Chondroitin sulfate proteoglycan 4: An attractive target for antibody-based immunotherapy

Abstract

Multifunctional molecules involved in tumor progression and metastasis have been identified as valuable targets for immunotherapy. Among these, chondroitin sulfate proteoglycan 4 (CSPG4), a significant tumor cell membrane-bound proteoglycan, has emerged as a promising target, especially in light of advances in chimeric antigen receptor (CAR) T-cell therapy. The profound bioactivity of CSPG4 and its role in pivotal processes such as tumor proliferation, migration, and neoangiogenesis underline its therapeutic potential. We reviewed the molecular intricacies of CSPG4, its functional attributes within tumor cells, and the latest clinical-translational advances targeting it. Strategies such as blocking monoclonal antibodies, conjugate therapies, bispecific antibodies, small-molecule inhibitors, CAR T-cell therapies, trispecific killer engagers, and ribonucleic acid vaccines against CSPG4 were assessed. CSPG4 overexpression in diverse tumors and its correlation with adverse prognostic outcomes emphasize its significance in cancer biology. These findings suggest that targeting CSPG4 offers a promising avenue for future cancer therapy, with potential synergistic effects when combined with existing treatments.

1. Introduction

Research into ever-evolving tumor-host interactions has elucidated the mechanisms via which tumor cells resist therapeutic interventions. Findings from such studies have invigorated the quest to identify multifunctional molecules that are pivotal to tumor progression and metastatic processes, making them invaluable targets for immunotherapeutic interventions. Capitalizing on immunotherapy against such molecular players presents potential approaches for neutralizing the multifaceted evasion mechanisms of tumor cells.

Advances in chimeric antigen receptor (CAR) T-cell therapy, as demonstrated in recent findings, exhibit favorable therapeutic outcomes in hematological malignancies.¹⁾ However, translating these successes into solid tumors remains challenging. A critical determinant of this translation is the judicious selection of target antigens, ensuring specificity, minimal off-target effects, and broad applicability across tumor types. Within this complex molecular framework, we focused on a salient tumor cell membrane-bound proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular-weight melanoma-associated antigen (HMW-MAA),²⁾ melanoma-associated chondroitin sulfate proteoglycan (MCSP), and neuron-glial antigen 2 (NG2).³⁾

CSPG4 is a molecule with profound bioactivity and a component of the CSPG family, alongside cluster of differentiation (CD) 44. CSPGs are involved across a spectrum of malignancies, steering pivotal processes such as tumor proliferation, orchestrated migration, and the intricate process of neoangiogenesis. CSPG4 upregulation is frequently correlated with poor prognostic outcomes in patients presenting with diverse tumors.⁴⁾

Given its characteristics and the need for effective targets in solid tumors, CSPG4 has emerged as a promising candidate for CAR T-cell therapy and other immunotherapeutics.

In this review, we examine the multifaceted mechanisms of CSPG4, exploring its functional attributes within tumor cells and its potential in targeted therapeutic strategies.

2. CSPG4 structure and distribution in malignant cells

Over the past 40 years, numerous studies have analyzed the structure and functions of CSPG4, also known as HMW-MAA,²⁾ NG2, or MCSP⁵⁾ (Fig. 1). CSPG4 is a type I transmembrane proteoglycan characterized by a core protein to which several chondroitin sulfate (CS) glycosaminoglycan chains are covalently attached.⁴⁾ The structural complexity of CSPG4 arises from its multi-domain core protein, which facilitates a plethora of interactions with extracellular matrix components, growth factors, and cell surface receptors.⁶⁾ These interactions play pivotal roles in multiple cellular processes, influencing not only cell-to-cell communication but also the broader cellular environment.

Fig. 1

Structure and location of chondroitin sulfate proteoglycan 4 (NG2/CSPG4). Schematic representation of the proposed structure and functions of CSPG4 in cancer. CSPG4 has three extracellular domains: Domain (D) 1, D2, and D3. D1 consists of two laminin G-like domains (L1 and L2) proposed to interact with the extracellular matrix (ECM). D2 consists of 15 CSPG repeats containing chondroitin sulfate chain decoration.

The extracellular CSPG4 domain is vast and predominantly rich in CS chains. These chains endow CSPG4 with a unique ability to interact with a diverse set of molecules, thereby modulating numerous signaling pathways.⁷⁾ At a deeper molecular level, the intracellular domain of CSPG4 harbors specific phosphorylation sites. Activation of these sites can lead to the initiation of several intracellular cascades, which in turn have a profound impact on cell growth, migration, and survival mechanisms.⁸⁾

CSPG4 expression is not limited to malignant cells but is also found in endothelial cells of the tumor vasculature, underscoring its multifaceted role in tumor biology (Table 1).

