Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer vaccine induces apoptosis and...

Cancer Immunol Immunother (2009) 58:1117–1128
DOI 10.1007/s00262-008-0634-y
SHORT COMMUNICATION

Anti-ganglioside anti-idiotypic monoclonal antibody-based cancer vaccine induces apoptosis and antiangiogenic eVect in a metastatic lung carcinoma

Y. Diaz · A. Gonzalez · A. Lopez · R. Perez · A. M. Vazquez · E. Montero

Received: 5 April 2008 / Accepted: 17 November 2008 / Published online: 10 December 2008© Springer-Verlag 2008

Abstract Anti-idiotype monoclonal antibody (mAb)1E10 was generated by immunizing BALB/c mice with anAb1 mAb which recognizes NeuGc-containing ganglio-sides, sulfatides and some tumor antigens. 1E10 mAbinduces therapeutic eVects in a primary breast carcinomaand a melanoma model. However, the tumor immunitymechanisms have not been elucidated. Here we show thataluminum hydroxide-precipitated 1E10 mAb immunizationinduced anti-metastatic eVect in the 3LL-D122 Lewis Lungcarcinoma, a poorly immunogenic and highly metastaticmodel in C57BL/6 mice. The therapeutic eVect was associ-ated to the increment of T cells inWltrating metastases, thereduction of new blood vessels formation and the increaseof apoptotic tumor cells in lung nodules. Interestingly,active immunization does not induce measurable antibodiesto the 1E10 mAb, the NeuGc-GM3 or tumor cells, whichmay suggest a diVerent mechanism which has to be eluci-dated. These Wndings may support the relevance of thistarget for cancer biotherapy.

Keywords Cancer vaccine · Lung carcinoma metastases · Anti-idiotype monoclonal antibody · NeuGc-containing ganglioside · Apoptosis · Angiogenesis

AbbreviationsAg AntigenAb(s) AntibodiesELISA Enzyme-linked immunosorbent assayId IdiotypemAb(s) Monoclonal antibody(ies)NeuGc Neu-glycolylsc Subcutaneous

Introduction

Active immunotherapy is one of the most promising Welds incancer research. One strategy to generate an eVectiveimmune response against tumor-associated antigens involvesthe use of anti-idiotypic antibodies (Ab2), bearing the “inter-nal image” of the antigen. The use of Ab2 as vaccines isbased on Jerne’s idiotypic network theory, which postulatethat the immunoglobulin idiotype repertoire must mimic theuniverse of non-self and self-antigens [1]. DiVerent Ab2shave been generated and some of them have demonstrated toinhibit tumor growth in preclinical studies [2, 3].

Anti-idiotypic vaccines directed to tumor-associated anti-gens have shown their capability to induce antitumor spe-ciWc humoral and cellular responses, and in some cases adelayed tumor progression and an improved survival [4–8].

We have previously reported the generation of an Ab2monoclonal antibody (mAb) to a murine Ab1, named P3,which binds NeuGc-containing gangliosides, sulfated gly-colipids and antigens expressed in diVerent murine andhuman tumor cells [9–12]. This IgG1 Ab2 mAb, designated

Y. Diaz · A. Gonzalez · A. Lopez · E. Montero (&)Experimental Immunotherapy Department, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cubae-mail: [email protected]

R. PerezResearch and Development, Center of Molecular Immunology, Havana, Cuba

A. M. VazquezAntibody Engineering Department, Center of Molecular Immunology, P.O. Box 16040, Havana 11600, Cuba

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1E10, was able to induce in mice and monkeys anti-anti-idiotypic antibodies (Ab3) bearing P3 mAb idiotopes, but itwas unable to generate Ab3 antibodies with the same anti-gen speciWcity as the P3 mAb [10, 13, 14]. In contrast,1E10 mAb was capable to generate a very strong and spe-ciWc antibody response against NeuGc-containing ganglio-sides in chickens [14]. Similar results have been obtained incancer patients immunized with aluminum hydroxide(Alum)-precipitated 1E10 mAb [11, 12, 15]. In these twospecies, humans and chickens, N-glycolilated gangliosidesare non-self antigens [16–18]. Previous results showed thattreatment with 1E10 Ab2 mAb induced a strong antitumoractivity in mice bearing melanoma or a breast carcinoma[13], but so far the mechanism by which this mAb exertsthis antitumor eVect has not been elucidated. These resultsin syngeneic and allogeneic models of diVerent origin dem-onstrate the potential of 1E10 mAb to activate mechanismsleading to an antitumor immunity, potentially beyond thedemonstrated anti-idiotypic antibody response [10].

