Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2021-002509 on 25 May 2021. Downloaded from T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia 1,2 2 1 Sayed Shahabuddin Hoseini, Mallika Vadlamudi, Madelyn Espinosa- Cotton , Hoa Tran,1 Yi Feng,1 Hong- fen Guo,1 Hong Xu,1 Irene Cheung,1 1 Nai- Kong V Cheung To cite: Hoseini SS, ABSTRACT membrane proximal immunoglobulin C Vadlamudi M, Espinosa- Background Acute myeloid leukemia (AML) remains (IgC) domain. Current anti-CD33 antibodies Cotton M, . T cell engaging et al one of the most challenging hematological malignancies. in clinical trials, including the only Food bispecific antibodies targeting Despite progress in therapeutics, majority of patients CD33 IgV and IgC domains and Drug Administration- approved antibody succumb to this neoplasm. CD33 is a proven therapeutic for the treatment of acute gemtuzumab ozogamicin (GO), target the target, given its expression on most AML cells. Almost myeloid leukemia. Journal for IgV domain. all anti-CD33 antibodies target the membrane distal ImmunoTherapy of Cancer Single nucleotide polymorphism (SNP) 2021;9:e002509. doi:10.1136/ immunoglobulin V (IgV) domain of the CD33 extracellular jitc-2021-002509 domain. rs12459419 C>T in exon 2 of CD33 antigen Methods In this manuscript, we present data on three eliminates the expression of the IgV domain Accepted 18 April 2021 bispecific antibodies (BsAbs) against the CD33 IgV and and reduces the expression of the full-length membrane proximal immunoglobulin C (IgC) domains. protein. This SNP, which occurs in almost We use in vitro binding and cytotoxicity assays to show 50% of AML cases, renders the patients unre- the effect of these BsAbs on AML cell lines. We also use sponsive to GO, a drug that targets the IgV immunodeficient mice-bearing leukemias from cell lines domain.3 To tackle this splice variant hetero- and patient-derived xenografts to show the effect of these geneity, we generated T cell engaging bispe- BsAbs in vivo. cific antibodies (BsAbs) targeting the IgV and In vitro, the IgV-targeting BsAb had higher binding Results IgC domains and compare their function in to AML cell lines using flow cytometry and delivered more http://jitc.bmj.com/ potent cytotoxicity in T-cell- dependent cytotoxicity assays; vitro and potency in vitro in xenograft models importantly, the IgC domain-targeting outperformed the IgV of human AML. domain- targeting BsAb in medullary and extramedullary leukemia animal models. Conclusions These data support further clinical MATERIALS AND METHODS development of this BsAb for first-in- human phase I Antibody humanization on September 25, 2021 by guest. Protected copyright. clinical trial. The murine M195 anti-CD33 antibody was humanized as explained earlier.4 The INTRODUCTION murine My96 anti- CD33 was humanized by Leukemia is the most common cancer in chil- grafting the heavy chain complementarity dren. Despite advances in the treatment of determining region (CDR) sequences onto © Author(s) (or their acute lymphoblastic leukemia, patients with the human framework IGHV1-46*01 and employer(s)) 2021. Re- use acute myeloid leukemia (AML) have a much IGHJ4*01 and the light chain CDR sequences permitted under CC BY-NC. No worse prognosis, with a 3- year overall survival onto the human framework IGKV4-1*01 and commercial re- use. See rights rate of about 20%.1 The outcome of adult IGKJ4*01. The murine HIM3-4 anti-CD33 and permissions. Published by BMJ. patients with AML is even poorer, with only was humanized by grafting the heavy chain 1 6% of patients older than 65 surviving 2 years CDR sequences onto the human framework Pediatrics, Memorial Sloan 2 Kettering Cancer Center, New after diagnosis. There is an obvious unmet IGHV1-2*02 and IGHJ4*01 and the light York, New York, USA need for better AML therapies. chain CDR sequences onto the human frame- 2Ymabs Therapeutics, Nutley, CD33, a member of the sialic acid–binding work IGKV1-33*01 and IGKJ2*01. New Jersey, USA immunoglobulin- like (Ig- like) lectin family, The CD3xCD33 BsAbs were designed Correspondence to is expressed on most AML. The CD33 extra- using heavy chain variable region fragment/ Dr Nai- Kong V Cheung; cellular domain is composed of a membrane light chain variable region fragment (VH/ cheungn@ mskcc. org distal immunoglobulin V (IgV) and a VL) domains from huM195, huMy96, and Hoseini SS, et al. J Immunother Cancer 2021;9:e002509. doi:10.1136/jitc-2021-002509 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-002509 on 25 May 2021. Downloaded from Figure 1 Bispecific antibodies (BsAbs) binding to the IgV or the IgC domains of CD33. (A, B) Schematic of the three CD3xCD33 T cell engaging BsAbs against the IgV (BC133 and BC269) and IgC (BC275) extracellular domains of CD33. (C) 293T cells were transiently transfected with DNA plasmids containing the gene for full-length human CD33 or the splice variant and