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Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with a better prognosis in high-grade serous ovarian cancer (HGSC). However, it is largely unknown how this prognostic benefit of TIL relates to current standard treatment of surgical resection and (neo-)adjuvant chemotherapy. To address this outstanding issue, we compared TIL infiltration in a unique cohort of patients with advanced-stage HGSC primarily treated with either surgery or neoadjuvant chemotherapy. Experimental Design: Tissue microarray slides containing samples of 171 patients were analyzed for CD8+ TIL by IHC. Freshly isolated CD8+ TIL subsets were characterized by flow cytometry based on differentiation, activation, and exhaustion markers. Relevant T-cell subsets (CD27+) were validated using IHC and immunofluorescence. Results: A prognostic benefit for patients with high intratumoral CD8+ TIL was observed if primary surgery had resulted in a complete cytoreduction (no residual tissue). By contrast, optimal ( Conclusions: Our findings indicate that treatment regimen, surgical result, and the differentiation of TIL should all be taken into account when studying immune factors in HGSC or, by extension, selecting patients for immunotherapy trials. Clin Cancer Res; 22(3); 714–24. ©2015 AACR.

Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L) covalently bound to antigenic synthetic long peptides (SLP) are attractive vaccine modalities, which can induce robust CD8+ T-cell immune responses. Previously, we have shown that the mechanism underlying the power of TLR-L SLP conjugates is improved delivery of the antigen together with a dendritic cell activation signal. In the present study, we have expanded the approach to tumor-specific CD4+ as well as CD8+ T-cell responses and in vivo studies in two nonrelated aggressive tumor models. We show that TLR2-L SLP conjugates have superior mouse CD8+ and CD4+ T-cell priming capacity compared with free SLPs injected together with a free TLR2-L. Vaccination with TLR2-L SLP conjugates leads to efficient induction of antitumor immunity in mice challenged with aggressive transplantable melanoma or lymphoma. Our data indicate that TLR2-L SLP conjugates are suitable to promote integrated antigen-specific CD8+ and CD4+ T-cell responses required for the antitumor effects. Collectively, these data show that TLR2-L SLP conjugates are promising synthetic vaccine candidates for active immunotherapy against cancer. Cancer Immunol Res; 2(8); 756–64. ©2014 AACR.

The therapeutic synthetic long peptide (SLP) vaccine ISA101 selectively enlarges the pool of tumor-specific T cells recognizing the human papillomavirus type 16 (HPV16) antigens E6 and E7. Vaccine monotherapy for HPV16-induced pre-malignant anogenital lesions is effective, inducing durable complete regressions in approximately 50% of treated patients. However, cancer treatment requires appropriate countermeasures to overcome the suppression of T-cell activation, expansion and effector function imposed by suppressive myeloid cell populations, regulatory T cells and co-inhibitory molecule expression. We recently showed that carboplatin/paclitaxel chemotherapy can normalize the abnormally high levels of immune suppressive myeloid cells, allowing the development of much stronger therapeutic ISA101-induced tumor immunity1. Here we show the effects of ISA101 vaccination during chemotherapy in 77 patients with advanced, recurrent or metastatic cervical cancer in a dose assessment study. Study design involved a single arm dose escalation study with four different ISA101 doses (20, 40, 100 and 300 µg per peptide) with or without immunomodulator pegylated interferon alpha (Pegitron), given concomitantly with standard of care chemotherapy (carboplatin, AUC 6; paclitaxel, 175 mg/m2). Objective regressions were observed in 43% of 72 evaluable patients. The depletion of myeloid suppressive cells by carboplatin/paclitaxel was associated with detection of low level spontaneous HPV16-specific immunity in 26 of the 61 tested patients. Patients mounted type 1 T-cell responses to the vaccine across all doses. No significant differences were observed between the different doses and strength of vaccine-induced T cell responses; the results were therefore combined. Patients with an above median HPV16-specific T cell immune response to ISA101, measured by a validated IFNγ-Elispot, displayed a significantly prolonged median overall survival (OS) of 16.8 months compared to those with a lower HPV-specific immune response (OS 11.2 months, logrank p=0.012). Importantly, this effect was not due to differences in general immune status, as measured by T-cell reactivity to unrelated common microbial recall antigens. In conclusion, our study demonstrates that chemo-immunotherapy can be exploited to the benefit of patients with cervical cancer and warrants confirmation of the benefit of this type of chemo-immunotherapy in a randomized controlled study in HPV16-induced cancer patients. 1Welters, M. J. et al. Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses. Sci Transl Med 8, 334ra352, doi:10.1126/scitranslmed.aad8307 (2016). Citation Format: Cornelis J M Melief, Marij J. Welters, Ignace Vergote, Judith R. Kroep, Gemma G. Kenter, Nelleke Ottevanger, Wiebren A. Tjalma, Hannelore Denys, Mariette van Poelgeest, Hans W. Nijman, Anna K. Reyners, Thierry Velu, Frederic Goffin, Roy Lalisang, Willem-Jan Krebber, Leon Hooftman, Sonja Visscher, Brent A. Blumenstein, Richard B. Stead, Winald Gerritsen, Sjoerd van der Burg. A strong HPV-specific T-cell response after chemo-immunotherapy for advanced cervical cancer is associated with prolonged survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT002.

