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¶储, 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)
cancer vaccine
A
key step in the development of generic cancer vaccines is the implementation of vaccination strategies allowing for the consistent induction of immune responses to tumor antigens. In this respect, the choice of appropriate antigens, based on both the frequency and the specificity of their expression in cancer tissues, is of paramount importance. The group of cancer/testis antigens (CTA) (1, 2), including the NY-ESO-1 antigen (3), is emerging among the most promising candidates. Because many CTA are not expressed on the surface of cancer cells but rather intracellularly, it is important that vaccination induces specific CD8⫹ T cells able to directly recognize antigen-expressing tumor cells. Recombinant proteins can be produced in large scale and at relatively low cost, are commonly used in the development of antiviral vaccines, and are therefore attractive candidate antitumor vaccines. The potential of tumor antigen recombinant protein vaccines, however, relies on their ability not only to elicit antibody (Ab) and CD4⫹ T cell responses but also to efficiently prime naive CD8⫹ T cells through cross-priming (4), which generally is inefficient during spontaneous immune responses to tumor antigens (5). Professional antigen-presenting cells (APCs) detect pathogens through a variety of receptors such as the Toll-like receptors (TLR), which recognize pathogen-associated molecular patterns including CpG dinucleotides within defined flanking sequences (CpG ODN) (6). Synthetic CpG ODN able to trigger TLR9 are potent vaccine adjuvants, stimulating T www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703395104
helper type 1 (Th1)-type immunity (7). In humans, they can directly activate B lymphocytes and plasmacytoid dendritic cells and also indirectly activate myeloid dendritic cells (mDCs), increasing antigen cross-presentation and stimulating adaptive immune responses (8–10). In this study, we have assessed the immune response elicited by repeated vaccination with a NY-ESO-1 recombinant protein (rNY-ESO-1) administered with CpG 7909 in a water–oil emulsion with Montanide ISA-51. We show that cancer patients receiving this vaccine developed integrated Ab and CD4⫹ T cell responses to NY-ESO-1 at an early phase of the vaccination protocol. A fraction of the patients also developed specific CD8⫹ T cell responses at a later time point. Assessment of the correlation between the development of Ab and T cell responses suggested that the presence of sufficient levels of NY-ESO-1specific antibodies was determinant for the cross-priming of CD8⫹ T cells to occur in vivo. In line with this concept, we found that in vitro cross-presentation of NY-ESO-1 protein to vaccineinduced CD8⫹ T cells by dendritic cells was enhanced by vaccine-induced Ab. Results Serological Response to Vaccination with rNY-ESO-1, CpG 7909, and Montanide ISA-51. Study patients received four s.c. injections of
NY-ESO-1/Montanide/CpG vaccine at 3-week intervals. The study included two arms receiving 100 or 400 g of rNY-ESO-1 per injection, together with 2.5 mg of CpG emulsified in Montanide. CpG 7909 belongs to the CpG-B class, which potently stimulates B cells (11). All patients developed significant serological responses to NY-ESO-1 (Fig. 1A). Specific IgG responses became significant after the second injection and further increased after the third and fourth injections, without generally reaching a plateau. Specific IgG titers were variable among patients: at the last time point, NY-ESO-1 reciprocal Ab titers had reached an average 42.429 ⫾ 29.080 in the 400 g group and 24.091 ⫾ 10.737 in the 100 g group. Serological responses to an Author contributions: D.V., L.J.O., and M.A. designed research; D.V., N.E.S., V.T., N.B., S.A., 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., E.W.H., R.V., G.R., and L.J.O. contributed new reagents/analytic tools; D.V. and M.A. analyzed data; and D.V. and M.A. wrote the paper. The authors declare no conflict of interest. Abbreviations: CTL, cytotoxic T lymphocyte; APC, antigen-presenting cell; TLR, Toll-like receptors; Th, T helper; mDC, myeloid dendritic cell; PBMC, peripheral blood mononuclear cell; ADCC, antibody-dependent cellular cytotoxicity. †To
whom correspondence may be sent at the present address: Institut National de la Sante´ et de la Recherche Me´dicale, Unite 601, Centre de Lutte Contre le Cancer Rene´ Gauducheau, Boulevard Jacques Monod, 44800 Saint Herblain, France. E-mail:
[email protected] or
[email protected].
储To
whom correspondence may be addressed. E-mail:
[email protected].
© 2007 by The National Academy of Sciences of the USA
PNAS 兩 May 22, 2007 兩 vol. 104 兩 no. 21 兩 8947– 8952
IMMUNOLOGY
The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8ⴙ T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4ⴙ Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8ⴙ T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8ⴙ T cells, indicated that elicitation of NY-ESO-1-specific CD8ⴙ T cell responses by crosspriming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.
