Nanomedicines As Multifunctional Modulators of Melanoma Immune Microenvironment

REVIEW

www.advtherap.com Nanomedicines as Multifunctional Modulators of Melanoma Immune Microenvironment

Barbara Carreira, Rita C. Acúrcio, Ana I. Matos, Carina Peres, Sabina Pozzi, Daniella Vaskovich-Koubi, Ron Kleiner, Mariana Bento, Ronit Satchi-Fainaro,* and Helena F. Florindo*

these tumors. New treatments approved Melanoma is the most destructive and deadly among skin cancers. Patients over the last decade have improved the sur- presenting the most disseminated form of this disease have very low survival vival of patients with melanoma, but the rates (≈15%) and highly restricted therapeutic alternatives. In recent years, global incidence rate of this disease is con- [1] the area of cancer immunotherapy has witnessed remarkable developments stantly rising. Melanoma is potentially treatable when in the management of many cancers, including melanoma. In fact, diagnosed at early stages. However, the immunotherapy unveiled as a feasible therapeutic alternative for late-stage prognosis of patients suffering from the melanoma patients, specifically using immune checkpoint therapies. advanced stage of this disease is highly However, despite the exciting outcomes, only a small percentage of patients limited. In this situation, melanoma com- respond to these therapies, and severe immune-related adverse reactions monly spreads to the lungs, gastrointesti- have been often reported. As such, most of preclinical and clinical studies nal tract, skin, and brain. Melanoma brain metastases constitute the third most com- currently explore melanoma tumor biology and immunology to guide the mon cause of intracranial tumors, affect- development of combinational immunotherapies aiming at relevant clinical ing around 10% of patients with ma- efficacy and minimal toxicity. Herein, the current knowledge on melanoma lignant melanoma, who usually have a biology and immunology is discussed, focusing on nanotechnology as a median survival of less than 4 months. crucial strategy for the development of combinatorial approaches able to Current melanoma treatment options are based on tumor stage and localiza- specifically modulate the function of key players responsible for melanoma tion. Therapeutic approaches available for evolution and evasion of host immune-mediated attacks. Finally, the major this disease include surgery, for the exci- challenges toward the clinical implementation of these emergent targeted sion of the lesions, radiation, chemother- nanomedicines for immunotherapy are further discussed, with particular apy, and targeted therapies. In the last focus on melanoma genomics, predictive biomarkers, clinical trial design, and few years, advanced knowledge has been clinical regulation of nanomedicines. generated about melanoma immune microenvironment, revealing the dynamic interaction between tumor, immune, and stromal cells. Immunotherapeutic-based 1. Introduction strategies constitute the new fourth pillar of cancer healthcare, being directed to the regulation of these tumor specific immune- Melanoma is a malignancy of melanocytes and represents 1% mediated pathways and targets, aiming at an improved efficacy, of skin cancers, being the most aggressive and lethal among while overcoming toxicity due to off-target effects. Checkpoint blockade therapy using monoclonal antibodies (mAb) against cytotoxic T lymphocyte-associated antigen 4 B. Carreira, Dr. R. C. Acúrcio, Dr. A. I. Matos, Dr. C. Peres, M. Bento, (CTLA-4) or programmed cell death 1 (PD-1)/programmed cell- Prof. H. F. Florindo ligand 1 (PD-L1) has indeed led to durable responses, as a Research Institute for Medicines (iMed.ULisboa) [2] Faculty of Pharmacy, University of Lisbon monotherapy or in combination. However, a significant part Av. Prof. Gama Pinto, Lisboa 1649-003, Portugal of individuals affected by the disseminated form of melanoma E-mail: hflorindo@ff.ulisboa.pt fails to develop a therapeutic response to these checkpoint in- S. Pozzi, D. Vaskovich-Koubi, R. Kleiner, Prof. R. Satchi-Fainaro hibitors, often acquiring resistance to these therapies.[3] There- Department of Physiology and Pharmacology, Sackler Faculty of Medicine fore, despite these exciting breakthroughs, the discovery and de- Tel Aviv University Tel Aviv 6997801, Israel velopment of new alternative immunotherapeutic options and/or E-mail: [email protected] combinations are urgently needed to promote effectiveness and patient response rates. The ORCID identification number(s) for the author(s) of this article Recent developments in the fields of nanotechnology, chem- can be found under https://doi.org/10.1002/adtp.202000147 ical biology, biotechnology, as well as tumor immunology and DOI: 10.1002/adtp.202000147 biology, are opportune to address this unmet medical need by

Adv. Therap. 2020, 2000147 2000147 (1 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com enabling the rational design of new strategies to target spe- ciﬁc cancer aggressiveness-related pathways, while regulating the function of tumor immune evasion-associated players.[4] This review discusses the potential of nanomedicines to target and modulate the function and frequency of multiple key players of the melanoma microenvironment to overcome tumor immune evasion, and thereby dramatically improve the safety and eﬃcacy of immunotherapies against advanced/metastatic melanoma. Im- portantly, it will discuss the major challenges toward the clinical implementation of these emerging nanotechnology-based immunotherapies.

2. Concept of Immunology in Cancer The modulation of host immune response toward the destruction of cancer cells is a major topic within biomedical science. It is now widely accepted that the host immune system can identify and eliminate malignant cells. However, while progressing, cancer cells develop mechanisms to escape immunosurveillance barriers.[5] The Chen and Mellman cancer-immunity cycle[6] nicely addresses this complex immune response induced against cancer. This immunoediting theory includes three intercon- Figure 1. Melanoma and host immune system interactions. Natural killer nected phases: i) immunosurveillance-mediated cancer cell elim- (NK) cells have distinct inhibitory and activating receptors, whose sig- ination; ii) equilibrium resultant from the cancer-immune cell nals control the active state of these innate immune system cells. Once activated, for example, following their binding to tumor-related ligands, interplay; and iii) immune escape. The transition between these NK cells release cytotoxic molecules that will cause the destruction of phases is adjusted by tumor cells’ activity and the immune sys- those malignant cells, in addition to cytokines that will attract dendritic tem status.[7] cells (DC) and other phagocytes. These will recognize and capture tumor During elimination, the first stage of the cancer-immunity cy- cells, further presenting their tumor-associated antigens (TAA) to T and cle, both innate and adaptive arms of the immune system act in B cells, thus leading to the activation of the adaptive arm of the immune a concerted manner to recognize and destroy tumor cells. This is system. Cytotoxic granules will be released from cytotoxic T cells (CTL) at tumor cell surface once the T cell receptor (TCR) of CD8+ T cells recognize a process based on two distinct but interconnected phases. The the TAA bound to major histocompatibility complex (MHC) molecules on first fast-nonspecific early response is driven by the innate arm of DC surface. CD4+ T cells that recognize TAA-MHC class II complex on the immune system, which includes the involvement of dendritic the surface of these antigen-presenting cells (APC), will further activate B cells (DCs), macrophages, natural killer (NK) cells, and granu- cells, leading to the release of tumor-specific antibodies. The cells of the locytes. A late phase encompasses the recognition and further adaptive immune system will reactivate the function of the innate immu- internalization of tumor-associated antigens (TAA) by antigen- nity components via receptor/ligand systems, soluble factors, as cytokines and chemokines, and/or increased amounts of antigens that are released presenting cells (APCs), primarily DC, being processed and sub- following tumor cell death. Adapted with permission.[11] Copyright 2019, sequently loaded into major histocompatibility complex (MHC) Elsevier. molecule and subsequently presented to T-lymphocyte populations in the lymph nodes (Figure 1). In this phase, CD8+ effector T lymphocytes are the major players in tumor cell clearance, assisted and complemented by various other populations of immune cells, including NK, CD4+ effector T cells, as well immunity-mediated events. However, tumor cells gradually as B cells, which act together either within tumor microenviron- downregulate the expression of MHC class I and MHC class II ment (TME) or at the peripheral tissues to effectively eliminate all molecules, blocking the binding of antigens, which will then be melanoma cells.[5a] Once the elimination phase is accomplished, released in high quantities. However, during this phase, the tu- malignant cells are eradicated before they become clinically de- mor cells are subjected to a constant selected immune pressure, tectable cancers, constituting an endpoint for the immunoediting and those malignant cells with higher ability to evade this con- process.[5b,8] trol, will escape and become a clinically relevant disorder.[9] This The equilibrium phase or tumor dormancy period results from tumor immune evasion constitutes the third phase of the cancer- the continuous editing of newly derived tumor cells by adaptive immunity cycle, and multiple tumor progression-related path- immune cells, thereby controlling tumor growth and immuno- ways and mechanisms are developed by these malignant cells genicity. This is the longest phase, during which the tumor cells to avoid immune cell-mediated recognition and eradication.[8b,10] are allowed to reside in patients’ body sometimes for decades be- For this reason, the complex TME and the interactions between fore they restart to grow and become clinically detectable.[5b,8b] its major cellular and molecular components, have been actively In this phase, distinct components of the adaptive immu- addressed aiming at the identification of these escape pathways nity are required, including interleukin-12 (IL-12), interferon- used by tumor cells to disrupt this cancer-immune cycle and gamma (IFN-𝛾), CD8+,andCD4+ T cells, in contrast to innate progress.

3. Melanoma Tumor Antigens and Related peptides combined with chemotherapy have been explored Immunity against melanoma.[27] Neo-antigens are not found on normal tissues, and therefore Melanoma is a highly immunogenic tumor able to regulate the their recognition is not controlled by central T cell tolerance.[28] adaptive immune response toward an effective control of tumor Indeed, tumor-infiltrating T cells specific for tumor neo-antigens cell proliferation and progression.[12] Melanoma immunogenic- has been related to extensive tumor immune-mediated destruc- ity is related to antigen expression and to the overall cross talk tion, being therefore highly promising to control cancer develop- between immune, stroma, and tumor cells, including the im- ment in humans. munomodulatory factors secreted by these different players. The The use of neoantigens as ideal targets for immune response mechanisms underlying the regulation of the immune response against tumors is not new. However, their discovery and screen- against melanoma are still unclear, but the spontaneous tumor ing only recently became possible due to the improvement of regression sometimes observed in patients may be explained by sequencing technologies able to detect all coding mutations the high levels of tumor cell somatic mutations. This high tumor within tumor cells, coupled with new machine learning tools able mutational burden leads to an increased expression of modified to predict mutated peptides presenting higher affinity to HLA surface epitopes.[13] molecules.[12,29] The use of neoantigens for vaccination against There are different types of cancer antigens, which are broadly tumors can expand neoantigen-specific T cells already present classified in three major categories: i) cancer-testis antigens within TME, while also eliciting the development of additional (CTA) found on several tumors and adult testis that do not ex- T-lymphocyte populations, contributing to an enhanced tumor press human leukocyte antigen (HLA) molecules, while missing control.[30] The detection of neoantigens has also been employed in any other normal cells,[14] ii) TAA found on cancer and normal to predict patient response to immunotherapeutic regimens, cells, and iii) tumor-derived neo-antigens derived from mutated including to immune checkpoint blockers (e.g., anti-PD-1 and genes.[15] anti-CTLA-4 mAb).[31] Indeed, it was reported that patients with CTA may promote the uncontrolled growth and subsequent metastatic cancers with high mutation loads benefit from anti- survival of tumor cells, similarly to what is known to be their ef- CTLA-4 therapy for longer periods, at least to some extent due to fect on human germline cells. The melanoma antigen (MAGE), the induction of new T cells primed against neoantigens.[32] This B-M antigen-1 (BAGE), G antigen (GAGE), and NY-ESO-1 are the can be a possible explanation for the relation between neoanti- most explored CTA, among the 80 families already reported.[16] gens and increase of progression-free survival (PFS) in anti- NY-ESO-1 immunogenicity is considered to be the highest CTLA-4 mAb-treated individuals with melanoma.[33] among the known CTA, leading to relevant humoral responses [17] MAGEA against various tumors. 1 antigens are found on sev- 4. Tumor Microenvironment Role in the eral cancers, and their expression is correlated with increased Pathogenesis of Melanoma tumorigenesis and poor prognosis.[18] MAGEA 1 peptides form complexes with HLA molecules, being thereby recognized by au- TME anatomical and physiological features are distinct from tologous CTL, likewise to peptides from the BAGE family.[19] Al- healthy tissues, presenting an abnormal architecture of blood though this family was reported to have a relevant expression in vessels and lower pH. Increased angiogenesis ensures a contin- metastatic melanoma, there are no published recent works ex- uous delivery of oxygen and nutrients to cancer cells and evolves ploring this CTA. The GAGE family has been associated with following an orchestrated engagement of various proteins, in- poor prognosis, being a diagnostic biomarker for melanoma.[20] cluding matrix metalloproteinases (MMP), proteins from the ex- Central and peripheral tolerance result in the selection of tracellular matrix, and pro-angiogenic proteins.[34] The acidic pH T cells which T cell receptor (TCR) recognizes and binds is caused by the increased metabolism and proliferation rates of with low affinity to TAA epitopes on normal cells. These tumor cells, which dictate superior levels of glycolysis to fulfill proteins do not result from mutations, and are expressed their needs. As a result, a greater amount of lactic acid is pro- by melanoma cells, as well as at different stages of non- duced, affecting the surrounding areas. Acidosis is further exac- malignant melanocyte development.[21] TAA include the gly- erbated by both carbon dioxide (CO2), which is the final product coprotein (gp)100, tyrosinase, tyrosine-related protein (TRP)- of cell respiration, and the hypoxic environment in the core of 1/TYRP-1 and TRP-2/ TYRP-2, and the melanocytic melan- solid tumor.[35] A/MART-1. Tyrosinase constitutes a relevant diagnostic marker Tumor progression has been mostly associated with molecular by being found on melanosomes of up to 90% of melanomas pathways and infiltrating cells in the TME.[36] Indeed, tumor (primary and metastatic).[22] TRP-1 and TRP-2 are found on the survival and progression are highly dependent on different majority of melanomas.[23] On the one hand, the TRP-2 overex- TME-infiltrating immune cell populations, and their commu- pression has been correlated to chemotherapy- and radiotherapy- nication with tumor and stromal cells. The TME is constituted resistant metastatic melanomas.[24] But, on the other hand, by noncancer and neoplastic cells. Noncancer cell types include TRP-2 has also been associated to humoral and cellular auto- stromal cells, such as adipocytes, epithelial and endothelial immunities, triggered following its recognition on melanocyte cells, fibroblasts, and smooth muscle cells. Collectively, these surface by TCR with high affinities.[25] Melan-A or MART-1, different stromal cells are important for tumor proliferation and as well as the gp100 or Pmel17 (pre-melanosomal protein) are metastasis. In addition, the cell-to-cell communications within only found on skin and retina melanocytes, being present in the tumor stroma mediated by a range of soluble components, such majority of melanomas (primary lesions and advanced disease), as growth factors, cytokines, or chemokines, are equally essential and recognized by autologous CTL.[26] DC pulsed with gp100 for cancer progression. The release of soluble factors is generally

Adv. Therap. 2020, 2000147 2000147 (3 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com triggered by the TME characteristic hypoxic environment, result- are associated with a higher survival rate, while FOXP3+ CD4+ ing in the migration of different cell populations into the TME, Treg TIL are related to poor prognosis.[46] as fibroblasts, macrophages, or neutrophils, thus potentiating Within TME, the various T cell populations have distinct cancer growth. On the opposite trend, the higher infiltration of crucial roles that are dictated by the expression of different certain subpopulations of immune cells, such as DC and CTL, factors, including receptors and/or stimulatory or inhibitory has been correlated with improved immunogenic cancer cell ligands at the surface of immune, stromal, or tumor cells. To be- death, as it will be further discussed below. come anti-tumor effector cells, naïve T cells need to be activated by the co-stimulatory signals related to the MHC-TCR complex and the CD28 expressed on T cells. The latter depends on DC 4.1. Dendritic Cells and TAA presentation, but its isolated stimulation is not enough to trigger a complete T-cell activation, which in that situation DC are the central point between the innate and the adaptive will become anergic. As an example, TIL can express the tumor immune systems,[37] constituting a complex immunomodula- tolerance-related CCL21 chemokine, which attracts naïve T cells tory network, orchestrated toward an integrated effective im- that cannot recognize TAA due to the absence of co-stimulatory mune response, specifically directed against antigens. DCs are molecules.[47] considered professional APC by being the most effective in in- Despite the high immunogenicity of melanoma, the elevated ducing specific T-cell responses against infection or malignant antigen burden is associated with the exhaustion and dysfunction diseases. DC are crucial for TAA capture and presentation.[37b] of CD8+ T cells, which is translated by the loss of their effector These APC are present in many tissues, where they internalize functions. and process antigens (exogenous or endogenous) that are subse- These dysfunctional T cells trigger the upregulation of check- quently complexed with MHC molecules, and further presented point molecules, such as CTLA-4, PD-1, T cell immunoglobulin to T cells via TCR recognition.[38] In addition to antigen recogni- and mucin domain-3 (TIM-3), and lymphocyte activation gene 3 tion and presentation by these APC, the accumulation of effec- (LAG-3),[48] which promotes tolerance.[49] Moreover, melanoma tor immune cells in the TME is also a critical factor to trigger cells enhance the expression of PD-L1, a co-inhibitory molecule effective immune-mediated responses able to control melanoma responsible for hampering the activation of T cells, also con- progression.[39] The number of DC in metastatic melanoma is tributing to T-cell anergy and exhaustion.[50] generally lower compared to non-metastatic melanoma, estab- The presence of T cells within tumor stroma was recently as- lishing a direct correlation between the number of DC within sociated with the establishment of tertiary lymphoid tissues, a TME and a lower risk of tumor recurrence.[40] However, DC lymph node-like structure comprising activated DC, T cells, and can have a skewed double role within the TME, translated B cells.[51] There is limited knowledge regarding germinal cen- into an immunosuppressive phenotype. This immature state is ters and Follicular T cell (Tfh) role in tumor immunity, but the characterized by the decreased expression of the co-stimulatory presence of these tertiary lymphoid structures within the TME, molecules (CD80 and CD86) and DC inability to present anti- which includes antibody-secreting cells in germinal centers, Tfh gens, being triggered by the expression of immunosuppres- cells, macrophages, mature DC, and high endothelial venules sive factors, such as vascular endothelial growth factor (VEGF), (HEV), in addition to infiltrating CD4+ and CD8+ T cells, were re- transforming growth factor beta (TGF-𝛽), or indoleamine-pyrrole cently correlated with improved prognosis against melanoma[52] 2,3-dioxygenase (IDO) secreted by melanoma cells.[41] DC with and sarcoma.[53] immature phenotype suppress other immune cells through the expression of inhibitory factors (e.g.,PD-L1), immunosuppressive cytokines or regulatory T cell (Tregs).[42] 4.3. B Lymphocytes In melanoma, CD123+ plasmocytoid DC (pDC) are correlated with poor prognosis and early relapse. pDC were shown Similarly to T lymphocytes, B cells are additional key compo- to promote a T helper (Th) 2 phenotype by inducing the expres- nents of the acquired immunity. In contrast to the undeniable sion of the OX40 ligand and the inducible T cell costimulatory role of TIL, the impact of B lymphocytes in melanoma is still ligand (ICOSL) that drive tumor progression.[43] Furthermore, controversial.[54] On the one hand, in some studies, B lympho- high levels of immunosuppressive cytokines (e.g., IL-6, IL-10) cytes have been related to carcinogenesis by inducing the secre- and miRNA favor melanoma cell progression through the sig- tion or appearance of pro-inflammatory factors. Additionally, renal transducer and activator of transcription 3 (STAT-3) pathway, sistance to BRAF and MEK inhibitors has been correlated to high impairing DC function.[44] levels of TME-infiltrating B cells, which can be supported by the ability of B cells to trigger the release of insulin-like growth factor 1 (IGF-1), a factor involved in the resistance of melanoma to 4.2. Tumor-Inflitrating Lymphocytes these tyrosine kinase inhibitors.[55] On the other hand, the presence of B cells within TME has Solid tumors, as melanoma, have high levels of tumor-infiltrating been associated with improved treatment outcomes, particularly lymphocytes (TIL), which is widely accepted as the most im- in tumors where tertiary lymphoid structures have been identi- portant prognosis factor for cancer patient survival.[45] Several fied, as melanoma. It has been hypothesized that these tertiary subpopulations of lymphocytes infiltrate the tumor, and each lymphoid structures actively modulate the immune response to- one of them has a different role. CTL, Tregs, and memory T cells ward tumor cell elimination. When these structures became ma- are the three TIL subsets mostly found on the TME. CD8+ TIL ture, they promoted the formation of germinal centers[56] and

B cell responses, thus suggesting the stimulation of humoral prevalence of one TAM phenotype over the other. This complex anti-tumor responses against melanoma and metastasis.[57] Fur- interplay is not fully understood, but the type I IFN pathway is thermore, high levels of TCF7+ naïve and memory T cells were widely accepted to be crucial for the stimulation of the anti-cancer reported in tumors rich in B cells, suggesting the im- innate immunity. portance of tertiary lymphoid structures within melanoma TAM regulate the expression of VEGFA, being therefore im- microenvironment, by promoting the expression of different T- plicated in melanoma angiogenesis. These innate cells also drive cell populations.[58] As so, different studies associated melanoma the hypoxia-inducible factor (HIF)2a expression, contributing to inflammation to the prevalence of B lymphocytes and tertiary low levels of oxygen within TME.[72] Moreover, TAM regulate lymphoid structures, which correlated to positive responses to the secretion of adrenomedullin, a vasodilator responsible for tu- immune-checkpoint inhibition.[58,59] mor progression.[73] This TAM function is the basis of emerging melanoma therapeutic approaches, where TAM deregulation counteracts angiogenesis and thereby inhibits tumor growth. Ad- 4.4. Myeloid-Derived Suppressor Cells ditionally, macrophages also release IDO that inhibits the immune response by decreasing the tryptophan levels. IDO also Myeloid-derived suppressor cells (MDSC) constitute a mixed promotes Treg migration to the tumor site, enhancing the im- subset of immunosuppressive myeloid cells driven by inflamma- munosuppressive environment. tory mediators and found accumulated within tumor stroma.[60] These myeloid cells inhibit the NK cell-mediated cytotoxic function and cause T-cell anergy and apoptosis[61] through different 4.6. Natural Killer Cells mechanisms. These include the expression of PD-L1 and Fas lig- and (FasL), being also able to improve the induction of Tregs, thus NK cells are also involved in orchestrating host immunity toward contributing to an enhanced immunosuppressive TME.[62] the destruction of melanoma. These innate cells recognize and at- Metastatic melanoma individuals showed high levels of MDSC tack cancer cells expressing low levels of MHC class I molecules, in the peripheral blood, which is associated with tumor progres- to a greater extent than T cells.[74] Moreover, NK cells once acti- sion and decreased overall survival (OS).[63] The high levels of vated upregulate expression of the natural cytotoxicity receptors, MDSC were also shown to impair the efficacy of immunotherapy- like NKG2D, NKp30, NKp44, or NKp46, fostering their interac- based treatments. These patients with high levels of MDSC were tion with their respective ligands on tumor cells.[75] Besides, per- less responsive to ipilimumab, and also to nivolumab given to forins and granzymes released within TME by NK cells in the ipilimumab-refractory individuals.[64] presence of antigens, improves DC and T-cell activation against cancer cells.[76] By secreting cytokines, NK contribute indirectly to 4.5. Tumor-Associated Macrophages the immune-surveillance, and can induce the maturation of DC, playing an additional role in the adaptive immune response.[77] Cells of the innate immune system can adopt opposite roles NK T-lymphocytes (NKT) and 𝛾𝛿 T cells hinder essential func- within melanoma microenvironment. On the one hand, innate tions in regulating immune responses within TME, by being immune cells are able to directly mediate anti-tumor cytotoxic- involved in tumor inhibitory mechanisms. NKT lymphocytes ity by promoting CTL recruitment and activation, while on the combine attributes of both T and NK cells, being the ability to other hand, they may fuel the immunosuppressive environment induce tumor apoptosis through their endogenous TCR their maby secreting suppressive molecules or inhibiting the interactions jor advantage over T cells. These TCR are elicited by glycolipids between anti-tumor effector immune cells. (e.g., 𝛼-galactosylceramide) via non-polymorphic MHC class Ib Tumor-associated macrophages (TAM) can have different phe- molecule CD1b on APC.[78] Then, stimulated NKT cells are able notypes with distinct polarization status.[65] The classification to rapidly secrete immunomodulatory cytokines via Th1, includ- of TAM is quite intricate, being essentially determined by ing IFN-𝛾, which is a crucial mediator for inhibiting angiogen- the TME conditions and cell–cell interactions.[66] M1 and M2 esis and triggering effective immune cell anti-tumor functions, macrophages are engaged with the pathogenesis of inflamma- by stimulating the downstream stimulation of DC, T and B lym- tory and cancer diseases.[67] phocytes, as well as NK cells.[79] The stimulation of DC by 𝛼- The M1 macrophages are characterized by the secretion galactosylceramide has been explored as a promising strategy to of high levels of pro-inflammatory cytokines, and overexpres- prime NKT to induce a strong anti-tumor response in advanced sion of cell surface factors necessary for antigen presentation melanoma.[80] (MHC class II, CD80, CD86). M1 macrophages thus elicit a The 𝛾𝛿 T lymphocytes constitute a distinctive T-cell subset that Th1-immune cell activity and thereby promote an anti-tumor links the innate immunity to the adaptative immunity.[81] De- response,[68] while the M2 phenotype regulates tissue repair pending on the environmental conditions, 𝛾𝛿 T cells can display and stimulates Th2-immune responses, displaying pro-tumoral different phenotypes with distinct functions. These cells can pro- properties.[67,69] M2 TAM can secrete numerous cytokines, such mote anti-tumor responses by secreting cytokines (IFN-𝛾, IL-2, as IL-1, IL-6, IL-10, and TGF-𝛽, promoting tumor cell pro- IL-17, and tumor necrosis factor alpha—TNF-𝛼) and directly in- liferation and metastasis.[70] Therefore, the levels of infiltrat- ducing tumor cytotoxicity. Nevertheless, these cells may also con- ing M2 TAM and M2/M1 ratio are key prognosis factors for tribute to TME immunosuppression, where the exposure to in- melanoma.[71] hibitory cytokines (TGF-𝛽, and IL-10) drives their polarization to These two phenotypes of TAM are not static, and different the 𝛾𝛿 T regulatory subtypes.[82] In melanoma, the higher expres- tumor-related but also environmental factors may dictate the sion of 𝛾𝛿 T cells was related to improved clinical responses in

Adv. Therap. 2020, 2000147 2000147 (5 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com individuals that received ipilimumab.[83] However, 𝛾𝛿 T cells col- paracrine or autocrine effects (e.g., IL-8 in primary melanoma, lected from melanoma patients showed a modified expression IL-6 and IL-10 in advanced melanoma), which support the of immune checkpoints, which contributes at least in part to proliferation and migration of these malignant cells.[98] IL-10 immune escape and tumor progression. Therefore, the skewed secreted by melanoma cells downregulates MHC molecules, melanoma microenvironment is able to impair the anti-tumor thus limiting the antigen presentation and subsequent activation function of 𝛾𝛿 T cells to escape immunosurveillance.[84] of T-cell effector functions.[99] The chemokines and their receptors are also an intrinsic part of the immune cell trafficking, having a crucial function in 4.7. Cancer-Associated Fibroblasts inflammation and tumorigenesis.[100] Chemokines within the TME can be released by both host and cancer cells. There, Fibroblasts are another cell population well-represented within chemokines stimulate the expression and activation of different the TME, with several functions and phenotypic characteristics. immune cells, as TIL, neutrophils, DC, and macrophages, which In melanoma, fibroblasts participate in the angiogenesis process can have anti-tumor or pro-tumor functions. In melanoma, by their conversion to pericytes or myofibroblasts.[85] The release CCL2, CCL3, or CCL21 have been related to increased levels of MMP by stromal fibroblasts has been shown to protect ma- TIL[101] and improved responses to immune checkpoint[102] and lignant cells from the NK-induced cytotoxicity.[86] The interplay adoptive T cell therapies.[103] In addition to this primary func- between MMP and their tissue inhibitors of metalloproteinases tion as chemoattractant agents, chemokines, as CCL22, CXCL1, (TIMP) is related to melanoma regression and improved patient CXCL2, or CXCL3, are also involved in tumor progression,[104] prognosis.[87] angiogenesis,[105] and metastasis.[106] Cancer-associated fibroblasts (CAF) result from the differenti- The significant efforts that have been done to uncover the di- ation of fibroblasts upon their activation by tumor cells.[88] Acti- verse tumor progression-related mechanisms within melanoma vated CAF promote tumor progression and invasion by releasing TME, already guided the development of alternative com- multiple growth factors and chemokines (e.g., TGF-𝛽, VEGF, ba- binatorial immunotherapies, aiming at the modulation of sic fibroblast growth factor (bFGF), IGF-1). The TGF-𝛽 is respon- different components implicated in tumor growth and immune sible for the alteration of the extracellular matrix (ECM), promot- evasion. Additionally, the characterization of the melanoma mi- ing cancer cell proliferation, invasion, and metastasis.[89] croenvironment fostered the recognition of biomarkers, driv- ing the development of more precision therapies, including targeted nanotechnology-based medicines to prevent off-targeted 4.8. Neutrophils effects. These emerging immune-based strategies and precision nanomedicines designed to overcome melanoma development Neutrophils are among the first cells to respond to inflam- are addressed in the subsequent sections. mation, infection, and injury. In tumors, as melanoma, they constitute a substantial portion of immune cell infiltrates.[90] However, their role in cancer is still controversial. Some stud- 5. Immune-Mediated Strategies to Regulate Tumor ies report that tumor-associated neutrophils are anti-tumoral, Immune Microenvironment to Overcome by inducing a direct cytotoxicity against tumor cells, and pre- Melanoma Evasion Mechanisms venting metastasis.[91] Other studies suggest that neutrophils are The immune system is fundamental to overcome melanoma pro-tumoral supporting tumor progression, migration and inva- evasion mechanisms and therefore control tumor progression. sion, by modulating other immunosuppressive cells.[92] It has Thus, several immunotherapeutic approaches to regulate the been reported that these pro-tumoral neutrophils support the TME have been successfully developed. Cancer immunotherapy preparation of premetastatic niches in distant secondary organs is now a clinical reality that has revolutionized the treatment of for the engraftment of primary tumor cells.[93] In melanoma, different cancers, including melanoma with exciting outcomes. neutrophils play a crucial role in inflammation, promoting an- Extensive research has been devoted to establish single or com- giotropism and tumor metastasis.[94] binatorial regimens of immune-based therapies for melanoma, This double phenotype is modulated by the TME.[95] For exam- either through “active”[107] or “passive”[108] approaches. These in- ple, the presence of TGF-𝛽 induces a N2 pro-tumoral phenotype, clude immune checkpoint modulators, cancer vaccines, adoptive whereas the IFN-𝛽 expression or inhibition of TGF-𝛽 promotes a T cell therapy, and adjuvant treatments based on cytokines, such N1 anti-tumoral phenotype.[96] as IL-2 and IFN-𝛼 (Figure 2). Despite the exciting outcomes, most patients do not benefit from cancer immunotherapies approved so far, and significant scientific efforts have been made to clarify 4.9. Cytokines and Chemokines the mechanisms beyond resistance to create broad and effective immune-related therapeutic solutions. Pro-inflammatory cytokines (IL-1, IL-6, IL-8) or anti- inflammatory (IL-4, IL-10, IFN-𝛼) mediators drive the inflammatory response during cancer development. However, cytokines 5.1. Active Immunotherapeutic Approaches may have a dual functionality. Cytokines favor chronic inflammation and oxidative stress in carcinogenesis.[97] Moreover, Active cancer immunotherapy, also known as cancer vaccination, melanoma cells secrete growth factors, such as bFGF, Platelet- relies on the direct stimulation of patient immune system to in- derived growth factor (PDGF), and TGF-𝛽 and cytokines with duce an antigen-specific response against tumor cells. Vaccines

