ROPN1 Antibody

What is ROPN1 and what is its expression pattern in normal tissues?

ROPN1 (Ropporin-1) is a 212-amino acid protein that belongs to the family of Cancer Germline Antigens (CGAs). In healthy tissues, ROPN1 expression is highly restricted, with significant expression limited to the testis and, to a lesser extent, the epididymis . This restricted expression pattern is crucial for its potential as an immunotherapy target, as both testis and epididymis are immune-privileged sites that lack MHC molecules and are generally ignored by T cells . Multiple gene expression databases have confirmed the absence of ROPN1 in other healthy organs, making it an ideal candidate for targeted therapies with minimal risk of on-target toxicity in normal tissues .

What is the relationship between ROPN1 and ROPN1B?

ROPN1 and ROPN1B are paralog proteins that share more than 95% amino acid sequence homology . Due to this high degree of similarity, antibodies developed against one protein may cross-react with the other. Both proteins have been classified as testis-selective Cancer Testis Antigens (CTAgs) . In research settings, it is often necessary to refer to them collectively as ROPN1/B when antibody specificity cannot definitively distinguish between the two . Despite their structural similarities, there may be functional differences that remain to be fully characterized.

What cancer types demonstrate significant ROPN1 expression?

ROPN1 expression has been documented in multiple cancer types, with particularly high prevalence in:

In comparative analyses, ROPN1 shows significantly higher expression rates than other established immunotherapy targets such as NY-ESO1 (expressed in only 16% of TNBC and 63.5% of melanoma patients) and MAGE-A4 (expressed in only 25% of TNBC patients) .

How is ROPN1 detected in experimental and clinical samples?

Detection of ROPN1 in research and clinical samples typically employs multiple complementary approaches:

• Gene expression analysis: RNA sequencing or qPCR for ROPN1 transcript detection

• Protein detection methods:

  • Multispectral immunohistochemistry (IHC) for tissue localization

  • Western blotting for protein size validation

  • Mass spectrometry for peptide identification

For IHC applications, researchers have successfully employed tissue microarrays to analyze ROPN1 expression patterns across multiple patient samples simultaneously . When performing IHC, nuclear SOX10 or cytoplasmic MLANA markers are often used as melanoma-specific controls to ensure proper sample quality and interpretation .

How can researchers validate ROPN1 antibody specificity?

Validating antibody specificity for ROPN1 requires a multi-step approach:

• Positive and negative controls: Use testicular tissue as a positive control and multiple non-expressing tissues as negative controls

• Knockout/knockdown validation: Test antibody reactivity in ROPN1 knockout cell lines or after siRNA knockdown

• Peptide blocking: Pre-incubate antibody with purified ROPN1 protein or peptide to demonstrate specific binding

• Cross-reactivity assessment: Test reactivity against ROPN1B to determine specificity between the paralogs

• Multiple antibody validation: Use antibodies targeting different epitopes of ROPN1 to confirm consistent staining patterns

Due to the high homology between ROPN1 and ROPN1B, it's critical to determine whether an antibody recognizes one or both proteins. Epitope mapping and sequence alignment analysis can help determine the likelihood of cross-reactivity .

What is the relationship between ROPN1 expression and cancer progression?

ROPN1 expression appears to correlate with cancer progression and potentially with immune responses:

• In melanoma patients, increased antibody reactivity against ROPN1A/B has been observed in more advanced disease stages, suggesting a relationship between ROPN1 expression and disease progression .

• In triple-negative breast cancer, a previous study indicated that ROPN1 activates RhoA signaling via rhophilin-1 (RHPN1), promoting cell migration, invasion, and metastatic potential .

• High ROPN1 expression levels have been associated with poor prognosis in triple-negative breast cancer .

Importantly, ROPN1 expression has been observed to be consistent between primary tumors and metastatic lesions. In TNBC, the majority of lymph node metastases showed ROPN1 expression in >50% of tumor cells, with patterns highly concordant with those observed in primary tumors .

How does ROPN1 expression compare with other cancer-testis antigens?

ROPN1 demonstrates several advantages compared to other established cancer-testis antigens used in immunotherapy development:

This comparative data shows that ROPN1 has uniquely favorable expression characteristics . Additionally, in melanoma samples expressing both ROPN1B and NY-ESO-1, ROPN1B was often detected ubiquitously throughout tumor cores, whereas NY-ESO-1 appeared more scattered and diffuse . In samples with co-expression, cells with exclusive ROPN1B positivity increased tumor cell coverage by an average of 29.3% (range: 9.0% to 73.0%) .

