Targeting MICA/B with cytotoxic therapeutic antibodies leads to tumor control.

Background: MICA and MICB are tightly regulated stress-induced proteins that trigger the immune system by binding to the activating receptor NKG2D on cytotoxic lymphocytes. MICA and MICB are highly polymorphic molecules with prevalent expression on several types of solid tumors and limited expression in normal/healthy tissues, making them attractive targets for therapeutic intervention. Methods: We have generated a series of anti-MICA and MICB cross-reactive antibodies with the unique feature of binding to the most prevalent isoforms of both these molecules. Results: The anti-MICA and MICB antibody MICAB1, a human IgG1 Fc-engineered monoclonal antibody (mAb), displayed potent antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) of MICA/B-expressing tumor cells in vitro. However, it showed insufficient efficiency against solid tumors in vivo, which prompted the development of antibody-drug conjugates (ADC). Indeed, optimal tumor control was achieved with MICAB1-ADC format in several solid tumor models, including patient-derived xenografts (PDX) and carcinogen-induced tumors in immunocompetent MICAgen transgenic mice. Conclusions: These data indicate that MICA and MICB are promising targets for cytotoxic immunotherapy.

A comprehensive approach to gene expression profiling in immune cells.

With the advent of whole-transcriptome studies and the growing need for public repositories, it has become essential to combine multiple heterogeneous datasets for immune cells. In this chapter, we describe the implementation of a compendium of 10,833 genes for 975 samples, corresponding to 52 resting immune cell types. We begin by describing the datasets, and their selection, in particular. We then explain the methodology implemented to create a qualified compendium: the processing of each array (quality control, normalization and bias correction), integration (merging rules, global normalization and batch removal) and validation. Finally some examples of use will be detailed. The utility and limitations of the compendium are also discussed, as an introduction to the next version.

Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity.

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. VIDEO ABSTRACT.

Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies.

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.

Kinetics of Cytotoxic Lymphocytes Reconstitution after Induction Chemotherapy in Elderly AML Patients Reveals Progressive Recovery of Normal Phenotypic and Functional Features in NK Cells.

NK cells are defective in acute myeloid leukemia (AML) at diagnosis. Here, we studied the kinetic of expression of the major activating and inhibitory receptors of NK, CD8 T, and γδ T cells in patients undergoing chemotherapy (CT) for the treatment of AML (n = 29). We showed that NK cells are the main affected population at diagnosis and that expression of activating receptors is partially restored within a few weeks after CT. CD8 T cells and γδ T cells are only weakly affected at diagnosis. Killer cell immunoglobulin-like receptor expression by NK cells, but not NKG2A and CD85j, was downregulated. Interestingly, the development of NK cells appeared altered as the most immature CD56bright NK cells were seriously underrepresented. Finally, we showed that NK cell functions were only partially restored 6 weeks after CT as degranulation capabilities of NK cells recovered, whereas cytokine production remained low. Our data point out NK cells as antitumor effectors peculiarly hampered by leukemic cells. This study may indicate a timeline when NK-mediated therapies or other immunotherapies could be performed, particularly for patients excluded of hematopoietic stem cell transplantation.

Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells.

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.

Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates.

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.

Anti-KIR antibody enhancement of anti-lymphoma activity of natural killer cells as monotherapy and in combination with anti-CD20 antibodies.

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.

What lessons can be learned from γδ T cell-based cancer immunotherapy trials?

During the last several years, research has produced a significant amount of knowledge concerning the characteristics of human γδ T lymphocytes. Findings regarding the immune functions of these cells, particularly their natural killer cell-like lytic activity against tumor cells, have raised expectations for the therapeutic applications of these cells for cancer. Pharmaceutical companies have produced selective agonists for these lymphocytes, and several teams have launched clinical trials of γδ T cell-based cancer therapies. The findings from these studies include hematological malignancies (follicular lymphoma, multiple myeloma, acute and chronic myeloid leukemia), as well as solid tumors (renal cell, breast and prostate carcinomas), consisting of samples from more than 250 patients from Europe, Japan and the United States. The results of these pioneering studies are now available, and this short review summarizes the lessons learned and the role of γδ T cell-based strategies in the current landscape of cancer immunotherapies.

