Publications by authors named "Jeffrey S Miller"

257 Publications

Novel cell and immune engagers in optimizing tumor specific immunity post autologous transplant in multiple myeloma.

Transplant Cell Ther 2021 Oct 8. Epub 2021 Oct 8.

Blood and Marrow Transplant Program, Department of Medicine, University of Minnesota; Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota.

Autologous transplant is an important component of treatment of multiple myeloma. The post ASCT setting offers a unique opportunity to increase myeloma specific immunity through enhancement of T and NK cell responses. The vast array of therapeutics which are being developed for myeloma including cell-based therapies, dendritic vaccines, bispecific antibodies, IL-15 agonists give us the opportunity to increase tumor specific immunity. Maintenance therapies including Immunomodulatory drugs, proteasome inhibitors and daratumumab exhibit a significant anti myeloma response by modulating the immune system. Lenalidomide promotes an anti-tumoral immune microenvironment, while daratumumab can potentially cause NK cell fratricide. Hence understanding the effects of commonly used maintenance drugs on the immune system is important. In this review, we look at current and emerging therapeutics and their integration post ASCT in the context of immune reconstitution to improve clinical responses in myeloma.
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http://dx.doi.org/10.1016/j.jtct.2021.10.001DOI Listing
October 2021

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy.

Cell Stem Cell 2021 Sep 9. Epub 2021 Sep 9.

University of Minnesota, Department of Medicine, Minneapolis, MN 55455, USA. Electronic address:

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer.
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http://dx.doi.org/10.1016/j.stem.2021.08.013DOI Listing
September 2021

Activation Status Dictates the Function of Unlicensed Natural Killer Cells.

Blood Adv 2021 Sep 8. Epub 2021 Sep 8.

University of California, Davis, Sacramento, California, United States.

Natural Killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class I molecules resulting in differential responses upon activation in a process called "licensing" or "arming". NK cells expressing receptors that bind self-MHC are considered licensed due to augmented effector lytic function capability compared to unlicensed subsets. However, we demonstrated unlicensed NK subsets instead positively regulate the adaptive T cell response during viral infections due to localization and cytokine production. We demonstrate here that the differential effects of the two types of NK subsets is contingent on the environment using viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) MCMV leads to a loss of licensing-associated differences as compared to mice with low-dose infection, as the unlicensed NK subset no longer localized in lymph nodes (LN), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled with the phenotypes of both human and mouse NK cells in a HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to effects of subset depletion in T-replete models, the licensed NK cell subsets still dominated anti-viral responses post-HSCT. Overall, our results highlight the intricate tuning of the NK cells and how it impacts overall immune responses with regard to licensing patterns, as it is dependent on the level of stimulation and their activation status.
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http://dx.doi.org/10.1182/bloodadvances.2021004589DOI Listing
September 2021

Putting On the Gas and Taking Off the Brakes: A Novel Combinatorial Strategy to Enhance Tumor-Infiltrating Lymphocytes.

Cancer Immunol Res 2021 Oct 7;9(10):1110. Epub 2021 Sep 7.

Masonic Cancer Center, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, Minnesota.

The advent of checkpoint blockade and use of cytokines to enhance immune responses have changed the field of immunotherapy. Yet, these approaches are not without drawbacks including systemic toxicities and acquired therapeutic resistance. In this issue, Xu and colleagues describe a novel biological molecule composed of a PD-1-targeting antibody linked to a mutated IL15 that induces better targeting of IL15 to tumor-infiltrating lymphocytes (TIL) to decrease systemic toxicities and enhance antitumor responses.
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http://dx.doi.org/10.1158/2326-6066.CIR-21-0556DOI Listing
October 2021

Correction to: CD16xCD33 Bispecific Killer Cell Engager (BiKE) as potential immunotherapeutic in pediatric patients with AML and biphenotypic ALL.

Cancer Immunol Immunother 2021 Sep 7. Epub 2021 Sep 7.

Department of Pediatric Oncology, Hematology and Clinical Immunology, Centre for Child and Adolescent Health, Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225, Düsseldorf, Germany.

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http://dx.doi.org/10.1007/s00262-021-03040-0DOI Listing
September 2021

A HER2 Tri-Specific NK Cell Engager Mediates Efficient Targeting of Human Ovarian Cancer.

Cancers (Basel) 2021 Aug 8;13(16). Epub 2021 Aug 8.

Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

Clinical studies validated antibodies directed against HER2, trastuzumab, and pertuzumab, as useful methodology to target breast cancer cases where HER2 is expressed. The hope was that HER2 targeting using these antibodies in ovarian cancer patients would prove useful as well, but clinical studies have shown lackluster results in this setting, indicating a need for a more comprehensive approach. Immunotherapy approaches stimulating the innate immune system show great promise, although enhancing natural killer (NK) function is not an established mainstream immunotherapy. This study focused on a new nanobody platform technology in which the bispecific antibody was altered to incorporate a cytokine. Herein we describe bioengineered CAM1615HER2 consisting of a camelid VHH antibody fragment recognizing CD16 and a single chain variable fragment (scFv) recognizing HER2 cross-linked by the human interleukin-15 (IL-15) cytokine. This tri-specific killer engager (TriKE) showed in vitro prowess in its ability to kill ovarian cancer human cell lines. In addition, we demonstrated its efficacy in inducing potent anti-cancer effects in an in vivo xenograft model of human ovarian cancer engrafting both cancer cells and human NK cells. While previous approaches with trastuzumab and pertuzumab faltered in ovarian cancer, the hope is incorporating targeting and cytokine priming within the same molecule will enhance efficacy in this setting.
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http://dx.doi.org/10.3390/cancers13163994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394622PMC
August 2021

CD16xCD33 Bispecific Killer Cell Engager (BiKE) as potential immunotherapeutic in pediatric patients with AML and biphenotypic ALL.

Cancer Immunol Immunother 2021 Aug 16. Epub 2021 Aug 16.

Department of Pediatric Oncology, Hematology and Clinical Immunology, Centre for Child and Adolescent Health, Medical Faculty, Heinrich Heine University, Moorenstraße 5, 40225, Düsseldorf, Germany.

Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33 acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33 AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33 cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation.
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http://dx.doi.org/10.1007/s00262-021-03008-0DOI Listing
August 2021

Activation of ADAM17 by IL-15 Limits Human NK Cell Proliferation.

Front Immunol 2021 22;12:711621. Epub 2021 Jul 22.

Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN, United States.

Natural killer (NK) cells are innate cytotoxic lymphocytes that can recognize assorted determinants on tumor cells and rapidly kill these cells. Due to their anti-tumor effector functions and potential for allogeneic use, various NK cell platforms are being examined for adoptive cell therapies. However, their limited persistence is a current challenge. Cytokine-mediated activation of these cells is under extensive investigation and interleukin-15 (IL-15) is a particular focus since it drives their activation and proliferation. IL-15 efficacy though is limited in part by its induction of regulatory checkpoints. A disintegrin and metalloproteinase-17 (ADAM17) is broadly expressed by leukocytes, including NK cells, and it plays a central role in cleaving cell surface receptors, a process that regulates cell activation and cell-cell interactions. We report that ADAM17 blockade with a monoclonal antibody markedly increased human NK cell proliferation by IL-15 both and in a xenograft mouse model. Blocking ADAM17 resulted in a significant increase in surface levels of the homing receptor CD62L on proliferating NK cells. We show that NK cell proliferation by IL-15 and the augmentation of this process upon blocking ADAM17 are dependent on CD62L. Hence, our findings reveal for the first time that ADAM17 activation in NK cells by IL-15 limits their proliferation, presumably functioning as a feedback system, and that its substrate CD62L has a key role in this process . ADAM17 blockade in combination with IL-15 may provide a new approach to improve NK cell persistence and function in cancer patients.
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http://dx.doi.org/10.3389/fimmu.2021.711621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339566PMC
July 2021

Bi-specific and Tri-specific NK Cell Engagers: The New Avenue of Targeted NK Cell Immunotherapy.

Mol Diagn Ther 2021 09 29;25(5):577-592. Epub 2021 Jul 29.

Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, USA.

Natural killer (NK) cell-mediated cancer immunotherapy has grown significantly over the past two decades. More recently, multi-specific engagers have been developed as cancer therapeutics to effectively arm endogenous NK cells to more potently induce specific cytolytic responses against tumor targets. This review explores the bi- and tri-specific NK/tumor engagers that are emerging as a new generation of immunotherapeutics. These molecules vary in configuration, but they typically have small molecular weights and domains that engage specific tumor antigens and NK cell-activating receptors such as CD16, NKp30, NKp46, and NKG2D. They have demonstrated compelling potential in boosting NK cell cytotoxicity against specific tumor targets. This highly adaptable off-the-shelf platform, which in some formats also integrates cytokines, is poised to revolutionize targeted NK cell immunotherapy, either as a monotherapy or in combination with other effective anti-cancer therapies.
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http://dx.doi.org/10.1007/s40291-021-00550-6DOI Listing
September 2021

Anti-NKG2C/IL-15/anti-CD33 killer engager directs primary and iPSC-derived NKG2C NK cells to target myeloid leukemia.

