Cellular & Molecular Immunology (2012) 9, 20–26 ß 2012 CSI and USTC. All rights reserved 1672-7681/12 $32.00 www.nature.com/cmi

REVIEW

Targeting and utilizing primary tumors as live vaccines: changing strategies

Xuanming Yang, Eric D Mortenson and Yang-Xin Fu

Tumor metastases and relapse are the major causes of morbidity and mortality in cancer. Although surgery, chemotherapy and/or radiation therapy can typically control primary tumor growth, metastatic and relapsing tumors are often inaccessible or resistant to these treatments. An adaptive immune response can be generated during these conventional treatments of the primary tumor, and presumably both the primary tumor and secondary metastases share many of the same or similar antigenic characteristics recognized by the immune system. Thus, when established, this response should be able to control metastatic growth and tumor relapse. This review summarizes the mechanisms by which antitumor immune responses are generated, and recent findings supporting the hypothesis that many therapies targeting primary tumors can generate antitumor adaptive immune responses to prevent metastases and tumor relapse. Cellular & Molecular Immunology (2012) 9, 20–26; doi:10.1038/cmi.2011.49; published online 21 November 2011

Keywords: chemotherapy; immunotherapy; ; radiotherapy; tumor vaccine

INTRODUCTION tissues.3–5 Thus, overcoming tumor-associated immune-suppressive Conventional treatments such as surgery and chemotherapy are effec- mechanisms to induce potent antitumor immunity is the first step for tive means of eliminating or reducing primary tumor growth, but have effective cancer immunotherapy.5–7 Many mechanisms are involved not proved effective in eradicating metastases. Because metastatic dis- in tumor-associated immune suppression of naive and previously ease may not respond similarly to chemotherapies used in treating the activated T cells. First, tumors create an environment insufficient primary tumor,1 recent research has focused on the importance of for expansion and maturation of effector tumor-specific T cells. generating an antitumor immune response to control metastatic dis- Second, effector T cells must be capable of reaching the site of the ease. Presumably, both the primary tumor and secondary metastases malignancy, and many tumors have physical and chemical barriers share many of the same or similar antigenic characteristics recognized preventing infiltration. Finally, tumors downregulate by the immune system; yet, there has been no clear strategy formulated receptors or find other means to prevent within the in which a patient’s own tumor tissues is used to generate an antitu- tumor microenvironment from appropriately executing effector func- mor adaptive immune response for the eradication of metastases and tions to destroy cancer cells. Multiple mechanisms employed by prevention of relapse. Once tumors metastasize, curing the disease is tumors to hinder the immune response at each of these steps have difficult and rare. This review summarizes recent findings supporting been identified. the hypothesis that targeting primary tumors with both conventional and new therapies can potentially generate antitumor adaptive THE CURRENT STRATEGIES FOR CANCER IMMUNOTHERAPY immune responses that prevent metastases and relapse. Active immunization and adoptive T-cell transfer therapy are cur- Due to the vast number of genetic changes associated with carcino- rently the main cancer immunotherapy strategies used. Both of these genesis, tumors express many neo-antigens and mutated antigens. strategies are designed to overcome deficiencies in priming of tumor Indeed, much evidence exists to indicate that many cancers are anti- antigen-specific T cells in cancer-bearing hosts. Cancer vaccines rely genic and thus recognizable by the .2 Mere on immunization of patients with antigenic peptides, or DNA recognition by adaptive immunity, however, is insufficient, given that expressed by the tumor directly or delivered by some other vehicle these antigenic cancers rarely regress spontaneously. Effective antitu- such as dendritic cells (DCs) or virus. Despite its relative simplicity mor immunity depends on both the presence of tumor-reactive and safety, vaccine treatments have shown very limited success.8 lymphocyte precursors and means by which these precursor lympho- Although tetramer or ELISPOT assays revealed the generation of cytes can be activated. Most T cells with high affinity to tissue-specific in vivo antitumor T cells in vaccinated patients,9–11 clinical respon- antigens are deleted in the thymus or later tolerized in the peripheral ses observed from these trials were few.8 This was consistent with

