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Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319–29. ©2017 AACR.

Figure Legends for Supplementary Figures S1-S4.

Supplementary Figure S1. Lung cancer cell lines do not express NE and P3 gene transcripts. Supplementary Figure S2. CTL proliferate in the presence of NE-exposed lung cancer cells. Supplementary Figure S3. HLA class I presentation of mutant neoantigens is enhanced by NE and P3. Supplementary Figure S4. Immunoproteasome expression by lung cancer cells is unaltered by serine proteases.

Background: The checkpoint inhibitors atezolizumab (anti-PD-L1) and pembrolizumab (anti-PD-1) are FDA approved for the treatment of patients with PD-L1 positive, metastatic triple negative breast cancer in combination with chemotherapy. The response rates to checkpoint blockade in hormone receptor positive, HER2 negative metastatic breast cancer have been less encouraging (RR 12% for pembrolizumab in KEYNOTE-028 and RR 2.8% for avelumab in the phase 1b trial JAVELIN). We conducted a feasibility trial and enrolled patients with Stage II-III hormone receptor positive, HER2 negative breast cancer to treatment with 2 cycles of durvalumab (anti-PD-L1) plus tremelimumab (anti-CTLA-4) prior to standard neoadjuvant chemotherapy and breast surgery.Methods: Eligible patients were treated with durvalumab at a dose of 1500 mg and tremelimumab at a dose of 75 mg, both administered intravenously, on days 1 and 28. Pre- and post-treatment tumor biopsies and blood samples were available for 5 out of 8 patients and were analyzed by CyTOF, IHC, and NanoString. Patients then received standard neoadjuvant chemotherapy prior to breast surgery. The target enrollment was 20 patients. Results: After 8 patients were enrolled and treated on protocol, the trial was stopped early due to toxicity, slow accrual, and delays in patients receiving standard neoadjuvant chemotherapy. The median age of the patients was 55 (range 39-66) and all 8 patients were women. All patients had ER/PR positive, HER2 negative invasive ductal carcinoma (2 with lobular features, 1 with focal mucinous features) and all patients had clinical Stage II disease. Five patients received both cycles of durvalumab/tremelimumab and the remaining 3 patients only received the first cycle. One patient discontinued therapy out of concern for progression, though repeat breast and lymph node biopsies were benign and she went on to have a pathologic complete response after standard neoadjuvant chemotherapy. The remaining 2 patients discontinued treatment after the first cycle due to Grade 3 colitis (Patient #1) and Grade 3 thyroiditis/adrenal insufficiency (Patient #8). Both patients required treatment with steroids and experienced delays in receiving standard therapy due to these adverse events (AEs). The other AEs reported were all Grade 1 or 2. A post-durvalumab/tremelimumab breast ultrasound was performed in 7 of 8 patients and the percentage change in volume of each patient’s primary breast mass was: -55%, +104% (biopsy benign), +65%, -30%, +90%, -8%, and -53%. Patient #1 (colitis) had chemotherapy administered adjuvantly and Patient #8 (thyroiditis, adrenal insufficiency) declined chemotherapy. Only 1 of 8 patients had a pathologic complete response at the time of surgery. IHC, CYTOF, and gene expression analyses showed an increase in immune cell subsets in tumor stroma post neoadjuvant immunotherapy. Conclusions: We conducted a feasibility trial of neoadjuvant durvalumab plus tremelimumab administered prior to standard neoadjuvant chemotherapy in patients with hormone receptor positive/HER2 negative Stage II or III breast cancer. The trial was stopped early after 2 of 8 patients experienced Grade 3 immune-related adverse events (colitis in 1 patient and thyroiditis/adrenal insufficiency in another patient). Citation Format: Haven R. Garber, Sreyashi Basu, Sonali Jindal, Akshara Singareeka Raghavendra, Lumarie Santiago, Beatriz E. Adrada, Padmanee Sharma, James P. Allison, Jennifer Litton. Durvalumab and tremelimumab before surgery in patients with hormone receptor positive, HER2 negative stage II-III breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-14.