Table 1. CSPG4 expression in solid tumors

Cancer type	CSPG4 expressing tumor tissues	Clinical correlation	References
CSPG4, chondroitin sulfate proteoglycan 4; TNBC, triple-negative breast cancer.
Oral squamous cell carcinoma	61%	Prognostic relevance	9)
Pancreatic ductal adenocarcinoma	100%	No prognostic relevance	10)
Hepatocellular carcinoma	63.6%	Prognostic relevance	11)
Breast cancer	72.7% (TNBC)	Prognostic and recurrence relevance	4),12)
Ovarian cancer	36.68%	Prognostic relevance	13)
Melanoma	\(>\) 70%	Prognostic relevance	14)
Glioblastoma	20–100%	Prognostic relevance	15)

In melanoma cells, CSPG4 expression is significantly elevated and correlates with increased tumor aggressiveness and poor prognosis. Beyond melanomas, CSPG4 overexpression extends to various cancers, such as basal-like breast cancers, glioblastoma multiforme, and pancreatic carcinomas.^9)-15)

The association of breast cancer with CSPG4, particularly in the context of triple-negative breast cancer (TNBC), has garnered significant attention. Focusing on TNBC is crucial because it represents a critical gap in targeted therapies. TNBCs, comprising 15% of all breast cancers, lack expression markers for estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2), rendering them resistant to current hormone and HER2-targeted treatments. Their aggressive nature and lack of available targeted therapies underscore the pressing need for innovative treatment avenues. CSPG4 expression has been documented in primary TNBC lesions and metastatic tumor cells, including cancer stem cells.⁴⁾

Although not exclusive to basal breast cancers such as TNBCs, CSPG4s might be linked to adverse outcomes and recurrence.¹²⁾ Current research indicates an association between carbohydrate sulfotransferase-11 (CHST11) expression (encoded by CHST11), which is responsible for decorating CSPG4 with CS, and the metastatic behavior of TNBC cells.¹⁶⁾ CHST11 overexpression in aggressive breast cancers bolsters the interaction between p-selectin and CSPG4, which is consistent with the proposed role of CSPG4 as an intermediary between the extracellular matrix and intracellular signaling pathways and its function as the metastatic driver, P-selectin. P-selectin is suggested to enable cancer cells to counteract immune responses, facilitate binding to endothelial cells, and promote hematogenous spread.¹⁷⁾ Comprehensive research is pivotal to decoding the implications of CSPG4 in breast cancer evolution and progression.

Head and neck squamous cell carcinomas (HNSCCs) typically manifest a grim prognosis, with a 5-year survival rate of 40–50%. Warta et al.⁹⁾ identified a significant correlation between CSPG4 overexpression in HNSCC cells and poor prognosis in patients with low CSPG4 expression. With a dearth of biomarkers for HNSCC survival prediction, CSPG4 presents promise as a potential prognostic indicator in future studies.

There is a significant contrast in CSPG4 expression between malignant and normal cells. Although some normal cells manifest CSPG4 expression, the levels are notably lower than those in their malignant counterparts.¹⁸⁾ This differential overexpression in cancer cells, juxtaposed with its muted presence in normal cells, accentuates the attractiveness of CSPG4 as a therapeutic target. Emerging studies have highlighted the potential of CSPG4-specific CAR T cells. These cells present a promising therapeutic avenue, particularly against melanomas, targeting not only CSPG4-expressing tumor cells but also addressing the challenges of CSPG4-induced resistance in melanoma treatments.¹⁹⁾

The intricate structure and distribution of CSPG4 on malignant cells indicate its profound importance in cancer biology. Given its widespread overexpression across various cancer types and its multifunctional nature, CSPG4 is a molecule of notable clinical and therapeutic interest.

3. Functional properties of CSPG4

CSPG4 is not merely a multifunctional proteoglycan but also central to various cellular processes that span both physiological development and pathological domains. Furthermore, CSPG4 presents considerable influence, particularly in oncology (Fig. 2).³⁾

Fig. 2

Chondroitin sulfate proteoglycan 4 (NG2/CSPG4) functionality. Potential contributions of CSPG4 in cancer growth, vascularization, dissemination, and metastasis. These may provide opportunities for therapeutic interventions targeting CSPG4.

