Multivalent immunoglobulin-based bioactive assemblies
An antibody and specific technology, applied in the field of multivalent biologically active assemblies, can solve problems such as instability, reduced scFv binding affinity, and impure product form.
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[0350] Preparation of Antibody Fragments
[0351] Certain embodiments of the claimed methods and / or compositions may involve antibody fragments. Such antibody fragments can be obtained by pepsin or papain digestion of full-length antibodies using conventional methods. For example, antibody fragments can be prepared by cleavage of antibodies with pepsin to provide F(ab') 2 fragment. This fragment can be further cleaved using a sulfhydryl reducing agent, and optionally a blocking group for the sulfhydryls generated by disulfide bond cleavage, to produce Fab' monovalent fragments. Alternatively, enzymatic digestion using papain yields two monovalent Fab fragments and one Fc fragment. See US Patent No. 4,036,945; US Patent No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys., 89:230; Porter, 1959, Biochem. J., 73:119; et al., 1967, METHODS IN ENZYMOLOGY, p. 422 (Academic Press), and Coligan et al., eds., 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).
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Embodiment 1
[0390] Example 1. General strategy for making modular Fab subunits
[0391]Fab modules can be prepared as fusion proteins containing DDD or AD sequences. Design independent transgenic cell lines for each Fab fusion protein. When prepared, the modules can be purified or kept in the supernatant of the cell culture, if desired. After preparation, (Fab-DDD) 2 Any combination of modules and Fab-AD modules can be used to generate bispecific trivalent Fab (bsTF).
[0392] The plasmid vector pdHL2 has been used to generate a variety of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6. A bicistronic mammalian expression vector mediates the synthesis of IgG heavy and light chains. The vector sequences of many different IgG-pdHL2 constructs are mostly identical, only in the variable region (V H and V L ) sequences are different. These IgG expression vectors can be converted into Fa...
Embodiment 2
[0437] Embodiment 2: expression vector
[0438] Construction of h679-Fd-AD1-pdHL2
[0439]h679-Fd-AD1-pdHL2 is an expression vector used to make h679Fab, in which AD1 is conjugated to the carboxy-terminus of the CH1 domain of Fd through a flexible Gly / Ser peptide spacer consisting of 14 amino acid residues. By replacing the SacII / EagI fragment of the pdHL2-based vector containing the variable region of h679 with the CH1-AD1 fragment (cut out from the CH1-AD1-SV3 shuttle vector by SacII and EagI), the vector was transformed into h679-Fd- AD1-pdHL2.
[0440] Construction of C-DDD1-Fd-hMN-14-pdHL2
[0441] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for making a stable dimer comprising two copies of the fusion protein C-DDD1-Fab-hMN-14 in which DDD1 is A peptide spacer was linked to the CH1 carboxyl terminus of hMN-14 Fab. Plasmid vector hMN14(I)-pdHL2 (which had been used to make hMN-14IgG) was digested with SacII and EagI restriction endonucleases to remove the CH1-CH3 d...
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