into the rational design of next-generation CAR molecules indicate that T cells expressing OR-gate CARs are indeed insen- will continue to facilitate the development of more effective and sitive to the loss of CD19 on target cells, proliferate robustly in predictable cell-based therapeutics. response to either CD19 or CD20 stimulation, do not exhibit Adoptive T-cell therapy has been hailed as one of the most altered cell-type differentiation patterns compared with single- promising advancements in cancer therapy in recent years, and input CAR T cells, and retain robust target-cell lysis capability CD19 CAR T-cell therapy holds the spotlight as the most upon repeated antigen stimulation. successful adoptive T-cell therapy to date. The work reported CARs are frequently thought of as modular proteins with here presents an effective and clinically applicable solution to sensor domains (i.e., the scFv) that can be easily changed to the challenge of antigen escape, which has been observed in alter receptor specificity. However, results from a previous report multiple clinical trials of CD19 CAR T-cell. The CD19-OR- (21) and our own investigation (Supplementary Fig. S1) CD20 CAR is fully compatible with current T-cell manufactur- revealed that efficient CD19 and CD20 targeting by single-input ing processes, does not impose extra burden in the form of large CARs requires distinct receptor structures. These divergent pre- viral packaging and transduction payloads, and enables a single ferences may be a consequence of structural differences between T-cell product to target two clinically validated antigens asso- the two antigens: CD19 is an immunoglobulin-like molecule ciated with B-cell leukemia and lymphoma. Finally, the design that belongs to a family of single-pass transmembrane proteins principles highlighted in this study can be further utilized to that project outward from the cell membrane (37); CD20 is a construct novel CARs for additional antigen pairs to broaden multipass transmembrane protein that lies close to the cell the applicability and increase the efficacy of T-cell therapy for surface (38). To achieve the optimal conjugation distance cancer. between a T cell and its target, the receptor needs to be adjusted to match the size of the target antigen, resulting in the need for a Disclosure of Potential Conflicts of Interest shorter CAR when targeting antigens with extensive extracellular M.C. Jensen reports serving as a scientific advisory board member and as a domains and vice versa. Although this hypothesis is consistent consultant for Juno Therapeutics, Inc., of which he is a scientific cofounder with

506 Cancer Immunol Res; 4(6) June 2016 Cancer Immunology Research

Bispeciﬁc T Cells Prevent B-cell Antigen Escape

ownership interest (including patents). No potential conﬂicts of interest were Acknowledgments disclosed by the other authors. The authors thank Lisa Rolczynski for her technical assistance.

Authors' Contributions Conception and design: M.C. Jensen, Y.Y. Chen Grant Support Development of methodology: E. Zah, A. Silva-Benedict, M.C. Jensen, Y.Y. Chen This research was funded by the NIH (5DP5OD012133, grant to Y.Y. Chen; Acquisition of data (provided animals, acquired and managed patients, 2R01CA136551-06A1, grant to M.C. Jensen). provided facilities, etc.): E. Zah, M.-Y. Lin, Y.Y. Chen The costs of publication of this article were defrayed in part by the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, payment of page charges. This article must therefore be hereby marked computational analysis): E. Zah, M.C. Jensen, Y.Y. Chen advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Writing, review, and/or revision of the manuscript: E. Zah, M.C. Jensen, this fact. Y.Y. Chen Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.C. Jensen, Y.Y. Chen Received September 10, 2015; revised February 19, 2016; accepted March 6, Study supervision: Y.Y. Chen 2016; published OnlineFirst April 8, 2016.

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Downloaded from cancerimmunolres.aacrjournals.org on September 26, 2021. © 2016 American Association for Cancer Research. Addendum Cancer Immunology Research ADDENDUM: T Cells Expressing CD19/CD20 Bispeciﬁc Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells Eugenia Zah, Meng-Yin Lin, Anne Silva-Benedict, Michael C. Jensen, and Yvonne Y. Chen

