New Solution for Multi-Pass Membrane Protein Antibodies—mRNA Antigen

Explore the Future of Antibody Research with mRNA Technology

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Overview

There are approximately 6,000 membrane proteins within the human proteome, and a substantial portion of them remains unexplored as potential targets for cancer diagnosis and treatment. Monoclonal antibodies play a pivotal role in identifying cell surface targets in tumors and find utility both as therapeutic agents and in diagnostic validation. Notably, among protein drug targets, 60% are membrane proteins, encompassing diverse categories such as GPCRs, ion channels, receptors, membrane-associated enzymes, solute carriers, and transporters.

Despite the considerable promise of antibodies in targeting membrane proteins for cancer diagnosis and treatment, several challenges persist. These include difficulties in accessing epitopes, which is reflected in the limited number of approved biologics for such targets. Additionally, progress in the development of transmembrane proteins as therapeutic agents is hindered by ongoing issues such as limited expression, in vitro insolubility, unstable purification processes, and the complexities of maintaining their native conformation.

GenScript proposes the use of mRNA as a viable antigen format for antibody development targeting these membrane proteins. The following cases provide insight into our experiences with this approach, showcasing its potential in addressing the current obstacles in this field.

Antibody Services

mRNA as Novel Immunization Strategy for Membrane Protein Targets

Immunogen format	Weaknesses	Strengths	Recommended index
mRNA	Instability of mRNA, mRNA delivery	Direct translation of mRNA in the cytoplasm, spontaneous protein PTM in mammalian cells, natural conformation, no need for adjuvant	★★★★★
DNA	low expression level since DNA must cross the nuclear membrane for transcription, high cost for DNA immunization		★
Protein	High technical limitation of full length GPCRs expression and purification in vitro	High immunogenicity	★★
Peptide	Only recognize linear epitopes, not conformational epitopes, epitope omission	Low synthesis cost	★★★★
VLP	Non-specific antibody generation, little difference between positive and negative screening	Higher abundance of target antigen compared with overexpressed cells	★★★
Overexpressed cell line	Low immunogenicity due to the very low percentage of GPCR of interest on the entire membrane		★★
Nanodisc	Low success rate of GPCRs assembly with MSPs and phospholipid to form Nanodisc	Similar to natural cell membrane structure	★★★

Workflow

Case Study

Case 1: 7TM protein hCCR9

Goal: Generate mouse monoclonal antibodies specific to 7-transmembrane protein hCCR9

Immunogen: hCCR9-mRNA-LNP

Screening Material: hCCR9-overexpressing CHO-K1

Challenges: No available purified protein & failed in DNA immunization

Results: Successfully generate 9 hCCR9-positive clones and 7 validated clones (client selected)

After one round of cell fusion

19 FACS positive parental clones

9 FACS positive stable hybridoma cell line & NGS results (with varies IgG1, IgG2a, IgG2b and IgG3 isotypes antibodies identified)

7 validated clones delivered

Isotype identification of 9 hybridoma clones

Isotype	IgG1, k	IgG2a, k	IgG2b, k	IgG3, k
Clone	58D7, 69D11, 80E9	69C5	50C11, 50G8, 53B4, 56B3	77F5

FACS EC50 of 7 recombinant antibody

	50C11-1	50G8-1	53B4-1	58D7-1	69C5-1	77F5-1	80E9-1
EC50	0.7974	2.121	0.7245	0.1847	0.6506	0.4246	0.266
R square	0.9997	0.9996	0.9987	0.9922	0.9997	0.9808	0.9899

FACS Results of 7 validated hybridoma clones

Negative control cells

Positive overexpressed cells

Case 2: 4TM protein hCLD-18.2

Goal: Generate mouse monoclonal antibodies specific to 4-transmembrane protein hCLD-18.2