A core function of CSPG4 is its role in cell adhesion and migration. It enhances cell-extracellular matrix interactions, acting as a pivotal co-receptor for integrins and influencing diverse physiological processes, including tissue regeneration and wound healing.⁴⁾ Moreover, CSPG4 plays a role in pathology; its influence on cell adhesion acts as a catalyst for tumor invasion and metastasis, exacerbating the aggressive process of several malignancies.⁴⁾ The molecular interplay between galectin-3 and CSPG4 further refines cellular migratory pathways, adding another layer of complexity to cellular movement dynamics.⁶⁾ Recent investigations have also highlighted the potential role of CSPG4 in modulating tumor microenvironments, offering novel perspectives on tumor-host interactions and therapy responsiveness (Fig. 3).⁸⁾

Fig. 3

Signaling pathways. Chondroitin sulfate proteoglycan 4 (CSPG4) activates two major signaling cascades via its cytoplasmic domain: (i) the focal adhesion kinase (FAK) integrin signaling pathway and (ii) the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK1/2) pathway. (i) The scaffold protein syntenin physically interacts with Src, and simultaneously, integrin engagement recruits and stimulates the autophosphorylation of FAK. Syntenin then promotes FAK/Src complex formation to initiate phosphorylation of p130cas and PI3K. p130Cas phosphorylation initiates activation of the small Rho GTPase Rac, leading to cytoskeletal reorganization. PI3K phosphorylation activates AKT, which regulates the transcriptional activity of NF- \(\kappa\) B, leading to survival chemoresistance. (ii) CSPG4 activates MAPK/ERK1/2 signaling via receptor tyrosine kinase (RTK)-dependent and independent mechanisms. CSPG4 activation in turn activates small GTPase Ras, which stimulates MEK phosphorylation and then ERK1/2 phosphorylation. ERK1/2 then upregulates MITF and c-Met, leading to enhanced EMT. ERK1/2 also has other targets and, along with Rac, promotes migration, proliferation, and angiogenesis.

In terms of cell proliferation, the interaction of CSPG4 with growth factors can potentially incite aberrant cell proliferation, a hallmark of numerous cancers.⁷⁾ One interaction in particular involves the platelet-derived growth factor receptor- \(\beta\) , which orchestrates a host of intracellular signaling events, thereby influencing cell cycle dynamics.²⁰⁾ Emerging studies have emphasized the implications of these interactions in therapy resistance, shedding light on the challenges and opportunities in designing therapeutic interventions.¹⁸⁾

CSPG4 plays a pivotal role in angiogenesis. Tumors rely heavily on an intricate vascular network to facilitate growth. Here, CSPG4 supports neovascularization, facilitating the growth of these lifelines.²¹⁾ Its interaction with the vascular endothelial growth factor underscores its pivotal role in angiogenesis, a process vital for tumor sustenance.²²⁾ Moreover, recent studies have indicated the potential of CSPG4 to facilitate a conducive environment for tumor progression, reinforcing its centrality in oncology.

CSPG4 plays various roles in the tumor microenvironment. Although it can construct immune cell infiltration patterns and potentially dampen antitumor immune responses,⁴⁾ its association with a niche group of cells, known as cancer stem cells, is more complex. These cells, often implicated in therapy resistance and recurrence, accentuate the therapeutic significance of CSPG4.⁴⁾ Furthermore, the influence of CSPG4 on the immune landscape, especially its potential to modulate T-cell responses, offers significant prospects in the field of immunotherapy.²³⁾

Numerous studies have delved into the interplay between CSPG, its degradation byproducts, and immune system components. In particular, CS stimulates monocytes to release interleukin (IL)-1 \(\beta\) and induce B cell proliferation in vitro.²⁴⁾ Aoyama et al.²⁵⁾ further confirmed the effect of CS on B-cell proliferation, demonstrating its ability to enhance murine B cell proliferation in vitro via protein kinase C translocation and protein kinase B activation kinase. The potential role of CS in dendritic cell maturation was highlighted when human monocyte-derived dendritic cells, cultured in vitro with CS, extracellular matrix components, and human granulocyte-macrophage colony-stimulating factor (GM-CSF), differentiated more rapidly than those cultured with GM-CSF and IL-4.²⁶⁾ Another study, using splenocytes from ovalbumin-immunized mice, revealed that CS stimulated the secretion of type 1 T helper-type cytokines, including interferon- \(\gamma\) , IL-2, and IL-12, while suppressing type 2 T helper-type cytokines (IL-5 and IL-10).²⁷⁾ Furthermore, injecting BALB/c mice with CS and other glycosaminoglycans induces autoimmune conditions, such as rheumatoid arthritis, by recruiting CD4 \(^+\) T cells.²⁸⁾ Conversely, low-molecular-weight disaccharide fragments of CSPG showed potential control of inflammatory responses in various animal models by reducing inflammatory T-cell migration and activation.²⁹⁾

