Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming

Danila Valmori*†, Naira E. Souleimanian*, Valeria Tosello*, Nina Bhardwaj‡, Sylvia Adams‡, David O’Neill‡, Anna Pavlick‡, Juliet B. Escalon‡, Crystal M. Cruz‡, Angelica Angiulli‡, Francesca Angiulli‡, Gregory Mears§, Susan M. Vogel§, Linda Pan¶, Achim A. Jungbluth¶, Eric W. Hoffmann¶, Ralph Venhaus¶, Gerd Ritter¶, Lloyd J. Old¶ʈ, and Maha Ayyoub*†

*Ludwig Institute Clinical Trial Center, Columbia University, New York, NY 10032; ‡New York University School of Medicine, New York, NY 10016; §Division of Medical Oncology, Columbia University Medical Center, New York, NY 10032; and ¶Ludwig Institute for Cancer Research, New York, NY 10158

Contributed by Lloyd J. Old, April 12, 2007 (sent for review February 22, 2007) The use of recombinant tumor proteins is a realistic helper type 1 (Th1)-type immunity (7). In humans, they can approach for the development of generic cancer , but the directly activate B lymphocytes and plasmacytoid dendritic cells ,(potential of this type of vaccines to induce specific CD8؉ and also indirectly activate myeloid dendritic cells (mDCs responses, through in vivo cross-priming, has remained unclear. In increasing antigen cross-presentation and stimulating adaptive this article, we report that repeated vaccination of cancer patients immune responses (8–10). with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG In this study, we have assessed the immune response elicited ODN 7909, a potent stimulator of B cells and T helper type 1 by repeated vaccination with a NY-ESO-1 recombinant protein (Th1)-type immunity, resulted in the early induction of specific (rNY-ESO-1) administered with CpG 7909 in a water–oil emul- integrated CD4؉ Th cells and antibody responses in most vacci- sion with Montanide ISA-51. We show that cancer patients nated patients, followed by the development of later CD8؉ T cell receiving this developed integrated Ab and CD4ϩ T cell responses in a fraction of them. The correlation between antibody responses to NY-ESO-1 at an early phase of the vaccination and T cell responses, together with the ability of vaccine-induced protocol. A fraction of the patients also developed specific CD8ϩ antibodies to promote in vitro cross-presentation of NY-ESO-1 by T cell responses at a later time point. Assessment of the dendritic cells to vaccine-induced CD8؉ T cells, indicated that correlation between the development of Ab and T cell responses elicitation of NY-ESO-1-specific CD8؉ T cell responses by cross- suggested that the presence of sufficient levels of NY-ESO-1- priming in vivo was associated with the induction of adequate specific antibodies was determinant for the cross-priming of ϩ levels of specific antibodies. Together, our data provide clear CD8 T cells to occur in vivo. In line with this concept, we found evidence of in vivo cross-priming of specific cytotoxic T lympho- that in vitro cross-presentation of NY-ESO-1 protein to vaccine- ϩ cytes by a recombinant vaccine, underline the induced CD8 T cells by dendritic cells was enhanced by importance of specific antibody induction for the cross-priming to vaccine-induced Ab. occur, and support the use of this type of formulation for the further development of efficient cancer vaccines. Results Serological Response to Vaccination with rNY-ESO-1, CpG 7909, and cancer vaccine Montanide ISA-51. Study patients received four s.c. injections of NY-ESO-1/Montanide/CpG vaccine at 3-week intervals. The ␮ key step in the development of generic cancer vaccines is study included two arms receiving 100 or 400 g of rNY-ESO-1 IMMUNOLOGY Athe implementation of vaccination strategies allowing for per injection, together with 2.5 mg of CpG emulsified in Mon- the consistent induction of immune responses to tumor . tanide. CpG 7909 belongs to the CpG-B class, which potently In this respect, the choice of appropriate antigens, based on both stimulates B cells (11). All patients developed significant sero- the frequency and the specificity of their expression in cancer logical responses to NY-ESO-1 (Fig. 1A). Specific IgG responses tissues, is of paramount importance. The group of cancer/testis became significant after the second injection and further in- antigens (CTA) (1, 2), including the NY-ESO-1 antigen (3), is creased after the third and fourth injections, without generally emerging among the most promising candidates. Because many reaching a plateau. Specific IgG titers were variable among CTA are not expressed on the surface of cancer cells but rather patients: at the last time point, NY-ESO-1 reciprocal Ab titers had reached an average 42.429 Ϯ 29.080 in the 400 ␮g group and intracellularly, it is important that vaccination induces specific Ϯ ␮ CD8ϩ T cells able to directly recognize antigen-expressing tumor 24.091 10.737 in the 100 g group. Serological responses to an cells. Recombinant proteins can be produced in large scale and

