Reticulocyte Survival Research Articles

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 erythrocytesHere 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. [Display omitted] DisclosuresPanicker:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Parry:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Gilbert:Truenorth Therapeutics, Inc.: Employment, Equity Ownership. Jaeger:Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

Basic pharmacodynamic models for agents that alter the lifespan distribution of natural cells.

A new class of basic indirect pharmacodynamic models for agents that alter the loss of natural cells based on a lifespan concept are presented. The lifespan indirect response (LIDR) models assume that cells (R) are produced at a constant rate (k(in)), survive during a certain duration T(R), and finally are lost. The rate of cell loss is equal to the production rate but is delayed by T(R). A therapeutic agent can increase or decrease the baseline cell lifespan to a new cell lifespan, T(D), by temporally changing the proportion of cells belonging to the two modes of the lifespan distribution. Therefore, the change of lifespan at time t is described according to the Hill function, H(C(t)), with capacity (E(max)) and sensitivity (EC(50)), and the pharmacokinetic function C(t). A one-compartment cell model was examined through simulations to describe the role of pharmacokinetics, pharmacodynamics and cell properties for the cases where the drug increases (T(D) > T(R)) or decreases (T(D) < T(R)) the cell lifespan. The area under the effect curve (AUCE) and explicit solutions of LIDR models for large doses were derived. The applicability of the model was further illustrated using the effects of recombinant human erythropoietin (rHuEPO) on reticulocytes. The cases of both stimulation of the proliferation of bone marrow progenitor cells and the increase of reticulocyte lifespans were used to describe mean data from healthy subjects who received single subcutaneous doses of rHuEPO ranging from 20 to 160 kIU. rHuEPO is about 4.5-fold less potent in increasing reticulocyte survival than in stimulating the precursor production. A maximum increase of 4.1 days in the mean reticulocyte lifespan was estimated and the effect duration on the lifespan distribution was dose dependent. LIDR models share similar properties with basic indirect response models describing drug stimulation or inhibition of the response loss rate with the exception of the presence of a lag time and a dose independent peak time. The current concept can be applied to describe the pharmacodynamic effects of agents affecting survival of hematopoietic cell populations yielding realistic physiological parameters.

Reticulocytes II: Reexamination of the In Vivo Survival of Stress Reticulocytes

Very young reticulocytes are released into the circulation in response to the stress of anemia. These stress reticulocytes have shortened in vivo survival when transfused into normal recipients, and are generally considered to be abnormal because they have skipped a terminal cell division. We reevaluated one aspect of their abnormality: that of in vivo survival. Using methodology that accounted for all cells transfused, in vivo survival of both normal and stress reticulocytes was investigated in both normal and anemic recipients. The experiments demonstrate that: (1) survival of reticulocytes is normal only when normal reticulocytes are injected into nonanemic animals; (2) intrinsic properties of stress reticulocytes lead to their immediate removal from the circulation by normal recipients to a significantly greater extent than by anemic recipients; and (3) both stress and normal reticulocytes are removed at an accelerated rate over time by anemic recipients. Taken together, the data indicate that in the course of becoming anemic, an adaptation occurs that allows cells produced during anemia to circulate considerably longer in anemic animals than they could in normal nonanemic animals. Other studies disclosed that increased reticulocyte survival in anemic animals could not be attributed to reticuloendothelial overload, but is induced by adaptation of the spleen, decreasing its removal of stress reticulocytes.

Reticulocytes II: Reexamination of the in vivo survival of stress reticulocytes

Very young reticulocytes are released into the circulation in response to the stress of anemia. These stress reticulocytes have shortened in vivo survival when transfused into normal recipients, and are generally considered to be abnormal because they have skipped a terminal cell division. We reevaluated one aspect of their abnormality: that of in vivo survival. Using methodology that accounted for all cells transfused, in vivo survival of both normal and stress reticulocytes was investigated in both normal and anemic recipients. The experiments demonstrate that: (1) survival of reticulocytes is normal only when normal reticulocytes are injected into nonanemic animals; (2) intrinsic properties of stress reticulocytes lead to their immediate removal from the circulation by normal recipients to a significantly greater extent than by anemic recipients; and (3) both stress and normal reticulocytes are removed at an accelerated rate over time by anemic recipients. Taken together, the data indicate that in the course of becoming anemic, an adaptation occurs that allows cells produced during anemia to circulate considerably longer in anemic animals than they could in normal nonanemic animals. Other studies disclosed that increased reticulocyte survival in anemic animals could not be attributed to reticuloendothelial overload, but is induced by adaptation of the spleen, decreasing its removal of stress reticulocytes.