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 epitopes were determined by using patients’ immune sera and a panel of 30-aa-long peptides spanning the protein sequence.
unrelated recombinant protein (MAGE-A4) generally were low in both groups (4.957 ⫾ 3.594 in the group receiving 400 g and 2.735 ⫾ 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 vaccine, we assessed the isotype of vaccine-induced IgG by using isotype-specific antibodies. As illustrated in Fig. 1B, the vaccine preferentially induced specific IgG of types 1 and 3, which are associated, in humans, with type 1 immune responses. We mapped the linear B cell epitopes recognized by the vaccineinduced Ab with a panel of peptides spanning the protein sequence (12). The analysis identified a dominant region located in the central part of the protein and comprising amino acids 80– 110, which was recognized strongly by Abs from all patients (Fig. 1C). Consistent with the previous identification of B cell epitopes located in the N-terminal half of the protein and recognized by specific Abs from cancer patients with spontaneous serological responses to NY-ESO-1 (12, 13), the immune sera from vaccinated patients reacted to peptides in the amino acids 1–70 region of the protein. A lower level of reactivity was detected toward sequences located in the C-terminal half of the protein, between amino acids 160 and 180. Assessment of T Cell Responses After in Vitro Stimulation. We
initially assessed the induction of specific T cells after in vitro stimulation of pre- and postimmune samples with a pool of overlapping peptides spanning the protein sequence, followed by quantification of specific IFN-␥-producing cells by intracellular staining (14, 15) (Fig. 2A). The response was considered significant if the frequency of T cells detected in at least one postvaccine sample exceeded by 3-fold that found in the baseline sample. CD4⫹ T cell responses were detectable in 17 of 18 patients and developed early, in general becoming detectable already at week 4 (Fig. 2B). CD8⫹ T cell responses were detectable in 9 of 18 patients (4/7 in the 400 g group and 5/11 in the 100 g group) and generally developed later. We used stimulated cultures to map the protein regions recognized by CD4⫹ and CD8⫹ T cells, by using single peptides in the NY-ESO-1 pool (Fig. 2C). Similar to what was found previously in patients with spontaneous responses (14–16), the majority of 8948 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703395104
Fig. 2. Assessment of CD4⫹ and CD8⫹ T cell responses after in vitro stimulation. (A) The presence of specific CD4⫹ and CD8⫹ T cells in stimulated cultures from vaccinated patients was assessed by intracellular staining with anti-IFN-␥ 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 IFN-␥-producing cells in CD4⫹ or CD8⫹ T cells. As an example, data are shown for one responder patient. (B) Summary of the results obtained for all patients. Values correspond to the percentage of CD4⫹ or CD8⫹ T cells specifically producing IFN-␥ in response to stimulation with the peptide pool. (C) Cultures were stimulated with single peptides in the NY-ESO-1 pool. The proportion of IFN-␥-producing cells was assessed by intracellular staining as above. Symbols are as given in the Fig. 1 key.
CD4⫹ T cells recognized sequences located in two distinct regions of the protein, corresponding to peptides 81–100 and 119–143. We found minor reactivity toward some other peptides, including 151–170. The sequences recognized by CD8⫹ T cells mostly were located between amino acids 81 and 110, as indicated by the reactivity of the majority of these cells to the overlapping peptides 81–100 and 91–110. The relative reactivity to these two peptides varied among patients, indicating the presence of multiple epitopes in this region, consistent with previous results in patients with spontaneous responses as well as in patients immunized with other NY-ESO-1 full-length vaccines (15, 17, 18). Ex Vivo Assessment of T Cell Responses. Next, total pre- and
postimmune peripheral blood mononuclear cell (PBMC) samples were stimulated directly ex vivo with the NY-ESO-1 peptide pool and assessed for the presence of specific T cells by using intracellular cytokine staining together with cell-surface staining to assess phenotype. NY-ESO-1-specific CD4⫹ and CD8⫹ T cells were detectable ex vivo after vaccination but not before vaccination (Fig. 3A) We detected significantly increased levels of NY-ESO-1-specific T cells in postvaccination samples from 12 of the 18 patients in the case of CD4⫹ T cells and in 7 patients in Valmori et al.
