Re-directed immunotherapy
A therapeutic agent, targeted technology, applied in immunoglobulin, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the elusive clinical success, etc. question
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Embodiment 1
[0282] Example 1: Stimulation of T cells by Cetuximab-NLVMVATV (SEQ ID NO: 21) conjugate
[0283] overview
[0284] We exposed breast cancer cells to an agent containing Cetuximab conjugated to HLA-B7 peptides with and without a cleavage site. Subsequent exposure to T cells led to the generation of a T cell response when the breast cancer cells were exposed to an agent containing the cleavage site.
[0285] result
[0286] MDA.MB.231 cells, which are often used as a model of breast cancer, were transduced with the MMP14 gene to ensure the expression of MMP14 protein in the cells. Unconjugated (1) or coupled with RPHERNGFTVL (SEQ ID No:32), HLA-B7 peptide (2), coupled with NLVPMVATV (SEQ ID No:21) without MMP14 cleavage sequence (3) or with Target cells (1×10 5 ) after staining, the stained cells were incubated overnight. The next day, cells were washed, and NLV-specific T cells were added to the culture (1 × 10 4 ), and incubated overnight. Supernatants were collected...
Embodiment 2
[0288] Example 2: Stimulation of CD4 by Retuximab-DYSNTHSTRYV (SEQ ID NO:55) conjugate + T cells
[0289] overview
[0290] We exposed B-lymphoblastoid cells (B-LCL) to an agent containing Retuximab conjugated to the cytomegalovirus class II HLA-restricted peptide DYSNTHSTRYV (SEQ ID NO:55) with and without a cleavage site. Subsequently, when B-LCL cells are exposed to agents containing cleavage sites, exposure to CD4 + T cells lead to the generation of a T cell response.
[0291] result
[0292] Conjugate unrelated mismatched peptide RPHERNGFTVL (SEQ ID No:32) without protease cleavage sequence, HLA-B7 peptide (1), and irrelevant mismatched class I HLA peptide VLEEETSVML (SEQ ID No:316) with protease cleavage sequence , HLAA-A2 peptide (2), the related peptide DYSNTHSTRYV (SEQ ID No:55) (3) without the protease cleavage sequence or the related peptide DYSNTHSTRYV (SEQ ID No:55) (4) including the protease cleavage sequence, Rituximab will After staining of B-LCL cel...
Embodiment 3
[0294] Example 3: Standard Operating Procedure for Chemically Conjugating Cysteinylated Peptides and Antibodies
[0295] 1. Dissolve cysteinylated peptide in DMSO to a final concentration of 5 mg / ml.
[0296] 2. Weigh 1 mg of sulfosuccinimide 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (thio-SMCC) and dissolve in 500 μl of phosphate-buffered saline (PBS )middle.
[0297] a. Other heterobifunctional cross-linking agents such as thiosuccinimide 6-(3'-[2-pyridyl dithio]-propionamido) hexanoate ( Thio-LC-SPDP ) and N-[β-maleimide propionic acid] hydrazide, trifluoroacetate ( BMHP ).
[0298] 3. Add 20 μl antibody (1 mg / ml, 20 μg antibody) to the dissolved thio-SMCC and incubate at room temperature for 1 hour.
[0299] 4. The protein G column (GE Healthcare) was first washed by centrifuging the column at 13,000 rpm for 30 seconds to remove ethanol (storage buffer).
[0300] 5. Add 500 μl PBS and mix protein G beads well, then centrifuge at 13,000 rpm for 30 seconds. Th...
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