We have completed a chemo-immunotherapy study in 70 patients with late stage HPV16+ cervical cancer. Three HPV16-SLP vaccine doses, consisting of 13 overlapping peptides covering the amino acid sequence of the oncogenic proteins E6 and E7 of HPV16, were given 2 weeks after the second, third and fourth cycle of standard chemotherapy (carboplatin, AUC 6; paclitaxel 175 mg/ m2). The vaccine was administered subcutaneously as an emulsion in adjuvant Montanide ISA-51. Cohorts of 12 patients were vaccinated with each of 4 dose levels (20, 40, 100 and 300 µg/ per peptide) of 13 overlapping HPV16 synthetic long peptides (HPV16-SLP) together covering the E6 and E7 protein sequence. Two additional cohorts of 6 patients each were vaccinated with the most promising doses of 40 and 100 µg/ peptide. A marked and significant positive correlation was observed between the strength of the vaccine-induced T cell immune response and overall survival. No such correlation was observed between the strength of the T cell response against common recall antigens and survival. In addition a remarkably high proportion of patients survived beyond 2 years after the start of therapy. We also completed a combination study of this vaccine with checkpoint blocker nivolumab (anti-PD-1) in patients with incurable HPV16+ oropharyngeal cancer. Of 22 treated patients, 2 achieved a durable complete clinical response (CR) by resist criteria and 6 a partial response (PR). The overall response rate was 36%, approximately twice that reported for nivolumab as monotherapy in a similar category of patients with oropharyngeal cancer. This combination treatment was associated with robust HPV16 E6/E7-specific T cell responses. Citation Format: Cornelis J.M. Melief. Combination immunotherapy of cancer caused by human papilloma virus [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA26.

Purpose: Blockade of CTLA-4 by antibodies has potentiated antitumor T-cell responses in both preclinical models and clinical trials. However, treatment with CTLA-4 blocking antibodies is associated with autoimmune and inflammatory side effects. In this study, we propose a novel administration method for CTLA-4 blocking antibodies as monotherapy. Experimental Design: We use different preclinical mouse models of cancer to investigate the local administration of CTLA-4 blocking antibody and its effect on cancer progression and the antitumor T-cell response. Results: By injecting the antibodies in a subcutaneous slow-release delivery formulation in the tumor area, we show that an eight-fold lower dose of antibody is as effective in inducing tumor eradication as systemic delivery. A lower dose and slow release of the antibody results in thousand-fold decreased levels of antibody in the serum, reducing adverse events and the risk of autoimmunity. The main target and effector cells of the CTLA-4 blockade treatment in the studied tumor models are tumor-specific endogenous CD8+ T cells that are capable of eradicating also distant tumors, whereas CD4+ T cells do not play a prominent role in the antibody-mediated tumor eradication. Conclusions: Injecting CTLA-4 blocking antibody in a slow-release formulation close to the tumor is an effective way of activating the antitumor T-cell response. This administration method is associated with very low serum levels of antibody, which decreases the risk of treatment-induced side effects. These results call for exploration of a similar delivery principle in clinical settings. Clin Cancer Res; 19(19); 5381–9. ©2013 AACR.

