A Novel Full-Human CD22-CAR T Cell Therapy with Potent Activity Against Cd22low B-ALL

Tan et al. Blood Cancer Journal (2021) 11:71 https://doi.org/10.1038/s41408-021-00465-9 Blood Cancer Journal

CORRESPONDENCE Open Access A novel full-human CD22-CAR T cell therapy with potent activity against CD22low B-ALL Yue Tan1,2, Haodong Cai3,ChuoLi2, Biping Deng4, Weiliang Song5, Zhuojun Ling5,GuangHu6, Yongkun Yang6, Panpan Niu6, Guangrong Meng6,WeiCheng6, Jinlong Xu5, Jiajia Duan5, Zelin Wang5, Xinjian Yu7, Xiaoming Feng 2,8, Jianfeng Zhou 3 andJingPan 1

Dear Editor, Furthermore, since the immunogenicity of full-human CD22-targeted chimeric antigen receptor (CD22-CAR) antibodies tends to be reduced compared with humanized – T cells have been proven to be effective in treating or chimeric constructs9 11, the usage of fully human- patients with B acute lymphoblastic leukemia (B-ALL) derived CAR constructs might be a better strategy, which who were unsuitable to receive CD19-CAR T cell ther- can further lower the risk of developing immune – apy1 3. However, a considerable proportion of patients responses against the secondarily infused CAR T cells. still relapsed after CD22-CAR T cell therapy3,4, with This study aims to develop a new CD22-CAR construct diminished or decreased levels of CD22 expression on with low immunogenicity and potent activity for treating blasts. For patients who had not completely lost CD22 B-ALL patients who failed from prior CD22-CAR T cell expression on blasts, CD22-CAR T cell re-treatment may therapies. Full-human anti-CD22 single chain fragment apply as a salvage regimen. But unfortunately, in a pre- variant(scFv)s were screened from a full-human scFv yeast vious trial on a humanized CD22-CAR (termed CD22- display library. The screened anti-CD22 scFvs were fused CARYK002) T cell therapy, a second CD22-CARYK002 to the intracellular 4-1BB co-stimulatory and CD3ζ sig-

1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; infusion produced no or suboptimal anti-leukemia naling domains to create a panel of CD22-BBz variants response and CAR T cell expansion (Table S1 and (Fig. 1a). The activities of the different CD22-BBz variants Fig. S1). Because the immunogenicity of humanized were tested with NFAT reporter assay in Jurkat cells in antibodies or CARs has been suggested in previous stu- response to CD22high Raji, CD22low JVM-2, and CD22- – dies5 8, the poor effect of the second CD22-CARYK002 K562 cells (Fig. 1b), and CD22-BBz 80, 27, and 36 were infusion might be due to the patient’s immune response identified as constructs that could transmit strong against CD22-CARYK002 transgene. On the other hand, antigen-specific activation signals in T cells (Figs. 1c and CD22 downregulation might also represent a mechanism S2). We then evaluated the effector function of different of resistance to a second CAR T-cell therapy3. Therefore, CD22-CAR variants via CD107a degranulation and cyto- a new CD22-CAR with no cross immunogenicity with toxicity assay and identified CD22-BBz 80 with superior CD22-CARYK002, and with strong activity against CD22low effector activity against CD22low target cells (Fig. 1d, e). cells, may be effective for the second treatment of patients The membrane proteome array (MPA) showed that who failed from previous CD22-CARYK002 T cell therapy. CD22-BBz 80 had a high specificity to the target antigen (Fig. 1f). The in vivo anti-leukemia effect of CD22-BBz 80 was confirmed in NPG mice injected with 1 × 106 Correspondence: Xiaoming Feng ([email protected])or Jianfeng Zhou ([email protected])orJingPan([email protected]) Nalm6-Luc cells (Fig. S3). Thus, CD22-BBz 80 (termed 1State Key Laboratory of Experimental Hematology, Boren Biotherapy Translational CD22-CARFH80) was identified to have potent and Laboratory, Boren Clinical Translational Center, Department of Hematology, Beijing antigen-specific anti-leukemia activity and was used in the Boren Hospital, Beijing 100070, China 2State Key Laboratory of Experimental Hematology, National Clinical Research subsequent clinical study. Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, A single-center, open-label, phase I clinical trial Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin was conducted to evaluate the safety and efficacy of 300020, China FH80 + Full list of author information is available at the end of the article CD22-CAR T cells in 8 children with CD22 or These authors contributed equally: Yue Tan, Haodong Cai, Chuo Li

