Antibody

Abstract

The present invention relates to antibodies that bind to TREM1 and inhibit its interaction with one or more of its natural ligands. Specific examples of such antibodies are provided. Therapeutic uses of the antibodies and methods for producing such antibodies are also provided.

Description

[Technical field]

[0001] The present invention relates to anti-TREM1 antibodies and their use in the treatment of neurological disorders, more particularly for the treatment of amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. [Background technology]

[0002] Triggering receptors expressed on myeloid cells (TREMs) are receptors that contain immune activating and immune inhibitory isoforms encoded by MHC gene clusters mapping to human chromosome 6p21 and mouse chromosome 17. TREMs are members of the immunoglobulin (Ig) superfamily that are expressed primarily in cells of the myeloid lineage, including monocytes, neutrophils, and dendritic cells in the periphery, and microglia in the central nervous system (CNS). Triggering receptor expressed on myeloid cells-1 (TREM1), also known as differentiation cluster 354, was the first member of the TREM family to be identified and shares limited homology with other receptors in the Ig superfamily. TREM1 is a transmembrane glycoprotein with a single Ig-like domain, a transmembrane region with a (+)-charged lysine residue that interacts with a negatively charged aspartic acid on its signaling partner, DAP12, and a short cytoplasmic tail that lacks a signaling domain.

[0003] TREM1 activation through interaction with its proposed ligands peptidoglycan recognition protein 1 (PGLYRP1), high mobility group B1 (HMGB1), soluble CD177, heat shock protein 70 (HSP70), and extracellular cold-inducible RNA-binding protein (eCIRP) is proposed to induce the formation of "head-to-tail" homodimers. Dimer cross-linking triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on recruited DAP12, which enables signaling and function by providing docking sites for spleen tyrosine kinase (SYK) and its downstream signaling partners, including zeta chain-associated protein kinase 70 (ZAP70), casitasb lineage lymphoma (Cbl), son of sevenless (SOS), and growth factor receptor-bound protein 2 (GRB2). These interactions trigger downstream signaling through the phosphatidylinositol 3-kinase (PI3K), phospholipase-C-γ2 (PLC-γ2) and ERK pathways. These events are followed by calcium mobilization and activation of transcription factors including ETS-containing protein (ELK1), nuclear factor of activated T cells (NFAT), AP1, c-fos, c-Jun and NF-κB. This pathway is shared with another member of the TREM family, TREM2.

[0004] Unlike TREM1, which is clearly an immune activator, TREM2 can act both pro- and anti-inflammatory when bound to high- and low-affinity ligands, respectively. Under homeostatic conditions, TREM2 interaction with low-affinity ligands maintains pathways that keep homeostasis in check (Konishi H., et al. Frontiers Cellular Neuroscience 2018).

[0005] In neuroinflammatory neurodegenerative conditions, intracellular factors, also known as damage-associated molecular patterns (DAMPs), (among others TREM1 ligands), spill out from dying neurons and activate microglial surveillance via TREM1 and other pattern recognition receptors. TREM1-DAMP interactions override TREM2 activity, resulting in microglial / innate immune activation, direct neurotoxicity, and disruption of synaptic structures through aberrant phagocytosis. Beyond its "Yin and Yang" dynamic with TREM2 in pathway regulation, TREM1 has a unique and distinct function as an enhancer of other key regulators of the innate immune response, including the Toll-like (TLR) and NOD-like receptor families. Amplification of these receptors occurs either by TREM1-induced overexpression of TLRs, their downstream nodes such as MYD88 and IKk, or by direct cross-linking via the TREM1-ligand complex formed between TLR agonists and TREM1 ligands, as in the case of PGN (TLR2 / TLR4 stimulator) and PGLYRP1 (TREM1 ligand).

[0006] The outcome of TREM1 multipathway activation is the amplification of innate immune / microglial proinflammatory responses, including cytokine and chemokine release, upregulation of costimulatory molecules / antigen presentation, and abnormal phagocytic activity downstream (Buchon et al., 2000). These processes are a common denominator of pathobiology in various neurodegenerative, neurodevelopmental, and autoimmune central nervous system disorders. Human genetics, including genome-wide association studies (GWAS), have implicated several nodes downstream of the TREM1 / TREM2 pathway, such as TREM2, DAP12, Syk, PLCγ2, and TLR, as risk genes in various neurodegenerative diseases.

[0007] No. 9,000,127 provides anti-TREM1 antibodies that disrupt the interaction of TREM1 with its ligands. The disclosed antibodies are provided for the treatment of individuals with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease.

[0008] WO 2017 / 152102 discloses antibodies that bind to TREM1 protein and modulate or enhance one or more TREM1 activities. Summary of the Invention

[0009] The present invention addresses the need for new treatments for neurological disorders by providing anti-TREM1 antibodies with the functional and structural properties described herein.

[0010] In particular, the present invention provides CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 and a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 and a heavy chain variable region comprising The present invention provides an antibody that binds to human TREM1, comprising:

[0011] The invention will now be described with reference to the following drawings. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the humanization of the antibody 12172 light chain. The various mutants generated for that chain are also shown. The CDR sequences are underlined.

[0013] [Diagram 2] Figure 1 shows the humanization of the heavy chain of antibody 12172. The various mutants generated for that chain are also shown. The CDR sequences are underlined.

[0014] [Figure 3A]Crystal structure of human TREM1 bound to PGLYRP1 and 12172 rabbit Fab. Crystal structure of human TREM1 bound to PGLYRP1. TREM1 residues with atoms within 4 Angstroms of any atom in PGLYRP1 are highlighted in black. [Figure 3B] Crystal structure of human TREM1 bound to PGLYRP1 and 12172 rabbit Fab. Crystal structure of human TREM1 bound to 12172 rabbit Fab. TREM1 residues with atoms within 4 Angstroms of any atom in 12172 are highlighted in black.

[0015] [Figure 4] Figure 13. Thermal stability of different variants of the 12172 antibody. Thermograms of 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) measured in a common pre-formulation storage buffer pH 7.4.

[0016] [Diagram 5] FIG. 1 shows inhibition of TREM1-mediated release of TNF-α, IL-6 and IL-1β by 12172 gL2gH11 hIgG4P from primary human monocytes.

[0017] [Figure 6] FIG. 1 shows increased release of IL-1R antagonist from primary human monocytes by various 12172 mutant and reference antibodies.

[0018] [Figure 7] FIG. 1 shows the effect of 12172 gL2gH11 hIgG4P and 0318-IgG1.3f in increasing IL-1RA release from unstimulated primary human monocytes.

[0019] [Figure 8] FIG. 1 shows the efficacy of 12172 gL2gH11 hIgG4P on TNF-α and IL-6 release from healthy control and Alzheimer's Disease (AD) PBMCs.

[0020] [Figure 9] FIG. 13 shows the efficacy of 12172 gL2gH11 hIgG4P on TNF-α and IL-6 release from healthy control and ALS PBMCs.

[0021] [Figure 10] FIG. 13 shows the efficacy of 12172 gL2gH11 hIgG4P on pro-inflammatory cytokine and chemokine release from ALS and AD PBMCs.

[0022] [Figure 11A] Volcano plot showing differentially expressed genes (DEGs) (considering an FDR of 0.05) following treatment of human monocytes with 12172 antibody variants and stimulation with TREM1-ligand complexes (compared to isotype control). [Figure 11B] Volcano plot showing differentially expressed genes (DEGs) (considering an FDR of 0.05) following treatment of human monocytes with 12172 antibody variants and stimulation with TREM1-ligand complexes (compared to isotype control). [Figure 11C] Volcano plot showing differentially expressed genes (DEGs) (considering an FDR of 0.05) following treatment of human monocytes with 12172 0318-IgG1.3f(11C) and stimulation with the TREM1-ligand complex (compared to isotype control).

[0023] [Figure 12A] Volcano plot showing DEGs (considering an FDR of 0.05) following treatment of human monocytes with 12172 antibody variants and stimulation with apoptotic human iPSC-derived motor neurons (compared to isotype control). [Figure 12B] Volcano plot showing DEGs (considering an FDR of 0.05) following treatment of human monocytes with 12172 antibody variants and stimulation with apoptotic human iPSC-derived motor neurons (compared to isotype control). [Figure 12C] Volcano plot showing DEGs (considering an FDR of 0.05) following treatment of human monocytes with 12172 0318-IgG1.3f (12C) and stimulation with apoptotic human iPSC-derived motor neurons (compared to isotype control).

[0024] [Figure 13] Figure 12172 gL2gH11 hIgG4P does not affect E. coli clearance by human neutrophils and monocytes in vitro. Data are representative of three individual donors for 12172 gL2gH11 hIgG4P v isotype and one donor with 0318-IgG1.3f (Ab318) molecule for comparison. Statistical analysis, one-way ANOVA Dunnet's post-hoc test was performed to compare anti-TREM1 antibodies to isotype control**** (p<0.0001).

[0025] [Figure 14] FIG. 13 shows the efficacy and potency of 12172 antibody variants in blocking SYK activation in hTREM1 / hDAP-12 Flp-In293 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Abbreviation Table 1. Abbreviations used throughout this specification [Table 1]

[0027] Table 2. Amino acid abbreviations [Table 2]

[0028] definition The following terms are used throughout the specification.

[0029] The term "acceptor human framework" is used herein as a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes.

[0030] The term "affinity" refers to the strength of all non-covalent interactions between the antibody and the target protein. Unless otherwise indicated, as used herein, the term "binding affinity" refers to the inherent 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 for its binding partner can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0031] The term "affinity maturation" in the context of antibodies refers to an antibody that has one or more changes in a hypervariable region, compared to a parent antibody that does not possess such changes, which changes result in an improvement in the affinity of the antibody for antigen.

[0032] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies, so long as they exhibit the desired antigen-binding activity. The term antibody, as used herein, relates to whole (full-length) antibodies (i.e., containing two heavy and two light chain elements) and functionally active fragments thereof (i.e., molecules that contain an antigen-binding domain that specifically binds to an antigen (also called antibody fragments or antigen-binding fragments)). The characteristics described herein with respect to antibodies also apply to antibody fragments, unless the context dictates otherwise. The term "antibody" encompasses monovalent, i.e., antibodies that contain only one antigen-binding domain (e.g., one-arm antibodies that contain a full-length heavy chain and an interconnected full-length light chain, also called "half antibody"), and multivalent, i.e., antibodies that contain two or more antigen-binding domains, e.g., bivalent antibodies.

[0033] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay.

[0034] The term "antibody-dependent cellular cytotoxicity" or "ADCC" is a mechanism of cell death induction that depends on the interaction of antibody-coated target cells with lytic effector cells, such as natural killer cells, monocytes, macrophages, and neutrophils, via Fc gamma receptors (FcγR) expressed on the effector cells.

[0035] The term "antigen-binding fragment" as used herein refers to a functionally active antibody-binding fragment, including, but not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies, scFv, Fv, bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3),209-217). As used herein, "binding fragment" refers to a fragment that can bind to a target peptide or antigen with sufficient affinity to characterize the fragment as specific for the peptide or antigen.

[0036] The term "antibody variant" refers to a polypeptide, e.g., an antibody comprising a VH and / or VL having the desired characteristics described herein and having at least about 80% amino acid sequence identity with the VH and / or VL of a reference antibody. Such antibody variants include, for example, antibodies in which one or more amino acid residues are added to or deleted from the VH and / or VL domains. Typically, the antibody variant will have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, with an antibody described herein. Optionally, the variant antibody will have no more than one conservative amino acid substitution compared to the antibody sequences provided herein, or alternatively no more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to the antibody sequences provided herein. In embodiments, "antibody variant" refers to an antibody or antigen-binding fragment thereof comprising a VH and / or a VL, wherein the non-CDR regions of the antibody or antigen-binding fragment thereof have at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to an antibody described herein.

[0037] The term "antigen-binding domain" as used herein refers to a portion of an antibody that comprises part or all of one or more variable domains that specifically interact with a target antigen, such as part or all of a pair of variable domains VH and VL. A binding domain may comprise a single domain antibody. Each binding domain may be monovalent. Each binding domain may comprise no more than one VH and one VL.

[0038] The term "bispecific" or "bispecific antibody" as used herein refers to an antibody with two antigen specificities.

[0039] The term "complementarity determining region" or "CDR" generally refers to the fact that an antibody comprises six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). The CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, AM J Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, "CDR-H1" as used herein is intended to refer to residues 26-35 as described by a combination of the Kabat numbering system and the topological loop definition of Chothia. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system. Unless otherwise indicated, the CDR residues and other residues in the variable domain (e.g. FR residues) are numbered herein according to Kabat.

[0040] The term "chimeric" antibody refers to an antibody in which the variable domains (or at least a portion thereof) of the heavy and / or light chains are derived from a particular source or species, and the remaining portions of the heavy and / or light chains (i.e., the constant domains) are derived from a different source or species. (Morrison; PNAS 81, 6851 (1984)). Chimeric antibodies can, for example, contain non-human variable domains and human constant domains. Chimeric antibodies are typically produced using recombinant DNA methods. A subcategory of "chimeric antibodies" is the "humanized antibody."

[0041] 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 several 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.

[0042] The term "complement dependent cytotoxicity" or "CDC" refers to a mechanism of cell death in which the Fc effector domain of a target-binding antibody binds and activates complement component C1q, which then activates the complement cascade, resulting in target cell death.

[0043] As used herein, the terms "constant domain(s)" or "constant region" are used interchangeably to refer to the domain(s) of an antibody outside the variable region. The constant domain is identical in all antibodies of the same isotype, but differs from one isotype to another. Typically, the constant region of the heavy chain is formed from the N-terminus to the C-terminus by CH1-hinge-CH2-CH3-optionally CH4, which includes three or four constant domains.

[0044] The term "competing antibody" or "cross-competing antibody" shall be interpreted to mean that the claimed antibody binds to either (i) the same location on the antigen that the reference antibody binds, or (ii) a location on the antigen where the antibody sterically hinders binding of the reference antibody to the antigen.

[0045] The term "derivative" as used herein is intended to include reactive derivatives, e.g., thiol-selective reactive groups such as maleimides. The reactive group may be linked to the polymer directly or via a linker segment. It will be understood that the residue of such a group will, in some cases, form part of the product as the linking group between the antibody fragment and the polymer.

[0046] The term "derived from" in the context of generating a variable sequence refers to the fact that the sequence used, or a sequence very similar to the sequence used, is obtained from original genetic material, such as the light or heavy chain of an antibody.

[0047] The term "diabody" as used herein refers to two Fv pairs with two inter-Fv linkers, a first VH / VL pair and a further VH / VL pair, such that the VH of the first Fv is linked to the VL of the second Fv and the VL of the first Fv is linked to the VH of the second Fv.

[0048] As used herein, the term "DiFab" refers to two Fab molecules linked via the C-terminus of the heavy chains.

[0049] The term "DiFab'" as used herein refers to two Fab' molecules linked via one or more disulfide bonds in their hinge regions.

[0050] The term "dsscFv" or "disulfide-stabilized single-chain variable fragment" as used herein refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains and also containing an interdomain disulfide bond between VH and VL (see, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012; WO 2007109254).

[0051] The term "DVD-Ig" (also known as double-V domain IgG) refers to a full-length antibody having four additional variable domains, one at the N-terminus of each heavy chain and each light chain.

[0052] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0053] As used herein, the term "effector molecule" includes, for example, anti-neoplastic agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, in particular radioactive iodides, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

[0054] The term "epitope" or "binding site" in the context of an antibody refers to the site (or portion) on an antigen to which the paratope of an antibody binds or recognizes. Epitopes can be formed both from contiguous amino acids (often referred to as "linear epitopes") or from discontinuous amino acids formed by tertiary folding of a protein (often referred to as "conformational epitopes"). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least 3, and more usually at least 5-10 amino acids in a unique spatial conformation. Epitopes usually consist of chemically active surface groups of molecules such as amino acids, sugar side chains, etc., and usually have specific 3D structure and charge characteristics.

[0055] "EU index" or "Kabat-like EU index" or "EU numbering scheme" refers to the EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85). It is generally used when referring to residues in antibody heavy chain constant regions (e.g., as reported in Kabat et al.). Unless otherwise specified, the EU numbering scheme is used to refer to residues in antibody heavy chain constant regions described herein.

[0056] The term "Fab" as used herein refers to an antibody fragment comprising a light chain fragment containing the VL (variable light) domain and the constant domain (CL) of the light chain, and a VH (variable heavy) domain and the first constant domain (CH1) of the heavy chain. Dimerization of Fab' according to the present disclosure produces F(ab')2, where, for example, dimerization may be via a hinge.

[0057] The term "Fab'-Fv" as used herein is analogous to FabFv, where the Fab portion is replaced by Fab'. The format may be provided as a PEGylated version thereof.

[0058] As used herein, the term "Fab'-scFv" refers to a Fab' molecule in which the scFv is added to the C-terminus of the light or heavy chain.

[0059] As used herein, the term "Fab-dsFv" refers to a FabFv in which an intra-Fv disulfide bond stabilizes the additional C-terminal variable region. The format may be provided as a PEGylated version thereof.

[0060] The term "Fab-Fv" as used herein refers to a Fab fragment in which the variable regions are added to the C-terminus of each of the following CH1 of the heavy chain and CL of the light chain. The format may be provided as a PEGylated version thereof.

[0061] As used herein, the term "Fab-scFv" refers to a Fab molecule in which an scFv is added to the C-terminus of the light or heavy chain.

[0062] The terms "Fc", "Fc fragment", and "Fc region" are used interchangeably to refer to the C-terminal region of an antibody that includes the constant region of the antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant domains, CH2 and CH3, of IgA, IgD and IgG, or the last three constant domains of IgE and IgM, and the flexible hinge N-terminal to these domains. The human IgG1 heavy chain Fc region is defined herein to include residue C226 to its carboxyl terminus, with numbering according to the EU index. In the context of human IgG1, the lower hinge refers to positions 226-236, the CH2 domain refers to positions 237-340, and the CH3 domain refers to positions 341-447, according to the EU index. Corresponding Fc regions of other immunoglobulins can be identified by sequence alignment.

[0063] The term "framework" or "FR" refers to variable domain residues other than the hypervariable region 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 following order in a VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0064] The term "full-length antibody" as used herein refers to an antibody having a structure substantially similar to that of a natural antibody or an antibody having a heavy chain including an Fc region as defined herein. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH) composed of three constant domains CH1, CH2 and CH3 or four constant domains CH1, CH2, CH3 and CH4, depending on the Ig class. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0065] The term "Fv" refers to two variable domains of a full-length antibody, eg, a cognate pair or affinity matured variable domains, i.e., a VH and VL pair.

[0066] The term "highly similar" as used in the context of amino acid sequences is intended to refer to amino acid sequences that are 95% or more similar over their entire length, such as 96, 97, 98 or 99% similar.

[0067] The term "human antibody" refers to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0068] The term "human consensus framework" refers to 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 derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as 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 as in Kabat et al., supra. In some embodiments, for VH, the subgroup is subgroup III as in. In some embodiments, for VH, the subgroup is subgroup IV as in Kabat et al.

[0069] The term "humanized" antibody refers to an antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. Typically, the heavy and / or light chains contain one or more CDRs (including one or more modified CDRs, if desired) from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) and are grafted onto the heavy and / or light chain variable region framework of an acceptor antibody (human antibody) (see, e.g., Vaughan et al, Nature Biotechnology, 16, 535-539, 1998). The advantage of such humanized antibodies is that they retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity to humans. Instead of transferring the entire CDR, only one or more of the specificity determining residues from any one of the CDRs described hereinabove can be transferred into the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0070] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally distinct loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contacts").

[0071] The term "IC50" as used herein refers to the half-maximal inhibitory concentration, which is a measure of the effectiveness of a substance, such as an antibody, in inhibiting a particular biological or biochemical function. IC50 is a quantitative measure of how much of a particular substance is needed to inhibit a given biological process by 50%.

[0072] "Identity" between amino acids in sequences indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.

[0073] As used herein, the term "IgG-scFv" refers to a full-length antibody having an scFv at the C-terminus of each heavy or light chain.

[0074] As used herein, the term "IgG-V" refers to a full-length antibody with a variable domain at the C-terminus of each heavy or light chain.

[0075] The term "IgG1 LALA" or "hIgG1 LALA" refers to a variant of the wild-type human IgG1 isoform in which the amino acid substitutions L234A / L235A in the IgG1 constant region have been introduced.

[0076] The term "IgG4P" or "hIgG4P" refers to a mutant of the wild-type human IgG4 isoform in which amino acid 228 (according to EU numbering) is replaced by proline, as described, for example, in Angal et al., Molecular Immunology, 1993, 30(1), 105-108.

[0077] The term "isolated" as used throughout this specification means that the antibody or polynucleotide, as the case may be, is present in a physical environment different from that in which it may occur in nature. The term "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from its natural environment or that has been synthetically produced. Isolated nucleic acid can include synthetic DNA, cDNA, genomic DNA, or any combination thereof, produced, for example, by chemical processing.

[0078] The term "Kabat residue designation" or "Kabat" refers to the residue numbering scheme commonly used for antibodies. It does not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering corresponding to shortening of structural components or insertions into components, whether framework or complementarity determining regions (CDRs) of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by alignment of the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. For details, see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Unless otherwise indicated, Kabat numbering is used throughout this specification.

[0079] The term "KD" as used herein refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). Kd and Ka refer to the dissociation and association rates, respectively, of a particular antigen-antibody interaction. The KD value of an antibody can be determined using methods well established in the art.

[0080] The term "monoclonal antibody" (or "mAb") refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual of a monoclonal antibody preparation is identical except for possible mutations that may be present in small amounts (e.g., naturally occurring mutations). Nevertheless, certain differences in protein sequence linked to post-translational modifications (e.g., truncation of the heavy chain C-terminal lysine, deamidation of asparagine residues, and / or isomerization of aspartate residues) may exist between the various different antibody molecules present in the composition. In contrast to a polyclonal antibody preparation, each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0081] The term "multiparatopic antibody" as used herein refers to an antibody described herein that contains two or more different paratopes that interact with different epitopes from either the same antigen or two different antigens. The multiparatopic antibodies described herein can be biparatopic, triparatopic, or tetraparatopic.

[0082] The term "multispecific" or "multispecific antibody" as used herein refers to an antibody described herein having at least two binding domains, i.e., two or more binding domains, e.g., two or three binding domains, where the at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). Multispecific antibodies described herein include monovalent and multivalent, e.g., bivalent, trivalent, tetravalent, multispecific antibodies. For example, an antibody can comprise a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv that binds to a therapeutic target and one scFv or dsscFv that extends half-life, e.g., by binding to albumin). Such antibodies are described in WO 2015 / 197772.

[0083] The term "neutralizing" (or "neutralize") in the context of antibodies refers to an antibody that is able to inhibit or attenuate the biological signaling activity of its target (target protein).

[0084] The term "paratope" refers to the region of an antibody that recognizes and binds to an antigen.

[0085] The term "percent (%) sequence identity (or similarity)" with respect to polypeptide and antibody sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical (or similar) to the amino acid residues in the compared polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0086] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, 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.

[0087] The term "polyclonal antibody" refers to a mixture of different antibody molecules that bind to (or otherwise interact with) two or more epitopes of an antigen.

[0088] The term "prevent" in the context of antibodies is used interchangeably herein with the term "inhibit" to refer to the effect that antibodies according to the invention have on a particular biological process or molecular interaction.

[0089] The term "scDiabody" refers to a diabody that contains an intra-Fv linker such that the molecule contains three linkers to form a normal scFv in which the VH and VL termini are each linked to one of the variable regions of an additional Fv pair.

[0090] As used herein, the term "Sc diabody-CH3" refers to two sc diabody molecules, each linked via a hinge at the CH3 domain.

[0091] The term "ScDiabody-Fc" as used herein refers to two sc diabodies, each attached, for example via a hinge, to the N-terminus of the CH2 domain of the constant region fragment -CH2CH3.

[0092] The term "single-chain variable fragment" or "scFv" as used herein refers to a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains.

[0093] As used herein, the term "ScFv-Fc-scFv" refers to four scFvs, one of each attached to the N-terminus and C-terminus of both heavy chains of a CH2CH3 fragment.

[0094] As used herein, the term "scFv-IgG" refers to a full-length antibody having an scFv at the N-terminus of each heavy or light chain.

[0095] The term "similarity" as used herein indicates that at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids that may be substituted for one another include, but are not limited to, the following: - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); - lysine, arginine and histidine (amino acids with basic side chains); - Aspartate and glutamate (amino acids with acidic side chains); - asparagine and glutamine (amino acids with amide side chains); and -Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0096] The term "single domain antibody" as used herein refers to an antibody fragment consisting of a single monomeric variable domain. Examples of single domain antibodies include VH or VL or VHH or V-NAR.

[0097] The term "specific" as used herein in the context of antibodies is intended to refer to an antibody that recognizes only the antigen for which it is specific, or an antibody that has a significantly higher binding affinity, e.g., at least 5, 6, 7, 8, 9, 10-fold higher, for the antigen for which it is specific compared to binding to an antigen for which it is non-specific.

[0098] The term "sterically blocking" or "sterically hindering" as used herein is intended to refer to a means of blocking an interaction between a first protein and a second protein by binding of a third protein to the first protein, which prevents the second protein from binding to the first protein due to unfavorable van der Waals or electrostatic interactions between the second and third proteins.

