Background:

Target B is a tumor-associated transmembrane protein, which is generally overexpressed in 75% of cancers such as pancreatic, breast, ovarian, and lung tumors. It is evidenced that target B displays significant correlation with increased propensity of cancer metastasis and poor prognosis in clinic. To date, there already exist many small molecular drugs against this particular target. However, some intrinsic characteristics of small molecules (i.e. poor drug-like properties, unsatisfactory pharmacokinetic behaviors, DDI, etc.) still remain as a problem. However, monoclonal antibody therapies provide a promising alternative to address these challenges.

Case design:

➢ Immunogen design & preparation

Since target B is a membrane protein with complicated structures and short extracellular loops, overexpressing cell lines or membrane fractions derived thereof are often used for immunization. However, there is a disadvantage that the unpurified immunogens can raise immune responses targeting other protein impurities. Besides, membrane fractions lose their out-side-out orientation in vivo, causing immune responses against intracellular epitopes as well. To minimize these off-target responses, we use side-by-side DNA immunization as well.

➢ Immunization scheme design & conduction

For the generation of a target B-specific immune response, three CAMouse™ models were immunized. DNA injections were repeated a total of four times with 2-week interval. In order to increase the potency, a special adjuvant was used.

Pre-immunization and post-immunization sera were collected from three mice to evaluate the immune response by titration ELISA. As shown in table 1, good antiserum titers were observed in all three CAMouse™ models, among which Mouse 3 reached 1/102,400. We used Mouse 3 for phage display immune library construction. Moreover, another 5-10 CAMouse™ of different strains/genetic backgrounds can serve as backup plans in case initial immunization outcome failed to meet the requirements.

Table 1. Titering results

[Green]: Titer
a. Blank: PBS
b. Negative: pre-immunized sera (1:1000)
c. Coating: target B in VLP format
d. Primary antibody: antisera
e. Secondary antibody: HRP-goat anti-mouse IgG (Fcr)

➢ Immune library construction

Mouse 3 was sacrificed, and its spleen was collected and subjected to RNA isolation. The purity, concentration, and QC of the total RNA were tested before amplifying antibody genes. Subsequently, PCR was conducted to amplify the V_H, V_λ, and V_κ genes, respectively. And then, the scFv cassette was assembled by over-lapping PCR, and packaged into phagemid vectors.
Comprehensive QC validations were conducted to check the library quality. As shown in Figure 1, sequencing analysis was performed in 20 random clones picked from the library. It is suggested that all 20 clones carried scFv insertion, and 28 of them are unique ones. By estimation, the constructed library has an adequate size of 3.5×10⁸.

➢ Library screening

Multiple rounds of biopanning were performed against target B to isolate high-specific, high-affinity binders. In this case, we conducted 5 rounds of biopanning to successively remove low affinity and nonspecifically binder phages. As shown in table 2, the percentage of phage yield increased from 7.50×10^-5 to 1.80×10^-2, representing good enrichment of ~200 fold.

Table 2. Selective enrichment of phage

By the end, 40 clones were picked up and validated via phage ELISA (table 3). All 40 clones displayed positive binding activities towards target B.

Table 3. ELISA validation of 40 clones

13 Clones: Only bind to the positive panning target B
27 Clones: Bind to both the positive panning target B and control protein

➢ Antibody DNA sequencing

Afterwards, antibody sequences will be obtained via hybridoma mRNA sequencing. Our robust sequencing platform can reveal full-length antibody sequences in 2-3 weeks. With sequence analysis, we can sort out the unique clones and proceed to recombinant expression stage. Meanwhile, positive hybridoma clones will be frozen for long-term storage.

➢ Soluble expression of scFv

The 12 candidates identified were cloned into the soluble expression vector. With rich experience in recombinant antibody expression (full-length, sdFv, Fab), our team can also engraft antibody variable regions with desired Fc portion of different isotypes (IgG1-4, IgM, etc.) and hosts (human, mouse, rabbit, etc.). Soluble scFv of the 12 candidates were successfully expressed in E. coli. According to ELISA results shown in table 4, 11 antibodies showed higher binding activity to target B compared with control protein.

Table 4. QC soluble scFv ELISA

➢ Antibody purification

For downstream in vitro / in vivo studies, scaling-up production of antibody candidates with high purity would be needed. We are professional in recombinant antibody expression (scFv/Fab/IgG) using a variety of systems. Both transient and stable transfection are available. Of note, our comprehensive cell bank provides many featured cell lines for your choice. We also offer GMP-grade manufacture strain establishment service upon request.

Conclusions:

In this case, we tailored a one-stop work plan for phage display library construction and screening using transgenic CAMouse™ models, and successfully discovered 12 good binders. Equipped with seasoned expert teams and world-leading tech platform, we conducted the all-round antibody production campaign from upstream immunogen design to the downstream antibody production.

Please note: we only produce antibodies for research purposes. Our products and services cannot be used directly on humans.