U266 cross-presents PR1 after exposure to NE/P3 from PMN co-culture

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Purpose:Inefficient homing of adoptively transferred cytotoxic T lymphocytes (CTLs) to tumors is a major limitation to the efficacy of adoptive cellular therapy (ACT) for cancer. However, through fucosylation, a process whereby fucosyltransferases (FT) add fucose groups to cell surface glycoproteins, this challenge may be overcome. Endogenously fucosylated CTLs and ex vivo fucosylated cord blood stem cells and regulatory T cells were shown to preferentially home to inflamed tissues and marrow. Here, we show a novel approach to enhance CTL homing to leukemic marrow and tumor tissue.Experimental Design:Using the enzyme FT-VII, we fucosylated CTLs that target the HLA-A2–restricted leukemia antigens CG1 and PR1, the HER2-derived breast cancer antigen E75, and the melanoma antigen gp-100. We performed in vitro homing assays to study the effects of fucosylation on CTL homing and target killing. We used in vivo mouse models to demonstrate the effects of ex vivo fucosylation on CTL antitumor activities against leukemia, breast cancer, and melanoma.Results:Our data show that fucosylation increases in vitro homing and cytotoxicity of antigen-specific CTLs. Furthermore, fucosylation enhances in vivo CTL homing to leukemic bone marrow, breast cancer, and melanoma tissue in NOD/SCID gamma (NSG) and immunocompetent mice, ultimately boosting the antitumor activity of the antigen-specific CTLs. Importantly, our work demonstrates that fucosylation does not interfere with CTL specificity.Conclusions:Together, our data establish ex vivo CTL fucosylation as a novel approach to improving the efficacy of ACT, which may be of great value for the future of ACT for cancer.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo.Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386–96. ©2018 AACR.

Supplementary Tables S1-S4

Purpose: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Experimental Design: We obtained pretreatment core-needle biopsies from 105 patients with stage I–III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. Results: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. Conclusions: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

BACKGROUND: Current treatment for early stage triple negative breast cancer (TNBC) includes neoadjuvant systemic chemotherapy (NAST), which is used to assess disease biology and the need for adjuvant treatment in case of residual disease at the time of surgery, also known as residual cancer burden (RCB). Patients with TNBC who experience RCB-0 (pathologic complete response [pCR]) or RCB-I after NAST have an excellent prognosis whereas patients with significant residual disease (RCB-II or RCB-III) are at a high risk of relapse. Standard NAST for TNBC achieves pCR in 30-50% of cases. NAST typically consists of anthracycline-based chemotherapy followed or preceded by a taxane +/- carboplatin. Disease progression (PD) is uncommon in TNBC patients receiving NAST and little is known regarding outcomes in patients who have PD during the initial phase of NAST. METHODS: Total 316 TNBC patients were evaluated from two prospectively accrued clinical trials of NAST (NCT02276443 and NCT01334021). The ARTEMIS trial (NCT02276443) aims to improve pCR rates by adding targeted therapy to chemotherapy as the second phase of NAST for those patients who do not experience at least a 70% volumetric reduction after 4 cycles of doxorubicin/cyclophosphamide (AC). Unique histopathologic features including % stromal tumor-infiltrating lymphocytes (sTIL), presence of mesenchymal histology (high vimentin expression by IHC), and androgen receptor expression are used to guide second phase therapy. RESULTS: 31 TNBC patients had PD while receiving AC as the first phase of NAST (10%; 95% CI= 6.69-13.31%). 9 of 31 patients proceeded to standard chemotherapy and all had RCB II/III disease. 22 of 31 patients were enrolled to targeted therapy trials. 6 were treated with the EGFR inhibitor panitumumab + carboplatin/paclitaxel, 9 with atezolizumab + nab-paclitaxel, and 7 with everolimus, bevacizumab, and liposomal doxorubicin (DAE). Of these 22 patients, 3 (13.6%) had pCR/RCB-0, 1 (4.5%) RCB-I and 18 (81.8%) had RCB II/III. All 4 patients who experienced RCB-0/I had T2N0 disease at diagnosis. 2 had sTIL < 5% and 2 patients had 70% sTIL. CONCLUSION: PD is uncommon while receiving NAST. Patients with TNBC and progression on initial NAST with AC are unlikely to achieve pCR or RCB-I status despite subsequent standard chemotherapy. Combination chemotherapy with targeted therapy on clinical trial resulted in a numerically higher rate of pCR+RCB-I (18%) as salvage therapy, but this was not statistically significant and requires confirmation in larger trials. Citation Format: Haven Garber, Gaiane Rauch, Beatriz Adrada, Rosalind Candelaria, Elizabeth Mittendorf, Alastair Thompson, Jennifer Litton, Senthil Damodaran, Bora Lim, Banu Arun, Naoto Ueno, Vicente Valero, Nuhad Ibrahim, Rashmi Murthy, Debu Tripathy, Helen Piwnica-Worms, Fraser Symmans, Lei Huo, Stacy Moulder. Residual cancer burden in patients with early stage triple negative breast cancer who progress on anthracycline-based neoadjuvant chemotherapy in an ongoing clinical trial (ARTEMIS) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-16-09.