In this work, we evaluated the anti-metastatic eVect of1E10 mAb in Alum adjuvant (1E10/Alum) in the 3LL-D122Lewis Lung carcinoma model. 1E10/Alum immunizationwas tested in two diVerent settings distinguished by the fre-quency of the immunizations, the amount of vaccine and theinitiation of the vaccine schedule related to the tumor chal-lenge. Independently to the immunization schedule, 1E10/Alum promoted a signiWcant reduction of spontaneous lungmetastases. The therapeutic eVect was associated to the incre-ment in the number of T cells inWltrating metastases, a reduc-tion of new blood vessels formation and the increment ofapoptotic tumor cells in lung nodules. Interestingly, activeimmunization does not induce measurable antibodies to the1E10 mAb, the NeuGc-GM3 or tumor cells, which may sug-gest a diVerent mechanism which has to be elucidated.

Materials and methods

Animals

Female C57BL/6 inbred mice, 8–10 weeks old, were pur-chased from the Center for Laboratory Animal Production(CENPALAB, Havana, Cuba). Animals were housed underconventional conditions with free access to water and foodand maintained in accordance with the guidelines stipulatedby Animal Subject Committee Review Board of the Centerof Molecular Immunology (CIM) and CIM’s InstitutionalAnimal Care and Use Committees.

Gangliosides

Gangliosides are named according to the nomenclature ofSvennerholm (1964) NeuAcGM3 and NeuGcGM3 were

kindly provided by Dr L.E. Fernández, Vaccine Depart-ment, Center of Molecular Immunology (Havana, Cuba).NeuAcGM3 and NeuGc-GM3 gangliosides were puriWedfrom dog and horse erythrocytes, respectively, as described[19]. Homogeneity and purity of gangliosides is more than95% as determined by thin layer chromatography and den-sitometry [17].

Aluminum hydroxide-precipitated 1E10 mAb (1E10/Alum)

1E10 mAb (IgG1, �) was puriWed from mouse ascites byaYnity chromatography on Protein A-CL Sepharose 4 Bcolumn (Amersham Pharmacia Biotech, Uppsala,Sweden). The purity of the isolated immunoglobulin wasmore than 97% as determined by SDS-PAGE, high-pres-sure liquid chromatography, and isoelectric focusing. Forthis study puriWed 1E10 mAb was aseptically mixed at aWnal concentration of 1 mg/ml with 5 mg/ml of aluminumhydroxide as adjuvant (Superfos Biosector, Frederikssund,Denmark). The mixture was gently stirred for 3 h at roomtemperature. The aluminum hydroxide-precipitated mAbwas aliquot and stored at 4°C until use. Aluminum hydrox-ide-precipitated iorC5 (iorC5/Alum), a murine mAb thatrecognizes an antigen preferentially expressed in humancolorectal cells [9], was used as isotypic and allotypiccontrol.

Cell line

Lewis Lung carcinoma clone D122 (3LL-D122) is low-immunogenic and high-metastatic in syngeneic C57BL/6mice [20]. 3LL-D122 cells were cultured in DMEM (GibcoLife Technologies, Grand Island, NY, USA) supplementedwith 10% heat inactivated fetal bovine serum (HyClone,UT, USA), 2-mM L-glutamine, 100 U/ml penicillin,100 �g/ml streptomycin and 50 �M 2-mercaptoethanol. Forinjection into mice, cell suspensions were prepared bydetaching cells from culture Xasks with trypsin-EDTA(0.05%), followed by washing and resuspension in phos-phate-buVered saline (PBS), pH 7.4. Tumor cells werecounted and viability was greater than 95%, as assayed bytrypan blue exclusion test.

