Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal expressed on cancer cells, to escape immune surveillance and macrophage-mediated clearance. Prior preclinical studies have shown that CD47 is critical to red blood cell (RBC) homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns that potential on-target anemia could result from the use of anti-CD47 agents via multiple mechanisms. Nonetheless, several clinical trials have demonstrated that magrolimab can be safely administered as monotherapy, with an initial lower “priming” dose yielding transient anemia with compensatory reticulocytosis and no anemia observed at subsequent higher maintenance doses. However, the mechanism underlying this observed protection has not been fully defined. Aims: To describe manageable anemia in patients with HR MDS treated with magrolimab in combination with azacitidine (AZA) in a phase 1 clinical trial (NCT03248479) and further investigate the underlying mechanisms in preclinical models. Methods: In a multicenter prospective study, complete blood counts (CBCs), peripheral blood, and bone marrow (BM) were collected at prespecified time points from patients with HR MDS (n=57) treated with magrolimab in combination with AZA. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for expression of CD47 on RBCs and white blood cells (WBCs). Magrolimab was initially administered as a priming dose (1 mg/kg) followed by an initial weekly maintenance dose (30 mg/kg) before transitioning to maintenance dosing every 2 weeks. AZA 75 mg/m2 was administered on days 1-7 of each 28-day cycle. Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice. Results: Combination treatment with magrolimab and AZA resulted in tolerable anemia that correlated with rapid, near-complete loss of CD47 in RBCs but not WBCs. The initial 1-mg/kg priming dose was sufficient for this CD47 loss, which persisted with subsequent 30-mg/kg maintenance doses. Both findings are consistent with prior clinical observations of magrolimab monotherapy in patients with solid tumors and magrolimab in combination with rituximab in patients with lymphoma. Our preclinical studies with mouse models revealed that CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 cross-linking between RBCs and non-RBCs. Summary/Conclusion: Overall, these results support the idea that on-target magrolimab-mediated anemia is mitigated by a near-complete loss of RBC CD47. Patients with HR MDS treated with magrolimab in combination with AZA had tolerable anemia with the use of priming and maintenance doses.
Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.
A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the lambda phage vector lambda gt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had beta-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome.
The object of the study was to determine whether alpha beta T cells can develop from hemopoietic stem cells in the absence of the thymus. C57BL/6 (Ly-5.1 and Thy-1.2) mice were thymectomized or sham-thymectomized at 4 wk of age, and received lethal whole body irradiation 2 wk later. These mice were reconstituted with an i.v. injection of 500 highly purified hemopoietic stem cells (Mac-1-, B220-, TER-119-, CD3-, CD4-, CD8-, Thy 1low, SCA-1+) obtained from the bone marrow of C57BL/6 (Ly-5.2 and Thy-1.1) donors. A similar percentage of Ly-5.2+ alpha beta T cells (donor) was found in the marrow of thymectomized recipients, sham-thymectomized recipients, and normal donor mice at least 3 mo after stem cell transplantation. The percentage of Ly-5.2+ alpha beta T cells in the spleens of sham-thymectomized and normal donor mice was similar. The percentage in the spleens of thymectomized recipients was reduced by about 50%, and approximately one-half of the latter T cells expressed the CD4-CD8- alpha beta+ phenotype. A purified population of Ly-5.2+ alpha beta- cells obtained from the marrow of thymectomized recipients was incubated in vitro for 48 h without exogenous growth factors. After the incubation procedure a proportion of the marrow cells acquired alpha beta TCR surface receptors. The results show that alpha beta T cells can develop from hemopoietic stem cells in the absence of the thymus.
Radiolabeled anti-Moloney antiserum in a paired radiolabel technique detected ≈ 5 X 10 4 Moloney antigen-specific sites/LSTRA Moloney lymphoma cell. The calculated average association constant (KA ) for this radiolabeled antibody on LSTRA cells was 7.8–20 X 107 liters/mole.