Immunogen: hCLD18.2-mRNA-LNP

Screening Material: hCLD18.2-overexpressing CT26

Challenges: No available purified protein & failed in DNA immunization

Results: Successfully generate 386 hCLD18.2-positive parental clones

After one round of cell fusion

386 FACS positive parental clones

90 Positive subclones for NGS sequencing and 84 subclones showing sequence-specific

83/84 Antibody recombinant expression for FACS-positive

Serum titer after CLD18.2-LNP immunization

Negative control cells

Positive overexpressed cells

FACS results of top 20 positive parental clones

Clone #	Median (+) MFI	Median (-) MFI	Median (+) MFI/ Median (-) MFI
39G6	11621	34	341.8
40C8	9621	34	283.0
40C4	9418	34	277.0
39D5	8701	34	255.9
38D9	8491	35	242.6
21E6	8727	37	235.9
11F8	7965	36	221.3
38A1	7314	34	215.1
37E3	7404	35	211.5
38F6	7051	34	207.4
40C11	7051	34	207.4
40B1	6903	34	203.0
40H8	6716	35	191.9
36E12	6635	35	189.6
38H3	6358	34	187.0
40C12	6534	35	186.7
39C1	6000	34	176.5
40H5	6055	35	173.0
36C2	5856	34	172.2
26C2	6000	36	166.7

Case 3: 7TM protein hPTX

Goal: Generate mouse monoclonal antibodies that are specific to 7-transmembrane protein hPTX

Immunogen: hPTX-mRNA-LNP

Screening Material: hPTX-overexpressing 293FT

Challenges: No available purified protein & highly PTM

Results: Successfully generate 21 hPTX-positive parental clones

After one round of cell fusion

21 FACS positive parental clones

21 Positive subclones for NGS sequencing and 84 subclones showing sequence-specific

19/21 Antibody recombinant expression for FACS-positive

Serum titer after hPTX-LNP immunization

Negative control cells

Positive overexpressed cells

FACS results of top representative positive subclones

Negative control cells

Positive overexpressed cells

Case 4: FACS performance comparison of antibody discovered through mRNA and protein immunization

Immunogen	ELISA-Positive	FACS-Positive	FACS EC50
18.2-Protein	111	36 (only 3 showed strong signal)	Shown in figure (6A6, 2D7, 4H3)
18.2-mRNA-LNP	N/A	386	Shown in figure (11F8C2, 40A2E12, 40H8H4, 15E11C3, 28B8C10)

Additional Resources

Q: How to choose various immune strategies?

A: First, select the form of the antigen based on the ease of preparing the antigen. Then, choose based on downstream applications.
Q: For mRNA-LNP immunization, how long does the immune cycle generally take? How do we evaluate the immune effect, for instance, how high can the titer of rabbit serum reach?

A: Generally, for seven-transmembrane targets with less than 85% homology, the immunization cycle is 4 weeks for mice and 6 weeks for rabbits. The evaluation is done using FACS, with rabbit serum diluted at 1:100, resulting in a (+/-) MFI of 60.
Q: MHC is likely to present different epitopes. How to select the most suitable B cell monoclonal clone?

A: The presentation of MHCII epitopes is determined by the APC cell itself. We can specify the selection scheme based on downstream applications. For instance, if the customer needs FACS-positive clones, we use overexpressing cell lines for FACS selection, picking the clones with the highest MFI value for the customer.
Q: Has GenScript compared the immune effects of different forms of mRNA (such as self-amplifying, circular, and linear mRNA)?

A: We have compared self-amplifying and linear forms and found a direct correlation between immune effects and mRNA purity. At the present stage, linear mRNA is recommended for single antibody preparation, while self-amplifying mRNA is recommended for multiple antibodies.
Q: Are there any principles for mRNA sequence design? What are the advantages of mRNA immunization compared to traditional protein?

A: The basic principles involve optimizing 5' cap, UTR, CDS, and PolyA in mRNA sequence design. The most significant advantage of mRNA immunization is that the number of FACS-positive clones obtained is much higher than with protein immunization, and their affinity is also higher.