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Abstract Malignant melanoma （MM） is a kind of highly-invasive and highly-metastatic tumor， which is insensitive to radiochemotherapy. It is also one of the most invasive tumors in skin cancer with limited treatment options. Due to its rapid increase in incidence in recent years and the limitations of monotherapy， it is necessary to use combination therapy. The combination therapy can not only enhance the treatment effect， but also avoid the development of drug resistance as much as possible. This paper focused on the combined therapy of targeted traditional therapies， immunotherapy with radiotherapy and chemotherapy or other traditional therapies， as well as the research status and new developments of these combined therapies.
Key words Melanoma; Targeted Therapy; Nanotechnology; Immunotherapy
Received： August 15， 2020 Accepted： October 23， 2020
Supported by Public Welfare Technology Application Research Program of Zhejiang Province （LGF18H160034， LGD20H300001， 2015C37125）; 2019 National Undergraduate Innovation Training Program （20190354034）; 2018 Zhejiang Science and Technology Innovation Activity Program （Emerging talent Program） （2018R417029）; 2018 Jiaxing Key Technology Innovation Team; 2018 SRT Key Project of Jiaxing University （85178428）.
Yiwen GU （1997-）， female， P. R. China， devoted to research about nano targeting and sustained and controlled release drug delivery system.
*Corresponding author. E-mail： [email protected].
Malignant melanoma （MM） is a malignant tumor originating from the embryonic neural crest， which is highly malignant and has a poor prognosis. It can occur in the skin， digestive tract， eyeball and reproductive system， but most common in the skin[1]. In recent years， the incidence and fatality rate of MM has continued to rise， surpassing any other cancer type. The fatality rate accounts for about 80%， and it is one of the deadliest forms of skin cancer[2]. When melanoma is diagnosed early， surgical resection is possible to cure it. However， in the case of advanced MM， complicated surgical procedures and drug treatments must be performed， and clinicians often face difficult treatment challenges and it is difficult to achieve long-term response. The main methods of conventional treatment of MM are surgery， radiotherapy and chemotherapy， but there are problems such as large trauma， obvious adverse reactions， and poor patient tolerance. Furthermore， MM is not very sensitive to postoperative adjuvant radiotherapy and chemotherapy. Therefore， the above methods cannot significantly improve the prognosis of patients， and the efficacy on MM is not satisfactory[3]. Currently， the main available medical therapies for melanoma are immune checkpoint suppression therapy， tumor vaccine， gene therapy such as talimogene laherparepvec-T-VEC， T cell directed therapy， and other non-specific methods （radiotherapy， cytotoxic chemotherapy， photothermal therapy， photodynamic therapy， etc.）. In the immune checkpoint inhibition therapy， programmed cell death 1 （PD-1） or programmed cell death ligand 1 （PD-L1） inhibitors （nivolumab， pembrolizumab） are used alone or in combination with anti-cytotoxic T lymphocyte associated protein 4 （CTLA-4） and antibody （ipilimumab）. With BRAF （v-raf murine sarcoma virus oncogene homolog B1） and MEK （mitogen-activated protein kinase） inhibitors （vemurafenib， dabrafenib）， patients carrying BRAF gene V600 mutation and tyrosine protein kinase （KIT） inhibitor （Imatinib） were treated with Braftovi （encorafenib）， trametinib， cobimetinib， and Mektovi （binimetinib）. Since the prognostic benefit of patients is not clear， these methods are only used in a small number of patients[4]. Despite continuous research and development of new anti-tumor therapies， MM is still a disease whose incidence continues to rise. The efficacy of currently available MM therapies is limited by a number of disadvantages. The interaction of drugs with biological fluids and cell membranes， as well as heterogeneous tumor immunology and vasculature， may be the cause of drug resistance[5-6].
In recent years， more and more scientists have devoted themselves to the development and research of MM treatment methods. Since the FDA approved the marketing of some monoclonal antibody drugs and BRAF inhibitors， targeted therapy and immunotherapy are the two main research directions. Both treatments have their pros and cons. Targeted therapy has obvious curative effect but short effective period， and is easy to produce drug resistance， while immunotherapy has long effective period but slow response. Therefore， combining the two with some conventional treatment methods to treat MM is an effective way to improve the anti-tumor effect， increase the validity period， and reduce drug resistance. This paper reviewed the research progress of several combination treatments on MM.
