Marrow-infiltrating Lymphocytes Research Articles

Expansion of tumor-infiltrating and marrow-infiltrating lymphocytes from pediatric malignant solid tumors

The expansion of tumor-infiltrating lymphocytes (TIL) for adoptive cellular therapy is under investigation in many solid tumors of adulthood. Marrow-infiltrating lymphocytes (MIL) have demonstrated antitumor reactivity preclinically. Successful expansion of TIL/MIL has not been reported across pediatric solid tumor histologies. The objective of this study was to demonstrate successful expansion of TIL from pediatric solid tumors for translation in an adoptive cell therapy (ACT) treatment strategy. A prospective study of TIL/MIL expansion was performed on solid tumors of pediatric patients undergoing standard-of-care procedures. TIL/MIL expansions were performed in the presence of high-dose interleukin 2. To demonstrate a full-scale expansion to clinically-relevant cell doses for TIL therapy, initial TIL culture was followed by a rapid expansion protocol for select patients. Expanded specimens were analyzed for phenotype by flow cytometry and for anti-tumor reactivity by the interferon-gamma release assay. Eighteen tumor samples were obtained. Initial TIL cultures were successfully generated from 14/18 samples (77.7%). A median of 5.52 × 107 (range: 2.5 × 106-3.23 × 108) cells were produced from initial cultures, with 46.9% expressing a CD3 phenotype (46.9%). Eight samples underwent rapid expansion, demonstrating a median 458-fold expansion and a CD3 phenotype of 98%. Initial MIL cultures were successfully generated from five samples, with a predominantly CD3 phenotype (45.2%). Sufficient tumor tissue was only available for seven TIL samples to be tested for reactivity; none demonstrated responsiveness to autologous tumor. TIL and MIL expansion from pediatric solid tumors was successful, including the full-scale expansion process. This data supports translation to an ACT-TIL treatment strategy in the pediatric population and thus a Phase I trial of ACT-TIL in pediatric high-risk solid tumors is planned.

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM. Methods: CD3 + T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments. Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was > 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 + T cells, while the proportion of CD4 + T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 + primary myeloma cells. Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.

A Journey through the Inter-Cellular Interactions in the Bone Marrow in Multiple Myeloma: Implications for the Next Generation of Treatments.

Simple SummaryHere, we describe the main interactions of multiple myeloma cells in the bone marrow with non-hematological and hematological immune cells that impact the disease’s progression and treatment resistance. Non-hematological cells in the bone marrow can secrete molecules that accelerate disease progression. On the other side, these interactions compromise immune cell activity, leading to immune evasion and disease progression. Deep knowledge of these interactions can lead to the design of improved treatments for multiple myeloma.Tumors are composed of a plethora of extracellular matrix, tumor and non-tumor cells that form a tumor microenvironment (TME) that nurtures the tumor cells and creates a favorable environment where tumor cells grow and proliferate. In multiple myeloma (MM), the TME is the bone marrow (BM). Non-tumor cells can belong either to the non-hematological compartment that secretes soluble mediators to create a favorable environment for MM cells to grow, or to the immune cell compartment that perform an anti-MM activity in healthy conditions. Indeed, marrow-infiltrating lymphocytes (MILs) are associated with a good prognosis in MM patients and have served as the basis for developing different immunotherapy strategies. However, MM cells and other cells in the BM can polarize their phenotype and activity, creating an immunosuppressive environment where immune cells do not perform their cytotoxic activity properly, promoting tumor progression. Understanding cell–cell interactions in the BM and their impact on MM proliferation and the performance of tumor surveillance will help in designing efficient anti-MM therapies. Here, we take a journey through the BM, describing the interactions of MM cells with cells of the non-hematological and hematological compartment to highlight their impact on MM progression and the development of novel MM treatments.

P-086: Antitumor activity of activated marrowinfiltrating lymphocytes in patients with multiple myeloma

P-086: Antitumor activity of activated marrowinfiltrating lymphocytes in patients with multiple myeloma

Abstract P2-14-07: Marrow-infiltrating lymphocytes as adoptive immunotherapy for breast cancer

Abstract Background: The bone marrow harbors tumor-antigen specific T cells in patients with breast cancer (1). Marrow-infiltrating lymphocytes (MILs®) are the product of activating and expanding bone-marrow T cells (2). In multiple myeloma, transfer of MILs as adoptive cell therapy has demonstrated anti-tumor activity (3). The use of adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) has been used successfully for hormone-receptor positive (HR+) metastatic breast cancer (4). Expansion of MILs was previously shown to be feasible in breast cancer, non-small cell lung cancer (NSCLC), prostate, head and neck, glioblastoma and multiple myeloma (5). Using an expanded cohort, we demonstrate that tumor-specific MILs can be expanded from bone marrow in patients with breast cancer. Methods: Bone marrow aspiration and blood samples were collected from 8 patients with metastatic and high-risk early-stage breast cancer, including 3 patients with HR+/HER2-, 2 patients with HER2+, and 3 patients with triple negative subtypes, at Providence Cancer Institute (Portland, OR). MILs and peripheral blood lymphocytes were activated and expanded from patient samples during a 10-day proprietary process. T cell lineage-specific markers CD3, CD4 and CD8 were characterized by flow cytometry pre- and post- expansion. Tumor-specific T cells were quantitated in expanded MILs and peripheral blood lymphocytes using a previously described cytokine-secretion assay (3). Briefly, they were defined as the IFNy-producing population by flow cytometry. Autologous antigen-presenting cells (APCs) were pulsed with lysates from allogeneic cancer cell lines and co-cultured with activated MILs or peripheral blood lymphocytes. APCs pulsed with irrelevant mis-matched cancer cell line lysates or media alone were used as negative controls. Results: In all harvested samples, MILs expansion successfully resulted in a selective increase in CD3+ T cells. Cytokine-producing CD4+ and CD8+ T cells were detected in all expanded MILs samples, but not in any of the matched activated and expanded peripheral blood lymphocytes. MILs were successfully expanded in HR+/HER2-, HER2+, and triple negative subtypes without apparent differences in activity as measured by cytokine production. Conclusion: MILs isolated from this expanded cohort of metastatic and high-risk early stage breast cancer patients demonstrated cytokine production in response to pulsed cancer cell line lysates, consistent with our experience with other solid (5) and hematological (3) malignancies. A phase II trial to evaluate MILs in combination with a checkpoint inhibitor is underway in patients with anti-PD1/PDL1-refractory NSCLC (NCT04069936). These preclinical data demonstrate that expanding MILs is feasible and could be evaluated therapeutically in breast cancer.

