Immune surveillance and immunotherapy: Lessons from carbohydrate mimotopes

Vaccine 27 (2009) 3405–3415

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Immune surveillance and immunotherapy: Lessons from carbohydrate mimotopes Anastas Pashov, Behjatolah Monzavi-Karbassi, Thomas Kieber-Emmons ∗ Department of Pathology and Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, United States of America

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Article history: Available online 5 February 2009 Keywords: Cancer immunotherapy Carbohydrate mimotopes

a b s t r a c t The immune system plays an intricate role in tumorigenesis, therefore cancer immunotherapy borrows concepts both from autoimmunity and vaccinology. Due to tumor-induced immune suppression, the adjuvant setting seems most suitable for immunotherapy, which optimally targets multiple tumor associated antigens after removal of the bulk of the tumor. The responses elicited need not match the intensity of those against pathogens. Retrospective studies suggest that cancer patients’ survival correlates with low-titer anti-tumor IgM antibodies. Carbohydrate mimetic peptides (CMPs) prove instrumental as immunogens by boosting similar persistent IgM anti-tumor responses, engaging the innate/adaptive immunity interface and promoting cytotoxic responses and epitope spreading. © 2009 Elsevier Ltd. All rights reserved.

The expanding repertoire of immunotherapeutic strategies, including passive, adoptive, active specific and adjuvant are all aimed at reducing disease recurrence and improving survival. In contrast to pathogen vaccines, which are used within the prophylactic setting, the majority of cancer vaccine strategies are directed to the therapeutic setting. The size of the malignant cell population that has to be eliminated, the associated immunosuppressive effects of the tumor, the diversion of inflammatory and immune processes by the organized stroma of the tumor, the antigenic diversity of the tumor cell population leading to immune escape, all present barriers that predestine the failure of most attempts for active immunotherapy. Consequently, tumor-induced immune suppression necessitates adhering to the adjuvant mode of immunotherapy, i.e. it may be efficient mostly after the removal of large tumor masses. To this end, immunotherapy aimed at inducing or enhancing tumor-specific immunity that may control or eradicate remaining tumor cells may be an appealing method. Likewise, to realize long-term disease-free survival, it will be necessary to develop complementary therapies that are non-cross-resistant with chemotherapy. Tumor immunotherapy may have to borrow approaches from autoimmunity due to the intricate role of the immune system in tumorigenesis. Understanding immune tolerance as active, threshold dependent and redundant processes helps to rationalize the reshaping, rather than breaking, of tolerance as a tumor

∗ Corresponding author at: Department of Pathology, University of Arkansas for Medical Sciences, 4301 West Markham St. Slot #824, Little Rock, AR, United States of America. Tel.: +1 501 526 5930 fax: +1 501 526 5934. E-mail address: [email protected] (T. Kieber-Emmons). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.074

immunotherapy objective. Instead of aiming at specific targets, the emerging concept of tumor vaccine is rather of immunomodulators that target the interface of innate and adaptive immunity [1]. An aspect of tumor immunology, challenging our definitions of tolerance, is that escape from immune surveillance usually occurs during persistent antitumor immune responses; selecting the expression of new tumor antigens and necessitating an approach to target a broad spectrum of antigens. By default antitumor responses end up hijacked and diverted by the tumor but, properly modulated, they can be amplified and manipulated to suppress the tumor, presenting substantial opportunities for immunotherapy. Among the valid clinical targets are the tumor associated carbohydrate antigens (TACAs). The clinical relevance of TACAs is demonstrated by their restricted expression on tumor cells compared with normal tissue, by the higher metastatic potential of tumors expressing high levels of certain types of TACAs, and by the negative impact of high expression levels of specific TACAs on disease prognosis and patient survival [2–4]. In infectious diseases, antibodies play a role in preventing blood-borne dissemination of the infection through immune mechanisms associated with disease eradication. Our most successful pathogen vaccines in the last decade such as Haemophilus influenzae Type b (Hib), are carbohydrate-based. One perceived objective of immunotherapy is to build intense, sustained immunity to cancer cells. Is this possible in view of the nature of TACAs? Why not replicate carbohydrate-targeting vaccines in cancer for eradication of bloodborne dissemination of tumor cells, e.g. minimal residual disease or adjuvant setting? As TACA, carbohydrate epitopes have the unique advantage of broad distribution, spanning both different molecular species and various tumor types. This does not prevent them from

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carrying tumor specific characteristics—mostly through altered levels of expression. The central importance of TACA in humoral immunotherapy is well supported. In retrospective studies patient survival significantly correlates with carbohydrate-reactive IgM levels [5,6]. Natural (mostly glycolipid reactive) IgM are proapoptotic for tumor cells [7,8]. Although the origin of these antibodies is not clear they seem to belong to the class of natural antibodies because they are not affinity matured and are encoded by distinct germ-line restricted gene families [9]. In this context induction of IgM reactive antibodies to TACA expressing tumor cells is akin to the role played by circulating IgM antibodies as a first line immune surveillance mechanism for eradication of blood-borne pathogens. Following nature’s lessons, the renaissance of natural IgM antibodies opens a new area of cancer therapeutics and diagnostics. But other functional mechanisms have been described too. Anti-ganglioside antibodies are suggested to eliminate suppressive gangliosides, shed by the tumor cells [10]. Mice transgenic for anti-GD2 monoclonal IgM antibody were more resistant to GD2 expressing tumor and the effectors were found to be natural killer (NK) cells [11]. This finding indicates the potential of ganglioside specific IgM responses to mobilize effectively innate mechanisms of tumor surveillance. Finally, anti-TACA antibodies might also lend to crosspresentation of antigens, facilitating cellular responses. Crosspresentation of antigens is essential for the response of cytotoxic T cells to tumors [12]. It has been known for many years that pre-existing antibody responses can regulate immune responses following subsequent antigen challenge. Consequently, the ability of TACA reactive natural antibodies to modify immune responses may be related to the ability of these antibodies to form immune complexes in vivo rather than through a complement receptor-dependent pathway. Although the exact mechanism by which immune complexes influence immune responses is insufficiently understood, a number of possibilities have been proposed (reviewed in Ref. [13]). Antigen complexed to IgG antibodies has been shown to promote deposition of antigen in lymphoid follicles, localizing antigen to follicular dendritic cells (DC), which can efficiently prime immune responses. Follicular DC have also been shown to retain IgM immune complexes [14]. TACA directed tumor vaccines not only enforce a natural antitumor antibody response but also conform with a novel view of immunotherapy as control of a complex immune processes more related to the interventions in autoimmunity as opposed to a purely vaccinological view of tumor vaccines. A better understanding of the immunoregulatory aspect of anti-ganglioside responses, for example, will help us to understand the link between TACA immunization, innate immunity and cellular immunity. T cell independent type 2 (TI-2) responses are sometimes referred to as innate immune memory because of the rapid dynamics, but unlike typical memory responses they are short-lived. Although not conforming to the current paradigm, long-term IgM responses have been observed [15]. A major task is the unlocking of the long-term antiTACA IgM responses by recruiting appropriate bystander T cell help, cytokine treatment and appropriate Toll Like rectptor (TLR) ligands. Another aspect of TI-2 responses is their integration in the anti-tumor immunity indirectly through antigen presentation and immunomodulation. In line with a more systemic view, combined modality therapy will prove indispensable involving also anti-regulatory T cells (Treg) treatment and careful design of the protocols in terms of adjuvant therapy. With all these considerations immunotherapy and tumor vaccines remain the most natural, safe and desirable among the adjuvant therapies. To generate sustained immunity to TACAs and to broaden our understanding of carbohydrate and tumor immunology, we have developed immunogens based on carbohydrate mimetic peptides (CMPs)—a strategy whose clinical promise is supported by our pre-