Department of Pathology and Committee on Immunology, University of Chicago, Chicago, IL, USA Correspondence: Dr XM Yang, Department of Pathology and Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. E-mail: [email protected] or Dr Y-X Fu, Department of Pathology and Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. E-mail: [email protected] Received 10 August 2011; revised 27 September 2011; accepted 28 September 2011 Utilizing primary tumor as in situ vaccine XM Yang et al 21 observations in murine models where the presence of large numbers of Indeed, the number of studies demonstrating that antibody therapy antigen-specific T cells was insufficient to mediate tumor regression in can induce both cellular and humoral immunity is rapidly increasing. tumor-bearing mice.12,13 There are a multitude of explanations for For instance, Trastuzumab treatment can induce antitumor cytotoxic this observation: an inadequate number or avidity of immune cells, T lymphocytes (CTLs) in patients.21 Similarly, it was demonstrated an inability of lymphocytes to migrate into the tumor microenviron- that a mouse anti-epidermal receptor antibody could ment, or a reduced activation of quiescent or precursor lymphocytes control metastasis in a CD4 and CD8 T cell-dependent manner.22 may all prevent successful vaccination. In addition, tolerance mechan- However, the mechanisms by which antibody therapy generates activ- isms induced by the tumor, including the lack of costimulation leading ate adaptive immunity has remained largely unclear. Our current to anergy induction and/or active suppressive mechanisms, create a findings demonstrated that anti-neu antibody treatment induced anti- great barrier to productive vaccination. These obstacles must be tumor adaptive immunity and this is essential for the therapeutic overcome if cancer vaccines are to be effective in mediating cancer effect in a syngenic mouse tumor model. This study revealed an essen- regression. tial role for the adaptive immune system in the therapeutic effect of Adoptive transfer therapies in which tumor-infiltrating lympho- antibody treatment on HER2/neu tumors. Given the fact that the cytes are isolated from the tumor, or peripheral blood in some cases, antibody mediated blockade of oncogenic signals and the induction expanded in an antigen-specific way, and adoptively transferred back of ADCC had been previously demonstrated, we proposed that both into the patient,14 have been effective in a small number of highly of these outcomes lead to a significant release of danger signals selected melanoma patients.15 Although many tumor-specific and (such as High-mobility group 1 (HMGB1)) to activate DCs -associated antigens have been identified, few of these antigens in and promote production. Furthermore, FcR-mediated sig- human tumors have been confirmed to be rejecting antigens. In addi- naling and phagocytosis could induce additional cytokine and danger tion, the rejecting antigen may vary greatly among different tumors. signal production, leading to MyD88-enhanced cross-presentation for Moreover, the best adjuvants for ex vivo expansion of cytotoxic T cells more effective activation of the adaptive immune system for enhanced are still uncertain. Therefore, the limited knowledge regarding the tumor control. Most importantly, the adaptive immunity induced by rejecting tumor antigens for T-cell expansion and the potential inab- targeting primary tumors could protect from tumor rechallenge which ility to physically isolate or expand these T cells, is preventing this is indicative of a response to metastatic disease and relapse.23 Recently, therapy from being applied to a wide variety of cancers. As a result, it was reported that the therapeutic effect of anti-HER2/neu did not understanding how immune responses are generated in vivo during require CD41 T cells, but did depend on both type I and type II conventional antitumor therapies and how to enhance these res- .24 In other models of oncogenic