Mechanisms of IL-21 Enhancement of Rituximab Efficacy in a Lymphoma Xenograft Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1404-1404
Author(s):  
Steve D. Hughes ◽  
Ken Bannink ◽  
Cecile Krejsa ◽  
Mark Heipel ◽  
Becky Johnson ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Antibody Therapy ◽  
Xenograft Model ◽  
Tumor Model ◽  
Cell Types ◽  
Current Evidence

Abstract Interleukin 21 (IL-21) is an IL-2 family cytokine produced by activated CD4+ T cells. Potent effects of IL-21 have been observed on the growth, survival, and functional activation of T cells, B cells, and natural killer (NK) cells. A Phase I clinical trial of IL-21 in metastatic melanoma and renal cell carcinoma is currently in progress. We recently reported that IL-21 significantly enhanced rituximab mediated clearance of CD20+ lymphoma cell lines both in vitro and in vivo, and that these effects were potentially mediated through IL-21 enhancement of NK cell capacity to effect antibody dependent cellular cytotoxicity (ADCC). Specifically, NK cells treated with IL-21 showed increased cytotoxicity, granzyme B and IFNg production. Current studies aim to further evaluate the mechanisms by which IL-21 enhances ADCC. A number of observations suggest a multi-factorial basis for IL-21 synergy with rituximab. In a xenograft tumor model, SCID mice were injected IV with HS Sultan cells on day 0. Treatment with recombinant murine IL-21 (mIL-21; starting day 1) combined with rituximab (starting day 3) resulted in significantly increased survival (70% vs. 20% on day 100), compared to rituximab alone. In separate studies, the spleens of mice treated with mIL-21 showed increased numbers of activated macrophages and granulocytes. As macrophages and granulocytes can participate in ADCC, IL-21 synergy with rituximab in vivo may be partly dependent on its activation of these cell types. We have also evaluated whether direct effects of IL-21 on lymphoma cells contribute to enhancement of rituximab efficacy. The xenogeneic B lymphoma models in which IL-21 plus rituximab exhibited enhanced survival are highly aggressive and these models were not shown to respond to treatment with mIL-21 alone. In vitro studies were performed to determine if IL-21 could potentiate the growth inhibitory and pro-apoptotic effects of rituximab. In the absence of effector cells synergistic interaction was not observed. In addition, we tested the ability of IL-21 to enhance cytotoxicity when combined with antibodies targeting non-hematopoietic tumor cells (e.g. trastuzumab). Human NK cells treated with IL-21 displayed significantly increased cytotoxicity in ADCC assays using trastuzumab to target breast cancer cells expressing varying levels of HER-2 antigen. In summary, the current evidence suggests that IL-21 can enhance antibody-mediated tumor cell lysis through activation of multiple effectors of ADCC. Thus IL-21 may prove to be broadly applicable to monoclonal antibody therapy of cancer.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

Optimal NK Cell Expansion Depends on Accessory Cells, Synergy between Physiologic Concentrations of IL-2 and IL-15, and Umbilical Cord Blood (UCB) NK Cell Precursors Expand Better Than Adult NK Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3642-3642 ◽  
Author(s):  
Purvi Gada ◽  
Michelle Gleason ◽  
Valarie McCullar ◽  
Philip B. McGlave ◽  
Jeffrey S. Miller
Keyword(s):  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cell Expansion ◽  
Nk Cell ◽  
High Dose ◽  
Unexpected Result ◽  
Accessory Cells