liminary studies. We have observed that CMP immunization leads to a significant tumor growth inhibition in therapeutic and prophylactic mouse models even when only low titer anti-TACA antibodies are elicited [16–18]. CMPs can prime for memory responses to TACAs [19], which might be related to the B1b cell compartment. CMPs can be manipulated in ways that TACA cannot. CMPs can be engineered to induce CD8+ T cells cross-reactive with tumorassociated glycopeptides and/or to induce CD4+ T cells that benefit the further expansion of CD8+ T cells and B cells [18,20]. Thus, CMPs have the potential to generate a multifaceted TACA-reactive immune response. The ability to induce a humoral carbohydrate cross-reactive response, a CD4+ T helper (Th) response, and a CD8+ cytotoxic T-lymphocyte (CTL) response with one simple inoculation is a novel approach to vaccination. The relative specificity of CMPs and the unusual dual immunological character (peptide epitopes/carbohydrate mimotopes) makes CMP novel tools to understand and manipulate immune responses to tumor cells. Several lessons have been learned from our preclinical studies which we summarize here. First CMPs induce low titer broad-spectrum antitumor responses; second, CMPs see B cells differently than TACA; third, CMPs may bridge the activity of NK cells; fourth, CMPs provide insight into tolerance and selfreactivity; and fifth, CMPs can mediate sustained immunity and be effective in combination therapy. 1. Lesson 1: When is enough enough Two important challenges lie ahead in developing immunotherapeutics. First we need to discover antigenic formulations that target multiple antigens associated with tumor cells. Monovalent vaccines, which target a single antigen, may not supply the longterm immunity necessary to prevent relapse as tumor cells adapt by changing antigen expression. Single-antigen vaccines may have a role but in advanced disease, if the targeted antigen is critical to tumor growth. Clinically useful tumor vaccines will have to immunize rather against multiple immunogenic antigens, targeting the important ones involved in malignant transformation. Several classes of antigens have been suggested as suitable targets and they may actually reflect significant host–tumor interactions as defined by analysis of the reactivity of high-titer circulating tumorassociated antibodies [21]. These include cancer testis antigens [22], differentiation antigens [23], over expressed tumor-associated antigens [24], mutated self-antigens and tumor-specific splice variants. Boosting responses to an array of similar targets probably underlies the increased survival in recurrent metastatic melanoma after active immunotherapy with a polyvalent allogeneic cell vaccine [25]. Carbohydrate design strategies are emphasizing the multivalent presentation of carbohydrate immunogens as a means to induce responses to a number of TACAs [26]. These polyvalent design strategies still argue for an immune response to see each carbohydrate constituent as a separate epitope. Inducing a response to each carbohydrate is seen as fulfilling the need for broad spectrum targeting by inducing a polyclonal response to tumor cells. Another facet of developing broad-spectrum vaccines, which is less well developed, is the idea of taking advantage of polyspecific antigen recognition. Polyspecific invariant pattern recognition receptors (PRR) are characteristic of innate immunity [27]. Many PRR, like macrophage scavenger receptor, TLR2 [28], TLR4 [29], DC-SIGN [30], CD9 [31], and NKG2D [32] recognize multiple, structurally diverse, ligands as signals of dangerous changes of the internal environment. A substantial part of the circulating antibodies are polyspecific [33] and are reactive with carbohydrate antigens. The structural degeneracy of carbohydrate determinants and their broad expression makes them an obvious choice as broadspectrum targets. We have suggested the possibility to build the