Purpose: The tumor-associated self-antigen p53 is commonly overexpressed in cancer, including colorectal cancer, and can serve as a target for immunotherapy. The safety and immunogenicity of a p53 synthetic long peptide (p53-SLP) vaccine were investigated in patients treated for metastatic colorectal cancer. Experimental Design: Ten patients were vaccinated twice with a set of 10 overlapping p53-SLP in a phase I/II trial. Both the safety and the breadth, magnitude, and polarization of vaccine-induced p53-specific T cells was evaluated in blood samples drawn before and after vaccination by IFN-γ enzyme-linked immunospot, proliferation, cytokine secretion, and multiparameter flow cytometry. The migratory capacity of p53-specific T cells was evaluated by assessing their presence in a biopsy of the second vaccination site. Results: Toxicity was limited to grade 1/2, mostly at the vaccination site. p53-specific T-cell responses were induced in 9 of 10 colorectal cancer patients as measured by IFN-γ enzyme-linked immunospot, proliferation, and cytokine bead array. In 6 of 9 tested patients, p53-specific T-cell reactivity persisted at least 6 months. Furthermore, p53-specific T cells isolated from the vaccination site were characterized as CD4+ T cells producing both T-helper types 1 and 2 cytokines on stimulation with p53 peptide and p53 protein. Multiparameter flow cytometry revealed that only a minor population of the p53-specific CD4+ T cells was optimally polarized. Conclusions: The p53-SLP vaccine is safe and capable to induce p53-specific T-cell responses in patients treated for colorectal cancer. New trials should focus on improving the polarization of the p53-SLP vaccine-induced T-cell response.

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16+)–induced or HPV18+-induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4+, CD8+, and regulatory T cells as well as by calculation of the ratio of CD8+/CD4+ T cells and CD8+/regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6- and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN−) or presence (LN+) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8+ T-cell tumor infiltration, a higher CD8+/CD4+ T-cell ratio, and higher CD8+/regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR+) or absence (IR−) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN− patients displaying a concomitant systemic tumor-specific immune response (LN−IR+). CD8+ T-cell infiltration in LN−IR− patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN− patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis. [Cancer Res 2007;67(1):354–61]

Modulation of the immune response by established tumors may contribute to the limited success of therapeutic vaccination for the treatment of cancer compared with vaccination in a preventive setting. We analyzed the contribution of the CD4+ T-cell population to the induction or suppression of tumor-specific CD8+ T cells in a tumor model in which eradication of tumors crucially depends on CD8+ T cell–mediated immunity. Vaccine-mediated induction of protective antitumor immunity in the preventive setting (i.e., before tumor challenge) was CD4+ T cell dependent because depletion of this T-cell subset prevented CD8+ T-cell induction. In contrast, depletion of CD4+ cells in mice bearing established E1A+ tumors empowered the mice to raise strong CD8+ T-cell immunity capable of tumor eradication without the need for tumor-specific vaccination. Spontaneous eradication of tumors, which had initially grown out, was similarly observed in MHC class II–deficient mice, supporting the notion that the tumor-bearing mice harbor a class II MHC–restricted CD4+ T-cell subset capable of suppressing a tumor-specific CD8+ T-cell immune response. The deleterious effects of the presence of CD4+ T cells in tumor-bearing hosts could be overcome by CD40-triggering or injection of CpG. Together these results show that CD4+ T cells with a suppressive activity are rapidly induced following tumor development and that their suppressive effect can be overcome by agents that activate professional antigen-presenting cells. These observations are important for the development of immune interventions aiming at treatment of cancer.

The clinical significance of tumor-infiltrating dendritic cells has been reported in a variety of human solid tumors as shown by the correlations found between the presence of tumor-infiltrating dendritic cells and clinical prognosis. In this study, we evaluated whether there is an association between the presence and maturation status of tumor-infiltrating dendritic cells, T lymphocytes, and clinical course in 104 primary tumor samples of patients with colorectal cancer. Dendritic cells were identified with four different markers (S-100, HLA class II, CD208, and CD1a) in double immunohistochemistry, with laminin as second marker to support the exact localization. Tumor-infiltrating dendritic cells showed a distinct infiltration pattern based on their maturation status. CD1a-positive dendritic cells resided in the advancing tumor margins in relatively high numbers, whereas mature CD208-positive dendritic cells were sparsely present in the tumor epithelium but mainly distributed in the tumor stroma and advancing tumor margin. Furthermore, high infiltration of CD1a-positive dendritic cells in the tumor epithelium was significantly correlated to the infiltration of CD4 lymphocytes (P = 0.006). Patients with relatively high numbers of mature CD208-positive infiltrating dendritic cells in the tumor epithelium had a shorter overall survival (P = 0.004). In addition, patients with relatively high numbers of CD1a-positive dendritic cells in the advancing margin of the tumor had a shorter disease-free survival (P = 0.03). We found that tumor-infiltrating dendritic cells had preferential infiltration sites within a tumor, affected local tumor cell-immune cell interactions, and correlated to the clinical prognosis of colorectal cancer patients.

