Antibody Fc variant

Abstract

The present invention relates to antibodies and uses thereof comprising Fc variants that exhibit reduced or undetectable binding to Fc receptors and reduced or undetectable effector function. Such variants are beneficial for patients suffering from diseases that may be treated with antibodies where it is desirable to reduce antibody-induced effector function.

Description

[Technical Field]

[0001] Array List This application contains an array list electronically filed in ASCII format, the entirety of which is incorporated by reference in this application. The ASCII copy (created on October 26, 2021) is named PAT058983-WO-PCT_SL.txt and has a size of 42,827 bytes.

[0002] The present invention relates to polypeptides containing variants of the Fc region, as well as compositions and methods of use thereof. [Background technology]

[0003] Monoclonal antibodies are highly effective biotherapeutic agents. A key aspect of antibodies is their ability to bind to antigens. Therapeutic antibodies can sequester targets upon binding. For example, they can prevent ligand-receptor interactions and downstream signaling without requiring any effector function. Other therapeutic antibodies recruit immune effector cells via Fc simultaneously with binding to antigens. To date, all approved recombinant monoclonal antibodies are human IgG subclasses capable of engaging both humoral and cellular immunity in the immune system. Cellular immune responses largely arise from the interaction between antibodies and Fc gamma receptors (FcγR). Intracellular signaling via the activating receptors FcγR1A, 2A, and 3A is regulated via phosphorylation of the immune receptor tyrosine-based activation motif (ITAM), resulting in effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and inflammation through the induction of cytokine secretion. Antibody-mediated complement activation is regulated via Fc interactions with complement component C1q and can trigger complement-dependent cytotoxicity (CDC).

[0004] The Fc region of an antibody exhibits limited variability and is responsible for achieving the physiological role that the antibody performs. The effector functions attributable to the antibody's Fc region vary depending on the antibody class and subclass, and include triggering various biological responses through the binding of the antibody to specific Fc receptors on cells via the Fc region. These receptors are expressed in a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, macrogranular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. Once an Fc / FcγR complex is formed, these effector cells are recruited to the binding antigen site, typically resulting in intracellular signaling events and important subsequent immune responses, such as inflammatory mediator release, B cell activation, endocytosis, phagocytosis, and cytotoxic attack. In addition, the overlapping region on the molecule's Fc domain also controls the activation of complement-mediated, cell-independent cytotoxicity, also known as complement-dependent cytotoxicity (CDC).

[0005] In many situations, binding and effector function stimulation mediated by the Fc region of immunoglobulins are highly beneficial, or even essential, for binding to tumor antigens on the cell surface of malignant transformed cells, for example, with CD20 antibodies. However, in other cases, reducing or even completely eliminating effector function may be more advantageous. This is especially true for antibodies designed to deliver drugs (e.g., toxins and isotopes) to target cells, in which case Fc / FcγR-mediated effector function brings healthy immune cells closer to the lethal payload, leading to the depletion of normal lymphoid tissue along with the target cells (Hutchins, et al., PNAS USA 92(1995)11980-11984; White, et al., Annu Rev Med 52(2001)125-145). In such cases, using antibodies that inadequately recruit complement or effector cells would yield significant benefits (see also Wu, et al., Cell Immunol 200(2000)16-26; Shields, et al., J. Biol Chem 276(9)(2001)6591-6604; U.S. Patent No. 6,194,551; U.S. Patent No. 5,885,573 and PCT International Publication No. 04 / 029207).

[0006] When an mAb is intended to engage with a cell surface receptor to prevent receptor-ligand interaction (e.g., an antagonist, e.g., a cytokine antagonist), it may be desirable to reduce or eliminate effector function, e.g., to prevent targeted cell death or undesirable cytokine secretion. Other examples where reduction of effector function may be necessary include preventing the interaction between an antibody-drug conjugate and FcγR from resulting in off-target cytotoxicity. The need to reduce or eliminate effector function was recognized with the first approved mAb, anti-CD3ε mAb, OKT3, muromonab, intended to prevent T cell activation in tissue transplant patients receiving donor kidneys, lungs, or hearts (Chatenoud and Bluestone, 2007). Many patients who took muromonab experienced adverse events, including the induction of pro-inflammatory cytokines (e.g., cytokine storms), which were partially attributed to the interaction between muromonab and FcγR (Alegre et al., 1992). To mitigate this unintended effector function, human IgG1 mutants L234A / L235A have been generated (Xu et al., 2000), which have shown reduced inflammatory cytokine release. In particular, reduced antibody affinity to the FcγRII receptor would be advantageous for antibodies that induce platelet activation and aggregation via FcγRII receptor binding. Such induction may lead to serious side effects of such antibodies.

