Tumor vaccines: Toward multidimensional anti-tumor therapies

ABSTRACT

For decades, immunotherapies have offered hope for patients with advanced cancer. However, they show distinct benefits and limited clinical effects. Tumor vaccines have the potential to prime tumor-antigen-specific T cells and induce broad subsets of immune responses, ultimately eradicating tumor cells. Here, we classify tumor vaccines by their anti-tumor mechanisms, which include boosting the immune system, overcoming tumor immunosuppression, and modulating tumor angiogenesis. We focus on multidimensional tumor vaccine strategies using combinations of two or three of the above mechanisms, as these are superior to single-dimensional treatments. This review offers a perspective on tumor vaccine strategies and the future role of vaccine therapies in cancer treatment.

KEYWORDS:

• Tumor vaccines
• anti-tumor therapy
• immune system
• cytotoxic T cells
• immunosuppression
• tumor angiogenesis
• multidimensional

Introduction

Although surgical resection, chemotherapy, and radiotherapy still play important roles in tumor treatment, the powerful therapeutic ability of tumor vaccines has attracted the attention of scholars. Tumor vaccines use tumor antigens to activate the immune system, provoking both specific cellular immunity and a humoral immune response to prevent tumor growth and ultimately eradicate tumor cells.¹ Many studies have shown that tumor vaccines can not only prevent or slow the occurrence and development of tumors and prolong survival, but can also prevent the growth, angiogenesis, and even metastasis of established tumors. The core mechanism of tumor vaccine therapy is the induction of an active immune response to antigens or cells by delivering the target antigen or cells to the body, thereby inducing cytotoxic T cells and serum antibodies to specifically kill target cells and antigens, and establishing an immune microenvironment for tumor inhibition. Th1 cytokines (IFN- γ, TNF- α, IL-2) tend to show high levels during this process.^2,3 The intensity of the cellular immune response or humoral immune response produced by the immune system is limited by many factors, including the immunogenicity of the tumor vaccine, the treatment of tumor vaccine by antigen-presenting cells, the immune tolerance of the body to the tumor vaccine, the integrity of the immune system, and the malignancy of the tumor. In order to overcome inherent resistance, researchers have developed tumor vaccines with different target cells and various antigen forms and have combined the use of adjuvants and DC ligands to modify or co-express DC-activating molecules to enhance the immune response to tumor vaccines. The most direct way to induce an immune response to a tumor is to deliver proteins characteristic of tumor cells or lysates of tumor cells to the immune system. However, tumor-specific antigens are often difficult to identify because the vast majority of molecular antigens of tumor cells are common to human cells, meaning that the immune system cannot recognize them as foreign antigens. Moreover, tumor cells have high mutagenicity, which creates limitations and challenges for tumor vaccines that directly target tumor cells. Therefore, we focus on the potential and development of tumor vaccines that activate the tumor immune microenvironment and inhibit tumor angiogenesis. Tumor vaccines induce the activation of effector T cells that kill tumor cells, weaken the functional activity of Treg in tumors, and enhance tumor immunity, making it difficult for tumor cells to continue to grow. Additionally, tumor vaccines inhibit tumor angiogenesis, assist tumor treatment, and allow clinical possibilities to prevent tumor growth and prolong survival. Tumor angiogenesis provides the key source of nutrition for tumor cells to survive, thus blocking it is critical. As tumor development and metastasis is a complex process involving multiple environments and cells, single-treatment tumor vaccines still face the problems of drug resistance, tumor cell variation and compensatory mechanisms. Therefore, to take full advantage of therapeutic tumor vaccines, greater efforts are needed to explore and develop multidimensional tumor vaccines. Different forms of tumor vaccines such as adenovirus vaccines, DNA vaccines, mRNA vaccines, protein vaccines, polypeptide vaccines, and whole-cell vaccines show different degrees of anti-tumor immune response. The selection of antigen type is important to allow multidimensional targeting by tumor vaccines.

Tumor vaccines based on boosting the immune system

Boosting the immune system is an important goal in cancer immunotherapy. To achieve this goal, several immunotherapies have been developed. In this section, we focus on the research status of tumor vaccines that induce and enhance the anti-tumor effects of CD8⁺ and CD4⁺ T cells. Figure 1 illustrates the anti-tumor mechanism of tumor vaccine strategies based on boosting the immune system.

Figure 1. Tumor vaccine strategies based on boosting the immune system. Different types of tumor vaccines are inoculated and presented by DC to activate the CD8⁺ T cells, CD4⁺ T cells and B cells to kill tumor cells directly or indirectly. Abbreviations: DC: dendritic cells; ICAM-1: intercellular cell adhesion molecule-1; CTL: cytotoxic T cells; MHC: Major histocompatibility complex; IFN-γ: interferon γ; GzmB: granzyme B; PFN: perforin; TAMs: tumor-associated macrophages.

Cross-presentation

The cross-presentation pathway is an antigen presentation pathway by antigen-presenting cells (APC) to exogenous antigens, that is, exogenous antigens form complexes with MHC class I molecules after being treated with APC and are then presented to CD8⁺ T cells.⁴ Through the cross-presentation pathway, exogenous antigens in tumor vaccines can be presented to CD8⁺ T cells, thus inducing them to kill the target cells. APCs, particularly dendritic cells (DCs), are essential for presenting tumor antigens on HLA-I to CD8⁺ T cells.^5,6

Many studies have been devoted to promoting the cross-presentation of DCs to induce the cytotoxic functions of tumor-specific CD8⁺ T cells. Enhancing the cross-presentation of antigens promotes the initiation of tumor immune responses. DCs are immature before being exposed to antigens, and only mature DCs can effectively initiate antigen-specific T cell responses. There are two ways to promote DC maturation: one is antigenic stimulation and the other is adjuvant. Dc-based cancer vaccines are typically loaded with antigens to immature DC, combined with the use of adjuvants. Dc-based cancer vaccines have been well discussed in other articles.⁷ One notable result was SiPuleucel-T, the first vaccine approved by the FDA in 2010 for the treatment of early-stage asymptomatic metastatic trend-resistant prostate cancer. In a randomized placebo-controlled trial involving 512 men with metastatic castration-resistant prostate cancer, a dendritic cell-based vaccine (sipuleucel-T) significantly improved overall survival in men with metastatic castration-resistant prostate cancer.⁸ The vaccine extracts APCs from the patient’s peripheral blood and pulses them with a fusion protein consisting of GM-CSF and human prostatic acid phosphatase (PAP), stimulating them and inducing them to mature before being injected back into the patient to trigger a systemic antigen-specific T cell response.^9–11 However, host factors, such as race population, and the lack of prostate specific antigen (PSA) or radiographic response in clinical trials negatively impact the clinical benefit of sipuleucel-T.¹² Cytoplasmic transduction peptides (CTP) were successfully used to mediate the localization of BCR-ABL protein-derived antigen peptides in the cytoplasm of dendritic cells, which stimulated a cross-antigen presentation reaction in dendritic cells, thereby presenting exogenous antigens to CD8⁺ T lymphocytes and inducing a specific cellular immune response in chronic myeloid leukemia, ultimately killing BP210 target cells.¹³ Based on the existing anti-tumor vaccine of DC cells, this experiment introduced CTP into the immunotherapy of CML to carry out DC antigen loading, thus stimulating the anti-CML effect. This provides a theoretical basis for dendritic cell immunotherapy. DC subsets are usually divided into five categories according to their developmental origin: conventional DC (including type 1 conventional DC (cDC1) and type 2 conventional DC (cDC2)), monocyte derived DC (MoDC), plasma cell-like DC (pDC) and a new type of DC (DC3).¹⁴ Choosing the right DC subgroup is crucial. In a mouse melanoma model, CD103⁺ DCs were the only APCs used to prime tumor-specific CD8⁺ T cells, and injection of a cytokine-adjuvant vaccine led to the major expansion of CD103⁺ DC precursor cells and immature CD103⁺ DC cells at the tumor site, thus promoting anti-tumor immunity.¹⁵ CD103⁺ DCs has been proved to be the main antigen presenting cell involved in the initiation of anti-tumor T cells.¹⁶ Good adjuvants stimulate DC antigen cross presentation and DC maturation at the same time.There are two main DC cross pathways related to vaccine adjuvant action: the vacuolar pathway and the cytosolic pathway.¹⁷ It is feasible to enhance the effectiveness of CD8⁺ T cell response by using different vaccine adjuvants that participate in these two pathways. Studies on the administration of adjuvants and chemokines and on targeting strategies to activate, recruit, and deliver antigens to cross-presenting dendritic cells are summarized in detail in.¹⁸ In addition, various types of nanoparticle vaccines to improve cross-presentation have been developed and studied. Several research teams have designed nanoparticle vaccines that contain interferon gene stimulator (STINT) agonists to stimulate interferon-I production, leading to more antigen cross-stimulation and stronger downstream CD8⁺ T cell responses.¹⁹ Boosting the cross-presentation of antigens in tumor vaccines to CD8⁺ T cells is a promising strategy for cancer immunotherapy, however, the selection of the best antigen, suitable DC subsets and adjuvants has always been the direction of researchers’ efforts.

Cytotoxic CD8⁺ T cells

The discovery of tumor-associated antigens (TAA) has led to the development of therapeutic tumor vaccines based on synthetic peptides, DNA, DCs or recombinant proteins in an attempt to enhance the anti-tumor immune response, especially that of tumor antigen-specific cytotoxic T cells (CTL). Cytotoxic CD8⁺ T cells (CD8-CTLs) play an important role in tumor immunity.

