Leukemia Antigen Research Articles

Prognostic role and therapeutic susceptibility of cathepsin in various types of solid tumor and leukemia: A systematic review.

Cathepsins (CTSs) are multifunctional proteins that can play prominent roles in cancer progression and metastasis. In this systematic review, we compared the prognosis of CTS subtypes overexpression in leukemia and solid tumors, and investigated the effect of different factors on CTS prognosis. We systematically searched published articles indexed in PubMed, Scopus, Cochrane library, ISI Web of Science, and EmBase databases from February 2000 until January 2020. Among the selected leukemia and solid tumors studies, overexpression of CTS subtypes in newly diagnosed and treated patients were with poor prognosis in 43 studies (79.6%) and with good prognosis in 9 studies (16.6%). However, there were 2 studies (3.8%) with either good or poor prognosis, depending on conditions and caner stage and host cell. The relation between CTS and human leukocyte antigen (HLA) in leukemia and solid tumors was mentioned in 7 studies (13%). Overexpression of CTS subtypes in all new case patients had contributed to the induction of poor prognosis. It seems that CTS subtypes, based on the type of cancer and its stage, the type of host cells, and the probable relation with HLA, breed good or poor prognosis in patients with cancer. Therefore, monitoring the overexpression of CTS subtypes and determining the effect of each of these factors on CTS prognosis could be helpful in predicting cancer prognosis both in newly diagnosed or under treatment patients. They could also be useful in finding ways for improving the efficiency of contemporary therapeutic strategies in various types of leukemia and solid tumors.

Direct Integration of Anti-CD19 Chimeric-Antigen-Receptor (CAR) T Cells Early after Post-Transplantation Cyclophosphamide (PTCy) in Murine MHC-Haploidentical Hematopoietic Cell Transplantation (HCT) Exerts Anti-Tumor Effects without Causing Graft-Versus-Host Disease (GVHD)

Anti-CD19 CAR T cells can induce remissions in many patients. Yet, relapses can occur and so some patients may proceed to HCT either prior to or after relapse to try to achieve long-term cure. Even so, relapse limits the success of HCT. We recently showed that the suppressive mechanisms induced immediately after PTCy prevent new donor T cells from causing GVHD. Thus, we hypothesized that donor anti-CD19 CAR T cells given early after PTCy would not cause GVHD but may retain an anti-tumor effect; the premise is that the CAR T cells would induce an initial deep remission, while the polyclonal graft-versus-tumor (GVT) T-cell response would ensure long-term cure. To test this approach, we modified our T-cell-replete murine MHC-haploidentical HCT model (B6C3F1→B6D2F1) by giving 1 × 10⁶ E2a-PBx leukemia cells 7 days prior to lethal irradiation and HCT [Figure 1A]. Importantly, E2a-PBx, as a pre-B-cell ALL cell line on a B6 background, is syngeneic with host and donor; thus, in this model only anti-CD19 CAR T cells can treat the leukemia (there is no allo response against it). T-cell-enriched B6C3F1 donor splenocytes were stimulated with anti-CD3/CD28 beads, transduced with an anti-CD19 CAR vector with a CD28 costimulatory domain, and infused at 1 × 10⁶ transduced T cells per mouse on day +5 (24 hours after PTCy). Mice receiving leukemia, HCT, and PTCy all died of leukemia when receiving non-transduced in vitro expanded T cells, but cleared leukemia when receiving CAR T cells [Figure 2A]; this effect occurred without clinical or histopathologic GVHD or toxicity. Leukemia relapse did occur in some CAR-treated mice and was more frequent in mice treated with PTCy (∼25% of mice). Relapse events after PTCy were associated with CD19+ extramedullary tumors but partial loss of CD19 in hematopoietic organs. Measurable CAR T cells were found but had increased expression of co-inhibitory molecules, suggesting that CAR T-cell exhaustion and leukemia antigen loss may be two mechanisms of escape. Attempts to assess the impact of having both CAR and allogeneic T cells able to treat the leukemia (to mirror the intended clinical use) in a C3H→B6D2F1 HCT model were limited by permanent clearance of leukemia by the allogeneic response alone. We also explored the impact of PTCy on GVT immunity by giving CAR T cells on day 0 prior to PTCy [Figure 1B]; leukemia was cleared by mice despite PTCy and did not relapse [Figure 2B]. CAR T cells persisted after PTCy and actually increased in numbers in the bone marrow from days +3 to +7. There was no increase of GVHD in CAR-treated mice clinically or histopathologically. Our data suggest that CAR T cells can be safely and effectively administered either prior to or after PTCy, permitting an anti-tumor response without GVHD. Furthermore, these studies show that tumor-specific T cells survive PTCy, consistent with our recent findings that alloreactive T cells persist after PTCy.

Targeting Two Antigens Associated with B-ALL with CD19-CD123 Compound Car T Cell Therapy.

The recent FDA approval of the first CAR immunotherapy marks a watershed moment in the advancement toward a cure for cancer. CD19 CAR treatment for B cell acute lymphocytic leukemia has achieved unprecedented remission rates. However, despite success in treating previously relapsed and refractory patients, CD19 CAR faces similar challenges as traditional chemotherapy, in that malignancy can adapt and overcome treatment. The emergence of both antigen positive and negative blasts after CAR treatment represents a need to bolster current CAR approaches. Here, we report on the anti-tumor activity of a CAR T cell possessing 2 discrete scFv domains against the leukemic antigens CD19 and CD123. We determined that the resulting compound CAR (cCAR) T cell possesses consistent, potent, and directed cytotoxicity against each target antigen population both in vitro and in vivo. Our findings indicate that targeting CD19 and CD123 on B-ALL cells may be an effective strategy for augmenting the response against leukemic blasts and reducing rates of relapse.