Abstract Allogeneic NK cells may play a therapeutic role in treating patients with AML. We have previously shown that high dose cyclophosphamide (120 mg/kg × 1 day) and fludarabine (125 mg/m2 × 5 days) can clear lymphoid space and induce a surge of endogenous IL-15 to expand haploidentical NK cells obtained from CD3-depleted lymphapheresis products from adult donors. In this initial study, 5 of 19 patients achieved remissions and in vivo NK cell expansion. Limitations of this therapy includeinability of NK cells to expand in most patients,development of PTLD (in one patient) andinadequate disease control.We hypothesized that contaminating T cells could compete for NK cell expansion, that B-cells may contribute to PTLD, and that a 2-step NK cell purification method using CD3 depletion followed by CD56 selection (CliniMacs) may overcome these problems. We tested this in 9 patients with advanced AML. The purified NK cells, activated with 1000 U/ml IL-2 (16–20 hours), were infused 48 hours after the last fludarabine dose. Patients then received subcutaneous IL-2 (10 MU) every other day × 6 doses to expand NK cells in vivo. None of the 9 pts treated on this protocol achieved remission or exhibited evidence of in vivo expansion. Several studies were designed to investigate this unexpected result. First, we found that the more extensive processing resulted in approximately 1/3 the NK cell recovery compared to CD3 depletion alone (38±% viable NK cells vs. 91±2% respectively). In addition, we questioned whether the contaminating B cells and monocytes that were removed in the 2-step depletion strategy had served a critical role in NK cell activation or expansion. Cytotoxicity assays performed against K562 targets showed that the killing was about 3-fold higher with the purified (CD3-CD56+) product compared the CD3-depleted product alone (P=0.001 at E:T of 6.6:1). Proliferation, measured by a 6-day thymidine assay, was higher in proportion to the higher NK cell content. The only difference between the two NK products was their expansion after 14 days of culture, where the CD3-depleted product, with contaminating B-cells and monocytes, gave rise to greater NK cell expansion (14 ±3-fold) compared to the 2-step purified product (4.5±0.9, n=6, P=0.005). If this finding holds true in vivo, the co-infusion of accessory cells may be required for NK cell expansion. We next developed in vitro assays using very low concentrations (0.5 ng/ml) of IL-2 and IL-15 to understand their role in expansion. IL-2 or IL-15 alone induced low proliferation and the combination was synergistic. Lastly, UCB, a rich source of NK cell precursors, was compared to adult NK cells. In a short term proliferation assay, CD56+ NK cells stimulated with IL-2 + IL-15 expanded better from adult donors (61274±12999, n=6) than from UCB (20827± 6959, n=5, P=0.026) but there was no difference after 14 days in expansion culture suggesting that the only difference is in kinetics. However, UCB depleted of T-cells (enriching for NK cell precursors) exhibited higher fold expansion over 14 days under different culture conditions conducive to NK cell progenitors. In conclusion, NK cell expansion in vitro depends on cell source, IL-2 and IL-15 (increased in vivo after lymphoid depleting chemotherapy) as well as accessory cells. The role of these factors to enhance in vivo expansion is under clinical investigation to further exploit the NK cell alloreactivity against AML targets.

scholarly journals T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy.

1996 ◽  
Vol 183 (5) ◽  
pp. 2361-2366 ◽  
Author(s):  
J C Becker ◽  
J D Pancook ◽  
S D Gillies ◽  
K Furukawa ◽  
R A Reisfeld
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Tumor Model ◽  
Melanoma Metastases ◽  
Tumor Eradication

Induction of a T-cell mediated antitumor response is the ultimate goal for tumor immunotherapy. We demonstrate here that antibody-targeted IL2 therapy is effective against established pulmonary and hepatic melanoma metastases in a syngeneic murine tumor model. The effector mechanisms involved in this tumor eradication are not dependent on NK cells, since the therapeutic effect of antibody-IL2 fusion protein was not altered in NK cell-deficient mice. In contrast, T cells are essential for the observed antitumor effect, since therapy with antibody IL2 fusion proteins is unable to induce tumor eradication in T cell-deficient SCID mice. In vivo depletion studies characterized the essential effector cell population further as CD8 + T cells. Such CD8 + T cells, isolated from tumor bearing mice after antibody-directed IL2 therapy, exerted a MHC class I-restricted cytotoxicity against the same tumor in vitro. These data demonstrate the ability of antibody-targeted IL2 delivery to induce a T cell-dependent host immune response that is capable of eradicating established melanoma metastases in clinically relevant organs.

The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 1015-1023 ◽  
Author(s):  
Remko Schotte ◽  
Marie-Clotilde Rissoan ◽  
Nathalie Bendriss-Vermare ◽  
Jean-Michel Bridon ◽  
Thomas Duhen ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Cdna Subtraction ◽  
Common Precursor ◽  
Functional Features

Abstract Human plasmacytoid dendritic cells (pDCs), also called type 2 dendritic cell precursors or natural interferon (IFN)–producing cells, represent a cell type with distinctive phenotypic and functional features. They are present in the thymus and probably share a common precursor with T and natural killer (NK) cells. In an effort to identify genes that control pDC development we searched for genes of which the expression is restricted to human pDC using a cDNA subtraction technique with activated monocyte-derived DCs (Mo-DCs) as competitor. We identified the transcription factor Spi-B to be expressed in pDCs but not in Mo-DCs. Spi-B expression in pDCs was maintained on in vitro maturation of pDCs. Spi-B was expressed in early CD34+CD38− hematopoietic progenitors and in CD34+CD1a− thymic precursors. Spi-B expression is down-regulated when uncommitted CD34+CD1a− thymic precursors differentiate into committed CD34+CD1a+ pre-T cells. Overexpression of Spi-B in hematopoietic progenitor cells resulted in inhibition of development of T cells both in vitro and in vivo. In addition, development of progenitor cells into B and NK cells in vitro was also inhibited by Spi-B overexpression. Our results indicate that Spi-B is involved in the control of pDC development by limiting the capacity of progenitor cells to develop into other lymphoid lineages.

