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A substance, CD30 technology, applied in biochemical equipment and methods, chemical equipment and methods, microorganisms, etc., can solve the elusive problems of clinical success
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Embodiment 1
[0223]Example 1: Stimulation of T cells by antibody peptide epitope conjugates (APEC)
[0224] The inventors have shown that by targeting T cell antigens to specific cell surface targets, T cell antigens can be internalized and presented on the surface of cells such that T cell responses are initiated.
[0225] figure 2 A demonstrates T cell recognition of target cells labeled with substances. Anti-CD70 antibodies were conjugated with peptide (PDDYSNTHSTRYV) or without peptide (DMSO) and used to label target cells. Cells labeled with antibodies without peptide were not recognized by T cells, as evidenced by the absence of IFN-γ in the culture supernatant after overnight incubation of target cells and T cells. Cells labeled with antibodies conjugated to immunogenic peptides were strongly recognized by T cells due to the presence of IFN-γ in the culture supernatant. These results indicate that the peptide has been released from the antibody and presented on the cell surface ...
Embodiment 2
[0236] Example 2: Standard Operating Procedure for Chemical Conjugation of Cysteinylated Peptides to Antibodies
[0237] 1. Dissolve cysteinated peptides in DMSO to a final concentration of 10 mg / ml.
[0238] 2. Weigh 1 mg of sulfosuccinimide 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) and dissolve it in 200 μl of phosphate-buffered saline (PBS )middle.
[0239] a. Other heterobifunctional crosslinkers can be used instead of sulfo-SMCC, such as sulfosuccinimide-6-(3'-[2-pyridyldithio]-propionamido)hexanoate (sulfo base-LC-SPDP) and N-[β-maleimidopropionic acid] hydrazide, trifluoroacetate (BMPH) and others.
[0240] 3. Add 50 μl of antibody (10 mg / ml, 500 μg antibody) to the dissolved sulfo-SMCC and incubate at room temperature for 30 minutes.
[0241] 4. The ZebaSpin desalting column (7 kDa molecular weight) (Thermo Fisher) was first washed by centrifuging the column at 1,500 g for 1 minute to remove ethanol (storage buffer).
[0242] 5. Add 300 μl of PBS ...
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