Patients with hereditary mutations in BRCA1 or BRCA2 (gBRCA1/2) and breast cancer have distinct tumor biology, and encompass a predilection for brain metastasis (BM). We looked into baseline risk of BMs among gBRCA1/2 patients. Patients with gBRCA1/2, stage I-III invasive breast cancer seen between 2000–2017 with parenchymal BMs. Among gBRCA1 with distant breast cancer recurrence, 34 of 76 (44.7%) were diagnosed with brain metastases compared to 7 of 42 (16.7%) patients with gBRCA2. In the comparator group, 65 of 182 (35.7%) noncarrier triple-negative breast cancer (TNBC) and a distant recurrence experienced BM’s. In a competitive risk analysis using death as a competing factor, the cumulative incidence of BMs was similar between gBRCA1 and noncarrier TNBC patients. The time from primary breast cancer diagnosis to detection of BMs was similar between gBRCA1 and noncarrier TNBC patients (2.4 vs 2.2 years). Survival was poor after BMs (7.8 months for gBRCA1 patients vs. 6.2 months for TNBC noncarriers). Brain was a more common site of initial distant recurrence in gBRCA1 patients versus TNBC noncarriers (26.3% vs. 12.1%). Importantly, the presence of BMs, adversely impacted overall survival across groups (HR 1.68 (95% CI 1.12–2.53), hazard ratio for death if a patient had BMs at the time of initial breast cancer recurrence vs. not). In conclusion, breast cancer BMs is common and is similarly frequent among gBRCA1 and noncarrier patients with recurrent TNBC. Our study highlights the importance of improving the prevention and treatment of BMs in patients with TNBC, gBRCA1 carriers, and noncarriers.

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319–29. ©2017 AACR.

Cathepsin G is broadly expressed in acute myeloid leukemia and is an effective immunotherapeutic target

Background aims PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood–derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci. Methods We first determined the frequency of PR1-CTLs in HLA-A2 + UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells. Results PR1-CTLs are detected at an average frequency of 0.14% within the CD8 + population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8 + population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines. Conclusions This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.