Mol Ther 2021 Jun 24. Epub 2021 Jun 24.

Hematology, Oncology, and Transplantation, Mayo Clinic, Minneapolis, MN, USA; Department of Medicine, University of Minnesota, Minneapolis, MN, USA. Electronic address:

Natural killer (NK) cells mediate the cytolysis of transformed cells and are currently used as an adoptive cellular therapy to treat cancer. Infection with human cytomegalovirus has been shown to expand a subset of "adaptive" NK cells expressing the activation receptor NKG2C that have preferred functional attributes distinct from conventional NK cells. Because NKG2C delivers a strong activating signal to NK cells, we hypothesized that NKG2C could specifically trigger NK-cell-mediated antitumor responses. To elicit a tumor-directed response from NKG2C NK cells, we created an anti-NKG2C/IL-15/anti-CD33 killer engager called NKG2C-KE that directs NKG2C cells to target CD33 cells and tumor-associated antigen expressed by acute myelogenous leukemia cells. The NKG2C-KE induced specific degranulation, interferon-γ production, and proliferation of NKG2C-expressing NK cells from patients who reactivated cytomegalovirus after allogeneic transplantation. The NKG2C-KE was also tested in a more homogeneous system using induced pluripotent stem cell (iPSC)-derived NK (iNK) cells that have been engineered to express NKG2C at high levels. The NKG2C-KE triggered iNK-cell-mediated cytotoxicity against CD33 cells and primary AML blasts. The NKG2C-KE-specific interaction with adaptive NK and NKG2C iNK cells represents a new immunotherapeutic paradigm that uniquely engages highly active NK cells to induce cytotoxicity against AML through redirected targeting.
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http://dx.doi.org/10.1016/j.ymthe.2021.06.018DOI Listing
June 2021

Following Transplantation for Acute Myelogenous Leukemia, Donor Better Protects against Relapse than .

J Immunol 2021 Jun 11. Epub 2021 Jun 11.

Department of Structural Biology, Stanford University, Stanford, CA;

In the treatment of acute myelogenous leukemia with allogeneic hematopoietic cell transplantation, we previously demonstrated that there is a greater protection from relapse of leukemia when the hematopoietic cell transplantation donor has either the / genotype or a genotype having two or more gene segments. In those earlier analyses, genotyping could only be assessed at the low resolution of gene presence or absence. To give the analysis greater depth, we developed high-resolution sequence-based typing that defines all the alleles and distinguishes the expressed alleles from those that are not expressed. We now describe and analyze high-resolution genotypes for 890 donors of this human transplant cohort. and are the common haplotypes, with having evolved from by deletion of the , , , and genes. We observed a consistent trend for to provide stronger protection against relapse than This correlation indicates that protection depends on the donor having inhibitory KIR2DL2 and/or activating KIR2DS2, and is enhanced by the donor lacking inhibitory KIR2DL1, 2DL3, and 3DL1. High-resolution KIR typing has allowed us to compare the strength of the interactions between the recipient's HLA class I and the KIR expressed by the donor-derived NK cells and T cells, but no clinically significant interactions were observed. The trend observed between donor and reduced relapse of leukemia points to the value of studying ever larger transplant cohorts.
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http://dx.doi.org/10.4049/jimmunol.2100119DOI Listing
June 2021

Infusion reactions in natural killer cell immunotherapy: a retrospective review.

Cytotherapy 2021 07 9;23(7):627-634. Epub 2021 May 9.

Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota, USA. Electronic address:

Background Aims: The use of natural killer (NK) cells as a cellular immunotherapy has increased over the past decade, specifically in patients with hematologic malignancies. NK cells have been used at the authors' institution for over 15 years. Most patients have a reaction to NK cell infusion. The authors retrospectively analyzed the reactions associated with NK cell infusions to characterize the types of reactions and investigate why some patients have higher-grade reactions than others.