Combination of Targeted Therapy with Other Methods
Combination therapy with multiple small-molecule targeted drugs
Molecular targeted drugs are a large class of anti-tumor targeted drugs， and their targets are signal transduction molecules of tumor cells. After analyzing the new drugs approved by the FDA in the past 5 years， it is found that the approved anti-tumor drugs are mainly targeted drugs， and they are transferred from single-target drugs to multi-target drugs[7]. Since 2010， the FDA has successively approved vemurafenib， dabrafenib， and trametinib as small-molecule targeted drugs for the treatment of MM， and in 2014 approved the combination of dabrafenib and trametinib for treatment of advanced MM that cannot be removed by surgery and that has been transferred. The combination therapy of multiple targeted drugs has become an important direction in the treatment of MM. In addition to trametinib and dabrafenib， which have been approved by the FDA to treat MM in combination， the combined application of many other targeted drugs has also made some progress. Cobimetinib （GDC-0973） is an oral small molecule inhibitor of MEK kinase， which can specifically bind to the catalytic center of MEK kinase （IC50=4.2 nmol/L）， thereby inhibiting phosphorylation activation and reducing cell proliferation. Preclinical studies have shown that GDC-0973 can effectively inhibit the growth of tumor cells with BRAF mutations. An IB clinical trial is evaluating the safety and effectiveness of GDC-0973 combined with vemurafenib in the treatment of MM patients. Studies have shown that of the 128 patients included in the trial， it was safe to treat metastatic MM with two maximum tolerated doses （60 mg for GDC-0973; 960 mg for vemurafenib）， which resulted in an objective response in patients who had never been treated with a BRAF inhibitor before， and 10% of them achieved complete remission （CR）[8].
The combination of PD-1/PD-L1 inhibitor and closed and complementary checkpoint CTLA-4 has obtained strong preclinical evidence， and has been proven to be very effective in patients with advanced MM in phase I-III trials[9].
Combination of targeted therapy with chemotherapy
Angiogenesis is an important link in the process of MM invasion and metastasis， which involves vascular endothelial growth factor （VEGF）， angiostatin， fibrin growth factor and many other cytokines[10]. Among them， VEGF， as the strongest known vascular penetrant and endothelial cell-specific mitogen， plays an important role in endothelial cell proliferation， migration and vascular construction[11]. Salven et al.[12] found that 32% of primary MM expressed VEGF， but in metastatic MM， the expression rate of VEGF was as high as 91%.
Therefore， anti-angiogenesis targeted drug therapy is expected to become a new solution for the treatment of MM， and targeted VEGF drugs combined with cytotoxic drugs are more effective than targeted drugs alone in treating MM patients. Bevacizumab is an artificial monoclonal IgG antibody that directly acts against VEGF. It has a significant anti-angiogenic effect， and is especially clinically beneficial to solid tumors such as kidney cancer， colorectal cancer， breast cancer and non-small cell lung cancer. However， bevacizumab monotherapy has a limited effect in the treatment of MM. The phase II experiment of MM showed that the median progression-free survival of patients under bevacizumab monotherapy was 3 months， and the median overall survival time was only 8.5 months， while when bevacizumab was combined with capecitabine or paclitaxel， most patients’ disease was stable for more than 8 weeks， and the median overall survival time was 12 months[13]. The anti-tumor effect of recombinant human vascular endothelial inhibitor is achieved by inhibiting the proliferation of vascular endothelium induced by VEGF， and it also has a synergistic effect in combination with chemotherapy drugs. Clinical trials have found that the combined application of endostar with dacarbazine and cisplatin is effective for metastatic MM without gene mutations， and the adverse events are controllable[14]. Combination of targeted therapy with radiotherapy
In recent years， targeted therapy combined with radiotherapy has made certain developments in the treatment of MM， especially in stage III and IV MM， and has become a new treatment mode. Ipilimumab injection[15] is the first drug that can definitely prolong the life of patients with metastatic MM. As the most representative monoclonal antibody drug， it can not only effectively block cytotoxic T lymphocyte antigen CT-LA-4 molecules， and significantly improve the survival rate of patients with advanced MM， effectively extending the life cycle by 3.5 months[16].
However， clinically， the total remission rate of ipilimumab alone for the treatment of MM is still low， only 5%-15%[17]. The latest study found that targeted therapy combined with radiotherapy can simultaneously control local and distant metastases of MM， and prolong progression-free survival[18]. Hodi et al.[19] reported that after ipilimumab monoclonal antibody combined with radiotherapy in patients with stage III and IV MM， the patient’s survival increased from 6.4 months to 10 months， indicating that the combination of the two treatments can enhance the treatment effect. Drake et al.[20] also reported that the combination of the two has a clear synergy. In addition， some studies have also shown that while receiving ipilimumab in patients with stage III and IV MM， palliative radiotherapy can cause distant effects at the far end of the radiotherapy site， and the potential effects of targeted immunotherapy and radiotherapy are increased， thereby reducing tumor metastases[21-23].
