The murine FMC63 anti-CD19 antibody18 was humanized by grafting the heavy chain complementarity determining region (CDR) sequences onto the human framework IGHV4-4*08-IGHJ4*01, and the light chain CDR sequences onto the human framework IGKV1-33*01-IGKJ2*01. The CDRs of the heavy and light chains of murine anti-CD33 My96 antibody19 were grafted onto human IgG1 frameworks based on their homology with human frameworks IGHV1-46*01- IGHJ6*01 for VH, IGKV4-1*01-IGKJ4*01 for VL, respectively. All VH and VL domains have a combined average humanness of more than 85% (table 1).

The anti-CD19 BsAb (BC250) and anti-CD33 BsAb (BC269) were designed using VH/VL domains from the corresponding humanized antibodies and huOKT3 scFv20 fused to the C-terminus of the light chain of a human IgG1 based on a method described previously.21 22 To remove glycosylation and complement binding, the N297A and K322A mutations in the Fc region were made, respectively. Several anti-CD19 BsAb humanized variants with different affinities for CD19 were generated during a single humanization campaign and named as BC253, BC254, and BC255. BsAbs were expressed by transient transfection of Expi293F cells (ThermoFisher Scientific - Waltham, MA, USA) and purified using protein-A affinity columns using ÄKTA fast protein liquid chromatography (GE Healthcare - Chicago, IL, USA). Purity was assessed by high performance liquid chromatography. Blinatumomab was obtained from the Memorial Sloan Kettering Cancer Center (MSKCC) pharmacy.

Leukemia cells were cultured in RPMI1640 (Corning Scientific - Corning, NY, USA) supplemented with 10% fetal bovine serum (Life Technologies - Carlsbad, CA, USA) at 37°C in a 5% CO₂ humidified incubator. Cytotoxicity assay was performed as described before.23

NALM6-luciferase cells were provided by Renier J. Brentjens at MSKCC. CD19-negative NALM6-luciferase cells were provided by David Scheinberg (MSKCC). These cells were transduced with human CD33 gene whose construct was provided by Trinidad Hernández-Caselles (IMIB-University of Murcia).24

BV173-luciferase cells were provided by R.J. O’Reilly (MSKCC). Raji and Daudi cells were transduced with retroviral virus to express click beetle luciferase (kindly provided by Vladimir Ponomarev (MSKCC). JIH-5 cells were bought from DSMZ (Leibniz, Germany). MOLM13-luciferase cells were provided by David Scheinberg (MSKCC).

Anti-human antibodies against CD3, CD4, CD8, CD19, and CD33 (Biolegend - San Diego, CA, USA) were used to stain cells. Stained cells were processed with a FACScalibur instrument (BD Biosciences - San Jose, CA, USA) and analyzed with FlowJo software (FlowJo, Ashland, Oregon, USA).

All mouse experiments were performed in compliance with the Institutional Animal Care and Use Committee guidelines. The immunodeficient NOD.Cg-Prkdc^scid IL-2R-γ^tm1Wjl/SzJ (NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and provided with Sulfatrim food. For tumor challenge experiments, 6–10-week-old mice were inoculated with leukemic cells intravenously with (0.5–1×10⁶ cells). For intravenous-leukemia model, activated human T cells (ATC) that were in culture for 7–9 days were admixed with the BsAb and were intravenously injected weekly for 1–3 weeks starting 3–6 days after tumor inoculation. BC119 (CD3xGD2 BsAb) was used as a control since GD2 is not expressed by any of the leukemia cell lines used. One thousand international unit of interleukin 2 (IL-2) was subcutaneously injected two times per week to support T cell persistence. Tumor growth was monitored using the IVIS bioluminescence imager.

Five 12-week-old female C57BL/6 mice were injected retroorbitally with 100 µg BC250. Blood was collected at 4, 8, 24, 48, 72, and 96 hours after BC250 administration. Immediately after collection blood was centrifuged at 800g for 10 min to separate serum and serum was frozen at −80°C. Serum antibody concentration was measured using ELISA. Briefly, huCD3 was adsorbed onto 96-well microtiter plates to capture the serum BC250 which was detected with peroxidase-conjugated mouse anti-human IgG1 (Jackson ImmunoResearch - West Grove, PA, USA) using o-phenylenediamine dihydrochloride as substrate.