Pre-Transplant Exhausted Marrow Infiltrating T Lymphocytes in MRD Negative Patients with Acute Leukemia Predict Early Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (alloSCT) is curative for patients with high-risk acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite this, relapse remains the leading cause of treatment failure. The use of flow cytometry to identify aberrant leukemia-associated immunophenotypes is an established method of detecting minimal residual disease (MRD). MRD status immediately prior to transplant remains one of the single best predictors of post-transplant outcome. Unfortunately, not all MRD(-) patients maintain remission post-transplant. Therefore, we sought to find additional predictors of post-transplant relapse. T cell exhaustion occurs following continuous exposure to antigen and leads to sustained up-regulation of multiple inhibitory receptors, progressive loss of function, and expression of a distinct transcriptional state. Bachireddy et al recently reported that exhausted CD8+ marrow infiltrating lymphocytes (MILs) were a novel predictor of response to donor lymphocyte infusion (DLI) for patients with relapsed chronic myelogenous leukemia (CML). Based on this principle, we hypothesized that exhausted CD8+MILs may also be predictive of outcome for MRD(-) patients prior to alloSCT.We matched pre-transplant samples from 17 MRD(-) patients who maintained remission for greater than 2 years post-transplant to 11 MRD(-) patients who suffered relapse within 2 years of transplant. Patients had either ALL or AML and did not differ by demographic details. MILs were evaluated for exhaustion using 2 multicolor flow cytometry panels which collectively included a viability dye, T cell markers (CD3 and CD8) surface inhibitory receptors (PD-1, Tim-3, and TIGIT), transcription factors (T-bet and Eomes), and intracellular cytokine staining (IL-2, TNF-α, and IFN-γ) in response to CD3/CD28 bead stimulation.There was no difference in the percentage of total T cells (CD3), the CD8+ T cells (CD3+CD8+), or CD8+ T cell subsets between the 2 groups. While there was also no difference in the mean fluorescent intensity (MFI) of Tim-3, both PD-1 and TIGIT were significantly elevated on the CD8+ MILs of MRD(-) patients that eventually relapsed (p=0.01 and p=0.03, respectively). Among CD8+ MILs, cells with the highest PD-1 expression (PD-1High) were significantly elevated in the relapse group (p=0.006), as was co-expression of multiple inhibitory receptors on single cells (p=0.01) (Figure A,B). In contrast, the proportion of CD8+ MILs lacking all 3 of these inhibitory receptors was significantly higher among the MRD(-) group that maintained remission (p=0.01) (Figure C). Interestingly, CD8+ MILs in the remission group with dual PD-1+TIGIT+ expression or expressing PD-1High were more likely to be central memory cells (both p=0.05), while these same immunophenotypes were significantly more often effector memory cells in patients who went on to relapse (p=0.03 and p=0.02, respectively).As noted above, inhibitory receptor expression alone does not define exhaustion. We further assessed the CD8+ inhibitory receptor expressing MILs in the relapse group for other phenotypic evidence of exhaustion by evaluating their function and transcription factor expression. Cells with either PD-1+TIGIT+ dual expression or PD-1High were directly compared to cells that lacked inhibitory receptors. The dual PD-1+TIGIT+ cells had impaired ability to produce TNF-α and IFN-γ (both with p<0.0001), while PD-1High cells produced significantly less TNF-α (p<0.0001), IFN-γ (P<0.0001) and IL-2 (p=0.0001). Moreover, both sets of MILs had significantly decreased polyfunctional cytokine production compared to cells without these surface receptors (p < 0.0001) (Figure D). Furthermore, transcription factor expression was also altered among inhibitory receptor expressing cells. They exhibited both increased early exhaustion as evidenced by high T-bet expression in PD-1 expressing cells as well as terminal exhaustion indicated by significantly elevated Eomes expression on PD-1High expressing cells (Figure E,F).Collectively, this study suggests that the presence of exhaustion in CD8+ MILs in acute leukemia patients who are MRD(-) prior to transplant may be at higher risk for post-transplant relapse. If validated, this data may be used to further risk-stratify patients and/or direct pre- or post-transplant therapy, including the potential use of checkpoint blockade. [Display omitted] DisclosuresBachanova:Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Wagner:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Inverse relationship between oligoclonal expanded CD69− TTE and CD69+ TTE cells in bone marrow of multiple myeloma patients