Chemotherapy/ targeted therapy are both known to trigger evolution of treatment resistant clones that can lead to relapse. Allogeneic stem cell transplant (alloSCT) for refractory Chronic Lymphocytic Leukemia (CLL) patients is associated with better outcomes. We hypothesized that allogeneic T-cell immunotherapies, including alloSCT and donor lymphocyte infusion (DLI) would impact tumor evolution through the application of selective immunologic pressure with reciprocal changes in the T-cell compartment. Here, we tested a cohort of 24 heavily pre-treated CLL patients treated. Treatments consisted of alloSCT alone, or with follow-up DLI, which are two established mediators of effective Graft versus Leukemia (GVL). Our cohort included 11 patients who relapsed (denoted as non-responder, NR) after alloSCT and 13 patients who had complete response (CR) after alloSCT, with 11/13 patients showing durable CR with a median post-transplant overall survival (OS) of 9.8 years. We mapped the evolutionary trajectories of tumor cells by whole exome sequencing (WES) of sort purified CLL in post-transplant relapsed patients. To investigate changes in immune repertoire and gene expression post-transplant, CD3 positive T-cells from peripheral blood and bone marrows of CLL patients at complete donor chimerism were analyzed both at bulk and at the single cell level. We found evidence of subclonal leukemic evolution in the majority of our CLL patient cohort after nonmyeloablative HLA-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. In all of the 11 patients with longitudinal post-alloSCT samples available, we observed branched CLL evolution in 4 patients, linear evolution in 4 patients, and no evolution in 3 patients. These data suggest that differential sensitivity of leukemic subclones to allogeneic T cell killing may underlie the branched and linear evolution that we observed, and therefore can shape leukemic subclonal architecture after transplant. Of note, we found that clonal CLL was more responsive to alloSCT in comparison to CLL with subclonal disease architecture.To identify T-cells with GVL potential, we first cataloged potential neoantigens by screening mutated regions in CLL with in silico HLA binding prediction models. Neoantigen specific T-cells were then sorted from longitudinal peripheral blood samples using tetramers, followed by identification of GVL specific TCR in both bulk and single cell setting. We were able to identify T-cells that coevolved with specific tumorigenic lesions in a subset of CLL patients. Taken together, our results suggest that donor-derived antigen-specific T-cells mediate clonal selection of CLL with concurrent changes in allogeneic T-cells, and that these changes can be monitored in longitudinal patient samples. Citation Format: Celine Kerros, John P. Miller, Xizeng Mao, Haven R. Garber, Hannah C. Beird, Jianhua Zhang, Jason Roszik, Paul Leonard, Li Zhao, Sahil Seth, Pei Lin, Huandong Sun, William G. Wierda, Issa F. Khouri, Karen Clise-Dwyer, Andrew Futreal, Shoudan Liang, Koppikar Priya, Jeffrey Molldrem. Deep profiling of T-cell repertoire and tumor heterogeneity in chronic lymphocytic leukemia patients following allogeneic T-cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1516.

Purpose: Inefficient homing of adoptively transferred cytotoxic T lymphocytes (CTLs) to tumors is a major limitation to the efficacy of adoptive cellular therapy (ACT) for cancer. However, through fucosylation, a process whereby fucosyltransferases (FT) add fucose groups to cell surface glycoproteins, this challenge may be overcome. Endogenously fucosylated CTLs and ex vivo fucosylated cord blood stem cells and regulatory T cells were shown to preferentially home to inflamed tissues and marrow. Here, we show a novel approach to enhance CTL homing to leukemic marrow and tumor tissue. Experimental Design: Using the enzyme FT-VII, we fucosylated CTLs that target the HLA-A2–restricted leukemia antigens CG1 and PR1, the HER2-derived breast cancer antigen E75, and the melanoma antigen gp-100. We performed in vitro homing assays to study the effects of fucosylation on CTL homing and target killing. We used in vivo mouse models to demonstrate the effects of ex vivo fucosylation on CTL antitumor activities against leukemia, breast cancer, and melanoma. Results: Our data show that fucosylation increases in vitro homing and cytotoxicity of antigen-specific CTLs. Furthermore, fucosylation enhances in vivo CTL homing to leukemic bone marrow, breast cancer, and melanoma tissue in NOD/SCID gamma (NSG) and immunocompetent mice, ultimately boosting the antitumor activity of the antigen-specific CTLs. Importantly, our work demonstrates that fucosylation does not interfere with CTL specificity. Conclusions: Together, our data establish ex vivo CTL fucosylation as a novel approach to improving the efficacy of ACT, which may be of great value for the future of ACT for cancer.

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. J. Clin. Invest. 111: 639–647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)–expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti–low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells’ capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.

T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. β2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies. Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples. Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivo. Conclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386–96. ©2018 AACR.

