How to Choose an Anti-CTLA-4 Antibody for Your Research

CTLA-4 Introduction

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a co-inhibitory receptor predominantly expressed on T cells. CTLA-4 maintains immune homeostasis during T-cell activation. CTLA-4 signaling results in immunosuppression and restricted T cell function, and its deficiency results in autoimmunity. Through a process called co-stimulation, the activation of T cells is dependent on the binding of CD28 to its ligands, CD80 (B7-1) and CD86 (B7-2). CTLA-4 preferentially binds to CD80/CD86 and transmits inhibitory signals to prevent CD28-mediated T-cell activation. CTLA-4 is constitutively expressed by regulatory T cells (Tregs), which prevents T cells from killing other cells, including cancer cells. Anti-CTLA-4 antibodies block CTLA-4 binding with CD80 and CD86, and the blockade of CTLA-4 enhances anti-tumor immune responses. The major biological effects and the underlying mechanism of action of CTLA-4-blocking antibodies are illustrated in the figure below:

CTLA-4-blocking antibodies (represented as α-CTLA-4 in the diagram) can promote antibody-dependent cellular cytotoxicity (ADCC), especially when bound to an Fc receptor on an antigen-presenting cell (APC). Because CD4+CD25+ Tregs express higher amounts of CTLA-4 than conventional T cells, they are more prone to anti-CTLA-4 antibody-induced ADCC than conventional T cells. Additionally, anti-CTLA-4 antibodies can bind to CTLA-4 on the surface of the Tregs and prevent it from counter-regulating the CD28-mediated co-stimulatory pathways. Concurrently, anti-CTLA-4 antibodies can also promote T cell responses by blocking CTLA-4 on the surface of conventional T cells as they undergo activation. (Source: Waldman et. al., 2020)

Anti-CTLA-4 Antibody Reactivity

The first step in deciding which anti-CTLA-4 antibody is appropriate for your research project is to identify the species reactivity required for your experimental model. Bio X Cell offers anti-mouse, anti-human, and anti-rat CTLA-4 antibodies. Which antibody product to choose would depend on the species of your experimental model. For example, if you are working with mice expressing murine CTLA-4 protein, you want to choose an anti-mouse CTLA-4 antibody. If you are using human cells in culture or humanized mice expressing human CTLA-4, then you want to choose an anti-human CTLA-4 antibody.

Anti-Mouse CTLA-4 Antibody Clones

Bio X Cell offers anti-mouse CTLA-4 antibodies from three distinct hybridoma clones: 9H10, 9D9, and UC10-4F10-11. Anti-mouse CTLA-4 (CD152) clone 9D9 (Quezada et al. 2006) is a mouse monoclonal antibody that functions in vivo through the depletion of intra-tumoral Tregs and it is also documented to neutralize CTLA-4 in vivo. Anti-mouse CTLA-4 (CD152) clone: 9H10 (Krummel and Allison 1995), on the other hand, is a Syrian hamster IgG that promotes T cell co-stimulation through blocking CTLA-4 binding to its ligands, allowing for CD28 binding. It is reported to neutralize CTLA-4 in vitro as well as in vivo. Importantly, 9H10 also depletes intra-tumoral Tregs, and in a mouse melanoma model, it was shown to have slightly stronger depletion efficacy than 9D9 (Simpson et. al., 2013). The anti-mouse CTLA-4 (CD152) clone UC10-4F10-11 (Walunas et al. 1994) is an Armenian hamster IgG that is great for neutralizing CTLA-4 in vitro as well as in vivo. All three of these antibodies are useful for the detection of murine CTLA-4 via Western blot, while UC10-4F10-11 can be used for flow cytometry as well. Accordingly, the main differences between these antibodies lie in their CTLA-4 blocking capacities, their listed or reported (i.e., published) applications, and their host and isotype.

The next step in choosing an anti-CTLA-4 antibody is to perform a literature search for publications to find an example of published data that uses an experimental model or approach similar to your own. Due to their respective underlying rational reasons, there may be one clone that is more often used for your specific mouse model and experimental setup.

Bio X Cell provides a list of selected references for each antibody we offer, which can be found on the product page. Additional references can be found using search engines such as Google Scholar and PubMed. Searching for the clone-name of the antibody in combination with experiment-specific search terms, for example, “9H10 xenograft tumors BALB/c,” is the best approach to finding applicable references. These references will help you select the antibody that best fits your research.