Antitumor experiments

C57BL/6 mice (six to eight mice per group) were chal-lenged subcutaneous (sc) into one hind footpad with2 £ 105 of 3LL-D122 cells (designated as day 0 of theexperiment). We tested two immunization schedules: (1)mixed setting; animals were immunized before and afterthe challenge with tumor, on days ¡28, ¡14, 7 and 21, with50 �g of 1E10/Alum, iorC5/Alum or only with the adju-vant, in 0.2 ml of PBS sc; (2) therapeutic setting; mice were

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treated after tumor challenge, on days 3, 10, 17, 24, 31 and38, sc with 100 �g of 1E10/Alum or only with the adjuvant.Primary tumors were surgically removed when reached adiameter of 8–9 mm (around days 25–29). Mice were sacri-Wced 21 days later and their lungs were removed, weighted,Wxed in Bouin’s solution and the number of surface lungmetastases was counted under a dissecting microscope.Sera were collected pretreatment and 14 days after the Wrstthree doses and 21 days after the last dose. Mice treatedwith iorC5/Alum or the adjuvant alone in PBS (PBS/Alum)were used as controls. Experiments were repeated at leastthree times.

Antibody binding assay

Reactivity to 1E10 (Ab3) in sera from immunized mice wasdetermined by ELISA as described previously [21]. BrieXy,96-well microtiter plates (high binding; Costar, Cambridge,MA, USA) coated with 10 �g/ml of F(ab�) 1E10 mAb wereincubated with animal sera. The second antibody, alkaline-phosphatase-conjugated goat anti-mouse IgG + IgM (Fcfragment speciWc; Sigma, St Louis, MO, USA), was addedand the reaction was developed with p-nitrophenylphos-phate substrate (Sigma) in diethanolamine buVer, pH 9.8.Absorbance was measured at 405 nm in an ELISA reader(Organon Teknika, Salsburg, Austria). iorC5 F(ab�)2 frag-ments were used as controls. The highest serum dilutiongiving optical density values ¸ 0.2 and being at least twotimes the value corresponding to the pre-immune serum atthe same dilution was considered as the antibody titer.

Binding of mice Ab3 to puriWed gangliosides wasdetermined using an ELISA assay described previously[21]. BrieXy, gangliosides (4 �g/ml) in 50 �l of methanolwere dried in 96-well microtiter plates (Polysorp, Nunc,Denmark). Plates were blocked with 1% BSA in 0.05-MTris–HCl buVer, pH 7.8, and incubated with serum sam-ples. The biotin-goat anti-mouse IgG + IgM (Fc fragmentspeciWc; Sigma) was added and the streptavidin conju-gate was developed with p-nitrophenylphosphate sub-strate (Sigma) in diethanolamine buVer, pH 9.8. Toconsider that a serum sample had a positive reaction to aparticular ganglioside, values of absorbance had tobe ¸ 0.2 and at least two times the absorbance valuegiven by incubating the serum in wells containing nogangliosides.

Flow cytometry analysis

The recognition of 3LL cells by the sera of tumor-free ortumor-bearing mice, treated with four doses of 50 �g of1E10/Alum, iorC5/Alum or PBS/Alum, were analyzed byXow cytometry. BrieXy, 3LL-D122 cells were incubated at4°C for 25 min with diVerent mice sera dilutions, then

washed in PBS and subsequently incubated with PE-conju-gated goat anti-mouse immunoglobulins (Dako, Denmark)for 25 min at 4°C. Cells were resuspended and expressionof molecules of interest was monitored by FACScan andanalyzed using the WinMDI 2.8 Program.

Histological and immunohistochemical analyses

For histological and immunohistochemical studies, frag-ments of metastatic lungs were formalin-Wxed andparaYn-embedded. Five-micrometer tissue sections werecut, air-dried on glass slides, deparaYnized and rehy-drated. Tissue sections were stained with hematoxylin andeosin under standard procedures for detection of mitoticbodies.