at relatively low cost, are commonly used in the development of Author contributions: D.V., L.J.O., and M.A. designed research; D.V., N.E.S., V.T., N.B., S.A., antiviral vaccines, and are therefore attractive candidate anti- D.O., A.P., J.B.E., C.M.C., A.A., F.A., G.M., S.M.V., A.A.J., and M.A. performed research; L.P., tumor vaccines. The potential of tumor antigen recombinant E.W.H., R.V., G.R., and L.J.O. contributed new reagents/analytic tools; D.V. and M.A. protein vaccines, however, relies on their ability not only to elicit analyzed data; and D.V. and M.A. wrote the paper. antibody (Ab) and CD4ϩ T cell responses but also to efficiently The authors declare no conflict of interest. prime naive CD8ϩ T cells through cross-priming (4), which Abbreviations: CTL, cytotoxic T lymphocyte; APC, antigen-presenting cell; TLR, Toll-like generally is inefficient during spontaneous immune responses to receptors; Th, T helper; mDC, myeloid ; PBMC, peripheral blood mononuclear cell; ADCC, antibody-dependent cellular cytotoxicity. tumor antigens (5). Professional antigen-presenting cells (APCs) †To whom correspondence may be sent at the present address: Institut National de la detect pathogens through a variety of receptors such as the Sante´ et de la Recherche Me´dicale, Unite 601, Centre de Lutte Contre le Cancer Rene´ Toll-like receptors (TLR), which recognize pathogen-associated Gauducheau, Boulevard Jacques Monod, 44800 Saint Herblain, France. E-mail: molecular patterns including CpG dinucleotides within defined [email protected] or [email protected]. flanking sequences (CpG ODN) (6). Synthetic CpG ODN able ʈTo whom correspondence may be addressed. E-mail: [email protected]. to trigger TLR9 are potent vaccine adjuvants, stimulating T © 2007 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703395104 PNAS ͉ May 22, 2007 ͉ vol. 104 ͉ no. 21 ͉ 8947–8952 Downloaded by guest on September 25, 2021 Fig. 1. Serological responses. (A) Serological responses were assessed by ELISA at baseline and at the indicated study week after vaccination. (B) The isotype of vaccine-induced IgG (week 12, serum dilution of 1:100) was assessed by ELISA using isotype-specific antibodies. (C) Linear B cell were determined by using patients’ immune sera and a panel of 30-aa-long pep- tides spanning the protein sequence.

unrelated recombinant protein (MAGE-A4) generally were low in both groups (4.957 Ϯ 3.594 in the group receiving 400 ␮g and 2.735 Ϯ 2.519 in the group receiving 100 ␮g).