In addition, early research identified CSPGs within human natural killer (NK) cell granules, and their release during NK cell-mediated tumor cell cytotoxicity.³⁰⁾ Another study involving primary cultured human macrophages found CSPGs as metabolic byproducts of macrophages, with an increase in secreted CSPG4 following lipopolysaccharide stimulation.³¹⁾

A recent immune monitoring study revealed the presence of CSPG4-reactive CD4 \(^+\) T cells in both healthy individuals and those with melanoma. However, no significant correlation between T-cell responses against a human leukocyte antigen-DR isotype-presented CSPG4 peptide and patient tumor burden was observed. Notably, a smaller proportion of patients with melanoma exhibited T-cell reactivity to CSPG4 than that in healthy volunteers.³²⁾ Other in vivo research indicated that LPS induced murine CSPG4 ortholog expression in rat microglia cells. Further in vitro tests demonstrated that NG2 ribonucleic acid (RNA) silencing in lipopolysaccharide-treated microglia inhibited messenger RNA (mRNA) expression of certain proinflammatory cytokines, emphasizing the potential regulatory role of NG2 in cytokine expression.³³⁾

In conclusion, CSPGs, their carbohydrate chains, and degradation products, including CSPG4, seem to modulate various immune cell functions. However, the precise roles of CSPG4 in cancer immunology remain largely uncharted. As such, further studies are essential to determine if interactions between CSPG4, its components, and the immune system can be harnessed to improve CSPG4-targeted immunotherapy outcomes or counteract potential adverse immunomodulatory effects.

Beyond the confines of oncology, the role of CSPG4 extends to the central nervous system, including conditions such as multiple sclerosis and other neuro-inflammatory disorders.³⁴⁾ Additionally, the potential implications of CSPG4 in neural plasticity and repair have been the subject of recent scientific inquiries, broadening our understanding of this proteoglycan.

In summary, CSPG4, with its multifarious functional attributes and impact across a spectrum of pathological conditions, is a molecule of paramount therapeutic potential. Its intricate roles in cancer progression and emerging significance in shaping treatment landscapes place it at the forefront of contemporary cancer research.

4. Clinical-translational advances

CSPG4 offers innovative approaches to the rapidly evolving field of cancer therapeutics. Greater insight into its molecular architecture, coupled with its multifaceted role in oncogenesis, has catalyzed a myriad of clinical-translational endeavors. A select group of monoclonal antibody (mAb) clones recognizes CSPG4. Among these, the murine clone 225.28, established by one of the authors (K.I.), is frequently mentioned in the literature.³⁵⁾ Initial studies showcased the antitumor efficacy of methotrexate-conjugated murine 225.28 in human melanoma xenografts in nude mice.³⁵⁾ Although this conjugated mAb outperformed methotrexate alone, the efficacy of the mAb in isolation remains to be explored. In severe combined immunodeficiency disease (SCID) mice with melanoma tumors, treatment with murine mAb 225.28 resulted in smaller tumors; moreover, it led to altered expression of tumor suppressor genes and metastasis-related genes.³⁶⁾

Beyond melanoma, this mAb inhibited various cancer cell activities and downregulated tumor-promoting pathways in vitro. It also curbed tumor growth in human TNBC cell line-derived lung metastasis models and reduced post-surgical tumor recurrence in SCID mice.⁴⁾ Furthermore, mAb 225.28 enhanced the efficacy of the chemotherapy agent Cytarabine against 11q23 acute lymphoblastic leukemia cells.³⁷⁾ An engineered version of this mAb with a human Fc region demonstrated its ability to mediate antibody-dependent cellular phagocytosis and restrict tumor growth in a melanoma NOD SCID \(\gamma\) murine model.³⁸⁾ Notably, the choice of Fc domain and antibody isotype is crucial in therapeutic mAb design, as evidenced by the contrasting effects of anti-CSPG4 immunoglobulin (Ig) G4 and its IgG1 analog. The original murine anti-CSPG4 clone, 225.28, exhibited direct cancer cell inhibition, prompting discussions on the impact of mAb modifications on their therapeutic properties.

CSPG4 has been targeted in other malignancies using different antibody clones. For instance, the murine mAb clone, TP41.2, was used against malignant mesothelioma, displaying in vitro antitumor effects and prolonging the survival of mesothelioma-bearing SCID mice.³⁹⁾ Another study used scFv-FcC21, which inhibited the growth and migration of a TNBC cell line in vitro and reduced lung metastasis in SCID mice.⁴⁰⁾ When conjugated to polyethylene glycol, an anti-mouse CSPG4/NG2 antibody clone restricted tumor growth in glioblastoma-engrafted athymic rats.⁴¹⁾ The therapeutic efficacy was attributed to tumor-infiltrating macrophages, although the exact mechanism remains elusive. Notably, the antigenic determinants of these mAbs are speculated to recognize the CSPG4 core protein, irrespective of the presence of CS. However, this hypothesis warrants further validation.^42),43)

In summary, although certain anti-CSPG4 antibody clones have shown therapeutic potential across various cancer types and models, most studies utilized antibodies with mouse Fc regions. Given the promising results against CSPG4-expressing tumors, further research, especially with constructs engineered with human Fc regions, is imperative.