Methods: A retrospective chart review of NK cell infusions was performed at the authors' institution under nine clinical protocols from 2008 to 2016. An infusion reaction was defined as any symptom from the time of NK cell infusion up to 4 h after infusion completion. The severity of infusion reactions was graded based on Common Terminology Criteria for Adverse Events, version 4. Two major endpoints of interest were (i) infusion reaction with any symptom and (ii) grade ≥3 infusion reaction. Multivariable logistic regression models were used to investigate the association between variables of interest and outcomes. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained for each variable.

Results: A total of 130 patients were receiving NK cell infusions at the authors' institution. The most common reported symptom was chills (n = 110, 85%), which were mostly grade 1 and 2, with only half of patients requiring intervention. There were 118 (91%) patients with infusion reactions, and only 36 (28%) were grade 3. There was one life-threatening grade 4 reaction, and no death was reported due to infusion reaction. Among grade ≥3 reactions, cardiovascular reactions (mainly hypertension) were the most common, and less than half of those with hypertension required intervention. NK cell dose was not associated with any of the grade 3 infusion reactions, whereas monocyte dose was associated with headache (grade ≤3, OR, 2.17, 95% CI, 1.19-3.97) and cardiovascular reaction (grade ≥3, OR, 2.13, 95% CI, 1.13-3.99). Cardiovascular reaction (grade ≥3) was also associated with in vitro IL-2 incubation and storage time. Additionally, there was no association between grade ≥3 infusion reactions and overall response rate (OR, 0.75, 95% CI, 0.29-1.95).

Conclusions: The majority of patients who receive NK cell therapy experience grade 1 or 2 infusion reactions. Some patients experience grade 3 reactions, which are mainly cardiovascular, suggesting that close monitoring within the first 4 h is beneficial. The association of monocytes with NK cell infusion reaction relates to toxicities seen in adoptive T-cell therapy and needs further exploration.
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http://dx.doi.org/10.1016/j.jcyt.2021.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217213PMC
July 2021

Cellular Immunotherapy-Highlights from TCT 2021.

Transplant Cell Ther 2021 07 26;27(7):527-532. Epub 2021 Apr 26.

Division of Hematology, Oncology, and Transplantation, University of Minnesota Cancer Center, Minneapolis, Minnesota.

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http://dx.doi.org/10.1016/j.jtct.2021.04.015DOI Listing
July 2021

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion.

Cytotherapy 2021 08 20;23(8):704-714. Epub 2021 Apr 20.

Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota Cancer Center, Minneapolis, Minnesota, USA. Electronic address:

Background Aims: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 10 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg.

Results: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset.

Discussion: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.
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http://dx.doi.org/10.1016/j.jcyt.2021.02.118DOI Listing
August 2021

Early Adaptive Natural Killer Cell Expansion Is Associated with Decreased Relapse After Autologous Transplantation for Multiple Myeloma.

Transplant Cell Ther 2021 04 17;27(4):310.e1-310.e6. Epub 2020 Dec 17.

Department of Medicine, University of Minnesota, Minneapolis, Minnesota.

Adaptive natural killer (NK) cells are long-lived and exhibit properties of immunologic memory against cytomegalovirus (CMV). We previously reported that expansion of adaptive NK cells after CMV reactivation in recipients of allogeneic hematopoietic cell transplantation (HCT) was associated with a lower rate of relapse of acute myelogenous leukemia. In the present study, we examined the impact of adaptive NK cell expansion in a cohort of 110 individuals who underwent autologous HCT (AHCT) for a lymphoid malignancy (lymphoma or multiple myeloma [MM]). In this cohort, higher absolute numbers of adaptive NK cells (>1.58/μL) at day 28 post-AHCT were associated with significantly decreased risk of relapse in patients with MM. No significant association was seen in patients with lymphoma. Further stratification of MM patients by CMV serostatus found a strong protective effect of adaptive NK cells only in CMV-seropositive individuals. These findings suggest that strategies to increase adaptive NK cells after AHCT may be a therapeutic option in patients with MM.
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http://dx.doi.org/10.1016/j.jtct.2020.10.023DOI Listing
April 2021

Endoscopic Robotic Retrieval of a Migrated Subclavian Vein Stent from the Right Ventricle.

Innovations (Phila) 2021 Mar-Apr;16(2):201-203. Epub 2021 Mar 23.

13713773422646 Department of Surgery, Division of Cardiothoracic Surgery, Emory Saint Joseph's Hospital, Emory University School of Medicine, Atlanta, GA, USA.

