FDA-approved drug screening in patient-derived organoids demonstrates potential of drug repurposing for rare cystic fibrosis genotypes.

BACKGROUND: Preclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF who carry the most prevalent F508del-CFTR mutation, but a large unmet need remains to identify new treatments for all pwCF. METHODS: We used 76 PDIOs not homozygous for F508del-CFTR to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays. The most promising hits were verified in a secondary FIS screen. Based on the results of this secondary screen, we further investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators. RESULTS: In the primary screen, 30 hits were characterized that elevated CFTR function. In the secondary validation screen, 19 hits were confirmed and categorized in three main drug families: CFTR modulators, PDE4 inhibitors and tyrosine kinase inhibitors. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs where residual CFTR function is either present, or created by additional compound exposure. Additionally, upon CFTR modulator treatment we show rescue of CF genotypes that are currently not eligible for this therapy. CONCLUSION: This study exemplifies the feasibility of high-throughput compound screening using PDIOs. We show the potential of repurposing drugs for pwCF carrying non-F508del genotypes that are currently not eligible for therapies. ONE-SENTENCE SUMMARY: We screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.

Density separation of spleen cells increases fusion frequency and yield of Ig-producing hybridomas.

The efficiency of hybridoma formation and growth after cell fusion can be much improved by fractionation of the mouse splenocytes. A simple procedure is described in which splenocytes with a specific gravity of more than 1.065 g/cm3 are selected by centrifugation on a Percoll gradient. The resulting cell suspension is largely depleted of macrophages and fibroblasts while the cell viability is improved. In fusion experiments performed with these cells, overgrowth of hybridomas by macrophages, fibroblasts and P-cells is avoided. The fusion efficiency and the frequency of immunoglobulin-secreting hybridomas is increased compared with fusions carried out with unfractionated spleen cells.

Colony-forming cells in chronic granulocytic leukemia--II. Analysis of membrane markers.

Membrane markers and functional properties in vitro of blast cells from the peripheral blood of 2 patients with chronic granulocytic leukemia were studied. Buffy-coat cells were enriched for colony-forming cells by density centrifugation (d less than or equal to 1.062 g cm-3). Upon culture, a large proportion of the (cryopreserved) low-density cells from both patients formed hemopoietic colonies that were heterogeneous with respect to size and cellular composition. Expression of membrane markers on the cells, which had the morphology of undifferentiated blasts, was studied using flow cytometry with a panel of monoclonal antibodies. A striking heterogeneity was observed in that variable numbers of cells were found to express myelomonocytic, megakaryocytic and erythroid membrane markers. Antigenic properties of colony-forming cells were studied by sorting of cells with a fluorescence activated cell sorter. Low numbers of cells (10, 4 and 1, respectively) were sorted directly into the wells of Terasaki microtest plates. With this system, it was shown that myeloid colony-forming cells from patient 1 were exclusively present in HLA-DR-positive cell fractions. Colony formation from the level of a single sorted cell was documented. Sorting of cells labeled with anti-blood-group-H antibody showed that small erythroid colony-forming cells from patient 2 were blood-group-H antigen-positive. These cells did not express HLA-DR. The other colony-forming cells from this patient and essentially all colony-forming cells from patient 1 were HLA-DR-positive and blood-group-H-negative. Although only 2 patients were tested, our studies clearly demonstrate that low-density cell fractions from the blood of patients with CGL provide distinct advantages for the study of membrane properties of hemopoietic cells and of hemopoietic differentiation in general.

Constancy of cross-reactivity patterns in sera of individual mice during the anti-H-2 response.

H-2 alloimmunization evokes antibodies of differing cross-reactivity patterns among individual mice of one inbred strain. We investigated whether this is due to unknown variations at the time of bleeding or whether it remains constant for an individual mouse during long-term immunization. At various times during immunization, serum was collected from individual mice and it was tested on a panel of mouse and human lymphocytes. Sera from three donor-recipient combinations were examined. In some sera, a broadening of the cross-reactivity pattern occurred after prolonged immunization, but no major change in specificity was observed. We conclude that the differences in cross-reactivity pattern among sera from individual mice remain constant during the immunization period and do not reflect variations in the condition of the animals at the time of bleeding.

Naturally occurring cytotoxic human antibodies recognize H-2-controlled murine lymphocyte antigens.

Human sera contain cytotoxic naturally occurring (CyNa) antibodies which discriminate between lymph node cells from mice differing only at the H-2 complex. Sera from three healthy subjects (normal human sera, NH sera) and one serum from a patient with multiple sclerosis reacted with cells expressing Db, Kd, Kk, and Kp molecules, respectively. However, the following observations suggested that the binding specificity of these CyNa antibodies is to antigens that are distinct from the classical H-2 antigens: (i) the NH sera did not contain cytotoxic anti-HLA antibodies, (ii) redistribution (capping) of H-2 antigens did not induce resistance to lysis for CyNa antibodies, and (iii) individual variation was demonstrated in the expression of the murine lymphocyte antigens detected by the human CyNa antibodies. The reason for this variation appeared to be different for individual NH serum. A maternal effect influenced the expression of the murine lymphocyte antigen detected by one NH serum (anti-H-2b). The differences detected by another NH serum (anti-H-2p) appeared to be inherited, as shown by progeny testing. We hypothesize that the human CyNa antibodies may be directed against antigens controlled or modified by murine viruses (milk borne or endogenous), whose expression is under the influence of the H-2 complex, and that their production might have been stimulated by the products of human genes homologous to murine viruses.

Anti-MHC immunity detected prior to intentional alloimmunization. III. Natural autoreactive H-2-specific antibodies.

Cell fusion was performed between spleen cells from young BALB/cBy (H-2d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2 specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2Kd, Dd, and H-2s antigens, gave weak cross-reactions with H-2Kk, Dq, H-2r, and H-2v antigens and was negative with H-2b, H-2f, H-2p, and H-2Ld antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2d cells and by studies on H-2d-transfected mouse L cells, the target molecules for McAb By-1 were H-2Kd and H-2Dd molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.