For endothelial cell detection by CD31 (platelet-endo-thelial cell adhesion molecule-1) staining, endogenousperoxidase activity was blocked by hydrogen peroxidase(0.3%) for 30 min at room temperature. Later, tissue sec-tions were treated with pepsin for 30 min at 37°C andblocked with PBS containing 5% of BSA for 15 min.Sections were incubated with an anti-CD31 mAb(Pharmingen, San Diego, CA, USA), for 90 min and thenwashed with Tris buVered saline, pH 7.4. Binding of theprimary antibody was detected using a biotinylated goatanti-mouse immunoglobulin secondary antiserum(Jackson Laboratories Bar Harbor, ME, USA), followedby the avidin–biotin–peroxidase system (Dako, Denmark)with 3,3-diaminobenzidine (Sigma, St Louis, MO, USA)as the chromogen. Sections were counterstained withhematoxylin.

Apoptosis was performed using Apoptag™ Plus Kit(Oncor, Canada), according to manufacturer’s instructions.Methyl green was used as counterstaining.

To determine the inWltrated lymphocyte population inlung metastasis, frozen sections were Wxed in acetone andincubated with biotinylated rat anti-mouse CD4 and CD8antisera (Pharmingen). The binding of biotinylated antibod-ies was detected as described above.

For all the techniques, 12 independent lung metastasesWelds from two mice from each group were counted.

Statistical analysis

The mean and the error of the values obtained in each groupwere calculated. Each experiment was repeated at leasttwice. Statistical diVerences between groups were deter-mined. Kruskal–Wallis with Dunn’s multiple comparisonstest was used to compare three groups and Mann–Whitneytest for the analysis of two groups. Chi-square was per-formed to compare the incidence of lung metastasisbetween the treatment groups. Analyses were performedwith Graph Pad Prism, version 4.0.

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Results

1E10/Al immunization reduces the incidence of spontaneous lung metastases in the murine 3LL-D122 Lewis Lung carcinoma model

To investigate if 1E10/Alum had the capacity to inhibit thespontaneous lung colonization of 3LL-D122 cells, micewere injected sc with this vaccine preparation. Mice treatedwith four doses of 50 �g of 1E10/Alum based on the sched-ule 1 (see Sect. “Materials and methods” for details),showed a signiWcant reduction of both lung weight and thenumber of macrometastases (mean: 1E10/Alum: 1.8, iorC5/Alum: 14.3 an PBS/Alum: 18.2); compared to controlstreated with an irrelevant isotype matched mAb or Alumalone (P < 0.05, Kruskal–Wallis test; Fig. 1a and Table 1).However, that treatment did not have an incidence on theprimary tumor growth (Fig. 1a).

Moreover following a therapeutic setting, animals weretreated six times with 100 �g of 1E10/Alum (schedule 2),beginning 3 days after tumor challenge. Despite that treat-ment did not modify the incidence the primary tumor vol-ume or lung weight (Fig. 1b), there was a signiWcantreduction in the number of micrometastasis and the micro-metastasis surface in the lung tissue (Fig. 1c) of vaccinatedanimals compared to the control group (P < 0.05, Mann–Whitney test).

The histopathological study of the lungs from controlanimals (PBS/Alum or iorC5/Alum) revealed a completeloss of lung parenchyma due to a massive invasion bytumor cells. There is a high incidence of lung metastaticnodules associated to a lack of visible alveolar spaces with-out a particular localization (Fig. 2a). In contrast, in tissuesfrom mice treated with 1E10/Alum, a reduced number oflung metastases and a smaller size of metastatic nodules inlung sections were observed. The alveolar spaces were sim-ilar to normal lung histology and small tumor nodules weredetected mainly in the proximity of the pleura (Fig. 2b).

In summary the vaccination with 1E10/Alum induced asigniWcant anti-metastatic eVect in the 3LL-D122, a poorlyimmunogenic and highly metastatic version of the LewisLung carcinoma, evidenced by a reduction in lung weightand/or a reduction in the number of spontaneous macro- ormicrometastasis.