[0007] While certain subclasses of human immunoglobulins lack specific effector functions, no known naturally occurring immunoglobulins lack all effector functions.

[0008] Silencing effector function can be achieved by mutations in the Fc region of antibodies, as described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69). See also Heusser et al.'s international publication pamphlet 2012065950. Among the four IgG subclasses, each has a different ability to induce immunoeffector function. For example, IgG1 and IgG3 recruit complement more effectively than IgG2 and IgG4 (Tao et al., 1993). In addition, IgG2 and IgG4 have a very limited ability to induce ADCC (Brezski et al., 2014). Therefore, some researchers have employed a cross-subclass approach to reduce effector function. In a further refinement of the cross-subclass approach, An et al. generated an IgG2 mutant from IgG4 by point mutation (i.e., H268Q / V309L / A330S / P331S). This mutant reduced effector function (An et al., 2009). A similar approach has reported a mutant containing the IgG2-to-IgG4 cross-subclass mutation V309L / A330S / P331S combined with the non-germline mutation V234A / G237A / P238S / H268A, which resulted in undetectable CDC, ADCC, and ADCP (Vafa et al., 2014). Other researchers have replaced amino acids in the Fc region with different amino acid residues so that the antibody modifies C1q binding and / or reduces or eliminates complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Patent No. 6,194,551 by Idusogie et al. Examples of silent Fc IgG1 antibodies include LALA mutants containing L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody is the DAPA (D265A, P329A) mutation (U.S. Patent No. 6,737,056).Another silent IgG1 antibody contains the N297A mutation, resulting in aglycosylated / nonglycosylated antibodies. Other alternative approaches have been reported for manipulating or mutating key residues in the Fc region responsible for effector function. See, for example, PCT International Publication 2009 / 100309 (Medimmune), International Publication 2006 / 076594 (Xencor), U.S. Patent Application Publication 2006 / 0134709 (Macrogenics), U.S. Patent No. 6,737,056 (Genentech), U.S. Patent Application Publication 2010 / 0166740 (Roche), and International Publication 2019068632 (Janssen). [Overview of the project]

[0009] There is an unmet need for therapeutic antibodies that possess significantly reduced or diminished effector function, along with long shelf life, even longer in vivo and in vitro half-lives, robust recombinant expression levels, and suitability for large-scale manufacturing and purification, as well as excellent pharmacokinetic properties such as stability in formulation. In addition, there is a need for highly silent Fc-containing proteins and antibodies that retain the N297 glycosylation site. Antibody glycosylation, particularly fucosylation, the presence of terminal galactose, and high mannose or sialylation all play a role not only in effector function such as the ADCC activation pathway, but also in regulating protein stability and half-life (reviewed in Boune S et al., Antibodies, 9, 22, 2020). The N297 position of IgG1 is often mutated to, for example, alanine or glutamine to eliminate N-glycosylation and downregulate effector function (Bolt S, Routledge E, Lloyd I, et al (1993) Eur J Immunol. 1993;23:403-411).

[0010] Retaining N297 and thus preserving Fc N-glycosylation may be beneficial for stabilizing Fc-containing protein molecules, particularly for stabilizing proper pairing of antibody heavy chains, increasing solubility and stability, and reducing protein aggregation tendencies, as well as promoting increased shelf life, in vitro and in vivo half-lives of silent Fc-containing therapeutic protein molecules. The half-lives of some glycoproteins can be enhanced by sialic acid, and sialylation acts as a cap to hide penaltymate galactose residues recognized by the hepatic asialoglycoprotein receptor (ASGPR). Therefore, maintaining the N-glycosylation attachment site to arginine 297 of the Fc portion of the molecule is necessary for silent Fc-containing molecules to have an extended half-life via hypersialylation (which can be mediated through expression systems and cell lines that provide specific hypersialylation).

[0011] The present invention provides a binding molecule, such as an antibody, containing an IgG1 Fc variant, which has unexpectedly drastically reduced and / or undetectable binding to all Fc gamma receptors and drastically reduced and / or undetectable binding to C1q, preferably resulting in drastically reduced or even undetectable effector function (including ADCC, CDC, and ADCP) while maintaining normal N-glycosylation ability.

