RAMIHM: An Innovative Technology Accelerating Monoclonal Antibody Development

RAMIHM: An Innovative Technology Accelerating Monoclonal Antibody Development

Monoclonal antibodies (mAbs) are essential tools in precision therapy, playing a pivotal role in fields like antiviral treatments and cancer immunotherapy. However, traditional antibody development relies on recombinant protein immunization, which is time-consuming, inefficient, and significantly limited when addressing complex targets (e.g., multi-transmembrane proteins) or rapidly mutating viral antigens. Overcoming these technical bottlenecks to rapidly discover effective antibodies remains a major challenge in biopharmaceutical research.

A study published in Cell Chemical Biology introduces a novel technology called RAMIHM (RAPID mRNA Immunization of Humanized Mice). By integrating mRNA immunization, humanized mouse models, and single-cell B-cell receptor sequencing (scBCR-seq), RAMIHM demonstrates exceptional efficiency and versatility in generating fully human monoclonal antibodies. This article provides an in-depth analysis of RAMIHM, from its technical principles to experimental results, and explores its potential in advancing antibody development.

The RAMIHM Workflow

RAMIHM is a cutting-edge antibody development platform that combines mRNA antigen delivery, humanized mouse models, and single-cell B-cell receptor sequencing (scBCR-seq) to efficiently generate fully human monoclonal antibodies. In this study, RAMIHM was successfully applied to develop antibodies against the SARS-CoV-2 Omicron variant and cancer targets, showcasing its remarkable adaptability and efficiency.

The core workflow of RAMIHM consists of three main steps:

• mRNA Antigen Delivery and Expression
• Immunization of Humanized Mice
• Single-Cell Sequencing and Antibody Screening

1. mRNA Antigen Delivery and In Vivo Expression

The first step in RAMIHM involves the design and delivery of mRNA antigens to ensure efficient in vivo expression of target antigens and activation of the immune system. This step includes the following key processes:

(1) mRNA Design and Optimization
The research team optimized the mRNA sequence encoding the target antigen (e.g., Omicron spike protein or GPRC5D multi-transmembrane protein) based on its gene of interest (GOI). The optimization included refining the 5' and 3' untranslated regions (UTRs) and poly(A) tail sequences to enhance mRNA translation efficiency and stability.

(2) mRNA Encapsulation
The synthesized mRNA was encapsulated in lipid nanoparticles (LNPs), which protect the mRNA from degradation and enable precise delivery via endocytosis.

(3) In Vivo Expression
After delivery, the mRNA is translated into the target protein within the mouse, presented in its native conformation, and triggers the immune system to produce specific antibodies.

Figure 1. Schematic of RAMIHM-Induced Humoral Immune Response and Antibody Discovery

2. Selection of Humanized Mouse Models and Immunization Design

RAMIHM utilizes genetically engineered humanized mouse models with optimized immune systems capable of generating fully human antibodies. These mice carry human immunoglobulin heavy and light chain genes, enabling them to mimic human immune responses and produce high-affinity, highly specific antibodies. This provides a solid foundation for direct clinical translation of the resulting antibodies.

In the experimental design, incremental immunization was employed to further enhance antibody generation efficiency. The mice received lipid nanoparticle-encapsulated mRNA (LNP-mRNA) injections at different doses (10 µg or 20 µg) on days 0, 2, 4, 7, and 11. This repeated incremental immunization not only significantly boosted the strength of the antibody response but also increased the diversity of the antibody repertoire. After the immunization phase, peripheral blood, spleen, and bone marrow samples were collected for subsequent screening and analysis.

3. Single-B-Cell Sequencing (scBCR-seq) and Antibody Screening

Single-B-cell sequencing is the core step of the RAMIHM platform, enabling precise analysis of B-cell antibody gene sequences for rapid and efficient antibody screening. In the study, the research team isolated memory B cells and plasma cells from the spleen, bone marrow, and peripheral blood samples of immunized mice. Using single-cell sequencing, they analyzed 3,502 B cells and successfully obtained 2,558 paired heavy and light chain sequences. Among these, 93.3% of the antibodies exhibited high specificity to the target antigen. These antibodies were then cloned and expressed in mammalian cells for functional validation.

Figure 2. Schematic of Human IgG1 Monoclonal Antibody Reconstruction, Production, and Validation

Experimental Data and Key Results

The RAMIHM technology was successfully applied in this study to develop antibodies targeting the SARS-CoV-2 Omicron subvariant and cancer-related targets, demonstrating its remarkable versatility and efficiency.