Statistical analyses were performed using Graphpad Prism software. Comparison between two groups were performed using a Student’s t-test and comparisons between multiple groups were determined by analysis of variance (ANOVA) with Tukey correction and multiple comparison analysis. A p value of <0.05 was considered statistically significant. Error bars denote the SEM. Survival analysis was performed using Log-rank test.

BsAb engaging T cells toward CD19 on ALL were made using the IgG(L)-scFv platform (figure 1A). The humanness of published humanized CD19 antibodies is summarized in table 1. These BsAb bound to CD19(+) ALL cells, carrying out ADTC at femtomolar EC50 concentrations (figure 1B,C). Antibody binding avidity, measured by flow cytometry correlated with ADTC potency (EC50) in cytotoxicity assays (figure 1D,E). Clone BC250 demonstrated the highest binding and potency against CD19(+) leukemia and was chosen for further testing (figure 1D).

To test the in vivo potency of BC250, NSG mice were inoculated intravenously with CD19(+) NALM6 ALL cells. After 3 days, treatment was started with weekly intravenous injections of 10 million human ATC co-administered with decreasing doses of BC250 two times per week. One hundred nanogram of BC250 in the presence of ATC was curative while 100 ng of control BsAb specific for GD2 (which is not expressed on NALM6) or ATC alone had no effect. Survival of the BC250-treated mice was also significantly improved (figure 2).

To evaluate the potency of BC250 across various CD19(+) lymphoid malignancies, NSG mice were inoculated intravenously with B-cell lymphoma cell lines Daudi or Raji, or the B cell precursor leukemia cell line BV173. Treatment was started after 3 days (Raji) or 14 days (Daudi and BV173). While the combination of human ATC with BC250 treated leukemic mice, control GD2 BsAb was completely ineffective similar to the no treatment group (figure 3 and online supplemental figure 1). The dose of T cells required for leukemia control varied with tumor model depending on the doubling time of xenografts. For BV173 cells, a single dose of 9 million T cells followed by six doses of BC250 was enough to cure leukemia, while three doses of T cells and BC250 were needed to control Daudi and Raji cells in mice. Furthermore, flow cytometry assessment of kidney and liver showed CD19(+) leukemia/lymphoma (Daudi and BV173) in untreated and in control BsAb/ATC-treated mice; in contrast, no tumor cells were detected in the BC250/ATC groups (figure 3B,D).

Blinatumomab (Blincyto) is a tandem scFv bispecific T cell engager (BiTE) against CD19 and the only Food and Drug Administration (FDA)-approved BsAb against ALL and lymphoma (figure 4A). The potency of BC250 and blinatumomab were compared in ADTC (figure 4B). In vitro, BC250 and blinatumomab appear to have very similar potencies (EC50, 0.65 vs 3 pM), although blinatumomab demonstrated slightly higher maximum killing at the higher antibody concentrations (figure 4B). To compare the potency of the drugs in vivo, NSG mice inoculated with NALM6 human ALL cells were treated with BsAbs in combination with ATC. To compensate for the monovalent CD19/CD3 binding of blinatumomab versus bivalent binding of BC250 to CD19/CD3, a twofold higher molar dose of blinatumomab was administered. In addition, to compensate for the short in vivo half-life of blinatumomab, both antibodies were injected daily. As shown in figure 4C,D, as low as 5 fmol of BC250 could reduce leukemia growth and improve mice survival while 10 fmol of blinatumomab did not have any benefit over the T cell-only control group (figure 4D top panels, p<0.0001 and p=0.006 for tumor signal at days 10 and 17; p=0.0034 for survival). For the second dose group (50–100 fmol), there was a significant benefit of BC250 over blinatumomab for both leukemia burden and survival (figure 4D middle panel, p=0.001 for tumor signal at days 10 and 17; p=0.0027 for survival). Higher doses of both BsAbs could significantly reduce the leukemia growth, however, BC250 seemed superior in reducing the leukemia growth and survival extension (figure 4D lower panel, p=0.01 for tumor signal at day 10; p=0.062 for survival).