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Blood Cancer Journal Tan et al. Blood Cancer Journal (2021) 11:71 Page 2 of 6

a c 1.4 Full-human Raji EF1α CD8(H/TM) 4-1BB CD3ζ T2A tEGFR anti-CD22 scFv JVM-2 1.2 K562 Medium 1.0 b 0.8 Raji 0.6

low JVM-2 (CD22 ) 0.4

0.2 K562 reporter luminescence value (RLU) NFAT % of Max 10-1 100 101 102 0.0 CD22 14 24 35 40 36 76 80 93 100 121 Clone Index

d 80 Raji Reh Nalm6JVM-2 K562Jurkat U266 Medium

60 59.0 57.4 57.3 58.8 58.5 51.5 48.5 40.9 40.9 40 38.1 36.2 37.4

24.4 20 CD107a degranulation rate (%) 14.8 11.7 11.4 10.0 8.0 9.1 8.7 4.7 1.8 0 0 0 0 1.3 1.7 1.2 0 0.8 1.4 0.9 0 0.7 1.1 0 0.6 1.2 0 0 0 6 7 1 lls 7 1 lls 0 6 7 1 lls 0 6 1 0 6 7 6 7 1 lls 0 7 1 0 6 7 1 s 8 3 2 e 36 2 5 2 e 3 5 8 3 51 ells 80 2 5 8 36 8 3 2 z c z z z 3 z c z 8 z z z z c z 3 z z z z z z 5 B Bz T B B T ce B B Bz 5T B B B T cells B B Bz T B B T ce B Bz 2 Bz 5T cells Bz B T cell -B -B -BBz 80 -BBz -B BBz 8B B -B -B -BBz 27 B BBz-B -B -BB -B -B -B -B -BB 2 2 2-B 2 2- 2- 2-B 2- 2-B 2- 2-BBz2-B 2 2 2 2 2 2 2 22 2 22-BBz 5 2 2 22 2 2 22 22 22-B 2 2 2 22- 22-BBz22-B 2 2 2 2 D D D D D D D D D2 D D D D D22 D22 D D2 D D D22-BB CD22-BBzC CD C CD2 C C C CD2 CD CD2 CD C C CD22C C CD CD C C CD2 C C C C C C C C CD C

ef8000 Reh-ffLuc 25000 Nalm6-ffLuc 600 CD22-BBz 80 CD22-BBz 80 CD22 500 CD22-BBz 36 20000 6000 CD22-BBz 27 400 T Cells 15000 4000 300 10000

elative light unit 200

Relative light unit 2000 **** R *** *** ***

5000 Normalized Target Binding 100 ****** **** ***** * ** * **** 0 0 ** 0 ne :1 e 1 :1 1 Membrane Proteome Array o 1:1 2 n 5: 1 2: al 0.5:1 0. t alo e et rg rg ta ta

g CD22-CARYK002 CD22-CARFH80 CD22-CARYK002 CD22-CARFH80 Pretreatment Pretreatment Pretreatment Pretreatment

