Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: to use or not to use?

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Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

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Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: To use or not to use?

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Marius M. Strioga a,∗ , Adas Darinskas a,1 , Vita Pasukoniene a,2 , Agata Mlynska a,2 , Valerijus Ostapenko b,3 , Virgil Schijns c,d,e,4 a

Department of Immunology, Center of Oncosurgery, Institute of Oncology, Vilnius University, Vilnius, Lithuania Section of Breast Surgery, 3rd Department of Surgery, Center of Oncosurgery, Institute of Oncology, Vilnius University, Vilnius, Lithuania c Immune Intervention, Cell Biology & Immunology group, Wageningen University, Wageningen, the Netherlands 8 9 Q2 d Epitopoietic Research Corporation (ERC), Namur, Belgium e De Elst 1, 6708 WD, Wageningen, the Netherlands 10 6 7

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Article history: Received 27 January 2014 Received in revised form 29 April 2014 Accepted 1 May 2014 Available online xxx

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Keywords: Tumor-associated antigens Xenogeneic vaccination Cross-reactivity Cancer vaccines Tumor immunotherapy

Accumulation of firm evidence that clinically apparent cancer develops only when malignant cells manage to escape immunosurveillance led to the introduction of tumor immunotherapy strategies aiming to reprogramm the cancer-dysbalanced antitumor immunity and restore its capacity to control tumor growth. There are several immunotherapeutical strategies, among which specific active immunotherapy or therapeutic cancer vaccination is one of the most promising. It targets dendritic cells (DCs) which have a unique ability of inducing naive and central memory T cell-mediated immune response in the most efficient manner. DCs can be therapeutically targeted either in vivo/in situ or by ex vivo manipulations followed by their re-injection back into the same patient. The majority of current DC targeting strategies are based on autologous or allogeneic tumor-associated antigens (TAAs) which possess various degrees of inherent tolerogenic potential. Therefore still limited efficacy of various tumor immunotherapy approaches may be attributed, among various other mechanisms, to the insufficient immunogenicity of self-protein-derived TAAs. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species was suggested to overcome the natural immune tolerance to self TAAs. Xenoantigens are supposed to differ sufficiently from self antigens to a degree that renders them immunogenic, but at the same time preserves an optimal homology range with self proteins still allowing xenoantigens to induce cross-reactive T cells. Here we discuss the concept of xenogeneic vaccination, describe the cons and pros of autologous/allogeneic versus xenogeneic therapeutic cancer vaccines, present the results of various pre-clinical and several clinical studies and highlight the future perspectives of integrating xenovaccination into rapidly developing tumor immunotherapy regimens. © 2014 Published by Elsevier Ltd.

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Abbreviations: BCG, Bacillus Calmette-Guérin; BMDC, bone marrow-derived dendritic cell; CTA, cancer/testis antigen; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T lymphocyte antigen-4; DC, dendritic cell; DNA, deoxyribonucleic acid; DTH, delayed-type hypersensitivity; hCG␤, human chorionic gonadotropin ␤ chain; HGP, human glioma membrane protein; HLA, human leukocyte antigen; hTH, human tyrosine hydroxylase; IFN-␥, interferon-␥; Ig, immunoglobulin; IL(-4,-12), interleukin(-4,-12); LAMP1, lysosome-associated membrane protein-1; LLC, Lewis lung carcinoma; MHC, major histocompatibility complex; NK, natural killer (cell); NSIT, non-specific immunotherapy; OS, overall survival; PAP, prostatic acid phosphatase; PBMC, peripheral blood mononuclear cells; PD-1, programmed death-1; PFS, progression-free survival; PMED, particlemediated epidermal delivery; PSA, prostate-specific antigen; RGP, rat glioma membrane protein; SIT, specific immunotherapy; TAA, tumor-associated antigen; Th, CD4+ T helper cell; TNF-␣, tumor necrosis factor-␣; VEGFR-2, vascular endothelial growth factor receptor-2. ∗ Corresponding author at: Department of Immunology, Center of Oncosurgery, Institute of Oncology, Vilnius University, P. Baublio Str. 3b-321, LT-08406, Vilnius, Lithuania. Tel.: +370 601 69 551; fax: +370 5 2 720 164. E-mail addresses: [email protected], [email protected] (M.M. Strioga), [email protected] (A. Darinskas), [email protected] (V. Pasukoniene), [email protected] (A. Mlynska), [email protected] (V. Ostapenko), [email protected] (V. Schijns). 1 Tel.: +370 5 2 190 932. 2 Tel.: +370 5 2 190 931. 3 Tel.: +370 5 2 786 814. 4 Tel.: +31 (0) 317 483922. http://dx.doi.org/10.1016/j.vaccine.2014.05.006 0264-410X/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Strioga MM, et al. Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: To use or not to use? Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.05.006

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Fig. 1. A general classification of tumor immunotherapy approaches. Various tumor immunotherapy strategies can be grouped into tumor antigen-specific and non-specific. Specific immunotherapy (SIT) targets one or several particular tumor-associated antigens (TAAs), i.e. it aims at attacking malignant cells in a specific manner. Non-specifc immunotherapy (NSIT) modulates the immune response “in general”, without discriminating between antigen specificity of the modulated immune responses. Both SIT and NSIT can be further subdivided into active and passive. By the simpliest definition, active immunotherapy induces the generation of immune effector mechanisms, whereas passive immunotherapy provides “ready-to-use” immune effectors. The main difference between active and passive immunotherapies is that the former educates the immune system to recognize tumor cells and induces, or at least is expected to induce, immune memory, whereas the latter substitutes the immune system until the supplied effectors are “consumed”. However such a strict distinction between specific active and passive immunotherapy is quite indefinite and should be revised, since some reports have shown a vaccinal effect of tumor antigen-specific monoclonal antibodies [80–82]. Along similar lines, an amplification of antitumor T cells after “passive” T cell therapy was reported [83]. Immune-targeted therapies intensify or shape immune responses, e.g. by blocking immune checkpoint molecules, such as cytotoxic T lymphocyte antigen-4 (CTLA-4, inhibited by ipilimumab, tremelimumab) or programmed death-1 (PD-1, inhibited by nivolumab), which either dampen the evolving immune reponses (CTLA-4) or interfere with the effector functions of immune effectors (PD-1) [84]. Intravesical instillation of Bacillus Calmette-Guérin (BCG) vaccine for the treatment of superficial urinary bladder carcinoma [85] is not a “true” therapeutic cancer vaccination. The BCG vaccine is an attenuated live bacterial (Mycobacterium bovis) vaccine which elicits immune responses specific to Mycobacteria (M. tuberculosis, M. bovis). Its antitumor activity can be conceptualized as a “bystander effect” in terms that immune responses against bacteria generate an “immunologic bustle” at the site of BCG “infection” thereby resulting in the mobilization of immune effectors which accidentally “notice” a nearby existing malignant process.