A 54-year-old woman with end-stage renal disease on hemodialysis with access through a right arm arteriovenous fistula presented with right arm swelling. Venography demonstrated right subclavian vein stenosis. A balloon angioplasty of the stenotic vein was unsuccessful, and she subsequently underwent stent placement with balloon angioplasty. Ten days following the procedure, she developed acute shortness of breath. Transthoracic echocardiogram demonstrated the migration of the venous stent into the right ventricle. Using an endoscopic robotic approach, the stent was successfully extracted from the beating heart.
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http://dx.doi.org/10.1177/1556984520986656DOI Listing
March 2021

First-in-human phase 1 trial of induced regulatory T cells for graft-versus-host disease prophylaxis in HLA-matched siblings.

Blood Adv 2021 03;5(5):1425-1436

Blood and Marrow Transplant Program.

Human CD4+25- T cells cultured in interleukin 2 (IL-2), rapamycin, and transforming growth factor β (TGFβ) along with anti-CD3 monoclonal antibody-loaded artificial antigen-presenting cells generate FoxP3+ induced regulatory T cells (iTregs) with potent suppressive function. We performed a phase 1, single-center, dose-escalation study to determine the safety profile of iTregs in adults with high-risk malignancy treated with reduced-intensity conditioning and mobilized peripheral blood stem cells (PBSCs) from HLA-identical sibling donors. Sixteen patients were enrolled and 14 were treated (2 productions failed to meet desired doses). One patient each received 3.0 × 106/kg, 3.0 × 107/kg, and 3.0 × 108/kg iTregs with corresponding T-conventional-to-iTreg ratios of 86:1, 8:1, and 1:2. After 3 patients received 3.0 × 108/kg in the presence of cyclosporine (CSA) and mycophenolate mofetil (MMF) with no dose-limiting toxicities, subsequent patients were to receive iTregs in the presence of sirolimus/MMF that favors Foxp3 stability based on preclinical modeling. However, 2 of 2 developed grade 3 acute graft-versus-host disease (GVHD), resulting in suspension of the sirolimus/MMF. An additional 7 patients received 3.0 × 108/kg iTregs with CSA/MMF. In the 14 patients treated with iTregs and CSA/MMF, there were no severe infusional toxicities with all achieving neutrophil recovery (median, day 13). Of 10 patients who received 3.0 × 108/kg iTregs and CSA/MMF, 7 had no aGVHD, 2 had grade 2, and 1 had grade 3. Circulating Foxp3+ iTregs were detectable through day 14. In summary, iTregs in the context of CSA/MMF can be delivered safely at doses as high as 3 × 108/kg. This trial was registered at www.clinicaltrials.gov as #NCT01634217.
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http://dx.doi.org/10.1182/bloodadvances.2020003219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948291PMC
March 2021

Low-density PD-1 expression on resting human natural killer cells is functional and upregulated after transplantation.

Blood Adv 2021 02;5(4):1069-1080

Blood and Marrow Transplant Program and.

Expression of programmed cell death protein 1 (PD-1) on natural killer (NK) cells has been difficult to analyze on human NK cells. By testing commercial clones and novel anti-PD-1 reagents, we found expression of functional PD-1 on resting human NK cells in healthy individuals and reconstituting NK cells early after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Peripheral blood samples from healthy individuals and transplant recipients were stained for PD-1 expression using the commercial anti-PD-1 clone PD1.3.1.3, fluorescein isothiocyanate (FITC)-labeled pembrolizumab, or an FITC-labeled single-chain variable fragment (scFv) reagent made from pembrolizumab. These reagents identified low yet consistent basal PD-1 expression on resting NK cells, a finding verified by finding lower PD-1 transcripts in sorted NK cells compared with those in resting or activated T cells. An increase in PD-1 expression was identified on paired resting NK cells after allo-HSCT. Blockade of PD-1 on resting NK cells from healthy donors with pembrolizumab did not enhance NK function against programmed death-ligand 1 (PD-L1)-expressing tumor lines, but blocking with its scFv derivative resulted in a twofold increase in NK cell degranulation and up to a fourfold increase in cytokine production. In support of this mechanism, PD-L1 overexpression of K562 targets suppressed NK cell function. Interleukin-15 (IL-15) activity was potent and could not be further enhanced by PD-1 blockade. A similar increase in function was observed with scFv PD-1 blockade on resting blood NK cells after allo-HSCT. We identify the functional importance of the PD-1/PD-L1 axis on human NK cells in which blockade or activation to overcome inhibition will enhance NK cell-mediated antitumor control.
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http://dx.doi.org/10.1182/bloodadvances.2019001110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903227PMC
February 2021

Quantitative serum PCR argues against long-term persistence of HHV-6 viremia after umbilical cord blood transplantation.