[0012] Accordingly, the present invention relates to a binding molecule comprising a human IgG1 Fc variant of the wild-type human IgG1 Fc region and one or more antigen-binding domains, wherein the Fc variant comprises amino acid substitutions selected from combinations of substitutions: L234A, L235A, G237A (LALAGA), L234A, L235A, S267K, P329A (LALASKPA), D265A, P329A, S267K (DAPASK), G237A, D265A, P329A (GADAPA), G237A, D265A, P329A, S267K (GADAPASK), L234A, L235A, P329G (LALAPG), and L234A, L235A, P329A (LALAPA), where the amino acid residues are numbered according to the Kabat EU index.

[0013] In one embodiment of the present invention, the Fc variant comprises the nucleic acid sequence of sequence numbers 8, 10, 12, 16, 18, 20, or 22 listed in Table 1 below, or any sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0014] A particularly preferred embodiment of the present invention comprises a human IgG1 Fc variant of the wild-type human IgG1 Fc region and one or more antigen-binding domains, wherein the Fc variant includes preferred amino acid substitutions L234A, L235A, S267K, P329A (LALASKPA) in SEQ ID NO: 15, or substitutions G237A, D265A, P329A, S267K (GADAPASK) in SEQ ID NO: 21, and the amino acid residues are numbered according to the Kabat EU index.

[0015] In one embodiment of the present invention, the Fc variant includes the sequence of SEQ ID NO: 21 (see Table 1 below) or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0016] In one embodiment of the present invention, the Fc variant includes the sequence of Sequence ID No. 15 (see Table 1 below) or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0017] In one embodiment of the present invention, the binding molecule is a human or humanized IgG1 monoclonal antibody.

[0018] In one embodiment of the present invention, the binding molecule has reduced or undetectable binding affinity to the Fc gamma receptor or C1q compared to a polypeptide containing the wild-type human IgG1 Fc region (optionally measured by surface plasmon resonance using a Biacore T200 instrument), the Fc gamma receptor is selected from the group consisting of Fc gamma RIA, Fc gamma RIIIa V158 variant and Fc gamma RIIIa F158 variant, and the binding is reduced by 50%, 80%, 90%, 95%, 98%, 99% or undetectable compared to the wild type.

[0019] In one embodiment of the present invention, the modified Fc-containing binding molecule binds to at least one antigen, the antigen being a cell surface antigen.

[0020] In one embodiment of the present invention, the modified Fc-containing binding molecule binds to at least one antigen, which is a secreted or soluble antigen.

[0021] In one aspect of the present invention, the modified Fc-containing binding molecule has reduced or undetectable effector function compared to a polypeptide containing the wild-type human IgG1 Fc region.

[0022] In one embodiment of the present invention, the modified Fc-containing binding molecule has the ability to bind to an antigen without triggering detectable antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

[0023] In one embodiment of the present invention, the modified Fc-containing binding molecule is a multispecific antibody containing binding domains for two or more antigens.

[0024] In one embodiment of the present invention, the modified Fc-containing binding molecule is a bispecific antibody containing binding domains for two antigens.

[0025] In one embodiment of the present invention, the modified Fc-containing binding molecule further includes a knob in the hole mutation.

[0026] Another aspect of the present invention is a method for treating a disease in an individual, wherein the effector function of the binding molecule is reduced or undetectable in the individual compared to the effector function induced by a polypeptide containing the wild-type human IgG1 Fc region, and the method comprises administering the binding molecule disclosed herein to the individual. In another aspect, the present invention provides a modified Fc-containing binding molecule for use in human therapy.

[0027] In some embodiments of the present invention, the effector function to be reduced or diminished is antibody-dependent cell-mediated cytotoxicity (ADCC) in an organism. In some embodiments of the present invention, the effector function to be reduced or diminished is antibody-dependent cellular phagocytosis (ADCP) in an organism. In some embodiments of the present invention, the effector function to be reduced or diminished is complement-dependent cytotoxicity (CDC) in an organism.

[0028] In addition, this specification discloses compositions comprising the Fc-modifying binding molecule according to the present invention. In one embodiment of the present invention, a composition further comprising a pharmaceutically acceptable carrier.

