Background: Preparing humanized antibodies on the basis of mouse-derived antibodies is one
of the mainstream antibody drug technologies. Sanyou
Bio provides Magnetic & Immunization & Tandem (MIT) mouse monoclonal antibody customization service to address problems appeared from
conventional mouse-derived antibody (hybridoma monoclonal antibody) preparation
technologies, such as long preparation period, heavy screening workload, and
small amount of candidate antibodies.
Methods: MIT mouse monoclonal antibody preparation technology integrates
four technologies, namely, diversified mouse immunization, arrayed phage
antibody library construction, magnetic bead high-throughput screening, and
reconstruction and expression of CHO eukaryotic cells with comprehensive
screening methods through physicochemical and biochemical analysis.
Advantages: It takes only 3–6 weeks (starting from the end of mouse immunization) to obtain
mouse monoclonal antibodies which have good specificity, high affinity, clear
sequence and have been verified through eukaryotic expression system.
Cases: From 2016 to December 2021, Sanyou
Bio has completed hundreds of projects through this MIT mouse monoclonal antibody preparation platform. In general,
40–60 lead antibody clones with unique sequence, good specificity, and verified
affinity can be obtained for each target.
Service Overviews
Background: Preparing humanized antibodies on the basis of mouse-derived antibodies is one of the mainstream antibody drug technologies. Sanyou Bio provides Magnetic & Immunization & Tandem (MIT) mouse monoclonal antibody customization service to address problems appeared from conventional mouse-derived antibody (hybridoma monoclonal antibody) preparation technologies, such as long preparation period, heavy screening workload, and small amount of candidate antibodies.
Methods: MIT mouse monoclonal antibody preparation technology integrates four technologies, namely, diversified mouse immunization, arrayed phage antibody library construction, magnetic bead high-throughput screening, and reconstruction and expression of CHO eukaryotic cells with comprehensive screening methods through physicochemical and biochemical analysis.
Advantages: It takes only 3–6 weeks (starting from the end of mouse immunization) to obtain mouse monoclonal antibodies which have good specificity, high affinity, clear sequence and have been verified through eukaryotic expression system.
Cases: From 2016 to December 2021, Sanyou Bio has completed hundreds of projects through this MIT mouse monoclonal antibody preparation platform. In general, 40–60 lead antibody clones with unique sequence, good specificity, and verified affinity can be obtained for each target.
Service Contents
Service
Service Details
Client Provides
Deliverables and Standards
Time
Customization of MIT mouse monoclonal antibodies
Mouse immunization, immune library construction, high-throughput screening, eukaryotic expression verification
Target name, target antigen, or cell line
Deliverables:
1. Dozens of lead antibody molecules
2. Sample antibodies as needed
Delivery standards:
1. Antibody affinity ranking
2. Molecules pass affinity kinetics or FACS verification
Approximately 3.5 months
Humanization of mouse-derived antibodies
Humanization design, eukaryotic expression verification
Mouse-derived antibody sequences, target proteins and control antibodies, cell lines for identification, etc.
Humanized sequences, proteins, plasmids, assay reports
4-6 weeks
Service Highlights
1. Clear Antibody Sequence Reducing Redundant Screening
Genetic engineering and phage display technology are used to obtain the gene sequence of the antibody directly, reducing unnecessary screening.
2. Three Months Faster than Hybridoma to Speed up Drug R&D
This service takes only 2 months for library construction and screening, which is nearly 3 months faster than the screening process of hybridoma technology and can speed up the R&D progress significantly.
3. Delivery of Dozens to Hundreds of High-Affinity Lead Antibody Molecules
Dozens to hundreds of candidate molecules can be obtained for each target using an arrayed multi-library approach and a multi-species library screening strategy. In this way, the candidate antibodies have higher affinity at pM level typically.
4. Immunization through Multiple Sites\Species to Increase Species Cross-activity of Candidate Antibodies
With up to 4 mouse strains, multiple immunization approaches, species of immunogens, and screening methods, it’s easier to increase species cross-activity of antibody molecules.
5. Ensure Drug Developability through Eukaryotic Expression System and Cellular Level Verification
The candidate molecules are expressed through eukaryotic system and followed by physicochemical characteristics and cellular function testing to ensure the delivered lead antibody has good drug developability.
Case Stastics
1. Number of lead antibodies
A large number of antibody clones can be obtained from MIT antibody library. MIT antibody library was validated by screening 12 targets, and a total of 637 antibody clones with unique sequences were obtained, as shown in Fig. 2, with a median number of 55 clones obtained.
Fig. 1 Antibody number per project
2. Higher affinity of the obtained antibodies
The affinity of antibodies obtained from MIT antibody library can usually reach the pM level. The antibodies in Fig. 3 and Fig. 4 are from two different projects, and as can be seen from the figures, both projects yielded multiple molecules with affinity comparable to or significantly superior to that of the reference antibodies (Benchmark). Fig. 5 shows the affinity comparison of monoclonal antibodies from different sources. The affinity of the antibody clones (B3, B4, and C4) from the MIT library is significantly higher than that of the hybridoma.
