Background：Bispecific antibodies with common light chain can reduce the mispair of light and heavy chains, and are highly consistent with natural IgG structure. Compared with various bispecific antibody formats that do not exist in human body, bispecific antibodies with common light chain have better druggability and higher safety. In order to obtain common light chain antibodies with good druggability, high affinity and high diversity, Sanyou Bio has launched a sub-trillion human recombinant common light chain antibody library screening service.
Method：Common light chain Fab-phage display libraries were constructed through the combination of fully human light chain sequences with good druggability and fully human heavy chain sequences with high diversity. The common light chain antibodies can be generated rapidly by high-throughput panning, screening, eukaryotic expression and identification.
Advantages：Common light chain antibodies targeting different epitopes of the same target or different targets can be generated in one step, and the obtained fully human common light chain antibodies can be directly used for bispecific antibody construction.
Cases：Sanyou Bio has completed the verification of dozens of projects through the human recombinant common light chain library. On Average, hundreds of leads with unique sequence and high affinity can be obtained for each target.
Preferable light chain, better druggability
The light chain with better druggability was selected from thousands of human light chain germline sequences. Expression in eukaryotic cells for validation of full-length activity and druggability.
Diverse heavy chains, more candidates
The well-chosen light chain germline was combined with ten billions humanized heavy chain sequences, which has been validated with dozens of targets, hundreds of leads can be obtained for each target averagely.
Multiple panning strategies, high quality candidates
Multiple panning strategies including cross-panning with Fc/His-tag antigen, cross-panning with solid-phase and liquid-phase antigens, cross-panning with over-expressed cell line and recombinant protein antigens, and competition or blocking panning were adopted to obtain preferable candidates efficiently.
Fully human antibody, high safety
Fully human antibody can be obtained by phage display technology directly and with high safety. It has a structure consistent with natural IgG antibodies, which increases stability and extends the half-life of bispecific antibodies.
Accurate pairing of light and heavy chains enables broad applications
Preferred light chains coupled with heavy chain KIH technology increase the yield of target antibodies without additional engineering design. It is suitable for developing bispecific, dual-target, or dual-epitope ADC antibodies for treatment or detection.
Segments of stages
1. Preferred light chains combined with diverse heavy chain sequences
As shown in Fig. 1, the light chains from IGKV3-11, IGKV3-20 and IGKV1-39 families have the highest proportion among the. light-chain germline of natural human antibodies. Moreover, the heavy chains from IGHV3, IGHV4 and IGHV1 families are the most abundant in natural human heavy-chain germline in Fig. 2. SanyouBio conducts thorough screening of thousands of light chains, ultimately selecting light chain combinations that are prevalent in nature, along with high-frequency heavy chain subtypes. They have constructed a library of over trillions of recombinant common light chains, following natural principles and exhibiting high antibody diversity
Fig. 1 Frequency distribution of VL germline
Fig. 2 Frequency distribution of VH germline
2. A large number of lead antibodies
The screening from Sanyou‘s library of trillions of recombinant common light chains yields a large number of lead molecules. As shown in Fig. 3, the common light chain library was screened and verified via 7 different targets, a total of 4942 unique clones of common light chain antibodies with distinct sequences were obtained, and the mean number of clones was 706.
Fig. 3 Antibody number per project
3. High diversity of lead antibodies
Leads from the sub-library of ST-CLC always have high diversity. As shown in Fig. 4, evolutionary tree analysis of heavy chain sequences of a representative project. The results showed that the sequences of heavy chain from each leads were significantly different, which indicated that the heavy chain sequences of the the sub-library of ST-CLC exhibit extremely high diversity.
Fig. 4 Statistics of Binder diversity
4. High antibody protein expression quantity
Fetorbio was used to detect the expression quantity of candidate antibodies(CLC) in the common light chain library and those on the market or under development(mAb). The mean expression quantity of the former and the latter was 123.9 μg/mL and 74.23 μg/mL, respectively. The results showed that candidate antibodies in the common light chain library have a greater eukaryotic protein expression quantity.
Fig. 5 Comparison of expression quantity
5. High antibody protein affinity
The binding activity of candidate antibodies from the human antibody library(hRAL) and common light chain library(CLC) was determined by ELISA. As shown in Fig. 6, 94.44% of candidate antibodies from the human antibody library had EC50 values between 0.01–0.1, while 78.95% of candidate antibodies from the common light chain library had EC50 values between 0.001–0.01. The results showed that candidate antibodies in the common light chain library have higher affinity.
