Mechanically interlocking complexes
A technology of mechanical interlocking and complex, applied in the direction of carrier-antigen complex structure, fusion polypeptide, animal/human protein, etc.
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[0134] For the preparation of suitable antibodies of the invention and for use in accordance with the invention, e.g., recombinant, monoclonal or polyclonal antibodies, a number of techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975) ; Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pages 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991) ; Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd Ed. 1986)). Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas, and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be prepared from hybridomas or plasma cells. Random combinations of hea...
Embodiment 1
[0303] Based on the unique peptide binding site found in the Fab arm of cetuximab, it was sought to develop non-covalent pathways to efficiently functionalize monoclonal antibodies (mAbs). To enhance the affinity of the peptide-Fab interaction, structural and biophysical approaches were used to introduce unnatural amino acids in the peptide and generate specific mutations within the Fab and achieve high-affinity peptide-Fab complexes. Based on the structure of the high affinity complex, it was observed that the guanidinium nitrogen (NH1 / 2) of arginine 8 in the peptide is accessible from the opposite side of the Fab. Peptides were prepared with arginine 8 modified to include a short polyethylene linker and terminal azide. The modified peptide was allowed to bind to the Fab and an interlocking mechanical junction was created using copper-free click chemistry.
[0304] Using copper-free click chemistry, Alexa fluor 647 was locked to trastuzumab, resulting in non-covalent mechani...
Embodiment 2
[0314] Two interlocked drug conjugates are produced.
[0315] Auristatin and mertansine will be individually conjugated to the free lysine of the 8-azido-meditope, the conjugates will be interlocked to the meditope-permissive trastuzumab, and will be characterized using BT474 and SKBR3 Cell growth inhibition / death by interlocking cytotoxins of cell lines. Cell growth inhibition will be compared directly to T-DM1 (trastuzumab emtansine). Next, tumor growth inhibition in animals will be tested.
[0316] Generation of interlocked DOTA for imaging and radioimmunotherapy.
[0317] DOTA will be conjugated directly to DIBO (dibenzocyclooctyne) and DOTA-DIBO will be interlocked via the 8-azido-mesitope to the meditope-permissive anti-CEA mAb. DOTA will be loaded with 64Cu and used to image LS-174T xenografts in immunodeficient mice or MC38.CEA syngeneic tumors in C57Bl.CEA transgenic mice. These images will be compared to anti-CEA directly conjugated to DOTA. Success here would a...
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Abstract
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