Compositions and methods for immunotherapy

Abstract

The present invention provides compositions and methods for engaging, coupling or binding to CD16 and CD112R to preferentially activate NK cells for use in the treatment of cancer.

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 936,176, filed November 15, 2019, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on November 12, 2020, is named 2020-11-12_01219-0006-00-PCT_ST25.txt and is 1,501,833 bytes in size. [Background technology]

[0003] Both the innate and adaptive arms of the immune system utilize highly specialized immune cells to patrol the body and search for signs of malignancy. Innate immunity provides a first line of defense and rapid response using mechanisms such as non-specific, naturally occurring barrier and destructive peptides. Natural killer (NK) cells are a type of lymphocyte that are part of the innate immune system and can recognize and destroy virus-infected cells and tumor cells using granzymes stored in their cytoplasm.

[0004] Adaptive immunity develops over time in response to an antigen and provides long-lasting immunity. CD8 +Cytotoxic lymphocytes (CTLs), also known as T cells, are part of the adaptive immune response and recognize virus- and tumor-derived antigens presented by antigen-presenting cells (APCs). CTLs are activated by interaction with APCs, e.g., dendritic cells or macrophages. APCs present tumor antigens in the context of MHC molecules to the T cell receptor (TCR) on the surface of the T cell. During this cognate interaction, the APCs provide costimulatory signals that lead to T cell activation, T cell proliferation, and the reduction or elimination of cells expressing the antigen via cytotoxic mechanisms.

[0005] Administration of anti-CD112R immunotherapy offers the opportunity to increase, enhance and sustain immune responses. CD112R is an inhibitory receptor expressed primarily by T cells and NK cells, which competes with CD112 for binding to the activating receptor CD226. CD112 interacts with CD112R with higher affinity than with CD226, thereby effectively controlling CD226-mediated cell activation. Anti-CD112R antibodies that block the interaction with CD112 promote immune cell activation by increasing the interaction of CD226 with CD112 while limiting inhibitory signaling directly downstream of CD112R. In in vitro studies, anti-CD112R antibodies have been shown to increase the proliferation, activation and cytotoxicity of immune effector cells.

[0006] CD112R mRNA expression has been detected in several cancer tissues and is based on predictive analysis using the TCGA (The Cancer Genome Atlas) dataset. Its expression is strongest in tumors rich in T and NK cells. In addition to being expressed on myeloid cells, expression of the CD112R ligand, CD112, is routinely elevated on tumor cells of different cellular origins. Given these circumstances, engagement of CD112R on tumor-infiltrating immune cells has the strong potential to negatively regulate local immune responses within the tumor microenvironment.

[0007] Despite the success of anti-CD112R immunotherapy, there is a need for improved therapies for treating cancers, as well as therapies for treating cancers that are resistant to PD-1 / PD-L1. Therapeutic treatment with agents that couple CD112R and CD16 provides an opportunity to down-regulate inhibitory signaling that presumably occurs when immune cells expressing CD112R engage with CD112 on tumor and / or myeloid cells within the tumor microenvironment, and that has the potential to enhance, increase and maintain anti-tumor immune responses. Provided herein are compositions and methods for use in treating cancer by coupling and / or simultaneously binding and / or engaging with CD112R and CD16. [Brief description of the drawings]

[0008] [Figure 1A] FIG. 1A is a schematic diagram showing the tumor microenvironment. [Figure 1B] FIG. 1B shows that CD112R expression is increased on activated NK cells and T cells. [Diagram 2] FIG. 2 shows that CD112R is upregulated on NK cells and CD8+ T cells infiltrating CT26 tumors. [Figure 3A] FIG. 3A is a schematic diagram showing clone 35 coupling CD112R and CD16 on NK cells. [Figure 3B] FIG. 3B shows the results of an NK activation assay using anti-CD112R antibodies: clone 35 (IgG1 / IgG4), clone 38 (IgG1 / IgG4), and clone 44 (IgG1 / IgG4). [Figure 4A] FIG. 4A shows that overexpression of CD112R inhibits T cell activation. [Figure 4B]FIG. 4B shows that clone 35 enhances T cell activation compared to isotype control. [Figure 5A] FIG. 5A shows the results of an in vitro NK cell activity assay. [Figure 5B] FIG. 5B shows tumor volume following administration of anti-CD112R antibody (clone 46) in mouse IgG1 (similar to human IgG4) and mouse IgG2a (similar to human IgG1). [Figure 6A] FIG. 6A shows that anti-CD112R activity is dependent on NK cells and T cells. [Figure 6B] FIG. 6B shows immune memory upon rechallenge in anti-CD112R treated mice. [Figure 7] 7A and 7B show clone 35 compared to clone 35.4 in an NK activation assay in two donors. [Figure 8] 8A-8B show granzyme B+ levels (FIG. 8A) and percent interferon-γ+ levels (FIG. 8B) after treatment with anti-CD112R (clone 46), anti-TIGIT, or a combination of anti-CD112R (clone 46) and anti-TIGIT. [Figure 9] FIG. 9 shows tumor volume in the CT26 model cancer for anti-CD112R (clone 46) antibody in mIgG1 / hIgG4 format versus mIgG2a / hIgG1 format. [Figure 10] 10A to 10B show the results of an in vivo study on changes in tumor volume upon administration of clone 46 in the mIgG2a / hIgG1 format. [Figure 11]11A-11C show the results of further experiments showing 4-1BB induction in NK cells co-cultured with K562 cells in the presence of 1 μg / mL of clone 35 or clone 35-Fab compared to hIgG1 isotype control antibody or hIgG1 isotype control Fab (n=5 donors) (FIG. 11A); 4-1BB induction in NK cells co-cultured with K562 cells in the presence of 1 μg / mL of clone 35, clone 35-IgG4 or clone 35 with IgG1 mutation (clone 35-N297A) compared to hlgG1 isotype control (n=5) (FIG. 11B); and the effect of 1 μg / mL of clone 35 on NK cell activation after co-culture with K562 cells in the presence of 2 μg / mL of anti-CD16, anti-CD32 or mIgG1 control Fab molecules (n=5) (FIG. 11C) (*P<0.05, **P<0.01; paired t-test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] I. Definition In this application, unless stated otherwise, the use of "or" means "and / or." In the context of multiple dependent claims, the use of "or" refers to the preceding multiple independent or dependent claims only in the alternative. The terms "including," "including," and "having" may be used interchangeably herein.