What are the optimal methods for identifying ROPN1 epitopes for antibody development?

Identifying optimal ROPN1 epitopes involves several complementary approaches:

• In silico prediction: Use algorithms like NetMHCpan to predict HLA-binding peptides from the ROPN1 sequence

• Immunopeptidomics: Employ mass spectrometry to identify naturally processed and presented ROPN1 peptides on MHC molecules

• Filtering strategies:

  • Assess protease cleavage sites

  • Evaluate TAP transporter affinities

  • Measure direct HLA binding through in vitro assays

• Cross-reactivity screening: Use tools like Expitope software to ensure peptide uniqueness to ROPN1/B

From ROPN1's 212 amino acids, over 200 theoretical HLA-A2-restricted peptides can be predicted. Through systematic filtering, researchers identified 11 peptides with significant HLA-A2 binding in a dose-dependent manner . The peptide FLYTYIAKV has been identified as a particularly promising epitope for TCR-based approaches .

How can ROPN1 be targeted in immunotherapy approaches?

Research has demonstrated multiple promising approaches for targeting ROPN1 in immunotherapy:

• T-cell receptor (TCR) engineered T-cells:

  • TCRs specific for ROPN1 epitopes (especially FLYTYIAKV) have been successfully engineered

  • These TCR-T cells have demonstrated effective killing of three-dimensional tumor organoids

  • FLY-1A TCR T-cells have shown superior efficacy compared to conventional treatments like cisplatin and Sacituzumab govitecan in TNBC models

• Vaccination strategies:

  • ROPN1 peptides can potentially be used as cancer vaccines

  • High immunogenicity observed in patient studies supports this approach

• Combination approaches:

  • ROPN1-targeted therapies could be combined with immune checkpoint inhibitors

  • Anti-PD-1 or anti-CTLA-4 therapies might synergize with ROPN1-specific approaches

  • Multi-antigen targeted immunotherapy combining ROPN1 with other cancer antigens may provide additive benefits

What is the immunogenic potential of ROPN1 in cancer patients?

ROPN1 demonstrates strong immunogenicity in cancer patients:

• In a study of 104 melanoma patients, antibodies against ROPN1A/B were detected in 71.2% (74/104) of patients .

• This compares favorably to NY-ESO-1, which showed antibody responses in 63.5% (66/104) of the same patient cohort .

• Distribution of ROPN1A/B antibody responses in melanoma patients:

  • Exclusive to ROPN1A/B: 19.2% (20/104)

  • Co-reactivity with NY-ESO-1: 51.9% (54/104)

  • No reactivity to either: 17.3% (18/104)

• In advanced melanoma (stages III and IV), 74.4% (64/86) of patients were seropositive for ROPN1A/B .

• No gender-related differences were observed in antibody reactivity rates:

  • Females: 75.0% (30/40)

  • Males: 69.8% (44/63)

This high rate of naturally occurring antibody responses indicates that ROPN1 is processed and presented to the immune system during cancer development, making it a promising target for immunotherapeutic interventions.

What controls should be included when studying ROPN1 expression?

When studying ROPN1 expression, researchers should include several critical controls:

• Tissue controls:

  • Positive control: Testicular tissue (known to express ROPN1)

  • Negative controls: Multiple healthy tissues (lung, liver, kidney, etc.)

• Cellular markers in multi-color IHC:

  • For melanoma: Nuclear SOX10 or cytoplasmic MLANA as tumor markers

  • For TNBC: Cytokeratin markers to identify tumor cells

• Comparative antigen controls:

  • NY-ESO-1 staining to compare expression patterns

  • Other cancer-testis antigens relevant to the tumor type

• Antibody controls:

  • Isotype control antibodies

  • Secondary antibody-only controls

  • Peptide competition/blocking controls

In studies of melanoma samples, nuclear SOX10 or cytoplasmic MLANA expression was detected in nearly all tumor cores (60/61 and 60/61, respectively), serving as effective positive controls for melanoma cells .

How can researchers distinguish between ROPN1 and ROPN1B in experimental settings?

• mRNA detection:

  • Design PCR primers targeting the few divergent regions between ROPN1 and ROPN1B

  • Use RNA-seq with specific bioinformatic pipelines to distinguish between the two transcripts

• Protein detection:

  • Develop antibodies targeting unique epitopes where sequences differ

  • Use mass spectrometry to identify peptides unique to each protein

• Functional studies:

  • Use isoform-specific knockdown approaches

  • Complement with isoform-specific rescue experiments

• Expression pattern analysis:

  • Compare tissue distribution patterns, which may reveal subtle differences

For many research applications, it may be appropriate to refer to them collectively as ROPN1/B when absolute specificity cannot be ensured .

