Leukemia Antigen Research Articles

ROR1 Specific T Cell Clones from Healthy Individuals Show Common T Cell Receptor Motifs

BackgroundImmunotherapy for CLL with new antibodies or T-cells with modified TCR relies on attractive targets. ROR1 is such a promising target since it is highly overexpressed in CLL. Chimeric antigen receptor engineered T cells and antibodies directed against the extracellular part of ROR1 have already been developed and tested in vitro or in animal models, but still there is no MHC-class I presented peptide serving as target structure for CD8+ T cells (with or without a genetically modified T cell receptor) available.AimThe aim of this study was (1) to identify an immunogenic MHC-class I presented ROR1 peptide, (2) to generate respective ROR1 peptide specific CD8+ T cell clones, and (3) to analyze the nucleotide sequence of the CDR3 region of the expressed alpha and beta T cell receptor chain.ResultsIn mass spectrometric-based analyses of the HLA-ligandome a HLA-B*07 presented ROR1 peptide was identified in primary CLL cells of two patients. Six T cell clones specific for this particular ROR1-peptide were generated from single CD8+ T cells from 2 healthy individuals with 3 T cell clones generated from each donor. Functionality and specificity of those T cell clones were tested in cytotoxicity assays. All 6 dextramer+ CD8+ T cell clones lysed peptide loaded and HLA-B*07+ transduced K562 cells (kindly provided by Lorenz Jahn, [Jahn et al., Blood, 2015 Feb 5;125(6):949-58]). Two selected clones (XD8 and XB6) were tested for their cytotoxic potential against 2 ROR1+ HLA-B*07+ tumor cell lines (with the ROR1 peptide identified by mass spectrometry for both of them) and against 2 primary CLL cell samples. Tested clones showed a significant lysis of the respective target cells. CDR3 regions of the alpha and beta T cell receptor chain were sequenced on a single cell level. The CDR3 alpha region from each of the 3 ROR1 specific T cell clones from donor A showed some similarities to T cell clones derived from donor B (Table 1).ConclusionFor the first time a MHC-class I presented ROR1 peptide antigen is reported. ROR1 positive CLL cells can be targeted by specific HLA-B*07 restricted CTLs. Respective CD8+ T cell clones with anti-leukemic activity from 2 donors share some amino acid motifs of the CDR3 alpha and beta regions. In conclusion, this information provides the possibility of generating ROR1 specific CD8+ T cells with genetically modified T cell receptors for immunotherapy and for tracking those cells after administration with next generation sequencing in peripheral blood samples of patients. Furthermore, data suggest the existence of public TCR motifs in leukemia antigen specific CTLs, which needs to be proven in follow-up experiments with larger cohorts of donors and patients. Finally, the presented strategy to identify leukemia specific peptide antigens for CD8+ T cells might be an attractive method for similar projects. [Display omitted] DisclosuresMiddeke:Sanofi: Honoraria. Schetelig:Sanofi: Honoraria.

Donor Lymphocyte Infusions Correct Deficiency of Naive T Cells and Improve T-Cell Competence after Allogeneic Haematopoietic Stem Cell Transplantation with Lymphocyte Depletion

Lymphocyte-depletion effectively reduces risk of graft versus host disease (GvHD) after allogeneic haematopoietic stem cell transplantation (allo-HSCT), but risk of infections and malignant disease relapse remains high. We have previously reported that pre-emptive donor lymphocyte infusions (pDLI) given to patients after allo-HSCT for myeloid malignancies to reverse falling donor T-cell chimerism improve overall and relapse-free survival. GvHD rates after pDLI were not high and grade rarely severe. To investigate the basis for better outcome after pDLI, we have assessed recovery of lymphocyte subsets, T-cell receptor (TCR) diversity and T-cell functional competence after allo-HSCT with fludarabine and busulphan in cohorts of 59 patients (median age 59) given alemtuzumab for lymphocyte-depletion and 34 patients (median age 58) given anti-thymocyte globulin (ATG).Lymphocytes were significantly less depleted with ATG compared to alemtuzumab (Day 30: Median 3.9 x 108/liter versus 2.3x108/liter, P=0.03) but numbers for both ATG and alemtuzumab remained significantly below the normal range (median 2.34x109/liter for 11 aged-matched healthy volunteers) for at least one year (Day 360 P<0.005: Median 8.35 x 108/liter after ATG; median 1.04 x 109/liter after alemtuzumab). Lymphocyte subset composition was similar after ATG or alemtuzumab, and abnormal. Notable, the T-cell population comprised only memory and effector T cells early after HSCT. These cells expressed significantly higher levels of Ki67 than T cells from healthy volunteers (Day 30 P<0.005: Median CD4 T cells 41.3% Ki67+ after ATG, 66% after alemtuzumab compared to 2.51% for healthy volunteers; median CD8 T cells 18.5% Ki67+ after ATG, 50.8% after alemtuzumab compared to 2.58% for healthy volunteers). This marker is indicative of homeostatic proliferation likely driven by increased levels of IL7 and IL15 detected in the serum of patients early after HSCT compared to healthy volunteers (Day 30 P=0.066 and P<0.005 respectively). Higher frequency of T cells expressing the proliferation marker in patients treated with alemtuzumab was associated with high frequencies of T cells expressing the PD1 marker, indicative of exhaustion (Day 30 P<0.005: Median CD4 T cells 84.0% PD1+ after alemtuzumab compared to 6.35% for healthy volunteers; median CD8 T cells 49.1% PD1+ after alemtuzumab compared to 12.3% for healthy volunteers). Expression of PD1 by T cells was near normal in patients treated with ATG.Naïve T cells were typically absent for at least six months after HSCT following lymphocyte depletion with ATG or alemtuzumab, and any subsequent recovery was poor. In contrast, the naïve T-cell population increased rapidly in patients after pDLI (n=18). Six of these patients received pDLI early after HSCT (at 3-5 months) and naïve T-cell recovery was significantly enhanced at six months compared to patients that did not receive pDLI (Day 180 P<0.005: Median 19.25% naïve CD4 T cells compared to 1.36%; median 23.5% naïve CD8 T cells compared to 3.48%).Naïve T cells are the main source of repertoire diversity and responsible for responses to antigens not previously encountered. Analysis of the TCR β chain repertoire of five patients by deep sequencing revealed that pDLI boosts repertoire diversity. For example, unique TCR β chain sequences increased 31-fold in 150 days after pDLI compared to a 2-fold increase during a similar period for another patient that did not receive DLI. Furthermore, instances of emergence of public clonotypes specific for CMV or EBV that were not detected before DLI were seen in virus-positive patients whose donors were virus-negative. Emergence and rapid expansion of donor-derived clonotypes to frequencies up to 6.75% suggests that naïve T cells present in the DLI had been primed upon encounter with virus in the patient. In vitro stimulation with overlapping 15-mer peptide libraries for CMV antigens and EBV antigens followed by assessment of activation marker expression and interferon-γ, MIP-1β, and TNF-α production showed that virus-specific T-cell responses increased in magnitude and poly-functionality after DLI.These findings show that DLI replenishes naïve T cells and restores ability to respond to viral antigens previously unseen. By inference, this may extend to leukaemia antigens and underlie the reduced rate of malignant disease relapse seen in patients given pDLI. DisclosuresNo relevant conflicts of interest to declare.

