Combination therapy of antibodies against human csf-1r and antibodies against human pd-l1
a technology of csf-1r and pdl1, which is applied in the field of conjugation therapy of antibodies against human csf-1r and antibodies against human pdl1, can solve the problems of significant unmet medical needs, refractory, exhaustion or tolerance to foreign antigens, etc., and achieves efficient antiproliferative activity
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example 1
Inhibition of CSF-1-Induced CSF-1R Phosphorylation in NIH3T3-CSF-1R Recombinant Cells
[0565]4.5×103 NIH 3T3 cells, retrovirally infected with an expression vector for full-length CSF-1R, were cultured in DMEM (PAA Cat. No. E15-011), 2 mM L-glutamine (Sigma, Cat. No. G7513, 2 mM Sodium pyruvate, 1× nonessential amino acids, 10% FKS (PAA, Cat. No. A15-649) and 100 μg / ml PenStrep (Sigma, Cat. No. P4333 [10 mg / ml]) until they reached con-fluency. Thereafter cells were washed with serum-free DMEM media (PAA Cat. No.E15-011) supplemented with sodium selenite [5 ng / ml] (Sigma, Cat. No. S9133), transferrin [10 μg / ml] (Sigma, Cat. No. T8158), BSA [400 iug / ml] (Roche Diagnostics GmbH, Cat. No. 10735078), 4 mM L-glutamine (Sigma, Cat. No. G7513), 2 mM sodium pyruvate (Gibco, Cat. No. 11360), 1× nonessential amino acids (Gibco, Cat: 11140-035), 2-mercaptoethanol [0.05 mM] (Merck, Cat. No. M7522), 100 μg / ml and PenStrep (Sigma, Cat. No. P4333) and incubated in 30 μl of the same medium for 16 hour...
example 2
Growth Inhibition of NIH3T3-CSF-1R Recombinant Cells in 3D Culture Under Treatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo®-Assay)
[0567]NIH 3T3 cells, retrovirally infected with either an expression vector for full-length wildtypc CSF-1R (SEQ ID NO: 62) or mutant CSF-1R L301S Y969F (SEQ ID NO: 63), were cultured in DMEM high glucose media (PAA, Pasching, Austria) supplemented with 2 mM L-glutamine, 2 mM sodium pyruvate and non-essential amino acids and 10% fetal bovine serum (Sigma, Taufkirchen, Germany) on poly-HEMA (poly(2-hydroxyethylmethacrylate)) (Polysciences, Warrington, Pa., USA)) coated dishes to prevent adherence to the plastic surface. Cells are seeded in medium replacing serum with 5 ng / ml sodium selenite, 10 mg / ml transferrin, 400 μg / ml BSA and 0.05 mM 2-mercaptoethanol. When treated with 100 ng / ml hu CSF-1 (active 149 aa fragment of human CSF-1 (aa 33-181 of SEQ ID NO: 86); Biomol, DE, Cat. No. 60530) wtCSF-1R (expressing cells form dense spheroids that gr...
example 3
Inhibition of Human Macrophage Differentiation Under Treatment with Anti-CSF-1R Monoclonal Antibodies (CellTiterGlo®-Assay)
[0568]Human monocytes were isolated from peripheral blood using the RosetteSep™ Human Monocyte Enrichment Cocktail (StemCell Tech.—Cat. No. 15028). Enriched monocyte populations were seeded into 96 well microtiterplates (2.5×104 cells / well) in 100 μl RPMI 1640 (Gibco—Cat. No. 31870) supplemented with 10% FCS (GIBCO—Cat. No. 011-090014M), 4 mM L-glutamine (GIBCO—Cat. No. 25030) and 1× PenStrep (Roche Cat. No. 1 074 440) at 37° C. and 5% CO2 in a humidified atmosphere. When 150 ng / ml huCSF-1 was added to the medium, a clear differentiation into adherent macrophages could be observed. This differentiation could be inhibited by addition of anti-CSF-1R antibodies. Furthermore, the monocyte survival is affected and could be analyzed by CellTiterGlo® (CTG) analysis. From the concentration dependent inhibition of the survival of monocytes by antibody treatment, an IC50 ...
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