Alternative pathway-specific antibodies for the treatment of hemolytic disease
An antibody, paroxysmal technology, applied in blood diseases, cardiovascular system diseases, antibodies, etc., can solve problems such as lack of calcium ions
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Embodiment 1
[0335] Cell Assays to Demonstrate Inhibition of AP in Subjects Using Exemplary Compounds of a Class of Antibodies of the Invention activation
[0336] To assess the ability of exemplary compounds of the invention to inhibit AP activation in an in vivo-like system, an erythrocyte hemolysis assay was used. Rabbit red blood cells (rRBC) were incubated with normal human serum (NHS) in AP enabling buffer. The presence of rRBCs ("foreign bodies") preferentially induces the activation of APs, resulting in the deposition of C5b-9 on erythrocytes and ultimately cell lysis. The extent of cell lysis was detected based on light scattering at an optical density of 700 nm. Exemplary compounds of a class of antibodies of the invention inhibit rRBC hemolysis in a dose-dependent manner, as figure 2 and 3 shown.
[0337] Rabbit red blood cells (rRBC) were introduced into 10% human serum (with Mg 2+ / EGTA) indicates the introduction of a foreign cell surface that initiates the alternat...
Embodiment 2
[0342] Antibodies of the invention do not inhibit the classical pathway
[0343] To test the activity of the antibody for CP inhibition, antibody-sensitized sheep red blood cells (sRBC) were incubated in 1% normal human serum in CP buffer (Ca 2+ / Mg 2+ ) incubate. These sRBCs activate CP, which induces cell membrane lysis. Lysis of the cell membrane results in a progressive decrease in the light scattered by the cells. When the alternative pathway specific antibody of the present invention was mixed with sRBC in 1% NHS at 37°C and contained Ca 2+ and Mg 2+ No hemolysis was observed during the time period from the onset of hemolysis to the end of maximal hemolysis when incubated in a buffered agent ("CP buffer"). This indicates that the alternative pathway specific antibodies of the invention do not affect CP hemolytic activity in NHS ( Figure 5 ), and is not expected to impair the expected contribution of CP to host defense against pathogens.
[0344] The monoclonal ...
Embodiment 3
[0348] The antibodies of the invention do not inhibit the amplification process necessary for the full potential of the classical pathway
[0349]Candidate antibodies are tested for any inhibition of any amplification process that would affect the full potential of the canonical pathway using specifically referred assays. In this test, antigen-sensitized sheep red blood cells (sRBCs) were incubated in 10% normal human serum in CP buffer (Ca 2+ / Mg 2+ ) incubate. These sRBCs activate CP through antibody-antigen binding complexes, which induce lysis of the cell membrane. Lysis of cell membranes results in progressively less light scattered by intact cells. When the alternative pathway-specific antibody of the present invention is mixed with sRBC at 37°C in the presence of Ca 2+ and Mg 2+ When incubated in 10% normal human serum (NHS) with buffer ("CP buffer"), no hemolysis was observed during the time from the onset of hemolysis to the end of maximal hemolysis ( figure ...
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