TNT009 Prevents Erythrocyte C3 Fragment Opsonization and Rescues Reticulocytes from Destruction in Patients with Cold Agglutinin Disease

Autoantibody mediated classical complement pathway (CP) activation has been hypothesized to drive hemolytic anemia in cold agglutinin disease (CAD) patients. Red blood cell (RBC) destruction is believed to occur as a result of C3 opsonin mediated extravascular hemolysis in the liver. We recently reported that TNT009, a humanized monoclonal antibody targeting the CP specific serine protease C1s, rapidly restores hemoglobin levels in severely anemic CAD patients. Here we describe the pharmacodynamic changes in the complement profile of patients to provide a mechanistic understanding of the hematological responses and therapeutic benefit observed following TNT009 treatment.

In a Phase 1b trial, we enrolled 5 female CAD patients with severe anemia, one of whom had a lymphoplasmacytic lymphoma with >70% bone marrow infiltration and no measurable CP activity prior to dosing. This patient did not respond to TNT009 while on study and will be omitted from subsequent analyses. Patients were given an initial 10 mg/kg test dose of TNT009 on Day 1, followed by four weekly doses of 60 mg/kg on Day 2 or Day 5. Patients were followed for 4 weeks following the last dose (washout). Plasma and serum samples were collected throughout the study to measure TNT009 concentrations and to monitor serological markers of anemia and hemolysis. Additionally, futhan-containing plasma samples were collected to assess the levels of CP specific components including C1s, C1s-C1INH, and C1q by ELISA. RBCs were collected to monitor cell surface complement deposition (C3 fragments) via flow cytometry. Finally, C4 levels and an ELISA-based readout of CP activity were examined as measures of the pharmacodynamic effect of TNT009.

Baseline levels of circulating C4 were either low or undetectable in CAD patients. Accordingly, serum CP activity was reduced compared to normal human serum samples. Following the first 60 mg/kg TNT009 dose, CP activity was immediately and completely inhibited within 15 minutes of dosing in all patients and remained inhibited for 3 weeks after the last dose. During this period of inhibition, C4 levels rose from a median circulating concentration of <90 mcg/mL (range: <70 - 145) to 251 mcg/mL (range: 238 - 353; p < .001). Plasma C1s levels, on the other hand, decreased from a median plasma concentration of 53.3 mcg/mL (range: 49.4 - 60.3) to a nadir of <3.13 mcg/mL, the lower limit of quantification (LLOQ), (p < .001). Similarly, C1s-C1INH decreased from a median value of 4.5 mcg/mL (range: 4.2 - 5.3) to a nadir of <0.16 mcg/mL (LLOQ; p < .001). Notably, circulating plasma C1q levels were unaffected.

Classical pathway inhibition led to a significant increase in reticulocytes in all patients by, on average, 69% within 24 hours of dosing (p < .05). Interestingly, within 1 week after the first TNT009 dose, reticulocyte counts returned to pre-treatment levels and continued to decrease throughout the study, as expected when hemoglobin normalizes. Similarly, within 24 hours of the first dose of TNT009, bilirubin levels dropped from a median value of 2.1 mg/dL (range 1.6 - 3.8) to 0.7 mg/dL (range 0.6 - 1.2), resulting in an average reduction of 66% from baseline levels (p < .05) and returning to pre-treatment levels following washout. Finally, we monitored in vivo complement activation by staining for C3 fragment deposition on RBCs. In general, we observed a gradual reduction in the percentage of C3 fragment positive RBCs over the course of the study from a median value of 49% (range: 37 - 81) to 29% (range: 20 - 38) before washout of TNT009. The decrease in opsonized RBCs was concomitant with the rise in hemoglobin (example shown in Figure 1).

Figure 1: Elevation of hemoglobin is associated with a decrease in C3 fragment coated erythrocytes

Here we report that TNT009 administration depletes circulating C1s and immediately halts in vivo CP activity, normalizing plasma C4 levels in CAD patients. The abrupt increase in reticulocyte count within 24 hours of dosing suggests that cold agglutinin mediated complement activation affects reticulocyte survival, preventing their maturation into erythrocytes. The observed reduction in C3 opsonized RBCs suggests that TNT009 ameliorates anemia by preventing complement mediated hepatic RBC sequestration, supported by the immediate normalization of circulating bilirubin levels. These results provide a mechanistic interpretation of the therapeutic effects of TNT009 in CAD patients.

Figure 1.

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Figure 1.

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