Indreshpal Kaur

Cord-blood engraftment with ex vivo mesenchymal-cell coculture.

BACKGROUND Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells

One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion techniques were compared: (1) CD133+ selection followed by ex vivo liquid culture and (2) co-culture of unmanipulated CB with bone-marrow-derived mesenchymal stem cells (MSCs). Ex vivo culture was performed in medium supplemented with granulocyte colony-stimulating factor, stem cell factor and either thrombopoietin or megakaryocyte growth and differentiation factor. Expansion was followed by measuring total nucleated cell (TNC), CD133+ and CD34+ cell, colony-forming unit and cobblestone area-forming cell output. When compared to liquid culture, CB-MSC co-culture (i) required less cell manipulation resulting in less initial HPC loss and (ii) markedly improved TNC and HPC output. CB-MSC co-culture therefore holds promise for improving engraftment kinetics in CB transplant recipients.

Antigen presenting cell-mediated expansion of human umbilical cord blood yields log-scale expansion of natural killer cells with anti-myeloma activity.

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56+/CD3−) and less than 1% CD3+ cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Phase 1 clinical trial using mbIL21 ex-vivo expanded donor-derived NK cells after haploidentical transplantation

Relapse has emerged as the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT). To test the hypothesis that natural killer (NK) cells can decrease the risk of leukemia relapse, we initiated a phase 1 dose-escalation study of membrane-bound interleukin 21 (mbIL21) expanded donor NK cells infused before and after haploidentical HSCT for high-risk myeloid malignancies. The goals were to determine the safety, feasibility, and maximum tolerated dose. Patients received a melphalan-based reduced-intensity conditioning regimen and posttransplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. NK cells were infused on days -2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 105/kg to 1 × 108/kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients engrafted with donor cells. Seven patients (54%) developed grade 1-2 acute GVHD (aGVHD), none developed grade 3-4 aGVHD or chronic GVHD, and a low incidence of viral complications was observed. One patient died of nonrelapse mortality; 1 patient relapsed. All others were alive and in remission at last follow-up (median, 14.7 months). NK-cell reconstitution was quantitatively, phenotypically, and functionally superior compared with a similar group of patients not receiving NK cells. In conclusion, this trial demonstrated production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, and was associated with significantly improved NK-cell number and function, lower viral infections, and low relapse rate posttransplant.

Phase I study of cord blood‐derived natural killer cells combined with autologous stem cell transplantation in multiple myeloma

Multiple myeloma (MM) is a disease with known immune dysregulation. Natural killer (NK) cells have shown preclinical activity in MM. We conducted a first‐in‐human study of umbilical cord blood‐derived (CB) NK cells for MM patients undergoing high dose chemotherapy and autologous haematopoietic stem cell transplantation (auto‐HCT). Patients received lenalidomide (10 mg) on days −8 to −2, melphalan 200 mg/m2 on day −7, CB‐NK cells on day −5 and auto‐HCT on day 0. Twelve patients were enrolled, three on each of four CB‐NK cell dose levels: 5 × 106, 1 × 107, 5 × 107 and 1 × 108 CB‐NK cells/kg. Ten patients had either high‐risk chromosomal changes or a history of relapsed/progressed disease. There were no infusional toxicities and no graft‐versus‐host disease. One patient failed to engraft due to poor autologous graft quality and was rescued with a back‐up autologous graft. Overall, 10 patients achieved at least a very good partial response as their best response, including eight with near complete response or better. With a median follow‐up of 21 months, four patients have progressed or relapsed, two of whom have died. CB‐NK cells were detected in vivo in six patients, with an activated phenotype (NKG2D+/NKp30+). These data warrant further development of this novel cellular therapy.

Comparison of two methodologies for the enrichment of mononuclear cells from thawed cord blood products: The automated Sepax system versus the manual Ficoll method

BACKGROUND AIMS Umbilical cord blood (CB) is being used as a source of hematopoietic stem cells (HSCs) and immune cells to treat many disorders. Because these cells are present in low numbers in CB, investigators have developed strategies to expand HSCs and other immune cells such as natural killer (NK) cells. The initial step in this process is to enrich mononuclear cells (MNCs) while depleting unwanted cells. The manual method of MNC enrichment is routinely used by many centers; however, it is an open system, time-consuming and operator dependent. For clinical manufacturing, it is important to have a closed system to avoid microbial contamination. METHODS In this study, we optimized an automated, closed system (Sepax) for enriching MNCs from cryopreserved CB units. RESULTS Using Sepax, we observed higher recovery of total nucleated cells (TNC), CD34+ cells, NK cells and monocytes when compared to manual enrichment, despite similar TNC and CD34+ viability with the two methods. Even though the depletion of red blood cells, granulocytes and platelets was superior using the manual method, significantly higher CFU-GM were obtained in MNCs enriched using Sepax compared to the manual method. This is likely related to the fact that the automated Sepax significantly shortened the processing time (Sepax: 74 - 175 minutes versus manual method: 180 - 290 minutes). The use of DNAse and MgCl2 during the Sepax thaw and wash procedure prevents clumping of cells and loss of viability, resulting in improved post-thaw cell recovery. DISCUSSION We optimized enrichment of MNCs from cryopreserved CB products in a closed system using the Sepax which is a walk away and automated processing system.