Transpl Infect Dis 2021 06 8;23(3):e13555. Epub 2021 Jan 8.

Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, MN, USA.

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http://dx.doi.org/10.1111/tid.13555DOI Listing
June 2021

iPSC-derived NK cells maintain high cytotoxicity and enhance in vivo tumor control in concert with T cells and anti-PD-1 therapy.

Sci Transl Med 2020 11;12(568)

Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA.

The development of immunotherapeutic monoclonal antibodies targeting checkpoint inhibitory receptors, such as programmed cell death 1 (PD-1), or their ligands, such as PD-L1, has transformed the oncology landscape. However, durable tumor regression is limited to a minority of patients. Therefore, combining immunotherapies with those targeting checkpoint inhibitory receptors is a promising strategy to bolster antitumor responses and improve response rates. Natural killer (NK) cells have the potential to augment checkpoint inhibition therapies, such as PD-L1/PD-1 blockade, because NK cells mediate both direct tumor lysis and T cell activation and recruitment. However, sourcing donor-derived NK cells for adoptive cell therapy has been limited by both cell number and quality. Thus, we developed a robust and efficient manufacturing system for the differentiation and expansion of high-quality NK cells derived from induced pluripotent stem cells (iPSCs). iPSC-derived NK (iNK) cells produced inflammatory cytokines and exerted strong cytotoxicity against an array of hematologic and solid tumors. Furthermore, we showed that iNK cells recruit T cells and cooperate with T cells and anti-PD-1 antibody, further enhancing inflammatory cytokine production and tumor lysis. Because the iNK cell derivation process uses a renewable starting material and enables the manufacturing of large numbers of doses from a single manufacture, iNK cells represent an "off-the-shelf" source of cells for immunotherapy with the capacity to target tumors and engage the adaptive arm of the immune system to make a "cold" tumor "hot" by promoting the influx of activated T cells to augment checkpoint inhibitor therapies.
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http://dx.doi.org/10.1126/scitranslmed.aaz5618DOI Listing
November 2020

A trispecific killer engager molecule against CLEC12A effectively induces NK-cell mediated killing of AML cells.

Leukemia 2021 06 23;35(6):1586-1596. Epub 2020 Oct 23.

Division of Adult and Pediatric Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, USA.

The low 5-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using natural killer (NK) cells, we have developed a trispecific killer engager (TriKE) molecule containing a humanized anti-CD16 heavy chain camelid single-domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK-cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK-cell specific proliferation, enhanced NK-cell activation, and killing of both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in preclinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.
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http://dx.doi.org/10.1038/s41375-020-01065-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189652PMC
June 2021

Therapeutic effect of TRC105 and decitabine combination in AML xenografts.

Heliyon 2020 Oct 13;6(10):e05242. Epub 2020 Oct 13.

Dept. of Medicine, University of Minnesota, Minneapolis, MN, USA.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, often characterized by poor prognosis following standard induction therapy. The hypomethylating agent decitabine (DAC) is an alternative treatment for elderly and relapsed/refractory AML patients, yet responses following DAC monotherapy are still modest. The transforming growth factor-β (TGF-β) receptor CD105 (endoglin) is expressed in various hematopoietic malignancies, and high CD105 expression correlates with poor prognosis in AML patients. Using a xenograft model, we have recently demonstrated that targeting CD105 AML blasts with the TRC105 monoclonal antibody inhibits leukemia progression. Here we investigated whether administration of TRC105 along with DAC could represent a novel therapeutic option for relapsed/refractory AML. Our data show that the DAC/TRC105 combination results in a more durable anti-leukemic effect in AML xenografts compared to DAC monotherapy. Moreover, the DAC/TRC105 combination enhanced reactive oxygen species (ROS) activity, which correlated with reduced leukemia burden. RNA-sequencing studies suggest that TRC105 may alter TGF-β activity in AML blasts. Taken together, these findings provide rationale for the clinical evaluation of TRC105 in combination with DAC in AML patients.
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http://dx.doi.org/10.1016/j.heliyon.2020.e05242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566100PMC
October 2020

The Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of acute leukemia.