[0010] The following terms: "CD112R", "PVR-related immunoglobulin domain containing", "CD112 receptor", "poliovirus receptor-related immunoglobulin domain containing protein", "poliovirus receptor-related immunoglobulin domain containing", "nectin-2 receptor", "C7orfl5" and "transmembrane protein PVRIG" are all used interchangeably and refer to native human CD112R unless otherwise specified (e.g., mouse CD112R, cynomolgus monkey CD112R, etc.). The term includes full-length unprocessed CD112R and any form of CD112R that results from processing within the cell. The term encompasses naturally occurring variants of human CD112R, such as splice variants or allelic variants. External IDs for the CD112R gene include Entrez Gene: 79037, Ensembl: ENSG00000213413, OMIM: 617012 and UniProtKB: Q6DKI7.

[0011] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein. Certain specific and exemplary embodiments for measuring binding affinity are described below.

[0012] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) which optionally result in an improvement in the affinity of the antibody for an antigen, compared to a parent antibody that does not possess such alterations.

[0013] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (so long as they exhibit the desired antigen-binding activity).

[0014] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody, where the portion of the intact antibody binds to the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0015] The term "block" is used herein in the context of an interaction between two or more molecules to refer to the inhibition or prevention of an interaction between two or more molecules, where the inhibition or prevention of the interaction between the two or more molecules is complete or near complete under at least one condition. "Near complete" inhibition is about 70-99.9% percent inhibition, and "complete" inhibition is 100%. For example, a molecule is said to "block" an interaction between two or more other molecules if it completely or near completely inhibits such interaction at a particular concentration in a dose-dependent manner.

[0016] The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also called benign tumors), pre-malignant or malignant. Cancer cells can be solid cancer cells or leukemic cancer cells. The term "tumor" is used herein to refer to one or more cells that make up a cancer. The term "tumor growth" is used herein to refer to proliferation or growth by one or more cells that make up a cancer, resulting in a corresponding increase in size or extent of the cancer.

[0017] "CD16", also known in the art as FcγRIII, is frequently found on the surface of natural killer (NK) cells, neutrophils, monocytes and macrophages.

[0018] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0019] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0020] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive (sequential) administration in any order. "Concurrent binding" (and repetitions thereof) refers to a composition that is capable of binding to one or more targets at the same time at any one time. Concurrent binding does not require binding to one or more targets at the same time at all times.

[0021] The term "cytotoxic agent" as used herein refers to an agent that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes, such as nucleolytic enzymes and fragments thereof; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin (including fragments and / or variants thereof); and various anti-tumor or anti-cancer agents disclosed below.

[0022] "Effector function" refers to the biological activities attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0023] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at the dosages required and for a period of time, to achieve a desired therapeutic or prophylactic result.

[0024] The term "Fc region" as used herein is used to define a C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. (Numbering in this paragraph follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.)

[0025] "Framework", "framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences generally appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0026] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0027] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny regardless of the number of transfers. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0028] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or that corresponds to that of an antibody derived from a non-human source that utilizes a human antibody repertoire, or that corresponds to other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0029] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen can be isolated using the VH or VL domain from an antibody that binds the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0030] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for VL, the subgroup is subgroup kappa I in Kabat et al., supra. In some embodiments, for VH, the subgroup is subgroup III in Kabat et al., supra.

[0031] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain ("complementarity determining region" or "CDR") that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).

[0032] In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG1. In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG4. In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG4 and there is a single mutation from serine 228 to proline (S228P). In some embodiments, the antibody is provided according to the sequence listing and the isotype is human IgG4 and there are two mutations from serine 228 to proline (S228P) and leucine 235 to glutamic acid (L235E). The sequences of human IgG1 and IgG4 are shown in the sequence listing as SEQ ID NOs: 40000 and 40001, respectively. The sequences of human IgG4 with one or two mutations are shown in SEQ ID NOs: 40002 and 40003, respectively. Throughout, where antibody or clone numbers are provided, the antibodies are in IgG1 format. Where a ".4" is appended to the antibody or clone number (e.g., "clone 35.4"), the antibody is an IgG4 antibody having a constant region comprising SEQ ID NO: 40002. The S228P mutation occurs at position 228 in the literature. The S→P mutation occurring in clone 35.4 could be at position 229, but is still referred to herein as S228P. In general, all exemplified antibodies described herein comprise a human kappa light chain.

[0033] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0034] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0035] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0036] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies, e.g., variant antibodies that contain naturally occurring mutations or that arise during production of the monoclonal antibody preparation, which are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci. Such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0037] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0038] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0039] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and does not consider any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, for example, any algorithm required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, together with user documentation, has been filed with the US Copyright Office, Washington DC, 20559, and is registered under US Copyright No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, such as Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0040] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or versus a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or versus a given amino acid sequence B) is calculated as follows: 100 x fraction (X / Y) where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all amino acid sequence % identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0041] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient contained therein is in a form such that it is effective, and which does not contain additional ingredients that are unacceptable and deleterious to the subject to which the pharmaceutical formulation is administered.