Combination of targeting genes with viral therapy
Combining targeting genes with viral therapy was first proposed by Academician Liu Xinyuan， and it is a new anti-cancer strategy that combines the advantages of gene therapy and viral therapy. Tumor-specific proliferating adenoviruses carrying therapeutic genes will not cause damage or have little impact to normal cells and tissues， but only replicate in a large number of specific tumor cells， retaining the ability of the virus to kill cancer cells. Meanwhile， it is accompanied by the replication of viruses， and the expression of anti-oncogenes in tumor cells also greatly increases， thereby further improving the therapeutic effect[24]. Zheng et al.[25] co-transfected human embryonic kidney cell HEK293 cells with a plasmid carrying ZD55-IL-18 and plasmid pBHGE3 containing the right arm of adenovirus to obtain the recombinant adenovirus ZD-55-IL-18， which can express IL-18 gene efficiently in MM cells and inhibit the growth of MM cells. Experiments have shown that the combination of targeting genes and viruses can increase the sensitivity of tumor cells to chemotherapeutics and improve the efficiency of chemotherapy. Jiang et al.[26] combined ZD55-IL-24 and DTIC to treat MM in vitro experiments and achieved better therapeutic effects than single use. What’s more worth mentioning is that targeting genes-viruses can also be combined with RNA interference technology. Zheng et al.[27] found in in vivo and in vitro experiments on kidney cancer using conditionally proliferating adenovirus （ZD-hTERT） loaded with small interfering RNA of human telomeras ereverset ranscrip tase （hTERT） gene that the virus replicated specifically in tumor cells and effectively silenced the hTERT gene of kidney cancer， thereby inhibiting cell proliferation and promoting its apoptosis. It suggests that we can combine RNAi technology to construct small interfering RNAs related to MM oncogenes to insert into the viral genome， providing new ideas for the treatment of MM. Combination of Immunotherapy with Other Methods
Combination of immunotherapy and chemotherapy
Recently， it has been discovered that MM has high immunogenicity. Therefore， immunotherapy for the occurrence， development and metastasis of MM has also become a research hotspot. Interleukin 2 （IL-2） is a lymphokine， which has anti-tumor effects and enhances the body’s immune function. As of 2011， high-dose interleukin 2 （HD-IL-2） is still one of the only two FDA-approved drugs for the systemic treatment of stage IV MM， and the other is dacarbazine. A prospective study by Kaufmann et al.[28] found that the therapeutic effects of temozolomide alone and temozolomide combined with interferon-α were different. The results showed that the overall response rate of the combination therapy was significantly higher than that of temozolomide alone， and the adverse reactions were tolerable. It is worthy of further research and clinical promotion.
The method of interleukin 2 combined with chemotherapy can prolong the survival time of patients， and the effective rate of the combination of the two drugs is significantly higher than that of chemotherapy or biological therapy alone. However， some scholars have found that[29]， after high-dose interferon is used for treatment of patients with high-risk recurrence factors， the toxic side effects brought by it can significantly increase the recurrence rate of the disease. Meanwhile， there are reports in the literature that the combined treatment of interleukin-2， histamine and interferon can reduce the dosage of interferon， thereby reducing the production of toxic and side effects， and the safety， tolerability and therapeutic effect are all improved to a certain degree[30].
Combination of immunotherapy and nanotechnology
Anti-tumor vaccine is one of the current research hotspots. Its main principle is to achieve anti-tumor effect mainly by initiating tumor-specific cytotoxic T lymphocyte （CTL） response. Tumor vaccines can activate the patient’s own immune system， effectively inhibit tumor growth， prevent tumor metastasis and recurrence， and will not damage other tissues. Its anti-tumor specificity and immune memory are unmatched by other methods[31]. However， tumor antigen vaccines are easily degraded， difficult to be effectively delivered to lymph nodes， and have low immunogenicity. Even if they are delivered to immune cells， they will quickly degrade and cause an effective immune response. In order to overcome the deficiencies of tumor antigen vaccines and make tumor antigen vaccines play a better effect， the application of nanotechnology has played an increasingly important role in the development of tumor antigen vaccines[32]. Researchers such as Conniot J. of Tel Aviv University[33] skillfully combined passive and active immunotherapy. Nanoparticles， that were made of biodegradable polymers and had a size between 160 and 190 nm， were wrapped with two peptides expressed in melanoma cells： Melan-A/MART-1 （26-35 （A27L）） class I major histocompatibility complex （MHCI） restricted peptide （MHCI-ag） and Melan-A/MART-1 （51-73） MHCII restricted peptide （MHCII-ag）. The nanoparticles were collected with mannose receptors to enable them to target ligand-mediated active dendritic cells. After the nano-vaccine was injected into the mouse model， it was found to be effective and preventive. It prevented the development of melanoma in healthy immunized mice， and significantly slowed down the progression of the disease even in mice with advanced melanoma.