Most peptide vaccines are based on epitope peptides that stimulate CD8⁺ T cells to target tumor-associated antigens (TAA) or tumor-specific antigens (TSA).²⁰ Short peptides (8–11 amino acids) can directly bind to human leukocyte antigen class I molecules to enhance MHC-I-restricted antigen-specific CD8⁺ T cellular immunity. When combined with effective adjuvants, long-peptide vaccines can usually produce more extensive immunity than short-peptide vaccines by inducing responses from both CD8-CTLs and CD4⁺ helper T cells. A study on the immunity induced by MHC I epitopes of tumor antigen SIM2 showed that a single peptide containing multiple SIM2 epitopes can be used to induce CD8⁺ CTL responses, providing a peptide-based vaccine formula for immunotherapy for various cancers.²¹ The first generation of tumor vaccines is based on non-mutant TAAs, which are also known as shared antigens, as they are expressed in many tumors. They include MART-1, gp100, tyrosinase, Trp-2, NY-ESO-1, MAGE-A3, and Her2/neu or telomerase proteins. They have been shown to be immunogenic and able to induce clinical responses in advanced cancer patients. Alpha-fetoprotein (AFP), a tumor-specific antigen, is an important index for the clinical diagnosis of hepatocellular carcinoma. AFP-specific CD8⁺ T cells activated by DCs showed significant anti-tumor immune activity in vitro.²² In an invasive B16F10 melanoma model, CD8⁺ T cells were strongly activated after a single immunization with a liposome-assisted mRNA vaccine. In a melanoma model of transgenic mice, the vaccine induced the proliferation of not only CD8⁺ T cells but also killer cells, which prolonged the overall survival time.²³ An individualized mRNA neoantigen vaccine, in combination with atezolizumab and mFOLFIRINOX induces substantial T cell activity in patients with surgically resected PDAC that correlates with delayed recurrence.²⁴ Preclinical studies demonstrated that a DNA vaccine (pTVG-AR, MVI-118) encoding the androgen receptor ligand-binding domain (AR LBD) augmented antigen-specific CD8⁺ T cells, delayed prostate cancer progression and emergence of castration-resistant disease, and prolonged survival of tumor-bearing mice. PTVG-AR was demonstrated to be safe and immunologically active in patients with mCSPC in a phase I clinical trial. And a prospective evaluation will be carried out in future trials.²⁵ A key challenge for nucleic acid vaccines is how to protect them from degradation before reaching the target. To this end, scientists are using a variety of nanomaterials for delivery to protect them from degradation. Lipid Nanoparticles and Liposomes has become the preferred drug delivery system (DDS, drug delivery system) for gene therapy and other complex injection drug products, representing the future of nanomedicine.²⁶ Studies have shown that, as a new type of viral vector, CMV (Cytomegalovirus) can induce different CD8⁺ T cell responses, and the tumor-specific CMV vector vaccine can induce a large number of CMV-specific CD8⁺ T cells.^27,28 Adenovirus-based vectors play an important role in promoting T cell broadening, expansion, and differentiation to maintain anti-tumor response. Development of virus-based vector vaccines, researchers have developed a fast process for producing viral vectors encoding 60 unique patient-specific neoantigens and recently initiated a clinical trial in patients with metastatic melanoma and non – small cell lung cancer.²⁹ A promising ISV (in situ vaccination) strategy is to use oncolytic virus, which can not only induce immunogenic cell death to load tumor Ag, but also stimulate pro-inflammatory signals to activate DC.³⁰ This strategy can be quickly translated into clinical practice. At present, it has entered the phase I clinical trial (NCT03889275) (NCT04613492). Advances in rapid genome sequencing have led to the development of personalized cancer vaccines (PCV) for tumor-specific mutations known as new antigens. Early studies in mice and phase I clinical trials have proven the feasibility of PCV to produce T-cell responses to new antigens. New antigen peptides were ligated to TLR7/8 agonists (SNP-7/8a) by a self-assembled nanoparticle vaccine. Intravenous administration of SNP-7/8a produced TCF1⁺PD-1⁺CD8⁺ T cells with anti-tumor ability after anti-PD-L1 treatment and mediated anti-tumor effects dependent on immune checkpoint inhibition.³¹ A nanoparticle (NP) vaccine significantly enhanced the activation of dendritic cells and antigen cross-presentation, resulting in the strong activation of new-antigen-specific CD8⁺ T cells. After a single subcutaneous injection, the frequency of CD8⁺ T cells in systemic circulation was as high as 23 ± 7%. An NP vaccine combined with STING agonist treatment eliminated tumors in MC-38 colon cancer and B16F10 melanoma mice and established long-term immune memory. These studies present a new treatment strategy based on combined nano-immunotherapy for personalized tumor immunotherapy.³² A retrospective study of seven patients with advanced pancreatic cancer showed that patients with longer overall survival had higher titers of interferon-γ and CD8⁺ effect memory T cells in their peripheral blood after vaccination with a new antigen peptide vaccine, indicating that this new antigen peptide vaccine had the potential to activate of specific T cell subsets to kill cancer cells.³³ The gene mutation of tumor is very important in the process of T cell activation, because the gene mutation can produce new antigens, which are more easily recognized and attacked by the immune system. Because of this, the higher the mutation frequency of tumor cells, the more the number of new antigens produced, and tumors with this characteristic tend to respond better to immunotherapy. Many studies have shown that the new antigen vaccine has the potential to induce anti-tumor immunity.³⁴

Effective CD4⁺ T cell responses

Tumor reactive CD4⁺ helper T cells

CD4⁺ T cells are well known for their auxiliary effects. For example, CD4⁺ T cells are involved in the regulation of anti-tumor immunity by helping to initiate and enhance the cytotoxic activity of CD8⁺ T cells. Although CD8⁺ T cells were initially considered to be the primary acting cells, it is now clear that CD4⁺ T cells also play a key role in the anti-tumor response. Tumor-reactive CD4⁺ helper T cells (Th1) produce interferon-γ, tumor necrosis factor-α, and IL-2, which play key roles in cell-mediated anti-tumor immunity. Newly identified telomerase reverse transcriptase (TERT)-derived peptides are known as universal cancer peptides (UCP). UCP vaccines can induce high affinity and tumor-specific CD4⁺ Th1 polarization.³⁵ By combining synthetic peptides with TLR agonists and OX40/CD40 co-stimulation, a major CD4⁺ T cell response can be produced. This vaccine strategy is effective in overcoming immune tolerance to self-tumor-associated antigens and had a significant anti-tumor effect in malignant melanoma mouse models.³⁶ The animal and clinical trials of tumor vaccines based on boosting immune response are listed in Tables 1 and 2 respectively.

Table 1. Experimental studies of tumor vaccines based on boosting the immune response.

Vaccine type	Regimen	Adjuvant	Tumor model	Outcome	Ref.
Peptide vaccines	CTP-mediated peptides	—	Mouse Leukemia model (protective and therapeutic immunization strategies).	Induced a more effective cellular immune response.	Yang^Citation13
	NanoSTING-vax (a synthetic cancer nanovaccine)	—	MC38 murine colorectal adenocarcinoma model (protective and therapeutic immunization strategies).	Enhanced the CD8^+ T cell responses to a diversity of peptide antigens.	Shae et al.^Citation19
	SIM2-derived peptides	—	Immunization models.	Induced an effective CD4^+ and CD8^+ T-Cell response.	Kissick et al.^Citation21
	TriVax vaccine (peptide, TLR ligand, and CD40 mAb, administered i.v.)		Mouse model of malignant melanoma (therapeutic immunization strategies).	Induced more robust antigen-specific CD4^+ T-cell responses than conventional adjuvants.	Kumai et al.^Citation36
mRNA vaccines	Lipid nanoparticles containing mRNA coding for the tumor-associated antigens gp100 and TRP2	LPS	Murine melanoma model (therapeutic immunization strategies).	Inducing a strong cytotoxic T cell response. Significantly extended the overall mice survival.	Oberli et al.^Citation23
	A lymph-node – targeting mRNA vaccine based on lipid nanoparticles (113-O12B/mOVA)	LNP	Murine B16F10-OVA melanoma model(protective and therapeutic immunization strategies)	O12B/mOVA Elicited a Robust CD8 T Cell Response and Protective Effect on the B16F10-OVA Tumor Model.	Chen et al.^Citation37
	Circular RNA vaccine(CART-circOVA vaccine)		Murine B16-F10-OVA melanoma model(protective immunization strategies)	Observed potent and persistent T cell responses in mice following immunization. The circRNA vaccine group showed a significant tumor growth inhibition compared to the untreated group	Amaya et al.^Citation38
Cell based cancer vaccines	AFP peptide-pulsed or lenti-AFP-engineered methods dendritic cells	—	Hepatocellular carcinoma (in vitro study).	AFP-specific T cells could efficiently kill HepG2 cells	Liu et al.^Citation22
Viral vector vaccine	Murine CMV to express a modified B16 melanoma antigen gp100	—	Aggressive B16 lung metastatic melanoma model (protective and therapeutic immunization strategies).	Induced a potent gp100-specific CD8^+ T-cell response and strongly protected mice from highly metastatic lung melanoma.	Qiu et al.^Citation27
	Mouse CMV vector expressing RAE-1γ	—	A mouse model for human melanoma (protective and therapeutic immunization strategies).	Promote the induction of KLRG1 CD8^+ T cells with augmented effector functions	Tršan et al.^Citation28
	Genetic vaccines based on adenoviruses derived from nonhuman Great Apes (GAd)	—	Murine MC38 colorectal adenocarcinoma model (therapeutic immunization strategies)	GAd vaccine combined with αPD-1 treatment enhances immunogenicity and expands neoantigen-specific CD8^+ T cells in the tumor	D’Alise et al.^Citation29
	Flt3L-NDV ISV	—	GFP A20 tumor model(therapeutic immunization strategies)	NDV efficiently induced cDC1 activation and tumor Ag cross-presentation, and that Flt3L induced robust amplification of tumor-specific T cell responses and long-term tumor control. Flt3L-NDV ISV therapy efficiently induced CD8 T cells reactive to neoepitopes identified by tumor exome and RNA sequencing.	Svensson-Arvelund et al.^Citation30

CTP: cytoplasmic transduction peptide; AFP: alpha-fetoprotein; LPS: lipopolysaccharide; LNP: lipid nanoparticle; OVA: ovalbumin; CMV: cytomegalovirus; NDV: oncolytic Newcastle disease virus; ISV: in situ vaccination.

Table 2. Clinical trials of tumor vaccines based on boosting the immune response.