[0042] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation or composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0043] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed either prophylactically or during the clinical pathology course. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or slow disease progression.

[0044] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0045] II. Compositions and Methods Compositions are provided for use in methods of simultaneously engaging, coupling or binding to CD16 and CD112R. In some embodiments, methods for treating cancer include administering one or more compositions capable of simultaneously coupling, engaging and / or binding to CD112R and CD16. In some embodiments, the composition includes one agent capable of simultaneous binding. In some embodiments, the composition includes two or more agents that bind simultaneously by administration at or near the same time.

[0046] In some embodiments, the composition is a multispecific antibody that binds to CD112R and CD 16. In some embodiments, the composition comprises two agents, one agent that engages, couples or binds to CD112R and another agent that engages, couples or binds to CD16.

[0047] In some embodiments, a) a method of treating cancer by preferentially activating NK cells; and / or b) a method for enhancing the activation of NK cells; and / or c) A method that enhances NK cell activation but not T cell activation The present invention provides a composition for use in a method comprising administering a composition that engages, couples or binds to CD16 and CD112R.

[0048] In some embodiments, the composition is a multispecific antibody, which binds to and / or blocks and / or activates CD16 and CD112R.

[0049] In some embodiments, the compositions comprise a CD16 agonist and an agent that binds to and / or activates CD112R.

[0050] In some embodiments, the composition comprises an anti-CD16 antibody.

[0051] In some embodiments, the composition comprises an anti-CD112R antibody.

[0052] In some embodiments, the composition comprises an anti-CD16 antibody and an anti-CD112R antibody.

[0053] 1. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one binding specificity is for CD112R and another binding specificity is for CD16. In certain embodiments, one binding specificity is for CD112R, one binding specificity is for CD16, and another binding specificity is independently selected from one or more of PD-1, PD-L1, CTLA-4, Lag-3, TIM-3, TIGIT, CD96, PVRL1, PVRL2, PVRL3, PVRL4, CD155, STING, CD47, CD39, and IL-27. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD112R. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0054] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1993)). (1994)); and by preparing trispecific antibodies (see, for example, Tutt et al. J. Immunol. 147: 60 (1991)).

[0055] Engineered antibodies (including "octopus antibodies") having three or more functional antigen binding sites are also included herein (see, eg, US 2006 / 0025576 A1).

[0056] The antibodies or fragments herein also include "dual acting FAbs" or "DABs" that contain an antigen binding site that binds to CD112R and another distinct antigen (see, e.g., US2008 / 0069820).

[0057] 2. Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0058] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications where in vivo antibody half-life is important, but where certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985)) and U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be utilized (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model, such as that described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0059] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0060] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0061] In a specific embodiment, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

[0062] In some embodiments, changes are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0063] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which are involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions which improve binding of the Fc region to FcRn. Such Fc variants include Fc variants having a substitution at one or more of Fc region residues 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (e.g., U.S. Patent No. 7,371,826).

[0064] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351 for other examples of Fc region variants.

[0065] In some embodiments, the antibodies are provided according to the sequence listing and are of isotype human IgG1. In some embodiments, the antibodies are provided according to the sequence listing and are of isotype human IgG4. In some embodiments, the antibodies are provided according to the sequence listing and are of isotype human IgG4 and there is a single mutation at serine 228 to proline (S228P).

[0066] 3. Antibody derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional nonproteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when more than one polymer is attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations, including, but not limited to, the specific property or function of the antibody to be improved, or whether the antibody derivative will be used in therapy under certain conditions.

[0067] In another embodiment, a conjugate of an antibody and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to a temperature that kills cells proximal to the antibody-nonproteinaceous moiety.

[0068] B. Pharmaceutical Preparations Pharmaceutical formulations of the compositions described are provided and may be used in the methods described herein. In some embodiments, the pharmaceutical formulations are prepared in the form of a lyophilized formulation or aqueous solution by mixing the active ingredient with one or more optional pharma- ceutically acceptable carriers, diluents and / or excipients (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) to obtain the desired degree of purity. Pharmaceutically acceptable carriers, diluents, and excipients are generally nontoxic to recipients at the dosages and concentrations employed, and may include, for example, sterile water; buffers, such as phosphates, citrates, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes, such as Zn-protein complexes; and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharma- ceutically acceptable carriers herein further include insterstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs (including rHuPH20) and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0069] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which includes a histidine-acetate buffer.

[0070] The pharmaceutical formulations or compositions herein may also contain two or more active ingredients, as necessary for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0071] The active ingredient may be encapsulated in microcapsules, for example microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose- or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively; colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0072] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0073] Pharmaceutical formulations or compositions to be used for in vivo administration are generally sterile. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0074] C. Therapeutic Uses and Methods Compositions are provided for use in methods of simultaneously engaging, coupling or binding to CD16 and CD112R. In some embodiments, methods for treating cancer include administering one or more compositions capable of simultaneously coupling, engaging and / or binding to CD112R and CD16. In some embodiments, the composition includes one agent capable of simultaneous binding. In some embodiments, the composition includes two or more agents that bind simultaneously by administration at or near the same time.

[0075] In some embodiments, the composition is a multispecific antibody that binds to CD112R and CD 16. In some embodiments, the composition comprises two agents, one agent that engages, couples or binds to CD112R and another agent that associates, links or binds to CD16.