4.1. Blocking mAbs.

mAbs tailored against CSPG4 have emerged as a significant oncology treatment. Early preclinical and clinical studies have showcased the efficacy, with mAbs selectively targeting CSPG4, thereby mitigating its oncogenic functions.^{3),4),35),36),44),45)} Recent advances have highlighted the therapeutic potential of mAbs, especially when integrated within combined strategies, heralding new therapeutic paradigms.⁸⁾

4.2. Antibody-dependent and conjugate therapy against CSPG4.

Utilizing the strength of the immune system, innovative strategies involve conjugating anti-CSPG4 antibodies with cytotoxic agents, marking CSPG4-abundant tumor cells for targeted destruction. This approach amplifies therapeutic precision and safeguards healthy tissues.^46),47) Emerging data have accentuated the versatility of this approach across a spectrum of tumor types, underscoring its broad therapeutic potential.⁴⁸⁾

4.3. Bispecific antibodies: bridging immunity and targeted therapy.

Bispecific antibodies, crafted to engage two distinct molecular targets, serve as conduits between CSPG4 and immune effector cells (Fig. 4). This dual-engagement strategy amplifies antitumor responses, blending the precision of targeted therapy with immunotherapy effectiveness.⁴⁹⁾ Recent studies have reported on the promising outcomes of this modality, especially in challenging tumor microenvironments.

Fig. 4

Bispecific antibody therapy. Bispecific T-cell engager antibodies redirect cytotoxic T cells toward chondroitin sulfate proteoglycan 4 (CSPG4)-overexpressing cells.

4.4. Small-molecule inhibitors: the next generation of therapeutics.

The efficacy of anti-CSPG4 mAbs in enhancing the growth-inhibitory impact of vemurafenib, a very rapidly accelerated fibrosarcoma murine sarcoma viral oncogene homolog B1 (BRAF) with V600E mutation (BRAFV600E) inhibitor, on cultured melanoma cell lines underscores the role of the proteoglycan in facilitating chemoresistance.⁵⁰⁾ There is also evidence pointing toward the potential role of CSPG4 in drug resistance.

Chemoresistance is a pivotal challenge in treating melanomas and other malignancies. Although many patients initially show positive responses to therapy, over time, resistance emerges and the cancer advances. Notably, the drug resistance of melanomas appears to be closely tied to the use of specific single-target inhibitors, such as those targeting BRAFV600E. The expression of CSPG4 correlates with multidrug resistance in experimental models of glioblastomas and melanomas, an effect tied to its interaction with the integrin-triggered activation of phosphatidylinositol 3-kinase pathways.⁵¹⁾ Collectively, these findings highlight the promise of targeting CSPG4 in adjunctive therapy for melanoma and certain other tumors, either through immunotherapeutic strategies or the creation of CSPG4-specific small-molecule inhibitors.

4.5. CSPG4 CAR T-cell therapy.

CAR T cells offer a notable avenue in T cell-based therapeutics, leveraging mAbs currently under evaluation for efficacy against CSPG4. These CAR T cells undergo genetic modification to express a chimeric receptor derived from the targeting fragment of a monoclonal antibody (single-chain fragment variable) specific to the desired antigen (Fig. 5). This modification facilitates the redirection of cytotoxic T cells toward tumor cells.^52),53) Their significant clinical efficacy in treating acute lymphoblastic leukemia has garnered substantial interest in CAR T-cell strategies.¹⁾

Fig. 5

Chimeric antigen receptor (CAR) T-cell therapy. CAR T cells redirect genetically modified T cells toward chondroitin sulfate proteoglycan 4 (CSPG4)-overexpressing cells.

An initial investigation regarding the potential of anti-CSPG4 CAR T cells demonstrated their in vitro cytolytic capabilities against a range of solid tumor cell lines, such as breast cancer, melanoma, mesothelioma, glioblastoma, and osteosarcoma.⁵⁴⁾ Using mouse cell line xenographs, subsequent research has highlighted the promising efficacy of anti-CSPG4 CAR T cells against melanoma, breast cancer, and head and neck cancer, both in vitro and in vivo.⁵⁵⁾

4.6. Trispecific killer engagers (TriKEs): a novel therapeutic class.

TriKEs are used in a new therapeutic approach to cancer treatment. They are designed to connect NK cells to tumor cells and enhance the cytotoxic potential of NK cells against tumor cells that abundantly express CSPG4. This innovative modality presents a potential new approach to cancer therapeutics (Fig. 6).