the case of CD8⫹ T cells (Fig. 3B). It is noteworthy that 5 of 6 patients in whom CD4⫹ T cell responses were not detectable ex vivo were among nonresponders or low responders after in vitro stimulation. In addition, 6 of 7 patients with CD8⫹ T cell responses detectable ex vivo were among the 9 patients in whom CD8⫹ T cell responses were detectable after in vitro stimulation. Vaccine-induced CD4⫹ T cells mostly were composed of effector-memory (CD45RA⫺ CCR7⫺) and, to a lower extent, centralmemory (CD45RA⫺ CCR7⫹) 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. Vaccineinduced CD8⫹ T cells also were composed mostly of effectormemory T cells, did not contain many central-memory T cells, and contained instead a significant proportion of terminally differentiated effectors (CD45RA⫹CCR7⫺). The cytokine profile of vaccine-induced CD4⫹ and CD8⫹ T cells was clearly of Th1 type, because we failed to detect any significant levels of IL-10-, IL-4-, or IL-17-secreting cells (data not shown). Recognition of NY-ESO-1 by Vaccine-Induced T Cells and Ab-Aided Cross-Presentation. To address the ability of vaccine-induced
CD4⫹ and CD8⫹ T cells to recognize exogenously and endog-
Valmori et al.
Fig. 4. Recognition of NY-ESO-1 by vaccine-induced T cells and Ab-aided cross-presentation. (A) Vaccine-induced CD4⫹ and CD8⫹ T cell populations enriched by cytokine-secretion-guided sorting were assessed by intracellular IFN-␥ staining after stimulation in the absence or presence of the NY-ESO-1 peptide pool. (B) Recognition of autologous mDCs by CD4⫹ and CD8⫹ T cells was assessed 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 CD8⫹ T cells was determined upon transfection of COS-7 cells with plasmids encoding the alleles of the patient. Recognition of endogenous NYESO-1 was assessed upon cotransfection of COS-7 cells with plasmids encoding NY-ESO-1 and HLA-B35 or by transfecting the tumor cell lines NA8-MEL (NY-ESO1⫺) and HT1080 (NY-ESO-1⫹) with a plasmid encoding HLA-B35. (D) Crosspresentation, by autologous mDCs, of rNY-ESO-1 (10 g/ml) or immune complexes formed either with a murine NY-ESO-1-specific monoclonal Ab (ES121, 10 g/ml) or with pre- and postimmune serum at the indicated dilution to vaccineinduced CD8⫹ T cells. In B and C, antigen recognition was assessed by measurement of IFN-␥ secretion in the culture supernatant.
enously processed NY-ESO-1 antigen, we isolated them from peptide-stimulated cultures by IFN-␥-guided cell sorting (Fig. 4A). We used the isolated populations to assess the recognition of autologous mDCs incubated with the rNY-ESO-1 or electroporated with a plasmid encoding NY-ESO-1 (Fig. 4B). The rNY-ESO-1 was recognized efficiently by CD4⫹ T cells but poorly by CD8⫹ T cells. In contrast, CD8⫹ T cells but not CD4⫹ T cells recognized the endogenous antigen. These results suggest that, in this setting, autologous professional APCs are likely to initiate the immune response by priming CD4⫹ Th cells after processing and presentation of the rNY-ESO-1 protein. To address the ability of CD8⫹ T cells to recognize tumor cells, we PNAS 兩 May 22, 2007 兩 vol. 104 兩 no. 21 兩 8949
IMMUNOLOGY
Fig. 3. Ex vivo assessment of CD4⫹ and CD8⫹ T cell responses. (A) Dot plots gated on CD3⫹ cells are shown for a high-responder patient. Numbers in the upper right quadrant are the percentages of IFN-␥-secreting cells among CD4⫹ T cells (Upper) and CD8⫹ 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 their standard deviation. The baseline sample from patient N4, who had detectable NY-ESO-1-specific CD8⫹ T cells before vaccination as shown in Fig. 2 and Table 1, was excluded from this calculation. Frequencies of IFN-␥secreting cells above this value were considered significant. (C) Phenotype and cytokine production of vaccine-induced CD4⫹ and CD8⫹ T cells.