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mimicry of diverse carbohydrate antigens using a small set of amino acids [34,35]. Most probably these residues interact with carbohydrate binding sites on antibodies and lectins through a degenerate alphabet of simple bonding patterns, mostly of hydrogen bonds and pi stacking that are common or similar to those of carbohydrate antigens. Many of the bonds established between a CMP and a template antibody are outside the target carbohydrate footprint and increasing the affinity of the interaction with the template may lead to peptide highly specific for the template but not a better CMP. Therefore, refining the specificity of the CMPs with respect to the mimicked structures (but not for the templates) may be impossible [36] and it may be advisable rather to use a (controlled) level of degeneracy of recognition to provide structures that elicit a repertoire of antibodies of predetermined polyspecificity (multiple antigen mimotopes (MAM)) [35,37]. It is possible that such carbohydrate mimics elicit a broader range of anti-carbohydrate antibodies than predicted from the initial characterization of these motifs [38]. We have described CMP reactivity with normal human IgG repertoire demonstrating that these fractions contain antibodies cross-reactive with multiple carbohydrate antigens [35,37]. Recently we have developed a CMP (P10s - WRYTAPVHLGDG)) capable of inducing mostly weak anti-GD2 IgM responses crossreactive with a number of other gangliosides; includingGD3, GM2, GD1a. [39]. P10s was derived from a sequence (P10 - GVVWRYTAPVHLGDG) selected by panning a peptide library on the GD2 binding monoclonal antibody ME36.1 [40]. The P10 peptide can mediate antitumor responses [41]. This sequence was further optimized by molecular modeling so that its binding interface overlapped better with the ME36.1 paratope, thus yielding P10s [39]. The carbohydrate cross-reactivity of this mimotope was mapped on the human pre-immune IgG repertoire (IVIg) using glycan arrays. Table 1 illustrates the magnitude of polyreactivity including specificities that are relevant to immune surveillance. Some of them probably can be boosted upon immunization with P10s. The set of cross-reactive glycans included several tumor associated antigens, short chain gangliosides but not the nominal antigen for the template antibody, ME36.1. Some non-self-reactivities, like anti-␣Gal epitope or anti-Globo-H, were very high in the starting mixture as they are normally highly represented in the IgG repertoire and although they are not highly enriched they are very strong among the cross-reactivities of anti-P10s IgG. Other reactivities are very weak in the normal IgG repertoire (like anti-GD3) being well tolerated self-antigens. A high reactivity to these antigens in the P10s fraction leads to very high enrichment ratios. Like in the case of serum titers, these reactivities cannot be attributed solely to affinity or concentration but are representative of the biologically relevant cross-reactivity of P10s. The second important challenge is to understand the therapeutically effective range of antibody or T cells reactivity for antitumor protection, which might be related to mechanisms of action. There is a prevailing view that more is better whether we speak of antibodies or T cell frequencies. Experience shows that after carbohydrate conjugate immunization protection from Haemophilus influenzae type b falls at a much slower rate than the protective antibody titers [42]. Unfortunately, unlike some pathogen infections in which the pathogen is cleared naturally, levels of protective antibody titer or T cell frequency is not readily determined in cancer patients. Low affinity natural IgM antibodies are indispensable for anti-pathogen responses [43]. The accepted protective antibody level for long term protection upon vaccination with the carbohydrate-based vaccines against Haemophilus influenzae Type b is 1 ␮g/ml and recent work has suggested serum concentration of 0.35 ␮g/ml to be point estimate of clinical efficacy against invasive meningococcal disease [44]. The physiological meaning of these quantities, of course, depends of the avidity and “quality” [45,46] of the antibodies, i.e. antibody concentration and even titer are insuf-

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Table 1 Highly enriched anti-carbohydrate IgG reactivities from human IgG in the fraction, affinity purified on CMP P10s immunoadsorbent—results from glycan array binding assay. Human IgG preparation for intravenous use (IVIg) was dialyzed and passed through a P10s affinity column. The retained antibodies were eluted and dialyzed. Both IVIg and anti-P10s fraction were tested on the glycan array (Consortium for Functional Glycomis) at 0.1 mg/ml. The enrichment ratio is the fold increase in the reactivity in the P10s specific fraction relative to non-fractionated IVIg. The magnitude of the reactivity of all these specificities in anti-P10s fraction was comparable to those of specific monoclonal antibodies tested on the array in concentration of 10−7 to 10−8 M. Glycan

Enrichment ratio

Lactose Fucosyl-Lactose Sulfated Fucosyl-Lactose sLNLeX Cellobiose Sulfated Lactose ([3OSO3]Gal␤1-4Glc␤) Blood Group B Glucose Sulfated Lactose ([3OSO3]Gal␤1-4[6OSO3]Glc␤) Sulfated Lactose ([6OSO3]Gal␤1-4Glc␤) Sialyllactose LNnT N. meningitidis epitope Tn␤ LNTriose Maltose CD60a, GD3 GT3 GM3 Isomaltotriose TriLex Blood group A(type 2) Mannose Man-6-P Galactose CD77, GB3 Le X sLNbiLex GM2 TF␣ Tn␣ ␣Gal epitope LeY sLeX LeY GD2 STn Globo-H

28.94 24.76 18.40 17.78 17.55 17.41 16.36 16.30 15.83 15.49 15.17 13.49 13.40 13.03 12.00 11.72 10.58 10.03 8.94 8.93 7.96 6.24 6.19 6.12 6.12 5.95 5.94 5.57 5.26 5.09 4.85 4.44 4.27 4.09 3.39 3.02 1.94

ficient as correlates of protection. Furthermore, a role for natural antibodies as an innate anti-cancer surveillance mechanism is suggested but underappreciated so far [7]. The fact that survival rates of cancer patients are correlated with low-titer [5,6] and presumably low-affinity TACA-reactive antibodies argues that more robust antibody responses may not be necessary. From a different perspective, cancer vaccines functionally resemble the process of autoimmune-mediated tissue damage [47]. Carbohydrate-targeting tissue rejection is best typified by the role of preexisting antibodies directed to the ␣-Gal antigen as a major barrier in porcine-to-human xenotransplantation [48]. The tissue rejection mediated by ␣-Gal-reactive antibodies demonstrates the feasibility of targeting TACAs for tumor therapy because tumorinduced antibody responses resemble autoimmune responses [49]. Early studies suggested that cytotoxic antibody titers were as low as 1:32 in the rejection of primary renal allografts in sheep [50]. In our own studies, we observe that low-titer IgM and IgG antibodies induced by CMPs suffice to inhibit tumor growth in vivo [16,18]. The role of carbohydrate reactive antibodies in tumor antigen presentation and the possible pitfalls in this hypothesis are illustrated by recent studies of the role of anti-␣ − Gal antibodies in tumor immunotherapy. Attempts to increase the immunogenicity of tumor cells by expressing the ␣ − Gal epitope, which is