In an effort to identify cellular proteins that may be involved in the Abelson murine leukemia virus (A-MuLV) transformation process, we have isolated a hybridoma antibody (6C3) that detects a tumor-associated antigen in all A-MuLV-induced pre-B-cell lymphomas. The 6C3 antibody immunoprecipitates two molecules of Mr 160,000 and Mr 125,000 from metabolically labeled A-MuLV tumors. The two proteins recognized by the 6C3 antibody are distinct from the A-MuLV-transforming protein in that they lack viral gag determinants and are neither phosphoproteins nor protein kinases. The 6C3 proteins can be detected in all A-MuLV pre-B-cell lymphomas and some nonviral B lymphomas but are not detected on any other tumor or normal cell, including A-MuLV-transformed fibroblast lines. Thus, the 6C3 proteins may represent the products of novel cellular genes whose expression is induced, stabilized, or amplified in B-cell tumors of both viral and nonviral origin. Further evidence in support of this hypothesis is provided by the finding that 6C3 antigen expression correlates with autonomous cell growth and the transformed phenotype in both normal bone marrow cultures and those infected with A-MuLV.
We report the protein purification and the cloning and characterization of a cDNA encoding the proteins that bind with high affinity to cyclophilin C (Cyp-C) in the absence of cyclosporin A. Transfection of this cDNA into COS cells directs the production of a glycoprotein of 77 kDa that binds to Cyp-C in the absence, but not the presence, of cyclosporin A. Homology comparisons reveal that this protein and gene, termed CyCAP for Cyp-C-associated protein, possess a cysteine-rich domain (scavenger receptor cysteine-rich domain) found in a variety of cell-surface molecules; the rest of the sequence is apparently specific. This result raises the possibility that Cyp-C serves as a mediator or regulator of an as-yet-unidentified signal or cellular process initiated via the Cyp-C-associated protein.
Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.
The monoclonal antibody MEL-14 has been used in conjunction with immunohistology and multiparameter immunofluorescence to identify and characterize homing receptor-bearing thymocytes at various stages of embryonic and neonatal development. MEL-14hi thymocytes first appear at day 14 of gestation and come to represent about 40% of day 15 fetal thymocytes. Thereafter, the proportion of MEL-14hi thymocytes rapidly declines such that by birth (usually the 20th day of embryonic development) only about 2% of thymocytes are MEL-14hi. Although newborn thymocytes resemble adult thymocytes in this respect, the phenotypic characteristics of fetal and neonatal MEL-14hi thymocytes suggest that this unique subset undergoes a gradual transition from containing exclusively phenotypically immature cells in early gestation to containing predominantly phenotypically mature cells by young adulthood. Thus, virtually none of day 15 MEL-14hi fetal thymocytes are peanut agglutinin (PNA)lo, Ly-1hi, or either Lyt-2-/L3T4+ or Lyt-2+/L3T4-, whereas in the weeks that follow a steadily greater proportion of MEL-14hi thymocytes come to express this mature pattern (roughly 70% at 4 wk of age). Most day 15 MEL-14hi fetal thymocytes appear to express the functional homing receptor molecule, since day 15 fetal thymocytes bind to peripheral lymph node high endothelial venules about 40 to 50% as well as do adult mesenteric node lymphocytes, whereas adult thymocytes bind only about 5% as well. We have also identified a population of outer cortical MEL-14hi Lyt-2-/L3T4- lymphoblasts that appears during thymus regeneration 5 to 6 days after the administration of hydrocortisone. These lymphoblasts express the same phenotype as cells that constitute 40% of the day 15 fetal thymus and only 0.4% of normal adult thymocytes, implying that this particular subset may make up a significant fraction of thymocytes whenever there is a requirement for rapid expansion of the intrathymic and/or peripheral T cell pools. Taken together, these results are consistent with the notion that expression of the MEL-14-defined homing receptor may be closely linked to important intrathymic events that may occur early in T cell development and yet still have an overriding impact on the selection of those thymocytes that will serve as precursors of thymus emigrants.
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