The expression of CD73 on pathological B-cells is associated with shorter overall survival of patients with CLL.

CD73 is a membrane-bound enzyme that catalyzes the extracellular conversion of adenosine monophosphate to adenosine. Adenosine is thought to play a role in promoting tumor growth and survival together with suppressing the host immune responses, which contribute to the multistep process of tumorigenesis. Here, we studied the expression of this antigen in chronic lymphocytic leukemia (CLL). The expression of CD73 was analyzed by multiparametric flow cytometry on normal and pathological B-cells from peripheral blood and bone marrow samples from 71 patients with CLL. Pathological B-cells expressed significantly lower levels of CD73 than normal B-cells (p<0.01). Patients with splenomegaly showed a higher expression of CD73 on pathological B-cells than patients without splenomegaly (p<0.05). The expression of CD73 also correlated with beta-2-microglobulin levels (p<0.05). Clinically, patients with higher levels of CD73 versus those with lower expression presented with shorter overall survival (median OS of 65 vs. 113 months, p<0.05). Our data indicate that CD73 may play a role in CLL pathophysiology, is correlated with poor clinical and biological prognostic factors and may be of potential value as a prognostic marker and therapeutic target.

A biomaterial-based vaccine eliciting durable tumour-specific responses against acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a malignancy of haematopoietic origin that has limited therapeutic options. The standard-of-care cytoreductive chemotherapy depletes AML cells to induce remission, but is infrequently curative. An immunosuppressive AML microenvironment in the bone marrow and the paucity of suitable immunotherapy targets limit the induction of effective immune responses. Here, in mouse models of AML, we show that a macroporous-biomaterial vaccine that delivers the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the Toll-like-receptor-9 agonist cytosine-guanosine oligodeoxynucleotide and one or multiple leukaemia antigens (in the form of a defined peptide antigen, cell lysates or antigens sourced from AML cells recruited in vivo) induces local immune-cell infiltration and activated dendritic cells, evoking a potent anti-AML response. The biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated established AML even in the absence of a defined vaccine antigen. Biomaterial-based AML vaccination can induce potent immune responses, deplete AML cells and prevent disease relapse.

Characterization of Changes in the T-Cell Receptor Repertoire in Patients with Acute Myeloid Leukemia with Durable Remission Following Allogeneic Stem Cell Transplant

Introduction: Allogeneic hematopoietic stem-cell transplantation (HSCT) is curative for many patients with advanced hematologic cancers, including adverse-risk acute myeloid leukemia (AML). This is principally through the induction of a graft-versus-leukemia (GVL) immune effect, mediated by donor T-cells. The incredible diversity and specificity of T-cells is due to rearrangement between V, D, and J regions and the random insertion/deletion of nucleotides, taking place in the hypervariable complementarity determining region 3 (CD3) of the T-cell receptor (TCR). Massively parallel sequencing of CDR3 allows for a detailed understanding of the T-cell repertoire, an area relatively unexplored in AML. Therefore, we sought out to characterize the T-cell repertoire in AML before and after HSCT, specifically for those with a durable remission. Methods: We identified 45 bone marrow biopsy samples, paired pre- and post-HSCT, from 14 patients with AML in remission for &gt; 2 years as of last follow-up. We next performed immunosequencing of the TCRβ repertoire (Adaptive Biotechnologies). DNA was amplified in a bias-controlled multiplex PCR, resulting in amplification of rearranged VDJ segments, followed by high-throughput sequencing. Resultant sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region. We next employed various metrics to characterize changes in the TCR repertoire: (1) clonality (range: 0-1; values closer to 1 indicate a more oligoclonal repertoire), it accounts for both the number of unique clonotypes and the extent to which a few clonotypes dominate the repertoire; (2) richness with a higher number indicating a more diverse repertoire with more unique rearrangements); (3) overlap (range: 0-1; with 1 being an identical T-cell repertoire). All calculations were done using the ImmunoSeq Analyzer software. Results: The median age of patients included in this cohort was 58 years (range: 31-69). Six patient (43%) had a matched related donor, and 8 (57%) had a matched unrelated donor. Baseline characteristics are summarized in Figure 1A. Six samples were excluded from further analysis due to quality. TCR richness did not differ comparing pre- and post-HSCT, with a median number pre-HSCT of 3566 unique sequences (range: 1282-22509) vs 3720 (range: 1540-12879) post-HSCT (P = 0.7). In order to assess whether there was expansion of certain T-cell clones following HSCT, we employed several metrics and all were indicative of an increase in clonality (Figure 2B). Productive clonality, a measure of reactivity, was significantly higher in post-transplant samples (0.09 vs 0.02, P = 0.003). This is a measure that would predict expansion of sequences likely to produce functional TCRs. The Maximum Productive Frequency Index was higher post-HSCT indicating that the increase in clonality was driven by the top clone (most prevalent per sample). Similarly for the Simpson's Dominance index, another marker of clonality which was higher post-HSCT (0.01 vs 0.0009, P = 0.04). In order to determine whether this clonal expansion was driven by TCR clones shared among patients, we compared the degree of overlap in unique sequences among pre and post-HSCT samples. We found there was very little overlap between samples in the pre and the post-transplant setting and no change in the Morisita and Jaccard Overlap Indices. Conclusions: In conclusion, we show in this analysis an increase in clonality of T-cells following HSCT in patients with AML. This is likely related to the GVL effect after recognition of leukemia antigens by donor T cells and subsequent expansion of these T-cells. These expanded T-cell clonotypes were unlikely to be shared by patients in this cohort, likely reflecting the variety of antigens leading to the GVL effect. This could have direct implications on TCR-mediated immune-therapies given the likely need for a personalized, patient-specific design for these therapies. Figure 1 Disclosures Kantarjian: BMS: Research Funding; Novartis: Research Funding; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Astex: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Takeda: Honoraria; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Short:Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Cortes:Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Sun Pharma: Research Funding; BiolineRx: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding. Molldrem:M. D. Anderson &amp; Astellas Pharma: Other: Royalties.