AbstractCD8+CD57+ terminal effector T (TTE) cells are a component of marrow-infiltrating lymphocytes and may contribute to the altered immune responses in multiple myeloma (MM) patients. We analyzed TTE cells in the bone marrow (BM) and peripheral blood (PB) of age-matched controls and patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed (ND) MM using flow cytometry, mass cytometry, and FlowSOM clustering. TTE cells are heterogeneous in all subjects, with BM containing both CD69− and CD69+ subsets, while only CD69− cells are found in PB. Within the BM-TTE compartment, CD69− and CD69+ cells are found in comparable proportions in controls, while CD69− cells are dominant in MGUS and SMM and predominantly either CD69− or CD69+ cells in NDMM. A positive relationship between CD69+TTE and CD69−TTE cells is observed in the BM of controls, lost in MGUS, and converted to an inverse relationship in NDMM. CD69−TTE cells include multiple oligoclonal expansions of T-cell receptor/Vβ families shared between BM and PB of NDMM. Oligoclonal expanded CD69−TTE cells from the PB include myeloma-reactive cells capable of killing autologous CD38hi plasma cells in vitro, involving degranulation and high expression of perforin and granzyme. In contrast to CD69−TTE cells, oligoclonal expansions are not evident within CD69+TTE cells, which possess low perforin and granzyme expression and high inhibitory checkpoint expression and resemble T resident memory cells. Both CD69−TTE and CD69+TTE cells from the BM of NDMM produce large amounts of the inflammatory cytokines interferon-γ and tumor necrosis factor α. The balance between CD69− and CD69+ cells within the BM-TTE compartment may regulate immune responses in NDMM and contribute to the clinical heterogeneity of the disease.

Phase IIa study of marrow infiltrating lymphocytes (MILs), an adoptive T cell therapy, alone or in combination with nivolumab in non-small cell lung cancer (NSCLC).

TPS9630 Background: Primary or secondary resistance to anti-PD-1 may be due to loss of T cell function. Persistent antigen stimulation can lead to impaired CD8+ T cell function, which often results in acquired resistance to PD-1 inhibition. It is unclear whether reinvigoration of tumor infiltrating cells or recruitment of novel T cells impart the activity of anti-PD-1 therapy. The bone marrow is a reservoir for antigen experienced memory T cells. We have previously shown that MILs can be generated for patients with hematologic malignancies and solid tumors including patients with NSCLC. MILs are the product of the activation and expansion of bone marrow T cells with a polyantigenic memory phenotype that recognize tumor antigens, are cytotoxic to autologous tumor and are able to persist over a long period of time. In a pre-clinical study of NSCLC, MILs were able to be expanded in all patients tested. Furthermore, all of the NSCLC products tested showed specificity to shared NSCLC antigens. The combination of adoptive cell therapy (ACT) with checkpoint inhibitors (CPIs) has distinctive positive effects on CD8 and CD4 T cell subsets, with the possibility for complete tumor control. We hypothesize that patients with NSCLC who have relapsed on anti-PD-1 treatment could benefit from an infusion of non-exhausted, central memory-enhanced, antigen specific T cells i.e. MILs which can delay the induction of tumor-associated anergy and augment the overall effectiveness of immunotherapy. Methods: Patients with advanced NSCLC who have progressed following prior anti-PD-1 therapy, with sufficient bone marrow reserve and an ECOG 0-1 are eligible. In eligible patients, bone marrow (200 mL) will be harvested and processed. Patients will undergo lymphodepletion (fludarabine 300 mg/m2/day and cyclophosphamide 30 mg/m2/day on days -5,-4,-3) followed by infusion of MILs on day 0. In Part 1, up to 6 patients will be administered MILs alone on day 0. In Part 2, approximately 20 subjects will be administered MILs on day 0 followed by NIVO 480 mg Q4W starting on day 1. The objectives of the study are to assess safety of MILs alone and in combination with NIVO, as well as efficacy. The first patient was treated in December 2019. Clinical trial information: NCT04069936 .

Superior Efficacy of CAR-T Cells Using Marrow-Infiltrating Lymphocytes (MILsTM) As Compared to Peripheral Blood Lymphocytes (PBLs)