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non-self for humans and the old world primates, were often successful [48,51]. These studies also pointed to a complexity of elicited responses, ranging from beneficial to deleterious and dependent on the participation or not of T cell help and different B cell subpopulations that respond to the same carbohydrate antigen [52]. An unexpected consequence of the presence of high titer anti-␣ − Gal antibodies in humans is the discovery of an instance of spontaneous peptide/carbohydrate mimicry with significance for tumor immunology. McKenzie and co-workers have suggested that MUC-1 contains a natural CMP spanning the major T cell epitope underdevelopment for CTL induction and have shown that anti-␣ − Gal antibodies recognize MUC1 peptides [53]. Incidentally, this crossreactivity may account for the occasional description of ␣ − Gal expression on human tissues. An observation, even more relevant to other CMP studies, is that the presence of anti-␣ − Gal antibodies and respectively the tolerance to this epitope shape the responses to the mimicking MUC1 peptide directing it to antibody or CTL compartments respectively [54]. The amount of circulating Gal reactive IgG would suggest that such antibodies would lend to cross-presentation of MUC expressing tumors. Recently the cross-presentation to Nyeso-1 has been suggested for T cell activation [55], yet studies with MUC-1 have not been as clear. Cross-presentation by IgGs may be limited to the IgG1 isotype. Our P10s CMP shares homology with this MUC-1 peptide and in our studies it seems that the major isotype that is reactive with P10s is IgG2. On the one hand this suggests that our CMP is a carbohydrate mimic, as IgG2 are carbohydrate reactive antibodies. But this also provides insight into why MUC-1 may not lead to efficient cross-presentation to activate CTLs as human IgG2 antibodies are not efficient at cross-presenting. Reactivity with anti-␣ − Gal antibodies has to be controlled for all CMP because of the high polyreactivity of these responses and, whenever discovered, has to be accounted for in the mechanistic studies and with respect to translation of mice model-based results. This is an excellent example of the considerable skewing of the interpretation of CMP immunization data, which may follow focusing on mimicry of one major glycan. Thus, the naive human repertoire contains B-cell clones that may produce an effective TACA response by an appropriate immunization. Because of the low probability for human IgG1 to originate from B1 cells, finding antimimic preimmune IgG1 antibodies raises the chances that B2 clones exist that can be boosted by a vaccine. Furthermore, the binding of isolated IgG from naïve immune repertoire to TACA expressing cell lines, we recently observed, demonstrates that the specificity of the antibodies might be immunologically relevant. As to the effector mechanisms, the degree of polyreactivity probably leads to qualitatively distinct outcomes as compared to highly specific immunity. Major consequences could be related to presentation, T cell phenotype, cross-presentation of CTL epitopes, and epitope spreading. Among the concequences the least probable, though, seems autoimmune pathology as illustrated in the glactosyltransferase KO mice in the case of MUC1 [54] and the preclinical study of the P10s CMP (manuscript in preparation). Differences in the levels of expression of carbohydrate epitopes on normal and malignant cells is viewed as the major prerequisite. A tolerance mechanism in this case would imply quantitative limits on the expansion of the antibody responses to TACA, which are readily observable. 2. Lesson 2: The same kind of difference Because TACAs are T-cell-independent antigens and selfantigens, their conjugation to immunologic carrier protein is perceived essential to recruit T cell help for antibody generation. Representative examples of TACA-based conjugate vaccines in clinical development include those directed toward gangliosides [56–58], Globo-H [59], LeY [60], and the STn antigen [61]. How-

ever, TACA-based vaccines are not living up to their promise. The question is: why? On the one hand there is still insufficient knowledge on the mechanisms that control tolerance to self-TACA. It may depend on mechanisms different from those, working in tolerance to protein epitopes. Conjugation of TACAs does not ensure successful vaccine because conjugation strategies do not uniformly enhance carbohydrate immunogenicity [62,63]. Furthermore, even after conjugation, a lack of amplification of the TACA-reactive humoral response necessitates constant boosting with vaccine. The relative lack of memory for IgG carbohydrate responses is believed to be secondary to the inability of carbohydrate to associate with MHC class II molecules and thus a failure to recruit cognate CD4+ T cell help [64]. We hypothesize that tolerance to TACA may be related to compartmentalization of the repertoires that generate TI responses to self- or non-self-carbohydrate antigens. One consequence of this hypothesis is that immunologic carriers in TACA-based vaccines may play another role besides recruiting T cell help. They may redirect or perhaps high-jack the immunogen from one compartment to another [65]. Thymus independent responses originate in the splenic marginal zone (MZ) or the B1b cell compartment and are markedly short lived. Until recently, the existence of a B lymphocyte compartment capable of sustaining prolonged TACA reactive IgM responses has been a highly speculative hypothesis but now these long lasting IgM TI type 2 (TI-2) responses are particularly attributed to B1b cells. Understanding the targeting of B1b responses (or other TI-2 memory compartment) maybe the key to developing efficient TACA directed vaccines. The design of immunogens eliciting long-lasting anti-TACA IgM responses would greatly improve the therapeutic utility of TACAtargeted vaccines. Unlike unconjugated carbohydrate antigens, we have shown that unconjugated MAP-CMPs prime for subsequent memory of unconjugated carbohydrate antigens, facilitating long-term surveillance through recall of carbohydrate immune responses. This effect is a major advantage that would minimize the need for constant boosting. Furthermore, we observed that CMPs mediate cognate B and T cell interactions as CMPs can induce antibodies in hosts deficient in thymus independent responses. Which B cell subsets are involved in long lasting TACA immunity? Although the mechanisms by which immunological memory is maintained after infection or vaccination are related to TD responses [66], similar mechanisms may also apply to cancer vaccines that target TACAs. Splenic marginal zone (MZ) and B1 B cells endowed with a “natural memory” provide a bridge between the very early innate and the later appearing adaptive immune response [67]. With this respect, the problem of developing longlasting TACA directed IgM levels most probably comes down to over-stimulation of the natural IgM memory. Some human splenic MZ B cells carry somatic mutations, and mutated antibodies can be raised after immunization with TI polysaccharide vaccine [68]. Somatically mutated ␮ heavy chain transcripts in human peripheral blood (PB) B-lymphocytes suggest that T-dependent (TD) B-cell memory might not be restricted to class-switched cells [69]. It was suggested recently that IgM memory B cells are generated in the spleen and control S. pneumoniae infections [70,71]. As previously reported [72], pneumococcal polysaccharide vaccination is associated with a significant reduction in the risk of pneumococcal bacteremia. It probably depends on IgM memory B cells, the origins and characteristics of which were highlighted recently [15,70,73]. We have shown that CMPs can prolong carbohydrate reactive IgM responses in prime and boost strategies to augment effective tumor specificities [19]. We further demonstrated that CMPs direct the generation of TACA reactive antibodies in immunodefficient Xid mice that have a point mutation in Bruton’s tyrosine kinase (btk) [74]. Functionally, xid mice have reduced levels of natural IgM and fail to respond to TI-2 antigens, such as TACA. They lack peritoneal