Fig. 2 Affinity ranking of MIT-056
Fig. 3 Affinity Ranking of MIT-051
Fig. 4 Comparison of affinities (MIT vs Hybridoma)
3. Analysis of binding and blocking activity in in vitro assays
Most of the antibody molecules obtained from MIT antibody library have good cellular-level activity. Fig. 6 shows an example project using FACS to analyze the binding activity of antibodies to cell surface proteins, and Fig. 7 shows an example project using FACS to analyze the blocking activity of antibodies to the binding between receptor and ligand. As shown in the figure, all the lead antibody molecules exhibited superior binding and blocking activities to the reference antibody (Benchmark 1).
Fig. 5 Binding affinity determination by FACS
Fig. 6 Blocking activity determination by FACS
4. In vitro efficacy validation
In vitro efficacy of antibody molecules obtained from MIT antibody library is superior. In phagocytosis experiments, blocking the "don't eat me" signal by antibodies promotes phagocytosis of target cells by macrophages. As shown in Fig. 8, the phagocytosis of target cells (CCRF-CEM) by macrophages was significantly enhanced as detected by FACS after treatment with the candidate antibody (E4A), indicating that the candidate antibody has good blocking activity on the "don't eat me" signaling pathway.
Fig. 7 Phagocytosis of CCRF-CEM cells by macrophages
5. In vivo efficacy validation
In vivo efficacy of antibody molecules obtained from MIT antibody library is superior. The results of in vivo anti-tumor efficacy validation of the candidate molecules in the CDX model of NOD/SCID mice are shown in Fig. 9. For the reference and treatment groups, the drug was administered three times a week for three weeks. The results showed that the anti-tumor effect of the candidate antibody E4A was significantly superior to that of the reference antibody (Benchmark1) .
Fig. 8 Tumor growth inhibition
6. Drug Developability Parameters
After construction of full-length sequence of the molecules obtained through MIT mouse monoclonal antibody preparation technology, the expression level and physicochemical characteristics are analyzed. Table 2 shows a comprehensive drug developability assessment that covers multiple aspects such as purity and concentration determination, primary structure analysis, affinity, and affinity kinetics.
Table 1 Comprehensive drug developability assessment
Category
Test
Test method
Purity and concentration detection
Purity identification
SDS-PAGE/SEC/CE-SDS
Purity and concentration detection
Concentration identification
Protein A-HPLC/UV280
Primary structure analysis
Molecular weight analysis
LC/MS
Primary structure analysis
Isoelectric point
iCIEF
Primary structure analysis
Hydrophobicity identification
HIC-HPLC
Primary structure analysis
Charge heterogeneity determination
CEX
Primary structure analysis
Peptide mapping analysis
LC-UV-MS/MS
Primary structure analysis
N-glycan mapping analysis
LC/MS
Affinity and affinity kinetics
Affinity test
ELISA
Affinity and affinity kinetics
Affinity kinetics test
BLI/SPR
Affinity and affinity kinetics
Cellular binding assay (if any)
FACS
7. Progress of Projects of the Same Type
Table 2 . Progress of projects of the same type
Project No.
Target
Indication
Project Difficulty
Molecule Form
Source of Library
Progress
BC008
CLND18.2
Blood cancers/solid tumors
Multi-pass transmembrane protein
Bispecific antibody
Mouse+alpaca immune library
IND
SYHD001
Not disclosed
Solid tumors and blood cancers
Cross-membrane receptor tyrosine kinase
ADC
Mouse immune library
Pre-clinical
SY-14
BCMA
Blood cancers
Endocytic activity
ADC
Mouse immune library
Pre-clinical
LK001
Not disclosed
Head and neck cancers
1 AA difference between human and monkey species
Monoclonal/bispecific/ trispecific antibody
Mouse+alpaca immune library
In vitro drug efficacy
SY09
4-1BB
Solid tumors
4-1BB toxicity
Monoclonal/bispecific/ trispecific antibody
Immune library+ human source library
PCC completed
MY001
Not disclosed
Solid tumors
Cross species and blocking of 3 types of receptors
Monoclonal/bispecific/
trispecific antibody
Immune library+ human source library
Pre-PCC completed
YL001
Not disclosed
Pancreatic cancer
Growth factors
Full-length antibody
Mouse immune library
Pre-PCC completed
GY001
Not disclosed
Solid tumors
Immune inhibitors
Bi-specific antibody
Mouse library+ alpaca immune library
Pre-PCC completed
SY10
OX40
Solid tumors
Co-stimulation targets
Monoclonal/bispecific/ trispecific antibody
Mouse immune library
Pre-PCC completed