Isotype of Vaccine-Induced IgG and Mapping of B Cell Epitopes. To investigate further the serological responses induced by the Fig. 2. Assessment of CD4ϩ and CD8ϩ T cell responses after in vitro stimu- vaccine, we assessed the isotype of vaccine-induced IgG by using lation. (A) The presence of specific CD4ϩ and CD8ϩ T cells in stimulated cultures isotype-specific antibodies. As illustrated in Fig. 1B, the vaccine from vaccinated patients was assessed by intracellular staining with anti-IFN-␥ preferentially induced specific IgG of types 1 and 3, which are monoclonal Ab after stimulation in the absence or presence of the NY-ESO-1 peptide pool. Numbers in the upper right quadrant are the percentages of associated, in humans, with type 1 immune responses. We ϩ ϩ mapped the linear B cell epitopes recognized by the vaccine- IFN-␥-producing cells in CD4 or CD8 T cells. As an example, data are shown induced Ab with a panel of peptides spanning the protein for one responder patient. (B) Summary of the results obtained for all patients. Values correspond to the percentage of CD4ϩ or CD8ϩ T cells specifically sequence (12). The analysis identified a dominant region located producing IFN-␥ in response to stimulation with the peptide pool. (C) Cultures in the central part of the protein and comprising amino acids 80– were stimulated with single peptides in the NY-ESO-1 pool. The proportion of 110, which was recognized strongly by Abs from all patients (Fig. IFN-␥-producing cells was assessed by intracellular staining as above. Symbols 1C). Consistent with the previous identification of B cell epitopes are as given in the Fig. 1 key. located in the N-terminal half of the protein and recognized by specific Abs from cancer patients with spontaneous serological ϩ responses to NY-ESO-1 (12, 13), the immune sera from vacci- CD4 T cells recognized sequences located in two distinct nated patients reacted to peptides in the amino acids 1–70 region regions of the protein, corresponding to peptides 81–100 and of the protein. A lower level of reactivity was detected toward 119–143. We found minor reactivity toward some other peptides, ϩ sequences located in the C-terminal half of the protein, between including 151–170. The sequences recognized by CD8 T cells amino acids 160 and 180. mostly were located between amino acids 81 and 110, as indi- cated by the reactivity of the majority of these cells to the Assessment of T Cell Responses After in Vitro Stimulation. We overlapping peptides 81–100 and 91–110. The relative reactivity initially assessed the induction of specific T cells after in vitro to these two peptides varied among patients, indicating the stimulation of pre- and postimmune samples with a pool of presence of multiple epitopes in this region, consistent with overlapping peptides spanning the protein sequence, followed by previous results in patients with spontaneous responses as well quantification of specific IFN-␥-producing cells by intracellular as in patients immunized with other NY-ESO-1 full-length staining (14, 15) (Fig. 2A). The response was considered signif- vaccines (15, 17, 18). icant if the frequency of T cells detected in at least one postvaccine sample exceeded by 3-fold that found in the baseline Ex Vivo Assessment of T Cell Responses. Next, total pre- and sample. CD4ϩ T cell responses were detectable in 17 of 18 postimmune peripheral blood mononuclear cell (PBMC) sam- patients and developed early, in general becoming detectable ples were stimulated directly ex vivo with the NY-ESO-1 peptide already at week 4 (Fig. 2B). CD8ϩ T cell responses were pool and assessed for the presence of specific T cells by using detectable in 9 of 18 patients (4/7 in the 400 ␮g group and 5/11 intracellular cytokine staining together with cell-surface staining in the 100 ␮g group) and generally developed later. We used to assess phenotype. NY-ESO-1-specific CD4ϩ and CD8ϩ T cells stimulated cultures to map the protein regions recognized by were detectable ex vivo after vaccination but not before vacci- CD4ϩ and CD8ϩ T cells, by using single peptides in the nation (Fig. 3A) We detected significantly increased levels of NY-ESO-1 pool (Fig. 2C). Similar to what was found previously NY-ESO-1-specific T cells in postvaccination samples from 12 of in patients with spontaneous responses (14–16), the majority of the 18 patients in the case of CD4ϩ T cells and in 7 patients in