Background The type of T cell used in generating chimeric antigen receptor (CAR) T cells is an important choice. Evidence suggests that T cells that are early in the effector/memory differentiation pathway with more stemness and greater potential to persist are better than more differentiated T cells with less stemness that are more readily exhausted and have less potential to persist. Marrow-infiltrating Lymphocytes (MILsTM) is a novel form of adoptive T cell therapy composed of patient-autologous, polyclonal CD4 and CD8 T cells that are activated and expanded from the bone marrow. Genetically unmodified MILsTM have demonstrated antitumor activity in patients with multiple myeloma and are being developed for several other tumor types, including non-small cell lung cancer and other solid tumors. Distinguishing features of bone marrow T cells used to produce MILsTM include their memory phenotype, inherent tumor antigen-specificity, higher CD8:CD4 ratio and ability to persist long-term when compared to peripheral blood lymphocytes (PBLs) which is the T cell source used to produce currently approved CAR-T therapies. Based on these differences, we hypothesize that MILsTM provide a more robust and better fit platform for CAR-T therapy compared to PBLs. Using a CD38-specific, 4-1BB/CD3z-signaling CAR as an initial model, we have demonstrated the feasibility of producing CAR-modified MILsTM (CAR-MILsTM) and showed that CAR-MILsTM demonstrate superior killing in vitro compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs). Herein, we build on our previous data and add a second BCMA-specific CAR model. We use the two multiple myeloma model systems to compare cytolytic potential, functionality, and expression of phenotypic markers of memory, stemness and exhaustion between patient-matched CAR-MILsTM and CAR-PBLs. Methods Matched pairs of CAR-MILsTM and CAR-PBLs were produced from the bone marrow and blood of multiple myeloma patients. Two different in vitro cytotoxicity assays, the RTCA xCelligence real-time impedance and FACS assays, were used to evaluate antigen-specific killing of target tumor cells. Functionality of CD4 and CD8 CAR-T cells, at the single-cell level, was evaluated by measuring the secretion of 32 cytokines and chemokines following in vitro antigen-specific stimulation using IsoPlexis IsoCode chips and analyzed using IsoPeak. Expression of markers of T cell memory (CD45RO &amp; CCR7/CD62L), stemness (CD27) and exhaustion (PD1 &amp; TIM3) on CAR-MILsTM and CAR-PBLs prior to and following antigen-specific stimulation was evaluated by flow-cytometry (FACS). Results CAR-MILsTM demonstrated superior killing of tumor target cells in vitro, regardless of the antigen specificity of the CAR, when compared to matched CAR-PBLs and this superiority persisted even upon repeated antigen encounter - a factor that may be critical in guaranteeing better anti-tumor efficacy and persistence. CAR-MILsTM demonstrated increased polyfunctionality (secretion of 2+ cytokines per cell) and an increased polyfunctional strength index (PSI) following antigen-stimulation compared to CAR-PBL in both CD4 and CD8 T cells. The enhanced PSI in CAR-MILsTM was predominately mediated by effector, stimulatory and chemoattractive proteins associated with antitumor activity including Granzyme B, IFNg, IL-8, MIP1a and MIP1b. Coincidentally, increased PSI and enhanced secretion of these same proteins was reported to be associated with improved clinical responses in patients with Non-Hodgkin lymphoma treated with CD19-specific CAR-T therapy. Expression of memory markers on CD4 and CD8 T cells were similar in CAR-MILsTM and CAR-PBLs both prior to and following antigen-stimulation. Although expression of CD27, PD1 and TIM3 were similar at baseline, CAR-MILs maintained higher levels of CD27 and lower levels of PD1 and TIM3 compared to CAR-PBLs following antigen-stimulation in both CD4 and CD8 T cells. Conclusions Collectively, our data suggest that CAR-MILsTM have several advantages over CAR-PBLs, including increased cytolytic potential, enhanced polyfunctionality, increased stemness and less exhaustion. Based on these differences and the inherent antitumor properties of MILsTM, we speculate that CAR-MILsTM would be more potent and effective than currently approved CAR-T products derived from PBLs. Disclosures Lutz: WindMIL Therapeutics: Employment, Equity Ownership. Jana:WindMIL Therapeutics: Employment, Equity Ownership. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. DeOliveira:WindMIL Therapeutics: Employment, Equity Ownership. Zhou:Isoplexis: Employment, Equity Ownership. Mackay:Isoplexis: Employment, Equity Ownership. Borrello:Aduro: Patents &amp; Royalties: intellectual property on allogeneic MM GVAX; BMS: Consultancy; WindMIL Therapeutics: Equity Ownership, Patents &amp; Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau. Noonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties; Aduro: Patents &amp; Royalties: intellectual property on allogeneic MM GVAX.

Progress of cellular immunotherapy in multiple myeloma

Multiple myeloma (MM) is a kind of refractory diseases, characterized by clonal proliferation of malignant plasma cells. Thanks to the wide application of new drugs such as proteasome inhibitors and immunomodulators, the survival time of MM patients has been prolonged obviously. Cellular immunotherapies like genetically modified T cell, activated marrow-infiltrating lymphocytes, dendritic cell vaccines, natural killer cells and donor lymphocyte infusion have made great progresses in the field of MM management recently, following the stable advance in clinical trials. This review discusses the recent progress of cellular immunotherapy for MM. Key words: Multiple myeloma; Immunity, cellular; Treatment outcome

Expansion and Characterization of Iovance Marrow Infiltrating Lymphocytes: A Potential Novel Therapeutic Strategy for the Treatment of Acute Myeloid Leukemia

▪Background: Acute myeloid leukemia (AML) is a poor-prognosis malignancy arising from hematopoietic stem/progenitor cells. To date, novel immunotherapies such as checkpoint inhibitors, vaccines and adoptive cell therapy (ACT) using CAR T cells have demonstrated only modest success for the treatment of patients who are ineligible for marrow transplantation and have minimal residual disease; additional approaches are warranted (Beyar-Katz O and Gill S, Clin Cancer Res 2018). ACT with tumor infiltrating lymphocytes (TIL) has emerged as an effective treatment for patients with metastatic melanoma (Goff SL et al, J Clin Oncol 2016), likely owing to the heterogeneous population of tumor-reactive T cells that comprises the TIL products. As demonstrated for solid cancers, such tumor-reactive T cells are preferentially found in the tumor microenvironment (Gros A et al JCI 2014; Thommen DS et al Nat Med 2018). By avoiding the highly immunosuppressive tumor microenvironment, ex vivo activation of those cells rescues them from tolerance and anergic status. We hypothesized that, in the case of AML for which the bone marrow represents the tumor microenvironment, tumor antigen-specific T cells could be recovered from the patient bone marrow to produce a highly effective therapeutic product that is cytotoxic to AML tumor cells. We present findings related to the ex vivo expansion of Iovance marrow infiltrating lymphocytes (MIL) for the treatment of AML patients.Methods: Immune cell and non-immune cell fractions were sorted from bone marrow mononuclear cells. Immune cell fractions loaded with sonicated non-immune cell fractions were expanded for 14 days in the presence of αCD3/αCD28 beads and interleukin-2 (IL-2) to generate MIL products. Phenotypic and functional characteristics of the cells were determined by flow cytometry and enzyme-linked immunospot assay (ELISpot).Results: MIL were generated from isolated bone marrow mononuclear cells (n=2) with a mean expansion fold of 86 (range 78-93). Equal percentage of CD4+ and CD8+ T cell subsets constituted the MIL products. Phenotypic analysis of the cells showed that the majority of T cell subsets are effector memory and CD28 positive. Low percentages of the T cell subsets were positive for immunosuppressive markers PD-1 and LAG3. ELISpot analysis demonstrated that MIL were readily activatable and produced normal levels of IFNγ in response to CD3/CD28 stimulation. Antigen specificity of MIL is being investigated.Conclusion: We demonstrated the feasibility of MIL expansion from bone marrow mononuclear cells from AML patients. MIL are functionally active and mostly comprised of effector memory T cells. Confirmation of tumor cell antigen specificity will determine whether MIL may deploy a robust anti-tumor activity in vivo. DisclosuresKaryampudi:Iovance Biotherapeutics: Employment, Equity Ownership. Frank:Iovance Biotherapeutics: Employment, Equity Ownership. Blaskovich:Iovance Biotherapeutics: Equity Ownership. Chartier:Iovance Biotherapeutics: Equity Ownership.

