EGFR: A Key Driver of Cell Growth and Cancer Progression

• Gene Information: EGFR encodes a transmembrane receptor tyrosine kinase belonging to the ErbB receptor family. EGFR plays a central role in regulating cell proliferation, survival, differentiation, and tissue repair and is frequently dysregulated in cancer.
• Protein Expression: EGFR is widely expressed in epithelial tissues, including the skin, lung, gastrointestinal tract, liver, and kidney. It is also detected in various epithelial-derived tumors, where overexpression, amplification, or activating mutations can drive tumor growth and disease progression.
• Signaling Pathway: EGFR is activated by ligands such as EGF and TGF-α, inducing receptor dimerization and activation of downstream pathways including RAS/RAF/MEK/ERK, PI3K/Akt/mTOR, JAK/STAT, and PLCγ/PKC. These signaling cascades promote cell proliferation, survival, migration, angiogenesis, and tumor progression.
• Therapeutic Inhibition: Current EGFR-targeted therapies include monoclonal antibodies and tyrosine kinase inhibitors (TKIs). Emerging approaches such as bispecific antibodies, ADCs, and combination therapies are being developed to overcome resistance and enhance anti-tumor efficacy.

• Human EGFR is specifically and correctly expressed in B-hEGFR mice.

Human EGFR was detectable in B-hEGFR mice by RT-PCR and sequencing. Liver tissues were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hEGFR mice (H/H). (A) Primers were designed to detect human and mouse EGFR. (B) Human EGFR mRNA was detectable in B-hEGFR mice, but not in wild-type mice. Sequencing of PCR products confirmed that the amplified sequences were consistent with database reference sequences.

• Human EGFR expression was detected in the brain, lung, liver, kidney, skin, uterus, esophagus, and ovary of homozygous B-hEGFR mice, but not in the heart, spleen, or thyroid. The antibodies were species-specific and did not bind to mouse EGFR in wild-type C57BL/6 mice.
• The results demonstrated that the EGFR expression profile in B-hEGFR mice closely resembles that in normal human tissues (Table 1).

Immunohistochemical analysis of organs in B-hEGFR mice. Major organs were collected from wild-type C57BL/6 mice and homozygous B-hEGFR mice (2 females, 8 weeks old), and analyzed by IHC using anti-EGFR antibody (Invitrogen, MA5-49312). The arrow indicates tissue cells with positive EGFR staining (brown). "+" and "-" indicate positive and negative tissues, respectively.

• Approximately 300 ng/mL of human sEGFR is detectable in the plasma of B-hEGFR mice.
• The relative expression levels of Egfr in the liver and skin were higher in B-hEGFR mice than in wild-type mice.

(A) Strain specific soluble EGFR expression analysis in homozygous B-hEGFR mice by ELISA. Plasma was collected from homozygous B-hEGFR mice (H/H) (male, n=3, 7-week-old). The expression levels of human sEGFR in B-hEGFR mice were analyzed by ELISA using the anti-human EGFR kit (RD, DEGFR0). Values are expressed as mean ± SEM.

(B) qPCR analysis of EGFR expression in homozygous B-hEGFR mice. RNA was isolated from the kidney, liver, skin, and spleen of wild-type (WT, +/+) and homozygous B-hEGFR (HO, H/H) mice (female, 6 weeks old, n=3 per group). cDNA libraries were generated by reverse transcription, followed by qPCR using mouse Egfr-specific primers. The relative expression levels of Egfr in the liver and skin were higher in B-hEGFR mice than in wild-type C57BL/6 mice.

• No significant differences were observed.

Complete blood count (CBC) of B-hEGFR Mice. Values are expressed as mean ± SD.

• No significant differences were observed.

Blood biochemical parameters of B-hEGFR Mice are shown. Values are expressed as mean ± SD.

Establishment of a B-Tg(hEGFR) MC38 model and in vivo efficacy study of Cetuximab analog-MMAE. B-Tg(hEGFR) MC38 cells were implanted subcutaneously into homozygous B-hEGFR mice (female, 6-8 weeks-old, n = 6). When the average tumor volume reached approximately 50-150 mm³, mice were randomized and subsequently administered the Cetuximab analog-MMAE (in house) via intravenous injection.

Efficacy of a Cetuximab analog-MMAE in B-hEGFR mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, anti-human EGFR antibody (in house) inhibited B-Tg(hEGFR) MC38 tumor growth in homozygous B-hEGFR mice, demonstrating that the B-hEGFR mice provide a powerful preclinical model for in vivo evaluation of EGFR-targeting ADC. Values are expressed as mean ± SEM.

Establishment of a toxicity evaluation model for EGFR-targeted antibody drugs in B-hEGFR mice. Anti-human EGFR antibody cetuximab biosimilar (Bio X Cell, 907623J2) or panitumumab (Takeda, 549661) were intravenously injected into B-hEGFR mice (male, 14-15 weeks-old, n=6). Mice were weighed twice a week, 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 the nasal and oral skin, abdominal skin, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum, and then subjected to pathological analysis.

Note: This experiment is a collaboration with the client.

Establishment of a toxicity evaluation model for EGFR-targeted antibody drugs in B-hEGFR mice. (A) Body weight and body weight changes during treatment. The results showed that 45.0 mg/kg cetuximab biosimilar and 30.0 mg/kg panitumumab resulted in significant weight loss in B-hEGFR mice. Values are expressed as mean ± SEM.

Note: This experiment is a collaboration with the client.