Fig. 6 Comparison of antibody affinity
6. Comprehensive evaluation system for drug developability
In the panning service of the sub-library of ST-CLC, the expression level and the biochemical and physicochemical properties of candidates will be comprehensively analyzed after full-length antibody construction. As shown in Table 1, it includes the purity and concentration determination, primary structure analysis, affinity and affinity kinetics, etc.
Table 1 Drug developability of Antibody from ST-CLC
Development of anti-5T4 dual-epitope bispecific antibody
5T4 is a highly-glycosylated transmembrane glycoprotein encoded by the TPBG gene, with 8 extracellular leucine-rich repeat domains flanked by cysteine-rich domains. 5T4 is rarely expressed in normal tissues in adults, but highly expressed in most tumor cells, such as 95% of non-small cell lung cancers, kidney cancers, or pancreatic cancers. In addition, the expression level of 5T4 is directly associated with the low cure rate of colon cancers and gastric cancers.
1.1. Competition pattern
To date, there are 5 anti-5T4 antibody drugs under clinical research worldwide, among which 3 are active, including ADC developed by Asana Bio and Byondis and CD3 bispecific antibody developed by Genmab.
1.2 MOA of antibodies
5T4 and CXCR4 are co-localized at the cell membrane on tumor cells. They can promote the response to CXCL12 and activate the ERK/AKT signaling pathway, leading to tumor metastasis and diffusion. Antibodies can specifically recognize tumor cells and exert tumor killing effects by activating killer immune cells (bispecific antibodies) or conjugating toxins (ADCs).
2. Key result of epitope clustering and structural modeling
2.1. Cell-level binding activity
The majority of candidate molecules obtained through screening from Sanyou super trillion common light chain library exhibited favorable cell-level binding activity. Fig. 1 demonstrates the affinity activity of the candidate molecules on 5T4 overexpressing cells, showing that the affinity of candidate antibodies Ab1 and Ab2 is comparable to that of the control antibody.
Fig. 1 Binding affinity determination by FACS
2.2. Epitope grouping and structure simulation
BLI was used to respectively combine two preferred molecules screened from the common light chain library with BM in pairs for epitope competition. The results are shown in Fig. 2. Ab1 competed with BM to bind to the 5T4 antigen protein, while Ab2 did not compete with the other two antibodies to bind to the 5T4 target protein. The results suggested that Ab1 and BM bind to the same group of epitopes, while Ab2 binds to another group of epitopes.
Fig. 2 Epitope grouping and structural simulation
2.3. Affinity kinetics analysis of the dual-epitope bispecific antibody
The binding activity of the common light chain dual-epitope bispecific antibody (CLC01) to 5T4 antigen protein was determined by BLI. The results are shown in Fig. 3. The binding dissociation constant of the combined common light chain dual-epitope bispecific antibody CLC01 was increased to 6.42E-10. The results showed that the common light chain dual-epitope bispecific antibody has a significantly improved affinity to the antigen protein.
Fig. 3 Affinity testing of antibodies
2.4. Cell binding activity of bispecific antibodies with common light chains at dual epitopes
The affinity activity of the common light chain dual-epitope bispecific antibody (CLC01) to 5T4 positive tumor cells was determined by FACS. The results are shown in Fig. 4. Compared with monoclonal antibodies, the combined CLC01 exhibited a higher MFI upper plateau value in its binding with tumor cells, which reached 27,939. The results showed that the common light chain dual-epitope bispecific antibody exerts a notably enhanced activity to tumor cells by FACS.
Fig. 3 Affinity testing of antibodies
2.5. In vitro efficacy analysis
The internalization of the common light chain dual-epitope bispecific antibody (CLC01) was tested by Fab Zap. The results are shown in Fig. 5. The cell viability inhibition rate IC50 of CLC01 increased to 0.0016 nM, significantly superior to those of the monoclonal antibodies Ab1 (IC50: 0.0029 nM) and Ab2 (IC50: 0.0032 nM). The common light chain dual-epitope bispecific antibody shows a greatly improved internalization efficiency.