blockade, however, it was ponses, is of great importance. reported that tumor regression did require CD41 T cells.25 Thus, the detailed mechanism of CD41 T-cell activation and the role of these CONVENTIONAL TREATMENTS ON PRIMARY TUMORS cells in antibody-mediated tumor regression are unclear. INDUCE IMMUNE RESPONSES Total control or elimination of tumor metastases by antibody Antibody therapy alone is rare. Most antibodies only partially delay the disease pro- The first antibody approved for cancer treatment was Rituximab in cess. Thus, improving the efficiency of antibody therapies is a great 1997. Now, there are more than 10 antibodies approved by the Food challenge in this field of study. Many companies have developed and Drug Administration (FDA) for cancer therapy.16 The current different strategies to increase the ability of oncogenic blockade and dogma is that antibodies inhibit tumor growth by oncogenic signal ADCC. We think another important aspect is enhancing the adapt- blockade and/or antibody-dependent cellular cytotoxicity (ADCC).16 ive immunity trigged by antibody therapy through a combination Many of these antibodies target the oncogenic receptor on the surface of immune modulators. For instance, it has been shown that the of the tumor cell, such as epidermal growth factor receptor in lung, therapeutic effect is greatly improved when antibodies are conju- 26,27 head and neck, and colorectal cancer or human epidermal growth factor gated to immune modulators. receptor 2 (HER2) in breast cancer. Usually, inhibition of tumor growth occurs by inducing oncogenic blockade through competition with the Local ablative radiation therapy (RT) natural ligands, impairing the receptor oncogenic signal pathway, or The antitumor effect of RT depends on the dose and treatment sched- inducing tumor cell . Another important effect of antibodies is ule. Our review focused on the use of ablative RT, during which a high ADCC mediated through Fc–Fc receptor (FcR) interactions, which may dose of radiation is precisely delivered to small tumors. The current induce FcR1 cells such as natural killer cells and macrophages to kill mechanistic explanation for the clinical efficacy of local ablative RT tumor cells. In cancer patients, FcR polymorphisms directly affect thera- centers on the induction of lethal DNA damage directly to tumor cells peutic responses to antibodies,17 and in mice lacking FcR, cancer thera- or tumor-associated stroma. Although RT has traditionally been peutic antibodies lose their effect on tumor growth.18 Recently, it was viewed as immunosuppressive due to the inherent sensitivity of lym- reported that the apoptosis-inducing ability of an antibody is greatly phocytes to radiation-induced death, RT has been demonstrated to dependent upon the FcR expressed on host cells.19 enhance tumor-specific immune responses.28,29 Ionizing radiation Most conclusions regarding antibody effector mechanisms were increases production of inflammatory , such as tumor- drawn from in vitro cell culture studies and xenograft tumor models, necrosis factor (TNF), IL-1a and IL-6, by human tumor cells in both of which ignore adaptive immunity. Thus, until recently, deter- vitro.30,31 Local irradiation of a single tumor site can reduce the size mining whether antibodies can induce adaptive immunity against the of non-irradiated metastases that are located at a distant site, a phe- tumor and whether this is essential for the therapeutic effect was nomenon known as the ‘abscopal effect’, which is mediated by the unclear. Now, however, transgenic mice expressing oncogenes and immune system. In addition, radiation can modulate the peptide rep- mouse tumor cell lines expressing human oncogenic have been ertoire and enhance major histocompatibility complex class I express- developed, both of which allow for the investigation of the role of ion by tumor cells, and alter their phenotype resulting in heightened adaptive immunity in cancer therapy.20 susceptibility to T-cell killing.32,33