[0099] The term "subject" or "individual" in the context of therapy and diagnosis generally refers to mammals. 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). More specifically, an individual or subject is a human.

[0100] As used herein, the term "tandem scFv" refers to at least two scFvs linked via a single linker such that there is a single inter-Fv linker.

[0101] The term "tandem scFv-Fc" as used herein refers to at least two tandem scFvs, each attached, for example via a hinge, to the N-terminus of the CH2 domain of the constant region fragment -CH2CH3.

[0102] The term "target" or "antibody target" as used herein refers to the target antigen to which an antibody binds.

[0103] The term "tetrabody" as used herein refers to a format similar to a diabody which contains four Fvs and four inter-Fv linkers.

[0104] The term "therapeutically effective amount" refers to the amount of an antibody that, when administered to a subject for treating a disease, is sufficient to effect such treatment of the disease. The therapeutically effective amount varies depending on the antibody, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0105] As used herein, the term "tribody" (also referred to as Fab(scFv)2) refers to a Fab fragment with a first scFv attached to the C-terminus of the light chain and a second scFv attached to the C-terminus of the heavy chain.

[0106] The term "trispecific or trispecific antibody" as used herein refers to an antibody with three antigen-binding specificities. For example, an antibody is an antibody with three antigen-binding domains (trivalent) that bind independently to three different antigens or three different epitopes on the same antigen, i.e., each binding domain is monovalent for each antigen. One example of a trispecific antibody format is TrYbe.

[0107] Terms such as "prevent" or "prevention" refer to achieving a prophylactic effect in that a disease or its symptoms are completely or partially prevented. Thus, preventing encompasses halting the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0108] The terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic with respect to partial or complete cure of a disease and / or adverse effects resulting from a disease. Thus, treatment encompasses (a) inhibiting the disease, i.e., halting its development; (b) relieving the disease, i.e., causing regression of the disease.

[0109] The term "TrYbe" as used herein refers to a tribody comprising two dsscFvs.dsFab, as used herein, refers to a Fab with an intravariable region disulfide bond.

[0110] 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 variable domains of the heavy (VH) and light (VL) chains of full-length antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form the variable region. By convention, the CDRs in the heavy chain variable region of an antibody are called CDR-H1, CDR-H2 and CDR-H3, and the CDRs in the light chain variable region are called CDR-L1, CDR-L2 and CDR-L3. They are numbered consecutively from the N-terminus to the C-terminus of each chain. The CDRs are conventionally numbered according to the system devised by Kabat.

[0111] 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." The term "vector" includes "expression vectors."

[0112] The term "VH" refers to the variable domain (or sequence) of the heavy chain.

[0113] As used herein, the term "V-IgG" refers to a full-length antibody that has a variable domain at the N-terminus of each heavy or light chain.

[0114] The term "VL" refers to the variable domain (or sequence) of the light chain.

[0115] TREM1 The term "TREM1" refers to "Triggering Receptor Expressed on Myeloid Cells 1" (also known as TREM-1 and CD354), a receptor expressed on monocytes, macrophages, neutrophils and other cell types. Primary ligands for TREM1 include peptidoglycan recognition protein 1 (PGLYRP1), which belongs to the family of peptidoglycan (PGN)-binding proteins (PGRPs). The term "TREM1" includes any variant or isoform of TREM1 that is naturally expressed by a cell.

[0116] Three isoforms of human TREM1 have been identified. Isoform 1 (Accession No. NP61113.1; SEQ ID NO: 1) consists of 234 amino acids and represents the canonical sequence. Isoform 2 (Accession No. NP1229518.1; SEQ ID NO: 2) consists of 225 amino acids and differs from the canonical sequence in amino acid residues 201 to 234, which code for the transmembrane domain and part of the cytoplasmic domain. Isoform 3 (Accession No. NP001229519; SEQ ID NO: 3) consists of 150 amino acids and is soluble. It lacks amino acid residues 151 to 234, which code for the transmembrane domain, the cytoplasmic domain, and part of the extracellular domain. Amino acid residues 138 to 150 also differ from the above canonical sequence.

[0117] Methods for identifying anti-TREM1 antibodies To identify antibodies that interact with different amino acid residues on TREM1 than the PGLYRP1 ligand and neutralize one or more of the TREM1 activities, a special screening and testing strategy had to be developed that included binding to TREM1 and measurement of the functional properties of the test antibodies, as well as high-throughput measurements of the structural aspects of the binding (target epitope residues). By establishing the TREM1 residues involved in the interaction with PGLYRP1, this method makes it possible to perform rapid tests and to select antibodies for further development that would bind to a site on TREM1 different from PGLYRP1. Such antibodies could offer the additional advantage of preventing the binding of other potential ligands that interact with different sites on TREM1.

[0118] Thus, there is provided a method for identifying an antibody that interacts with an amino acid residue on TREM1 that is distinct from PGLYRP1 and that neutralizes the activity of human TREM1, the method comprising: a) immunizing an animal with cells transiently expressing human TREM1; b) recovering B cells from the animal; and c) detecting antibodies produced by the B cells; i. binds to human TREM1 with an affinity of at least 1 nM; and ii. blocks PGLYRP1-mediated signaling in a THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cell assay; and iii. binds to a site on human TREM1 that is distinct from PGLYRP1; Selecting based on their capabilities; Includes.

[0119] To identify antibodies that bind to sites different from PGLYRP1, a method was developed that uses arrays of mutant TREM1 proteins that allow rapid testing of binding sites on the TREM1 protein (residues of TREM1 involved in interaction with a test antibody). The same method is used to determine the binding site of a PGLYRP1 ligand. Such a method identifies the amino acid residues on TREM1 that form the binding site for a test antibody (or PGLYRP1): a) obtaining 3D structural information of TREM1; b) using the obtained 3D structural data to identify amino acid residues that are within accessible surface regions; c) for each identified amino acid, selecting one or two amino acids that are within a predefined distance from the identified amino acid and within an accessible surface area, whereby such combinations of amino acid residues form a patch of two or three amino acids; d) from the large number of potential patches generated, selecting a representative set of patches that cover most of the accessible surface area of ​​TREM1 while minimizing the number of patches that may misfold the TREM1 protein; e) generating a set of mutant proteins, each of the mutant proteins comprising a mutant sequence of the target protein, each of the mutant sequences comprising a single mutated patch of an amino acid identified in step (c), each of the amino acids of the patch being replaced by another amino acid; f) determining the binding properties of each mutant protein; g) identifying patches that exhibit reduced binding properties of an antibody to a corresponding mutant TREM1 protein that contains such a patch, whereby residues in such patches are identified as being part of the binding site of the antibody;

[0120] In a preferred embodiment, the antibody is selected as interacting with residues E26, E27, K28, Y29, E30, L31, K32 and Q35 of human TREM1 (wherein the numbering is according to SEQ ID NO:1).

[0121] To identify amino acid residues for generating mutant forms of TREM1, 3D structural data needs to be obtained for TREM1. Such data is available in the form of a PDB structure (PDB code: 1SMO, chain A). Alternatively, such structural data can be obtained using techniques known to those skilled in the art. Such techniques include X-ray analysis or NMR data. Preferably, such 3D data is of sufficient spatial resolution to allow identification of target residues.

[0122] In particular, the predetermined distance between the residues of each patch is 4, 5, 6, or 7 Å. Preferably, such distance is 6 Å. Preferably, alanine and glycine are not selected for substitution. Depending on the relevance of Cys residues in the 3D structure, they may or may not be selected for substitution. Cys is often involved in the formation of SS bonds in proteins and is important for tertiary structure. Gly is a very flexible amino acid, and replacing it with a larger amino acid such as Ala may also have structural effects. Optionally, Pro residues can be excluded from the analysis, since they are often involved in secondary structure formation.

[0123] More specifically, amino acids within the accessible surface area are selected based on the calculated solvent-accessible surface area of ​​their side chains. Standard methods for calculating solvent accessibility can be applied. In a typical example, a probe of 1.4 Å is used in the calculation (a simplified version of an H2O molecule where such a probe has a similar size to an H2O molecule). In such a calculation, atoms of the amino acid residues that contact the probe are classified as surface-accessible atoms. The surface accessibility of each amino acid is calculated as A 2 Next, the actual exposed surface area (Å 2 ) and theoretical possible surface exposure (Å 2) is calculated. Depending on the desired accuracy and the size of the protein, different cutoffs can be selected. Such cutoffs can be selected from 0.5, 0.2, preferably such cutoffs are 0.05-0.1, more preferably such cutoffs are 0.07. Such a filtration step is useful to eliminate potentially misfolded proteins.

[0124] Further steps can be performed to reduce the amount of misfolded TREM1 protein in the final array. For example, residues that cause the breaking of more than one hydrogen bond between any of the original residues (2 or 3 residues) of each mutation patch and the rest of the protein are preferably avoided. Similarly, the breaking of salt bridges should also be preferably avoided. Furthermore, mutated residues that expose large hydrophobic regions of the protein are also avoided. In another embodiment, residues that cause the breaking of more than two hydrogen bonds in the protein are also avoided. Similarly, the breaking of salt bridges should also be preferably avoided.

[0125] Thus, in a preferred embodiment of the method, the method excludes or filters out 1) patches that result in hydrogen bond breaking (preferably a maximum of 2 broken bonds are allowed), and 2) salt bridges (preferably a maximum of 1 broken bond is allowed), and 3) exposure of large hydrophobic patches (preferably a maximum of 15 Å2 exposed hydrophobic surface is allowed). The distance threshold for defining patches can be set between 6 Å and 6.5 Å, and the minimum side chain surface exposure can be set at 7%.

[0126] Optionally, further granularity can be achieved by performing molecular dynamics simulations with any widely used simulation package (e.g., AMBER, GROMACS, DESMOND, etc.) with subsequent analysis of interaction persistence. Hydrogen bonds and salt bridges present in most of the simulation trajectories can be considered "essential" and should not be disrupted by Ala mutations, whereas bonds observed in only a small portion of the simulations are likely to have little effect on protein stability.

[0127] Furthermore, after all patches of residues have been identified, such redundancies are eliminated by eliminating patches that generate redundancies. This step is optional since it may be beneficial to have some redundancy in the coverage of accessible surface areas, but having such redundancy may lead to technical difficulties in subsequently generating mutant clones. Therefore, such redundancy should be considered in the context of protein size, complexity and technical limitations in designing the corresponding mutant proteins.

[0128] Ideally, the above steps are performed on the entire protein surface to ensure that the maximum surface-accessible area is covered by the identified patches. It would be preferable to avoid having some portion of the surface-accessible area not covered by such patches. The aim is to cover the solvent-accessible surface while minimizing the number of misfolded proteins generated.

[0129] For example, if a patch of two substitutions is used but does not cover the entire surface accessible region, an additional patch of three substitutions can be designed. Larger patches of more than three substitutions can also be used, but more than three substitutions may lead to misfolding of mutant TREM1 protein. Therefore, preferably, a patch containing two or three Ala substitutions is used. If necessary, an additional single Ala substitution can also be selected. However, this may not provide the desired sensitivity compared to two or three substitutions.

[0130] Arrays of mutant TREM1 proteins with two or three Ala patches following this strategy are provided as examples.

[0131] The resulting sequence of mutant TREM1 protein is then generated for experimental testing. The typical method of generating such is by cloning the sequence into a suitable expression vector. As a control, the wild-type sequence of the target protein of interest is also cloned.

[0132] The array of mutant TREM1 proteins can be produced using techniques known to those skilled in the art. Any suitable expression system for expressing proteins in target cells can be used. Preferably, a mammalian cell system is used for the expression of cloned mutant peptides. Mammalian cells allow mutant polypeptides to be secreted from such cells, making such peptides easier to test. Any mammalian cell or cell line can be used as long as it allows sufficient expression of each mutant peptide. In such mammalian systems, a suitable expression vector can be used. Many mammalian expression vectors are commercially available. Typically, such vectors will include a constitutive promoter, such as the cytomegalovirus (CMV) promoter.

[0133] Each of the mutant TREM1 proteins could be fused to an Fc region, preferably a human Fc domain. The use of the Fc domain in such fusion proteins offers practical advantages such as greater robustness in detection and ease of capturing such fusion proteins on surfaces. If necessary, one or more linker sequences can be introduced into the fusion protein sequence between the Fc domain and the target mutant protein, for example a triple Ala linker.

[0134] Preferably, such fusion proteins containing a human Fc domain are expressed in mammalian Expi293 cells, or any other cell capable of producing sufficient concentrations of the protein.

[0135] Optionally, potentially misfolded TREM1 proteins can be removed from the array by pre-screening the array with a polyclonal antibody against TREM1 (targeting multiple epitopes) or any commercially available monoclonal antibody of a known epitope suitable for an ELISA assay (such an antibody recognizes a structural epitope).

[0136] Finally, the binding properties of the antibody to each mutant target protein on the array are measured. Such measurements can be performed using any suitable method available. Preferably, such measurements are performed using high-throughput methods.

[0137] The affinity of a molecule of interest, as well as the degree to which such a molecule inhibits binding to a target protein, can be determined by those skilled in the art using conventional techniques, such as those described by Scatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949)) or by surface plasmon resonance (SPR) using a system such as BIAcore. For surface plasmon resonance, the mutant protein is immobilized on a solid phase and exposed to a ligand and / or a molecule of interest in a mobile phase along a flow cell. Binding of the ligand to the immobilized target changes the local refractive index, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to obtain the apparent rate constants of the association and dissociation phases of the binding reaction. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al, Cancer Res. 53:2560-65 (1993)).

[0138] Alternative platforms that use technology similar to SPR are offered by Cartera (carterra-bio.com), such as the Carterra LSA Platform, a high-throughput antibody characterization platform that combines flow-printed microfluidics with high-throughput surface plasmon resonance (SPR) detection technology.

[0139] Other types of platforms include technologies that utilize cell surface expression arrays. An example of such a platform is LigandTracer (ligandtracer.com), which is particularly suitable for tracking protein binding to cell surface receptors, allowing on- and off-rates and affinities to be measured.

[0140] For ease of measurement, each mutant protein in the array can be fused to a molecule or protein and captured on a surface for easy detection of binding properties.

[0141] Preferably, binding to each mutant protein is determined using the label-free technique Biolayer Interferometry (BLI), an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized proteins on a biosensor chip and an internal reference layer. Changes in the number of molecules bound to the biosensor chip result in a shift in the interference pattern that can be measured in real time (REF).

[0142] Typically, an array of 30 to 60 cloned mutant proteins is used. However, the size of such an array depends on the size of the target protein and the desired coverage of the solvent-accessible area. Preferably, the mutant proteins are provided on a 96-well plate or a 384-well plate. Generally, BLI instruments can handle 96-well plates or 384-well plates for measurement.

[0143] When using BLI technology, typically each sensor is exposed to a solution containing a molecule of interest (such as an antibody or ligand) whose binding site has been determined. The advantages of BLI technology are that it is almost as sensitive as a regular BIACore, has high throughput (96 clones can be tested simultaneously), and uses disposable sensor chips, so there is no need to regenerate the surface and reuse the chip as is usually done with BIACore.

[0144] Different measurements of the binding of the test antibody to the mutant TREM1 protein can be used to determine which mutant proteins show reduced binding. Typically, a dissociation constant or binding constant is measured. Typically, a complete loss of binding or how quickly the molecule of interest leaves the mutant protein can be measured. When measuring the binding properties of an antibody, appropriate controls are generally used. Generally, the binding properties are compared to the parent sequence of the target protein (wild type, WT). Typically, the majority of mutant proteins have the same K dMutant proteins showing differences in binding should be considered. Typically, any dissociation constant difference of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more compared to wild type TREM1 is considered. Preferably, any difference of at least 3-fold is considered significant. Mutant TREM1 proteins resulting in low noise to signal resolution are ignored or remeasured.

[0145] If desired, mutant proteins containing patches of different sizes can be used on the array, for example patches of two or three substitutions. Mutant proteins containing single substitutions can also be further tested for binding properties if greater precision is required, as long as they provide sufficient sensitivity to obtain a measurable effect.

[0146] Antibody that binds to TREM1 The present invention provides anti-TREM1 antibodies that bind to human TREM1 (target polypeptide) and have the functional and structural properties further described herein.

[0147] Antibodies in the context of the present invention include whole antibodies and functionally active antibody fragments, i.e., molecules that contain an antigen-binding domain that specifically binds to an antigen (also called antigen-binding fragments). The characteristics described herein also apply to antibody fragments, unless the context dictates otherwise. Antibodies can be (or can be derived from) polyclonal, monoclonal, multivalent, multispecific, bispecific, fully human, humanized, or chimeric.

[0148] Furthermore, the antibodies described are specific antibody types and do not limit the scope of the invention.

[0149] The antibodies used according to the present invention may be monoclonal or polyclonal, preferably monoclonal. The antibodies used according to the present invention may be chimeric, CDR-grafted (any suitable receptor variable region framework sequence may be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including, for example, mouse, primate and human framework regions), nanobody, human or humanized antibodies. For the production of both monoclonal and polyclonal antibodies, the animals used to produce such antibodies are typically non-human mammals, such as goats, rabbits, rats or mice, although antibodies may also be produced in other species.

[0150] Polyclonal antibodies may be produced by routine methods such as immunization of a suitable animal with an antigen of interest. Blood can then be removed from such animals and the antibodies produced can be purified.

[0151] Monoclonal antibodies can be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Several exemplary methods for making monoclonal antibodies are described herein.

[0152] For example, monoclonal antibodies can be prepared using hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0153] Antibodies can also be produced using single lymphocyte antibody techniques by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected for the production of specific antibodies, for example, by the methods described in WO9202551, WO2004051268 and WO2004106377.

[0154] Antibodies generated against a target polypeptide can be obtained by administering the polypeptide to an animal, preferably a non-human animal, using well-known conventional protocols, if immunization of the animal is required (see, for example, Handbook of Experimental Immunology, DM Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many animals can be immunized, such as rabbits, mice, rats, sheep, cattle, camels, or pigs. However, mice, rabbits, pigs, and rats are commonly used.

[0155] Monoclonal antibodies can also be generated using various phage display methods known in the art, including those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182:41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280). In a particular phage display method, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) or randomly recombined in a phage library and screened for antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol, 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227:381-388 (1992).Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and U.S. Pat. No. 2005 / 0079574, U.S. Pat. No. 2005 / 0119455, U.S. Pat. No. 2005 / 0266000, U.S. Pat. No. 2007 / 0117126, U.S. Pat. No. 2007 / 0160598, U.S. Pat. No. 2007 / 0237764, U.S. Pat. No. 2007 / 0292936, and U.S. Pat. No. 2009 / 0002360.

[0156] Antibodies can be screened using assays that measure binding to target polypeptides and / or that measure the ability of antibodies to block specific interactions. An example of a binding assay is, for example, an ELISA that uses a fusion protein of the target polypeptide immobilized on a plate and uses a conjugated secondary antibody to detect the antibody bound to the target. An example of a blocking assay is a flow cytometry-based assay that measures blocking of ligand protein binding to the target polypeptide. A fluorescently labeled secondary antibody is used to detect the amount of such ligand protein that binds to the target polypeptide.

[0157] Antibodies can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics.

[0158] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments.

[0159] The antibody may be a full-length antibody. More specifically, the antibody may be of the IgG isotype. More specifically, the antibody may be an IgG1 or IgG4.

[0160] The constant region domain of the antibody may be selected taking into account the proposed function of the antibody molecule, if any, particularly the effector function that may be required. For example, the constant region domain may be a human IgA, IgD, IgE, IgG or IgM domain. In particular, when the antibody molecule is intended for therapeutic use and antibody effector function is required, human IgG constant region domains, particularly IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, IgG2 and IgG4 isotypes may be used. It will be understood that sequence variants of these constant region domains may also be used. It will also be known to those skilled in the art that antibodies may undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody as well as the cell culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) by the action of carboxypeptidases (described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Thus, the C-terminal lysine of the antibody heavy chain may be absent.

[0161] Alternatively, the antibody is an antigen-binding fragment.

[0162] For a review of specific antigen-binding fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), and see WO 93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458. Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives are disclosed in U.S. Patent No. 5,869,046.

[0163] Antigen-binding fragments and methods for making them are well known in the art, see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181; Adair and Lawson, 2005. Therapeutic antibodies. Drug Design Reviews-Online 2(3):209-217. The Fab-Fv format was first disclosed in WO 2009 / 040562, its disulfide-stabilized version, Fab-dsFv, first disclosed in WO 2010 / 035012, and the TrYbe format in WO 2015 / 197772.

[0164] Various techniques have been developed to produce antibody fragments. Such fragments can be derived through proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117(1992) and Brennan et al, Science 229:81(1985)). However, antibody fragments can also be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167(1992)).

[0165] F(ab')2 fragments can be directly isolated from recombinant host cell culture. The antibody can be a single chain Fv fragment (scFv), as described in WO 93 / 16185; U.S. Pat. No. 5,571,894; U.S. Pat. No. 5,587,458. The antibody fragment can also be a "linear antibody", as described, for example, in U.S. Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

[0166] The antibody may be a Fab, Fab', F(ab')2, Fv, dsFv, scFv, or dsscFv. The antibody may be a single domain antibody or nanobody, such as a VH or VL or VHH or VNAR. The antibody may be a Fab or Fab' fragment as described in WO2011 / 117648, WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171.

[0167] The antibody may be a disulfide-stabilized single-chain variable fragment (dsscFv).

[0168] The disulfide bond between the variable domains VH and VL may be between two of the residues listed below. ·V H 37+V L 95, e.g., Protein Science 6, 781-788 Zhu et al (1997); ·V H 44+V L 100, see, e.g., Weatherill et al., Protein Engineering, Design & Selection, 25(321-329), 2012; ·V H 44+V L 105, e.g., J Biochem.118,825-831 Luo et al(1995); ·V H 45+V L 87, e.g., Protein Science 6, 781-788 Zhu et al (1997); ·V H 55+V L 101, see e.g. FEBS Letters 377 135-139 Young et al (1995); ·V H 100+V L 50, see for example Biochemistry 29 1362-1367 Glockshuber et al (1990); ·V H 100b+V L 49, see for example Biochemistry 29 1362-1367 Glockshuber et al (1990); ·V H 98+V L 46, see for example Protein Science 6, 781-788 Zhu et al (1997); ·V H 101+V L 46, see for example Protein Science 6, 781-788 Zhu et al (1997); ·V H105+V L 43, see, for example, Proc. Natl. Acad. Sci. USA Vol. 90 pp. 7538-7542 Brinkmann et al. (1993); or Proteins 19, 35-47 Jung et al. (1994); ·V H 106+V L 57, e.g. FEBS Letters 377 135-139 Young et al (1995) and the corresponding position or positions in the variable region pair located within the molecule.

[0169] A disulfide bond can be formed between positions VH44 and VL100.

[0170] The antigen-binding fragments described herein may also be characterized as monoclonal, chimeric, humanized, fully human, multispecific, bispecific, etc., and it will be understood by those of skill in the art that the discussion of these terms also relates to such fragments.

[0171] multispecific antibodies The antibody of the present invention may be a multispecific antibody.

[0172] Examples of multispecific antibodies or antigen-binding fragments thereof that are also contemplated for use in the context of the present disclosure include bivalent, trivalent or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, bibodies and tribodies (see, e.g., Holliger and Hudson, 2005, Nature Biotech 23(9):1126-1136; Schoonjans et al. 2001, Biomolecular Engineering, 17(6), 193-202).

[0173] A variety of multispecific antibody formats have been produced. Although various classifications have been proposed, multispecific IgG antibody formats generally include bispecific IgG, adducted IgG, multispecific (e.g., bispecific) antibody fragments, multispecific (e.g., bispecific) fusion proteins, and multispecific (e.g., bispecific) antibody conjugates, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106. Mol Immunol. 67(2015):95-106.

[0174] The antibody can be a bispecific antibody.In one aspect, the antibody comprises two antigen-binding domains, one binding domain binds to TREM1, and the other binding domain binds to another antigen, i.e., each binding domain is monovalent for each antigen.In one embodiment, the antibody is a tetravalent bispecific antibody, i.e., the antibody comprises four antigen-binding domains, for example, two binding domains bind to TREM1, and the other two binding domains bind to another antigen.In one embodiment, the antibody is a trivalent bispecific antibody.

[0175] Techniques for producing bispecific antibodies include, but are not limited to, CrossMab technology (Klein et al. Engineering therapeutic bispecific antibodies using CrossMab technology, Methods 154 (2019) 21-31), Knobs-in-holes engineering (e.g., WO 1996027011, WO 1998050431), DuoBody technology (e.g., WO 2011131746), Azymetric technology (e.g., WO 2012058768). Further techniques for producing bispecific antibodies are described, for example, in Godar et al., 2018, Therapeutic bispecific antibody formats: a patent applications review (1994-2017), Expert Opinion on Therapeutic Patents, 28: 3, 251-276. Bispecific antibodies include CrossMab antibodies, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-lgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body and orthogonal Fab, among others.

[0176] The antibody construct may be a trispecific antibody.

[0177] The antibody may be a multiparatopic antibody.

[0178] In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises no more than one VH and one VL.