8948 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703395104 Valmori et al. Downloaded by guest on September 25, 2021 Fig. 3. Ex vivo assessment of CD4ϩ and CD8ϩ T cell responses. (A) Dot plots gated on CD3ϩ cells are shown for a high-responder patient. Numbers in the upper right quadrant are the percentages of IFN-␥-secreting cells among CD4ϩ T cells (Upper) and CD8ϩ T cells (Lower). (B) Summary of the results obtained for all patients. Symbols are as given in the Fig. 1 key. For each sample, values obtained in the absence of the NY-ESO-1 peptide pool were subtracted. Dashed lines represent the mean values of baseline samples plus three times Fig. 4. Recognition of NY-ESO-1 by vaccine-induced T cells and Ab-aided their standard deviation. The baseline sample from patient N4, who had cross-presentation. (A) Vaccine-induced CD4ϩ and CD8ϩ T cell populations en- ϩ detectable NY-ESO-1-specific CD8 T cells before vaccination as shown in Fig. riched by cytokine-secretion-guided sorting were assessed by intracellular IFN-␥ ␥ 2 and Table 1, was excluded from this calculation. Frequencies of IFN- - staining after stimulation in the absence or presence of the NY-ESO-1 peptide secreting cells above this value were considered significant. (C) Phenotype and ϩ ϩ pool. (B) Recognition of autologous mDCs by CD4 and CD8 T cells was assessed IMMUNOLOGY ϩ ϩ cytokine production of vaccine-induced CD4 and CD8 T cells. after incubation with serial dilutions of rNY-ESO-1 or after electroporation with a plasmid encoding NY-ESO-1. (C) The MHC class I allele restricting antigen recognition by CD8ϩ T cells was determined upon transfection of COS-7 cells with ϩ the case of CD8 T cells (Fig. 3B). It is noteworthy that 5 of 6 plasmids encoding the alleles of the patient. Recognition of endogenous NY- ϩ patients in whom CD4 T cell responses were not detectable ex ESO-1 was assessed upon cotransfection of COS-7 cells with plasmids encoding vivo were among nonresponders or low responders after in vitro NY-ESO-1 and HLA-B35 or by transfecting the tumor cell lines NA8-MEL (NY-ESO- stimulation. In addition, 6 of 7 patients with CD8ϩ T cell 1Ϫ) and HT1080 (NY-ESO-1ϩ) with a plasmid encoding HLA-B35. (D) Cross- responses detectable ex vivo were among the 9 patients in whom presentation, by autologous mDCs, of rNY-ESO-1 (10 ␮g/ml) or immune com- CD8ϩ T cell responses were detectable after in vitro stimulation. plexes formed either with a murine NY-ESO-1-specific monoclonal Ab (ES121, 10 ϩ ␮g/ml) or with pre- and postimmune serum at the indicated dilution to vaccine- Vaccine-induced CD4 T cells mostly were composed of effec- ϩ Ϫ Ϫ induced CD8 T cells. In B and C, antigen recognition was assessed by measure- tor-memory (CD45RA CCR7 ) and, to a lower extent, central- ment of IFN-␥ secretion in the culture supernatant. memory (CD45RAϪ CCR7ϩ) T cells (19) (Fig. 3C). A large part of these cells secreted IFN-␥ only, whereas lower proportions either cosecreted IFN-␥ and IL-2 or secreted IL-2 only. Vaccine- enously processed NY-ESO-1 antigen, we isolated them from induced CD8ϩ T cells also were composed mostly of effector- peptide-stimulated cultures by IFN-␥-guided cell sorting (Fig. memory T cells, did not contain many central-memory T cells, 4A). We used the isolated populations to assess the recognition and contained instead a significant proportion of terminally of autologous mDCs incubated with the rNY-ESO-1 or electro- differentiated effectors (CD45RAϩCCR7Ϫ). The cytokine pro- porated with a plasmid encoding NY-ESO-1 (Fig. 4B). The file of vaccine-induced CD4ϩ and CD8ϩ T cells was clearly of rNY-ESO-1 was recognized efficiently by CD4ϩ T cells but Th1 type, because we failed to detect any significant levels of poorly by CD8ϩ T cells. In contrast, CD8ϩ T cells but not CD4ϩ IL-10-, IL-4-, or IL-17-secreting cells (data not shown). T cells recognized the endogenous antigen. These results suggest that, in this setting, autologous professional APCs are likely to Recognition of NY-ESO-1 by Vaccine-Induced T Cells and Ab-Aided initiate the immune response by priming CD4ϩ Th cells after Cross-Presentation. To address the ability of vaccine-induced processing and presentation of the rNY-ESO-1 protein. To CD4ϩ and CD8ϩ T cells to recognize exogenously and endog- address the ability of CD8ϩ T cells to recognize tumor cells, we

Valmori et al. PNAS ͉ May 22, 2007 ͉ vol. 104 ͉ no. 21 ͉ 8949 Downloaded by guest on September 25, 2021 Table 1. Patient characteristics, antigen dose, summary of vaccine-related toxicity, and vaccine-induced immune responses Ab§ CD4¶ CD8 Before study Antigen Toxicity‡ Patient Disease* Stage status† dose, ␮g (grade) Before After Before After Before After

N14 IIB NED 100 L(1), S(1) ϪϩϪϪϪϪ N11 Melanoma IIIB NED 100 L(1), S(1) ϪϩϪϩϪϪ C3 I NED 100 L(2) ϪϩϪϩϪϪ C4 Breast cancer IIA NED 100 L(1), S(1) ϪϩϪϩϩϪϪ N10 Melanoma IVA NED 400 L(1) ϪϩϪϩϪϪ C7 Melanoma III NED 100 L(1), S(1) ϪϩϩϪϩ Ϫ Ϫ N13 Melanoma IIIA NED 400 L(1), S(1) ϪϩϩϪϩ Ϫ ϩ N5 Melanoma IVB NED 400 L(1) ϪϩϩϪϩ Ϫ Ϫ C1 Sarcoma II NED 400 L(2) ϪϩϩϪϩϩϪ Ϫ N4 Melanoma IVA NED 100 L(1), S(1) ϩϩϪϩϩϩϩ C5 Sarcoma III M (lung) 100 L(2), S(2) ϪϩϩϪϩϩϪ Ϫ N2 Melanoma IIIB NED 100 L(1), S(1) ϪϩϩϪϩϩϪ ϩ N7 Melanoma IIIB NED 100 L(1), S(1) ϪϩϩϪϩϩϪ ϩ N9 IIIC NED 400 L(1), S(1) ϪϩϩϪϩϩϪϩϩ N3 Melanoma IIIB NED 100 L(1), S(1) ϪϩϩϪϩϩϩϪϩϩ C6 Sarcoma IIIB NED 400 L(1), S(1) Ϫ ϩϩϩ Ϫ ϩϩ Ϫ ϩϩ N8 Melanoma IIC NED 100 L(1), S(1) Ϫ ϩϩϩ Ϫ ϩϩ Ϫ ϩϩ C2 Breast cancer IIA NED 400 L(2), S(2) Ϫ ϩϩϩ Ϫ ϩϩ Ϫ ϩϩϩ