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1. Introduction Unequivocal evidence reveals bidirectional interactions between cancer and the immune system. A properly functioning immune system can prevent/control tumor development and progression without toxicity to normal tissues [1]. Components of both innate and adaptive immunity are involved in fighting cancer [2], however T helper (Th)1-type adaptive immune responses, supporting the generation of CD8+ cytotoxic T lymphocytes (CTLs) and promoting natural killer (NK) cell functional activity, are the key effectors in antitumor immunity [3–6]. Normally the immune system manages to control malignant cells which may potentially emerge throughout the lifetime of an individual, although this has not yet been directly observed in vivo. The immune system either eliminates cancer cells or keeps them in a long-term dormancy without clinically evident disease [7,8]. However, about 13 million of people are newly diagnosed with clinically apparent cancer annually [9]. In these patients some cancer cells managed to break the immune barrier through various immune escape mechanisms that can be broadly grouped into immunoselection and immunosubversion [10,11]. Immunoselection is characterized by (i) an altered expression pattern of tumor antigens (e.g. mutations in epitope regions, downregulation of MHC molecule expression, etc.) rendering them invisible for immune effectors, or/and (ii) neutralization of cytotoxic mechanisms exerted by functional immune effectors, e.g. protease-mediated inhibition of granzyme B and avoidance of apoptosis by malignant cells [12]. Hence, immunoselection results in an emergence of cancer cell clone(s), which are poor targets for an immune attack, because they become either

undetectable or resistant to the effectors of antitumor immunity. Immunosubversion is characterized by tumor-mediated expansion and accumulation of various immunosuppressive components (e.g. regulatory T cells, myeloid-derived suppressor cells, M2-type macrophages, etc.), which inhibit functional activity of cytotoxic immune effectors and promote cancer growth [10,11]. It is believed that the cancer-induced immunosubversion plays a central role in tumor immune evasion [1]. When the capability of the immune system to control cancer fails, kick-starting antitumor immunity by therapeutic means is a reasonable approach for cancer treatment and is defined as tumor immunotherapy. It includes many strategies (Fig. 1), among which tumor antigen-specific active immunotherapy or therapeutic cancer vaccination is one of the most promising approaches. It is based on targeting dendritic cells (DCs), which are the most potent professional antigen-presenting cells in the body, capable of inducing naive and central memory T cell-mediated immune responses most efficiently [13]. DCs can be targeted either in vivo/in situ by co-delivering tumor-associated antigens (TAAs) and immune adjuvants or ex vivo by isolating DC precursors, mainly monocytes, from peripheral blood, manipulating them in vitro (loading with TAAs and inducing DC maturation), and then reinjecting the “fully equipped” DCs back into the same patient [14]. There are many strategies for DC targeting both in situ and ex vivo, such as the use tumor antigen-derived peptides, recombinant DNA, etc. (reviewed in [15]). Currently these strategies are mainly based on providing DCs with tumor epitopes derived from either autologous or allogeneic TAAs which can be broadly grouped into tumor unique and shared TAAs (see Fig. 2 for a more detailed description).


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tumor-specific antigens. However this specificity is relative because these mutated antigens are counterparts of normal proteins and they may potentially be found in any altered, but yet non-malignant cell. Most likely, only a particular set of cumulating mutations (cancer mutanome) drive malignant transformation of a normal cell. • Shared TAAs. As the definition says, these TAAs are normal (nonmutated) self proteins shared between malignant and normal cells. The only difference is that in cancer cells shared TAAs have an altered expression pattern – they are either overexpressed or aberrantly expressed. Greatly excessive expression of such otherwise normal proteins is believed to be responsible for breaking the immune tolerance to self proteins, at least in part due to their increased presentation to the components of adaptive immunity [20]. There are three types of shared TAAs:

Fig. 2. Types of tumor-associated antigens (TAAs). Classification of protein TAAs, based on their tissue/viral origin and mutational status. Non-protein TAAs, including carbohydrates and (glyco)lipids, which constitute of distinct category of TAAs, are not included in the figure.

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1.1. Description of tumor-associated antigens All TAAs, except for oncoviral, are derived from self proteins, which are expressed by normal cells constantly or at some particular periods of human life, e.g. during the perinatal period. Hence all tumor antigens are tumor-associated rather than exquisitely tumor-inherent [16]. Any normal protein which, owing to random somatic genetic modifications, is mutated or inappropriately expressed (over- or aberrantly expressed) in a cancer cell becomes a TAA. All protein TAAs can be grouped into several types (Fig. 2): • Unique TAAs (neo-antigens, mutational antigens) [17,18]. This group of TAAs includes any mutated self-protein, which is not (or at least should not) be present in normal cells. Unique TAAs are products of random somatic point mutations and are therefore expressed uniquely by individual tumors. Some of unique TAAs may be directly linked to cancer development (e.g. altered proteins encoded by mutated proto-oncogenes, tumor suppressor genes, DNA repair or apoptosis-related genes) and are relatively resistant to immunoselection owing to their indispensible role in maintaining the neoplastic state [19]. Other unique TAAs may have no direct or even indirect association with malignant transformation and their appearance is a result of a general genetic instability in cancer cells. Unique TAAs may be regarded as