Fig. 6

Trispecific killer engagers (TriKEs). TriKEs form an antigen-specific immunologic synapse between natural killer (NK) and tumor cells, thereby triggering NK cell-mediated tumor cell lysis.

Emerging translational studies have highlighted the versatility of TriKEs across various tumor contexts, emphasizing their therapeutic range. These studies suggest that TriKEs tailored against CSPG4 can amplify the cytotoxic potential of NK cells against CSPG4-abundant tumor cells. This is significant because CSPG4 has been associated with the malignant progression of melanomas and the growth, survival, migration, invasion, and metastasis of malignant melanomas.¹⁴⁾ CSPG4 is also found in other tumor types, making it a potential target for cancer therapy. The development of therapies targeting CSPG4, such as TriKEs, is based on the understanding that CSPG4 plays an important role in tumorigenesis at multiple levels.⁵⁶⁾ It interacts with various proteins and signaling pathways, promoting integrin function and signaling and participating in signal transduction as a co-receptor. By targeting CSPG4, TriKEs disrupt these interactions and signaling pathways, leading to enhanced cytotoxicity against tumor cells.⁵⁷⁾

Overall, TriKEs tailored against CSPG4 hold promise as a new therapeutic approach in cancer treatment because they have the potential to amplify the cytotoxic potential of NK cells against CSPG4-abundant tumor cells, offering a broader range of therapeutic options for cancer.

4.7. Anti-idiotypic antibodies (anti-ids) targeting CSPG4.

Initial techniques aimed at CSPG4 involved using anti-ids. These anti-ids specifically target the binding sites of other anti-CSPG4 antibodies, replicating the antibody binding site of the tumor antigen. Their primary function is to act as immunogens or vaccines.^56),58) Clinical data revealed that patients with melanoma who developed anti-CSPG4 antibodies after receiving the anti-id mAb MK2-23 showed improved survival rates and regression of metastases.³⁾ However, MK2-23 was never approved for therapeutic use due to challenges in standardization and safety issues when co-administered with the adjuvant Bacille Calmette-Guerin. This adjuvant was crucial for inducing potent adaptive immune responses.⁵⁹⁾ Attempts to address these concerns involved fusing MK2-23 with human IL-2⁵⁹⁾ or creating deoxyribonucleic acid (DNA) vaccines that encode MK2-23 scFv.⁶⁰⁾ In 1990, Mittelman et al.⁶¹⁾ presented their findings from two clinical trials on the use of the mouse-derived mAb, MF11-30, for treating stage IV malignant melanoma. This mAb, which mimics human HMW-MAA, was administered to patients subcutaneously at varying doses. The first trial with 16 patients began with a dose of 0.5 mg per injection, increasing to 4 mg. No toxicity or allergic reactions were observed in this phase, and minor responses were noted in three patients. In the second trial, 21 patients received a steady dose of 2 mg per injection, based on the efficacy observed in the first trial. Of these, 19 completed the trial, with an average treatment duration of 34 weeks. This trial also reported no toxicity or allergic reactions. A significant number of patients developed antibodies against the mAb, with one patient achieving complete remission lasting 95 weeks. These trials suggest that the mouse anti-idiotypic mAb, MF11-30, might be a promising tool for active, specific immunotherapy in melanoma.

Vaccine strategies, such as mimotope vaccination studies, have been explored using conformational CSPG4 epitopes recognized by the 763.74 or 225.28S anti-CSPG4 mAb clones. Such studies report successful CSPG4-specific antibody induction in vaccinated animals and highlight the promising anti-melanoma activity of these antibodies, which is evident in their direct growth inhibition in vitro and in mouse effector cell antibody-dependent cellular cytotoxicity assays.^62)-64) While anti-id techniques and mimotope vaccines are not currently under the spotlight, these preliminary studies underscore the potential of CSPG4 as a promising target for vaccine-based cancer immunotherapy.

4.8. RNA vaccination.

DNA vaccination has emerged as a promising strategy for treating various diseases, including canine malignant melanoma. This is especially true when targeting antigens such as CSPG4.⁵⁶⁾ A primary advantage of DNA vaccination is its high stability, allowing for cost-effective and rapid production in large quantities.⁶⁵⁾ These vaccines use circular DNA constructs (plasmids) that encode specific tumor-associated antigens (TAAs). Due to their bacterial origin, these plasmids can stimulate the innate immune system via Toll-like receptor 9 interaction,⁶⁶⁾ enhancing the antigen-specific immune response. This interaction facilitates an inflammatory environment, triggering an adaptive immune response.⁶⁷⁾ Upon introduction into the body, these plasmids lead to the transfection of resident cells, such as dendritic cells, promoting TAA expression.⁶⁸⁾ This process stimulates both the cellular and humoral aspects of the immune system, targeting TAA-positive cancer cells.