[0029] In addition, this specification discloses isolated polynucleotides encoding binding molecules containing modified IgG1 Fc sequences according to the present invention. In one embodiment of the present invention, the isolated polynucleotide comprises the modified IgG1 Fc sequence of SEQ ID NO: 16, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto. In another embodiment of the present invention, the isolated polynucleotide comprises the modified IgG1 Fc sequence of SEQ ID NO: 22, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology thereto.

[0030] In addition, this specification discloses a vector comprising a polynucleotide encoding the modified Fc-containing binding molecule of the present invention.

[0031] In addition, this specification discloses a host cell comprising a vector or polynucleotide capable of encoding and expressing the modified Fc-containing binding molecule of the present invention. [Brief explanation of the drawing]

[0032] [Figure 1-1] Figures 1A and 1B show a schematic overview of the biacore measurement cycle. [Figure 1-2] (As stated above.) [Figure 2-1] Figure 2: Representative sensorgrams and response and concentration plots are shown. Figure 2A shows representative sensorgrams and response plots for WT, LALAPA-IgG1, LALAGA-IgG1, LALAPG-IgG1, DAPA-IgG1, LALASKPA-IgG1, DAPASK-IgG1, GADAPA-IgG1, GADAPASK-IgG1, and DANAPA-IgG1. Figure 2B shows the sensograms and binding kinetics of WT, LALAPA-IgG1, LALAGA-IgG1, LALAPG-IgG1, DAPA-IgG1, LALASKPA-IgG1, DAPASK-IgG1, GADAPA-IgG1, GADAPASK-IgG1, and DANAPA-IgG1 for FcγR3A V158, and Figure 2C shows the sensograms and binding kinetics of WT, LALAPA-IgG1, LALAGA-IgG1, LALAPG-IgG1, DAPA-IgG1, LALASKPA-IgG1, DAPASK-IgG1, GADAPA-IgG1, GADAPASK-IgG1, and DANAPA-IgG1 for C1q. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 2-4] (As stated above.) [Figure 2-5] (As stated above.) [Figure 2-6] (As stated above.) [Figure 2-7] (As stated above.) [Figure 2-8] (As stated above.) [Figure 2-9](As stated above.) [Figure 2-10] (As stated above.) [Figure 2-11] (As stated above.) [Figure 2-12] (As stated above.) [Figure 2-13] (As stated above.) [Figure 2-14] (As stated above.) [Figure 2-15] (As stated above.) [Figure 2-16] (As stated above.) [Figure 2-17] (As stated above.) [Figure 2-18] (As stated above.) [Figure 2-19] (As stated above.) [Figure 2-20] (As stated above.) [Figure 2-21] (As stated above.) [Figure 2-22] (As stated above.) [Figure 2-23] (As stated above.) [Figure 2-24] (As stated above.) [Figure 2-25] (As stated above.) [Figure 2-26] (As stated above.) [Figure 2-27] (As stated above.) [Figure 2-28] (As stated above.) [Figure 2-29] (As stated above.) [Figure 2-30] (As stated above.) [Figure 2-31] (As stated above.) [Figure 2-32] (As stated above.) [Figure 2-33] (As stated above.) [Figure 3-1] Figure 3: Figure 3A shows the activation T cell nuclear factor (NFAT) pathway activity of wild-type and mutant antibodies. Figure 3B shows the NFAT pathway activity of wild-type and mutant antibodies (cells sensitized by the addition of INFγ). [Figure 3-2] (As stated above.) [Modes for carrying out the invention]

[0033] General matters To make the present invention more easily understandable, certain terms are defined throughout the detailed description. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the present invention relates.

[0034] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings:

[0035] As used herein, the term “binding molecule” refers to a target molecule, for example, a molecule that binds to an antigen and has reduced or undetectable binding affinity to the Fc receptor or C1q. As used herein, “reduced binding affinity to the Fc gamma receptor or C1q” refers to a reduction of at least 20% in binding affinity to the Fc gamma receptor or C1q compared to a control (e.g., a polypeptide having a wild-type Fc region); as used herein, “significantly reduced binding affinity to the Fc gamma receptor or C1q” refers to a reduction of at least 50% in binding affinity to the Fc gamma receptor compared to a control; and as used herein, “undetectable binding affinity to the Fc gamma receptor or C1q” refers to a binding affinity to the Fc gamma receptor or C1q below the detection limit of the assay used. In some embodiments, binding affinity is measured by surface plasmon resonance using a Biacore T200 instrument.

[0036] The binding molecules of this disclosure include antibodies, antibody variants, antibody fragments, and antibody antigen-binding portions (which can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs) (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The binding molecules also include nanobodies, Fabs, DARPins, avimers, afibodies, and anticarins.