*NY-ESO-1 expression in the tumor was not an entrance criterion for this study. However, 10 of 18 patients were tested for NY-ESO-1 expression by immunohistochemistry. Patients N4 and N9 were NY-ESO-1ϩ. Patients C3, C4, C5, C6, N2, N3, N7, and N13 were NY-ESO-1Ϫ. †NED, no evidence of disease; M, metastatic disease. ‡Adverse events with possible, probable, or definite relationship to study drug. L, local toxicity at injection site, included redness, discomfort, itching, induration, erythema, and pain. S, systemic toxicity, included fever, chills, fatigue, cold-like symptoms, sweats, muscle aches, and joint pain. §Ab (week 12 reciprocal serum titer): Ϫ, Յ100; ϩ, Ͼ100 to 20,000; ϩϩ, Ͼ20,000 to 40,000; ϩϩϩ, Ͼ40,000. ¶CD4 and CD8 (percentage of IFN-␥ϩ cells after in vitro stimulation): Ϫ, Յ0.3%; ϩ, Ͼ0.3% to 1%; ϩϩ, Ͼ1% to 10%; ϩϩϩ, Ͼ10%.

identified the MHC class I allele restricting antigen recognition vaccine formulation used in this study may be a determining of CD8ϩ T cells from one responder patient and assessed its factor for the induction of CD8ϩ T cells, through Ab-aided ability to recognize antigen-expressing tumor cells transfected cross-priming in vivo. with the appropriate allele. Transfection of COS-7 cells with an HLA-B35-encoding plasmid, but not with plasmids encoding Discussion other patient’s HLA alleles, resulted in peptide presentation to NY-ESO-1 is a nonmutated self-antigen frequently expressed in CD8ϩ T cells (Fig. 4C). In addition, both COS-7 cells cotrans- human tumors that often induces spontaneous immune responses fected with NY-ESO-1 and HLA-B35 and NY-ESO-1- in patients bearing antigen-expressing tumors. We have selected expressing tumor cells transfected with HLA-B35 were recog- NY-ESO-1 as a prototype tumor antigen for the development of nized specifically by CD8ϩ T cells. generic cancer vaccines and, under the sponsorship of the Cancer Vaccine Collaborative (www.cancerresearch.org), are assessing the Enhancement of Antigen Cross-Presentation by Vaccine-Induced Ab. immunogenicity of NY-ESO-1-based candidate vaccines in early- The data above provided evidence that vaccination resulted in phase clinical trials. Among the immunogens tested is the rNY- the consistent induction of CD4ϩ T cell and Ab responses in most ESO-1 protein, in various formulations (17, 18). In this study, we patients, as well as of specific CD8ϩ T cells in a fraction of them have obtained direct evidence that immunization with rNY-ESO-1 (summarized in Table 1). The in vivo kinetic of the responses administered with Montanide and CpG induces integrated Ab and clearly showed that CD4ϩ T cells and Abs developed early, CD4ϩ T cell responses in the majority of vaccinated patients and whereas CD8ϩ T cells generally developed later. In addition, the cross-primes CD8ϩ T cells in a significant fraction of them. Al- development of CD8ϩ T cell responses generally correlated with though NY-ESO-1 expression in tumor specimens could not be medium to high titers of NY-ESO-1 Ab. This, together with the assessed for all patients, we failed to detect any correlation between relatively poor recognition of the rNY-ESO-1 alone after cross- antigen expression and responsiveness to vaccination (Table 1). It presentation by dendritic cells, suggested an involvement of is noteworthy that, with the exception of one patient, no significant NY-ESO-1 Ab in the in vivo cross-priming of CD8ϩ T cells, serological reactivity to NY-ESO-1 was detectable before vaccina- through formation of immune complexes. To address this point, tion, indicating the ability of the vaccine to prime specific naı¨ve B mDCs from a responder patient were pulsed with the NY-ESO-1 cells. This finding is in line with the recent proposal by Ruprecht et recombinant protein for 2 h and then assessed for antigen al. that triggering of TLR9 by CpG on human naı¨ve B cells acts as recognition by autologous CD8ϩ T cells. Under these conditions, a third signal, required, in addition to B cell receptor stimulation no significant cross-presentation was detected (Fig. 4D). How- and CD4ϩ T cell help, for their full differentiation into IgG- ever, dendritic cell incubation with immune complexes gener- secreting cells (20). Montanide may contribute significantly to the ated by mixing NY-ESO-1 recombinant protein with the NY- immunogenicity of the formulation, through its known effects, such ESO-1-specific monoclonal Ab ES121 resulted in efficient as depot effect, slow antigen release, and recruitment of APCs at antigen cross-presentation. In addition, preincubation of NY- the injection site. In support of this data, in a separate study, ESO-1 protein with postvaccine serum but not with prevaccine administration of NY-ESO-1 protein and CpG without Montanide serum, at various dilutions, also resulted in efficient antigen was less immunogenic (E. Jager, personal communication). Ad- cross-presentation. Together, these results are in line with the ministration of CpG in combination with Montanide, however, is concept that the induction of NY-ESO-1-specific Ab by the likely to be responsible for the high Ab titers detected in this study