[0179] Loaded IgGs classically comprise full-length IgGs that have been engineered by adding additional antigen-binding domains or antigen-binding fragments to the N-terminus and / or C-terminus of the IgG heavy and / or light chains. Examples of such additional antigen-binding fragments include sdAb antibodies (e.g. VH or VL), Fv, scFv, dsscFv, Fab, scFab and additional IgG antibody formats, in particular DVD-IgG, IgG(H)-scFv, scFv-(H)lgG, IgG(L)-scFv, scFv-(L)IgG, lgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgC(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, lgG-2scFv, scFv4-Ig, Zybody and DVI-IgG (four-in-one), as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106.

[0180] Multispecific antibody fragments include nanobodies, nanobody-HSA, BiTE, diabodies, DART, TandAb, scDiabody, sc-Diabody-CH3, Diabody-CH3, Triple Body, Miniantibody, minibody, Tri Bi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc; and intrabody, as described, for example, in Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015):95-106. Mol Immunol. 67(2015):95-106.

[0181] Multispecific fusion proteins include Dock and Lock, ImmTAC, HSAbody, scDiabody-HSA, and Tandem scFv-Toxin.

[0182] Multispecific antibody conjugates include IgG-IgG; Cov-X-body; and scFv1-PEG-scFv2.

[0183] Further multispecific antibody formats are described, for example, in Brinkmann et al, The making of bispecific antibodies, mAbs, 9:2, 182-212 (2017), in particular Figure 2, e.g. tandem scFv, triplexes, Fab-VHH, taFv-Fc, scFv4-Ig, scFv2-Fcab, scFv4-IgG. Biobodies, tribodies and methods for producing them are disclosed, for example, in WO 99 / 37791.

[0184] The antibody for use in the present invention may be a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g. one scFv or dsscFv binding to a therapeutic target and one scFv or dsscFv extending half-life, e.g. by binding to albumin). Such antibody fragments are described in WO 2015 / 197772. Another preferred antibody for use in the present invention fragment comprises a Fab linked to only one scFv or dsscFv, e.g. as described in WO 2013 / 068571 and Dave et al., Mabs, 8(7)1319-1335 (2016).

[0185] Another antibody for use in the present invention is the knobs-in-holes antibody ("KiH"). This is a multispecific antibody format consisting of heavy chain homodimers for heterodimerization (e.g., for efficient production of bispecific, multispecific or mono-armed antibodies). In general, such techniques involve introducing a protrusion ("knob") at the interface of a first polypeptide (such as the first CH3 domain in the first antibody heavy chain) and a corresponding cavity ("hole") at the interface of a second polypeptide (such as the second CH3 domain in the second antibody heavy chain), such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide (such as the first CH3 domain in the first antibody heavy chain) with a larger side chain (e.g., arginine, phenylalanine, tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created at the interface of the second polypeptide (such as the second CH3 domain in the second antibody heavy chain) by replacing large amino acid side chains with smaller ones (e.g., alanine, serine, valine, or threonine). The protuberances and cavities can be made by modifying the nucleic acid encoding the polypeptides, for example, by site-directed mutagenesis or peptide synthesis. Further details regarding "knobs-in-holes" technology are described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; WO 2009 / 089004; U.S. Patent Application Publication No. 2009 / 0182127; Marvin md Zu, Acta Pharmacologica Sincia (2005) 26(6):649-658; Kontermann Acta Pharmacologica Sincia (2005) 26:1-9; Ridgway et al, Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

[0186] Humanized, human and chimeric antibodies and methods for producing such antibodies The antibodies of the present invention can be, but are not limited to, humanized antibodies, fully human antibodies, or chimeric antibodies.

[0187] In one embodiment, the antibody is humanized. More particularly, the antibody is a chimeric, human, or humanized antibody.

[0188] In certain embodiments, the antibody provided herein is a chimeric antibody. Examples of chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0189] In one embodiment, the antibody is a humanized antibody.

[0190] Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs are derived. It will be appreciated that only the specificity determining residues of the CDRs need to be transferred rather than the entire CDRs (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34).

[0191] Suitably, humanised antibodies according to the invention have variable domains which comprise human acceptor framework regions as well as one or more CDRs and optionally further comprise one or more donor framework residues.

[0192] Thus, in one embodiment, a humanized antibody is provided in which the variable domains comprise human acceptor framework regions and non-human donor CDRs.

[0193] When CDRs or specificity determining residues are grafted, any suitable receptor variable region framework sequence may be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including murine, primate and human framework regions.

[0194] Examples of human frameworks that may be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al.). For example, KOL and NEWM may be used for the heavy chain, REI may be used for the light chain, and EU, LAY and POM may be used for both the heavy and light chains. Alternatively, human germline sequences may be used, which are available at www.imgt.org. In an embodiment, the acceptor framework is IGKV1-9 human germline and / or IGHV3-66 human germline. In an embodiment, the human framework comprises 1-5, 1-4, 1-3 or 1-2 donor antibody amino acid residues.

[0195] In the humanized antibodies of the invention, the receptor heavy and light chains do not necessarily have to be derived from the same antibody, but may, if desired, comprise composite chains having framework regions derived from different chains.

[0196] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art.

[0197] A human antibody includes a heavy or light chain variable region or a full-length heavy or light chain that is the "product" or "derived" from a particular germline sequence, if the variable region or full-length chain of the antibody is obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest, or screening a human immunoglobulin gene library displayed on a phage with the antigen of interest. A human antibody or fragment thereof that is the "product" or "derived" from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to that of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the sequence of the human antibody (i.e., maximum % identity). A human antibody that is the "product" or "derived" from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or deliberate introduction of site-specific mutations. However, the selected human antibody will typically be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene when compared to the germline immunoglobulin amino acid sequence of another species (e.g., mouse germline sequence) and will contain amino acid residues that identify the human antibody as human. In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0198] Structural features of antibodies The antibody of the present invention comprises a binding domain. The binding domain generally comprises six CDRs, three from the heavy chain and three from the light chain. In one embodiment, the CDRs are within a framework and together form a variable region. Thus, an antibody has a binding domain specific for an antigen, the binding domain comprising a light chain variable region and a heavy chain variable region.

[0199] In one embodiment, the antibody comprises a heavy chain and a light chain, the heavy chain comprising a CH1 domain and the light chain comprising a CL domain, either kappa or lambda.

[0200] As demonstrated by the examples of the present invention, different variants of the variable regions of the heavy and light chains were generated and tested for their binding affinity. These variants contain the same set of CDR sequences and show a similar range of binding affinities. An overview of the different structural elements of the selected antibody variants is shown in Table 3.

[0201] Table 3. Amino acid sequences of anti-TREM1 antibodies [Table 3]

[0202] In one embodiment, the invention provides an antibody that binds to human TREM1, CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 The present invention provides an antibody comprising a light chain variable domain comprising at least one of:

[0203] In one embodiment, the invention provides an antibody that binds to human TREM1, CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 The present invention provides an antibody comprising a light chain variable domain comprising:

[0204] In one embodiment, the invention provides an antibody that binds to human TREM1, CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 The present invention provides an antibody comprising a heavy chain variable domain comprising at least one of:

[0205] In one embodiment, the invention provides an antibody that binds to human TREM1, CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 The present invention provides an antibody comprising a heavy chain variable domain comprising:

[0206] The antibody molecules of the present invention may each comprise a complementary light chain or a complementary heavy chain.

[0207] Thus, in one embodiment, the invention provides an antibody that binds to human TREM1, CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 and a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 and a heavy chain variable region comprising The present invention provides an antibody comprising:

[0208] In one embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence shown in SEQ ID NO:29 or SEQ ID NO:33.

[0209] In one embodiment, an antibody of the invention comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO:57 or SEQ ID NO:79.

[0210] In one embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:33 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:57.

[0211] In an alternative embodiment, an antibody of the invention comprises a light chain variable region comprising the sequence set forth in SEQ ID NO:29 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:79.

[0212] In one embodiment, the antibody of the invention comprises: CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 and a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 and a heavy chain variable region comprising The antibody is a full-length antibody comprising:

[0213] In one embodiment, the antibody of the invention comprises: CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 and a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 and a heavy chain variable region comprising and IgG1 LALA, including

[0214] In another embodiment, the antibody of the invention is an IgG1 LALA comprising a light chain comprising the sequence set forth in SEQ ID NO:35 and a heavy chain comprising the sequence set forth in SEQ ID NO:65.

[0215] In another embodiment, an antibody of the invention is an IgG1 LALA comprising a light chain comprising the sequence set forth in SEQ ID NO:31 and a heavy chain comprising the sequence set forth in SEQ ID NO:87.

[0216] In another embodiment, IgG4P is preferred. Several variants of the 12172 antibody described herein have been tested in multiple assays to determine their physicochemical properties, and they all showed a very similar development profile with the IgG4P variant having less favorable properties than the other variants. However, the IgG4P variant exhibits surprising biological properties not observed in the other variants, and is therefore a preferred variant for applications where such properties are beneficial. For example, in the treatment of conditions where such properties provide a therapeutic effect.

[0217] IgG4P contains a Ser-228-Pro mutation in the hinge region to improve hinge stability, numbering according to EU numbering (Ser-241-Pro according to Kabat numbering) (Angal S et al, (1993), Mol Immunol, 30(1), 105-108).

[0218] Thus, in one embodiment, the antibody of the invention comprises: CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 and a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 and a heavy chain variable region comprising and IgG4P, including

[0219] In yet another embodiment, the antibody of the invention is an IgG4P comprising a light chain comprising the sequence set forth in SEQ ID NO:35 and a heavy chain comprising the sequence set forth in SEQ ID NO:59.

[0220] In a more specific embodiment, the antibody of the invention is an IgG4P comprising a light chain comprising the sequence set forth in SEQ ID NO:31 and a heavy chain comprising the sequence set forth in SEQ ID NO:81.

[0221] Functional characterization of anti-TREM1 antibodies In one embodiment, the antibody of the invention is a neutralizing antibody. Preferably, the antibody according to the invention neutralizes one or more TREM1 activities.

[0222] The antibodies of the present invention specifically bind to human TREM1, more specifically to a specific region within the extracellular domain of human TREM1. In some embodiments, the antibodies specifically bind to distinct or minimally overlapping sites on TREM1 to which TREM1 ligands (e.g., PGLYRP1) bind. In some embodiments, the antibodies are antagonistic antibodies, i.e., they inhibit or suppress the activity of TREM1 on cells. Such cells may be monocytes, macrophages, and / or neutrophils. In some embodiments, the antibodies specifically bind to TREM1 allosterically rather than orthosterically to a single ligand, and thus may provide more effective inhibition of binding of other ligands that bind to sites on TREM1 that are different from PGLYRP1.

[0223] As demonstrated by the examples, PGLYRP1 binds to an epitope on TREM1, the epitope comprising residues selected from the list consisting of E27, D42-E46, A49, Y90-L95 and F126 of human TREM1 (SEQ ID NO: 1) as measured with a contact distance of less than 4 Å.

[0224] In one particular embodiment, the invention provides antibodies that bind to a region on TREM1 distinct from the binding site of PGLYRP1, such that binding still prevents the interaction between TREM1 and PGLYRP1.

[0225] In some embodiments, the anti-TREM1 antibody exhibits very weak binding to cynomolgus monkey TREM1, hi some embodiments, the anti-TREM1 antibody exhibits no detectable binding to mouse, rat, pig or dog TREM1.

[0226] In some embodiments, the anti-TREM1 antibodies reduce the release of multiple cytokines and chemokines from activated human monocytes, such as CCL-3, CCL-20, CXCL-9, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-6, IL-10, IL-12p40, IL-15, IL-18, IL-27, TNF-α, and TNF-β.

[0227] In some embodiments, the anti-TREM1 antibody is IgG4P and significantly increases the release of IL-1R antagonist (IL-1RA), an anti-inflammatory negative regulator of the IL-1 pathway, from primary human monocytes.

[0228] The antibody according to the invention is specific for human TREM1.

[0229] In some embodiments, the antibody binds to human TREM1 with sufficient affinity and specificity. In particular embodiments, the antibody binds to human TREM1 with a KD of about any one of 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, lnM, 0.5 nM, including any range between these values. In one embodiment, the antibody according to the invention binds to human TREM1 with a KD of less than 600 pM. In a more specific embodiment, the antibody according to the invention binds to human TREM1 with a KD of 300-1200 pM, more preferably 300-600 pM.

[0230] The affinity of an antibody, as well as the extent to which it inhibits binding, can be determined by one of skill in the art using conventional techniques, such as those described in Scatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949) or by surface plasmon resonance (SPR) using a system such as BIAcore. In surface plasmon resonance, the target molecule is immobilized on a solid phase and exposed to a ligand in a mobile phase that moves along a flow cell. Binding of the ligand to the immobilized target changes the local refractive index, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in the intensity of the reflected light. The rate of change of the SPR signal can be analyzed to obtain apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these values ​​gives the apparent equilibrium constant (affinity) (see, e.g., Wolff et al, Cancer Res. 53:2560-65 (1993)).

[0231] Preferably, the antibody according to the invention is specific for human TREM1.

[0232] The disclosure herein regarding antibodies, particularly with regard to binding affinity and specificity, and activity, is also applicable to antigen-binding fragments and antibody-like molecules.

[0233] Antibodies that bind to the same epitope The antibodies may compete with or bind to the same epitope as defined above for the light chain, heavy chain, light chain variable region (LCVR), heavy chain variable region (HCVR) or CDR sequences for binding to TREM1.

[0234] In particular, the invention provides an antibody that competes for binding to TREM1 with, or binds to the same epitope as, an antibody comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NOs: 11 / 12 / 13 / 14 / 15 / 16. The antibody may compete for binding to TREM1 with, or bind to the same epitope as, an antibody comprising the LCVR and HCVR sequence pair of SEQ ID NOs: 29 / 79. The antibody may compete for binding to TREM1 with, or bind to the same epitope as, an IgG4P comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequence combination of SEQ ID NOs: 11 / 12 / 13 / 14 / 15 / 16.

[0235] In some embodiments, the anti-TREM1 antibody binds to an epitope on human TREM1, the epitope comprising residues E26, E27, K28, Y29, E30, L31, K32 and Q35, where numbering is according to SEQ ID NO: 1. Such epitopes can be determined using methods disclosed herein, including designing an array of mutant TREM1 proteins and measuring binding of the antibody to mutant TREM1 proteins that contain two or three of the residues mutated to smaller amino acids, such as Ala.

[0236] In one embodiment, the invention provides an IgG4P antibody that binds to an epitope of human TREM1, wherein the epitope comprises residues E26, E27, K28, Y29, E30, L31, K32 and Q35 of human TREM1 (SEQ ID NO:1).

[0237] In one embodiment, the invention provides an anti-TREM1 antibody that binds to an epitope on TREM1, wherein the epitope includes at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all of residues selected from the list consisting of E26, E27, K28, Y29, E30, L31, K32, Q35, T36, D38, K40, D42, R97, D127, T134 and G136 of human TREM1 (SEQ ID NO: 1) as measured with a contact distance of less than 4 Å.

[0238] In one embodiment, the invention provides an IgG4P antibody that binds to an epitope of human TREM1, wherein the epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or all of residues selected from the list consisting of E26, E27, K28, Y29, E30, L31, K32, Q35, T36, D38, K40, D42, R97, D127, T134 and G136 of human TREM1 (SEQ ID NO: 1) as measured with a contact distance of less than 4 Å.

[0239] In some embodiments, the invention provides anti-TREM1 antibodies that bind to an epitope distinct from PGLYRP1. In some embodiments, the invention provides anti-TREM1 antibodies that bind to an epitope on TREM1, the epitope comprising no more than one or two residues selected from the list consisting of E27, D42-E46, A49, Y90-L95 and F126 of human TREM1 (SEQ ID NO:1) as measured with a contact distance of less than 4 Å.

[0240] The epitope can be identified by any suitable binding site mapping method known in the art in combination with any one of the antibodies provided by the present invention. A particular method is provided by the present disclosure that relies on an array of mutant TREM1 proteins to establish which of the mutant residues are important for binding of a particular antibody. Using such a method, it is possible to identify an antibody that binds to essentially the same amino acid residues as the antibody provided by the present invention. Other examples of epitope mapping methods include screening peptides of various lengths derived from the full-length target protein for binding to the antibody of the present invention or a fragment thereof, and identifying a fragment that can specifically bind to the antibody that contains the sequence of the epitope recognized by the antibody. The target peptide can be synthetically produced. The peptide that binds to the antibody can be identified, for example, by mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by the antibody of the present invention. Typically, when epitope determination is performed by X-ray crystallography, the amino acid residues of the antigen within 4 Å of the CDR are considered to be the amino acid residues part of the epitope. Once identified, an epitope can be useful for preparing fragments that bind to the antibodies of the invention and, if desired, can be used as an immunogen to obtain additional antibodies that bind to the same epitope.

[0241] In one embodiment, the epitope of the antibody is determined by x-ray crystallography.

[0242] Whether an antibody binds to the same epitope as a reference antibody or competes for binding with a reference antibody can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody of the present invention, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the present invention, or the reference antibody causes a conformational change in the antigen, thus preventing the binding of the test antibody.

[0243] To determine whether an antibody competes for binding with a reference antibody, the above binding methodology is carried out in two different experimental settings. In the first setting, the reference antibody is bound to the antigen under saturating conditions, and then the binding of the test antibody to the antigen is evaluated. In the second setting, the test antibody is bound to the antigen under saturating conditions, and then the binding of the reference antibody to the protein / peptide is evaluated. In both experimental settings, if only the first (saturating) antibody can bind to the protein / peptide, it is concluded that the test antibody and the reference antibody compete for binding to the antigen. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope, or may cause a conformational change that results in a lack of binding.

[0244] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0245] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same part of the antigen as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0246] Antibody variants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions, insertions and / or deletions. Sites of interest for substitution mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest and screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0247] In certain embodiments, amino acid sequence variants of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the anti-TREM1 antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the anti-TREM1 antibody (e.g., within one or more CDR and / or framework sequences, or within the VH and / or VL domains). Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired properties.

[0248] In certain embodiments of the variant VH and VL sequences provided herein, each HVR is unaltered or contains no more than one, two or three amino acid substitutions.

[0249] It will be understood that one or more amino acid substitutions, additions and / or deletions may be made to the CDRs provided by the invention without significantly altering the ability of the antibody to bind to TREM1 and neutralize TREM1 activity. The effect of any amino acid substitution, addition and / or deletion can be readily tested by one skilled in the art, for example by determining inhibition of TREM1 binding and TREM1 interaction with its natural ligand, using the methods described herein, particularly those exemplified in the examples.

[0250] As a result, in certain embodiments of the variant VH and VL sequences, each CDR contains no more than one, two or three amino acid substitutions, where such amino acid substitutions are conservative and the antibody retains its binding properties to TREM1.

[0251] Thus, the present invention provides an anti-TREM1 antibody comprising one or more CDRs selected from CDR-L1 (including SEQ ID NO: 11), CDR-L2 (including SEQ ID NO: 12), CDR-L3 (including SEQ ID NO: 13), CDR-H1 (including SEQ ID NO: 14), CDR-H2 (including SEQ ID NO: 15) and CDR-H3 (including SEQ ID NO: 16), wherein one or more amino acids in one or more CDRs are replaced with another amino acid, e.g. a similar amino acid as defined herein below.

[0252] In one embodiment, the invention provides an anti-TREM1 antibody comprising CDR-L1 (comprising SEQ ID NO: 11), CDR-L2 (comprising SEQ ID NO: 12), CDR-L3 (comprising SEQ ID NO: 13), CDR-H1 (comprising SEQ ID NO: 14), CDR-H2 (comprising SEQ ID NO: 15) and CDR-H3 (comprising SEQ ID NO: 16), wherein, for example, one or more amino acids in one or more CDRs are replaced with another amino acid, such as a similar amino acid as defined herein below.

[0253] In one embodiment, the invention provides an anti-TREM1 antibody CDR-L2 (comprising SEQ ID NO: 12), in which the first amino acid of SEQ ID NO: 12 is replaced with another amino acid. More particularly, K is replaced with S.

[0254] In one embodiment, an anti-TREM1 antibody of the invention comprises a light chain variable domain comprising three CDRs, wherein the sequence of CDR-L1 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 11, CDR-L2 comprises a sequence having at least 70%, 80%, 90%, 95%, or 98% identity or similarity to the sequence set forth in SEQ ID NO: 12, and / or CDR-L3 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 13.

[0255] In one embodiment, an anti-TREM1 antibody of the invention comprises a heavy chain variable domain comprising three CDRs, wherein the sequence of CDR-H1 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 14, CDR-H2 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 15, and / or CDR-H3 comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 16.

[0256] In one embodiment, an anti-TREM1 antibody of the invention comprises a light chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:29.

[0257] In one embodiment, an antibody of the invention comprises a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO:79.

[0258] In one embodiment, an anti-TREM1 antibody of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to that set forth in SEQ ID NO:29, and / or the heavy chain variable region comprises a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to that set forth in SEQ ID NO:79.

[0259] In one embodiment, the anti-TREM1 antibody of the invention comprises CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences comprising SEQ ID NOs: 11 / 12 / 13 / 14 / 15 / 16, respectively, and the remainder of the light chain variable region and heavy chain variable region have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to SEQ ID NOs: 29 and 79, respectively.

[0260] In one embodiment, the anti-TREM1 antibody of the invention is an IgG4P comprising a light chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 31, and a heavy chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to the sequence set forth in SEQ ID NO: 81.

[0261] In one embodiment, the anti-TREM1 antibody of the invention is an IgG4P comprising the CDR-L1 / CDR-L2 / CDR-L3 / CDR-H1 / CDR-H2 / CDR-H3 sequences as shown in SEQ ID NOs: 11 / 12 / 13 / 14 / 15 / 16, respectively, and the remainder of the light and heavy chains having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to SEQ ID NOs: 31 and 81, respectively.

[0262] In one embodiment, an antibody of the invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:29, in which one or more residues at positions 1, 2, 3, 18 and 50 are substituted by another amino acid, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO:79, in which one or more residues at positions 23, 48, 49, 71, 73, 75 and 78 are substituted by another amino acid.

[0263] Sequence Identity and Similarity The degree of identity and similarity between sequences can be easily calculated. "Sequence identity %" (or "sequence similarity %") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.); (2) determining the number of positions that contain identical (or similar) amino acids (e.g., identical amino acids are present in both sequences, similar amino acids are present in both sequences) to obtain the number of matched positions; (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to obtain the sequence identity % or sequence similarity %.

[0264] The method of aligning sequences for comparison is well known in the art. The optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0265] Preferred examples of algorithms suitable for determining percent sequence identity and percent sequence similarity include the BLAST and BLAST 2.0 algorithms described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). Polypeptide sequences can also be compared using FASTA using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provide alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.

[0266] In certain embodiments, substitutions, insertions or deletions may be made in one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind to its target.

[0267] For example, conservative changes can be made in the CDRs that do not substantially reduce binding affinity. Such modifications may be made outside the antigen contact residues in the CDRs.

[0268] Conservative substitutions are shown in Table 4, along with more substantial "exemplary substitutions."

[0269] Table 4. Examples of amino acid substitutions [Table 4]

[0270] Substantial modification of the biological properties of antibody variants can be achieved by selecting substitutions that differ significantly in their effect on the structure of the polypeptide backbone in the area of ​​the substitution, on the charge or hydrophobicity of the molecule at the target site, or on maintaining the bulk of the side chain. Amino acids can be grouped according to the similarity of the properties of their side chains (ALLehninger, Biochemistry second ed., pp. 73-75, Worth Publishers, New York (1975)).

[0271] One type of substitution variant involves substituting one or more CDR region residues of a parent antibody (humanized or human antibody). Generally, the resulting variant(s) selected for further study have altered specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or substantially retain specific biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated using affinity maturation techniques, e.g., based on phage display. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0272] For example, alterations (e.g., substitutions) can be made to the CDRs to improve antibody affinity. Such changes can be made at HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or at residues that contact the antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from secondary libraries is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.

[0273] One method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is alanine scanning mutagenesis (Cunningham and Wells (1989) Science, 244:1081-1085). In this method, a residue or a number of target residues are identified and substituted with alanine to determine whether the interaction of the antibody with the antigen is affected. Alternatively, or in addition, an X-ray structure of an antigen-antibody complex can be used to identify contact points between the antibody and its antigen. Mutants can be screened to determine whether they contain the desired properties.

[0274] Constant region mutants In some 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) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0275] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).).

[0276] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions that improve binding of the Fc region to FcRn.

[0277] In certain embodiments, 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 of the Fc region (EU numbering of residues).

[0278] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 234, 235, 237, 238, 265, 269, 270, 297, 327, and 329 (see, e.g., 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, where amino acid residues are numbered according to the EU numbering system.

[0279] In order to confirm the reduction / depletion of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362, U.S. Pat. No. 5,821,337. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model, such as that disclosed in Clynes et al. Proc. Nat l Acad. Sci. USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that the antibody is unable to bind Clq and thus lacks CDC activity. See, e.g., Clq and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 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 MI 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)).

[0280] The constant region domains of the antibody molecule of the present invention, if any, may be selected taking into account the proposed function of the antibody molecule, in particular the effector functions that may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant region domains, in particular IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes may be used. It will be understood that sequence variants of these constant region domains may also be used.

