B-HLA-A2.1/hMAGEA3 MC38

Basic Information

Description

• Origin: The MC38 cell line is derived from C57BL/6 murine colon adenocarcinoma cells. The cell line is a commonly used murine model for colorectal carcinoma.
• Background Information: HLA-A belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. MAGEA3 is a member of the MAGEA gene family. The MAGEA genes are clustered at chromosomal location Xq28. They have been implicated in some hereditary disorders, such as dyskeratosis congenita.
• Gene targeting strategy: The B2M gene (Exon2 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS, HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2Db gene containing the α3, transmembrane and cytoplasmic domains and MAGEA3 CDS. Human HLA-A2.1 is highly expressed on the surface of B-HLA-A2.1/hMAGEA3 MC38. MAGEA3 is detectable by western blot.
• Application: B-HLA-A2.1/hMAGEA3 MC38 tumor models can be used for preclinical evaluation of cancer vaccines.
• Notes:

1. Inoculated cell lines can be suspended with DMEM stock solution.
2. Before implementing the project, it is recommended to perform tumor growth experiments. The recommended cell inoculation amount is between 1E6-1E7.
3. In the experiment, it is necessary to ensure that the number of animals inoculated subcutaneously is at least 1.6 times the actual grouping number.

Protein expression analysis

HLA-A2.1 and MAGEA3 expression analysis in B-HLA-A2.1/hMAGEA3 MC38 cells by flow cytometry and western blot, respectively. Single cell suspensions from wild-type MC38 and B-HLA-A2.1/hMAGEA3 MC38 #1-H01 cultures were detected with species-specific anti-HLA-A2.1 antibody (Biolegend, 343306) and anti-MAGEA3 (abcam, AB223162), respectively. Human HLA-A2.1 was detected on the surface of B-HLA-A2.1/hMAGEA3 MC38 cells but not wild-type MC38 cells(A). Human MAGEA3 was detected in the B-HLA-A2.1/hMAGEA3 MC38 cells but not wild-type MC38 cells(B).

Tumor growth curve & Body weight changes

Subcutaneous tumor growth of B-HLA-A2.1/hMAGEA3 MC38 cells. B-HLA-A2.1/hMAGEA3 MC38 cells (1×10⁶) were subcutaneously implanted into B-HLA-A2.1 mice (male, 7-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 × long diameter × short diameter². Results indicate that B-HLA-A2.1/hMAGEA3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Protein expression analysis of tumor tissue

HLA-A2.1 and MAGEA3 expression evaluated in B-HLA-A2.1/hMAGEA3 MC38 tumor cells by flow cytometry and western blot, respectively. B-HLA-A2.1/hMAGEA3 MC38 cells were subcutaneously transplanted into B-HLA-A2.1 mice (male, 7-week-old, n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed with species-specific anti-HLA-A2.1 antibody (Biolegend, 343306) and anti-MAGEA3 (abcam, AB223162), respectively. Human HLA-A2.1 was highly expressed on the surface of tumor cells(A). Human MAGEA3 was detected in the tumor cells(B). Therefore, B-HLA-A2.1/hMAGEA3 MC38 cells can be used for in vivo efficacy studies evaluating cancer vaccines.

In vivo efficacy of anti-human HER2 antibody-drug conjugate (ADC)

Antitumor activity of anti-human HER2 ADC (Trastuzumab analog-MMAE, in-house) in B-h4-1BB/hHER2 mice. (A) Anti-human HER2 ADC inhibited B-hHER2 MC38 plus tumor growth in B-h4-1BB/hHER2 mice. Murine colon cancer B-hHER2 MC38 plus cells were subcutaneously implanted into homozygous B-h4-1BB/hHER2 mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100-150 mm³, at which time they were intraperitoneally injected with anti-human HER2 ADC Trastuzumab analog-MMAE (in-house) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, 10mg/kg anti-human HER2 ADC was efficacious in controlling tumor growth in B-h4-1BB/hHER2 mice, demonstrating that B-hHER2 MC38 plus provide a powerful preclinical model for in vivo evaluation of anti-human HER2 ADC. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human HER2 ADC DS8201

Antitumor activity of anti-human HER2 ADC DS8201 in B-h4-1BB/hHER2 mice. (A) Anti-human HER2 ADC inhibited B-hHER2 MC38 plus tumor growth in B-h4-1BB/hHER2 mice. Murine colon cancer B-hHER2 MC38 plus cells were subcutaneously implanted into homozygous B-h4-1BB/hHER2 mice (female, 9-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intraperitoneally injected with anti-human HER2 ADC DS8201 (purchased from Daiichi Sankyo) or Trastuzumab analog-MMAE (in-house) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, Both anti-human HER2 ADC were efficacious in controlling tumor growth in B-h4-1BB/hHER2 mice, demonstrating that B-hHER2 MC38 plus provide a powerful preclinical model for in vivo evaluation of anti-human HER2 ADC. Values are expressed as mean ± SEM.