J Immunother Cancer 2020 10;8(2)

Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Acute leukemia is a constellation of rapidly progressing diseases that affect a wide range of patients regardless of age or gender. Traditional treatment options for patients with acute leukemia include chemotherapy and hematopoietic cell transplantation. The advent of cancer immunotherapy has had a significant impact on acute leukemia treatment. Novel immunotherapeutic agents including antibody-drug conjugates, bispecific T cell engagers, and chimeric antigen receptor T cell therapies have efficacy and have recently been approved by the US Food and Drug Administration (FDA) for the treatment of patients with acute leukemia. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to develop a clinical practice guideline composed of consensus recommendations on immunotherapy for the treatment of acute lymphoblastic leukemia and acute myeloid leukemia.
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http://dx.doi.org/10.1136/jitc-2020-000810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574947PMC
October 2020

NK-Cell-Mediated Targeting of Various Solid Tumors Using a B7-H3 Tri-Specific Killer Engager In Vitro and In Vivo.

Cancers (Basel) 2020 Sep 18;12(9). Epub 2020 Sep 18.

Department of Medicine, Division of Hematology, Oncology, and Transplantation, University of Minnesota, Minneapolis, MN 55455, USA.

We improved the bispecific antibody platform that primarily engages natural killer (NK) cells to kill cancer cells through antibody-dependent cellular cytotoxicity (ADCC) by adding IL-15 as a crosslinker that expands and self-sustains the effector NK cell population. The overall goal was to target B7-H3, an established marker predominantly expressed on cancer cells and minimally expressed on normal cells, and prove that it could target cancer cells in vitro and inhibit tumor growth in vivo. The tri-specific killer engager (TriKE) was assembled by DNA shuffling and ligation using DNA encoding a camelid anti-CD16 antibody fragment, a wild-type IL-15 moiety, and an anti-B7-H3 scFv (clone 376.96). The expressed and purified cam1615B7H3 protein was tested for in vitro NK cell activity against a variety of tumors and in vivo against a tagged human MA-148 ovarian cancer cell line grafted in NSG mice. cam1615B7H3 showed specific NK cell expansion, high killing activity across a range of B7-H3+ carcinomas, and the ability to mediate growth inhibition of aggressive ovarian cancer in vivo. cam1615B7H3 TriKE improves NK cell function, expansion, targeted cytotoxicity against various types of B7-H3-positive human cancer cell lines, and delivers an anti-cancer effect in vivo in a solid tumor setting.
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http://dx.doi.org/10.3390/cancers12092659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564091PMC
September 2020

Exploring the NK cell platform for cancer immunotherapy.

Nat Rev Clin Oncol 2021 02 15;18(2):85-100. Epub 2020 Sep 15.

Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN, USA.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.
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http://dx.doi.org/10.1038/s41571-020-0426-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316981PMC
February 2021

Ascorbic Acid Promotes Demethylation during Early NK Cell Differentiation.

J Immunol 2020 09 5;205(6):1513-1523. Epub 2020 Aug 5.

Department of Medicine, University of Minnesota, Minneapolis, MN 55455; and

Variegated expression of killer Ig-like receptors (KIR) in human NK cells is a stochastic process exclusive to subsets of mature NK cells and CD8 T cells. Allele-specific expression is maintained by DNA methylation within the proximal promoter regions. Because genes are densely methylated in NK cell progenitors, there is an implied stage of human NK cell development in which DNA demethylation takes place to allow for active transcription. When and how this process occurs is unknown. In this study, we show that proximal promoters are densely methylated in less mature CD56 NK cells and are progressively demethylated in CD56 NK cells as they mature and acquire KIR. We hypothesized that ten-eleven translocation (TET) enzymes, which oxidize 5mC on DNA could mediate promoter demethylation. The catalytic efficiency of TET enzymes is known to be enhanced by ascorbic acid. We found that the addition of ascorbic acid to ex vivo culture of sorted CD56 NK cells increased the frequency of KIR expression in a dose-dependent manner and facilitated demethylation of proximal promoters. A marked enrichment of the transcription factor Runx3 as well as TET2 and TET3 was observed within proximal promoters in CD56 NK cells cultured with ascorbic acid. Additionally, overexpression of TET3 and Runx3 promoted KIR expression in CD56 NK cells and NK-92 cells. Our results show that promoter demethylation can be induced in CD56, and this process is facilitated by ascorbic acid.
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http://dx.doi.org/10.4049/jimmunol.2000212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484163PMC
September 2020

Unraveling exhaustion in adaptive and conventional NK cells.

