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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

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
    1. 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
    1. 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
    1. 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
    1. 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
    1. 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 Studies
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