[0281] In some embodiments, the antibody is IgG1 LALA, a variant of the wild-type human IgG1 isoform that introduces the amino acid substitutions L234A / L235A (according to EU numbering) in the IgG1 constant region.

[0282] In some embodiments, the antibody is IgG4P, a mutant of the wild-type human IgG4 isoform in which amino acid 228 (according to EU numbering) has been replaced by proline, e.g., as described in Angal et al., Molecular Immunology, 1993, 30(1), 105-108.

[0283] Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0284] Humanized, human and chimeric antibodies The antibodies of the present invention can be, but are not limited to, humanized antibodies, fully human antibodies, or chimeric antibodies.

[0285] In one embodiment, the antibody is humanized. More specifically, the anti-TREM1 antibody is a chimeric antibody, a human antibody or a humanized antibody.

[0286] In certain embodiments, the antibodies provided herein are chimeric antibodies. Examples of chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody.

[0287] A chimeric antibody is composed of elements derived from two different species such that the elements retain the characteristics of the species they are derived from. Generally, a chimeric antibody will contain a variable region from one species and an analogous constant region from another species, such as mouse, rat, rabbit, or human.

[0288] In certain embodiments, the chimeric antibody is a humanized antibody.

[0289] It will be appreciated that it is necessary to only transfer the specificity determining residues of the CDRs rather than the entire CDRs (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs are derived.

[0290] Suitably, humanised antibodies according to the invention have variable domains which comprise human acceptor framework regions as well as one or more CDRs and optionally further comprise one or more donor framework residues.

[0291] In one embodiment, the antibody is a humanized antibody, and the variable domains comprise human acceptor framework regions and non-human donor CDRs.

[0292] When CDRs are grafted, any suitable receptor variable region framework sequence may be used, taking into account the class / type of the donor antibody from which the CDRs are derived, including murine, primate and human framework regions.

[0293] Examples of human frameworks that may be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al.). For example, KOL and NEWM may be used for the heavy chain, REI may be used for the light chain, and EU, LAY and POM may be used for both the heavy and light chains. Alternatively, human germline sequences may be used, which are available at www.imgt.org. In an embodiment, the acceptor framework is IGKV1-9 human germline and / or IGHV3-66 human germline. In an embodiment, the human framework comprises 1-5, 1-4, 1-3 or 1-2 donor antibody amino acid residues.

[0294] In the humanized antibodies of the invention, the receptor heavy and light chains do not necessarily have to be derived from the same antibody, but may, if desired, comprise composite chains having framework regions derived from different chains.

[0295] In some embodiments, the antibody is a human antibody. Human antibodies can be produced using a variety of techniques known in the art. More specifically, the anti-TREM1 antibody comprises a human antibody heavy chain constant region and a human light chain constant region.

[0296] A human antibody includes a heavy or light chain variable region or a full-length heavy or light chain derived from a particular germline sequence, if the variable region or full-length chain of the antibody is obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest, or screening a human immunoglobulin gene library displayed on a phage with the antigen of interest. A human antibody derived from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with that of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is closest in sequence to the sequence of the human antibody (i.e., maximum % identity). A human antibody derived from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or deliberate introduction of site-specific mutations. However, the selected human antibody will typically be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene when compared to the germline immunoglobulin amino acid sequence of another species (e.g., mouse germline sequence) and will contain amino acid residues that identify the human antibody as human. In certain cases, a human antibody may be at least 60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene.

[0297] Human antibodies can be produced by a number of methods known to those skilled in the art. Human antibodies can be produced by the hybridoma method using human myeloma or mouse-human heteromyeloma cell lines (Kozbor, J Immunol; (1984) 133:3001; Brodeur, Monoclonal Isolated Antibody Production Techniques and Applications, pp51-63, Marcel Dekker Inc, 1987). Alternative methods include the use of phage libraries or transgenic mice, both of which utilize human variable region repertoires ((Winter G; (1994) Annu Rev Immunol 12:433-455, Green LL, (1999) J Immunol Methods 231:1 1-23). ​​Human antibodies can be produced, for example, in mice in which the mouse immunoglobulin variable genes, and optionally constant region genes, have been replaced by their human counterparts, as described, for example, in U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and 5,770,429.

[0298] Effector molecules If desired, the antibody according to the invention may be conjugated to one or more effector molecule(s). In one embodiment, the antibody is not bound to an effector molecule.

[0299] It will be understood that the effector molecule may comprise a single effector molecule or two or more such molecules linked to form a single moiety that can be bound to the antibody of the present invention. If it is desired to obtain an antibody fragment linked to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked to the effector molecule directly or via a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Particular chemical procedures include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 03 / 031581. Alternatively, where the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA procedures, for example as described in WO 86 / 01533 and EP 0 392 745.

[0300] Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that is detrimental to (e.g., kills) cells. Examples include combrestatins, dolastatins, epothilones, staurosporines, maytansinoids, spongiostatins, rhizoxins, halichondrins, roridin, hemiasterin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof.

[0301] Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicin or duocarmycin), and antimitotic agents (e.g., vincristine and vinblastine).

[0302] Other effector molecules may include chelated radionuclides such as 111In and 90Y, Lu177, bismuth-213, californium-252, iridium-192 and tungsten-188 / rhenium-188; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

[0303] Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin, proteins such as insulin, tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, thrombotic or antiangiogenic agents such as angiostatin or endostatin, or biological response modifiers such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins.

[0304] Other effector molecules may include detectable substances useful, for example, for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase, suitable prosthetic groups include streptavidin, avidin, and biotin, suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol, suitable bioluminescent materials include luciferase, luciferin, and aequorin, and suitable radionuclides include 125I, 131I, 111In, and 99Tc.

[0305] In another example, the effector molecule may increase the half-life of the antibody in vivo and / or reduce the immunogenicity of the antibody and / or enhance delivery of the antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins, or albumin binding compounds such as those described in WO 2005 / 117984.

[0306] When the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, such as an optionally substituted linear or branched polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

[0307] Particular optional substituents which may be present on the above synthetic polymers include one or more hydroxy, methyl or methoxy groups.

[0308] Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol) poly(vinyl alcohol) or derivatives thereof, in particular optionally substituted poly(ethylene glycol), such as methoxypoly(ethylene glycol) or derivatives thereof.

[0309] Exemplary naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

[0310] In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof.

[0311] The size of the polymer can vary as desired, but generally ranges from 500 Da to 50,000 Da, e.g., 5,000 to 40,000 Da, e.g., 20,000 to 40,000 Da, in average molecular weight. The polymer size can be selected based on, among other things, the intended use of the product, e.g., its ability to localize to a particular tissue, such as a tumor, or its ability to extend circulation half-life (for a review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, if the product is intended to leave the circulation and penetrate into tissues, e.g., for use in treating tumors, it may be advantageous to use a low molecular weight polymer, e.g., with a molecular weight of about 5,000 Da. For applications in which the product remains in the circulation, it may be advantageous to use a high molecular weight polymer, e.g., with a molecular weight in the range of 20,000 Da to 40,000 Da.

[0312] Suitable polymers include polyalkylene polymers such as poly(ethylene glycol) or especially methoxypoly(ethylene glycol) or derivatives thereof, especially those having a molecular weight in the range of about 15,000 Da to about 40,000 Da.

[0313] In one example, the antibody according to the invention is conjugated to a poly(ethylene glycol) (PEG) moiety. In a particular embodiment, the antigen-binding fragment according to the invention and the PEG molecule can be attached via any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, such as any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids can be naturally present in the antibody fragment or can be engineered into the fragment using recombinant DNA methods (see, for example, U.S. Pat. No. 5,219,996; U.S. Pat. No. 5,667,425; WO 98 / 25971; WO 2008 / 038024). In one example, the antibody molecule of the invention is a modified Fab fragment, where the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow for the attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region that includes one or more cysteine ​​residues to which the effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

[0314] Suitably, the PEG molecule is covalently attached via a thiol group of at least one cysteine ​​residue located in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently attached to the sulfur atom of a cysteine ​​residue located in the fragment. The covalent bond is generally a disulfide bond, or in particular a sulfur-carbon bond. When a thiol group is used as the attachment point, an appropriately activated effector molecule can be used, for example, a thiol-selective derivative such as maleimide and cysteine ​​derivatives. An activated polymer can be used as a starting material in the preparation of the above polymer-modified antibody fragment. An activated polymer can be any polymer containing a thiol-reactive group, such as an α-halo carboxylic acid or ester, e.g., iodoacetamide, imide, e.g., maleimide, vinyl sulfone or disulfide. Such starting materials can be obtained commercially (e.g., from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials using conventional chemical procedures. Particular PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

[0315] In one embodiment, the antibody is PEGylated, i.e. is a modified Fab fragment, Fab' fragment or diFab to which PEG (poly(ethylene glycol)) has been covalently attached, e.g. according to the methods disclosed in EP 0 948 544 or EP 1 090 037 (see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545). In one example, PEG is attached to a cysteine ​​in the hinge region. In one example, the PEG-modified Fab fragment has a maleimide group covalently attached to a single thiol group in the modified hinge region. A lysine residue may be covalently attached to the maleimide group, and a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da may be attached to each amine group on the lysine residue. Thus, the total molecular weight of the PEG attached to the Fab fragment may be about 40,000 Da.

[0316] In one embodiment, the antibody is a modified Fab' fragment having at the C-terminus of its heavy chain a modified hinge region comprising at least one cysteine ​​residue to which an effector molecule is attached. Suitably the effector molecule is PEG and is attached using the methods described in WO 98 / 25971 and WO 2004072116 or WO 2007 / 003898. Effector molecules may be attached to the antibody fragment using the methods described in International Patent Applications WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171.

[0317] In one embodiment, the antibody is not conjugated to an effector molecule.

[0318] Polynucleotides and Vectors The present invention also provides isolated polynucleotides encoding an antibody or portion thereof according to the invention (e.g., an amino acid sequence ID number listed in Table 5). Isolated polynucleotides according to the invention can include synthetic DNA, produced, for example, by chemical processing, cDNA, genomic DNA, or any combination thereof.

[0319] Table 5. Amino acid sequences of anti-TREM1 antibodies and their corresponding nucleic acid sequences. [Table 5]

[0320] Examples of suitable sequences are provided herein. Thus, in one embodiment, the invention provides an isolated polynucleotide encoding an antibody comprising the sequence set forth in SEQ ID NO: 34, 58, 36, 64, 66, 60, 62, 30, 80, 32, 86, 88, 82 or 84.

[0321] In one embodiment, the invention provides an isolated polynucleotide encoding the heavy chain of an IgG1 LALA or IgG4P antibody of the invention comprising the sequence as shown in SEQ ID NO: 88 or 82, respectively.

[0322] Also provided is an isolated polynucleotide encoding the light chain of an IgG1 LALA or IgG4P antibody of the invention comprising the sequence as shown in SEQ ID NO:32.

[0323] In another embodiment, the invention provides isolated polynucleotides encoding the heavy and light chains of an IgG4P antibody of the invention, wherein the polynucleotide encoding the heavy chain comprises the sequence set forth in SEQ ID NO: 82 and the polynucleotide encoding the light chain comprises the sequence set forth in SEQ ID NO: 32.

[0324] The present invention also provides a cloning or expression vector comprising one or more polynucleotides described herein. In one example, a cloning or expression vector according to the present invention comprises one or more isolated polynucleotides comprising a sequence selected from SEQ ID NOs: 34, 58, 36, 64, 66, 60, 62, 30, 80, 32, 86, 88, 82, or 84.

[0325] Standard techniques of molecular biology can be used to prepare DNA sequences encoding the antibodies of the present invention or antigen-binding fragments thereof. The desired DNA sequence can be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques can be used as needed.

[0326] General methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art, see in this regard "Current Protocols in Molecular Biology", 1999, FMA USubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0327] Host cells for producing antibodies and antigen-binding fragments thereof Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more isolated polynucleotide sequences according to the invention or one or more isolated polynucleotide sequences encoding an antibody of the invention. Any suitable host cell / vector system can be used for the expression of the polynucleotide sequences encoding the antibody or antigen-binding fragment thereof of the invention. Bacterial, such as E. coli, and other microbial systems may be used, or eukaryotic, such as mammalian, host cell expression systems may be used. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

[0328] In further embodiments, a host cell is provided that comprises such a nucleic acid(s) or vector(s). In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector that comprises a nucleic acid encoding an amino acid sequence comprising the VL of an anti-TREM1 antibody and an amino acid sequence comprising the VH of an anti-TREM1 antibody, or (2) a first vector that comprises a nucleic acid encoding an amino acid sequence comprising the VL of an anti-TREM1 antibody and a second vector that comprises a nucleic acid encoding an amino acid sequence comprising the VH of an anti-TREM1 antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, the host cell is a prokaryotic cell, such as an E. coli cell. In one embodiment, a method of producing an anti-TREM1 antibody is provided, comprising culturing a host cell that comprises a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0329] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0330] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including "humanized" fungal and yeast strains in which the glycosylation pathway results in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0331] Types of Chinese Hamster Ovary (CHO cells) suitable for use in the present invention may include CHO and CHO-K1 cells, including dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells, which may be used with a DHFR selectable marker, or CHOK1-SV cells, which may be used with a glutamine synthetase selectable marker. Other cell types used for expression of antibodies include lymphoid cell lines, such as NS0 myeloma cells and SP2 cells, COS cells. Host cells may be stably transformed or transfected with the isolated polynucleotide sequences or expression vectors according to the present invention.

[0332] Antibody manufacturing process The present invention also provides a method for producing an antibody according to the invention, which comprises culturing a host cell according to the invention under conditions suitable for producing an antibody according to the invention, and isolating the antibody.

[0333] The antibody may contain only heavy or light chain polypeptides, in which case only heavy or light chain polypeptide coding sequences need to be used to transfect the host cell. For the production of antibodies or antigen-binding fragments thereof containing both heavy and light chains, the cell line can be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding light and heavy chain polypeptides may be used.

[0334] Thus, a process is provided for culturing a host cell, expressing the antibody, isolating the antibody, and optionally purifying the antibody to provide an isolated antibody, in one embodiment, the process further comprises conjugating an effector molecule to the isolated antibody.

[0335] The invention also provides a method for producing an antibody according to the invention which comprises culturing a host cell containing a vector of the invention under conditions suitable to result in expression of protein from DNA encoding the antibody molecule of the invention, and isolating the antibody molecule.

[0336] An antibody molecule may contain only heavy or light chain polypeptides, in which case only heavy or light chain polypeptide coding sequences need to be used to transfect the host cell. For the production of products containing both heavy and light chains, a cell line can be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding both light and heavy chain polypeptides may be used.

[0337] The antibodies according to the invention are expressed at good levels from the host cells and therefore the properties of the antibodies are likely to be optimized for commercial processing.

[0338] purified antibody In one embodiment, a purified antibody, such as a humanized antibody, in particular an antibody according to the invention, is provided which is substantially purified from endotoxins and / or host cell proteins or DNA, in particular free or substantially free from endotoxins and / or host cell proteins or DNA.

[0339] Substantially free of endotoxin is generally intended to refer to an endotoxin content of 1 EU / mg of antibody product or less, for example 0.5 or 0.1 EU / mg of product.

[0340] Substantially free of host cell proteins or DNA is generally intended to refer to a host cell protein and / or DNA content of less than 400 μg per mg of antibody product, as appropriate, such as less than 100 μg / mg, in particular less than 20 μg / mg.

[0341] Therapeutic Uses of Antibodies The antibodies, formulations or pharmaceutical compositions of the invention may be administered for prophylactic and / or therapeutic treatments.

[0342] The present invention provides an anti-TREM1 antibody of the present invention or a pharmaceutical composition thereof for use as a pharmaceutical.

[0343] In prophylactic applications, antibodies, formulations or compositions are administered to a subject at risk of a disorder or condition as described herein in an amount sufficient to prevent or reduce the subsequent effects of the condition or one or more of its symptoms.

[0344] In therapeutic applications, antibodies are administered to a subject already suffering from a disorder or condition described herein in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms, or an increase in the frequency or duration of symptom-free periods.

[0345] The subject to be treated may be an animal. Preferably, the pharmaceutical composition according to the invention is adapted for administration to human subjects.

[0346] The invention provides a method of treating a disorder or condition as described herein in a subject in need thereof, the method comprising administering to the subject an antibody according to the invention, wherein such antibody is administered in a therapeutically effective amount.

[0347] The present invention also provides an antibody of the invention for use in the treatment of a disorder or condition as described herein.

[0348] Treatment indications The antibodies of the invention may be used in the treatment, prevention or amelioration of any symptoms associated with TREM1 activity, for example any condition that results in whole or in part from signaling through TREM1.

[0349] TREM1 and its multiple pathways are implicated in many neurological, neurodevelopmental, psychiatric, systemic and autoimmune inflammatory conditions. Some examples of conditions that can be treated using the antibodies and compositions of the present invention include amyotrophic lateral sclerosis, Alzheimer's disease (AD), Parkinson's disease (PD), tauopathy diseases, dementia, frontotemporal dementia, vascular dementia, mixed dementia, multiple system atrophy, epilepsy (including tuberous sclerosis complex and focal cortical dysplasia), Huntington's disease, spinal cord injury, traumatic brain injury, chronic traumatic encephalopathy, ischemic stroke, multiple sclerosis, autoimmune neuritis, schizophrenia, autism spectrum disorder, major depressive disorder, bipolar disorder, genetic conditions, or any combination thereof.

[0350] The antibodies and compositions of the invention can be used to treat neurological disorders, more specifically, amyotrophic lateral sclerosis (ALS) or Alzheimer's disease.

[0351] Diagnostic Uses of Antibodies and Antigen-Binding Fragments Thereof

[0352] The present invention also provides the use of the antibodies of the present invention as diagnostically active agents or in diagnostic assays, for example, to diagnose a disease or its severity.

[0353] The diagnosis may preferably be performed on a biological sample. A "biological sample" encompasses various sample types obtained from an individual and can be used in diagnostic or monitoring assays. The definition includes blood, such as cerebrospinal fluid, plasma and serum, and other liquid samples of biological origin, such as urine and saliva, solid tissue samples, such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in some way after their procurement, for example, by treatment with a reagent, solubilization, or enrichment for certain components, such as polynucleotides.

[0354] The diagnostic test may preferably be performed on a biological sample that is not in contact with the human or animal body. Such diagnostic tests are also called in vitro tests. In vitro diagnostic tests may rely on in vitro methods to detect TREM1 in a biological sample obtained from a subject.

[0355] Pharmaceutical and diagnostic compositions The antibodies of the invention may be provided in a pharmaceutical composition, which is usually sterile and may further comprise a pharma- ceutical acceptable adjuvant and / or carrier.

[0356] Since the antibodies of the invention are useful in the treatment, diagnosis and / or prevention of disorders or conditions as described herein, the present invention also provides pharmaceutical or diagnostic compositions comprising an antibody or antigen-binding fragment thereof according to the invention in combination with one or more pharma- ceutically acceptable carriers, excipients or diluents.

[0357] In particular, the antibody or antigen-binding fragment thereof is provided as a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0358] These compositions may contain, in addition to the therapeutically active ingredient(s), pharma- ceutically acceptable excipients, carriers, diluents, buffers, stabilizers, or other materials known to those skilled in the art, such materials should be non-toxic and should not interfere with the efficacy of the active ingredients.

[0359] Compositions (including pharmaceutical formulations) comprising an anti-TREM1 antibody of the invention, or a polynucleotide comprising a sequence encoding an antibody of the invention, are also provided. In certain embodiments, the composition comprises one or more antibodies of the invention, or one or more polynucleotides comprising a sequence encoding one or more antibodies of the invention. These compositions may further comprise suitable carriers well known in the art, such as adjuvants including pharma- ceutically acceptable excipients and / or buffers.

[0360] Pharmaceutical compositions of the antibodies of the present invention are prepared by mixing such antibodies having the desired purity with one or more optional pharma- ceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution.

[0361] Examples of the above mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0362] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides, and other carbohydrates, including 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 (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial 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 Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0363] The active ingredient may be encapsulated in microcapsules (e.g., hydroxymethylcellulose, or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0364] Sustained-release preparations may also 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.

[0365] Formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0366] The pharmaceutical compositions of the invention may include one or more pharma- ceutically acceptable salts.

[0367] Pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, buffered water and saline. Examples of other carriers include ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and their appropriate mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. In many cases, it is desirable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0368] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0369] In one embodiment, the antibody of the invention is the only active ingredient. In another embodiment, the antibody of the invention is combined with one or more additional active ingredients. Alternatively, the pharmaceutical composition comprises the antibody of the invention as the only active ingredient and may be administered separately to a patient in combination (e.g., simultaneously, sequentially or separately) with other agents, drugs or hormones.

[0370] The exact nature of the carrier or other material may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular and intraperitoneal. For example, solid oral forms may contain, together with the active substance, diluents such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium stearate or calcium stearate, and / or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disaggregating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbates, lauryl sulfate, and non-toxic and pharmacologically inert substances generally used in pharmaceutical formulations. Such pharmaceutical preparations can be manufactured in known manner, for example by mixing, granulating, tableting, sugar-coating or film-coating processes.

[0371] Oral formulations contain commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95%, preferably 25%-70%, of the active ingredient. If the pharmaceutical composition is lyophilized, the lyophilized material may be reconstituted (e.g., suspension) prior to administration. Reconstitution is preferably carried out in a buffer solution.

[0372] The solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile aqueous isotonic saline solutions.

[0373] Preferably, the pharmaceutical or diagnostic composition comprises a humanized antibody according to the invention.

[0374] Determination of Therapeutically Effective Amounts and Dosages The antibody and pharmaceutical composition according to the present invention can be appropriately administered to a patient to determine the therapeutically effective amount required. For any antibody, the therapeutically effective amount can be estimated first either in cell culture assays or animal models, usually rodents, rabbits, dogs, pigs or primates. Animal models can also be used to determine the appropriate concentration range and administration route. Such information can then be used to determine the useful dosage and administration route in humans.

[0375] The exact therapeutically effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the subject's age, weight and sex, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance / response to treatment. The compositions may be conveniently provided in unit dose forms containing a predetermined amount of the active agent of the present disclosure per dose. Dose ranges and regimens for any of the embodiments described herein include, but are not limited to, doses ranging from 1 mg to 1000 mg unit dose.

[0376] The appropriate dosage of the antibody or pharmaceutical composition of the present invention can be determined by those skilled in the art. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level depends on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0377] Suitable doses can be, for example, in the range of about 0.01 μg / kg to about 1000 mg / kg body weight of the patient being treated, typically about 0.1 μg / kg to about 100 mg / kg body weight.

[0378] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0379] Administration of pharmaceutical compositions or formulations The antibodies or formulations or compositions thereof described herein can be administered for prophylactic and / or therapeutic treatments.

[0380] The antibody or pharmaceutical composition of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Examples of routes of administration of the antibody or pharmaceutical composition of the present invention include intravenous, intramuscular, intradermal, intraocular, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Alternatively, the antibody or pharmaceutical composition of the present invention may be administered via a parenteral route, such as a topical, epidermal or mucosal route of administration. The antibody or pharmaceutical composition of the present invention may be for oral administration.

[0381] Suitable forms for administration include those suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion, in intravenous, inhalable or subcutaneous form. If the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain additional agents such as suspending agents, preservatives, stabilizers and / or dispersing agents. Alternatively, the antibody or antigen-binding fragment thereof according to the invention may be in a dry form for reconstitution with an appropriate sterile liquid before use. Solid forms suitable for dissolution or suspension in a liquid vehicle before injection may also be prepared.

[0382] Once formulated, the pharmaceutical compositions of the invention can be administered directly to a subject.Thus, there is provided herein the use of an antibody or antigen-binding fragment thereof according to the invention for the manufacture of a medicament.

[0383] Products and Kits The present disclosure also provides a kit comprising an anti-TREM1 antibody of the invention and instructions for use. The kit may further comprise one or more additional reagents, such as an additional therapeutic or prophylactic agent, as discussed above.

[0384] The present invention provides the use of an antibody according to the present invention or a pharmaceutical composition thereof for the manufacture of a medicament.

[0385] The invention also provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of a disorder or condition as described herein.

[0386] In certain embodiments, the article of manufacture or kit comprises a container containing one or more antibodies of the invention or compositions described herein. In certain embodiments, the article of manufacture or kit comprises a container containing a nucleic acid(s) encoding one(s) of the antibody(s) or compositions described herein. In some embodiments, the kit comprises a cell or cell line that produces an antibody described herein.

[0387] In certain embodiments, the article of manufacture or kit comprises 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, such as glass or plastic. The container holds the composition, either by itself or in combination with another therapeutically, prophylactically, and / or diagnostically effective composition, and may have a sterile access port. At least one agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used for treating a disorder or condition described herein.

[0388] It should be noted that the above embodiments are illustrative rather than limiting of the present invention, and that those skilled in the art can design many alternative embodiments without departing from the scope of the claims. In the claims, any reference signs placed between parentheses shall not be construed as limiting the scope of the claims.

