• HER2 (ERBB2) is a receptor tyrosine kinase belonging to the EGFR family and is a well-established therapeutic target in HER2-positive cancers, particularly breast and gastric cancers. Multiple anti-HER2 monoclonal antibodies and antibody-drug conjugates have been developed; however, their efficacy, on-target toxicity and tissue distribution cannot be fully evaluated in conventional wild-type mouse models due to species differences.
• In HER2 humanized mice, human HER2 mRNA and protein are expressed across multiple organs with a distribution pattern similar to that observed in normal human tissues. Comprehensive immunophenotyping demonstrates that replacement of mouse Her2 with human HER2 does not alter immune cell development, differentiation, or distribution in spleen, lymph nodes, or peripheral blood.
• Together, these characteristics make HER2 humanized mice a robust and physiologically relevant in vivo platform for preclinical efficacy assessment, toxicity evaluation, and translational research of HER2-targeted biologics.

Key Advantages

• Human-like HER2 tissue expression profile: HER2 protein distribution in major organs closely resembles that of normal human tissues, supporting translational relevance.
• Preserved immune system integrity: Comprehensive FACS analysis confirms normal leukocyte and T cell subpopulation distributions in spleen, lymph nodes, and blood.
• Validated toxicity sensitivity: Dose-dependent toxicity and organ-specific effects are detectable following administration of anti-human HER2 antibodies.
• Suitable for safety and risk assessment: Normal blood routine and liver biochemistry parameters support baseline physiological stability.
• High relevance for biologics development: Enables evaluation of on-target, off-tumor toxicity of HER2-directed antibodies and antibody-drug conjugates.

Validation

• Human HER2 expression: Human HER2 mRNA detected exclusively in homozygous HER2 humanized mice. Mouse Her2 mRNA detected only in wild-type mice. IHC analysis demonstrates human HER2 protein expression across multiple tissues in HER2 humanized mice. Expression pattern is consistent with normal human tissue distribution.
• Immune profiling: Normal leukocyte and T cell subset composition in spleen, lymph nodes, and peripheral blood. No impact on immune cell development or distribution.
• Safety and Hepatotoxicity: Normal complete blood count (CBC). Normal serum ALT and AST levels, indicating preserved liver function.
• Toxicological evaluation: Dose-dependent body weight loss and organ-specific toxicities observed following administration of anti-human HER2 antibodies.

Application

• Preclinical toxicity evaluation of anti-HER2 antibodies: Assessment of dose-dependent, on-target toxicities in a human-relevant context.
• Safety profiling of HER2-targeted biologics: Including monoclonal antibodies and antibody-drug conjugates.
• Target-mediated risk assessment: Identification of sensitive tissues expressing human HER2.
• Translational drug development support: Bridging preclinical findings with clinical safety considerations.
• Regulatory-supportive in vivo studies: Providing humanized target expression data for IND-enabling studies.

HER2

• The exons 2-17 of mouse Erbb2 gene that encode extracellular domain and transmembrane domain and were replaced by human counterparts in HER2 humanized mice. The genomic region of mouse Erbb2 gene that encodes cytoplasmic portion was retained. The promoter, 5' UTR and 3' UTR regions of the Erbb2 gene were also retained. The chimeric ERBB2 expression was driven by endogenous mouse Erbb2 promoter, while mouse Erbb2 gene transcription and translation will be disrupted.

Strain specific analysis of HER2 mRNA expression in wild-type C57BL/6 mice and homozygous HER2 humanized mice by RT-PCR. Small intestine, heart, eye, tongue, lung, liver, kidney and stomach RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous HER2 humanized mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human HER2 primers. Sequencing of the short-form PCR products confirmed that the amplified sequences were consistent with database reference sequences.

Immunohistochemical (IHC) analysis of HER2 protein expression in wild-type mice and HER2 humanized mice. The mammary gland, colon and stomach tissues were collected from wild-type mice and homozygous HER2 humanized mice (H/H) and analyzed by IHC with anti-HER2 antibody (Abcam, ab214275). The arrow indicates tissue cells with positive HER2 staining (brown).

Immunohistochemical (IHC) analysis of HER2 protein expression in wild-type mice and HER2 humanized mice. Fourteen major tissues were collected from wild-type mice and homozygous HER2 humanized mice and analyzed by IHC with anti-mouse HER2 antibody (ab214275) and anti-human HER2 antibody (ab16662).

Growth curve of HER2 humanized mice. Three-week-old HER2 humanized mice were grouped by sex (10 males and 10 females). Body weight was measured weekly for 13 weeks on the same day each week. The minimum and maximum body weights shown in the table were calculated from the mean ± SD.

Complete blood count (CBC) of HER2 humanized mice. Values are expressed as mean ± SD.

Blood biochemical parameters of HER2 humanized mice are shown. Values are expressed as mean ± SD.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood of HER2 humanized mice. Splenocytes, peripheral blood, and Lymph nodes were isolated from female C57BL/6 and HER2 humanized mice (female, 8-week-old, n = 3). Single live cells were gated on the CD45⁺ population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood of HER2 humanized mice. Splenocytes, peripheral blood, and Lymph nodes were isolated from female C57BL/6 and HER2 humanized mice (female, 8-week-old, n=3). Single live cells were gated on the TCRβ⁺ T-cell population and analyzed by flow cytometry as indicated. Values are expressed as mean ± SEM.

In vivo toxicity test of anti-human HER2 antibody (by the clients) in HER2 humanized mice. Different doses of anti-human HER2 antibody Ab1 were injected into HER2 humanized mice via a single intravenous injection. The body weight of HER2 humanized mice decreased in a dose-dependent manner. In the high-dose group, one mouse died. Histopathological analysis revealed no significant abnormal changes in liver, but no immature follicles were seen in ovaries, and the myeloid cells in the bone marrow increased and the red blood cell count decreased. This suggests that HER2 humanized mice can be used to assess the toxicity of anti-human HER2 antibodies. Data were obtained from a partner. Values ​​are expressed as mean ± SEM.

