CST BLOG: Lab Expectations

The official blog of Cell Signaling Technology (CST) where we discuss what to expect from your time at the bench, share tips, tricks, and information.

Application-Specific Antibody Validation: Finding an IHC-Validated Claudin-6 mAb

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Developing research antibodies is an extensive process that takes advantage of an animal’s adaptive immune response to produce reagent probes that bind to molecules of interest. Antibody scientists harness this unique ability of the adaptive immune system to identify and manufacture antibody reagents for use in various immunoassays, which represent some of the most powerful technologies available for studying biological samples.

However, identifying and developing an antibody reagent to a relevant target that binds with both specificity and sensitivity can take monthsor even yearsof research, experimentation, and validation. Antibody scientists at CST actively stay abreast of ongoing therapeutic trends and developments in our understanding of disease. Product development scientists use their insights and predictions to inform target selection and begin the search for highly specific, sensitive antibodies to hundreds of promising targets years in advance.

Related Resources: Hallmark of Validation eBook

Cell Therapy Challenges for Solid Tumors

Targeted immunotherapies including chimeric antigen receptor (CAR) T cells, antibody-drug conjugates (ADC), mRNA vaccines, T-cell redirecting bispeific antibodies, and cell-activating bispecific antibodies have been successful at treating blood cancers like leukemia. However, developing cell therapies to treat solid tumors found in cancers such as breast, lung, pancreatic, ovarian, and prostate remains elusive. Despite the fact that cancers of this type make up the overwhelming majority, as of 2023, there are no successfully engineered cell therapies that target solid tumors.

Immunotherapies work by teaching the body’s immune system to target cells that contain a designated marker. The therapy relies on the identification of cellular markers that are present in tumors, but, importantly, are not found in healthy tissue. Finding the right molecular targets is one of the biggest roadblocks that scientists face during the research and development of new therapeutics. Whenever a potential new target is identified, the race begins to develop the antibody research tools needed to study the novel target.

Moroever, immunohistochemical (IHC) analysis of FFPE tissue samples is a critical component for solid tumor research. IHC is a challenging application for antibody development to begin with: the complexity of tissue samples and their specialized preparation–as compared to other techniques like western blot (WB) and immunofluorescence (IF)–requires scientists to approach antibody validation for IHC carefully, to ensure specificity and sensitivity. Here’s the story of how our talented team of scientists at CST developed an IHC-validated antibody to Claudin-6, a promising drug target in immuno-oncology that has gained prominence in recent years.

The Race for IHC-Validated Claudin-6 Research Antibodies

Claudin-6 (CLDN6), a member of the claudin family of tight junction proteins, is a novel target that has gained prominence in recent years. Expressed during embryonic and fetal development but normally transcriptionally silenced in adult tissues, it is re-expressed on the surface of several epithelial cancers including ovarian, testicular, and endometrial cancer. The absence of Claudin-6 in normal adult tissue and its presence in tumors makes it a good target for immuno-oncology research.

While Claudin-6 was first discovered in the 1990s, it wasn’t until recently that interest in the target exploded. Currently, BioNTech, Xencor, NovaRock, Amgen, I-Mab, Chugai, Daiichi Sankyo, and AbbVie all have research programs exploring its therapeutic potential.

CST scientists were following early research exploring the oncologic function of this exciting target, and initiated antibody development campaigns in an effort to identify Claudin-6-specific antibodies that could be utilized by researchers studying this target. The use of specific and sensitive antibodies during the research phase is critical to the later development of safe and successful immunotherapies so that engineered T cells do not target healthy tissue. For example, although Claudin-6 is rarely expressed in adult tissue, a closely related protein, Claudin-9 (CLDN9), is structurally similar to Claudin-6 and is expressed in high levels throughout the body.

In addition to specificity and sensitivity, a successful antibody for Claudin-6 needs to work in IHC assays due to the widespread use of this technique in immuno-oncology research.

Identification and Validation of an Anti-Claudin-6 Monoclonal Antibody

CST scientists initiated multiple projects before a successful monoclonal antibody for Claudin-6 was identified that worked in multiple applications, including IHC.

To begin the search, a binary model system was utilized that demonstrated overexpression of the target in the cell line OVCAR-3 (+), and low or negative expression of the cell line DU 145 (-). Next, western blotting using hundreds of antibody samples was conducted to look for antibodies that detected proteins of the correct molecular weight—23 κDa, in the case of Claudin-6—in OVCAR-3 derived lysates, and showed no signal in the DU 145 derived lysates.

Susan Kane, PhD, Principal Scientist at CST, picks up the story, “Once the monoclonal antibody candidates are deemed promising by WB, we test them in the applications of interest using a similar binary system, as well as other validation strategies to confirm antibody specificity and sensitivity, such as screening expression levels in tissues of interest in relevant disease states.”

Using CST® Hallmarks of Antibody Validation strategies, the team was able to validate a monoclonal antibody for WB, immunoprecipitation (IP), and immunofluorescence (IF-IC): Claudin-6 (E2S5M) Rabbit mAb #62831.

22-BCH-99750-NOV23 FIG1

Figure 1. Some of the validation data for product Claudin-6 (E2S5M) Rabbit mAb #62831. Left: IF analysis of OVCAR-3 and DU 145 cells using #62831. Right: WB analysis of extracts from OVCAR3 and DU 145 cells using #62831 (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, DU 145 is low or negative for Claudin-6 expression.

 

However, all of the initial clones identified by the WB screen failed during IHC validation, as shown in Figure 2. “Screening antibody clones for many applications, but most especially IHC, is an arduous process,” explains Susan. “For IHC, the intricacy of testing grows due to the inherent complexity of tissue. Our IHC scientists often look at hundreds of clones for a given target, searching through staining patterns on slides to identify similarities, differences, discrepancies, and oddities, etc.”