Development of Novel Anti-CD10 Target Modules for Redirection of Universal CAR T Cells Against CD10-Positive Malignancies

The common acute lymphoblastic leukemia antigen CD10 is a marker for several hematological malignancies, including acute lymphoblastic leukemia as well as T and B cell lymphomas, Burkitt lymphomas, and some solid tumors like renal cell carcinomas, pancreatic tumors and melanomas. Because of its tumor related expression pattern, CD10 is an attractive target for adoptively transferred T cells that are genetically modified to express chimeric antigen receptors (CARs). Recently, conventional CAR T cell therapy targeting CD19-positive hematological malignancies was clinically approved because of its impressive effectiveness in patients. However, CAR T cells can also cause severe side effects like on-target, off-tumor reactions, tumor lysis syndrome and cytokine release syndrome. Most critically, activity of conventional CAR T cells cannot be controlled, once they are applied in patients. As CD10 is also widely expressed on normal tissues, CAR T cell reactivity has to be controllable in order to stop CAR T cell therapy in case of on-target, off-tumor toxicities occur. Especially for this purpose, we have recently established a switchable, modular and universal CAR platform technology, named UniCAR system, which can be repeatedly turned on and off. In contrast to conventional CARs, that directly recognize a tumor-associated antigen (TAA) on the tumor cell surface via their extracellular single-chain variable fragment (scFv), the UniCAR system is structured in a modular manner of two components. The first component are T cells genetically engineered to express UniCARs and the second component are target modules (TMs). Most importantly, UniCARs cannot directly bind to a TAA because their extracellular scFv is directed against the peptide epitope E5B9 which is not present on the surface of living cells. Consequently, UniCAR armed T cells are per se inert. They can be redirected towards tumor cells only via a TM. TMs consist of a scFv targeting a TAA and the epitope E5B9 recognized by UniCARs allowing a cross-linkage of UniCAR T cells with tumor cells which results in T cell activation. As TMs have a very short half-life, UniCAR T cell activity can be controlled by dosing of the TM. Once the TM is administered, UniCAR T cells can be switched on, but once the TM injection is stopped and the TM is eliminated, UniCAR T cells are switched off immediately. Here, we show proof of concept for functionality of the UniCAR system targeting CD10-positive malignancies. Therefor, a novel anti-CD10 TM was constructed which is able to redirect UniCAR T cells to eliminate CD10-expressing tumor cells. In summary, we have established a universal, switchable, modular UniCAR platform technology that can be used to target CD10-positive malignancies. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents &amp; Royalties.

T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (&gt;120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

Characterization of BCR-ABL Specific CD4+ T Cells and Their Role in Controlling Ph+ Acute Lymphoblastic Leukemia

Background: Patients with BCR-ABL+ acute lymphoblastic leukemia (Ph+ ALL) can experience significant disease reduction after induction chemotherapy, but subsequently incur adverse long-term outcomes, due to the presence of minimal residual disease (MRD). This suggests a need for novel therapeutic strategies to eliminate MRD. Checkpoint blockade has been considered of little benefit in ALL due to the overall low frequency of somatic mutations. However, the protein product of the BCR-ABL translocation region forms a tumor associated antigen (TAA). Prior studies have demonstrated that patients with Ph+ ALL harbor CD8+ and CD4+ T-cells specific for peptides derived from this junctional region, presented in MHCI or MHCII contexts. This suggests that relapse in Ph+ ALL may be countered by immunotherapeutic strategies that expand sufficient numbers of BCR-ABL-specific effector T-cells. The current study uses a murine model of Ph+ ALL to characterize effector cell types and mechanisms responsible for leukemia control and eradication. We also investigated the impact of commonly used chemotherapy agents on effector cell function, to determine the viability of a combination immunotherapy and vaccination approach at eliminating MRD after induction. Methods: Bone marrow cells from p19ARF-null C57BL/6 mice were transduced with a BCR-ABL expressing retrovirus. Transformed cells were injected into immunocompetent B/6 mice, resulting in a uniformly fatal leukemia in 20-25 days. We used a peptide: MHC-II tetramer to label endogenous CD4+ T-cells specific for a BCR-ABL-derived peptide ("BAp") presented in an MHC II I-Ab context (hereafter referred to as "BAp"-specific T-cells). Results: Previously, we found that BAp-specific CD4+ T-cells were elicited during leukemia development but were limited by regulatory-T-cells (Tregs) that were cross-reactive with endogenous ABL protein. Nonetheless, a heterologous vaccination strategy using BAp peptide, combined with dual CTLA-4 and PD1 checkpoint blockade, was able to extend survival and eradicate leukemia in a subset of mice. Prolonged survival correlated with the robust expression of an ensemble of antigen-presentation molecules by host leukemia cells, as well as the presence of polyfunctional, cytotoxic CD4+ BAp-specific T-cells. To formally evaluate the relative contributions of CD4+ vs. CD8+ T-cells, we next established leukemia and allowed it to progress after the selective depletion of CD4+, CD8+, or both CD4+ and CD8+ T-cells. All mice were also treated with nilotinib and PD-L1 checkpoint blockade. Depletion of CD4+ T-cells led to markedly shortened survival. In contrast, depletion of CD8+ T-cells had no effect, and the majority of mice survived long-term. As this confirmed that CD4+ T-cells were critical for leukemia control, we next characterized the impact of common induction chemotherapy regimens on this subset. Combinations of cytotoxic chemotherapies mimicking either a standard intensity Hyper-CVAD-like regimen or a reduced-intensity regimen were administered to naïve mice, followed by vaccination with BAp. Mice treated with a Hyper-CVAD-like regimen generated fewer numbers of BAp-specific CD4+ T-cells in response to subsequent vaccination. Moreover, scRNAseq analysis of activated CD4+ T-cells demonstrated that standard intensity regimens induced dysfunctional gene expression signatures. These changes were not observed after treatment with a reduced intensity regimen. Dysfunctional signatures were characterized by the upregulation of senescence-associated genes including p21, as well as decreased expression of T-stem-central-memory (Tscm) genes including TCF7 and Tox2. Altogether these findings suggest that Hyper-CVAD-like chemotherapy inhibits proliferation as well as the self-renewal of CD4+ T-cells responding to leukemia antigens. Conclusions: CD4+ T-cells specific for peptides derived from the BCR-ABL protein are responsive to PD-L1 checkpoint blockade, and are critical for Ph+ ALL clearance. Standard intensity induction chemotherapy leads to significant dysfunction of these cells. Ongoing experiments are exploring the mechanisms underlying this dysfunction, and methods to overcome it via BCR-ABL-based peptide vaccination combined with checkpoint blockade. Validation experiments utilizing human samples are also underway and will be reported. Disclosures No relevant conflicts of interest to declare.