The Distribution of T Memory Stem Cells in Cord Blood, Peripheral Blood from Healthy Individuals and Patients with Leukemia/Lymphoma

Human T memory stem cells (TSCM) are a unique memory T cell subset with enhanced self-renewal capacity and the ability to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. The distribution of TSCM cells in umbilical cord blood and leukemia/lymphoma remain unclear. In this study, we investigated the distribution of TSCM and other memory T (TM) subsets in the cord blood (CB) (n = 11), peripheral blood (PB) of lymphoblastic leukemia/lymphoma (LL) (n = 7), Myeloid leukemia (ML) (n = 7) as well as healthy individual (HI) (n = 16) by multicolor flow cytometry.There were high percentage of CD4+ TSCM and CD8+ TSCM in CB group than that in HI-PB, however, fewer CD4+T central memory (TCM), CD4+T effector memory (TEM), CD8+ TEM and CD8+ terminal effector (TE) cells in CB group than in HI-PB (Figure 1). When compared LL or ML group with HI group, there was an increase in the CD4+ TSCM in the ML group, while the frequencies of TCM, TEM, or TE were similar. However, the percentage of memory T cell subsets in patient group was quite disperse, for example, CD4+ TSCM could range from 0.35% to 6.52% (LL) and from 0.67% to 3.71%(ML), the percentage of CD8+ TSCM could even reach to 10.43% and 7.62% in LL and ML group respectively, the same dispersion also exist in other T cell subsets of patient group but not in the blood and cord blood from HI. According to individually analyze distribution of T cell subsets in each patients, a higher percentage of CD4+ TSCM was detected in a B-ALL patient in the LL group comparing with HI, combined with significant lower percentage of CD4+ TCM, TEM and TE (Figure 2 A, L3), similar distribution pattern of CD8 T cell subsets also detected in the same B-ALL patient (Figure 2B, L3). Interesting, a case with BCR-ABL+ B-ALL include in this study showed a relative normal distribution pattern (Figure 2 A and B, L5). There were four patients in LL group showed a relative higher percentage of CD8+ TCM (Figure 2 B, L1, L2, L4 and L6), while two of them followed with quite lower TE in PB (Figure 2 B, L2 and 6). In the ML group, relative higher CD4+ TSCM combined with lower TEM was also detected in a case with AML-M5 (Figure 2 C, ML-3). In terms of CD8 T cell subsets distribution in ML group, we found two patients with AML-M5 possessing a higher CD8+ TSCM proportion compare with HI and other patients in ML group (Figure 2 D, ML-2 and 3).In conclusion, we characterized the distribution of different memory T cell subsets in healthy CB and PB, the reason of high percentage of TSCM in CB is needed further investigation. In addition, the higher CD4+ TSCM detected in ML group and individual cases of LL group may related to the leukemia antigens they encountered, whether such a characteristic would impact their prognostic worth further investigation. Overall, the distribution of TSCM and other memory T cell subsets in blood between healthy and leukemia is not shown obviously difference, while, the large difference among individual patients reflect the various immunosuppression degree and immune reaction ability to tumor antigens. To characterize the relationship between distribution pattern of TSCM and TM cells and immune status of patients will benefit to developing more suitable individually immune treatment strategies.Acknowledgments: The study was supported by grants from the National Natural Science Foundation of China (Nos. 81270604, U1301226, and 81400109), the China Postdoctoral Science Foundation (No. 2013M540685), Guangdong Natural Science Foundation (Nos. S2013040016151 and S2013020012863), the Foundation for High-level Talents in Higher Education of Guangdong, China (No. [2013]246-54), and the Guangzhou Science and Technology Project Foundation (201510010211). [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impaired mammary development in tamoxifen-treated prepubertal heifers is associated with altered development and morphology of myoepithelial cells