[0076] In some embodiments, a) a method of treating cancer by preferentially activating NK cells; and / or b) a method for enhancing the activation of NK cells; and / or c) A method that enhances NK cell activation but not T cell activation The present invention provides a composition for use in a method comprising administering a composition that engages, couples or binds to CD16 and CD112R.

[0077] In a further aspect, the present invention provides methods of treating diseases and / or disorders in which blocking CD112R is desired. In some embodiments, methods are provided for enhancing, increasing and / or maintaining an anti-tumor immune response in a subject having a tumor, comprising administering one or more agents that couple to, engage with or block CD16 and CD112R. In some embodiments, the tumor is cancerous. In some embodiments, methods are provided for treating cancer in a subject having cancer, comprising administering one or more agents that couple to, engage with or block CD16 and CD112R.

[0078] The compositions described herein can be used, for example, to treat cancer. In some embodiments, a method of treating cancer is provided that includes administering an effective amount of one or more compositions that engage, couple or bind to CD16 and CD112R.

[0079] The cancer can be a cancer involving a solid tumor or a hematological malignancy (eg, a liquid tumor).

[0080] Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colon cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (e.g., glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, cancer), germ cell tumors, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic melanoma, e.g., cutaneous melanoma or intraocular melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) ) tumors, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers (including asbestos induced cancers), virus-associated cancers or cancers of viral origin (e.g., tumors related to or derived from human papillomavirus (HPV)); and hematological malignancies derived from either of the two major blood cell lineages (i.e., myeloid cell lines (producing granulocytes, erythrocytes, platelet cells, macrophages and mast cells) or lymphoid cell lines (producing B cells, T cells, NK cells and plasma cells)), e.g., all types of leukemias, lymphomas and myelomas: e.g., acute, chronic, lymphocytic and / or myeloid leukemias, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia ( CLL), chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (M1), myeloblastic leukemia (M2, with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma;Lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies (e.g., B-cell lymphoma), T-cell lymphoma, lymphoplasmacytic lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, hematoimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T Lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma myeloma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma with Waldenström's macroglobulinemia (LPL); myeloma, e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma; chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin (e.g., fibrosarcoma and rhabdomyosarcoma); central and peripheral nervous tumors of mesenchymal origin, such as fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, such as melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma; hematopoietic tumors of lymphoid lineage, such as tumors of T and B cells, including but not limited to T cell cytotoxicity, such as T-prolymphocytic leukemia (T-PLL) (including small cell and gyrus cell types); large granular lymphocytic leukemia (LGL) of T cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / mature T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head or neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma;and any combination of the above cancers. The methods described herein may also be used for the treatment of metastatic cancer, unresectable refractory cancer (e.g., cancer refractory to previous immunotherapy, e.g., immunotherapy with CTLA-4 or PD-1 blocking antibodies), and / or recurrent cancer;

[0081] In certain embodiments, the compositions described herein are administered to subjects with cancer that has responded inadequately to a previous treatment, e.g., a previous treatment with an immuno-oncology or immunotherapy agent, or that has progressed during a previous treatment with an immuno-oncology or immunotherapy agent. In some embodiments, the cancer is refractory or resistant to a previous treatment, either the cancer is inherently refractory or resistant (e.g., refractory to a PD-1 pathway antagonist) or the resistant or refractory state is acquired. For example, the compositions described herein can be administered to subjects who do not respond or do not respond adequately to a first therapy, or whose disease has progressed after a treatment, e.g., an anti-PD-1 pathway antagonist treatment, alone or in combination with another therapy (e.g., an anti-PD-1 pathway antagonist therapy). In other embodiments, the compositions described herein are administered to subjects who have not previously received (i.e., been treated with) an immuno-oncology agent, e.g., a PD-1 pathway antagonist.

[0082] D. Combination The compositions of the invention can be used alone or in combination with other agents in a therapy, for example, the compositions of the invention can be co-administered with at least one additional therapeutic agent (e.g., further comprising administering a second therapy).

[0083] In some embodiments, targeting additional independent inhibitory pathways or combinations thereof has the potential to result in even greater enhanced immune cell activation than monotherapy.

[0084] In some embodiments, the additional therapeutic agent or second agent is a chemotherapeutic agent, an opsonizing agent, a regulatory T cell ("Treg") depleting agent, an antagonist of a target other than CD112R, or an agonist of a target other than CD112R. In certain embodiments, the second agent is a chemotherapeutic agent described herein or any known chemotherapeutic agent. In some embodiments, the second agent is an opsonizing agent, and the opsonizing agent is an antibody other than an anti-CD112R antibody that targets cancer cells or tumor cells. In some embodiments, the second agent is a Treg depleting agent described herein or any known Treg depleting agent. In some embodiments, the second agent is an antagonist of a target other than CD112R. In some embodiments, the second agent is an agonist of a target other than CD112R.

[0085] In some cases, the second agent targets an independent inhibitory pathway, e.g., a pathway involving PD-1, PD-L1, CTLA-4, Lag-3, or TIM-3. In some embodiments, the second agent antagonizes one or more of PD-1, PD-L1, CTLA-4, Lag-3, and TIM-3. Antagonists suitable for use in the combination therapies described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal and bispecific antibodies), and multivalent agents. In one embodiment, the antagonist is a fusion protein, e.g., an Fc fusion protein, e.g., AMP-244. In some embodiments, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

[0086] An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibody that includes the CDRs or variable regions of one of the following antibodies: 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11 described in WO2006 / 121168. In certain embodiments, the anti-PD-1 antibody is MK-3475 (lambrolizumab), described in WO2012 / 145493, AMP-514, or PDR001, described in WO2012 / 145493. Additional known PD-1 antibodies and other PD-1 inhibitors include those described in WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2011 / 066389, WO2011 / 161699, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Publication No. 2009 / 0317368. Any of the anti-PD-1 antibodies described in WO2013 / 173223 may also be used. Anti-PD-1 antibodies that compete for binding with one of these antibodies and / or bind to the same epitope on PD-1 as one of these antibodies may also be used in combination treatments.