Combination of immunotherapy and gene therapy
Compared with free molecular drugs， IL-2 or anti-CD137 immunoliposome delivery system can achieve the same anti-tumor activity， but the systemic toxicity is low or even non-existent. Zhang et al.[34] showed that the combined application of IL-2 and anti-CD137 quickly accumulated on the surface of liposomes in melanoma tumors， thereby minimizing systemic exposure and reducing the risk of toxicity.
Molecular Targeted Therapy Combined with Immune Checkpoint Inhibition Therapy
Molecular targeted therapy has a high overall remission rate but is prone to the production of drug resistance. However， the biggest problem faced by immune checkpoint inhibition therapy is the low remission rate， but it is not easy to develop drug resistance. The results of some clinical trials show that after the combined application of molecular targeted therapy and immune checkpoint inhibition therapy， patients will have sustained clinical benefits[35-37].
The results of a phase II clinical trial showed that in newly treated patients with metastatic MM with BRAF gene V600E/K mutations， by combined application of PD-L1 antibody atezolizumab， BRAF gene inhibitor vemurafenib and MEK inhibitior cobimetinib， the ORR reached 76%， and the adverse reactions in the test were controllable and all reversible[38].
Another clinical study divided patients with metastatic MM with BRAF gene mutations into three groups. The 26 patients in group A were given anti-PD-L1 treatment， combined with BRAF gene inhibitor Dabrafenib and MEK inhibitor Cobitinib until the disease progressed; the 20 patients in group B were given anti-PD-L1 treatment and cobitinib treatment at the same time until the disease progressed; and the 19 patients in group C were first treated with cobimetinib for 4 weeks， then given anti-PD-L1 treatment and cobimetinib treatment for 2 weeks， and then anti-PD-L1 treatment alone until the disease progressed[38]. This study allowed patients who had received immunosuppressive therapy （anti-CTLA-4， anti-PD-1， anti-PD-L1）， but excluded those who had received targeted drug therapy. The treatment results showed that the ORRs of patients in groups A， B， and C were 69%， 21%， and 13%， respectively， and the corresponding disease control rates （including complete remission， partial remission， and stable disease without progression） were 100%， 79% and 80%， respectively; and in the next 50 weeks of follow-up， only 90% of patients in group A received sustained clinical benefit[39]. Conclusions
The clinical effects of various monotherapy for MM patients are not very satisfactory. Targeted therapy and immunotherapy have obvious therapeutic effects on MM， but these methods are limited in the process of implementation. For example， patients receiving targeted therapy will soon develop resistance， while immunotherapy patients are only partially effective. The combined use of different treatment methods has achieved good results in preclinical and clinical trials. How can we improve the safety and maximize the anti-tumor effect is the main direction of future research. As people’s understanding of the pathogenesis of MM and biotechnology continues to deepen， combined treatment options are constantly being enriched in the medical treasure house. It is believed that with the development of tumor treatment， the combined treatment of MM will surely enter a new era.
References
[1] MILLER KD， SIEGEL RL， LIN CC， et al. Cancer treatment and survivorship statistics[J]. CA： A Cancer Journal for Clinicians， 2016， 66（4）： 271-289.
[2] KEE DM， MCARTHUR G. Targeted therapies for cutaneous melanoma[J]. Hematology/Oncology Clinics of North America， 2014， 28（3）： 491-505.
[3] SUZUKI S， ISHIDA T， YOSHIKAWA K， et al. Current status of immunotherapy[J]. Japanese Journal of Clinical Oncology， 2016， 46（3）： 191-203.