[0037] As used herein, the term “antibody” refers to a polypeptide of the immunoglobulin family that can reversibly and specifically bind to a corresponding antigen in a non-covalent manner. For example, naturally occurring IgG antibodies are tetramers containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), and interspersed with more conserved regions called framework regions (FRs). VH and VL, respectively, consist of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors (such as various cells of the immune system (e.g., effector cells) and components of the classical complement system).

[0038] The term “antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (for example, anti-Id antibodies against the antibodies of this disclosure), and their functional fragments or fusions. Antibodies may be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibodies according to the present invention comprise at least one modified and silentized IgG1 Fc fragment.

[0039] The "complementarity-determining domain" or "complementarity-determining region" ("CDR") interchangeably refers to the hypervariable regions of the VL and VH. The CDR is the target protein binding site of the antibody chain, and it possesses specificity for such target proteins. Three CDRs (numbered sequentially from the N-terminus, CDR1-3) are present in each human VL or VH, collectively constituting approximately 15-20% of the variable domain. CDRs can be named according to their region and order. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. Structurally, the CDR is complementary to the target protein epitope and therefore directly contributes to binding specificity. The remaining range of the VL or VH, the so-called framework region, shows little variation in its amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York, 2000).

[0040] The location of the CDR and framework area can be determined using various well-known definitions in the art, such as Kabat, Chothia, IMGT, AbM, and combined definitions (see, for example, Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J.Mol.Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J.Mol.Biol., 227:799-817 (1992); Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); Al-Lazikani et al., J.Mol.Biol., 273:927-748 (1997)). The definition of antigen-binding sites is also described in: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); MacCallum et al., J.Mol.Biol., 262:732-745 (1996); and Martin et al., Proc.Natl.Acad Sci.USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In the Kabat and Chothia combined numbering scheme, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in VH, e.g., mammalian VH, e.g., human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL, e.g., mammalian VL, e.g., human VL.Under IMGT, CDR amino acid residues in the VH region are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and CDR amino acid residues in the VL region are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to "Kabat"). Under IMGT, the CDR region of an antibody can be determined using the IMGT / DomainGap Align program. The IMGT tool is available on the World Wide Web (www.imgt.org).

[0041] Both the light and heavy chains are divided into structurally homologous and functionally homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it goes without saying that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, transplacental motility, Fc receptor binding, and complement binding. According to notation rules, the numbering of constant region domains increases distal to the antigen-binding site or amino terminus of the antibody. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 domain and CL domain actually contain the carboxyl-terminal domains of the heavy and light chains, respectively.

[0042] As used herein, the terms “antigen-binding domain” and “antigen-binding fragment” are interchangeable and refer to one or more portions of an antibody that possess the ability to specifically interact with an antigen epitope (e.g., by binding, steric hindrance, stabilization / destabilization, or spatial distribution). Examples of binding fragments include, but are not limited to, single-chain Fv(scFv), disulfide-bridged Fv(sdFv), F(ab)2 fragment, Fab fragment, F(ab')2 fragment, F(ab') fragment, monovalent fragments consisting of VL, VH, CL, and CH1 domains; bivalent fragments containing two Fab fragments bound by disulfide bridges at the hinge region; Fd fragments consisting of a VH domain and a CH1 domain (and optionally the hinge region); Fv fragments consisting of the VL and VH domains of a single arm of the antibody; dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546, 1989); and isolated complementarity-determining regions (CDRs), or other epitope-binding fragments of the antibody.

[0043] Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together by a synthetic linker using recombination methods. This allows the two domains to be paired, with the VL and VH regions forming a single protein chain that creates a monovalent molecule (known as single-chain Fv ("scFv"); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment." In many cases, V H and V L A peptide linker is present between the domains. In a preferred embodiment, the scFv of this disclosure has a general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V LThey contain a COOH group. These antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for practical use in the same manner as intact antibodies.

[0044] Antigen-binding fragments may also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments may be grafted into polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0045] The antigen-binding fragment may be incorporated into a single-chain molecule containing a pair of tandem Fv segments (VH-CH1-VH-CH1), which, together with a complementary light chain polypeptide, form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995; and U.S. Patent No. 5,641,870).