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B1 B lymphocytes, but show only a 30–50% reduction in other B lymphocyte populations. We observe that depending on formulation, CMPs can target repertoire compartments inaccessible to native TACA. Respectively, it is possible that CMPs can stimulate B cell compartments that bridge innate and adaptive immunity differently as compared to TACA. Positive selection signal strength compartmentalizes germline repertoires in functionally distinct subpopulations. Signals through B cell receptors (BCR) are crucial for the survival of the B cells at multiple checkpoints in their development [75]. Although tonic signaling from BCR (independent of antigen interaction) may be important, there is evidence that this differentiation signal depends also on the presence of self-antigens [76]. The strength of this signal defines also the fate of the B cell, making it dependent on the germline structure of its antigen receptor [76,77]. Interaction of B cells with self-antigens (as well as non-self in the later stages) shapes the B cell’s commitment to one of 4 major mature subpopulations (at least in mice). They populate defined anatomical compartments: follicular (FO) B2 cells (CD23+ ), splenic marginal zone (MZ) B2 cells (CD21+ ) as well as B1a (CD11b+ CD5+ ) and B1b (CD11b+ CD5− ) cells-abundant in body cavities. Apart from FO cells being responsible for TD responses, apparently TI-2 responses depend mostly on MZ and B1 cells while natural antibodies secretion—mostly on B1a cells [78–80]. The ease of identification of the populations by anatomical and phenotypic criteria warrants their use, at least as a first approximation, in studies of repertoire compartments related to types of immune responses and tolerance. In Fig. 1, a schematic presentation relates the self-reactivity of BCR, signal strength and the resulting B cell fate. A large part of the B1 cells in mice are selected by self-antigens from the CD19+ CD45− precursor in the fetal liver and bone marrow and turn to B1a when the signal is stronger or B1b when it is weaker [81–83]. B1b, but not B1a, seem to be formed also in the adult bone marrow. Another group of B cells originates from CD19− CD45R+ precursors in adult bone marrow and differentiates in the spleen under the action of weak BCR (self- or non-self) signals to MZ B2 cells [84]. The lack of N-nucleotides added in the V–J junction of CDR3 of MZ B cell BCR

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distinguishes them form the FO population and further supports the repertoire selection-based origin of the MZ compartment [85]. A population of adult B2 cells with high affinity to self-antigens does not exist—they are purged at earlier stages. Instead, probably a signal delivered by rare non-self-antigens in the highly organized follicular compartment leads to the differentiation of FO B2 cells. To preserve immunological homeostasis, the self-reactivity of the enumerated subpopulations is inversely proportional to their capacity to mount dynamic antibody responses. Thus, B1a cells mostly produce natural antibodies that provide innate, background antibody activity. They can be stimulated mostly by “danger signals” through TLRs in a non-specific manner. B1b cells can be boosted under certain conditions in a TI manner and the same seems to be true of MZ B cells [86,87]. All these populations are involved in the TI-2 response to carbohydrates, but the degree of self-reactivity, respectively, determines different response dynamics within the paradigm of the TI-2 responses. This is paralleled by differences in the repertoire [88–91]. A well-studied example is a particular epitope on phosphatidyl choline in mice recognized predominantly by antibodies with the VH11Vk9 gene pairing, which is expressed only in B1 cells [92,93]. Although the notion of B1 repertoire paucity was challenged by Kantor et al., in an unbiased Vh-D-Jh analysis, the authors confirm the distinction between B1 and B2 cells [94]. Moreover, a clear distinction between B1b and B1a as well as B2 cells in VH family use was observed (Fig. 2). The small population of B1b cells proved to have the least biased repertoire with respect to VH usage, which may reflect lower selection pressure. Approximately 20% of mature naive B cells in peripheral blood of healthy donors express low-affinity self-reactive antibodies, which are often polyspecific [95]. It seems that “seeing” the self-antigen landscape through different affinity windows selects for different subsets of the germline repertoires. The reason carbohydrate reactivities are confined to the compartments characterized with at least some degree of self-reactivity may be due to the degeneracy of carbohydrate recognition (high cross-reactivity) and potential for autoimmunity. This degeneracy is reflected also in the phenomenon of peptide mimicry. B1a, B1b, MZ and FO B2 have also

Fig. 1. B cell differentiation is dependent on BCR signal strength (based on [83,145]). Immune tolerance is partial in the innate antibody repertoire. The correlation between compartments/subpopulations and tolerance (resp. immunogenicity) is due to the BCR signal strength control of B cell differentiation. Immune tolerance sets the boundaries of usefulness of antigenic mimics. On the one hand, there is the danger of inducing deleterious autoimmunity, on the other—the inability to break tolerance is considered to preclude clinical relevance.

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Fig. 2. Ternary plot of the proportional representation of different VH families in B1a, B1b and MZ B cells (based on data from [94]). The closer a point lies to a vertex the higher the association with this population. The points in the middle have approximately equal proportions in all 3 populations. Note that together VH1,2,3 and 5 were found respectively in approx. 88%, 83% and 69% of the B2, B1a and B1b cells, while the less represented families contribute more to the specificity of the repertoires. Nevertheless, the most represented VH1 also shows some bias (approx. 50%, 40% and 30% of the B2, B1a and B1b clones).

functional specialization as they are cooperating on a systemic level. For instance, IgM specific for Streptococcus polysaccharide epitopes are lacking in asplenic mice and MZ B cells and B1b, although important for TI-2 responses, seem insufficient to restore this TI-2 response without B1a cells [95]. Nevertheless, among the populations listed, it seems the most interesting clinically are the B1b cells because they have been implicated in long-term IgM responses. It is possible that their targeting is possible by addressing characteristic specificities. 3. Lesson 3: CMPs provide insight into tolerance and self-reactivity A selective response to structures that do not belong to the internal environment is the essence of immunity. The understanding of self-/non-self-distinction has gone through multiple hypotheses including clonal selection, active tolerance through suppressive recognition, even network theories [96–100]. These concepts focus mainly on the recognition of protein epitopes by T and B cells. Yet, carbohydrate recognition plays a major role in the defense against bacterial infections [101] and may be also in tumor surveillance. TACAs are often self or highly cross-reactive with self-antigens. Is it possible for CMP to overcome tolerance and induce stronger immune responses to TACA? Carbohydrate recognition by T cells is rare and in general carbohydrate antigens are thymus independent (TI). The typical case are the TI-2 antigens which do not have inbuilt mitogenic activity. In the absence of cognate T cell help, tolerance seems maintained by the B cell repertoire selection, a requirement for optimal cross-linking of BCR as well as permissive signals through pattern recognition receptors (TLR - “danger signal”), complement receptor (CD21), from BAFF through TACI (CD267), etc. [102]. Another challenge to tolerance is the inherent polyreactivity [103] related to the degenerate recognition of carbohydrate epitopes based on a limited diversity of functional groups arranged in highly homologous spatial arrangements. These characteristics underlie high crossreactivity between tolerated highly accessible self-carbohydrate structures like gangliosides and blood group antigens and pathogen associated carbohydrates [104], which leads to high level of physiological autoreactivity. T cell control may still be operative when