A phase 1 trial of the anti-inhibitory KIR mAb IPH2101 for AML in complete remission.

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-1ß 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.

NK cell MHC class I specific receptors (KIR): from biology to clinical intervention.

The natural killer (NK) cell effector response towards infected cells or tumoural cells is guided by the integration of activating and inhibitory signals sensed by NK cell surface receptors. Major histocompatibility complex class I specific inhibitory receptors expressed by NK cells have two distinct roles: while allowing self tolerance, they are also needed for the acquisition of NK cell functional competence, a process termed education. In the context of allotransplantation, NK cell alloreactivity, arising from the expression on donor NK cells of inhibitory killer Ig-like receptors (KIRs) that do not recognize human leukocyte antigen from the patient, has shown clinical benefit for leukaemia patients. Based on these genetic studies, a blocking antibody directed against KIRs, as well as allogeneic NK cell infusions are now tested in clinical trials in various oncology indications. They offer promising immunotherapeutic approaches for the treatment of cancer patients.

IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

Multiple myeloma (MM) patients who receive killer cell Ig-like receptor (KIR) ligand-mismatched, T cell-depleted, allogeneic transplantation may have a reduced risk of relapse compared with patients who receive KIR ligand-matched grafts, suggesting the importance of this signaling axis in the natural killer (NK) cell-versus-MM effect. Expanding on this concept, IPH2101 (1-7F9), an anti-inhibitory KIR mAb, enhances NK-cell function against autologous MM cells by blocking the engagement of inhibitory KIR with cognate ligands, promoting immune complex formation and NK-cell cytotoxicity specifically against MM cell targets but not normal cells. IPH2101 prevents negative regulatory signals by inhibitory KIR, whereas lenalidomide augments NK-cell function and also appears to up-regulate ligands for activating NK-cell receptors on MM cells. Lenalidomide and a murine anti-inhibitory NK-cell receptor Ab mediate in vivo rejection of a lenalidomide-resistant tumor. These mechanistic, preclinical data support the use of a combination of IPH2101 and lenalidomide in a phase 2 trial for MM.

Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.

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-ß1, 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.

Natural killer cell-based therapies.

Allotransplantation of natural killer (NK) cells has been shown to be a key factor in the control and cure of at least some hematologic diseases, such as acute myeloid leukemia or pediatric acute lymphocytic leukemia. These results support the idea that stimulation of NK cells could be an important therapeutic tool in many diseases, and several such approaches are now in clinical trials, sometimes with conflicting results. In parallel, recent advances in the understanding of the molecular mechanisms governing NK-cell maturation and activity show that NK-cell effector functions are controlled by complex mechanisms that must be taken into account for optimal design of therapeutic protocols. We review here innovative protocols based on allotransplantation, use of NK-cell therapies, and use of newly available drug candidates targeting NK-cell receptors, in the light of fundamental new data on NK-cell biology.

Bromohydrin pyrophosphate-stimulated Vgamma9delta2 T cells expanded ex vivo from patients with poor-prognosis neuroblastoma lyse autologous primary tumor cells.

Gamma/delta T cells (Vgamma9delta2) contribute to innate immunity and exert natural cytotoxicity against a variety of tumors. Using a synthetic phosphoantigen (Bromohydrin Pyrophosphate, BrHPP), we amplified Vgamma9delta2 T cells in vitro from neuroblastoma patients. In the presence of BrHPP and low doses of IL-2, robust proliferation of Vgamma9delta2 T cells was obtained from peripheral blood mononuclear cells (PBMC) harvested at diagnosis. Moderate proliferation was observed from PBMC harvested after stem cell transplantation, whereas modest levels of Vgamma9delta2 T cells were obtained from PBMC harvested after induction therapy. Proliferation was observed after a single in vitro stimulation with BrHPP. After 21 days in culture, Vgamma9delta2 T cells represented more than 80% of cultured cells (a 50-fold expansion from baseline). Moreover, BrHPP-amplified Vgamma9delta2 T cells from patients-expressed activation markers and were able to lyse allogeneic and autologous neuroblasts. This cytotoxic activity was gammadelta T-cell receptor-dependent. Clinical trials using BrHPP are warranted in patients with poor-prognosis neuroblastoma, either to expand patient-derived Vgamma9delta2 T cells ex vivo or by direct administration to in vivo to boost the pool of resident Vgamma9delta2 T cells in vivo.