[0046] As used herein, the terms “monoclonal antibody” or “monoclonal antibody composition” refer to polypeptides, including antibodies and antigen-binding fragments having substantially the same amino acid sequence or originating from the same genetic source. The term also includes preparations of antibody molecules with a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Methods for producing monoclonal antibodies using phage display technology are known in the art (Proetzel, G., Ebersbach, H. (Eds.) Antibody Methods and Protocols. Humana Press ISBN 978-1-61779-930-3; 2012).

[0047] As used herein, the term "human antibody" includes antibodies in which both the framework and CDR regions have variable regions derived from human sequences. Furthermore, if the antibody contains a constant region, the constant region also derives from a human sequence, such as, for example, a human germline sequence, a mutant version of a human germline sequence, or a human sequence such as an antibody-containing consensus framework sequence derived from human framework sequence analysis, as described, for example, Knappik et al., J.Mol.Biol.296:57-86, 2000. In a preferred embodiment, the conjugate molecule of this disclosure is a human antibody.

[0048] The human antibodies of this disclosure may contain amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo, or conservative substitutions that promote stability or production).

[0049] "Humanized" antibodies are antibodies that maintain the reactivity of non-human antibodies while reducing their immunogenicity in humans. This can be achieved, for example, by retaining the non-human CDR region and replacing the rest of the antibody (i.e., the framework portion of the constant and variable regions) with its human counterpart. See, for example, Morrison et al. 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855; Morrison and Oi, 1988, Adv. Immunol., 44:65-92; Verhoeyen et al. 1988, Science, 239:1534-1536; Padlan 1991, Molec. Immun., 28:489-498; and Padlan 1994, Molec. Immun., 31:169-217. Other examples of human manipulation techniques include, but are not limited to, the Xoma technique disclosed in U.S. Patent No. 5,766,886. In some embodiments, the binding molecule of this disclosure is a humanized antibody or a chimeric antibody.

[0050] The chimeric or humanized antibodies of this disclosure can be prepared based on the sequences of the murine monoclonal antibodies prepared as described above. The DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and can be manipulated using standard molecular biology techniques to include non-murine (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, the murine variable region can be ligated to the human constant region using methods known in the art (see, for example, U.S. Patent No. 4,816,567, granted to Cabilly et al.). To produce a humanized antibody, the murine CDR region can be inserted into the human framework using methods known in the art. See, for example, U.S. Patent No. 5,225,539, granted to Winter, and U.S. Patents No. 5,530,101, 5,585,089, 5,693,762, and 6,180,370, granted to Queen et al.

[0051] As used herein, the terms “recognize” or “bind” mean that a binding molecule, antibody, or its antigen-binding fragment finds and interacts with (e.g., binds to or recognizes) its epitope, whether linear, discontinuous, or conformator. The term “epitope” refers to a site on an antigen to which the antibody or antigen-binding fragment of this disclosure specifically binds. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids arranged by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained immediately after exposure to a denaturing solvent, while epitopes formed by tertiary folding are typically lost immediately after treatment with the denaturing solvent. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a specific spatial conformation. Methods for determining the spatial conformation of an epitope include the techniques described in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)), or electron microscopy. A "paratope" is the part of an antibody that recognizes the epitope of an antigen.

[0052] The phrases "specifically binds" or "selectively binds" when used in the context of describing the interaction of an antigen (e.g., a protein) with an antibody, an antibody fragment, or an antibody-derived binder refer to a binding reaction that is determinative by the presence of the antigen in a heterogeneous population of proteins and other biological agents, e.g., in a biological sample, e.g., blood, serum, plasma, or tissue sample. Thus, under specified particular immunoassay conditions, an antibody or binder having a particular binding specificity binds to a particular antigen at least 2-fold over background and substantially does not bind in large amounts to other antigens present in the sample. In one embodiment, under specified immunoassay conditions, an antibody or binder having a particular binding specificity binds to a particular antigen at least 10-fold over background and substantially does not bind in large amounts to other antigens present in the sample. Specific binding to an antibody or binder under such conditions may require that the antibody or agent be selected for its specificity for that particular protein. If desired or appropriate, this selection can be achieved by removing antibodies that cross-react with the molecule from other species (e.g., mouse or rat) or other subtypes. In addition, in some embodiments, an antibody or antibody fragment that cross-reacts with a particular desired molecule is selected.