the immunogenic particle is a complex structure containing both protein and sugar epitopes. Recruiting T cell help in responses to conjugated bacterial carbohydrates is one example [105,106]. The role of ␣-Gal epitope in tissue rejection in species, that are not capable of synthesizing it, was found to depend crucially on the presence of non-self-peptide epitopes on the carrier [107]. The timing of T cell help seems crucial for the functionality of the induced antibodies [52]. Unexpectedly, the isotype correlating with rejection in mice was IgG3 (related to TI-2 responses) while accommodating antibodies were IgG2b. IgM and IgG1 were present in both cases. One possible explanation is that different repertoires of crossreactivities are induced from different subpopulations of B cells. It is conceivable that the presence of protein self-epitopes that stimulate Treg would affect the outcome too. Tolerance to the carbohydrate moiety also affects such responses. Recently the results with a heptavalent vaccine of cancer associated carbohydrate antigens conjugated to a strong xenogenic carrier—KLH were reported [58]. Success, similar to the one seen in bacterial antigens, was achieved only for those that are seen as non-self like Tn, TF and Globo H while TACA that are (partially) accessible in the adult organism like GM2 and LeY were still nonimmunogenic. The low response to the self-carbohydrate is not due to a lack of self-reactive B cell clones. Previously it has been shown that autologous blood group antigen reactive IgG and IgM antibodies are present but “controlled” possibly by idiotypic interactions [108]. It is interesting to speculate that the B cell compartment responsible for TI-2 responses has evolved to meet two contradictory requirements: to provide efficient protection against encapsulated bacteria (impaired TI responses lead to an increased risk of sepsis) and at the same time limit the danger of autoimmunity to cross-reactive self-carbohydrate antigens. The observed delay in TI2 responses development in infants is an evolutionary disadvantage because infections with capsulated bacteria show high mortality in this period of the life [109]. May be this penalty is paid for a complex maturation of the compartment required to ensure TI-2 responses. It seems to depend on the precise selection of a repertoire of B cells, activating them and establishing a pool of their memory like progeny to ensure the dynamic properties and the repertoire profile of TI-2 responses as a gray area between self- and non-self. The lack of high titer IgG responses and affinity maturation are compensated by extremely rapid yet measured IgM response. As for the lower affinity of these antibodies—it is a desirable property, as long as their avidity is above certain threshold ensuring efficient neutralization, opsonization, complement fixation, and immunomodulatory functions. The danger of auto-immunity would pose an upper threshold defining a functional window for the titer of these antibodies. It seems logical that a similar precise titer window would be easier to maintain by relatively lower affinity antibodies through control of concentration. The paradox of higher responsiveness to conjugated vaccines in infants with immature marginal zones [110] indicates that TI-2 responses are not merely restricted by the structure of the antigens, but are an actively controlled compartmentalization of highly cross-reactive carbohydrate immunity. In the case of TACA the compartment producing TI-2 responses may be very limited to tumor associated antigens but may produce also stronger responses to carbohydrate antigens that are seen as non-self (e.g. from embryonic origin). Our results show that CMPs suppress tumor growth even when inducing only weak IgM responses to TACA [18]. Previously arguments have been provided for the role in immune surveillance of IgM [7,111] and IgM as opposed to IgG responses were found to correlate with patient survival in a polyvalent melanoma vaccine trial [6]. It is possible that the efficiency window of TI-2 responses discussed above is shifted for TACA on tumor cells due to higher epitope density. Thus,

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titers that are not functional for normal levels of expression are sufficient for immune targeting of tumor cells [112,113]. Another aspect of CMP immunization is related to the intrinsic polyspecificity both of TACA reactive antibodies and of CMP-induced IgM. Since mimicry does not present exactly the same binding interface, the repertoire of polyreactive antibodies induced by CMP will overlap but will not be identical to those induced by TACA. A subtle shift in the repertoire may be responsible for different characteristics of the anti-TACA response. Overall the impression is that CMPs induce TACA cross-reactive responses (e.g. to sLeX, LeY, GD2, or GD3,) that are weak and strictly TI-2 type. The accompanying strong TD antipeptide response, which is occasionally observed, does little to help the anti-TACA response. But it may prove essential for indirect effects of CMP immunization. Peptide mimics of carbohydrates have the potential to carry T cell epitopes overlapping the mimotope. Mimotopes of TACA elicit also cellular responses, which may be essential for the anti-tumor effects of CMP immunization [17,114]. The specificity of these cellular responses remains unclear. It may well be unrelated to TACA as specificity. Moreover, NK responses are also augmented, e.g. after immunization with the GD2 mimic P10s (manuscript in preparation). It is possible that the accompanying antipeptide T cell responses modulate the TI-2 responses and also benefit from them promoting epitope spreading. For instance, it has been shown that unlike B2 cells, B1 and marginal zone B cells are efficient antigen presenting cells [115–117] skewing the T cell balance to Th1 and TH17 responses [118]. A special case of collaboration between different branches of the immune system is the role of antibodies in the induction of cellular responses. Not only antibodies may facilitate cross-priming of CTL epitopes but they can play a role both in breaking or reinforcing of tolerance [119,120]. Although IgG is considered the major isotype involved in cross-priming, participation of IgM and C3d containing immune complexes and CD21 on the B cells stimulated after CMP immunization may have an additional role. These observations prompt a new view of the role of CMP in antitumor immunity and surveillance. Carbohydrate mimics may interact not only with B cell subpopulations involved in innate immunity (B1 and marginal zone B cells) but also with pattern recognition receptors PRR (as mimics of pathogen associated recognition patterns). The parallel in the specificity of natural and anti-carbohydrate antibodies and some PRR indicates a similar role for these two compartments of innate recognition. Finally, immune tolerance in general has been related to the balance of positive and negative signals from C-type lectins and toll-like receptors [121]. Thus, CMP immunization may lead to a complex cascade of immunoregulatory signals involving but not restricted to the IgM responses they induce. 4. Lesson 4: CMPs may bridge the activity of NK cells NK cells are critical players in tumor surveillance both by their tumoricidal activity [122,123] as well as by their regulatory function. They establish a cross-talk with DCs, some B cell subpopulations and may promote a Th1-mediated immunity [124–128]. A somewhat unexpected cooperation in anti-tumor activity between anti-TACA responses and NK has been observed by Kawashima et al. [11]. In mice transgenic for an anti-GD2 IgM antibody, GD2 tumors are rejected and the effector cells proved to be NK. CMP P10s has an antitumor activity that is affected by but not dependent on T cells, since it could be reproduced in nude mice (manuscript in preparation). At the same time, the tumors in immunized animals had strong NK cell infiltration and purging the NK cells by anti-asialoGM1 antibody abrogated the antitumor effect. The mechanism of NK activation following P10s mimotope immunization is not clear yet. It is accompanied by interstitial IgM infiltration into the tumor but neither direct killing (by CDC, CDCC or apoptosis) nor inhibition