Cellular & Molecular Immunology Utilizing primary tumor as in situ vaccine XM Yang et al 22

Regarding the specific immune cell types involved in these pro- highlight that some chemotherapeutic drugs reduce regulatory T-cell cesses, RT can induce more lymphocytes trafficking to tumor sites (Treg) numbers, although the detailed mechanism of this action and draining lymph node, which includes CD41, CD81, CD11b1 remains unknown. These drugs include cyclophosphamide (Cy),38–40 and CD11c1 cell populations.28 Moreover, Apetoh et al. demonstrated fludarabine41,42 and gemcitabine,43 among which, Cy is the best that RT also increases DC maturation and cross-presentation.34 In this described. In mouse models, it was reported that Cy depletes study, the authors revealed that RT induced the release of HMGB1, CD41CD251 Tregs,38 and in vitro studiesrevealedthatCyreduces which in turn activated DCs through the TLR4–MyD88 signal path- the suppressive function of Tregs by decreasing the expression of way leading to a strong antitumor CTL response. Collectively, these the transcription factors GITR and FoxP3.39 In addition, data from data suggest that the antitumor effect may act through DC cross- a recent clinical showed a decrease in Treg numbers in peri- presentation to activate T cells. Constant with this hypothesis, our pheral blood after low-dose Cy treatment. Very importantly, this studies clearly demonstrated that RT generates strong CD81 T cell- study reported that a combination of Cy and fludarabine with dependent immunity leading to tumor reduction and reduced relapse total-body irradiation prior to adoptive CTL immunotherapy for of the primary tumor. Furthermore, we observed that type I interfer- melanoma, increased objective response rates from 50% to 70%.44 ons produced by tumor-infiltrating myeloid cells are required However, the mechanism by which low doses of Cy or other to endow tumor-infiltrated DCs with T-cell cross-priming capacity chemotherapy drugs selectively deplete Tregs, remains unclear. A and are essential for tumor reduction following RT.35 Impressively, recent study by Zhao et al. suggested that the low adenosine tri- when combined with immune therapy, RT is more effective at erad- phosphate (ATP) levels in Tregs may account for the selective icating metastasis than either therapy alone.29 Therefore, proper loca- depletion of this population. Compared to other cell types, Tregs lized RT given at appropriate doses and scheduling may tip the balance express low intracellular ATP due to the downregulation of miR- in favor of antitumor immunity both through endogenous priming 142-3p and upregulation of CD39. Low levels of ATP affect the mechanisms and in combination with immunotherapy.29 activity of glutamate cysteine ligase, which is required for glu- tathione synthesis. Since conjugation with glutathione is an Chemotherapy important route for detoxification from Cy therapy, the low intra- cellular level of ATP will eventually cause the selective cytotoxic Chemotherapy was designed to kill fast-dividing tumor cells by tar- 45 geting essential metabolic steps, such as DNA replication and tran- effect on Tregs. scription. This therapy has been very successful in reducing tumor It is conceivable that some chemotherapies can trigger danger sig- burden and is the standard treatment for many types of cancer. nals to bridge innate and adaptive immunity. In addition, different doses of chemotherapeutic drugs have distinct effects in tumor and Although chemotherapy is often viewed as a strategy that specifically 40 affects tumor cells, accumulating evidence indicates that these cyto- autoimmune disease models. Therefore, future studies examining toxic drugs also affect cells of the immune system. For instance, our how different cytotoxic drugs affect the immune system are necessary. data revealed that administration chemotherapy in conjunction with Understanding the proper timing and dosing of chemotherapy for a anti-neu antibody treatment abrogated resistance to tumor rechal- combination with immunotherapy will be necessary to optimize syn- lenge.23 In other models, however, tumor cell death induced by cyto- ergy between these therapies resulting in better antitumor activity. toxic drugs induced antitumor immunity, which is critical for the therapeutic effect of chemotherapy.34,36 TARGETED IMMUNOTHERAPY TO PRIMARY TUMOR TISSUES Therefore, the effects of chemotherapy extend beyond inducing CAN GENERATE IMMUNITY AGAINST DISTAL TUMOR tumor cell death. Of special note is a study by Obeid et al., demonstrat- TISSUES ing that the translocation of calreticulin determines the immunogeni- Creating lymphoid tissue inside the tumor to improve recognition city of dying tumors induced by cytotoxic drugs.37 Anthracyclins Although cancer cells express mutated or unique proteins recognizable induce the translocation of calreticulin to the cell surface, which will as foreign antigens by the immune system,2 malignant cells are sur- enhance the phagocytosis of tumor cell by DCs and induce antitumor rounded by non-malignant stroma to form a complex multicellular immunity. This process can be suppressed by blockade or knockdown ‘organ’ resembling self. Thus, the induction of immunity against nor- of calreticulin. However, other DNA replication targeting drugs, such mal, non-mutated differentiation antigens expressed by tumors as mitomycin C and etoposide, were unable to induce a strong anti- resembles autoimmunity. It has been reported recently that the orga- tumor immune response since they could not induce the translocation nized tertiary lymphoid structure (TLS) is necessary and sufficient to of calreticulin. In addition, if supplied with exogenous calreticulin induce autoimmune destruction of pancreatic islets.46 Indeed, de novo during mitomycin C and etoposide treatment, the immunogenicity organization of a TLS is known to precede the development of a of the dying tumor cell was restored and antitumor immunity was number of human autoimmune diseases and animal models.47,48 induced. The same research group also showed that the danger signal These observations suggest that lymphoid neogenesis within the target molecules, such as HMGB1, released by dying tumor cells could activ- tissue may have a critical role in initiating and maintaining immune ate DCs through TLR4–MyD88 signal pathway on DCs, which is responses against persistent antigens. TLSs are not supplied by afferent essential for processing and cross-presentation of antigen from dying lymph vessels and are not encapsulated, which implies that they are tumor cells. Most importantly, breast cancer patients carrying a TLR4 directly exposed to signals such as stimulating antigens and cytokines loss-of-function allele relapse more quickly after chemotherapy than from the environment. This unique structure could potentially result those carrying the normal TLR4 allele. This observation provides in unrestricted access of DCs and lymphocytes to the TLS, favoring insights into designing more effective chemotherapy strategies for immune activation. different patients based on the polymorphisms and mutation of Clues to understanding the signals that lead to TLS formation come genes.34 from the study of signaling pathways involved in secondary lymphoid Chemotherapy also influences the constitution of the tumor micro- tissue organogenesis. Studies in mutant mice and blocking experi- environment. Data from both preclinical studies and clinical trails ments have identified a key requirement for TNF family members,