Prepubertal mammary development involves elongation and branching of ducts and stromal tissue remodeling. This process is closely linked with ovarian and pituitary hormones, growth factors, and local regulators. Accumulating evidence suggests that the myoepithelial cells also play a role in ductal development in addition to their well-recognized importance in the milk ejection reflex. Following reports that myoepithelial cells changed in correspondence with decreased mammary growth after ovariectomy of prepubertal heifers, we evaluated myoepithelial cells in mammary tissue collected from prepubertal heifers treated with the antiestrogen tamoxifen. Briefly, heifers were given placebo (n=7) or tamoxifen (n=8; 0.3mg/kg per day) beginning on d 28 of life until the animals were euthanized on d 120. Tissues were collected from each of 3 zones (near the gland cistern, midway between the gland cistern and mammary fat pad, and at the interface of the parenchyma and mammary fat pad). Samples were processed to measure expression of transformation-related protein 63 (p63), smooth muscle actin, and common acute lymphoblastic leukemia antigen. We found that smooth muscle actin and common acute lymphoblastic leukemia antigen were expressed in the cytoplasm and p63 in the nuclei of myoepithelial cells. In concert with a 50% impairment in mammary growth after tamoxifen, we found that the number of myoepithelial cells around developing mammary ducts was reduced. But the average intensity of p63 expression per nucleus was not affected. We used the very distinct and exclusive staining of p63 in myoepithelial cell nuclei to capture hundreds of nuclear images for subsequent analysis using CellProfiler software. From this image analysis, we found that the area of myoepithelial cell nuclei and perimeter distances were reduced by tamoxifen. When nuclei were classified based on nuclear shape (eccentricity), we found differences in area, perimeter, and patterns of p63 expression based on Zernike number evaluations as well as treatment differences within each shape classification. These data provide support to the concept that myoepithelial cells are also the involved in mammary development in the prepubertal bovine mammary gland and that use of multispectral imaging combined with image analysis software can provide quantitative data to better understand the complex cellular interactions that ultimately regulate mammary morphogenesis in the bovine.

Abstract 1497: Development of a novel TCR-like bi-specific T-cell engager targeting endogenous PR1/HLA-A2 leukemia antigen

Abstract PR1 (VLQELNVTV) is a HLA-A2 restricted peptide derived from endogenous tumor associated antigens proteinase-3 and neutrophil elastase that are aberrantly expressed in myeloid leukemia blasts. Previously, we have developed a high affinity T-cell receptor-like monoclonal antibody (h8F4) specific for PR1/HLA-A2, and demonstrated the effective killing of myeloid leukemia blasts via antibody dependent cellular cytotoxicity (ADCC). To improve the therapeutic efficacy of h8F4, we adopted a novel antibody engineering strategy, bi-specific T-cell engaging (BiTE) antibody that can bring T cells in close proximity to leukemia blasts through the binding of both PR1/HLA-A2 and CD3, resulting in T-cell mediated cytolysis of leukemia blasts. Here we successfully generated h8F4-BiTE proteins using a eukaryotic expression system and investigated binding characteristics, target specificities, and subsequent T-cell activation with the h8F4-BiTE. The h8F4 BiTE showed binding affinities in the nM range for PR1/HLA-A2 and CD3 moiety of T cells. Within 18 hours of h8F4-BiTE engagement with PR1 pulsed T2 cells, polyclonal T cells from healthy donors up-regulated surface expression of CD69 and down-regulated CD3 in a PR1/HLA-A2 specific and concentration dependent manner, suggesting successful T-cell activation. In conclusion, we developed a novel h8F4 BiTE antibody that recognizes both the CD3 on T cells and endogenous PR1/HLA-A2 complexes on myeloid leukemia, and demonstrated high affinity bindings and subsequent T cell activation. We envision our novel TCR-like BiTE antibody will provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies. Citation Format: Amanda Cernosek Herrmann, Jin S. Im, Sijie Lu, Anna Sergueeva, Jeffrey Molldrem. Development of a novel TCR-like bi-specific T-cell engager targeting endogenous PR1/HLA-A2 leukemia antigen. [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 1497.

ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation.

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

Dysregulation of iCD8α cells promotes susceptibility to intestinal inflammation

Abstract iCD8α cells are a population of innate immune cells present in the intestinal intraepithelial lymphocyte (IEL) compartment, characterized by the expression of CD8αα. We have shown that iCD8α cells play a critical role during immune responses in the intestinal mucosa, however, little is known about the role of iCD8α cells during inflammatory processes of the intestines. The thymus leukemia (TL) antigen is predominantly expressed by intestinal epithelial cells and preferentially interacts with CD8aa. We have shown that CD8αα+ IEL from TL-deficient mice have increased cytokine production and proliferation, indicating that TL controls CD8αα+ IEL effector functions. Rag-deficient mice facilitate the study of innate immune cells, and are an excellent model for the study of iCD8α cells because these cells are the only CD8αα+ IEL present in these mice. Here we present evidence showing that TL regulates the functions of iCD8α cells. Using the anti-CD40 antibody model of colitis, we show that TL−/−Rag-2−/− mice lose more weight, have higher mortality, have more colon damage, and present higher levels of pro-inflammatory cytokines (IFN-γ, IL-6 and OPN) than colons from Rag-2−/− mice. Interestingly, we show that colons and iCD8α cells from TL-deficient mice produce more granzyme than colons or iCD8α cells from TL-competent mice. Moreover, stimulation of iCD8α cells with the pro-inflammatory cytokine IL-12 in vitro induces granzyme secretion. Therefore, we propose that the increased colon inflammation observed in TL-deficient mice is due to: a) dysregulation of iCD8α cells by the lack of TL, and b) increased granzyme secretion by iCD8α cells which may serve as a pro-inflammatory signal.

Emerging roles of NudC family: from molecular regulation to clinical implications.

Nuclear distribution gene C (NudC) was first found in Aspergillus nidulans as an upstream regulator of NudF, whose mammalian homolog is Lissencephaly 1 (Lis1). NudC is conserved from fungi to mammals. Vertebrate NudC has three homologs: NudC, NudC-like protein (NudCL), and NudC-like protein 2 (NudCL2). All members of the NudC family share a conserved p23 domain, which possesses chaperone activity both in conjunction with and independently of heat shock protein 90 (Hsp90). Our group and the others found that NudC homologs were involved in cell cycle regulation by stabilizing the components of the LIS1/dynein complex. Additionally, NudC plays important roles in cell migration, ciliogenesis, thrombopoiesis, and the inflammatory response. It has been reported that NudCL is essential for the stability of the dynein intermediate chain and ciliogenesis via its interaction with the dynein 2 complex. Our data showed that NudCL2 regulates the LIS1/dynein pathway by stabilizing LIS1 with Hsp90 chaperone. The fourth distantly related member of the NudC family, CML66, a tumor-associated antigen in human leukemia, contains a p23 domain and appears to promote oncogenesis by regulating the IGF-1R-MAPK signaling pathway. In this review, we summarize our current knowledge of the NudC family and highlight its potential clinical relevance.