[0087] In some embodiments, the anti-PD-L1 antibody useful in combination therapy is BMS-936559 (referred to as 12A4 in WO 2007 / 005874 and US Patent No. 7,943,743) or an antibody that comprises the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, as described in PCT Publication No. WO 07 / 005874 and US Patent No. 7,943,743. In certain embodiments, the anti-PD-L1 antibody is MEDI4736 (also known as durvalumab and anti-B7-H1), MPDL3280A (also known as atezolizumab and RG7446), MSB0010718C (also known as avelumab, WO2013 / 79174) or rHigM12B7. Any of the anti-PD-L1 antibodies described in WO2013 / 173223, WO2011 / 066389, WO2012 / 145493, U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Publication No. 2009 / 145493 may also be used. Anti-PD-L1 antibodies that compete with and / or bind to the same epitope as any of these antibodies may also be used in the combination treatment.

[0088] In certain embodiments, compositions of the present disclosure may be used in conjunction with a CTLA-4 antagonist, such as an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is Yervoy® (ipilimumab or antibody 10D1, described in PCT Publication No. WO01 / 14424), tremelimumab (formerly ticilimumab, CP-675,206), or any of the following publications: WO98 / 42752, WO00 / 37504, U.S. Pat. No. 6,207,156, Hurwitz et al. (1998) Pro. Natl. Acad. Sci. USA 95(17): 10067-10071, Camacho et al. (2004) J. Clin. Oncology 22(145): antibodiestract No. 2505 (antibody CP-675206), and Mokyr et al. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodies described in WO2013 / 173223 may also be used.

[0089] In some embodiments, the compositions of the present disclosure are used in combination with LAG-3 (also referred to herein and elsewhere as LAG3) antagonists. Examples of anti-LAG3 antibodies include antibodies that include the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, as described in U.S. Patent Publication Nos. US2011 / 0150892, WO10 / 19570, and WO2014 / 008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other art-recognized anti-LAG-3 antibodies that can be used include IMP731 and IMP-321, as described in US2011 / 007023, WO08 / 132601, and WO09 / 44273. Anti-LAG-3 antibodies that compete with and / or bind to the same epitope as any of these antibodies can also be used in combination treatments.

[0090] In some embodiments, targeting two or more of TIGIT, CD96 and CD112R receptors simultaneously increases CD226-mediated signaling over anti-CD112R monotherapy. Thus, in some embodiments, the second agent is an antagonist of TIGIT and / or CD96. Antagonists suitable for use in the combination therapies described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal and bispecific antibodies) and multivalent agents.

[0091] In some embodiments, PVR gene family members are upregulated on tumor cells and can exhibit intrinsic tumor-promoting properties. Additional targeting of PVR gene family members in combination with anti-CD112R antibodies results in enhanced tumor susceptibility over monotherapy. Thus, in some embodiments, the second agent is selected from one or more of PVRL1, PVRL2, PVRL3, PVRL4 and CD155 antagonists. Antagonists suitable for use in the combination therapy described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal and bispecific antibodies) and multivalent agents.

[0092] STING agonists induce innate immune cell activation and increase T cell priming and immune cell recruitment to the tumor microenvironment. Targeting STING agonists in combination with CD112R has the potential to further increase T and NK cell recruitment and activation.

[0093] Increased phagocytosis via anti-CD47 antibodies can result in increased presentation of cancer-derived antigens by macrophages to T cells. Combined treatment with anti-CD47 antibodies and anti-CD112R antibodies, such as those provided herein, provides an opportunity to enhance cancer antigen-specific T cell responses and is fully encompassed herein.

[0094] Adenosine inhibits the activation of T cells and NK cells through adenosine receptors expressed on immune cells. Anti-CD39 antibodies inhibit the production of adenosine by preventing the hydrolysis of adenosine triphosphate (ATP). Combined treatment of anti-CD39 antibodies with anti-CD112R antibodies, such as those provided herein, provides an opportunity to further enhance CD112R therapy by inhibiting adenosine-mediated cell signaling in immune cells.

[0095] Cytokines can effectively regulate the activation of T cells and NK cells. IL-27 is an immunosuppressive cytokine that inhibits T cell and NK cell-mediated responses. Anti-IL-27 antibodies provide an opportunity to enhance CD112R therapy by limiting the signaling of immunosuppressive cytokines in immune cells. Thus, combination therapy with anti-IL-27 antibodies and anti-CD112R antibodies, such as those provided herein, is provided.

[0096] The compositions herein may also be provided prior to, substantially simultaneously with, or following other therapeutic modalities, such as surgery, chemotherapy, radiation therapy, or administration of a biological agent (e.g., administration of another therapeutic antibody). In some embodiments, the cancer has recurred or progressed after a therapy selected from surgery, chemotherapy and radiation therapy, or a combination thereof. For example, the CD112R antibodies described herein may be administered as adjuvant therapy when there is a risk that micrometastases may be present and / or to reduce the risk of recurrence.

[0097] For the treatment of cancer, the combination may be administered in conjunction with one or more additional anti-cancer agents, such as chemotherapeutic agents, growth inhibitory agents, anti-cancer vaccines (e.g., gene therapy vaccines), anti-angiogenic agents, and / or anti-tumor compositions.

