In the process of developing therapeutic antibodies, recombinant proteins play an extremely important bridging role. Whether in early antibody discovery, affinity optimization, functional validation, or later pharmacokinetics/toxicology studies, suitable target proteins or antigen proteins are indispensable. Especially when choosing different species sources (human/mouse/monkey/dog), recombinant proteins can be used for cross-species homology verification, cross-reactivity testing, and mechanism-of-action studies in animal models. Proteins derived from these species not only support the full pipeline from animal immunization to humanized antibody design, but also play an irreplaceable role in efficacy and safety evaluation [1]. As the antibody drug market rapidly expands and technology platforms continually improve, the importance of recombinant proteins from different species in antibody discovery and optimization has received increasing attention [2].
1. Types of Recombinant Proteins and Expression Systems
Recombinant proteins come in various forms, commonly including: full-length proteins or their extracellular domains (ECD), fusion proteins, antigen fragments or peptides, and membrane protein reconstructs (e.g., as nanodiscs or vesicles). In antibody drug development, the choice of form often depends on the antigen’s structural characteristics, whether binding assays (such as SPR or BLI) are needed, and the requirements for subsequent animal models.
For expression systems, common options include mammalian cells (e.g., CHO, HEK293), insect cell systems, yeast systems (e.g., Pichia), and prokaryotic systems (e.g., E. coli). The correct choice of expression system strongly influences downstream antibody binding performance, stability, and immunogenicity. If one needs to closely mimic human glycosylation or conformation, mammalian systems are preferred because they can produce more natural glycosylation, folding, and activity. However, their drawbacks are higher cost and sometimes lower yield, especially for multi-pass membrane proteins – a major reason why many multi-pass drug targets (like GPCRs or ion channels) remain challenging [3].
2. Specific Roles of Recombinant Proteins from Different Species (Human / Mouse / Monkey / Dog) in Antibody Development
2.1 Human Recombinant Proteins: The “Core Standard” for Antibody Discovery
Human recombinant proteins are the central reference standard in antibody drug development. Since most therapeutic antibodies ultimately target human proteins, high-purity, correctly folded human proteins are required throughout both the early immunization and later screening stages to ensure that selected antibodies have clinical relevance.
Main applications include:
- Immunization and screening: During immunization in animals such as mice, rabbits, or llamas, human recombinant proteins are often used as antigens to generate antibodies specific to the human target.
- Affinity and specificity assays: In binding experiments (ELISA, BLI, SPR), human proteins serve as antigen targets to confirm whether candidate antibodies have high affinity toward the human target.
- Cell functional validation: In human cell-based models, human recombinant proteins can be used as control stimulants or ligands to verify antibody function (e.g., receptor blocking, pathway inhibition).
Human recombinant proteins expressed in mammalian cell systems (such as HEK293 or CHO) are especially useful because they better preserve natural conformation and glycosylation, thereby improving the precision of antibody screening.
2.2 Mouse Recombinant Proteins: The Basis for Immunization Modeling and Antibody Screening
Mouse-derived recombinant proteins play multiple roles in antibody development. Since mice are traditional immunization hosts, mouse proteins can be used for immunization, hybridoma generation, and cross-reactivity detection in early in vivo models. If an antibody cannot bind the mouse target, it may be ineffective in mouse models. However, one must be careful: mouse proteins can differ significantly from human proteins in sequence, structure, and glycosylation, which may lead to antibodies that work in mice but fail in humans, or vice versa. Previous studies have noted that “species cross-reactivity” is a critical factor [4].
Key applications:
- Immunogenicity studies: When human proteins are used for immunization, researchers often check whether the resulting antibodies cross-react with the mouse homolog to assess species differences.
- In vivo efficacy models: Many antibody drugs are first validated in mouse disease models (e.g., tumor suppression or immune modulation), so it’s critical to ensure that the antibody can recognize the mouse target, or to design a version that binds the mouse protein.