• products of “silent” genes. Expression of “silent” genes in adult (postnatal) organism is normally shut down (with some minor tissue-specific exceptions), since their encoded proteins are needed only during embryonic and/or fetal development. However their expression can be re-activated (e.g. due to DNA demethylation and/or histone modifications) in cancer cells. There are two closely related categories of TAAs encoded by awaked “silent” genes – cancer/testis antigens (CTAs) [21] and oncofetal antigens [22,23]. In an adult organism CTAs are normally expressed only in immune privileged organs including as testis and placenta, and can be aberrantly expressed in cancer cells. CTAs possess a high immunogenic potential since they are “unknown” for the immune system and hence are not tolerized [21]. Oncofetal antigens may be normally expressed at very low levels in normal tissues (e.g. ␣-fetoprotein in the liver) and can be overexpressed in some cancers or during various non-malignant pathologies [23]. The overexpressed oncofetal antigens are less immunogenic than CTAs, except for a 37 kDa immature Laminin Receptor Protein, defined as the true Oncofetal Antigen (OFA) by some authors [24]. OFA is expressed in an immunogenic manner during embryogenesis and early fetogenesis and re-expressed universally by tumors, whereas in adult healthy tissues it is present in a non-immunogenic mature 67 kDa form [24]. • differentiation antigens. This group of shared TAAs shows tissue-specific and sometimes differentiation stage-dependent expression pattern. Their expression is generally increased in malignant cells, originating from a particular tissue. Examples include tyrosinase, Melan-A/MART-1, gp100, expressed in normal melanocytes and overexpressed in melanoma cells [25]. • overexpressed widely-occuring antigens (universal tumor antigens). These shared TAAs are widely expressed in many normal tissues at very low to moderate levels and overexpressed in a variety of histologically different types of tumors. Proteins like Her2/neu [26], Wilm’s tumor-1 [27], telomerase [28], and survivin [29] fall into this category. It should be also noted that TAAs (both unique and shared) may be non-protein in origin (not shown in the Fig. 2). Some TAAs include carbohydrates [30] or (glyco)lipids [31,32]. Although these non-protein TAAs are not recognized by conventional MHCI-restricted cytotoxic CD8+ T cells, they are targets for other components of antitumor immune response, including natural killer (NK) cells, NK T cells, and ␥␦ T cells. Oncoviral TAAs are non-self, non-human proteins, which are expressed only by malignant cells, whose tranformation was induced by latent infection with oncogenic viruses, such as human papilloma viruses, human herpes virus 7 (Kaposi sarcoma virus), and others. Oncoviral TAAs are highly immunogenic, however viruses, are capable of inhibiting spontaneous host immune responses directed toward viral antigens-carrying cancer cells. The


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mechanisms include virus-mediated defects in antigen processing or presentation by tumor cells, virus-mediated generation of an immunosuppressive microenvironment in the tumor tissue [33]. 2. Immunogenicity of autologous and allogeneic tumor-associated antigens Since all TAAs, except of oncoviral origin, are unexceptionally derived from self proteins, they possess a various degree of inherent tolerogenicity/low immunogenicity potential, depending of the type of TAA. This is especially true for shared TAAs, which are abnormally expressed normal (non-mutated), intrinsically tolerogenic self proteins. The immunogenicity of shared TAAs (except for cancer/testis antigens) relies only on their abnormal expression pattern (overexpression) in cancer cells; an excess expression of a normal protein exceeds the threshold for T cell activation, thereby potentially breaking the tolerogenicity barrier [20]. This suggests that T cell tolerance may be in part determined by a level of antigen presentation on the surface of DCs rather than by the identity of the antigen [34]. However the immunogenicity of overexpressed TAAs is also subject to their presentation to T cells in an immunostimulatory context, i.e. besides delivery of antigen-specific signal 1, correct provision of additional signals, including costimulatory signal 2, polarizing signal 3, and homing signal 4, is obligatory for optimal immunogenic T cell activation and generation of fully functionally-competent CTLs (reviewed in [14]). Mutated self proteins (unique TAAs) and reactivated cancer/testis antigens are “unknown” to the immune system. Therefore these TAAs have signs of “non-self” or “altered self” and should possess higher immunogenicity than overexpressed but nonmutated shared TAAs. Nevertheless it can be presumed that the immunogenicity of mutated proteins may still greatly depend on the level of their mutational status, i.e. on how “foreign” is a particular mutation and whether it is sufficient for the induction of a robust antitumor immune response. Furthermore, immune recognition of autologous TAAs may not translate into effective antitumor immunity capable of controlling tumor growth both in animals [35] and cancer patients, as observed in therapeutic cancer vaccination trials, demonstrating clinical responses only in a subset of patients [36–38]. Many immune suppressive mechanisms [39] may contribute to the failure of cancer vaccination in the non-responding patients. Insufficient immunogenicity (or natural tolerogenicity) of self-protein-derived TAAs may be among these mechanisms. 3. If self TAAs are too weak, may be xenogeneic TAAs are strong enough? The failure of the immune system to control advanced tumors and the limited efficacy of various tumor immunotherapy approaches may be attributed, at least in part, to the concept that self-protein-derived TAAs possess various degrees (depending on a TAA type) of immune tolerogenic potential. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species, to overcome the immune tolerance to self TAAs has been suggested [34,35,40,41]. Indeed, many genes are highly evolutionarily conserved with various degrees of similarities among different species [42,43]. Nevertheless small interspecies sequence variations between homologous human and animal proteins exist, since their amino acid sequences are similar, but not identical. These structural differences may confer increased immunogenicity to xenoproteins based on cross-reactivity between epitopes of a xenogeneic TAA and its homologous human counterpart [35,44,45]. Hence, the immune system senses xenoantigens as “altered self”, which differ sufficiently from self antigens to render them immunogenic, but at the same time preserve an optimal homology range

with self proteins to a degree which allows them to induce crossreactive T cells capable of recognizing tumor cells [35,45,46]. There is evidence that xenoepitopes can bind host major histocompatibility complex (MHC) molecules more strongly than epitopes derived from native homologous proteins, resulting in the formation of more sustained xenogeneic peptide/MHC complexes. Ultimately this leads to more potent xenoantigen-induced T cell responses, cross-reactive with self-protein-derived TAAs [44].