Figure 2. Examples of different immune-based approaches against melanoma. Different immunotherapies can be used to eliminate melanoma, including therapeutic vaccination, antibody-based immunomodulation, dendritic cell (DC)-based vaccination, cytokine and chemokine-based therapies, chimeric antigen receptor (CAR) T cell therapy, or adoptive cell treatments. Therapeutic vaccines consist in the delivery of peptides containing both major histocompatibility (MHC) class I and MHC class II epitopes to ensure that both CD4+ T cells and CD8+ T cells are activated. Antibody-based modulation can be achieved using immune checkpoint inhibitors or agonists to decrease tumor-associated immune suppression and enhance cytotoxic T lymphocytes (CTL) response. DC-based vaccination depends on DC populations that are able to prime CD4+ T lymphocytes and CD8+ T lymphocytes. Adoptive cell therapy includes T lymphocytes or natural killer (NK) cells to provide help at the tumor microenvironment (TME). Transferred CTL should be expanded in conditions related to the delivery of help signals. CAR T cell therapies can involve cells that express the signaling pattern of suitable co-stimulatory receptors to provide help signals. Cytokine treatment relies on the administration of chemical signals that stimulate T cells. are powerful weapons commonly associated to the control and munogenicity of the identified TAA will be improved follow- even eradication of certain infectious diseases, mainly due to ing their combination with adjuvants, such as toll-like recep- the induction of B-lymphocyte responses and related neutraliz- tor (TLR) ligands (e.g., CpG,[109a] Poly (I:C),[111] Monophos- ing antibodies. In cancer treatment, active immunotherapy re- phoryl Lipid A (MPLA)[112]), or other adjuvants, as for exam- lies mostly on the administration of TAA, or neoantigens, alone ple montanide[113] or retinoic acid-inducible protein 1 (RIG-I) or combined with adjuvants, toward the activation of a systemic ligands.[114] Several studies have demonstrated that the use of antigen-specific immune response with a major involvement of a single platform to deliver mixtures of antigens and adjuvants DC on CTL responses.[109] Nevertheless, the tertiary-lymphoid- is the key to unlock effective humoral and cellular anti-tumor like structures recently reported in tumors have driven researcher responses.[109a] In fact, studies have demonstrated that the simul- attentions to the potential role of germinal center responses, taneous delivery of antigens and adjuvants to the same APC is follicular T lymphocytes and related high-affinity antibodies on essential to prime tumor-specific immune responses.[115] There- achieving orchestrated humoral and cellular immune responses fore, an ideal cancer vaccine should be an “all in one,” combin- against cancer. ing the most immunogenic TAA with the most effective adju- Several engineered molecules designed to enhance immune- vant on an optimal delivery platform. Based on their safety and based mechanisms are available, from proteins and peptides, to selectivity, nanomedicines emerge as one of the most suitable antibodies and oligonucleotides. These molecules will be fun- delivery platforms for cancer vaccines. DC-based cancer vacci- damental to advance host responses to cancer immunother- nation as melanoma therapeutics will be further discussion in apy. Also, the identification of several defined T-cell TAA Section 6. or neoantigens, along with the recognition that specific T cells can mediate the elimination of tumors, have encour- aged cancer vaccine development.[109,110] However, the clini- 5.2. Passive Immunotherapeutic Approaches cal translation of these vaccine candidates shows that their efficacy requires the rational design of approaches able to Passive immunotherapy is currently the most used cancer protect these biological molecules while allowing their trans- immune-based treatment, being centered on the modulation port across biological barriers, in addition to overcome estab- of different receptors following the administration of different lished tumor-related evasion mechanisms. Moreover, the im- molecular agents or cells. Examples include the manipulation of

Table 1. FDA and EMA approved monoclonal antibodies for melanoma. mAb Brand name Type Target Indication Approval

Ipilimumab Yervoy Human CTLA-4 Melanoma FDA, EMA Renal cell carcinoma FDA, EMA Colorectal cancer FDA Nivolumab Opdivo Human PD-1 Melanoma FDA, EMA NSCLC FDA, EMA Small cell lung cancer FDA Renal cell carcinoma FDA, EMA Hodgkin lymphoma FDA, EMA SCCHN FDA, EMA Urothelial carcinoma FDA, EMA Colorectal cancer FDA Hepatocellular carcinoma FDA Pembrolizumab Keytruda Humanized PD-1 Melanoma FDA, EMA NSCLC FDA, EMA SCCHN FDA, EMA Hodgkin lymphoma FDA, EMA Urothelial carcinoma FDA, EMA Colorectal cancer FDA Gastric cancer FDA Cervical cancer FDA

CTLA-4, cytotoxic T-lymphocyte-associated protein 4; EMA, European Medicines Agency; FDA, Food and Drug Administration; NSCLC, non-small cell lung cancer; PD-1, programmed cell death protein 1; SCCHN, squamous cell carcinoma of the head and neck. immune effector cells ex vivo, such as CTL, or the recombinant of immune checkpoint molecules may be altered, preventing mAb. effector immune cells from mounting a successful anti-tumor immune response.[2a,124] For this reason, molecules regulating immune checkpoint activation have drawn considerable interest 5.2.1. Monoclonal Antibodies in cancer immunotherapy,[2a] especially those directed to CTLA-4 and PD-1 co-inhibitory signals.[125] The high selectivity and specificity of mAb have been greatly ex- Three mAb targeting immune checkpoint molecules are ap- ploited in cancer. Since the invention of the hybridoma tech- proved by the American and European regulatory authori- nology in the early 1980s, mAb have been directed toward ties, and are used in clinical practice against melanoma: ipil- a wide range of tumor targets, including tumor cell surface imumab (anti-CTLA-4 mAb), nivolumab (anti-PD-1 mAb) and proteins,[116] antigens associated with the tumor stroma,[117] anti- pembrolizumab (anti-PD-1 mAb), which are currently consid- gens on tumor-associated vasculature,[118] secreted growth fac- ered as the standard of care in melanoma therapy (Table 1).[121a] tors or cytokines,[119] and more recently, co-stimulatory pathways Ipilimumab, an anti-CTLA-4 mAb, was the first ICI newly- and co-inhibitory molecules found on several cell surface, the so approved medicine in 13 years for the treatment of metastatic called immune checkpoints.[120] melanoma,[120a,126] enabling a significant improvement in their Since the approval of the first mAb for cancer treatment (Ritux- OS. CTLA-4 is a T-cell co-inhibitory molecule involved in the imab) by the American and European regulatory agencies (EMA regulation of the early phases of the activation of these lympho- and FDA), mAb have emerged as the most rapidly expanding cat- cytic cells, upon ligation to CD86 or CD80 on APC.[127] Although egory of biopharmaceuticals with over 30 mAb being approved CTLA-4 is expressed by activated CTL, its major physiologic role and introduced in cancer clinical practice.[121] seems to be through distinct effects on the two major subsets of + Immune-Checkpoint Blockade: In the last decade, the in- CD4 T-cells, by downmodulating Th cell activity and enhancing troduction of therapeutic mAb targeting immune checkpoint Treg suppressive activity.[128] The blockade of this immune check- molecules was a breakthrough in cancer immunotherapy. In- point molecule with ipilimumab significantly improved the OS stead of attacking directly the malignant cells, these mAb en- in individuals affected by the disseminated form of melanoma in hance the anti-tumor immune response by targeting immune two distinct phase III clinical trials.[120a,129] In a follow-up analysis regulatory pathways.[2a,122] Such mAb are collectively known as of individuals enrolled in different phase II and phase III clinical “immune checkpoint inhibitors” (ICI) and those have radically trials, it was observed that those receiving ipilimumab presented altered the therapeutic landscape available at that time to treat an OS of 11.4 months and 22% three-year survival rate.[130] patients with advanced melanoma. The interaction between PD-1 and its naturally binding Immune checkpoint receptors are membrane molecules molecules, PD-L1, and PD-L2, prevents T-cell activation, which found mainly (but not exclusively) on T lymphocytes and NK allows the maintenance of immune homeostasis and avoids cells, which interact with specific molecules expressed by APC autoimmune reactions in healthy tissues.[131] However, this or other cells (e.g., tumor cells).[123] In the TME, the expression pathway is also used by tumor cells as a potent immune escape

Adv. Therap. 2020, 2000147 2000147 (8 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com mechanism.[132] Indeed, the expression of immune checkpoint mAb plus anti-PD-1 mAb enhanced the immune-mediated anti- ligands, such as PD-L1 and PD-L2, is commonly elevated in tumor response, causing regression of the tumor, while TIM-3 cancer cells, preventing T-cell activation and thus, supporting inhibitors alone did not induce relevant effects.[144] This ICI is tumor growth.[133] The blockade of PD-1/PD-L1 interaction by currently being studied in clinical trials against several metastatic ICI reverts T-cell suppression, increasing the stimulation and cancers, including melanoma, as monotherapy or in associa- proliferation of effector T lymphocytes, and subsequent immune tion with conventional anti-cancer therapies or with other ICI, response against cancer cells.[132,134] ICI-mediated disruption of such as anti-LAG-3 and anti-CTLA-4, but specially with PD-1/PD- the PD-1/PD-L1 pathway has proved to be the most successful L1 inhibitors (e.g., NCT02817633, NCT03489343, NCT03680508, immunotherapeutic strategy to date.[125] NCT03099109, NCT02608268). Following ipilimumab, nivolumab and pembrolizumab are Promotion of T-Cell Activation Pathways: Although im- also ICI approved by EMA and the FDA for melanoma treat- munotherapeutic strategies involving immune checkpoint ment, alone and combined with other conventional and tar- modulators have been initially directed to inhibitory immune geted therapies. These anti-PD-1 molecules enhanced the OS checkpoints, recently special attention has been given to the mod- of melanoma patients, although response rates were higher ulation of stimulatory immune checkpoint pathways for cancer in PD-L1-positive patients.[120b] Phase III CheckMate 067 clin- treatment. The targeting of co-stimulatory immune checkpoints ical trial (NCT01844505) revealed that individuals with ad- with agonistic antibodies aims to mimic the activity of the native vanced melanoma receiving nivolumab combined with ipili- ligand, thereby activating its receptor. This is an attractive way mumab presented improved OS, PFS, and objective response of boosting anti-tumor immune responses and might be used rated, when compared with the ones receiving ipilimumab or to potentiate existing responses or act as adjuvants for cancer nivolumab alone.[108a] In a phase Ib clinical trial (KEYNOTE-029, vaccines.[145] A great diversity of co-stimulatory receptors has NCT02089685), advanced melanoma patients receiving pem- been targeted with mAb in clinical trials for cancer indications, brolizumab combined with ipilimumab presented an average namely CD28, OX40 (CD134), 4-1BB (CD137), and CD40. 61% response rate, which is comparable to that observed with CD28 is found on the surface of T lymphocytes and engages ipilimumab plus nivolumab.[135] with the CD80 (B7-1) and CD86 (B7-2) proteins. The interaction Despite their success and the initial clinical benefit observed in of CD28 with its ligands provides the secondary signal required cancer patients, patients receiving PD-1 and CTLA-4 ICI present for the stimulation of T lymphocytes, following the recognition of low frequencies of sustained responses.[136] Additionally, severe cognate MHC-antigen complex by TCR, activating downstream immune-related adverse events (irAEs) have been reported in pathways that promotes the function, proliferation, and survival many patients undergoing this therapeutic approach, which can of these immune cells.[146] CD28 is considered as the most pow- be explained by the rupture of the immune homeostasis and the erful stimulatory immune checkpoint on T lymphocytes, as it reduction of the peripheral tolerance to antigens expressed by is permanently expressed on the surface of these cells, in con- healthy tissues.[137] In this context, additional ICI, such as mAbs trast to the other co-stimulatory receptors.[123] Similarly to CD28, targeting LAG-3, TIM-3, and others, are currently under clinical CTLA-4 also targets the CD80/CD86 proteins, but with superior investigation as potential therapeutic targets for antibody therapy, affinity. The first agonist mAb targeting this receptor, TGN1412, either alone or in combination with other immunotherapeutic was initially designed to manage autoimmune diseases and B approaches. cell chronic lymphocytic leukemia.[147] Although the outstand- LAG-3 is an immune checkpoint receptor protein found on ing results obtained in both in vitro and animal studies, the first activated T and B lymphocytes, NK cells, and DC, which inter- phase I clinical trial performed in 2006 was met with unaccept- acts with MHC class II molecules.[120c] The LAG-3-MHC class able clinical toxic effects. Unexpectedly, theralizumab (TGN1412) II interaction improves Treg suppressor activity, and regulates intravenously administered to six healthy volunteers led to severe the clonal expansion CD8+ T lymphocytes.[138] LAG-3 inhibitors adverse effects by inducing the rapid release of cytokines follow- have exhibited positive results in cancer therapy in preclinical ing the activation of T cells (cytokine release syndrome), which assays, especially when combined with PD-1 ICI. In a preclini- ended up with multiple organ failure within hours.[148] Despite a cal study, the simultaneous inhibition of LAG-3 and PD-1 with temporary withdrawal of anti-CD28 agonistic antibodies, a new mAbs synergistically improved the infiltration of CD8+ TIL and phase I trial (NCT03006029) is being conducted in individuals decreased the levels of Treg cells in the TME, successfully con- with metastatic or unresectable solid malignancies. trolling tumor growth in a mouse model.[138b,139] mAbs targeting OX40 is a co-stimulatory receptor transiently induced in LAG-3 are under clinical development against different types of activated T lymphocytes, NK cells, neutrophils, and endothe- cancer, including melanoma, either isolated or combined with lial cells, and is downregulated after antigen clearance. OX40 chemotherapeutic drugs or additional ICI (e.g., NCT03250832, ligand, OX40L (CD52) is also found on activated APC and T NCT04082364, NCT01968109, NCT03459222, NCT02966548, cells.[149] The OX40-OX40L interaction provides critical co- and NCT02658981). stimulatory signals to activated T lymphocytes, inducing their TIM-3 is a checkpoint receptor found on T cells (Th1, Th17, development, trafficking, and survival, but also increases pro- Tregs, CD8 + T lymphocytes), DC, monocytes, and NK cells.[140] inflammatory cytokine secretion.[150] In addition, the activation It interacts with galectin-9 that is overexpressed in different of the OX40-OX40L signaling pathway is also able to overcome tumors.[141] Engagement of TIM-3 pathway induces the func- the tumor-infiltrating Treg function by hindering Foxp3, TGF- tional inhibition and apoptosis of T-cell,[142] but also enhances 𝛽, and IL-10 signaling.[151] Due to its higher expression on the immune-suppressive activity of Tregs.[143] The treatment of CD4+ thanonCD8+ T cells, several studies have pointed out CT26-bearing BALB/c mice with the combination anti-TIM-3 a dominant role of OX40 in CD4+ T-cell immunity.[152] Many

Adv. Therap. 2020, 2000147 2000147 (9 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com preclinical experiments have reported the promising anti-tumor way leads to the overexpression of MHC and CD80/CD86 effect triggered by the activation of OX-40 in several murine co-stimulatory molecules,[161] but also of other TNF superfamily models, including melanoma,[107a,153] glioma,[154] renal[155] and ligands, as 4-1BBL and OX40L on DC surface,[172] promoting DC colon carcinoma,[155,156] prostate,[157] ovarian,[158] urothelial[159] and T-lymphocyte stimulation, respectively. Moreover, increased and breast cancer,[160] as monotherapy or combined with many levels of critical T-cell stimulatory cytokines were also found in other agents, namely immune checkpoint modulators, and animal studies. It includes the IL-12, which is important to drive active immunotherapeutic approaches. Encouraging results the Th polarization toward Th1 adaptive immunity, as well as for were also achieved in early clinical trials using agonist anti- the induction of CD8+ lymphocytes.[161] OX40 mAb. In addition to being generally well-tolerated in Numerous preclinical studies and clinical trials have assessed cancer patients, anti-OX40 mAb have shown to promote the the efficacy of agonist CD40 antibodies. In murine models for stimulation and expansion of T-cell populations in late-stage pancreatic and bladder cancers, the administration of the ag- cancer patients[120d]. Clinical trials assessing the efficacy of onist CD40 mAb induced strong systemic CTL activation and anti-OX40 antibodies, such as GSK3174998 (NCT02528357), tumor regression.[173] Early clinical trials suggest safety for ag- INCAGN01949 (NCT03241173, NCT04387071), and PF- onistic CD40 antibodies with encouraging anti-tumor effects, 04518600 (NCT03971409, NCT03217747, NCT03092856), namely in melanoma patients, and tumor regression in patients are in progress alone or in combination with conventional with pancreatic cancer or mesothelioma. Examples of anti-CD40 anti-tumor treatments or with other immune checkpoint agents, mAb that are currently under clinical development against ad- such as pembrolizumab, nivolumab, avelumab, and ipilimumab vanced melanoma include APX005M combined with nivolumab for different advanced cancer types, including melanoma. (NCT03123783), nivolumab and cabiralizumab (NCT03502330), 4-1BB is a molecule found on activated T lymphocytes and and pembrolizumab (NCT02706353); CDX-1140 combined with NK cells, being induced by the 4-1BBL on APC.[120e,161] Activa- 6MHP- and BRAF585-614-V600E-based peptide vaccine and ad- tion of this signaling pathway promotes T lymphocyte expan- juvants (NCT04364230). sion, cytokine secretion, as well as survival,[162] while its effect on NK cells is related to improved antibody-dependent cellular cytotoxicity (ADCC).[163] Contrary to OX40, several studies have 5.2.2. Adoptive Cell Therapy pointed out a stronger outcome of 4-1BB in CD8+ T-lymphocyte immunity due to its higher expression on CD8+ than on CD4+ Besides immune checkpoint modulation, other immunothera- T cells.[164] Agonistic 4-1BB mAb led to tumor clearance and peutic approaches have been explored to promote immune effec- durable anti-tumor immunity against several cancers under pre- tor functions, and therefore boost effective anti-tumor responses clinical settings, alone and in combination with other agents.[165] against multiple cancers. Among immunotherapies, adoptive The anti-4-1BB mAb triggered antigen-specific memory T cells, cell therapy (ACT; T and NK cell therapies) has been one of which overcome tumor recurrence and metastasis, following the the most prominent strategies against melanoma, leading to removal of primary melanoma.[166] In addition, synergistic anti- durable and complete regressions, including in patients affected tumor effects were obtained in different types of cancer mouse by the disseminated form of this disease. models when agonistic anti-4-1BB were combined with other Adoptive T-cell therapy is a highly individualized treatment, antibodies, as reviewed by Chu et al..[167] Currently, numerous which requires the extraction of T lymphocytes from patients and clinical trials are exploring agonistic anti-4-1BB antibodies, such their ex vivo activation to develop anti-tumor immune cells that as utomilumab and urelumab, in cancer patients, mainly com- will mediate tumor regression.[108b,174] ACT also benefits from the bined with other ICI. Although some hepatotoxicity was in- host manipulation before cell transfer, which provides a better duced by both urelumab and utomilumab, these antibodies led microenvironment and ultimately improves the anti-tumor im- to promising results in individuals with advanced solid tumors. munity. Adoptive T-cell therapy uses either native host T lympho- Utomilumab induced a strong immune-mediated immunity in a cytes that show anti-tumor activity (TIL) or genetically-modified phase I clinical study (NCT01307267), and was well tolerated in native cells with anti-tumor TCR or chimeric antigen receptors patients affected by progressive malignancies, such as metastatic (CAR). melanoma, lymphoma, lung and kidney cancers, head and neck TIL has been showing exciting outcomes on metastatic squamous cell carcinoma.[168] Moreover, anti-4-1BB antibodies melanoma patients. These patients presented long-term and to- have proven to act synergistically with other conventional and tar- tal tumor regression using this ACT-based treatment.[175] TIL are geted therapies, such as anti-cancer drugs, radiation, ICI (espe- amixtureofmainlyCD4+ and CD8+ T cells. In vitro studies in- cially, PD-1), vaccination, cytokines, and adoptive T-cell therapy volving human cells from resected melanomas showed the abil- in defeating cancer.[169] ity of the autologous TIL in specifically recognizing autologous In contrast to the previous co-stimulatory receptors that act tumors.[176] Later, clinical trials confirmed the tumor regression primarily on T-cell activation, CD40 improves DC stimulation, on metastatic patients treated with TIL.[177] The potent immune- which in turn, directly promotes innate myeloid cells and indi- mediated effect of this ACT-based approach even led to the re- rectly fosters anti-tumor T-cell activation.[170] This makes this gression of melanoma brain metastasis.[178] pathway complementary to direct T-cell stimulation by other Other ACT techniques involving the introduction of anti- agonist antibodies targeting for instance OX40 and 4-1BB. CD40 tumor receptors into normal T cells were designed to benefit is expressed broadly in hematopoietic and non-hematopoietic a higher number of cancer patients. These new ACT-based ap- tissues, while CD40L (CD154) is found on activated platelets proaches aimed to improve T-cell specificity by integrating genes and CD4+ T lymphocytes.[171] Activation of CD40/CD40L path- encoding either conventional 𝛼𝛽 TCR or CAR. Overall, these two

Adv. Therap. 2020, 2000147 2000147 (10 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com approaches present distinct structures and recognition patterns. mune system, and is critical to immune homeostasis.[188] IL-2 TCR have 𝛼 and 𝛽 chains that recognize only MHC-restricted is mainly secreted by CD4+ T cells, however it can also be se- antigens, in contrast to CAR, which are artificial receptors that creted by CD8+ T cells, activated DC, and NK cells.[189] In the late link the antibody heavy and light chains to intracellular signaling 90s, the FDA approved the administration of high-dose IL-2 in ad- chains (CD3-𝜁, CD28, 41BB), alone or combined with signaling vanced melanoma patients. The administration of IL-2 was able moieties. CAR are not limited to MHC molecules, as they just to induce durable, complete, and curative regressions in these pa- need to be presented at the cancer cell surface. tients. However, significant toxic events were reported, including Following the results achieved with F5 TCR mouse model,[179] pulmonary edema and liver cell damage derived from vascular tumor regression was also observed in humans after the adminis- leak syndrome.[190] Currently, there are limitations to the admin- tration of autologous T cells transduced with retrovirus encoding istration of IL-2 as monotherapy, and only patients who can sup- a TCR recognizing the MART-1 antigen.[180] TCR-directed thera- port this therapy are receiving it. pies against other melanoma antigens, as MAGE or NY-ESO-1, The considerable potential of IL-2 for cancer treatment has cre- were also developed and led to objective clinical responses.[175c] ated efforts to improve its therapeutic properties by combining These antigens are expressed in varying frequencies among dif- IL-2 with other treatments, including chemotherapy, radiation, ferent tumor histology. However, toxicities in the skin, inner ear, tyrosine kinase inhibitors, vaccines, other cytokines, immune and eye tissue-resident melanocytes were commonly reported. checkpoint modulators, and ACT.[191] In addition, IL-2 mutation As for CAR therapy, despite the exciting outcomes observed and/or chemical modification have been developed.[192] Overall, against hematologic malignancies, the same therapeutic effi- efforts to improve IL-2 therapy are focused on extending cytokine cacy was not obtained in solid tumors, including melanoma.[181] half-life, simplifying the manufacturing process, and modulat- The disappointing results are attributed to the highly immuno- ing its interaction with other receptors. However, there are in- suppressive TME, the lack on CAR migration from blood ves- herent challenges to the design of novel therapeutic IL-2 agents. sels to the tumor, and to the limited identification of a tar- For example, alterations on the amino acid sequence can signifi- get antigen. Currently, strategies to overcome these limitations cantly change the protein stability, alter its pharmacokinetics pro- have been developed, including the identification of melanoma file or ability to penetrate tissues. Additionally, some modifica- antigens,[182] the use of NKT cells caring a CAR specific for tions can alter the inherent immunogenicity of the engineered melanoma antigens,[183] or even the combination of CAR with protein, which can potentially lead to severe side effects.[192b] ICI.[184] Based on its similarity to IL-2, and its strong immune- Currently, many clinical trials are ongoing to evaluate stimulating capabilities, IL-15 has also been explored as an im- T-cell therapies, both alone (NCT04217473; NCT03649529; munotherapeutic strategy for late-stage melanoma. IL-15 and NCT02989064) and in combination with other treatments IL-2 present many similarities, including their binding to the (NCT03374839; NCT03374839; NCT03068624). Despite the ex- receptor, signaling and biological functions, however their im- citing ACT outcomes, the selective targeting of tumor cells and pact on innate and adaptive immune responses is considerably antigens has been limiting the clinical success of adoptive T-cell different.[193] Numerous preclinical and clinical studies were re- therapies. ported demonstrating the ability of IL-15 in promoting anti- tumor responses.[194]The systemic administration of IL-15 in patients with metastatic melanoma stimulated NK cell activity up to 5.2.3. Cytokines tenfold above baseline levels, and increased the levels of inflammatory cytokines.[193] Cytokine therapy has been intensely explored in cancer im- The intra-tumoral administration of IL-12 as anti-tumoral munotherapy and comprehensively reviewed elsewhere.[185] Cy- agent was assessed in vivo.[195] IL-12 is secreted by APC against tokines as a monotherapy have shown several shortcomings, in- pathogens, leading to the differentiation of CD4+ T cells into cluding their mode of administration, the large quantities needed Th1 lymphocytes.[195] It promoted T and NK cell proliferation, to obtain therapeutic effects, and the consequent induction improving synergistically and/or independently the cytotoxic ef- of severe immune-related adverse events. Currently, different fect of these two cell populations.[195] Besides, IL-12-stimulated approaches, involving cytokine engineering, antibody–cytokine cells improved the secretion of several cytokines, including fusion proteins, and cytokines combination with ICI or cancer granulocyte-macrophage colony-stimulating factor (GM-CSF), vaccines, are being developed to overcome cytokine therapy limi- IFN-𝛾, and TNF-𝛼. Initial preclinical studies revealed exciting tations. The most thoroughly studied examples (e.g., IFN-𝛼, IL-2, outcomes, however IL-12 failed in the clinical trials by the induc- IL-12, IL-15, and IL-21) in melanoma are described below. tion of immune-related toxic events in melanoma patients.[196] IFN-𝛼 was the first cytokine approved by the FDA in 1995, Recently, IL-21 holds a promising immune modulatory ac- as an adjuvant therapy for patients with metastatic melanoma. tivity. It acts on the innate and adaptive immune responses, IFN-𝛼 is an immunomodulator[186] and has been used either as and their role on autoimmune diseases and cancer has been monotherapy or as part of the chemotherapy regimen.[187] Cur- assessed. IL-21 per se has almost no impact on CD8+ T rently, there are many clinic investigations using IFN-𝛼 in com- cells. However, once cells are activated, it leads to CD8+ bination with tyrosine kinase inhibitors, immune checkpoint T-cell expansion and differentiation into a CTL or memory- inhibitors (NCT04093323, NCT02339324), and DC vaccination like phenotypes.[197] IL-21 supported NK-cell survival, includ- (NCT01973322) that are ongoing. ing their maturation and proliferation.[198] Besides, IL-21 is The most prominent cytokine in cancer immunotherapy is a key cytokine to induce B-cell differentiation and estab- the IL-2, which simultaneously stimulate and regulate the im- lish memory phenotype, thus promoting prolonged humoral

Adv. Therap. 2020, 2000147 2000147 (11 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com responses.[199] Preclinical[200] and clinical studies (NCT00514085, Evidences that arginase overexpression by CAF and NCT00336986, NCT00389285, NCT01489059, NCT00095108, MDSC predisposes a poor OS in cancer patients have been NCT00601861, and NCT01152788) involving the use of IL-21 as reported.[62,212] Recent studies revealed the role of arginase on anti-tumoral agent to treat melanoma patients are reported. melanoma-associated fibroblasts (MAF) and how these sup- Overall, cytokines (IL-2 and IFN-𝛼) approved as monother- press CD8+ T cells.[213] Therefore, alternative approaches have apy led to disappointing results. Currently, they are used as im- included the use of arginase inhibitors to promote immune munotherapeutic agents in combinatorial regimens. Combina- effector function against melanoma by defeating MAF and tions of cytokines with ICI and ACT therapies may improve the MDSC suppressor activity. outcome of these therapies and restore cytokine role in cancer immunotherapy. 6. Delivery Approaches Targeting Immune Cells as Melanoma Immunotherapies 5.2.4. Additional Therapies to Enhance Innate Immune Function DC constitute an attractive immunotherapeutic target to repro- Other immunotherapeutic strategies involving NK cells and gram the immune system against cancer due to their ability to MSDC hold promise as “off-the-shelf” therapies against take up and present TAA by different mechanisms, such as cross- melanoma,[201] and have been explored as an alternative to ACT. presentation, cross-dressing, antigen transfer, and MHC class II- NK cells rapidly sense and destroy virally infected cells and tu- restricted presentation. Moreover, the ability of DC to migrate be- mor cells by unique mechanisms using a set of stimulatory and tween non-lymphoid and lymphoid tissues, and their role on the inhibitory receptors,[202] which in addition to their limited reac- modulation of cytokines and chemokines secretion are also rele- tivity against healthy tissues, encourage their use as anticancer vant for anti-tumor immunity. agents. In vitro experiments and animal models unequivocally Although no DC-based cancer vaccine has received demonstrate the ability of NK cells as anti-tumoral agents in mul- FDA approval for melanoma, there are many undergoing tiple cancers.[203] preclinical[107a,214] and clinical studies (Table 2).[215] Among The first studies that reported the NK-mediated cytotoxicity DC vaccines, the most explored approach involves the reinfu- in melanoma patients involved the infusion of different propor- sion of ex vivo differentiated DC. Briefly, CD14+ monocytes tions of NK cell predecessors, the lymphokine-activated killer or CD34+ DC precursors are isolated from a person, loaded cells (LAK).[204] The anti-tumor activity of LAK cells resulted from with TAA and cultured in the presence of cytokines to induce the contribution of NK cells combined with the administration their differentiation and maturation. Matured TAA-loaded DC of IL-2, which improved their cytotoxic effect.[205] In general, the are subsequently administered back into the patient using studies with unmodified autologous NK cells have yielded disap- different routes of administration (intravenous, subcutaneous, pointing results.[206] Therefore, currently, different strategies are intradermal, intra-tumoral, or intra-lymphatic),[216] being able exploring engineered NK cells, mAb antagonizing KIR inhibitory to cross-prime T cells and produce anti-tumoral cytokines (e.g., receptors, and combinations of NK cells with infused anti-tumor IL-12).[217] However, ex vivo DC-based vaccines led to suboptimal T cells. Altogether, these approaches will provide knowledge fun- efficacy and consequently to low response rates. damental to advance the next generation of NK cell therapies and The basis for their failure can be explained by the generation of boost the development of new and effective immunotherapeutic DC transcriptionally and phenotypically different from the natu- approaches. rally occurring ones.[218] It has been reported the insufficient anti- MDSC have hampered immunotherapy outcomes in many gen presentation and related reduced T-cell priming, limited DC cancer patients, particularly in melanoma. Generally, high fre- migratory capacity and compromised cytokine release, in addi- quencies of MDSC led to decreased OS, PFS, and increased risk tion to their inability to overcome the immunosuppressive tumor for patients.[207] Besides, the presence of MDSC have impaired microenvironment.[219] In addition, ex vivo cell-based vaccines the efficacy of immune checkpoint blockade as ipilimumab (anti- constitute a tremendous complex, time-consuming, and costly CTLA-4), pembrolizumab (anti-PD-1), and nivolumab (anti-PD- strategy. 1).[64b,207] Several studies explore the combination of ex vivo DC- Currently, different approaches to overcome MDSC immuno- based cancer vaccines with other approaches to overcome suppressive activities and thus enhance anti-tumor immunity their suboptimal efficacy. Examples include their administra- have been explored. For example, certain clinical chemotherapeu- tion with immune checkpoint blockade (NCT01302496 and tic agents reduced the MDSC levels, even when administered at NCT03092453), or colony-stimulating factor-1 receptor (CSF1R) low doses.[208] A small clinical trial combining checkpoint block- inhibitors to induce TAM depletion, which have been shown to ade with vitamin-A derivative all-trans retinoic acid (ATRA) treat- directly suppress DC function.[220] Other studies have involved ment is ongoing for stage IV metastatic melanoma.[209] Several the combination of DC vaccines with certain chemotherapies approaches using modulators of the inflammatory pathways have (e.g., NCT02285413) that induce the release of TAA via im- been considered due their role on the induction and mainte- munogenic cell death, thus improving their availability to be nance of MDSC function.[210] However, the recent failure of IDO internalized and presented by APC. inhibitors in phase III showed that targeting MDSC induction Alternative approaches are being developed to deliver cancer may be more effective than targeting their suppressive function antigens and adjuvants to DC in vivo. Thus, peripheral lymph and products.[211] Nevertheless, strategies adopted to overcome nodes emerge as the key organ to target, as DC and T cells in- MDSC infiltration have been disappointing so far. teract to trigger adaptive immunity against tumors.[221] However,