Appreciation for genomic and immune heterogeneity in cancer has grown though the relationship of these factors to treatment response has not been thoroughly elucidated. To better understand this, we studied a large cohort of melanoma patients treated with targeted therapy or immune checkpoint blockade (n = 60). Heterogeneity in therapeutic responses via radiologic assessment was observed in the majority of patients. Synchronous melanoma metastases were analyzed via deep genomic and immune profiling, and revealed substantial genomic and immune heterogeneity in all patients studied, with considerable diversity in T cell frequency, and few shared T cell clones (, Melanoma: Tumor differences within a patient may explain heterogeneous responses Patients with metastatic melanoma display molecular and immune differences across tumor sites associated with differential drug responses. A team led by Jennifer Wargo from the University of Texas MD Anderson Cancer Center, Houston, USA, studied the radiological responses of 60 patients with metastatic melanoma, half of whom received targeted drug therapy and half of whom received an immune checkpoint inhibitor. The majority (83%) showed differences in responses across metastases. The group then profiled tumors in a subset, and found molecular and immune heterogeneity in different tumors within the same patient. Heterogeneity in mutational and immune profiles within tumors from individual patients could explain differences in treatment response. Knowing this, the authors emphasize the importance of acquiring biopsies from more than one tumor site in order to best tailor therapies to the features of metastatic cancer.

Pretransplant conditioning regimens critically determine outcomes in the setting of allogeneic stem cell transplantation (allo-SCT). The use of nucleoside analogs such as fludarabine (Flu) in combination with i.v. busulfan (Bu) has been shown to be highly effective as a pretransplant conditioning regimen in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS). Because leukemia relapse remains the leading cause of death after allo-SCT, we studied whether clofarabine (Clo), a nucleoside analog with potent antileukemia activity, can be used to complement Flu. In a preliminary report, we previously showed the safety and efficacy of Clo ± Flu with i.v. Bu in 51 patients with high-risk AML, CML, and MDS. The study has now been completed, and we present long-term follow-up data on the entire 70-patient population, which included 49 (70%), 8 (11%), and 13 (19%) patients with AML, MDS, and CML, respectively. Thirteen patients (19%) were in complete remission, and 41 patients (59%) received matched unrelated donor grafts. Engraftment was achieved in all patients. Sixty-three patients (90%) achieved complete remission. There were no deaths reported at day +30, and the 100-day nonrelapse mortality rate was 4% (n = 3). Thirty-one percent of patients (n = 22) developed grades II to IV acute graft-versus-host disease, and the median overall survival and progression-free survival times were 2.4 years and .9 years, respectively. Our results confirm the safety and overall and progression-free survival advantage of the arms with higher Clo doses and lower Flu doses, which was most prominent in the AML/MDS group.

Background aims The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity. Methods Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. Results Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro . Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. Conclusions Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro . Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.

There have been significant advances in the treatment of metastatic melanoma through targeted and immunotherapy, however a significant proportion of patients still progress on these regimens with many experiencing mixed responses. Intense research efforts to better understand resistance are underway, and multiple molecular resistance mechanisms to targeted therapy have been identified. The appreciation of genetic heterogeneity as a contributor to resistance to therapy has grown, though immune heterogeneity has been poorly characterized. The goal of the present study is to better understand the molecular and immune heterogeneity in synchronous melanoma metastases at the time of disease progression. In this study, we prospectively evaluated 32 tumors from 15 patients who were treatment-naïve (n = 4), or had received prior targeted (n = 4) or immunotherapy (n = 7). Whole exome sequencing demonstrated between 4-41% of non-synonymous exonic mutations (NSEM) were restricted to individual tumors within a patient. Deep profiling of infiltrating immune cell subsets by flow cytometry and immunohistochemistry analyses confirmed the immune infiltrate between synchronous metastases to be highly heterogeneous, specifically in regards to CD4 and CD8 T lymphocytes. In aggregate, 92% of these T cell clones were unique to distinct tumors within the same patient, with limited overlap with clones detected in the blood. NetMHC3.4 neoantigen prediction demonstrated a large fraction of predicted neoantigens were restricted to individual tumors, with over 10% of these presenting extremely high predicted affinity. Importantly, analysis of RECIST measurements of individual lesions within the same patient suggested this molecular and immune heterogeneity could contribute to differential tumor growth and response to therapy within the same patient. This has important clinical implications, and suggests a single tumor biopsy may not be sufficiently representative of the molecular and immune landscape of multiple tumors within the same individual. Citation Format: Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Xizeng Mao, Wei-Shen Chen, Hannah Cheung, Hong Jiang, Cara Haymaker, Mariana Petaccia De Macedo, Haven R. Garber, Pei-Ling Chen, Vancheswaran Gopalakrishnan, Jacob Austin-Breneman, Courtney W. Hudgens, Jason Roszik, Patrick Hwu, Scott E. Woodman, Lynda Chin, Michael A. Davies, Rodabe N. Amaria, Sapna P. Patel, Alexander J. Lazar, Michael T. Tetzlaff, Karen C. Dwyer, Ignacio I. Wistuba, Padmanee Sharma, James P. Allison, Jianhua Zhang, Andrew Futreal, Zachary A. Cooper, Jennifer A. Wargo. Genomic and immune heterogeneity in synchronous melanoma metastases is associated with differential tumor growth and response to therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2392.