[0389] The sequences encompassed by the present invention are shown in Tables 6 and 7:

[0390] Table 6. TREM1 sequence [Table 6]

[0391] Table 7. Sequences of 12172 antibodies and related variants

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

[0392] example Example 1: Production of TREM1 protein Human TREM1 IgV-like domain fused to an N-terminal hexahistidine SUMO (small ubiquitin-related modifier) ​​solubility tag (CID101907) was expressed in Escherichia coli BL21(DE3). Bacteria were harvested by centrifugation, resuspended in 100mM Tris pH8, 300mM NaCl, 250U benzonase, 1PI tab, and lysed by sonication. Lysate was clarified by centrifugation at 42,000RPM (Ti45), 4°C, 45min, and applied to a 5ml HiTrap Ni Chelating Column. The column was washed with 5CV of 100mM Tris, pH8, 300mM NaCl. Bound TREM1 protein was eluted with a 15 CV linear gradient 2-60% 100 mM Tris, pH 8, 300 mM NaCl, 500 mM imidazole buffer, followed by 4 CV of 100% buffer B. Fractions containing TREM1 were pooled, dialyzed in 100 mM Tris pH 8, 300 mM NaCl (10 kDa MWCO) and digested overnight at 4 °C with ULP-1. The cleaved protein was applied to a 5 ml HiTrap Ni Chelating Column and the flow-through fraction was collected and concentrated with a Vivaspin PES Turbo, 10 kDa MWCO concentrator. The cleaved IgV protein was then polished and buffer exchanged into 100 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM EDTA using a superdex s75 column (GE Healthcare). Final protein concentrations were determined by measuring absorbance at 280 nm with a Nanodrop UV spectrometer. Protein purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0393] Both human (CID101904) and cynomolgus monkey (CID101953) TREM1 extracellular domains (ECDs) containing the native N-terminal signal sequence and a C-terminal avidin affinity tag (AVI), a TEV protease cleavage site and an HKH affinity tag were expressed in mammalian HEK cells. The medium was passed through a tangential flow filtration (TFF) and applied to a HiTrap Ni Chelating 5 mL column and washed with 100 mM HEPES pH 7.0, 300 mM NaCl. Bound TREM1 protein was eluted with a 4 CV linear gradient of 2-60% then 100% 100 mM HEPES pH 7.0, 300 mM NaCl, 500 mM imidazole, respectively. Fractions containing TREM1 were pooled and concentrated with a Vivaspin PES Turbo, 10 kDa MWCO concentrator. The protein was then polished and buffer exchanged into 50 mM HEPES pH 7.0, 250 mM NaCl using a Sephacryl s300 column. Final protein concentration and purity were assessed as previously described.

[0394] Human PGLYRP1 containing the native N-terminal signal sequence and a non-cleavable C-terminal his-tag (CID101951) was expressed in mammalian HEK cells. The medium was applied to a HiTrap Ni Excel 2 x 5 mL column and washed with 100 mM HEPES pH 7.0, 300 mM NaCl. Bound PGLYRP1 protein was eluted with a 4 CV linear gradient of 0-60% then 100% 100 mM HEPES pH 7.0, 300 mM NaCl, 500 mM imidazole, respectively. Fractions containing PGLYRP1 were pooled and concentrated with a Vivaspin PES Turbo, 10 kDa MWCO concentrator. The protein was then polished and buffer exchanged into PBS pH 7.0 using a superdex S200 column. Final protein concentration and purity were assessed as previously described.

[0395] Example 2. Generation and selection of therapeutic anti-TREM1 antibody 12172 One female New Zealand White rabbit was cultured with 3 × 10 cells transiently expressing human TREM1 on the cell surface. 7Rabbits were immunized subcutaneously with 1000 rabbit fibroblasts. The cells were transfected by electroporation, and expression of TREM1 was confirmed by flow cytometry using an anti-TREM1 antibody (R&D FAB1278P). Concurrent with cell immunization, rabbits were injected subcutaneously at another site with an equal volume of complete Freund's adjuvant.

[0396] Rabbits were given two booster injections 14 days apart with rabbit fibroblasts transiently expressing human TREM1 on the cell surface. Prior to each immunization, a heparinized bleed (200 μl) was taken from an ear vein. Serum was collected from the blood draw after a 5-minute spin at 10,000 rpm in a benchtop centrifuge and frozen at -20°C. Termination occurred 14 days after the final boost, when single cell suspensions of spleen, lymph node, bone marrow and peripheral blood mononuclear cells were prepared and frozen in 10% DMSO / FCS at -80°C until required for B cell discovery purposes. Blood was also drawn at termination and serum was prepared as described above.

[0397] Memory B cell cultures were set up using the method described by Tickle et al. (2015) in J Biomol Screen 20(4):492-7, and supernatants were first screened for their ability to bind human and cynomolgus TREM1 in a cell-based assay on a TTP Labtech Mirror-ball system. The cell-based assay was a homogeneous multiplex assay using HEK293 cells transiently transfected with either human TREM1 or cynomolgus TREM1 DNA and counterscreened against HEK293 cells transiently transfected with unrelated DNA. Cells were stained with either Vybrant™ DIO or DIL label (ThermoFisher) and goat anti-rabbit Fc-AF647 conjugate as a revealing agent.

[0398] Approximately 7000 TREM1-specific positive hits were identified in the primary mirror-ball screen from a total of 20x200 plate B cell culture experiments. Positive supernatants from this assay were then progressed for further characterization by profiling on BIAcore to estimate off-rates.

[0399] Using fluorescence focusing and single-cell reverse transcription (RT) and PCR (RT-PCR), wells with dissociation rates resulting in less than 25% loss of binding during the 300 second dissociation step were progressed for V-region gene recovery.

[0400] Following reverse transcription (RT) and PCR of sorted cells, "transcriptionally active PCR" (TAP) products encoding the V-regions of the antibodies were generated and used to transiently transfect HEK-293 cells. The resulting TAP supernatants containing the recombinant antibodies were tested for their ability to bind human (CID101904; SEQ ID NO: 7) and cynomolgus (CID101953; SEQ ID NO: 8) TREM1 extracellular domains (generated as described in Example 1) by ELISA, to bind human sTREM1 with an affinity of at least 1000 pM in BIAcore, and to block PGLYRP1-mediated signaling in a THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cell assay.

[0401] Functionality was assessed by the ability of the antibodies to inhibit PGLYRP1 / PGN-mediated NF-κB signaling activation via human TREM1. To do this, THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cells were used (produced in UCB). These cells stably express human TREM1, human DAP12 and NF-κB luciferase reporter genes. PGLYRP1 complexed with soluble peptidoglycan (PGN) from Escherichia coli (E. coli) was used as the TREM1 ligand, which induces NF-κB activation by binding to TREM1. PGN, which does not bind to TREM1, also induces NF-κB activation, but to a lesser extent and via an alternative signaling pathway. Inhibition of luciferase activity demonstrates the functional blocking activity of the antibodies in this system.

[0402] THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cells were cultured in complete medium containing selection antibiotics (RPMI + 10% FBS + 50 μM 2-mercaptoethanol + 10 μg / ml blasticidin + 1 μg / ml puromycin + 200 μg / ml geneticin) using standard tissue culture techniques. Three days prior to assay setup, cells were plated at 10 × 10 in 50 ml complete medium (200,000 cells / ml) in a T175 flask. 6 Cells were seeded and placed flat in an incubator. On the day of the assay, cells were removed from the flask, transferred to a 50 ml Falcon and centrifuged at 300 × g for 5 min. The medium was removed and cells were resuspended in 5-10 ml of complete medium and counted. Cells were then diluted to 1 × 10 by adding the cell suspension to complete medium. 6Cells were resuspended at 10 μl / well and added to the assay plate (Corning#3570). Antibodies were serially diluted in complete medium in a 384-well dilution plate (Greiner#781281). The serial dilutions of antibody were then transferred to the assay plate (10 μl / well) and the assay plate was incubated for 1 hour at 37° C. / 5% CO2. Recombinant human PGLYRP1 (R&D Systems#2590-PGB) was complexed with PGN (Invivogen#tlrl-ksspgn) for 1 hour at room temperature in sterile DPBS. After 1 hour, the solution was diluted with complete medium and then transferred to the assay plate (10 μl / well) to give a final assay concentration of 2.5 μg / ml PGLYRP1 and 10 μg / ml PGN. Plate controls (no antibody added) included PGLYRP1 / PGN complex and PGN alone as assay maximum and minimum, respectively. The assay plates were then incubated at 37°C / 5% CO2 for 16 hours ± 2 hours. After incubation, luciferase activity was measured using the SteadyGlo Luciferase Assay System (Promega #E2520). Steady-Glo Reagent was prepared according to the manufacturer's instructions and 30 μl / well was added to the assay plate. The plates were then centrifuged at 200×g for 3 minutes and then incubated at room temperature for an additional 2 minutes for a total incubation time with SteadyGlo Reagent of 5 minutes. Luminescence was then measured using a Synergy Neo2 plate reader and the raw luminescence values ​​were used to determine the relative inhibition compared to control wells. 4PL Curve Fitting and IC 50 Values ​​were calculated using ActivityBase v9.4.

[0403] Heavy and light chain variable region gene pairs from the TAP products of interest were then cloned as rabbit IgG antibodies and re-expressed in a HEK-293 transient expression system. A total of 144 V regions were cloned. The recombinant cloned antibodies were then retested for their ability to bind human and cynomolgus TREM1 by ELISA, binding in BIAcore, and inhibition of PGLYRP1+PGN-mediated signaling in an NF-κB luciferase reporter cell assay. Following characterization of the ligand binding site of the known TREM1 ligand PGLYRP1 using a human TREM1 alanine mutant array (the same approach as described further below for the TREM1 inhibitory antibody), it was hypothesized that antibodies that bind to the same binding site would modulate TREM1 function by direct ligand blocking. To identify alternative antibody binding sites on TREM1 that confer function, antibodies that were proven to inhibit TREM1 activity in an NF-κB luciferase reporter cell assay were evaluated for epitope location using a human TREM1 alanine mutant array.

[0404] Arrays of human TREM1 IgV domain mutant clones were generated. They consisted of either 58 clones each with three adjacent surface residues mutated to alanine; 65 clones each with two adjacent surface residues mutated to alanine; or 63 clones each with a single surface residue mutated to alanine. All arrays contained wild-type human TREM1 clones. The sequences of the mutant human TREM1 array clones, including the wild-type, are shown in Tables 8, 9, and 10.

[0405] Table 8.3 - List of TREM1 protein sequences used to design the alanine mutant arrays [Table 8-1] [Table 8-2] [Table 8-3]

[0406] Table 9.2 - List of TREM1 protein sequences used to design the alanine mutant arrays [Table 9-1] [Table 9-2] [Table 9-3]

[0407] Table 10. List of TREM1 protein sequences used to design single alanine mutant arrays [Table 10-1] [Table 10-2] [Table 10-3]

[0408] Each of the above clones was expressed as a fusion protein consisting of the TREM1 IgV domain followed by a triple alanine linker fused to a human Fc domain. Each clone was captured on a sensor coated with an anti-human Fc antibody. The sensor was then immersed in a solution containing the antibody of interest. Binding kinetics were monitored using a Bio-Layer Interferometry (BLI) instrument (Octet RED384 or Octet HTX, ForteBio).

[0409] By monitoring the binding kinetics of the antibody to each mutant TREM1 clone and comparing them to the kinetics to the wild-type protein, the epitope could be deduced. An increase in the antibody dissociation rate constant or loss of antibody binding to the protein indicated that the mutated residue in that clone was important for antibody binding and therefore part of its epitope.

[0410] The 12172 antibody was selected as a potent inhibitor of TREM1 activity. Interestingly, an alanine scanning approach demonstrated that this molecule has an epitope distant from the identified PGLYRP1 ligand-binding site, which was subsequently selected as a lead molecule.

[0411] Using the methods described above and considering all three arrays, the important epitope residues of the 12172 antibody were determined to be residues E26, E27, K28, Y29, E30, L31, K32 and Q35 (wherein the numbering is according to SEQ ID NO:1).

[0412] Example 3: Humanization of antibodies Antibody 12172 was humanized by grafting the CDRs from the rabbit V-regions onto a human germline antibody V-region framework. To restore antibody activity, a number of framework residues from the rabbit V-regions were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (WO 91 / 09967). An alignment of the rabbit antibody (donor) V-region sequences with the human germline (receptor) V-region sequences, along with the designed humanized sequences, is shown in Figures 1 and 2. The CDRs grafted from the donor to the receptor sequence are as defined by Kabat (Kabat et al., 1987), except for CDR-H1 (see Adair et al., WO 91 / 09967), where the combined Chothia / Kabat definition is used.

[0413] The human V region IGKV1-9+IGKJ4 J region (IMGT, http: / / www.imgt.org / ) was selected as the recipient for the antibody 12172 light chain CDRs. All light chain framework residues in the humanized grafted variants were from the human germline gene, but the donor residues alanine (A1), valine (V2) and valine (V3) were retained except for 0, 1, 2 or 3 residues from the group including residues 1, 2 and 3 (with reference to SEQ ID NO:25), respectively (Figure 1 and Table 11).

[0414] The human V region IGHV3-66+IGHJ4J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the heavy chain CDR of antibody 12172. Like many rabbit antibodies, the VH gene of antibody 12172 is shorter than the selected human acceptor. When aligned with the human acceptor sequence, framework 1 of the VH region of antibody 12172 lacks the N-terminal residues that are retained in the humanized antibody (Figure 2). Framework 3 of the 12172 rabbit VH region also lacks two residues (75 and 76, with reference to SEQ ID NO: 45) in the loop between beta sheet strands D and E: in the humanized graft variant, the gap is filled with the corresponding residues (lysine 75, K75; asparagine 76, N76) from the selected human acceptor sequence (Figure 2). The heavy chain framework residues in the humanized graft variants are all from the human germline gene, except for one or more residues from the group including residues 23, 48, 49, 71, 73 and 78 (with reference to SEQ ID NO: 45), where the donor residues threonine (T23), isoleucine (I48), glycine (G49), lysine (K71), serine (S73) and valine (V78), respectively, were retained.

[0415] Genes encoding a number of variant heavy and light chain V region sequences were designed and constructed by an automated synthesis approach by ATUM (CA, USA). Further variants of the heavy and light chain V regions were generated by modifying the VH and VK genes by oligonucleotide-directed mutagenesis. For transient expression in mammalian cells, the humanized light chain V region genes were cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human Kappa chain constant region (Km3 allotype). Humanized heavy chain V region genes were cloned into the UCB human gamma 4 heavy chain expression vector pMhγ4PFL, which contains DNA encoding the human gamma 4 heavy chain constant region with the hinge stabilizing mutation S228P (Angal S., King DJ, Bodmer MW, Turner A., ​​Lawson ADG, Roberts G., Pedley B. and Adair JRA single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody. Mol. Immunol. 1993,30(1):105-8), or into the UCB gamma 1 LALA heavy chain expression vector pMhγ1 L234A L235A, which contains DNA encoding the human gamma 1 heavy chain constant region with the mutations L234A and L235A to reduce binding to the Fc gamma receptor (FcγR) (Canfield SM and Morrison SL The Binding affinity of Human IgG for its High Affinity Fc Receptor Is Determined by Multiple Amino Acids in the CH2 Domain and Is Modulated by the Hinge Region.J.Exp.Med.1991,173:1483-1491).Co-transfection of the resulting heavy and light chain vectors into Expi293™ suspension cells was achieved using ExpiFectamine™ 293 transfection reagent (A14525, ThermoFisher Scientific), resulting in expression of humanized recombinant IgG4P and IgG1 LALA antibodies.

[0416] The mutant humanized antibody chains and their combinations were expressed and evaluated for their binding affinity to human TREM1 compared to the parental antibody, their thermal stability by fluorescence-based thermal shift assay (described in Example 13) and their tendency to self-interaction by AC-SINS (affinity capture self-interaction nanoparticle spectroscopy as described in Example 17). Retention of donor residues I48, G49 and K71 of the VH framework in graft gH11 was essential for the highest affinity binding to human TREM1 as measured by surface plasmon resonance (Table 11). All light chain framework residues in graft gL2 were from human germline genes. Retention of VL donor residue V3 in graft gL6 reduced the tendency to self-interaction as measured by AC-SINS assay (Table 22).

[0417] Heat resistance (denaturation) is an indicator of conformational stability and long-term storage stability. Humanized IgG4P antibody has good thermal stability, with the midpoint (Tm) of unfolding of the Fab domain ranging from 73.5 to 74.6°C (Table 11).

[0418] Molecular self-interactions can lead to native-state aggregation and poor solubility, especially at the high protein concentrations used for subcutaneous administration of therapeutic mAbs. The net charge of antibody Fv domains has been shown to affect native-state aggregation of human IgG at pH 7.4 and pH 5.0 in an isotype-specific manner (Heads JT, Lamb R, Kelm S, Adams R, Elliott P, Tyson K, Topia S, West S, Nan R, Turner A, Lawson ADG. Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability. Protein Eng Des Sel. 2019 Dec 31; 32(6): 277-288. doi: 10.1093 / protein / gzz046. PMID: 31868219; PMCID: PMC7036597). To reduce the tendency of the humanized 12172 hIgG4P antibody to self-interaction, as indicated by the high Δλmax values ​​measured by AC-SINS assay (Table 12 in this example and Table 22 in Example 17), the net positive charge of the Fv domain was reduced by mutating positively charged residues to either neutral or negatively charged residues. These residues were selected using the rationale outlined by Heads et al. (2019) (WO 2019 / 234094). Residues 18 and 50 of the humanized light chain graft 12712 gL2 (SEQ ID NO: 29) were mutated from arginine (R18) to serine (S18) and lysine (K50) to serine (S50) in grafts gL9 and gL11, respectively. Residue 75 in humanized heavy chain graft 12172gH11 (SEQ ID NO:79) was mutated from lysine (K75) to either serine (S75), glutamine (Q75) or glutamic acid (E75) in grafts gH26, gH48 and gH49, respectively.The modified heavy and light chain genes were combined and transiently expressed in Expi293™ suspension cells and the recombinant IgG4P antibodies were evaluated for their binding affinity to human TREM1, thermal stability and tendency to self-interact (Table 12). The humanized 12172 charge variants retained affinity for human TREM1 and showed a reduced tendency to self-interact as indicated by a reduced Δλmax measured by AC-SINS assay.

[0419] Biophysical characterization of humanized 12172 gL2gH11 and 12172 gL6gH6 (in both hIgG4P and hIgG1LALA formats) was performed using different stress conditions to assess developability, as described in Examples 12 to 20. Additionally, all molecules were analyzed by liquid chromatography-mass spectrometry (LC-MS) to confirm that the predicted sequence molecular weight (MW) was consistent with experimental data.

[0420] The humanized 12172gL2gH11 IgG4P antibody demonstrated similar NF-κB inhibition as the rabbit parental 12172 antibody in the THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cell assay (described in Example 2) (see Table 13).

[0421] Table 11. Affinities and Tm of various 12172 antibody variants. [Table 11]

[0422] Table 12. Affinity, Tm and AC-SINS of various 12172 antibody variants (duplicate values ​​provided for some of the variants) [Table 12]

[0423] Table 13. Summary of potency, efficacy and slope values ​​for 12172gL2gH11 IgG4P and 12172 Rabbit IgG1 [Table 13]

[0424] Example 4. Expression and purification of rabbit 12172 Fab Co-transfection of heavy and light chain vectors CID102769 and CID102770 into Expi293™ suspension cells was achieved using ExpiFectamine™ 293 transfection reagent (A14525, ThermoFisher Scientific) resulting in expression of rabbit recombinant 12172Fab. The medium was filtered through a PALL 0.4 / 0.2 μm capsule filter and applied to Protein G GammaBind Plus resin (7 mL) precipitated on a XK16 / 60 column and washed with 10 CV 1× PBS pH 7.4. Bound Fab complex protein was eluted with 50 mL 100 mM glycine (pH 2.7) and immediately neutralized with 10% 1M Tris (pH 8). Fractions containing 12172Fab complex were pooled and concentrated with a Vivaspin PES 20, 10 kDa MWCO concentrator. It was then polished using a superdex s200 16 / 60 column (GE Healthcare) and buffer exchanged into 1x PBS pH 7.4. The final protein concentration and purity were assessed as previously described in Example 1.

[0425] Example 5.12 Expression and purification of 172 gL2gH11 / gL6gH6 hIgG4P and hIgG1 LALA Transient mammalian expression, CHOSXE culture Suspension CHOSXE cells were pre-adapted in CDCHO serum-free medium (Invitrogen) supplemented with 2 mM (100x) Glutamax.

[0426] Cells were maintained in logarithmic growth phase and cultured at 37°C in a shaking incubator (Kuhner AG, Bersfelden, Switzerland) with shaking at 190 rpm and supplemented with 8% CO2.

[0427] Electroporation transfer Prior to transfection, cell number and viability were determined using a Vi-Cell™ XR cell viability analyzer (Beckman Coulter) to determine the required number of cells (2.3 × 10) with 99% viability. 8 The pellet was then washed with Hyclone™ MaxCyte® buffer (Thermo Scientific) and centrifuged for an additional 15 minutes. The pellet was diluted with 2.3 x 10 8 Resuspended at 100 cells / ml.

[0428] Plasmid DNA purified using the Qiagen Plasmid Plus Giga Kit® was added at 400 μg / ml. After electroporation using a MaxCyte STx® flow electroporation instrument, cells were transferred to ProCHO™5 protein-free CHO medium (Lonza) containing 2 mM Glutamax, 0.75 mM sodium butyrate (n-butyric acid sodium salt, Sigma B-5887), a 100x solution of antibiotic mitotic inhibitors (1 in 500) and a bolus feed added on day 0.

[0429] The transfected cells were then transferred directly into vented flasks and cultured in a Kuhner Shaker Incubator set at 37°C, 8% CO2 and 190 rpm shaking. 24 hours after transfection, the temperature was lowered to 32°C and the cells were cultured for an additional 11-13 days.

[0430] On days 12-14, the cultures were transferred to centrifuge tubes and spun at 4000 rpm for 30 minutes before the supernatant was separated from the cells. The retained supernatant was further clarified by filtration through a 0.22 μm Sartobran® P Millipore cartridge followed by a 0.22 μm Millipak® Gamma Gold filter. Final expression titers were determined by Protein G quantitative HPLC assay using 1 mg / ml A33 hIgG1 as standard, 0.8 ml POROS™ G 20 μm column and an Agilent 1100 Series HPLC system. The clarified cell culture harvest was stored at 4° C. prior to purification.

[0431] Antibody purification and analysis Clarified cell culture harvest was warmed to room temperature before loading onto a 215 ml MabSelect™ SuRe™ column (Cytiva) pre-equilibrated in Hyclone™ Phosphate Buffered Saline (PBS) pH 7.4 using an AKTA Pure 25L chromatography system (Cytiva). After washing with PBS pH 7.4, bound material was eluted by lowering the pH to 3.4 (0.1 M sodium citrate buffer) for human IgG1 isotype or pH 3.7 (30 mM sodium acetate) followed by pH 3.6 (0.1 M sodium citrate buffer) for human IgG4P isotype. After elution, the column was stripped with 0.1 M citric acid pH 2.0 to remove tightly bound aggregates. Affinity capture elution peak fractions were pooled and neutralized to pH 5.5-7.5 by addition of 2 M Tris-HCl pH 8.5. Protein concentration was determined by reading absorbance at 280 nm using a Nanodrop and purity was determined by analytical size exclusion HPLC (method below).

[0432] The affinity pool was concentrated using centrifugal filtration devices (Centricon® Plus-70 or Amicon® Ultra-15) or pressurized stirred cell chambers (Amicon®) with 10 KDa or 30 KDa MWCO membranes depending on the volume for loading onto HiLoad Superdex 200 26 / 60 (Cytiva) or 50 / 60 prep grade columns (custom packed by Cytiva). The HiLoad Superdex200 26 / 60 or 50 / 60 columns were equilibrated in 50 mM sodium acetate, 125 mM sodium chloride buffer pH 5.0 before loading the samples using an AKTA chromatography system (Cytiva). An isocratic elution was performed and fractions were collected after 0.3 CV. Fractions containing monomer were identified by running fractions or pseudopools on analytical size exclusion HPLC. Fractions were pooled to obtain >98% monomer content. Pools were concentrated to 10-15 mg / ml using centrifugal filtration devices (Centricon® Plus-70 or Amicon® Ultra-15) or pressurized stirred cell chambers (Amicon®) with 10 KDa or 30 KDa MWCO membranes depending on volume, then harvested and 0.22 μm sterile filtered using a Stericup® filtration unit or Millex GV syringe filters.

[0433] Final protein concentration was determined by reading absorbance at 280 nm using a nanodrop. Monomer content was determined by analytical size-exclusion HPLC. Correct banding patterns were determined by SDS-PAGE using an Invitrogen Novex™ WedgeWell™ 4-20% Tris-Glycine and XCell SureLock™ minicell electrophoresis system and Coomassie staining. Endotoxin levels were determined using Charles River Endosafe® LAL Reagent Cartridge Technology and an Endosafe® nexgen-PTS reader, with levels below 1 EU / mg being of acceptable quality. Samples were analyzed by intact mass spectrometry to confirm heavy and light chain masses, expected modifications and identities.