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4. Preclinical data of therapeutic cancer xenovaccination

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The first data from in vivo experiments involving xenogeneic cancer vaccination were published in the late 90s of the last century [35,40,41,44]. Overwijk et al. [44] showed that vaccination of normal CD57BL/6 mice with a recombinant vaccinia virus, encoding the murine homologue of gp100, was non-immunogenic. However immunization of mice with human gp100-encoding virus elicited the generation of antigen-specific, interferon (IFN)-␥-secreting CTLs. Importantly, these human gp100-specific CTLs showed highly specific cross-reactivity with mouse homologue of gp100 in vitro, using various tumor targets. Furthermore, adoptive transfer of xenovaccination-induced and in vitro-restimulated CTLs into mice bearing 3-day-old pulmonary nodules of B16 melanoma resulted in dramatic tumor destruction. The increased immunogenicity of xenogeneic versus self gp100 was shown to be intrinsic to a particular MHC-I-restricted human 9-mer peptide epitope. The peptide was capable of binding mouse MHC-I more strongly compared with its murine counterpart owing to the differences in three NH2 terminal amino acid residues between these homologous epitopes. A sustained expression of stabilized MHC-I/xenopeptide complexes results in their higher amount on the DC surface, which is sufficient for the immunogenic T cell activation [44]. Weber et al. [35] demonstrated that genetic immunization of mice with a recombinant plasmid DNA, encoding a full-length xenogeneic human, but not syngeneic murine gp75, could break immune tolerance and induce a long-lasting antibody response against murine gp75. Moreover C57BL/6J mice immunized with human gp75 were significantly protected from lung metastases following intravenous injection of B16F10/LM3 melanoma cells 2 weeks after the last (5th) vaccination compared with control mice vaccinated with a null vector. In contrast, no significant protection could be observed in mice immunized with DNA, encoding syngeneic murine gp75. Furthermore, priming of C57BL/6J mice with xenogeneic human gp75 could break immune tolerance and induce a strong antibody response to subsequent single immunization with DNA, encoding syngeneic murine gp75 [35]. Wei et al. [40] vaccinated BALB/c, C57BL/6, and C3H mice with fixed proliferative xenogeneic human and bovine endothelial cells, namely primary and cultured human umbilical vein endothelial cells, human dermal microvascular endothelial cells, and bovine glomerular endothelial cells. Xenovaccination could induce a long-lasting complete immune protection from tumor growth after challenge with several solid tumor cell lines and showed a prominent therapeutic efficacy in established tumors. These effects were not observed in mice vaccinated with control vaccines, including mouse endothelial cells transiently infected with Simian vacuolating virus 40, human aorta vascular smooth muscle cells, and human B-lymphoblastoid cells transformed with Epstein-Barr virus. Survival of tumor-bearing mice treated with xenogeneic endothelial cell vaccines was significantly longer when compared with untreated mice or mice immunized with control vaccines. Xenovaccination had no direct cytotoxic activity on malignant cells. Control of tumor growth was associated with


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the inhibition of tumor angiogenesis as evidenced by a gradual decrease in tumor microvessel density. In these models the antitumor effect of xenovaccines was completely dependent on CD4+ 297 T cells and mediated by the induction of antibody rather than CTL 298 response to proliferating endothelial cells. These xenovaccination299 induced antibodies were cross-reactive with murine and bovine 300 endothelium and recognized only proliferating endothelial cells 301 with no recognition of normal quiescient endothelium owing to a 302 different antigen expression profile among these cells. No adverse 303 reactions to treatment were observed [40]. Very similar results with 304 regard to protective and therapeutic antitumor immunity were 305 obtained by the same group [29] using quail vascular endothelial 306 growth factor receptor-2 (VEGFR-2) protein for xenovaccination 307 of mice with several solid and hematopoietic tumor models. In 308 addition, an extensive examination of potential toxicity did not 309 reveal any marked adverse effects in xenovaccinated mice [47]. 310 Yet another study [48] further confirmed these results in terms of 311 safety, protective and therapeutic efficacy of xenogeneic rat, but not 312 syngeneic murine, ␣-fetoprotein-based vaccine in a mouse hepa313 tocellular carcinoma model. The obligatory requirement of CD4+ T 314 cells for the induction of antitumor immune responses was con315 firmed by CD4+ T cell depletion in vivo [48]. 316 Sioud and Sorensen [49] found that intramuscular immunizaQ4 317 tion of glioma BT4C-inoculated rats with syngeneic rat glioma 318 membrane proteins (RGP) did not induce significant reactivity 319 against cancer cells in vitro. However immunization with xeno320 geneic human glioma membrane proteins (HGP) induced both 321 antibody- and CTL-mediated tumor-specific immune responses 322 cross-reactive with human and rat glioma cells. In an in vivo 323 setting, there was a significant protection from tumor growth 324 in HGP-xenovaccinated rats compared with tumors growing in 325 RGP-immunized rats indicative of xenovaccine ability to elicit a 326 cross-reactive systemic immune response. The predominance of a 327 Th1-type immune response was evidenced by higher levels of IgG2a 328 versus IgG1 antibodies and a prominent CTL response. Histologi329 cal analysis revealed that tumors from syngeneic RGP-immunized 330 rats consisted of a homogenous mass of viable glioma cells. In 331 contrast, in xenogeneic HGP-immunized rats, glioma cells were 332 sparse and showed morphological changes characteristic of apo333 ptosis. Importantly, tumors from HGP-immunized rats displayed 334 a large number of infiltrating CD4+ and CD8+ T cells, whereas all 335 glioma samples from RGP-immunized rats exhibited sparse T cell 336 infiltration. Of note, no signs of autoimmune diseases were seen in 337 the HGP-immunized rats after 6 months of observation [49]. 338 Huebener et al. [50] investigated the prophylactic and thera339 peutic antitumor activity of xenovaccination with human tyrosine 340 hydroxylase (hTH) DNA in a primary and spontaneous metastatic 341 mouse neuroblastoma NXS2 NB model. The DNA vaccines were 342 based either on mammalian ubiquitin expression vectors, encod343 ing for full-length hTH protein (Vaccine A), or on hTH minigene 344 comprising the three major hTH-derived epitopes with high affin345 ity to MHC-I molecules (Vaccine B), or on pBudCE4.1 plasmid vector 346 encoding for full-length hTH and bioactive single chain interleukin 347 (IL)-12 (scIL-12) genes expressed by two independent expression 348 units (Vaccine C). All vaccines were delivered by an oral route 349 using as a carrier live attenuated Salmonella typhimurium bacte350 ria, which are rapidly taken-up by gastrointestinal tract DCs. In 351 the prophylactic setting Vaccines A and B induced a 50% reduc352 tion of primary tumor growth and prevented liver metastases in 353 the majority of animals. Vaccine C (hTH + scIL-12) was not supe354 rior to Vaccines A and B, because it did not further enhance the 355 antitumor immune response. This was attributed to a possible 356 role of unmethylated CpG motives in the plasmid DNA vaccine, 357 which effectively induced sufficient IL-12 secretion by DCs, and 358 to the crucial role of a mutated ubiquitin tag in DNA vaccine; 359 the tag ensures a very effective MHC-I epitope presentation after 360 295 296