8950 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703395104 Valmori et al. Downloaded by guest on September 25, 2021 and to the skewing of the induced immunological response toward support of this, postimmune sera from vaccinated patients were a Th1 type, attributable to the induction of proinflammatory able to promote efficiently cross-presentation of NY-ESO-1 to cytokines and type 1 IFNs, in contrast to incomplete Freund’s vaccine-induced CD8ϩ T cells in vitro. In addition to their titers, the adjuvant alone (equivalent to Montanide), which induced a Th2- induction of type 1 IgG Ab is likely of importance. Fc␥R is a family type bias (11, 21). Consistent with this concept, vaccine-induced of glycosylated membrane proteins that includes members that IgG predominantly were IgG1 and IgG3, the most prominent IgG differ largely with respect to their capacity to bind IgG (30, 31). For type 1 human subclasses, involved in functions such as complement example, Fc␥RI (CD64) binds monomeric IgG with high affinity, activation, mediation of antibody-dependent cellular cytotoxicity but it signals only if the latter is cross-linked by specific polymeric (ADCC), and opsonization (22). ligands. In contrast, Fc␥RII (CD32) binds monomeric IgG poorly ϩ After one round of in vitro stimulation, vaccine-induced CD4 T but binds immune complexes with very high affinity. IgG3 and IgG1 cell responses were detectable in most vaccinated patients, and preferentially interact with Fc␥RIIa, which is a potent leukocyte ϩ CD8 T cell responses were detectable in a fraction of them. In activator and can stimulate the release of high levels of inflamma- most cases, their reactivity was restricted to one or few peptides in tory cytokines. Thus, the Ab isotypes induced by our vaccine, in the protein, corresponding to immunodominant regions identified particular IgG3, may be effective at inducing dendritic cell activa- in our previous studies (14, 15). The immunodominant region tion and maturation, rapid internalization, concentration, and recognized by cross-primed CD8ϩ T cells (between amino acids 81 ϩ transport of the antigen to the appropriate endosomal/lysosomal and 110) was one of the two main regions also recognized by CD4 compartments, ultimately enhancing the induction of T cells. T cells and the most efficiently recognized by Ab. We recently have As in the case of NY-ESO-1, specific Ab responses to tumor- reported the identification of overlapping clusters of MHC class I associated antigens often develop during spontaneous immune and class II binding sequences in immunodominant regions of responses to tumors. The biological role of these Abs in the NY-ESO-1 that contain a high density of proteasomal cleavage sites development of the disease, however, is poorly understood. The (14, 15). This interesting phenomenon provides the molecular bases mechanism of action in vivo of some antitumor Abs currently used for the development of integrated Ab, Th, and cytotoxic T lym- for the treatment of breast cancer (anti-Her2/neu) or B cell phocyte (CTL) responses around short immunodominant regions. lymphomas (anti-CD20) remains unclear but generally is consid- Based on these findings, we have proposed that CTL epitopes in the ered to be through ADCC (32). Possibly complementary to ADCC, NY-ESO-1 immunodominant 81–110 region may be generated Ab-aided cross-priming of CTLs specific for tumor antigens also efficiently through cross-priming, a hypothesis now supported by could contribute to the antitumor effect of these therapies. In a the findings of the present study. In addition, the overlapping murine model of melanoma, passive transfer of Abs against the between regions concentrating B and T cell immunodominant melanoma antigen gp75 has been shown to be highly effective in epitopes, suggests a role for Ab-mediated protection during inhibiting tumor growth (33) in a Fc␥R-dependent manner (34). In antigen processing. a different mouse model, using EL-4 lymphoma cells transfected Both vaccine-induced CD4ϩ and CD8ϩ T cell populations ex- with the model antigen OVA, Dyall et al. (35) have provided further hibited ex vivo a clear Th1 profile and contained polyfunctional evidence for Fc␥R-mediated Ab-dependent tumor eradication and populations producing IL-2 and/or IFN-␥. They mostly exhibited an ϩ ϩ shown that CD8 T cells are the critical effector population in this effector-memory phenotype, although CD4 T cell populations system. Additional evidence for the role of antitumor Abs in contained detectable proportions of central memory cells and ϩ promoting cross-priming of tumor-specific CTLs has been provided CD8 T cell populations of so-called terminally differentiated effectors. The ex vivo frequencies of vaccine-induced NY-ESO-1- by an in vitro study with human cells in which Dhodapkar and specific T cells were variable among patients, and, with the excep- colleagues (36) demonstrated that coating myeloma cells with tion of one high-responder patient, they remained relatively low in anti-syndecan-1 Abs strongly enhanced cross-presentation and most patients. The ex vivo frequency of CD4ϩ T cells detected in our cross-priming of CTLs specific for the NY-ESO-1 and MAGE-A3 study is in the range of those recently reported after vaccination of antigens. Adding to these previous findings, our data indicate that nonhuman primates with HIV Gag protein, Montanide, and CpG Ab-aided cross-priming of NY-ESO-1-specific CTLs, and possibly ODN (C class) (23). Interestingly, in the study from Wille-Reece et of other human tumor antigens, could be achieved through active IMMUNOLOGY al. (23), TLR7/8 agonists stimulated higher responses than CpG immunization with recombinant proteins. ODN. In addition, in their study, further immunization with Along with Abs, other factors are likely to contribute, both Gag-encoding recombinant adenovirus resulted in a high increase directly and indirectly, to promote in vivo cross-priming after in the frequency of CD8ϩ T cells, suggesting that prime/boost vaccination with the formulation used in our study. Namely, regimens alternating rNY-ESO-1 and NY-ESO-1-encoding recom- although class B CpG may not be optimal for inducing the pro- ␣ binant viruses (currently in development as part of the Cancer duction of IFN- , the latter still could play a significant role by both stimulation of B cell activating factor (BAFF) (37) and activation of Vaccine Collaborative program) (18) could lead to an optimal ϩ expansion of specific CD8ϩ T cells. mDCs (38). Also, vaccine-induced CD4 T cells could play a role The role of CpG in the induction of primary and memory T cell in the activation of mDCs through CD40–CD40L interactions (28). responses has not been elucidated completely. Based on in vitro In conclusion, the vaccine formulation used in this study seems to experiments, Rothenfusser et al. (24) have proposed that class A be highly immunogenic and able to stimulate integrated Ab and T ϩ CpG could selectively enhance memory CD8ϩ T cell responses and cell responses, including tumor-reactive CD8 T cells, by cross- cytotoxicity, whereas class B CpG could be superior for priming priming in an Ab-aided fashion. On the other hand, the relatively ϩ CD8ϩ T cells. In support of this data, in a recent study, Speiser et delayed development of specific CD8 T cell responses requiring al. (25) reported evidence of induction of CD8ϩ T cell responses in repeated vaccine injections, along with their detection in only a melanoma patients, including some with no detectable preimmune fraction of vaccinated patients, suggests that additional injections, responses, by vaccination with a Melan-A peptide analog, Mon- a combination of Montanide/CpG with other TLR ligands, or the tanide, and CpG. In our study, priming of CD4ϩ T cells occurred use of prime/boost strategies alternating different NY-ESO-1- at an early phase of the vaccination protocol, in contrast to CD8ϩ based immunogens may be required to optimize their induction. T cell responses that became detectable only at a later stage. This Based on our data, administration of NY-ESO-1-specific Abs delayed appearance, which occurred concomitant with the titer of before or concomitant with vaccination or administration of pre- NY-ESO-1-specific Abs reaching high levels in responder patients, formed activating Ab–protein immune complexes in combination suggested a role for vaccine-induced Abs in promoting CD8ϩ T cell with adjuvant also may be promising approaches for the develop- cross-priming through Ab-aided cross-presentation (26–29). In ment of cancer vaccines of increasing efficacy.