[0098] In some embodiments, anti-inflammatory drugs, such as steroids or nonsteroidal anti-inflammatory drugs (NSAIDs), may be administered in combination.Hormones and steroids (including synthetic analogs), such as 17a-ethynylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX®, may also be administered to the subject in conjunction with or prior to treatment with the CD112R antibodies described herein, if it is desired to place abnormally proliferative cells in a quiescent state. When using the methods or compositions described herein, other agents used to modulate tumor growth or metastasis in a clinical setting, such as antimimetics, can also be administered as needed.

[0099] The above combination therapies include combined administration (wherein two or more therapeutic agents are included in the same or separate formulations or compositions) and separate administration, where administration of an antibody of the invention may precede, be concurrent with, and / or follow administration of the additional therapeutic agent(s). In some embodiments, administration of an anti-CD112R antibody and administration of the additional therapeutic agent(s) may be within about one month of each other, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days.

[0100] The compositions of the present invention (and any additional therapeutic agents) can be administered by any suitable means, such as parenteral, pulmonary and intranasal administration, and intralesional administration if localized treatment is desired. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous administration. They can be administered by any suitable route, such as injections, e.g., intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including but not limited to single doses or multiple doses over various time periods, bolus doses and pulse infusions.

[0101] The compositions of the present invention can be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the drug, the method of administration, the schedule of administration, and other factors known to medical practitioners. As used herein, a "split dose" is the division of a single unit dose or total daily dose into two or more doses, e.g., two or more administrations of a single unit dose. The composition can be administered as a "split dose".

[0102] The composition is optionally, but need not be, formulated with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs depends on the amount of the composition present in the formulation or composition, the type of disorder or treatment, and other factors described above. These are generally used in the same dosages and by the routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route empirically / clinically determined to be appropriate. In some embodiments, the composition is provided in a formulation for immediate release and the other drug is formulated for sustained release, or vice versa.

[0103] E. Manufactured products In another aspect of the invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the above disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, for example, glass or plastic. The container holds the composition alone or in combination with another composition that is effective for treating, preventing, and / or diagnosing a condition, and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition can be an antibody. The label or package insert indicates that the composition is used to treat a selected range of conditions. Additionally, the article of manufacture can include (a) a first container in which a composition comprising the antibody is contained, and (b) a second container in which a composition comprising an additional cytotoxic or therapeutic agent is contained. The article of manufacture of this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0104] It will be understood that any of the above articles of manufacture may include an immunoconjugate. EXAMPLES

[0105] III. Examples Example 1 CD112R is an inhibitory receptor expressed on NK cells and T cells. CD112R is a ligand of the cell adhesion molecule CD112 (PVRL2) expressed on tumor cells, and inhibits immune cell activation by binding to CD112, which competes with the activating receptor CD226. Binding of CD112 to CD112R induces downstream signaling via the immunoreceptor tyrosine based inhibitory motif (ITIM) in the cytoplasmic tail, inhibiting effector cell activation. Figure 1A shows a schematic diagram, and Figure 1B shows the binding of CD112R to NK cells and CD8 cells. + 1 shows CD112R expression data on T cells.

[0106] Expression of CD112R is upregulated in NK cells infiltrating mouse tumors. See Figure 2. Expression of CD112R was assessed by flow cytometry in immune cell populations from spleens and dissociated tumors of Balb / c mice implanted subcutaneously with CT-26 tumors. CD112R expression is expressed as fold over negative (isotype control).

[0107] Figure 3A shows a model of the therapeutic activity of clone 35 on NK cells. By engaging both CD16 and CD112R, clone 35 treatment promotes antitumor activity. Figure 3B shows that anti-CD112R antibodies with enhanced Fc effector function (hIgG1 Fc, clone 35, clone 38, and clone 44) lead to stronger NK cell degranulation than antibodies with reduced Fc effector function (hIgG4 Fc, clone 35.4, clone 38.4, and clone 44.4) in tumor cell coculture. In particular, Figure 3B shows enhanced NK cell-mediated degranulation in response to tumor cells in the presence of CD112R antibodies with enhanced Fc effector function compared to CD112R antibodies with reduced Fc effector function. Human NK cells and Raji CD112 cells were cocultured with CD107a PE antibody for 4 hours in the presence of CD112R antibodies with IgG1 or IgG4.1 (S228P) isotypes. After coculture, NK cell degranulation was determined by the frequency of CD107a-positive NK cells.

[0108] In Figure 4A, overexpression of CD112R inhibits TCR-mediated activation of Jurkat cells. Jurkat cells transduced with CD112R-ires-GFP (Jurkat CD112R) or with a GFP control vector (Jurkat GFP) were cocultured for 24 h with TCR-stimulator cells expressing membrane-bound anti-CD3 scFv and CD112 ligand. Activation was measured by ELISA and IL-2 secretion into the supernatant.

[0109] In Figure 4B, Clone 35 treatment increased TCR-mediated activation of Jurkat.CD112R. Jurkat cells overexpressing CD112R were co-cultured with TCR stimulator cells (Raji cells transduced with membrane-bound anti-CD3 scFv and CD112 ligand) for 24 h in the presence of Clone 35 or isotype control antibody. Jurkat cell activation was measured by ELISA by IL-2 secretion into the supernatant.

[0110] Figure 5A shows that clone 35-mediated NK cell activation is partially suppressed by CD16 blockade. PBMCs from a single donor were co-cultured with K562 target cells, clone 35, a CD16-blocking F(ab')2 antibody (Ancell, clone 3G8), a CD32-blocking F(ab')2 antibody (Ancell, clone 7.3) or medium alone for 24 hours. NK cell activation was assessed by upregulation of 4-1BB expression by flow cytometry.