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proteosomal degradation of antigens. Both in vitro assays and CD8+ T cell depletion experiments in vivo showed that antitumor activity of DNA xenovaccines was primarily mediated by Th1-type immune response and its associated CTL activity. Infiltration of CD4+ and CD8+ T cells was shown by immunohistochemical analysis of postvaccination tumor samples. Anti-TH antibodies were not detectable in sera taken at different time points after xenovaccination, indicating that a humoral immune response plays a subordinate role in this mouse neuroblastoma model [50]. Soong et al. [45] xenovaccinated C57BL/6 mice with plasmid DNA, encoding a full-length human p53 protein. The vaccine was administered intramuscularly followed by electroporation. Xenovaccination generated significantly higher levels of antibodies specific to murine p53 protein and exhibited an increase in the number of murine p53-specific, IFN-␥-producing CTLs compared with syngeneic vaccination. Xenovaccination-induced immune responses could effectively control the growth of subcutaneouslyinjected murine colon cancer MC38 cells both in prophylactic and therapeutic models. CD8+ T cells were identified as critical components of xenovaccine-induced antitumor effects, as evidenced by CD4+ , CD8+ T cell and NK1.1 cell depletion experiments in vivo [45]. Wei et al. [51] showed that the efficacy of xenovaccination may be further increased using a complex DNA xenovaccine construct. They investigated the antitumor efficacy of a combined antitumor and anti-angiogeneic therapeutic xenovaccination in a mouse Lewis lung carcinoma (LLC) model. The authors constructed a fusion gene DNA vaccine comprised of human DNA fragments encoding: (i) 37 amino acids from the C terminal end of human chorionic gonadotropin ␤ chain (hCG␤), (ii) five different HLArestricted CTL epitopes from human survivin, and (iii) the third and fourth extracellular domains of human VEGFR-2. The chimeric construct was inserted into the cDNA sequence between the luminal and transmembrane domains of human lysosome-associated membrane protein-1 (LAMP-1) to target the antigen sequences to the lysosomal compartment. This ensures an enhanced peptide presentation with MHC-II molecules leading to a facilitated CD4+ T cell activation. The authors found that combination of multiple antigenic targets (two universal TAAs – hCG␤ and survivin, and one widely expressed angiogenesis-associated product VEGFR-2) and the use of immune-enhancing LAMP-1 molecule act synergistically and show a dramatically enhanced antibody- and CTL-mediated antitumor immune responses compared with DNA xenovaccine constructs missing one to all three antigenic targets and/or the LAMP-1 sequence. Apoptotic and necrotic tumor cell death and decreased microvessel density was confirmed by immunohistochemical analysis of post-vaccination tumor tissue. Furthermore, the complex DNA xenovaccine-induced memory T cell response was long-lived as evidenced by the frequency of IFN-␥-secreting CD8+ T cells in isolated splenocytes, following DNA xenovaccine boost at 6 months after the primary xenovaccination. Again, no important adverse effects of xenovaccination were detected in major organs by immunohistochemical analysis [51]. 4.2. Veterinary studies Interesting results were provided by adjunctive therapeutic DNA xenovaccination studies in dogs with malignant melanoma. Xenogeneic plasmid DNA vaccines were based on genes, encoding human tyrosinase [52,53] and mouse tyrosinase or gp75 [54,55]. The vaccine preparations were administered intramuscularly using a carbon dioxide powered Biojector 2000 jet delivery device or a Vitajet spring-loaded needle-free injection device. Results from dog studies are of great importance for translation of preclinical data into clinical practice. Canine therapeutic melanoma model is more clinically relevant to human melanoma as compared with widely


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used rodent models. Both human and canine melanomas occur spontaneously in an outbred, immunocompetent host, share similar metastatic profiles, and are generally resistant to chemotherapy and radiation therapy [55]. Grosenbaugh et al. [53] evaluated safety and efficacy of human tyrosinase-encoding plasmid DNA xenovaccine as an adjunctive treatment for stage II or III oral malignant melanoma in dogs (n = 58) with the achieved locoregional disease control. Survival time until death attributable to melanoma was significantly improved for xenovaccinated dogs compared with historical controls (n = 53). Median survival time for historical controls was 324 days, whereas it could not be determined for xenovaccinated dogs, because fewer than 50% of animals in this group died of melanoma before the end of the observation period. Notably, there were no serious systemic adverse reactions observed. Local reactions were limited to acute wheal, hematoma formation, post-vaccination bruising, and mild signs of pain at the injection site [53]. This canine melanoma vaccine (Oncept® ) has received a full licensure from the United States Department of Agriculture [56]. Nevertheless a recent retrospective analysis of the efficacy of Oncept® for the adjunct treatment of canine oral malignant melanoma showed that dogs (n = 40, among them 30 with stage II and III disease) treated with the xenovaccine did not achieve a greater progression-free survival (PFS), diseasefree interval, or median survival time relative to dogs that did not receive the xenovaccine [57]. Therefore, further studies on clinical efficacy of Oncept® are obviously needed. Interestingly, the antitumor effects of Oncept® are mediated by a human and canine tyrosinase cross-reactive antibody response, which coincides with the observed clinical responses in some animals [52]. This is quite unexpected considering that tyrosinase is an intracellular melanosomal glycoprotein, and thus normally unaccessable for antibodies in intact tumor cells. The authors postulated that a low-level cell surface expression of canine tyrosinase is the target for xenovaccine-induced antibodies [52], an assumption supported by the data that another tyrosinase-related melanosomal glycoprotein gp75 is expressed on the surface of mouse tumor cells in the presence of IFN-␥ [58].

