• Gene Information: TROP2 (Trophoblast Cell Surface Antigen 2), also known as TACSTD2, is a transmembrane glycoprotein encoded on human chromosome 1. It is a well-established tumor-associated antigen and therapeutic target in multiple solid cancers.
• Protein Expression: TROP2 is expressed in various epithelial tissues and is frequently overexpressed in cancers such as breast, lung, and gastrointestinal tumors. Elevated TROP2 expression is often associated with poor prognosis and aggressive disease.
• Signaling Pathway: TROP2 activates downstream signaling pathways including PI3K/AKT, MAPK/ERK, and β-catenin pathways, promoting cell proliferation, survival, migration, and invasion.
• Therapeutic Targeting: Targeting TROP2 with antibodies, ADCs, bispecific antibodies, and other therapeutic modalities can selectively eliminate TROP2-expressing tumor cells and suppress tumor progression.

TROP2

• The exon 1 of mouse Trop2 gene that encodes the full-length protein was replaced by human TROP2 exon 1 in B-hTROP2 mice.

• Mouse TROP2 mRNA was detectable in wild-type C57BL/6 mice.
• Human TROP2 mRNA was detectable only in homozygous B-hTROP2 mice but not in wild-type mice.

Strain specific analysis of TROP2 mRNA expression in wild-type C57BL/6 mice and homozygous B-hTROP2 mice by RT-PCR. Skin RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hTROP2 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TROP2 primers.

• TROP2 was detected in the lung, kidney, stomach, skin, mammary gland, eye, and uterus of wild-type mice and B-hTROP2 mice due to the cross-reactivity of antibody, but not expressed in the heart, liver, spleen, brain, small intestine, colon, and testis (Figure 1).
• The results showed that humanized TROP2 did not change the expression site of TROP2 protein in mice, and the expression profile of TROP2 in B-hTROP2 mice was similar to that in normal human tissues (Table 1). B-hTROP2 mice can be used to evaluate the toxicity of TROP2 drugs.

Western blot analysis of TROP2 protein expression in homozygous B-hTROP2 mice. Fourteen major tissues were collected from wild-type mice and homozygous B-hTROP2 mice (2 males and 2 females, 8 week-old), and analyzed by western blotting with anti-TROP2 antibody (Abcam, ab214488).

• Human TROP2 was detected in lung, skin, stomach, oral mucosa, kidney, tongue, eyes and pancreas of B-hTROP2 mice.

Immunohistochemical (IHC) analysis of TROP2 protein expression in B-hTROP2 mice. Major were collected from homozygous B-hTROP2 mice (H/H) and analyzed by IHC with anti-human TROP2 antibody (Abcam, ab214488)

• No significant differences were observed compared with wild-type mice.

Complete blood count (CBC) of B-hTROP2 mice. Values are expressed as mean ± SD.

• No significant differences were observed compared with wild-type mice.

Blood biochemical parameters of B-hTROP2 mice are shown. Values are expressed as mean ± SD.

• The percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, and macrophages in homozygous B-hTROP2 mice are similar to those in C57BL/6 mice.
• Humanization of TROP2 does not affect normal immune cell development and distribution.

Analysis of leukocyte subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and B-hTROP2 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.

• The proportions of CD4⁺ T cells, CD8⁺ T cells, and Tregs in homozygous B-hTROP2 mice are comparable to those in C57BL/6 mice.
• Humanization of TROP2 does not affect normal T cell development, differentiation  and distribution.

Analysis of T-cell subpopulations by flow cytometry in immune organs and blood. Splenocytes, peripheral blood, and lymph nodes were isolated from female C57BL/6 and B-hTROP2 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.

• B6ADC showed acceptable safety at 44 mg/kg in TROP2-humanized mice but induced mortality at 66 mg/kg.

Safety and pharmacokinetics of bispecific nanobody-drug conjugate (B6ADC) (by the clients). (A) Body weight changes in BALB/c mice treated with B6ADC at doses of 44 mg/kg, 66 mg/kg, and 110 mg/kg (n = 4∼6/group). (B) Dose-dependent safety evaluation of B6ADC in TROP2-humanized mice and c-Met-humanized mice (n = 4/group). (C) Biochemical analysis of liver function (AST, ALT, and T-Bil) and kidney function (Cr and BUN) in TROP2-humanized mice and c-Met-humanized mice (n =4/group).

• TROP2 in a TROP2-humanized mouse model and found that CAR.TROP2 caused severe lung toxicity and systemic inflammation, suggesting direct TROP2 targeting by CAR-Ts could be detrimental.
• These data show that CAR.TROP2 could cause major toxicity by inducing strong inflammation in TROP2 positive lung.

Preclinical safety evaluation of TROP2-specific CAR-Ts using TROP2-humanized mouse model (by the clients). (D) Schematics of safety evaluation of mCAR.TROP2 T cells in TROP2-humanized (B-hTROP2) mouse model. (E) Changes in body weight of the B-hTROP2 mice infused with mCAR.TROP2 or Ctrl T cells at day 4 and 7 compared with day 0. (F) Representative micrographs of H&E staining on various organs harvested from B-hTROP2 mice infused with mCAR.TROP2 or Ctrl T cells for 7days; data shown are representative of two mice from Ctrl group and four mice from mCAR.TROP2 group; scale bars, 50µm.

In vivo toxicity test of Datopotamab Deruxtecan (Dato-DXd). Anti-human TROP2 antibody Datopotamab Deruxtecan (In house) was intravenously injected into B-hTROP2 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. Values are expressed as mean ± SEM.

• The decrease in RBC, WBC, and HGB reflected potential myelosuppression caused by off-target effects of the DXd payload on rapidly dividing hematopoietic stem cells in bone marrow.

In vivo toxicity test of Datopotamab Deruxtecan (Dato-DXd). At the end of the experiment, blood samples were collected for complete blood count test.