[0434] Analytical size exclusion HPLC A TSKgel G3000SWXL HPLC column (Tosoh) was equilibrated in Hyclone phosphate buffered saline (PBS) pH 7.4 using an Agilent 1100 or 1200 series HPLC. Samples of 20-50 μg were injected and run under isocratic elution conditions (PBS pH 7.4) at 1 ml / min for 16 min. Data were compared to BioRad molecular weight marker standards. Retention times and percentages were reported for impurities associated with monomer and high and low molecular weight products.

[0435] Example 6. Binding kinetics of 12172gL2gH11 hIgG4P to human and cynomolgus monkey TREM1 The kinetics of 12172gL2gH11 hIgG4P binding to human and cynomolgus TREM1 was measured by surface plasmon resonance on a Biacore T200 instrument and a Biacore 8k instrument at 25°C.

[0436] A goat anti-human IgG, Fc fragment specific antibody (F(ab')2 fragment, Jackson ImmunoResearch 109-006-098) was immobilized on a CM5 sensor chip via amine coupling chemistry to a level of approximately 5000 RU. The reference cell was treated similarly. After amine coupling was completed, all subsequent solutions, except for the capture solution, were run over the reference and sample cells in sequence, and the response of the reference cell was subtracted from the sample cell throughout the run.

[0437] Each analytical cycle consisted of capture of approximately 250 RU of 12172gL2gH11 hIgG4P onto the anti-Fc surface, injection of analyte for 180 seconds (25°C, flow rate 30 μl / min), dissociation of analyte for 600 seconds, followed by surface regeneration (by injection of 50 mM HCl for 60 seconds, injection of 5 mM NaOH for 30 seconds, and another 60 seconds of 50 mM HCl). Human TREM1 ECD analyte (in-house, His-tagged) was injected in 3-fold serial dilutions in HBS-EP+ running buffer (GE Healthcare) at concentrations ranging from 200 nM to 2.5 nM for the T200 and from 500 nM to 2 nM for the 8k. Cyno TREM1 ECD analytes (in-house, His-tagged) were injected in 3-fold serial dilutions in HBS-EP+ running buffer (GE Healthcare) at concentrations from 4100 nM to 17 nM, run only on the T200. Buffer blank injections were included to subtract instrument noise and drift.

[0438] Kinetic parameters were determined using a 1:1 binding model using Biacore T200 Evaluation software (version 3.0) or Biacore Insight Evaluation software (version 3.0) as appropriate. 12172gL2gH11 hIgG4P was shown to have an affinity of 0.52 nM for human TREM1 and 870 nM for cynomolgus TREM1. The kinetic parameters are summarized in Table 14.

[0439] Table 14. Kinetic parameters of 12172gL2gH11 hIgG4P binding to human and cynomolgus TREM1 [Table 14]

[0440] Example 7. Binding of 12172gL2gH11 hIgG4P to different species of TREM1 The kinetics of 12172gL2gH11 hIgG4P binding to different TREM1 species was measured by surface plasmon resonance on a Biacore T200 instrument at 25° C. Species tested were human, cynomolgus monkey, rhesus monkey, marmoset, rat, mouse, dog and pig.

[0441] A goat anti-human IgG, Fc fragment specific antibody (F(ab')2 fragment, Jackson ImmunoResearch 109-006-098) was immobilized on a CM5 sensor chip via amine coupling chemistry to a level of approximately 5000 RU. The reference cell was treated similarly. After amine coupling was completed, all subsequent solutions, except for the capture solution, were run over the reference and sample cells in sequence, and the response of the reference cell was subtracted from the sample cell throughout the run.

[0442] Each analytical cycle consisted of capture of approximately 250 RU of 12172gL2gH11 hIgG4P onto the anti-Fc surface, injection of analyte for 180 seconds (25° C., flow rate 30 μl / min), dissociation of analyte for 600 seconds, followed by surface regeneration (by injection of 50 mM HCl for 60 seconds, injection of 5 mM NaOH for 30 seconds, and another 60 seconds of 50 mM HCl). TREM1 ECD analyte (in-house, His-tagged) was injected in 3-fold serial dilutions into HBS-EP+ running buffer (GE Healthcare), with the highest concentrations shown in Table 15, and 3-fold serial dilutions performed down to a minimum concentration of 2 nM. A buffer blank injection was included to subtract instrument noise and drift.

[0443] Kinetic parameters were determined using a 1:1 binding model using Biacore Insight Evaluation software (version 3.0). This experiment showed that 12172 gL2gH11 hIgG4P has an affinity of 0.54 nM for human TREM1 (kinetic parameters summarized in Table 15). Minimal binding responses were observed for cynomolgus and rhesus monkeys, but were insufficient to determine binding kinetics. No binding of 12172 gL2gH11 hIgG4P to TREM1 of any other species was detected (summarized in Table 16).

[0444] Table 15. Kinetic parameters of 12172gL2gH11 hIgG4P binding to human TREM1 [Table 15]

[0445] Table 16. Report point data showing binding of TREM1 species to 12172gL2gH11 hIgG4P. BL indicates binding delay: the average signal 7.5-12.5 s before the end of TREM1 injection is subtracted from the average signal 7.5-12.5 s before the start of this injection. SE indicates early stability: the average signal 7.5-12.5 s after the end of TREM1 injection is subtracted from the average signal 7.5-12.5 s before the start of this injection. Theoretical Rmax is the signal generated if all captured antibody (150 kDa) is fully bound to two molecules of TREM1. SR_BL is BL divided by theoretical Rmax and SR_SE is SE divided by theoretical Rmax. [Table 16]

[0446] Example 8.12 Blockade of the TREM1 / PGLYRP1 interaction by 172gL2gH11 hIgG4P 12172gL2gH11 hIgG4P was demonstrated to block the interaction between human TREM1 and human PGLYRP1 at 25° C. by surface plasmon resonance on a Biacore T200 instrument.

[0447] Goat anti-human IgG, Fc fragment specific antibody F(ab')2 fragment, Jackson ImmunoResearch 109-006-098) was immobilized to a level of approximately 4000 RU on all four flow cells of an HC30M sensor chip (XanTec Bioanalytics) via amine coupling chemistry. The response of flow cell 1 was subtracted from the response of flow cell 2 throughout the run, and similarly the response of flow cell 3 was subtracted from the response of flow cell 4 throughout the run.

[0448] Each analytical cycle consisted of capture of approximately 100RU of TREM1-Fc (R&D 1278-TR Lot GZF0220071) onto the surface of flow cell 2, capture of approximately 150RU of 12172gL2gH11 hIgG4P onto the surface of flow cell 4, and TREM1 ECD analyte (in-house, His tagged) was flowed over the surfaces of flow cells 3 and 4 for 180 seconds. A mixture of PGLYRP1 (R&D 2590-PGB, NLC1520031) and PGN (Invivogen tlrl-ksspgn Lot KSS-41-01) was flowed over all surfaces for 180 seconds and binding was monitored, followed by a 300 second dissociation period. The surfaces were then regenerated (by injection of 50 mM HCl for 60 seconds, 5 mM NaOH for 30 seconds, and 50 mM HCl for another 60 seconds).

[0449] 12172gL2gH11 hIgG4P blocked the interaction between PGLYRP1 and TREM1 in the presence and absence of PGN (see Table 17).

[0450] Table 17. Relative responses at the binding late reporting time point. This is calculated during the infusion of PGLYRP1 and PGN: the average signal from 7.5 to 12.5 seconds before the end of the infusion is subtracted from the average signal from 7.5 to 12.5 seconds before the start of this infusion. This value is then subtracted from the value at an equivalent cycle where no ligand is captured. This shows that TREM1-Fc binds to 5 RU of PGLYRP1 alone and 12 RU of a mixture of PGLYPR1 and PGN, but PGLYRP1 does not bind to TREM1 captured on a surface coated with 12172gL2gH11 hIgG4P. [Table 17]

[0451] Example 9: Determination of the binding interface between human PGLYRP1 and human TREM1 by crystallography Crystallography Human TREM1 IgV domain was complexed with full-length human PGLYRP1 (1:1 molar ratio) at 15 mg / ml in PBS (pH 7.0) and incubated for 1 h at 4° C. Proteins were co-crystallized in hanging drops by vapor diffusion consisting of 0.2 ul of protein and 0.2 ul of reservoir Molecular Dimensions ProPlex screen D7 (15% (w / V) PEG6000, 100 mM trisodium citrate / sodium hydroxide pH 5.5). 20% ethylene glycol was used for cryoprotection.

[0452] X-ray diffraction, data collection, structure determination and refinement Cryogenic (100 K) X-ray diffraction data were collected remotely on an APS21-ID-F. Raw data frames were indexed, integrated, and scaled using XDS. The space group of the crystals was P21 with unit cell parameters of a=58.68 Å, b=98.73 Å, c=60.43 Å, α=90°, β=109.145°, γ=90° to a resolution of 2.55 Å. The quality parameters of the structure were good with an overall R-factor of the structure=17.9% and a free R-factor=22.9%. The protein complex structure was modeled in COOT and refined using PHENIX with the TLS protocol. Water molecules were added and confirmed by COOT.

[0453] Human TREM1 IgV domain (positions 21-139) (SEQ ID NO: 9) complexed with full-length PGLYRP1 (SEQ ID NO: 10). The stoichiometry of the crystal complex is 1:2, with a single hTREM1 molecule bound to a dimerized PGLYRP1 molecule despite an equimolar mix. Analysis of the intermolecular distance of 4 Å or less between the IgV-like domain of human TREM1 and hPGLYRP1 was performed using the program NCONT of the CCP4 suite of programs. The epitope has been crystallographically determined as follows: residues E27, D42-E46, A49, Y90-L95 and F126 (positions correspond to SEQ ID NO: 1). Figure 3A shows the structural mapping of the PGLYRP1 ligand-binding site on hTREM1:PDBPDB 1SMO.

[0454] Example 10: Determination of the 12172 rabbit parental Fab human TREM1 epitope by X-ray crystallography Crystallography X-ray crystallography was used to identify the exact epitope of the 12172 antibody. Human TREM1 IgV domain (SEQ ID NO: 9) was complexed with rabbit parental Fab (1:1 molar ratio) in PBS, pH 7.4 and applied to size exclusion. Chaperone Fab11994 was mixed with hTREM1:Fab complex at 15mg / ml and incubated for 1 hour at 4°C. Proteins were co-crystallized in hanging drop vapor diffusion consisting of 0.2ul protein and 0.1ul reservoir Molecular Dimensions ProPlex Screening A6 (25% (w / V) PEG1000, 200mM sodium chloride, 100m potassium phosphate dibasic / sodium phosphate monobasic pH 6.5). 20% ethylene glycol was used for cryoprotection.

[0455] X-ray diffraction, data collection, structure determination and refinement Cryogenic (100 K) X-ray diffraction data were collected remotely on an APS21-ID-F. Raw data frames were indexed, integrated, and scaled using XDS. The space group of the crystals was I222 with unit cell parameters a=91.98 Å, b=139.10 Å, c=224.81 Å, α=90°, β=90°, γ=90° to a resolution of 2.60 Å. The quality parameters of the structure were good with an overall R-factor of the structure=19.3% and a free R-factor of 22.6%. The protein complex structure was modeled in COOT and refined using PHENIX with the TLS protocol. Water molecules were added and confirmed by COOT.

[0456] The human TREM1 IgV domain (positions 21-139) (SEQ ID NO: 9) complexed with rabbit parental Fab (12172) (SEQ ID NO: 19 and 23) was obtained in the presence of a chaperone Fab molecule (11994). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), it was confirmed that the 11994 Fab chaperone used in the crystallography of the 12172 rabbit parental Fab does not affect the binding of 12172 to TREM1. The stoichiometry of the crystal complex is 1:1:1, with the Fab epitope binding on both sides of the hTREM1 molecule. Analysis of the intermolecular distance of 4 Å or less between the IgV-like domain of human TREM1 and Fab12172 was performed using the program NCONT of the CCP4 suite of programs. The epitope was determined crystallographically as follows: residues E26-K32, Q35, T36, D38, K40, D42, R97, D127, T134 and G136 (positions correspond to SEQ ID NO: 1). Figure 3B shows the structural mapping of the Fab 12172 epitope on hTREM1:PDBPDB 1SMO. This epitope was confirmed to be distinct from that of PGLYRP1 (see Example 9).

[0457] Example 11. Development of full length antibody 12172gL2gH11 hIgG4P-mammalian cell line. To demonstrate stable expression of 12172gL2gH11 hIgG4P, a stable expressing mammalian cell line was generated. The plasmid vector 12172_gL2_ckappa_gH11_IgG4(p) was transfected into a CHO cell line. The cell line was cloned and evaluated for suitability for the appropriate manufacturing process. The cell line was evaluated in a small-scale model of a production fed-batch bioreactor to assess the quality and quantity of the protein of interest and ensure that the optimal cell line was selected. A clonal CHO cell line was selected that expressed 12172gL2gH11 hIgG4P at an acceptable level and contained more than 95% monomer.

[0458] Example 12. Characterization of antibody molecules by liquid chromatography-mass spectrometry (LC-MS). The molecular weights (MW) of the resulting 12172 gL2gH11 (hIgG4P and hIG1 LALA) and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) antibody molecules were measured by LC-MS, both for the intact molecules (non-reduced) and for the separate heavy and light chains (reduced), using a Waters ACQUITY UPLC system equipped with a Xevo G2 Q-ToF mass spectrometer. Samples (approximately 5 μg) were reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) in 150 mM ammonium acetate at 37° C. for 40 min. For non-reduced (intact) measurements, samples were diluted to the same concentration in PBS pH 7.4 and incubated as above before analysis. The LC column was a Waters BioResolve™ RP mAb Polyphenyl, 450 Å, 2.7 μm held at 80° C. and equilibrated with 95% solvent A (water / 0.02% trifluoroacetic acid (TFA) / 0.08% formic acid) and 5% solvent B (95% acetonitrile / 5% water / 0.02% TFA / 0.08% formic acid) at a flow rate of 0.6 mL / min. Proteins were eluted with a gradient of 5% to 50% solvent B over 8.8 min, followed by washing and re-equilibration with 95% solvent B. UV data were acquired at 280 nm. MS conditions were as follows: ion mode: ESI positive ion, resolved mode, mass range: 400-5000 m / z and external calibration with NaI.

[0459] Data was analyzed using Waters MassLynx™ and MaxEnt Software.

[0460] As shown in Tables 18 and 19, the predicted MW from the sequences of the 12172 gL2gH11 (hIgG4P and hIgG1 LALA) and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) antibody molecules was consistent with the measured MW for the intact molecules and for the heavy and light chains by LC-MS.

[0461] Table 18. Intact (non-reduced) LC-MS data for 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) antibody molecules. [Table 18]

[0462] Table 19. Reduced LC-MS data for 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) antibody molecules. [Table 19]

[0463] Example 13. Thermal stability (Tm) measurements. The melting temperature (Tm) or temperature at the midpoint of unfolding was determined using (i) thermal shift assays or (ii) differential scanning calorimetry (DSC) to assess the conformational stability of the molecules and therefore their robustness to manufacturing and long-term stability.

[0464] Thermal shift assay Thermal shift assays were performed in early graft screening and selection (see Example 3).

[0465] The fluorescent dye SYPRO® Orange was used to monitor the protein unfolding process by binding to hydrophobic regions that become exposed as the temperature increases. The reaction mixture contained 5 μL of 30× SYPRO® Orange Protein Gel Stain (Thermofisher scientific, S6651) diluted from a 5000-fold concentration in test buffer. 45 μL of sample at 0.2 mg / mL in common preformulation storage buffer (pH 7.4) was added to the dye and mixed. 10 μL of this solution was dispensed in quadruplicate into a 384 PCR optical well plate and run on a QuantStudio7 real-time PCR system (Thermofisher™). The PCR system heating device was set to 20° C. and ramped to 99° C. at a rate of 1.1° C. / min. The charge-coupled device monitored the fluorescence change in the wells. The increase in fluorescence intensity was plotted and the inflection point of the slope was used to generate the apparent midpoint temperature (Tm). The data are shown in Tables 11 and 12 (see Example 3).

[0466] Differential Scanning Calorimetry (DSC) Thermal stability was assessed using differential scanning calorimetry. All samples were diluted to 10 μM in a common preformulation storage buffer (pH 7.4 or pH 5.0, total volume 400 μL), added to a 96-well plate, and centrifuged at 4,000 × g for 5 min to remove air bubbles. Plates were run on an automated MicroCal VP DSC (Malvern Panalytical) from 10 to 100 °C at a rate of 1 °C / min with a 15 min prescan thermostat, 8 s filtering period, and passive feedback mode. Data were buffer subtracted using manual baseline correction and the data were fitted to a non-two-state model in Origin 7.0. Transition midpoints (Tm) and onset of unfolding are shown in Table 20 below and Figure 4.

[0467] The IgG4P isotype fitted three transitions and the IgG1 LALA fitted two transitions, and the unfolding of Fab and CH3 could not be distinguished. Thermal stability was within the expected range for each isotype.

[0468] Table 20. Summary of thermal stability data for 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) in a common preformulation storage buffer pH 7.4. Tm1=CH2 unfolding, Tm2=Fab unfolding, Tm3=CH3 unfolding. [Table 20]

[0469] Example 14. Experimental isoelectric point (pI) determination. The experimental pI was found to be similar to the hIgG4P format for 12172 gL2gH11 and 12172 gL6gH6. This was also observed for the hIgG1 LALA molecule. The pI was within the range expected to be good for the manufacturing process and formulation buffers. The presence of different charged species was consistent with observations of other therapeutic molecules and was attributed to common post-translational modifications such as C-terminal heavy chain removal of lysines.

[0470] Example 15. Hydrophobic interaction chromatography (HIC). Hydrophobic interaction chromatography (HIC) was used to measure the hydrophobicity of 12172 gL2gH11 and 12172 gL6gH6 as hIgG4P formats. HIC separates molecules in order of increasing hydrophobicity. Molecules bind to a hydrophobic stationary phase in the presence of high concentrations of polar salts and desorb into the mobile phase as the concentration of salt decreases. The longer the retention time, the more hydrophobic the molecule.

[0471] All molecules showed low apparent hydrophobicity (retention time less than 15 min). There was no significant difference between the 12172 gL2gH11 and 12172 gL6gH6 hIgG4P molecules. Similarly, there was no significant difference in the hydrophobic retention times of the hIgG1 LALA samples. The hIgG4P molecules showed a slightly slower retention time compared to the corresponding hIgG1 LALA molecules.

[0472] Example 16. Solubility measurements using the polyethylene glycol (PEG) aggregation assay. A PEG aggregation assay was used as a mimic of high concentration solubility. PEG is a non-adsorbing, non-denaturing polymer, and due to its inert nature, it has been used to promote protein precipitation primarily through excluded volume effects. Samples were exposed to increasing concentrations of PEG3350, and the amount of sample remaining in solution was determined by plotting the absorbance at A280 nm. Measurement of the %PEG concentration at which half of the sample precipitated generated a PEG midpoint (PEGmdpnt) score. This score allows test molecules to be ranked based on their apparent native state aggregation propensity, with a lower PEGmdpnt score (e.g., ≦10) indicating a greater propensity for native state aggregation.

[0473] Stock 40% PEG3350 (Merck, 202444) solutions (w / v) were prepared in common preformulation storage buffers pH 7.4 and 5.0 and common preformulation buffer pH 5.5). Serial titrations were performed by an ASSIST PLUS liquid handling robot (INTEGRA 4505) to yield PEG3350 ranging from 40% to 15.4%. Sample preparation consisted of mixing antibody and PEG solutions in a 1:1 volumetric ratio to minimize non-equilibrium precipitation. 35 μL of PEG3350 stock solution was added by the liquid handling robot to a 96-well v-bottom PCR plate (A1-H1). 35 μL of 2 mg / mL sample solution was added to the PEG stock solution to yield a 1 mg / mL test concentration and final PEG3350 concentrations ranging from 20% to 7.7%. The solutions were mixed by automated slow repetitive pipetting and incubated at 37 °C for 0.5 h to redissolve non-equilibrium aggregates. The samples were then incubated at 20° C. for 24 hours. The sample plate was then centrifuged at 4000×g for 1 hour at 20° C. 50 μL of the supernatant was dispensed into a UV-Star®, half-area, 96-well, μClear®, microplate (Greiner, 675801). Protein concentrations were determined by UV spectrophotometry at 280 nm using a FLUOstar® Omega multidetection microplate reader (BMG LABTECH). The obtained values ​​were plotted using Graphpad Prism (version 7.04) and the PEG midpoint (PEG mdpnt ) scores were derived from the midpoint of a sigmoidal dose-response (variable slope) fit.

[0474] Data shown in Table 21 indicates that a higher PEG midpoint (%) equates to higher solubility.

[0475] Buffer-dependent solubility was observed for the molecules tested. In the common preformulation storage buffer pH 7.4, both 12172 gL2gH11 and 12172 gL6gH6 isotypes (hIgG4P and hIgG1 LALA) showed low PEG midpoint scores, indicating poor solubility at high concentrations. Increased PEG midpoint scores were observed in the common preformulation storage buffer pH 5 buffer. Notably, all samples showed substantially improved PEG midpoint scores when formulated in the common preformulation buffer pH 5.5. The hIgG1 LALA sample did not precipitate at the highest tested concentration of PEG3350 in this buffer.

[0476] Table 21. PEG aggregation assay data for 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) in common preformulation storage buffers pH 7.4 and 5.5, and common preformulation buffer pH 5.5. Higher PEG % midpoint = higher concentration solubility. NB* samples showed signs of aggregation at the lowest tested concentration of PEG3350 (7.7%), therefore an accurate PEG midpoint could not be generated. [Table 21]

[0477] Example 17. Assessment of protein-protein self-interactions using AC-SINS (Affinity Capture Self-Interaction Nanoparticle Spectroscopy). The AC-SINS assay was used to screen the developability of humanized molecules including 12172 gL2gH11 and 12172 gL6gH6 (as hIgG4P and hIgG1 LALA; see also Example 3) by determining protein-protein self-interaction propensity and thus inform potential aggregation stability. This was performed in a common pre-formulation storage buffer pH 7.4.

[0478] Goat anti-human Fcγ-specific capture antibody (Jackson ImmunoResearch) was buffer exchanged into 20 mM sodium acetate, pH 4.3, diluted to 0.4 mg / mL, and 50 μL was added to 450 μL of citrate-stabilized 20 nm gold nanoparticles (TedPella, USA) and left at room temperature overnight. The bound nanoparticles were blocked with 55 μL of PEG methyl ether thiol (average Mn=2,000 (Sigma#729140)) for 1 h, centrifuged at 21,000×g for 6 min, the supernatant removed, and resuspended in 20 mM sodium acetate, pH 4.3 to a final volume of 150 μL.

[0479] Antibody samples were diluted to 22 μg / mL in common preformulation storage buffer pH 7.4 (180 μL) and added to 20 μL of mock supernatant and 200 μL of nonspecific total IgG at 222 μg / mL (Jackson ImmunoResearch), vortexed briefly, and 72 μL was added to a 96-well plate. 8 μL of nanoparticles were added to each well (n=4). Absorbance was read from 500-600 nm on a BMG plate reader and fitted to a Lorentz curve (RShiny), and the common preformulation storage buffer alone was subtracted from the samples to obtain Δλmax.

[0480] The data is summarized in Table 22, where a higher Δλmax (nm) value equates to a higher protein-protein self-interaction propensity. The hIgG1 LALA molecules for both 12172 gL2gH11 and 12172 gL6gH6 were found to exhibit lower self-interaction than the corresponding hIgG4P molecules, as indicated by the lower Δλmax (nm). Furthermore, the 12172 gL6gH6 (hIgG4P and hIgG1 LALA) molecules exhibited slightly lower Δλmax (nm) values ​​than the 12172 gL2gH11 (hIgG4P and hIgG1 LALA) molecules.

[0481] Table 22. Self-interaction measurements (AC SINS) for 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1). Low values ​​= less protein-protein self-interactions. [Table 22]

[0482] Example 18. kD interaction parameter measurement (colloid stability) The kD interaction parameter is used to assess colloidal stability, with positive and negative values ​​relating to repulsive and attractive intermolecular forces, respectively.

[0483] Dynamic light scattering (DLS) was performed on a DynaPro III plate reader (Wyatt Technology Corp, Santa Barbara, CA, USA). Samples were either diluted in a common preformulation storage buffer pH 7.4 or buffer exchanged into a common preformulation storage buffer pH 5.0 and diluted from 7 mg / mL to 1 mg / mL in 1 mg / mL increments. The wells containing buffer were selected as the solvent offset and measurements were performed at 25°C, the laser power was set to 20% and auto attenuation was enabled. Each measurement was the average of three (5 x 3) 5, 5 second scans. The diffusion coefficients were measured (Dm) and the interaction parameters (kD) were calculated according to the following equation, where D0 represents the diffusion coefficient at infinite dilution: Dm = D0(1+K D C) Formula: D0 given by Debye plot at Y-intercept. Slope=kD*D0

[0484] Diffusion coefficients are measured as a function of protein concentration and kD, which is used to assess colloidal stability, with positive and negative values ​​indicating repulsive and attractive intermolecular forces, respectively. For samples that exhibit attractive / self-association, the diffusion coefficient increases as a function of protein concentration, which is reflected by negative kD values. The data are shown in Table 23.