Pancreatic cancer is the fourth-leading cause of death in the United States and one of the most aggressive known malignancies. New and innovative advances in treatment are desperately needed. One promising area of investigational treatment for pancreatic cancer involves the use of immunotherapy. The development of immunotherapy for pancreatic cancer has been hampered by difficulty in generating tumor-reactive lymphocytes from resected specimens and by a lack of appropriate target antigens expressed on tumor cells. Innovative strategies have been developed with the use of peripheral blood lymphocytes that are genetically engineered to express T-cell receptors targeting common tumor antigens, including cancer-testis antigens, such as the MAGE-A3 antigen. Cancer-testis antigens pose excellent targets for immunotherapy because they are expressed in cancer and in the testis, an immune-privileged site, but have limited expression in normal tissue. An additional advantage in targeting cancer-testis antigens for immunotherapy is that their expression can be selectively up-regulated in tumor cells via epigenetic regulation with chromatin remodeling agents. Current interest in targeting cancer-testis antigens in pancreatic cancer is well-founded because cancer-testis antigens have been shown to be expressed in pancreatic cancer as potential targets for therapy. In our studies, we validated the expression pattern of cancer-testis antigens in resected specimens of pancreatic cancer and tested the hypothesis that treatment of pancreatic cancer cells with chromatin remodeling agents would render them more sensitive to antigen-specific T lymphocytes. We focused predominately on the MAGE-A3 antigen because it is highly expressed in pancreatic cancer, and several immunotherapeutic strategies are in clinical trials targeting this specific antigen. The results of these studies have important translational implications and provide the rationale for combined treatment with chromatin remodeling agents and immunotherapeutic approaches for pancreatic cancer.

Next-generation sequencing (NGS) has revealed that the malignant subclones comprising a patient's cancer can possess tremendous genetic heterogeneity at different sites of disease and over time. In leukemia, chemotherapy can hasten subclonal evolution allowing for rare leukemic subclones with aggressive driver mutations to gain a competitive advantage and to predominate at relapse, often portending an inferior treatment response. The impact of immunotherapy on subclonal evolution is less well studied. To determine the effects of allogeneic stem cell transplant (alloSCT) and donor lymphocyte infusion (DLI) on subclonal evolution, we performed whole exome sequencing (WES) on longitudinal peripheral blood and bone marrow from 4 patients with CLL. Specifically, timepoints analyzed included pre-transplant, post-transplant relapse, and post-DLI relapse over a period of up to 8.5 years. B-CLL cells (CD19+CD5+) and normal T cells (CD3+) were sort-purified by fluorescence-activated cell sorting prior to genomic DNA extraction. Libraries for WES were constructed and sequenced to an average depth of 300x on an Illumina HiSeq 2000 using 76 bp paired-end reads. Somatic single nucleotide variants (sSNVs) and indels were called using MuTect and Pindel, respectively, and copy number changes were assessed using an in-house algorithm. In general, these patients had more nonsynonymous mutations per pre-alloSCT sample than reported in other CLL NGS studies (average 30.3; range 8-45), likely related to the significant amount of pre-transplant therapies. Heterogeneous patterns of linear and branched subclonal evolution were seen after alloSCT and DLI in both responders and non-responders. Mutations in several candidate CLL driver genes were seen in this cohort, including SF3B1, SAMHD1, BCOR, EGR2, TP53, and DDX3X. Interestingly, sSNVs in multiple recurrently mutated CLL or cancer census genes (e.g. MAP2K1) rose to levels of detection only after alloSCT or DLI, suggesting they may play a role in immune evasion. In addition, several subclonal genetic variants, including missense mutations in FAM126B and LTBP3, were no longer detected after alloSCT or DLI and may thus represent potential neoantigens. In one treatment-refractory patient, a somatic nonsynonymous clonal CHEK2 mutation was found in 8 longitudinal samples and may represent a novel unique driver mutation. Finally, in one patient who experienced a durable complete remission after DLI, concurrent CLL WES and T-cell receptor beta chain CDR3 NGS was performed, which demonstrated a rapidly evolving T-cell repertoire at the time of complete remission after DLI. For CLL, alloSCT and DLI offer a potentially curative treatment strategy and a better understanding of the genes that confer susceptibility or resistance to these immunotherapies may help unlock the mechanisms that underlie these durable responses. Citation Format: Haven R. Garber, Hannah Beird, Yu Cao, Jianhua Zhang, Rachel Sargent, Pei Lin, Sahil Seth, Xingzhi Song, Huandong Sun, Xizeng Mao, Lisa St John, Karen Clise-Dwyer, Gheath Alatrash, P. Andrew Futreal, Jeffrey J. Molldrem. Long-term subclonal evolution of CLL from immune selective pressure after allogeneic stem cell transplant and donor lymphocyte infusion. [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 LB-222. doi:10.1158/1538-7445.AM2015-LB-222

