Bispecific antibodies against HER2 and CD3
A bispecific antibody and antibody technology, applied in the direction of antibodies, antibody medical components, specific peptides, etc., can solve problems such as not identifying ligands
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Embodiment 1
[0784] Example 1 - Expression constructs for HER2 and HER2 variants
[0785] A fully codon-optimized construct was generated for expression of full-length HER2 (1255 aa, Swissprot P04626), the extracellular domain (ECD) of HER2 (Her2-ECDHis, aa1-653 with a C-terminal His6 tag), naturally occurring HER2 (Her2-delex16, derived from exon 16 deletion and lacking aa633-648), and a truncated form of the HER2 receptor (Her2-stumpy, aa648-1256). The construct contained appropriate restriction sites and an optimized Kozak sequence for cloning (Kozak, M., Gene 1999; 234(2): 187-208). The construct was cloned into the mammalian expression vector pEE13.4 (Lonza Biologics; Bebbington, C.R., et al., Biotechnology (N Y) 1992; 10(2):169-75) and fully sequenced to confirm the construct correctness.
Embodiment 2
[0786] Example 2 - Expression Constructs for Pertuzumab, C1, and F5
[0787]Complete codon-optimized constructs for the expression of the heavy chain (HC) and light chain (LC) of the IgG1 antibodies pertuzumab, C1 and F5 in HEK cells were generated. The variable regions encoded by these constructs are identical to those described for the Pertuzumab heavy and light chains in U.S. Patent No. 6,949,245 and for the C1 and F5 heavy and light chains in U.S. Patent No. 7,244,826 Those are the same. For C1 and F5, mammalian vectors p33G1f and p33K or p33L (pcDNA3.3 (Invitrogen)). For pertuzumab, mammalian expression vectors pG1f (pEE12.4 (Lonza Biologics) and pKappa (pEE6.4 (Lonza Biologics).
[0788] Trastuzumab( ) can be produced in the same manner, using the heavy and light chain sequences described in, eg, US Pat. No. 7,632,924.
[0789] The sequence disclosures of US Patent Nos. 6,949,245; 7,244,826; and 7,632,924 are hereby incorporated by reference in their entirety.
Embodiment 3
[0790] Example 3 - Transient expression in HEK-293 or CHO cells
[0791] Freestyle TM 293-F (a subclone of HEK-293 adapted to suspension growth and chemically defined Freestyle medium, (HEK-293F)) cells were obtained from Invitrogen and 293fectin (Invitrogen) was used according to the manufacturer's instructions with the appropriate plasmid DNA transfection. In the case of antibody expression, appropriate heavy and light chain expression vectors are co-expressed.
[0792] pEE13.4Her2, pEE13.4Her2-delex16, and pEE13.4Her2-stumpy were transfected into Freestyle MAX transfection reagent (Invitrogen) TM in CHO-S (Invitrogen) cell line. Expression of HER2 and Her2-delex16 was tested by FACS analysis as described below.
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