Figure 3. Examples of nanoparticle-based carriers. Polymeric nanoparticles are spherical entities commonly prepared using poly-lactic acid (PLA) and/or poly(l-lactic acid-co-glycolic acid) (PLGA)-based polymers. Polymeric micelles are self-assembling systems using amphiphilic block copolymers as main components. Dendrimers are hyperbranched systems presenting a main core, branching monomers, and functionalized peripheral groups. Hydrogel nanoparticles are polymeric networks that can incorporate large quantities of water or biological fluids. Liposomes are self-assembled bilayer membranes with an aqueous nucleus capable of incorporating hydrophilic molecules. These are mainly composed by phospholipids and cholesterol. Solid lipid nanoparticles are lipid emulsions where the liquid lipid has been substituted by a solid lipid. Phospholipid micelles are prepared by self-assembly surfactants formed by phospholipids. Metal and inorganic nanoparticles are submicron scale entities made of metals (e.g., gold, platinum, silver),and inorganic compounds (e.g., magnetite, silicon dioxide). Adapted under the term of the Creative Commons CC BY 4.0 license.[222] Copyright 2014, The Authors, published by Frontiers. the low antigen intracellular access together with antigen degra- lipid, and metal or inorganic nanocarriers (Figure 3). These sys- dation upon cellular entry has hindered their full potential. tems offer off-the-shelf product properties, that can be manufac- tured at industrial scale with low costs. One of the main advantages of nanotechnology is its versatility 6.1. Nanoparticle-Based Approaches for Dendritic Cell Trageting which led to the development of a wide variety of precise mul- and Modulation tifunctional nanoparticles specifically designed for their final application. For instance, nanoparticles were designed to protect Delivery systems based on particles can vary from nano to mi- biomolecules susceptible of being rapidly degraded in vivo, like croparticles, including virus-like particles or liposomes. Nanopar- peptides or nucleic acids. These bioactive molecules can be ticles are the most popular particulate-based platform, being used encapsulated in nanoparticles, embedded, or still conjugated or for a variety of applications, including as delivery systems for can- adsorbed onto nanocarrier surface. Moreover, the characteristics cer and gene therapy, as well as analytic approaches for theranos- of nanoparticles can also be optimized aiming at an adjusted re- tics, being used as biosensors or biomarkers for cell imaging. A lease profile of the active agent,[222] and to enhance the cytosolic wide variety of nanoparticle-based systems were developed over delivery of valuable bioactive agents implicated in the modulation the years, taking advantage of distinct core materials with specific of gene expression, as small interfering ribonucleic acid (siRNA) physicochemical characteristics. Therefore, nanoparticles can be and microRNA (miRNA), by enabling endo-lysosomal escape divided based on their main compositions, such as polymeric, of payloads. In addition to improve the bioavailability of these

Figure 4. Nanoparticulate cancer vaccines. Nanoparticle-based cancer vaccines can target dendritic cells (DC) in vivo, thus triggering the activation and maturation of these antigen-presenting cells (APC). The simultaneous transport of tumor-associated antigens (TAA) and adjuvants to a single DC guarantees their coordinated induction. TAA are presented to CD8+ and CD4+ naïve T cells through major histocompatibility complex (MHC) class I and class II molecules, respectively that recognize the antigens through T cell receptors (TCR). Stimulated CD8+ T cells differentiate into cytotoxic T lymphocytes (CTL), which trigger the tumor elimination, and memory T cells, that prevent metastasis. CD4+ T cells will differentiate into Th1 cells, which stimulate CTL function, as well as cells of the innate immune system, such as macrophages, NK, and granulocytes that synergistically have an anti-tumor activity. associated payloads, nanosystems can be engineered to guide teria and cells.[225] While small-sized particles are preferentially their delivery to a specific target, and/or modulate their function internalized by DC, larger particles are specially taken up by or frequency toward the regulation of the immune system macrophages. DC and macrophages phagocyte preferentially par- response. ticles less than 10 and 30 µm in size, respectively. However, the DC-targeted vaccine nano-platforms are being explored by of- ideal size to target lymph nodes and APC remain a utopian issue, fering the possibility for co-delivering TAA and immune modu- being a matter of debate. lators to a single cell in its natural environment (Figure 4).[115] Lymph node targeting can be achieved by two distinct path- This is fundamental to enhance antigen recognition and incor- ways that depend on particle size. At the one hand, smaller par- poration by cells, processing, and presentation, thus promoting ticles are more effective carriers, due to their ability to cross a successful T-lymphocyte priming and expansion.[107a,223] biological barriers and thereby reach the target sites. Usually, The internalization mechanism of nanoparticles by targeted smaller nanocarriers (<100 nm) reach directly the lymphoid or- immune cells will be deeply affected by numerous nanopar- gans, within 2–3 h post-administration, by traveling throughout ticle physicochemical characteristics, including hydrophobicity, the lymphatic drainage and interstitial flow, further interacting biodegradability, average mean diameter, morphology, and sur- with lymph node-resident DC.[226] On the other hand, larger car- face charge. Thus, the control of these essential parameters while riers (>500 nm) require a pathway dependent on cell transport, as designing these immunotherapeutic tools is crucial to direct they remain imprisoned in the skin until being phagocytosed by nanotechnology-based vaccines to DC-rich areas, as lymph nodes monocytes, as well as dermal and epidermal immature DC, be- and skin. ing further drained within 18 h to the lymph nodes.[227] Despite the faster lymph node targeting, small-sized nanoparticles can bypass the lymph nodes. Thus, it is important to reach a balanced 6.1.1. Size between size and retention time within lymph nodes.[228] Parti- cles ranging from 100–500 nm can trigger more extensive anti- The particulate antigen carriers have been shown to promote gen deliveries through both peripheral and lymph node-resident a broad antigen presentation by APC and to prime T func- immature DC by trafficking throughout both dependent and tion at higher extent when compared to soluble antigens.[224] independent cell transport-based pathways, respectively.[225,226] APC process antigens within a nanometer size range, as viral Nanocarriers ranging from 10–200 nm can target immune cells pathogens (20–100 nm), but also at the micrometer size, as bac- within TME by benefiting from of the enhanced permeability and

Adv. Therap. 2020, 2000147 2000147 (14 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com retention (EPR) effect, which allows nanoparticle extravasation to avoid the aggregation of hydrophobic particles and their recog- through the enlarged gap junctions or pores formed between en- nition as external agents by cells of the mononuclear phagocyte dothelial cells and promoted by the disorganized, irregular, and system.[239] In fact, this recognition process resultant from the leaky tumor vasculature.[229] Due to the insignificant lymphatic binding of opsonins is presented as one of the major barriers for drainage and high interstitial pressure on tumor core, nanoparti- nanoparticle-controlled delivery. Thus, the “PEGylation” process cles are highly retained and remain for long periods within tumor improves in vivo half-life circulation of hydrophobic and surface site, during which the therapeutic agents are released.[230] charged particles in the bloodstream.[239] The size of the nano-based systems also dictate their cellular internalization mechanisms and intracellular pathways.[231] Each endocytic route is indeed determined according to the size scale 6.1.3. Shape of the internalized nanoparticles. At the one hand, internalized particles ranging from 20 to 200 nm commonly follow the clas- Besides size, carrier shape is another physicochemical prop- sic endocytosis mediated by receptors, via clathrin- (<200 nm) erty with a relevant impact on cellular toxicity, internalization, or caveolae-endocytic vesicles (50–100 nm), thus endorsing the and biodistribution. Venkataraman et al.[240] supply a complete activation of cytotoxic immune response based on CTL and Th1 overview of the impact of differently shaped nanocarriers in the cells. On the other hand, the internalization of particles larger context of relevant biological events, such as biodistribution, as than 500 nm is mediated by phagocytosis or macropinocytosis, well as cellular uptake and viability. Accordingly, spherical par- promoting a preferential humoral immunity with the activation ticles have been shown to have improved blood circulation life- of Th2 cells and high antibody production.[231,232] time and organ distribution, as well as lower premature clear- Although size-dependent, subcutaneous, and intradermal are ance compared to non-spherical counterparts. Although it is not the most common administration routes to direct nanocarriers consensual, higher internalization levels have been reported for to circulating DC, while the intraperitoneal and intranodal in- spherical particles, when compared to carriers presenting non- jections mostly target DC population resident within the lymph spherical shapes. This can be explained by the lower average nodes. Less dependent on size, the intravenous administration is curvature radius of spherical particles when interacting with regularly followed to target DC in the blood and spleen.[230] cells.[241]

6.1.2. Charge 6.2. Major Targets at Immune Cell Surface to Potentiate Nano-Based Melanoma Immunotherapies Surface charge of particles and their hydrophobicity play a central role in their internalization by APC and in the nature of Pattern recognition receptors (PRR) are useful sites for immune the induced immune response.[233] An improved internaliza- cell targeting. PRR are cell surface receptors on innate immune tion of positively charged particles has been shown when com- cells that recognize danger signals as pathogen-associated molec- pared to negatively or neutrally charged particles as a result ular patterns (PAMP), such as nucleic acids, proteins, carbohy- of the establishment of ionic interactions with negative cel- drates, lipids, and lipoproteins. PRR are directly involved in trig- lular membranes.[233,234] Moreover, positively charged particles gering an ideal processing and presentation of antigens. Besides, showed the highest ability to escape from the endo-lysosomal PRR are also responsible for the expression of co-stimulatory route after being internalized.[222] Nevertheless, the interaction markers and the release of several pro-inflammatory cytokines, of positively charged particles with the lipid bilayer induces in leading to strong innate and adaptive immunities.[242] The de- situ fluidity changes on cellular membranes, leading to the desta- sign of nanostructured platforms based on the attachment of bilization of normal cell metabolism and toxicity.[235] It is also PRR ligands at their surface constitute an excellent approach well known that particles tend to interact with the so-called “pro- for vaccines design. It allows a specific immune cell targeting tein corona” in a biological fluid, being more susceptible to clear- and the modulation of nanocarrier internalization, by allowing ance from the bloodstream by the reticuloendothelial system.[236] the delivery of combinations of antigen and adjuvants within These are plasma proteins, mainly opsonins (e.g., C3, C4, and C5 the same platform.[115] PRR include the transmembrane TLR, C- complement proteins) and immunoglobulins that aggregate with type lectin receptors (CLR), mannose and scavenger receptors), cationic particles, particularly when these are administered intra- as well as other cytoplasmic proteins, as the leucine-rich repeat- venously, which can lead to complement activation, hypersensi- containing receptors (NLR), nucleotide-binding oligomerization tivity reactions, and overall toxicity.[235] Therefore, anionic or neu- domain (NOD), AIM2 (absent in melanoma 2)-like receptors, and tral particles are desirable by repulsing proteins of the interstitial the retinoic acid-inducible gene (RIG)-I like receptors (RLR).[243] milieu, thus exhibiting enhanced biocompatibility, and improved TLR are the most explored and best described class of PRR ability to reach the lymph nodes.[237] Nevertheless, anionic parti- mainly expressed by APC, such as DC, being also found on cles can also destabilize the normal cell membrane metabolism 𝛼𝛽T cells, Treg, 𝛾𝛿T cells, and NKT cells.[244] CLR, receptor fam- by promoting local gelation toxic events.[235] Moreover, charged ily particularly expressed by APC and involved in antigen in- particles present lower circulation times compared to the neutral ternalization, is well recognized as being able to bind to carbo- ones.[238] Particle surface coating with hydrophilic moieties, such hydrates. For example, ligands that can direct nanoparticles to as the non-ionic polymer poly(ethylene glycol) (PEG), has been CLR, including the mannose receptor CD206, DC-SIGN, and used not only to stabilize the formulation, but especially to cover DEC-205, represent an interesting strategy. DEC-205-labeled particle charge and enhance their hydrophilic character, in order nanoparticles, encapsulating a MART-127–35 melanoma peptide,

Adv. Therap. 2020, 2000147 2000147 (15 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com were internalized by human monocyte-derived DC and more suc- loaded M2NP increased the IFN-𝛾 secretion and CD8+ T-cell den- cessfully cross-presented to CD8+ T lymphocytes, in contrast to sity within TME, which contributed to a lower tumor growth rate the isotype control-coated nanoparticle.[245] Unger et al.[246] re- and increased survival of melanoma-bearing mice.[251] PLGA- ported an enhanced antigen presentation and priming of MART- based nanoparticle entrapping a TGF-𝛽 signaling inhibitor were 1-specific CD8+ T lymphocytes triggered by DC-SIGN-binding also synthesized to target T cells in circulation and within TME by X 𝛼 glycan lacto-N-fucopentose III (Le) -coated liposome-based vac- conjugating anti-CD8a F(ab’)2 and PD-1 antibody fragments to cine containing the MART-1 melanoma peptide, in the presence their surfaces. Exploiting this strategy, Schmid et al.[252] observed of lipopolysaccharide (LPS), compared to antigens in solution or a specific binding to CD8+ T cells in vitro and PD-1-expressing uncoated liposomes. Mannose-receptor targeting in APC is also CD8+ T cells in B16-bearing mice treated with TGF-𝛽 signal- associated to an enhanced internalization, antigen processing ing inhibitor-loaded PD-1-targeting nanoparticles compared to and presentation, inducing a more extensive immune response. these agents in solution. Kwong et al.[253] also reported the cure of Moreover, mannose also acts as an immune modulator by driv- most of the established primary B16F10 melanoma tumors when ing the antigen cross-presentation to CD8+ T lymphocytes.[247] treated intratumorally with PEGylated liposome-coupled IL-2Fc Several studies have indicated that antigen-associated mannosy- fusion protein + 𝛼CD137, a co-stimulatory marker overexpressed lated nanocarriers are effective in inducing anti-tumor immune on activated T cells. It triggered systemic anti-tumor immunity responses against tumors, including melanoma, compared to through the stimulation of T cells without any lethal off-targeted non-functionalized nanoparticle, by targeting mannose receptors effects triggered by the systemic effect of soluble agents. on APC surface. For instance, mannose-functionalized nanovaccines, incorporating MHC class I or MHC class II-restricted 6.3. General Strategies Used for the Surface Functionalization Melan-A peptides and the TLR ligands CpG and Poly(I:C), in- of Nanosystems duced strong Th1 immunity and the highest cancer progression delay in murine-bearing B16F10 tumors, under prophy- The active targeting is based on the design of nanoparticle- lactic and therapeutic settings.[223] Conniot et al.[107a] also show conjugated targeting moieties that will bind to specific receptors that the combination of DC-targeted mannosylated nanovac- overexpressed by targeted immune cells, thus inducing receptor- cines with anti-PD-1/anti-OX40 antibodies, and a Bruton Tyro- mediated endocytosis.[254] Different targeting moieties, such as sine Kinase (BTK) inhibitor (ibrutinib), potentiated an effector antibody fragments and whole antibodies, peptides, proteins, and T-cell stimulation, enabling a remarkable melanoma remission carbohydrates, have been used to promote a more effective and and prolonged survival in prophylactic and therapeutic combi- selective targeting of immune cells, as addressed above. nation regimens. Nanoparticle surface conjugation with anti- The surface functionalization of nanosystems has also been CD11c and anti-CD40 antibodies, have also been proven to en- used to modulate the nonspecific biodistribution and increase hance the in vivo DC targeted delivery and vaccine efficacy. A nanocarrier lifetime in vivo. The “PEGylation” process has been versatile nanovaccine based on antibody 𝛼CD11c-modified lipo- extensively described for this purpose. This strategy relies on somes developed for the DC-targeted delivery of the adjuvant the conjugation, grafting, or adsorption of hydrophilic PEG monophosphoryl lipid A (MPLA) and melanoma antigen Trp-2, molecules onto nanoparticle surface. The linkage of specific lig- revealed improved anti-tumor immune responses that inhib- ands to the surface of the nanoparticle by using PEG-grafted poly- ited tumor development in mice subsequently challenged with mers has been applied for the active targeting of particular tissues B16F10 cells, in addition to overcome lung metastasis, com- and intracellular compartments.[255] The use of PEG derivates pared to non-targeted nanovaccines.[248] Indeed, Rosalia et al.[249] (e.g., 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)- concluded that 𝛼CD40-mAb-labeled biodegradable poly(lactic-co- PEG and polycaprolactone (PCL)-PEG) containing functional tar- glycolic acid) (PLGA) nanoparticle enabled an efficient and selec- geting moieties (e.g., mannose) attached through the native ter- tive co-delivery of ovalbumin and Pam3CSK4/Poly(I:C) adjuvants + minal groups of PEG chains have been reported as suitable to to DC in vivo post-subcutaneous injection, which drove CD8 T- develop active-targeted carriers.[223,256] cell responses toward an improved tumor control and prolonged survival in B16-OVA-bearing mice. Moreover, Qian et al.[250] developed the lipid-based nanovaccine 𝛼-peptide-gp100-NP-CpG, 6.3.1. Computational Chemistry Toll for Site-Orientation of Ligands designed to target mature DC in the lymph nodes via the scavenger receptor class B1 (SR-B1) route, with potent therapeutic The process by which the nanocarriers overcome and interact effect against B16F10-bearing mice. The targets expressed at DC with several biological barriers at different sites (e.g., blood- surface are also commonly found on lymphocytes, macrophages, stream, tumor, cell surface, and subcellular sites) to achieve their and monocytes. In addition to APC, nanoparticles can also be biological target, is highly complex and can be affected by many directed to other immune cells within the TME. Dual-targeting variables, including platform type, nanocarrier physical parame- nanoparticles, coated with SR-B1 targeting 𝛼-peptide conjugated ters, and chemical surface.[257] to the M2-like TAM binding peptide (M2NP), were developed to Computational modeling has been a valuable tool in the drug specifically deliver a siRNA against the colony stimulating factor1 discovery process. In the last decades, the computational power receptor (anti-CSF1R), to deplete M2-like TAM. These targeted and software have improved the predictability and accuracy of siRNA-loaded M2NP could eliminate 52% of M2-like TAM and the real biological and chemical interactions, and ultimately the downregulate the expression of immune suppressive cytokines delivery processes. Importantly, computational simulations are (IL-10 and TGF-𝛽), as well as PD-1 and TIM-3 exhaustion mark- also valuable to explain and predict experiments, in addition to ers on tumor infiltrating CD8+ T cells. Moreover, these siRNA- assess the validation of the experimental results.

Figure 5. Surface modification of nanoparticles. These carriers can be functionalized with a variety of molecules to attain distinct goals. Selectively targeting can be attained through the functionalization of tumor markers or antibodies. Cell penetrating peptides can enhance nanoparticle uptake. Molecules like polyethylene glycol (PEG) or D-𝛼-Tocopherol polyethylene glycol 1000 succinate (TPGS) offer stealth characteristics to nano-based systems, preventing their recognition by phagocytic cells and enhancing their half-life. Imaging molecules (fluorescent probes or contrast agents), offer applicability of nanocarriers to theranostic or in vivo imaging. Immunogenic polymers (e.g., chitosan, poloxamers) or pathogen-associated molecular patterns (PAMP) (carbohydrates, lipids, nucleic acids) can be used as functionalization agents. Adapted under the term of the Creative Commons CC BY 4.0 license.[222] Copyright 2014, The Authors, published by Frontiers.

In detail, computational modeling can be used to screen the tion of ligand-decorated nanocarriers[107a,223,250,251] or afterward best nanosystem by providing optimized solutions in terms of through their direct coupling at nanocarrier surface.[245,246,248,249] geometrical and physicochemical properties.[257] Besides the con- The size of the ligand that will be attached to nanocarrier sur- tinuum modeling that is used to study nanoparticle transport face dictates the most suitable strategy. Bulky ligands (e.g., anti- through the vascular network, the molecular dynamics is com- bodies, antibody fragments, proteins, or polypeptides) are usu- monly used to simulate nanocarrier-cell interactions by clarify- ally linked to the surface of preformed nanoparticles to avoid the ing the internalization process and the impact of nanoparticle destabilization of the hydrophilic-lipophilic balance (HLB), and physicochemical features (size, shape, charge).[257] In the field the denaturation of their secondary structure in the presence of of molecular modeling, a molecular docking approach intents to organic solvents used during nanosystem formulation. In this sit- elucidate the preferred interaction among the ligand and the re- uation, the major drawbacks are mainly related with nanocarrier ceptor at the atomic level through i) the prediction of the pre- purification methods, including dialysis, filtration, or centrifuga- ferred ligand conformation, position, and orientation toward the tion, which can induce degradation or modification of these car- target binding site, and ii) the assessment of the strength of asso- riers. In addition, despite the use of advanced methods (nuclear ciation or binding affinity between the ligand and the receptor, us- magnetic resonance, surface plasmon resonance, X-ray photo- ing scoring functions.[258] Besides, the Monte Carlo approach was electron spectroscopy) it is generally hard to distinguish among reported to evaluate the highly selective binding of ligand-grafted the covalently bound or simple adsorbed ligands onto nanoparti- nanoparticle to specific receptors on both tumor and healthy cle surface.[261] cells.[259] Monte Carlo simulations were also used to prove that Small organic or peptide-based molecules can be either the nanosystem surface covered by the anti-ICAM-1 antibody is attached before or after nanoparticle production.[261] The chem- crucial for their binding to the mice pulmonary endothelium.[260] ical characterization of ligand conjugate before nanocarrier development allows the control of the ligand density at nanoparticle surface. However, the binding of target ligands to the raw 6.3.2. Bioconjugation Chemistry Principles for Material material can alter the nanocarrier physicochemical properties, Functionalization thus further requiring the optimization of the formulation conditions.[261] As previously reviewed, the surface or peripheral modification of Different strategies have been established to conjugate bioac- nanocarriers with bioactive ligands is crucial for immune cell tar- tive moieties to the nanocarrier surface. The carbodiimide chem- geting (Figure 5). Different strategies can be used to adsorb or at- istry is one of the most explored methods for biomolecule tach ligand molecules onto NP surface, either prior to the produc- functionalization. In this synthetic methodology, the coupling

Adv. Therap. 2020, 2000147 2000147 (17 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com of carboxylic acid derivatives to primary amines occurs via tides. Indeed, phage display technology had been successfully ap- N-hydroxysuccinimide (NHS) ester.[222] This strategy was em- plied to isolate peptides specifically targeting surface antigens at ployed after nanoparticle formulation following the reaction be- tumor cell surface or tumor-associated vasculature in mice.[266] tween the previously activated carboxyl-PLGA nanocarrier using Additionally, phage display revolutionized the discovery and en- 1-ethyl-3-[3- dimethylaminopropyl] carbodiimide hydrochloride gineering of antibodies, and human mAb are now produced with- (EDC)/NHS and 𝛼CD40-mAb[249] or the natural polymannose out immunization. molecule mannan.[262] It was also used prior to the production of Currently, there are a few phage library-derived antibodies the ligand-decorated nanocarrier through the synthesis of PLGA- in clinical use. Adalimumab was the first phage display-derived mannose, by conjugating the activated terminal PLGA carboxylic mAb approved for therapy. It is a human IgG1 antibody direct acid groups with the amine group of the mannosamine.[107a] to TNF-𝛼, for the treatment of inflammatory diseases.[267] Since The Michael addition is the second most used coupling strat- then, several antibodies were developed using this technique egy, being based on the thiol-maleimide chemistry. For instance, for different purposes, including cancer and autoimmune dis- thiol-modified targeting ligands (e.g., glycans or antibodies) are eases, targeting distinct growth and angiogenic factors, such as coupled directly to nanocarriers developed using maleimide- EGFR,[268] IGF-1R,[269] TGF-𝛽,[270] and VEGF-A.[271] TNF-related modified polymers or lipids via a thiol-ene reaction.[246,252,253,263] apoptosis inducing ligand (TRAIL), a TNF superfamily mem- However, some proteins present vestigial levels or poorly avail- brane, is also an appealing target for cancer therapy and numer- able native thiols. An alternative to overwhelm this thiol ab- ous phage-derived human mAb have been also developed.[272] sence is the reduction of existing disulfide bonds or the use Ribosome display has been investigated for the design of of heterobifunctional cross-linking molecules.[261,263] This cou- therapeutic and diagnostic tools for different applications, such pling approach allows an improved bioligand orientation man- as cancer, allergic disorders, and infectious, autoimmune, and agement without undesirable cross-linking or side reactions, and metabolic diseases.[273] This technique has been successfully the preservation of the biochemical protein properties.[261] The used for the selection and evolution of peptides, single-chain an- strongest non-covalent biological interaction between function- tibodies, enzymes, and ligand-binding proteins. Ribose display ally active avidin, introduced in the nanoparticle surface during can be combined with high-throughput protein microarrays to the formulation process, and biotinylated proteins that bind to assess protein-protein interactions.[273b] Moreover, massive pep- avidin with high affinity, has also been reported.[245] Moreover, tides can be screened simultaneously, leading the way for low- synthesis of both biotinylated and (strep)avidin-antibodies, has cost and high-throughput epitope screening, especially impor- also been described.[261] The “click chemistry” based on copper- tant for cancer immunotherapy.[274] catalyzed ligation is a method able to regulate the degree of functionalization at nanocarrier surface. The cycloaddition reaction is a highly efficient approach to achieve both selectivity and con- 6.4. Major Immune Cell Intracellular Targets for Optimal version. Moreover, this simple workup established in mild con- Immunotherapy ditions do not present byproduct. The main drawback of this reaction is related to the difficult removal of Cu-based catalyst Most bioactive molecules (e.g., proteins, enzymes, antibodies, or used during the reaction, which can be achieved with cooper lig- oligonucleotides) require their delivery within the intracellular ands or organic scavengers.[261] compartments to be effective.[229,275] The major intracellular targets for immunotherapeutic purposes include the cytosol, the endo/lysosomes, and the endoplasmic reticulum. However, the 6.3.3. Technologies for the Selection of Surface Ligands capacity of those therapeutic agents to target specifically their local of action within the cytoplasm constitutes a real challenge, as The selection of the right ligand for a specific cell target is cru- they need to cross the cell and organelle membranes.[229,275] cial for the efficacy of nano-based immunotherapies. In this man- Nanotechnology-based systems have been designed to deliver ner, display techniques are useful tools to select and isolate lig- therapeutics in a controlled manner and site-specifically to sub- ands with higher affinity to a specific target. These techniques cellular targets, to improve their therapeutic efficacy and re- can be divided in cell-based and cell-free techniques. Cell-based duce related adverse off-target effects. Beyond the previously display techniques use living cells directly or indirectly. Phage mentioned advantages of nanocarriers regarding their ability display and ribosome display technologies are examples of in- to entrap and protect distinct active molecules, thus improv- direct cell-based display and cell-free display techniques, respec- ing their circulation lifetime and bioavailability; the surface of tively. Despite the differences, all the display techniques allow the nanocarriers can be modified with targeting moieties to specif- association between the binding molecule with their encoding ically bind to target cell surface receptors.[229,276] In this case, sequence.[264] nanoparticles are taken up via receptor-mediated endocytosis Phase display technology is a methodology for screening and to release bioactive molecules at their specific subcellular des- identifying specific ligands for molecular targets. It has been tination within cell cytoplasm. Nanoparticle transport across used in different fields, including drug discovery and cancer treat- cell membrane can occur through different endocytic pathways, ment. This methodology can isolate high-affinity ligands for di- such as caveolae-or clathrin-mediated endocytosis, macropinocy- verse substrates, including peptides, proteins, and antibodies.[265] tosis, and phagocytosis. However, the majority of nanocarriers Despite other methods like yeast surface display or ribosomal follow the endo-lysosomal pathway, in which nanoparticles are display, phage display technology is the most applied technique entrapped within endosomes and subsequently degraded in the for the secretion of human antibodies and cancer-associated pep- lysosomes, and therefore do not release their content in the

Adv. Therap. 2020, 2000147 2000147 (18 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com cytosolic compartment.[229,275] Since the majority of formed en- poration of short peptide domain of 20–30 amino acids, named dosomes do not degrades spontaneously, the endosomal escape fusogenic peptides, within the nanocarriers. Peptides with fu- must be induced to promote a successful and extensive cytoso- sogenic properties, such as GALA/KALA sequence-based pep- lic release of therapeutics favoring their cytoplasmatic bioavail- tides and pH-dependent fusogenic peptide diINF-7 derived from ability and their successful therapeutic index in vivo.[229,275] influenza virus, have been reported to promote the endosomal Invasive (physical) and non-invasive (chemical) methods have escape for the cytoplasmic release of bioactive agents.[275] Accord- been explored to disrupt the endosomal membrane. The non- ingly, GALA-functionalized streptavidin-lactadherin-modified ex- invasive chemical alternatives have been preferred, since phys- osomes were engineered from transfected murine melanoma ical approaches, including sonoporation, microinjection, elec- B16BL6 cells to efficiently deliver endogenous TAA to DC cy- troporation, and biolistic transfections with gene guns, lead tosol, as an active immunotherapy.[279] GALA-exosomes were to cell membrane damage.[229,275,276] The chemical strategies able to promote the intracellular trafficking of tumor antigens use polymeric-, lipidic-, peptide-, inorganic-, and metallic-based and enhance the tumor antigen presentation through MHC class nanocarriers composed by molecules with high intrinsic capac- I molecules toward a cytotoxic immune phenotype desirable for ity to trigger membrane translocation, such as fusogenic lipids, an effective anti-tumor response.[279] cell-penetrating peptides (CPP), and pH-sensitive/independent In addition to fusogenic lipids or peptides, the use of CPP as fusogenic peptides, resulting in an improved cytosolic cargo well as cationic nanocarriers have been explored in melanoma delivery.[275] However, to avoid nanoparticle accumulation at par- immunotherapy. As reviewed by Silva et al.,[275] CPP are 20 ticular subcellular compartments without cargo release, targeted amino acid short cationic peptides, enriched of lysine and argi- delivery to the organelle of interest must be promoted to attain nine amino acids, also known as transduction domains.[275] an extensive biological response.[275] CPP, including the transactivating transcriptional activator (TAT) In the following section, several nanosystems and targeting peptide, polyarginine, as well as penetrating, transporting, and strategies designed in the last years for the intracellular delivery mitogen-activated protein, have been used to deliver different of bioactive molecules will be discussed. macromolecules both in vitro and in vivo, such as proteins, nucleic acids, drugs, and nanocarriers. While CPP conjugated to small agents are promptly taken up by cells via energy- 6.4.1. Cytoplasmic Compartment independent transduction through their electrostatic interaction with anionic cell membrane, CPP bound to bulky agents The efficacy of therapeutic agents relies on the targeting of spe- are internalized via macropinocytosis, or clathrin- and caveolin- cific receptors, either present in the cytosol or in an intracel- mediated endocytosis.[275] Accordingly, Nakamura et al.[280] in- lular organelle accessible through the cytosolic transport. Fu- corporated 𝛼-galactosylceramide into stearylated octaarginine- sogenic mechanisms have been explored for the cytosolic de- modified liposomes (R8-Lip) to act as a delivery system to NKT livery of different cargo molecules by nanocarriers containing cells for 𝛼GC therapy purposes. The size control of 𝛼GC/ R8- endosomolytic agents able to fuse and disrupt the endosomal Lip promoted an increased 𝛼GC presentation on CD1d by APC membrane. and NKT expansion, resulting in a positive therapeutic out- The use of pH-sensitive or fusogenic lipids for nanocarrier come against B16 melanoma. Mice immunized with positive (R)- fabrication is one example of those approaches, which can pro- DOTAP/Trp-2 peptide complexes showed increased T-cell IFN-𝛾 mote the interaction and destabilization of endosomal mem- secretion after re-stimulation with Trp-2, CTL activity in vivo, branes, under acidic conditions, facilitating the cytosolic delivery and high density of functionally active TIL, resulting in a sta- of payloads.[229,275] The pH-sensitive fusogenic liposomes have tistically significant growth delay of aggressive murine solid been explored as a potential melanoma immunotherapy. Markov melanoma.[281] et al.[277] reported the benefit of using liposomes composed by the helper-fusogenic lipid dioleoylphosphatidylethanolamine (DOPE) and the cationic lipid 2D3 to deliver RNA into murine 6.4.2. Endo-Lysosomal Compartment DC progenitors and immature DC, as prophylactic RNA-loaded DC vaccines to treat highly aggressive B16 metastatic melanoma Lysosomes are intracellular organelles involved in several tumors. The anti-metastatic effect of DC vaccination resulted in important cellular activities, including autophagy, exocytosis, the suppression of lung metastases and reduction of Th2-specific endocytosis, phagocytosis, cell plasma membrane recovery, cytokine levels (IL-10, IL-5, and IL-4).[277] Nakamura et al.[278] cholesterol homeostasis, and cell death signaling.[229,282] They incorporated the pH-sensitive-fusogenic cationic lipid YSK05 are considered as the main cellular clearance organelles, and in liposomes to enhance the cytosolic release of c-di-GMP the terminal of the endo/lysosomal pathway, where the degra- and to promote an efficient activation of NK cells to trigger dation of the endocytosed molecules occurs. As the lysosomes anti-cancer immune responses against lung metastases in a have an acidic interior due to the presence of several acidic hy- melanoma mouse model. The intravenous administration of c-di- drolases, these organelles are appealing targets for the design GMP/YSK05-Lip into mice induced MHC class I non-restricted of acidic-responsive delivery systems. For DC-based vaccines, anti-tumor response mediated by NK cells through the signifi- these nanocarrier responsive features are crucial since they help cant type I IFN secretion and the infiltration of NK cells in the in the release of TAA under endolysosomal acidic conditions. lungs of B16F10 melanoma-bearing mice.[278] The construction of pH-sensitive targeting systems by using Another mechanism used to destabilize the endosomal mem- acidic-responsive peptides, lipids, and/or polymers may induce brane and promote the cytosolic delivery encompasses the incor- the disassembly and release of loaded cargo within the acidic