Valmori et al. PNAS ͉ May 22, 2007 ͉ vol. 104 ͉ no. 21 ͉ 8951 Downloaded by guest on September 25, 2021 Materials and Methods absence or presence of the NY-ESO-1 peptide pool. Brefeldin A Clinical Study and Patient Population. The study protocol was was added2hafterthebeginning of the incubation. At the end of approved by the Institutional Review Boards of Columbia the incubation, cells were stained with antibodies directed against University and New York University Medical Centers. Patient surface markers (CD3, CD4, CD8, CD45RA, and CCR7), fixed, samples were obtained by means of written informed consent. permeabilized, and stained with cytokine-specific antibodies. The patients enrolled had histological diagnoses of cancer types known to express NY-ESO-1. However, demonstration of ex- Assessment of Antigen Recognition by Specific T Cells. NY-ESO-1- pression of NY-ESO-1 in the patient’s tumor specimen was not specific T cells were isolated from peptide-stimulated cultures by an inclusion criterion. The primary end point of the study was to cytokine-guided magnetic cell sorting (Miltenyi Biotec Inc.), stim- determine the safety and immunogenicity of the vaccine. Tox- ulated as polyclonal cultures in the presence of phytohemagglutinin, icity and adverse events were graded according to the National allogeneic irradiated PBMCs, and rhIL-2 (100 units/ml), and used Cancer Institute’s Common Terminology Criteria for Adverse in antigen recognition assays. mDCs were obtained by culture of Events scale (version 3.0; accessed December 12, 2003). isolated CD14ϩ cells during 5 days in the presence of rhIL-4 and rhGM-CSF. mDCs were either incubated overnight in the presence Assessment of Serological Responses. Serological responses were of the NY-ESO-1 protein and washed before use in recognition assessed by ELISA using NY-ESO-1 or MAGE-A4 recombinant assays or incubated for2hinthepresence of the rNY-ESO-1 or proteins produced in Escherichia coli (12, 39). Sera were assessed NY-ESO-1 immune complexes, washed, cultured overnight, and over a range of dilutions from 1/100 to 1/100,000. Titers were used in recognition assays. Where indicated, mDCs were trans- calculated as the serum dilution giving 50% of maximal optical fected with a NY-ESO-1-expressing plasmid by electroporation density obtained by using a standard positive serum. IgG isotypes (Amaxa, Gaithersburg, MD). COS-7 cells were transiently trans- were determined by ELISA using the same protocol and antibodies fected with the NY-ESO-1 encoding plasmid and/or the appropri- specific for IgG1 (BD Biosciences, San Jose, CA) and IgG2, IgG3, ate HLA class I expressing plasmids by using FuGENE (Roche and IgG4 (Southern Biotech, Birmingham, AL). Diagnostics, Basel, Switzerland). NA8-MEL and HT1080 tumor