Cervical cancer is the possible outcome of genital infection with high-risk human papillomavirus (HPV) and is preceded by a phase of persistent HPV infection during which the host immune system fails to eliminate the virus. Fortunately, the majority of genital HPV infections are cleared before the development of (pre)malignant lesions. Analysis of CD4+ T-helper (Th) immunity against the E2, E6, and E7 antigens of HPV16 in healthy women revealed strong proliferative E2- and E6-specific responses associated with prominent IFN-γ and interleukin 5 secretion. This indicates that the naturally arising virus-induced immune response displays a mixed Th1/Th2 cytokine profile. Of all HPV16+ cervical cancer patients, approximately half failed to mount a detectable immune response against the HPV16-derived peptides. The other half of the patients showed impaired HPV16-specific proliferative responses, which generally lacked both IFN-γ and interleukin 5. This indicates that the HPV16-specific CD4+ T-cell response in cervical cancer patients is either absent or severely impaired, despite a relatively good immune status of the patients, as indicated by intact responses against recall antigens. It is highly conceivable that proper CD4+ T-cell help is important for launching an effective immune attack against HPV because infection of cervical epithelia by this virus is, at least initially, not accompanied by gross disturbance of this tissue and/or strong proinflammatory stimuli. Therefore, our observations concerning the lack of functional HPV16-specific CD4+ T-cell immunity in patients with cervical cancer offer a possible explanation for the development of this disease.

The ability of dendritic cells (DCs) to boost an antigen-specific immune response is utilized in several cancer immunotherapy strategies including therapeutic vaccination using long peptides. Naked peptides are rapidly degraded and oil-based delivery strategies trap immune cells to unwanted sites or have inappropriate adjuvant properties. To enhance the uptake of cancer or viral T cell epitopes and subsequent activation of DCs we make use of circulating antibodies to mount cellular responses against tumor antigens of interest by conjugating a B cell epitope to a T cell epitope. The conjugation of the two greatly improves antigen uptake and concomitant activation of the same DC in contrast to naked long peptides which are not conjugated. Our identified B cell epitope of choice is derived from tetanus toxin and can be targeted by tetanus-specific antibodies boosted by a standard tetanus toxoid vaccine. We have applied a modified chandler loop model preserving intact cascade systems to characterize how the tetanus-peptide conjugated vaccine is targeted to human immune cells. The B cell-T cell conjugate is taken up by human monocytes and blood DCs in an antibody-dependent manner. Rather than FcγRs, the internalization of the antigen appears to be partly mediated through the classical pathway of the complement system. Tetanus-CMV conjugates, containing a T cell epitope from the pp65 protein of cytomegalovirus (CMV), strongly reactivates memory T cells when analyzed in blood from donors with CMV-specific T cells. The CMV-specific T cells rapidly produce IFNγ and TNF in response to the conjugate illustrating that the uptake of the conjugate leads to activation of antigen-specific T cells. Uptake as well as T cell activation occurs at low concentrations of the SLP conjugate, superior to a conjugate lacking the tetanus-sequence as well as to SLPs with or without additional adjuvant (LPS). Of importance, when the B and T cell epitopes are separate entities but mixed, CMV-specific T cells are not activated, illustrating the requirement of conjugating the two. Our data show that we have a unique delivery system for peptide based vaccines that can aid induction of human T cell responses, and may potentiate immune responses in cancer patients.We are now actively working on a prostate cancer vaccine candidate using this novel loading/adjuvant technology. Citation Format: Erika A. Fletcher, Wendy van Maren, Robert Cordfunke, Jasper Dinkelaar, Ricardo Castelli, Jeroen Codee, Gijs van de Marel, Cornelis J. Melief, Ferry Ossendorp, Jan Wouter Drijfhout, Sara Mangsbo. T cell responses to peptide-epitopes can be boosted by immune complexes of circulating anti-tetanus antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2017-1693

Therapeutic vaccination with a synthetic long peptide (SLP@) vaccine mediated the eradication of established human papillomavirus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papillomavirus. Subsequent phase I/II studies with an HPV16 SLP@ vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. The purpose of the current study was to test the clinical efficacy of this HPV16 SLP@ vaccine in HPV16-induced high-grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses, determined before and after vaccination. The vaccine was safe, as no side effects exceeding CTC grade 2 were observed. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions as compared to nonresponders. This study shows that in women with VIN3 objective clinical responses can be achieved by therapeutic vaccination with synthetic long peptides that is able to induce effective HVP16-specific T-cell responses. Literature: 1. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP, Essahsah F, Fathers LM, Drijfhout JW, Offringa R,Wafelman AR, Oostendorp J, Fleuren GJ, Burg van der SH, Melief CJ. Vaccination against Human Papillomavirus 16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009 Nov 5;361(19):1838-47. 2. Melief CJ, van der Burg SH. Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat Rev Cancer. 2008 May;8(5):351-60. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN01-03.