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Above we discussed the results of in situ xenovaccination, i.e. direct administration of cancer xenovaccines, mostly recombinant DNA-based, which are aimed at targeting DCs (epidermal, dermal, subcutaneous, muscular, depending on the administration route) in vivo [59]. However xenogeneic TAAs can also be used for ex vivo loading of precursor-derived DCs followed by the induction of DC maturation. For example, Ciesielski et al. [60] vaccinated C57BL/6 mice with bone marrow-derived DCs (BMDCs) transfected with cDNA, encoding murine or human survivin. Mice were first subcutaneously injected with human survivin-loaded BMDCs, and 4 days after the initial xenovaccination challenged with subcutaneous injection of syngeneic glioma GL261 cells. Additional vaccine boosters were given on days 7 and 14. Protective xenovaccination attenuated flank tumor growth leading to complete tumor-free survival in 12 of the 15 (80%) mice, whereas BMDCs transfected with empty parent vector provided no protective effect. Other groups of mice were vaccinated with murine or human survivintransfected BMDCs and then challenged with intracerebral GL261 tumor implants. Both murine and human survivin-based BMDC xenovaccines prolonged survival of mice, however the latter were significantly more effective. Notably, xenovaccination only delayed the growth of intracerebral tumor, as all mice died from tumor progression following primary challenge with intracerebral GL261 tumor implants. However mice that have initially resisted subcutaneous flank tumor challenge (tumor-free survival for more than 180 days), following xenovaccination with human survivin-transfected

BMDCs, were also able to resist subsequent rechallenge with intracerebral GL261 tumor implants and exhibited an extended (more than 60 days) survival with no detectable tumor after sacrifice. Immunofluorescence studies showed that xenogeneic vaccination induced abundant infiltration by CD8+ and CD4+ T cells, but not CD94+ NK cells in both subcutaneous flank and intracerebral tumors, however fever T cells were detected in the latter. This was attributed to an intact blood–brain barrier which could potentially limit the entry of immune effectors to the brain. CD8+ T cells were crucial immune effectors in this glioma xenovaccination model as evidenced by CD8+ T cell depletion experiments in vivo and adoptive transfer of CD3+ T cells isolated from xenovaccinated into naive mice which were subsequently challenged with GL261 gliomas. Using separate epitopes-carrying fragments of human survivin, it was shown that although each individual fragment produced some prolongation of survival, none was superior to either full-length or truncated (nearly full-length and with essential epitopes, but biologically inactive) survivin [60]. These results support the general concept that full-length multi-epitope vaccines have the advantage of providing a full range of epitopes for various MHC haplotypes.

5. Clinical experience with therapeutic cancer xenovaccines Several small-scale xenovaccination clinical studies have been performed to investigate feasibility, safety, immunological and clinical efficacy of xenogeneic cancer vaccines. Fong et al. [46] were the first to xenovaccinate 21 patients with progressive metastatic prostate cancer, who showed a rise in serum prostate-specific antigen (PSA) levels. The xenovaccine was based on autologous DCs, enriched from peripheral blood, and loaded with xenogeneic murine prostatic acid phosphatase (PAP). The vaccine was administered either intravenously, or intradermally, or intralymphatically. The patients were vaccinated twice, with a 4-week interval between two vaccine injections, and did not receive any other therapies during the vaccination study, unless they had further clinical progression at which point they were withdrawn from the study. Following vaccination, all patients developed a T cell proliferative response to murine PAP, and 11 of the 21 (52%) patients developed cross-reactive T cell proliferation in response to the homologous human PAP. Importantly, T cell proliferation could be measured for months following vaccination, indicating the induction of a memory T cell response. There was no difference in T cell proliferation in response to human PAP between patients vaccinated by the three different routes. Importantly, responses to human PAP were associated with IFN-␥ and tumor necrosis factor (TNF)-␣, but not IL-4 production, indicative of a vaccineinduced Th1-type antitumor immune response. The frequency of IFN-␥-secreting CD8+ T cells specific for murine PAP was similar in magnitude to the frequency of memory T cells against influenza. Notably, the frequency of T cells cross-reactive to human PAP was about half that for murine PAP in these patients. Following completion of xenovaccination 6 of the 21 (28.6%) patients experienced disease stabilization assessed by serum PSA measurements and confirmed by computed tomography and bone scans. All six clinical responders had developed human PAP-specific T cell response compared with five of the 15 (33%) non-responders. Although the induction of predominantly low-titer PAP-specific antibodies was observed, there was no correlation between the presence of these antibodies and the induction of T cell immunity or clinical response. This implies that antibody-mediated response had a minor, if any, role in the clinical effect. There was no correlation between clinical stabilization and baseline PSA, PAP level, lymphocyte count, prior treatment, or route of xenoantigen-loaded DC vaccine administration. Only minimal vaccination-associated side effects were observed, including two cases of DC vaccine


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infusion-related reactions, which manifested as self-limited rigors and fevers, one case of a transiently swollen, painful inguinal lymph node following vaccination, and three cases of grade 1 erythema at the injection site. None of the patients developed clinically evident autoimmune diseases despite the fact that five patients developed elevated antinuclear antibodies and one patient developed an elevated rheumatoid factor following vaccination [46]. DCs in this trial were enriched directly from the blood rather than generated from monocytes. However the DCs were loaded with xenoantigen (murine PAP) without subsequent induction of DC maturation. The authors did not provide full description about the phenotype and function of DCs they used. It is widely accepted that induction of a poper DC maturation is a critical factor, determining DC ability to stimulate effective immune responses and avoid induction of tolerance [61–63]. Therefore it can be speculated that application of mature DCs would have given considerably better results. Seledtsov et al. [64] treated 40 patients with stage III/IV melanoma using a xenogeneic poly-antigenic vaccine prepared from disrupted murine tumors (melanoma B16 and LLC cells). LLC-associated antigens were included in the vaccine to broaden the spectrum of immunizing antigen targets and to act as a local inflammation-stimulating adjuvant. In this phase I/II trial, an inducing course of xenovaccination consisted of 10 subcutaneous immunizations – five at weekly and five at fortnight intervals. Twenty-four hours after the first five vaccinations, a low dose of recombinant IL-2 was given subcutaneously. During subsequent consolidating treatment the patients were xenovaccinated monthly. After the inducing vaccinations a significant increase in skin immunoreactivity, assessed by delayed-type hypersensitivity (DTH) skin reaction to B16 melanoma, but not to LCC or mouse spleen non-tumor antigens, was found in 28 of the 42 (70%) patients. A statistically significant increase in proliferative reactivity of autologous peripheral blood mononuclear cells (PBMC) to xenoantigens was detected in all xenovaccinated patients. Most importantly, a marked increase of post-vaccination PBMC proliferative response to human (patient’s own) melanoma-associated TAAs was observed in two of the four evaluated patients, whose autologous tumors were obtained, with no reactivity to unrelated control alloantigens present in the lysate of autologous PBMC. The 3-year overall survival (OS) was evaluated in the 32 xenovaccinated patients with stage IV disease. The control group was composed retrospectively of highly-matched patients who received conventional therapy. Throughout the follow-up period the trial patients did not receive any other systemic therapy. The median OS of the xenovaccinated patients was significantly longer compared with the control group (13.8 months versus 5.8 months, p < 0.05) and it appeared to correlate with the degree of DTH reactivity. The 3-year OS of treated and control patients proved to be 25% and 2%, respectively. Among 32 stage IV melanoma patients, complete response, partial response and stable disease were achieved in 5 (16%), 2 (6%), and 14 (44%) individuals, respectively. An extensive evaluation of side effects (measurement of renal and hepatic function parameters in the serum, autoimmune markers, blood cell parameters) revealed no serious toxicities. During 24–48 h following xenovaccination 19 of the 40 (47%) patients experienced influenza-like symptoms manifesting as subfebrile fever and musculoskeletal discomfort, which was usually self-limiting. Local grade I–II toxicity (irritation at the injection site) was observed in 29 of the 40 (72%) xenovaccinated patients [64]. The Seledtsov group [65] also conducted a phase I/II clinical trial evaluating the toxicity, immunological and clinical responses (long-term OS) in stage IV colorectal cancer patients (n = 37) treated with a xenogeneic poly-antigenic vaccine compared with retrospective highly-matched (in terms of prognostic and clinical parameters) control group patients (n = 35), treated with conventional therapies. The median OS was 17 months versus 7 months

