Journal: Molecular Cancer Therapeutics
Article Title: Novel Chimeric CTLA-4 B-cell Epitope Peptide Vaccines Demonstrate Effective Antitumor Immunity with/without PD1/PDL1 Blockade in Multiple Syngeneic Murine Models of Breast and Colorectal Cancers
doi: 10.1158/1535-7163.MCT-24-0908
Figure Lengend Snippet: Combination CTLA-4 with PDL1 peptide vaccine in CT26-BALB/c tumor model. A, Six- to eight-week-old BALB/c mice were vaccinated with combination peptides as designed MVF-CTLA-4 (130) combination with PD1-Vaxx [MVF-PD1 (92)] for 3-week interval. A measure of 0.1 mg of each peptide cancer vaccine mixed with ISA 720 (1:1) used per mouse. Mice were boosted with the designed doses for every 3 weeks intervals. Blood was collected weekly for monitoring antibody titers. After 2 weeks of the third time immunization (3Y), mice were challenged with 1 × 10 5 CT26 per mouse, or 2 × 10 5 per mouse D2F2 tumor cells, or 5 × 10 5 per mouse 4T1 tumor cells. After tumor challenge, in the positive control group, we treated the mice with anti-mouse antibodies 9H10 plus 29F.1A12 twice a week for up to 3 weeks, and the negative control group was treated with PBS. Tumor volume was calculated as follows: Tumor volume (LWW) = (length × width × width)/2. B, Immunogenicity of MVF-CTLA-4 plus PD1-Vaxx peptides immunized BALB/c mice. Mice bleeds were collected weekly except the first collection was 3 weeks after primary immunization, and ELISA were used to detect antibody titers in sera. C, Antibody isotypes from mice bleed immunized with MVF-CTLA-4 (130) plus MVF-PD1 (92) combination peptide vaccine. D, Tumor burden (LWW) by days in BALB/c mice of each treatment group (10 mice/group) and tumor models of CT26, D2F2, and 4T1 BALB/c carcinoma, and two-way ANOVA was used to analyze the whole curves of tumor growth. Tumor burden (LWW) analysis in BALB/c mice of each treatment group and tumor models, and one-way ANOVA was used to analyze at each time point as indicated at days 9 and 12 for the CT26 model, days 12 and 14 for the D2F2 model, and days 9 and 12 for 4T1 model after tumor challenging. E, The log-rank (Mantel–Cox) test was used to compare the survival curves. ns, no significant; *, P < 0.05; **, P < 0.01, all vs. with the PBS control group.
Article Snippet: BALB/c mouse CT26 colon cancer cell line wild-type (WT; CT26 WT and CRL-2638, RRID: CVCL_7256) and 4T1 (CRL-2539, RRID: CVCL_0125) TNBC tumor cell lines were purchased in 2018 and 2020 from the ATCC.
Techniques: Positive Control, Negative Control, Immunopeptidomics, Enzyme-linked Immunosorbent Assay, Control
ATCC is a verified supplier 
ATCC manufactures this product 
![Combination CTLA-4 with PDL1 peptide vaccine in <t>CT26-BALB/c</t> tumor model. A, Six- to eight-week-old BALB/c mice were vaccinated with combination peptides as designed [MVF-PDL1 (130) with MVF-CTLA-4 (130)] for 3-week interval. A measure of 0.1 mg of each peptide cancer vaccine mixed with ISA 720 (1:1) used per mouse. Mice were boosted with the designed doses for every 3-week intervals. Blood was collected weekly for monitoring antibody titers. After 2 weeks of the third time immunization (3Y), mice were challenged with 2 × 10 5 per mouse D2F2 tumor cells or 5 × 10 5 per mouse 4T1 tumor cells. After tumor challenge, in the positive control group, we treated the mice with anti-mouse antibodies 10F.9G2 plus 9H10 twice a week for up to 3 weeks, and the negative control group was treated with PBS. Tumor volume was calculated as follows: Tumor volume (LWW) = (length × width × width)/2. B, Immunogenicity of MVF-CTLA-4 plus MVF-PDL1 (130) peptides immunized BALB/c mice. Mice bleeds were collected weekly except the first collection was 3 weeks after primary immunization, and ELISA was used to detect antibody titers in sera; C, Antibody isotypes from mice bleed immunized with MVF-CTLA-4 (130) plus MVF-PDL1 (130) combination peptide vaccine. D, Tumor burden (LWW) by days in BALB/c mice of each treatment group (10 mice/group) and tumor models of both D2F2 and 4T1 BALB/c mammary carcinoma, and two-way ANOVA was used to analyze the whole curves of tumor growth. Tumor burden (LWW) analysis in BALB/c mice of each treatment group and tumor models, and one-way ANOVA was used to analyze at each time point as indicated at days 12 and 14 for the D2F2 model and days 9 and 12 for the 4T1 model after tumor challenging. E, The log-rank (Mantel–Cox) test was used to compare the survival curves. ns, no significant; *, P < 0.05; **, P < 0.01, all vs. with the PBS control group.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2803/pmc12402803/pmc12402803__mct-24-0908_f5.jpg)