Cancer antigens of choice include mutant DNA sequences encoding neo-antigens, selected cancer testis antigens, and differentiation antigens, in addition to viral antigens. Neo-antigens and viral antigens are the most attractive, because the T cell repertoire against these antigens has not been blunted by central thymic tolerance. Optimal vaccine platforms include DNA and RNA vaccines as well as synthetic long peptides and selected prime-boost protocols involving recombinant viruses encoding cancer antigens. The most effective cancer vaccines deliver concentrated antigen to both HLA class I and II molecules of DCs, promoting both CD4 and CD8 T cell responses. Suboptimal antigen selection, suboptimal vaccine design, and an immunosuppressive cancer microenvironment are the root causes of the lack of clinical efficacy of cancer vaccines in many clinical trials. Drugs or physical treatments can mitigate the immunosuppressive cancer microenvironment and include chemotherapeutics, radiation, inhibitors of T cell checkpoints, indoleamine 2,3-dioxygenase (IDO) inhibitors, agonists of selected TNF receptor family members, and inhibitors of undesirable cytokines. The specificity of therapeutic vaccination combined with such immunomodulation offers an attractive avenue for the development of future cancer therapies. Clinical benefit of certain therapeutic cancer vaccines has been established, usually by noting prolonged progression free or overall survival. Reviews of the properties and prospects of cancer vaccines have been listed below (1-3). The addition of one or more immunomodulation strategies may further enhance the potential of therapeutic cancer vaccines, including of those that have been only marginally active due to the immunosuppressive cancer microenvironment. We have established clinical benefit of a cancer vaccine as monotherapy in patients with premalignant disease induced by high risk human papilloma virus. Regression of lesions was associated with a better vaccine-induced immune response against the target antigens of the vaccine. However in patients with cervical cancer the vaccine monotherapy failed to install clinically effective immunity. Recently we showed in a pilot trial that vaccine-induced immunity could be markedly enhanced by combination with standard of care chemotherapy for late stage recurrent or metastatic cervical cancer. Increased levels of myeloid-derived suppressor cells were shown to be depleted by the carboplatin and paclitaxel chemotherapy to the low levels of healthy donors, while T cell numbers in PBMC remained intact. Timed vaccination with a single dose of vaccine at the MDSC nadir during chemotherapy led to a robust vaccine-induced T cell response (4). In a subsequent chemo-immunotherapy trial with three vaccine doses, spaced by three weeks, and in cohorts of patients at four different vaccine doses, the initiation and maintenance of robust tumor-specific T cell responses was confirmed and a broad dose response relationship of tumor-specific T cell responses was noted. Preliminary data on clinical outcomes relative to HPV-specific immune responses will be discussed. In summary, therapeutic benefit of cancer vaccines can be maximized in patients with established cancers by improving vaccine design and by using vaccines in combination with standard chemotherapies. In addition cancer vaccines can establish and/or maintain tumor-specific T cells in combination with checkpoint blockade or other immunomodulatory therapies. Finally, cancer vaccines can also help to sustain and expand adoptively transferred tumor-specific T cells. References 1. Melero I, Gaudernack G, Gerritsen W, Huber C, Parmiani G, Scholl S, Thatcher N, Wagstaff J, Zielinski C, Faulkner I, Mellstedt H, Therapeutic vaccines for cancer: an overview of clinical trials. Nat Rev Clin Oncol. 2014;11(9):509-524. 2. Melief CJ,.van Hall T, Arens R, Ossendorp F, van der Burg SH. Therapeutic cancer vaccines. J Clin Invest. 2015 125(9):3401-3412. 3. van der Burg SH, Arens R, Ossendorp F, van Hall T, Melief CJ. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat Rev.Cancer. 2016 Apr;16(4):219-33. 4. Welters MJ, van der Sluis TC, van Meir H, Loof NM, van Ham VJ, van Duikeren S, Santegoets SJ, Arens R, de Kam ML, Cohen AF, van Poelgeest MI, Kenter GG, Kroep JR, Burggraaf J, Melief CJ, van der Burg SH. Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses. Sci Transl Med. 2016 Apr 8(334):334ra52. Citation Format: Cornelis J. M. Melief. Selection of cancer antigens and conditions for success of immunotherapy of cancer involving vaccination [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA02.