Ex Vivo Mesenchymal Precursor Cell–Expanded Cord Blood Transplantation after Reduced-Intensity Conditioning Regimens Improves Time to Neutrophil Recovery

We previously showed the safety of using cord blood (CB) expanded ex vivo in cocultures with allogeneic mesenchymal precursor cells (MPC) after myeloablative conditioning with faster recovery of neutrophils and platelets compared with historical controls. Herein, we report the transplantation outcomes of 27 patients with hematologic cancers who received 1 CB unit expanded ex vivo with MPCs in addition to an unmanipulated CB (MPC group) after reduced-intensity conditioning (RIC). The results in this group were compared with 51 historical controls who received 2 unmanipulated CB units (control group). The analyses were stratified for 2 RIC treatment groups: (1) total body irradiation 200 cGy + cyclophosphamide + fludarabine) (TCF), and (2) fludarabine + melphalan  (FM). Coculture of CB with MPCs led to an expansion of total nucleated cells by a median factor of 12 and of CD34+ cells by a median factor of 49. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 12 days in the MPC group, as compared with 16 days in controls (P = .02). The faster neutrophil engraftment was observed in both RIC groups. The cumulative incidence of neutrophil engraftment on day 26 was 75% with expansion versus 50% without expansion in patients who received FM as the RIC regimen (P = .03). Incidence of neutrophil engraftment was comparable in MPC and control groups if treated with TCF (82% versus 79%, P = .40). Transplantation of CB units expanded with MPCs is safe and effective with faster neutrophil engraftment even after RIC regimens.

Cytokines Produced by Dendritic Cells Administered Intratumorally Correlate with Clinical Outcome in Patients with Diverse Cancers

Purpose: Dendritic cells (DC) initiate adaptive immune responses through the uptake and presentation of antigenic material. In preclinical studies, intratumorally injected activated DCs (aDCs; DCVax-Direct) were superior to immature DCs in rejecting tumors from mice. Experimental Design: This single-arm, open-label phase I clinical trial evaluated the safety and efficacy of aDCs, administered intratumorally, in patients with solid tumors. Three dose levels (2 million, 6 million, and 15 million aDCs per injection) were tested using a standard 3 + 3 dose-escalation trial design. Feasibility, immunogenicity, changes to the tumor microenvironment after direct injection, and survival were evaluated. We also investigated cytokine production of aDCs prior to injection. Results: In total, 39 of the 40 enrolled patients were evaluable. The injections of aDCs were well tolerated with no dose-limiting toxicities. Increased lymphocyte infiltration was observed in 54% of assessed patients. Stable disease (SD; best response) at week 8 was associated with increased overall survival. Increased secretion of interleukin (IL)-8 and IL12p40 by aDCs was significantly associated with survival (P = 0.023 and 0.024, respectively). Increased TNFα levels correlated positively with SD at week 8 (P < 0.01). Conclusions: Intratumoral aDC injections were feasible and safe. Increased production of specific cytokines was correlated with SD and prolonged survival, demonstrating a link between the functional profile of aDCs prior to injection and patient outcomes. Clin Cancer Res; 24(16); 3845–56. ©2018 AACR.

Abstract B005: Cytokine production by intratumorally administered activated dendritic cells correlates with survival in a Phase I clinical trial in diverse cancers