Despite the potential and advantages of DNA vaccines, their translation into clinical benefits has been somewhat limited. Obstacles persist, including challenges related to vaccine administration methods, design, and the body’s inherent immune tolerance to many oncoantigens, such as CSPG4. Strategies to overcome these challenges, such as using DNA plasmids coding for xenogeneic proteins, have been explored with a degree of reported success.^69),70)

Building on the foundational knowledge of DNA vaccines, the focus has shifted toward the potential of RNA vaccines, especially given their recent success during the coronavirus disease 2019 (COVID-19) pandemic. RNA vaccines, similar to their DNA counterparts, offer several inherent benefits, such as the fact that they are well-tolerated, non-infectious, and induce a broad spectrum of immune responses.^71),72) The ease and speed of producing mRNA vaccines attribute them an advantage over traditional vaccine production methods. Furthermore, early clinical trials of mRNA-based cancer vaccines have demonstrated encouraging results, particularly when used with other treatments, such as checkpoint inhibitors.⁷³⁾

Two studies of lipid nanoparticle mRNA cancer vaccines (NCT0331313778 and NCT03897881) that encode multiple new antigens (mRNA-4157) are being evaluated in combination with pembrolizumab as adjuvant therapy in patients with high-risk cutaneous melanoma following complete resection. The same vaccine template (mRNA-4157) was investigated as monotherapy in patients with completely resected solid tumors (NCT03313778) and in combination with pembrolizumab in patients with unresectable solid tumors. The treatment induced neoantigen-specific T cells and no serious adverse events ( \(\geq\) grade 3). Here, 13 patients received mRNA-4157 monotherapy, and all but one remained cancer-free after treatment with a median follow-up of 8 months. Of the 19 evaluable patients who received combination therapy, one (5%) had a complete response before vaccination, two (11%) had a partial response, and five (36%) had stable disease.^74),75)

Considering these results, we suggest that a combination of mRNA vaccine and anti-programmed cell death protein 1 therapy may be more effective than mRNA vaccine therapy alone. Of particular note was the higher percentage of patients receiving combination therapy with partial or complete responses. Checkpoint inhibitors block immune checkpoints that cancer cells use to assist the immune system in attacking cancer cells. Therefore, when combined with mRNA vaccines, they may enhance the immune response against cancer cells. The results of the clinical trials described above indicate that this combination may be effective.

In essence, the successes and challenges of DNA vaccines have paved the way for RNA vaccine exploration and development. DNA vaccine research experience, combined with the inherent advantages of RNA vaccines, presents a promising future in the field of immunotherapy and disease treatment.

The antigenic determinants of the monoclonal antibodies described in the literature recognize the CSPG4 core protein independent of the presence of CS. Furthermore, the removal of the CS decoration from the core CSPG4 protein may not affect the reactivity mentioned above.⁷⁶⁾ Therefore, the anti-CSPG4 antibodies described here are likely to recognize the peptide sequence of the CSPG4 protein itself rather than its CS decoration or glycosylation. Antibody reactivity may not differ for individual cancer samples because they target the core protein of CSPG4, which is not expected to vary significantly among different cancer samples.

Similar to numerous other antigens, expression of CSPG4 is also detected in normal tissues.⁷⁶⁾ This observation contributes to the complexities surrounding its clinical application, despite the significant potential of CSPG4 as a therapeutic target. As in any therapeutic approach, treatment benefits should be balanced against side effect potential and severity. Therefore, evaluating and mitigating the extra-tumor toxic effects of CSPG4-specific targeted therapy is critical. Encouragingly, a phase I clinical trial investigating anti-CSPG4 radioimmunotherapy in melanoma patients with systemic administration of an \(\alpha\) particle-emitting radioisotope-conjugated mAb (9.2.27) reported some clinical benefits with no adverse events.⁷⁷⁾ Furthermore, CSPG4-based immunotherapy strategies may likely benefit from the development of new, effective therapeutic dosing regimens reviewed in this paper, which may contribute to limiting potential off-target effects and reducing treatment costs.