[4] DUMMER R， HAUSCHILD A， LINDENBLATT N， et al. Cutaneous melanoma： ESMO Clinical Practice Guidelines for diagnosis， treatment and follow-up[J]. Annals of Oncology： Official Journal of the European Society for Medical Oncology， 2019， 30（12）： 1884-1901.
[5] VINCENT P， DANIELA L， ELISE L， et al. Nanomedicine as a potent strategy in melanoma tumor microenvironment[J]. Pharmacological Research， 2017（126）： 31-53.
[6] POPESCU M， MUNTEANU A， ISVORANU G， et al. Dynamics of endothelial progenitor cells following sevoflurane preconditioning[J]. Roumanian Archives of Microbiology and Immunology， 2011， 70（3）： 109-113.
[7] SUN J， ZHU XH， WANG S， et al. Overview of targeted antitumor drugs approved by FDA in recent five years[J]. Chinese Journal of New Drugs， 2016， 25（1）： 1-6. （in Chinese）
[8] DRéNO B， RIBAS A， LARKIN J， et al. Incidence， course， and management of toxicities associated with cobimetinib in combination with vemurafenib in the coBRIM study[J]. Annals of Oncology： Official Journal of the European Society for Medical Oncology， 2017， 28（5）： 1137-1144.
[9] LARKIN J， CHIARION-SILENI V， GONZALEZ R， et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma[J]. The New England Journal of Medicine， 2015， 373（1）： 23-34.
Key words Melanoma; Targeted Therapy; Nanotechnology; Immunotherapy
Received： August 15， 2020 Accepted： October 23， 2020
Supported by Public Welfare Technology Application Research Program of Zhejiang Province （LGF18H160034， LGD20H300001， 2015C37125）; 2019 National Undergraduate Innovation Training Program （20190354034）; 2018 Zhejiang Science and Technology Innovation Activity Program （Emerging talent Program） （2018R417029）; 2018 Jiaxing Key Technology Innovation Team; 2018 SRT Key Project of Jiaxing University （85178428）.
Yiwen GU （1997-）， female， P. R. China， devoted to research about nano targeting and sustained and controlled release drug delivery system.
*Corresponding author. E-mail： [email protected].
Malignant melanoma （MM） is a malignant tumor originating from the embryonic neural crest， which is highly malignant and has a poor prognosis. It can occur in the skin， digestive tract， eyeball and reproductive system， but most common in the skin[1]. In recent years， the incidence and fatality rate of MM has continued to rise， surpassing any other cancer type. The fatality rate accounts for about 80%， and it is one of the deadliest forms of skin cancer[2]. When melanoma is diagnosed early， surgical resection is possible to cure it. However， in the case of advanced MM， complicated surgical procedures and drug treatments must be performed， and clinicians often face difficult treatment challenges and it is difficult to achieve long-term response. The main methods of conventional treatment of MM are surgery， radiotherapy and chemotherapy， but there are problems such as large trauma， obvious adverse reactions， and poor patient tolerance. Furthermore， MM is not very sensitive to postoperative adjuvant radiotherapy and chemotherapy. Therefore， the above methods cannot significantly improve the prognosis of patients， and the efficacy on MM is not satisfactory[3]. Currently， the main available medical therapies for melanoma are immune checkpoint suppression therapy， tumor vaccine， gene therapy such as talimogene laherparepvec-T-VEC， T cell directed therapy， and other non-specific methods （radiotherapy， cytotoxic chemotherapy， photothermal therapy， photodynamic therapy， etc.）. In the immune checkpoint inhibition therapy， programmed cell death 1 （PD-1） or programmed cell death ligand 1 （PD-L1） inhibitors （nivolumab， pembrolizumab） are used alone or in combination with anti-cytotoxic T lymphocyte associated protein 4 （CTLA-4） and antibody （ipilimumab）. With BRAF （v-raf murine sarcoma virus oncogene homolog B1） and MEK （mitogen-activated protein kinase） inhibitors （vemurafenib， dabrafenib）， patients carrying BRAF gene V600 mutation and tyrosine protein kinase （KIT） inhibitor （Imatinib） were treated with Braftovi （encorafenib）， trametinib， cobimetinib， and Mektovi （binimetinib）. Since the prognostic benefit of patients is not clear， these methods are only used in a small number of patients[4]. Despite continuous research and development of new anti-tumor therapies， MM is still a disease whose incidence continues to rise. The efficacy of currently available MM therapies is limited by a number of disadvantages. The interaction of drugs with biological fluids and cell membranes， as well as heterogeneous tumor immunology and vasculature， may be the cause of drug resistance[5-6].