The small leucine rich proteoglycan fibromodulin is overexpressed in human prostate epithelial cancer cell lines in culture and human prostate cancer tissue

Fibromodulin is a small leucine-rich proteoglycan important for extracellular matrix organization and essential for tissue repair in multiple organs. The main function of this proteoglycan is the regulation of collagen fibrillogenesis; however, more recently described roles for fibromodulin have expanded to include regulation of angiogenesis, reprogramming of human fibroblasts into pluripotent cells, modulation of TGF-β activity, inflammatory processes and association with metastatic phenotypes. Additionally, fibromodulin has been identified as a novel tumor-associated antigen in leukemia, lymphoma, and leiomyoma. Knowledge about its expression in the prostate is limited. Fibromodulin expression was analyzed in two different malignant and one non-tumorigenic prostatic cell lines in culture, and in benign and malignant human prostate tissue. Expression was analyzed by real time PCR, immunocytochemistry, and immunohistochemistry. DNA sequencing was performed on a PCR fragment amplified with primers specific for the FMOD gene from cDNA obtained from the cultured cell lines. Both immunostaining and real time PCR analysis of cell lines indicated that fibromodulin was differentially expressed in the cancerous cell lines compared to the non-tumorigenic cell line. Likewise, cancerous tissue expressed significantly higher levels of intracellular fibromodulin compared to matched, benign tissue from the same patients, as well as compared to tissue from patients with only benign disease. The expression of fibromodulin was higher in prostatic cancer cells (cell-lines and human tissue) than in normal/benign prostatic cells. Additional studies are required to determine the biological and clinical significance and whether this proteoglycan has a role in carcinogenesis of the prostate or in prostate cancer related inflammatory processes.

Adoptive immunotherapy utilizing cancer antigen-specific T-cell receptors.

Synthetic immunology based on rapidly-advancing gene-engineering and immunobiology has made novel anticancer adoptive immunotherapies, using gene-modified T lymphocytes to express cancer antigen-specific receptors, a reality. Various technological innovations have overcome recent difficulties and achieved clear and long-lasting clinical efficacy against tumors, while seeking more powerful effector gene-modified T cells has yielded serious treatment-related adverse events. In this article, along with introducing our clinical trial for a novel anti-leukemia adoptive immunotherapy regimen using gene-modified autologous lymphocytes to express leukemia antigen Wilms Tumor 1(WT1)-specific T cell receptor (TCR) against refractory acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS), we provide an overview of the current status of this emerging treatment option and discuss its future form in the context of neoantigens encoded by mutated genes in cancer cells and immune checkpoint inhibitors.

Latest in Clinical Application of CAR Cell Therapy for B-cell Malignancy and Transplantation

Unparalleled remission rates in patients with chemorefractory B-ALL treated with CD19-CAR T cells illustrate the potential for immunotherapy to eradicate chemoresistant cancer. CD19-CAR therapy is poised to fundamentally alter the clinical approach to relapsed B-ALL and ultimately may be incorporated into frontline therapy. Despite these successes, as clinical experience with this novel modality has increased, so has understanding of factors that limit success of CD19-CAR T cells for leukemia. These insights have implications for the future of cell based immunotherapy for leukemia and provide a glimpse of more global challenges likely to face the emerging field of cancer immunotherapy. Five challenges limiting the overall effectiveness of CD19-CAR therapy will be discussed: 1) T cell exhaustion is a differentiation pathway that occurs in T cells subjected to excessive T cell receptor signaling. A progressive functional decline occurs, manifest first by diminished proliferative potential and cytokine production, following by diminished cytolytic function and ultimately cell death. High leukemic burdens predispose CD19-CAR T cells to exhaustion as does the presence of a CD28 costimulatory signal, while a 4-1BB costimulatory signal diminishes the susceptibility to exhaustion. This biology is likely responsible for limited CD19-CAR persistence observed in clinical trials using a CD19-zeta-28 CAR compared to that observed using a CD19-zeta-BB CAR. 2) Leukemia resistance occurs in approximately 20% of patients treated with CD19-CAR and is associated with selection of B-ALL cells lacking CD19 targeted by the chimeric receptor. Emerging data demonstrates two distinct biologies associated with CD19-epitope loss. Isoform switch is characterized by an increase in CD19 isoforms specifically lacking exon 2, which binds the scFvs incorporated into CD19-CARs currently in clinical trials. Lineage switch is characterized by a global change in leukemia cell phenotype, and is associated with dedifferentiation toward a more stem-like, or myeloid leukemia in the setting of CD19-CAR for B-ALL. These insights raise the prospect that effectiveness of immunotherapy for leukemia may be significantly enhanced by targeting of more than one leukemia antigen. 3) CAR immunogenicity describes immune responses induced in the host that can lead to rejection of the CD19-CAR transduced T cells. Anti-CAR immune responses have been observed by several groups, and mapping is underway to identify the most immunogenic regions of the CAR, as a first step toward preventing this complication. 4) The most common toxicities associated with CD19-CAR therapy are cytokine release syndrome, neurotoxicity and B cell aplasia. Cytokine release syndrome is primarily observed in the setting of high disease burdens and efforts are underway to standardize grading and treatment algorithms to diminish morbidity. Increased information is needed to better understand the neurotoxicity observed in the context of this therapy. Although clinical data is limited, B cell aplasia appears to be adequately treated with IVIG replacement therapy. 5) Technical graft failure (e.g. inadequate expansion/transduction) is a challenge that has received limited attention, primarily since many trials have not reported the percentage of patients in whom adequate products could not be generated. We have observed that technical graft failure is often associated with a high frequency of contaminating myeloid populations in the lymphocyte product and selection approaches designed to eradicate myeloid populations have resulted in improved T cell expansion and transduction. These results suggest that optimization of lymphocyte selection may diminish the incidence of technical graft failure. Disclosures Mackall: Juno: Patents &amp; Royalties: CD22-CAR. Off Label Use: cyclophosphamide.