Monoclonal antibody (mAb) blockade of immune checkpoints such as PD-1 and CTLA-4 can stimulate therapeutic, T cell-dependent anti-tumor activity in mice and humans. We asked whether exome sequencing and epitope prediction algorithms could identify antigens in progressively growing tumors that render them susceptible to anti-PD-1- or anti-CTLA-4-induced immune elimination. Expressed mutations in progressively growing, T3 methylcholanthrene (MCA)-sarcomas were identified by exome sequencing and prioritized based primarily on potential MHC class I binding affinity and predicted peptide processing. The two best candidate tumor-specific mutant antigens (TSMA) were identified from mutations in Laminin α subunit 4 (Lama4) and a glucosyltransferase called Alg8. When tested in vitro, mutant Lama4 (mLama4) and mutant Alg8 (mAlg8) peptides were the only top predicted TSMA that stimulated T3-specific T cells. The relevance of these findings was validated by four in vivo studies: (i) mLama4 and mAlg8 peptides bound to H-2Kb were biochemically detected on the surface of T3 tumors, (ii) as shown by tetramer staining, CD8+ tumor infiltrating lymphocytes (TIL) expressing TCRs for mLama4 and mAlg8 accumulated over time in T3 tumors in checkpoint blockade treated, tumor-bearing mice, reaching maximum levels just prior to tumor rejection, (iii) vaccination of naïve wild type mice with mutant but not WT forms of Lama4 or Alg8 induced strong CD8+ T cell responses and (iv) prophylactic or therapeutic vaccination with mLama4 plus mAlg8 synthetic long peptides (SLP) resulted in rejection of T3 cells. The therapeutic protection provided by the SLP vaccine was equal to that afforded by checkpoint blockade therapy. Since we did not see evidence of T cell responses to any other TSMA, we then explored whether the immunodominant antigens masked responses to other TSMA. Using CRISPR/Cas9, we introduced point mutations to revert mLama4 and mAlg8 in T3 back to their nonimmunogenic wild type counterparts. T3 tumor cells with either mLama4 or mAlg8 reverted to wild type were still susceptible to anti-PD-1 or anti-CTLA-4 immune-mediated control with skewing of the T cell response in the TIL towards the remaining TSMA. Additionally, we have obtained T3 tumor cells with both TSMA reverted to wild type and are currently testing whether these “fixed” T3 tumor cells are still subject to anti-PD-1 or anti-CTLA-4 and whether SLP vaccines comprised of subdominant TSMA still present in fixed T3 tumor cells can protect mice against tumor growth. This system will allow us to address the many questions surrounding poorly understood phenomenon of immunodominance and how this may influence patient-specific personalized cancer immunotherapy directed against TSMA. Citation Format: Matthew M. Gubin, Jeffrey P. Ward, Takuro Noguchi, Xiuli Zhang, Cora Arthur, Willem-Jan Krebber, Gwenn E. Mulder, Cornelis J.M. Melief, William E. Gillanders, Maxim Artyomov, Elaine R. Mardis, Robert D. Schreiber. Tumor-specific mutant antigens in cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A001.

Previously we have shown that Synthetic Long Peptide (SLP) vaccination against Human Papilloma Virus (HPV) oncogenic proteins is safe and induces functional T-cell responses in mice and humans. Although vaccination induces potent clinical responses in mice and in patients with premalignant lesions, no clinical responses were observed in patients with cervical cancer. Here we show in a preclinical mouse model for HPV induced malignancies that SLP vaccination can be safely combined with clinically relevant chemotherapeutics to eradicate tumors. In mice that received either peptide vaccination or chemotherapy, only a temporary regression in tumor size was observed. Importantly, combined chemo-immunotherapy induced complete and sustained tumor eradication in nearly all mice. The chemotherapeutic agent cisplatin displayed the strongest synergy with SLP vaccination. Long term regression of tumors occurred at both the maximal tolerated dose of cisplatin and at 40% of that dose when combined with SLP vaccination, but at the later dose side effects such as body weight loss were absent. Upon vaccination tumors were highly infiltrated with Tumor Necrosis Factor alpha (TNFα) and Interferon gamma (IFN-γ) producing CD8+ CTLs. Tumor cells incubated with these cytokines and cisplatin in vitro strongly enhance their chemokine expression, compatible with the abundant leukocyte infiltration into the tumor upon combined chemo-immunotherapy. Accordingly, when combined with cisplatin treatment, SLP vaccine-induced CTLs appeared to migrate earlier from the tumor rim into the tumor beds. Moreover, analysis of the tumor cells in vivo showed that combined treatment significantly decreased the proliferative capacity of tumor cells compared to single treated or untreated tumors. Furthermore, we showed that TNFα enhanced cisplatin- induced, JNK dependent tumor cell apoptosis. This cell death was accompanied by an increased expression of pro-apoptotic molecules. The synergy between cisplatin and SLP vaccination was largely abolished by in vivo treatment with monoclonal antibody against TNFα. Together, our data show that combined peptide treatment with cisplatin leads to superior tumor eradication in the absence of T cell immunosuppression. Citation Format: Cornelis J. Melief, Tetje C. van der Sluis, Sjoerd H. van der Burg. Vaccine-induced TNF alpha producing T cells synergize with cisplatin in tumor eradication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2494. doi:10.1158/1538-7445.AM2015-2494