Vaccine type	Regimen	Clinical trial identifier	Conditions	Adjuvant	Phases	Outcome	Ref.
Peptide vaccine	CPV S-588410 (a novel CPV comprising five human leukocyte antigens (HLA)-A *24:02-restricted 9–10-mer peptides derived from five cancer testis antigens (DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1))	UMIN000023324	Esophageal cancer (n = 15)	MONTANIDE ISA51VG	Exploratory open‑label clinical study	Densities of CD8^+, CD8^+ Granzyme B^+, CD8^+ programmed death-1-positive (PD-1+) and programmed death-ligand 1-positive (PD-L1+) cells were higher in post- versus pre-vaccination tumor tissue.	Daiko et al.^Citation39
	iNeo-Vac-P01(Neoantigen peptides)	NCT03645148	Pancreatic cancer(n = 7)	GM-CSF	I	Indicated the potential of iNeo-Vac-P01 in inducing the activation of a specific subset of T cells to kill cancer cells	Chen et al.^Citation33
	NEO-PV-01 (consisting of up to 20 synthesized peptides of approximately 14–35 amino acids in length that are derived from an individual patient’s mutated tumor DNA)	NCT03380871	Lung Cancer (n = 38)	Poly-ICLC	Ib	NEO-PV-01 plus chemotherapy and anti-PD-1 induces neoantigen-reactive T cell responses that are neo-epitope specific, persistent, and show cytotoxic potential. A large majority of the vaccine-induced cytotoxic responses were CD4^+ T cells.	Awad et al.^Citation34
	VX-001 (targets the universal tumor antigen TElomerase Reverse Transcriptase (TERT))	NCT01935154	Non-small Cell Lung Cancer (n = 221)		II	Vx-001 could induce specific CD8 immune response but failed to meet its primary endpoint.	Gridelli et al.^Citation40
mRNA vaccines	Autogene cevumeran	NCT04161755	Pancreatic ductal adenocarcinoma (n = 16)	Lipoplex nanoparticles	I	The vaccine induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen.Vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8^+T cells.	Rojas et al.^Citation24
DNA vaccines	pTVG-AR (a DNA vaccine encoding the androgen receptor ligand-binding domain)	NCT02411786	Prostate Cancer (n = 40	GM-CSF	I	pTVG-AR was safe and immunologically active in patients with mCSPC. Association between immunity and PPFS suggests that treatment may delay the time to castration resistance, consistent with preclinical findings.	Kyriakopoulos et al.^Citation25
Cell based cancer vaccines	Sipuleucel-T (an active cellular immunotherapy, a type of therapeutic cancer vaccine, consisting of autologous peripheral-bloodmononuclear cells)	NCT00065442	Prostate cancer (n = 512)		III	Sipuleucel-T prolonged overall survival among men with metastatic castration-resistant prostate cancer.	Kantoff et al.^Citation8
	GBM6-AD (lysate of an allogeneic glioblastoma stem cell line)	NCT02549833	Low-grade gliomas (n = 17)	Poly-ICLC	I	Neoadjuvant vaccination induced upregulation of type-1 cytokines and chemokines and increased activated CD8 T cells in peripheral blood.	Hirokazu et al.^Citation41
	CpG-MCL vaccine	NCT00490529	Mantle cell lymphoma (n = 47)(MCL)		II	CpG-MCL vaccine induce CD8 T cell immune responses that are associated with a superior clinical outcome.	Frank et al.^Citation42
Viral vector vaccines	Nous-209 vaccine(Nous-209 is based on a heterologous prime/boost regimen composed of the Great Ape Adenovirus GAd20–209-FSP used for priming and Modified Vaccinia virus Ankara MVA-209-FSP used for boosting)	NCT04041310	Unresectable or metastatic deficient mismatch repair (dMMR) or MSI-H CRC, gastric, or gastro-esophageal junction (G-E junction) tumors(n = 10)		I	Nous-209 vaccination elicits a strong and broad neoantigen-specific T cell response in patients with dMMR tumors.	Svensson-Arvelund et al.^Citation30
	MEDI5395(a Recombinant Newcastle Disease Virus Encoding genes for GM-CSF)	NCT03889275	Advanced Solid Tumors(n = 39)		I	Unpublished
	MEDI9253(a Recombinant Newcastle Disease Virus Encoding Interleukin-12)	NCT04613492	Solid Tumors(n = 37)		I	Unpublished
	VRP-HER2（based on a novel alphaviral vector encoding a portion of HER2）	NCT01526473	HER2 Expressing Breast Cancer (n = 22)		I	VRP-HER2 was well tolerated in patients and vaccination induced HER2-specific T cells and antibodies.	Crosby et al.^Citation43

CPV: cancer peptide vaccines; GM-CSF: granulocyte-macrophage colony-stimulating factor; Poly-ICLC: poly-IC containing poly-lysine and carboxymethylcellulose; Poly-IC: a dsRNA mimic that functions as a pattern recognition receptor ligand; CpG: cytosine-phosphate- guanosine.

Cytotoxic CD4⁺ T cells

In addition to conventional CD4⁺ T cells including Th1, Th2, Th17, peripherally- induced regulatory T cells, T follicular helper T cells (Tfh),^44,45 there is a group of CD4 cells that express cytotoxic molecules such as perforin and granzyme, which are defined as cytotoxic CD4⁺ T cells (CD4-CTLs).⁴⁶

The earliest discovery of CD4-CTLs in cancer patients was reported in 2001⁴⁷; the number of CD4-CTLs expressing perforin was significantly increased in chronic B-cell lymphocytic leukemia patients compared with healthy controls.⁴⁷ Moreover, this group of cells can kill autogenous B-cell tumor cells dependent on the release of perforin without the involvement of FAS-FASL pathways or tumor necrosis factor.⁴⁸ In a melanoma mouse model, a small amount of tumor-reactive CD4⁺ T cells induced significant tumor regression, and the anti-tumor effect was more significant after blocking the co-suppressor molecule CTLA-4. This anti-tumor effect is achieved through MHC II molecule-restricted degranulation, which does not depend on apoptosis induced by the CD95-CD95L pathway or on the participation of CD8⁺ T cells, B cells, NK cells, or NKT cells. This suggests that CD4⁺ T cells have a strong killing effect.⁴⁹ Retrospective immunoanalysis of circulating T cells from melanoma patients treated with ipilimumab showed that an increase in the percentage of CD4-CTLs cells at 8–14 weeks is associated with positive clinical outcomes. CD4⁺ T cells established from peripheral blood mononuclear cells (PBMCs) of melanoma patients express granzyme B, perforin and LAMP (CD107a), and can lyse autologous melanoma cells in an MHC II-dependent manner.⁵⁰

The number of CD4-CTLs was increased in patients with early liver cancer but decreased significantly with the progression of the disease.⁵¹ Through analysis of a comprehensive RNA-seq gene expression data set of neuroblastoma, the effector cell of high-risk neuroblastoma was determined to be CD4+CD3+NKG2C/E+CRTAM+, also known as CD4-CTLs. In 2021, it was found that the expression of Fas is not related to the survival of patients with high-risk neuroblastoma. On the contrary, the expression of GZMA/GZMB in CD4-CTLs is related to longer survival time in patients with high-risk neuroblastoma. The expression of OX40-encoding costimulatory molecules in CD4-CTLs is also associated with better outcomes. A similar trend was observed in 4-1BB, although its expression level was lower than that of OX40. These observations suggest that the anti-neuroblastoma effect of CD4-CTLs depends on the perforin/granzyme pathway.⁵² In addition, CD4-CTLs were found in patients with other solid tumors, such as breast cancer⁵³ and osteosarcoma.⁵⁴

After adoptive transfer of CD4⁺Trp1⁺ cells to mice received 5 Gy of irradiation, the cells were rapidly expanded and differentiated into cytotoxic CD4⁺ T cells, which efficiently eliminated established melanoma tumors in a mouse model of advanced disease.⁴⁹ This study provided preliminary evidence of the clinical potential of CD4-CTL-based adoptive cellular therapy (ACT). A clinical trial reported that adoptive transferred amplified autologous CD4⁺ T cell clones with melanoma-associated antigen NY-ESO-1 specificity mediated a durable clinical response and caused an endogenous response to melanoma antigens other than NY-ESO-1.⁵⁵ Prostate cancer patients have received the AE37 vaccine (a HER2 hybrid peptide vaccine) to induce AE37-specific CD4-CTLs.⁵⁶ The anti-tumor effect of CD4-CTLs has also been shown in studies on dendritic cell vaccines combined with IL-12 for the treatment of liver cancer in mice. Overall, these studies show that CD4-CTLs play a role in anti-tumor therapy. Although the specific mechanism of their anti-tumor effect needs to be further explored, these findings emphasize the possibility of using CD4-CTLs in tumor immunotherapy.

Tumor vaccines based on overcoming tumor immunosuppression

Studies have identified multiple tumor vaccine strategies, but clinical adoption rates remain low due to the occurrence of immune-related adverse events. These events are related to immunosuppressive cells such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), tumor-associated macrophages (TAMs), and tumor-associated fibroblasts (CAFs) in the tumor microenvironment (TME).⁵⁷ This has led to an increasing interest in tumor vaccines that can overcome tumor immunosuppression. The anti-tumor mechanism of tumor vaccine strategies based on overcoming tumor immunosuppression is described in Figure 2.

Figure 2. Tumor vaccine strategies based on overcoming tumor immunosuppression. Tumor vaccines reshape the tumor microenvironment by remodeling, repolarizing, gene silencing or target immunosuppressive cells, turning immunosuppression into immunostimulation. [Figure reproduced from bleve A, durante B, sica A, consonni FM. Lipid metabolism and cancer immunotherapy: immunosuppressive myeloid cells at the Crossroad. Int J Mol Sci. 2020 Aug 14; 21(16): 5845. doi: 10. 3390/ijms21165845. PMID: 32823961; PMCID: PMC7461616] Abbreviations: MDSC: myeloid-derived suppressor cells; TAN: tumor-associated neutrophils.

