Multi-specific binding conjugate, related pharmaceutical compositions and use
a multi-specific binding and conjugate technology, applied in the field of medical immunology, can solve the problems of poor pharmacokinetic and physical properties of bispecific antibodies, and difficulty in manufactur
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example 2
Other Bispecific Binding Conjugates
[0107]Other bispecific binding conjugates i.e. anti-CD16-PLGA-anti-MUC1, anti-CD56-PLGA-anti-MUC1, anti-CD16-PLGA-anti-CD19, anti-CD56-PLGA-anti-CD19, anti-CD16-PLGA-anti-CD20 and anti-CD56-PLGA-anti-CD20 were prepared in a manner similar to that described in Example 1. The antibodies used therein are all commercially available monoclonal antibodies.
[0108]In addition, anti-CD56-PLA-anti-CD19, anti-CD56-PCL-anti-CD19, anti-CD16-PLA-anti-CD20, anti-CD16-PCL-anti-CD20, anti-CD16-PLA-anti-MUC1 and anti-CD16-PCL-anti-MUC1 were also prepared in a manner similar to that described in Example 1.
example 3
of Bispecific Binding Conjugates on Killing Cancer Cells
[0109]The ability of the bispecific binding conjugates prepared in Example 1 and Example 2 to kill tumor cells was evaluated.
[0110]Specifically, 5000 target cells per well were cultured for 12 h in 96-well plates and then the initial medium was discarded. The cytokine-free X-vivo 15 medium (purchased from lonza) was used to adjust the density of NK cells (induced by peripheral blood mononuclear cells from healthy blood donors), so that number of NK cells in a volume of 100 μl reaches 4 times larger than that of the target cells (effector to target ratio is 4:1). 100 μl suspension of NK cells was added in a cancer cell culture plate, and 10 μl prepared bispecific binding conjugate (bispecific binding conjugate content: 0.2 mg, with a total amount of monoclonal antibodies 0.2 μg) was added, incubated for 8 h in an incubator, and then CCK-8 reagent was added, incubated according to the reagent instruction. The absorbance at 450 nm...
example 4
t of Inhibiting Tumor In Vitro
[0115]Anti-CD3 and / or anti-CD16 and anti-MUC1 bispecific or trispecific binding conjugates connected by PLGA (i.e. anti-CD3-PLGA-anti-MUC1, anti-CD16-PLGA-anti-MUC1 or anti-CD3 plus anti-CD16-PLGA-anti-MUC1, hereinafter referred to as CD3-MUC1 group, CD16-MUC1 group and CD3 / CD16-MUC1 group, respectively) were prepared according to the methods of the previous Examples, wherein the antibodies used were all commercially available.
[0116]The ability of the bispecific or trispecific binding conjugates to assist NK to kill A549 cells was evaluated. Specifically, 50 ml of peripheral blood of healthy human was collected, monocytes (PBMC) were isolated, and then NK cytokines were added for induction and expansion culture. The percentage of NK cells was measured by culturing cells until day 21. The results were as follows: the ratio of NK cells was 86.9%, and the ratio of CD3+ T cells was 10.2% (see FIG. 8). 5000 A549 cells as target cells were cultured in each we...
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