Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display ...different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.
NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells ...accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.
IPH2101 is an anti-killer inhibitory receptor (anti-KIR) mAb that can block KIR-mediated inhibition of natural killer (NK) cells to enhance cytotoxicity against acute myeloid leukemia blasts. We have ...conducted a phase 1 study of IPH2101 in elderly patients with acute myeloid leukemia in first complete remission. Patients received escalating doses (0.0003-3 mg/kg) of IPH2101 following a 3 + 3 design. Safety, toxicity (primary end points), pharmacokinetics, outcome, and immunologic correlates were evaluated. Twenty-three patients (median age, 71 years), were enrolled. Adverse events were mild and transient, consisting mainly of infusion syndrome and erythema. The maximum tolerated dose was not reached, although full KIR saturation (> 90%) was sustained for more than 2 weeks at 1 and 3 mg/kg. There was a clear correlation between mAb exposure and KIR occupancy. Neither hematologic toxicity nor significant changes in the numbers and distribution of lymphocyte subsets, NK cell receptor expression, or in vitro cytotoxicity were seen. At the highest dose levels (0.3, 1, and 3 mg/kg), transient increases in TNF-α and MIP-1β serum concentrations and NK cell CD69 expression were observed. Overall and relapse-free survival in the present study compared favorably to reports in comparable patient populations. We conclude that IPH2101 administration is safe and can block KIR for prolonged periods of time with limited side effects. Registered with the European Union Drug Regulating Authorities Clinical Trials (EUDRACT) as 2005-005298-31.
Highlights ► NK cell activity is governed by balance of activating and inhibitory receptors. ► Different KIR molecules recognize groups of class I alleles. ► Their variegated expression leads to a ...repertoire of NK cells with different cytotoxicities. ► Allotransplantation demonstrated that KIR can be key for haematologic tumour control. ► Therapeutic approaches based on KIR blockade are currently tested in clinics.
Over the last decade, various new therapies have been developed to promote anti-tumor immunity. Despite interesting clinical results in hematological malignancies, the development of bispecific ...killer-cell-engager antibody formats directed against tumor cells and stimulating anti-tumor T cell immunity has proved challenging, mostly due to toxicity problems. We report here the generation of trifunctional natural killer (NK) cell engagers (NKCEs), targeting two activating receptors, NKp46 and CD16, on NK cells and a tumor antigen on cancer cells. Trifunctional NKCEs were more potent in vitro than clinical therapeutic antibodies targeting the same tumor antigen. They had similar in vivo pharmacokinetics to full IgG antibodies and no off-target effects and efficiently controlled tumor growth in mouse models of solid and invasive tumors. Trifunctional NKCEs thus constitute a new generation of molecules for fighting cancer.
Display omitted
Display omitted
•NK cell engagers are multifunctional Abs targeting tumor antigens, NKp46 and CD16•NKCEs bring tumor cells and NK cells together and trigger tumor-cell destruction•NKCEs can show killing potency superior to therapeutic Abs in vitro and in vivo•NKCEs may improve benefit-risk profile for cancer treatment compared to BiTEs
Trifunctional antibodies that engage natural killer cells by binding NKp46 and CD16, in addition to an antigen on cancer cells, show higher potency than current clinically available therapeutic antibodies.
Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor ...evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer.
Display omitted
•IPH5201 and IPH5301 block cell-borne and soluble CD39 and CD73, respectively•IPH5201 maintains immunogenic extracellular ATP•When used in combination with chemotherapy, IPH5201 promotes antitumor immunity•Targeting CD39 and CD73 synergistically promotes cancer patient T cell activation
The production of adenosine via CD39 and CD73 ectoenzymes participates in an immunosuppressive tumor microenvironment. Perrot et al. generated two antibodies, IPH5201 and IPH5301, targeting human CD39 and CD73, respectively. In vitro and in vivo data support the use of anti-CD39 and anti-CD73 mAbs in combination cancer therapies.