Inhibiting tregs

Tregs are a subset of T cells that have the function of suppressing excessive immune activation. They are present in all body tissues. Tregs in different organs exert different functions through different mediators; in addition to maintaining immune tolerance, Tregs also act as organ-specific suppressors of tumor immunity, thereby mediating tumor immune escape.^58,59 An increased number of Tregs is the primary factor minimizing the protective effect of tumor vaccines and reducing the number of immunosuppressive cells in the TME. Therefore, eliminating them or weakening their function can enhance the immune response to tumor vaccines.⁶⁰

IMU-131/HER-Vaxx is a therapeutic B-cell epitope vaccine consisting of three fused B-cell epitopes from the HER2/neu extracellular domain coupled to CRM197 and adjuvanted with Montanide. In a gastroesophageal adenocarcinoma (GEA) phase Ib experiment, 14 patients with either gastric or gastroesophageal junction adenoma were enrolled. The effectiveness of the cancer vaccine, evaluated by progression-free survival, and tumor response, belonged to the exploratory endpoints of this phase Ib trial. IMU-131/HER-Vaxx induced a HER2-specific Ab and cellular immune response after inoculation, with a significantly reduced number of regulatory T cells and the reductions of Tregs were often in accordance with tumor regression. This may be due to the expansion of the T effect population, resulting in a relative decrease in Tregs in peripheral blood. Vaccine-induced Ab has functions similar to trastuzumab.⁶¹ Trastuzumab is a widely used mAb targeting HER 2/neu EGF. The IMU-131/HER-Vaxx vaccine not only overcomes the shortcomings of passive antibody administration; it also overcomes the tumor-promoting effect of tumor immunosuppressive cells, making treatment more comprehensive.

A study of the CHP-MAGE-A4 cancer vaccine explored whether the treatment effect and immune response caused by cancer vaccines can be predicted by the percentage of CD4⁺CD25⁺Foxp3⁺ Tregs in peripheral blood mononuclear cells (PBMCs) after vaccination. The results show that the proportion of Tregs after vaccination seems to have predictive value for the prognosis of patients.⁶² It has been found that the FoxP3 transcription factor, a specific intracellular marker of Tregs, helps distinguish the phenotype of suppressive CD4⁺CD25⁺ T cells from that of effector T cells.⁶³ Foxp3 is expressed not only in CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells but also in immunosuppressive CD8⁺T cells. Therefore, immunotherapy targeting Foxp3 was developed to achieve tumor immunity.⁶⁴ In B16 melanoma, Foxp3 silenced GM-CSF gene-modified tumor vaccine,⁶⁵ prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine)⁶⁶ could target and inhibit Tregs in the TME to enhance the anti-tumor effect. Some studies have also found that the immunogenicity of tumor antigens can be enhanced when they are fused with segment C (Fc) of the immunoglobulin heavy chain to produce antigen-Fc fusion proteins such as Foxp3-Fc (IgG). Foxp3 silenced vaccine combined with other vaccines can stimulate higher immune responses than its single application.^67–69

Inhibiting other immunosuppression

In contrast to the Tregs-inhibiting tumor vaccines described above, some vaccines inhibit other immunosuppressors to generate anti-tumor immunity. A novel synthetic tyrosinase (Tyr) DNA vaccine improved the survival rate of melanoma mice by reducing the number of MDSCs in the TME and improving the inhibition environment.⁷⁰ Some studies have found that a dendritic cell-based hepatocellular carcinoma (HCC) neoadjuvant nano-vaccine can reshape TANs to enhance the anti-tumor immune response, thus enhancing the vaccine’s therapeutic effect.⁷¹ A DC-shA20-FAP-TRP2 vaccine in a B16 melanoma model targeted cancer-associated fibroblasts (CAFs) in addition to cancer cells and may enhance vaccine-induced immunity and effects.⁷² In other cases, such as in studies on DC and CAF fusion vaccines, FAD-modified transgenic tumor cell vaccines, etc., the accumulation of immunosuppressive cells in the TME was significantly reduced.^73,74 We list tumor vaccines that reduce tumor immunosuppression in Table 3.

Table 3. Tumor vaccines based on overcoming tumor immunosuppression.

Study type	Type of tumor	Phase	Substance	Outcome	Ref.
Experimental study	Melanoma	-	HCA587 protein + CpG ODN + ISCOM	The vaccination resulted in enhanced accumulation of TILs with enrichment of conventional CD4+ T cells but a reduced representation of Treg cells.	Chen et al.^Citation75
	HPV	-	HPV16 E7 43–77 peptide + CpG ODN	Increased cellular immunity was mediated by CD4+IFN-γ+ T cells and CD8+IFN-γ+ T cells, while decreased numbers of immunosuppressive cells including Tregs and MDSCs were induced by the vaccine.	Yang et al.^Citation76
	Melanoma	-	Vac + ASOcont + transfected irradiated B16 cell vaccine	This tumor cell vaccine combined with Foxp3 or CTLA4 gene silencing increased the efficacy of antitumor therapy.	Miguel et al.^Citation65
		-	Fragment C (Fc) portion of IgG + Foxp3	The expression of Foxp3-Fc (IgG) in vaccines in tumor therapies for removal of regulatory cells serves as a successful strategy to increase the efficiency of other immunotherapies.	Mousavi et al.^Citation67
	Melanoma	-	Human tyrosinase gene (Tyr) sequences	This vaccine reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5, and arginase II, which are factors important for MDSC expansion.	Yan et al.^Citation70
	HCC	-	SiPCCl2-hybridized mesoporous silica with coordination of Fe (III)-captopril	This neoantigen nano-vaccine remodels tumor-associated neutrophils to potentiate the anticancer immune response and enhance immunotherapy efficiency.	Wang et al.^Citation71
		-	Mouse A20 small hairpin interfering RNA sequence	This vaccine encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2).	Gottschalk et al.^Citation72
Clinical trial	HNSCC	I	Autologous monocyte-derived DCs loaded with selected wt p53 peptides	Two-year DFS was 88%; p53-specific T-cell and IFN-ded with selected wt p53 pept Treg frequency was consistently decreased.	Schuler et al.^Citation77
	GEA	Ib	HER2/neu fusion peptide P467+nontoxic diphtheria protein CRM197	IMU-131 was well tolerated and safe. The vaccine-induced HER2-specific Abs and cellular responses were dose-dependent. The numbers of FOXP3+ Tregs in patients were reduced following tumor regression.	Wiedermann et al.^Citation61
	Esophageal, stomach, or lung cancer	I/II	Cholesterol-bearing hydrophobized pullulan (CHP)+MAGE-A4 protein (CHP-MAGE-A4)	Treg ratios that remained low both before and after vaccination were associated with a good prognosis, and a low Treg/CD4 lymphocyte ratio 7 weeks after the initial vaccination was correlated with a better prognosis.	Wada et al.^Citation62

HPV: human papillomavirus; CpG ODN: CpG-containing oligodeoxynucleotides; HCA587: a cancer-testis antigen; ISCOM: immune- stimulating complex; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cell carcinoma; GEA: gastroesophageal adenocarcinomas; DC: dendritic cell; TILs: tumor-infiltrating lymphocytes.

Tumor vaccines based on anti-angiogenesis

Professor J Folkman proposed that the growth and metastasis of solid tumors depend on neovascularization,^78,79 which opens the door to study the mechanism of tumor growth and leads to extensive exploration of immune strategies against tumor angiogenesis. Immunotherapy against tumor angiogenesis can be divided into passive immunotherapy and active immunotherapy. The former focuses on angiogenesis-related molecules and their interaction with receptors. Using antibodies to neutralize pro-angiogenic factors and blocking pro-angiogenic factor receptors to reduce the proliferation and migration of tumor endothelial cells, which can quickly block tumor neovascularization. However, long-term anti-angiogenesis often requires repeated high-dose continuous administration. The latter induces specific immune responses directly targeting tumor endothelial cells and or tumor cells by inoculating the body with vaccines based on allogeneic endothelial cells, pro-angiogenic molecules or tumor-associated antigens involved in angiogenesis. The anti-tumor effects of these three anti-angiogenic vaccines last for a long time and have little side effects, showing excellent therapeutic advantages.⁸⁰ Among them, the third one has attracted the attention of scholars because of its combination of anti-tumor angiogenesis and anti-tumor mechanism that directly attacks tumor cells. The goal of tumor vaccines is usually to induce the immune system to produce adaptive immune responses targeting tumor cells expressing tumor-specific antigens or tumor-associated antigens in order to dissolve or kill these cells and eventually lead to tumor growth inhibition and tumor regression.^81–83 Different from most vaccines that rely on a single anti-tumor mechanism, tumor vaccines that inhibit tumor progression by multiple mechanisms are multi-dimensional targets for tumors and tumor microenvironment, and have strong and lasting anti-tumor ability. This part mainly describes the research progress and achievements of tumor vaccine based on tumor associated antigen involved in tumor angiogenesis in preclinical animal experiments and clinical trials, which is mediated by the combined anti-tumor mechanism of inhibition of intratumoral angiogenesis and direct destruction of tumor cells. To explore its prospect and importance as a tumor treatment strategy. It also emphasizes the therapeutic potential and advantages of this tumor vaccine with multiple mechanisms (anti-tumor angiogenesis, weakening tumor immunosuppression, enhancing anti-tumor immune function) to prevent tumor progression. Figure 3 shows the anti-tumor mechanism of tumor vaccine strategies based on anti-tumor angiogenesis.

Figure 3. Tumor vaccine strategies based on anti-angiogenesis. Tumor vaccines induce cellular and humoral immune responses targeting endothelial cells, and inhibit tumor angiogenesis through CTL-mediated killing of TAMs that secrete pro-angiogenic factors. Abbreviations: MMP-9: matrix metalloproteinase-9.