![Combination CTLA-4 with PDL1 peptide vaccine in CT26-BALB/c tumor model. A, Six- to eight-week-old BALB/c mice (10 mice/group) were vaccinated with MVF peptides [MVF-CTLA-4 (59) or (130) with MVF-PDL1 (36) or (130)] as indicated for 3-week interval. Each peptide cancer vaccine 100 μg mixed with ISA 720 (1:1) was immunized per mouse. Mice were boosted with the designed doses, and bleeds were collected weekly for monitoring antibody titers. After 2 weeks of the third time immunization (3Y), mice were challenged with 1 × 10 5 CT26 tumor cells on day 0. The tumor growth checked daily, especially 7 days after challenging, and tumor size was measured using calipers. For the positive control group, we treated the mice with anti-mouse CTLA-4 antibody (clone 9H10) plus anti-mouse PDL1 antibody (10F.9G2) twice a week for up to 3 weeks, whereas the negative control group was treated with PBS. B, Immunogenicity of combination MVF-CTLA-4 with MVF-PDL1 peptides immunized BALB/c mice. Mice bleeds were collected weekly except the first collection was 3 weeks after primary immunization, and ELISA was used to detect antibody titers in sera; C, Tumor burden (LWW) by days in BALB/c mice of each treatment group (10 mice/group) and tumor models, and two-way ANOVA was used to analyze the whole curves of tumor growth. Tumor burden (LWW) analysis in BALB/c mice of each treatment group and tumor models, and one-way ANOVA was used to analyze at each time point as indicated at days 14 and 16 after tumor challenging. D, The log-rank (Mantel–Cox) test was used to compare the survival curves. ns, no significant; *, P < 0.05; **, P < 0.01, all vs. with the PBS control group.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2803/pmc12402803/pmc12402803__mct-24-0908_f4.jpg)
![Combination CTLA-4 with PDL1 peptide vaccine in CT26-BALB/c tumor model. A, Six- to eight-week-old BALB/c mice were vaccinated with combination peptides as designed MVF-CTLA-4 (130) combination with PD1-Vaxx [MVF-PD1 (92)] for 3-week interval. A measure of 0.1 mg of each peptide cancer vaccine mixed with ISA 720 (1:1) used per mouse. Mice were boosted with the designed doses for every 3 weeks intervals. Blood was collected weekly for monitoring antibody titers. After 2 weeks of the third time immunization (3Y), mice were challenged with 1 × 10 5 CT26 per mouse, or 2 × 10 5 per mouse D2F2 tumor cells, or 5 × 10 5 per mouse 4T1 tumor cells. After tumor challenge, in the positive control group, we treated the mice with anti-mouse antibodies 9H10 plus 29F.1A12 twice a week for up to 3 weeks, and the negative control group was treated with PBS. Tumor volume was calculated as follows: Tumor volume (LWW) = (length × width × width)/2. B, Immunogenicity of MVF-CTLA-4 plus PD1-Vaxx peptides immunized BALB/c mice. Mice bleeds were collected weekly except the first collection was 3 weeks after primary immunization, and ELISA were used to detect antibody titers in sera. C, Antibody isotypes from mice bleed immunized with MVF-CTLA-4 (130) plus MVF-PD1 (92) combination peptide vaccine. D, Tumor burden (LWW) by days in BALB/c mice of each treatment group (10 mice/group) and tumor models of CT26, D2F2, and 4T1 BALB/c carcinoma, and two-way ANOVA was used to analyze the whole curves of tumor growth. Tumor burden (LWW) analysis in BALB/c mice of each treatment group and tumor models, and one-way ANOVA was used to analyze at each time point as indicated at days 9 and 12 for the CT26 model, days 12 and 14 for the D2F2 model, and days 9 and 12 for 4T1 model after tumor challenging. E, The log-rank (Mantel–Cox) test was used to compare the survival curves. ns, no significant; *, P < 0.05; **, P < 0.01, all vs. with the PBS control group.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2803/pmc12402803/pmc12402803__mct-24-0908_f6.jpg)