Table 1. Patient characteristics, antigen dose, summary of vaccine-related toxicity, and vaccine-induced immune responses
Patient N14 N11 C3 C4 N10 C7 N13 N5 C1 N4 C5 N2 N7 N9 N3 C6 N8 C2
Disease*
Stage
Before study status†
Melanoma Melanoma Breast cancer Breast cancer Melanoma Melanoma Melanoma Melanoma Sarcoma Melanoma Sarcoma Melanoma Melanoma Ovarian cancer Melanoma Sarcoma Melanoma Breast cancer
IIB IIIB I IIA IVA III IIIA IVB II IVA III IIIB IIIB IIIC IIIB IIIB IIC IIA
NED NED NED NED NED NED NED NED NED NED M (lung) NED NED NED NED NED NED NED
Ab§
CD4¶
CD8
Antigen dose, g
Toxicity‡ (grade)
Before
After
Before
After
Before
After
100 100 100 100 400 100 400 400 400 100 100 100 100 400 100 400 100 400
L(1), S(1) L(1), S(1) L(2) L(1), S(1) L(1) L(1), S(1) L(1), S(1) L(1) L(2) L(1), S(1) L(2), S(2) L(1), S(1) L(1), S(1) L(1), S(1) L(1), S(1) L(1), S(1) L(1), S(1) L(2), S(2)
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹⫹ ⫺ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
*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-1⫹. Patients C3, C4, C5, C6, N2, N3, N7, and N13 were NY-ESO-1⫺. †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): ⫺, ⱕ100; ⫹, ⬎100 to 20,000; ⫹⫹, ⬎20,000 to 40,000; ⫹⫹⫹, ⬎40,000. ¶CD4 and CD8 (percentage of IFN-␥⫹ cells after in vitro stimulation): ⫺, ⱕ0.3%; ⫹, ⬎0.3% to 1%; ⫹⫹, ⬎1% to 10%; ⫹⫹⫹, ⬎10%.
identified the MHC class I allele restricting antigen recognition of CD8⫹ T cells from one responder patient and assessed its ability to recognize antigen-expressing tumor cells transfected with the appropriate allele. Transfection of COS-7 cells with an HLA-B35-encoding plasmid, but not with plasmids encoding other patient’s HLA alleles, resulted in peptide presentation to CD8⫹ T cells (Fig. 4C). In addition, both COS-7 cells cotransfected with NY-ESO-1 and HLA-B35 and NY-ESO-1expressing tumor cells transfected with HLA-B35 were recognized specifically by CD8⫹ T cells. Enhancement of Antigen Cross-Presentation by Vaccine-Induced Ab.
The data above provided evidence that vaccination resulted in the consistent induction of CD4⫹ T cell and Ab responses in most patients, as well as of specific CD8⫹ T cells in a fraction of them (summarized in Table 1). The in vivo kinetic of the responses clearly showed that CD4⫹ T cells and Abs developed early, whereas CD8⫹ T cells generally developed later. In addition, the development of CD8⫹ T cell responses generally correlated with medium to high titers of NY-ESO-1 Ab. This, together with the relatively poor recognition of the rNY-ESO-1 alone after crosspresentation by dendritic cells, suggested an involvement of NY-ESO-1 Ab in the in vivo cross-priming of CD8⫹ T cells, through formation of immune complexes. To address this point, mDCs from a responder patient were pulsed with the NY-ESO-1 recombinant protein for 2 h and then assessed for antigen recognition by autologous CD8⫹ T cells. Under these conditions, no significant cross-presentation was detected (Fig. 4D). However, dendritic cell incubation with immune complexes generated by mixing NY-ESO-1 recombinant protein with the NYESO-1-specific monoclonal Ab ES121 resulted in efficient antigen cross-presentation. In addition, preincubation of NYESO-1 protein with postvaccine serum but not with prevaccine serum, at various dilutions, also resulted in efficient antigen cross-presentation. Together, these results are in line with the concept that the induction of NY-ESO-1-specific Ab by the 8950 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0703395104
vaccine formulation used in this study may be a determining factor for the induction of CD8⫹ T cells, through Ab-aided cross-priming in vivo. Discussion NY-ESO-1 is a nonmutated self-antigen frequently expressed in human tumors that often induces spontaneous immune responses in patients bearing antigen-expressing tumors. We have selected NY-ESO-1 as a prototype tumor antigen for the development of generic cancer vaccines and, under the sponsorship of the Cancer Vaccine Collaborative (www.cancerresearch.org), are assessing the immunogenicity of NY-ESO-1-based candidate vaccines in earlyphase clinical trials. Among the immunogens tested is the rNYESO-1 protein, in various formulations (17, 18). In this study, we have obtained direct evidence that immunization with rNY-ESO-1 administered with Montanide and CpG induces integrated Ab and CD4⫹ T cell responses in the majority of vaccinated patients and cross-primes CD8⫹ T cells in a significant fraction of them. Although NY-ESO-1 expression in tumor specimens could not be assessed for all patients, we failed to detect any correlation between antigen expression and responsiveness to vaccination (Table 1). It is noteworthy that, with the exception of one patient, no significant serological reactivity to NY-ESO-1 was detectable before vaccination, indicating the ability of the vaccine to prime specific naı¨ve B cells. This finding is in line with the recent proposal by Ruprecht et al. that triggering of TLR9 by CpG on human naı¨ve B cells acts as a third signal, required, in addition to B cell receptor stimulation and CD4⫹ T cell help, for their full differentiation into IgGsecreting cells (20). Montanide may contribute significantly to the immunogenicity of the formulation, through its known effects, such as depot effect, slow antigen release, and recruitment of APCs at the injection site. In support of this data, in a separate study, administration of NY-ESO-1 protein and CpG without Montanide was less immunogenic (E. Jager, personal communication). Administration of CpG in combination with Montanide, however, is likely to be responsible for the high Ab titers detected in this study Valmori et al.