Marrow-Infiltrating Lymphocytes (MILs) Provide a Robust Platform for CAR-T Cell Therapy

▪Adoptive cellular immunotherapy (ACT) using chimeric-antigen receptor (CAR)-modified T cells has shown unprecedented initial response rates in patients with advanced B cell malignancies and is emerging as a powerful tool for cancer treatment. However, there remains room for improvement in initial response rates, and relapse occurs in a significant fraction of those patients that do respond. Reasons for failure to respond are being elucidated. At least two mechanism of relapse - lack of persistence of infused CAR-T cells and the loss of antigen-expression by tumor cells - are well documented.We have developed a novel platform for ACT called Marrow-infiltrating Lymphocytes (MILs). MILs are a population of polyclonal T cells expanded from the bone marrow (BM) with distinct properties that set them apart from T cells expanded from peripheral blood lymphocytes (PBLs). MILs are enriched with memory T cells and contain tumor antigen-specific T cells that are typically not detected in PBLs. MILs are being developed for several tumor types, including both hematological and solid tumors.Distinguishing features of MILs compared to PBLs include their memory phenotype, inherent tumor antigen-specificity, and ability to persist long-term. As such, we hypothesized that MILs would provide a robust platform for CAR-T cell therapy with the potential to boost initial response rates and reduce the risk of relapse. Herein, data is presented demonstrating superior anti-tumor activity of CAR-modified MILs (CAR-MILs) compared to CAR-T cells generated from patient-matched PBLs (CAR-PBLs).We designed a 2nd generation 4-1BB-CD3z CD38 CAR using an scFv derived from Daratumumab. The CD38 CAR was linked to GFP by a T2a cleavage peptide so that GFP could be used as a marker of CAR expression. The CD38 CAR was expressed in a lentiviral construct and demonstrated equal transduction efficiencies and CAR expression levels in MILs and PBLs. The CAR modified MILs demonstrated superior antigen specific killing compared to PBLs against the 8226 myeloma (MM) cell line but not its isogenic CD38 knocked out line. More importantly, CAR-MILs retained their inherent tumor antigen-specificity and capacity to respond through their endogenous tumor antigen-specific T cell receptors - a property that PBL-CARs did not appear to possess. Tumor antigen-specific T cells were quantified by co-culturing MILs with autologous antigen-presenting cells pulsed with allogeneic multiple myeloma cell line lysates as a source of antigen and measuring IFNγ-production as a read-out. An equal or greater number of tumor antigen-specific IFNγ-producing T cells were measured in CD38 CAR-MILs compared to matched unmodified MILs.Finally, we compared the in vitro cytolytic potential of CD38 CAR-MILs to matched CD38 CAR-PBLs. Co-culture killing assays were performed at decreasing CAR-T effector: target (E:T) ratios. At high E:T ratios, CAR-MILs and CAR-PBLs lysed comparable percentages of CD38+ targets with similar kinetics. However, at lower E:T ratios, CAR-MILs killed a greater percentage of CD38+ targets with faster kinetics compared to CAR-PBLs.In conclusion, we have demonstrated 1) the feasibility of producing CAR-modified MILs, 2) that CAR-MILs retain their inherent tumor antigen-specificity and functional capacity - something that was lacking with CAR-PBLs, and 3) that CAR-MILs kill more efficiently in vitro than matched CAR-PBLs. These data suggest that CAR-MILs may provide both better antigen specific killing but more importantly by targeting the endogenous antigenic repertoire, they could also prevent or minimize the risk of relapse via antigen escape variants and thus increase the overall efficacy of adoptive CAR T cell therapy. DisclosuresLutz:WIndMIL Therapeutics: Employment, Patents & Royalties. Rudraraju:WIndMIL Therapeutics: Employment. DeOliveira:WIndMIL Therapeutics: Employment. Jana:WIndMIL Therapeutics: Employment. Weiss:WIndMIL Therapeutics: Employment. Borrello:WIndMIL Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Noonan:WIndMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Abstract 1757: Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4