127 Preclinical evaluation of NKX019, a CD19-targeting CAR NK Cell

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A140-A140
Author(s):  
Nadege Morisot ◽  
Sarah Wadsworth ◽  
Tina Davis ◽  
Nicole Dailey ◽  
Kyle Hansen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Nk Cells ◽  
Half Life ◽  
Nk Cell ◽  
Cytolytic Activity ◽  
Nsg Mice ◽  
Animal Care

BackgroundNatural killer (NK) cells are highly effective and fast-acting cytolytic cells capable of eradicating target cells with limited adverse effects such as cytokine release syndrome (CRS) or graft-versus-host disease. Chimeric antigen receptors (CARs)-engineered NK cells have been recently used against leukemia with encouraging clinical outcomes.1 The surface antigen CD19, expressed by B-lymphoblasts, represents an ideal CAR target against B cell acute lymphoblastic leukemia (B-ALL). We developed a highly potent CD19 -directed CAR NK cell therapy, NKX019, with an extended in vivo half-life aimed at killing CD19-expressing target.MethodsNK cells isolated from healthy PBMCs were expanded in the presence of NKSTIM cells, IL-2, IL-12, IL-18 and transduced with both a CD19-targeted CAR construct and a membrane-bound form of IL-15 (mbIL-15). Control (non-engineered) NK cells were produced in parallel. Cytotoxic activity of NKX019 against CD19+ B-ALL cell line (REH), pre-B ALL cell line (Nalm-6), allogeneic PBMCs was assessed using Incucyte® or flow cytometry. NSG mice bearing either Nalm-6.fluc (Nalm6) or REH.fluc (REH) tumor received different concentrations of NKX019 or control NK cells. In-life analysis of tumor-bearing and naïve NSG mice include: 1) bioluminescence imaging, 2) clinical observations, 3) serum cytokines and 4) CAR+ NK cell persistency.ResultsNKX019 showed enhanced cytolytic activity against REH and Nalm-6 tumor cells compared to control NK cells and CAR19+ T cells. The superiority of NKX019 over CAR19+ T cells was more pronounced at the earlier time point (24 hours) with near identical calculated EC50 observed at 72 hours for both cell types. Increased cytolytic activity of NKX019 was limited to CD19+ cells in bulk PBMCs. Consistent with our in vitro observations, NKX019 controlled Nalm-6 and REH tumor growth in doses as low as 2 × 106 cells/kg for up to 30 days with no apparent increase in cytokines commonly associated with CRS. Increased Nalm-6 tumor growth coincided with an apparent decrease in measurable NKX019 in the periphery. In tumor-naïve NSG mice, NKX019 was detectable in the blood for up to 9 weeks post-infusion consistent with its extended half-life.ConclusionsNKX019 expresses mbIL-15 and is produced in the presence of IL-12 and IL-18, resulting in enhanced in vitro expansion and longer in vivo half-life than non-engineered NK cells. NKX019 also exhibited advantages compared to CAR19+ T cells including faster cytotoxic kinetics and limited production of cytokines associated with CRS. A first-in-human trial of NKX019 in B cell malignancies is planned for 2021.Ethics ApprovalThe animal procedures described in this abstract were conducted in accordance with Explora BioLabs Animal Care and Use Protocol approved by Explora BioLabs Institutional Animal Care and Use Committee.ReferenceLiu, et al. 2020 NEJM

IL-15 Administration to In-Vivo Expand Adoptively Transferred NK Cells In Rhesus Macaques

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 960-960
Author(s):  
Rebecca Lopez ◽  
Stephanie Sellers ◽  
Cynthia E. Dunbar ◽  
Richard Childs
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Rhesus Macaques ◽  
Cell Transfer ◽  
Regulatory Phenotype