Valmori et al.
support of this, postimmune sera from vaccinated patients were able to promote efficiently cross-presentation of NY-ESO-1 to vaccine-induced CD8⫹ T cells in vitro. In addition to their titers, the induction of type 1 IgG Ab is likely of importance. Fc␥R is a family of glycosylated membrane proteins that includes members that differ largely with respect to their capacity to bind IgG (30, 31). For example, Fc␥RI (CD64) binds monomeric IgG with high affinity, but it signals only if the latter is cross-linked by specific polymeric ligands. In contrast, Fc␥RII (CD32) binds monomeric IgG poorly but binds immune complexes with very high affinity. IgG3 and IgG1 preferentially interact with Fc␥RIIa, which is a potent leukocyte activator and can stimulate the release of high levels of inflammatory cytokines. Thus, the Ab isotypes induced by our vaccine, in particular IgG3, may be effective at inducing dendritic cell activation and maturation, rapid internalization, concentration, and transport of the antigen to the appropriate endosomal/lysosomal compartments, ultimately enhancing the induction of T cells. As in the case of NY-ESO-1, specific Ab responses to tumorassociated antigens often develop during spontaneous immune responses to tumors. The biological role of these Abs in the development of the disease, however, is poorly understood. The mechanism of action in vivo of some antitumor Abs currently used for the treatment of breast cancer (anti-Her2/neu) or B cell lymphomas (anti-CD20) remains unclear but generally is considered to be through ADCC (32). Possibly complementary to ADCC, Ab-aided cross-priming of CTLs specific for tumor antigens also could contribute to the antitumor effect of these therapies. In a murine model of melanoma, passive transfer of Abs against the melanoma antigen gp75 has been shown to be highly effective in inhibiting tumor growth (33) in a Fc␥R-dependent manner (34). In a different mouse model, using EL-4 lymphoma cells transfected with the model antigen OVA, Dyall et al. (35) have provided further evidence for Fc␥R-mediated Ab-dependent tumor eradication and shown that CD8⫹ T cells are the critical effector population in this system. Additional evidence for the role of antitumor Abs in promoting cross-priming of tumor-specific CTLs has been provided by an in vitro study with human cells in which Dhodapkar and colleagues (36) demonstrated that coating myeloma cells with anti-syndecan-1 Abs strongly enhanced cross-presentation and cross-priming of CTLs specific for the NY-ESO-1 and MAGE-A3 antigens. Adding to these previous findings, our data indicate that Ab-aided cross-priming of NY-ESO-1-specific CTLs, and possibly of other human tumor antigens, could be achieved through active immunization with recombinant proteins. Along with Abs, other factors are likely to contribute, both directly and indirectly, to promote in vivo cross-priming after vaccination with the formulation used in our study. Namely, although class B CpG may not be optimal for inducing the production of IFN-␣, the latter still could play a significant role by both stimulation of B cell activating factor (BAFF) (37) and activation of mDCs (38). Also, vaccine-induced CD4⫹ T cells could play a role in the activation of mDCs through CD40–CD40L interactions (28). In conclusion, the vaccine formulation used in this study seems to be highly immunogenic and able to stimulate integrated Ab and T cell responses, including tumor-reactive CD8⫹ T cells, by crosspriming in an Ab-aided fashion. On the other hand, the relatively delayed development of specific CD8⫹ T cell responses requiring repeated vaccine injections, along with their detection in only a fraction of vaccinated patients, suggests that additional injections, a combination of Montanide/CpG with other TLR ligands, or the use of prime/boost strategies alternating different NY-ESO-1based immunogens may be required to optimize their induction. Based on our data, administration of NY-ESO-1-specific Abs before or concomitant with vaccination or administration of preformed activating Ab–protein immune complexes in combination with adjuvant also may be promising approaches for the development of cancer vaccines of increasing efficacy. PNAS 兩 May 22, 2007 兩 vol. 104 兩 no. 21 兩 8951