ϩ ϩ cell lines were transfected with an HLA-B35 expressing plasmid by Assessment of T Cell Responses. CD4 and CD8 cells were enriched electroporation (Amaxa). mDCs, COS-7 cells, or tumor cell lines from PBMCs by magnetic cell sorting using miniMACS (Miltenyi (105 per well) were incubated with the enriched T cell populations Biotec Inc., Bergisch Gladbach, Germany) and stimulated with (2 ϫ 104 per well) for 24 h, and IFN-␥ concentration was measured irradiated autologous APCs in the presence of the NY-ESO-1 in the culture supernatant by using ELISA as described in ref. 14. peptide pool (each 2 ␮M; NeoMPS Inc., San Diego, CA), rhIL-2 (10 IU/ml), and rhIL-7 (10 ng/ml). At day 8, cultures were tested for We thank Rose-Marie Holman and Sean Lemoine for regulatory and data ␥ intracellular IFN- secretion after stimulation, during 4 h, and in management support. We are grateful to Coley Pharmaceutical Group the absence or presence of the peptide pool or of individual (Wellesley, MA) for providing the CpG 7909. This study was supported by peptides (14, 15). For ex vivo assessments, cryopreserved total the Cancer Vaccine Collaborative program of the Ludwig Institute for PBMCs were thawed, rested overnight, and stimulated for7hinthe Cancer Research and the Cancer Research Institute (New York, NY).

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