Targeting CD44 Variant 5 with an Antibody–Drug Conjugate Is an Effective Therapeutic Strategy for Intrahepatic Cholangiocarcinoma | Cancer Research

AS is a key regulator of most hallmarks of cancer (30–32), which has recently been investigated as a novel source of neoantigens (33). In this study, immunohistochemical analysis of ICC TMA identified that AS for CD44 occurs predominantly in ICC tumor tissue, but not in normal human tissue, and the potential role of CD44v as a therapeutic target for ICC. is showing. To this end, we generated and tested a panel of CD44v antibodies, then developed CD44v5-targeted ADCs that were proven to induce potent cytotoxicity. in vitro Acts against CD44v5-expressing cells and elicits potent anti-tumor activity in ICC PDX tumors. These data provide a rationale for conducting CD44v5-targeted testing and support the clinical efficacy of her H1D8-DC for CD44v5-positive ICC patients.

Targeted therapy aims to deliver drugs to cancer cells based on targeting specific genes or proteins that are unique to cancer cells or the tumor microenvironment, while sparing normal cells. The approach has shown significant advantages in clinical cancer intervention. However, the choice of target antigen remains the most important determinant of targeted therapy. ICC has enabled recent advances in the development of targeted therapies based on comprehensive genetic analyses. Multiple clinical trials are underway targeting tumor-specific molecular and genetic abnormalities such as IDH mutations (NCT02073994 and NCT02273739) and FGFR2 fusions (NCT01752920). However, their low incidence still limits the clinical application of these targeted therapies. Recently, Mr. Carless and his colleague (49) provided a comprehensive picture of splicing phenomena, revealing approximately 251,000 exon-exon junctions that predominantly occur in tumors but not in normal tissues. Claudin-18 splice variant 2 (Claudin 18.2), a highly selective gastric lineage marker, has been identified as a pan-cancer target in primary and metastatic lesions (50), the first-in-class Claudin 18.2-targeted mAb zolbetuximab achieved significantly superior efficacy and safety compared to control clinical treatments (51). These data indicate that tumor-specific AS are an excellent source of neoantigens. We and others demonstrated elevated levels of CD44 splice variants in cancer cells (23, 31, 39, 52). Similar results were demonstrated in the ICC in this study, with an unexpectedly >50% incidence of CD44v5-positive tumors, much higher than those of IDH mutations (~15%) and FGFR2 fusions (~10%). was expensive. Furthermore, no significant CD44v5 expression was detected in normal human tissues, suggesting that the CD44v5 splice isoform has great potential as an ideal target for ICC clinical therapy.

CSCs are a crucial group of tumor cells with self-renewal capacity, responsible for tumorigenesis and malignant transformation. Increased amounts of CSCs serve as an early event in the carcinogenesis of cholangiocarcinoma. In most solid tumors CSCs account for less than 3% of the total tumor mass, whereas in ICCs CSCs account for more than 30% of the tumor mass (53), which indicates that CSCs may be a new target for ICC therapy. To date, many surface markers of CSCs in ICC have been identified, including CD133, CD24, EpCAM, CD117, and CD44. Encouragingly, mAb-based therapeutics targeting CSC-associated surface biomarkers show great clinical potential.

CD44 is physiologically expressed on several cell types as a general marker for CSCs. CD44 targeting and inhibition by neutralizing antibodies (54), peptidomimetic (55), aptamers (56), and pharmacological inhibitors (57) have shown potent antitumor activity in many preclinical studies, and several of these strategies are undergoing clinical trials (NCT01358903). A growing number of studies are validating that CD44 variants, rather than the CD44 canonical isoform, are more specifically expressed in various cancers (see37), especially advanced ones (39, 40, 41), which has great potential as a clinical prognostic indicator (58–60). Tumor cells that express the CD44v isoform but not the CD44s isoform have enhanced tumor-initiating potential (61) and resistance to ROS stress (52), thus promoting tumor growth (52) and the transition (39). Mechanistically, the extra domains formed by the mutated exons after assembly with distinct ASs endow CD44 with additional functions as they can interact with and sequester different growth factors as well as cytokines. (17). Targeting tumor-specific CD44v by an antibody-based approach (62), specific peptides (63), and shRNA (64) showed promising antitumor effects. Furthermore, we observed that suppression of CD44 AS from CD44s to CD44v enhanced immunotherapeutic efficacy in triple-negative breast cancer (twenty three). These studies indicate that CD44v may be a promising therapeutic target and shed light on ICC-targeted therapeutic options.