IMMUNOLOGY
and to the skewing of the induced immunological response toward a Th1 type, attributable to the induction of proinflammatory cytokines and type 1 IFNs, in contrast to incomplete Freund’s adjuvant alone (equivalent to Montanide), which induced a Th2type bias (11, 21). Consistent with this concept, vaccine-induced IgG predominantly were IgG1 and IgG3, the most prominent IgG type 1 human subclasses, involved in functions such as complement activation, mediation of antibody-dependent cellular cytotoxicity (ADCC), and opsonization (22). After one round of in vitro stimulation, vaccine-induced CD4⫹ T cell responses were detectable in most vaccinated patients, and CD8⫹ T cell responses were detectable in a fraction of them. In most cases, their reactivity was restricted to one or few peptides in the protein, corresponding to immunodominant regions identified in our previous studies (14, 15). The immunodominant region recognized by cross-primed CD8⫹ T cells (between amino acids 81 and 110) was one of the two main regions also recognized by CD4⫹ T cells and the most efficiently recognized by Ab. We recently have reported the identification of overlapping clusters of MHC class I and class II binding sequences in immunodominant regions of NY-ESO-1 that contain a high density of proteasomal cleavage sites (14, 15). This interesting phenomenon provides the molecular bases for the development of integrated Ab, Th, and cytotoxic T lymphocyte (CTL) responses around short immunodominant regions. Based on these findings, we have proposed that CTL epitopes in the NY-ESO-1 immunodominant 81–110 region may be generated efficiently through cross-priming, a hypothesis now supported by the findings of the present study. In addition, the overlapping between regions concentrating B and T cell immunodominant epitopes, suggests a role for Ab-mediated epitope protection during antigen processing. Both vaccine-induced CD4⫹ and CD8⫹ T cell populations exhibited ex vivo a clear Th1 profile and contained polyfunctional populations producing IL-2 and/or IFN-␥. They mostly exhibited an effector-memory phenotype, although CD4⫹ T cell populations contained detectable proportions of central memory cells and CD8⫹ T cell populations of so-called terminally differentiated effectors. The ex vivo frequencies of vaccine-induced NY-ESO-1specific T cells were variable among patients, and, with the exception of one high-responder patient, they remained relatively low in most patients. The ex vivo frequency of CD4⫹ T cells detected in our study is in the range of those recently reported after vaccination of nonhuman primates with HIV Gag protein, Montanide, and CpG ODN (C class) (23). Interestingly, in the study from Wille-Reece et al. (23), TLR7/8 agonists stimulated higher responses than CpG ODN. In addition, in their study, further immunization with Gag-encoding recombinant adenovirus resulted in a high increase in the frequency of CD8⫹ T cells, suggesting that prime/boost regimens alternating rNY-ESO-1 and NY-ESO-1-encoding recombinant viruses (currently in development as part of the Cancer Vaccine Collaborative program) (18) could lead to an optimal expansion of specific CD8⫹ T cells. The role of CpG in the induction of primary and memory T cell responses has not been elucidated completely. Based on in vitro experiments, Rothenfusser et al. (24) have proposed that class A CpG could selectively enhance memory CD8⫹ T cell responses and cytotoxicity, whereas class B CpG could be superior for priming CD8⫹ T cells. In support of this data, in a recent study, Speiser et al. (25) reported evidence of induction of CD8⫹ T cell responses in melanoma patients, including some with no detectable preimmune responses, by vaccination with a Melan-A peptide analog, Montanide, and CpG. In our study, priming of CD4⫹ T cells occurred at an early phase of the vaccination protocol, in contrast to CD8⫹ T cell responses that became detectable only at a later stage. This delayed appearance, which occurred concomitant with the titer of NY-ESO-1-specific Abs reaching high levels in responder patients, suggested a role for vaccine-induced Abs in promoting CD8⫹ T cell cross-priming through Ab-aided cross-presentation (26–29). In