22-BCH-99750-NOV23 FIG2

Figure 2. IHC analysis of paraffin-embedded OVCAR-3 cell pellet (left, positive) or DU 145 cell pellet (right, negative) using Claudin-6 (E2S5M) Rabbit mAb. Non-specific nuclear signal is observed in both cell pellets at the first dilution point. With titration, the non-specific signal is largely eliminated from the negative cells, but strong nuclear signal remains in some OVCAR-3 cells, while limited specific signal remains. Clone E2S5M was not validated for IHC.

Why did an antibody shown to work in WB, IP, and IF fail validation in IHC?

Because of the variation in sample preparation for different application protocols, independent validation in each application is the only way to ensure that an antibody will work in that application.1 This is especially important in IHC, where tissue processing can greatly affect antibody reactivity. Formalin fixation during the preparation of FFPE tissue affects the antigenicity of the target antigen due to the formation of methylene bridges, which can modify protein conformation and antigen binding sites (epitopes).2 While antigen retrieval techniques such as the use of proteases (trypsin, proteinase K) or heat can partially restore antigen reactivity, the fact remains that antibody performance in FFPE samples can vary greatly compared to other application types, especially cellular assays like immunocytochemistry (ICC).

“Part of the challenge with validating antibodies as rigorously as we do at CST is that we look for known expression in various tissue types,” explains Susan. “However, for cutting-edge targets like Caudin-6, where expected expression is still being researched and unusual results are being discovered, it can be challenging to truly validate an antibody up to our standards. To ensure that our IHC antibody was binding to Claudin-6 and only Claudin-6, we had to get creative and employ additional methods, such as LC-MS (liquid chromatography-mass spectrometry) proteomics, to confirm our results.”

In tandem with the search for an IHC-validated Claudin-6 antibody, another internal team at CST was already hard at work analyzing levels of Claudin-6 using LC-MS to identify FFPE tumor blocks that express Claudin-6. Using the wealth of proteomics data produced, the IHC team was able to leverage additional tissue samples determined to have high, medium, and low expression levels of Claudin-6 to find promising antibody clones. Once promising clones were identified after multiple rounds of testing on large numbers of diseased and normal tissues, staining patterns obtained in the mass-spec-characterized tissues helped to confirm that the clones were staining Claudin-6 specifically.

After going back to the drawing board multiple times and retesting antibodies that had failed at different development stages and for various reasons, the team finally struck (antibody) gold: a monoclonal antibody that was shown to work with specificity and sensitivity in IHC applications on FFPE tissue.

22-BCH-99750-NOV23 FIG3

Figure 3. IHC analysis of various paraffin-embedded human tissues: serous papillary carcinoma of the ovary (left-top), small cell carcinoma of the salivary gland (middle-top), papillary thyroid carcinoma (right-top), hepatocellular carcinoma (left-bottom), non-Hodgkin lymphoma (middle-bottom), and pulmonary sarcoma (right-bottom) using Claudin-6 (E7U2O) XP® Rabbit mAb.

22-BCH-99750-NOV23 FIG4

Figure 4. IHC analysis of various paraffin-embedded normal human tissues: placenta (left-top), appendix (middle-top), esophagus (right-top), brain (left-bottom), pancreas (middle-bottom), and tonsil (right-bottom) using Claudin-6 (E7U2O) XP® Rabbit mAb.

 

The final step in the process was to ensure that there was no cross-reactivity of clone E7U2O with structurally similar proteins, such as Claudin-9. Additional testing in WB and IHC was conducted to confirm that clone E7U2O reacted with Claudin-6 and only Claudin-6. This clone eventually became Claudin-6 (E7U2O) XP® Rabbit mAb #18932.

22-BCH-99750-NOV23 FIG5

Figure 5. IHC analysis of paraffin-embedded 293T cell pellet, untransfected (left) or Claudin-9 transfected (right), using Claudin-6 (E7U2O) XP® Rabbit mAb (top) or DYKDDDDK Tag (D6W5B) Rabbit mAb #14793 (bottom).

 

“The role of antibody development and application scientists at CST is to try to get an antibody candidate to fail,” explains Susan. “When we’ve tested every possibility we can think of, and the antibody still works as expected, then we know it’s truly specific and sensitive enough to release to customers. Finally getting an antibody clone like E7U2O to pass for IHC is better than birthday cake and balloons!

A highly specific monoclonal antibody against Claudin-6 for use in IHC is a critical tool for investigating new approaches in the treatment of adult solid tumors. The rigor with which CST antibodies are validated for IHC can increase confidence and reliability in the results produced, hopefully leading to new therapeutic possibilities on the road ahead.

Visit the CST® product catalog to learn more about Claudin-6 (E7U2O) XP® Rabbit mAb #18932.

22-bch-99750

Select References 

  1. Mariaville C, Martineau P. Antibody Identification for Antigen Detection in Formalin-Fixed Paraffin-Embedded Tissue Using Phage Display and Naïve Libraries
    Antibodies (Basel). 2021;10(1):4. Published 2021 Jan 14. doi:10.3390/antib10010004
  2. Bayer, M., Angenendt, L., Schliemann, C., Hartmann, W., & König, S. (2019). Are formalin-fixed and paraffin-embedded tissues fit for proteomic analysis? Journal of Mass Spectrometry, 55(8).
  3. Chen A. In the search for therapies for solid tumors, companies are turning to a novel target: claudin-6. STAT.  https://www.statnews.com/2023/01/18/cancer-therapies-solid-tumors-claudin-6-cldn6/. Published January 18, 2023. Accessed September 25, 2023.
    G06
Alexandra Foley
Alexandra Foley
Alexandra is a scientific marketing writer at CST and the editor of the Lab Expectations blog.

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