Anti-cd228 antibodies and antibody-drug conjugates
A technology of conjugates and antibodies, applied in drug combinations, antibodies, anti-tumor drugs, etc., can solve incurable problems
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Embodiment 1
[0554] Example 1: CD288 Expression in Cancer Cell Lines
[0555] CD228 copies on the cell surface of various cancer cell lines were determined using murine CD228 mAb as primary antibody and the DAKO QiFiKit flow cytometry indirect assay as described by the manufacturer (DAKO A / S, Glostrup Municipality, Denmark) Quantification of the number and evaluation with an Attune NxT flow cytometer. The resulting numbers of expressed CD228 molecules per cell are shown in Table 1.
[0556] Table 1: CD228 molecules per cell for various cell lines
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Embodiment 2
[0559] Example 2: Immunohistochemical analysis of CD228 expression
[0560] Tumor tissue arrays were obtained from commercial sources. Formalin-fixed and paraffin-embedded (FFPE) tumor tissues were purchased from Biomax Inc, USA. All samples were placed in Bond-MaxTM Processing was performed on an automatic stainer (Leica).
[0561] Use Bond TM Deparaffinization solution (Leica, Cat# AR9222) FFPE slides sectioned on glass slides were deparaffinized and rehydrated at 72°C. Use EDTA-based Bond at 95°C-100°C TM Antigen retrieval was performed with Epitope Retrieval Solution 2 (Leica, Cat. No. AR9640) for 20 min and then incubated with primary anti-CD228 antibody (Sigma; Cat. No. HPA004880). Isotype-matched rabbit IgG1 was used as a negative control for background staining. For automated IHC staining, we used the Refine DAB kit or alkaline phosphatase-based detection kit: Bond TM Polymer AP Red Detection Kit (Leica, Cat# DS9305). Slides were incubated with primary rabbit mo...
Embodiment 3
[0569] Example 3: Anti-CD228 Antibody Drug Conjugates
[0570] Various anti-CD228 antibodies were conjugated to the drug MMAE via the linker MDpr-PEG(12)-gluc, resulting in an average drug loading of about 8 per antibody. Conjugation methods are described in US Publication No. 2018 / 0092984. Tumor cells were incubated with CD228 antibody drug conjugate (ADC) for 96-144 hours at 37°C. Human IgG ADC was used as a negative control. Cell viability was measured using Cell Titer Glo according to the manufacturer's instructions. Fluorescence signals were measured on a Fusion HT fluorescence plate reader (PerkinElmer, Waltham, MA). Data were normalized to untreated cells and x50 values were calculated using Graph Pad software. Results are reported in Table 2 as IC 50 , the concentration of compound required to produce a 50% reduction in viability compared to vehicle-treated cells (control = 100%). Chimeric L49 (cL49) and mouse (mL49) ADCs outperformed all other anti-CD228 ADCs,...
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