Adv. Therap. 2020, 2000147 2000147 (19 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com microenvironment of lysosomes. In the lysosomes of APC, the Sneh-Edri et al.[285] developed and characterized a PLGA-based TAA bind to MHC class II molecules to form the TAA-MHC nanocarrier decorated with an endoplasmic reticulum targeting class II complex, which is presented to CD4+ T cells to trigger peptide to deliver encapsulating antigen peptides to DC, to de- Th2 humoral or Th1 cytotoxic immune responses.[230] Following velop a potential anti-cancer vaccine. Nanoparticles were effi- the previously mentioned strategies, nanocarriers can also escape ciently internalized by murine DC2.4 cells in vitro and induced a the endo-lysosomal route to reach the cytosol, allowing the estab- sustained antigen cross-presentation. lishment of the TAA-MHC class I complex, which is presented Nakagawa and co-workers reported amplified cellular immune + to CD8 T cells to trigger CTL activation, in a process named responses with activated CTL and gp10025–33 TAA-specific IFN- cross-presentation.[230] Moreover, targeting ligands including fo- ɣ-secreting cells in mice vaccinated with poly(ɣ-glutamic acid)- late, transferrin, VEGF, and low-density lipoprotein (LDL), can based nanoparticles (ɣ-PGA nanoparticles) entrapping an en- be used to target endo/lysosome surface receptors. The man- doplasmic reticulum-insertion signal sequence (Eriss) bound nose 6-phosphate is the main signal for lysosomes targeting.[275] to the TAA. No cellular immune responses were obtained In addition, TLR 3, TLR 7/8 or TLR 9 in the endosomal mem- in mice immunized with ɣ-PGA nanoparticle-antigen, com- branes can also be targeted by using Poly(I:C/A:U), Resiquimod plete Freund’s adjuvant (CFA)-antigen, CFA-Eriss-antigen, or (R848)/Imiquimod (R837), or CpG-ODN adjuvant molecules, Eriss-antigen.[286] ɣ-PGA nanoparticles were proved to be out- respectively.[230] standing vaccine delivery systems by boosting the fusion of en- In accordance, pH-sensitive mannose-modified poly (𝛽-amino doplasmic reticulum-endosome fusion for cross-presentation. ester)-stearylamine (man-PBAE-S) nanovaccines (T/M@mPS), Moreover, endoplasmic reticulum translocon sec61 seems to be co-entrapping TAA Trp-2 and the TLR 4 agonist MPLA, especially important to retro-translocate TAA from the merged endoplas- in combination with anti-PD-L1 immune checkpoint therapy, im- mic reticulum–endosome to the cytoplasm for MHC class I cross- proved the anti-cancer effect and prolonged the median survival presentation.[287] These data provide an elegant approach for in B16F10-bearing mice, in both therapeutic and prophylactic peptide-based cancer immunotherapy, as it promotes an active settings. T/M@mPS were able to target and induce DC matu- endoplasmic reticulum-translocation of antigenic peptides into ration, which presented the antigen to T lymphocytes, inducing APC by adding an Eriss. Moreover, the authors presented another their stimulation, expansion and boosting antigen-specific cyto- peptide-vaccine strategy that combined Eriss and fusogenic lipo- toxic immune responses against melanoma.[283] somes (FL) to improve in vivo tumor protective immunity. FL- Mockey et al.[284] reported a specific and significant encapsulated Eriss+ peptide allowed the maintenance of peptide- tumor growth restrain in B16F10-bearing mice that re- presentation activity within APC for the longest period (at least ceived systemic injections of MART-1 mRNA-loaded in 140 hours), which correlated with a stronger anti-tumor immune PEGylated-histidylated/polylysine/l-histidine-(N,N-di-n- response, when compared to Eriss+/Eriss− peptides in solution hexadecylamine)ethylamide-based liposomes (histidylated or FL-entrapped Eriss− peptides.[288] lipopolyplexes). Moreover, the anti-tumor immune response Other endoplasmic reticulum-targeting signals have also been was improved by the histidylated lipopolyplexes containing both explored in cancer immunotherapy. For instance, the endoplas- MART-1 mRNA, and MART-1-LAMP1 mRNA developed to mic reticulum-targeting sequences of adenovirus E3/19K pro- encode the antigen, allowing its presentation in the context of tein and KDEL peptide were fused with TAA to facilitate TAA MHC class II molecules through the LAMP1-based lysosomal presentation via MHC class I pathway to CD8+ T lymphocytes. sorting signal. These observations indicate an improved protec- Accordingly, a higher protective and long-term anti-tumor im- tion against B16 melanoma growth when targeting MART-1 to munity was induced when melanoma-bearing mice were immu- both MHC class I and MHC class II presentation routes. nized with a pEKL6 plasmid containing TAL6 fused with the ER-targeting sequence (adenovirus E3/19K protein), when compared to full-length TAL6 (pL6) immunization.[289] Perez-Trujillo 6.4.3. Endoplasmic Reticulum et al.[290] also described a strong therapeutic anti-tumor response induced by a DNA-based vaccine encoding E6 and E7 antigens of The endoplasmic reticulum is a continuous and compartmen- human papillomavirus type 16 fused with import and retention talized membrane network, found from the nuclear mem- signals of the endoplasmic reticulum (SP-E6E7m-KDEL), as the brane throughout the cytoplasm, which controls the transfer of immunization against E6 and E7 antigens fused to human cal- membrane-bound and secretory proteins, and regulates several reticulin (hCRT-E6E7m). Thus, endoplasmic reticulum-targeting signaling pathways and stress responses, such as cell homeosta- sequence fused with TAA holds promise in cancer therapy by trig- sis, calcium balance, and lipid/protein biosynthesis.[275,282] The gering strong CTL immune responses. endoplasmic reticulum stress, caused by calcium and biosynthesis homeostasis disturbance, ischemia, hypoxia, free radi- cal exposure, has been associated to several pathologies, such 7. Preclinical Studies of Emerging Therapies: as cardiovascular and neurodegenerative diseases, cancer and Current Challenges diabetes.[282] For cancer vaccination purposes, TAA must reach the major MHC class I-loaded organelle, the endoplasmic reticu- 7.1. Predictive Preclinical Melanoma Models for the Translation lum, to improve cross-presentation efficacy.[282] Thus, the devel- of Immunotherapeutic Systems opment of novel vaccine nanocarriers able to promote an efficient and targeted delivery of TAA to this organelle where the antigen Although some of the immunotherapeutic agents presented im- cross-presentation process occurs has been highlighted. pressive preclinical results, most of them failed later in clinic

Adv. Therap. 2020, 2000147 2000147 (20 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com settings. This may be explained by the unsuccessful translation important to assess whether these models represent the best pre- of data obtained from the pre-clinical models to humans. Ani- clinical tools to assess the efficacy of cancer immunotherapeutics. mal models have been the major preclinical tool employed for Especially due to the lack of an integral immunity, PDX models the evaluation of novel diagnostic and therapeutic anti-cancer are not suitable preclinical models to study immune-modulatory therapies before clinical testing. Therefore, the development of treatments.[297] To overcome this issue, an early phase technol- proper preclinical models is fundamental to successfully trans- ogy was developed to create humanized mouse models hold- late immune-based cancer therapies into the clinic. These models ing the major human immune players based on the introduc- must recapitulate the human cancer progression, while encom- tion of CD34+ hematopoietic stem cells previously irradiated with passing the distinct subpopulations of the major host immunity gamma rays.[298] However, this humanized model takes 3 months players. to develop, in addition to being an expensive alternative. Mouse models, and more specifically the immunocompe- Great efforts have been made to develop clinically relevant tent syngeneic models, constitute the preclinical tool most used and reliable murine models that mimic the melanoma genetic for the evaluation of biological effect of immunotherapies, due heterogeneity and patient immunotherapeutic responses. Im- to their wide availability and experimental reproducibility. The munocompetent genetically engineered mouse models (GEMM) widely used syngeneic melanoma models rely on the subcu- have been developed to explore melanoma genetic mutations.[299] taneous injection of the well-established B16F1 and B16F10 For instance, both familial and sporadic melanomas have melanoma cells in immunocompetent C57BL/6 mice. While shown genetic signatures with somatic mutations in genes B16F1-bearing models mimic tumorigenesis and less metastatic highly involved in tumorigenesis and metastasis. For in- disease, B16F10 is a highly aggressive and metastatic melanoma stance, BRAFV600E mutation, which is present in 50–65% model.[291] However, B16 models undergo a completely dissimi- of human melanomas, heavily impacts melanomagenesis and lar set of factors and mechanisms related to invasion, dissemina- development.[299] Melanocyte-specific inducible Cre recombi- tion, and metastasis, when compared to human melanoma and, nase (Tyr:Cre-ERT2, BRAFV600E) GEMM, as a human ame- therefore, do not mimic the human genetic diversity.[292] For in- lanotic/oligomelanotic malignant melanoma, was established stance, B16 models are poorly immunogenic due to the absence to demonstrate the importance of BRAFV600E mutation on of MHC class I molecules on tumor cell surface[293] and do not melanomagenesis.[300] Perna and collaborators generated a present activating BRAF mutations found in at least 50% of hu- conditionally expressed BRAFV618E GEMM (correspondent man melanomas[292b]. Moreover, the aggressiveness of B16 cells BRAFV600E in humans) to confirm that oncogenic BRAF alone impairs their use in prolonged in vivo assays. In general, these is enough to initiate melanomagenesis.[301] However, additional models are frequently poorly predictive of clinical challenges, be- genetic alterations are required to synergize with BRAFV600E ing the average rate of successful translation less than 8%.[294] mutation in order to accelerate melanoma progression.[302] Ac- These models do not recapitulate the cancer genomic instability cordingly, p16INK4a or p16INK4A/p19ARF loss, as well as, and heterogenous microenvironment. Besides, the tumors im- phosphatase and tensin homolog (PTEN) tumor suppressor planted on these models generate poorly differentiated malig- gene silencing dramatically enhances the metastatic behavior of nancies and do not go through the natural stages of tumor pro- BRAFV600E melanoma GEMM.[303] Since a reasonable num- gression (premalignant transformation, tumor development, and ber of uncultured primary (20%) and metastatic melanoma progression).[295] The aggressive tumor growth observed in these (55%) tumors does not have the PTEN phosphatase, leading to models limits the time window to evaluate the efficacy of certain AKT signaling and BRAF activation,[304] BRAFV600E;PTEN−/− immunotherapeutic approaches, which impairs their study at tu- metastatic melanoma GEMM was developed to validate the mor early stages.[295] key role of these signaling pathways associated to this genetic As an alternative, patient-derived xenograft (PDX) mouse makeup.[305] BRAF mutations are commonly limited to RAS models, derived from the surgical subcutaneously implant and mutations, being both associated with the MAPK signaling ac- growth of a tumor in mice, have been developed for cancer tivation in more than 85% of malignant melanomas.[306] Hu- immunotherapy research. Also known as “xeno-patients,” these man melanomas harbors mutations that active the RAS path- “mini-human-in-mouse” models are more predictive of clinical way. While Harvey rat sarcoma viral oncogene homolog (HRAS) disease compared to tumor cell-based xenografts.[296] In contrast and Kirsten rat sarcoma viral oncogene homolog (KRAS) muta- to syngeneic models, PDX mouse models mimic the tumor com- tions are only found in 2% of melanomas, the neuroblastoma plex architecture and genomic heterogeneity, as well as its natural rat sarcoma viral oncogene homolog (NRAS) mutations account microenvironment components,[295] which are crucial to evalu- for 15–25% of these malignant cells.[307] NRAS (NRASQ61R, ate immunotherapeutic agents, such as cancer vaccines and im- NRASQ61K, NRASG12D) mutated GEMM have also been devel- mune checkpoint modulators. Nevertheless, PDX mouse models oped similarly to BRAFV618E models.[308] Moreover, the coop- are also associated with diverse drawbacks, namely: i) cost and eration of NRAS mutation with other genetic alterations, for in- time required for their development; ii) the success rate of hu- stance, p16INK4a/p19ARF knockout,[308] p16Ink4 or liver kinase man tumor tissue engraftment into the mouse is variable, lead- B1 (Lkb1) or CDKN2A depletion was also confirmed to increase ing to the lack of standardization and reproducibility; iii) short melanomagenesis, metastasis, and aggressiveness.[309] Although lifespan, which precludes, for instance, long survival assays; iv) the wide use of GEMM, the generation of inbred mouse strains not suitable to study early stages of tumor development; v) do not is an expensive and labor-intensive process and usually presents represent a complete and functional immune system, lacking im- defective reproductive fitness.[310] portant immune cell subsets (T and B lymphocytes, NK cells, or The impact of the TME on tumor progression has motivated myeloid cells). Given these issues and scientific limitations, it is the use of GEMM in immunocompetent mice, as well as primary

Adv. Therap. 2020, 2000147 2000147 (21 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com human tumor xenografts in humanized mouse models (hu- labeled with [111In]. These results suggest that anti-PD-L1 imag- manized PDX models), as priority models for cancer immuno- ing has the potential to optimize the treatment dose, which can therapy. be adjusted until an adequate amount reaches the tumor.[321b] PET advantage as an imaging method for melanoma diagnosis over computed tomography (CT), as well as magnetic resonance 7.2. Non-Invasive Strategies for Personalized Immune-Oncology imaging (MRI), is the ability to identify small metabolic changes Treatments before anatomical or structural changes could be detected.[315] Its main disadvantage is the lack of a sufficient spatial resolution. Significant improvements have been accomplished in the design Therefore, fused anatomical images from CT or MRI, and func- of effective immunotherapies for melanoma, which led to in- tional images from PET provides a non-invasive and sensitive creased survival rates of advanced melanoma patients.[130,311] As way to monitor and predict the clinical response of melanoma the variety of cancer treatments continue to progress, the devel- patients to immunotherapy.[316,323] 18F-FDG PET/CT scans have opment of non-invasive strategies for assessing the response of been used for predicting early responses to ICI treatments by each patient to the given therapy is needed.[312] Imaging stud- patients with advanced melanoma.[324] This study showed en- ies are crucial for monitoring immune-associated adverse events, hanced 18F-FDG internalization in the early phase of ICI therapy, as well as to analyze tumor responses and progression follow- which could be correlated with immune-mediated events within ing immunotherapies.[313] Non-invasive imaging for immune tumor site that might lead to favorable results.[325] Similarly to monitoring to support immunotherapy follow-up is a developing PET/CT, PET/MRI is a metabolic-anatomic imaging technique, field, and additional advanced research needs to be conducted, however, it is more accurate when high soft-tissue contrast for di- yet it holds a great promise for a successful anti-melanoma im- agnosis is required.[326] Although this technique is not as widely munotherapy. The main available non-invasive imaging tech- used as PET/CTin the clinic, it has been shown promising results nologies that study tumor response in vivo offer advantages in in clinical trials as a diagnostic tool for melanoma brain metasta- different areas. Therefore, combinations between two methods sis and for monitoring early treatment response in these patients often constitute the preferred approach in the clinic.[314] who undergo ICI.[327] Positron emission tomography (PET) is a type of nuclear CT is characterized by high temporal and spatial resolution, imaging, which detects gamma rays from positron-emitting iso- being however difficult to distinguish between different soft tis- topes and provides a functional or metabolic assessment of sues that have similar densities.[328] Thus, CT imaging might diseases.[315] PET is a highly sensitive imaging tool that is of- not be ideal as a standalone imaging method for anti-tumor im- ten used for the detection of melanoma metastasis, by the munotherapies follow-up.[329] Studies have shown that standard identification of biochemical alterations in the early stage of CT imaging may be inadequate to differentiate the impact of dis- the disease.[316] Melanoma is known for its increased glucose tinct immunotherapeutic tools, due to unconventional or delayed metabolism that can be detected by 18F-fluorodeoxyglucose up- patterns of response.[325] However, therapy combination of ICI take in PET imaging (FDG-PET).[316] This implies that the tumor and anti-angiogenesis agents led to tumor density changes that metabolic state may be monitored effectively according to defined can be detected by CT imaging.[330] Moreover, as T cell-based ther- prognostic parameters.[317] Furthermore, cancer immunotherapy apy holds a good potential for cancer treatment, it also has clinical has a distinct metabolic fingerprint,[318] as upon activation, im- challenges.[331] T-cell distribution, migration, and kinetics in vivo mune cells undergo specific metabolic changes and upregulate remains unclear, and advanced understanding on these mech- the glycolysis pathway.[319] Therefore, the prediction of an effec- anisms could be a key to unlock the total success of this im- tive anti-tumor immunity might be possible by detecting these munotherapy. A novel T cell therapy uses the visualization of CT immunotherapy-associated metabolic changes via FDG-PET.[318] attempt to overcome those challenges, by labeling T cells that ex- Interestingly, FDG-PET detected a residual metabolic activity in press melanoma-specific TCR with gold nanoparticles, as a CT individuals with metastatic melanoma that presented a sustained contrast agent.[329] response to immune checkpoint antibodies, which might be an MRI is considered as standard imaging for melanoma indication of immune cell infiltration to the lesion rather than metastases.[332] It can produce a non-invasive assessment of a melanoma.[320] Therefore, although FDG-PET has the potential number of tumor characteristics, as it provides superior visual- to predict melanoma progression and early detection, as a sole ization in soft tissue imaging, which can be used qualitatively and method it could lead to false positive results.[320] quantitatively to predict treatment outcome.[333] MRI can pro- A different way to exploit PET for in vivo melanoma imag- vide high-resolution data due to a superior soft-tissue contrast, ing includes immunotherapies and immune cell components’ about the post-injection location and biodistribution of magneti- labeling with radioisotopes for activity monitoring.[321] Apre- cally labeled cells with superparamagnetic iron oxide (SPIO).[334] clinical study investigated and quantified the migratory abilities Moreover, nanoparticles with SPIO core and biocompatible coat of MDSC subpopulations in B16F10 melanoma-bearing mice, can activate DC by delivering TAA, while acting as an imaging by utilizing a 64Cu-labeled CD11b monoclonal antibody for PET agent for in vivo study by MRI.[335] Another imaging system for imaging.[321a] Another study used a64Cu-labeled PD-1 antibody to cell tracking and molecular imaging is fluorine-19 (19F) MRI.[336] assess the status of TIL in vivo as predictive biomarker to anti-PD- Cells labeled with probes based on perfluorocarbon (PFC) could 1/PD-L1 ICI efficacy.[322] Moreover, single-photon emission com- be detected by 19F-MRI. This method enables the characteriza- puted tomography (SPECT) imaging was used to distinguish the tion of labeled adaptive immune cell populations, as well as the most responsive individuals to anti-PD-L1 treatment and moni- quantification of cell localization, migration, and clearance.[337] toring its bio-distribution. To that end, anti-PD-L1 treatment was A research on B16 OVA melanoma mouse model monitored and

quantified tumor infiltration of Tova cells that were labeled in vitro tentially a powerful and quantitative tool for the assessment of with PFC up to 3 weeks post-transfer.[338] immunotherapies in melanoma animal studies. Scintigraphy is another nuclear imaging technique based on the detection of gamma radiation. The information provided by this imaging technic depends on the integrity, dispersion, 8. Clinical Melanoma Combinational or release characteristics of the chosen radiolabeled delivery Immunotherapies system.[339] Scintigraphy can be used for the detection of malignant melanoma, bone metastases, early detection of lymph node Since its discovery in 1826,[349] melanoma has been studied for metastases of melanoma, and Immunoscintigraphy.[340] Further- more than two centuries. Conventional pharmacological strate- more, it was previously demonstrated that this technique can gies struggled to delay tumor progression and improve patients’ identify immune system components, such as lymphocytes and survival. Acquired resistance, non-responders, and high muta- DC by radioisotopes. A study of 30 patients with cutaneous le- tion rate represent the major challenges for melanoma therapy. sions demonstrated that 99mTc-IL-2 scintigraphy constitutes a Only in the last two decades, new molecular targets involved in promising technique to address TIL in melanoma.[341] The ac- the oncogenic signaling were discovered, offering new insights tivity of T lymphocytes against melanoma cells was evaluated for novel therapeutic approaches.[350] by in vivo binding of 99mTc-IL-2 to the cluster designation (CD) Table 2 summarizes the most representative studies from 25 antigen (IL-2 receptor) expressed by lymphocytes. That result the last decade. Conventional therapies, such as radiotherapy suggests that 99mTc-Interleukin-2 scintigraphy can be used as a and chemotherapy, were combined with targeted-therapies, co- prognostic tool for the stratification of patients based on their inhibitory/stimulatory checkpoint molecules and vaccines, in or- probability to respond to IL-2 immunotherapy and for monitor- der to directly affect the tumor, while sensitizing the patient ing immune response following immunotherapy treatment. Ad- immunity against malignant cells, and further increase patient ditionally, many studies, on animal models and patients, have survival. been shown that scintigraphy can be a useful technique for moni- In addition to the immunotherapies and targeted therapies toring and trafficking DC.[342] Moreover, it was demonstrated that used for the management of melanoma discussed above, the tal- scintigraphy imaging can visualize in patients and mouse mod- imogene laherparepvec (T-VEC) oncolytic-based therapy is an ad- els the distribution patterns of 111In-oxine labeled DC following ditional (immune)therapeutic approach showing great promis- vaccination.[343] Those results suggest that the immune effect of ing under clinical settings (Table 2). T-VEC is an oncolytic viral potential immunotherapies for melanoma treatment can be eval- melanoma treatment based on attenuated herpes simplex viruses uated by using this imaging method. type I (HSV-1), which has the ability to attack tumors by two dis- Non-invasive bioluminescence imaging (BLI) is one of the tinct but complementary ways. On the one hand, T-VEC is able currently available technologies to quantify the tumor bur- to directly induce a cytotoxic effect through the selective lysis of den in vivo at real-time. Moreover, this technique can be cancer cells, but on the other hand can also promote an anti- used to evaluate and monitor immune responses to different tumor immune response due to the integration of the GM-CSF immunotherapies.[314,344] BLI does not have a very high resolu- gene into the viral genome, which induces local inflammation tion and depends on foreign gene expression in the cells that and antigen presentation.[351] are being evaluated, which limits its use to endogenous tumors. Nevertheless, this technique enables a fast quantification of the tumor response to a specific treatment, with a very short 9. Major Challenges Toward the Clinical image collection time, imaging of multiple animals simultane- Implementations of Emerging Nano-Based [345] ously, and relatively high sensitivity. Furthermore, a previ- Targeted Immunotherapies ous study demonstrated that BLI can potentially evaluate primary melanoma tumor progression and even detect melanoma metas- 9.1. Patient-Related Challenges tasis by using bioluminescent tumor cells.[344a] The immune response in BLI is usually monitored by an in- Melanoma is very heterogeneous, reflecting the molecular direct methodology based on imaging the bioluminescence (BL) changes occurred during the development of this disease. of tumor cells to assess the cytotoxic effect.[344,346] However, few Decades of standard research and unfruitful clinical trials anal- preclinical studies demonstrated a direct way to identify immune ysis show that most of the chemotherapeutic drugs were not ef- response by using immune cells transduced with BL. A preclini- fective against metastatic melanoma. Some patients showed par- cal study that evaluated the functional efficacy of cancer antigen- tial responses to dacarbazine and other chemotherapeutic agents, specific T cells, characterized the CTL trafficking kinetics in liv- but no long-term benefits have ever been observed.[352] ing tumor-bearing animals via BL imaging of MART-1 specific Compared to other tumor types, melanoma has an extraordi- CTL transduced with luciferase gene (MART-1-Luc CTL).[314] An- narily high frequency of acquired mutations, which should be other study has shown the trafficking pattern of ex vivo expanded considered while selecting the treatment most suitable for a spe- DC, transduced to express luciferase, in mice after allogeneic cific patient.[13a] Personalized medicine has provided some suc- bone marrow transplantation by BL imaging.[347] Additionally, it cessful results against solid malignancies, such as melanoma, was shown that BLI can be used for monitoring the trafficking of changing the natural evolution of this fatal disease. Fluc-transduced DC after Imiquimod, TLR-7 agonist, a treatment The molecular analysis of melanoma demonstrated that about that enhances the DC survival, while priming tumor-specific T 50% of these cancer cells contain a BRAF oncogene mutation, cells.[348] These studies provide validation that BLI imaging is po- being the V600E mutation the most frequent one.[353] Moreover,

Table 2. Examples of the most relevant clinical trials involving melanoma combinational immunotherapies.

Study number Year Phase Molecular target Combination therapies (ICM-TT) Stage disease Administration Status

NCT02587650 2015 I BRAF/NRAS wild-type Capmatinib Melanoma stage III-IV Oral Terminated Ceritinib Entrectinib Regorafenib NCT03754179 2018 I-II BRAF V600 Dabrafenib Melanoma stage III-IV Oral Recruiting Trametinib Hydroxychloroquine NCT03430947 2018 II BRAF Vemurafenib Melanoma stage IV Oral Recruiting MEK Cobimetinib BRAF V600 mutation Brain metastases NCT03772899 2018 I PD-1 Fecal microbial transplantation (**) Advanced melanoma IV Recruiting PD-1 inhibitors (FDA) NCT03470922 2018 II-III PD-1 Relatlimab Metastatic or IV Recruiting Nivolumab Unresectable melanoma NCT03673332 2018 IV PD-1 Monoclonal antibodies Advanced or metastastic IV Recruiting melanoma NCT03396952 2018 II PD-1 Pembrolizumab Advanced or unresectable IV Recruiting CTLA-4 Ipilimumab melanoma Oral Aspirin NCT03235245 2017 II PD-1 Ipilimumab Nivolumab Metastatic melanoma BRAF IV Recruiting BRAF V600 Encorafenib Binimetinib V600 mutation Oral NCT03329846 2017 III PD-1 Nivolumab Metastatic or IV Active IDO BMS-986205 unresectable melanoma Oral NCT03278665 2017 I-II PD-1 Pembrolizumab Advanced melanoma Oral Recruiting HDAC 4SC-202 IV NCT03313206 2017 II PD-1 Pembrolizumab Resectable head/neck melanoma IV Recruiting DNA damage Surgery Radiotherapy NCT03685890 2018 I-II PD-1 Nivolumab Early metastatic melanoma IV Recruiting DNA damage Chemotherapy NCT03276832 2017 Early I PD-1 Pembrolizumab Melanoma stage IIIB-IV Topically Recruiting INF-𝛾 stimulator Imiquimod IV NCT03021460 2017 II PD-1 Pembrolizumab Melanoma stage III-IV IV Recruiting BTK Ibrutinib Oral NCT03229278 2017 II PD-1/PD-L1 Pembrolizumab Nivolumab Advanced or metastastic Oral Recruiting Cell cycle Trigriluzole melanoma IV NCT03207867 2017 II PD-1 PDR001 Solid tumor including melanoma Oral Recruiting Adenosine A2a-r agonist NIR178 IV NCT03325101 2017 I-II PD-1 Pembrolizumab Melanoma stage III-IV IT Recruiting T cells mDC IV Cryosurgery NCT03047928 2017 I-II PD-1 Nivolumab Metastatic melanoma IV Recruiting T cells PD-L1/IDO peptides NCT03645928 2018 II PD-1 Pembrolizumab Metastatic melanoma IV Recruiting T cells Liﬁleucel LN145/ LN-145-S1 NCT04068181 2019 II PD-1 Pembrolizumab Advanced or unresectable IV Recruiting T cells T-VEC melanoma IT NCT03384836 2017 I-II PD-1 Pembrolizumab Advanced or unresectable IV Recruiting Beta-blockers Propranolol Hydrochloride melanoma NCT03502330 2018 I PD-1 Nivolumab Advanced melanoma IV Recruiting CD40 APX005M CSF1R Cabiralizumab NCT03635983 2018 III PD-1 Nivolumab Metastatic or unresectable IV Recruiting CD122 NKTR-214 (IL-2 PEG) melanoma (Continued)

Table 2. Continued.

Study number Year Phase Molecular target Combination therapies (ICM-TT) Stage disease Administration Status

NCT04152863 2019 II PD-1 Pembrolizumab Advanced or metastastic IT Recruiting ICAM V937 melanoma IV NCT04165967 2019 I PD-1 Nivolumab Melanoma IV Not yet re- DNA damage Chemotherapy SC cruiting T cells TIL IL-2 NCT03092453 2017 I PD-1 Pembrolizumab Advanced melanoma IV Recruiting T cells mDC DNA damage Cyclophosphamide NCT02680184 2016 I PD-1 Pembrolizumab Advanced melanoma IV Active CpGA DNA TLR9 CMP-001 NCT03425461 2018 I PD-1/PD-L1 Nivolumab Melanoma stage III-IV IV Recruiting CTLA-4 Ipilimumab SEMA4D Anti-SEMA4D (VX15/2503) NCT03289962 2017 I PD-L1 Atezolizumab Locally advanced or metastatic IV Recruiting APC RO7198457 (mRNA) melanoma NCT04123470 2019 I-II PD-L1 Atezolizumab Advanced or unresectable IT Recruiting T cells Delolimogene mupadenorepvec melanoma IV NCT03420963 2018 I DNA damage NK Cells Recurrence of primary IV Recruiting Cyclophosphamide melanoma Etoposide NCT04357509 2020 I T cells ScTIL Melanoma IV Not yet recruiting NCT03649529 2018 Early I gp100 AP GPA-TriMAR lentivirus (CAR T) Melanoma IV Recruiting NCT03068624 2017 I IL-2 Aldesleukin Metastatic uveal melanoma SC Active T cells CD8+ SLC45A2-speciﬁc T Lymphocytes IV Cyclophosphamide Ipilimumab NCT01622933 2012 I MART-1 DC Vaccine + IFN Stage III-IV or unresectable ID Completed MAGE-A6 AdVTMM2/DC Vaccination melanoma T cells NCT01876212 2013 II HLA-2A DC vaccine Metastatic melanoma ID Completed Src TKs Dasatinib NCT01973322 2013 II T cells (1) DC Vaccine + RT Advanced melanoma SC Recruiting DNA damage (2) DC Vaccine + IFN-𝛼 ID Both 1 and 2 + RT NCT02285413 2014 II Gp100/tyrosinase DC vaccination Stage III-IV melanoma Topically Completed DNA damage cisplatinum IV

NCT03617328 2018 I-II MHC-II 6MHP Melanoma stage III-IV SC Recruiting CD27 Montanide ISA-51 ID polyICLC Varlilumab NCT03747744 2018 I myDC Talimogene laherparepvec (T-VEC) Non-visceral melanoma IT Recruiting CD1c (BDCA-1)+ myDC metastasis NCT03655756 2018 Early I Emm55 streptococcal pDNA Resistance melanoma IT Recruiting antigen Unresectable melanoma NCT04364230 2020 I-II Neoantigen 6MHP vaccine Primary and advanced SC Not yet re- BRAF V600E NeoAg-mBRAF melanoma ID cruiting PolyICLC (TLR3 agonisT) CDX-1140 (CD40 agonist) NCT04335890 2020 I T cells (CD4 CD8) IKKb-RNA mDC Metastatic uveal melanoma IV Not yet re- (gp100, tyrosinase, PRAME, MAGE-A3, cruiting IDO)

APC, antigen presenting cell; BTK, Bruton’s tyrosine kinase; CAR, chimeric antigen receptor; CSF1R, colony stimulating factor 1 receptor; CTLA-4, cytotoxic T-lymphocyte- associated protein 4; DC, dendritic cell; HDAC, histone deacetylase; ICAM, intercellular adhesion molecule; IDO, indoleamine-pyrrole 2,3-dioxygenase; ID, intradermal; IFN-𝛾, interferon gamma; IL-2, interleukin-2; IV, intravenous; IT, intra-tumoral; NK, natural killer; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PEG, polyethylene glycol; SC, subcutaneous; TIL, tumor inﬁltrating lymphocytes; TLR9, toll-like receptor 9.

