(19) TZZ _ T (11) EP 2 287 195 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 23.02.2011 Bulletin 2011/08 C07K 16/28 (2006.01) G01N 33/52 (2006.01) (21) Application number: 10178924.6 (22) Date of filing: 01.07.2005 (84) Designated Contracting States: • Romagne, François AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 13600 La Ciotat (FR) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Wagtmann, Peter, Andreas, Nicolai, Reumert SK TR 2960 Rungsted Kyst (DK) • Svendsen, Ivan (30) Priority: 06.01.2005 DK 200500025 2765 Smorum (DK) 01.07.2004 PCT/DK2004/000470 • Zahn, Stefan 01.07.2004 PCT/IB2004/002464 2750 Ballerup (DK) • Svensson, Anders (62) Document number(s) of the earlier application(s) in 21746 Malmö (DK) accordance with Art. 76 EPC: • Thorolfsson, Matthias 05758642.2 / 1 791 868 2920 Charlottenlund (DK) • Berg Padkaer, Soren (71) Applicants: 3500 Vaerlose (DK) • Novo Nordisk A/S • Kjaergaard, Kristian 2880 Bagsvaerd (DK) 2750 Ballerup (DK) • Innate Pharma • Spee, Pieter 13009 Marseille (FR) 3450 Allerod (DK) • Universita di Genova • Wilken, Michael 16132 Genova (IT) 3390 Hundested (DK) (72) Inventors: (74) Representative: Gallois, Valérie et al • Moretta, Alessandro Cabinet BECKER & ASSOCIES 16133 Genova (IT) 25, rue Louis Le Grand • Della Chiesa, Mariella 75002 Paris (FR) 16132 Genova (IT) • Andre, Pascale Remarks: 13006 Marseille (FR) This application was filed on 23-09-2010 as a • Gauthier, Laurent divisional application to the application mentioned 13008 Marseille (FR) under INID code 62. (54) Pan-KIR2DL NK-receptor antibodies and their use in diagnostik and therapy (57) Compositions and methods for regulating an im- monoclonal antibody 1-7F9 or 14F1. Described also are mune response in a subject are described. More partic- fragments and derivatives of such antibodies, as well as ularly, described are human antibodies that regulate the pharmaceutical compositions comprising the same and activity of NK cells and allow a potentiation of NK cell their uses, particularly for use in therapy, to increase NK cytotoxicity in mammalian subjects, and antibodies hav- cell activity or cytotoxicity in subjects. ing antigen-binding properties similar to those of human EP 2 287 195 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 287 195 A2 Description FIELD OF THE INVENTION 5 [0001] The present invention relates to human antibodies, as well as fragments and derivatives thereof, which cross- react with and/or block two or more inhibitory KIR receptors present on the surface of NK cells, and potentiate NK cell cytotoxicity in mammalian subjects or in a biological sample. The invention also relates to methods of making such antibodies, fragments, variants, and derivatives; pharmaceutical compositions comprising the same; and the use of such molecules and compositions, particularly in therapy, to increase NK cell activity or cytotoxicity in subjects. 10 BACKGROUND OF THE INVENTION [0002] Natural killer (NK) cells are a sub-population of lymphocytes, involved in immunity and in the host immune surveillance system. 15 [0003] NK cells are mononuclear cell that develop in the bone marrow from lymphoid progenitors, and morphological features and biological properties typically include the expression of the cluster determinants (CDs) CD16, CD56, and/or CD57; the absence of the alpha/beta or gamma/delta TCR complex on the cell surface; the ability to bind to and kill target cells that fail to express "self" major histocompatibility complex (MHC)/human leukocyte antigen (HLA) proteins; and the ability to kill tumor cells or other diseased cells that express ligands for activating NK receptors. NK cells are 20 characterized by their ability to bind and kill several types of tumor cell lines without the need for prior immunization or activation. NK cells can also release soluble proteins and cytokines that exert a regulatory effect on the immune system; and can undergo multiple rounds of cell division and produce daughter cells with similar biologic properties as the parent cell. Upon activation by interferons and/or cytokines, NK cells mediate the lysis of tumor cells and of cells infected with intracellular pathogens by mechanisms that require direct, physical contacts between the NK cell and the target cell. 25 Lysis of target cells involves the release of cytotoxic granules from the NK cell onto the surface of the bound target, and effector proteins such as perforin and granzyme B that penetrate the target plasma membrane and induce apoptosis or programmed cell death. Normal, healthy cells are protected from lysis by NK cells. [0004] Based on their biological properties, various therapeutic and vaccine strategies have been proposed in the art that rely on a modulation of NK cells. However, NK cell activity is regulated by a complex mechanism that involves both 30 stimulating and inhibitory signals. [0005] Briefly, the lytic activity of NK cells is regulated by