Background: Activated, autologous dendritic cells (aaDC) can be used to induce anti-tumor immune responses. A unique method of applying aaDC is through intratumoral injection, where the tumor cells serve as the source of antigen required for an adaptive anti-tumor response. A local effect may also occur as a result of cytokine production by the injected DC which makes the tumor more susceptible to a pre-existing or an induced immune attack. Methods: Forty patients with locally advanced or metastatic solid tissue cancers were treated in a dose escalation trial in which aaDC were injected percutaneously under image guidance into a single tumor. Subjects had a median of 3 tumors (range 1 - 5) and had received an average of 3.1 prior treatments. To generate the aaDC, autologous monocytes were converted ex vivo into DC which were then activated. All batches of DC were released based on pre-specified criteria which included immunophenotyping and a T cell-stimulation assay, as well as sterility and endotoxin levels. Cytokine levels produced by the activated DC during manufacturing were measured and patient outcomes were correlated to these expression levels. Results: All three doses levels were well tolerated. The main adverse events related to treatment were grade 1 and 2 fevers. Twenty-one patients achieved stable disease (SD) 8 weeks after initiating treatment, and this was found to correlate with survival (p = 0.01). Levels of certain cytokines, such as such IL-8 and IL-12 p40, and TNFα were substantially elevated in vitro and IL-8 and IL-12 p40 production were predictive of survival (p = 0.001 and p = 0.008 resp.). TNFα levels also correlated with SD at week 8 (p = 0.01). More than 70% of patients tested were found to have significant T cell responses, and/or de novo or significantly enhanced PD-L1 expression in the tumor post treatment, with a trend towards improved survival (p = 0.1). Conclusions: Study outcomes such as stabilization of disease and survival correlated with high DC cytokine levels, in the absence of meaningful toxicity. The DCVax treatment may be mediated through direct cytotoxic effects, as well as modulation of the tumor microenvironment to increase tumor infiltration by T cells, and attraction of inflammatory cells to the tumor. The development of PD-L1 expression likely reflects an induced immune response. Citation Format: Vivek Subbiah, Ravi Murthi, Robert Prins, Kyle Hendricks, Chitra Hosing, Lori Noffsinger, Mary McGuire, Robert Brown, Aung Naing, David Hong, Siqing Fu, Anthony Conley, Indreshpal Kaur, Sarah Campion, Marnix Bosch. Cytokine production by intratumorally administered activated dendritic cells correlates with survival in a Phase I clinical trial in diverse cancers [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B005.

Abstract 2499: Clinical and immunopathological effects following Image-guided intratumoral injection of activated, autologous dendritic cells in patients with advanced solid cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Preclinical models have shown that activated DCs can effectively clear both injected (local) and non-injected (distal) inoperable tumor lesions. Based on this strong rationale a Phase I trial was designed to exploit this finding in late stage solid tumors. Methods: We designed a phase I study to determine the safety of intratumoral injection of activated DC by administering the product repeatedly until disease progression or unacceptable toxicity, in advanced cancers. Endpoints included maximum tolerated (MTD), dose limiting toxicities (DLT), safety, and immune response criteria (iRC) and RECIST. The treatment consisted of image-guided intratumoral (IT) injections of autologous, activated dendritic cells (DCVax-Direct,). Injections were given on days 0, 7 and 14, followed by booster immunizations at weeks 4, 8 and 16. Three dose levels, at 2 million, 6 million or 15 million DC per injection, were tested. Concomitant, serial biopsies were performed at the time of the vaccination in the absence of toxicity /progression. Systemic immune responses were tracked through evaluation of T cell subsets and cytokines in circulation, and through T cell receptor (TCR) sequencing in the tumor tissue and in the periphery. Results: To date, 40 patients (men, n = 18; women, n = 22), median age 53.7 (range 30 - 73) years, median of 3.1 (1 - 6) prior therapies (including 1 patient with prior immune therapy) were enrolled. Seventeen patients were treated at the 2 million dose level, 19 at the 6 million dose level, and 3 at 15 million. The MTD has not been reached and there were no dose limiting toxicities. Two patients experienced Grade 3/4 drug related toxicities : one case of fever and dehydration and one case of systemic inflammatory response syndrome. Patterns of immunological reactivity were assessed by pathological scoring on tumor biopsies for 29 patients, and included increasing necrosis in 62% of patients and emergence or amplification of infiltrating T cells in 55%. In-depth study was carried out in a patient with therapy-refractory, de-differentiated liposarcoma whose imaging at 12 weeks post initial injection revealed necrosis in the primary tumor and stable lung metastases. Biopsies demonstrated an intra-tumoral inflammatory response consisting of lymphocytes, macrophages and TIA-1 expressing cells, suggesting cytolytic activity. TCR sequencing revealed the presence of shared TCR sequences in the tumor as well as in circulation, demonstrating a systemic anti-tumor response. Analysis of T cell subsets in circulation demonstrated normalization of the CD4/CD8ratio. Conclusion: Immunotherapy with partially activated DCs injected IT demonstrate early signals of immune reactivity even in late stage patients with cancer. Preliminary data support the generation of anti-tumor effects following IT injection of the partially activated, autologous DC’s. Citation Format: Vivek Subbiah, Ravi Murthy, David S. Hong, Robert E. Brown, Robert Prins, Chitra Hosing, Mary McGuire, Aung Naing, Siquing Fu, Tina Chou, Quan Lin, Richard P. Guevarra, Anthony Conley, Indreshpal Kaur, Funda Meric-Bernstam, Marnix Bosch. Clinical and immunopathological effects following Image-guided intratumoral injection of activated, autologous dendritic cells in patients with advanced solid cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2015-2499