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(p < 0.05) in xenovaccinated and control group patients, respectively. A 2-year OS was 27% and 3% in the xenovaccine-treated and control group patients, respectively. Durable clinical response (complete, partial response or stable disease) of at least 6 months was observed in 23 of the 37 (62%) xenovaccinated patients [66]. However patients in the trial group almost completely lost their survival advantage at 3.5 years after the initiation of xenovaccination [65]. No grade III/IV toxicities or evidence of systemic autoimmune disorders were observed. Xenovaccination led to a significant increase in serum concentrations of both IFN-␥ and IL-4, suggesting the concomitant induction of balanced Th1- and Th2-type immune responses, likely involved in generating effective antitumor immunity [66]. The same group [65] also therapeutically xenovaccinated 16 patients with stage IV renal cell carcinoma and compared their survival with retrospective clinically comparable control group. The median OS in the trial patients was 20 months compared with 8 months in the control group (p < 0.05). Importantly, the patients in the trial group maintained survival benefits throughout the 5-year follow-up period [65]. The Wolchok group [67] conducted a phase 1 randomized cross-over trial of melanoma patients vaccinated with plasmid DNA, encoding either a xenogeneic mouse or a syngeneic human gp100. The vaccine was injected at three different doses (100, 500 or 1500 ␮g) intramuscularly, using a Biojector200 jet delivery device, every 3 weeks for three doses followed by another three doses with the DNA vaccine from another species. Five of the 18 (28%) evaluable patients developed an increase in the frequency of gp100280–288 (but not the gp100209–217 ) peptide tetramerreactive CD8+ T cells with effector memory phenotype (CCR7low , CD45RAlow , CD28low/int , CD27high ) either at the time of cross-over and/or upon completion of all six vaccinations. One patient had an increase in the frequency of antigen-specific CD8+ IFN-␥+ T cells after immunization with the gp100 DNA vaccine. There were two other patients with the T cell increase that was more than three standard deviations higher than baseline value. Interestingly, these patients had an increase in polyfunctional cytokine/chemokine responses, including various combinations of IFN-␥, macrophage inflammatory protein-1␤, TNF-␣, and surface CD107a expression. There was no significant difference between the development of gp100-specific tetramer positivity or increase in intracellular IFN␥ production and PFS, mOS, the three different vaccine doses, or the sequence of vaccination with the human and mouse gp100 DNA vaccines. Interestingly, it was a non-statistically significant trend (p = 0.11) towards increased tetramer positivity in patients who received two lower vaccine doses (100 and 500 ␮g; 5 of 12 patients) compared with those who received the highest vaccine dose (1500 ␮g; zero of six patients). There was no dose-limiting toxicity documented. Only grade 1/2 toxicities were observed, including local reaction at the injection site (most common; in 63% of patients), fatigue and mialgias in two patients each, and fever, chills and pruritis in one patient each [67]. Another pilot study by the same group [68] compared the efficacy of a xenogeneic murine gp100-encoding DNA, in terms of induction of antigen-specific immune responses, and safety. The vaccine was administered intramuscularly (i.m., as a standard technique) or using a gold particle-mediated epidermal delivery (PMED) system (also known as gene gun) in disease-free high-risk melanoma patients (n = 27). Vaccine administered by PMED had a potentially enhanced ability to induce tumor antigen-specific immune responses at a significantly lower DNA dose (1000 ␮g versus 2 ␮g of DNA vaccine in the i.m. and PMED groups, respectively). In particular, 4 of the 27 (15%) assessed patients experienced an increase in gp100 tetramer-reactive CD8+ T cells, with effector memory phenotype, compared with baseline, corresponding to 1 and 3 patients in the i.m. and PMED groups, respectively. Three


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responding patients were positive for gp100280–288 alone, and one patient was positive for both gp100280–288 and gp100209–217 . Xenovaccination was generally well-tolerated with 10 (59%) patients in the i.m. arm and 6 (35%) patients in the PMED arm without any side effects. One dose-limiting toxicity incident was observed in a patient within the PMED arm developing an acute hypersensitivity reaction after the first injection consistent with previously undiagnosed gold sensitivity as considered by dermatologist. The most common toxicity otherwise was grade 1 local injection site reactions in four and five patients in the i.m. and the PMED arm, respectively. Other potentially vaccine-associated toxicities included fatigue, watery eyes, and abdominal pain in two (6%) patients each, and nausea, arthralgia, myalgia, pruritus, and rash in one (3%) patient each. One patient with a prior history of gout had an exacerbation after the third vaccine injection, and another previously irradiated patient experienced radiation recall after the first injection. There was no PFS advantage when comparing both arms of the study, nor when comparing patients who had a detectable immune response to treatment. However, it should be noted that this pilot trial was not powered to analyze survival [68]. Very similar results were obtained in a recent phase I clinical study [69] which investigated the efficacy of a xenogeneic murine tyrosinase-encoding DNA vaccine in patients with malignant melanoma (n = 21). The xenovaccine was administered at doses of 200 ␮g (three patients), 500 ␮g (three patients), or 1500 ␮g (15 patients) by electroporation every 3 weeks for a total of five doses. In 6 of the 15 (40%) patients at the 1500 ␮g dose the DNA xenovaccination resulted in the development of human tyrosinase-reactive CD8+ T cell response detected by tetramer or intracellular IFN-␥ expression analysis. One patient showed an epitope spreading as evidenced by a robust and rapid expansion of CD4+ and CD8+ T cell responses specific to xenovaccine-unrelated cancer-testis antigen NY-ESO-1, which showed seropositivity at baseline. Interestingly, this patient did not develop a response to xenovaccine-related tyrosinase epitopes. No antigen-specific CTL responses were detected in 200 or 500 ␮g dose cohort patients. After a median follow-up of 40.9 months mOS has not been reached [69].