Adv. Therap. 2020, 2000147 2000147 (25 of 39) © 2020 Wiley-VCH GmbH www.advancedsciencenews.com www.advtherap.com up to 10% of melanoma cells contain a mutation on the NRAS agents substantially increased the OS and PFS of melanoma pa- gene, an upstream gene of BRAF, in the RAS/RAF/MEK/ERK tients bearing this mutation.[362] The combination of dabrafenib cascade. Therefore, the high mutation load frequency of this with the MEK1/2 inhibitor trametinib also improved the OS of pathway highlighted the relevance of this signaling cascade for this subset of patients.[363] Currently, this is the recommend com- melanoma proliferation, thus constituting an attractive thera- binational therapy for BRAF-mutant melanoma patients. In ad- peutic target.[354] Since then, many other relevant mutational dition, some patients also have mutations or deletions of tumor signatures have been discovered, allowing better prognosis and suppressor genes, such as TP53, PTEN,andCDKN2A, but their melanoma patient stratification. clinical relevance remains unclear.[305,364] Despite the remarkable success of molecularly targeted and After BRAF, NRAS oncogene has been found to be mutated immunotherapies for advanced melanoma, such as the ICI, sev- in 20% of melanoma cases.[357] Currently, targeted therapies for eral patients do not attain sustained tumor regression. Primary NRAS-mutant patients are based on MEK1/2 inhibitors, but a low and acquired resistances remain a major obstacle to successfully percentage of patients respond to this therapy.[365] As for NF1- treat this skin cancer. Therefore, it is crucial to understand the loss subset, this alteration activates the RAS pathway leading to biology behind these despair clinical outcomes, by comparing MAPK inhibitors sensitivity.[366] Further findings are required responder and non-responder data to identify the biomarkers to elucidate the response of NF1-loss melanoma patients to potentially useful to guide clinicians in selecting an adequate tar- currently available targeted therapies to allow a better stratifi- geted therapy.[355] cation of these patients into effectiveness treatment subgroups. Although data are still very limited, there is no doubt that Triple wild-type melanoma patients have a frequent mutation on the identification of gene signatures or predictive biomarkers of KIT oncogene.[357] For these patients, tyrosine kinase inhibitors response to immunotherapy will certainly advance the clinical (imatinib and desatinib) are being explored, but just a minor frac- management of melanoma. tion of patients respond to these targeted drugs.[367] BRAFV600 mutation is an example of a predictive validated Even though important advances were made in melanoma ge- biomarker for melanoma. Other emerging biomarkers have been nomics, the identification of driver mutations is not easy, and identified over the years during clinical trials, including tu- most mutations identified so far were not functionally validated mor mutation burden linked to neoantigen expression, concomi- regarding their pathogenic or therapeutic relevance. A recent tant molecular alterations, immune checkpoint expression (PD- study hypothesized that in highly mutated tumor genomes, many L1 or LAG-3), immune cell infiltration (CD8+ T cells), IL-17 somatic driver mutations might be instead recurrent passenger expression, immune-related gene expression profile, and TCR mutations, which can have an impact on the response to the sequencing.[356] matched targeted therapy.[368] Besides these challenges, genetic alterations have allowed not only the stratification of patients into treatment subsets but are 9.1.1. Genomics also being linked to primary and acquired resistance to targeted therapies. For instance, resistance to BRAF inhibitors associated Large-scale sequencing initiatives taking advantage of next- with the loss of the PTEN gene, that induces a constitutive acti- generation sequencing (NGS) tools have prompted the discov- vation of PI3K/AKT pathway leading to tumor progression.[369] ery of new clinically relevant genetic alterations.[357] NGS allow The MITF gene is another resistance regulator. High levels of the simultaneous single-gene analysis of different genes, which this gene are associated to resistance to both BRAF and MEK optimizes the use of tissue samples and reduces its costs. This inhibitors.[369,370] Other mechanisms of resistance are related to sequencing analysis can be applied to a pre-specific group of the reactivation of the MAPK/ERK pathways.[371] genes (targeted gene panels), which in melanoma are restricted Gene expression profiling can also provide crucial insights re- to BRAF, NRAS,andKIT oncogenes.[358] This option is generally garding genomic changes after immune checkpoint therapies in the most applied in clinical settings, either to select the best treat- melanoma.[372] The high levels of mutation load in melanoma ment, or to stratify patients into ongoing clinical trials. NGS can seem to be correlated to higher T-cell infiltration and thus better also be applied for coding regions of all genome base pairs (whole responses to immunotherapy with anti-PD-1 or anti-CTLA-4.[373] exome sequencing) or can interrogate the whole tumor genome However, these studies are not consensual, and the genetic in- (whole genome sequencing).[359] stability impact on ICI efficacy it is still not fully understood.[374] New sequencing projects directed to melanoma are ongo- The major problem is the definition of “low” and “high” tumor ing, following the Human Genome Project that set the pace mutation loads. for large-scale DNA studies by identifying BRAF and NRAS Hence, genetic profiling is crucial for the identification of rel- oncogenes.[360] “The Australian Melanoma Genome Project” is evant pathogenic events and have enable the stratification of pa- currently addressing the whole tumor genome of 500 melanoma tients based on their individual mutational and gene signatures, patients to allow the discovery of new targeted therapies and strat- helping clinicians to make increasingly rational treatment deci- ify patients for currently available treatments.[361] sions. Based on the already established driver mutations, it is possible to categorize melanoma in four genomic groups: BRAF- mutated, NRAS-mutated, NF1-loss, and triple wild-type.[357,361] 9.1.2. Predictive Biomarkers The first mutation exploited for targeted therapies was the BRAFV600E/K, with the development of the specific BRAF Tremendous efforts are being done toward the discovery of inhibitors, vemurafenib, dabrafenib, and encorafenib. These new response predictive biomarkers to melanoma treatment.

However, tumor monitoring is required to provide an indication As discussed in Section 6, ligands can be linked to the sur- of treatment response, and development of acquired resistance face of nanoparticle to promote their interactions with targeted since the early stages. cells. Functionalized nanocarriers exhibiting such modifications Liquid biopsies are a promising, non-invasive assessment are more susceptible to be internalized following their interac- methods that can be used to detect and monitor melanoma.[375] tion with the appropriate receptor found on targeted cells.[381] Based on this method, it is possible to monitor different parame- However, there is limited information concerning the ligand den- ters, such as treatment response, toxicity and resistance, through sity required to enhance nanoparticle cellular internalization, as a single blood sampling of on-treatment biomarkers. The cir- well as regarding the impact of the targeting moieties distri- culating tumor DNA (ctDNA) detection and quantification have bution on nanocarrier interaction with cells. Additionally, non- arisen as a valuable biomarker of cancer load and response to functionalized nano-systems can still be internalized by different treatment in melanoma, as in many other cancer types. As well- uptake mechanisms, as phagocytosis or macropinocytosis.[382] It known, exosomes containing nucleic acids are released by differ- is evident that the surface charge, size, and shape are impor- ent types of cells.[376] Tumor cells can release amount of circulat- tant features dictating the internalization of nanoparticles,[382] ing DNA significantly superior to those related to non-tumoral but studies on how these physicochemical parameters control cells, which can be correlated to the tumor phase, and to the rate cell uptake pathways, which may also vary according to targeted of tumor cell death.[377] Besides this applicability, ctDNA analysis cell, are scarce. Furthermore, the cellular uptake of NP becomes can also be used for monitoring on-treatment response in cases an even more complex situation to be characterized if the protein were clinical efficacy is still not evident. corona is added to this equation.[383] Moreover, once internalized, Most of these analyses are set for the detection of BRAFV600 the intracellular trafficking of nanomedicines specifies their the mutated ctDNA in metastatic melanoma patients, and its corre- fate and thus their final efficacy. This is highly dependable on lation with OS, progression-free diseases, and response to BRAF the internalization pathways and nanoparticle properties, that are and/or MEK inhibitors (as extensively reviewed in ref. [356]). modulated to at least enable endo-lysosomal escape.[382,384] De- Additionally, crucial data suggest that ctDNA analysis can be spite being an important theoretical aspect, the control of this used for a wider range of biomarkers, in ICI-treated patients phenomenon is not fully understood. presenting BRAF, NRAS, or KIT mutations.[378] Although some In 2004, the National Institute of Health – Nanotechnology improvements were made in the ctDNA analysis, large-scale Characterization Laboratory (NIH-NCL) set a group of character- prospective studies assessing the value of monitoring ctDNA in istics and methods to be evaluated looking at nanoparticle clinical melanoma patients are needed. translation.[385] The two main categories were divided between Another possibility to predict outcomes raises from the un- the biological and non-biological studies. The non-biological as- derstanding of the immune responses following the treatment says are related to the characterization of the inherent physic- with ICI. These therapeutic approaches induce specific immune- ochemical characteristics of nanomedicines (average mean related alterations within TME, and the follow-up of these diameter, surface charge, composition, morphology, concentra- changes can offer an initial indication of disease outcomes (as ex- tion, cargo loading, and impurities). The biological assays in- tensively reviewed in ref. [356]). On-treatment monitoring of the volve the understanding of the interactions established between TME is focused on the expression of different cell populations, nanoparticles and biological in vivo components. For this, dif- including white blood cells, absolute lymphocyte count, and T ferent in vitro, and in vivo experiments are suggested, including cell subsets (CD3+,CD8+,CD4+), as well as other markers, like hemocompatibility tests (red blood cell lysis, cytokine secretion), lactate dehydrogenase (LDH) and C-reactive protein (CRP). As study of interferences with biochemical cascades (complement, stated for ctDNA profiling, on-treatment monitoring of immune- coagulation).[386] related alterations in the tumor and tumor stroma requires ad- Suitable material properties and administration routes (e.g., ditional validation and defined guidelines before becoming a parental administration) with a stable overall clinical benefit- clinical reality. risk, have facilitated access to regulatory appraisal and to clinical translation.[115] Despite all difficulties, some nanomedicines to treat cancer, including Abraxane, Doxil, Eligard, Kadcyla, My- 9.2. Nanosystem-Related Challenges ocet, were already approved by the FDA and EMA.[387]

As previously stated, nanotechnology is a promising approach to improve medical therapies. However, operational barriers involv- 9.3. Clinical Trial Design ing the characterization and the wider understanding of their in vivo behavior have hindered the progress of nanomedicine. The The full potential of nano-based targeted immunotherapies will bench to bedside translation is still hard to achieve for many of only be fulfilled by innovative clinical trials centered on stratified these nano-based products. One key factor to reduce this trans- patients, regardless of disease (Figure 6). To help to determine the lational gap is related to a proper physicochemical characteriza- best immunotherapy candidates, study design, patient inclusion tion of nanoparticles, which will validate the batch to batch repro- and exclusion criteria, and clinical endpoints will be planned con- ducibility but also advance the global knowledge about the impact sidering previously acquired data related to genomics and predic- of nanoparticle properties on their biological effect and ultimate tive biomarkers. This approach will guide the rational selection in vivo fate.[379] An additional limitation faced by researchers in- of the combinatorial treatments most suitable to overcome resis- volves the categorization of nanomedicines in different classes tance, one of the major problems of current immunotherapeutic considering the current pharmaceutical regulations.[380] approaches.

Figure 6. Trial design based on precision medicine. A) Current approaches investigate limited molecular biomarkers prior to initiation of standard therapy in heterogenous patient population, resulting in modest and variable responses. B) Basket trial aims to pair therapies based on genomic proﬁle regardless of origin of the tumor. Following treatment initiation, on-treatment biopsies are continuously obtained to evaluate the success of current therapy. Based on results, patients either continue with therapy or switch to an alternative treatment regimen.

Regarding trial design, basket trials will allow clinicians to sive and ineffective treatments and preventing dangerous side design the clinical study centered on a specific molecular alter- effects. Most immunotherapeutic approaches include the use of ation. In this way, it is possible to assess immunotherapy regi- a single test to define the right medicine for a specific subset of mens in different cancer types, in patients identified by predictive patients. However, economic, and operational challenges seem biomarkers based on the highest probability to respond.[388] Sim- to be the major hindrances to advance precision medicine into ilarly, adaptive trials increase enrolment into stratified groups clinical practice. Screening patients for such personalized thera- with relevant response rates using interim analyses throughout pies and producing drugs that target specific individuals or small the entire study, in order to re-arrange cohort sizes to ensure patient populations is very costly. If we look deeper to types of statistical significance.[389] Appropriate new clinical trials need ultra-personalized therapy, as gene therapy and CAR T cells for to be designed considering combination dose-escalation stud- cancer, the issue of how society will manage to afford these treat- ies, where sequential combination could be administered follow- ments gains another proportion due to the remarkably high ex- ing monotherapy within the same subject. Additional novel trial penses of these individualized treatments. Many of these costs schedules are needed for assessing the outcome of therapies per- are not covered by public health systems, or by insurance compa- mutations and bifurcated arms for single-therapies and combi- nies, with patients supporting higher percentages of their medi- nations with dose escalation schedules.[390] cal costs. Therefore, these enormously expensive types of therapy Besides trial design, patient recruitment can also be improved can exacerbate inequities in the access to medicines, restricting based on genomic alterations. For this, automated tools could be them to those in the highest socioeconomic stratum. established to help clinicians to consider patients for a specific Despite all these associated costs, we are facing an acceler- trial based on their molecular alterations, and/or disease progres- ated development of personalized medicines. Drug developers sion, or even when new trials are open.[391] and payers need to devise innovative solutions to fund future re- As for immunotherapy endpoints, early-phase trials must be search thinking about the accessibility of these therapies. It is considered to address dose-response as well as dose-toxicity pro- necessary to demonstrate the value of precision medicine to the files of new ICI, using predictive biomarkers criteria. Although people responsible for distributing funds for healthcare and re- OS continues to be the gold-standard endpoint, it can take many search. There will be a lag between the investment on personal- years before a phase III study provides these data. Efforts are be- ized medicine, and the data suggesting their role in health im- ing made to address if surrogate endpoints predictive of OS, as provement. But at the end, the potential for improving patients PFS, overall response rate (ORR), or duration of response (DoR), outcomes will be of note, as it is already possible to state in the can be used. For late-stage studies, new endpoints are needed, improvement of the OS of cancer patients being treated with new such as criteria related to immune-based response and sustained tools designed taking into account their personal genetic pro- response rate, which continues to be under development.[392] file and predictive biomarkers. Precision medicine has an im- In the era of big data analysis and rapidly emerging immune- mense impact on patient outcomes, but research needs to be related clinical trials for cancer treatment, efforts need to be strategically conducted to overcome cost-related challenges and made to avoid redundancy and prioritize the most promising harness the full range of benefits. immunotherapies. 10. Conclusions 9.4. Demonstration of Increased Cost-Value Advances in melanoma treatment have been achieved using targeted-therapy drugs and immune-based therapies. Despite the Precision medicine is gaining relevance within scientific stud- overall improvement observed in these therapies, the treatment ies and clinical practice. Identifying the best treatment for the of melanoma continues to be a major challenge with many pa- right patient at the correct time, benefits not only patients but tients developing resistance or being nonresponsive to therapy. also healthcare systems, by preventing the prescription of expen- One of the causes of therapy resistance is related to the hostile

TME favoring tumor growth and progression. Major progress has [2] a) D. M. Pardoll, Nat. Rev. Cancer 2012, 12, 252; b) S. L. Topalian, C. been reached in understanding key cellular and molecular mech- G. Drake, D. M. Pardoll, Cancer Cell 2015, 27, 450. anisms related to the management of the melanoma microenvi- [3] P. Sharma, S. Hu-Lieskovan, J. A. Wargo, A. Ribas, Cell 2017, 168, ronment. This information is fundamental to support the design 707. of novel approaches to modulate distinct TME players involved [4] J. D. Martin, H. Cabral, T. Stylianopoulos, R. K. Jain, Nat. Rev. Clin. Oncol. 2020, 17, 251. in melanoma proliferation and dissemination. [5] a)G.P.Dunn,A.T.Bruce,H.Ikeda,L.J.Old,R.D.Schreiber,Nat. Nanotechnology may be the key to unlock the development Immunol. 2002, 3, 991; b) R. D. Schreiber, L. J. Old, M. J. Smyth, of chemo-, and immuno-based therapeutics against melanoma. Science 2011, 331, 1565. Nano-based systems simultaneously allow the modulation of dis- [6] D. S. Chen, I. Mellman, Immunity 2013, 39,1. tinct immune cells, while controlling the delivery of molecu- [7] G. Palmieri, M. Ombra, M. Colombino, M. Casula, M. Sini, A. lar agents. Moreover, they also can act as tracking agents us- Manca, P. Paliogiannis, P. A. Ascierto, A. Cossu, Front. Oncol. 2015, ing different probes for cell-labeling applications. However, their 5, 183. translation to clinical settings is still a challenge, requiring manu- [8] a) M. W. Teng, S. F. Ngiow, A. Ribas, M. J. Smyth, Cancer Res. 2015, facturing optimization toward the formulation of a reproducible 75, 2139; b) M. D. Vesely, M. H. Kershaw, R. D. Schreiber, M. J. platform, as well as the understanding and prediction of nanopar- Smyth, Annu. Rev. Immunol. 2011, 29, 235. [9] C.M.Koebel,W.Vermi,J.B.Swann,N.Zerafa,S.J.Rodig,L.J.Old, ticle biological influence in the host immunity. Moreover, ap- M. J. Smyth, R. D. Schreiber, Nature 2007, 450, 903. propriate preclinical models for validation of the complex im- [10] D. Mittal, M. M. Gubin, R. D. Schreiber, M. J. Smyth, Curr. Opin. munotherapeutic approaches are still required, to fill the gap be- Immunol. 2014, 27, 16. tween bench to bedside translation of the most promising emerg- [11] M. Marzagalli, N. D. Ebelt, E. R. Manuel, Semin. Cancer Biol. 2019, ing combinatorial therapies. Personalized medicine emerges as 59, 236. a central point for the design of prospective clinical cohorts based [12] C. Greenman, P. Stephens, R. Smith, G. L. Dalgliesh, C. Hunter, on the stratification of patients in accordance with their genomics G. Bignell, H. Davies, J. Teague, A. Butler, C. Stevens, S. Edkins, S. and biomarkers profiles. O’Meara, I. Vastrik, E. E. Schmidt, T. Avis, S. Barthorpe, G. Bhamra, G. Buck, B. Choudhury, J. Clements, J. Cole, E. Dicks, S. Forbes, K. Gray, K. Halliday, R. Harrison, K. Hills, J. Hinton, A. Jenkinson, D. Acknowledgements Jones, et al., Nature 2007, 446, 153. [13] a) L. B. Alexandrov, S. Nik-Zainal, D. C. Wedge, S. A. Aparicio, S. R.S.-F. and H.F.F. thank the following funding agencies for their generous support: The MultiNano@MBM project supported by The Is- Behjati, A. V. Biankin, G. R. Bignell, N. Bolli, A. Borg, A. L. Børresen- raeli Ministry of Health and The Fundação para a Ciência e Tecnologia- Dale, S. Boyault, B. Burkhardt, A. P. Butler, C. Caldas, H. R. Davies, C. Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES) un- Desmedt, R. Eils, J. E. Eyfjörd, J. A. Foekens, M. Greaves, F. Hosoda, der the framework of EuroNanoMed-II (ENMed/0051/2016); “la Caixa” B. Hutter, T. Ilicic, S. Imbeaud, M. Imielinski, N. Jäger, D. T. Jones, Foundation under the framework of the Healthcare Research call 2019 D. Jones, S. Knappskog, M. Kool, et al., Nature 2013, 500, 415; (LCF/PR/HR19/52160021; NanoPanther), CaixaImpulse CF01-00014 (Co- b)E.Tran,S.Turcotte,A.Gros,P.F.Robbins,Y.C.Lu,M.E.Dudley, Vax). In addition, R.S.-F. thanks the generous financial support from the J. R. Wunderlich, R. P. Somerville, K. Hogan, C. S. Hinrichs, M. R. European Research Council (ERC), PoC Grant Agreement no. 862580 – Parkhurst, J. C. Yang, S. A. Rosenberg, Science 2014, 344,641. 3DCanPredict and ERC Advanced Grant Agreement no. [835227] – [14] M. J. Scanlan, A. O. Gure, A. A. Jungbluth, L. J. Old, Y. T. Chen, Im- 3DBrainStrom, the Israel Science Foundation (1969/18), the Melanoma munol. Rev. 2002, 188, 22. Research Alliance (Established Investigator Award n°615808 to R.S.-F.), [15] A. Passarelli, F. Mannavola, L. S. Stucci, M. Tucci, F. Silvestris, On- the Israel Cancer Research Fund (ICRF) Professorship award (n° PROF- cotarget 2017, 8, 106132. 18-682), the Morris Kahn Foundation, and The NOFAR incentive pro- [16] O. L. Caballero, Y. T. Chen, Cancer Sci. 2009, 100, 2014. gram on COVID-19 by the Israel Innovation Authority supported by Merck [17] E. F. Velazquez, A. A. Jungbluth, M. Yancovitz, S. Gnjatic, S. Adams, Group. D.V. is grateful to the Rothschild Foundation (IL) for funding her D. O’Neill, K. Zavilevich, T. Albukh, P. Christos, M. Mazumdar, A. Ph.D. scholarship. B.C. is supported by the FCT-MCTES (Ph.D. Fellowship Pavlick, D. Polsky, R. Shapiro, R. Berman, J. Spira, K. Busam, I. Os- SFRH/BD/131969/2017). man, N. Bhardwaj, Cancer Immun. 2007, 7, 11. [18] a) P. van der Bruggen, C. Traversari, P. Chomez, C. Lurquin, E. De Conflict of Interest Plaen, B. Van den Eynde, A. Knuth, T. Boon, Science 1991, 254, 1643; b) P. Chomez, O. De Backer, M. Bertrand, E. De Plaen, T. Boon, S. The authors declare no conflict of interest. Lucas, Cancer Res. 2001, 61, 5544. [19] a) C. Farina, P. van der Bruggen, P. Boël, G. Parmiani, M. Sensi, Int. Keywords Immunol. 1996, 8, 1463; b) M. Ruault, P. van der Bruggen, M. E. Brun,S.Boyle,G.Roizès,A.DeSario,Eur. J. Hum. Genet. 2002, 10, cancer vaccines, immunotherapy, nanotechnology, tumor immune 833. microenvironment [20] a) A. V. Bazhin, N. Wiedemann, M. Schnölzer, D. Schadendorf, S. B. Eichmüller, Cancer Lett. 2007, 251, 258; b) M. F. Gjerstorff, H. J. Received: June 23, 2020 Ditzel, Tissue Antigens 2008, 71, 187. Revised: August 4, 2020 [21] a) C. Barrow, J. Browning, D. MacGregor, I. D. Davis, S. Sturrock, A. Published online: A. Jungbluth, J. Cebon, Clin. Cancer Res. 2006, 12, 764; b) F. S. Hodi, Clin. Cancer Res. 2006, 12, 673. [22] N. G. Ordóñez, Hum. Pathol. 2014, 45, 191. [23] S. G. Schaed, V. M. Klimek, K. S. Panageas, C. M. Musselli, L. But- [1] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, A. Jemal, terworth, W. J. Hwu, P. O. Livingston, L. Williams, J. J. Lewis, A. N. Ca—Cancer J. Clin. 2018, 68, 394. Houghton, P. B. Chapman, Clin. Cancer Res. 2002, 8, 967.

[24] B. J. Pak, W. Chu, S. J. Lu, R. S. Kerbel, Y. Ben-David, Cancer Metas- [47] J. D. Shields, I. C. Kourtis, A. A. Tomei, J. M. Roberts, M. A. Swartz, tasis Rev. 2001, 20, 27. Science 2010, 328, 749. [25] D. N. Khalil, N. Suek, L. F. Campesato, S. Budhu, D. Redmond, R. [48] H. Li, A. M. van der Leun, I. Yofe, Y. Lubling, D. Gelbard-Solodkin, A. M. Samstein, C. Krishna, K. S. Panageas, M. Capanu, S. Houghton, C. J. van Akkooi, M. van den Braber, E. A. Rozeman, J. Haanen, C. U. D. Hirschhorn, R. Zappasodi, R. Giese, B. Gasmi, M. Schneider, A. Blank, H. M. Horlings, E. David, Y. Baran, A. Bercovich, A. Lifshitz, Gupta, J. J. Harding, J. A. Moral, V. P. Balachandran, J. D. Wolchok, T. N. Schumacher, A. Tanay, I. Amit, Cell 2019, 176, 775. T. Merghoub, J. Clin. Invest. 2019, 129, 3435. [49] A. Constantinidou, C. Alifieris, D. T. Trafalis, Pharmacol. Ther. 2019, [26] a) P. G. Coulie, V. Brichard, A. Van Pel, T. Wölfel, J. Schneider, C. 194, 84. Traversari, S. Mattei, E. De Plaen, C. Lurquin, J. P. Szikora, J. C. Re- [50] a) Z. Gatalica, C. Snyder, T. Maney, A. Ghazalpour, D. A. Holterman, nauld, T. Boon, J. Exp. Med. 1994, 180, 35; b) Y.Kawakami, S. Eliyahu, N. Xiao, P. Overberg, I. Rose, G. D. Basu, S. Vranic, H. T. Lynch, D. K. Sakaguchi, P. F. Robbins, L. Rivoltini, J. R. Yannelli, E. Appella, S. D. Von Hoff, O. Hamid, Cancer Epidemiol. Biomarkers Prev. 2014, A. Rosenberg, J. Exp. Med. 1994, 180, 347. 23, 2965; b) M. Ahmadzadeh, L. A. Johnson, B. Heemskerk, J. R. [27] K. Fukuda, T. Funakoshi, T. Sakurai, Y. Nakamura, M. Mori, K. Wunderlich, M. E. Dudley, D. E. White, S. A. Rosenberg, Blood 2009, Tanese, A. Tanikawa, J. Taguchi, T. Fujita, M. Okamoto, M. Amagai, 114, 1537. Y. Kawakami, Melanoma Res. 2017, 27, 326. [51] J. L. Messina, D. A. Fenstermacher, S. Eschrich, X. Qu, A. E. [28] E. Gilboa, Immunity 1999, 11, 263. Berglund, M. C. Lloyd, M. J. Schell, V. K. Sondak, J. S. Weber, J. J. [29] T. Blanchard, P. K. Srivastava, F. Duan, Clin. Dermatol. 2013, 31, 179. Mulé, Sci. Rep. 2012, 2, 765. [30] T. N. Schumacher, R. D. Schreiber, Science 2015, 348, 69. [52] R. Cabrita, M. Lauss, A. Sanna, M. Donia, M. Skaarup Larsen, S. Mi- [31] a) S. Champiat, C. Ferté, S. Lebel-Binay, A. Eggermont, J. C. So- tra, I. Johansson, B. Phung, K. Harbst, J. Vallon-Christersson, A. van ria, Oncoimmunology 2014, 3, e27817; b) A. Snyder, V. Makarov, T. Schoiack, K. Lovgren, S. Warren, K. Jirstrom, H. Olsson, K. Pietras, Merghoub, J. Yuan, J. M. Zaretsky, A. Desrichard, L. A. Walsh, M. A. C. Ingvar, K. Isaksson, D. Schadendorf, H. Schmidt, L. Bastholt, A. Postow, P. Wong, T. S. Ho, T. J. Hollmann, C. Bruggeman, K. Kan- Carneiro, J. A. Wargo, I. M. Svane, G. Jonsson, Nature 2020, 577, nan, Y. Li, C. Elipenahli, C. Liu, C. T. Harbison, L. Wang, A. Ribas, J. 561. D. Wolchok, T. A. Chan, N. Engl. J. Med. 2014, 371, 2189. [53] F. Petitprez, A. de Reynies, E. Z. Keung, T. W. Chen, C. M. Sun, J. [32] D. T. Le, J. N. Uram, H. Wang, B. R. Bartlett, H. Kemberling, A. D. Calderaro, Y. M. Jeng, L. P. Hsiao, L. Lacroix, A. Bougouin, M. Mor- Eyring, A. D. Skora, B. S. Luber, N. S. Azad, D. Laheru, B. Biedrzycki, eira, G. Lacroix, I. Natario, J. Adam, C. Lucchesi, Y. H. Laizet, M. R. C. Donehower, A. Zaheer, G. A. Fisher, T. S. Crocenzi, J. J. Lee, S. Toulmonde, M. A. Burgess, V. Bolejack, D. Reinke, K. M. Wani, W. L. M. Duffy, R. M. Goldberg, A. de la Chapelle, M. Koshiji, F. Bhaijee, Wang, A. J. Lazar, C. L. Roland, J. A. Wargo, A. Italiano, C. Sautes- T.Huebner,R.H.Hruban,L.D.Wood,N.Cuka,D.M.Pardoll,N. Fridman, H. A. Tawbi, W. H. Fridman, Nature 2020, 577, 556. Papadopoulos, K. W. Kinzler, S. Zhou, T. C. Cornish, et al., N. Engl. [54] J. Da Gama Duarte, J. M. Peyper, J. M. Blackburn, Mamm. Genome J. Med. 2015, 372, 2509. 2018, 29, 790. [33] P. Kvistborg, D. Philips, S. Kelderman, L. Hageman, C. Ottensmeier, [55] R. Somasundaram, G. Zhang, M. Fukunaga-Kalabis, M. Perego, C. D. Joseph-Pietras, M. J. Welters, S. van der Burg, E. Kapiteijn, O. Krepler, X. Xu, C. Wagner, D. Hristova, J. Zhang, T. Tian, Z. Wei, Q. Michielin, E. Romano, C. Linnemann, D. Speiser, C. Blank, J. B. Haa- Liu, K. Garg, J. Griss, R. Hards, M. Maurer, C. Hafner, M. Mayer- nen, T. N. Schumacher, Sci. Transl. Med. 2014, 6, 254ra128. höfer, G. Karanikas, A. Jalili, V. Bauer-Pohl, F. Weihsengruber, K. Rap- [34] a) Y. Matsumura, H. Maeda, Cancer Res. 1986, 46, 6387; b) F. Yuan, persberger, J. Koller, R. Lang, C. Hudgens, G. Chen, M. Tetzlaff, L. M. Dellian, D. Fukumura, M. Leunig, D. A. Berk, V. P. Torchilin, R. K. Wu, D. T. Frederick, et al., Nat. Commun. 2017, 8, 607. Jain, Cancer Res. 1995, 55, 3752. [56] K. Sili¸na, A. Soltermann, F. M. Attar, R. Casanova, Z. M. Uckeley, [35] S. Romero-Garcia, J. S. Lopez-Gonzalez, J. L. Báez-Viveros, D. H. Thut, M. Wandres, S. Isajevs, P. Cheng, A. Curioni-Fontecedro, P. Aguilar-Cazares, H. Prado-Garcia, Cancer Biol. Ther. 2011, 12, 939. Foukas, M. P. Levesque, H. Moch, A. Line,¯ M. van den Broek, Cancer [36] a) T. F. Gajewski, Mol. Oncol. 2012, 6, 242; b) Y. Ma, G. V. Shurin, Z. Res. 2018, 78, 1308. Peiyuan, M. R. Shurin, J. Cancer 2013, 4, 36. [57] a) A. Cipponi, M. Mercier, T. Seremet, J. F. Baurain, I. Théate, J. van [37] a) J. Banchereau, J. Fay, V. Pascual, A. K. Palucka, Ann. N. Y. Acad. den Oord, M. Stas, T. Boon, P. G. Coulie, N. van Baren, Cancer Res. Sci. 2003, 987, 180; b) K. Palucka, J. Banchereau, Nat. Rev. Cancer 2012, 72, 3997; b) S. R. Selitsky, L. E. Mose, C. C. Smith, S. Chai, K. 2012, 12, 265. A. Hoadley, D. P. Dittmer, S. J. Moschos, J. S. Parker, B. G. Vincent, [38] O. Preynat-Seauve, E. Contassot, P. Schuler, L. E. French, B. Huard, Genome Med. 2019, 11, 36. Melanoma Res. 2007, 17, 169. [58] R. Cabrita, M. Lauss, A. Sanna, M. Donia, M. Skaarup Larsen, S. Mi- [39] J. F. Miller, M. Sadelain, Cancer Cell 2015, 27, 439. tra, I. Johansson, B. Phung, K. Harbst, J. Vallon-Christersson, A. van [40] M. Tucci, A. Passarelli, F. Mannavola, C. Felici, L. S. Stucci, M. Cives, Schoiack, K. Lövgren, S. Warren, K. Jirström, H. Olsson, K. Pietras, F. Silvestris, Front. Oncol. 2019, 9, 1148. C. Ingvar, K. Isaksson, D. Schadendorf, H. Schmidt, L. Bastholt, A. [41] J. M. Tran Janco, P. Lamichhane, L. Karyampudi, K. L. Knutson, J. Carneiro, J. A. Wargo, I. M. Svane, G. Jönsson, Nature 2020, 577, Immunol. 2015, 194, 2985. 561. [42] D. E. Speiser, P. C. Ho, G. Verdeil, Nat. Rev. Immunol. 2016, 16, 599. [59] J. Griss, W. Bauer, C. Wagner, M. Simon, M. Chen, K. Grabmeier- [43] C. Aspord, M. T.Leccia, J. Charles, J. Plumas, Oncoimmunology 2014, Pfistershammer, M. Maurer-Granofszky, F. Roka, T. Penz, C. Bock, 3, e27402. G. Zhang, M. Herlyn, K. Glatz, H. Läubli, K. D. Mertz, P. Petzelbauer, [44] a) A. Covre, S. Coral, A. M. Di Giacomo, P. Taverna, M. Azab, M. T. Wiesner, M. Hartl, W. F. Pickl, R. Somasundaram, P. Steinberger, Maio, Semin. Oncol. 2015, 42, 506; b) F. Mannavola, M. Tucci, C. S. N. Wagner, Nat. Commun. 2019, 10, 4186. Felici, S. Stucci, F. Silvestris, Expert Rev. Clin. Immunol. 2016, 12, 79. [60] F. Veglia, M. Perego, D. Gabrilovich, Nat. Immunol. 2018, 19, 108. [45] M. Antohe, R. I. Nedelcu, L. Nichita, C. G. Popp, M. Cioplea, A. [61] M. Z. Noman, G. Desantis, B. Janji, M. Hasmim, S. Karray, P. Brinzea, A. Hodorogea, A. Calinescu, M. Balaban, D. A. Ion, C. Di- Dessen, V. Bronte, S. Chouaib, J. Exp. Med. 2014, 211, 781. aconu, C. Bleotu, D. Pirici, S. A. Zurac, G. Turcu, Oncol. Lett. 2019, [62] D. I. Gabrilovich, Cancer Immunol. Res. 2017, 5,3. 17, 4155. [63] a) H. Jiang, C. Gebhardt, L. Umansky, P. Beckhove, T. J. Schulze, J. [46] M. J. Gooden, G. H. de Bock, N. Leffers, T. Daemen, H. W. Nijman, Utikal, V. Umansky, Int. J. Cancer 2015, 136, 2352; b) K. R. Jordan, Br. J. Cancer 2011, 105, 93. R. N. Amaria, O. Ramirez, E. B. Callihan, D. Gao, M. Borakove, E.