Identification of Targetable Tumor Associated Proteins in Adult T-Acute Lymphoblastic Leukemia/Lymphoma (T-ALL/LBL) Including a Novel CC-Chemokine 4 (CCR4)-Positive T-ALL/LBL with Unique Immunophenotype

Introduction There have been few recent therapeutic advances in T-acute lymphoblastic leukemia/lymphoma (T-ALL/LBL), a rare and aggressive malignancy. Intensive chemotherapy +/- allogeneic transplantation (AlloHCT) can effect cure, realized by only a minority of adults. In distinction to B-ALL, there have been no safe/effective targeted therapies developed in T-ALL/LBL. We therefore performed a focused tissue array to identify novel tumor proteins, including CCR4; evaluated together with established immunophenotypic (IP) T-ALL/LBL subtypes [early-T-precursor (ETP), early non-ETP (Pre/Pro-T), common &amp; late thymocyte]. We evaluated the clinical phenotype and outcome together with clinical and epidemiologic data in patients diagnosed at the 3-site Mayo Clinic Cancer Center (MCCC: Rochester, MN; Phoenix, AZ; and Jacksonville, FL). Methods Following IRB approval, we identified 50 consecutive T-ALL/LBL patients from 1997 -2019 diagnosed at the 3-site MCCC. Available remnant leukemia/lymphoma paraffin blocks were identified (n=28). Using immunohistochemistry (IHC) we evaluated expression of leukemic antigens of interest (CCR4, CD47, BCL2, BCL6, PD-L1, CD38 and CD123); these were selected based on clinical availability of novel targeted agents, approved or in development for other indications. Tissue array studies were performed and interpreted by a single hematopathologist (L.J) blinded to clinical data, using positive and negative controls, according to standard techniques. We also evaluated the clinical and epidemiological parameters, including different IP subtypes [ETP: CD1a-, sCD3- &amp; CD8-, Pre/Pro-T: CD1a-, sCD3- &amp; CD8+ or CD8- (11 marker T-ALL IP score was used to distinguish CD8- ETP vs. Pre/Pro-T), thymic: CD1a+ and mature: CD1a- &amp; sCD3+] (Khogeer et al., Br J Haematol., 2019) &amp; the presence of the unique MLLT10-PICALM fusion (n=6), as well as therapy and outcome. Comparisons of characteristics and outcomes of interest were made using a Wilcoxon rank sum test (continuous characteristics) or Fisher's exact test (categorical characteristics), unadjusted Cox proportional hazards regression models (overall survival), or unadjusted logistic regression models (complete remission after first line treatment). Results We identified the expression of novel targetable tissue proteins in all adult T-ALL/LBL cases [Table 1]. BCL2, CD38 &amp; CD47 were expressed in majority of the cases, and CCR4 [Fig. 1] was expressed in 11/28 (39.3%) cases. CCR4 expression was significantly more common in the Pre/Pro-T (75%) vs. other IP subtypes (19%) (p=0.011); there was no other association of CCR4 expression with clinical characteristics/outcomes. We also observed differences in T-ALL vs. T-LBL, specifically T-ALL patients were more likely to be male (71% vs. LBL 38.9%, p=0.038). Surprisingly, T-LBL patients were more likely to have a family history of leukemia/lymphoma (41.2% vs. T-ALL 0%, p&lt;0.001). In addition, a significantly different distribution of IP subtype in T-LBL was noted [Table 2]. Not surprisingly, T-LBL patients were more likely to receive 1st line Hyper-CVAD therapy (88.2% v. T-ALL 43.3%, p=0.005), &amp; had a longer delay from diagnosis to initiation of therapy (median 14 vs. 4 days, p=0.012), however this did not appear to impact clinical outcome. In contrast, we observed an important association of IP subtype with outcome after therapy, including complete remission (p=0.035), &amp; a trend suggesting differences in survival (p=0.069) [Table 3]. However, MLLT10-PICALM was not associated with any unique clinical features or with outcome (death, p=0.85; CR, p=0.98), although 4/6 patients underwent AlloHCT, suggesting an important role of AlloHCT in this subgroup. No other significant association of epidemiologic risk factors with phenotype or outcome was noted. Conclusion Using focused tissue array we have identified expression of tumor associated proteins in T-ALL/LBL including CCR4 (Pre/Pro-T IP) &amp; others, which may be targets of therapeutic agents like mogamulizumab. Furthermore, our analysis demonstrates an important clinical impact of IP subtype in T-ALL/LBL, as well as important clinical &amp; therapeutic differences in T-ALL vs. LBL. This study will help guide development of targeted clinical trials in T-ALL/LBL (CCR4 trial already in development). Further analyses are planned to determine the clinical &amp; epidemiologic association of genetic lesions in this cohort. Disclosures Foran: Agios: Honoraria, Research Funding.