In vivo toxicity test of anti-human HER2 antibody (by the clients) in HER2 humanized mice. Different anti-human HER2 antibodies were injected into HER2 humanized mice through a single intravenous injection. Mice in different antibody treatment groups exhibited varying rates of weight loss. In antibody Ab1 treatment group, all mice were euthanized due to rapid weight loss. Mild toxicity was observed in the bone marrow and ovaries through histopathological analysis. This suggests that HER2 humanized mice can be used to assess the toxicity of anti-human HER2 antibodies. Data were obtained from a partner. Values ​​are expressed as mean ± SEM.

In vivo toxicity test of the HER2-targeted antibody-drug conjugate (ADC) DS-8201 (Enhertu) in HER2 humanized mice. Anti-human HER2 ADC DS8201 (Enhertu, Commercialized) was intravenously injected into HER2 humanized mice (Male, 8 weeks old, n=3). Mice were weighed every two days, and their condition was observed daily. At the end of the experiment, blood samples were collected for complete blood count test. Additionally, tissue samples were collected from spleen, thymus,  small intestine and testis and then subjected to pathological analysis. Values are expressed as mean ± SEM.

In vivo toxicity test of DS-8201 (Enhertu) in HER2 humanized mice. At the end of the experiment, blood samples were collected for complete blood count test. Additionally, tissue samples were collected from spleen, thymus,  small intestine and testis and then subjected to pathological analysis.

Establishment of an MC38 model in HER2 humanized mice and in vivo efficacy study of anti-HER2 ADC. Murine colon cancer B-hHER2 MC38 plus cells (Cat# 322353) were subcutaneously implanted into homozygous HER2 humanized mice (female, 10 weeks-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human HER2 ADC DS8201 (purchased from AstraZeneca/Daiichi Sankyo) indicated in panel.

Efficacy of anti-human HER2 ADC in HER2 humanized mice. (A) Tumor growth curves. (B) Body weight changes during treatment. (C) In vivo efficacy of anti-human HER2 antibodies-individual tumor growth curves. Values are expressed as mean ± SEM.

Establishment of MC38 model in HER2 humanized mice and in vivo efficacy study of anti-HER2 antibody (Trastuzumab analog-MMAE, in-house). Murine colon cancer B-hHER2 MC38 plus cells (Cat# 322353) were subcutaneously implanted into homozygous HER2 humanized mice (female, 7-8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human HER2 ADC Trastuzumab analog-MMAE (in-house) as indicated in panel. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human HER2 ADC-individual tumor growth curves in HER2 humanized mice.

Establishment of an MC38 model and in vivo Efficacy of HER2 ADC combined with anti-mPD-1 antibody in HER2 humanized mice. Murine colon cancer B-hHER2 MC38 plus cells (Cat# 322353) were subcutaneously implanted into homozygous HER2 humanized mice (female, 7-8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human HER2 ADC Trastuzumab analog-MMAE (in-house) or intraperitoneally injected anti-mPD-1 antibody (in-house).

In vivo efficacy of HER2 ADC combined with anti-mPD-1 antibody-individual tumor growth curves in HER2 humanized mice.

Establishment of a B16-F10 model and in vivo efficacy study of HER2 ADC DS8201 (purchased from Daiichi Sankyo) in HER2 humanized mice. Murine melanoma B-hHER2 B16-F10 cells (Cat# 322393) were subcutaneously implanted into homozygous HER2 humanized mice (female, 9-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human HER2 ADC DS8201 (purchased from Daiichi Sankyo) as indicated in panel. (A) Tumor growth curves. (B) Body weight changes during treatment in vivo. (C) Efficacy of HER2 ADC DS8201: individual tumor growth curves.

Establishment of a B16-F10 model and in vivo efficacy study of HER2 ADC DS8201 (purchased from Daiichi Sankyo) in HER2 humanized mice. Murine melanoma B-hHER2 B16-F10 cells (Cat# 322393) were subcutaneously implanted into homozygous HER2 humanized mice (female, 9-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human HER2 ADC DS8201 (purchased from Daiichi Sankyo) as indicated in panel. (A) Tumor growth curves. (B) Body weight changes during treatment in vivo. (C) Efficacy of HER2 ADC DS8201: individual tumor growth curves.

Q1: What is the main application of HER2 humanized mice (B-hHER2)?

HER2 humanized mice are primarily used for preclinical toxicity and safety evaluation of anti-HER2 antibodies and biologics, enabling assessment of on-target effects in a human-relevant in vivo setting.

Q2: How is HER2 expression validated in HER2 humanized mice (B-hHER2)?

HER2 expression in HER2 humanized mice is validated at both the mRNA and protein levels using RT-PCR and immunohistochemistry. Human HER2 expression is detected exclusively in homozygous HER2 humanized mice, with tissue distribution patterns similar to those observed in normal human tissues.

Q3: Does humanization of HER2 affect the immune system of the mice?

No. Comprehensive flow cytometry analyses demonstrate that leukocyte and T cell subpopulation distributions in spleen, lymph nodes, and peripheral blood of HER2 humanized mice are comparable to those of wild-type C57BL/6 mice, indicating preserved immune system development and homeostasis.

Q4: What types of drugs can be evaluated using HER2 humanized mice (B-hHER2)?

HER2 humanized mice can be used to evaluate the safety and toxicity of anti-HER2 monoclonal antibodies, antibody–drug conjugates (ADCs), and other HER2-targeted biologics that specifically recognize human HER2.