Bispecific anti-LRRC15 and CD3 epsilon antibodies
A LRRC15, multi-specific technology, applied in the direction of antibodies, antibody medical components, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of no expression
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[0224] Preparation of Conjugated Compounds
[0225] Multispecific binding compounds of the invention can be prepared by methods known in the art. For example, binding compounds and antigen-binding fragments thereof can also be produced by recombinant DNA techniques by expressing the encoding nucleic acid in a suitable eukaryotic or prokaryotic host, including, for example, mammalian cells (eg, CHO cells), E. coli or yeast.
[0226] Pharmaceutical compositions, uses and methods of treatment
[0227] Another aspect of the invention provides pharmaceutical compositions comprising one or more multispecific binding compounds of the invention in admixture with a suitable pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier is, for example, but not limited to, an adjuvant, a solid carrier, water, a buffer, or other carriers used in the art to support therapeutic components, or combinations thereof.
[0228] In one embodiment, the pharmaceut...
Embodiment 1
[0245] Example 1: Library construction and phage display
[0246] A humanized / optimized library was constructed based on the following principles. Mouse CDRs will not be altered to maintain binding to human and cynomolgus CD3ε. The homology of the human framework to the mouse framework was analyzed. Diversity of framework differences based on mouse and human framework amino acids. Furthermore, 890 framework family antibodies were analyzed to preserve covariant amino acid diversity, assuming that amino acids maintained throughout framework evolution could lead to improved stability. The combinatorial diversity of VH was 1.26E7 and that of VL was 2.56E2.
[0247] This diversity is encoded in an Ultramer (IDT) that includes a designer amino acid at each position. In the splicing PCR reaction, overlapping conserved regions contribute to the construction of the V region. Full-length VL and VH segments were rescued by end-primers using high-fidelity PCR. The ScFv library ins...
Embodiment 2
[0249] Embodiment 2: vector construction
[0250] The vector pcDNA3.4TOPO (Invitrogen) was ligated to a short polylinker containing EcoRI, XhoI and NotI. The resulting plasmid was digested with EcoRI and NotI restriction enzymes and purified by gel electrophoresis. For heavy chain cloning, the prepared vector was assembled with the appropriate DNA fragment encoding the VH region and the human IgG1 AAA fragment (CH1 to CH3 domains) using the Gibson method. The variable light chain region was constructed by a similar method using gblock to assemble the VK region with a gblock fragment encoding a constant Kappa (Ck). The plasmid expressing ScFv-Fc used the gblock fragment (IDT) to encode the scFv and the PCR fragment to encode the antibody hinge to CH3 domain of IgGl. Symmetrical forms scFv-Fc, type 2 and type 5 ( image 3 , panels A, C and F) contain the IGHG1 Fc sequence with three mutations that disrupt Fcg receptor interaction and complement fixation: L234A, L235A and G2...
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