0.24 99.8 0.71 99.3 0.36 99.6 3.86 85.4

Pt01 Pt04

0 0 CD45 0 0 0 0 CD19 3.39 7.39 CD19 CD45 CD19 CD45 CD45 CD19 CD19 CD22 CD19 CD22 CD19 CD22 CD19 CD22

0 100 0 75.5 0 100 0 100

Pt02 Pt05

0 0 0 24.5 0 0 0 0 CD19 CD19 CD45 CD45 CD19 CD19 CD45 CD45 CD10 CD22 CD10 CD22 CD10 CD22 CD10 CD22

0 0 14.7 47.2 0 100 0 100

Pt03 Pt08

0 100 17.0 21.1 0 0 0 0 CD45 CD19 CD45 CD19 CD19 CD45 CD19 CD45 CD10 CD22 CD10 CD22 CD81 CD22 CD81 CD22 h Pt03 Pt04 400 Tumor cells 600 Tumor cells Non-B cells Non-B cells CD22-CARYK002 300 CD22-CARYK002 Pretreatment Pretreatment 400

200

CD22-CARFH80 CD22-CARFH80 200 Pretreatment 100 Pretreatment % of Max % of Max Mean Fluorescence Intensity

Mean Fluorescence Intensity 0 CD22 0 CD22 CD22-CARYK002 CD22-CARFH80 CD22-CARYK002 CD22-CARFH80 Pretreatment Pretreatment Pretreatment Pretreatment

Fig. 1 (See legend on next page.)

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(see figure on previous page) Fig. 1 Development of a novel full-human CD22-CARFH80 construct and leukemic CD19/CD22 expression in patients before enrolling in CD22-CARFH80 therapy. a Schematic of the recombinant lentiviral vectors encoding the full-human CD22-BBz variants. The expression of CAR transgene is under the control of the elongation factor 1α promoter. CD8 (H/TM), CD8α hinge and transmembrane domains; EGFR, epidermal growth factor receptor; T2A, Thoseaasigna virus 2A. b Histogram of CD22 expression on Raji, JVM-2, and K562 cells, determined by flow cytometry. c Nuclear factor of activated T cells (NFAT) reporter assay in T cells transduced with different CD22-BBz variants, after co-culturing with CD22high Raji, CD22low JVM-2, and CD22- K562 cells, or cultured in medium alone in triplicates. d Bar chart showing CD107a expression in specific CD22-BBz T cell clones after co-culturing with CD22high Raji, Nalm6, Reh cells, CD22low JVM-2, CD22− K562, Jurkat, and U266 cells, as determined by flow cytometry. CD107a degranulation rates were calculated as the percentages of CD107a+ cells among CAR+CD8+ T cells and indicated above each bar. e Cytolytic effects of specific CD22-BBz T cell clones against Nalm6 and Reh cells during co-culturing in triplicates at E:T ratios of 2:1, 1:1, 0.5:1, and 0:1. % specific lysis = (spontaneous relative light unit (RLU) − test RLU)/(spontaneous death RLU) × 100. A two-tailed, unpaired two-sample t-test was used for statistical analysis. The brown, green, and blue asterisks indicate the comparison of the cytolytic effects of CD22-BBz 80 with those of CD22-BBz 36, CD22-BBz 27, or control T cells, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. f Membrane proteome array for CD22-BBz. g Dot plots showing the proportions of blasts (red) and non-tumor cells (black) among all mononuclear cells from patients at the indicated time points, and contour plots showing CD22 and CD19 expression on blasts (red) before CD22-CARYK002 and CD22-CARFH80 T cell therapy, determined by flow cytometry with a population of CD22-negative non-B cells with similar cell size in the same staining tube (blue) as a negative control for evaluating CD22 expression level. The blasts were defined based on the combined analysis of multiple markers. The number represents the proportion of blasts (red) in the four quadrants. h Histogram and mean fluorescence intensity of CD22 cell-surface expression in patients 03 and 04.