stained with BC133, BC269, BC275, and an irrelevant control antibody. (D) Several CD33(+) and CD33(−) cell lines were stained with BC133, BC269 and BC275. A secondary anti-human Fc fluorochrome-conjugated antibody was used to stain the cells. AML, acute myeloid leukemia; GMFI, geometric mean fluorescence intensity; IgC, immunoglobulin C; IgV, immunoglobulin V; MFI, mean fluorescence intensity. huHIM3-4 antibodies and huOKT3 scFv fused to the C T cell activation, proliferation, and cytokine release terminus of the light chain of a human IgG1 as previously Buffy coats were purchased from the New York Blood described and were named BC133, BC269, and BC275, Center. Peripheral blood mononuclear cells (PBMCs) respectively.5 The N297A and K322A mutations were were isolated from buffy coats using the lymphocyte generated to eliminate Fc glycosylation and complement separation medium (Corning, Cat # 25-072-CV). T 6 binding, respectively. cells were purified from PBMCs using the EasySep Human T Cell Isolation Kit (Stem Cell Technolo- Surface plasmon resonance http://jitc.bmj.com/ gies, Cat # 17951). For T cell proliferation assays, Human CD33 (Sino Biological, Cat # 12238- H08H) was carboxyfluorescein succinimidyl ester (CFSE)-labeled immobilized on a CM5 chip. Serial twofold dilutions (Thermo Scientific, Cat # C34554) effector cells were of the BsAbs (starting at 100 nM) were flowed over the mixed with AML cells with an E:T ratio of 10 with chip using a Biacore T200 system. Binding kinetics were different concentrations of BC275 for 4 days before measured at 37°C. The sensorgrams were fitted with a being assessed by flow cytometry (Attune, Thermo two- state binding model. on September 25, 2021 by guest. Protected copyright. Scientific). The percentage of cells with diluted CFSE Cytotoxicity assay (51Cr release assay) signal was used as the proliferation readout. For T Leukemia cells were cultured in RPMI 1640 medium cell activation and proliferation assays, effector cells (Cellgro) supplemented with 10% heat-inactivated fetal were mixed with AML cells (E:T of 10) and dilutions bovine serum (FBS) (Life Technologies) at 37°C in a 5% of BC275 for 1 day (early activation marker CD69 CO2 humidified incubator. Standard chromium release and cytokine release assay) or 4 days (late activation cytotoxicity assays were performed using activated human marker CD25). For the activation assays, cells were T cells as described before.4 assessed using a flow cytometer using anti-human Table 1 Binding kinetics of CD3xCD33 bispecific antibodies to human CD33 ka (1/Ms) kd (1/s) ka2 (1/s) kd2 (1/s) KD (M) BC269 7.84E+05 6.58E−04 3.12E−03 4.30E−04 1.02E−10 BC133 9.85E+05 7.97E−03 2.06E−03 9.02E−04 2.47E−09 BC275 3.37E+05 1.36E−03 3.72E−03 7.13E−04 6.49E−10 ka2, equilibrium association constant 2; ka, equilibrium association constant; kd, equilibrium dissociation constant; kd2, equilibrium dissocation constant 2; KD (M), equilibrium dissociaiton constant molarity. 2 Hoseini SS, et al. J Immunother Cancer 2021;9:e002509. doi:10.1136/jitc-2021-002509 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-002509 on 25 May 2021. Downloaded from Table 2 Humanness of the CD33 binding moieties of the (ATCC Cat # CRL-9591), K562 (ATCC Cat # CCL-243), BsAbs U937 (ATCC Cat # CRL-1593.2), OCIAML2 (DSMZ Cat # ACC 99), Raji (ATCC Cat # CCL-86), Daudi (ATCC Cat BC133 BC269 BC275 # CCL-213), HDLM-2 (DSMZ Cat # ACC 17), CML-T1 VL humanness 76.6 91 88.3 (DSMZ Cat # ACC 7), MOLT4 (ATCC Cat # CRL-1582), VH humanness 76.5 86 85.7 NALM6 (gift from Dr David Scheinberg’s Lab), BV173 VH, heavy chain variable region fragment; VL, light chain variable (DSMZ Cat # ACC 20). The cells were cultured in RPMI region fragment. 1640 (Gibco) supplemented with 10%–20% FBS (Gibco) at 37°C in a 5% CO2 humidified incubator. Cryopreserved bone marrow (BM) and cord blood (CB)-derived CD34(+) CD69 (Biolegend, Cat # 310906), CD25 (Biolegend, hematopoietic stem and progenitor cells (HSPCs) were Cat # 356104), CD4 (Biolegend, Cat # 344612), and purchased from ZenBio (Cat # SER- BMCD34- F and SER- CD8 (Biolegend, Cat # 344722). The cytokine concen- CD34- 1F, respectively). tration in the supernatant (after 24 hours of culture) The human CD33 plasmids, gifts from Trinidad was measured using the LegendPlex Human Th1 Hernández- Caselles of the IMIB-University of Murcia,7 Panel (Biolegend, Cat # 741035). were cloned into lentiviral vectors. NALM6 cells were Cell lines and analysis by flow cytometry transduced with a lentiviral vector expressing the human The following cell lines were used in this paper: Kasumi-1 CD33 gene. 293T cells (Thermo Fisher Scientific) were (ATCC Cat # CRL-2724, RRID:CVCL_0589), MOLM-13 transiently transfected with DNA plasmids containing (DSMZ Cat # ACC-554, RRID:CVCL_2119), SKM1 the full- length or splice variant (truncated) human CD33 (DSMZ Cat # ACC 547, RRID:CVCL_0098), HL60 (ATCC genes.