RESUMO
Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.
RESUMO
Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.
RESUMO
OBJECTIVE: To explore the relationship between clinical features, peripheral blood cell count, coagulation function, gene mutation and hemorrhagic events and thrombotic events in essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis(PMF) patients. METHODS: Clinical data of 78 patients with ET, PV, and PMF who were admitted to the Second Affiliated Hospital of Chongqing Medical University between September 2019 and August 2020 were retrospectively analyzed. Information about sex, age, gene mutation, peripheral blood cell count, coagulation function, and hemorrhagic and thrombotic events was included, and the influence of these data on the occurrence of hemorrhagic and thrombotic events was estimated. RESULTS: Among the 78 patients with myeloproliferative neoplasms, there were 47 cases of ET, 15 cases of PV, and 16 cases of PMF.A total of 10 patients (12.82%) experienced hemorrhagic events and 27 (34.62%) experienced thrombotic events. Male,patients aged ≥ 60 years, and patients with a JAK2V617F mutation were more likely to experience thrombotic events (P<0.05). Patients with thrombotic events had higher platelet (PLT) counts and fibrinogen (FIB) levels than patients without hemorrhagic-thrombotic events (P<0.05).White blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) level, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and international normalized ratio (INR) showed no statistical difference between patients with thrombotic events and patients without hemorrhagic-thrombotic events (P>0.05). There was also no significant difference in the above-mentioned indexes between patients with hemorrhagic events and patients without hemorrhagic-thrombotic events (P>0.05). Among JAK2V617F positive myeloproliferative neoplasm patients, male patients were more likely to have thrombotic events (P<0.05), and patients with thrombotic events had higher platelet counts than those without hemorrhagic-thrombotic events (P<0.05). There was no significant difference in age, white blood cell count, red blood cell count, hemoglobin level, PT, APTT, FIB, TT or INR between patients with thrombotic events and patients without hemorrhagic-thrombotic events (P>0.05). CONCLUSION: Sex, age, JAK2V617F mutation and platelet count have a certain value for predicting thrombosis in patients with myeloproliferative neoplasms.