Abstract Abstract 960 Immunotherapy using natural killer (NK) cells is currently being explored as a treatment option for patients with advanced malignant diseases. Although pilot clinical trials have shown adoptive NK cell transfer can result in tumor regression in humans with cancer, additional insight from animal models is needed to optimize methods to enhance the function and in vivo persistence of these adoptively infused lymphocytes. In contrast to mice, rhesus macaques have orthologues to most of the human MHC class I and II genes and possess NK cells expressing KIRs that are phenotypically and functionally similar to human NK cells, thus providing an excellent model to evaluate adoptive NK cell therapy. To characterize their in vivo longevity and tissue trafficking following adoptive infusion, we developed a method to expand large numbers of rhesus NK cells in vitro. NK cells enriched from peripheral blood mononuclear cells by depleting CD3+ cells using immunomagnetic beads were expanded in vitro with autologous plasma and a human EBV-LCL feeder cell line using culture conditions identical to those used for human NK cell expansion. Expanded rhesus NK cells were both phenotypically and genotypically similar to their human counterparts; NK cell cultures expanded up to 1000 fold within 2–3 weeks, were greater than 99% CD3 negative, and had a large proportion of CD16/CD56 double positive cells. In addition, expanded NK cells up-regulated receptors involved in tumor killing, including NKG2D, Granzyme B, TRAIL and Fas-ligand and were highly cytotoxic to K562 cells. Adoptive transfer of (3.2×107 – 1×108) CFSE-labeled ex vivo expanded rhesus NK cells has been well tolerated without any overt toxicities noted to date. Remarkably, despite the infusion of large cell numbers, CFSE labeled NK cells were detectable in the peripheral blood, lymph nodes, and bone marrow compartments at very low levels for only a few hours following infusion. Combining adoptive transfer of ex vivo expanded NK cells with IL-15 administration (rhesus recombinant IL-15 10 ug/kg s.c. × 5 days) resulted in only a minimal and transient 24 hour increase in the number of detectable CFSE labeled NK cells in the circulation and bone marrow. Although IL-15 administration did not substantially expand the number of circulating CFSE labeled NK cells that were adoptively transferred, it did result in a substantial increase in circulating numbers of endogenous NK and T-cells (4.74 fold and 5.2 fold increase in CD3-/CD56+ NK cells and CD3+ T-cells respectively). Surprisingly, IL-15 administration also resulted in a significant expansion of circulating T-regs (CD4-/CD25+/CD127Dim/FOXP3 +) which have previously been shown to suppress NK cell effector function in vitro and vivo; T-cells with a regulatory phenotype expanded 4.54 fold. Expansion of circulating T-regs occurred both when IL-15 was administered alone or in conjunction with adoptive NK cell transfer. Conclusions: IL-15 administration in macaques at the doses used in this study did not expand circulating numbers of adoptively transferred ex-vivo expanded NK cells, although it did significantly expand the numbers of circulating endogeneous NK cells. Remarkably, IL-15 administration was also associated with a significant expansion of T-cells with a regulatory phenotype. We are currently evaluating whether lympho-depletion followed by adoptive NK cell transfer can be used as a method to prevent the expansion of T-regs associated with IL-15 administration. Disclosures: No relevant conflicts of interest to declare.

Primary B-CLL Resistance to NK Cell Cytotoxicity can be Overcome In Vitro and In Vivo by Priming NK Cells and Monoclonal Antibody Therapy

2012 ◽  
Vol 32 (3) ◽  
pp. 632-646 ◽  
Author(s):  
Caroline Veuillen ◽  
Thérèse Aurran-Schleinitz ◽  
Rémy Castellano ◽  
Jérôme Rey ◽  
Françoise Mallet ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Nk Cells ◽  
Nk Cell ◽  
Antibody Therapy ◽  
Monoclonal Antibody Therapy ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity

scholarly journals A Novel Strategy for Off-the-Shelf T Cell Therapy Which Evades Allogeneic T Cell and NK Cell Rejection

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1711-1711
Author(s):  
Yong Zhang ◽  
Surbhi Goel ◽  
Aaron Prodeus ◽  
Utsav Jetley ◽  
Yiyang Tan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Class Ii ◽  
Class I

Abstract Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of "off-the-shelf" T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a "missing-self" signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed. Methods. Here, we developed an immunologically stealth "off-the-shelf" T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo. Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an "off-the-shelf" CAR or TCR-T cell solution for a large proportion of the population. Conclusions. We have successfully developed a differentiated "off-the-shelf" approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. Disclosures Zhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.

scholarly journals 722 INBRX-121 is an NKp46-targeted detuned IL-2 with antitumor activity as a monotherapy or in combination with multiple cancer immunotherapy modalities