Expression of JL1 Antigen in Acute Leukemia and Myelodysplastic Syndrome

Background: The JL1 antigen is a novel epitope of CD43, a cell surface glycoprotein of mucin family. JL1 is a differentiation antigen expressed on stage II double positive (CD4+CD8+) human cortical thymocytes. The antigen is not expressed on mature peripheral blood cells or other normal tissues. The anti-JL1 monoclonal antibody binds to human leukemia MOLT-4 cells with 5,100-9,600 binding sites per cell. Preclinical studies have shown the cytotoxic effects of anti-JL1-based immunotoxin against JL1-positive leukemic cells, sparing most normal tissues other than thymocytes and some bone marrow mononuclear cells. Phase I clinical trial of new anti-leukemic agent with an anti-JL1 antibody (Leukotuximab; DiNonA, Korea) is now underway. In this study, we prospectively investigated the JL1 expression in patients with acute leukemia and myelodysplastic syndrome (MDS).Patients & methods: Flow cytometric analysis for the JL1 expression on leukemic blasts was performed using a FACSCanto II (Becton-Dickinson, Sunnyvale, CA, USA). The percent expression of JL1 antigen among leukemic blasts was recorded. Positive JL1 expression was defined if 20% or more leukemic blasts expressed the antigen. Association of JL1 expression with clinical, pathologic, and genetic characteristics was analyzed. Influence of JL1 expression on clinical outcomes of patients was also explored.Results: Between March 2014 and June 2015, a total of 245 adult patients with acute myeloid leukemia (AML, n=170), acute lymphoblastic leukemia (ALL, n=52), and MDS (n=23) were enrolled in this study. Positive JL1 expression was observed in 96 (57.1%) patients with AML, 28 (51.9%) with ALL, and 5 (21.7%) with MDS (P =0.006), while three normal controls showed negative JL1 antigen expression. Interestingly, JL1 expression was positive in all 14 patients with AML M3 with a median expression of 94.3% (range, 60.3-97.8%). In contrast, only 13 (39.4%) of 33 patients with AML with myelodysplasia-related changes (MRC) had positive JL1 expression. In AML patients, positive JL1 expression was significantly associated with CD34- (P =0.003), HLA-DR- (P =0.019), PML-RARA + (P =0.001), FLT3-ITD + (P =0.026), mutated NPM1 (P =0.003), and complex karyotype (3 or more clonal chromosomal abnormalities) (P =0.020). Cytarabine plus anthracycline based chemotherapy was given to 117 patients with AML, and the complete remission (CR) rate was significantly different between 63 JL1 expression positive patients and 54 negative patients (84.1% vs. 59.3%, P =0.003). Positivity of JL1 expression was not significantly associated with overall survival in all patients with AML (median survival, JL1 positive vs. negative, 20.6 vs. 18.2 months, P =0.489). In ALL patients, positive JL1 expression was significantly associated with CD13- (P =0.032) and the CR rate was not significantly different by JL1 expression. JL1 expression was measured twice or more in 85 patients during their clinical courses and positivity of JL1 expression was not changed in 61 (71.8%) (P =0.307). Five MDS patients progressed to AML and JL1 expression was changed in only one patient (JL1 positive to JL1 negative).Conclusion: JL1 was expressed in around 50% of patients with AML or ALL while less frequent expression of JL1 was observed in MDS and AML with MRC. JL1 expression was significantly associated with some immunophenotypic and genetic features, especially PML-RARA +. JL1 expression was significantly associated with the CR rate of AML patients. Expression of JL1 seems to be stable during clinical courses. Our data suggest that immunotherapeutic approach targeting JL1 antigen may be feasible in significant proportion of patients with acute leukemia and MDS. DisclosuresKim:Dinona Institute, Dinona Inc.: Employment. Yoon:Dinona Institute, Dinona Inc.: Employment. Jung:Dinona Institute, Dinona Inc.: Employment.

Induction of Cancer Testis Antigen Expression in Circulating Acute Myeloid Leukemia Blasts Following Hypomethylating Agent Monotherapy