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of tumor cell migration and adhesion to ECM or anti-angiogenic effect of the serum were observed. It has been speculated that anti-ganglioside antibodies may block the triggering of inhibitory Sialic acid Ig like-lectin (Siglec) receptors on NK [11]. It has been shown that murine NK cells express Siglec-E, which has the ability to bind disialogangliosides. [129,130]. Yet in vitro experiments failed to demonstrate increased cytotoxicity for the tumor cell line used (MDA-MB-231) in the presence of sera from immunized mice. It is less likely that P10s modified directly NK activity as animals were immunized in a prophylactic manner 7–10 days prior tumor challenge, and that it was shown that MAP peptides have a halflife leading to clearance by that time. Furthermore, incubation of P10s peptide with mouse NK cells did not increase killing of MDAMB-231 in an in vitro NK cells cytotoxicity assay. Therefore, it was hypothesized that the elusive mechanism of tumor suppression after CMP immunization may actually involve B cells that have been activated/recruited in the process rather than their weak antibody responses. B cell subpopulations that would normally harbor carbohydrate specificities (B1 and marginal zone B cells) may secrete IL-10 upon activation [131,132]. The mainstream view is that IL10 is one of the major mediators of immune suppression in the tumor microenvironment. This is especially true with respect to the CTL responses. The fact that some B cells secrete large amounts of IL-10 (and these subpopulations largely overlap with the carbohydrate specific B cells) has been interpreted as a mechanistic basis for their immunosuppressive function (some of them are labeled Bregs) and are generally considered detrimental in the anti-tumor responses. Yet, IL-10 has been found in another setting to contribute to tumor suppression [133] by activating NK cells [134] and sensitizing tumor cells to NK killing by reducing the expression of MHC [135]. It has been hypothesized that the IL-10 effect depends on the cytokine context, e.g.—IL-2 may promote its anti-tumor and suppress its tumor promoting properties [134]. It is possible that CMP immunization activated IL-10 secreting B cells in a compartment bringing together B and NK cells (spleen, regional lymph node or the tumor) and thus led to the activation of NK cells. The B cell/IL-10/NK circuit may have contributed especially in the nude mice model in the absence of the detrimental effects of IL-10 on the cytotoxic responses. The ambiguous role of IL-10 in tumor/immune system interactions is an example of a more general principle. The interaction of the tumors and the immune system is a network of bidirectional effects and feedback circuits that are related to tissue remodeling rather than to rejection of non-self. As a homeostatic mechanisms, the regulatory network, this system represents, may be adapted to tend to equilibrium, e.g. in favor of tissue and organ building, rather than of immunological destruction of the tumor. This notion has two consequences essential in the context of TACA immunization: (a) applying strictly vaccinological approaches may be inappropriate because of the dissimilarity between the immunological dynamics of pathogen infection and tumor development; and (b) immunomodulatory treatment based on a single or few regulatory elements cannot be efficient because of the stability of the system. It is possible that CMPs are efficient because of their inbuilt polyspecificity [38], through which they target immunologically multiple TACA. These specificities are related to a part of the B cell repertoire that is involved more intimately in innate immunity and, thus, in immune surveillance. Furthermore, the carbohydrate mimicry may be instrumental in the above context by rendering CMPs the character of ligands of diverse carbohydrate binding proteins and pattern recognition receptors with immunomodulatory function. The potential of these peptides to carry T cell epitopes further enlarges the circle of possible interactions. Unfortunately, the exact consequences of so complex interactions are hardly comprehensible, threatening to keep the development of such immunogens/immunoregulators largely empirical. This is in