The fate of leukemia-specific CD8+ T cell clones is dictated by antigen specificity and the mechanism of cognate antigen presentation

Abstract We previously demonstrated that high-affinity leukemia-specific CD8+ T cells are deleted in leukemia-bearing hosts in a manner that depends on cross-presentation of the antigen by splenic Batf3-lineage dendritic cells. Here, we generated a T cell receptor transgenic mouse strain (Tg101) that expresses TCR-α and -β chains from a CD8+ T cell clone specific for a native H-2Kb-restricted antigen on C1498 leukemia cells. In naïve mice, Tg101 T cells demonstrate no evidence of self-reactivity, arguing that the Tg101 antigen is leukemia-specific. In leukemia-bearing mice, high-affinity 2C CD8+ T cells specific for the H-2Kb-restricted model antigen, SIYRYYGL, encounter antigen rapidly (day 3–4) via its cross-presentation by splenic CD8a+ DCs, proliferate briefly, and are largely deleted by day 12. Conversely, antigen encounter by Tg101 CD8+ T cells in mice with leukemia is delayed (day 8–10), and primarily occurs in the liver - a prominent site of leukemia progression. Subsequently, Tg101 T cells expand, upregulate expression of co-inhibitory receptors, including PD-1, LAG-3, TIM-3 and TIGIT, and produce effector cytokines poorly, consistent with the acquisition of a dysfunctional state. Surprisingly, in vivo antigen encounter by Tg101 T cells occurs independently of CD11c+ cells, indicating that the Tg101 antigen is poorly cross-presented. Rather, the antigen is directly presented to Tg101 T cells by C1498 cells. Collectively, our data reveal that CD8+ T cell tolerance is effectively generated in leukemia-bearing animals. However, the nature of the leukemia antigen and the context in which it is presented are critical factors in dictating the underlying mechanism of tolerance that ensues. A search for the Tg101 antigen is ongoing.

Fucosylation Enhances the Efficacy of Adoptively Transferred Antigen-Specific Cytotoxic T Lymphocytes.

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. 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. 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. 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.

Pediatric Survivorship: Considerations Following CAR T-Cell Therapy.

This article presents an overview of pediatric relapsed and refractory acute lymphoblastic leukemia (ALL) and chimeric antigen receptor (CAR) T-cell therapy in pediatric patients. Acute and chronic post-CAR T-cell effects and considerations are discussed, along with survivorship considerations. A case study illustrates the identification and management of physiologic and psychosocial sequelae. B-cell aplasia, hypogammaglobulinemia, infections, and cumulative effects of CAR T-cell therapy and other treatments are a concern in the pediatric population. Unique to pediatric and young adult survivors of childbearing potential are implications for post-treatment fertility. Financial toxicities and psychosocial needs require a family-centered approach to interventions that address the impact of CAR T-cell therapy not only on the patient, but also on caregivers and siblings.

A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy.

Role of CD10 Marker in Differentiating Malignant Thyroid Neoplasms from Benign Thyroid Lesions (Immunohistochemical & Histopathological Study).

BACKGROUND:CD10 was initially recognised as a cell–surface antigen expressed by acute lymphoblastic leukaemias, and hence it’s early designation as Common Acute Lymphoblastic Leukemia Antigen (CALLA). Also, it has been proven to be reactive in various non-lymphoid cells and tissue and different types of neoplasms.AIM:To evaluate the immunohistochemical expression of CD10 in malignant thyroid neoplasms and different benign lesions and to assess whether CD10 can be used as a malignancy marker in thyroid pathology or not.MATERIAL AND METHODS:A total of 83 archived, formalin fixed, paraffin embedded tissue blocks of 83 cases of malignant thyroid neoplasms and different benign lesions. The samples were immunohistochemically analysed for CD10 expression. A p-value of less than 0.05 was considered statistically significant.RESULTS:CD10 was expressed in 91% of the studied malignant thyroid neoplasms and 58% of benign thyroid lesions. It was expressed in 26 of 28 (92.9%) conventional papillary carcinomas, ten of 10 (100%) follicular variants of papillary carcinoma, seven of nine (77.8%) minimally invasive follicular carcinomas, two of three (66.7%) widely invasive follicular carcinomas, and seven of 7 (100%) undifferentiated carcinomas, seven of 11 (66.7%) adenomatous nodules and eight of 15 (53.3%) follicular adenomas. No statistically significant correlations were detected between CD10 expression and patients’ age, sex, lymph node metastasis, tumour stage and capsular invasion.CONCLUSION:CD10 shows strong sensitivity (91.2%) and moderate specificity (42.3%) in the diagnosis of malignancy overall and shows strong sensitivity (86.4%) and moderate specificity (42.3%) in the diagnosis of malignancy in the follicular-patterned lesions. So, CD10 might be useful in differentiating malignant from benign thyroid lesions (good positive test) and in the diagnosis of follicular variant of papillary carcinoma.

Generation of Suicide Gene-Modified Chimeric Antigen Receptor-Redirected T-Cells for Cancer Immunotherapy.