Background: The development of immunotherapeutic strategies for the treatment of leukemia has shown considerable promise but targeting suitable myeloid leukemia antigens remains a significant challenge. Cancer testis antigens (CTA) have been identified as promising targets for immunotherapy in solid tumors, but dense promoter methylation silences their expression in acute myeloid leukemia, limiting their potential as targets. Patients with acute myeloid leukemia are frequently treated with hypomethylating agents (HMAs) and previous studies have established that exposure of leukemia cell lines to HMAs induces expression of CTAs. In this study, we tested the hypothesis that patients receiving HMAs exhibit increased expression of CTAs.Methods: AML patients receiving decitabine were enrolled under an IRB-approved protocol (Roswell Park) or with approval of the Ethics Committee (University of Freiburg). Peripheral blood samples were serially collected prior to decitabine treatment and two to four times per week during their first cycle of decitabine therapy (20 mg/m2 per day for 10 days).Results: We analyzed expression and demethylation of CTA genes in peripheral blood samples serially isolated from AML patients (n = 5) during and following their first cycle of decitabine therapy (20 mg/m2 per day for 10 days). These patients demonstrated induction of MAGEA1 (1/5 patients), XAGE1 (3/5), MAGEA3/A6 (3/5), and NY-ESO-1 (5/5). Western blot analysis demonstrated increased expression of NY-ESO-1 protein following decitabine treatment. The induction of NY-ESO1 mRNA was confirmed in an independent group of AML patients (5/7, treated at University of Freiburg) receiving decitabine. Since NY-ESO-1 is an established cancer immunotherapy target with clinically translatable vaccines in development, we further examined induced NY-ESO-1 expression in a larger cohort of AML patients. We performed sodium bisulfite pyrosequencing to quantify changes in NY-ESO-1 promoter methylation pre-decitabine to the post-decitabine nadir time point for each patient (n = 22). There was a statistically significant decrease in NY-ESO-1 promoter methylation (p < 0.001). Prior to decitabine treatment, 18% (4/22) of samples exhibited detectable levels of NY-ESO-1 mRNA as measured by quantitative PCR. Following decitabine therapy, 78% (17/22) of samples had detectable levels of NY-ESO-1 mRNA. Treatment with decitabine was associated with a significant increase in NY-ESO-1 expression when comparing pre-treatment expression to the maximum expression at any time interval post decitabine (p < 0.0001). We then tested whether levels of NY-ESO-1 induction were different in patients who demonstrated a clinical response compared with those who did not. Overall, 7/22 patients (32%) demonstrated a clinical response to decitabine. 6/7 patients who clinically responded to decitabine demonstrated a significant increase in NY-ESO-1 mRNA (p < 0.03). Crucially, NY-ESO-1 mRNA levels were also significantly increased in 11 out of the 15 patients that did not demonstrate a response to decitabine (p = 0.001). To test whether AML blasts expressing NY-ESO-1 could induce a T-cell response, we stimulated HLA compatible NY-ESO-1- specific CD8+ T cells with AML blasts isolated from HLA-A*0201+ AML patients before and after decitabine treatment. T-cell responses were determined by intracellular cytokine staining (IFN-γ, TNF-α and IL-2) and the expression of CD107a/b, a marker for T-cell degranulation. Co-culture of AML blasts harvested post-decitabine, resulted in increased levels of IFN-γ, TNF-α, IL-2, and CD107a/b in HLA-A*0201/NY-ESO-1157-165 tetramer+ CD8+ T-cells in three of the four patients studied, compared to T-cells co-cultured with AML blasts from the same four patients obtained prior to decitabine exposure.Conclusion: We observed enhanced expression of NY-ESO-1 in AML patients receiving decitabine and this induction was sufficient to produce a cytotoxic response in HLA-compatible antigen specific T-cells. A majority of patients who did not respond to decitabine still exhibited an increase NY-ESO-1 mRNA, suggesting that immunotherapies that target NY-ESO-1 have the potential to be effective even in patients who have not demonstrated a prior response to decitabine. These data support the combination of decitabine with immunotherapeutic approaches targeting NY-ESO-1 and other CTAs in myeloid cancer. DisclosuresGriffiths:Alexion Pharmaceuticals: Honoraria; Astex: Research Funding; Celgene: Honoraria. Off Label Use: Decitabine is in routine clinical use in the United States for the management of unfit/elderly patients with AML. Patients received decitabine as standard of care off label in the hospital per Bloom et al PNAS 2010.

Fucosylated Antigen-Specific Cytotoxic T Lymphocytes Demonstrate Enhanced Killing of Leukemia Targets

IntroductionImmunotherapy using cytotoxic T lymphocytes (CTL) has shown efficacy in the management of leukemia. However the efficacy of CTL, whether they are engineered and adoptively transferred or administered as part of allogeneic stem cell transplantation, must be balanced by their off-target toxicities, which at times can be lethal. Fucosylation, which is mediated by fucosyl transferases, is a process by which fucose sugar groups are added to cell surface receptors. Fucosylated T cells have been shown to preferentially home to inflamed tissues, including bone marrow. In view of recent data showing that fucosylation with fucosyltransferase (FT)-VI facilitates homing of regulatory T cells (T-regs) to inflamed tissues and cord blood engraftment into the bone marrow, we hypothesized that fucosylation could enhance the efficacy of CTL that target leukemia antigens. In this study, we tested whether ex vivo fucosylation of CTL that target the HLA-A2 restricted leukemia peptides, CG1 (derived from cathepsin G) and PR1 (derived from neutrophil elastase and proteinase 3), with the novel enzyme FT-VII enhances their migration and anti-leukemia functions.Experimental designCG1- and PR1-CTL were generated using standard methodologies. Fucosylation was achieved by incubating T cells with FTVII enzyme and GDP fucose (Targazyme). To study migration, fucosylated and non-fucosylated CTL were passed through chambers coated with a HUVEC barrier and migrated CTL were detected using cell fluorescence. To examine CTL surface markers, cells were stained for standard co-stimulatory and adhesion molecules and were analyzed using flow cytometry. Calcein AM cytotoxicity assays were used to determine the effects of fucosylation on CTL killing of target cells. In vitro effects of fucosylation on leukemia-CTL specificity was accomplished using standard CFU assays. For in vivo assessment of fucosylation on activity of CTL, NSG mice were engrafted with U937-A2 human acute myeloid leukemia (AML) cells or primary AML and were treated with intravenous injections of 5.0 x 105 fucosylated or non-fucosylated CTL. Mice were followed twice weekly and were sacrificed for bone marrow and tissue analysis at prespecified time points or when they became moribund.ResultsFucosylated CG1-CTL and PR1-CTL showed approximately 2-fold higher migration through the HUVEC cell barrier compared to non-fucosylated CTL. Analysis of T cell surface expression of chemokine/adhesion molecules showed an approximately a 5-fold increase in CD49d and CD195, and a 50% increase in CXCR1 and CXCR3 following fucosylation. Fucosylation enhanced the cytotoxicity of leukemia specific-CTL against primary HLA-A2+ leukemia and HLA-A2+ U937 cells at increasing effector to target ratios. For primary patient AML, we show enhanced leukemia killing by fucosylated-PR1-CTL in comparison with non-fucosylated-PR1-CTL at the 20:1 effector to target (E:T) ratio (25-fold higher killing ) and the 10:1 E:T ratio (4-fold higher killing). Similar results were seen using the U937-A2 AML cell line favoring fucosylated-CG1-CTL: 20-fold higher killing at 20:1 E:T ratio and a 9-fold higher killing at the 10:1 E:T ratio. In vitro CFU assays using HLA-A2+ healthy donor bone marrow showed no change in the specificity of the antigen specific CTL following fucosylation. Specifically we show 283 and 295 colonies in the fucosylated and non-fucosylated CG1-CTL groups, respectively (P >0.05). These were also compared to irrelevant peptide HIV-CTL, which demonstrated 286 and 269 CFUs in the fucosylated and non-fucosylated HIV-CTL groups, respectively (P >0.05). In vivo experiments using CG1-CTL against primary AML showed 5-fold higher killing of AML by fucosylated CTL vs. non-fucosylated CTL. Similar results were also seen using U937-A2 AML targets.ConclusionFucosylation with FT-VII enhances the efficacy of leukemia-targeting CTL against primary human AML and AML cell lines. These data demonstrate a novel approach to enhance the efficacy of antigen specific CTL that could be used in adoptive cellular immunotherapy approaches for leukemia. DisclosuresNo relevant conflicts of interest to declare.