We previously developed a synthetic long overlapping HPV16 E6/E7 peptides (HPV16-SLP) vaccine that was clinically active in patients with HPV16+ high-grade vulvar intraepithelial neoplasia. Complete lesion regression was related to a strong vaccine-prompted HPV16-specific effector T-cell response. However, in patients with HPV16-induced metastatic cervical cancer the vaccine-induced T-cell responses were weaker and did not result in clear clinical benefit. A new study on the immune constitution of PBMC of such patients revealed increased numbers of myeloid cells, low T-cell reactivity against common microbial recall antigens and a lower stimulatory capacity of antigen presenting cells, indicating an immunosuppressive status. When these patients were subsequently treated with standard carboplatin-paclitaxel chemotherapy we observed that at a specific time point during chemotherapy this tumor-biased immune profile was normalized to a profile similar to that found in healthy subjects. Experiments in an HPV16+ mouse tumor model showed that the tumor-induced abnormally high myeloid cell level was also normalized by this chemotherapy, not only among PBMC but also locally in the tumor. Residual tumor-present myeloid cells phenotypically mirrored activated non-suppressive antigen presenting cells. Moreover, combined HPV16 SLP vaccination and chemotherapy effectively led to a higher cure rate of mice with established tumors. Together these data indicated that standard chemotherapy has an immune stimulatory effect by deletion of suppressive myeloid cells in the mouse tumor model and in patients with metastatic cervical cancer . Therefore, a clinical trial was started in which such patients were treated with standard chemotherapy in combination with HPV16 SLP (ISA101) vaccination. Comprehensive immune monitoring confirmed the beneficial effect of myeloid cell depletion associated with a robust induction of HPV16-specific T-cell responses that were sustained throughout several cycles of chemotherapy. The data of these studies will be reported. Currently, we have started a multicenter clinical trial (ISA-HPV-01-12) in which vaccination with the HPV16 SLP ISA101 vaccine is combined with carboplatin-paclitaxel and type I interferon to assess clinical and immunological outcome in a group of HPV16+ metastatic cervical cancer patients. Citation Format: Sjoerd H. Van Der Burg, Tetje C. van der Sluis, Helene van Meir, Judith R. Kroep, Gemma G. Kenter, Mariette I.E. van Poelgeest, Koos Burggraaf, Cornelis J.M. Melief, Marij J.P. Welters. Synergistic effects of properly timed HPV16 synthetic long peptide vaccination during standard carboplatin-paclitaxel chemotherapy in animals and in patients with metastatic cervical carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2014-2938