Abstract Marrow-infiltrating lymphocytes (MILs) primed to tumor antigens have been described in patients with hematologic malignancies and in metastatic disease arising from carcinomas. The presence of tumor-reactive MILs in these patients has suggested the possibility of their utilization in T-cell immunotherapeutic approaches. Inherent in this approach are considerations that mediate MIL interactions with the microenvironment and how these may be governed for adoptive or active immunotherapy. Both E-selectin and CXCR4 are known to regulate the homing and retention of T cells to the bone marrow. GMI-1271 and GMI-1359 are potent, small-molecule glycomimetic antagonists of E-selectin and E-selectin/CXCR4, respectively. GMI-1359 is a potent small-molecule glycomimetic dual antagonist targeting E-selectin and CXCR4. In the present studies tumor-specific MILs were established in BALB/c mice that had been induced to reject the syngeneic CT26 colon carcinoma via treatment with anti-CTLA-4 T cell checkpoint antibody, and the subsequent effects of antagonizing E-selectin and/or CXCR4 with GMI-1271 or GMI-1359 on the mobilization and distribution of these bone marrow-derived tumor-specific CD8+ T cells were determined. CT26-immune mice were treated for three days with saline, GMI-1271 (40 mg/kg), or GMI-1359 (40 mg/kg) and 12 hours following the last injection, the phenotype and functional activity of CD8+ T cells were determined in bone marrow and peripheral blood. Additional controls included CT26-immune mice treated with G-CSF (0.125 mg/kg) and tumor-naïve mice treated with saline. Treatment of mice with GMI-1271 and to a greater extent with GMI-1359 led to an approximate 3-4 fold increase in CD8+CD62L+CD44- naïve and CD8+CD62L+CD44+ central memory T cells in peripheral blood. This was not observed following treatment of tumor-immune mice with G-CSF. Treatment of mice with GMI-1271 or GMI-1359 did not affect distribution of CD8+CD62L-CD44+ effector memory T cells in peripheral blood. The increase in percentages of CD8+ naïve and central memory T cells in peripheral blood following treatment with GMI-1271 or GMI-1357 functionally correlated with increased production of IFN-γ ex vivo in response to irradiated CT26 tumor cells or the immunodominant CT26 peptide, AH-1. Collectively these results demonstrate the mobilization or redistribution of marrow-infiltrating tumor-specific CD8+ T cells into peripheral blood as a consequence of E-selectin and/or E-selectin and CXCR4 antagonism. Once in the periphery, these MILs could (1) be collected for adoptive immunotherapy approaches or (2) serve as a systemic augmentation of T cells for combination with immune stimulants as a foundation to boost active immunotherapy. Citation Format: William E. Fogler, Theodore A. Smith, Rachel K. King, John L. Magnani. Mobilization of tumor-primed, marrow-infiltrating lymphocytes into peripheral blood with inhibitors of E-selectin or E-selectin and CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1757.

Cells to prevent/treat relapse following allogeneic stem cell transplantation.

Relapse of cancer remains one of the primary causes of treatment failure and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). A multitude of approaches have been used in the management of posttransplant relapse. This review focuses on recent data with cellular therapies designed to treat or prevent posttransplant relapse of hematologic malignancies, although many of these therapeutic approaches also have applications to solid tumors and in the nontransplant setting. Currently available cell therapies include second transplant, natural killer cells, monocyte-derived dendritic cell vaccines, and lymphocytes via donor lymphocyte infusion, antigen-primed cytotoxic T lymphocytes, cytokine-induced killer cells, marrow-infiltrating lymphocytes, and chimeric antigen receptor T cells. These treatment options offer the prospect for improved relapse-free survival after HSCT.

Progress of cellular and vaccine immunotherapy in multiple myeloma

Allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion for multiple myeloma (MM) can induce graft-versus-myeloma immunity and long-term survival, but limited efficacy and associated toxicities have prevented its widespread application. Cellular immunotherapies and vaccines are explored to induce more specific, reliable, and potent antimyeloma immune responses with less treatment-related risk. Advances in molecular biology, basic and applied immunology, have led to several promising approaches such as genetically engineered T cells with chimeric antigen receptors and T-cell receptors targeting myeloma-specific epitopes, vaccine primed ex vivo expanded autologous T cells, expanded marrow-infiltrating lymphocytes, and plasma cell/dendritic cell fusion vaccines. The combination of these emerging therapies to immunomodulatory drugs and inhibitors of programmed death-1 T-cell regulatory pathways could improve the outcome for MM patients. This article reviews the latest progress of cellular and vaccine immunotherapy for MM at the 58th American Society of Hematology (ASH) Annual Meeting, and discusses how these therapies might integrate and synergize with existing treatment paradigms. Key words: Multiple myeloma; Cellular immunotherapy; Vaccine immunotherapy; American Society of Hematology Annual Meeting

Immunotherapy in multiple myeloma

Therapeutic approaches in multiple myeloma (MM) are increasingly focused on restoring antitumor immunity. Immunomodulators have a variety of effects on the immune microenvironment, and are frequently incorporated into multidrug regimens. The monoclonal antibodies daratumumab and elotuzumab have been granted accelerated approval for use in the relapsed or refractory setting, and several other antibodies including immune checkpoint inhibitors are currently being evaluated for the treatment of MM. Antimyeloma vaccines have been developed, and may be useful in maintaining remission. The role of allogeneic stem cell transplantation continues to be an area of active research, as reduced intensity conditioning (RIC) regimens have significantly decreased treatment-related complications. Other immunotherapeutic approaches in development include marrow infiltrating lymphocytes, T cell receptor modified T cells (TCRts), and chimeric antigen receptor (CAR) T cells. Here we review the existing data on immunotherapy in MM, and discuss some promising areas of research which may impact the future of myeloma therapy.

