The rabbit (Oryctolagus cuniculus) is an important species worldwide used as a laboratory animal and model for larger species [1], [2]. Owing to their specific physiological characteristics similar to humans, rabbits have become indispensable models in biomedical research, widely utilized in vaccine development, as well as studies on infectious diseases, ophthalmic disorders [3], and reproductive physiology [4]. Notably, antibody production represents the most prevalent application of rabbits in biomedical research [5]. Rabbits possess approximately 50 times larger spleens than those of mice, exhibit a moderate body size, and are amenable to farming and breeding, making them ideal candidates for antibody production [6], [7]. While most monoclonal antibodies (mAbs) are currently derived from mice [8], [9], the unique immune system of the rabbit endows rabbit monoclonal antibodies (RabMAbs) with increasing value in biomedical research, clinical diagnostics, and therapeutic applications [10], [11]. Unlike most monoclonal antibodies with dissociation constants (Kd) at the nanomolar or sub-nanomolar level (10−7-10−9M), RabMAbs typically exhibit picomolar-level(10−10-10−12M) Kd values, demonstrating their significantly higher antigen-binding affinity (Table 1) [12], [13]. Currently, the U.S. Food and Drug Administration (FDA) has approved the use of several therapeutic rabbit-derived monoclonal antibodies, with information on the corresponding antibody drugs provided in Table 2.

Compared with mouse monoclonal antibodies, RabMAbs not only exhibit greater affinity and specificity but also show superior overall performance [22], [23]. First, while the primary antibody production mechanism of rabbit shares similarities with that in humans and mice, rabbits also possess an additional somatic gene conversion (SGC) mechanism during the secondary antibody maturation stage, in addition to somatic hypermutation (SHM) [24], [25]. This dual mechanism enables certain RabMAbs to recognize antigenic epitopes inaccessible to murine mAbs and to mount robust immune responses against small molecules and semi-antigens (haptens), thereby expanding the diversity of the rabbit antibody repertoire [6], [22]. Bystryn et al. compared the melanoma-associated antigens (MAAs) on the cell surface recognized by rabbit antibodies and murine antibodies. They found that certain human cell-surface MAAs identifiable by rabbit anti-melanoma sera could not be detected by murine monoclonal antibodies. This result demonstrates a difference in the MAAs recognized by rabbit and murine antibodies, and further implies that the immunogenicity of an antigen in humans may not necessarily be immunogeneic in mice [26]. Second, the simple and stable structure of rabbit IgG confers a considerable advantage for reagent storage and experimental result consistency [27], [28]. These characteristics render RabMAbs a highly promising candidate for antibody engineering (Fig. 1).

Currently, the production scale and application scope of RabMAbs remain less extensive than those of mouse monoclonal antibodies, primarily due to dual constraints from patents and screening technologies [29]. On one hand, key patents related to rabbit hybridoma techniques and genetic engineering methods are predominantly held by a few companies or institutions (e.g., Epitomics), resulting in high barriers to access and utilization [30]. On the other hand, rabbit B cells exhibit low in vitro fusion efficiency, and hybridomas tend to be unstable, limiting the success rate of antibody screening. Although phage display technology has been applied to RabMAb screening, it entails high technical requirements for library construction and screening, requiring library amplification and multiple rounds of panning, which renders the process time-consuming [31].

In recent years, single B-cell technology has emerged as a pivotal tool in screening rabbit monoclonal antibodies [32], [33]. By strategically circumventing the reliance on myeloma cells and navigating around cell fusion-related patent constraints, this approach enables high-throughput analysis and sorting of primary B cells in a short time, thereby significantly enhancing the efficiency and throughput of antibody screening [33], [34], [35]. Single B-cell screening methodologies generally diverge into two paradigms: (1) targeting clone identification via the B-cell receptor (BCR) profiling, which requires in vitro culture and differentiation of B cells [36], [37], and (2) directly selecting antibody-secreting plasma cells, a state-of-the-art strategy where early-stage plasma cell enrichment serves as a critical step [38]. In humans and mice, this enrichment depends on conserved surface markers (e.g., CD138 [39], CD38 [40]). However, due to the absence of specific surface markers on rabbit plasma cells, obtaining a sufficient quantity of plasma cells necessitates first enriching memory B cells via BCR, then placing them in an in vitro culture system to induce differentiation into plasma cells for subsequent single-cell screening (Table 3). Notably, this workflow not only increases material and labor costs but also yields plasma cells of low quantity and quality compared to those directly sorted from the blood. Therefore, regardless of the technical path used, a deeper understanding of rabbit B cell development remains fundamental as it directly influences the success rate of obtaining high-performance antibodies.

This review synthesizes the unique B cell development and antibody production mechanisms in rabbits, particularly focusing on the dynamic changes of the surface markers, as well as key events during rabbit antibody development, thus underpinning the inherent advantages of rabbit monoclonal antibodies. These insights provide a theoretical foundation for optimizing rabbit single-B antibody screening. Building on this foundation, we discuss the technologies for developing rabbit monoclonal antibodies (mAbs), exploring how to further enhance the screening and identification of rabbit mAbs. The overarching goal is to offer a reference framework for establishing a high-throughput, high-precision rabbit mAb screening platform, thereby facilitating the iterative upgrade of rabbit mAb screening technology, expanding production scales, and broadening their application fields.