Table 1: Detailed Comparison of anti-mouse CTLA-4 Antibody Clones

All of Bio X Cell’s InVivoPlus™ antibodies, including these anti-CTLA-4 clones, are rigorously tested to ensure that they are murine pathogen-free, have an endotoxin level of <1EU/mg, and a protein aggregation level of <5%. These clones are also available in our cost-effective InVivoMAb™ format. These alternatives are: 9D9 (BE0164), 9H10 (BE0131), and UC10-4F10-11 (BE0032). A detailed explanation of the differences between our InVivoPlus™ and InVivoMAb™ formats can be viewed here.

Recombinant anti-mouse CTLA-4 Antibody Clones

Bio X Cell also offers recombinant chimeric versions of our three anti-mouse CTLA-4 antibody clones. RecombiMAb™ antibodies are provided as chimeric antibodies with mouse constant regions instead of the typical rat or hamster constant regions. This ensures reduced immunogenicity in sensitive mouse models. Select RecombiMAb™ antibodies are also available in multiple Fc-silenced versions. These Fc-silenced versions have mutations in the Fc fragment of the antibody, rendering them unable to bind to endogenous Fcγ receptors.

Table 2: Detailed Comparison of Recombinant anti-mouse CTLA-4 Antibody Clones

Anti-Human CTLA-4 Antibody Clones

Bio X Cell also offers multiple anti-human CTLA-4 antibodies for use in humanized mouse models or immunoassays. These include clones BN13 and an Ipilimumab biosimilar antibody.

Table 3: Detailed Comparison of anti-human CTLA-4 Antibody Clones

Anti-Rat CTLA-4 Antibody Clone

Bio X Cell also offers an anti-rat CTLA-4 antibody, clone WKH203 (Lin and Hunig 2003), for use in experiments involving rat models. The WKH203 monoclonal antibody was generated against a purified rat CTLA-4hIg fusion protein, and this antibody neutralizes CTLA-4 in vitro.

Recombinant CTLA-4-Ig Fusion Protein

For investigating the functional properties of CTLA-4, we also offer a recombinant CTLA-4-Ig fusion protein. CTLA-4-Ig is a fusion protein consisting of the extracellular domain of human CTLA-4 linked to the Fc domain of human IgG. Our recombinant CTLA-4-Ig (hum/hum) is equivalent to the CTLA-4-Ig that was originally created at Bristol-Myers Squibb (BMS), i.e., BMS-188667), which was published by Linsley et al. (1999) in the Journal of Experimental Medicine (JEM). CTLA-4-Ig has been used in human, mouse, rat, and monkey species-based samples in various functional experiments. We offer CTLA-4-Ig in InVivoMAb™ as well as InVivoPlus™ versions.

Table 4: Details of InVivoMAb™ and InVivoPlus™ versions of Recombinant CTLA-4-Ig (hum/hum)

* Additional quality control measures for our InVivoPlus™ products include advanced binding validation, murine pathogen screening, protein aggregation screening, and ultra-low endotoxin levels. The superior quality of our InVivoPlus™ products will meet and exceed the strict demands and rigorous standards required for in vivo research. Learn more about the InVivoPlus™ difference here.

References

• Krummel MF, Allison JP. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J Exp Med. 1995 Aug 1;182(2):459-65. doi: 10.1084/jem.182.2.459.
• Lin CH, Hunig T. Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist. Eur J Immunol . 2003 Mar;33(3):626-38. doi: 10.1002/eji.200323570.
• Linsley P S, Brady W, Urnes M et al. CTLA-4 is a second receptor for the B cell activation antigen B7 J Exp Med . 1991 Sep 1;174(3):561-9. doi: 10.1084/jem.174.3.561.
• Quezada SA, Karl S Peggs et al. CTLA-4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J Clin Invest . 2006 Jul;116(7):1935-45. doi: 10.1172/JCI27745. Epub 2006 Jun 15.
• Simpson TR, Li F, Montalvo-Ortiz W et. al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med . 2013 Aug 26;210(9):1695-710. doi: 10.1084/jem.20130579
• Waldman AD, Fritz JM, Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol . 2020 Nov;20(11):651-668. doi: 10.1038/s41577-020-0306-5.
• Walunas TL, Lenschow DJ, Bakker CY et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity . 1994 Aug;1(5):405-13. doi: 10.1016/1074-7613(94)90071-x.

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