Manthey, V. F. Borges, M. D. McCarter, Cancer Immunol. Im- Larbi, B. Schilling, D. Schadendorf, J. D. Wolchok, C. U. Blank, G. munother. 2013, 62, 1711. Pawelec, C. Garbe, B. Weide, Eur. J. Cancer 2016, 64, 116. [64] a) M. Sade-Feldman, J. Kanterman, Y. Klieger, E. Ish-Shalom, M. [84] P. Girard, J. Charles, C. Cluzel, E. Degeorges, O. Manches, J. Plumas, Olga, A. Saragovi, H. Shtainberg, M. Lotem, M. Baniyash, Clin. Can- F. De Fraipont, M.-T. Leccia, S. Mouret, L. Chaperot, C. Aspord, On- cer Res. 2016, 22, 5661; b) J. Weber, G. Gibney, R. Kudchadkar, B. Yu, coImmunology 2019, 8, 1601483. P. Cheng, A. J. Martinez, J. Kroeger, A. Richards, L. McCormick, V. [85] S. Gurzu, M. A. Beleaua, I. Jung, Rom. J. Morphol. Embryol. 2018, 59, Moberg, H. Cronin, X. Zhao, M. Schell, Y. A. Chen, Cancer Immunol. 23. Res. 2016, 4, 345; c) C. Gebhardt, A. Sevko, H. Jiang, R. Lichten- [86] L. Ziani, T. B. Safta-Saadoun, J. Gourbeix, A. Cavalcanti, C. Robert, berger, M. Reith, K. Tarnanidis, T. Holland-Letz, L. Umansky, P. Beck- G. Favre, S. Chouaib, J. Thiery, Oncotarget 2017, 8, 19780. hove, A. Sucker, D. Schadendorf, J. Utikal, V. Umansky, Clin. Cancer [87] S. Zurac, M. Neagu, C. Constantin, M. Cioplea, R. Nedelcu, A. Bas- Res. 2015, 21, 5453. tian, C. Popp, L. Nichita, R. Andrei, T. Tebeica, C. Tanase, V. Chitu, C. [65] A. Mantovani, F. Marchesi, A. Malesci, L. Laghi, P. Allavena, Nat. Caruntu, M. Ghita, C. Popescu, D. Boda, B. Mastalier, N. Maru, C. Rev. Clin. Oncol. 2017, 14, 399. Daha, B. Andreescu, I. Marinescu, A. Rebosapca, F. Staniceanu, G. [66] a) Q. C. Cai, H. Liao, S. X. Lin, Y. Xia, X. X. Wang, Y. Gao, Z. X. Lin, J. Negroiu, D. A. Ion, D. Nikitovic, G. N. Tzanakakis, D. A. Spandidos, B. Lu, H. Q. Huang, Med. Oncol. 2012, 29, 2317; b) M. M. Escribese, A. M. Tsatsakis, Oncol. Lett. 2016, 11, 3354. M. Casas, A. L. Corbí, Immunobiology 2012, 217, 1233; c) H. Shime, [88] L. Ziani, S. Chouaib, J. Thiery, Front. Immunol. 2018, 9, 414. M. Matsumoto, H. Oshiumi, S. Tanaka, A. Nakane, Y. Iwakura, H. [89] a) B. Erdogan, D. J. Webb, Biochem. Soc. Trans. 2017, 45, 229; b) S. Tahara, N. Inoue, T. Seya, Proc. Natl. Acad. Sci. U. S. A. 2012, 109, Hsu, J. L. Borke, J. B. Lewis, B. Singh, A. C. Aiken, C. T. Huynh, G. 2066. S. Schuster, G. B. Caughman, D. P. Dickinson, A. K. Smith, T. Osaki, [67] A. Sica, M. Erreni, P. Allavena, C. Porta, Cell. Mol. Life Sci. 2015, 72, X. F. Wang, Cell Proliferation 2002, 35, 183. 4111. [90] M. Shen, P. Hu, F. Donskov, G. Wang, Q. Liu, J. Du, PLoS One 2014, [68] J. MacMicking, Q. W. Xie, C. Nathan, Annu. Rev. Immunol. 1997, 15, 9, e98259. 323. [91] Z. Granot, E. Henke, E. A. Comen, T. A. King, L. Norton, R. Benezra, [69] R. Cornelissen, L. A. Lievense, A. P. Maat, R. W. Hendriks, H. C. Cancer Cell 2011, 20, 300. Hoogsteden, A. J. Bogers, J. P. Hegmans, J. G. Aerts, PLoS One 2014, [92] S. B. Coffelt, M. D. Wellenstein, K. E. de Visser, Nat. Rev. Cancer 9, e106742. 2016, 16, 431. [70] a) S. K. Biswas, A. Mantovani, Nat. Immunol. 2010, 11, 889; b) J. [93] a) Y.Liu, Y.Gu, Y.Han, Q. Zhang, Z. Jiang, X. Zhang, B. Huang, X. Xu, Condeelis, J. W. Pollard, Cell 2006, 124, 263. J. Zheng, X. Cao, Cancer Cell 2016, 30, 243; b) J. Park, R. W. Wysocki, [71] G. Erdag, J. T. Schaefer, M. E. Smolkin, D. H. Deacon, S. M. Shea, Z. Amoozgar, L. Maiorino, M. R. Fein, J. Jorns, A. F. Schott, Y. L. T. Dengel, J. W. Patterson, C. L. Slingluff, Jr., Cancer Res. 2012, 72, Kinugasa-Katayama, Y. Lee, N. H. Won, E. S. Nakasone, S. A. Hearn, 1070. V. Küttner, J. Qiu, A. S. Almeida, N. Perurena, K. Kessenbrock, M. S. [72] G. Botti, M. Cerrone, G. Scognamiglio, A. Anniciello, P. A. Ascierto, Goldberg, M. Egeblad, Sci. Transl. Med. 2016, 8, 361ra138. M. Cantile, J. Immunotoxicol. 2013, 10, 235. [94] J. Landsberg, T. Tüting, R. L. Barnhill, C. Lugassy, J. Invest. Dermatol. [73] P. Chen, Y. Huang, R. Bong, Y. Ding, N. Song, X. Wang, X. Song, Y. 2016, 136, 372. Luo, Clin. Cancer Res. 2011, 17, 7230. [95] Z. Granot, Z. G. Fridlender, Cancer Res. 2015, 75, 4441. [74] F. Garrido, I. Algarra, A. M. García-Lora, Cancer Immunol. Im- [96] L. Andzinski, N. Kasnitz, S. Stahnke, C. F. Wu, M. Gereke, M. von munother. 2010, 59, 1601. Köckritz-Blickwede, B. Schilling, S. Brandau, S. Weiss, J. Jablonska, [75] T. Lakshmikanth, S. Burke, T. H. Ali, S. Kimpfler, F. Ursini, L. Ruggeri, Int. J. Cancer 2016, 138, 1982. M. Capanni, V. Umansky, A. Paschen, A. Sucker, D. Pende, V. Groh, [97] T. A. Gonda, S. Tu, T. C. Wang, Cell Cycle 2009, 8, 2005. R. Biassoni, P. Höglund, M. Kato, K. Shibuya, D. Schadendorf, A. [98] M. Surcel, C. Constantin, C. Caruntu, S. Zurac, M. Neagu, J. Im- Anichini, S. Ferrone, A. Velardi, K. Kärre, A. Shibuya, E. Carbone, F. munol. Res. 2017, 2017,1. Colucci, J. Clin. Invest. 2009, 119, 1251. [99] W. Yao, Y. Li, L. Zeng, X. Zhang, Z. Zhou, M. Zheng, H. Wan, Oncol. [76] A. Marcus, B. G. Gowen, T. W. Thompson, A. Iannello, M. Ardolino, Rep. 2019, 42, 370. W. Deng, L. Wang, N. Shifrin, D. H. Raulet, Adv. Immunol. 2014, 122, [100] N. Nagarsheth, M. S. Wicha, W. Zou, Nat. Rev. Immunol. 2017, 17, 91. 559. [77] R. Wehner, K. Dietze, M. Bachmann, M. Schmitz, J. Innate Immun. [101] N. Jacquelot, C. P. M. Duong, G. T. Belz, L. Zitvogel, Front. Immunol. 2011, 3, 258. 2018, 9, 2480. [78] a) D. I. Godfrey, S. Stankovic, A. G. Baxter, Nat. Immunol. 2010, 11, [102] a) R.-R. Ji, S. D. Chasalow, L. Wang, O. Hamid, H. Schmidt, J. 197; b) B. J. Wolf, J. E. Choi, M. A. Exley, Front. Immunol. 2018, 9, Cogswell, S. Alaparthy, D. Berman, M. Jure-Kunkel, N. O. Siemers, 384. J. R. Jackson, V. Shahabi, Cancer Immunol. Immunother. 2012, 61, [79] T. Kawano, J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. 1019; b) B. D. Shields, F. Mahmoud, E. M. Taylor, S. D. Byrum, D. Ueno, R. Nakagawa, H. Sato, E. Kondo, H. Koseki, M. Taniguchi, Sengupta, B. Koss, G. Baldini, S. Ransom, K. Cline, S. G. Mackin- Science 1997, 278, 1626. tosh, R. D. Edmondson, S. Shalin, A. J. Tackett, Sci. Rep. 2017, 7, [80] M. A. Exley, P. Friedlander, N. Alatrakchi, L. Vriend, S. Yue, T. Sasada, 807. W. Zeng, Y. Mizukami, J. Clark, D. Nemer, K. LeClair, C. Canning, H. [103] D. Bedognetti, T. L. Spivey, Y.Zhao, L. Uccellini, S. Tomei, M. E. Dud- Daley, G. Dranoff, A. Giobbie-Hurder, F. S. Hodi, J. Ritz, S. P. Balk, ley, M. L. Ascierto, V. De Giorgi, Q. Liu, L. G. Delogu, M. Sommariva, Clin. Cancer Res. 2017, 23, 3510. M. R. Sertoli, R. Simon, E. Wang, S. A. Rosenberg, F. M. Marincola, [81] M. B. Brenner, J. McLean, D. P. Dialynas, J. L. Strominger, J. A. Smith, Br. J. Cancer 2013, 109, 2412. F. L. Owen, J. G. Seidman, S. Ip, F. Rosen, M. S. Krangel, Nature [104] M. Bar-Eli, Pathobiology 1999, 67, 12. 1986, 322, 145. [105] M. Caunt, L. Hu, T. Tang, P. C. Brooks, S. Ibrahim, S. Karpatkin, Can- [82] H. Chen, W. He, Cell. Mol. Immunol. 2018, 15, 411. cer Res. 2006, 66, 4125. [83] K. Wistuba-Hamprecht, A. Martens, K. Haehnel, M. Geukes Foppen, [106] J. Klarquist, K. Tobin, P. Farhangi Oskuei, S. W. Henning, M. F. Fer- J. Yuan, M. A. Postow, P. Wong, E. Romano, A. Khammari, B. Dreno, nandez, E. R. Dellacecca, F. C. Navarro, J. M. Eby, S. Chatterjee, S. M. Capone, P. A. Ascierto, I. Demuth, E. Steinhagen-Thiessen, A. Mehrotra,J.I.Clark,I.C.LePoole,Cancer Res. 2016, 76, 6230.

[107] a) J. Conniot, A. Scomparin, C. Peres, E. Yeini, S. Pozzi, A. I. Matos, W. S. Din, T. Davis-Smith, D. M. Anderson, S. D. Gimpel, T. A. Sato, R. Kleiner, L. I. Moura, E. Zupanˇciˇc, A. S. Viana, Nat. Nanotechnol. C. R. Maliszewski, C. I. Brannan, N. G. Copeland, N. A. Jenkins, Eur. 2019, 14, 891; b) T. Gargett, M. N. Abbas, P. Rolan, J. D. Price, K. M. J. Immunol. 1993, 23, 2631. Gosling, A. Ferrante, A. Ruszkiewicz, I. I. C. Atmosukarto, J. Altin, [121] a) R.-M. Lu, Y.-C. Hwang, I.-J. Liu, C.-C. Lee, H.-Z. Tsai, H.-J. Li, H.-C. C. R. Parish, M. P. Brown, Cancer Immunol. Immunother. 2018, 67, Wu, J. Biomed. Sci. 2020, 27, 1; b) H. Kaplon, M. Muralidharan, Z. 1461; c) O. Gasser, K. J. Sharples, C. Barrow, G. M. Williams, E. Schneider, J. M. Reichert, mAbs 2020, 12, 1703531. Bauer, C. E. Wood, B. Mester, M. Dzhelali, G. Caygill, J. Jones, C. M. [122] Y. Diesendruck, I. Benhar, Drug Resist. Update. 2017, 30,39. Hayman, V. A. Hinder, J. Macapagal, M. McCusker, R. Weinkove, G. [123] K. Marhelava, Z. Pilch, M. Bajor, A. Graczyk-Jarzynka, R. Zagozdzon, F. Painter, M. A. Brimble, M. P. Findlay, P. R. Dunbar, I. F. Hermans, Cancers 2019, 11, 1756. Cancer Immunol. Immunother. 2018, 67, 285. [124] F. Marcucci, C. Rumio, A. Corti, Biochim. Biophys. Acta Rev. Cancer [108] a) J. Larkin, V. Chiarion-Sileni, R. Gonzalez, J.-J. Grob, P. Rutkowski, 2017, 1868, 571. C. D. Lao, C. L. Cowey, D. Schadendorf, J. Wagstaff, R. Dummer, P. F. [125] M. Bhandaru, A. Rotte, Methods Mol. Biol. 2019, 1904, 83. Ferrucci, M. Smylie, D. Hogg, A. Hill, I. Márquez-Rodas, J. Haanen, [126] G. T. Gibney, L. M. Weiner, M. B. Atkins, Lancet Oncol. 2016, 17, M. Guidoboni, M. Maio, P. Schöffski, M. S. Carlino, C. Lebbé, G. e542. McArthur, P. A. Ascierto, G. A. Daniels, G. V. Long, L. Bastholt, J. [127] C. E. Rudd, A. Taylor, H. Schneider, Immunol. Rev. 2009, 229, 12. I. Rizzo, A. Balogh, A. Moshyk, F. S. Hodi, J. D. Wolchok, N. Engl. [128] K. S. Peggs, S. A. Quezada, C. A. Chambers, A. J. Korman, J. P. Alli- J. Med. 2019, 381, 1535; b) S. A. Rosenberg, N. P. Restifo, Science son, J. Exp. Med. 2009, 206, 1717. 2015, 348, 62. [129] C. Robert, L. Thomas, I. Bondarenko, S. O’Day, J. Weber, C. Garbe, [109] a) R. Bos, K. L. Marquardt, J. Cheung, L. A. Sherman, OncoImmunol- C. Lebbe, J.-F. Baurain, A. Testori, J.-J. Grob, N. Engl. J. Med. 2011, ogy 2012, 1, 1239; b) B. Engels, V. H. Engelhard, J. Sidney, A. Sette, 364, 2517. D. C. Binder, R. B. Liu, D. M. Kranz, S. C. Meredith, D. A. Rowley, H. [130] D. Schadendorf, F. S. Hodi, C. Robert, J. S. Weber, K. Margolin, O. Schreiber, Cancer Cell 2013, 23, 516. Hamid, D. Patt, T. T. Chen, D. M. Berman, J. D. Wolchok, J. Clin. [110] D. E. Speiser, P. Romero, Semin. Immunol. 2010, 22, 144. Oncol. 2015, 33, 1889. [111] A. Kumar, J. Zhang, F. S. Yu, Immunology 2006, 117, 11. [131] A. Lee, M. B. Djamgoz, Cancer Treat. Rev. 2018, 62, 110. [112] S. G. Reed, S. Bertholet, R. N. Coler, M. Friede, Trends Immunol. [132] F. Le Du, B. L. Eckhardt, B. Lim, J. K. Litton, S. Moulder, F. Meric- 2009, 30, 23. Bernstam, A. M. Gonzalez-Angulo, N. T. Ueno, Oncotarget 2015, 6, [113] A. Azizi, S. Aucoin, H. Tadesse, R. Frost, M. Ghorbani, C. Soare, T. 12890. Naas, F. Diaz-Mitoma, Genet Vaccines Ther. 2005, 3,7. [133] S. M. Toor, E. Elkord, Immunol. Cell Biol. 2018, 96, 888. [114] M. M. Linehan, T. H. Dickey, E. S. Molinari, M. E. Fitzgerald, O. [134] C. Robert, A. Ribas, J. D. Wolchok, F. S. Hodi, O. Hamid, R. Kefford, Potapova, A. Iwasaki, A. M. Pyle, Sci. Adv. 2018, 4, e1701854. J. S. Weber, A. M. Joshua, W.-J. Hwu, T. C. Gangadhar, A. Patnaik, [115] J. M. Silva, M. Videira, R. Gaspar, V.Préat, H. F. Florindo, J. Controlled R. Dronca, H. Zarour, R. W. Joseph, P. Boasberg, B. Chmielowski, C. Release 2013, 168, 179. Mateus, M. A. Postow, K. Gergich, J. Elassaiss-Schaap, X. N. Li, R. [116] a) W. Ebel, E. L. Routhier, B. Foley, S. Jacob, J. M. McDonough, R. Iannone, S. W. Ebbinghaus, S. P. Kang, A. Daud, Lancet 2014, 384, K. Patel, H. A. Turchin, Q. Chao, J. B. Kline, L. J. Old, Cancer Immun. 1109. 2007, 7, 6; b) J. Burton, D. Mishina, T. Cardillo, K. Lew, A. Rubin, D. [135] G. V. Long, V. Atkinson, J. S. Cebon, M. B. Jameson, B. M. Fitzharris, Goldenberg, D. Gold, Clin. Cancer Res. 1999, 5, 3065s. C. M. McNeil, A. G. Hill, A. Ribas, M. B. Atkins, J. A. Thompson, [117] a) R.-D. Hofheinz, S.-E. Al-Batran, F. Hartmann, G. Hartung, D. Lancet Oncol. 2017, 18, 1202. Jäger, C. Renner, P. Tanswell, U. Kunz, A. Amelsberg, H. Kuthan, [136] a) S. Spranger, H. K. Koblish, B. Horton, P. A. Scherle, R. Newton, Onkologie 2003, 26, 44; b) D. A. Reardon, M. R. Zalutsky, D. D. T. F. Gajewski, J. Immunother. Cancer 2014, 2, 3; b) A. J. Minn, E. J. Bigner, Expert Rev. Anticancer Ther. 2007, 7, 675. Wherry, Cell 2016, 165, 272. [118] a) R. A. Burger, M. F. Brady, M. A. Bookman, G. F. Fleming, B. J. [137] a) J. A. Marin-Acevedo, R. M. Chirila, R. S. Dronca, Mayo Clin. Monk, H. Huang, R. S. Mannel, H. D. Homesley, J. Fowler, B. E. Proc. 2019, 94, 1321; b) N. Vigneron, Biomed. Res. Inst. 2015, 2015, Greer, N. Engl. J. Med. 2011, 365, 2473; b) K. P. Papadopoulos, R. 9485501. K. Kelley, A. W. Tolcher, A. R. Razak, K. Van Loon, A. Patnaik, P. L. [138] a) A. C. Anderson, N. Joller, V. K. Kuchroo, Immunity 2016, 44, 989; Bedard, A. A. Alfaro, M. Beeram, L. Adriaens, C. M. Brownstein, I. b) J. B. Williams, B. L. Horton, Y. Zheng, Y. Duan, J. D. Powell, T. F. Lowy, A. Kostic, P. A. Trail, B. Gao, A. T. DiCioccio, L. L. Siu, Clin. Gajewski, J. Exp. Med. 2017, 214, 381. Cancer Res. 2016, 22, 1348. [139] a) R.-Y. Huang, C. Eppolito, S. Lele, P. Shrikant, J. Matsuzaki, K. [119] a) W. Zhang, M. Gordon, H. J. Lenz, Ann. Med. 2006, 38, 545; b) W. Odunsi, Oncotarget 2015, 6, 27359; b) S. R. Woo, M. E. Turnis, M. V. Dougall, M. Chaisson, Clin. Calcium 2006, 16, 627. Goldberg, J. Bankoti, M. Selby, C. J. Nirschl, M. L. Bettini, D. M. Gra- [120] a) F. S. Hodi, S. J. O’Day, D. F. McDermott, R. W. Weber, J. A. Sos- vano, P. Vogel, C. L. Liu, S. Tangsombatvisit, J. F. Grosso, G. Netto, man, J. B. Haanen, R. Gonzalez, C. Robert, D. Schadendorf, J. C. M. P. Smeltzer, A. Chaux, P. J. Utz, C. J. Workman, D. M. Pardoll, A. Hassel, N. Engl. J. Med. 2010, 363, 711; b) A. I. Daud, J. D. Wol- J. Korman, C. G. Drake, D. A. Vignali, Cancer Res. 2012, 72, 917. chok, C. Robert, W.-J. Hwu, J. S. Weber, A. Ribas, F. S. Hodi, A. M. [140] a) C. Donini, L. D’Ambrosio, G. Grignani, M. Aglietta, D. Sangiolo, Joshua, R. Kefford, P. Hersey, R. Joseph, T. C. Gangadhar, R. Dronca, J. Thorac. Dis. 2018, 10, S1581; b) C. Solinas, P. De Silva, D. Bron, K. A. Patnaik, H. Zarour, C. Roach, G. Toland, J. K. Lunceford, X. N. Willard-Gallo, D. Sangiolo, Semin. Oncol. 2019, 46, 372. Li, K. Emancipator, M. Dolled-Filhart, S. P. Kang, S. Ebbinghaus, O. [141] X. Zhou, L. Sun, D. Jing, G. Xu, J. Zhang, L. Lin, J. Zhao, Z. Yao, H. Hamid, J. Clin. Oncol. 2016, 34, 4102; c) M. V. Goldberg, C. G. Drake, Lin, Front. Physiol. 2018, 9, 452. in Cancer Immunology and Immunotherapy, Vol. 344 (Ed: G. Dra- [142] A. Sánchez-Fueyo, J. Tian, D. Picarella, C. Domenig, X. X. Zheng, C. noff), Springer-Verlag, Heidelberg, Berlin, Germany 2011, Ch. 12; A. Sabatos, N. Manlongat, O. Bender, T. Kamradt, V. K. Kuchroo, d) B. D. Curti, M. Kovacsovics-Bankowski, N. Morris, E. Walker, L. Nat. Immunol. 2003, 4, 1093. Chisholm, K. Floyd, J. Walker, I. Gonzalez, T. Meeuwsen, B. A. Fox, [143] A. S. Gautron, M. Dominguez-Villar, M. de Marcken, D. A. Hafler, T. Moudgil, W. Miller, D. Haley, T. Coffey, B. Fisher, L. Delanty-Miller, Eur. J. Immunol. 2014, 44, 2703. N. Rymarchyk, T. Kelly, T. Crocenzi, E. Bernstein, R. Sanborn, W. J. [144] a) J. Fourcade, Z. Sun, M. Benallaoua, P. Guillaume, I. F. Luescher, C. Urba, A. D. Weinberg, Cancer Res. 2013, 73, 7189; e) R. G. Goodwin, Sander, J. M. Kirkwood, V. Kuchroo, H. M. Zarour, J. Exp. Med. 2010,

207, 2175; b) K. Sakuishi, L. Apetoh, J. M. Sullivan, B. R. Blazar, V. [170] L. Diehl, A. Den Boer, E. Van der Voort, C. Melief, R. Offringa, R. K. Kuchroo, A. C. Anderson, J. Exp. Med. 2010, 207, 2187. Toes, J. Mol. Med. 2000, 78, 363. [145] I. Melero, S. Hervas-Stubbs, M. Glennie, D. M. Pardoll, L. Chen, Nat. [171] C.-L. Wang, Y.-T. Wu, C.-A. Liu, M.-W. Lin, C.-J. Lee, L.-T. Huang, K. Rev. Cancer 2007, 7, 95. D. Yang, Pediatrics 2003, 111, e140. [146] J. S. Boomer, J. M. Green, Cold Spring Harb. Perspect. Biol. 2010, 2, [172] R. H. Vonderheide, Annu. Rev. Med. 2020, 71, 47. a002436. [173] a) G. J. van Mierlo, A. T. den Boer, J. P. Medema, E. I. van der Voort, [147] K. E. Brown, Diseases 2018, 6, 41. M. F. Fransen, R. Offringa, C. J. Melief, R. E. Toes, Proc. Natl. Acad. [148] B. Schraven, U. Kalinke, Immunity 2008, 28, 591. Sci.U.S.A.2002, 99, 5561; b) G. L. Beatty, E. G. Chiorean, M. P. Fish- [149] A. D. Weinberg, N. P. Morris, M. Kovacsovics-Bankowski, W. J. Urba, man, B. Saboury, U. R. Teitelbaum, W. Sun, R. D. Huhn, W. Song, D. B. D. Curti, Immunol. Rev. 2011, 244, 218. Li, L. L. Sharp, Science 2011, 331, 1612; c) S. M. Mangsbo, S. Broos, [150] a) I. Gramaglia, A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, E. Fletcher, N. Veitonmäki, C. Furebring, E. Dahlén, P. Norlén, M. M. Croft, J. Immunol. 2000, 165, 3043; b) R. H. Arch, C. B. Thomp- Lindstedt, T. H. Tötterman, P. Ellmark, Clin. Cancer Res. 2015, 21, son, Mol. Cell. Biol. 1998, 18, 558. 1115. [151] J. T. Vetto, S. Lum, A. Morris, M. Sicotte, J. Davis, M. Lemon, A. [174] E. Chru´sciel, Z. Urban-Wójciuk, Ł. Arcimowicz, M. Kurkowiak, J. Weinberg, Am. J. Surg. 1997, 174, 258. Kowalski, M. Gliwinski,´ T. Marjanski,´ W. Rzyman, W. Biernat, R. Dzi- [152] T. So, N. Ishii, Adv. Exp. Med. Biol. 2019, 1189, 53. adziuszko, C. Montesano, R. Bernardini, N. Marek-Trzonkowska, [153] S. Fujiwara, H. Nagai, N. Shimoura, S. Oniki, T. Yoshimoto, C. Cancers 2020, 12, 683. Nishigori, J. Invest. Dermatol. 2014, 134, 1884. [175] a) S. Pilon-Thomas, L. Kuhn, S. Ellwanger, W. Janssen, E. Royster, [154] N. Jahan, H. Talat, W. T. Curry, Neuro. Oncol. 2018, 20, 44. S. Marzban, R. Kudchadkar, J. Zager, G. Gibney, V. K. Sondak, J. We- [155] R. E. Sadun, W.-E. Hsu, N. Zhang, Y.-C. Nien, S. A. Bergfeld, H. ber, J. J. Mulé, A. A. Sarnaik, J. Immunother. 2012, 35, 615; b) L. G. Sabzevari, M. C. Lutsiak, L. Khawli, P. Hu, A. L. Epstein, J. Im- Radvanyi, C. Bernatchez, M. Zhang, P. S. Fox, P. Miller, J. Chacon, R. munother. 2008, 31, 235. Wu, G. Lizee, S. Mahoney, G. Alvarado, M. Glass, V. E. Johnson, J. D. [156] S. A. Ali, M. Ahmad, J. Lynam, C. S. McLean, C. Entwisle, P. Loudon, McMannis, E. Shpall, V. Prieto, N. Papadopoulos, K. Kim, J. Homsi, E.Choolun,S.E.B.McArdle,G.Li,S.Mian,R.C.Rees,Vaccine 2004, A. Bedikian, W. J. Hwu, S. Patel, M. I. Ross, J. E. Lee, J. E. Gershen- 22, 3585. wald, A. Lucci, R. Royal, J. N. Cormier, M. A. Davies, R. Mansaray, [157] W. L. Redmond, M. J. Gough, A. D. Weinberg, Eur. J. Immunol. 2009, O. J. Fulbright, et al., Clin. Cancer Res. 2012, 18, 6758; c) S. A. Rosen- 39, 2184. berg, J. C. Yang, R. M. Sherry, U. S. Kammula, M. S. Hughes, G. Q. [158] a) Z. Guo, X. Wang, D. Cheng, Z. Xia, M. Luan, S. Zhang, PLoS One Phan, D. E. Citrin, N. P. Restifo, P. F. Robbins, J. R. Wunderlich, K. E. 2014, 9, e89350; b) H. Wei, L. Zhao, I. Hellstrom, K. E. Hellstrom, Morton,C.M.Laurencot,S.M.Steinberg,D.E.White,M.E.Dudley, Y. Guo, OncoImmunology 2014, 3, e28248. Clin. Cancer Res. 2011, 17, 4550. [159] R. Alvim, L. Nogueira, P. M. Georgala, A. Somma, K. Nagar, J. [176] S. A. Rosenberg, M. T. Lotze, L. M. Muul, S. Leitman, A. E. Chang, Thomas, S. Ledbai, C. Hudges, A. J. Scherz, S. M. Zanganeh, K. Kim, S. E. Ettinghausen, Y. L. Matory, J. M. Skibber, E. Shiloni, J. T. Vetto, J. Coleman, J. Clin. Oncol. 2020, 38, e17004. C. A. Seipp, C. Simpson, C. M. Reichert, N. Engl. J. Med. 1985, 313, [160] a) S. N. Linch, M. J. Kasiewicz, M. J. McNamara, I. F. Hilgart- 1485. Martiszus, M. Farhad, W. L. Redmond, Proc. Natl. Acad. Sci. U. S. [177] L. M. Muul, P. J. Spiess, E. P. Director, S. A. Rosenberg, J. Immunol. A. 2016, 113, E319; b) K. Garrison, T. Hahn, W.-C. Lee, L. E. Ling, A. 1987, 138, 989. D. Weinberg, E. T. Akporiaye, Cancer Immunol. Immunother. 2012, [178] J. J. Hong, S. A. Rosenberg, M. E. Dudley, J. C. Yang, D. E. White, J. 61, 511. A. Butman, R. M. Sherry, Clin. Cancer Res. 2010, 16, 4892. [161] T. Futagawa, H. Akiba, T. Kodama, K. Takeda, Y. Hosoda, H. Yagita, [179] H. W. H. G. Kessels, M. C. Wolkers, M. D. van den Boom, M. A. van K. Okumura, Int. Immunol. 2002, 14, 275. den Valk, T. N. M. Schumacher, Nat. Immunol. 2001, 2, 957. [162] C. Chester, M. F. Sanmamed, J. Wang, I. Melero, Blood 2018, 131, [180] R. A. Morgan, M. E. Dudley, J. R. Wunderlich, M. S. Hughes, J. C. 49. Yang, R. M. Sherry, R. E. Royal, S. L. Topalían, U. S. Kammula, N. P. [163] R. Houot, H. Kohrt, R. Levy, OncoImmunology 2012, 1, 957. Restifo, Z. Zheng, A. Nahvi, C. R. De Vries, L. J. Rogers-Freezer, S. [164] W. W. Shuford, K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny, A. Mavroukakis, S. A. Rosenberg, Science 2006, 314, 126. A. W. Siadak, T. J. Brown, J. Emswiler, H. Raecho, C. P. Larsen, T. C. [181] K. J. Curran, R. J. Brentjens, J. Clin. Oncol. 2015, 33, 1703. Pearson, J. A. Ledbetter, A. Aruffo, R. S. Mittler, J. Exp. Med. 1997, [182] a) U. Uslu, G. Schuler, J. Dörrie, N. Schaft, Exp. Dermatol. 2016, 25, 186, 47. 872; b) E. M. V. Forsberg, M. F. Lindberg, H. Jespersen, S. Alsén, R. [165] a) I. Melero, W. W. Shuford, S. A. Newby, A. Aruffo, J. A. Ledbetter, O.Bagge,M.Donia,I.M.Svane,O.Nilsson,L.Ny,L.M.Nilsson, K. E. Hellström, R. S. Mittler, L. Chen, Nat. Med. 1997, 3, 682; b) H. J. A. Nilsson, Cancer Res. 2019, 79, 899. E. Kohrt, A. D. Colevas, R. Houot, K. Weiskopf, M. J. Goldstein, P. [183] A. Heczey, D. Liu, G. Tian, A. N. Courtney, J. Wei, E. Marinova, X. Lund, A. Mueller, I. Sagiv-Barfi, A. Marabelle, R. Lira, J. Clin. Invest. Gao, L. Guo, E. Yvon, J. Hicks, H. Liu, G. Dotti, L. S. Metelitsa, Blood 2014, 124, 2668. 2014, 124, 2824. [166] H. Narazaki, Y. Zhu, L. Luo, G. Zhu, L. Chen, Blood 2010, 115, [184] a) L. J. Rupp, K. Schumann, K. T. Roybal, R. E. Gate, C. J. Ye, W. A. 1941. Lim, A. Marson, Sci. Rep. 2017, 7, 737; b) B. Simon, D. C. Harrer, [167] D.-T. Chu, N. D. Bac, K.-H. Nguyen, N. L. B. Tien, V. V. Thanh, V. T. B. Schuler-Thurner, N. Schaft, G. Schuler, J. Dörrie, U. Uslu, Exp. Nga, V. T. N. Ngoc, A. Dao, D. Thi, L. N. Hoan, Int. J. Mol. Sci. 2019, Dermatol. 2018, 27, 769. 20, 1822. [185] T. A. Waldmann, Cold Spring Harb. Perspect. Biol. 2018, 10, a028472. [168] N. H. Segal, A. R. He, T. Doi, R. Levy, S. Bhatia, M. J. Pishvaian, R. [186] a) S. Parlato, S. M. Santini, C. Lapenta, T. Di Pucchio, M. Logozzi, Cesari, Y. Chen, C. B. Davis, B. Huang, Clin. Cancer Res. 2018, 24, M. Spada, A. M. Giammarioli, W. Malorni, S. Fais, F. Belardelli, Blood 1816. 2001, 98, 3022; b) T. Zhang, H. C. Sun, H. Y. Zhou, J. T. Luo, B. L. [169] a) A. R. Sanchez-Paulete, S. Labiano, M. E. Rodriguez-Ruiz, A. Azpi- Zhang, P. Wang, L. Wang, L. X. Qin, N. Ren, S. L. Ye, Q. Li, Z. Y. Tang, likueta, I. Etxeberria, E. Bolanos, V. Lang, M. Rodriguez, M. A. Aznar, Cancer Lett. 2010, 290, 204. M. Jure-Kunkel, I. Melero, Eur. J. Immunol. 2016, 46, 513; b) D. S. [187] a) M. B. Atkins, J. Hsu, S. Lee, G. I. Cohen, L. E. Flaherty, J. A. Sos- Vinay, B. S. Kwon, BMB Rep. 2014, 47, 122. man, V. K. Sondak, J. M. Kirkwood, J. Clin. Oncol. 2008, 26, 5748;