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A751-A751
Author(s):  
Florian Sulzmaier ◽  
Heather Kinkead ◽  
Anya Polovina ◽  
Nadja Kern ◽  
Angelica Sanabria ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Cell Expansion ◽  
Nk Cell ◽  
Human Lymphocytes ◽  
Multiple Cancer

BackgroundNatural Killer (NK) cells play a pivotal role in cancer immunosurveillance due to their potent cytolytic activity and NK cell-centric therapies have emerged as safer alternatives to targeting T cells.1 2 Interleukin 2 (IL-2) drives NK cell expansion and activity, but its therapeutic utility is limited by rapid clearance, expansion of immunosuppressive regulatory T cells, and by severe dose-limiting toxicities.3 INBRX-121 overcomes these liabilities through specific targeting of an affinity-detuned IL-2 variant to cells expressing NKp46.MethodsAn IL-2 variant was engineered to eliminate binding to CD25 and to have attenuated affinity for CD122. This detuned cytokine was fused to a high-affinity single-domain antibody targeting NKp46 to generate INBRX-121. The ability of INBRX-121 to target IL-2-like signaling specifically to NKp46-expressing cells was evaluated in vitro using human lymphocytes by measuring STAT5 signaling and cytotoxic activity in tumor cell co-cultures. Characterization of the pharmacokinetic/pharmacodynamic relationship of INBRX-121 was completed in non-human primates across escalating dose levels, while anti-tumor activity as a monotherapy and in combination with Rituximab or PD-1 checkpoint blockade was tested in Raji xenografts and syngeneic CT-26 mouse models, respectively.ResultsINBRX-121 induces a STAT5 signal equal to that of wild-type IL-2 in human lymphocytes but shows an NK cell-centric activity profile. Cells targeted by INBRX-121 have increased proliferative capacity and improved cytotoxicity in antibody-dependent and -independent tumor cell killing assays. INBRX-121 shows prolonged pharmacokinetic exposure in vivo and is well-tolerated in mice and cynomolgus monkeys. The NKp46-specific IL-2 stimulus in these models results in a robust, dose-dependent NK cell expansion. As predicted by its in vitro activity, INBRX-121 also enhances the cytotoxic capacity of NK cells in vivo measured via elevated intracellular levels of Granzyme B. In a Raji xenograft model, INBRX-121 slows tumor growth as a single agent and synergizes with Rituximab to induce complete tumor regression. Similarly, co-treatment with INBRX-121 improves the incomplete suppression of CT-26 tumor growth by a PD-1 blocking antibody to yield complete responses that show immunological memory upon re-challenge.ConclusionsINBRX-121 offers a unique approach to overcoming the limitations of current IL-2 therapeutics. NKp46-targeting of a detuned IL-2 variant helps to avoid IL-2-mediated toxicity while enhancing the antitumor activities of NK cells. Through its novel therapeutic concept INBRX-121 provides a promising treatment option for multiple cancer indications both as a monotherapy and in combination with a variety of frontline agents.ReferencesShimasaki N, Jain A, Campana D. NK cells for cancer immunotherapy. Nat Rev Drug Discov 2020;19:200–218.Liu S, Galat V, Galat Y, Lee Y, Wainwright D, Wu J. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 2021;14:7.Overwijk W, Tagliaferri M, Zalevsky J. Engineering IL-2 to give new life to T Cell immunotherapy. Annu Rev Med 2021;72:281–311.Ethics ApprovalAll animal studies were conducted in accordance with AAALAC regulations and were approved by the IACUC for Explora BioLabs (#SP17-010-013) and BTS Research (20-015 Enrollment 05).

scholarly journals Maintenance with Histamine and IL-2 Induces a Marked Expansion of Activated CD56bright NK Cells in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1422-1422
Author(s):  
Angelica Cuapio ◽  
Mirte Post ◽  
Sabine Cerny-Reiterer ◽  
Markus Osl ◽  
Volker Huppert ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
Leukemic Cells ◽  
Cell Numbers ◽  
Before And After ◽  
Cell Subpopulations ◽  
Maintenance Of Remission