Natural killer cells are key cells of the innate immune system. Natural killer cell receptor repertoires are diversified by a stochastic expression of killer-cell-immunoglobulin-like receptors and ...lectin-like receptors such as NKG2 receptors. All individuals harbor a subset of natural killer cells expressing NKG2A, the inhibitory checkpoint receptor for HLA-E. Most neoplastic and normal hematopoietic cells express HLA-E, the inhibitory ligand of NKG2A. A novel anti-human NKG2A antibody induced tumor cell death, suggesting that the antibody could be useful in the treatment of cancers expressing HLA-E. We found that immunodeficient mice, co-infused with human primary leukemia or Epstein-Barr virus cell lines and NKG2A(+) natural killer cells, pre-treated with anti-human NKG2A, were rescued from disease progression. Human NKG2A(+) natural killer cells reconstituted in immunodeficient mice after transplantation of human CD34(+) cells. These natural killer cells are able to kill engrafted human primary leukemia or Epstein-Barr virus cell lines by lysis after intraperitoneal administration of anti-human NKG2A. Thus, this anti-NKG2A may exploit the anti-leukemic action of the wave of NKG2A(+) natural killer cells recovering after hematopoietic stem cell transplants or adoptive therapy with natural killer cell infusions from matched or mismatched family donors after chemotherapy for acute leukemia, without the need to search for a natural killer cell alloreactive donor.
Summary
T‐cell‐mediated immunotherapy is a promising therapeutic option for multiple myeloma (MM). Gamma‐delta T cells (γδ T cells) recognize phosphoantigens and display strong anti‐tumour ...cytotoxicity. The synthetic agonist Phosphostim (bromohydrin pyrophosphate, BrHPP) has been shown to selectively activate Vγ9Vδ2 T cells. This study aimed to evaluate the expansion capacity and anti‐myeloma cell cytotoxicity of circulating γδ T cells from MM patients at different time points throughout the disease, using Phosphostim and interleukin 2 (IL‐2). Circulating γδ T cell counts in patients with newly diagnosed MM or in relapse did not differ from those in healthy donors. A 14‐d culture of peripheral blood mononuclear cells with Phosphostim and IL‐2 triggered a 100‐fold expansion of γδ T cells in 78% of newly diagnosed patients. γδ T cells harvested at the time of haematopoietic progenitor collection or in relapsing patients expanded less efficiently. Expanded γδ T cells killed 13/14 myeloma cell lines as well as primary myeloma cells, but not normal CD34 cells. Their killing efficiency was not affected by 2‐d IL‐2 starvation. This study demonstrated the ability of Phosphostim and IL‐2 to expand γδ T cells from MM patients, and the efficient and stable killing of human myeloma cells by gd T cells.
Natural killer (NK) cells recognize the absence of self MHC class I as a way to discriminate normal cells from cells in distress. In humans, this “missing self” recognition is ensured by inhibitory ...receptors such as KIR, which dampen NK cell activation upon interaction with their MHC class I ligands. We show here that NK cells lacking inhibitory KIR for self MHC class I molecules are present in human peripheral blood. These cells harbor a mature NK cell phenotype but are hyporesponsive to various stimuli, including MHC class I-deficient target cells. This response is in contrast to NK cells that express a single inhibitory KIR specific for self MHC class I, which are functionally competent when exposed to the same stimuli. These results show the involvement of KIR-MHC class I interactions in the calibration of NK cell effector capacities, suggesting its role in the subsequent “missing self” recognition.
Natural killer (NK) cells mediate antilymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal ...antibody (mAb) used to treat patients with B-cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of the efficacy of NK cells by spontaneous cytotoxicity. Here, using a killer-cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with major histocompatibility complex (MHC) class I antigens on lymphoma cells by anti-KIR antibodies prevents a tolerogenic interaction and augments NK-cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK-cell–mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in KIR transgenic and syngeneic murine lymphoma models. These results support a therapeutic strategy of combination rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor-targeting therapy with an NK-cell agonist, thus stimulating the postrituximab antilymphoma immune response.
•Blockade of inhibitory KIRs with MHC class I antigens on lymphoma cells by anti-KIR antibodies augments NK-cell spontaneous cytotoxicity.•In combination with anti-CD20 mAbs, anti-KIR induces enhanced NK cell–mediated, rituximab-dependent cytotoxicity against lymphoma.
PDF