In recent years， more and more scientists have devoted themselves to the development and research of MM treatment methods. Since the FDA approved the marketing of some monoclonal antibody drugs and BRAF inhibitors， targeted therapy and immunotherapy are the two main research directions. Both treatments have their pros and cons. Targeted therapy has obvious curative effect but short effective period， and is easy to produce drug resistance， while immunotherapy has long effective period but slow response. Therefore， combining the two with some conventional treatment methods to treat MM is an effective way to improve the anti-tumor effect， increase the validity period， and reduce drug resistance. This paper reviewed the research progress of several combination treatments on MM.
Combination of Targeted Therapy with Other Methods
Combination therapy with multiple small-molecule targeted drugs
Molecular targeted drugs are a large class of anti-tumor targeted drugs， and their targets are signal transduction molecules of tumor cells. After analyzing the new drugs approved by the FDA in the past 5 years， it is found that the approved anti-tumor drugs are mainly targeted drugs， and they are transferred from single-target drugs to multi-target drugs[7]. Since 2010， the FDA has successively approved vemurafenib， dabrafenib， and trametinib as small-molecule targeted drugs for the treatment of MM， and in 2014 approved the combination of dabrafenib and trametinib for treatment of advanced MM that cannot be removed by surgery and that has been transferred. The combination therapy of multiple targeted drugs has become an important direction in the treatment of MM. In addition to trametinib and dabrafenib， which have been approved by the FDA to treat MM in combination， the combined application of many other targeted drugs has also made some progress. Cobimetinib （GDC-0973） is an oral small molecule inhibitor of MEK kinase， which can specifically bind to the catalytic center of MEK kinase （IC50=4.2 nmol/L）， thereby inhibiting phosphorylation activation and reducing cell proliferation. Preclinical studies have shown that GDC-0973 can effectively inhibit the growth of tumor cells with BRAF mutations. An IB clinical trial is evaluating the safety and effectiveness of GDC-0973 combined with vemurafenib in the treatment of MM patients. Studies have shown that of the 128 patients included in the trial， it was safe to treat metastatic MM with two maximum tolerated doses （60 mg for GDC-0973; 960 mg for vemurafenib）， which resulted in an objective response in patients who had never been treated with a BRAF inhibitor before， and 10% of them achieved complete remission （CR）[8].
The combination of PD-1/PD-L1 inhibitor and closed and complementary checkpoint CTLA-4 has obtained strong preclinical evidence， and has been proven to be very effective in patients with advanced MM in phase I-III trials[9].
Combination of targeted therapy with chemotherapy
Angiogenesis is an important link in the process of MM invasion and metastasis， which involves vascular endothelial growth factor （VEGF）， angiostatin， fibrin growth factor and many other cytokines[10]. Among them， VEGF， as the strongest known vascular penetrant and endothelial cell-specific mitogen， plays an important role in endothelial cell proliferation， migration and vascular construction[11]. Salven et al.[12] found that 32% of primary MM expressed VEGF， but in metastatic MM， the expression rate of VEGF was as high as 91%.
Therefore， anti-angiogenesis targeted drug therapy is expected to become a new solution for the treatment of MM， and targeted VEGF drugs combined with cytotoxic drugs are more effective than targeted drugs alone in treating MM patients. Bevacizumab is an artificial monoclonal IgG antibody that directly acts against VEGF. It has a significant anti-angiogenic effect， and is especially clinically beneficial to solid tumors such as kidney cancer， colorectal cancer， breast cancer and non-small cell lung cancer. However， bevacizumab monotherapy has a limited effect in the treatment of MM. The phase II experiment of MM showed that the median progression-free survival of patients under bevacizumab monotherapy was 3 months， and the median overall survival time was only 8.5 months， while when bevacizumab was combined with capecitabine or paclitaxel， most patients’ disease was stable for more than 8 weeks， and the median overall survival time was 12 months[13]. The anti-tumor effect of recombinant human vascular endothelial inhibitor is achieved by inhibiting the proliferation of vascular endothelium induced by VEGF， and it also has a synergistic effect in combination with chemotherapy drugs. Clinical trials have found that the combined application of endostar with dacarbazine and cisplatin is effective for metastatic MM without gene mutations， and the adverse events are controllable[14]. Combination of targeted therapy with radiotherapy
In recent years， targeted therapy combined with radiotherapy has made certain developments in the treatment of MM， especially in stage III and IV MM， and has become a new treatment mode. Ipilimumab injection[15] is the first drug that can definitely prolong the life of patients with metastatic MM. As the most representative monoclonal antibody drug， it can not only effectively block cytotoxic T lymphocyte antigen CT-LA-4 molecules， and significantly improve the survival rate of patients with advanced MM， effectively extending the life cycle by 3.5 months[16].