Reticulocytosis, hypochromia, and microcytosis: an unusual presentation of the preleukemic syndrome

Abstract Two patients exhibiting a highly unusual preleukemic syndrome with marked reticulocytosis, hypochromia, and microcytosis are reported. This red cell phenotype has been investigated by means of HbF, HbA2, and i-antigen activity dosages, immunofluorescence labeling of F cells, reticulocyte survival, globin chain synthesis, and electron microscopy study. The marked reticulocytosis is explained by a delayed disappearance of the reticulum. Serum iron is normal, and a thalassemic syndrome is excluded because of a balanced alpha/non-alpha globin chain synthesis. Electron microscopy studies are consistent with a defect in iron uptake by erythroid cells. All the hematologic data and investigations are similar to those observed for the Belgrade laboratory rat. It is hypothesized that the low expression of HbF and i- Ag associated with microcytosis are related to a prolongation of erythroid maturation as reflected by abnormal reticulocyte survival.

Reticulocytosis, hypochromia, and microcytosis: an unusual presentation of the preleukemic syndrome

Two patients exhibiting a highly unusual preleukemic syndrome with marked reticulocytosis, hypochromia, and microcytosis are reported. This red cell phenotype has been investigated by means of HbF, HbA2, and i-antigen activity dosages, immunofluorescence labeling of F cells, reticulocyte survival, globin chain synthesis, and electron microscopy study. The marked reticulocytosis is explained by a delayed disappearance of the reticulum. Serum iron is normal, and a thalassemic syndrome is excluded because of a balanced alpha/non-alpha globin chain synthesis. Electron microscopy studies are consistent with a defect in iron uptake by erythroid cells. All the hematologic data and investigations are similar to those observed for the Belgrade laboratory rat. It is hypothesized that the low expression of HbF and i-Ag associated with microcytosis are related to a prolongation of erythroid maturation as reflected by abnormal reticulocyte survival.

Preleukaemia and reticulocytosis: a case report with in vitro evidence for abnormal reticulocyte maturation.

A case of preleukaemia with marked reticulocytosis and a slightly reduced red cell 51Cr survival is presented. An in vitro 'reticulocyte survival' test suggested that the apparent reticulocytosis was due to delayed maturation of reticulocytes. The globin synthesis ratio suggested an abnormal ribonucleic acid. At autopsy, in addition to the findings of acute leukaemia, mesenteric lipodystrophy was discovered. This abnormality has been seen in patients with malignant diseases, especially lymphomas, but heretofore has not been reported in acute leukaemia.

Hemoglobin Abraham Lincoln, beta32 (B14) leucine leads to proline. An unstable variant producing severe hemolytic disease.

An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t(1/2) using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[(14)C]leucine indicated a t(1/2) of 7.2 days. When stroma-free hemolysates were heated at 50 degrees C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37 degrees C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant beta-chain by precipitation with p-hydroxymercuribenzoate. Purification of the beta-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for beta32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at beta31 to impair heme stabilization.

Reticulocyte Survival in Sickle Cell Anemia: Effect of Cyanate

Abstract Cyanate prevents sickling in vitro and apparently prolongs the survival of 51Cr-tagged sickle erythrocytes in vivo. Cautious interpretation is required because the effects of cyanate on 51Cr binding to sickle and fetal hemoglobin-containing red cells are unknown, and comparison of the effect of cyanate on sickle red cell survival to control red cell survival must be performed sequentially. We have studied the survival of sickle reticulocytes utilizing radioactive amino acids that are incorporated into hemoglobin. Two informed adult patients with sickle cell disease were studied. In each study, two 50-ml samples of blood were incubated separately with 14C- and 3H-leucine for 2 hr, after which 50 mM cyanate was added to one aliquot for 1 hr. The cells were then washed and reinfused. Frequent venous samples were obtained, and the specific activities of 14C and 3H in the hemoglobin were followed. The t ½ of the carbamylated cells was tripled, but remained below normal. This method provides a generally useful measurement of the influence of drugs bound to red cells on reticulocyte lifespan. The labels are incorporated into the hemoglobin molecule of the reticulocyte, and simultaneous comparison of the survivals of the same cohort of drug-treated and control cells is achieved.

Observations on the survival of circulating reticulocytes.

Summary. The survival of reticulocytes carrying HL‐A antigens is reduced in patients with circulating cytotoxic HL‐A antibodies. The survival of mature red cells is also reduced in these patients but to a lesser degree. Both mature red cells and reticulocytes survive longer in patients without circulating cytotoxic antibodies than reticulocytes (but not mature red cells) in a cytotoxic environment. The significance of these findings is discussed.