(1) Development of Neutralizing Antibodies for the Omicron Subvariant

The rapid spread and immune evasion of the Omicron variant present significant challenges to existing antibody therapies. Through mRNA immunization and single-cell B-cell sequencing (scBCR-seq), the research team quickly screened and validated several highly effective candidate antibodies. Using optimized mRNA design and delivery strategies, robust antibody responses against Omicron BA.1 and BA.5 were successfully induced in humanized mice. Key experimental results are as follows:

• Antibody Screening Efficiency

From post-immunization mouse samples, 15 antibody clones were successfully screened via single-cell sequencing, with 93.3% of these antibodies exhibiting significant antigen specificity against the Omicron RBD (receptor-binding domain).

The selected antibodies were cloned and expressed in mammalian cells for functional validation.

• Neutralization Capability

The antibody MB.02 demonstrated broad neutralization capability, with IC50 values of 0.094 µg/mL against Omicron BA.1 and 0.121 µg/mL against BA.5, indicating its effectiveness across multiple Omicron subvariants. MB.08 exhibited even greater specificity against Omicron BA.1, with an IC50 value of 0.044 µg/mL, making it the most potent candidate among those screened.

(2) Application to Complex Targets

In cancer immunotherapy, RAMIHM showcased significant advantages in addressing complex targets like the multi-transmembrane protein GPRC5D. As a multi-transmembrane protein, GPRC5D is challenging to prepare as a high-quality antigen using traditional recombinant protein immunization methods, limiting efficient antibody development. RAMIHM overcomes this limitation by enabling in vivo expression of the full-length antigen via mRNA, preserving the native conformation of GPRC5D and enhancing its immunogenicity. This provides a breakthrough solution for developing antibodies against complex targets.

Key immunization results for GPRC5D include:

Antibody Screening and Specificity : Using scBCR-seq, multiple candidate antibodies were successfully screened from the memory B cells and plasma cells of humanized mice. Among these, 90% of antibody clones demonstrated specific recognition of GPRC5D-expressing K562 cells, confirming the efficiency and precision of RAMIHM in developing antibodies for complex targets.

Antibody Diversity : Sequencing analysis revealed that antibodies targeting GPRC5D exhibited longer HCDR3 regions, indicating structural diversity and adaptability for binding complex targets. This diverse antibody repertoire enhances the coverage of complex antigens.

Experimental Validation : The selected antibodies were cloned and expressed in mammalian cells for functional validation. High-specificity antibodies exhibited strong binding capacity in binding assays, effectively recognizing GPRC5D and showing potential for clinical translation.

Core Advantages of mRNA Immunization

The RAMIHM platform achieved efficient antibody development for both the SARS-CoV-2 Omicron subvariant and complex cancer targets. Compared to traditional methods, mRNA immunization offers significant advantages in development timeline, antigen quality, and adaptability.

Significantly Shortened Development Cycle : Unlike traditional antibody development methods that rely on recombinant protein expression and purification, mRNA immunization eliminates the need for complex protein production steps. Synthesized mRNA can be directly delivered, reducing preparation time. For example, in this study, full-length Omicron spike protein was delivered via mRNA, and high-potency neutralizing antibodies were successfully screened within 14 days—a process that could take months using traditional approaches.

Higher Antigen Quality : mRNA can precisely encode complete target antigen sequences, including multi-transmembrane proteins or highly mutated viral antigens. Translation of the mRNA in cells generates antigens in their native conformations, ensuring high fidelity between the immunized antigen and the actual target. Additionally, LNPs effectively protect mRNA from degradation, ensuring its activity in vivo.

Greater Adaptability : The flexible design of mRNA sequences allows for rapid adaptation to different targets, particularly complex proteins and rapidly mutating viral strains.

mRNA Immunization vs Traditional Recombinant Protein Immunization

Summary

The RAMIHM technology, by integrating mRNA immunization, transgenic humanized mouse models, and high-throughput single-cell sequencing, not only significantly enhances antibody development efficiency but also broadens the scope of antibody discovery. Whether addressing rapidly mutating viral antigens or challenging multi-transmembrane proteins that are difficult to handle with traditional methods, RAMIHM demonstrates remarkable technical advantages. It provides an innovative solution for the rapid discovery of therapeutic antibodies.

Reference

Ren, Ping, Lei Peng, Luojia Yang, et al. "RAMIHM Generates Fully Human Monoclonal Antibodies by Rapid mRNA Immunization of Humanized Mice and BCR-Seq." *Cell Chemical Biology* 30, no. 1 (2023): 1-12. https://doi.org/10.1016/j.chembiol.2022.12.005.