Animal experimental studies on tumor vaccines based on anti-angiogenesis

After it was found that tumor angiogenesis is the driving factor of tumor growth and metastasis, more attention has been paid to the tumor-associated antigen involved in tumor angiogenesis. This antigen protein is overexpressed on the surface of tumor cells and produced by endothelial cells.⁸⁴ The specific toxic T lymphocyte response and or humoral immune response produced by the tumor vaccine based on this antigen stimulate the immune system not only against tumor cells, but also target endothelial cells, both of which synergistically antagonize tumor growth and invasion. The National Cancer Institute pilot project tests well-reviewed criteria to develop a priority list of cancer vaccine target antigens,⁸⁵ suggesting the possibility of vascular endothelial growth factor receptor 2 (VEGFR2) and survivin, which are highly expressed in tumor cells, as promising tumor vaccine target antigens. This view is supported by experimental studies on the delivery of these target antigens into the body through various antigen delivery platforms.

In the delivery platform of molecular vaccines, DNA vaccines have the advantage of not being limited by major histocompatibility complex (MHC) types.⁸² We found that the injection of DNA vaccine encoding survivin into melanoma-bearing mice not only induced specific toxic T lymphocyte responses targeting melanoma cells expressing the antigen, but also inhibited angiogenesis in tumor tissue, thus promoting its protective effect on highly invasive melanoma.^86,87 The strong anti-tumor ability shown by this DNA vaccine which encodes a protein encourages scholars to explore the anti-tumor ability of DNA vaccines that encode multiple proteins. In fact, DNA vaccines encoding VEGFR2 and many other different tumor antigens have produced significant tumor treatment, protection and survival benefits in mouse tumor models such as metastatic melanoma,^88,89 lung cancer⁹⁰and cervical cancer.⁹¹ Among them, the DNA vaccine encoding VEGFR2 and mouse MHCI heavy chain (H-2D^b)⁸⁸ or IL-12⁸⁹ enhanced the immune system’s recognition of VEGFR2 antigen and CTL response, and reduced the intratumoral blood vessel density. Whether it was DNA vaccine encoding VEGFR2 and HPV16E6E7 antigen⁹¹/MUC1 antigen⁹⁰ or nanoliposomal VEGFR2 peptide vaccine,⁹² it could reduce tumor vascular density and kill tumor cells by promoting VEGFR2-specific and E6E7/MUC1 antigen-specific CTL response. Importantly, these vaccines also increased the release of IFN- γ and IL-4 to enhance anti-tumor immunity mediated by Th1 and Th2 immune responses. In the delivery platform of cellular vaccine and carrier vaccine, the main carriers for delivering immune antigens are antigen-presenting cells-dendritic cells (DC), tumor cells and viruses, bacteria and fungi. It was found that DC vaccine loaded with Flk1,^93,94 the 4T1 breast cancer cell vaccine infected by adenovirus encoding VEGFR2,⁹⁵ the oral attenuated Salmonella typhimurium vaccine carrying Flk1⁹⁶ and the adenovirus vaccine encoding TERT andVEGFR-2^97,98 induced the immune system to produce a strong immune response targeting tumor antigen through the efficient antigen delivery ability of DC and the strong immunogenicity of tumor cells and viruses, respectively. Tumor angiogenesis was inhibited by enhancing humoral and cellular immune responses mediated by Flk1 antibody and Flk1-specific CTL respectively, which ultimately inhibited tumor growth, metastasis and prolonged the survival time of tumor-bearing mice.

In addition, the view that tumor vaccine treatment needs to rely on a variety of anti-tumor mechanisms to achieve excellent tumor inhibition was also confirmed in vaccines based on other angiogenic molecules. After immunization with MUC1 peptide vaccine with mouse VEGF (mVEGF165b) as adjuvant,⁹⁹ VEGF164 modified lung cancer cell vaccine,¹⁰⁰ VE-cadherin loaded DC vaccine¹⁰¹ and VEGFR3 attenuated Salmonella typhimurium oral vaccine,¹⁰² in addition to activating antigen specific CTL and antibody immune response mediated anti-tumor angiogenesis and tumor cell destruction. It also reduced the proportion of myelogenous suppressor cells and Treg cells in tumor tissue or increased the tumor infiltration of CD68⁺ monocytes / macrophages to improve the tumor immune microenvironment. It was also found that Th1 and Th2 anti-tumor immune responses were induced. These vaccines have significant tumor treatment and protection effects as well as survival benefits, suggesting that we should further explore the anti-tumor mechanism of tumor vaccines in order to develop tumor vaccines with multi-dimensional mechanisms to mediate anti-tumor effects. Table 4 lists a summary of preclinical animal experimental studies of tumor vaccines based on anti-angiogenesis.

Table 4. List of animal experimental studies on tumor vaccines based on anti-angiogenesis.

Vaccine types	Vaccine	Model	Adjuvant	Outcome	Ref.
Peptide Vaccines	Nanoliposomal VEGF-R2 peptide vaccine	Murine B16F10 melanoma model (therapeutic immunization strategies)	-	Results represented that VEGFR-2 peptide nanoliposomal formulations (Lip-V1) substantially activated CD4^+ and CD8^+ T cell responses against B16F10 cells and significantly boosted the production of IFN-γ and IL-4. The formulation led to a significant decrease in tumor volume and enhanced survival in mice, which could be a promising therapeutic vaccination approach capable of eliciting strong antigen-specific immunologic and anti-tumor responses.	Zahedipour et al.^Citation92
	MUC1 peptide vaccine	Murine breast cancer model（protective and therapeutic immunization strategies）	mVEGF165b	Co-immunized mVEGF165b with MUC1 peptide vaccine increased the VEGF antibody titers and MUC-1-specific IFN-γ-producing CD8^+ T cells, thus to decreasing the frequency of regulatory T cells (Tregs) in both preventive and therapeutic scenarios. mVEGF165b may be an ideal immunization adjunct to enhance the immune efficacy of peptide-based tumor vaccines by overcoming immune tolerance.	Zhang et al.^Citation99
DNA Vaccines	DNA plasmids encoding MUC1,VEGFR2, GM-CSF	Murine Lewis carcinoma model (preventive strategies)	GM-CSF	The anti-tumor effect of MUC1-VEGFR2 was greater than that of MUC1 or VEGFR2 alone. The anti-tumor effect of MUC1-VEGFR2 combined with GM-CSF was greater than that of MUC1-VEGFR2. The DNA vaccine induced humoral and cellular MUC1-specific immune responses in mice and increased IFN-γ and IL-4 release. The DNA vaccine inhibited the growth of MUC1-expressing tumors and prolonged mouse survival.	Ruan et al.^Citation90
	DNA plasmids encoding HPV 16 E6E7 fusion protein,VEGFR2 (pVAX1-E6E7-F2A-VEGFR2)	Murine TC-1-E6E7 Cervical cancer model (preventive strategies)	-	The plasmid harboring the E6E7 fusion gene and VEGFR2 showed stronger inhibition of tumor growth and angiogenesis than the plasmid expressing E6E7 or VEGFR2 alone by inducing more robust CTLs targeting E6E7 or VEGFR2，Th1 (IFN-γ)and Th2 (IL-4)cell response, which indicated that the combination of E6E7 and VEGFR2 could exert a synergistic antitumor effect.	Gao et al.^Citation91
	CAVE:Semliki Forest Virus (SFV) replicon DNA vaccine expressing murine VEGFR2 and IL12 CAVA: SFV replicon DNA vaccine targeting survivin and β-hCG antigens	Murine B16F10-β-hCG melanoma model (preventive strategies)	-	Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. The intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group.	Yin et al.^Citation89
	SCT-KDR2 DNA: SCT-encoding VEGFR2 antigen peptide and mouse MHC class I heavy chain H-2D^b	Murine B16 melanoma model 3LL Lewis lung carcinoma metastasis mouse model (preventive strategies)	-	SCT-KDR2 DNA was able to successfully break self-immunological tolerance and induce robust cytotoxic T-lymphocyte (CTL) response to VEGFR2, leading to marked suppression of tumor cell-induced angiogenesis and metastasis in murine models of B16 melanoma and 3LL Lewis lung carcinoma.	Chen et al.^Citation88
	DNA plasmids encoding survivin	Murine B16 melanoma model (preventive strategies)	-	Human survivin DNA vaccine delivered by intradermal electroporation (EP) generated Survivin-specific CD8^+ cytotoxic T lymphocyte (CTL) responses against B16 and peptide-pulsed RMA-S cells, cross-reactive with the mouse epitope surv20–28, suppressed angiogenesis in vivo and elicited protection against highly aggressive syngeneic B16 melanoma tumor challenge.	Lladser et al.^Citation87
	DNA plasmids encoding survivin	Murine immune model	GM-CSF	The DNA vaccine induced a survivin-specific humoral through generating IgG2a, as well as cellular immune response in recipient by activating cytotoxic Interferon-gamma (IFN-g) secreting CD8^+ T cells.	Lladser et al.^Citation86
Cell Based Vaccines	4T1 murine mammary cancer cell vaccine (irradiated and infected adenovirus encoding the VEGFR2 gene)	Murine 4T1 breast cancer models (therapeutic immunization strategies)	-	AdVEGFR2-infected whole-cancer cell vaccines inhibited subsequent tumor growth and pulmonary metastasis compared with challenge inoculations. Angiogenesis was inhibited, and the number of CD8^+ T lymphocytes was increased within the tumors.	Yan et al.^Citation95
	Soluble flk1 and IFN-γ fusion gene-transfected DC(DC-sflk1-IFN-γ)	Murine immune model Murine B16 Metastasis Model Lewis Lung Metastasis Model (preventive strategies)	-	DC-sflk1-IFN-γ induced a CD8^+ cytotoxic T cell responses to flk1, leading to profound inhibition of tumor-cell-induced angiogenesis and metastasis.	Pan et al.^Citation94
	Dendritic cells pulsed with a soluble flk1 protein (DC-flk1)	Murine immune model Murine B16 Metastasis Model Lewis Lung Metastasis Model (preventive strategies)	-	DC-flk1 generated flk1-specific neutralizing antibody and CD8^+ cytotoxic T cell responses, breaking tolerance to self-flk1 antigen. DC-flk1 suppressed tumor-induced angiogenesis and inhibited development of pulmonary metastases in DC-flk1–immunized mice challenged with B16 melanoma or Lewis lung carcinoma cells. DC-flk1 prolonged the survival of mice challenged with Lewis lung tumors.	Li et al.^Citation93
	DC-VEC:Dendritic cell vaccine (loading VE-cadherin)	Murine 4T1 breast carcinoma and CT26 murine colon carcinoma model (preventive strategies and therapeutic immunization strategies)	-	DC-VEC vaccine exhibited CTLs against VE-cadherin and suppressed angiogenesis. DC-VEC vaccine decreased VEGF-A, TGF-β1 and the percentage of myeloid-derived suppressor cells and regulatory T cells, but increased IL-4, IFN-γ CD4^+ and CD8^+ T cells.	Zhou et al.^Citation101
	VEGF164-modified LL/2 lung cancer cell vaccine (LL/2-VEGF164)	Murine lung cancer model (protective strategies）	-	VEGF164-modified tumor vaccine induced humoral immune response and CTLs against the parental tumor cells. LL/2-VEGF164 vaccine modulated host antitumor immune response via increasing infiltration of CD68-positive monocytes/macrophage and hold therapeutic potential for cancer.	Kan et al.^Citation100
Viral Vector Vaccines	Mannan modified adenovirus encoding VEGFR2	Murine LL/2 (Lewis lung carcinoma), B16 (melanoma) and CT26 (colon carcinoma) modell (preventive strategies and therapeutic immunization strategies) Murine Lewis lung carcinoma metastasis model(preventive strategies)	-	Oxidized mannan-modified adenovirus expressing VEGFR-2 vaccination led to greater IFN-γ and VEGFR-2-specific CTLs to stimulate both protective and therapeutic immune response in a mice model, which resulted in the suppression of angiogenesis and an increase in CD8^+ cells in tumor tissues. The data suggest that the combination of cancer immunity and anti-angiogenesis via modified mannan is a promising strategy in tumor prophylaxis and therapy.	Zhang et al.^Citation97
Attenuated Bacteria Vaccines	Orally administered DNA vaccine encoding FLK1 (pcDNA3.1-FLK1ECD) carried by attenuated Salmonella typhimurium	Murine 3LL Lewis Lung Carcinoma model and pulmonary metastasis model (preventive strategies)	-	pcDNA3.1-FLK1ECD was effective at protective antitumor immunity in Lewis lung carcinoma models in mice by breaking immune tolerance to FLK1 self-antigen and inducing FLK1-specific humoral and cellular immune responses against endothelial cells and FLK1-positive target cells to inhibit tumor neovascularization. pcDNA3.1-FLK1ECD resulted in tumor suppression and prolonged survival and inhibited pulmonary metastases in mice.	Zuo et al.^Citation96
	Orally administered DNA vaccine encoding murine VEGFR-3 carried by attenuated S. typhimurium SL3261	Murine LL/2 (Lewis lung carcinoma) model (preventive strategies)	-	The oral VEGFR-3-based vaccine inhibited lung carcinoma growth and suppressed lymphangiogenesis via increasing the levels of T helper type 1(Th1) cell intracellular cytokine expression (IL-2, IFN-γ, and TNF-α) and VEGFR-3-specific antibody, increasing CD4^+ or CD8^+ T cell numbers.	Chen et al.^Citation102