Therapeutic vaccination of persistent virus infections and associated diseases including (pre-)cancer so far has largely evaded success, mainly due to the fact that insufficiently consistent and robust effector T cell responses were induced by the commonly used vaccine constructs and formulations such as recombinant viruses and bacteria, recombinant proteins, DNA constructs, Dendritic Cell (DC) vaccines and exact HLA class I binding peptides (short peptides). Problems have been severe antigenic competition from vector sequences by recombinant viruses and bacteria, insufficiently powerful T cell generation by DNA constructs, insufficient homing to lymph nodes of injected DC and antigen presentation of short peptides by non-professional antigen presenting cells in vivo, causing tolerance instead of immunity. Much more robust and consistent T cell responses can be obtained by vaccination with long (28-35 amino acid long) synthetic peptides. Such immunogens are more efficiently processed and presented than intact proteins by DC and only DC can efficiently perform this task. Moreover, only concentrated antigen of choice is offered and antigenic competition therefore plays no role. Phase I/II studies with an HPV16 SLP® vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. We then tested the clinical efficacy of this HPV16 SLP® vaccine in HPV16-induced high grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In this phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses. The vaccine was safe. At 3 months after the last vaccination, an objective response was observed in 12/20 (60%). Five patients showed a complete and durable regression of the lesions. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions compared to non-responders. Patients with large lesions were less likely to experience a complete clinical response than patients with small lesions and we ascribe this to a larger proportion of vaccine induced HPV-specific regulatory cells in the patients with large lesions. In a preclinical mouse model we have shown marked synergism in eradication of established HPV16 E6/E7+ tumors between platinum-based chemotherapy and HPV16 SLP vaccination, associated with a decrease in myeloid cells in the blood and in the tumor. In a pilot clinical study, end stage cervical cancer patients, treated with carbotaxol, showed no T cell immunodeficiency and showed robust T cell effector responses to a single dose of HPV16 SLP® vaccine. These findings set the stage for a randomized phase II study of chemo-immunotherapy in this category of patients, in which chemotherapy alone will be compared with chemo-immunotherapy. In conclusion, treatment with the HPV16 SLP vaccine is effective in patients with established VIN disease. The SLP platform lends itself for development of therapeutic vaccines against many other chronic infections and non-viral cancers. In patients with cancer, it is attractive to combine this type of vaccination with chemotherapy and with immunomodulatory drugs such as type I interferon and checkpoint-control blocking monoclonal antibodies. Citation Format: Cornelis J. M. Melief. Synthetic vaccine for immunotherapy of high risk HPV infections. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY27-01. doi:10.1158/1538-7445.AM2013-SY27-01

Therapeutic vaccination of persistent virus infections and associated diseases including (pre-)cancer so far has largely evaded success, mainly due to the fact that insufficiently consistent and robust effector T cell responses were induced by the commonly used vaccine constructs and formulations such as recombinant viruses and bacteria, recombinant proteins, DNA constructs, Dendritic Cell (DC) vaccines and exact HLA class I binding peptides (short peptides). Problems have been severe antigenic competion from vector sequences by recombinant viruses and bacteria, insufficiently powerful T cell generation by DNA constructs, insufficient homing to lymph nodes injected DC and antigen presentation of short peptides by non-professional antigen presenting cells in vivo, causing tolerance instead of immunity. Much more robust and consistent T cell responses can be obtained by vaccination with long (28–35 amino acid long) synthetic peptides. Such immunogens are more efficiently processed and presented than intact proteins by DC and only DC can efficiently perform this task. Moreover only concentrated antigen of choice is offered and antigenic competition therefore plays no role. In earlier work we showed that therapeutic vaccination with a synthetic long peptide (SLP®) vaccine mediated the eradication of established human papilloma virus type 16 (HPV16)-positive tumors in mice and controlled wart growth and latent virus infection in rabbits persistently infected with cottontail rabbit papilloma virus. Subsequent phase I/II studies with an HPV16 SLP® vaccine, consisting of 13 long peptides covering the HPV16 E6 and E7 antigens, in patients with advanced HPV16-positive cervical cancer, revealed that this vaccine was safe and highly immunogenic. We then tested the clinical efficacy of this HPV16 SLP® vaccine in HPV16-induced high grade vulvar intraepithelial neoplasia (VIN3), a premalignant epithelial disorder, spontaneous regression of which occurs in less than 2% of patients and in which recurrence after standard treatment is high. In a phase 2 trial, 20 women with VIN3 were vaccinated three times sc in the limbs with a mix of the HPV16 E6 and E7 synthetic long peptides formulated in Montanide ISA-51. The endpoints were objective clinical responses, defined as reduction of at least 50% in lesion size (partial response) or complete regressions, and HPV16-specific T-cell responses. The vaccine was safe. At 3 and 12 months after the last vaccination an objective response was observed in 12/20 (60%) and 15/19 (79%) patients respectively. Nine of them showed a complete and durable regression of the lesions at 12 months and at 24 months. The strength of the vaccine-induced HPV16-specific T-cell response was significantly higher in the group of patients with a complete regression of their lesions compared to non-responders. Patients with large lesions were less likely to experience a complete clinical response than patients with small lesions and we ascribe this to a larger proportion of vaccine induced HPV-specific regulatory cells in the patients with large lesions. In conclusion, treatment with the HPV16 SLP vaccine is clearly effective in patients with established VIN disease. The SLP platform lends itself for development of therapeutic vaccines against many other chronic infections and non-viral cancers. In patients with cancer, it is attractive to combine this type of vaccination with immunogenic forms of cancer chemotherapy and with immuno-modulatory drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL04-04.