However， clinically， the total remission rate of ipilimumab alone for the treatment of MM is still low， only 5%-15%[17]. The latest study found that targeted therapy combined with radiotherapy can simultaneously control local and distant metastases of MM， and prolong progression-free survival[18]. Hodi et al.[19] reported that after ipilimumab monoclonal antibody combined with radiotherapy in patients with stage III and IV MM， the patient’s survival increased from 6.4 months to 10 months， indicating that the combination of the two treatments can enhance the treatment effect. Drake et al.[20] also reported that the combination of the two has a clear synergy. In addition， some studies have also shown that while receiving ipilimumab in patients with stage III and IV MM， palliative radiotherapy can cause distant effects at the far end of the radiotherapy site， and the potential effects of targeted immunotherapy and radiotherapy are increased， thereby reducing tumor metastases[21-23].
Combination of targeting genes with viral therapy
Combining targeting genes with viral therapy was first proposed by Academician Liu Xinyuan， and it is a new anti-cancer strategy that combines the advantages of gene therapy and viral therapy. Tumor-specific proliferating adenoviruses carrying therapeutic genes will not cause damage or have little impact to normal cells and tissues， but only replicate in a large number of specific tumor cells， retaining the ability of the virus to kill cancer cells. Meanwhile， it is accompanied by the replication of viruses， and the expression of anti-oncogenes in tumor cells also greatly increases， thereby further improving the therapeutic effect[24]. Zheng et al.[25] co-transfected human embryonic kidney cell HEK293 cells with a plasmid carrying ZD55-IL-18 and plasmid pBHGE3 containing the right arm of adenovirus to obtain the recombinant adenovirus ZD-55-IL-18， which can express IL-18 gene efficiently in MM cells and inhibit the growth of MM cells. Experiments have shown that the combination of targeting genes and viruses can increase the sensitivity of tumor cells to chemotherapeutics and improve the efficiency of chemotherapy. Jiang et al.[26] combined ZD55-IL-24 and DTIC to treat MM in vitro experiments and achieved better therapeutic effects than single use. What’s more worth mentioning is that targeting genes-viruses can also be combined with RNA interference technology. Zheng et al.[27] found in in vivo and in vitro experiments on kidney cancer using conditionally proliferating adenovirus （ZD-hTERT） loaded with small interfering RNA of human telomeras ereverset ranscrip tase （hTERT） gene that the virus replicated specifically in tumor cells and effectively silenced the hTERT gene of kidney cancer， thereby inhibiting cell proliferation and promoting its apoptosis. It suggests that we can combine RNAi technology to construct small interfering RNAs related to MM oncogenes to insert into the viral genome， providing new ideas for the treatment of MM. Combination of Immunotherapy with Other Methods
Combination of immunotherapy and chemotherapy
Recently， it has been discovered that MM has high immunogenicity. Therefore， immunotherapy for the occurrence， development and metastasis of MM has also become a research hotspot. Interleukin 2 （IL-2） is a lymphokine， which has anti-tumor effects and enhances the body’s immune function. As of 2011， high-dose interleukin 2 （HD-IL-2） is still one of the only two FDA-approved drugs for the systemic treatment of stage IV MM， and the other is dacarbazine. A prospective study by Kaufmann et al.[28] found that the therapeutic effects of temozolomide alone and temozolomide combined with interferon-α were different. The results showed that the overall response rate of the combination therapy was significantly higher than that of temozolomide alone， and the adverse reactions were tolerable. It is worthy of further research and clinical promotion.
The method of interleukin 2 combined with chemotherapy can prolong the survival time of patients， and the effective rate of the combination of the two drugs is significantly higher than that of chemotherapy or biological therapy alone. However， some scholars have found that[29]， after high-dose interferon is used for treatment of patients with high-risk recurrence factors， the toxic side effects brought by it can significantly increase the recurrence rate of the disease. Meanwhile， there are reports in the literature that the combined treatment of interleukin-2， histamine and interferon can reduce the dosage of interferon， thereby reducing the production of toxic and side effects， and the safety， tolerability and therapeutic effect are all improved to a certain degree[30].