Targeted therapy against oncogenic BRAF (BRAFV600E) is a promising new therapeutic approach for the treatment of melanoma with up to 80% of patients with stage IV melanoma responding to treatment. Despite these results, the median duration of response is only 8.5 months. Investigators propose combining this therapy with other targeted agents to address redundancy and signaling through different oncogenic pathways in hopes to improve the durability of response. An alternate approach is to combine BRAF-targeted agents with immunotherapy based on evidence that oncogenic BRAF contributes to immune escape. We recently reported data supporting this hypothesis, showing that treatment of melanoma cell lines with MEK or selective BRAFV600E inhibition results in increased expression of melanocyte differentiation antigens (MDAs) which is associated with enhanced recognition by antigen-specific T cells. The purpose of these studies was to validate these findings by assaying expression of MDAs in patients with metastatic melanoma treated with BRAFV600E inhibition. We also sought to test immune cell infiltrate in tumors to analyze host immune response. Patients with metastatic melanoma undergoing treatment with a selective BRAFV600E inhibitor were consented on a tissue procurement protocol approved through the IRB at the Massachusetts General Hospital. Patients underwent treatment with the BRAFV600E inhibitor (PLX4032) per protocol (960 mg given twice daily). Tumor biopsies and blood draws were performed pre-treatment and 10-14 days after initiation of treatment. RNA was harvested and expression of MDAs was assayed by quantitative RT-PCR. The level of MITF, the master transcriptional regulator of melanocyte which controls MDA expression, was also assayed. Immunohistochemistry was performed on formalin-fixed, paraffin embedded tissue for MDAs, as well as CD4 and CD8. The effect of BRAFV600E inhibition on T lymphocyte function was analyzed by assaying fold-expansion and viability in blood samples collected before and during treatment. Treatment with a selective inhibitor of BRAFV600E resulted in significantly increased levels of MDAs (up to 95-fold) which was associated with higher levels of MITF. In addition, CD8 T cell infiltrate also increased after treatment. Of note, T cell function was preserved after treatment with a specific inhibitor of BRAFV600E. These data provide in vivo correlates to our in vitro findings that selective BRAFV600E inhibition enhances MDA expression in melanoma and suggest a possible improved host immune response. The studies provide further support for potential synergy between BRAF-targeted therapy and immunotherapy in the treatment of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 958. doi:10.1158/1538-7445.AM2011-958

Meeting abstracts Despite recent advances in the treatment of metastatic melanoma through targeted and immunotherapy, the majority of patients do not achieve a durable response. Research efforts to better understand responses are underway, and numerous molecular mechanisms of resistance to targeted