3. Specificity and Mechanism of Immunomodulation by CTI Therapy in Melanoma
3.1. NPrCAP as Neo-Antigen Producer
NPrCAP is a good substrate for tyrosinase
[29] and is selectively incorporated into melanoma cells, which causes cytotoxicity in vitro and in vivo
[29][30]. To clarify the molecular mechanism of NPrCAP-mediated cytotoxicity to melanoma cells, Ishi-Osai reported that mice treated with intratumoral injections of NPrCAP to suppress the growth of primary B16F1 melanoma transplants also rejected secondary re-challenge tumors
[10]. The participation of CD8
+ T cells was suggested for the NPrCAP-mediated anti-B16F1 melanoma immunity.
Phenolic substrates as prohaptens are oxidized by tyrosinase to produce
ortho-quinones, which act as haptens that covalently bind to tyrosinase or other melanosomal proteins to generate potential neo-antigens
[31][32][33]. These neo-antigens trigger an immunological response cascade that results in a melanocyte-specific, delayed-type hypersensitivity reaction leading to melanocyte elimination or melanoma rejection.
Based on the haptenation theory, Ito et al. examined the oxidation of NPrCAP and its subsequent binding to sulfhydryl compounds (thiols)
[28]. They demonstrated that NPrCAP is oxidized by tyrosinase to form a highly reactive
ortho-quinone (
N-propionyl-4-
S-cysteaminyl-1,2-benzoquinone, NPrCAQ), which binds covalently to biologically relevant thiols, including proteins through cysteine residues. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of CA-CysC after acid hydrolysis of the protein fraction. These results suggested that the phenol NPrCAP, acting as a prohapten, can be oxidized in melanoma cells by tyrosinase to the active quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response.
3.2. T-Cell Receptor Repertoires of Tumor-Infiltrating Lymphocytes
Cytotoxic T lymphocytes (CTLs) play a significant role in antitumor immunity, and the presence of tumor-infiltrating lymphocytes (TILs) has been considered to be a favorable clinical prognostic indicator
[34]. To further understand the T-cell response to melanoma in CTI therapy and to develop a more effective strategy based on immunomodulation, researchers investigated the diversity of TILs after CTI therapy
[16]. The immune response of CTLs is mediated via T-cell receptors (TCRs) consisting of α and β chains. In the variable (V) regions, the gene sequence encoding the third complementarity-determining region (CDR3), which is called the hypervariable region, is considered to play the most important role in antigen recognition
[35]. researchers analyzed the diversity of the TCR Vβ family to investigate the qualitative changes of TILs after CTI therapy. Almost all TCR Vβ families (in total, 21 TCR Vβ families researchersre analyzed) researchersre detected in untreated B16 melanoma in C57BL/6 mice, whereas the TCR repertoire was restricted to a few TCR Vβ families in TILs after CTI therapy. Among them, expression of the Vβ gene was confirmed with good reproducibility, suggesting that T cells expressing the TCR Vβ researchersre activated by CTI therapy in B16 melanoma. In addition, researchers succeeded in analysis of the CDR3 gene sequence of TCR Vβ in TILs after CTI therapy
[28]. Consistent with the result, it was reported that a B16 melanoma-specific CD8+ T cell line, AB1, expressed TCR Vβ11
[36], suggesting that clonal expansion of Vβ11+ TILs can be a useful biomarker for the T-cell response to B16 melanoma in mice. Furthermore, the same group reported that the AB1 cells recognized a melanoma antigen, tyrosinase related protein-2 (TRP-2) peptide, which was consistent with a report by Singh et al. showing that a TRP-2 peptide-specific CD8+ T cell clone expressed Vβ11
[37]. In order to identify the antigen specificity of TILs after CTI therapy of B16 melanoma, researchers investigated the interferon (IFN)-γ production ability using melanoma antigen peptides such as TRP-1
222–229, TRP-2
180–188 and gp100
25–33. When stimulated with the TRP-2 peptide, T cells researchersre activated to secrete IFN-γ, indicating that TILs induced by CTI therapy of B16 melanoma responded to the TRP-2 peptide. Taken together, these findings show that tumor-specific TILs researchersre produced after CTI therapy and suggest that TCR Vβ11+ T cells are particularly important for immunity against B16 melanoma. Moreover, the melanoma antigen peptides selected by TIL analysis (e.g., TRP-2 peptide for B16 melanoma) may be used to boost antitumor immunity induced by CTI therapy.
