Polyclonal Anti-Kel (KELGAM) Prevents Alloimmunization to Transfused Kel RBCs in a Murine Model

Introduction: Polyclonal anti-D preparations are among the most successful prophylactic immunotherapies in existence today. The mechanism(s) of action of polyclonal anti-D are not well understood, and no monoclonal therapy developed to date has been as effective as polyclonal preparations at preventing pregnancy associated RhD sensitization. Furthermore, no prophylactic therapies exist to prevent alloimmunization to non-RhD antigens. We have recently developed a transgenic mouse model of RBC alloimmunization to KEL, with sensitization to KEL occurring through pregnancy as well as through transfusion. We hypothesized that polyclonal anti-KEL (“KELGAM”) would prevent KEL RBC alloimmunization, and herein test this hypothesis in a controlled transfusion setting.

Materials and Methods: Polyclonal anti-KEL (KELGAM) was generated in IgHa congenic mice following repeat transfusions of transgenic KEL RBCs. This polyclonal anti-KEL was then passively infused into naïve C57BL/6 (IgHb) mice, at a dose that led to maximal clearance of KEL RBCs. 50 microliters of lipophilically labeled KEL RBCs were transfused into these passively immunized mice, with evaluation by flow cytometry of anti-KEL RBC binding, complement positivity, transfused RBC clearance, and active recipient anti-KEL responses. Controls included animals transfused in the absence of passively infused anti-KEL, animals given a 3^rd party polyclonal antibody prior to KEL RBC transfusion, and animals given anti-KEL but transfused with RBCs expressing a 3^rdparty RBC antigen. 16 weeks post-transfusion, animals initially treated with or without KELGAM prior to KEL RBCs were transfused again with KEL RBCs, with responses compared to that of naïve animals.

Results: In 3 of 4 experiments (n=3-5 mice/group/experiment), mice treated with IgHa KELGAM prior to KEL RBC transfusion generated no detectable anti-KEL glycoprotein IgHb alloantibodies by 4 months post-transfusion. In contrast, 100% of control mice transfused with KEL RBCs in the absence of KELGAM or transfused with KEL RBCs in the presence of a 3^rdparty antibody generated anti-KEL glycoprotein IgHb alloantibodies (adjusted MFI >100 using sera at a 1:2 dilution). Approximately 50% of DiI labeled KEL RBCs cleared within 24 hours in the KELGAM group, whereas KEL RBCs didn’t begin clearing in the control groups until 5-7 days post-transfusion at which time the recipients began to generate anti-KEL. The DiI labeled KEL RBCs had surface bound IgG and C3b until 24 hours post-transfusion, after which time neither bound antibody, C3b, nor KEL antigen could be detected. In contrast, DiI labeled KEL RBCs in the control groups had detectable KEL antigen on their surface until 5 days post-transfusion, at which time the recipients began to generate anti-KEL. Animals initially treated with KELGAM prior to transfusion were able to generate anti-KEL upon a second exposure to KEL RBCs 4 months later, with similar responses to naïve animals seeing KEL RBCs for the first time.

Conclusions: Polyclonal anti-KEL (KELGAM) effectively eliminates primary anti-KEL glycoprotein alloantibody responses to transfused KEL RBCs. However, the mechanism of action of KELGAM may be different from that of polyclonal anti-D. The dose of KELGAM used for these studies saturates all detectable KEL antigen sites on the transfused RBCs. Distinct from what has been described with polyclonal anti-D, the transfused RBCs remaining in circulation from 24 hours after KELGAM treatment until day 28 post-transfusion modulate KEL antigen expression such that they no longer bind to KELGAM during in vitro testing. Furthermore, the addition of an excess of KELGAM does not lead to KEL RBC clearance rates beyond 50%. KELGAM transfusion recipients are not tolerized to the KEL antigen, as re-challenge with KEL RBCs months later leads to robust anti-KEL responses. Ongoing studies are further investigating whether KELGAM recipients may experience antibody mediated immune deviation, whether KELGAM is capable of suppressing responses to other antigens co-expressed on the same RBC, and whether KELGAM is effective at eliminating pregnancy induced anti-KEL alloimmunization. A goal of these studies includes the eventual translation of this knowledge to the setting of hemolytic disease of the fetus and newborn in humans.