In this article (Cancer Immunol Res 2016;4:498–508), which confirmed that donor A CAR T cells could survive and expand appeared in the June 2016 issue of Cancer Immunology Research,we in vivo (1), they may not have proliferated well enough to reported the design and optimization of bispecific, OR-gate completely eliminate the tumor xenograft. chimeric antigen receptors (CARs) that can trigger robust T-cell activation in response to target cells that present either CD19 or CAR T cells with high post-thaw viability efficiently eradicate CD20 (1), thus preventing malignant B cells from escaping T-cell established tumor xenografts therapy through loss of CD19 expression. In our original study, To investigate this hypothesis, we repeated the in vivo study although OR-gate CAR T cells could significantly delay tumor using donor C cells, which showed superior viability in culture expansion and prolonged survival, tumors were not eradicated. after thawing (Fig. 1C). As in the previous study, 5 105 Raji Tumor persistence was not due to the loss of both CD19 and cells were delivered by tail-vein injection and allowed to engraft CD20 antigens, nor due to the inability of the adoptively trans- prior to treatment with 10 106 CAR T cells from donor C. ferred T cells to survive or expand in vivo (1). We concluded that Animals were injected with either purely wild-type (WT; þ þ the failure to eradicate tumors was due to suboptimal tumor or T- CD19 /CD20 ) Raji cells or a mixture of 75% WT and 25% cell dosing and/or the aggressiveness of the Raji tumor model. CD19 -mutant Raji tumors. In contrast to the results obtained As the Raji tumor model had been used in other studies at with donor A cells, we observed complete clearance of WT dosages comparable with those employed in our own experi- tumors treated with both single-input and OR-gate CAR T cells ments (2), complete tumor clearance should be possible in this (Fig. 2A and B). Only OR-gate CAR T cells, however, could xenograft model using adoptively transferred CAR T cells. Here, eradicate mixed (75% WT, 25% CD19 ) Raji tumors (Fig. 2A we report follow-up studies showing that the viability levels of and B). Animals engrafted with mixed Raji cells and treated with CAR T cells, both immediately after thawing and after 1–2 days of single-input CD19 CAR T cells eventually succumbed to tumor in vitro culture, are an important indicator of the CAR T cells' growths that consisted almost exclusively of the CD19 -mutant antitumor capability in vivo. Healthy T cells with OR-gate CARs phenotype (Fig. 2C), confirming the selective expansion of could efficiently eradicate established lymphoma xenografts even antigen-negative clones when treated with single-input CAR T if the lymphoma cells had spontaneously lost CD19 expression, cells. Thus, unlike single-input CD19 CAR T cells, OR-gate CAR T whereas single-input CD19 CAR T cells succumbed to the selective cells limited the escape of CD19 -mutant tumors. expansion of CD19 mutants. OR-gate CARs prevent the spontaneous emergence of CD19 Stability of CAR T-cell viability varies across donors mutant tumors The in vivo protocol employed in our study called for the Having confirmed the ability to eradicate both WT and tail-vein injection of luciferase-expressing Raji tumor cells, fol- CD19-knockout tumors using high-quality CAR T cells, we next lowed by the tail-vein injection of CAR T cells upon confirmation investigated whether OR-gate CAR T cells would have superior of tumor establishment, which was defined as clear tumor signal resistance compared with single-input CD19 CAR T cells against observed on two consecutive days via bioluminescence imaging the spontaneous loss of CD19 expression by tumor cells. Specif- (BLI). The CAR T cells used in these studies were previously frozen ically, we investigated whether antigen loss is dependent on the and thawed on the day of injection, and cells with >70% viability timing of CAR T-cell treatment relative to the size of tumor were considered suitable for adoptive transfer. To better charac- burden, and whether the OR-gate CAR T cells could safeguard terize the CAR T cells (from donor A) used in our initial animal against the spontaneous emergence of CD19 mutants. study reported in (1), we thawed additional stocks of the same Mice were injected with 5 105 WT Raji cells, which were donor's cells (donor A) and compared them with CAR T cells allowed to grow either until first confirmation of engraftment derived from other healthy donor blood samples (donors B and (i.e., yielding clear tumor signal observed on 2 consecutive C) for cell viability, T-cell subtype distribution, and exhaustion days via BLI, which occurred on day 6 in this set of studies), or marker expression. T-cell subtype distribution, exhaustion marker until the tumor signal reached a 6-fold increase in radiance expression, and viability at the time of thawing were similar for all intensity compared with the first group (which occurred on day donors (Fig. 1). However, cell viability of CAR T cells from donors 9). Animals were then treated with 10 106 donor C T cells A and B drastically declined after 24 hours in culture, whereas expressing either the single-input CD19 CAR or the OR-gate donor C cells remained >70% viable, indicating that donor A cells CAR. Consistent with previous results, animals treated at the may not have been of optimal quality (Fig. 1C). Although we had earlier time point with either single-input or OR-gate CAR T cells were cleared of tumor engraftment within 6 days of T-cell injection (Fig. 3A). In contrast, all animals treated with doi: 10.1158/2326-6066.CIR-16-0108 CAR T cells on day 9 after tumor injection showed continued 2016 American Association for Cancer Research. tumor progression for 3 days before tumor size began to

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A B 120 120 100 100 TCM (CD45RA-, CCR7+) No marker 80 80 TE/EM (CD45RA-, CCR7-) One marker 60 60 T (CD45RA+, CCR7-) 40 EMRA 40 Two markers % Positive % Positive

20 Tnaive (CD45RA+, CCR7+) 20 Three markers

% ofOverall population 0 0 Donor Donor Donor Donor Donor Donor A B C A B C C 90 80 70 Donor A 60 Donor B 50 40 Donor C

% Viable % Viable 30 20 10 0 0 24 48 Hours post thaw

Figure 1. Comparison of CAR T-cell preparations from multiple donors. A, T-cell subtype distribution patterns. B, exhaustion markers. Cells were stained for Tim-3, Lag-3, and PD-1. Values shown in A and B are means of three technical replicates 1 SD. C, viability changes during cell culture. Data shown are representative of at least two independent experiments performed on the cells from each of the three donors.