[0111] Figure 5B shows stronger tumor growth inhibition in mice treated with an engineered anti-CD112R antibody with enhanced Fc effector function (mouse IgG2a, clone 46) compared to an engineered anti-CD112R antibody with reduced Fc effector function (mouse IgG1, clone 46.mG1). Graph is a summary of three experiments, N=44-45 per group.

[0112] The in vivo efficacy of CD112R blockade was evaluated in a CT26 syngeneic mouse tumor model after depletion of NK cells or CD8 T cells. Mice were treated twice weekly for 3 weeks starting after randomization with Asialo-GM1 antibody (Biolegend; Cat. No. 146002; Dose: 100uL / mouse; i.p.) and anti-CD8a antibody (Bioxcell; Cat. No. BE0085; 200μg / mouse; i.p.) to deplete NK cells and CD8 T cells, respectively. 7-week-old BALB / cAnNTac female mice (Taconic Biosciences, Cat. No. BALB-F) were treated with 0.2×10 IgG in 0.1mL of 50% Geltrex (GIBCO, Cat. No. A1432-02) and 50% RPMI-1640 serum-free medium (GIBCO, Cat. No. A10491-01). 6 Cells CT26.WT (ATCC, Cat. No. CRL-2638) were implanted subcutaneously into the right flank. Mice with palpable tumors were randomized 4 days after implantation and treated intraperitoneally with clone 46 (anti-CD112R mouse IgG2a; 12.5 mg / kg; i.p.) twice a week for 3 weeks, starting on the day of randomization. If tumors were within the IACUC limit size (<2000 mm3 Tumor volumes were measured with a caliper every 2-3 days until tumor volumes (mm 3 ) was calculated as follows: width (mm) × [length (mm)] 2 x 0.5. The results are shown in Figure 6A. The graph shows the mean tumor volume for each treatment group as a function of time. These results show that the therapeutic effect of anti-CD112R is significantly reduced after depletion of NK cells or CD8 T cells.

[0113] Anti-tumor immunity was evaluated in anti-CD112R-treated mice that showed a complete response from the initial CT26.WT tumor challenge. For the initial challenge, 7-week-old BALB / cAnNTac female mice (Taconic Biosciences, Cat. No. BALB-F) were dosed with 0.2 × 10 6 Cells CT26.WT (ATCC, Catalog No. CRL-2638) were implanted subcutaneously into the right flank. Mice with palpable tumors were randomized 4 days after implantation and treated intraperitoneally as shown in Table 1 below, twice weekly for 3 weeks starting from the day of randomization.

[0114] [Table 1]

[0115] Tumors were within the IACUC limit of size (<2000 mm 3 Tumor volumes were measured with a caliper every 2-3 days until tumor volumes (mm 3 ) was calculated as follows: width (mm) × [length (mm)] 2 ×0.5.

[0116] All surviving mice 50 days after implantation that lacked discernible tumors were considered survivors / complete responders. Complete responder mice (n=8) in the anti-CD112R treatment group received 1×10 6 The mice were re-challenged by inoculation of 1 × 10 CT26.WT cells (ATCC, Cat. No. CRL-2638) in the left flank (a 5-fold increase from the primary inoculum). As controls, age-matched naïve Balb / c female mice (n=5) were also inoculated with 1 × 10 CT26.WT cells in 0.1 mL of 50% Geltrex and 50% RPMI-1640 serum-free medium. 6 CT26.WT cells were inoculated into the left flank. Mice received no further treatment. Tumors were allowed to grow beyond the IACUC limit size (<2000 mm 3 Tumor volumes were measured every 2-3 days until the tumor volume reached 100 mm. 3 ) was calculated as follows: width (mm) × [length (mm)] 2 × 0.5. The results are shown in Figure 6B.

[0117] To evaluate the effect of CD112R antibodies on NK cell activation, clone 35 (hIgG1) and clone 35.4 (hIgG4) were evaluated by PBMC-tumor cell coculture. Upregulation of CD137(4-1BB) has been previously established as a marker of NK cell activation (Baessler et al. (2010) Blood 115(15); Andre et al. (2018) Cell 175, 1731-1743), and upregulation of CD137(4-1BB) was measured in NK cells from PBMCs cocultured with K562 target cells (chronic myeloid leukemia cell line, ATCC#CCL-243) together with anti-CD112R antibodies or isotype control antibodies.

[0118] Briefly, frozen PBMCs isolated from buffy coats of healthy donors were thawed, washed, resuspended in DMEM + 10% FBS + 1% penicillin-streptomycin (D10), and plated in 96-well flat-bottom plates at 5 × 10 cells per well. 5 K562 cells were plated at a concentration of 10x10 per well and left at 37°C for 4 hours before adding target cells and antibodies. In the first experiment (Figure 7A), antibodies were then diluted in D10 and added to each well at a starting concentration of 10 μg / mL in a 10-fold serial dilution. In the second experiment (Figures 8C-8D), a single concentration (1 μg / mL) of anti-CD112R or IgG1 isotype control antibody was added to each well. In both experiments, each condition was run in duplicate. K562 cells were then harvested, washed, resuspended in D10 and added at 5 × 10 per well. 4 Cells were added to each well at a concentration of 100 mM NaCl. The final volume of each well was 200 μL. Plates were then incubated at 37° C. for 16 hours. After 16 hours, cells were transferred to V-bottom plates and washed twice with PBS+2% FBS. Cells were stained with anti-CD3 FITC (Biolegend, #300306), anti-NKp46 BV421 (Biolegend, #331914) and anti-CD137 APC (Biolegend, #309810) in PBS+2% FBS for 30 minutes at 4° C. Cells were then washed twice and resuspended in PBS+2% FBS. Data were acquired using a LSRFortessa X-20 (BD Biosciences) flow cytometer and analyzed with FlowJo software (Tree Star). NK cell activation was defined as the frequency of CD137+ cells within the CD3-NKp46+ lymphocyte gate.