5. Conclusion

CSPG4 has both immunologic and nonimmunologic functions and it is commonly overexpressed in human tumor tissues. This expression positively correlates with cancer severity and poor outcomes. Therefore, CSPG4 presents a unique and interesting target for future cancer immunotherapies. One such promising therapeutic strategy is the use of CAR T cells against CSPG4. Rather than administering these CAR T cells alone, combining them with a chemotherapeutic regimen or checkpoint inhibitors may achieve synergistic antitumor effects.

Simultaneously, identifying CSPG4 receptor(s) and comprehending their role in immune responses and cancer development is crucial. Such knowledge can aid in effective therapeutic agent design, ultimately aiming for complete and lasting cancer treatments.

In conclusion, the clinical-translational advances targeting CSPG4 underscore its potential as a therapeutic target. Although many of these strategies are in their nascent stages, the preliminary results are promising, paving the way for a new era in cancer therapy.

Acknowledgments

We would like to thank Editage (www.editage.jp) for the English language editing.

Figure 5 was illustrated by Dr. Keisuke Koyama.

Notes

Edited by Hiroyuki MANO, M.J.A.

Correspondence should be addressed to: T. Kurokawa, Department of Medical Epigenomics Research, Fukushima Medical University, 1 Hikariga-oka, Fukushima 960-1295, Japan (e-mail: [e-mail]).

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Non-standard abbreviation list

anti-id

anti-idiotypic antibody

BRAFV600E

very rapidly accelerated fibrosarcoma murine sarcoma viral oncogene homolog B1 (BRAF) with V600E mutation

CAR

chimeric antigen receptor

CD

cluster of differentiation

CHST11

carbohydrate sulfotransferase-11

CS

chondroitin sulfate

CSPG4

chondroitin sulfate proteoglycan 4

DNA

deoxyribonucleic acid

GM-CSF

granulocyte-macrophage colony-stimulating factor

HER2

human epidermal growth factor receptor 2

HMW-MAA

high molecular-weight melanoma-associated antigen

HNSCC

head and neck squamous cell carcinoma

Ig

immunoglobulin

IL

interleukin

mAb

monoclonal antibody

MCSP

melanoma-associated chondroitin sulfate proteoglycan

mRNA

messenger ribonucleic acid

NG2

neuron-glial antigen 2

NK

natural killer

RNA

ribonucleic acid

SCID

severe combined immunodeficiency disease

TAA

tumor-associated antigen

TNBC

triple-negative breast cancer

TriKE

trispecific killer engager

Profile

Tomohiro Kurokawa was born in Tokyo, Japan, in 1981. He graduated from the University of Tsukuba in 2005 and received his PhD degree from the University of Tsukuba in 2016 under the supervision of Prof. Nobuhiro Ohkohchi. Then he worked as a research fellow in the laboratory of Prof. Soldano Ferrone and Prof. Cristina Ferrone in Massachusetts General Hospital, Harvard Medical School (2017–2019). He worked at IMSUT Hospital at the Institute of Medical Science, The University of Tokyo as an assistant professor (2019–2020). He was appointed as a chief of surgery in Jyoban Hospital of Tokiwa Foundation from 2021 and as an associate professor from 2022. He received the Best Presentation Award for Young Scientists in the 79th Annual Meeting of the Japan Society of Coloproctology in 2016 and Excellence in Surgery Research, 7th Annual Harvard Medical School Surgery Research Day in 2018. His current research focus is in gastrointestinal surgery and oncology.

Kohzoh Imai was born in Hokkaido Prefecture in 1948 and graduated from Sapporo Medical University in 1972. He majored in internal medicine, especially oncology and gastroenterology. He received his PhD degree in 1976 and worked as a postdoctoral fellow funded by an NIH Fogarty International Fellowship at Scripps Clinic and Research Foundation between 1978 and 1981. He became Professor of Medicine at Sapporo Medical University in 1994. He was elected President of Sapporo Medical University in 2004 and served a 6-year term. Then, he was appointed as Director of IMSUT Hospital at the Institute of Medical Science, The University of Tokyo from 2010 to 2014. He was then appointed as Head of the Medical Research Platform Office at the same university between 2015 and 2019, and the Director of Kanagawa Cancer Center Research Institute from 2014 to 2016. He was then invited to become a Guest Professor of Hokkaido University. He is currently a core member of the Japan Agency for Medical Research and Development (AMED). He has developed the diagnostic method for digestive tract cancer utilizing the methylation of genes expressed in cancer cells. He has also dedicated himself to translational research on treatment with siRNAs targeting PRDM14 in cancer cells. This nucleic acid-based drug is a novel approach for cancer and is expected soon to be applied clinically in patients with cancer following a promising proof of concept in mouse models and other animal experiments. For his accomplishments, he received the Medal with Purple Ribbon, the ISOBM Award from International Society of Oncology and Biomarkers (ISOBM), and further awards in Japan.