ROR1 Antibodies: Research Progress and Clinical Translation of a Novel Tumor Therapeutic Target  

Biological Characteristics and Tumor Relevance of ROR1 Receptor

Receptor tyrosine kinase-like orphan receptor 1 (ROR1), a key member of the ROR receptor family, is a type I transmembrane protein widely expressed during embryonic development but minimally detectable in healthy adult tissues. Its molecular structure includes an extracellular immunoglobulin-like domain rich in cysteine residues, a coiled-coil region, and an intracellular tyrosine kinase-like domain. Although its kinase activity remains unconfirmed, ROR1 activates multiple pro-survival signaling pathways upon binding to ligands such as Wnt5a. Tumor genomic studies reveal aberrant ROR1 overexpression in various hematologic malignancies and solid tumors. Approximately 90% of chronic lymphocytic leukemia (CLL) cases exhibit ROR1 overexpression, with mRNA levels up to 1,000-fold higher than in normal B cells. In solid tumors like breast, lung, and ovarian cancers, ROR1 expression correlates with cancer stem cell properties, epithelial-mesenchymal transition (EMT), and metastatic potential. Triple-negative breast cancer patients with ROR1 positivity show a 35% lower five-year survival rate compared to ROR1-negative patients. This tumor-restricted expression pattern makes ROR1 an attractive therapeutic target.

Development Strategies and Structural Optimization of ROR1 Antibodies

ROR1-targeted antibody development focuses on three main designs: naked antibodies, antibody-drug conjugates (ADCs), and bispecific antibodies. First-generation naked antibodies (e.g., cirmtuzumab, UC-961) target ROR1’s immunoglobulin-like domain, inhibiting Wnt5a binding and inducing receptor internalization. Epitope mapping identifies the cysteine-rich domain (CRD) as the most therapeutically relevant region, critical for receptor dimerization and signaling. Humanization via CDR grafting and framework optimization reduces immunogenicity while maintaining nanomolar (KD ≈1–5 nM) binding affinity.

ADCs significantly enhance ROR1 antibodies’ therapeutic potential. Preclinical ROR1-ADCs employ cleavable linkers (e.g., vc-PABC) conjugated to microtubule inhibitors (MMAE or DM1), with drug-to-antibody ratios (DAR) of 3.5–4.0 balancing potency and toxicity. In vitro, these ADCs achieve picomolar IC50 against ROR1+ cells while sparing ROR1- cells (>1,000-fold selectivity). To improve tumor penetration, next-generation ROR1-ADCs use smaller scFv fragments linked to topoisomerase I inhibitors, demonstrating deeper tissue distribution and superior efficacy in pancreatic cancer PDX models.

Bispecific antibodies enhance immune cell recruitment. ROR1×CD3 bispecifics redirect T cells to tumors, showing activity even in low-ROR1-expressing solid tumors. Optimized 2:1 formats (two ROR1-binding domains, one CD3-binding domain) ensure specificity while minimizing cytokine release syndrome (CRS). Preclinical data indicate T-cell activation at 0.1–1 μg/mL, inducing tumor lysis with minimal on-target/off-tumor effects.

Mechanisms of Action and Preclinical Studies of ROR1 Antibodies

Naked ROR1 antibodies act via three mechanisms:

1. Signal blockade: Inhibiting Wnt5a-induced ROR1/ROR2 heterodimerization suppresses non-canonical Wnt signaling, reducing AKT (Ser473) and ERK (Thr202/Tyr204) phosphorylation by >60% within 24 hours, leading to cell-cycle arrest and apoptosis.
2. ADCC: Fc engineering (e.g., enhanced FcγRIIIa binding) boosts NK-mediated cytotoxicity from 20% to 80%.
3. Receptor internalization: Promotes ROR1 degradation.

ADCs deliver cytotoxic payloads directly. Upon internalization, lysosomal cleavage releases the payload, sustaining tumor-effective concentrations for >72 hours and increasing DNA damage markers (γH2AX) 5–8-fold. Some ROR1-ADCs exhibit "bystander effects," killing adjacent ROR1- cells—critical for heterogeneous tumors. In vivo, doses as low as 1 mg/kg reduce tumor volume by >50% with good tolerability.

Bispecific antibodies excel in immune activation. By bridging ROR1+ tumors and CD3+ T cells, they form immune synapses, inducing rapid T-cell infiltration (within 6 hours) and apoptosis (60–70% at 24 hours). Notably, they also elicit immune memory, protecting 90% of animals from tumor rechallenge.

Clinical Progress and Efficacy Evaluation

Multiple ROR1 antibodies are in clinical trials for hematologic and solid tumors:

• Cirmtuzumab (UC-961): In CLL Phase I (NCT02222688), monotherapy achieved 15% ORR, but combined with ibrutinib, ORR rose to 83% (10% CR). A pivotal Phase II trial (NCT03420183) is ongoing.
• VLS-101 (ROR1-MMAE): In Phase I/II (NCT03833180), ORR was 33% (DCR 67%) in heavily pretreated solid tumors (median prior lines: 4). Grade ≥3 TRAEs (45%) included neutropenia and neuropathy.
• NVG-111 (ROR1×CD3): Early-phase data (NCT04763083) show tumor marker declines at 5–15 μg doses, with manageable CRS (Grade 1–2). CD8+ T-cell activation correlates with response.

Resistance Mechanisms and Combination Strategies

Resistance emerges via:

• Target loss: ROR1 promoter methylation or transmembrane domain mutations.
• ADC resistance: Lysosomal dysfunction or MDR1 upregulation.
• Immune evasion: T-cell exhaustion or PD-L1-rich microenvironments.

Promising combinations:

• Immunotherapy: ROR1 bispecifics + anti-PD-1 increase complete response rates from 30% to 80% in preclinical models.
• Epigenetic modulators: HDAC inhibitors restore ROR1 expression.
• Signal inhibitors: AKT/ERK blockade synergizes with ROR1 targeting. An Ib/II trial (NCT04504916) combines ROR1-ADC with mTOR inhibitors, showing 2.5-month PFS improvement.

Biomarker-guided approaches:

• ctDNA: Monitors ROR1 dynamics.
• 89Zr-DFO-cirmtuzumab PET: Non-invasively assesses target expression (NCT03977103).
• AI-powered pathology: Predicts combination therapy responders.

Future Directions and Technological Breakthroughs

Next-gen antibody engineering:

• Site-specific ADCs (e.g., THIOMAB): Improve homogeneity.
• Dual-epitope antibodies: Target CRD + KNG domains for stronger signaling blockade.
• Prodrug antibodies: Activated by tumor-specific proteases (e.g., MMP-2) to reduce toxicity.

Cell therapies:

• ROR1-CAR-T/ CAR-NK: Fourth-gen CAR-Ts with cytokine (e.g., IL-12) expression show enhanced persistence in solid tumors. Local ROR1-CAR-NK injections control ovarian cancer ascites without GVHD. A first-in-human ROR1-CAR-T trial (NCT05274451) reported a metastatic breast cancer patient with >6-month response.

Diagnostics:

• Liquid biopsy: Detects ROR1+ exosomes (sensitivity: 0.01%).
• Immuno-PET: 68Ga-labeled nanobodies detect ≤100 ROR1+ cells.
• Microfluidic CTC analysis: Enables real-time ROR1 monitoring.

Within 5–10 years, optimized ADCs/bispecifics may gain accelerated approval (e.g., for ROR1-high TNBC), while CAR-Ts address solid tumor challenges. Personalized strategies integrating multi-omics data (transcriptome, proteome, microenvironment) will maximize patient benefit from this innovative target.

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