Unfortunately, little progress has been made in CD44v-targeted therapy due to the high heterogeneity of CD44 splicing variants and technical difficulties in detecting, analyzing and manipulating CD44v. Theoretically, over 800 membrane-bound CD44 isoforms could be generated due to differences in variant exon usage. However, so far only a few of them have been identified. Interestingly, the expression pattern of each CD44v isoform appears to vary by cancer type (37). Todaro and his friends (31) revealed that CD44v6 predicts poor prognosis in colon cancer. CD44v8–10 is a specific marker for gastric CSC (65), whereas CD44v3 is specifically expressed in head and neck cancer CSCs (66, 67). The explanation for this phenomenon is still unknown. This is due to the lack of commercially available well-characterized CD44 and various CD44v identifying mAbs with standardized protocols. In this study, we investigated CD44 splicing across normal and ICC tissues based on the TCGA database and revealed for the first time that CD44v5 shares a distinct co-expression signature with other variants and CD44 in ICC (Supplementary Figure S1E). . Using an established mAb (H1D8) with high specificity and affinity to further characterize CD44v5 expression in ICC clinical specimens, the key role of CD44v5 as a potential tumor-selective target in ICC is demonstrated. Not only was it corroborated, it also provided suitable mAb candidates for the detection of CD44v5. Establishment of an ADC targeting CD44v5.

To date, more than 10 ADCs have been approved by the FDA for clinical cancer treatment, but many ADC candidates have favorable benefit and risk profiles, largely due to toxicity and limited efficacy at tolerated doses. could not be provided (48). Bivatuzumab mertansine, the first and only commercially available ADC targeting CD44v6, consists of an IgG-based humanized mAb and the tubulin inhibitor mertansine (DM1) linked by a disulfide linker. Discontinued due to severe on-target/off-tumor cutaneous toxicity (twenty one). Unlike vivatuzumab, the humanized anti-CD44v5 mAb H1D8 established in this study did not show significant staining in skin and other normal tissues. Moreover, H1D8-DCs with dipeptide linkers were remarkably stable under physiological conditions even after repeated freeze-thaw cycles (Supplementary Figure S10A) and were precisely hydrolyzed in the presence of lysosomal CTSB. Most importantly, we observed that CTSB was overexpressed not only in ICC (Supplementary Figure S9), but also in various other malignancies such as breast, lung, prostate and colorectal cancers. is(44). Low expression of CTSB limited the cytotoxicity of H1D8-DCs in the human keratinocyte cell line HaCaT (Supplementary Fig. S8). Consistently, no significant systemic toxicity was observed in xenograft mouse models (figure. 5G–I and 6D, and Supplementary Table S3). More importantly, CD44v5 antigen expression was not reduced after his H1D8-DC treatment in cancer cell lines or PDX-formed ICC mouse models, resulting in sustained anti-tumor effects of H1D8-DC. It’s what I was told. However, only one animal showed complete regression, whereas most other animals showed stable disease, indicating that H1D8-DC needs further improvement. The main reason may be that the target cells of H1D8 were CD44v5-positive CSCs. Many strategies have been established to target and eliminate CSCs, such as CSC signaling pathway inhibition.68), epigenetic regulators (69), and ADC (70, 71). Nevertheless, this remains a major challenge. Inherent resistance to cytotoxic agents (72) and increased proliferation capacity (Figure 6B) may contribute to the limited efficacy of H1D8-DC. Therefore, there is an urgent need to screen novel payloads that can induce potent cytotoxicity against CSCs. In addition to intrinsic resistance, heterogeneous expression of CSC markers also contributes to the limited efficacy of CSC-targeted ADCs (72). Similarly, CD44v5 was found not to be expressed in some tumor cells (Figure 6B and E.), potentially promoting tumor cell escape from H1D8-DC-induced cytotoxicity. More importantly, due to deficiencies in the immune system, ADC-induced indirect anti-tumor effects via antibody-dependent cytotoxicity, antibody-dependent cell phagocytosis, and induced immunogenic cell death were suppressed by PDX. This is something that the model cannot adequately explain. Furthermore, the pivotal role of ADC-induced CSC clearance in the remodeling of the immunosuppressive tumor microenvironment (73) are also ignored. therefore, bona fide The efficacy of H1D8-DC needs to be investigated systemically through further clinical trials. Overall, this study provides insight into CD44v5 as a potential therapeutic target for ICC and a rationale for further clinical investigation of CD44v5-targeted H1D8-DC-based approaches in ICC therapy.