IL-2: interleukin-2; IFN-γ: interferon-γ; TNF-α: tumor necrosis factor-α; FLK1: fetal liver kinase 1; KDR: kinase insert domain receptor; IL-4: interleukin-4; VEGF-A: vascular endothelial growth factor A; TGF-β1: transforming growth factor beta-1; Tregs: regulatory T cells; MDSCs: myeloid-derived suppressor cells; DC: dendritic cell; VE-cadherin: vascular endothelial-cadherin; GM-CSF: granulocyte macrophage colony stimulating factor; MUC1: melanoma-associated antigen 3 and mucin 1; HPV: human papillomavirus; TEM8: tumor endothelial marker 8; MBD2: murine beta-defensin 2; MUC1: a T-cell epitope dominant peptide vaccine from Mucin.

Clinical trials of tumor vaccines based on anti-angiogenesis

In view of the strong anti-tumor growth and metastasis ability of VEGFR2 and survivin tumor vaccines in various preclinical tumor-bearing mice models, clinical studies based on this vaccine were also continuing to evaluate the feasibility and effectiveness of this vaccine in the treatment of tumor patients. In the past decade, there has been a tendency to carry out clinical research on VEGFRs multi-epitope peptide vaccines. The peptide epitopes of these vaccines come from VEGFRs with or without multiple tumor antigens. Although Elpamotide (VEGFR2 peptide vaccine) showed good safety and tolerance in phase I trials for patients with pancreatic cancer,^103,104 its combination with gemcitabine did not achieve overall survival benefits in phase II/III trials.¹⁰⁵ However, it was gratifying that studies have found that VEGFR2 and tumor antigen multi-epitope peptide cocktail vaccine have achieved initial success in a variety of patients with recurrent, metastatic or refractory solid tumors.^106–115 The polypeptide vaccine regimens used in patients were well tolerated and there were virtually no serious systemic adverse events associated with treatment. Further studies have shown that the dipeptide epitope vaccine of VEGFR2 and VEGFR1 enhances the therapeutic efficacy of temozolomide (TMZ) in patients with high-grade gliomas,¹¹⁶ suggesting that multi-epitope peptide vaccine might be a promising tumor therapy. Surprisingly, the II phase trial of multi-epitope peptide cocktail vaccine prepared by VEGFR1/2 and a variety of tumor antigens (such as KIF20A, CDCA1, URLC10, TTK, RNF43, TOMM34, FOXM1, DEPDC1, MELK, HJURP, KOC1, HIG2) in patients with pancreatic cancer,^117,118 advanced colon cancer,^119,120 refractory cervical cancer and ovarian cancer¹²¹ showed that more patients remained stable for a long time. And in these studies, we observed that patients with simultaneous immune response to multiple antigens had longer disease-free survival and total survival time, especially when combined with gemcitabine drugs. These data suggested that multi-epitope peptide vaccine was a promising choice for the treatment of advanced solid tumors and could be considered in combination with other antineoplastic drugs or immune checkpoint inhibitors (ICIs) to find the best combination for clinical cancer patients.¹²² However, there were also survivin peptide vaccines that produced excellent clinical results in clinical trials conducted by patients with various solid tumors. The survivin peptide vaccine strategy alone was safe in patients with colon cancer, but the immune response was not sufficient to produce visible clinical effects.¹²³ In order to improve the immunogenicity of the vaccine, the strategy of vinvin peptide vaccine combined with different adjuvants was proposed. The addition of adjuvants did enhance the immune response of vinvin peptide vaccine in patients with advanced breast cancer,¹²⁴ colon cancer¹²⁵ and pancreatic cancer,^126,127 mainly in the overall survival of patients was significantly improved.

In addition to the VEGFR2 molecular vaccine, the attenuated salmonella vaccine encoding VEGFR2 plasmid had also conducted phase I clinical trials in pancreatic cancer patients, but the reactivation or induction of VEGFR reactive Treg might lead to the failure of angiogenesis inhibition in some patients.^128,129 The above tumor vaccines based on VEGFR2 molecules could effectively control the progress of patients with various refractory solid tumors, supporting the conclusion of previous animal experiments that they had a strong ability to treat and protect tumors. Table 5 lists a summary of clinical trials and clinical applications of tumor vaccines based on anti-angiogenesis.

Table 5. List of clinical trials of tumor vaccines based on anti-angiogenesis.