Blinatumomab is a 54 KD protein whose molecular weight is below the kidney clearance (70 kD) and therefore it has a poor PK with an elimination half-life (T_1/2) of 2.1 hours (blinatumomab FDA label). To assess the mechanism of BC250’s superior in vivo potency, we assessed the PK of BC250 in mice (figure 5A). The T_1/2 of BC250 in this experiment was ~19 hours, approximately nine times the T_1/2 of blinatumomab. BC250 remained detectable in two of five mice for up to 96 hours post injection. To investigate other potential mechanisms of BC250’s superior potency, we conducted an in vivo armed-T cell study. Activated T cells were mixed with either BC250 or blinatumomab for 20 min in a small volume of media (50 µL) and the unbound antibody was washed away. Equimolar doses of BC250 and blinatumomab were chosen, corrected for the bivalency of BC250 versus the univalent structure of blinatumomab, meaning that two times as many moles of blinatumomab were used compared with BC250 to arm the T cells. Armed-T cells were injected into mice that had been inoculated with NALM6 leukemia 10 days prior. BC250 armed-T cells reduced tumor burden 4.7-fold and 3.8-fold by days 21 (p=0.03) and 25 (p=0.017), respectively, and extended the survival of the leukemic mice (7/10 BC250 mice alive on day 47 versus 0/10 blinatumomab mice, p<0.0001) (figure 5D). These data demonstrate that the superiority of BC250 over blinatumomab cannot be entirely attributed to its more favorable PK and suggest that bivalent binding of BC250 versus monovalent binding of blinatumomab plays an important role in vivo.

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous type of acute leukemia with the expression of myeloid and lymphoid markers on a single type of blast cells (biphenotypic acute leukemia, BAL) or a discrete admixed population of myeloid and lymphoid blasts (bilineal leukemia).25 B/myeloid leukemia is the most common type of MPAL.2 JIH-5 is a human B lymphoid BAL cell line with homogenous expression of CD19 on all cells and variable expression of CD33 on subpopulations (figure 6A). We previously reported a humanized T cell-engaging BsAb against CD33 (BC133) based on the CD3 clone M195 with potent anti-AML function in vitro and in vivo.17 23 However, the CD3xCD33 BsAb we reported in this manuscript (BC269) has 24-fold higher affinity to CD33 and improved cytotoxicity in ADTC assays (fivefold lower EC50, figure 6B) and a better humanness score (humanness score of 93% vs 76.6% for VL and 85.7% vs 76.5% for VH for BC269 vs BC133, respectively) (table 2). To test the effect of combined BsAb therapy, the susceptibility of this cell line to monotherapy or combination therapy with anti-CD19 (BC250) and anti-CD33 (BC269) BsAbs was assessed in ADTC. The maximum killing of BC269 was about 50% of the maximum killing of BC269/BC250 combination therapy, consistent with the partial expression of CD33 on BAL cells (figure 6C). Since CD19 was expressed on all JIH-5 cells, BC250 monotherapy was as effective as BC250/BC269 combination therapy. To test the combination therapy of BsAbs against BAL, NSG mice were inoculated with a mixture of CD19(−)CD33(+) and CD19(+)CD33(−) NALM6 cells. Whereas treatment with either BC250 or BC269 in the presence of ATC did not prevent tumor growth, combination therapy of the same two BsAbs showed strong anti-leukemia responses (figure 6D,E).

Another type of MPAL is acute bilineal leukemia where discrete admixed populations of myeloid and lymphoid blasts coexist in the same patient. In ADTC assays where target cells were composed of a mixture of CD19(+) NALM6 and CD33(+) MOLM13 cells, the combination of BC250 and BC269 showed an additive effect at higher antibody concentrations (figure 7A). In a xenograft model of bilineal leukemia generated by intravenous injection of equal numbers of CD19(+) ALL and CD33(+) AML cells, the combination of CD19-BsAb and CD33-BsAb suppressed tumor growth, in contrast to groups treated with either BC250 or BC269 alone (figure 7B,C).