Chimeric antigen receptor (CAR)-redirected T-cells are a powerful tool for the treatment of several type of cancers; however, they can cause several adverse effects including cytokine release syndrome, off-target effects resulting in potentially fatal organ damage or even death. Particularly, for CAR T-cells redirected toward acute myeloid leukemia (AML) antigens myelosuppression can be a challenge. The previously validated inducible Caspase9 (iC9) suicide gene system is one of the approaches to control the infused cells in vivo through its activation with a nontherapeutic chemical inducer of dimerizer (CID). We performed a preclinical validation using a model of CD33+ AML, and generated iC9 CAR T-cells co-expressing a CAR targeting the AML-associated antigen CD33 and a selectable marker (ΔCD19). ΔCD19 selected (sel.) iC9-CAR.CD33 T-cells were effective in controlling leukemia growth in vitro, and could be partially eliminated (76%) using a chemical inducer of dimerization that activates iC9. Moreover, to completely eliminate residual cells, a second targeted agent was added. Future plans with these methods are to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant. Additional strategies that we are currently validating include (1) the modulation of the suicide gene activation, using different concentrations of the inducing agent(s), to be able to eliminate CAR T-cells modified by a regulatable gene, ideally aiming at preserving a proportion of the infused cells (and their antitumor activity) for mild to moderate toxicities, or (2) the co-expression of an inhibitory CAR aiming at sparing normal cells co-expressing an antigen not shared with the tumor.

Expression and Clinical Significance of Leukemia Stem Cell Antigen in Acute Myeloid Leukemia

To analyze the expression characteristics of leukemia stem cell (LSC) antigen in acute myeloid leukemia (AML) and to explore the correation of LSC-specific antigens with the subtypes, cytogenetics and clinical efficacy of AML. A total of 61 newly diagnosed patients with AML (except M3) hospltalized in Department of Hematology of our hopital were selected from January 2013 to March 2016. The immun phenotypes and expression of Tim-3, CD96 and CD123 on leucamia cells were detected by direct immunofluorescenct flow cytometry. 61 patients were divided into positive expression and megative expression groups according to expression of Tim-3, CD96 and CD123; the correlation of LSC antigen expression level with high WBC count, chromosome and therapeutic efficacy was analyzed. Among 61 newly diagnosed patients with AML (except M3), the expression rate of Tim-3, CD96 and CD123 was 52.45%, 44.26% and 55.73% respectively. The expression rates of Tim-3, CD96 and CD123 between the AML subtypes and total patients was not stetistically different (P>0.05). The high WBC count occurred more easily in AML (except MS) patients with positive expression of Tim-3, CD96 and CD123, but compared with AML patients with negative espression, the difference was not statstically significant (P>0.05). The proportion of chromosone karyotype with poor prognosis detected in patients with positive expression of Tim-3 and CD96 was higher than that in patients with negative expreesion (P<0.05); while the preoprtion of chromosome karyotype with poor prognosis detected in patients with positive and negative expression of CD123 was not significantly different (P>0.05). After 2 courses of chemotherapy, the complete remission (CR) rate in patients with positive expression of Tim-3, CD96 and CD123 was significantly lower than that in patients with negative expression of Tim-3, CD96 and CD123 (P<0.05), the comparison of OS time in patients with positive and negative expression of Tim-3 and CD96 showed the statistical difference (P>0.05), while the difference of OS time in patients with positive and negative expression of CD123 was not significant (P>0.05). The expression levels of Tim-3, CD96 and CD123 in newly diagnosed AML (except M3) sybtype patients are not significantly different form those in total patients. The high WBC count ocours more easily in patients with positive expression of Tim-3, CD96 and CD123. After 2 course of chemotherapy, the CR rate in patients with positive expression of Tim-3, CD96 and CD123 was significantly lower than that in patients with negative expression. The proportion of chromsome karyotype with poor prognosis detected in patients with positive expression of Tim-3 and CD96 is high, moreover, OS time in patients with positive expression of Tim-3 and CD96 is shorter than that in patients with negative expression.

Pembrolizumab Enhances the Anti-Leukemia Activity of Antigen Specific Cytotoxic T Lymphocytes