Vaccine types	regimen	Clinical trial identifier	Conditions	Adjuvant	Phases	Outcome	Ref.
Peptide Vaccines	7 peptides epitopes derived from FOXM1,MELK, HJURP,VEGFR1,VEGFR2,URLC10,HIG-2	UMIN 000003860 UMIN 000003862 UMIN 000003902 UMIN 000003903	Cervical cancer (CC)or ovarian cancer(OC) (n = 66)	Montanide ISA-51VG	II	The vaccination activated CTLs to attack and destroy the tumor cells without severe adverse events, which was safe and effective for disease control. Response rate in OC and CC were 22.9% and 20%, respectively. Further clinical study of peptide vaccines in combination with ICIs and/or an anti-VEGF antibody such as bevacizumab will be mandatory.	Takeuchi et al.^Citation121
	7 peptides epitopes derived from FOXM1,MELK, HJURP,VEGFR1,VEGFR2,RNF43,TOMM34,	UMIN000007801	Colorectal cancer (n = 30)	Montanide ISA-51VG	Ib	The 7-peptide vaccine used with leucovorin (UFT/LV) was safety Patients who exhibited positive CTLs to all seven peptides had longer overall survival. 3 patients showed PR,15 patients showed SD,12 patients showed PD.	Okuno et al.^Citation111
	6 peptides epitopes derived from FOXM1,LY6K, DEPDC1,KIF20A,VEGFR1, VEGFR2	-	High-grade gliomas (HGGs) (n = 10)	Montanide ISA-51VG	Pilot Study	The median overall survival time in all patients was 9.2 months. 2 patients demonstrated SD and 1 patient achieved CR. Six peptides vaccine was well tolerated without any severe systemic adverse events and induced robust glioma oncoantigen (GOA)/glioma angiogenesis-associated antigen (GAAA)-specific T-lymphocyte responses in recurrent/progressive HGG patients.	Kikuchi et al.^Citation114
	5peptides epitopes derived from RNF43,TOMM34,KOC1,VEGFR1, VEGFR2	UMIN000001791	Colorectal cancer (CRC) (n = 89)	Montanide ISA-51	II	Therapeutic epitope peptides vaccine increased VEGFR1 and 2-specific robust CTLs response, which did not correlate with OS. VEGFR2 IgG responses may be an important immunological biomarker in the early course of treatment for CRC patients.	Kanekiyo et al.^Citation120
	5peptides epitopes derived from RNF43,TOMM34,KOC1,VEGFR1, VEGFR2	UMIN000001791	Colorectal cancer (CRC) (n = 96)	Montanide ISA-51	II	Five therapeutic epitope peptides vaccine appeared to be effective in a subset of patients, and warranted a randomized phase III study. A delayed response was observed in subgroup with a NLR of < 3.0. Biomarkers such as NLR might be useful for selecting patients with a better treatment outcome by the vaccination. RR and the median OS was 62.0%,60.9% and 20.7 months,24.0 months in the HLA-A *2402-matched and -unmatched groups, respectively.	Hazama et al.^Citation119
	5peptides epitopes derived from RNF43,TOMM34,KOC1,VEGFR1, VEGFR2	UMIN000004948	Colorectal cancer (n = 18)	Montanide ISA-51	I	The vaccine treatment using multiple peptides was well tolerated without any severe treatment-associated systemic adverse events. Dose-dependent induction of peptide-specific cytotoxic T lymphocytes was observed. Patients, in which we detected induction of cytotoxic T lymphocytes specific to 3 or more peptides, revealed significantly better prognosis.	Hazama et al.^Citation110
	5peptides epitopes derived from FOXM1,MELK, HJURP,VEGFR1,VEGFR2	UMIN000003999	Cervical cancer (n = 21)	Montanide ISA-51	I	Five peptides vaccination was safety and induced high T-cell responses to FOXM1 and MELK in patients with cervical cancer, but showed modest clinical efficacy. All vaccine-related adverse events were tolerable.	Hasegawa et al.^Citation112
	4peptides epitopes derived from RNF43,TOMM34,VEGFR1, VEGFR2	UMIN000004452	Colorectal cancer (n = 10)	Montanide ISA-51VG	I	Results showed that 1 patient had PR, 7 patients had SD and 2 patients had PD. The peptide vaccine therapy together with UFT+LV was safe and induced peptide-specific CTLs recognizing RNF43, TOMM34 and VEGFR2 in patients with PR and SD, thus further study is needed. Patients who had a strong CTL reaction had a tendency to have longer progression-free survival and overall survival.	Matsushita et al.^Citation107
	4peptides epitopes derived from URLC10,CDCA1/TTK,VEGFR1, VEGFR2	NCT00633724 NCT00874588	Non-small cell lung cancer (NSCLC) (n = 15)	Montanide ISA-51	I	Peptide vaccine was well tolerated with no severe treatment-associated adverse events and enhanced peptide-specific T cell responses against at least one peptide in 13 of the 15 patients.	Suzuki et al.^Citation109
	4peptides epitopes derived from KIF20A,CDCA1,VEGFR1, VEGFR2	UMIN000004337	Pancreatic cancer (n = 9)	Montanide ISA-51	I	The multi-peptide vaccine was well-tolerated without grade 3 or 4 adverse events, induced robust peptide-specific T-cell responses against KIF20A,CDCA1,VEGFR1,VEGFR2 and showed clinical benefits. Median PFS and OS were 90 and 207 d, respectively.	Okuyama et al.^Citation108
	3peptides epitopes derived from KIF20A,VEGFR1,VEGFR2 (OCV-C01)	UMIN000007991 UMIN000020366	Pancreatic ductal carcinoma (n = 30)	Montanide ISA-51VG	II	OCV-C01 combined with gemcitabine was tolerable and DFS of patients with positive KIF20A expression in resected pancreatic cancer specimen may potentially be prolonged with frequent KIF20A-specific CTL response by receiving this immunotherapy treatment. A randomized controlled trial was essential to demonstrate the clinical benefits of OCV-C01 for surgically resected pancreatic cancer patients	Miyazawa et al.^Citation117
	3peptides epitopes derived from KIF20A,VEGFR1,VEGFR2	UMIN000008082	Pancreatic cancer (n = 68)	Montanide ISA-51	II	The therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions. The patients with peptide-specific CTL induction against either KIF20A or VEGFR1 showed significantly better OS.	Suzuki et al.^Citation118
	2peptides epitopes derived from VEGFR1–1084, VEGFR2–169	UMIN000013381 UMIN000023565	High-grade glioma (WHO grade III or IV) (n = 4)	Montanide ISA-51VG	I and II	VEGFR1 and 2-specific CTLs attacked not only tumor vessels but also tumor cells and regulatory T cells expressing VEGFRs even in recurrent tumors. VEGFR1 and 2 peptide vaccination may possibly enhance the effects of temozolomide (TMZ).	Tamura et al.^Citation116
	2peptides epitopes derived from VEGFR1–1084, VEGFR2–169	UMIN000023565	Schwannomas (n = 7)	-	exploratory clinical study	VEGFR1 and 2-peptide vaccination was safety and induced VEGFR1 and VEGFR2-specific CTLs to directly kill a wide variety of cells associated with tumor growth, including tumor vessels, tumor cells, and VEGFR2-expressing Tregs. Tumor volume reductions of ≥ 20% are observed in two patients, including one in which bevacizumab had not been effective. There are no severe adverse events related to the vaccine.	Tamura et al.^Citation115
	2peptides epitopes derived from VEGFR1–1084, VEGFR2–169	-	Glioblastoma (n = 8)	Montanide ISA-51VG	Pilot Study	This treatment was well-tolerated, safety and induced positive CTLs responses against at least one of the targeted VEGFR epitopes targeting tumor vasculatures in high grade gliomas. The median overall survival time in all patients was 15.9 months. Two achieved progression-free status lasting at least 6 months.	Shibao et al.^Citation113
	2peptides epitopes derived from VEGFR1–1084, VEGFR2–169	UMIN000005007	Gastric adenocarcinoma (n = 22)	Montanide ISA-51VG	I and II	The combination therapy was well tolerated and highly effective in advanced or recurrent gastric cancer. Substantial specific CTL for both peptides was frequently induced even under chemotherapy. Cancer vaccination combined with standard chemotherapy warrants further analysis as a promising strategy for the treatment of advanced cancer.	Masuzawa et al.^Citation106
	VEGFR peptide vaccine	UMIN000013381 UMIN000012774 UMIN000005545	Glioma (n = 13)	-	Pilot Study	1 patient had a Grade 4 pulmonary embolism and 2 patients had Grade 2 cerebral infarctions in the group of bevacizumab (Bev)+ VEGFR peptide vaccine VEGFRs-targeted vaccination may coexist with Bev for malignant gliomas.	Tamura et al.^Citation122
	Elpamotide (VEGFR2)	UMIN000001664	Pancreatic cancer (n = 153)	Montanide ISA-51VG	II/III	Combination therapy of elpamotide with gemcitabine was well tolerated and failed to obtain overall survival benefits. Patients who experienced severe injection site reaction might have better survival.	Yamaue et al.^Citation105
	Elpamotide (VEGFR2–169)	UMIN000008336	Advanced refractory solid tumors (n = 10)	-	I	Elpamotide was well tolerated, biologically active and induced an immune response that targets angiogenesis in the clinical setting.	Okamoto et al.^Citation104
	VEGFR2–169	NCT00622622	Pancreatic cancer (n = 21)	Montanide ISA-51VG	I	VEGFR2–169 peptide vaccine in combination with gemcitabine was well tolerated and induced peptide-specific CTL. The proportion of Treg cells after the vaccination was significantly reduced. A randomized, controlled clinical trial will be essential to demonstrate the clinical benefits of the VEGFR2–169 peptide for advanced pancreatic cancer patients treated with gemcitabine.	Miyazawa et al.^Citation103
	Survivin	UMIN000012146	Pancreatic adenocarcinoma (n = 83)	Montanide ISA‐51 VG	II	No significant improvement in PFS was observed. Compared with placebo group, the SVN‐2B plus IFNβ group increased survivin 2B‐specific CTLs and showed better overall survival, significant immunological reaction after vaccination, but did not have improved PFS. A longer SVN‐2B plus IFN-β vaccination protocol might confer survival benefit.	Shima et al.^Citation127
	Survivin	UMIN000000905	Pancreatic cancer (n = 6)	IFA and IFNα	I	More than 50% of the patients had positive clinical and immunological responses. The vaccination protocol of survivin-2B80–88 plus IFA and IFNa was very effective and useful in immunotherapy for this type of poor-prognosis neoplasm.	Kameshima et al.^Citation126
	Survivin	—	Colon cancer (n = 13)	IFA and IFNα	-	The effect of survivin-2B80–88 plus IFA was not significantly different from that with survivin-2B80–88 alone, treatment with the vaccination protocol of survivin-2B80–88 plus IFA and IFNa resulted in clinical improvement and enhanced immunological responses of patients via increasing survivin-2B80–88 peptide-specific CTL The vaccination of colon cancer patients with survivin-2B80–88 plus IFA and IFNa could be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers.	Kameshima et al.^Citation125
	Survivin	—	Breast cancer (n = 14)	Montanide ISA −51	I	The survivin-2B peptide vaccination was well tolerated. When it mixed with IFA, the frequency of peptide-specific CTL effectively increased, although neither vaccination could induce efficient clinical responses. The addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.	Tsuruma et al.^Citation124
	Survivin	-	Colorectal cancer (n = 15)	-	I	No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19–9) decreased transiently during the period of vaccination. One patient revealed an increase in peptide-specific CTL frequency Survivin-2B peptide-based vaccination was safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.	Tsuruma et al.^Citation123
Attenuated Bacteria Vaccines	VXM01 (Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2)	NCT01486329	Pancreatic cancer (n = 45)	-	I	VXM01 elicited a productive, complex VEGFR2 specific T effector cell response in patients with pancreatic cancer that correlates with anti-angiogenic activity and had a favorable safety profile.	Schmitz-Winnenthal et al.^Citation129
	VXM01 (Salmonella typhi strain Ty21a encoding the human full-length VEGFR-2)	NCT01486329	Pancreatic cancer (n = 45)	-	I	The results of this study shall provide the first data regarding the safety and immunogenicity of the oral anti-VEGFR-2 vaccine V×M01 in cancer patients. They will also define the recommended dose for phase II and provide the basis for further clinical evaluation, which may also include additional cancer indications.	Niethammer et al.^Citation128

FOXM1: forkhead box M1; MELK: maternal embryonic leucine zipper Kinase; HJURP: holliday junction recognition protein; URLC10: up regulating lung cancer 10 gene; HIG-2: hypoxia-inducible gene 2; VEGFR1/2: vascular epithelial growth factor receptor 1 and 2; HLA: human leukocyte antigen; IFA: incomplete Freund’s adjuvant; CTLs: cytotoxic T lymphocytes; ICIs: immune checkpoint inhibitors; VEGF: vascular epithelial growth factor; KOC1: kinase of the outer chloroplast membrane 1; TOMM34: translocase of outer mitochondrial membrane 34; RNF43: ring finger protein 43; OS: overall survival; DFS: disease-free survival; NLR: neutrophil/lymphocyte ratio; PR: partial response; SD: stable disease; PD: progressive disease; DCs: dendritic cells; HUVEC: human umbilical vein endothelial cell; PFS: progression-free survival; TTK: TTK protein kinase; CDCA1: cell division cycle associated gene 1; ORR: objective response rate; CR: complete response; PR: partial response; RR: response rates; LY6K: lymphocyte antigen 6 family member K; DEPDC1: DEP domain containing 1; KIF20A: CT antigens kinesin family member 20A; CDCA1: cell division cycle-associated 1; PFS: progression-free survival; Teff: T effector; Tregs: regulatory T cells.