1E10/Alum immunization induces apoptosis without aVecting cell mitosis of spontaneous lung metastases in the murine 3LL-D122 Lewis Lung carcinoma model

To determine whether the therapeutic eVect was associatedto the tumor cell apoptosis in 3LL-D122 pulmonary metas-tases, sections of lung tissues from C57BL/6 mice immu-nized with 1E10/Alum, iorC5/Alum or adjuvant were

examined using the Apoptag Plus Kit. As shown inFigs. 2c, d and 3a, tumors from mice treated with 1E10/Alum displayed a signiWcant increment of apoptotic cellscompared to the control groups (P < 0.05, Kruskal–Wallisor Mann–Whitney test).

The inXuence of 1E10/Alum treatment in tumor mitosiswas also examined. The analysis of mitotic cell counts inlung metastatic tissues did not show diVerences betweenthe control groups and the animals that received 1E10/Alum (Fig. 3b). Therefore, 1E10/Alum treatment promotedapoptosis of lung metastatic cells without interfering withthe normal mitosis process of tumor cells.

1E10/Alum immunization promotes an antiangiogenic eVect in spontaneous lung metastases of the murine 3LL-D122 Lewis Lung carcinoma model

Immunohistochemical analysis by CD31 staining of meta-static lungs from mice injected with 1E10/Alum, in bothtested schedules, revealed a signiWcantly lower mean ofmicrovessel counts compared with those from animalstreated with the irrelevant isotype mAb precipitated inAlum or only with the adjuvant alone (Figs. 2e, f, 3c;P < 0.05, Kruskal–Wallis test).

1E10/Al immunization increases the T cell inWltration in spontaneous lung metastases of the murine 3LL-D122 Lewis Lung carcinoma model

Tumor tissues from 3LL-D122 Lewis Lung carcinomabearing mice displayed a limited leukocyte inWltration.However, lung sections from 1E10/Alum treated miceshowed higher intra-tumor inXammatory inWltrates thancontrols. To measure the lymphocyte inWltration in metas-tases, lung cryostat sections from animals immunizedwith 1E10/Alum, iorC5/Alum or Alum alone, werelabeled with anti-CD4 and anti-CD8 antibodies and ana-lyzed by immunohystochemistry. The examinationrevealed a signiWcant increase in the number of CD4+ andCD8+ cells inWltrating lung metastases from mice immu-nized with 1E10/Alum (Figs. 2g, h, 4; P < 0.05, Kruskal–Wallis test).

1E10/Al immunization does not induce measurable antibodies to the 1E10 mAb, the NeuGc-GM3 or the 3LL-D122 Lewis Lung carcinoma cells

Sera from mice immunized with four doses of 1E10/Alumwere analyzed by an indirect ELISA against the F(ab�)2

fragments of the anti-Id mAb or against NeuGc-GM3ganglioside. No reactivity of the sera against these anti-gens was detected. To deWne whether 1E10/Alum treat-ment is able to induce antibodies capable to recognize the

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3LL-D122 tumor cells, sera were analyzed by Xow cytom-etry. 1E10/Alum treated-normal mice did not recognizethe tumor cells, however, a weak sera reactivity withtumor cells was detected in animals immunized with

1E10/Alum and challenged with 3LL-D122 tumor cells,but it was not substantially diVerent from the observed inmice challenged with tumor cells in control groups(Fig. 5).

Fig. 1 EVect of 1E10/Alum treatment on primary tumor and sponta-neous lung colonization produced by 3LL-D122 cells. Mean diameterof primary tumor from animals challenged sc with 2 £ 105 3LL-D122cells into a hind footpad (a and b, left panel) and the correspondinglung weight (a and b, right panel) evaluated 21 days after the primarytumor removal were determined. a Mice were injected sc four timesevery 2 weeks with 50 �g of 1E10/Alum, iorC5/Alum and PBS/Alum,the Wrst two doses were administered before the tumor inoculation.

b Mice were immunized six times with 100 �g of the vaccine starting3 days after the tumor challenge and weekly afterward (seeSect. “Materials and methods” for details). c Metastatic lung tissuefrom animals vaccinated under a therapeutic regime were staining withhematoxylin and eosin; tumor micrometastases were counted (leftpanel) and measured their surface (right panel). Experiments wererepeated three times and a representative result is shown. Results areexpressed as the mean § SEM (*P < 0.05)

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Discussion

Most preclinical studies for the evaluation of antitumoragents are frequently based on the use of rapidly growingtransplantable mouse tumors which usually progress aslocalized solid masses. This kind of experimental scenariolargely diVers from the real situation in patients bearingmalignant tumors, where in most cases are the metastasesresponsible for cancer lethality [22]. Therefore, we focusour present preclinical studies on the anti-metastatic capac-ity of 1E10/Alum in a model of spontaneous lung metasta-ses generated after the surgical removal of the primary3LL-D122 Lewis Lung carcinoma inoculated subcutane-ously in C57BL/6 mice.