Donor Lymphocytes Depleted of Alloreactive T-Cells (ATIR101) Reduce Transplant Related Mortality and Improve Overall Survival in Haploidentical HSCT for Patients with AML and ALL, Using an Immunosuppressant-Free Transplant Regimen

IntroductionHaploidentical family donors are becoming an effective alternative for patients in need of an hematopoietic stem cell transplantation (HSCT) but lacking an HLA-matched donor. However, to prevent graft-versus-host disease (GVHD), intensive in-vivo or ex-vivo T-cell depletion is often utilised, resulting in slow immune reconstitution, frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival.To overcome these limitations, we have developed a strategy that allows additional donor lymphocytes to be infused post-HSCT without the risk of inducing severe GVHD and maintaining the ability to react against infections and leukemic cells.Patients and MethodsIn this open-label, multi-centre phase 2 clinical trial (CR-AIR-007; NCT01794299), 23 patients with high-risk AML or ALL were enrolled. Patients underwent myeloablative conditioning and were given a CD34+ selected stem cell graft from a haploidentical family donor. In addition, donor lymphocytes from the same donor were processed using a selective photodepletion technology, creating a donor lymphocyte product depleted of alloreactive T-cells (ATIR101). ATIR101 was to be given to patients at 28-32 days post-HSCT at a fixed dose of 2x106 CD3+ cells/kg. No post-transplant GVHD prophylaxis was administered.The primary endpoint of the study is transplant-related mortality at 6 months post-HSCT. Patients are followed to determine disease-free and overall survival.Patients undergoing a similar T-cell depleted haploidentical HSCT without ATIR infusion (CR-AIR-006; NCT02188290) were used as controls. Most centers providing the historic control data also participate in this phase 2 trial with ATIR101.ResultsTwenty-three patients, median age of 41 years (range 21 - 64), have been enrolled into this study. Seventeen patients, 7 male and 10 female, had AML, 12 in CR1 and 5 in CR2 at the time of HSCT. Six patients, 5 male and 1 female, had ALL, 4 in CR1 and 2 in CR2 at the time of HSCT.Conditioning regimen consisted of a] TBI (1200 cGy; n=10) or b] melphalan (120 mg/m2; n=12), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x 5d) and ATG (2.5mg/kg x 4d). No patient experienced graft rejection, with neutrophil and platelet engraftment achieved at a median of 11 and 12 days, respectively (range 8-18, range 9-35). 21 patients to date received ATIR101, at a fixed dose of 2x106 CD3+ cells/kg at a median of 28 days (range: 28-73 days) post-transplant. Median follow-up is 270 Days post-HSCT.No patient developed grade III-IV acute GVHD. Two cases of grade II acute GVHD were reported thus far. The onset of these two acute GVHD cases was delayed, at day 173 and day 247 post-HSCT, 145 and 219 post ATIR respectively.No patient died within the first 100 days post-HSCT. There are 3 deaths as a result of transplant related mortality within 6 months post-HSCT (primary endpoint).When compared to the matched historic control group (N=35), TRM was significantly lower in patients given ATIR101 after a T-cell depleted haplotransplant with a 6-month TRM for HSCT + ATIR101 of 17% versus 34% for HSCT only (Figure 1a).Figure 1a: Kaplan Meier of Transplant related mortality: HSCT + ATIR101 vs HSCT alone (p=0.0075), data as per 3 August 2015.Thus far, in CR-AIR-007 study, only one patient experienced a relapse, which occurred at 90 days post-HSCT and resulted in death of the patient on day 122.The overall survival of patients given ATIR101 is significantly improved, compared to the control group, with a 1-year survival of 67% in the HSCT + ATIR101 group and 20% in the control group (Figure 1b).Figure 1b: Kaplan Meier of Overall Survival: HSCT + ATIR101 vs HSCT alone (p=0.0032), data as per 3 August 2015.ConclusionsAddition of ATIR101 to a T-cell depleted haploidentical HSCT protocol significantly improves transplantation outcome, with reduced TRM and improved OS. Specifically, no patient died during the first 100 days post-HSCT. Administration of ATIR101 at the dose of 2x106 cells/kg does not induce severe GVHD, confirming the efficacy of alloreactive T-cell elimination from the donor lymphocyte infusion. We attribute the reduced TRM (mainly infections in the control group) to the additive immunological protection afforded by ATIR101. Moreover, the low number of relapses observed thus far is most encouraging and supports the preservation within ATIR101 of T-cells able to recognize leukemic antigens. [Display omitted] [Display omitted] DisclosuresRoy:Kiadis Pharma: Consultancy, Research Funding; Novartis: Honoraria. Rudiger:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Rovers:Kiadis Pharma: Employment.

Plasma Neprilysin Concentrations: A New Prognostic Marker in Heart Failure?