J Leukoc Biol 2020 10 29;108(4):1361-1368. Epub 2020 Jul 29.

Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA.

Immune exhaustion in T cells significantly impacts their ability to control malignancies and infections, and its discovery has led to revolutionary therapies for cancer in the form of checkpoint blockade. NK cells, like T cells, are lymphocytes that recognize virally infected and malignantly transformed cells. However, it remains unclear if NK cells are similarly susceptible to exhaustion. In this review, the aims are to summarize what is currently known and to identify key areas of variability that skew the scientific literature on NK cell exhaustion. A lack of consensus on the defining features of NK cell dysfunctional states such as senescence, suppression, and exhaustion has made a comparison between studies difficult. There are also significant differences in the biology of NK cell subsets with long-lived, adaptive NK cells sharing an epigenetic signature closer to memory CD8  T cells than to conventional NK cells. Very different checkpoint receptor expression and effector functions have been shown in adaptive versus conventional NK cells chronically exposed to activating signals. Adaptive NK cells develop in individuals with cytomegalovirus (CMV) infection and well over half of the human population worldwide is CMV seropositive by adulthood. Despite this high prevalence, most studies do not account or control for this population. This may contribute to some of the variability reported in the literature on checkpoint receptor expression on NK cells. In this review, the protective role that exhaustion plays in T cells will also be discussed and the evidence for a similar phenomenon in NK cells will be examined.
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http://dx.doi.org/10.1002/JLB.4MR0620-091RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722010PMC
October 2020

Potent Cytolytic Activity and Specific IL15 Delivery in a Second-Generation Trispecific Killer Engager.

Cancer Immunol Res 2020 09 13;8(9):1139-1149. Epub 2020 Jul 13.

Division of Hematology, Oncology, and Transplantation, Department of Medicine, University of Minnesota, Minneapolis, Minnesota.

Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484162PMC
September 2020

Vacuum-assisted extraction of ilio-caval and right heart masses: A 5-year single center experience.

J Card Surg 2020 Aug 19;35(8):1787-1792. Epub 2020 Jun 19.

Department of Cardiothoracic Surgery, Emory University, Atlanta, Georgia.

Purpose: Despite paucity of data, there exists growing popularity of catheter-based extraction methods for intravascular thrombi and vegetations. We describe a large single center experience with vacuum-assisted extraction techniques (VAET) for right-sided intravascular and cardiac masses.

Methods: We retrospectively reviewed the perioperative course of patients undergoing VAET between 2014 and 2019. Primary outcomes were survival and freedom from recurrent bacteremia. Procedural success was a composite definition of survival, majority of mass extraction, absence of recurrent bacteremia, and valve function not requiring further intervention during index hospitalization.

Results: Of the entire cohort (n = 58), 48% and 52% underwent VAET for vegetations and sterile thrombi, respectively. Of those with positive cultures, the most common organism isolated was Staphylococcus aureus (48%). Preoperative active bacteremia was present in 36% (21/58) and of these patients, 76% (16/21) had neither recurrent nor persistent bacteremia post-op. The majority of masses (67%, 38/58) were debulked with an average reduction in size of 42%. Conversion to open surgery occurred in 3.5% (2/58). Intraoperative and 30-day survival were 98% (57/58) and 90% (28/31), respectively. Overall success was 86% (50/58). The prevalence of moderate/severe tricuspid regurgitation was 37% pre-op and 61% post-op. Average length of intensive care unit and overall hospital stay was 5.6 and 16 days, respectively.

Conclusions: In this single center experience, VAET was conducted safely with a high degree of success and freedom from short-term recurrent bacteremia. This minimally invasive procedure is an attractive alternative to traditional open techniques for removal of right-sided intravascular and cardiac masses.
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http://dx.doi.org/10.1111/jocs.14711DOI Listing
August 2020

Human CD83-targeted chimeric antigen receptor T cells prevent and treat graft-versus-host disease.

J Clin Invest 2020 09;130(9):4652-4662

Department of Immunology, Moffitt Cancer Center, Tampa, Florida, USA.

Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4+ T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT - GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83+ AML, warrants clinical investigation.
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http://dx.doi.org/10.1172/JCI135754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456225PMC
September 2020
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