CTLA-4 (cytotoxic T-lymphocyte antigen 4) is a transmembrane glycoprotein and an immune checkpoint receptor expressed on the surface of cytotoxic T lymphocytes. CTLA-4 is homologous to the immune co-stimulatory receptor CD28. Among the known immune checkpoint proteins that have been reported so far, the CTLA-4 pathway is one of the most well-studied immune checkpoint pathways.
Figure 1. CTLA-4 is a co-inhibitory receptor of CD28.
In the normal immunization process, the effective activation of T cells is the premise and guarantee for T cells to exert their immune effects. If the activation of T cells is incomplete or if T cells are in the state of incompetent immune response, they cannot play the role of killing tumor cells. Activation of T cells requires simultaneous stimulation by two signals, i.e., the synergy of the first and second signals. The first signal is transmitted by the binding of peptide antigen-MHC complexes, which are expressed on the surface of APC cells, to their corresponding receptors expressed on the surface of T cells. The second signal is transmitted by the binding of costimulatory molecules, which are also expressed on the surface of APC cells, to their corresponding receptors expressed on the surface of T cells.
The most important costimulatory molecules identified so far are B7-1 and B7-2, and their corresponding T cell surface receptor is CD28. The binding of B7-1/B7-2 to CD28 transmits the stimulatory second signal, which works synergistically with the first signal to stimulate the activation of T cells. Recent studies have shown that CTLA4 and CD28 competitively bind to the B7 protein family, and the affinity of CTLA4 for B7 protein is more than 20 times higher than the affinity of CD28 for B7 protein. The binding between CTLA4 and B7 protein transmits an inhibitory signal in the early stage of the immune response, thus leading to the inhibition of T cell activation. The immune checkpoint CTLA4 is equivalent to a brake mechanism generated by evolution. CTLA4 plays an important role in maintaining the immune homeostasis and preventing the occurrence of excessive immune responses. By activating the CTLA4 signaling pathway, tumor cells can avoid the anti-tumor effects exerted by the immune system. The function of immune checkpoint inhibitors is to release the "brake" pressed by the tumor cells, thus allowing the immune cells to kill tumor cells. In clinical trials, anti–CTLA-4 monoclonal antibody therapy has been associated with a 10%–21% response rate in patients with advanced metastatic melanoma.
Humanized CTLA4 Mouse
The humanized mouse models of immune checkpoints allow researchers to study drugs that only recognize human immune checkpoints. Humanized mice offer the possibility of investigating checkpoint inhibitors that target human immune checkpoints, and allow more accurate prediction of drug efficacy and possible side effects (e.g., autoimmunity, pro-inflammatory response, etc.) prior to the start of clinical trials. Shanghai Model Organisms has developed a series of humanized mouse models for known human immune checkpoints, including the humanized mouse model of CTLA-4, to test drugs that target human immune checkpoints.
Figure 2. Generation strategy of humanized CTLA4 mice. On the C57BL/6J genetic background, the protein coding sequences for human CTLA4 were inserted into the ATG position of the mouse Ctla4 gene, so that the expression of endogenous Ctla4 in the mouse was replaced by the expression of fully humanized CTLA4 protein.
Flow cytometry (FACS) analysis data of humanized CTLA4 mouse
Figure 3. Expression of CTLA4 in the activated spleen lymphocytes of humanized CTLA4 mice is detected by FACS. The spleen lymphocytes of homozygous humanized CTLA4 mice were activated by anti-CD3 and anti-CD28 for 72 hours, and then collected for staining. The expression of humanized CTLA4 was detected by FACS. The results showed that the active expression of humanized CTLA4 can be detected in both activated CD4+ and CD8+ T lymphocytes collected from homozygous humanized CTLA4 mice. (Completed in collaboration with CrownBio).
In vivo validation in a MC38 tumor-bearing model of humanized CTLA4 mouse
Figure 4. In vivo anti-tumor effect of Ipilimumab, a humanized anti-CTLA4 antibody, in a humanized mouse model of CTLA4 (data were obtained in cooperation with Innovent Biologics, Inc.).
Grouping, dosing and testing:
MC38 colorectal cancer cells were inoculated subcutaneously under the right back of CTLA-4 transgenic mice. After tumor formation, the mice were randomly divided into 2 groups and were then treated with 10 mg/kg of Ipilimumab or an equal volume of normal saline. The drug was administered twice a week for a total of 4 administrations. The tumor volume was measured before each drug administration.
The anti-human-CTLA4 drug Ipilimumab significantly inhibited the growth of MC38 tumors in CTLA-4 mice, and the tumor suppressive effect was detected at 1 week after drug administration, along with a significant anti-tumor effect after 2 weeks of drug administration (as shown above). At the same time, the tumors completely disappeared in 2 animals of the Ipilimumab treatment group, while the tumor continued to grow in the control group.
Figure 5. In vivo anti-tumor effect of Ipilimumab, a humanized anti-CTLA4 antibody, in a humanized mouse model of CTLA4 (data were obtained in cooperation with PharmaLegacy).
In vivo validation of anti-tumor efficacy in a MC38 tumor-bearing model of humanized CTLA4 mice. Homozygous humanized CTLA4 mice were inoculated with MC38 colon cancer cells. After the tumors grew to 100 mm3, the animals were randomly assigned into a control group and a treatment group (n=9). The results showed: Yervoy, a drug targeting human CTLA4, showed a very significant anti-tumor effect (p<0.001), demonstrating that the humanized CTLA4 mice are a good in vivo model for validating the efficacy of antibodies targeting human CTLA4.
Know More Humanized Mouse Models
1.Sanmamed M F, Chester C, Melero I, et al. (2016) “Defining the optimal murine models to investigate immune checkpoint blockers and their combination with otherimmunotherapies.” AnnOncol 27(7):1190-1198.doi:10.1093/annonc/mdw041.
2.Wolchok J D and Saenger Y (2008) “The mechanism of anti-CTLA-4 activity and the negative regulation of T-cell activation.” Oncologist 13 Suppl 4:2-9. doi: 10.1634/theoncologist.