CD22low B-ALL who failed in prior CD19 and CD22-CAR (Pt 04 and 08) remained in remission for 9 and 5 months, − therapies. The median age was 9 (range, 5–16) years. Six while one (Pt 02) had a CD22 relapse and died of tumor patients (75%) had hematological relapses as confirmed by progression 6 months after infusion. Three patients bone marrow morphology. One patient (pt 01) had per- (Pt 01, 03, and 07) were bridged to HSCT as consolidation + sistent positive measurable residual disease (MRD ) after CAR T cell infusion (the donors and pre- detected by flow cytometry and the other patient (Pt 06) conditioning regimen are detailed in Table S4), and − was MRD in bone marrow but had diffused extra- among them, two (Pt 01 and 07) remained in remission 10 medullary disease (Table S2). Four patients (50%; Pt 01, and 5 months after infusion, and one (Pt 03) had a relapse − 04, 06, and 08) had previously undergone allogeneic with a mixture of CD22low and CD22 blasts 3 months hematopoietic stem cell transplantation (allo-HSCT 3 of 4 after HSCT (Fig. S6). once, 1of 4 twice). All patients had received at least two The expansion of CD22-CARFH80 T cells peaked from lines of treatments including chemotherapies, allo-HSCT, days 11 to15 after infusion with a median count of 19.8 CD19, or CD22 CAR T therapies. All patients (100%) (range, 1.01–408) × 106/L and a median percentage of 30 + failed from prior versions of CD22-CAR therapies (two (range, 1–45) % among CD3 T cells in the blood (Fig. 2c, d). refractory, six relapsed Fig. S4 and Table S2). The CD22 The non-responding patient (Pt 05) had a significantly expression level on blasts was not obviously reduced in six lower peak CAR T cell expansion (1.01 × 106/L; 1% among + patients after prior CD22-CARYK002 therapies, while CD3 T cells) than other patients, despite her cultured patients 03 and 04 displayed lower CD22 expression on CAR T cell viability (76.5%) transduction efficiency (44%) blasts than that before the CD22-CARYK002 treatments and infused dose (3.49 × 106/kg) being at intermediate to (Fig. 1g, h). After receiving lymphodepleting chemother- high levels among all patients (Table S5). apy with fludarabine (30 mg/m2/day) and cyclopho- B cell aplasia and hypogammaglobulinemia could be sphamide (250 mg/m2/day), all patients received 1 (range, used as a measurement of the active surveillance of CAR 0.68 to 9.4) × 106 CD22-CARFH80 T cells per kilogram T cells12. The three patients who achieved remission and body weight (/kg) (Table S3). received no further treatments all exhibited B cell aplasia Seven (87.5%) of eight patients had a response to CD22- and hypogammaglobulinemia until the observation end CARFH80 T cells. Six patients (75%) achieved MRD- point (Fig. 2e), suggesting a prolonged persistence of CAR complete remission, including two (Pt 03, 04) with a low T cells. level of CD22 expression at enrollment; one patient (Pt CRS occurred in 7/8 (87.5%) patients, including six with 06) achieved partial remission in his extramedullary dis- grade 1 CRS (75%) and 1 with grade 3 CRS (12.5%). The ease, but unfortunately, she succumbed to the infection median time to onset was 3 days (range, 1–12 days), and on day 42 after infusion (Fig. 2a, b and Fig. S5). One non- the median duration was 10 days (range, 2–19 days). The response patient achieved remission after receiving ino- non-responding patient exhibited no signs of CRS, in tuzumab, but died due to transplantation complication at accordance with his minimal CAR T cell expansion. 5 months. The six remission patients were followed up Neurologic toxicities occurred in 2/8 (25%) patients, with a median time of 6 months. Of the three patients including 1 with grade 2 (12.5%) and 1 with grade 3 (Pt 02, 04, and 08) who received no further treatment, two (12.5%). The detailed manifestations and managements

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a b Pt06 Before treatment Before treatment