Combination of immunotherapy and nanotechnology
Anti-tumor vaccine is one of the current research hotspots. Its main principle is to achieve anti-tumor effect mainly by initiating tumor-specific cytotoxic T lymphocyte （CTL） response. Tumor vaccines can activate the patient’s own immune system， effectively inhibit tumor growth， prevent tumor metastasis and recurrence， and will not damage other tissues. Its anti-tumor specificity and immune memory are unmatched by other methods[31]. However， tumor antigen vaccines are easily degraded， difficult to be effectively delivered to lymph nodes， and have low immunogenicity. Even if they are delivered to immune cells， they will quickly degrade and cause an effective immune response. In order to overcome the deficiencies of tumor antigen vaccines and make tumor antigen vaccines play a better effect， the application of nanotechnology has played an increasingly important role in the development of tumor antigen vaccines[32]. Researchers such as Conniot J. of Tel Aviv University[33] skillfully combined passive and active immunotherapy. Nanoparticles， that were made of biodegradable polymers and had a size between 160 and 190 nm， were wrapped with two peptides expressed in melanoma cells： Melan-A/MART-1 （26-35 （A27L）） class I major histocompatibility complex （MHCI） restricted peptide （MHCI-ag） and Melan-A/MART-1 （51-73） MHCII restricted peptide （MHCII-ag）. The nanoparticles were collected with mannose receptors to enable them to target ligand-mediated active dendritic cells. After the nano-vaccine was injected into the mouse model， it was found to be effective and preventive. It prevented the development of melanoma in healthy immunized mice， and significantly slowed down the progression of the disease even in mice with advanced melanoma.
Combination of immunotherapy and gene therapy
Compared with free molecular drugs， IL-2 or anti-CD137 immunoliposome delivery system can achieve the same anti-tumor activity， but the systemic toxicity is low or even non-existent. Zhang et al.[34] showed that the combined application of IL-2 and anti-CD137 quickly accumulated on the surface of liposomes in melanoma tumors， thereby minimizing systemic exposure and reducing the risk of toxicity.
Molecular Targeted Therapy Combined with Immune Checkpoint Inhibition Therapy
Molecular targeted therapy has a high overall remission rate but is prone to the production of drug resistance. However， the biggest problem faced by immune checkpoint inhibition therapy is the low remission rate， but it is not easy to develop drug resistance. The results of some clinical trials show that after the combined application of molecular targeted therapy and immune checkpoint inhibition therapy， patients will have sustained clinical benefits[35-37].
The results of a phase II clinical trial showed that in newly treated patients with metastatic MM with BRAF gene V600E/K mutations， by combined application of PD-L1 antibody atezolizumab， BRAF gene inhibitor vemurafenib and MEK inhibitior cobimetinib， the ORR reached 76%， and the adverse reactions in the test were controllable and all reversible[38].
Another clinical study divided patients with metastatic MM with BRAF gene mutations into three groups. The 26 patients in group A were given anti-PD-L1 treatment， combined with BRAF gene inhibitor Dabrafenib and MEK inhibitor Cobitinib until the disease progressed; the 20 patients in group B were given anti-PD-L1 treatment and cobitinib treatment at the same time until the disease progressed; and the 19 patients in group C were first treated with cobimetinib for 4 weeks， then given anti-PD-L1 treatment and cobimetinib treatment for 2 weeks， and then anti-PD-L1 treatment alone until the disease progressed[38]. This study allowed patients who had received immunosuppressive therapy （anti-CTLA-4， anti-PD-1， anti-PD-L1）， but excluded those who had received targeted drug therapy. The treatment results showed that the ORRs of patients in groups A， B， and C were 69%， 21%， and 13%， respectively， and the corresponding disease control rates （including complete remission， partial remission， and stable disease without progression） were 100%， 79% and 80%， respectively; and in the next 50 weeks of follow-up， only 90% of patients in group A received sustained clinical benefit[39]. Conclusions
The clinical effects of various monotherapy for MM patients are not very satisfactory. Targeted therapy and immunotherapy have obvious therapeutic effects on MM， but these methods are limited in the process of implementation. For example， patients receiving targeted therapy will soon develop resistance， while immunotherapy patients are only partially effective. The combined use of different treatment methods has achieved good results in preclinical and clinical trials. How can we improve the safety and maximize the anti-tumor effect is the main direction of future research. As people’s understanding of the pathogenesis of MM and biotechnology continues to deepen， combined treatment options are constantly being enriched in the medical treasure house. It is believed that with the development of tumor treatment， the combined treatment of MM will surely enter a new era.
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