6. Is xenogeneic vaccination superior to autologous antigen vaccination? The idea of xenovaccination arose with the aim to overcome immune tolerance to self-protein-derived TAAs, in order to induce more effective immunological and clinical responses to therapeutic vaccination in cancer patients. However, data from animal studies, and more importantly, from several early-phase clinical trials show that despite the induction of cross-reactive immune responses, therapeutic cancer xenovaccination [46,67–69] is not necessarily superior to vaccination approaches based on autologous TAAs both in terms of immune and clinical responses [36,70–72]. Furthermore, Schreurs et al. [73] reported that although xenovaccination of mice with DNA, encoding human gp100, could induce crossreactive CTLs specific to mouse gp100, this cross-reactivity was not sufficient to eradicate murine melanoma B16 tumors, expressing murine gp100, in vivo. Along similar lines, a recent study by Johnson et al. [74] demonstrated that xenovaccination of Lewis rats with DNA, encoding human PAP, could induce immune responses specific to human PAP with no cross-reactivity to rat PAP, despite high amino acid sequence homology between some human and rat PAP peptides. Moreover, even the selective elimination of the immunodominant human PAP epitope (which could potentially overwhelm responses to less potent but cross-reactive epitopes) did not result in the generation of cross-reactive responses to the native rat protein. Also there was no evidence of epitope

spreading to other rat PAP-derived peptides following immunization with human PAP [74]. Nevertheless we account that these results should not discourage from the idea of using xenogeneic vaccination for cancer immunotherapy. First, the identification of defined mimetic peptides (mimo-epitopes) rather than selection of xenovaccine targets based on protein homology between species is critical for eliciting more potent cross-reactive immune responses. This is certainly achievable with continually developing bioinformatic approaches. Second, it is possible that xenovaccination may act as an icebreaker breaching the glacier of inherent immune tolerance to selfderived TAA epitopes. Xenovaccine-induced antitumor immune responses, which are cross-reactive with autologous epitopes from self TAAs, may possess by themselves an insufficient potential for the recognition and eventual killing of autologous tumor cells in the patient. However a full-scale reactivation and augmentation of xenovaccine-induced, cross-reactive memory T cell responses may be achieved by booster vaccinations with autologous tumor antigens. Third, even if xenovaccine-induced immune responses are specific only to foreign epitopes with no cross-reactivity to autologous antigens [74], this may still be advantageous. In particular, the development of a correct Th1-polarized antitumor immune response to xenoepitopes may create a proper milieu or a by-stander immunologic microenvironment, which synergistically accompanies the generation of natural or autologous vaccination-induced antitumor immune responses. Of course, in such a scenario, the by-stander immune response to xenoantigens should not be too robust and dominant, in order not to overwhelm immune responses to self-TAAs. Fourth, a comprehensive immune monitoring (evaluation of immune parameters before, during and after treatment) of patients receiving xenovaccination were missing in the majority of early-phase clinical trials discussed above and limit drawing final conclusions about the efficacy of xenogeneic vaccines. These data are of critical importance, since analysis of dynamic immune changes in clinically responding and non-responding patients enables to reveal potential immunosuppressive components that interfere with the development of effective antitumor immune response. This immune responsedampening barrier can be overcome by a reasonable combination of auto-, allo- and xenovaccines together with suitable immune modulators (e.g. immune checkpoint inhibitors such as ipilimumab or/and nivolumab), which release the immune brakes that coevolved during tumor progression, may be a key to a more effective cancer immunotherapy, a notion which is supported by some clinical results [67,69,75]. Finally, accumulating evidence indicates that the choice of an appropriate route of administration of therapeutic cancer vaccines has a great impact on the clinical efficacy of vaccination [3,76–79]. In addition to the route of administration, the number and frequency of vaccine injections, the dose of antigen(s) or DCs per vaccine vary greatly among various clinical trials and are yet to be fully elucidated. In conclusion, based on all these arguments, we support the idea that different prime/boost strategies that exploit alternating immunization schedules, using vaccines based on native and allo- or xenogeneic TAAs, as well as a reasonable combination of therapeutic vaccination with other cancer treatment strategies (chemotherapy, hormone therapy, targeted therapies) are a means of increasing the immunogenicity and clinical efficacy of therapeutic cancer vaccination. These critical aspects remain to be resolved in well-designed, large-scale, comparative clinical trials.

Conflict of interest A. Darinskas works at a biotechnology company “Froceth” (Vilnius, Lithuania), V. Schijns is a chief scientific advisor of


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a biotechnology company “Epitopoietic Research Corporation” (Namur, Belgium). Other authors declare no conflicts of interest.

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[Herbal medicines: when to use or not to use?].

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Use of vaccines as probes to define disease burden.

Prostate cancer vaccines: the long road to clinical application.

Immune suppression in tumors as a surmountable obstacle to clinical efficacy of cancer vaccines.

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To use or not to use? Compulsive behavior and its role in smartphone addiction.

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To Use or Not to Use: Male Partners' Perspectives on Decision Making About Prenatal Diagnosis.

Innovative use of porcine factor VIII:C for immune tolerance induction.

Therapeutic vaccines for cancer: an overview of clinical trials.

The FDA guidance on therapeutic cancer vaccines: the need for revision to include preventive cancer vaccines or for a new guidance dedicated to them.

Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: hope or reality from clinical studies.

To use or not to use hydroxyethyl starch in intraoperative care: are we ready to answer the 'Gretchen question'?

Immune adjuvants as critical guides directing immunity triggered by therapeutic cancer vaccines.

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Femoral Traction Splints in Mountain Rescue Prehospital Care: To Use or Not to Use? That Is the Question.

Ethics of trial drug use: to give or not to give?

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To Share or Not to Share: Ethical Acquisition and Use of Medical Data.

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