Cellular & Molecular Immunology Utilizing primary tumor as in situ vaccine XM Yang et al 23 mainly a1b2 (LTa1b2), and to some extent, TNF, in the molecules inside the tumor.68 These experiments demonstrate that development and organization of lymph nodes and spleen microarch- introduction of the lymphoid-like structure into the tumor stroma itecture.49–52 Binding of LTa1b2 and TNF to their respective recep- can be highly effective in enhancing antigen recognition and may be an tors, LTbR and TNFR1, induce the expression of and effective strategy for cancer immunotherapy. adhesion molecules, which directly mediate lymphocyte migration Generation of TLS within the tumor allows naive T cells to be and homing.53 The first evidence that TLS formation could involve primed within the tumor itself, and this has several advantages. the same signaling pathways that regulate lymphoid organogenesis First, the efficiency and specificity of priming will be increased due came from studies of transgenic mice.48 The ectopic expression of to the heightened tumor antigen load in situ relative to that collected in LTa and LTb in pancreatic islets induced the formation of in situ the draining lymphoid tissues.69 Second, a broader repertoire of TLSs.54–57 Extrapolating from this, stimulation of LTbR or TNFR tumor-specific naive T cells is recruited to the site of tumor antigens, expressed by tumor stromal cells might promote the formation of leading to a more comprehensive response.59,70 It has been demon- lymphoid-like structures inside the tumor tissues for its destruction. strated that some tumor antigens may not be efficiently cross- Systemic TNFR signaling is of course too toxic, as evidenced by presented due to the antigen bias in T-cell cross-priming.71 other systemic TNF treatments.58 Soluble LTa can signal through Furthermore, no additional migration steps are required for CTLs the TNFR resulting in the upregulation of chemokines. To avoid sys- to reach the site of effector function, which leads to the appearance temic toxic effects, recombinant LTa has been conjugated with a of activated tumor-reactive T cells in a short period of time. In support tumor-specific antibody to be delivered specifically to the tumor tis- of this reasoning, we demonstrated in our experimental system using sue.59 Targeting of recombinant LTa to the tumor elicits an efficient the tumor cell line Ag104Ld–LIGHT that certain antigens are pre- immune response associated with induction of peripheral lymphoid- sented to and activate naive T cells within the tumor via a direct like tissue containing L-selectin1 naive T cells and major histocom- presentation mechanism.71 The same tumor antigen would have patibility complex class II1 antigen-presenting cells.59 Secondary otherwise been missed by the host immune response, had draining lymphoid tissue (SLC or CCL21) is among the chemokines lymphoid tissues been the sole location, and cross-presentation the controlled by LTbR and TNFR signaling.53 It is normally expressed in only means, of activation. Finally, T-cell responses may react more high endothelial venules and in T-cell zones of spleen and lymph nodes readily to the shifting tumor antigen expression profile in situ. T-cell and strongly attracts naive T cells and DCs.60 The expression of SLC stimulation in the absence of costimulation can induce anergy and inside the tumor resulted in a substantial, sustained influx of T cells apoptosis of antigen-specific T cells.6,72,73 Costimulation has also been within the mass as well as retention of DCs at the tumor site. By shown to greatly enhance tumor-specific T-cell function during the recruiting T cells and DCs, SLC in the tumor may lead to extranodal effector phase.74 Earlier studies show that LIGHT provides CD28- priming and inhibition of tumor growth.61 independent costimulation,64 which may be essential for the selective Besides, LTa1b2, LTbR is activated by LIGHT, another member of and effective activation, expansion and maintenance of tumor-specific the TNF family (a name derived from: homologous to , T cells among infiltrating naive T cells in Ag104Ld–LIGHT tumors. shows inducible expression, and competes with herpes simplex virus Thus, the introduction of a lymphoid-like milieu, including a com- glycoprotein D for herpes virus entry mediator, a receptor expressed bination of chemokines, adhesion molecules and costimulatory prop- by T lymphocytes).62 LIGHT is a ligand that signals through two erties inside tumor tissues, may allow LIGHT to be a potent antitumor receptors: LTbR expressed on stromal cells and herpesvirus entry immunostimulatory