The anti-metastatic eVect of 1E10/Alum immunizationwas evaluated in two diVerent settings distinguished by thefrequency of the immunizations, the amount of vaccine andthe initiation of the vaccine schedule related to the tumorchallenge. In a mixed schedule mice were injected sc fourtimes every 2 weeks with 50 �g of 1E10/Alum where theWrst two doses were administered before the tumor inocula-tion. Additionally, in a fully therapeutic setting mice wereimmunized six times with 100 �g of the vaccine starting3 days after the tumor challenge and weekly afterward,resulting closer to the clinical scenario.

Independently to the immunization schedule, 1E10/Alum promoted a signiWcant reduction of spontaneous lungmetastases (Fig. 1). The sc administration of 1E10/Alum,before and after the implantation of 3LL-D122 lung carci-noma cells in mice, produced a dramatic decrease in thenumber of macro- and micrometastases in the animal lungs.These results agree with our previous Wnding that intraperi-toneal vaccination with this anti-idiotypic antibody coupledwith a carrier protein in Freund’s adjuvant, or its intravenous

administration alone, was capable to reduce the spontane-ous lung metastases induced by the murine F3II mammarycarcinoma or the experimental lung metastases produced bythe B16 melanoma cells, respectively [10].

In order to explore the role of the immune response inthe anti-metastatic activity elicited by 1E10/Alum, we Wrstevaluated the association of induced antibodies to this phe-nomenon. Ab3 response was not detected in the sera ofmice treated with 1E10/Alum when analyzed by an indirectELISA against F(ab�)2 fragments of the anti-Id mAb oragainst NeuGc-GM3 ganglioside. Moreover, an enhance-ment of the reactivity of the mouse sera against the 3LL-D122 tumor cells was not demonstrable by Xow cytometry(Fig. 5 and data not shown). These results are in agreementwith those previously reported by our group treating micewith this anti-idiotypic mAb [10, 13]. Taking these resultsinto account, the anti-metastatic eVect induced by 1E10/Alum seems not to be directly linked to a potential antibodyresponse induced by this vaccine.

In contrast, immunohistochemical analyses demon-strated an increase in the number of CD4+ and CD8+ cellsinWltrating lung metastases from 1E10/Alum treated mice.Involvement of CD8+ and CD4+ cells in the antitumoractivity of diVerent vaccines has been documented inmouse tumor models [23, 24]. In addition, the developmentof cellular immune responses in cancer patients due to thevaccination with anti-idiotypic antibodies mimickingtumor-associated antigens has been well demonstrated [7,25–27]. On the other hand, it is important to remark the factthat gangliosides based cancer vaccine (using the intactmolecule or anti-idiotypic Ab) usually induce antibodyresponse but not cellular immunity [28, 29]. The glycolipidnature of gangliosides may limit their presentation in theclassical major histocompatibility complex molecules [30].However, the recent Wnding of CD1-restricted human T cellspeciWc for GM1 gangliosides, which are able to secreteIFN� and TNF� cytokines [31], and the presentation of theGD3 ganglioside by CD1d to mouse NKT cells [32], mayunveil the potential development of T cell response to thiskind of antigens.

Recently, the development of CD8 T cells response pro-tecting against the melanoma B16 challenge, immunizingwith a GM3-based ganglioside vaccine has been docu-mented. In addition, T cells from immunized animals rec-ognized the dendritic cells pulsed with melanoma extract,secreting IFN� [33]. Altogether, these emerging resultsmay suggest a direct involvement of the cellular immuneresponse to gangliosides in the antitumor immunity. In par-ticular, patients with advanced breast cancer immunizedwith 1E10/Alum induced a NeuGc-speciWc T cell IFN�response [34]. However, the direct mechanism for antitu-mor eVect of tumor-inWltrating T cell promoted by 1E10/Alum requires to be further dissected.