Pt01

Pt02 Day20 Pt03

Pt04 Inotuzumab

Patient ID Pt05 Extramedullary diseases Partial response Pt06 Leukemic status Day41 At remission Pt07 HSCT Death Pt08 CD22 partial positive relapse CD22 negative relapse 0123456789 10 11 Months after CAR T cell infusion

cdD7 D11 D14 D20 D30 34.3% 83.0% 34.8% 28.2% 11.8% Pt01 Pt05 ）

/L 600 6 Pt02 Pt06 Pt04 500 Pt03 Pt07 Pt04 Pt08 400

300 0.35% 0.2% 1.72% 0.66% 0.29%

200 Pt05 100 CAR T Cells Expansion in PB (×10 0 CD22 CAR 0 5 10 15 20 25 30 CD3 Days after CAR T cell infusion

100 ） + 80 e 60 Pt02 L 40 Pt04 L D CD22+ B cell<2% Pt06 L Low Ig in serum Patient ID CAR T Cells (% inCD3 20 L D Death Pt08 L Duration of B cell aplasia 0 0 5 10 15 20 25 30 012345 678910 Days after CAR T cell infusion Months after CAR T cell infusion

Pt01 CRS Grade 0 ICANS Grade 1 Grade 2 Grade 3 4 Pt02 CRS ICANS Mannitol and furosemide Dex (10mg/kg/d; IV) Pt03 CRS Dex (7.5mg/kg/d; IV) 3 ICANS Dex (5mg/d; IT) Low-flow nasal cannula Pt04 CRS (3L/minute) Grade 2 ICANS Low-flow nasal cannula (6L/minute) CRS T Tocilizumab (80mg/d) Patient ID Pt05 ICANS 1

Pt06 CRS ICANS 0

n e ss / r a io r e el s e P a T e o r v s n C m Pt07 CRS v oxi c le e k I e e p ns s izu a F y te e d n e ICANS H S e us ted l ed po s a a y ICE es io v r H r sc tor w le b Pt08 CRS p n o E re e e ICANS D co M c of 01234567891011121314151617181920 CRS ICANS Days after CAR T cell infusion

Fig. 2 (See legend on next page.)