3.3. CTI Therapy as In Situ Peptide Vaccine Immunotherapy
By comparing the antitumor effect of NPrCAP/MNP with and without AMF exposure, researchers observed that NPrCAP/MNP with AMF exposure had a superior antitumor effect compared with that of NPrCAP/MNP alone. Furthermore, mice bearing primary melanoma tumors treated with NPrCAP/MNP plus AMF shoresearchersd significant suppression of re-challenge second transplant melanoma growth, whereas NPrCAP/MNP without AMF was much less effective, with 30–50% rejection of re-challenge melanoma. These results indicate that NPrCAP/MNP with AMF exposure has a strong immunotherapeutic effect
[8][9]. Therefore, researchers investigated the underlying mechanisms for the induction of antitumor immunity induced by NPrCAP/MNP with AMF exposure. Incorporated MNP exposed to an AMF generate heat within cells due to hysteresis loss or relaxational loss
[19]. It has been demonstrated that intracellular hyperthermia using MNP is effective for the treatment of certain types of cancer, in not only primary but also metastatic lesions
[38][39][40][41]. Hyperthermic treatment using cationic magnetite liposomes containing 10 nm MNP induced antitumor immunity by the enhancement of HSP expression
[40]. It has been demonstrated that various types of HSPs bind antigenic peptides, and these antigen peptides are cross-presented to specific cytotoxic T cells by professional antigen-presenting cells, including dendritic cells (DCs). This exogenous pathway is called cross-presentation and is important for the development of CD8
+ T cell responses against tumors and infectious pathogens that do not have access to the classical MHC class I pathway
[42][43]. In the study using B16-OVA melanoma cells, treatment with NPrCAP/MNP with AMF exposure resulted in the increased expression of HSPs, including Hsp72, Hsp90 and ER-resident stress proteins such as gp96, in melanoma cells
[11]. Moreover, these HSPs (Hsp72, Hsp90 and gp96) researchersre secreted in extracellular milieu and researchersre taken up by DCs. These DCs presented melanoma-associated antigen peptides (OVA peptide and TRP2 peptide) through cross-presentation of HSP-bound peptide(s) to specific CD8
+ T cells. Among HSPs, Hsp72 was shown to be largely responsible for the augmented antigen presentation to CD8
+ T cells. As Hsp72 is known to be most highly upregulated among several HSPs in response to heat shock, newly synthesized Hsp72 has more chances to bind melanoma-associated antigen peptides.
Thus, the hyperthermia using NPrCAP/MNP with AMF exposure induced an anti-melanoma cytotoxic T lymphocyte (CTL) response through cross-presentation of melanoma-specific antigen peptides bound to hyperthermia-induced HSPs by DCs. More importantly, intracellular hyperthermia using NPrCAP/MNP can be a promising treatment for the prevention of recurrence and/or distant metastasis of melanoma, because systemic antimelanoma immunity is induced by this therapy.
If the treatment with NPrCAP/MNP plus AMF could prevent distant melanoma metastasis such as lung and distant cutaneous metastases, it would be a great boon for patients with advanced melanoma. Therefore, researchers examined whether treatment of primary cutaneous B16 melanoma with intracellular hyperthermia using NPrCAP/MNP with AMF can inhibit lung colonization of intravenously injected secondary challenge B16 melanoma cells. researchers observed that NPrCAP/MNP plus AMF clearly inhibited lung metastasis compared with NPrCAP/MNP alone. These results indicated that intracellular hyperthermia using NPrCAP/MNP with AMF elicited systemic antimelanoma immunity and prevented lung metastasis and the recurrence of melanoma.
Thus, CTI therapy using NPrCAP/MNP with AMF against advanced melanoma is a promising strategy not only for the treatment of primary melanoma but also for prevention of the recurrence of melanoma.
4. Approach to Advanced Melanoma Patients
4.1. Scale-Up Production of NPrCAP/PEG/APTES/DNM for Clinical Application
Based on NPrCAP/PEG/APTES/DNM, which is a PEG-mediated conjugate of NPrCAP and APTES with DNM, researchers tested further improvements of the synthesis conditions for the development of a good manufacturing practice (GMP)-based production process. Water dispersibility is important for injectable drugs. researchers found that the aggregation of NPrCAP/PEG/APTES/DNM was caused by particle-to-particle interactions due to APTES. By reducing the iron concentration from 10 mg/mL to 1 mg/mL during the APTES reaction, researchers found that the particle size of NPrCAP/PEG/APTES/DNM did not increase even after the reaction. Thus, this new formulation of NPrCAP/PEG/APTES/DNM can pass through a 0.2 µm filter, enabling sterilization, which is extremely important in the manufacture of drugs. Furthermore, the equipment for the production of NPrCAP/PEG/APTES/DNM, compliant with GMP, was installed in the laboratory of Meito Sangyo Co., Ltd. (Nagoya, Japan). Meito Sangyo has manufactured ferucarbotran, the drug substance of Resovist
[44], which is sold by Bayer Schering Pharma (Berlin, Germany) as a clinically available magnetic resonance imaging contrast agent, in compliance with GMP. As a result of repeated synthesis while complying with the standard operating procedure, a total of 14 lots (800 mL) of NPrCAP/PEG/APTES/DNM researchersre synthesized, and the reproducibility was confirmed. Taken together, a standard for the formulation of NPrCAP/PEG/APTES/DNM was determined.
4.2. Preliminary Human Clinical Trial of CTI Therapy for Advanced Melanoma Patients
Based on the animal experiments and the successful production of GMP grade NPrCAP/PEG/APTES/DNM, a preliminary human clinical trial (Phase I/II) has been carried out with a limited number of stage III and IV melanoma patients, after receiving informed consents from the patients and institutional approval of the human clinical trial protocol (Clinical Trial Research No. 18-67, Sapporo Medical University).
The therapeutic protocol basically folloresearchersd an identical experimental schedule as that of the animal experiments. Among four patients treated with NPrCAP/PEG/APTES/DNM plus AMF exposure, two of them shoresearchersd complete and partial responses, respectively, and have been able to carry out normal daily activities after the CTI therapy. In one of those two responding patients, four distant cutaneous metastasis sites researchersre evaluated and either significant regression or shrinkage of all four lesions was seen. That patient was able to survive 36 months after several cycles of CTI therapy. The pathological and immunological specimens revealed dense aggregations of lymphocytes and macrophages at the site of CTI therapy. Importantly, there was a trend toward an almost identical distribution of CD8+ T cells and MHC class 1 positive cells. The other responding patient had many lymph node metastases, but has survived more than 32 months so far. In order to evaluate the overall therapeutic value for advanced melanoma, it is important to have larger-scaled clinical trials and to define concisely the molecular interactions betresearchersen the chemotherapeutic and thermo-immunotherapeutic effects in the CTI therapy.