Table 1 EVect of 1E10/Alum immunization on 3LL-D122 spontane-ous metastases

Mice were injected sc four times every 2 weeks with 50 �g of 1E10/Alum, iorC5/Alum and PBS/Alum, the Wrst two doses were adminis-tered before the sc inoculation with 2 £ 105 3LL-D122 cells into a hindfootpad. At day 48, mice were sacriWced and lung macrometastaseswere counted

*** P < 0.001, Chi-squared test

Treatments (number of animals)

Lung macrometastases

Percent of animals per group

Mean § SEM

0–4 ¸5

PBS/Alum (14) 27 73 14.3 § 4.8

iorC5/Alum (6) 33 67 18.2 § 7.4

1E10/Alum (15)*** 93 7 1.8 § 0.4

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Nevertheless, some direct immunological or indirectmechanisms could be working. For instance, the reductionof lung metastasis by 1E10/Alum vaccine could be relatedwith the migration process of both the endothelial cellsforming new blood vessels, and the tumor cells forming

the metastasis. Both angiogenesis and tumor invasion canbe considered as invasive processes, in which cells areactivated and moved away from their initial location, bymodifying the adhesiveness with the extracellular matrix,expressing new adhesion molecules and degrading the

Fig. 2 Histopathology of 3LL-D122 lung metastases after 1E10/Alum immunization. Lungs were surgically removed on day 48 after 3LL-D122 cells inoculation from mice injected sc four times every 2 weeks with 50 �g of 1E10/Alum (right pan-el), iorC5/Alum or PBS/Alum (left panel), the Wrst two doses were administered before the tu-mor inoculation. a and b ParaYn sections stained with hematoxy-lin and eosin (£5). c and d Immunostaining with the Apoptag Plus Kit (£25, arrows indicate cells in apoptosis). e and f Immunostaining with an anti-CD31 antibody (£25, arrows indicate microvessels). g and h CD4 immunostaining of tissue cryosections (£25, arrows indicate CD4+ cells)

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extracellular matrix components by active proteases secre-tion. This process requires a complex cross-talk betweenendothelial and tumor cells, extracellular matrix compo-nents, and cellular elements of the host microenvironment.The interference between the components of the extracel-lular matrix and tumor or endothelial cells could lead tothe inhibition of this migration process and inhibit themetastasis or the angiogenesis process [35]. The inhibitionof the Wbronectin and integrin association using syntheticpeptides mimicking one of the extracellular matrix compo-nents has resulted in a successful approach [36]. The 1E10mAb contains a CDR3 motif also seen in integrin mole-cules [37], so the development of antibodies against thismotif could block the interaction between cells and itsextracellular matrix and prevent the migration process of

both tumor and endothelial cells. The inhibition in themigration process of endothelial cells could prevent theformation of new blood vessels, causing tumor cell death,a process that can attract diVerent elements of the immunesystem. Among the six essential alterations in cell physiol-ogy that collectively dictate malignant growth are sus-tained angiogenesis, and evasion of apoptosis [38].Therapies able to induce antiangiogenic and proapoptoticeVects have been developed, and some of them are nowbeen tested in diVerent stage clinical trials in cancerpatients [39–42]. Nevertheless, due to the complexity andthe number of diVerent molecules involved in these pro-cesses in diVerent types of tumor cells, it is rather improb-able that a single therapy would control the tumorprogression [38].