NK cell activation by KIR-binding antibody 1-7F9 and response to HIV-infected autologous cells in viremic and controller HIV-infected patients.

Natural killer (NK) cells may be protective in HIV infection and are inhibited by killer cell immunoglobulin-like receptors (KIRs) interacting with MHC class I molecules, including HLA-C. Retention of HLA-C despite downregulation of other MHC class I molecules on HIV infected cells might protect infected cells from NK cell recognition in vitro. To assess the role of inhibitory HLA-C ligands in the capacity of NK cells to recognize autologous infected T cells, we measured NK cell degranulation in vitro in viremic patients, controllers with low viremia, and healthy donors. No difference in NK cell response to uninfected compared to HIV-1(IIIB) infected targets was observed. Activation of NK cells was regulated by KIRs, because NK cell degranulation was increased by 1-7F9, a human antibody that binds KIR2DL1/L2/L3 and KIR2DS1/S2, and this effect was most pronounced in KIR haplotype B individuals.

In vivo tumor cell rejection induced by NK cell inhibitory receptor blockade: maintained tolerance to normal cells even in the presence of IL-2.

Missing-self-reactivity can be mimicked by blocking self-specific inhibitory receptors on NK cells, leading to increased rejection of syngeneic tumor cells. Using a mouse model, we investigated whether Ab-mediated blocking of inhibitory receptors, to a degree where NK cells rejected syngeneic tumor cells, would still allow self-tolerance toward normal syngeneic cells. Ly49C/I inhibitory receptors on C57BL/6 (H-2(b)) NK cells were blocked with F(ab')(2) fragments of the mAb 5E6. Inhibitory receptor blockade in vivo caused rejection of i.v. inoculated fluorescence-labeled syngeneic lymphoma line cells but not of syngeneic spleen cells, BM cells or lymphoblasts. The selective rejection of tumor cells was NK cell-dependent and specifically induced by Ly49C/I blockade. Moreover, selective tumor rejection was maintained after treatment with 5E6 F(ab')(2) for 9 wk, arguing against the induction of NK cell anergy or autoreactivity during this time. Combination therapy using 5E6 F(ab')(2) together with high dose IL-2 treatment further increased lymphoma cell rejection. In addition, combination therapy reduced growth of melanoma cell line tumors established by s.c. inoculation 3 days before start of treatment. Our results demonstrate that inhibitory receptor blockade does not result in attack on normal cells, despite potent reactivity against MHC class I-expressing tumors.

Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells.

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

Genetic and antibody-mediated reprogramming of natural killer cell missing-self recognition in vivo.

Natural killer (NK) cells are lymphocytes of the innate immune system able to recognize and kill tumors lacking self-MHC class I molecules. This "missing-self" recognition is mediated by the lack of engagement of MHC class I-specific inhibitory NK cell receptors that include the killer cell Ig-like receptors (KIR) in humans and Ly49 molecules in mice. A promising immunotherapeutic strategy against MHC class I(+) cancer cells is to block NK cell inhibitory receptors using monoclonal antibodies (mAb). However, interactions between MHC class I molecules and their inhibitory receptors are also required for the acquisition of NK cell functional competence, a process referred as to "education." In addition, inhibitory receptors are involved in self-tolerance on educated NK cells. Here, we developed a preclinical mouse model in which all NK cells are educated by a single transgenic inhibitory receptor, human KIR2DL3, through the engagement with its HLA-Cw3 ligand. This approach revealed that NK cells could be reprogrammed to control the development of mouse syngenic tumors in vivo. Moreover, in vivo anti-KIR mAb treatment induced the killing of HLA(+) target cells without breaking self-tolerance. Finally, the long-term infusion of anti-KIR mAb neither abolished NK cell education nor tumor cell recognition. Therefore, these results strongly support the use of inhibitory receptor blockade in cancer patients.