▪Many aggressive hematologic malignancies are exquisitely responsive to immunotherapy. While allogeneic (allo) hematopoietic stem cell transplantation (SCT) is the prime example of anti-leukemia immunotherapy, it is highly toxic, limiting its application to patients with aggressive disease and a good performance status. Although targeting leukemia-associated antigens (LAA) with antigen specific cytotoxic T lymphocytes (CTL) and chimeric antigen receptor (CAR) T cells has proven to be a successful immunotherapeutic option, the immune response generated with these approaches often fails to fully eradicate the underlying malignancy. The expression of immune inhibitory molecules and their ligands by myeloid leukemia cells and leukemia-specific T cells could contribute to the failure of cellular immunotherapy approaches for acute myeloid leukemia (AML). Specifically, blocking the PD1/PD-L1 pathway using antibodies was shown to enhance the graft versus leukemia response in murine leukemia transplant models, and promising results with immune checkpoint blockade have been demonstrated in clinical trials for patients with AML. We therefore investigated the role of blocking the PD1/PD-L1 pathway in combination with adoptive cellular immunotherapy in the setting of AML. We used pembrolizumab in combination with CTLs that target the HLA-A2 restricted myeloid leukemia antigens CG1, derived from cathepsin G, and PR1, derived from neutrophil elastase and proteinase 3.Using a standard calcein AM in vitro cytotoxicity assay, we co-cultured AML targets, including U937 HLA-A2+ (U937-A2) AML cell line and primary patient AML, with CG1-CTL and PR1-CTL. AML cells were loaded with calcein AM and then incubated with CTL at increasing effector to target ratios. Pembrolizumab or isotype antibody was added to the cultures. After 4 hours, calcein AM was measured to determine cell viability. Our results demonstrated that U937-A2 cells and patient primary AML samples can be effectively lysed by CG1/PR1 CTL. In vitro lysis of AML by CTL was enhanced after the addition of pembrolizumab in comparison with the AML cells that were co-cultured only with CTL. Killing was dose-dependent and decreased at the lower effector:target ratios. We then tested whether anti-leukemia activity can be enhanced in vivo. For these experiments, NOD/SCID gamma (NSG) mice were engrafted with U937-A2 cells or patient AML intravenously (IV) via tail vein at a dose of 1 x 106 to 1 x 107 cells. After confirming engraftment (1-5% PB HLA-A2+/human (h) CD45+ cells), CG1-CTL and PR1-CTL (0.5 x 106) were administered to mice IV via tail vein and pembrolizumab or isotype antibody [100ug/mouse] was given intraperitoneally 4 times over 2 weeks. Mice were monitored for clinical graft versus host disease (GVHD) and AML 3 times/week. Mice were sacrificed at approximately 2 weeks following treatment and bone marrow (BM) was processed and analyzed for residual AML (Human[h] CD45+/mouse[m] CD45- cells) by flow cytometry. Our data demonstrate a decrease in U937-A2 (Figure) and primary AML after treatment with CG1/PR1-CTL. This decrease was enhanced when pembrolizumab was administered to the CTL-treated mice in comparison with mice treated with pembrolizumab only, CTL only, or CTL+ isotype. Furthermore, there was no increased toxicity or GVHD after the addition of pembrolizumab. Together these data highlight the potential for combination immune checkpoint blockade and antigen specific CTL to eradicate AML in vitro and in vivo. [Display omitted] DisclosuresDaver:Incyte: Consultancy; BMS: Research Funding; ImmunoGen: Consultancy; ARIAD: Research Funding; Sunesis: Consultancy; Alexion: Consultancy; Novartis: Research Funding; Daiichi-Sankyo: Research Funding; Sunesis: Research Funding; Incyte: Research Funding; Karyopharm: Research Funding; Otsuka: Consultancy; Novartis: Consultancy; Kiromic: Research Funding; Karyopharm: Consultancy; Pfizer: Consultancy; Pfizer: Research Funding.

Ex-Vivo Stimulation with DC/AML Fusion Vaccine in the Presence of Cytokines Leads to an Activated T Cell Memory Phenotype and Enhanced Cytotoxicity with Potential for Use As an Adoptive Cellular Therapy

Introduction: Adoptive cellular transfer using engineered cells in hematologic malignancies often rely on single antigens as targets but antigen downregulation has been described as a mechanism of tumor resistance. Furthermore, identification of optimal tumor-specific antigens that are immunogenic and have tolerable off-target effects remain an area of active investigation. Our group has developed a novel vaccine using whole patient-derived AML cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. Here we describe the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo and provide a framework for therapeutic exploration of vaccine stimulated T cells as a cellular therapy.Methods/Results:Vaccine was generated with C57BL/6J bone marrow matured DCs and syngeneic TIB-49 AML cells as previously described. T cells were obtained from C57BL/6J splenocytes after magnetic bead isolation and cultured with vaccine plus IL-15 and IL-7. Vaccine stimulated T cells showed increased immune activation as measured by multicolor flow cytometric analysis. Compared to unstimulated T cells, there was 8-fold increase in CD3+CD69+ expression, which is detected upon TCR ligation. Likewise, there was a 12-fold and 3-fold increase in CD137 expression on CD4+ and CD8+ cells, which is up-regulated upon antigen recognition.In addition, a shift from naïve to memory T cell phenotype occurred, which has important implications for response to adoptive cell transfer. Amongst CD4+ cells, the CD44+CD62L- subset comprised 71% after vaccine stimulation. Amongst CD8+ cells, there was increase in both effector and memory subsets. Furthermore, there was enhanced Th1 polarization in ex-vivo culture with vaccine, as IFN-γ expression was increased 2-fold and 5-fold in CD4+ and CD8+ subsets respectively. A corresponding 1.5-fold increase in cytotoxicity was detected using a standard granzyme B CTL assay.The efficacy of adoptive therapy with vaccine stimulated T cells was demonstrated in a xenograft model in which NSG mice were engrafted with patient derived AML cells following sublethal total body irradiation. T cells autologous to the primary tumor were obtained at the time of disease remission and DC/AML fusions were generated. T cells were cultured with or without fusion vaccine at 1:10 ratio. Intracellular IFN-γ expression increased 9-fold and 11-fold on CD8+ and CD4+ cells respectively with ex-vivo vaccine stimulation compared to unstimulated T cells. On day 14 after tumor inoculation, mice were injected with 1.5 x 106 T cells that had been cultured with or without fusion vaccine. Untreated mice were used as a control. The mice were sacrificed two weeks later at which time bone marrow and spleen cells were harvested.AML engraftment in bone marrow was significantly reduced in mice treated with ex -vivo vaccine stimulated T cells compared to control as measured by presence of hCD45+hCD33+ leukemia cells with mean levels of 18% and 38% respectively (n=4). Increased marrow infiltration of human CD8+ T cells was detected in animals treated with ex vivo stimulated T cells and corresponded with lower AML burden. In contrast, both control mice and those given unstimulated T cells demonstrated low hCD8+ marrow T cell infiltration and higher disease burden. We propose this was due to tumor-specific T cell activation of vaccine educated T cells as evidenced by their increased levels of hCD8+IFN-γ expression following exposure to autologous tumor lysate at the time of splenocyte harvesting.Conclusion: In the current study we have shown the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo, as demonstrated by increased CD69, CD137 and IFN-γ expression, and enhance memory and effector subsets when co-cultured in the presence of cytokines. Results of treatment with vaccine stimulated T cells to date have shown reduced engraftment of primary AML in NSG mice. This provides a framework to evaluate the therapeutic use of adoptive transfer of ex-vivo fusion vaccine stimulated T cells in AML. Further in vitro work incorporates marrow-infiltrating T cells and circulating tumor-specific T cells to assess efficacy in active disease. In vivo models are also ongoing including both xenograft and an immunocompetent murine model and subsequent survival analyses will be reported. DisclosuresRosenblatt:Celgene: Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding.