Fig. 3 Proapoptotic and antian-giogenic eVect of 1E10/Alum immunization in 3LL-D122 lung metastases. Mice were injected sc four times every 2 weeks with 50 �g of 1E10/Alum, iorC5/Alum and PBS/Alum, the Wrst two doses were administered be-fore the tumor inoculation (left panel), or animals were immu-nized six times with 100 �g of the vaccine starting 3 days after the tumor challenge and weekly afterward (right panel, see Sect. “Materials and methods” for details). Lung metastasis analyzes was performed by immunohistochemistry. a Apop-tosis was measured with the Apoptag Plus Kit. b Tumor mitosis was counted in hematox-ylin–eosin stained samples. c Number of CD31+ microvessels was analyzed using an anti-CD31 antibody. Data represent the mean § SEM in 12 independent lung metastases Welds from two mice from each group. Representative data from one experiment are shown (**P < 0.01 and ***P < 0.001)

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Interestingly, immunohistochemistry analysis of lungmetastatic tissues from vaccinated mice showed a signiW-cant increase in the number of apoptotic tumor cells whencompared with control animals (Fig. 3). This result associ-ated with the fact that only vaccinated mice had a decreasein the number of lung metastases suggest that apoptosisseems to be a potential mechanism related with the antitu-mor activity of 1E10/Alum vaccine. A similar eVect hasbeen found in tumor tissues from patients treated with theanti-Id mAb 105AD7 [43].

The higher expression levels of NeuGc-GM3 ganglio-side level in spontaneous metastasis (A. Gonzalez et al., inpreparation), could be related with the observed eVective-ness of 1E10/Alum vaccine only on lung metastasis but noton primary tumors. Similarly the T cell inWltration was onlyobserved in lung metastasis from 1E10/Alum immunizedanimals but not in their primary tumors (Fig. 4). We maypropose that the enhanced tumor apoptosis observed after1E10/Alum immunization lead to an increase in antigenspresentation, triggering an antigen spreading, which wouldinvolve in the response to other tumor antigens, besides theNeuGc-GM3 ganglioside, which has been evidenced inother experimental conditions [44].

That would explain the sustained antitumor eVect in thisstringent lung cancer model as well as the lack of a demon-strable speciWc response to the ganglioside itself. It would

be expected based on the fact that active and passive immu-nity to GM3 induce tumor cell death [33, 45], which in turnmay enhance antigen presentation and antitumor eVectinvolving new tumor speciWcities, which is currently underevaluation.

The tumor mitosis did not diverge between controls and1E10/Alum vaccinated animals (Fig. 3), suggesting that theobserved antitumor eVect is not associated to an antiprolif-erative process of this vaccine. The increase in apoptosisdetected in the pulmonary metastases from mice treatedwith 1E10/Alum could be due to its capacity to inhibit angi-ogenesis. In fact, the observation that antiangiogenic mole-cules can induce apoptosis of tumor cells has beenpreviously reported [46, 47]. On the other hand, tumor-inWltrating T cells and their cytotoxic released cytokinescould be implicated in the process [48, 49].

These preclinical evidences for the antitumor mecha-nisms of a cancer vaccine deWned by an anti-gangliosideanti-idiotypic mAb may support the relevance of this targetfor cancer biotherapy. However, the correspondence withthe mechanisms responsible for the therapeutic eVectobserved in patients has to be elucidated [12, 50, 51].Finally, due to the reported preclinical and clinical Wndingsthat the combinations of antiangiogenic, proapoptotic andantiproliferative agents with conventional drugs improvethe antitumor eYcacy of the latter [40, 52–55], future

Fig. 4 1E10/Alum immuniza-tion promotes the lymphocyte inWltration in lung metastases. Mice were injected sc four times every 2 weeks with 50 �g of 1E10/Alum, iorC5/Alum and PBS/Alum, the Wrst two doses were administered before the tumor inoculation (left panel), or animals were immunized six times with 100 �g of the vaccine starting 3 days after the tumor challenge and weekly afterward (right panel, see Sect. “Materials and methods” for details). Lung metastasis analyzes was performed by immunohistochemistry. a CD4+ and b CD8+ cells inWltrating lung metastasis. The bars repre-sent the mean § SEM of total cells per Weld (*P < 0.05 and ***P < 0.001)

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experiments combining 1E10/Alum with cytotoxic drugs indiVerent murine tumor models are encouraged.

Acknowledgments We thank Dr L.E. Fernández (Vaccine Depart-ment, Center of Molecular Immunology) for generously providinggangliosides. This study was supported by Recom-Bio S.L. and by theCuban Government.

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