Persistence of Non Gene-Modified Adoptively Transferred Marrow Infiltrating Lymphocytes (MILs) More Than Five Years Post Transfer

Hallmarks of effective adoptive T cell therapy include: 1) the ability to access and expand T cells of interest 2) measurable anti-tumor efficacy of these cells upon reinfusion and 3) T cell persistence over time. In examining the long-term follow-up of patients treated with MILs, we show evidence of persistence of adoptively transferred MILs for over five years post transfer.In a clinical trial conducted in 2007, Prevnar naïve myeloma patients were immunized against the modified diphtheria toxin, CRM197 with the Prevnar vaccine administered 2 weeks prior to collecting marrow infiltrating lymphocytes (MILs) from bone marrow. Patients received a CD34-selected (T cell depleted) autologous stem cell transplant (SCT) and had activated and expanded MILs infused 3 days later. As such, 99% of infused T cells were Prevnar-primed, ex vivo expanded MILs. This approach enabled us to track the fate of the non gene-modified MILs by examining CRM197 specific T cells. As originally reported, persistence at one year of both CRM197 and myeloma antigen-specific T cells correlated with clinical responses.1Long-term follow up was performed in a subset of the patients that had undergone bone marrow biopsies for clinical purposes. The results showed that patients who had been in a complete response at the end of the clinical trial maintained a greater number of both CRM197 and myeloma antigen-specific T cells over time for up to 7 years after the clinical trial. Patients who did not achieve a complete response and subsequently relapsed showed a loss of both CRM197 as well as myeloma antigen- specific T cells in most cases. Considering that the patients received a T cell depleted stem cell graft, the CRM197-specific T cells that were measured were likely a result of the Prevnar-primed MILs.From these results, we conclude that: 1) adoptively transferred CRM-197 specific MILs showed persistence up 7 years following their infusion. 2) the magnitude of CRM197 and myeloma antigen-specific T cell responses correlated with clinical responsiveness. Taken together, these data demonstrate several unique attributes of MILs which are critical for successful adoptive T cell therapyAdoptive transfer of activated marrow-infiltrating lymphocytes induces measurable antitumor immunity in the bone marrow in multiple myeloma. Noonan KA, Huff CA, Davis J, Lemas MV, Fiorino S, Bitzan J, Ferguson A, Emerling A, Luznik L, Matsui W, Powell J, Fuchs E, Rosner GL, Epstein C, Rudraraju L, Ambinder RF, Jones RJ, Pardoll D, Borrello I. Sci Transl Med. 2015 May 20;7(288). DisclosuresNoonan:WindMIL Therapeutics: Employment, Equity Ownership, Patents & Royalties. Rudraraju:WindMIL Therapeutics: Employment, Equity Ownership. Lutz:WindMIL Therapeutics: Employment, Equity Ownership. Borrello:BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau; WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding.

Marrow Infiltrating Lymphocytes and Vaccines in Myeloma

Despite several decades of research with limited clinical successes, immunotherapy is now establishing a permanent presence in the clinical arena for several malignancies including multiple myeloma. The ability to understand the mechanisms mediating tumor-induced tolerance as well as the availability of reagents to target them have facilitated the clinical development of several immune-based strategies. The efficacy of adoptive cell therapies has already been demonstrated by the clinical benefit observed with allogeneic bone marrow transplantation where the immune-mediated graft vs. tumor effect accounts for the deeper and more durable clinical responses when compared to autologous stem cell transplantation. Recently, adoptive T cell approaches have focused on ex vivo manipulation of cells with the goal of increasing their tumor specificity. To date, the approaches can be divided into three groups: unmodified cells; T cells expressing transgenic T cell receptors; and cells modified with chimeric antigen receptors (CARs). Each has its own benefits. Historically, adoptive T cell therapy utilized peripheral blood lymphocytes in large part because of their ease of access. Early studies in myeloma demonstrated their ability to augment immune reconstitution and effectively transfer vaccine-specific responses. However, their lack of tumor specificity has prompted our group to evaluate the role of marrow infiltrating lymphocytes (MILs). These cells possess a broad endogenous tumor specificity and are enriched for central memory T cells that make them a more suitable source of cells for adoptive T cell approaches. The properties and potential benefits of this approach will be discussed. Another immunotherapeutic approach with potential appeal is the use of myeloma vaccines to address the issue of minimal residual disease. In light of the fact that patients with myeloma rarely have long-term sustained remissions, it is reasonable to integrate a novel approach that is capable of eradicating the remaining disease burden by generating a broad, myeloma-specific, systemic immune response. We have developed a cellular, GM-CSF-based, myeloma vaccine which has been administered in the combination with the immunomodulatory agent, lenalidomide, to patients in a near complete remission (negative M-spike but with a positive immunofixation). We have demonstrated priming of a tumor-specific immune response which correlates with a durable clinical benefit. With the ever-increasing development of immune-based approaches, combining them with various agents in different disease setting to improve the overall outcomes of myeloma patients. Disclosures Borrello: Celgene: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding; WindMIL Therapeutics: Equity Ownership, Patents &amp; Royalties, Research Funding.