Establishment of a toxicity evaluation model for EGFR-targeted antibody drugs in B-hEGFR mice. (B) Complete blood cell count detection at the endpoint of the experiment. 45.0 mg/kg cetuximab biosimilar and 30.0 mg/kg panitumumab resulted in an increase in the number of neutrophils and monocytes, while there were no significant changes in blood parameters in the other treatment groups. The increase in​ neutrophils and monocytes in the peripheral blood may be induced by the activation of the immune system and mild inflammatory responses following high-dose antibody treatment. Values are expressed as mean ± SD. Significance was determined by one-way ANOVA test. *p<0.05, **p<0.01.

Note: This experiment is a collaboration with the client.

Establishment of a toxicity evaluation model for EGFR-targeted antibody drugs in B-hEGFR mice. (C) Pathological diagrams of various tissues. The histopathological examination in muzzle skin revealed article-related alteration in cetuximab biosimilar 45 mg/kg dose group (1/6, minimal), which showed squamous epithelial cells proliferation and subcutaneous infiltration of inflammatory cells. The similar article-related changes were also found in panitumumab group (5/6, minimal). Squamous epithelial cells proliferation in stomach limiting ridge were observed in mice administrated cetuximab biosimilar at the dose of 12.5 mg/kg (4/6, minimal) and 45 mg/kg groups (2/6, minimal; 4/6, slight), which were considered test-article and dose related. The similar article-related changes were also found in panitumumab group (3/6, minimal; 3/6, slight). Neither cetuximab biosimilar nor panitumumab showed any toxicity in the abdominal skin, duodenum, jejunum, ileum, cecum, colon, and rectum of mice (The data is not shown).

Note: This experiment is a collaboration with the client.

Toxicokinetic analysis of anti-human EGFR antibody in B-hEGFR mice. Anti-human EGFR antibody cetuximab biosimilar (Bio X Cell, 907623J2) or panitumumab (Takeda, 549661) were intravenously injected into B-hEGFR mice (male, 14-15 weeks-old, n=2). Blood samples were collected at 1h, 8h, 24h, 48h, 96h, and 168h after the first and fourth doses, and then the drug concentrations in the blood were measured.

Note: This experiment is a collaboration with the client.

Establishment of a toxicity evaluation model for the EGFR-targeting ADC Cetuximab-MMAE in B-hEGFR mice. The Cetuximab-MMAE (in house) was intravenously injected into B-hEGFR mice (female, 5-7 weeks-old, n=12). Mice were weighed twice a week, 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 skin, eyes, liver, ovary, uterus, heart, lungs, kidneys, thyroid gland, esophagus, and stomach, and then subjected to pathological analysis.

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (A) Body weight and (B) Survival rate (%) during treatment. Abnormal death were observed following three doses in the absence of preceding body weight changes, suggesting potential accumulated acute toxicity.

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (C) Complete blood count after the second cetuximab-MMAE treatment (80 mg/kg). Treatment with 80 mg/kg cetuximab-MMAE resulted in increased neutrophils (NEUT) and decreased eosinophils (EO), indicative of systemic inflammation. An elevated reticulocyte percentage (RET%) suggested compensatory hematopoiesis, while MMAE potentially contributed to bone marrow dysfunction. Values are expressed as mean ± SD. Significance was determined by one-way ANOVA test. *p

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (D) Representative images of skin and ocular toxicities following repeated cetuximab-MMAE treatment. Before the second dose, progressive hair loss was noted around the muzzle, eyes, and on parts of the abdomen and ears; severity further increased prior to the third dose. Periocular swelling, profuse ocular discharge, and corneal opacity were additionally observed. Yellow arrows indicate regions of skin toxicity characterized by hair loss, whereas red arrows indicate ophthalmic abnormalities. Ophthalmic findings prior to the second and third cetuximab-MMAE doses—periocular swelling, ocular discharge, and corneal opacity—are summarized in the table.

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (E) Pathological changes in oronasal skin. Compared to the vehicle control (G1), B-hEGFR mice treated with Cetuximab-MMAE at 80 mg/kg (G2) exhibited significant skin toxicity. Key histopathological findings included squamous epithelial proliferation (hyperplasia) and inflammatory cell infiltration in the subcutis. Additional observations such as ulceration and crust formation are detailed in the accompanying incidence table.

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (F) Pathological changes in the eye. Compared to the vehicle control (G1), mice treated with Cetuximab-MMAE at 80 mg/kg (G2) exhibited significant ocular toxicity. Histopathological examination revealed corneal stromal neutrophilic infiltration and retinal atrophy involving the ganglion and inner nuclear layers. Detailed incidence data for these abnormalities are provided in the accompanying table.

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (G) Pathological changes in reproductive organs and liver. Compared to the vehicle control (G1), mice treated with Cetuximab-MMAE at 80 mg/kg (G2) exhibited significant tissue alterations. Key findings included liver extramedullary hematopoiesis, profound atrophy of ovarian interstitial glands, and mild uterine atrophy. Statistical analysis of these pathological abnormalities is presented in the accompanying table.

In vivo toxicity evaluation of Cetuximab-MMAE in B-hEGFR mice. (H) Representative H&E staining images of other organs and tissues, indicating no obvious damage and abnormalities.

Toxicokinetic analysis of anti-human EGFR antibody in B-hEGFR mice. As shown in the figure, with the increase of dosage, the blood concentration of cetuximab biosimilar also increases. This indicates that the relationship between the blood concentration of cetuximab biosimilar and dosage is linear within the experimental dosage range. The existence of this linear relationship may be due to the absence of saturation during the absorption, distribution, metabolism, and excretion of drugs in mice.

Note: This experiment is a collaboration with the client.