various cell surface receptors that transduce either positive or negative intracellular signals upon interaction with ligands on the target cell. The balance between positive and negative signals transmitted via these receptors determines whether or not a target cell is lysed (killed) by a NK cell. NK cell stimulatory signals can be mediated by Natural Cytotoxicity Receptors (NCR) such as NKp30, NKp44, and 35 NKp46; as well as NKG2C receptors, NKG2D receptors, certain activating Killer Ig-like Receptors (KIRs), and other activating NK receptors (Lanier, Annual Review of Immunology 2005;23:225-74). NK cell inhibitory signals can be mediated by receptors like Ly49, CD94/NKG2A, as well as certain inhibitory KIRs, which recognize major histocompat- ibility complex (MHC) class I-molecules (Kärre et al., Nature 1986;319:675-8; Öhlén et al, Science 1989;246:666-8). These inhibitory receptors bind to polymorphic determinants of MHC class I molecules (including HLA class I) present 40 on other cells and inhibit NK cell-mediated lysis. [0006] KIRs, sometimes also referred to as Killer Inhibitory Receptors, have been characterized in humans and non- human primates, and are polymorphic type 1 trans-membrane molecules present on certain subsets of lymphocytes, including NK cells and some T cells. KIRs interact with determinants in the alpha 1 and 2 domains of the MHC class I molecules and, as described above, distinct KIRs are either stimulatory or inhibitory for NK cells. 45 [0007] The nomenclature for KIRs is based upon the number of extracellular domains (KIR2D and KIR3D having two and three extracellular Ig-domains, respectively) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). The presence or absence of a given KIR is variable from one NK cell to another within the NK population present in a single individual. Among humans, there is also a relatively high level of polymorphism of KIR genes, with certain KIR genes being present in some, but not all individuals. The expression of KIR alleles on NK cells 50 is stochastically regulated, meaning that, in a given individual, a given lymphocyte may express one, two, or more different KIRs, depending on the genoptype of the individual. The NK cells of a single individual typically express different combinations of KIRs, providing a repertoire of NK cells with different specificities for MHC class I molecules. [0008] Certain KIR gene products cause stimulation of lymphocyte activity when bound to an appropriate ligand. The activating KIRs all have a short cytoplasmic tail with a charged transmembrane residue that associates with an adapter 55 molecule having an Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) which transduce stimulatory signals to the NK cell. By contrast, inhibitory KIRs have a long cytoplasmic tail containing Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM), which transduce inhibitory signals to the NK cell upon engagement of their MHC class I ligands. The known inhibitory KIRs include members of the KIR2DL and KIR3DL subfamilies. Inhibitory KIRs having two Ig domains (KIR2DL) 2 EP 2 287 195 A2 recognize HLA-C allotypes: KIR2DL2 (formerly designated p58.2) and the closely related, allelic gene product KIR2DL3 both recognize "group 1" HLA-C allotypes (including HLA-Cw1, -3, -7, and - 8), whereas KIR2DL1 (p58.1) recognizes "group 2" HLA-C allotypes (such as HLA-Cw2, -4, - 5, and -6). The recognition by KIR2DL1 is dictated by the presence of a Lys residue at position 80 of HLA-C alleles. KIR2DL2 and KIR2DL3 recognition is dictated by the presence of an 5 Asn residue at position 80 in HLA-C. Importantly, the great majority of HLA-C alleles have either an Asn or a Lys residue at position 80. Therefore, KIR2DL1, -2, and -3 collectively recognize essentially all HLA-C allotypes found in humans. One KIR with three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by HLA-Bw4 alleles. Finally, KIR3DL2 (p140), a homodimer of molecules with three Ig domains, recognizes HLA-A3 and -A11. [0009] Although multiple inhibitory KIRs and/or other MHC class I-specific inhibitory receptors (Moretta et al, Eur J 10 Immunogenet. 1997;24(6):455-68; Valiante et al, Immunol Rev 1997;155:155-64; Lanier, Annu Rev Immunol 1998;16: 359-93) may be co-expressed by NK cells, in any given individual’s NK repertoire there are cells that express only a single KIR, and thus are inhibited only by specific MHC class I alleles (or alleles belonging to the same group of MHC class I allotypes). Human MHC class I molecules often are referred to as Human Histocompatibility Antigen (HLA) class I. [0010] NK cell populations or clones that are KIR-ligand mismatched with respect to their targets, i.e., that express 15 KIRs which do not recognize any HLA molecule of a host, have been shown to mediate potent, life-saving anti-tumor responses after allogeneic bone-marrow transplantation in leukemia patients (Ruggeri et al., Science 2002,295: 2097-2100).