[0119] Results from two separate donors from two independent experiments are shown in Figures 7A-7B.

[0120] Figure 8 shows that intratumoral NK cell activation was increased 72 hours after a single dose of anti-CD112R and anti-TIGIT combination therapy compared to isotype control. Activation was assessed as the percentage of granzyme B+ (Figure 8A) and interferon-γ+ (Figure 8B).

[0121] Figure 9 shows stronger tumor growth inhibition in mice treated with an anti-CD112R antibody with enhanced Fc effector function (mouse IgG2a, clone 46) compared to an engineered anti-CD112R antibody with reduced Fc effector function (mouse IgG1, clone 46.mG1). Graph is a summary of three experiments, N=44-45 per group. Statistical analysis was performed by Mann-Whitney test at day 24.

[0122] Figure 10 shows a subset of mice that rejected CT26 tumors after treatment with anti-CD112R.mG2a and did not exhibit palpable tumors after day 50 post-inoculation. These mice rapidly rejected CT26 tumors upon re-challenge, indicating that treatment of tumor-bearing mice with CD112R antibodies with enhanced Fc effector function leads to the development of immunological memory and protective immunity in a subset of mice.

[0123] Example 2 In a series of follow-up experiments, NK cell activation was assessed following co-culture with multiple permutations of anti-CD112R antibodies and anti-CD112R Fabs in PBMC from additional donors. Results show that enhanced Fc effector function and CD16 engagement are both required for clone 35-mediated enhanced NK cell activation in in vitro assays (Figure 11A-C).

[0124] Figure 11A shows that clone 35-mediated NK cell activation is partially abolished in the absence of Fc scaffold (antibody Fab). PBMCs from five donors were co-cultured with K562 target cells for 24 hours with either full-length clone 35, clone 35 Fab, full-length isotype control or isotype control Fab. NK cell activation was assessed by flow cytometry by upregulation of 4-1BB (CD137) expression. Statistical analysis was performed by paired t-test analysis.

[0125] Figure 11B shows that clone 35-mediated NK cell activation is partially abolished in the absence of glycosylated Fc backbone. Glycosylation of the Fc backbone at residue 297 significantly enhances the ability of IgG1 antibodies to bind to Fc receptors. An engineered mutation of the asparagine residue at position 297 to alanine (N297A) blocks glycosylation of this residue and therefore significantly reduces the ability of the antibody Fc backbone to engage Fc receptors (Wang et al, Protein Cell 2018). In this experiment, PBMCs from five donors were co-cultured with K562 target cells with clone 35, non-glycosylated clone 35 (clone 35-N297A), clone 35 with reduced effector function (hIgG4, clone 35.4) or hIgG1 isotype control antibody for 24 hours. NK cell activation was assessed by flow cytometry by upregulation of 4-1BB (CD137) expression. Statistical analysis was performed by paired t-test analysis.

[0126] Figure 11C shows that clone 35-mediated NK cell activation is partially suppressed by CD16 blockade. PBMCs from five donors were co-cultured with K562 target cells and either clone 35 or a Fab antibody blocking CD16 (Ancell, clone 3G8), a Fab antibody blocking CD32 (Ancell, clone 7.3) or an isotype control for 24 hours. NK cell activation was assessed by flow cytometry by upregulation of 4-1BB (CD137) expression. Statistical analysis was performed by paired t-test analysis.

[0127] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

Table 2-5

Table 2-6

Claims

1. i) a method for treating cancer by preferentially activating NK cells; and / or ii) Methods for enhancing NK cell activation A composition for use in The method includes administering a composition that engages, couples or binds to CD16 and CD112R, the composition comprises an anti-CD112R antibody; the antibody is human IgG1, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL); the VH has the amino acid sequence of SEQ ID NO:712; the VL has the amino acid sequence of SEQ ID NO:718; The composition, wherein the human IgG1 has the amino acid sequence of SEQ ID NO: 40000.

2. The composition of claim 1 , wherein the cancer is a carcinoma, lymphoma, blastoma, sarcoma, or leukemia.

3. 3. The composition of claim 2, wherein the cancer is squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, gastric cancer, melanoma, or various types of head and neck cancer (including squamous cell carcinoma of the head and neck).

4. The composition of any one of claims 1 to 3, further comprising administering a second therapy.

5. The method of claim 4, wherein the second therapy is radiation therapy or surgery.

6. The composition of claim 4, wherein the second therapy is administration of a chemotherapeutic agent, an opsonizing agent, or a regulatory T cell depleting agent.

7. The composition of claim 4, wherein the second therapy is administration of an antagonist of PD-1, PD-L1, CTLA-4, Lag-3, or TIM-3.

8. The composition of claim 4 , wherein the second therapy is administration of an antagonist of TIGIT or CD96.

9. The composition of claim 4, wherein the second therapy is administration of an antagonist of PVRL1, PVRL2, PVRL3, PVRL4 or CD155.

10. The composition of claim 4 , wherein the second therapy is administration of a CD47 antagonist.

11. The method of claim 4 , wherein the second therapy is administration of a CD39 antagonist.

12. The method of claim 4, wherein the second therapy is administration of an antagonist of IL-27.

13. The composition of claim 4 , wherein the second therapy is administration of a STING agonist.

14. The composition of any one of claims 7 to 12, wherein the antagonist is an antibody.

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