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(see figure on previous page) Fig. 2 Clinical efficacy and safety of CD22-CARFH80 T cell therapy. a Swimmer plot showing the clinical responses, response duration, and outcome in individual patients treated with CD22-CARFH80 T cells. HSCT, hematopoietic stem cell transplantation. b Images showing the retreatment extramedullary disease detected by position emission tomography (PET), the reduction of intracranial mass detected by magnetic resonance imaging (MRI), and the reduction of chest wall mass detected by computed tomography (CT) during CAR T cell treatment in patient 6. The red dotted circle indicates the border of a leukemia mass. c Absolute number of CAR T cells (above panel) and the percentage of CAR T cells among CD3+ T cells (below panel) in the peripheral blood of eight patients who received CD22-CARFH80 T cell therapy. d Representative dot plot, detected by flow cytometry, showing the presence of CD22-CARFH80 T cells among CD3+ T cells in the peripheral blood of two patients, including Pt 4 who achieved complete remission and Pt 5 who did not respond to the therapy. e Swimmer plot showing the duration of B cell aplasia in the bone marrow and hypogammaglobulinemia in four patients who achieved complete remission but received no further treatments. Serum immunoglobulins and bone marrow non-malignant B cells were determined by immunoturbidimetry and flow cytometry, respectively. f Swimmer plot showing the grade, duration, and management CRS and ICANS in each patient. The color in the swimmer lane indicates the existence of a specific grade of CRS or ICANS at a specific time point during CAR T cell treatment. IT intrathecal injection IV intravenous injection CRS Cytokine-release syndrome ICANS immune effector cell-associated neurotoxicity syndrome. g Symptoms of CRS and ICANS in individual patients during CD22-CARFH80 T cell therapy. Horizontal lines indicate mean values of the grade of specific symptoms. of CRS, ICANS, and other toxicities suspected to be generated from a human phage library has been reported related to CAR T cells are shown in Fig. 2f, g and to produce convincing activity in B-ALL patients in a Table S6. We further compared the severity of CRS and phase I trial3. The specific property and value of different ICANS between CD22-CARFH80 and the prior CD22- CD22-CAR versions need to be further addressed in CARYK002 therapies in the same individual patients but future studies. Although future phase 2/3 trials are needed found no significant difference (P = 0.414 and 0.285, to verify and optimize CD22-CARFH80 therapy, our study Fig. S7). Serum cytokines indicative of systemic inflam- indicates an important value of CD22-CARFH80 as a sal- mation were detailed in Fig. S8. Most patients had dra- vage regimen for advanced B-ALL cases that are refrac- matic increases in IL-6, ferritin, and sCD25, while only a tory to prior versions of CD22-CAR therapies. small proportion of the patients had an obvious increase in TNF-α and IL-10. The patient (Pt 07) who developed Acknowledgements This work was supported by the National Key R&D Program of China grade 3 CRS and ICANS showed the highest peak levels (2019YFA0110200), the Tianjin Science Funds for Distinguished Young Scholars of IL-6, ferritin, and sCD25. (17JCJQJC45800), the CAMS Innovation Fund for Medical Sciences (CIFMS, In this study, we developed a novel full-human CD22- 2016-I2M-1–003), the Non-profit Central Research Institute Fund of Chinese FH80 low Academy of Medical Sciences (2018PT32034), and the National Natural Science CAR construct with superior activity against CD22 Foundation of China (81870090). target cells. In seven of eight B-ALL patients who were refractory or relapsed after previous CD19- and CD22- Author details FH80 CAR T cell therapies, CD22-CAR T cell therapy 1State Key Laboratory of Experimental Hematology, Boren Biotherapy Translational Laboratory, Boren Clinical Translational Center, Department of exhibited potent anti-tumor activity with a manageable 2 fi FH80 Hematology, Beijing Boren Hospital, Beijing 100070, China. State Key safety pro le. The high response rate with CD22-CAR Laboratory of Experimental Hematology, National Clinical Research Center for therapy implicates that there is no overt cross immuno- Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese FH80 Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, genicity between CD22-CAR and prior infused CD22- 3 YK002 FH80 China. Department of Hematology, Tongji Hospital, Tongji Medical College, CAR transgenes. Markedly, CD22-CAR therapy Huazhong University of Science and Technology, Wuhan 430030, China. low was even effective in two patients with CD22 blasts, 4Cytology Laboratory, Beijing Boren Hospital, Beijing 100070, China. 5Department of Hematology, Beijing Boren Hospital, Beijing 100070, China. while in the two patients who relapsed after CD22- 6 7 FH80 − Nanjing Iaso Biotherapeutics Co. Ltd, Nanjing 210000, China. Medical CAR therapy CD22 blasts were majorly present, Laboratory, Beijing Boren Hospital, Beijing 100070, China. 8Central Laboratory, low indicating that CD22 leukemia cells can be efficiently Fujian Medical University Union Hospital, Fuzhou 350001, China eliminated by CD22-CARFH80 T cells. Three patients without HSCT consolidation remained in B cell aplasia Author contributions and hypogammaglobulinemia until the cut-off date, Y.T., C.L., and J.P. contributed to clinical data collection, data analyses, and data FH80 interpretation. H.C. performed most preclinical experiments and analyses. B.D., indicating the continued presence of CD22-CAR Y.Y., P.N., G.M., and W.C. contributed to the CAR T cell manufacture. J.P., Z.L., T cells, but the follow-up time is not long enough, and the W.S., J.X., J.D., and Z.W. contributed to the clinical protocol. X.Y. was responsible immunogenicity and long-term persistent capability of for leukemic cell immunophenotyping. Y.T. conducted statistical analyses. Y.T., FH80 C.L., J.P., and X.F. wrote the manuscript. J.P., J.Z., and X.F. designed the clinical CD22-CAR need to be further investigated. Our trial, directed the study, and had the final responsibility to submit for previous version of CD22-CARYK002 was also very effi- publication. The authors read and approved the final manuscript. cient in inducing remission in B-ALL2. Whether CD22- CARFH80 outperforms CD22-CARYK002 in persistence or Data availability fi All data associated with this study are present in the paper or the long-term ef cacy still warrants future study. In addition, Supplementary Materials. Any data related to the clinical trial can be emailed a CD22-CAR containing a full-human scFv, termed m971, to the corresponding author.

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