Materials and Methods Clinical Study and Patient Population. The study protocol was
approved by the Institutional Review Boards of Columbia University and New York University Medical Centers. Patient samples were obtained by means of written informed consent. The patients enrolled had histological diagnoses of cancer types known to express NY-ESO-1. However, demonstration of expression of NY-ESO-1 in the patient’s tumor specimen was not an inclusion criterion. The primary end point of the study was to determine the safety and immunogenicity of the vaccine. Toxicity and adverse events were graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events scale (version 3.0; accessed December 12, 2003). Assessment of Serological Responses. Serological responses were assessed by ELISA using NY-ESO-1 or MAGE-A4 recombinant proteins produced in Escherichia coli (12, 39). Sera were assessed over a range of dilutions from 1/100 to 1/100,000. Titers were calculated as the serum dilution giving 50% of maximal optical density obtained by using a standard positive serum. IgG isotypes were determined by ELISA using the same protocol and antibodies specific for IgG1 (BD Biosciences, San Jose, CA) and IgG2, IgG3, and IgG4 (Southern Biotech, Birmingham, AL). Assessment of T Cell Responses. CD4⫹ and CD8⫹ cells were enriched from PBMCs by magnetic cell sorting using miniMACS (Miltenyi Biotec Inc., Bergisch Gladbach, Germany) and stimulated with irradiated autologous APCs in the presence of the NY-ESO-1 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 intracellular IFN-␥ secretion after stimulation, during 4 h, and in the absence or presence of the peptide pool or of individual peptides (14, 15). For ex vivo assessments, cryopreserved total PBMCs were thawed, rested overnight, and stimulated for 7 h in the 1. Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT (2002) Immunol Rev 188:22–32. 2. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ (2005) Nat Rev Cancer 5:615–625. 3. Chen YT, Scanlan MJ, Sahin U, Tureci O, Gure AO, Tsang SL, Williamson B, Stockert E, Pfreundschuh M, Old LJ (1997) Proc Natl Acad Sci USA 94:1914–1918. 4. Carbone FR, Kurts C, Bennett SR, Miller JF, Heath WR (1998) Immunol Today 19:368–373. 5. Ochsenbein AF, Sierro S, Odermatt B, Pericin M, Karrer U, Hermans J, Hemmi S, Hengartner H, Zinkernagel RM (2001) Nature 411:1058–1064. 6. Krieg AM (2003) Nat Med 9:831–835. 7. Chu RS, Targoni OS, Krieg AM, Lehmann PV, Harding CV (1997) J Exp Med 186:1623–1631. 8. Maurer T, Heit A, Hochrein H, Ampenberger F, O’Keeffe M, Bauer S, Lipford GB, Vabulas RM, Wagner H (2002) Eur J Immunol 32:2356–2364. 9. Heit A, Huster KM, Schmitz F, Schiemann M, Busch DH, Wagner H (2004) J Immunol 172:1501–1507. 10. Shinohara ML, Lu L, Bu J, Werneck MB, Kobayashi KS, Glimcher LH, Cantor H (2006) Nat Immunol 7:498–506. 11. Krieg AM (2002) Annu Rev Immunol 20:709–760. 12. Valmori D, Souleimanian NE, Hesdorffer CS, Ritter G, Old LJ, Ayyoub M (2005) Clin Immunol 117:24–30. 13. Zeng G, Aldridge ME, Wang Y, Pantuck AJ, Wang AY, Liu YX, Han Y, Yuan YH, Robbins PF, Dubinett SM, et al. (2005) Int J Cancer 114:268–273. 14. Ayyoub M, Souleimanian NE, Godefroy E, Scotto L, Hesdorffer CS, Old LJ, Valmori D (2006) Clin Immunol 118:188–194. 15. Valmori D, Levy F, Godefroy E, Scotto L, Souleimanian NE, Karbach J, Tosello V, Hesdorffer CS, Old LJ, Jager E, et al. (2007) Clin Immunol 122:163–172. 16. Zarour HM, Maillere B, Brusic V, Coval K, Williams E, Pouvelle-Moratille S, Castelli F, Land S, Bennouna J, Logan T, et al. (2002) Cancer Res 62:213–218. 17. Davis ID, Chen W, Jackson H, Parente P, Shackleton M, Hopkins W, Chen Q, Dimopoulos N, Luke T, Murphy R, et al. (2004) Proc Natl Acad Sci USA 101:10697–10702. 18. Jager E, Karbach J, Gnjatic S, Neumann A, Bender A, Valmori D, Ayyoub M, Ritter E, Ritter G, Jager D, et al. (2006) Proc Natl Acad Sci USA 103:14453–14458. 19. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A (1999) Nature 401:708–712.