What experimental models are suitable for studying ROPN1 function?

Several experimental models have proven effective for studying ROPN1 in cancer research:

• Cell line models:

  • TNBC cell lines with endogenous ROPN1 expression

  • Melanoma cell lines with confirmed ROPN1 expression

  • Engineered cell lines with controlled ROPN1 expression

• Three-dimensional models:

  • Patient-derived organoids that maintain ROPN1 expression

  • "Tumoroids" that better recapitulate tumor microenvironment

• In vivo models:

  • Patient-derived xenografts (PDXs) that maintain ROPN1 expression

  • Humanized mouse models for immunotherapy studies

Recent research has successfully employed three-dimensional tumor organoids to test ROPN1-targeted T-cell therapies. These organoids have demonstrated superior physiological relevance compared to traditional 2D cultures when evaluating the efficacy of TCR-engineered T-cells targeting ROPN1 .

What are the key unresolved questions about ROPN1 in cancer biology?

Despite significant progress, several important questions about ROPN1 remain to be fully addressed:

• Functional role in tumorigenesis:

  • Is ROPN1 expression merely a consequence of cancer-related epigenetic changes, or does it actively contribute to tumor development?

  • What signaling pathways are modulated by ROPN1 in different cancer types?

• Regulation of expression:

  • What mechanisms control ROPN1 upregulation in cancer cells?

  • Are there specific transcription factors or epigenetic modifications responsible?

• Relationship to treatment response:

  • Does ROPN1 expression correlate with response to standard treatments?

  • Could ROPN1 serve as a predictive biomarker for immunotherapy response?

• Functional differences between paralogs:

  • Do ROPN1 and ROPN1B have distinct functions in cancer cells?

  • Is there differential regulation of these paralogs during disease progression?

How might combination approaches enhance ROPN1-targeted therapies?

ROPN1-targeted therapies may benefit from strategic combinations:

• Immune checkpoint inhibitors:

  • Combining ROPN1-specific TCR-T cells with anti-PD-1/PD-L1 antibodies

  • Adding anti-CTLA-4 therapies to enhance T-cell activation

• Multi-antigen targeting:

  • Simultaneous targeting of ROPN1 and NY-ESO-1 could increase tumor cell coverage

  • In melanoma samples with co-expression, cells with exclusive ROPN1B positivity increased tumor cell coverage by 29.3% on average beyond NY-ESO-1 expressing cells

• Conventional therapies:

  • Sequencing with chemotherapy or radiation to enhance immunogenicity

  • Combination with targeted therapies relevant to specific cancer types

• Epigenetic modifiers:

  • Using drugs that upregulate ROPN1 expression to enhance immunotherapy efficacy

  • Combining with DNA methyltransferase inhibitors that may increase cancer-testis antigen expression

What are the most promising applications of ROPN1 antibodies in cancer research?

Based on current evidence, ROPN1 antibodies offer several valuable applications:

• Diagnostic and prognostic biomarker:

  • Identifying ROPN1-positive cancers that may be suitable for targeted therapies

  • Potential correlation with disease aggressiveness and outcome

• Patient stratification:

  • Selecting appropriate patients for ROPN1-targeted immunotherapies

  • Identifying those who might benefit from multi-antigen approaches

• Therapeutic monitoring:

  • Tracking changes in ROPN1 expression during treatment

  • Analyzing potential immune escape mechanisms

• Basic research applications:

  • Understanding the functional role of ROPN1 in cancer biology

  • Investigating potential signaling pathways and protein interactions

The high prevalence and homogeneous expression pattern of ROPN1 in both melanoma and TNBC make it a particularly valuable biomarker for these cancer types .

What protocols are recommended for optimal ROPN1 detection in research settings?

For optimal detection of ROPN1 in research settings, consider these methodological recommendations:

• Tissue processing:

  • Formalin-fixed paraffin-embedded (FFPE) tissues have proven suitable for ROPN1 IHC

  • Tissue microarrays can efficiently analyze multiple samples

• Immunohistochemistry:

  • Multispectral IHC provides advantages for co-expression studies

  • Include SOX10 or MLANA co-staining for melanoma studies

  • Use appropriate epitope retrieval methods

• Antibody selection:

  • Use well-validated antibodies with demonstrated specificity

  • Consider the potential cross-reactivity with ROPN1B based on research objectives

• Expression analysis:

  • Quantify both intensity and extent of expression

  • Report percentage of positive tumor cells for standardized comparison