[0485] The kD interaction parameters were shown to be less negative (more colloidally stable) for both hIgG4P and hIgG1 LALA molecules in a common preformulation storage buffer pH 5 compared to the data obtained in a common preformulation storage buffer pH 7.4. The hIgG1 LALA molecules were shown to be more stable than the corresponding hIgG4P molecules. Furthermore, the 12172 gL6gH6 (hIgG4P and hIgG1 LALA) molecules showed slightly greater colloidal stability than the 12172 gL2gH11 (hIgG4P and hIgG1 LALA). This data confirmed the data generated from the AC-SINS assay (see Example 17).

[0486] Table 23. kD interaction parameter data for 12172 gL2gH11 and 12172 gH6gL6 (hIgG4P and hIgG1 LALA). More negative values ​​= more attractive force (higher protein-protein self-interaction). [Table 23]

[0487] Example 19. Effect of mechanical stress on aggregate stability (aggregation assay). Proteins tend to unfold when exposed to an air-liquid interface, exposing their hydrophobic surfaces to a hydrophobic environment (air) and their hydrophilic surfaces to a hydrophilic environment (water). Agitation of the protein solution achieves a large air-liquid interface that can promote aggregation. This assay mimics the stresses molecules undergo during production (e.g., ultrafiltration) and serves to provide stringent conditions to try to distinguish between different antibody molecules.

[0488] Samples in common preformulation storage buffer pH 7.4 or pH 5 were stressed by vortexing using an Eppendorf Thermomixer Comfort™. Prior to vortexing, concentrations were adjusted to 1 mg / mL using appropriate extinction coefficients (1.42 and 1.43 for hIgG1 LALA and hIgG4P, respectively, 280 nm, 1 mg / mL, 1 cm path length) and absorbance at 595 nm obtained using a Varian Cary® 50-Bio spectrophotometer to establish a time 0 reading. Each sample was aliquoted into 1.5 mL conical Eppendorf® capped tubes (3×250 μL) and vortexed at 1400 rpm at 25° C. for 24 hours. Aggregation (turbidity) was monitored by measuring samples at 595 nm 3 hours and 24 hours after vortexing using a Varian Cary® 50-Bio spectrophotometer. Data are summarized in Table 24.

[0489] Both 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1 LALA) showed good aggregation stability in both buffers (common preformulation storage buffers pH 7.4 and pH 5) at 3 hours after vortexing, i.e. no turbidity was observed at 595 nm. At 24 hours, it was possible to distinguish molecules where 12172 gL2gH11 and 12172 gL6gH6 (hIgG1 LALA) showed greater aggregation stability than the corresponding hIgG4P molecules in both buffers. For hIgG4P molecules, greater aggregation stability was observed in the common preformulation storage buffer at pH 7.4 compared to pH 5. 12172 gL2gH11 and 12172 gL6gH6 (as hIgG4P and hIgG1 LALA) would be expected to be aggregation stable to shear stress conditions during manufacturing, e.g. ultrafiltration.

[0490] Table 24. Effect of stress at the air-liquid interface for 12172 gL2gH11 and 12172 gL6gH6 (hIgG4P and hIgG1) in common preformulation storage buffers pH 7.4 and pH 5 (turbidity at 595 nm). [Table 24]

[0491] Example 20.12 Viscosity evaluation at different concentrations for 172 gL2gH11 (hIgG4P and hIgG1 LALA). Low viscosity at high antibody concentrations is important for subcutaneous administration of therapeutic molecules, therefore, the viscosity at increasing concentrations in a common pre-formulation buffer (pH 5) was obtained and measured for 12172 gL2gH11 (hIgG4P and hIgG1 LALA) to assess suitability for subcutaneous administration.

[0492] Testing was performed by measuring (i) the initial concentration of the sample and (ii) viscosity, as detailed below.

[0493] Concentration of 12172 gL2gH11 (hIgG4P and hIgG1 LALA). 12mL of 12172 gL2gH11 hIgG4P (15.2mg / mL) and 11mL of 12172 gL2 gH11 IgG1 LALA (15.5mg / mL) in common preformulation storage buffer pH 5.0 were concentrated at 4000xg and 20°C using Vivaspin20 MWCO 30kDa centrifugal filters (Z14637, Sigma-Aldrich). Samples were centrifuged until a volume of approximately 750μL was obtained. The retentate was collected and the resulting antibody concentration was determined using UV absorbance measurements at 280nm (NanoDrop™ 1000). Extinction coefficients of 1.43mL / (mg cm) for 12172gL2gH11 hIgG4P and 1.42mL / (mg cm) for 12172 gL2gH11 IgG1 LALA were used.

[0494] The antibody samples were then diluted using a common pre-formulation storage buffer pH 5.0 to obtain a suitable concentration range for viscosity testing. The concentrations of the diluted antibodies were confirmed by re-measuring UV absorbance at 280 nm. The concentrations were found to be 158 mg / mL, 94 mg / mL and 52 mg / mL for 12172 gL2gH11 hIgG4P and 144 mg / mL, 100 mg / mL and 45 mg / mL for 12172 gL2gH11 hIgG1 LALA.

[0495] Viscosity measurements of 12172 gL2gH11 (hIgG4P and hIgG1 LALA). The viscosity at each concentration was measured using a Discovery Hybrid Rheometer-1 (DHR-1, TA Instruments) equipped with a Peltier plate and liquid cooling system for temperature control, and a 20 mm stainless steel parallel plate geometry for measurement. The sample (80 μL) was placed at the center of the Peltier plate, and the viscosity was measured from 2.87918 to 287.918 s -1 Viscosity ((mPa s, or cP)) was measured with a steady-state flow sweep procedure set at 20 °C at various shear rates of 100 s. When the values ​​for each shear rate point were constant (SD ± 5%), the measured viscosities were averaged. Different concentrations of both 12172 gL2gH11 hIgG4P and 12172 hIgG1 LALA molecules were measured using the instrument to observe the change in viscosity with respect to sample concentration. The results are summarized in Table 25.

[0496] Both 12172 gL2gH11 hIgG4P and 12172 hIgG1 LALA molecules showed an increasing trend between concentration and viscosity coefficient. The viscosity increased from 1.2 to 4.1 cP and for 12172 hIgG4P at concentrations between 52 and 158 mg / ml. Similarly, the viscosity of the IgG1 LALA molecule increased from 1.4 to 5.4 cP and for concentrations between 45 and 144 mg / ml. All these samples showed a constant viscosity coefficient (variability less than 5%) at different shear rates. The results showed that 12172 hIgG4P and 12172 hIgG1 LALA showed low viscosity levels at higher concentrations and therefore could be considered suitable for subcutaneous administration.

[0497] Table 25. Mean viscosity (centipoise (cP)) at 20°C for different concentrations of 12172 gL2gH11 (hIgG4P and hIgG1 LALA) in common pre-formulation storage buffers pH 5. [Table 25]

[0498] Example 21. Evaluation of functional activity of 12172 gL2gH11 hIgG4P using the human TREM1 THP1 NF-κB reporter cell line The aim of this study was to evaluate the ability of 12172 gL2gH11 hIgG4P to inhibit PGLYRP1 / PGN-mediated NF-κB signaling activation through human TREM1. To do this, THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cells were used. These cells stably express human TREM1, human DAP12 and NF-κB luciferase reporter genes. PGLYRP1 complexed with soluble peptidoglycan (PGN) from Escherichia coli (E. coli) was used as the TREM1 ligand, which induces NF-κB activation by binding to TREM1. PGN, which does not bind to TREM1, also induces NF-κB activation, but to a lesser extent and via an alternative signaling pathway. Inhibition of luciferase activity demonstrates the functional blocking activity of 12172 gL2gH11 hIgG4P in this system.

[0499] THP1 monocyte TREM1 / DAP12 NF-κB luciferase reporter cells were cultured in complete medium containing selection antibiotics (RPMI + 10% FBS + 50 μM 2-mercaptoethanol + 10 μg / ml blasticidin + 1 μg / ml puromycin + 200 μg / ml geneticin) using standard tissue culture techniques. Three days prior to assay setup, cells were plated at 10 × 10 in 50 ml complete medium (200,000 cells / ml) in a T175 flask. 6Cells were seeded and placed flat in an incubator. On the day of the assay, cells were removed from the flask, transferred to a 50 ml Falcon and centrifuged at 300 × g for 5 min. The medium was removed and cells were resuspended in 5-10 ml of complete medium and counted. Cells were then diluted to 1 × 10 by adding the cell suspension to complete medium. 6 Cells were resuspended at 1000 cells / ml and 10 μl / well was added to the assay plate (Corning#3570). 12172 gL2gH11 hIgG4P was serially diluted in complete medium in a 384-well dilution plate (Greiner#781281) to a final assay concentration range of 33.3 nM to 1.69 pM. Serial dilutions of 12172 gL2gH11 hIgG4P were then transferred to the assay plate (10 μl / well) and the assay plate was incubated for 1 hour at 37°C / 5% CO2. Recombinant human PGLYRP1 (R&D Systems#2590-PGB) was complexed with PGN (Invivogen#tlrl-ksspgn) in sterile DPBS for 1 hour at room temperature. After 1 hour, the solutions were diluted with complete medium and then transferred to the assay plate (10 μl / well) to give a final assay concentration of 2.5 μg / ml PGLYRP1 and 10 μg / ml PGN. Plate controls (no antibody added) included PGLYRP1 / PGN complex and PGN alone as assay maximum and minimum, respectively. The assay plate was then incubated at 37° C. / 5% CO2 for 16±2 hours. After incubation, luciferase activity was measured using the SteadyGlo Luciferase Assay System (Promega#E2520). Steady-Glo reagent was prepared according to the manufacturer's instructions and 30 μl / well was added to the assay plate. The plate was then centrifuged at 200×g for 3 minutes and then incubated at room temperature for an additional 2 minutes for a total incubation time with SteadyGlo reagent of 5 minutes. Luminescence was then measured using a Synergy Neo2 plate reader and raw luminescence values ​​were used to determine relative inhibition compared to control wells. 4PL Curve Fitting and IC 50 Values ​​were calculated using ActivityBase v9.4.

[0500] Table 26. Summary of potency, efficacy and slope values ​​for 12172 gL2gH11 hIgG4P [Table 26]

[0501] Example 22. Efficacy and potency of 12172 antibody in blocking TREM1-mediated pro-inflammatory cytokine and chemokine release from primary human monocytes To assess the ability of anti-TREM1 12172 mutant antibodies to block TREM1 signaling, the release of proinflammatory cytokines and chemokines from activated primary human monocytes was measured after 12172 antibody treatment. Monocytes were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) of healthy human donors by negative selection (Miltenyi, 130-117-337). Monocyte viability and purity were assessed by flow cytometry and exceeded ≥ 90%. Monocytes were plated at 5 × 10 4 Cells were seeded at a density of 1000 cells / well and stimulated with precomplexed peptidoglycan from Bacillus subtilis (PGN-BS; 3 μg / ml; Invivogen, tlrl-pgnb3) and recombinant human peptidoglycan recognition protein 1 (PGLYRP1; 1 μg / ml; R&D Systems, 2590-PGB) to activate TREM1. ( Cell supernatants were harvested after 24 hours for measurement of pro-inflammatory cytokine release (TNF-α, IL-6, IL-1β) by immunofluorescence assay (Cisbio).

[0502] As shown in Table 27, the 12172 gL2gH11 hIgG4P variant inhibited TNF-α (geometric mean IC 50 =15pM), IL-6 (geometric mean IC 50 =27pM) and IL-1β (geometric mean IC 50=5 pM) in primary human monocytes. As shown in Figure 5, the potency of 12172 gL2gH11 hIgG4P in primary human monocytes was observed across donors.

[0503] To further evaluate the ability of anti-TREM1 12172 mutant antibodies in blocking TREM1-mediated pro-inflammatory cytokine and chemokine release, supernatants from primary human monocytes treated with anti-TREM1 antibodies (1 nM) and activated with precomplexed PGN-BS / PGLYRP1 were quantitatively analyzed using two multiplex immunoassays: MILLIPLEX® Human Cytokine / Chemokine / Growth Factor Panel A (Merck Millipore, HCYTA-60K-PX48) and a custom LegendPlex panel (Biolegend).

[0504] As shown in Table 28, inhibition of TREM1 by different 12172 antibody variants strongly reduced the release of multiple cytokines and chemokines (CCL-3, CCL-20, CXCL-9, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-6, IL-10, IL-12p40, IL-15, IL-18, IL-27, TNF-α, TNF-β) from activated primary human monocytes (n=4 donors). The 12172 gL2gH11 hIgG4P variant was the most effective 12172 variant, with percent inhibition values ​​ranging from 57-110%. As shown in Figure 6, 12172 gL2gH11 hIgG4P significantly increased the release of IL-1R antagonist (IL-1RA), a negative regulator of the IL-1 pathway, from primary monocytes across donors. IL-1RA is a monogenic (mutation causing low levels of IL-1RA) associated with severe systemic autoimmune disease. A single nucleotide polymorphism in IL-1RN (encoding IL-1RA) has been identified in ALS patients. Higher circulating IL-1RA levels are significantly associated with a lower risk of ALS (Yuan et al. 2020 Eur J Neurol). IL-1RA levels are also significantly reduced in the cerebrospinal fluid of AD patients compared to healthy controls (Tarkowski et al. 2001 Dement Geriatr Cogn Disord). In contrast to 12172 gL2gH11 hIgG4P, the prior art anti-TREM1 antibody (0318-IgG1.3f) did not affect IL-1RA release from primary monocytes.

[0505] Table 27. Potency of different 12172 variants on TNF-α, IL-6 and IL-1β release [Table 27]

[0506] Table 28. Efficacy of different 12172 antibody variants on cytokine and chemokine release (inhibitory percentage) [Table 28]

[0507] Example 23: Efficacy and potency of 12172 gL2gH11 hIgG4P in increasing IL-1RA release from unstimulated primary human monocytes Observing that 12172 gL2gH11 hIgG4P significantly increased IL-1RA release from TREM1 ligand stimulated human monocytes, its effect on IL-1RA release from unstimulated human monocytes was also evaluated. Human monocytes were isolated and plated as described above in Example 22, antibodies were added for 24 hours, and then supernatants were collected for IL-1RA measurement using the IL-1RA Quantikine ELISA kit (R&D Systems).

[0508] As shown in Table 29 and Figure 7, 12172 gL2gH11 hIgG4P dose-dependently increased IL-1RA release in unstimulated primary human monocytes. In contrast to 12172 gL2gH11 hIgG4P, another prior art anti-TREM1 antibody (0318-IgG1.3f) did not affect IL-1RA release from unstimulated primary human monocytes.

[0509] Table 29: Efficacy and potency of 12172 gL2gH11 hIgG4P and 0318-IgG1.3f in increasing IL-1RA release from unstimulated primary human monocytes. [Table 29]

[0510] Example 24. Efficacy of 12172 gL2gH11 hIgG4P in blocking TREM1-mediated pro-inflammatory cytokine and chemokine release from PBMCs of Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS) patients Neurodegeneration and neuroinflammation in AD and ALS are associated with elevated levels of multiple pro-inflammatory cytokines and chemokines in the CSF and blood of patients. For example, levels of TNF-α, IL-6 and IL-1β are significantly increased in the blood of ALS patients (Hu et al. 2017 Sci Rep), while CCL-3, G-CSF and TNF-α are elevated in the CSF of ALS patients (Chen et al. 2018 Front Immunol), and all factors were observed to be reduced by TREM1 inhibition in human monocytes.

[0511] To assess the efficacy of anti-TREM1 12172 gL2gH11 hIgG4P in blocking TREM1 signaling in patient-derived cells, the release of pro-inflammatory cytokines and chemokines was measured in PBMCs from AD and ALS patients following TREM1 activation. PBMCs were isolated by density gradient centrifugation from whole blood of AD and ALS patients and matched healthy controls. PBMCs were plated in 96-well plates (Falcon) at 1 × 10 5 Cells were seeded at a density of 1000 cells / well, pretreated with 12172 gL2gH11 hIgG4P (1 nM) for 1 h, and stimulated with precomplexed peptidoglycan from Bacillus subtilis (PGN-BS; 3 μg / ml; Invivogen, tlrl-pgnb3) and recombinant human peptidoglycan recognition protein 1 (PGLYRP1; 1 μg / ml; R&D Systems, 2590-PGB) to activate TREM1. Cell supernatants were harvested after 24 h to measure proinflammatory cytokine and chemokine release using homogeneous time-resolved fluorescence technology (HTRF®; Cisbio) and MILLIPLEX® Human Cytokine / Chemokine / Growth Factor Panel A (Merck Millipore, HCYTA-60K-PX48).

[0512] As shown in Figure 8 and Table 30, 12172 gL2gH11 hIgG4P was effective in inhibiting TREM1-mediated TNF-α release (66% ± 15 inhibition) and IL-6 release (70% ± 17) from PBMCs of AD patients. As shown in Figure 9 and Table 31, 12172 gL2gH11 hIgG4P was also effective in inhibiting TREM1-mediated TNF-α release (72% ± 7 inhibition) and IL-6 release (69% ± 9) from PBMCs of ALS patients. As shown in FIG. 10, inhibition of TREM1 by 12172 gL2gH11 hIgG4P also strongly reduced the release of multiple cytokines and chemokines (CCL-3, CCL-4, CCL-20, CCL-22, CXCL-9, G-CSF, GM-CSF, GRO-α, IL-1α, IL-1β, IL-6, IL-10, IL-12p40, TNF-α) from ALS PBMCs (representative of n=4 donors) and AD PBMCs (representative of n=5 donors).

[0513] Table 30: Efficacy of 12172 gL2gH11 hIgG4P on TNF-α and IL-6 release from healthy control and AD PBMCs [Table 30]

[0514] Table 31: Efficacy of 12172 gL2gH11 hIgG4P on TNF-α and IL-6 release from healthy control and ALS PBMCs [Table 31]

[0515] Example 25. Transcriptome profile of human monocytes following stimulation with TREM1-ligand complexes or apoptotic iPSC-derived human motor neurons and treatment with 12172 antibody variants To further characterize the cellular profile of the anti-TREM1 12172 antibody variants, transcriptome analysis was performed on human monocytes stimulated with TREM1-ligand complexes or apoptosis-induced pluripotent stem cell (iPSC)-derived human motor neurons (ALS disease-associated ligand). Monocytes were isolated from cryopreserved peripheral blood mononuclear cells (PBMCs) of healthy human donors (n=8) by negative selection (Miltenyi, 130-117-337). Monocyte viability and purity were assessed by flow cytometry and exceeded ≥90%. Monocytes were plated at 2 × 10 per well in 6-well plates (Falcon). 6 Cells were seeded at a density of 1000 x g / ml and pretreated with 12172 antibody variants (1 nM) for 1 h. Monocytes were then stimulated for 4 h with (i) precomplexed peptidoglycan from Bacillus subtilis (PGN-BS; 3 μg / ml; Invivogen, tlrl-pgnb3) and recombinant human peptidoglycan recognition protein 1 (PGLYRP1; 1 μg / ml; R&D Systems, 2590-PGB) to activate TREM1 or (ii) UV-induced apoptotic iPSC-derived human motor neurons. RNA was isolated using RNeasy Plus Mini Kit (Qiagen) and RNA quality was assessed using Experion™ RNA Analysis Kit (Bio-Rad). Sequencing libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs) and samples were sequenced using an Illumina NovaSeq 6000.

[0516] As shown in Figure 11 (A and B) and Table 32, the highest number of significant differentially expressed genes (DEGs) was observed in the 12172 gL2gH11 and 12172 gL6gH6 hIgG4P formats after TREM1-ligand complex stimulation. Among the top DEGs, the two 12172 hIgG4P antibodies also showed similar transcriptome profiles (e.g., downregulation of HERC5, OAS1, DDX58, TNF-α). These profiles were in contrast to the prior art anti-TREM1 antibody (0318-IgG1.3f) (Figure 11C). Furthermore, as shown in Table 33, 111 genes were significantly upregulated and 121 genes were significantly downregulated in 12172 gL2gH11 hIgG4P-treated monocytes when compared to 0318-IgG1.3f-treated monocytes.

[0517] Table 32. Number of differentially expressed genes (DEGs) after treatment of human monocytes with 12172 antibody variants and stimulation with TREM1-ligand complexes (compared to ligand or isotype control), considering a false discovery rate (FDR) of 0.05. [Table 32]

[0518] Table 33. Number of DEGs considering an FDR of 0.05 between 12172 gL2gH11 hIgG4P and 0318-IgG1.3f after stimulation of human monocytes with TREM1-ligand complex. [Table 33]

[0519] As shown in Figure 12 (A and B) and Table 34, there were more DEGs after stimulation with apoptotic iPSC-derived human motor neurons compared to TREM1-ligand complex stimulation. Among the top DEGs, the two 12172 hIgG4P antibodies showed some overlap, including, for example, downregulation of CCR2 and upregulation of IL-1RN, the gene encoding IL-1RA. In contrast, the top DEGs differed from the prior art anti-TREM1 antibody (0318-IgG1.3f) (Figure 12C), where no significant upregulation of IL-1RN was observed. Furthermore, as shown in Table 35, 598 genes were significantly upregulated and 808 genes were significantly downregulated in 12172 gL2gH11 hIgG4P-treated monocytes when compared to 0318-IgG1.3f-treated monocytes.

[0520] Table 34. Number of DEGs considering an FDR of 0.05 after treatment of human monocytes with 12172 antibody variants and stimulation with apoptotic iPSC-derived human motor neurons (compared to ligand or isotype control). [Table 34]

[0521] Table 35. Number of DEGs considering an FDR of 0.05 between 12172 gL2gH11 hIgG4P and 0318-IgG1.3f after stimulating human monocytes with apoptotic iPSC-derived human motor neurons. [Table 35]

[0522] Example 26. Efficacy of 12172 antibody in blocking TREM1-mediated phagocytosis and production of reactive oxygen species (ROS) by primary human monocytes and neutrophils To assess the impact of blocking TREM1 signaling on antimicrobial immune responses, both phagocytosis and ROS production from activated primary human monocytes and neutrophils in whole blood were assessed by flow cytometry. To examine ROS production, dihydrorhodamine-123 (5 μg / ml) was added to blood (25 μl) from healthy human donors for 5 min, followed by preincubation with 12172 gL2gH11 hIgG4P or 0318-IgG1.3f antibodies (10 μg / ml) for an additional 30 min. Whole blood samples were then diluted with 1 × 10 6 After incubation with mCherry-expressing bacteria for 1 hour, samples were washed and stained with surface antibodies against CD45 and CD14 to identify neutrophils and monocytes by flow cytometry.

[0523] As shown in Figure 13, the 12172 gL2gH11 hIgG4P variant did not impair bacterial clearance by neutrophils or monocytes, nor did it affect their ability to produce reactive oxygen species. In contrast, analysis of 0318-IgG1.3f revealed a significant reduction in E. coli-induced ROS production by both neutrophils and monocytes. The phagocytic capacity of both immune cell types was not significantly affected by 0318-IgG1.3f.

[0524] Example 27: Efficacy and potency of 12172 antibody in blocking TREM1-mediated activation of spleen tyrosine kinase (SYK) Association of TREM1 with its adaptor protein DAP-12 leads to phosphorylation of DAP-12 and subsequent recruitment and phosphorylation of spleen tyrosine kinase (SYK; Carrasco et al. 2018 Cell Mol Immunol). SYK has previously been implicated in driving TREM1-mediated neuroinflammatory damage (Xu et al. 2019 Cell Death Dis) and is known to be activated following amyloid-β deposition and the formation of pathological tau species (Schweig et al. 2017 Acta Neuropathol Commun). To assess the ability of the anti-TREM1 12172 mutant antibody to block TREM1-mediated SYK activation, phosphorylated SYK (pSYK) levels were measured in Flp-In™ 293 cells stably expressing human TREM1 and human DAP-12. Cells were seeded at a density of 25,000 cells / well in 384-well plates (Greiner), pretreated with 12172 mutant or isotype antibodies for 1 h, and stimulated with precomplexed peptidoglycan from Escherichia coli (PGN-EC; 5 μg / ml; Invivogen, tlrl-pgnb3) and recombinant human peptidoglycan recognition protein 1 (PGLYRP1; 2.5 μg / ml; R&D Systems, 2590-PGB) to activate TREM1. Protein lysates were collected after 30 min for measuring pSYK levels using the AlphaLISA SureFire Ultra p-SYK (Tyr525 / 526) Assay Kit® (PerkinElmer).

[0525] As shown in Table 36 and Figure 14, all four 12172 mutant antibodies were efficient (Emax = 57-72%) and potent (357-1015 pM) in blocking SYK activation following TREM1 activation, whereas the A33 isotype antibody showed no activity.