A Novel Monoclonal Antibody Combination Plus DC/AML Fusion Vaccine Eradicates AML in an Immunocompetent Murine Model

Introduction: Acute myeloid leukemia (AML) is a lethal hematologic malignancy in which standard therapy is often inadequate in sustaining durable remissions. In part due to an immunosuppressive milieu characterized by the upregulation of checkpoint inhibitory pathways leading to T cell anergy, ineffective antigen (Ag) presentation, and increased numbers of myeloid derived suppressor cells (MDSCs). We have pioneered a novel vaccine that targets an array of leukemic antigens by fusing whole patient-derived AML cells to autologous dendritic cells (DCs) in the presence of polyethylene glycol (PEG). In a phase I/II clinical trial in AML, vaccination induced an expansion of tumor-specific T cells and resulted in prolonged remissions in a subset of patients. Checkpoint blockade is being investigated in combination with cancer vaccines to overcome the suppressive effects of the tumor microenvironment on cytotoxic tumor-specific T cells. Here we describe the addition of a novel combination of checkpoint inhibitors with DC/AML fusion vaccine in an immunocompetent murine model.Methods/Results: C57BL/6J mice underwent retro-orbital inoculation with 50,000 syngeneic luciferase and mCherry labeled TIB-49 AML cells. 24 hours later a cohort of mice was treated with 100,000 DC/AML irradiated fusion cells. An additional cohort was treated with 6 doses of a combination of anti-PD1/anti-TIM3/anti-RGMb mAbs administered IP every 3 days with and without vaccine. Control mice were treated with appropriate isotype control. AML burden was evaluated in peripheral blood (PB) 14 days post inoculation using flow cytometric analysis for mCherry expression. AML cells were present in PB of control mice and mice treated with check point inhibitors alone (mean 1.2% and 1.5% respectively). Lower disease burden was detected in mice treated with the DC/AML fusion vaccine (0.5%) or combination of fusion vaccine with check point inhibitors (0.5%).Furthermore, to measure tumor-specific T cell response, PB cells were cultured in the presence of autologous TIB-49 tumor lysate for 3 days and analyzed for intracellular IFN-γ expression using multicolor flow cytometric analysis. Mice treated with combination of DC/AML fusion vaccine and checkpoint inhibition showed a significant increase in IFN-γ expression by CD8+ T cells with mean values of 6.4%. Mice treated with vaccine, checkpoint inhibitors alone or IgG control demonstrated mean levels of 3.4%, 1.9% and 1.9% respectively (n=5; p=0.01).Additional immunologic assessment was performed from PB of surviving animals 37 days after tumor challenge. Combination treatment with DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs led to a statistically significant increase in CD4/CD44+/CD62L- memory T cells with concurrent decrease in naïve CD8/ CD44-/CD62L+ T cells as compared to single agent treatments. Furthermore, a statistically significant decrease in CD4+CD25+FOXP3+ Tregs was detected after combination treatment compared to single treatment groups.The mice were followed for survival and disease progression using BLI imaging. All control mice developed visible AML by luminescence following luciferin injection as well as symptomatic disease requiring euthanasia by day 31 after initial challenge with tumor cells (Figure 1). Mice treated with the anti-PD1/anti-TIM3/anti-RGMb mAbs alone demonstrated a survival benefit but all required euthanasia by day 44. 3 of 5 mice treated with vaccine alone remain disease free at more than 65 days. And strikingly, the entire cohort of mice treated with the combination of DC/AML fusion vaccine and PD1/anti-TIM3/anti-RGMb mAbs remain alive and disease free in this aggressive AML model.Conclusion: In the current study we have demonstrated the capacity of a combination of PD-1, TIM-3 and RGMb checkpoint inhibition to create an enhanced environment for immune response to the DC/AML fusion vaccine in an immunocompetent murine AML model. Treatment with this combination led to an increase in tumor specific T cell immunity, decrease in circulating Tregs and shift toward a memory phenotype. Most significantly, mice who received the combination treatment remain disease-free several months post inoculation. This synergistic approach has promising translational potential and a phase 1 clinical trial is planned. [Display omitted] DisclosuresRosenblatt:Bristol-Myers Squibb: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Stone:Astellas: Consultancy; Otsuka: Consultancy; Arog: Consultancy, Research Funding; Argenx: Other: Data and Safety Monitoring Board; Pfizer: Consultancy; Fujifilm: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Orsenix: Consultancy; Amgen: Consultancy; Merck: Consultancy; Agios: Consultancy, Research Funding; AbbVie: Consultancy; Novartis: Consultancy, Research Funding; Jazz: Consultancy; Cornerstone: Consultancy; Ono: Consultancy; Sumitomo: Consultancy. Freeman:Roche: Patents & Royalties; Merck: Patents & Royalties; Origimed: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Roche: Membership on an entity's Board of Directors or advisory committees; EMD-Serono: Patents & Royalties; AstraZeneca: Patents & Royalties.