molecule. mediator expressed on T cells.62,63 LIGHT is predominantly expressed on lymphoid tissues, especially on the surface of activated DCs and T Generation of CTLs in the LIGHT-mediated tumor environment cells. Signaling via its receptor herpesvirus entry mediator, LIGHT acts for treatment of metastases as a strong costimulatory molecule for T-cell activation.62,64,65 Our Generation of a potent antitumor adaptive immune response can data indicate that the interactions between LIGHT and LTbR restore reduce the primary tumor burden, and help control the metastatic lymphoid structures in the spleen of LTa2/2 mice.66 and relapsing tumors. We have shown that expression of LIGHT in In vivo data demonstrate that LIGHT, signaling through two recep- the tumor microenvironment can lead to rejection of both the tors, mediates a microenvironment sufficient to break immunological local and distal sites. In an effort to develop more clinically relevant tolerance to self-antigens. First, ectopic expression of LIGHT in the approaches, adenovirus vectors expressing LIGHT (Ad-LIGHT) have pancreatic islets resulted in the formation of lymphoid-like structures, been constructed to deliver LIGHT into the tumor tissue. The advan- and was necessary and sufficient for pancreatic islet destruction.46 In tages of adenovirus are: (i) high production of a non-replicable virus; addition, sustained expression of LIGHT on T cells leads to their (ii) ease of manufacture; (iii) activation of innate immunity; (iv) an activation, migration into peripheral tissues and the establishment ability to express its carrying in undividing cells; and (v) readily of lymphoid-like structures in situ.66,67 LIGHT also acts as a strong expression in most tumor cell lines. Administration of Ad-LIGHT into costimulatory molecule for T-cell activation, possibly by binding to the tumor tissue leads to the complete rejection or significant delay of the herpesvirus entry mediator on T cells.64 Therefore, LIGHT is an aggressive murine tumors.75 Local treatment with Ad-LIGHT ideal candidate for delivery inside the tumor to create TLSs, recruit T initiated priming of tumor-specific CD81 T cells directly in the prim- cells and subsequently expand antigen-specific clones already inside ary tumor, with subsequent exit of CTLs which homed to distal the tumor. In addition to the cross-presentation pathway, tumor- tumors to elicit immune-mediated eradication of spontaneous meta- reactive T cells can be activated via direct-presentation pathway in stases.75 This strategy is an example that the generation of immune the presence of antigens and costimulation. Indeed, LIGHT ectopi- responses in primary tumor tissues prior to surgical resection can cally expressed in the tumor can effectively recruit and activate naive T produce tumor-specific effector T cells sufficient to eradicate distant cells. The expression of LIGHT in the tumor environment induces metastases in a CD8-dependent fashion. an infiltration of naive T lymphocytes that correlates with an upre- Although local RT treatment cannot completely clear established gulation of both chemokine production and expression of adhesion tumors or established metastasis, additional immunotherapy can

Cellular & Molecular Immunology Utilizing primary tumor as in situ vaccine XM Yang et al 24

Figure 1 Schematic representation of the model of generating immune responses against metastasis and relapse tumor with shared antigens by targeting primary tumor. CTL, cytotoxic T lymphocyte; DC, dendritic cell.

amplify or sustain immune responses. To test this hypothesis, we have preexisting CTLs by radiation and chemotherapy. Thus, new strategies added Ad-LIGHT therapy after RT or antibody treatment and focused on utilizing the primary tumor as the site of CTL priming observed more profound immunity against tumor, including estab- prior to surgical resection could offer a great advantage. The future of lished metastasis.29 Similarly, antibody treatment can trigger an antitumor therapies requires redesigning of conventional treatment immune response that can be amplified by Ad-LIGHT treatment lead- and further evaluation on how to best combine these therapies with ing to a response superior to the single treatment. Most importantly, immunotherapy to achieve the best therapeutic effect ultimately complete regression of the primary tumor and heightened resistance resulting in the control of metastatic disease and tumor relapse. to a secondary challenge are observed, supporting the role this therapy has in treating relapse.23

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