Humanization of Clinically Used Murine CD45 Antibody for Optimized CD45-Targeted Radioimmunotherapy

Background: CD45-targeted radioimmunotherapy (RIT) has long been explored as augmentation of conditioning before hematopoietic cell transplantation (HCT). The CD45 monoclonal antibody (mAb) most exploited for this purpose is BC8. Validating this approach, BC8 labeled with iodine-131 (¹³¹I-apamistamab [Iomab-B]) followed by allogeneic HCT was recently shown to improve outcomes of older adults with relapsed/refractory AML relative to conventional care. However, as a murine mAb, BC8 has important limitations, including substantial infusion toxicities and development of human anti-mouse mAbs, which preclude BC8 redosing and future use of any other murine mab. To overcome these limitations, we humanized BC8 and tested its anti-tumor properties in vivo using the potent alpha-emitter, astatine-211 (²¹¹At), as payload.

Methods and Results: BC8 was humanized by grafting its complementarity-determining regions (CDRs) into the human variable domain germline heavy and light chain sequences with highest homology to BC8's sequences. Two variants were generated, one containing murine CDRs only and the other additionally including 2 murine residues within the non-CDR human light chain variable region and 4 murine residues within the non-CDR human heavy chain variable region. In competitive ELISA assays, CDR-grafted BC8 with murine back mutations (“HuBC8”) showed only minimally reduced binding to human CD45 compared to chimeric BC8 (ChiBC8) or murine BC8, and flow cytometrically determined binding of HuBC8 to CD45+ leukemia cell lines was similar to that of ChiBC8. For comparative in vivo testing, BC8, ChiBC8, and HuBC8 were conjugated with isothiocyanatophenethyl-ureido-closo-decaborate(2-) (B10-NCS), a boron cage molecule used for subsequent labeling with ²¹¹At as done in our early phase clinical trials with ²¹¹At-based RIT. For assessment of CD45+ cell targeting, NOD-Rag1^null IL2rɣ^null/J (NRG) mice were injected with CD45+ MOLM-13 cells in the flank to generate human AML cell flank tumors, followed by a single infusion of BC8 or HuBC8 labeled with 10 µCi of ²¹¹At. Tissues were harvested at 24 hours for analysis on a gamma counter and demonstrated similar tumor cell accumulation of radiolabeled BC8 and HuBC8. In vivo efficacy studies with ChiBC8, HuBC8, and non-binding isotype control mAbs (13R4; all human IgG4 frameworks) were performed by injection of 0.2x10⁶ luciferase-transduced MOLM-13 or ML-1 cells into tail veins of NRG mice to generate disseminated human AML. 2 days later, groups of 8 mice were treated with radiolabeled mAbs (40 µCi ²¹¹At/animal); one group did not receive any mAb. In both leukemia models, ChiBC8 and HuBC8 extended the survival of treated mice relative to 13R4 (P<0.0001), without significant difference between ChiBC8 and HuBC8 (for MOLM-13: median survival 39.5 days [13R4]) vs. 60 days [ChiBC8] vs. 59 days [HuBC8]; for ML-1: 73 days vs. 138 days vs. 114 days), demonstrating potent in vivo anti-tumor efficacy of ²¹¹At-NCS-HuBC8 RIT. Since Fc engineering to minimize Fc receptor interactions has been shown to improve RIT and its therapeutic ratio (i.e. tumor-to-normal cell targeting), we then compared ChiBC8/IgG1 with ChiBC8 using frameworks with reduced Fc binding properties (IgG4, IgG4PAA, IgG2m4, and IgG2σ). There were no significant differences in CD45+ cell targeting in NRG mice bearing MOLM-13 flank tumors. However, there were substantial differences in anti-leukemia efficacies between different antibody frameworks, with ChiBC8/IgG1 labeled with 20 µCi ²¹¹At leading to longest survival of NRG mice whereas ²¹¹At-NCS-ChiBC8 with IgG4 ProAlaAla, IgG2m4, or IgG2σ did not extend survival of mice beyond what was accomplished with ²¹¹At-NCS-13R4 (P<0.0001; median survival 28.5 days [13R4/IgG1]) vs. not reached [ChiBC8/IgG1] vs. 95.5 days [ChiBC8/IgG4] vs. 36 days [ChiBC8/IgG2m4] vs. 27.5 days [ChiBC8/IgG4PAA] vs. 25 days [ChiBC8/IgG2σ]). Increasing the specific activity further increased the anti-leukemia efficacy of ²¹¹At-NCS-HuBC8.

Conclusions:²¹¹At-NCS-HuBC8 shows similar in vivo CD45+ cell targeting properties and anti-leukemia efficacy as murine ²¹¹At-NCS-BC8. Our studies identify the antibody framework (with greatest efficacy so far observed with IgG1) and specific activity as critical factors for the efficacy of ²¹¹At-NCS-HuBC8. Together, these study support further development of HuBC8 as antibody for possible clinical RIT applications.

Sandmaier:Actinium Pharmaceuticals: Other: Attended Advisory Board Meeting; Royalty agreement with employer (Fred Hutch. King:Lassen Therapeutics: Current Employment. Walter:Jazz: Research Funding; Kura: Research Funding; Pfizer: Research Funding; VOR: Research Funding; Celgene/Bristol Myers Squibb: Research Funding; Janssen: Research Funding; Wugen, Inc.: Consultancy; Aptevo: Research Funding; Kite: Research Funding; ImmunoGen: Research Funding.