Bispecific heterodimeric diabodies and uses thereof
A bispecific, heterodimer technology, applied in the direction of antibodies, applications, specific peptides, etc., can solve problems such as poor stability characteristics, limited ineffective generation, etc.
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Embodiment 1
[0261] Generation of P-cadherin and bispecific heterodimeric diabodies
[0262] Bispecific heterodimeric diabodies were constructed to evaluate the recombinant production, purification and binding characteristics of each. Affinity-purified bispecific heterodimeric diabodies were produced by the recombinant expression system described herein. ELISA and SPR analysis also revealed that the covalent bispecific heterodimeric diabody had affinity for two target antigens, P-cadherin and CD-3, and was able to bind both antigens simultaneously.
[0263] A. Phage display
[0264] A phage display library consisting of scFv derived from non-immunized human donors was used to identify single chain fragment variable (scFv) portions that bind the extracellular domain (ECD) of P-cadherin using techniques known in the art. Binding of scFv expressed on the surface of phage was measured by standard ELISA techniques on P-Cadherin constructs and P-Cadherin expressing cells.
[0265] B. Sequ...
Embodiment 2
[0302] Construction of anti-P-cadherin / anti-human CD3 LP-DART
[0303] Anti-P-cadherin / anti-human CD3 DART proteins were amplified by PCR such that each DART protein contained restriction enzyme cloning sites at each end for cloning into mammalian expression vectors, see Table 11 and Figure 33A and 33B .
[0304] Table 11. Anti-P-Cadherin / anti-human CD3 DART proteins.
[0305]
[0306] Subsequently, a nucleic acid encoding a DART protein sequence is fused to each nucleic acid encoding a modified Fc construct such as a "button" Fc chain (SEQ ID NO: 63) and a "button" Fc chain (SEQ ID NO: 64), which Each is linked to the C-terminus of the DART with a cysteine linker such as GCPPCP (SEQ ID NO: 73), GGTGGCPPCP (SEQ ID NO 74), GEPKSSDKTHTCPPCP (SEQ ID NO 75) and GGTGGGEPKSSDKTHTCPPCP (SEQ ID NO 76).
[0307] After PCR amplification, DART and Fc chain nucleic acids were digested with BspEI and HindIII (5' DART cloning site) or EcoRI (3' Fc cloning site). Digested DNA was i...
Embodiment 3
[0309] Binding properties of P-cadherin scFv-Fc and DART proteins
[0310] P-cadherin scFv-Fc constructs (P-cadherin VH and VL fused to the Fc region of human IgG) and DART proteins were analyzed with P-cadherin constructs and with expression of P-cadherin using standard ELISA techniques. Cell-binding properties of cohesins. The data in Table 12 indicate that the phage-derived P-Cadherin scFv-Fc constructs bind strongly to recombinant human P-Cadherin and have no detectable binding to mouse P-Cadherin.
[0311] Table 12. Binding properties of P-cadherin scFv-Fc to P-cadherin protein constructs.
[0312] P-CAD Clone# huP-cad-ECD EC50 (nM) huP-cad-ECD Biotinylated EC50(nM) muP-cad-ECD EC50 (nM) 33 scFv-Fc 3.464 0.1012 NB 34 scFv-Fc 11.16 0.1943 NB 35 scFv-Fc 2.5 0.01762 NB PF03732010 1.936 0.0188 2.333 negative control NB NB NB
[0313] NB = no detectable binding.
[0314] The binding properties of P-cadherin s...
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