- Comparative functional research: Mouse proteins are often used to explore whether a target’s function is conserved across species; for example, to verify if a human antibody only recognizes human protein or also cross-reacts with mouse.
By using both human and mouse recombinant proteins early in the screening stage, researchers can optimize immunization strategies, evaluate cross-species specificity, and provide structural guidance for humanization.
2.3 Monkey Recombinant Proteins: Key for Pharmacology and Cross-Reactivity Studies
Monkey-derived recombinant proteins (especially from cynomolgus monkeys and rhesus monkeys) are highly valuable in antibody drug development because of their high similarity to human proteins in sequence and receptor structure. These non-human primates are widely used for preclinical evaluation.
Major uses:
- Cross-reactivity testing: Before entering preclinical studies, many antibodies need to be validated for binding to non-human primate targets. Monkey proteins help assess whether the antibody can bind to the monkey homolog – which determines whether toxicity or efficacy studies are feasible in that species.
- Pharmacology studies: If the antibody binds the monkey target, pharmacology models can be established in monkeys to simulate human-like responses, measuring parameters like blood drug concentration and target occupancy.
- Safety assessment: For highly specific antibodies, binding data to monkey proteins form the basis of safety evaluation, helping decide whether GLP (Good Laboratory Practice) toxicology studies in monkeys are justified.
Thus, monkey proteins are often used alongside human proteins to confirm cross-species binding, bridging early screening to non-clinical GLP toxicology studies.
2.4 Dog (Canine) Recombinant Proteins: Emerging Preclinical Research Models
Although dogs are less commonly used than monkeys in antibody development, they are increasingly employed in tumor immunology, inflammatory disease, and orphan disease research. Canine recombinant proteins are mainly used in:
- Spontaneous disease models: Dogs naturally develop tumors whose pathology and immune environments closely resemble human cancers, so dog target proteins support translational research.
- Safety and efficacy supplemental studies: When monkey models are not suitable or ethical constraints are high, canine models can serve as auxiliary species for pharmacokinetics and tissue-distribution studies.
- Cross-species immunology research: In veterinary therapeutic antibody development, canine proteins are crucial antigens for creating monoclonal antibodies against canine diseases (e.g., immunoinflammation or tumors).
Using dog-derived proteins expands the species reach of antibody development, enabling evaluation under more physiologically relevant disease conditions. This cross-species strategy (cross-species protein validation) improves the success rate of antibody screening and helps early identification of risks, thereby reducing costs of later failure.
In a words, in the full pipeline of human antibody drug development, combining recombinant proteins from different species builds critical “bridges” from animal immunization to clinical translation: Human proteins define the final therapeutic target and ensure clinical relevance; mouse proteins support immunization strategies and early in vivo screening; monkey proteins enable non-human primate pharmacology and safety studies; dog proteins offer complementary models for translational or veterinary research.
With advancements in expression systems (especially CHO, HEK293) and structural optimization, future antibody R&D will increasingly rely on high-homology, multi-species recombinant protein resources – achieving true continuity from animal models to clinical applications.
About DIMA Biotech: DIMA BIOTECH is a biotech company focusing on preclinical R&D products and services for druggable targets. They have used their mammalian cell expression platform to produce over 1,600 ECD (extracellular domain) recombinant proteins and more than 900 full-length membrane proteins, covering major target families (e.g., GPCRs, ion channels) in species such as human, mouse, monkey, and dog.
Reference:
[1] Antibody therapeutics by the numbers 2024. Nature Reviews Drug Discovery, 23(1): 1–4.
[2] Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020).
[3] Stephens, A.D., Wilkinson, T. Discovery of Therapeutic Antibodies Targeting Complex Multi-Spanning Membrane Proteins. BioDrugs 38, 769–794 (2024).
[4] Farady CJ, Sellers BD, Jacobson MP, Craik CS. Improving the species cross-reactivity of an antibody using computational design. Bioorg Med Chem Lett. 2009 Jul 15;19(14):3744-7.