Abstract In acute myeloid leukemia (AML), residual leukemic (stem) cells that escape initial chemotherapy are considered a major source of relapse. Clinical trials have used IL-2 for AML patients with the aim to reduce relapse rates by eliminating residual leukemic cells through activation of NK and T cells. However, monotherapy with IL-2 has led to disappointing results. Nevertheless, recent clinical trials have shown that the co-administration of IL-2 and histamine dihydrochloride (HDC) provides maintenance of remission in AML. Histamine suppresses the formation of reactive oxygen species (ROS) thereby protecting NK and T cells from ROS-induced dysfunction and apoptosis. In addition, IL-2 is considered to maintain the anti-leukemic activity of NK cells. However, the direct effect of this treatment on NK cell numbers and anti-AML activity has not been studied in detail so far. In this study, we analyzed the immunophenotype and function of NK cells in a cohort of 7 AML patients (FAB M2, n=1; M4, n=2; M4-eo with inv 16, n=2; M5, n=2) treated with HDC plus IL-2. All patients had received induction chemotherapy with daunorubicin (45 mg/m² i.v. days 1-3), cytosine arabinoside, ARA-C (2 x 100 mg/m² i.v., days 1-7) and etoposide (100 mg/m² i.v., days 1-5) as well as at least 3 cycles of consolidation chemotherapy with high dose or intermediate dose ARA-C (Sperr et al, Clin Cancer Res 2004;10:3965-3971 and Krauth et al, J Immunol 2006;176:1759-1768). After having achieved a complete hematologic remission, patients were treated with HDC (0.5 mg) plus recombinant IL-2 (0.9 x 106 IU) twice daily s.c. for 21 days per cycle. Blood was drawn before and during treatment with HDC plus IL-2. We found that after one week of treatment with HDC plus IL-2, NK cell numbers increased in peripheral blood (from 101.8 ± 28.25 cells/µl before therapy to 208.2 ± 38.27 cells/µl after therapy, p<0.05). In the NK cell fraction, we observed an astonishing increment of CD56bright NK cells in all treated patients (from 7.2±0.97% or 17.6±5.8 cells/µl before therapy to 38.8±4.4% or 104±19.4 cells/µl after therapy, p<0.05; see Fig.1A/B). The cytotoxic activity of the CD56bright cells, as determined by NK cell degranulation and target cell lysis using the cell line K562, showed a significant increase in comparison to cells obtained before treatment (p<0.05). This was associated with an increased expression of KIR as well as the activation markers NKp44 (see Fig.1C), NKp46, and CD25 on NK cells. Furthermore, we observed a significant increase in expression of CD56 on NK cells after treatment with HDC plus IL-2 in our AML patients (2.5 ± 0.55 fold increase in the mean fluorescence intensity of CD56, p=0.02), whereas CD16 expression did not change significantly. In addition, treatment with HDC+IL-2 also induced an increased proportion of circulating CD4+CD25highCD127low/neg regulatory T cells (Treg). Finally, in vitro stimulation of NK cells with histamine plus IL-2 mimicked the effects observed in vivo. Interestingly, the in vitro treatment was also associated with an increased expression of CD56 without altered expression of CD16, suggesting that this effect could be a specific and reliable indicator of in vivo responses of NK cells to HDC plus IL-2 therapy. In conclusion, treatment with HDC plus IL-2 causes a striking increase in CD56bright NK cells. These specifically expanded NK cells exhibit an activated phenotype with enhanced potential to kill leukemic cells. We propose that the maintenance of remission in AML patients treated with HDC plus IL-2 might, at least in part, be the result of an improved anti-leukemic NK cell function. Fig 1. Effect of HDC plus IL2 on NK cells of AML patients. A) Representative dot plots of the CD56bright and CD56dim NK cell subpopulations from an AML patient treated with histamine+IL2 before and after treatment. B) Absolute cell numbers of CD56bright NK cells of 7 AML patients before and after treatment, *** p<0.01. C) Follow-up of NKp44 and KIR expression after HDC plus IL-2 therapy. Shown are histograms for NKp44 and KIR expression on total CD56+ CD3- NK cells of one patient representative for the majority of patients tested. Fig 1. Effect of HDC plus IL2 on NK cells of AML patients. A) Representative dot plots of the CD56bright and CD56dim NK cell subpopulations from an AML patient treated with histamine+IL2 before and after treatment. B) Absolute cell numbers of CD56bright NK cells of 7 AML patients before and after treatment, *** p<0.01. C) Follow-up of NKp44 and KIR expression after HDC plus IL-2 therapy. Shown are histograms for NKp44 and KIR expression on total CD56+ CD3- NK cells of one patient representative for the majority of patients tested. Disclosures Sperr: MEDA Pharma GmbH & Co. KG: Speakers Bureau. Valent:MEDA Pharma GmbH & Co. KG: Speakers Bureau.

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