A Mock CD19 CAR OR-gate CAR WT Raji Mixed Raji WT Raji Mixed Raji WT Raji Mixed Raji

Day 5

Day 8

Day 11

B C 100

50 Raji/total Raji − 25 Average radiance %CD19 0

Mock

CD19 CAR Day post tumor injection Ratio at time of tumor injection

Figure 2. Wild-type (WT) and CD19-mutant lymphoma growth in vivo after injection of OR-gate CAR or single-input CD19 CAR T cells. A, tumor progression in NSG mice bearing WT or mixed (75% WT, 25% CD19) Raji xenografts. T cells were injected 5 days after tumor injection. B, radiance intensity of tumors engrafted in NSG mice; n ¼ 5 in all test groups. Reported values are the means of all surviving animals in each test group, with error bars indicating 1 SD. C, femoral bone marrow of mice bearing mixed Raji tumors and treated with mock-transduced or single-input CD19 CAR T cells analyzed by ﬂow cytometry. Raji cells were identiﬁed by the expression of EGFP, which was stably integrated into both Raji cell lines, and CD19 staining was performed to distinguish WT and CD19-mutant Raji populations. Results represent one independent trial.

640 Cancer Immunol Res; 4(7) July 2016 Cancer Immunology Research Bispeciﬁc T Cells Prevent B-cell Antigen Escape

A B Day post tumor injection Day post tumor injection 6 9 12 39 9 12 24 34 61

CD19 CAR CD19 CAR

OR-gate CAR OR-gate CAR

Min: 1E7 Scaling: Min: 1E5 Max: 2E6 Min: 2E4 Max: 1E5 Max: 2E8

T-cell 1.0E+05 injecon T-cell CD19 CAR injecon

OR-gate CAR

1.0E+04 Average radiance Average radianceAverage 1.0E+03 –1 10 20 30 40 Day post tumor injection Day post tumor injection

C D Pre-injection Tumor Unstained Stained Chest Liver

EGFP ffLuc 97.0 24.5 25.9

Chest CD19

Liver 68.5 73.1

CD20

Figure 3. Prevention of spontaneous CD19-mutant tumor outgrowth by OR-gate CAR T cells. A and B, NSG mice bearing WT Raji xenografts were treated with CAR T cells either (A) at the first sign of tumor engraftment (day 6 after tumor injection in this trial) or (B) after the tumor had expanded by 6 folds (day 9 after tumor injection). C, Raji tumor nodules were recovered from relapsed animals and their identity was verified by EGFP and firefly luciferase (ffLuc) expression via IVIS imaging. D, tumor nodule recovery from animals treated with CD19 CAR T cells. n ¼ 2 in all test groups. Radiance values reported in A and B are the means of two animals with error bars indicating the data range. Signal intensity scaling in images in A and B were adjusted as indicated for visual clarity. Results represent one independent trial. decline (Fig. 3B). Animals treated with OR-gate CAR T cells the risk of antigen escape with single-input CD19 CAR T cells completely cleared their tumors and showed no sign of relapse and the OR-gate CAR T cells' ability to safeguard against this at the time of this writing (through day 109). In contrast, tumor defense mechanism. animals treated with single-input CD19 CAR T cells experi- Patient relapse due to loss of CD19 antigen expression has been enced only temporary tumor regression before relapsing, ulti- observed in multiple clinical trials, and antigen escape has been mately succumbing to multifocal cancer growth (Fig. 3B). identified as a potential pitfall of CD19-directed therapies for B- Postmortem analysis revealed tumor engraftments in the liver cell malignancies (3, 4). The CD19/CD20 bispecific, OR-gate CAR and the chest cavity (Fig. 3C). The recovered Raji tumors, demonstrates the ability to address this critical medical challenge which were identified by their luciferase and EGFP expression and increase the efficacy of adoptive T-cell therapy for cancer. þ (Fig.3C),wereCD20 but had either no CD19 expression or significantly reduced CD19 expression (Fig. 3D), highlighting Published online July 1, 2016.

References 1. Zah E, Lin MY, Silva-Benedict A, Jensen MC, Chen YY. T cells expressing 3. Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feld- CD19/CD20 bi-speciﬁc chimeric antigen receptors prevent antigen escape man SA, et al. T cells expressing CD19 chimeric antigen receptors for acute by malignant B cells. Cancer Immunol Res 2016;4:498–508. lymphoblastic leukaemia in children and young adults: a phase 1 dose- 2. Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, escalation trial. Lancet 2015;385:517–28. et al. The nonsignaling extracellular spacer domain of chimeric antigen 4. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimeric receptors is decisive for invivo antitumor activity. Cancer Immunol Res antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2015;3:125–35. 2014;371:1507–17.

www.aacrjournals.org Cancer Immunol Res; 4(7) July 2016 641 Published OnlineFirst April 8, 2016; DOI: 10.1158/2326-6066.CIR-15-0231

T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells

Eugenia Zah, Meng-Yin Lin, Anne Silva-Benedict, et al.

Cancer Immunol Res 2016;4:498-508. Published OnlineFirst April 8, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-15-0231

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