b) A. A. Tarhini, J. Cherian, S. J. Moschos, H. A. Tawbi, Y. Shuai, [201] N. Shimasaki, A. Jain, D. Campana, Nat. Rev. Drug Discovery 2020, W. E. Gooding, C. Sander, J. M. Kirkwood, J. Clin. Oncol. 2012, 30, 19, 200. 322. [202] a) C. Guillerey, N. D. Huntington, M. J. Smyth, Nat. Immunol. 2016, [188] a) O. Boyman, C. D. Surh, J. Sprent, Expert Opin. Biol. Ther. 2006, 6, 17, 1025; b) M. G. Morvan, L. L. Lanier, Nat. Rev. Cancer 2016, 16, 1323; b) T. R. Malek, Annu. Rev. Immunol. 2008, 26, 453. 7. [189] F. Granucci, C. Vizzardelli, N. Pavelka, S. Feau, M. Persico, E. Virzi, [203] Y.H. Chang, J. Connolly, N. Shimasaki, K. Mimura, K. Kono, D. Cam- M. Rescigno, G. Moro, P. Ricciardi-Castagnoli, Nat. Immunol. 2001, pana, Cancer Res. 2013, 73, 1777. 2, 882. [204] a) E. A. Grimm, R. J. Robb, J. A. Roth, L. M. Neckers, L. B. Lachman, [190] a) A. L. Epstein, M. M. Mizokami, J. Li, P. Hu, L. A. Khawli, J. Natl. D. J. Wilson, S. A. Rosenberg, J. Exp. Med. 1983, 158, 1356; b) S. E. Cancer Inst. 2003, 95, 741; b) D. F. McDermott, M. B. Atkins, Expert Ettinghausen, J. G. Moore, D. E. White, L. Platanias, N. S. Young, S. Opin. Biol. Ther. 2004, 4, 455. A. Rosenberg, Blood 1987, 69, 1654. [191] a) U. B. Dandamudi, M. Ghebremichael, J. A. Sosman, J. I. Clark, D. [205] J. H. Phillips, L. L. Lanier, J. Exp. Med. 1986, 164, 814. F. McDermott, M. B. Atkins, J. P. Dutcher, W. J. Urba, M. M. Regan, [206] M. R. Parkhurst, J. P. Riley, M. E. Dudley, S. A. Rosenberg, Clin. Can- I. Puzanov, T. S. Crocenzi, B. D. Curti, U. N. Vaishampayan, N. A. cer Res. 2011, 17, 6287. Crosby, K. A. Margolin, M. S. Ernstoff, J. Immunother. 2013, 36, 490; [207] R. Weber, V. Fleming, X. Hu, V. Nagibin, C. Groth, P. Altevogt, J. b) G. Procopio, E. Verzoni, S. Bracarda, S. Ricci, C. Sacco, L. Ridolfi, Utikal, V. Umansky, Front. Immunol. 2018, 9, 1310. C. Porta, R. Miceli, N. Zilembo, E. Bajetta, Ann. Oncol. 2013, 24, [208] N. Mirza, M. Fishman, I. Fricke, M. Dunn, A. M. Neuger, T. J. Frost, 2967; c) D. J. Schwartzentruber, D. H. Lawson, J. M. Richards, R. R. M. Lush, S. Antonia, D. I. Gabrilovich, Cancer Res. 2006, 66, M. Conry, D. M. Miller, J. Treisman, F. Gailani, L. Riley, K. Conlon, 9299. B. Pockaj, K. L. Kendra, R. L. White, R. Gonzalez, T. M. Kuzel, B. [209] R. P. Tobin, K. R. Jordan, P. Kapoor, E. Spongberg, D. Davis, V. Curti, P. D. Leming, E. D. Whitman, J. Balkissoon, D. S. Reintgen, H. M. Vorwald, K. L. Couts, D. Gao, D. E. Smith, J. S. W. Borgers, S. Kaufman, F. M. Marincola, M. J. Merino, S. A. Rosenberg, P. Choyke, Robinson, C. Amato, R. Gonzalez, K. D. Lewis, W. A. Robinson, V. F. D. Vena, P. Hwu, N. Engl. J. Med. 2011, 364, 2119. Borges, M. D. McCarter, Front. Oncol. 2019, 9,1. [192] a) L. S. Cheung, J. Fu, P. Kumar, A. Kumar, M. E. Urbanowski, E. A. [210] Y. Liu, G. Wei, W. A. Cheng, Z. Dong, H. Sun, V. Y. Lee, S. C. Cha, D. Ihms, S. Parveen, C. K. Bullen, G. J. Patrick, R. Harrison, J. R. Mur- L. Smith, L. W. Kwak, H. Qin, Cancer Immunol. Immunother. 2018, phy, D. M. Pardoll, W. R. Bishai, Proc. Natl. Acad. Sci. U. S. A. 2019, 67, 1181. 116, 3100; b) D.-A. Silva, S. Yu, U. Y. Ulge, J. B. Spangler, K. M. Jude, [211] a) D. W. Beury, K. A. Carter, C. Nelson, P. Sinha, E. Hanson, M. C. Labão-Almeida, L. R. Ali, A. Quijano-Rubio, M. Ruterbusch, I. Le- Nyandjo, P. J. Fitzgerald, A. Majeed, N. Wali, S. Ostrand-Rosenberg, ung, T. Biary, S. J. Crowley, E. Marcos, C. D. Walkey, B. D. Weitzner, J. Immunol. 2016, 196, 3470; b) K. Garber, Science 2018, 360, 588. F. Pardo-Avila, J. Castellanos, L. Carter, L. Stewart, S. R. Riddell, M. [212] Y.Ino, R. Yamazaki-Itoh, S. Oguro, K. Shimada, T. Kosuge, J. Zavada, Pepper, G. J. L. Bernardes, M. Dougan, K. C. Garcia, D. Baker, Nature Y. Kanai, N. Hiraoka, PLoS One 2013, 8, e55146. 2019, 565, 186. [213] B. Érsek, P. Silló, U. Cakir, V. Molnár, A. Bencsik, B. Mayer, E. Mezey, [193] A. Mishra, L. Sullivan, M. A. Caligiuri, Clin. Cancer Res. 2014, 20, S. Kárpáti, Z. Pós, K. Németh, Cell. Mol. Life Sci. https://doi.org/10. 2044. 1007/s00018-020-03517-8. [194] a) K. C. Conlon, E. Lugli, H. C. Welles, S. A. Rosenberg, A. T. Fojo, [214] R. R. Meka, S. Mukherjee, C. R. Patra, A. Chaudhuri, Nanoscale 2019, J. C. Morris, T. A. Fleisher, S. P. Dubois, L. P. Perera, D. M. Stewart, 11, 7931. C. K. Goldman, B. R. Bryant, J. M. Decker, J. Chen, T. Y. A. Worthy, [215] a) H. Rehman, A. W. Silk, M. P. Kane, H. L. Kaufman, J. Immunother. W. D. Figg, C. J. Peer, M. C. Sneller, H. C. Lane, J. L. Yovandich, S. Cancer 2016, 4, 53; b) E. J. Lipson, W. H. Sharfman, S. Chen, T. L. P. Creekmore, M. Roederer, T. A. Waldmann, J. Clin. Oncol. 2015, McMiller, T. S. Pritchard, J. T. Salas, S. Sartorius-Mergenthaler, I. 33, 74; b) E. Huarte, J. Fisher, M. J. Turk, D. Mellinger, C. Foster, B. Freed, S. Ravi, H. Wang, B. Luber, J. D. Sproul, J. M. Taube, D. M. Wolf,K.R.Meehan,C.E.Fadul,M.S.Ernstoff,Cancer Lett. 2009, Pardoll, S. L. Topalian, J. Transl. Med. 2015, 13, 214. 285, 80; c) T. A. Stoklasek, K. S. Schluns, L. Lefrançois, J. Immunol. [216] K. Inaba, M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. 2006, 177, 6072; d) W. Xu, M. Jones, B. Liu, X. Zhu, C. B. Johnson, Muramatsu, R. M. Steinman, J. Exp. Med. 1992, 176, 1693. A. C. Edwards, L. Kong, E. K. Jeng, K. Han, W. D. Marcus, M. P. [217] B. Ruffell, D. Chang-Strachan, V. Chan, A. Rosenbusch, C. M. Ho, N. Rubinstein, P. R. Rhode, H. C. Wong, Cancer Res. 2013, 73, 3075. Pryer, D. Daniel, E. S. Hwang, H. S. Rugo, L. M. Coussens, Cancer [195] M. J. Smyth, M. Taniguchi, S. E. A. Street, J. Immunol. 2000, 165, Cell 2014, 26, 623. 2665. [218] a) S. Balan, V. Ollion, N. Colletti, R. Chelbi, F. Montanana-Sanchis, [196] J. A. Gollob, K. G. Veenstra, R. A. Parker, J. W. Mier, D. F. McDermott, H. Liu, T. P. Vu Manh, C. Sanchez, J. Savoret, I. Perrot, A. C. Doffin, E. D.Clancy,L.Tutin,H.Koon,M.B.Atkins,J. Clin. Oncol. 2003, 21, Fossum, D. Bechlian, C. Chabannon, B. Bogen, C. Asselin-Paturel, 2564. M. Shaw, T. Soos, C. Caux, J. Valladeau-Guilemond, M. Dalod, J. Im- [197] C. S. Hinrichs, R. Spolski, C. M. Paulos, L. Gattinoni, K. W. Kerstann, munol. 2014, 193, 1622; b) J. Helft, J. Böttcher, P. Chakravarty, S. D. C. Palmer, C. A. Klebanoff, S. A. Rosenberg, W. J. Leonard, N. P. Zelenay, J. Huotari, B. U. Schraml, D. Goubau, C. Reis e Sousa, Im- Restifo, Blood 2008, 111, 5326. munity 2015, 42, 1197. [198] M. T. Kasaian, M. J. Whitters, L. L. Carter, L. D. Lowe, J. M. Jussif, [219] a) P. Shinde, S. Fernandes, S. Melinkeri, V. Kale, L. Limaye, Sci. Rep. B. Deng, K. A. Johnson, J. A. S. Witek, M. Senices, R. F. Konz, A. L. 2018, 8, 5705; b) I. J. De Vries, D. J. Krooshoop, N. M. Scharenborg, Wurster, D. D. Donaldson, M. Collins, D. A. Young, M. J. Grusby, W. J. Lesterhuis, J. H. Diepstra, G. N. Van Muijen, S. P. Strijk, T. J. Immunity 2002, 16, 559. Ruers, O. C. Boerman, W. J. Oyen, G. J. Adema, C. J. Punt, C. G. [199] B. Rodríguez-Bayona, A. Ramos-Amaya, J. Bernal, A. Campos-Caro, Figdor, Cancer Res. 2003, 63, 12. J. A. Brieva, J. Immunol. 2012, 188, 1578. [220] N. J. Neubert, M. Schmittnaegel, N. Bordry, S. Nassiri, N. Wald, C. [200] a) M. Granzin, A. Stojanovic, M. Miller, R. Childs, V. Huppert, A. Martignier, L. Tillé, K. Homicsko, W. Damsky, H. Maby-El Hajjami, Cerwenka, OncoImmunology 2016, 5, e1219007; b) Y. Chen, F. Yu, Y. I. Klaman, E. Danenberg, K. Ioannidou, L. Kandalaft, G. Coukos, S. Jiang, J. Chen, K. Wu, X. Chen, Y. Lin, H. Zhang, L. Li, Y. Zhang, J. Hoves, C. H. Ries, S. A. Fuertes Marraco, P. G. Foukas, M. De Palma, Immunother. 2018, 41, 274. D. E. Speiser, Sci. Transl. Med. 2018, 10, eaan3311.

[221] D. J. Irvine, M. C. Hanson, K. Rakhra, T. Tokatlian, Chem. Rev. 2015, [253] B. Kwong, S. A. Gai, J. Elkhader, K. D. Wittrup, D. J. Irvine, Cancer 115, 11109. Res. 2013, 73, 1547. [222] J. Conniot, J. M. Silva, J. G. Fernandes, L. C. Silva, R. Gaspar, S. Broc- [254] N. Bertrand, J. Wu, X. Xu, N. Kamaly, O. C. Farokhzad, Adv. Drug chini, H. F. Florindo, T. S. Barata, Front. Chem. 2014, 2, 105. Deliv. Rev. 2014, 66,2. [223] J. M. Silva, E. Zupancic, G. Vandermeulen, V. G. Oliveira, A. Salgado, [255] T. Betancourt, J. D. Byrne, N. Sunaryo, S. W. Crowder, M. Kada- M. Videira, M. Gaspar, L. Graca, V. Préat, H. F. Florindo, J. Controlled pakkam, S. Patel, S. Casciato, L. Brannon-Peppas, J. Biomed. Mater. Release 2015, 198, 91. Res., Part A 2009, 91A, 263. [224] E. S. Trombetta, I. Mellman, Annu. Rev. Immunol. 2005, 23, 975. [256] a) H. Freichels, V. Pourcelle, R. Auzély-Velty, J. Marchand-Brynaert, [225] M. F. Bachmann, G. T. Jennings, Nat. Rev. Immunol. 2010, 10, 787. C. Jérôme, Biomacromolecules 2012, 13, 760; b) R. Yang, J. Xu, L. Xu, [226] S. N. Thomas, E. Vokali, A. W. Lund, J. A. Hubbell, M. A. Swartz, X. Sun, Q. Chen, Y. Zhao, R. Peng, Z. Liu, ACS Nano 2018, 12, 5121; Biomaterials 2014, 35, 814. c) Y. Guo, D. Wang, Q. Song, T. Wu, X. Zhuang, Y. Bao, M. Kong, Y. [227] V. Manolova, A. Flace, M. Bauer, K. Schwarz, P. Saudan, M. F. Bach- Qi, S. Tan, Z. Zhang, ACS Nano 2015, 9, 6918. mann, Eur. J. Immunol. 2008, 38, 1404. [257] X. Yu, I. Trase, M. Ren, K. Duval, X. Guo, Z. Chen, J. Nanomater. [228] S. Yan, W. Gu, Z. P. Xu, J. Colloid Interface Sci. 2013, 395,1. 2016, 2016, 1087250. [229] A. Jhaveri, V. Torchilin, Expert Opin. Drug Deliv. 2016, 13, 49. [258] X.-Y.Meng, H.-X. Zhang, M. Mezei, M. Cui, Curr. Comput. Aided Drug [230] A. I. Matos, B. Carreira, C. Peres, L. I. Moura, J. Conniot, T. Fourniols, Des. 2011, 7, 146. A. Scomparin, Á. Martínez-Barriocanal, D. Arango, J. P. Conde, J. [259] G. A. Duncan, M. A. Bevan, Nanoscale 2015, 7, 15332. Controlled Release 2019, 307, 108. [260] J. Liu, G. E. Weller, B. Zern, P. S. Ayyaswamy, D. M. Eckmann, V. R. [231] M. O. Oyewumi, A. Kumar, Z. Cui, Expert Rev. Vaccines 2010, 9, Muzykantov, R. Radhakrishnan, Proc. Natl. Acad. Sci. U. S. A. 2010, 1095. 107, 16530. [232] a) Z. Wang, C. Tiruppathi, R. D. Minshall, A. B. Malik, ACS Nano [261] J. Nicolas, S. Mura, D. Brambilla, N. Mackiewicz, P. Couvreur, Chem. 2009, 3, 4110; b) M. Ehrlich, W. Boll, A. Van Oijen, R. Hariharan, K. Soc. Rev. 2013, 42, 1147. Chandran, M. L. Nibert, T. Kirchhausen, Cell 2004, 118, 591. [262] S. Hamdy, A. Haddadi, A. Shayeganpour, J. Samuel, A. Lavasanifar, [233] A. Verma, F. Stellacci, Small 2010, 6, 12. Pharm. Res. 2011, 28, 2288. [234] J. K. Vasir, V. Labhasetwar, Biomaterials 2008, 29, 4244. [263] L. J. Cruz, P. J. Tacken, R. Fokkink, B. Joosten, M. C. Stuart, F. Al- [235] A. Albanese, P. S. Tang, W. C. Chan, Annu. Rev. Biomed. Eng. 2012, bericio, R. Torensma, C. G. Figdor, J. Controlled Release 2010, 144, 14,1. 118. [236] F. Alexis, E. Pridgen, L. K. Molnar, O. C. Farokhzad, Mol. Pharmaceu- [264] R. Li, G. Kang, M. Hu, H. Huang, Mol. Biotechnol. 2019, 61, 60. tics 2008, 5, 505. [265] K. Omidfar, M. Daneshpour, Expert Opin. Drug Discov. 2015, 10, 651. [237] E. Fröhlich, Int. J. Nanomed. 2012, 7, 5577. [266] a) A. Frenzel, T. Schirrmann, M. Hust, mAbs 2016, 8, 1177; b) B. P. [238] R. R. Arvizo, O. R. Miranda, D. F. Moyano, C. A. Walden, K. Giri, R. Gray, K. C. Brown, Chem. Rev. 2014, 114, 1020. Bhattacharya, J. D. Robertson, V. M. Rotello, J. M. Reid, P. Mukher- [267] Z. Kaymakcalan, P. Sakorafas, S. Bose, S. Scesney, L. Xiong, D. K. jee, PLoS One 2011, 6, e24374. Hanzatian, J. Salfeld, E. H. Sasso, Clin. Immunol. 2009, 131, 308. [239] L. J. Cruz, P. J. Tacken, R. Fokkink, C. G. Figdor, Biomaterials 2011, [268] H. J. de Haard, N. van Neer, A. Reurs, S. E. Hufton, R. C. Roovers, 32, 6791. P. Henderikx, A. P. de Bruïne, J. W. Arends, H. R. Hoogenboom, J. [240] S. Venkataraman, J. L. Hedrick, Z. Y. Ong, C. Yang, P. L. R. Ee, P. T. Biol. Chem. 1999, 274, 18218. Hammond,Y.Y.Yang,Adv. Drug Deliv. Rev. 2011, 63, 1228. [269] A. Weickhardt, R. Doebele, A. Oton, J. Lettieri, D. Maxson, M. [241] L. Florez, C. Herrmann, J. M. Cramer, C. P. Hauser, K. Koynov, K. Reynolds, A. Brown, M. K. Jackson, G. Dy, A. Adjei, G. Fetterly, X. Landfester, D. Crespy, V. Mailänder, Small 2012, 8, 2222. Lu, W. Franklin, M. Varella-Garcia, F. R. Hirsch, M. W. Wynes, H. [242] C. Maisonneuve, S. Bertholet, D. J. Philpott, E. De Gregorio, Proc. Youssoufian, A. Adjei, D. R. Camidge, J. Thorac. Oncol. 2012, 7, 419. Natl. Acad. Sci. U. S. A. 2014, 111, 12294. [270] M. W. den Hollander, F. Bensch, A. W. Glaudemans, T. H. Oude [243] T. Kawai, S. Akira, Int. Immunol. 2009, 21, 317. Munnink, R. H. Enting, W. F. den Dunnen, M. A. Heesters, F. A. [244] D. Wesch, C. Peters, H.-H. Oberg, K. Pietschmann, D. Kabelitz, Cell. Kruyt, M. N. Lub-de Hooge, J. Cees de Groot, J. Pearlberg, J. A. Gi- Mol. Life Sci. 2011, 68, 2357. etema, E. G. de Vries, A. M. Walenkamp, J. Nucl. Med. 2015, 56, [245] S. S. Saluja, D. J. Hanlon, F. A. Sharp, E. Hong, D. Khalil, E. Robin- 1310. son, R. Tigelaar, T. M. Fahmy, R. L. Edelson, Int. J. Nanomed. 2014, [271] a) J. L. Spratlin, R. B. Cohen, M. Eadens, L. Gore, D. R. Camidge, S. 9, 5231. Diab, S. Leong, C. O’Bryant, L. Q. Chow, N. J. Serkova, N. J. Meropol, [246] W. W. Unger, A. J. Van Beelen, S. C. Bruijns, M. Joshi, C. M. Fehres, N. L. Lewis, E. G. Chiorean, F. Fox, H. Youssoufian, E. K. Rowinsky, L. Van Bloois, M. I. Verstege, M. Ambrosini, H. Kalay, K. Nazmi, J. S. G. Eckhardt, J. Clin. Oncol. 2010, 28, 780; b) R. Steinbrook, N. Controlled Release 2012, 160, 88. Engl. J. Med. 2006, 355, 1409. [247] S. Burgdorf, A. Kautz, V. Böhnert, P. A. Knolle, C. Kurts, Science 2007, [272] a) C. Adams, K. Totpal, D. Lawrence, S. Marsters, R. Pitti, S. Yee, 316, 612. S. Ross, L. Deforge, H. Koeppen, M. Sagolla, D. Compaan, H. Low- [248] R. Liang, J. Xie, J. Li, K. Wang, L. Liu, Y. Gao, M. Hussain, G. Shen, man, S. Hymowitz, A. Ashkenazi, Cell Death Differ. 2008, 15, 751; b) J. Zhu, J. Tao, Biomaterials 2017, 149, 41. S. J. Hotte, H. W. Hirte, E. X. Chen, L. L. Siu, L. H. Le, A. Corey, A. [249] R. A. Rosalia, L. J. Cruz, S. van Duikeren, A. T. Tromp, A. L. Silva, Iacobucci, M. MacLean, L. Lo, N. L. Fox, A. M. Oza, Clin. Cancer Res. W. Jiskoot, T. de Gruijl, C. Löwik, J. Oostendorp, S. H. van der Burg, 2008, 14, 3450. Biomaterials 2015, 40, 88. [273] a) S. Ahangarzadeh, M. Bandehpour, B. Kazemi, Iran. J. Basic Med. [250] Y. Qian, H. Jin, S. Qiao, Y. Dai, C. Huang, L. Lu, Q. Luo, Z. Zhang, Sci. 2017, 20, 327; b) E. Heyduk, T. Heyduk, Anal. Biochem. 2014, Biomaterials 2016, 98, 171. 464, 73. [251] Y. Qian, S. Qiao, Y. Dai, G. Xu, B. Dai, L. Lu, X. Yu, Q. Luo, Z. Zhang, [274] Z. Li, T. Uzawa, H. Zhao, S.-C. Luo, H.-H. Yu, E. Kobatake, Y. Ito, J. ACS Nano 2017, 11, 9536. Biosci. 2014, 117, 501. [252] D. Schmid, C. G. Park, C. A. Hartl, N. Subedi, A. N. Cartwright, R. B. [275] A. L. Silva, L. I. Moura, B. Carreira, J. Conniot, A. I. Matos, C. Peres, Puerto, Y. Zheng, J. Maiarana, G. J. Freeman, K. W. Wucherpfennig, V. Sainz, L. C. Silva, R. S. Gaspar, H. F. Florindo, in Biomedical Appli- Nat. Commun. 2017, 8, 1747. cations of Functionalized Nanomaterials: Concepts, Development and

Clinical Translation, Vol. 1 (Eds: B. Sarmento, J. das Neves), Elsevier, [300] a) N. Dhomen, J. S. Reis-Filho, S. da Rocha Dias, R. Hayward, K. Sav- Amsterdam, The Netherlands 2018, Ch. 14. age, V. Delmas, L. Larue, C. Pritchard, R. Marais, Cancer Cell 2009, [276] L. Yan, J. Zhang, C. S. Lee, X. Chen, Small 2014, 10, 4487. 15, 294; b) M. Bosenberg, V. Muthusamy, D. P. Curley, Z. Wang, C. [277] O. V. Markov, N. L. Mironova, S. V. Sennikov, V. V. Vlassov, M. A. Hobbs, B. Nelson, C. Nogueira, J. W. Horner, R. DePinho, L. Chin, Zenkova, PLoS One 2015, 10, e0136911. Genesis 2006, 44, 262; c) I. Yajima, E. Belloir, Y. Bourgeois, M. Ku- [278] T. Nakamura, H. Miyabe, M. Hyodo, Y. Sato, Y. Hayakawa, H. Ha- masaka, V. Delmas, L. Larue, Genesis 2006, 44, 34. rashima, J. Controlled Release 2015, 216, 149. [301] D. Perna, F. A. Karreth, A. G. Rust, P. A. Perez-Mancera, M. Rashid, [279] M. Morishita, Y. Takahashi, M. Nishikawa, R. Ariizumi, Y. Takakura, F. Iorio, C. Alifrangis, M. J. Arends, M. W. Bosenberg, G. Bollag, Proc. Mol. Pharmaceutics 2017, 14, 4079. Natl. Acad. Sci. U. S. A. 2015, 112, E536. [280] T. Nakamura, D. Yamazaki, J. Yamauchi, H. Harashima, J. Controlled [302] P. M. Pollock, U. L. Harper, K. S. Hansen, L. M. Yudt, M. Stark, C. Release 2013, 171, 216. M. Robbins, T. Y. Moses, G. Hostetter, U. Wagner, J. Kakareka, Nat. [281] E. A. Vasievich, S. Ramishetti, Y. Zhang, L. Huang, Mol. Pharmaceu- Genet. 2003, 33, 19. tics 2012, 9, 261. [303] A. I. Hooijkaas, J. Gadiot, M. van der Valk, W. J. Mooi, C. U. Blank, [282] X. Ma, N. Gong, L. Zhong, J. Sun, X.-J. Liang, Biomaterials 2016, 97, Am. J. Pathol. 2012, 181, 785. 10. [304] a) H. Tsao, G. Yang, V.Goel, H. Wu, F. G. Haluska, J. Invest. Dermatol. [283] C. Zou, G. Jiang, X. Gao, W. Zhang, H. Deng, C. Zhang, J. Ding, R. 2004, 122, 337; b) J. M. Stahl, A. Sharma, M. Cheung, M. Zimmer- Wei, X. Wang, L. Xi, Nanomedicine 2019, 22, 102092. man, J. Q. Cheng, M. W. Bosenberg, M. Kester, L. Sandirasegarane, [284] M. Mockey, E. Bourseau, V. Chandrashekhar, A. Chaudhuri, S. G. P. Robertson, Cancer Res. 2004, 64, 7002. Lafosse, E. Le Cam, V. Quesniaux, B. Ryﬀel, C. Pichon, P. Midoux, [305] D. Dankort, D. P. Curley, R. A. Cartlidge, B. Nelson, A. N. Karnezis, Cancer Gene Ther. 2007, 14, 802. W.E.Damsky,Jr.,M.J.You,R.A.DePinho,M.McMahon,M.Bosen- [285] H. Sneh-Edri, D. Likhtenshtein, D. Stepensky, Mol. Pharmaceutics berg, Nat. Genet. 2009, 41, 544. 2011, 8, 1266. [306] G. Barutello, V. Rolih, M. Arigoni, L. Tarone, L. Conti, E. Quaglino, [286] K. Matsuo, T. Yoshikawa, A. Oda, T. Akagi, M. Akashi, Y. Mukai, Y. P. Buracco, F. Cavallo, F. Riccardo, Int. J. Mol. Sci. 2018, 19, 799. Yoshioka, N. Okada, S. Nakagawa, Biochem. Biophys. Res. Commun. [307] a) E. Hodis, I. R. Watson, G. V. Kryukov, S. T. Arold, M. Imielinski, J.- 2007, 362, 1069. P. Theurillat, E. Nickerson, D. Auclair, L. Li, C. Place, Cell 2012, 150, [287] Y. Mukai, T. Yoshinaga, M. Yoshikawa, K. Matsuo, T. Yoshikawa, K. 251; b) M. Colombino, M. Capone, A. Lissia, A. Cossu, C. Rubino, Matsuo, K. Niki, Y. Yoshioka, N. Okada, S. Nakagawa, J. Immunol. V. De Giorgi, D. Massi, E. Fonsatti, S. Staibano, O. Nappi, J. Clin. 2011, 187, 6249. Oncol. 2012, 30, 2522; c) C. Milagre, N. Dhomen, F. C. Geyer, R. [288] A. Hayashi, H. Wakita, T. Yoshikawa, T. Nakanishi, Y. Tsutsumi, T. Hayward, M. Lambros, J. S. Reis-Filho, R. Marais, Cancer Res. 2010, Mayumi, Y.Mukai, Y.Yoshioka, N. Okada, S. Nakagawa, J. Controlled 70, 5549. Release 2007, 117, 11. [308] a) J. Ackermann, M. Frutschi, K. Kaloulis, T. McKee, A. Trumpp, [289] Y.-P. Sher, S.-I. Lin, K. M. Chai, I.-H. Chen, S.-J. Liu, Am. J. Transl. Res. F. Beermann, Cancer Res. 2005, 65, 4005; b) M. Pedersen, H. V. 2019, 9, 2028. Küsters-Vandevelde, A. Viros, P. J. Groenen, B. Sanchez-Laorden, J. [290] J. J. Perez-Trujillo, R. Garza-Morales, J. A. Barron-Cantu, G. Figueroa- H. Gilhuis, I. A. van Engen-van Grunsven, W. Renier, J. Schieving, I. Parra, A. Garcia-Garcia, H. Rodriguez-Rocha, J. Garcia-Juarez, G. Niculescu-Duvaz, Cancer Discov. 2013, 3, 458. E. Muñoz-Maldonado, O. Saucedo-Cardenas, R. Montes-De-Oca- [309] a) C. E. Burd, W. Liu, M. V. Huynh, M. A. Waqas, J. E. Gillahan, K. S. Luna, Oncol. Lett. 2017, 13, 1569. Clark, K. Fu, B. L. Martin, W. R. Jeck, G. P. Souroullas, D. B. Darr, D. [291] K. Nakamura, N. Yoshikawa, Y. Yamaguchi, S. Kagota, K. Shinozuka, C. Zedek, M. J. Miley, B. C. Baguley, S. L. Campbell, N. E. Sharpless, M. Kunitomo, Life Sci. 2002, 70, 791. Cancer Discov. 2014, 4, 1418; b) W. Liu, K. B. Monahan, A. D. Pfef- [292] a) J. Nakayama, X.-C. Guan, M. Nakashima, T. Mashino, Y. ferle, T. Shimamura, J. Sorrentino, K. T. Chan, D. W. Roadcap, D. W. Hori, J. Dermatol. 1997, 24, 351; b) V. O. Melnikova, S. V. Bol- Ollila, N. E. Thomas, D. H. Castrillon, C. R. Miller, C. M. Perou, K. K. shakov, C. Walker, H. N. Ananthaswamy, Oncogene 2004, 23, Wong, J. E. Bear, N. E. Sharpless, Cancer Cell 2012, 21, 751; c) L. N. 2347. Kwong,J.C.Costello,H.Liu,S.Jiang,T.L.Helms,A.E.Langsdorf, [293] B. Seliger, U. Wollscheid, F. Momburg, T. Blankenstein, C. Huber, D. Jakubosky, G. Genovese, F. L. Muller, J. H. Jeong, R. P. Bender, Cancer Res. 2001, 61, 1095. G. C. Chu, K. T. Flaherty, J. A. Wargo, J. J. Collins, L. Chin, Nat. Med. [294] I. W. Mak, N. Evaniew, M. Ghert, Am. J. Transl. Res. 2014, 6, 2012, 18, 1503. 114. [310] O. F. Kuzu, F. D. Nguyen, M. A. Noory, A. Sharma, Cancer Growth [295] B. Olson, Y. Li, Y. Lin, E. T. Liu, A. Patnaik, Cancer Discov. 2018, 8, Metastasis 2015, 8, 81. 1358. [311] F. S. Hodi, S. J. O’Day, D. F. McDermott, R. W. Weber, J. A. Sosman, [296] S. Madhunapantula, T. Patel, C. Annageldiyev, A. Sharma, in Animal J. B. Haanen, R. Gonzalez, C. Robert, D. Schadendorf, J. C. Hassel, Models in Cancer Drug Discovery, Vol. 1 (Eds: A. Azmi, R. M. Moham- W. Akerley, A. J. van den Eertwegh, J. Lutzky, P. Lorigan, J. M. Vaubel, mad), Elsevier, Amsterdam, The Netherlands 2019, Ch. 17. G. P. Linette, D. Hogg, C. H. Ottensmeier, C. Lebbé, C. Peschel, I. [297] M. Hidalgo, F. Amant, A. V. Biankin, E. Budinská, A. T. Byrne, C. Cal- Quirt, J. I. Clark, J. D. Wolchok, J. S. Weber, J. Tian, M. J. Yellin, G. das, R. B. Clarke, S. de Jong, J. Jonkers, G. M. Mælandsmo, Cancer M. Nichol, A. Hoos, W. J. Urba, N. Engl. J. Med. 2010, 363,711. Discov. 2014, 4, 998. [312] B. W. Carter, P. R. Bhosale, W. T. Yang, Cancer 2018, 124, 2906. [298] T. Pearson, D. L. Greiner, L. D. Shultz, Curr. Protoc. Immunol. 2008, [313] a) J. D. Wolchok, A. Hoos, S. O’Day, J. S. Weber, O. Hamid, C. Lebbé, 81, 15. M. Maio, M. Binder, O. Bohnsack, G. Nichol, R. Humphrey, F. S. [299] a) J. A. Curtin, J. Fridlyand, T. Kageshita, H. N. Patel, K. J. Busam, Hodi, Clin. Cancer Res. 2009, 15, 7412; b) M. Nishino, H. Hatabu, F. H. Kutzner, K.-H. Cho, S. Aiba, E.-B. Bröcker, P. E. LeBoit, N. Engl. S. Hodi, Radiology 2019, 290,9. J. Med. 2005, 353, 2135; b) J. Caramel, E. Papadogeorgakis, L. Hill, [314] M. Durai, C. Krueger, Z. Ye, L. Cheng, A. Mackensen, M. Oelke, J. P. G. J. Browne, G. Richard, A. Wierinckx, G. Saldanha, J. Osborne, P. Schneck, Cancer Immunol. Immunother. 2009, 58, 209. Hutchinson, G. Tse, Cancer Cell 2013, 24, 466; c) C. Wellbrock, S. [315] W. Wei, E. B. Ehlerding, X. Lan, Q. Luo, W. Cai, Eur. J. Nucl. Med. Rana, H. Paterson, H. Pickersgill, T. Brummelkamp, R. Marais, PLoS Mol. Imaging 2018, 45, 132. One 2008, 3, e2734. [316] A. Zhu, D. Lee, H. Shim, Semin. Oncol. 2011, 38, 55.