[0526] Table 36. Efficacy and potency of 12172 mutant anti-TREM1 antibodies in blocking SYK activation in hTREM1 / hDAP-12 Flp-In293 cells (ND - not detectable) [Table 36] Example 28:12172 gL2gH11 hIgG4P cell surface TREM1 affinity measurement The kinetics of 12172 gL2gH11 hIgG4P binding to human or cynomolgus TREM1 expressed on live cells was measured at 25°C using LigandTracer. Two HEK293 polyclonal cell lines were developed in-house to express either human or cynomolgus TREM1, and parental normal adherent HEK293 were used as control cells. All three cell types were maintained in growth medium DMEM (Gibco, 21969-035) supplemented with fetal bovine serum (Invitrogen, 10082), GlutaMAX (Gibco, 35050061), and further included 0.5mg / ml Geneticin (Gibco, 10131-027) to maintain selection in the TREM1 polyclonal cell lines. The day before the experiment, 1.4 × 10 cells were plated onto each quarter of a LigandTracer MultiDish 2 × 2 (Ridgeview, 1-04-204-5) that had been pre-coated with poly-D-lysine (Gibco, A38904-01) according to the manufacturer's instructions. 6 Cells were seeded and incubated overnight at 37°C / 5%CO2. TREM1 expressing cells were seeded in 1 / 4 of each dish compartment and negative expressing control cells were seeded in the other. The next morning, the medium was replaced with exactly 1.8ml of fresh growth medium (without geneticin) in each dish compartment (half) and placed in the LigandTracer instrument. Rotation was started and a baseline reading was recorded for approximately 20 minutes or until stable. Rotation was stopped and AlexaFluor647 labeled 12172 gL2gH11 hIgG4P (in-house) was added to the expected 12172 gL2gH11 hIgG4P K DThe antibody was added at a concentration of 0.5 nM, a concentration close to the binding of the antibody to the cells. Rotation was resumed and fluorescence measurements were recorded, representing real-time binding of the antibody to the cells, until a curvature indicative of equilibrium was observed (which took about 2 hours). In this way, two more additions of the antibody were made, at 1.5 nM and 5 nM, respectively, about three times higher than the last one. Finally, all medium containing the antibody was removed and replaced with fresh medium. Rotation and measurements were continued until the dissociation signal had dropped by at least 10%, or overnight if dissociation was slow. Affinity measurements were analyzed and calculated within the LigandTracer "TraceDrawer" software (version 1.9.2). Raw data readings for binding of 12172 gL2gH11 hIgG4P to TREM1-expressing cells were first normalized by subtracting the equivalent readings from binding to control cells. The subtracted traces were evaluated using the 1:1 binding model of the software. If the 1:1 model was not appropriate for the data traces, alternative models were considered. 12172 gL2gH11 hIgG4P was shown to have an affinity of 16.5 pM for human TREM1 and approximately 300-fold weaker affinity for cynomolgus TREM1. Kinetic parameters are summarized in Table 37 and 38. 12172 gL2gH11 hIgG4P showed binding well represented by the 1:1 model. Five replicate experiments gave similar data, although the slow dissociation rate is difficult for the LigandTracer instrument to measure, toward the limits of the accurate range. Binding of 12172 gL2gH11 hIgG4P to cynomolgus TREM1 was significantly more complex and did not fit the 1:1 binding model. A 1:2 model, or a 1:1-2 state model, better represented the data and gave similar affinity values ​​(not all data shown), but further experiments are required to determine which of these alternative fits correctly describes the binding. In general, however, there was a clear reduction in affinity of 12172 gL2gH11 hIgG4P for cynomolgus TREM1 compared to human TREM1, approximately 300-fold.In conclusion, 12172gL2gH11 hIgG4P showed stronger affinity for cell surface human TREM1 compared to soluble human TREM1 ECD (Example 6) due to binding avidity on cells, and both methods (Biacore and LigandTracer) showed significantly weaker affinity of 12172gL2gH11 hIgG4P for cynomolgus TREM1 compared to human TREM1.

[0527] Table 37. Kinetic parameters for 12172 gL2gH11 hIgG4P binding to human TREM1. U values ​​represent the quality of the fit model to the data. Low U values ​​below 10% are considered a good fit, values ​​above 20% are considered a poor fit. [Table 37]

[0528] Table 38. Kinetic parameters for 12172 gL2gH11 hIgG4P binding to cynomolgus TREM1. The U value represents the quality of the fit model to the data. A low U value below 10% is considered a good fit, and a value above 20% is considered a poor fit. The 1:2 model generates two sets of affinity values ​​describing two contributing binding events. [Table 38]

[0529] All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein, are hereby incorporated by reference in their entirety, unless already present. [Sequence List Free Text]

[0530] Sequence Listing 17 <223> Rabbit Ab12172 VL domain Sequence Listing 18 <223> Rabbit Ab12172 VL domain Sequence Listing 19 <223> CID102770: Rabbit Ab12172 light chain (Fab) Sequence Listing 20 <223> Rabbit Ab12172 light chain Sequence Listing 21 <223> Rabbit Ab12172 VH region Sequence Listing 22 <223> Rabbit Ab12172 VH region Sequence Listing 23 <223> CID120769: Rabbit Ab12172 heavy chain (Fab) Sequence Listing 24 <223> Rabbit Ab12172 heavy chain (Fab) Sequence Listing 25 <223> 12172gL1 V area Sequence Listing 26 <223> 12172gL1 V area Sequence Listing 27 <223> 12172gL1 light chain Sequence Listing 28 <223> 12172gL1 light chain Sequence List 29-30 <223> 12172gL2 V area Sequence Table 31-32 <223> 12172gL2 light chain Sequence Table 33-34 <223> 12172gL6 V area Sequence Table 35-36 <223> 12172gL6 light chain Sequence List 37-38 <223> 12172gL9 V area Sequence List 39-40 <223> 12172gL9 light chain Sequence Table 41-42 <223> 12172gL11 V area Sequence Table 43-44 <223> 12172gL11 light chain Sequence table 45-46 <223> 12172gH1 V area Sequence list 47-48 <223> 12172gH1 heavy chain (IgG4P) Sequence list 49-50 <223> 12172gH2 V area Sequence List 51-52 <223> 12172gH2 heavy chain (IgG4P) Sequence List 53-54 <223> 12172gH4 V area Sequence List 55-56 <223> 12172gH4 heavy chain (IgG4P) Sequence List 57-58 <223> 12172gH6 V area Sequence List 59-60 <223> 12172gH6 heavy chain (IgG4P) Sequence table 61-62 <223> 12172gH6 heavy chain (IgG4P F234A L235A) Sequence table 63-64 <223> 12172gH6 heavy chain (IgG1) Sequence table 65-66 <223> 12172gH6 heavy chain (IgG1 L234A L235A) Sequence list 67-68 <223> 12172gH8 V area Sequence list 69-70 <223> 12172gH8 heavy chain (IgG4P) Sequence Table 71-72 <223> 12172gH9 V area Sequence Table 73-74 <223> 12172gH9 heavy chain (IgG4P) Sequence table 75-76 <223> 12172gH10 V area Sequence List 77-78 <223> 12172gH10 heavy chain (IgG4P) Sequence list 79-80 <223> 12172gH11 V area Sequence table 81-82 <223> 12172gH11 heavy chain (IgG4P) Sequence table 83-84 <223> 12172gH11 heavy chain (IgG4P F234A L235A) Sequence table 85-86 <223> 12172gH11 heavy chain (IgG1) Sequence list 87-88 <223> 12172gH11 heavy chain (IgG1 L234A L235A) Sequence table 89-90 <223> 12172gH12 V area Sequence table 91-92 <223> 12172gH12 heavy chain (IgG4P) Sequence table 93-94 <223> 12172gH26 V area Sequence table 95-96 <223> 12172gH26 heavy chain (hIgG4P) Sequence list 97-98 <223> 12172gH48 V area Sequence table 99-100 <223> 12172gH48 heavy chain (hIgG4P) Sequence table 101-102 <223> 12172gH49 V area Sequence Listing 103-104 <223> 12172gH49 heavy chain (hIgG4P) Sequence list 105-106 <223> Human IGKV1-9 IGKJ4 receptor framework Sequence list 107-108 <223> Human IGHV3-66 IGHJ4 receptor framework Sequence Listing 109 <223> Human TREM1 WT 01_WT_3A Sequence Listing 110 <223> Human TREM1 mutant 02_6 Sequence Listing 111 <223> Human TREM1 mutant 03_6_7 Sequence Listing 112 <223> Human TREM1 mutant 04_6_7_9 Sequence Listing 113 <223> Human TREM1 mutant 05_7_8_9 Sequence Listing 114 <223> Human TREM1 mutant 06_9_10_111 Sequence Listing 115 <223> Human TREM1 mutants 07_9_10_11 Sequence Listing 116 <223> Human TREM1 mutant 08_10_11_12 Sequence Listing 117 <223> Human TREM1 mutants 09_11_12_13 Sequence Listing 118 <223> Human TREM1 variants 10_16_118_119 Sequence Listing 119 <223> Human TREM1 variants 11_19_20_85 Sequence Listing 120 <223> Human TREM1 variants 12_22_24_26 Sequence Listing 121 <223> Human TREM1 variants 13_24_26_28 Sequence Listing 122 <223> Human TREM1 variants 14_26_28_29 Sequence Listing 123 <223> Human TREM1 variants 15_28_29_30 Sequence Listing 124 <223> Human TREM1 variants 16_29_30_31 Sequence Listing 125 <223> Human TREM1 variants 17_31_32_35 Sequence Listing 126 <223> Human TREM1 variants 18_30_34 Sequence Listing 127 <223> Human TREM1 variants 19_34_35_57 Sequence Listing 128 <223> Human TREM1 variants 20_35_36_56 Sequence Listing 129 <223> Human TREM1 variants 21_40_49_50 Sequence Listing 130 <223> Human TREM1 variants 22_43_44_47 Sequence Listing 131 <223> Human TREM1 variants 23_43_44_45 Sequence Listing 132 <223> Human TREM1 variants 24_44_45_46 Sequence Listing 133 <223> Human TREM1 variants 25_45_46_47 Sequence Listing 134 <223> Human TREM1 variants 26_43_46_47 Sequence Listing 135 <223> Human TREM1 variants 27_47_48_49 Sequence Listing 136 <223> Human TREM1 variants 28_47_49_50 Sequence Listing 137 <223> Human TREM1 variants 29_49_50_53 Sequence Listing 138 <223> Human TREM1 variants 30_50_53_55 Sequence Listing 139 <223> Human TREM1 variants 31_55_56_57 Sequence Listing 140 <223> Human TREM1 variants 32_34_56_57 Sequence Listing 141 <223> Human TREM1 variants 33_57_59_60 Sequence Listing 142 <223> Human TREM1 variants 34_59_60_61 Sequence Listing 143 <223> Human TREM1 variants 35_60_61_62 Sequence Listing 144 <223> Human TREM1 variants 36_61_62_63 Sequence Listing 145 <223> Human TREM1 variants 37_62_63_64 Sequence Listing 146 <223> Human TREM1 variants 38_63_64_65 Sequence Listing 147 <223> Human TREM1 variants 39_64_65_66 Sequence Listing 148 <223> Human TREM1 variants 40_60_74_76 Sequence Listing 149 <223> Human TREM1 variants 41_60_75_76 Sequence Listing 150 <223> Human TREM1 variants 42_60_76_77 Sequence Listing 151 <223> Human TREM1 variants 43_28_76_77 Sequence Listing 152 <223> Human TREM1 variant 44_28_77_78 Sequence Listing 153 <223> Human TREM1 variants 45_96_109_111 Sequence Listing 154 <223> Human TREM1 variants 46_98_100_109 Sequence Listing 155 <223> Human TREM1 variants 47_35_100_102 Sequence Listing 156 <223> Human TREM1 mutants 48_102_103_104 Sequence Listing 157 <223> Human TREM1 mutants 49_103_104_105 Sequence Listing 158 <223> Human TREM1 variants 50_104_105_106 Sequence Listing 159 <223> Human TREM1 variants 51_105_106_107 Sequence Listing 160 <223> Human TREM1 variants 52_106_107_108 Sequence Listing 161 <223> Human TREM1 variants 53_107_108_109 Sequence Listing 162 <223> Human TREM1 variants 54_109_110_111 Sequence Listing 163 <223> Human TREM1 variants 55_11_113_114 Sequence Listing 164 <223> Human TREM1 variant 56_10_12_114 Sequence Listing 165 <223> Human TREM1 variants 57_118_119_120 Sequence Listing 166 <223> Human TREM1 variants 58_16_119_120 Sequence Listing 167 <223> Human TREM1 WT 01_WT_2A Sequence Listing 168 <223> Human TREM1 mutant 02_2_3 Sequence Listing 169 <223> Human TREM1 mutant 03_3_4 Sequence Listing 170 <223> Human TREM1 mutant 04_4_5 Sequence Listing 171 <223> Human TREM1 mutant 05_5_104 Sequence Listing 172 <223> Human TREM1 mutant 06_5_106 Sequence Listing 173 <223> Human TREM1 mutant 07_5_107 Sequence Listing 174 <223> Human TREM1 mutant 08_7_9 Sequence Listing 175 <223> Human TREM1 mutant 09_7_107 Sequence Listing 176 <223> Human TREM1 variant 10_8_10 Sequence Listing 177 <223> Human TREM1 variant 11_11_12 Sequence Listing 178 <223> Human TREM1 variant 12_12_13 Sequence Listing 179 <223> Human TREM1 variants 13_12_15 Sequence Listing 180 <223> Human TREM1 variant 14_13_85 Sequence Listing 181 <223> Human TREM1 variants 15_16_81 Sequence Listing 182 <223> Human TREM1 variant 16_20_75 Sequence Listing 183 <223> Human TREM1 variants 17_24_25 Sequence Listing 184 <223> Human TREM1 variants 18_24_26 Sequence Listing 185 <223> Human TREM1 variants 19_24_27 Sequence Listing 186 <223> Human TREM1 variants 20_25_26 Sequence Listing 187 <223> Human TREM1 variants 21_26_27 Sequence Listing 188 <223> Human TREM1 variants 22_30_31 Sequence Listing 189 <223> Human TREM1 variant 23_30_53 Sequence Listing 190 <223> Human TREM1 variants 24_37_38 Sequence Listing 191 <223> Human TREM1 variants 25_37_39 Sequence Listing 192 <223> Human TREM1 variants 26_37_45 Sequence Listing 193 <223> Human TREM1 variants 27_37_47 Sequence Listing 194 <223> Human TREM1 variants 28_38_44 Sequence Listing 195 <223> Human TREM1 variants 29_38_90 Sequence Listing 196 <223> Human TREM1 variants 30_42_43 Sequence Listing 197 <223> Human TREM1 variants 31_42_45 Sequence Listing 198 <223> Human TREM1 variants 32_43_44 Sequence Listing 199 <223> Human TREM1 mutant 33_43_101 Sequence Listing 200 <223> Human TREM1 variant 34_44_45 Sequence Listing 201 <223> Human TREM1 variant 35_45_47 Sequence Listing 202 <223> Human TREM1 variants 36_47_65 Sequence Listing 203 <223> Human TREM1 variants 37_51_55 Sequence Listing 204 <223> Human TREM1 variants 38_56_57 Sequence Listing 205 <223> Human TREM1 variants 39_58_59 Sequence Listing 206 <223> Human TREM1 variants 40_59_60 Sequence Listing 207 <223> Human TREM1 variants 41_59_61 Sequence Listing 208 <223> Human TREM1 variants 42_59_66 Sequence Listing 209 <223> Human TREM1 variants 43_60_61 Sequence Listing 210 <223> Human TREM1 variants 44_60_62 Sequence Listing 211 <223> Human TREM1 variants 45_61_62 Sequence Listing 212 <223> Human TREM1 variants 46_61_66 Sequence Listing 213 <223> Human TREM1 variants 47_62_65 Sequence Listing 214 <223> Human TREM1 variants 48_65_66 Sequence Listing 215 <223> Human TREM1 variants 49_66_81 Sequence Listing 216 <223> Human TREM1 variants 50_72_73 Sequence Listing 217 <223> Human TREM1 variants 51_73_75 Sequence Listing 218 <223> Human TREM1 variants 52_81_82 Sequence Listing 219 <223> Human TREM1 variants 53_85_86 Sequence Listing 220 <223> Human TREM1 variants 54_98_99 Sequence Listing 221 <223> Human TREM1 variants 55_99_100 Sequence Listing 222 <223> Human TREM1 variant 56_100_101 Sequence Listing 223 <223> Human TREM1 variants 57_101_102 Sequence Listing 224 <223> Human TREM1 variants 58_102_103 Sequence Listing 225 <223> Human TREM1 mutants 59_103_104 Sequence Listing 226 <223> Human TREM1 mutant 60_104_106 Sequence Listing 227 <223> Human TREM1 mutant 61_106_107 Sequence Listing 228 <223> Human TREM1 variants 62_8_10_112 Sequence Listing 229 <223> Human TREM1 mutants 63_37_39_40 Sequence Listing 230 <223> Human TREM1 mutant 64_40 Sequence Listing 231 <223> Human TREM1 variant 65_112 Sequence Listing 232 <223> Human TREM1 WT 01_WT_1A Sequence Listing 233 <223> Human TREM1 mutant 02_1 Sequence Listing 234 <223> Human TREM1 mutant 03_2 Sequence Listing 235 <223> Human TREM1 mutant 04_4 Sequence Listing 236 <223> Human TREM1 mutant 05_5 Sequence Listing 237 <223> Human TREM1 mutant 06_6 Sequence Listing 238 <223> Human TREM1 mutant 07_7 Sequence Listing 239 <223> Human TREM1 mutant 08_8 Sequence Listing 240 <223> Human TREM1 mutant 09_9 Sequence Listing 241 <223> Human TREM1 variant 10_11 Sequence Listing 242 <223> Human TREM1 variant 11_14 Sequence Listing 243 <223> Human TREM1 variant 12_17 Sequence Listing 244 <223> Human TREM1 variant 13_19 Sequence Listing 245 <223> Human TREM1 variant 14_21 Sequence Listing 246 <223> Human TREM1 variant 15_23 Sequence Listing 247 <223> Human TREM1 variant 16_24 Sequence Listing 248 <223> Human TREM1 variant 17_25 Sequence Listing 249 <223> Human TREM1 variant 18_26 Sequence Listing 250 <223> Human TREM1 variant 19_29 Sequence Listing 251 <223> Human TREM1 variants 20_30 Sequence Listing 252 <223> Human TREM1 variant 21_38 Sequence Listing 253 <223> Human TREM1 variant 22_39 Sequence Listing 254 <223> Human TREM1 mutant 23_41 Sequence Listing 255 <223> Human TREM1 mutant 24_42 Sequence Listing 256 <223> Human TREM1 variant 25_44 Sequence Listing 257 <223> Human TREM1 variant 26_45 Sequence Listing 258 <223> Human TREM1 variant 27_50 Sequence Listing 259 <223> Human TREM1 variant 28_51 Sequence Listing 260 <223> Human TREM1 variant 29_52 Sequence Listing 261 <223> Human TREM1 variant 30_54 Sequence Listing 262 <223> Human TREM1 variant 31_55 Sequence Listing 263 <223> Human TREM1 variant 32_56 Sequence Listing 264 <223> Human TREM1 variant 33_57 Sequence Listing 265 <223> Human TREM1 variant 34_58 Sequence Listing 266 <223> Human TREM1 variant 35_59 Sequence Listing 267 <223> Human TREM1 mutant 36_60 Sequence Listing 268 <223> Human TREM1 variant 37_61 Sequence Listing 269 <223> Human TREM1 variant 38_63 Sequence Listing 270 <223> Human TREM1 variant 39_65 Sequence Listing 271 <223> Human TREM1 variant 40_71 Sequence Listing 272 <223> Human TREM1 variant 41_72 Sequence Listing 273 <223> Human TREM1 variant 42_78 Sequence Listing 274 <223> Human TREM1 variant 43_80 Sequence Listing 275 <223> Human TREM1 variant 44_81 Sequence Listing 276 <223> Human TREM1 variant 45_83 Sequence Listing 277 <223> Human TREM1 variant 46_84 Sequence Listing 278 <223> Human TREM1 variant 47_85 Sequence Listing 279 <223> Human TREM1 variant 48_87 Sequence Listing 280 <223> Human TREM1 variant 49_95 Sequence Listing 281 <223> Human TREM1 variant 50_97 Sequence Listing 282 <223> Human TREM1 variant 51_98 Sequence Listing 283 <223> Human TREM1 variant 52_99 Sequence Listing 284 <223> Human TREM1 variant 53_100 Sequence Listing 285 <223> Human TREM1 variant 54_101 Sequence Listing 286 <223> Human TREM1 variant 55_102 Sequence Listing 287 <223> Human TREM1 variant 56_103 Sequence Listing 288 <223> Human TREM1 variant 57_104 Sequence Listing 289 <223> Human TREM1 variant 58_106 Sequence Listing 290 <223> Human TREM1 variant 59_109 Sequence Listing 291 <223> Human TREM1 mutant 60_111 Sequence Listing 292 <223> Human TREM1 mutant 61_113 Sequence Listing 293 <223> Human TREM1 variant 62_114 Sequence Listing 294 <223> Human TREM1 variant 63_115

Claims

1. CDR-L1 comprising SEQ ID NO: 11, CDR-L2 comprising SEQ ID NO: 12, and CDR-L3 comprising SEQ ID NO: 13 and a light chain variable region comprising CDR-H1 comprising SEQ ID NO: 14, CDR-H2 comprising SEQ ID NO: 15, and CDR-H3 comprising SEQ ID NO: 16 and a heavy chain variable region comprising An antibody that binds to human TREM1.

2. The antibody of claim 1 , wherein the antibody inhibits or attenuates binding of TREM1 to one or more of its natural ligands.

3. The antibody of claim 2, which inhibits or attenuates binding of TREM1 to PGLYRP1.

4. The antibody of claim 1, having a dissociation equilibrium constant (KD) for human TREM1 of less than 600 pM.

5. The antibody of claim 1 , wherein the antibody binds to a different site on TREM1 than PGLYRP1.

6. The antibody of claim 1 , which binds to an epitope of human TREM1 comprising residues E26, E27, K28, Y29, E30, L31, and K32 of human TREM1 (SEQ ID NO:1).

7. The antibody of claim 1, wherein the antibody binds to an epitope of human TREM1 that includes five or more residues selected from E26, E27, K28, Y29, E30, L31, K32, Q35, T36, D38, K40, D42, R97, D127, T134 and G136 of human TREM1 (SEQ ID NO: 1), as determined by a distance of less than 4 Å contact distance between the antibody and TREM1.

8. The antibody of claim 7, wherein the binding is determined using X-ray crystallography.

9. The antibody of claim 1 , wherein the light chain variable region comprises the sequence set forth in SEQ ID NO:

29.

10. The antibody of claim 1 , wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:

79.

11. 2. The antibody of claim 1, wherein the light chain variable region comprises a sequence set forth in SEQ ID NO:29, or a sequence at least 90% identical thereto, and the heavy chain variable region comprises a sequence set forth in SEQ ID NO:79, or a sequence at least 90% identical thereto.

12. The antibody of claim 1, wherein each CDR contains up to three amino acid substitutions, and such amino acid substitutions are conservative.

13. The antibody of claim 1 , wherein the remaining portions of the light chain variable region and the heavy chain variable region have at least 90% identity to SEQ ID NOs: 29 and 79, respectively.

14. The antibody of claim 1 which is an antibody fragment.

15. The antibody fragment is Fab, Fab', F(ab') ２ 15. The antibody of claim 14, which is an Fv, dsFv, scFv, or dsscFv.

16. The antibody of claim 1 which is a full-length antibody.

17. 17. The antibody of claim 16 which is IgG1, IgG1 LALA, IgG4, IgG4P, or IgG4P FALA.

18. 17. The antibody of claim 16, which is an IgG4P comprising a light chain comprising the sequence set forth in SEQ ID NO:31 and a heavy chain comprising the sequence set forth in SEQ ID NO:

81.

19. The antibody of claim 1, wherein the antibody is IgG4P and the remaining portions of the light and heavy chains have at least 90% identity or similarity to SEQ ID NOs: 31 and 81, respectively.

20. 2. An antibody that cross-competes with the antibody of claim 1 for binding to a TREM1 epitope comprising residues E26, E27, K28, Y29, E30, L31, K32 and Q35 of human TREM1 (SEQ ID NO:1).

21. 1. An IgG4P antibody that binds to an epitope of human TREM1, said epitope comprising residues E26, E27, K28, Y29, E30, L31, K32 and Q35 of human TREM1 (SEQ ID NO:1).

22. An isolated polynucleotide encoding the antibody of any one of claims 1 to 21.

23. An expression vector carrying the polynucleotide of claim 22.

24. A host cell comprising the vector of claim 23.

25. A method for producing an antibody according to any one of claims 1 to 21, comprising culturing a host cell according to claim 24 under conditions allowing the production of said antibody, and recovering the produced antibody.

26. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 21 and a pharma- ceutically acceptable adjuvant or carrier.

27. 27. A pharmaceutical composition as claimed in claim 26 for use in the treatment of the human or animal body by therapy.

28. 27. A pharmaceutical composition according to claim 26 for use as a medicament.

29. Use of an antibody according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 26 for the manufacture of a medicament.

30. 27. A pharmaceutical composition according to claim 26 for use in the treatment or prevention of a neurological disorder.

31. Use of an antibody according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 26 for the manufacture of a medicament for treating or preventing a neurological disorder.

32. 31. The pharmaceutical composition of claim 30, wherein the neurological disorder is amyotrophic lateral sclerosis (ALS) or Alzheimer's disease.

33. The use of claim 31, wherein the neurological disorder is amyotrophic lateral sclerosis (ALS) or Alzheimer's disease.

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