Neprilysin, also known as neutral endopeptidase (NEP), endopeptidase 24.11, CD10, enkephalinase, and common acute lymphoblastic leukemia antigen (CALLA), is a membrane-bound metalloendopeptidase (EC 3.4.24.11) that has recently sprung to renewed prominence as a target in cardiovascular therapeutics. Numbered among the many substrates to NEP are multiple vasoactive peptides, including the cardiac natriuretic peptides, with important roles in regulating pressure and volume status in health and disease. In the 1990s, NEP inhibitors were combined with angiotensin converting enzyme inhibitor (ACEI) therapy with the hope of adding the expected multifaceted benefits of enhanced plasma and tissue natriuretic peptide levels to the proven benefits of ACEI-related suppression of adverse renin-angiotensin-aldosterone system activation. After initial promise from trials in hypertension and heart failure, drug development was halted due to an unacceptable incidence of angioneurotic edema. The current renewed interest in NEP reflects the recent success of a new combination, that of inhibition of NEP plus angiotensin II type 1 receptor blockade. The PARADIGM trial tested the NEP inhibitor sacubitril combined with the angiotensin receptor blocker valsartan (LCZ696) in chronic heart failure with reduced ejection fraction. The new treatment was significantly beneficial with respect to all important clinical endpoints and was associated with no excess of important adverse effects and in fact caused less renal failure and hyperkalemia than established evidence-based treatment with the ACEI enalapril. The results point to this strategy being the greatest advance in the pharmacotherapy of chronic heart failure in the last 20 years. In addition to its widespread tissue-based, membrane-bound form, NEP exists in a circulating nonbound form that retains catalytic activity. In the article published in Revista Espanola de Cardiologia, Bayes-Genis et al build on previous work to confirm their original finding that plasma NEP concentrations have

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis.

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.

Abstract 4705: Late ALL relapse following CD19 CAR immune-pressure demonstrates reversible pan-antigen loss

Abstract Despite remarkable remission rates in relapsed and refractory acute lymphoblastic leukemia (ALL) following CD19 chimeric antigen receptor (CAR)-T cells or CD3-CD19 bispecific constructs, relapse of CD19 negative leukemic blasts occurs in approximately 10-20% of cases. The mechanism of antigen loss under CD19-targeted immune pressure on pre-B ALL has yet to be elucidated. Although xenograft models have been excellent tools for pre-clinical studies, the development of xenogeneic GVHD has precluded the ability to study late leukemic relapse under CD19-targeted immunotherapeutic pressure. Thus, we performed such studies in a murine transplantable pre-B ALL (CD19+ B220+ CD22+ sIgM- CD127+ CD43+) in immunocompetent mice that induces lethality of recipients within 15-25 days without therapy. Treating ALL bearing mice with a murine CD19 CAR T cells (CART), resulted in prolonged remissions followed by CART dose-dependent relapses varying between 50 days to &amp;gt;200 days post treatment. All relapses were negative for CD19 expression with earlier relapses maintaining similar pre-B cell surface marker phenotype of the parent ALL. However, relapses longer than 100 days consistently showed a dramatic alteration in phenotype, with loss of CD22, B220 and CD127, stability of CD45 and CD43, and partial gain of cKIT, resembling a more immature B cell or common lymphoid progenitor. While early relapses had normal mRNA levels of CD19 despite loss of surface antigen, late antigen loss correlated with low CD19 mRNA, along with other alterations in pre-B cell signature on microarray. This late antigen negative leukemia took longer time to engraft in secondary recipients, compared to the parent cell line. However, serial transplantation without immune-pressure resulted in regain of CD19 and CD22 antigens with tertiary recipients demonstrating leukemia phenotype and time to engraftment similar to the parent cell line. Interestingly, in-vivo reacquisition of CD22 always preceded regain of CD19. To further study loss of B cell antigen, we co-cultured CAR T cells with both human and murine leukemia. Using CD19 CART, we observed rapid CART dose-dependent loss of surface CD19 followed by partial loss of surface CD22, but not other leukemia markers, in both systems. Interestingly, antigen loss was not seen when co-culturing leukemia with anti-CD22 CART. Suboptimal dose of CD22 CART in an NSG mouse model of human leukemia did generate a CD22-CD19- late relapse. We conclude that loss of CD19 may be an early isolated phenomenon, while prolonged immune-pressure drives an mRNA-regulated loss of CD19 accompanied by fundamental alterations in leukemia antigens. We also demonstrated that CD22 loss requires previous loss of CD19. Altogether, as antigen-directed immunotherapies become more common in the clinic, these findings have implications for the optimization of CAR therapy, particularly if antigen loss can be regulated therapeutically. Citation Format: Elad Jacoby, Yinmeng Yang, Chris D. Chien, Waleed Haso, Haiying Qin, Terry J. Fry. Late ALL relapse following CD19 CAR immune-pressure demonstrates reversible pan-antigen loss. [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 4705. doi:10.1158/1538-7445.AM2015-4705

Editorial: "Cancer Immunotherapy: Lights and Shadows".

Editorial: "Cancer Immunotherapy: Lights and Shadows".

A novel antibody-drug conjugate targeting SAIL for the treatment of hematologic malignancies.

Although several new therapeutic approaches have improved outcomes in the treatment of hematologic malignancies, unmet need persists in acute myeloid leukemia (AML), multiple myeloma (MM) and non-Hodgkin's lymphoma. Here we describe the proteomic identification of a novel cancer target, SAIL (Surface Antigen In Leukemia), whose expression is observed in AML, MM, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). While SAIL is widely expressed in CLL, AML, MM, DLBCL and FL patient samples, expression in cancer cell lines is mostly limited to cells of AML origin. We evaluated the antitumor activity of anti-SAIL monoclonal antibodies, 7-1C and 67-7A, conjugated to monomethyl auristatin F. Following internalization, anti-SAIL antibody–drug conjugates (ADCs) exhibited subnanomolar IC50 values against AML cell lines in vitro. In pharmacology studies employing AML cell line xenografts, anti-SAIL ADCs resulted in significant tumor growth inhibition. The restricted expression profile of this target in normal tissues, the high prevalence in different types of hematologic cancers and the observed preclinical activity support the clinical development of SAIL-targeted ADCs.