[317] Y. Sanli, J. Leake, A. Odu, Y. Xi, R. M. Subramaniam, AJR Am. J. b) S. Sau, H. O. Alsaab, K. Bhise, R. Alzhrani, G. Nabil, A. K. Iyer, J. Roentgenol. 2019, 212,1. Controlled Release 2018, 274, 24. [318] J. Schwenck, B. Schörg, F. Fiz, D. Sonanini, A. Forschner, T.Eigentler, [336] E. A. Tanifum, C. Patel, M. E. Liaw, R. G. Pautler, A. V. Annapragada, B. Weide, M. Martella, I. Gonzalez-Menendez, C. Campi, G. Sam- Sci. Rep. 2018, 8, 2889. buceti, F. Seith, L. Quintanilla-Martinez, C. Garbe, C. Pfannenberg, [337] a) F. Chapelin, C. M. Capitini, E. T. Ahrens, J. Immunother. Cancer M. Röcken, C. la Fougere, B. J. Pichler, M. Kneilling, Theranostics 2018, 6, 105; b) C. Fink, M. Smith, J. M. Gaudet, A. Makela, P. J. 2020, 10, 925. Foster, G. A. Dekaban, Mol. Imaging Biol. 2020, 22, 549. [319] a) C. S. Palmer, M. Ostrowski, B. Balderson, N. Christian, S. M. [338] C. Gonzales, H. A. Yoshihara, N. Dilek, J. Leignadier, M. Irving, P. Crowe, Front. Immunol. 2015, 6, 1; b) B. Ghesquière, B. W. Wong, Mieville, L. Helm, O. Michielin, J. Schwitter, PLoS One 2016, 11, A. Kuchnio, P. Carmeliet, Nature 2014, 511, 167. e0164557. [320] B. Y.Kong, A. M. Menzies, C. A. Saunders, E. Liniker, S. Ramanujam, [339] a) S. P. Newman, P. H. Hirst, I. R. Wilding, Eur. J. Pharm. Sci. 2003, A. Guminski, R. F. Kefford, G. V. Long, M. S. Carlino, Pigment Cell 18, 19; b) T.Marvola, J. Marvola, H. Kanerva, A. Ahonen, K. Lindevall, Melanoma Res. 2016, 29, 572. M. Marvola, Int. J. Pharm. 2008, 349, 24. [321] a) S. H. L. Hoffmann, D. I. Reck, A. Maurer, B. Fehrenbacher, J. E. [340] a) I. Ak, M. P. Stokkel, W. Bergman, E. K. Pauwels, Eur. J. Nucl. Med. Sceneay, M. Poxleitner, H. H. Öz, W. Ehrlichmann, G. Reischl, K. 2000, 27, 447; b) O. Alonso, M. Martínez, L. Delgado, A. De León, D. Fuchs, M. Schaller, D. Hartl, M. Kneilling, A. Möller, B. J. Pichler, C. De Boni, G. Lago, M. Garcés, F. Fontes, J. Espasandín, J. Priario, J. M. Griessinger, Theranostics 2019, 9, 5869; b) J. R. Nedrow, A. Josef- Nucl. Med. 2003, 44, 1561; c) S. E. Halpern, W.Haindl, J. Beauregard, sson,S.Park,S.Ranka,S.Roy,G.Sgouros,J. Nucl. Med. 2017, 58, P. Hagan, M. Clutter, D. Amox, B. Merchant, M. Unger, C. Mongovi, 1560; c) A. Natarajan, A. T. Mayer, R. E. Reeves, C. M. Nagamine, S. R. Bartholomew, Radiology 1988, 168, 529. S. Gambhir, Mol. Imaging Biol. 2017, 19, 903; d) S. Mall, N. Yusufi, R. [341] A. Signore, A. Annovazzi, R. Barone, E. Bonanno, C. D’Alessandria, Wagner, R. Klar, H. Bianchi, K. Steiger, M. Straub, S. Audehm, I. Laiti- M. Chianelli, S. J. Mather, U. Bottoni, C. Panetta, D. Innocenzi, F. nen, M. Aichler, C. Peschel, S. Ziegler, M. Mustafa, M. Schwaiger, Scopinaro, S. Calvieri, J. Nucl. Med. 2004, 45, 1647. C. D’Alessandria, A. M. Krackhardt, Cancer Res. 2016, 76, 4113. [342] a) S. Nair, C. McLaughlin, A. Weizer, Z. Su, D. Boczkowski, J. Dan- [322] A. Natarajan, A. T. Mayer, L. Xu, R. E. Reeves, J. Gano, S. S. Gambhir, null, J. Vieweg, E. Gilboa, J. Immunol. 2003, 171, 6275; b) R. Ridolfi, Bioconjug. Chem. 2015, 26, 2062. A. Riccobon, R. Galassi, G. Giorgetti, M. Petrini, L. Fiammenghi, M. [323] a) A. Dimitrakopoulou-Strauss, Cancer Immunol. Immunother. 2019, Stefanelli, L. Ridolfi, A. Moretti, G. Migliori, G. Fiorentini, J. Transl. 68, 813; b) H. Anwar, C. Sachpekidis, J. Winkler, A. Kopp-Schneider, Med. 2004, 2, 27; c) D. Blocklet, M. Toungouz, R. Kiss, M. Lamber- U. Haberkorn, J. C. Hassel, A. Dimitrakopoulou-Strauss, Eur. J. Nucl. mont, T. Velu, D. Duriau, M. Goldman, S. Goldman, Eur. J. Nucl. Med. Mol. Imaging 2018, 45, 376; c) C. Sachpekidis, H. Anwar, J. K. Med. Mol. Imaging 2003, 30, 440. Winkler, A. Kopp-Schneider, L. Larribere, U. Haberkorn, J. C. Hassel, [343] a) G. Lucignani, M. Rescigno, Eur. J. Nucl. Med. Mol. Imaging 2005, A. Dimitrakopoulou-Strauss, Cancer Immunol. Immunother. 2018, 32, 725; b) M. Trakatelli, M. Toungouz, D. Blocklet, Y.Dodoo, L. Gor- 67, 1261; d) K. Ito, R. Teng, H. Schöder, J. L. Humm, A. Ni, L. dower, M. Laporte, P. Vereecken, F. Sales, L. Mortier, N. Mazouz, M. Michaud, R. Nakajima, R. Yamashita, J. D. Wolchok, W. A. Weber, Lambermont, S. Goldman, P. Coulie, M. Goldman, T. Velu, Cancer J. Nucl. Med. 2019, 60, 335. Immunol. Immunother. 2006, 55, 469. [324] A. N. M. Wong, G. A. McArthur, M. S. Hofman, R. J. Hicks, Eur. J. [344] a) N. Craft, K. W. Bruhn, B. D. Nguyen, R. Prins, L. M. Liau, E. A. Nucl. Med. Mol. Imaging 2017, 44, 67. Collisson, A. De, M. S. Kolodney, S. S. Gambhir, J. F. Miller, J. Invest. [325] S. Y. Cho, E. J. Lipson, H. J. Im, S. P. Rowe, E. M. Gonzalez, A. Black- Dermatol. 2005, 125, 159; b) J. Ma, T. Shang, P. Ma, X. Sun, J. Zhao, ford, A. Chirindel, D. M. Pardoll, S. L. Topalian, R. L. Wahl, J. Nucl. M. Zhang, Invest. New Drugs 2019, 37, 1036. Med. 2017, 58, 1421. [345] a) G. D. Luker, J. P. Bardill, J. L. Prior, C. M. Pica, D. Piwnica-Worms, [326] a) C. Buchbender, T. A. Heusner, T. C. Lauenstein, A. Bockisch, G. D. A. Leib, J. Virol. 2002, 76, 12149; b) M. G. Hollingshead, C. A. Antoch, J. Nucl. Med. 2012, 53, 1244; b) N. F. Schwenzer, A. C. Pfan- Bonomi, S. D. Borgel, J. P. Carter, R. Shoemaker, G. Melillo, E. A. nenberg, Semin. Nucl. Med. 2015, 45, 322. Sausville, Eur. J. Cancer 2004, 40, 890. [327] N. C. Nguyen, M. K. Yee, A. M. Tuchayi, J. M. Kirkwood, H. Tawbi, J. [346] L. Zhu, S. Kalimuthu, P. Gangadaran, J. M. Oh, H. W. Lee, S. H. M. Mountz, Front. Oncol. 2018, 8, 18. Baek, S. Y. Jeong, S. W. Lee, J. Lee, B. C. Ahn, Theranostics 2017, 7, [328] S. B. Yu, A. D. Watson, Chem. Rev. 1999, 99, 2353. 2732. [329] R. Meir, K. Shamalov, O. Betzer, M. Motiei, M. Horovitz-Fried, R. [347] C. H. Schimmelpfennig, S. Schulz, C. Arber, J. Baker, I. Tarner, J. Yehuda, A. Popovtzer, R. Popovtzer, C. J. Cohen, ACS Nano 2015, 9, McBride, C. H. Contag, R. S. Negrin, Am.J.Pathol.2005, 167, 1321. 6363. [348] R. M. Prins, N. Craft, K. W. Bruhn, H. Khan-Farooqi, R. C. Koya, R. [330] a) M. Nishino, A. Giobbie-Hurder, N. H. Ramaiya, F. S. Hodi, J. Im- Stripecke, J. F. Miller, L. M. Liau, J. Immunol. 2006, 176,157. munother. Cancer 2014, 2, 40; b) A. Schraag, B. Klumpp, S. Afat, S. [349] T. Fawdington, A Case of Melanosis: With General Observations on the Gatidis, K. Nikolaou, T. K. Eigentler, A. E. Othman, Eur. J. Radiol. Pathology of this Interesting Disease, Hardpress Publishing, Stock- 2019, 121, 108688. bridge MA 2020, p. 1826. [331] S. A. Rosenberg, N. P. Restifo, Science 2015, 348, 62. [350] a) K. B. Tran, C. M. Buchanan, P. R. Shepherd, Curr. Pharm. Des. [332] D. M. King, Cancer Imaging 2006, 6, 204. 2020, 26, 396; b) J. V. Cohen, E. I. Buchbinder, Curr.Oncol.Rep.2019, [333] H. C. Thoeny, B. D. Ross, J. Magn. Reson. Imaging 2010, 32,2. 21, 106. [334] a) I. J. de Vries, W. J. Lesterhuis, J. O. Barentsz, P. Verdijk, J. H. van [351] H. Rehman, A. W. Silk, M. P. Kane, H. L. Kaufman, J. Immunother. Krieken, O. C. Boerman, W. J. Oyen, J. J. Bonenkamp, J. B. Boeze- Cancer 2016, 4, 53. man, G. J. Adema, J. W. Bulte, T. W. Scheenen, C. J. Punt, A. Heer- [352] a) M. R. Middleton, J. J. Grob, N. Aaronson, G. Fierlbeck, W. Tilgen, schap, C. G. Figdor, Nat. Biotechnol. 2005, 23, 1407; b) R. Tavaré, P. S. Seiter, M. Gore, S. Aamdal, J. Cebon, A. Coates, B. Dreno, M. Sagoo, G. Varama, Y. Tanriver, A. Warely, S. S. Diebold, R. South- Henz, D. Schadendorf, A. Kapp, J. Weiss, U. Fraass, P. Statkevich, worth, T. Schaeffter, R. I. Lechler, R. Razavi, G. Lombardi, G. E. M. Muller, N. Thatcher, J. Clin. Oncol. 2000, 18, 158; b) P. B. Chap- Mullen, PLoS One 2011, 6, e19662. man, L. H. Einhorn, M. L. Meyers, S. Saxman, A. N. Destro, K. S. [335] a) N. H. Cho, T. C. Cheong, J. H. Min, J. H. Wu, S. J. Lee, D. Kim, J. S. Panageas, C. B. Begg, S. S. Agarwala, L. M. Schuchter, M. S. Ern- Yang,S.Kim,Y.K.Kim,S.Y.Seong,Nat. Nanotechnol. 2011, 6, 675; stoff, A. N. Houghton, J. M. Kirkwood, J. Clin. Oncol. 1999, 17, 2745.

[353] T. Muranen, L. M. Selfors, D. T. Worster, M. P. Iwanicki, L. Song, F. [370] E. M. Van Allen, N. Wagle, A. Sucker, D. J. Treacy, C. M. Johannessen, C. Morales, S. Gao, G. B. Mills, J. S. Brugge, Cancer Cell 2012, 21, E. M. Goetz, C. S. Place, A. Taylor-Weiner, S. Whittaker, G. V.Kryukov, 227. E. Hodis, M. Rosenberg, A. McKenna, K. Cibulskis, D. Farlow, L. [354] A. Gorden, I. Osman, W. Gai, D. He, W. Huang, A. Davidson, Zimmer, U. Hillen, R. Gutzmer, S. M. Goldinger, S. Ugurel, H. J. A. N. Houghton, K. Busam, D. Polsky, Cancer Res. 2003, 63, Gogas, F. Egberts, C. Berking, U. Trefzer, C. Loquai, B. Weide, J. C. 3955. Hassel, S. B. Gabriel, S. L. Carter, G. Getz, et al., Cancer Discov. 2014, [355] a) S. L. Topalian, J. M. Taube, R. A. Anders, D. M. Pardoll, Nat. Rev. 4, 94. Cancer 2016, 16, 275; b) D. C. Flaherty, S. Lavotshkin, J. R. Jalas, H. [371] M. Griffin, D. Scotto, D. H. Josephs, S. Mele, S. Crescioli, H. J. Bax, Torisu-Itakura, D. D. Kirchoff, M. S. Sim, D. J. Lee, A. J. Bilchik, J. G. Pellizzari, M. D. Wynne, M. Nakamura, R. M. Hoffmann, K. M. Am. Coll. Surg. 2016, 223, 134. Ilieva, A. Cheung, J. F. Spicer, S. Papa, K. E. Lacy, S. N. Karagiannis, [356] A. Tarhini, R. R. Kudchadkar, Cancer Treat. Rev. 2018, 71,8. Oncotarget 2017, 8, 78174. [357] The Cancer Genome Atlas Network, Cell 2015, 161, 1681. [372] P. L. Chen, W. Roh, A. Reuben, Z. A. Cooper, C. N. Spencer, P. A. Pri- [358] M. Kunz, M. Hölzel, Cancer Metastasis Rev. 2017, 36, 53. eto, J. P. Miller, R. L. Bassett, V. Gopalakrishnan, K. Wani, M. P. De [359] E. R. Malone, M. Oliva, P. J. B. Sabatini, T. L. Stockley, L. L. Siu, Macedo, J. L. Austin-Breneman, H. Jiang, Q. Chang, S. M. Reddy, W. Genome Med. 2020, 12,8. S. Chen, M. T. Tetzlaff, R. J. Broaddus, M. A. Davies, J. E. Gershen- [360] F. S. Collins, M. Morgan, A. Patrinos, Science 2003, 300, 286. wald, L. Haydu, A. J. Lazar, S. P. Patel, P. Hwu, W. J. Hwu, A. Diab, [361] N. K. Hayward, J. S. Wilmott, N. Waddell, P. A. Johansson, M. A. I. C. Glitza, S. E. Woodman, L. M. Vence, I. I. Wistuba, et al., Cancer Field, K. Nones, A.-M. Patch, H. Kakavand, L. B. Alexandrov, H. Discov. 2016, 6, 827. Burke, V. Jakrot, S. Kazakoff, O. Holmes, C. Leonard, R. Sabari- [373] a) N. McGranahan, A. J. Furness, R. Rosenthal, S. Ramskov, R. Lyn- nathan, L. Mularoni, S. Wood, Q. Xu, N. Waddell, V. Tembe, G. M. gaa, S. K. Saini, M. Jamal-Hanjani, G. A. Wilson, N. J. Birkbak, C. T. Pupo, R. De Paoli-Iseppi, R. E. Vilain, P. Shang, L. M. S. Lau, R. A. Hiley, T. B. Watkins, S. Shafi, N. Murugaesu, R. Mitter, A. U. Akarca, Dagg, S.-J. Schramm, A. Pritchard, K. Dutton-Regester, F. Newell, J. Linares, T. Marafioti, J. Y. Henry, E. M. Van Allen, D. Miao, B. et al., Nature 2017, 545, 175. Schilling, D. Schadendorf, L. A. Garraway, V. Makarov, N. A. Rizvi, A. [362] R. J. Sullivan, K. T. Flaherty, Clin. Cancer Res. 2015, 21, 2424. Snyder, M. D. Hellmann, T. Merghoub, J. D. Wolchok, S. A. Shukla, [363] G. V. Long, D. Stroyakovskiy, H. Gogas, E. Levchenko, F. de Braud, J. et al., Science 2016, 351, 1463; b) M. Lauss, M. Donia, K. Harbst, R. Larkin, C. Garbe, T.Jouary, A. Hauschild, J. J. Grob, V.Chiarion Sileni, Andersen, S. Mitra, F. Rosengren, M. Salim, J. Vallon-Christersson, C. Lebbe, M. Mandalà, M. Millward, A. Arance, I. Bondarenko, J. B. T. Törngren, A. Kvist, M. Ringnér, I. M. Svane, G. Jönsson, Nat. Haanen, J. Hansson, J. Utikal, V. Ferraresi, N. Kovalenko, P. Mohr, Commun. 2017, 8, 1738; c) N. van Rooij, M. M. van Buuren, D. V. Probachai, D. Schadendorf, P. Nathan, C. Robert, A. Ribas, D. J. Philips, A. Velds, M. Toebes, B. Heemskerk, L. J. van Dijk, S. Behjati, DeMarini, J. G. Irani, M. Casey, et al., N. Engl. J. Med. 2014, 371, H. Hilkmann, D. El Atmioui, M. Nieuwland, M. R. Stratton, R. M. 1877. Kerkhoven, C. Kesmir, J. B. Haanen, P. Kvistborg, T. N. Schumacher, [364] A. S. McNeal, K. Liu, V. Nakhate, C. A. Natale, E. K. Duperret, B. C. J. Clin. Oncol. 2013, 31, e439. Capell, T. Dentchev, S. L. Berger, M. Herlyn, J. T. Seykora, T. W. Ridky, [374] W. Roh, P. L. Chen, A. Reuben, C. N. Spencer, P. A. Prieto, J. P. Miller, Cancer Discov. 2015, 5, 1072. V. Gopalakrishnan, F. Wang, Z. A. Cooper, S. M. Reddy, C. Gumbs, [365] a) N. Coupe, P. Corrie, M. Hategan, J. Larkin, M. Gore, A. Gupta, L. Little, Q. Chang, W. S. Chen, K. Wani, M. P. De Macedo, E. Chen, J. A. Wise, S. Suter, C. Ciria, S. Love, L. Collins, M. R. Middleton, Eur. L. Austin-Breneman, H. Jiang, J. Roszik, M. T. Tetzlaff, M. A. Davies, J. Cancer 2015, 51, 359; b) P. A. Ascierto, D. Schadendorf, C. Berk- J. E. Gershenwald, H. Tawbi, A. J. Lazar, P. Hwu, W. J. Hwu, A. Diab, ing, S. S. Agarwala, C. M. van Herpen, P. Queirolo, C. U. Blank, A. I. C. Glitza, S. P. Patel, et al., Sci. Transl. Med. 2017, 9, eaah3560. Hauschild, J. T. Beck, A. St-Pierre, F. Niazi, S. Wandel, M. Peters, A. [375] L. Calapre, L. Warburton, M. Millward, M. Ziman, E. S. Gray, Cancer Zubel, R. Dummer, Lancet Oncol. 2013, 14, 249; c) J. M. Kirkwood, Lett. 2017, 404, 62. L. Bastholt, C. Robert, J. Sosman, J. Larkin, P. Hersey, M. Middleton, [376] F. Properzi, M. Logozzi, S. Fais, Biomark. Med. 2013, 7, 769. M. Cantarini, V. Zazulina, K. Kemsley, R. Dummer, Clin. Cancer Res. [377] a) S. Jahr, H. Hentze, S. Englisch, D. Hardt, F. O. Fackelmayer, R. 2012, 18, 555. D. Hesch, R. Knippers, Cancer Res. 2001, 61, 1659; b) F. Diehl, K. [366] a) M. Ranzani, C. Alifrangis, D. Perna, K. Dutton-Regester, A. Schmidt, M. A. Choti, K. Romans, S. Goodman, M. Li, K. Thornton, Pritchard, K. Wong, M. Rashid, C. D. Robles-Espinoza, N. K. N. Agrawal, L. Sokoll, S. A. Szabo, K. W. Kinzler, B. Vogelstein, L. A. Hayward, U. McDermott, M. Garnett, D. J. Adams, Pigment Cell Diaz, Jr., Nat. Med. 2008, 14, 985. Melanoma Res. 2015, 28, 117; b) M. Krauthammer, Y. Kong, A. Bac- [378] S. J. Lee, C. H. Lee, S. H. Choi, S. H. Ahn, B. H. Son, J. W. Lee, J. chiocchi, P. Evans, N. Pornputtapong, C. Wu, J. P. McCusker, S. Ma, H. Yu, N. J. Kwon, W. C. Lee, K. S. Yang, D. H. Lee, D. Y. Han, M. S. E. Cheng, R. Straub, M. Serin, M. Bosenberg, S. Ariyan, D. Narayan, Choi, P. S. Park, H. K. Lee, M. S. Kim, J. Lee, B. H. Jeon, Oncol. Lett. M. Sznol, H. M. Kluger, S. Mane, J. Schlessinger, R. P. Lifton, R. Ha- 2017, 13, 3025. laban, Nat. Genet. 2015, 47, 996. [379] a) A. A. Khorasani, J. L. Weaver, C. Salvador-Morales, Int. J. [367] a) S. E. Woodman, J. C. Trent, K. Stemke-Hale, A. J. Lazar, S. Pricl, Nanomed. 2014, 9, 5729; b) M. A. Eaton, L. Levy, O. M. Fontaine, G. M. Pavan, M. Fermeglia, Y. N. Gopal, D. Yang, D. A. Podoloff, D. Nanomedicine 2015, 11, 983. Ivan, K. B. Kim, N. Papadopoulos, P. Hwu, G. B. Mills, M. A. Davies, [380] M. L. Etheridge, S. A. Campbell, A. G. Erdman, C. L. Haynes, S. M. Mol. Cancer Ther. 2009, 8, 2079; b) F. S. Hodi, P. Friedlander, C. L. Wolf, J. McCullough, Nanomedicine 2013, 9,1. Corless, M. C. Heinrich, S. Mac Rae, A. Kruse, J. Jagannathan, A. D. [381] V. J. Yao, S. D’Angelo, K. S. Butler, C. Theron, T. L. Smith, S. Marchiò, Van den Abbeele, E. F. Velazquez, G. D. Demetri, D. E. Fisher, J. Clin. J. G. Gelovani, R. L. Sidman, A. S. Dobroff, C. J. Brinker, A. R. M. Oncol. 2008, 26, 2046. Bradbury, W.Arap, R. Pasqualini, J. Controlled Release 2016, 240,267. [368] J. M. Hess, A. Bernards, J. Kim, M. Miller, A. Taylor-Weiner, N. J. [382] S. Behzadi, V. Serpooshan, W. Tao, M. A. Hamaly, M. Y. Alkawareek, Haradhvala, M. S. Lawrence, G. Getz, Cancer Cell 2019, 36, 288. E. C. Dreaden, D. Brown, A. M. Alkilany, O. C. Farokhzad, M. Mah- [369] F. Catalanotti, D. T. Cheng, A. N. Shoushtari, D. B. Johnson, K. moudi, Chem.Soc.Rev.2017, 46, 4218. S. Panageas, P. Momtaz, C. Higham, H. H. Won, J. J. Harding, T. [383] A. L. Barrán-Berdón, D. Pozzi, G. Caracciolo, A. L. Capriotti, G. Merghoub, N. Rosen, J. A. Sosman, M. F. Berger, P. B. Chapman, D. Caruso, C. Cavaliere, A. Riccioli, S. Palchetti, A. Laganà, Langmuir B. Solit, JCO Precis. Oncol. 2017, 1,1. 2013, 29, 6485.

[384] B. Yameen, W. I. Choi, C. Vilos, A. Swami, J. Shi, O. C. Farokhzad, J. [390] a) V. Anagnostou, M. Yarchoan, A. R. Hansen, H. Wang, F. Verde, Controlled Release 2014, 190, 485. E. Sharon, D. Collyar, L. Q. M. Chow, P. M. Forde, Clin. Cancer Res. [385] J.-B. Coty, C. Vauthier, J. Controlled Release 2018, 275, 254. 2017, 23, 4959; b) L. A. Emens, P. A. Ascierto, P. K. Darcy, S. De- [386] A. M. Nyström, B. Fadeel, J. Controlled Release 2012, 161, 403. maria, A. M. M. Eggermont, W. L. Redmond, B. Seliger, F. M. Mar- [387] V. Sainz, J. Conniot, A. I. Matos, C. Peres, E. Zupanoiˇ o,ˇ L. Moura, incola, Eur. J. Cancer 2017, 81, 116. L. C. Silva, H. F. Florindo, R. S. Gaspar, Biochem. Biophys. Res. Com- [391] K. E. Keenan, Z. Gimbutas, A. Dienstfrey, K. F. Stupic, J. Magn. Reson. mun. 2015, 468, 504. Imaging 2019, 50, 1948. [388] J. J. Tao, A. M. Schram, D. M. Hyman, Annu. Rev. Med. 2018, 69, 319. [392] P. A. Ott, F. S. Hodi, H. L. Kaufman, J. M. Wigginton, J. D. Wolchok, [389] K. Thorlund, J. Haggstrom, J. J. Park, E. J. Mills, BMJ 2018, 360, k698. J. Immunother. Cancer 2017, 5, 16.

Barbara Carreira received her B.S. (2013) in Biochemistry and her M.Sc. (2015) in Biotechnology from the Faculty of Sciences and Technology, NOVA University of Lisbon. In 2017, she was awarded a fellowship from the Portuguese Foundation for Science and Technology, to develop her Ph.D. under the supervision of Prof. Helena Florindo (University of Lisbon) and Prof. Ronit Satchi-Fainaro (Tel Aviv University). Since then, she has been developing nanomedicines to modulate melanoma-immune cell interactions to regulate the expression of tumor-associated immune evasion factors within the stromal microenvironment.

Ronit Satchi-Fainaro (Ph.D.) is a full professor at TelAviv University, Head of the Cancer Research & Nanomedicine Laboratory at the Department of Physiology & Pharmacology at the Sackler Faculty of Medicine, and The Lion Chair in Nanosciences and Nanotechnologies. She received her B.Pharm. from the Hebrew University in Jerusalem, her Ph.D. from the University of London and a Postdoctoral Research Fellowship at Harvard University and Children’s Hospital Boston. She joined Tel Aviv Univer- sity in 2006.

Helena F. Florindo (Pharm.D., Ph.D.) graduated in Pharmaceutical Sciences in 2003 and received her Ph.D. in Pharmaceutical Technologyin 2008 from the University of Lisbon. Currently she is assistant professor at the Faculty of Pharmacy, University of Lisbon, and the Head of the BioNanoSciences – Drug Delivery & Immunotherapy Research Group at the Research Institute for Medicines. Her research group focuses on the development of nanobiomaterial-based (immune)therapies for cancer, rationally designed to target and modulate the function of dendritic cells and other tumor evasion- related players within tumor microenvironment and metastases.