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a stark contrast with the initial intention to design rationally a vaccine but seems closer to an adequate tumor immunotherapy. 5. Lesson 5: CMPs can mediate sustained immunity and be effective in combination therapy Sustained protection following immunization is maintained both by effective titers of antibodies as well as a swift spike in specific immune activity when the antigen is reintroduced. The swiftness and the magnitude of the response depend on memory mechanisms involving specialized lymphocyte populations. As discussed in the previous section TI-2 anti-carbohydrate responses (including anti-TACA) apparently lack this property and their magnitude is constant. Although formally this means no memory, the rapid rise in IgM titers and the phenotype of the MZ and B1 cells prompted some authors to relate to TI-2 responses as to “natural memory” [87]. The lengthy process of establishing the marginal zone compartment may be the period of selecting and generating the necessary elements of this innate memory response [85]. With this consideration using anti-TACA responses in immunotherapy seems problematic since it is hardly possible to augment the magnitude of the response. The existence of memory type of increased and lasting IgM responses to TI-2 antigens in borreliosis related to the B1b subset is intriguing [15,136] but it remains to be demonstrated for TACA that are much closer or identical with self. That is why the finding that CMPs can prime for long lasting IgM responses to self-carbohydrate epitopes drew the attention to the peculiarities of CMP-induced TI-2 response. Since this prolonged IgM response occurred after boosting with the sugar form, it is possible that CMPs stimulate a repertoire of B cells that is not identical to the one stimulated by the carbohydrate although these repertoires overlap as they cross-react. Another possible explanation is the recruitment of T cell help but the same CMP (p106) failed to induce IgG responses in Xid mice unless it was conjugated to BSA [74]. On the other hand CMP p106 stimulates cellular responses which is dependent on CD4 and is responsible at least for the major part of the antitumor effect of this peptide [17,20]. The sequence of p106 (GGIYWRYDIYWRYDIYWRYD) has both I-Ad and H2-Kd binding motifs. It is possible that p106 induces CD4 T cell responses, which are heavily skewed towards Th1 response even in Th2 prone BALB/c unless p106 is conjugated to BSA [74,137]. IFN␥ can have both positive and negative effects on B cell function. In case of membrane immunoglobulin (but not CD40) stimulation IFN␥ promotes secretion of IgM and IL6 [138,139]. The fact that mostly pre-activated B cells are subject to this regulation and the dependence of TI-2 responses on NKMZ B cell interaction, in which NK are stimulated to secrete IFN␥ [125,140,141] indicate a role for Th1 cytokines in TI-2 responses. The cross-talk with the T cells may be ensured by the particular capacity of MZ B cells to present antigen to T cells and skew their differentiation to Th1 phenotype [142]. Thus, a hypothetic sequence of events starts with activation of B cells involved in TI-2 response, which also present the T cell epitopes on CMP to T cells, skewed in the process to Th1 responses. This milieu may promote further capture and presentation of tumor antigens carrying the TACA epitope mimicked by CMP. Ultimately this cascade may lead to a more efficient epitope spreading, cross-presentation and CTL responses. It is not clear how the cooperation with NK or Th1 cells relates to the promotion of long term IgM responses as well as what is the role of CD40 engagement. Recently it was shown that MZ B sell stimulation to produce anti-xenogenic IgM depended on NK cells and partially on CD40L but not on IFN␥ [143] and CD4+ T cells help for TI-2 responses was dependent on CD40 binding on hemopoietic non-B cells [144]. The regulation of TI-2 responses is still very poorly understood but it is possible that the circuitry involving Th1, NK and DC will prove central and CMPs that can independently stimulate Th1 responses may prove instrumental in this analysis.

6. Conclusion Carbohydrate mimetic peptides are an attractive alternative to carbohydrate-based immunogens that challenges our understanding of cancer immunotherapy. Although carbohydrate epitopes have been conjugated to protein carriers for more than 30 years, the overlapping of T cell epitopes with carbohydrate mimotopes creates a qualitatively new type of immunogens. It is possible that the observed type of response to CMP immunization is the outcome of complex recognition of highly biologically active peptide sequence: the carbohydrate mimicry with self-epitopes overlap T cell epitopes and a putative PRR recognition motif (enhanced by the repetitive structure). The applicability of TACA-based tumor vaccines depends heavily on the therapeutic strategy. It seems that inducing sustained immune responses to TACA would be useful only in the correct context of prevention of recurrence and metastasis. Presently, immunotherapy of large tumors is considered in most cases unwieldy because of the immunosuppressive environment they create. On the contrary, after removal of the bulk of the tumor mass and correction of the immune suppression, immunotherapy using CMP vaccines will rather help restore or reinforce a very natural mechanism of immune surveillance. Acknowledgements This work was supported by a Clinical Translational Award from the Department of Defense Breast Cancer Program (W81XWH-061-0542). Glycan specificity of IVIg fractions was performed by the Glycan Array Screening Core of the Consortium for Functional Glycomics. References [1] Pashov A, Monzavi-Karbassi B, Chow M, Cannon M, Kieber-Emmons T. Immune surveillance as a rationale for immunotherapy? Hum Vaccin 2007;3(5):224–8. [2] Dabelsteen E. Cell surface carbohydrates as prognostic markers in human carcinomas. J Pathol 1996;179(4):358–69. [3] Nakagoe T, Sawai T, Tuji T, Jibiki M, Nanashima A, Yamaguchi H, et al. Prognostic value of expression of sialosyl-Tn antigen in colorectal carcinoma and transitional mucosa. Digest Dis Sci 2002;47(2):322–30. [4] Slovin SF, Keding SJ, Ragupathi G. Carbohydrate vaccines as immunotherapy for cancer. Immunol Cell Biol 2005;83(4):418–28. [5] Takahashi T, Johnson TD, Nishinaka Y, Morton DL, Irie RF. IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients. J Invest Dermatol 1999;112(2):205–9. [6] Hsueh EC, Gupta RK, Qi K, Morton DL. Correlation of specific immune responses with survival in melanoma patients with distant metastases receiving polyvalent melanoma cell vaccine. J Clin Oncol 1998;16(9):2913–20. [7] Vollmers HP, Brandlein S. The “early birds”: natural IgM antibodies and immune surveillance. Histol Histopathol 2005;20(3):927–37. [8] Vuist WM, Van Schaik IN, Van Lint M, Brand A. The growth arresting effect of human immunoglobulin for intravenous use is mediated by antibodies recognizing membrane glycolipids. J Clin Immunol 1997;17(4):301–10. [9] Erttmann R. Treatment of neuroblastoma with human natural antibodies. Autoimmun Rev 2008;7(6):496–500. Epub 2008 Apr 15. [10] Ravindranath MH, Muthugounder S, Presser N, Ye X, Brosman S, Morton DL. Endogenous immune response to gangliosides in patients with confined prostate cancer. Int J Cancer 2005;116(3):368–77. [11] Kawashima I, Yoshida Y, Taya C, Shitara H, Yonekawa H, Karasuyama H, et al. Expansion of natural killer cells in mice transgenic for IgM antibody to ganglioside GD2: demonstration of prolonged survival after challenge with syngeneic tumor cells. Int J Oncol 2003;23(2):381–8. [12] Plautz GE, Mukai S, Cohen PA, Shu S. Cross-presentation of tumor antigens to effector T cells is sufficient to mediate effective immunotherapy of established intracranial tumors. J Immunol 2000;165(7):3656–62. [13] Ochsenbein AF, Zinkernagel RM. Natural antibodies and complement link innate and acquired immunity. Immunol Today 2000;21(12):624–30. [14] Youd ME, Ferguson AR, Corley RB. Synergistic roles of IgM and complement in antigen trapping and follicular localization. Eur J Immunol 2002;32(8):2328–37. [15] Alugupalli KR, Leong JM, Woodland RT, Muramatsu M, Honjo T, Gerstein RM. B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity 2004;21(3):379–90.

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