In summary, tumor vaccines based on anti-angiogenesis mediate anti-tumor effects by preventing tumor angiogenesis, weakening tumor immunosuppression (mainly targeting tumor immunosuppressive cells such as Treg and monocytes/macrophages), and enhancing anti-tumor immune response (inducing CTLs response of anti-tumor cells and activating Th1 and Th2 immune responses). These vaccines have shown satisfactory therapeutic effects in both preclinical tumor-bearing animal experiments and clinical trials. The preclinical tumor-bearing animal experiments clarified the specific mechanism of these vaccines in inhibiting tumor growth and metastasis, which provided theoretical support for their entry into clinical trials. The results of clinical trials confirmed the clinical benefits of these vaccines. In the future, the research of these vaccines should be focused on the clinical efficacy of their combination with different adjuvants or with various radiotherapy and chemotherapy strategies in advanced solid tumors.

Future perspectives

Although tumor vaccines with single targeting mechanisms are almost mature, their clinical application has not achieved the expected therapeutic benefits. We know that tumor cells grow in a complex tumor microenvironment and can escape anti-tumor immunity in a variety of ways, which may be an important reason for the limited effects of one-dimensional tumor vaccine therapy. However, we found that some vaccines exhibit multidimensional targeting of tumors by not only enhancing the immune response but also weakening tumor immunosuppression and inhibiting tumor growth. In the B16 melanoma mouse model, the accumulation of tumor-infiltrating lymphocytes (TILs) increased after the injection of an HCA587/MAGE-C2 protein vaccine.⁷⁵ The vaccine resulted in the enrichment of IFN-γ⁺CD4⁺ T cells and the reduction of Treg expression in the tumor microenvironment.¹³⁰ In a phase I trial to overcome immunosuppression in head and neck square cell carcinoma (HNSCC), inoculation with a dendritic cell p53 peptide vaccine increased patients’ p53-specific T-cell frequencies, and IFN-γ secretion was detected.⁷⁷ Treg frequencies were consistently decreased relative to pre-vaccination values. A two-year disease-free survival (DFS) rate of 88% was feasible, providing a basis for a confirmatory phase II vaccine test in HNSCC in the future. In addition, peptide vaccines targeting VEGF165b induced humoral immune responses to VEGF, thereby inhibiting tumor angiogenesis. More importantly, the vaccine also reduced Tregs in breast cancer tissues and directly reduced tumor immunosuppression.¹³¹ When a VEGFR peptide vaccine was used in neurofibromatosis type 2 (NF2)-derived schwannoma patients,¹¹⁵ tumor volume was reduced by more than 20% in two patients. Additionally, the number of Foxp3-positive cells was reduced and Foxp3 gene expression was lower in tumors.

Recent research advances have revealed significant differences in the efficacy of tumor vaccines at different tissue sites, which have been observed not only in primary tumors but also in metastatic sites. To extend the benefits of tumor vaccines, more comprehensive vaccine strategies are needed. Decades of progress have provided proof of principle that multidimensional tumor vaccines can indeed elicit systemic tumor regression.

These studies suggest that tumor vaccines can utilize a variety of mechanisms to inhibit tumor progression and that this has good therapeutic benefits for highly variable and invasive tumors. Therefore, we believe that follow-up research should focus more on the development of multidimensional tumor vaccines and explore more anti-tumor mechanisms to find potential tumor-dominant antigens. In Figure 4, based on the three anti-tumor mechanisms of tumor vaccines – boosting the immune system, overcoming immunosuppression, and modulating angiogenesis – we compare the differences between single-dimensional therapy and multidimensional tumor vaccine strategies.

Figure 4. Multidimensional tumor vaccine strategies versus single-dimensional therapy. Tumor growth inhibition is more potent when tumor vaccines mediate antitumor effects through two mechanisms, while maybe the inhibition is even the most potent when three mechanisms used. The three mechanisms of the multidimensional tumor vaccine strategies are mainly outlined here: (a) Tumor vaccines activate the immune system to kill tumor cells; (b)Tumor vaccines inhibit the tumor growth through overcoming the immunosuppression status; (c) Tumor vaccines activate the immune system to target the endothelial cells or TAMs, thus inhibit tumor angiogenesis.

Discussion

In addition to directly stimulating immune responses targeting tumor cells, tumor vaccines have also been developed to activate the immune system. Under pathological tumor conditions, tumor cells secrete a large number of immunosuppressive cytokines to transmit inhibitory signals to reduce the infiltration of CTLs into tumor tissue and prevent their proliferation and activation, as well as enhance the regulatory function of immunosuppressive cells such as Treg and macrophages, which help tumor cells to escape immune cell attack. Although many studies have recognized that the anti-tumor effect of tumor vaccines mainly depends on the immune response of CD8-CTLs, studies have also found that CD4⁺ T cells play an indispensable role in tumor therapy. High levels of CD4⁺ T cells are associated with high levels of serum antibodies because CD4⁺ T cells help activate the humoral immune response produced by B-cell antibodies. After tumor vaccination, CD4⁺ T cells can secrete a large amount of IFN- γ, which directly interferes with the growth of tumor cells and inhibits tumor angiogenesis.^132,133 In addition, some studies have found that vaccines can increase the expression of granzyme B in CD4⁺ T cells to produce a specific tumor immune response, which is also an important anti-tumor mechanism of CD4⁺ T cells.¹³⁰ In addition, given the important role of Tregs in tumor cell immune escape mechanisms, tumor treatment strategies should explore vaccines or drugs that inhibit Tregs in tumors. When tumor vaccines directly inhibit or deplete Tregs in tumor tissue, tumor growth is significantly inhibited. In addition, the anti-tumor effect of vaccines is enhanced when combined with the depletion of Tregs, indicating that Treg-targeted tumor vaccine strategies are worth considering. It is likely that Treg inhibition can effectively assist in combined therapy. We believe that this presents a new multi-directional way to stimulate the immune system: activating CD4⁺ T cells, improving the immune microenvironment of tumor tissue, and strengthening the effect of the humoral immune response to tumor growth. The advantages of immunization may be maximized through combined immunization strategies.

Judah Folkman and colleagues proposed for the first time that the growth of solid tumors in the avascular period was inhibited at the diameter of 2–3 mm. Therefore, tumor cells secreted tumor angiogenesis factor (TAF) to stimulate the rapid formation of new blood vessels and induce tumor to grow at an exponential rate.^78,79 These studies emphasized that tumor angiogenesis was a possible control point in tumor growth, and introduced the concept of anti-angiogenesis. Anti-angiogenesis refers to preventing new vessels sprouts from penetrating into an early tumor implant, which does not apply to vasoconstriction or infarction of vessels already connected to the tumor. Although anti-angiogenic-factor antibodies and immune-targeted angiogenesis inhibitors have shown a certain degree of anti-tumor effect, high drug dosages, long drug-use times, and adverse reactions are often not acceptable to patients. Thus, the high safety, high efficacy, and low toxicity of anti-angiogenic tumor vaccines can provide clinical confidence in their use. Both whole-cell anti-angiogenic tumor vaccines and vascular endothelial growth factor-targeted tumor vaccines can significantly reduce the angiogenesis of tumor tissue, block the blood perfusion of tumor tissue, and inhibit the growth and spread of tumor cells.

Tumor growth and progression is a complex process involving the whole and local immune state. Thus, a single-dimensional tumor vaccine strategy may have limitations to fight tumor cells. Multidimensional tumor vaccine therapies can play each role of different mechanisms, such as boosting the immune system, overcoming tumor immunosuppression, and modulation of tumor angiogenesis. Many studies have pointed to multidimensional tumor vaccine strategies to maximize the anti-tumor effect by boosting the immune system and inhibiting tumor growth. For example, HCA587 protein combined with CpG and ISCOM adjuvants can enhance immune system against tumor attack by inducing CD4⁺ T cells to express granzyme B¹³⁰ and IFN- γ to kill tumor cells, and reducing the levels of Tregs in tumor tissue, then inhibit the growth of melanoma cells and improve the survival of mice.⁷⁵ We have observed that single-dimension tumor vaccines show certain anti-tumor effects in both animal and clinical studies, but in many cases, they cannot prevent tumor progression and metastasis. Relatively speaking, when tumor vaccines have multidimensional anti-tumor mechanisms, they can inhibit tumor growth and slow malignant development thoroughly.

Conclusions

In this review, we have emphasized the role of multidimensional tumor vaccines in tumor immunotherapy. We have illustrated the importance of three anti-tumor mechanisms: boosting the immune system, overcoming tumor immunosuppression, and modulating tumor angiogenesis. Tumor vaccines can increase the infiltration of effector T lymphocytes into tumor tissue, reduce the inhibiting factors in the tumor microenvironment, and blocks the blood supply of tumor cells to inhibit tumor growth. When a tumor vaccine or a combination of tumor vaccines utilizes two or more of these mechanisms, it may more effectively eliminate tumor cells.

Author contributions

Conceptualization, J.C.; writing – original draft preparation, Y.T., H.C. and X.G.; writing – review and editing, J.C., Q.F., Y.T., H.C. and X.G. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China, grants 82160314 and 81960497 to J.C. The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.