Increased Frequencies of PD-1+ CD8+ Marrow-Infiltrating Lymphocytes Associated with Highly Clonal T-Lymphocyte Expansions in Relapsed and Refractory AML Patients but Not Healthy Adults

Immune checkpoint therapy, particularly inhibition of the PD-1 axis, has shown remarkable efficacy in multiple solid tumor types and some hematological malignancies. Clinical results using anti-PD1 immunotherapy in AML have not yet been reported. Immune checkpoint expression levels and magnitude and/or repertoire of tumor-infiltrating lymphocytes have all been proposed as potential predictive biomarkers of response to such targeted immunotherapy. We therefore examined the marrow infiltrating T-lymphocyte compartment of Relapsed/Refractory Acute Myeloid Leukemia (RR-AML) patients undergoing salvage chemotherapy and that of healthy donors (HD) to determine baseline data for PD-1 expression and T-lymphocyte clonality.Nine adult RR-AML patients recruited to the clinical trial NCT02527447 (Myeloid Malignancies Section, NHLBI, NIH) were analyzed. Cryopreserved bone marrow aspirate mononuclear cells (BMMCs) from these patients prior to salvage chemotherapy (Day 0) and HD (n=9) were assessed using two custom ten-color flow cytometry panels. Frequencies of naive (TN), central memory (TCM), effector memory (TEM), terminal effector (TEMRA), stem cell memory (TSCM), and activated T-lymphocyte subsets, and the expression of PD-1, TIM-3, CTLA-4, 4-1BB on CD8+ T-lymphocyte subpopulations were calculated. DNA extracted from bone marrow of 5 of the RR-AML patients and 5 HD was used for sequencing of the CDR3 region of TCRB gene to evaluate TCR repertoire (ImmunoSEQ, Adaptive Biotechnologies).Immunohistochemistry of pre-treatment bone marrow biopsy sections revealed 10-40% CD3+ cells, typically scattered with focal small aggregations. Flow cytometry of marrow aspirate demonstrated more activated CD8+ CD69+ T-lymphocytes in the marrow of RR-AML patients compared with HD (19.8% vs. 9.4%, p=0.031). Significantly more CD8+ TEMRA T-lymphocytes (60.8% vs. 45.2%, p=0.040) and a trend towards fewer CD8+ TEM T-lymphocytes (19.0% vs. 32.1%, p=0.062) were observed in RR-AML versus HD. An increased proportion of total CD8+ T-lymphocytes in RR-AML had PD-1 expression compared to HD (22.0% vs. 9.2%, p=0.008). This pattern held true for every CD8+ sub-population in AML versus HD: TN (14.0% vs. 4.0%, p=0.014); TCM (24.2% vs. 7.3%, p=0.003); TEMRA (19.6% vs. 10.5%, p=0.060) and TEM (37.3% vs. 16.2%, p=0.004) (Figure 1a). There were no significant differences in frequencies of CD8+ T-lymphocyte subsets or PD-1 expression between Day 0 and Day 28 in AML patients (in 4 patients tested). Furthermore, there was negligible PD-1 co-expression with other immune checkpoint markers and no differences in 4-1BB or CTLA4 expression in AML versus HD. However, we noted significantly higher TIM-3 expression on TN and TCM CD8+ T-lymphocytes in AML (6.5% vs. 2.2%, p=0.001, and 4.5% vs. 0.7%, p=0.021, respectively).Sequencing of productive TCRB gene rearrangements revealed significantly higher average marrow T-cell clonality in AML versus HD (0.232 vs. 0.086, respectively, n=5 AML, n=5 HD). When considering the top 10 most frequent clonotypes for each AML patient and HD, frequencies were found to be much higher in AML than HD (Figure 1b), consistent with large clonal T-lymphocyte expansions in the marrow of RR-AML patients. Furthermore, we identified several TCR clones that were found in AML patients but never seen in HD. Sequencing of an additional cohort of 20 newly diagnosed adult AML patients however discovered that only a subset (25%) of those patients had T cell clonal expansions above the upper limit of that seen in HD.AML is already known to be an immunogenic malignancy, as demonstrated by the measurable efficacy of allogeneic transplantation and donor lymphocyte infusion. Our data demonstrate that the bone marrow tumor microenvironment in RR-AML is enriched for PD-1+ CD8+ marrow-infiltrating lymphocytes, and that large clonal expansions of T-lymphocytes can be found in the bone marrow in these patients. This is suggestive that RR-AML patients, for whom cytotoxic chemotherapy is often suboptimal, may benefit from immune checkpoint blockade therapies, particularly PD-1 axis inhibition, to enable improved immunologic control of AML by autologous T-lymphocytes already resident in the tumor microenvironment. Based in part on these data, a clinical trial of anti-PD-1 immunotherapy in combination with a hypomethylating agent for treatment of relapsed and refractory AML patients will open at our institution. [Display omitted] DisclosuresHourigan:Sellas Life Sciences Group: Research Funding.

Cellular and vaccine immunotherapy for multiple myeloma.

Allogeneic hematopoietic cell transplantation and donor lymphocyte infusion for multiple myeloma (MM) can induce graft-versus-myeloma immunity and long-term survivorship, but limited efficacy and associated toxicities have prevented its widespread use. Cellular immunotherapies and vaccines seek to induce more specific, reliable, and potent antimyeloma immune responses with less treatment-related risk than is possible with allogeneic transplantation. Advances in molecular biology, and basic and applied immunology, have led to promising approaches such as genetically engineered T cells with chimeric antigen receptors and T-cell receptors targeting myeloma-specific epitopes, vaccine primed ex vivo expanded autologous T cells, expanded marrow-infiltrating lymphocytes, and plasma cell/dendritic cell fusion vaccines. The addition of these emerging therapies to immunomodulatory drugs and inhibitors of programmed death-1 T-cell regulatory pathways are poised to improve outcome for our patients with myeloma.