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absence or presence of the NY-ESO-1 peptide pool. Brefeldin A was added 2 h after the beginning of the incubation. At the end of the incubation, cells were stained with antibodies directed against surface markers (CD3, CD4, CD8, CD45RA, and CCR7), fixed, permeabilized, and stained with cytokine-specific antibodies. Assessment of Antigen Recognition by Specific T Cells. NY-ESO-1specific T cells were isolated from peptide-stimulated cultures by cytokine-guided magnetic cell sorting (Miltenyi Biotec Inc.), stimulated as polyclonal cultures in the presence of phytohemagglutinin, allogeneic irradiated PBMCs, and rhIL-2 (100 units/ml), and used in antigen recognition assays. mDCs were obtained by culture of isolated CD14⫹ cells during 5 days in the presence of rhIL-4 and rhGM-CSF. mDCs were either incubated overnight in the presence of the NY-ESO-1 protein and washed before use in recognition assays or incubated for 2 h in the presence of the rNY-ESO-1 or NY-ESO-1 immune complexes, washed, cultured overnight, and used in recognition assays. Where indicated, mDCs were transfected with a NY-ESO-1-expressing plasmid by electroporation (Amaxa, Gaithersburg, MD). COS-7 cells were transiently transfected with the NY-ESO-1 encoding plasmid and/or the appropriate HLA class I expressing plasmids by using FuGENE (Roche Diagnostics, Basel, Switzerland). NA8-MEL and HT1080 tumor cell lines were transfected with an HLA-B35 expressing plasmid by electroporation (Amaxa). mDCs, COS-7 cells, or tumor cell lines (105 per well) were incubated with the enriched T cell populations (2 ⫻ 104 per well) for 24 h, and IFN-␥ concentration was measured in the culture supernatant by using ELISA as described in ref. 14. We thank Rose-Marie Holman and Sean Lemoine for regulatory and data management support. We are grateful to Coley Pharmaceutical Group (Wellesley, MA) for providing the CpG 7909. This study was supported by the Cancer Vaccine Collaborative program of the Ludwig Institute for Cancer Research and the Cancer Research Institute (New York, NY). 20. Ruprecht CR, Lanzavecchia A (2006) Eur J Immunol 36:810–816. 21. Kumar S, Jones TR, Oakley MS, Zheng H, Kuppusamy SP, Taye A, Krieg AM, Stowers AW, Kaslow DC, Hoffman SL (2004) Infect Immun 72:949–957. 22. Papadea C, Check IJ (1989) Crit Rev Clin Lab Sci 27:27–58. 23. Wille-Reece U, Flynn BJ, Lore K, Koup RA, Miles AP, Saul A, Kedl RM, Mattapallil JJ, Weiss WR, Roederer M, et al. (2006) J Exp Med 203:1249–1258. 24. Rothenfusser S, Hornung V, Ayyoub M, Britsch S, Towarowski A, Krug A, Sarris A, Lubenow N, Speiser D, Endres S, et al. (2004) Blood 103:2162–2169. 25. Speiser DE, Lienard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F, Krieg AM, Cerottini JC, Romero P (2005) J Clin Invest 115:739–746. 26. Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P, et al. (1999) J Exp Med 189:371–380. 27. Nagata Y, Ono S, Matsuo M, Gnjatic S, Valmori D, Ritter G, Garrett W, Old LJ, Mellman I, You Z, et al. (2002) Proc Natl Acad Sci USA 99:10629–10634. 28. Schnurr M, Chen Q, Shin A, Chen W, Toy T, Jenderek C, Green S, Miloradovic L, Drane D, Davis ID, et al. (2005) Blood 105:2465–2472. 29. Matsuo M, Nagata Y, Sato E, Atanackovic D, Valmori D, Chen YT, Ritter G, Mellman I, Old LJ, Gnjatic S (2004) Proc Natl Acad Sci USA 101:14467–14472. 30. Hulett MD, Hogarth PM (1994) Adv Immunol 57:1–127. 31. van de Winkel JG, Capel PJ (1993) Immunol Today 14:215–221. 32. Houghton AN, Scheinberg DA (2000) Nat Med 6:373–374. 33. Hara I, Takechi Y, Houghton AN (1995) J Exp Med 182:1609–1614. 34. Clynes R, Takechi Y, Moroi Y, Houghton A, Ravetch JV (1998) Proc Natl Acad Sci USA 95:652–656. 35. Dyall R, Vasovic LV, Clynes RA, Nikolic-Zugic J (1999) Eur J Immunol 29:30–37. 36. Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV (2002) J Exp Med 195:125–133. 37. Ittah M, Miceli-Richard C, Gottenberg JE, Lavie F, Lazure T, Ba N, Sellam J, Lepajolec C, Mariette X (2006) Arthritis Res Ther 8:R51. 38. Hartmann E, Wollenberg B, Rothenfusser S, Wagner M, Wellisch D, Mack B, Giese T, Gires O, Endres S, Hartmann G (2003) Cancer Res 63:6478–6487. 39. Stockert E, Jager E, Chen YT, Scanlan MJ, Gout I, Karbach J, Arand M, Knuth A, Old LJ (1998) J Exp Med 187:1349–1354.
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