[0053] In some embodiments, the specific binding of an antibody or antigen-binding fragment of the present disclosure is at least 10 2 M -1 , at least 5×10 2 M -1 , at least 10 3 M -1 , at least 5×10<00​​​​​​​​​​​​​​​​​​​​​​​6 M -1 , at least 10 7 M -1 , at least 5 × 10 7 M -1 , at least 10 8 M -1 , at least 5 × 10 8 M -1 , at least 10 9 M -1 , at least 5 × 10 9 M -1 , at least 10 10 M -1 , at least 5 × 10 10 M -1 , at least 10 11 M -1 , at least 5 × 10 11 M -1 , at least 10 12 M -1 , at least 5 × 10 12 M -1 , at least 10 13 M -1 , at least 5 × 10 13 M -1 , at least 10 14 M -1 , at least 5 × 10 14 M -1 , at least 10 15 M -1 , or at least 5 × 10 15 M -1 The equilibrium constant (K A )(k on / k off It means a combination that has ).

[0054] In some embodiments, the specific binding of the antibody or antigen-binding fragment of this disclosure is 5 × 10 -2 Less than M, 10 -2 Less than M, 5 x 10 -3 Less than M, 10 -3 Less than M, 5 x 10 -4 Less than M, 10 -4 Less than M, 5 x 10 -5 Less than M, 10 -5 Less than M, 5 x 10 -6 Less than M, 10-6 less than M, 5 × 10 -7 less than M, 10 -7 less than M, 5 × 10 -8 less than M, 10 -8 less than M, 5 × 10 -9 less than M, 10 -9 less than M, 5 × 10 -10 less than M, 10 -10 less than M, 5 × 10 -11 less than M, 10 -11 less than M, 5 × 10 -12 less than M, 10 -12 less than M, 5 × 10 -13 less than M, 10 -13 less than M, 5 × 10 -14 less than M, 10 -14 less than M, 5 × 10 -15 less than M, or 10 -15 less than M or a dissociation rate constant (K D )(k off / k on ) that binds to the target antigen with an affinity at least twice that of its binding affinity to a non-specific antigen (e.g., HSA).

[0055] As used herein, the term "affinity" refers to the strength of the interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites via weak non-covalent forces; the more interactions, the stronger the affinity. As used herein, the term "high affinity" for an IgG antibody or a fragment thereof (e.g., a Fab fragment) refers to an antibody having a K -8 of 10 -9 M or less, 10 -10 M or less, or 10 -11 M or less, or 10 -12 M or less, or 10 -13 M or less. However, high affinity binding may be different for other antibody isotypes. For example, high affinity binding for an IgM isotype refers to an antibody having a K D of 10 -7 M or less or 10 -8 M or less. Affinity, e.g., K D D ​One suitable assay for measuring this involves using BIACORE technology, which allows for the measurement of the degree of interaction using surface plasmon resonance techniques (e.g., using a BIACORE 3000 instrument (BIACORE, Uppsala, Sweden, or Biacore T200, GE Healthcare)).

[0056] As used herein, the term "avidity" refers to a measure that provides information about the overall stability or strength of an antibody-antigen complex. It is controlled by three main factors: antibody epitope affinity; the binding titer of both the antigen and the antibody; and the structural arrangement of the interaction moiety. Ultimately, these factors define the specificity of the antibody, i.e., the likelihood that a particular antibody is binding to a precise antigen epitope.

[0057] The term "isolated antibody" refers to an antibody that is substantially free from other antibodies with different antigenic specificity. However, an isolated antibody that specifically binds to a particular antigen may exhibit cross-reactivity to other antigens. Furthermore, an isolated antibody may be substantially free from other cellular substances and / or chemicals.

[0058] The term “corresponding human germline sequence” refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined by comparison with all other known or inferred variable region amino acid sequences encoded by the human germline immunoglobulin variable region sequence. The corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence, as determined by comparison with all other evaluated variable region amino acid sequences. The corresponding human germline sequence may be the framework region only, the complementarity-determining region only, the framework and complementarity-determining region, the variable segment (as defined above), or any other combination of sequences or subsequences containing the variable region. Sequence identity may be determined using methods described herein, such as aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid sequence or amino acid sequence may have sequence identity with the reference variable region nucleic acid sequence or amino acid sequence of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0059] Various immunoassay formats may be used to select antibodies that are specifically immunoreactive to a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive to a protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that may be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal at least twice the background signal, and more typically at least 10 to 100 times the background signal.

[0060] The term “equilibrium dissociation constant (Kd, M)” refers to the dissociation rate constant (kd, time-1) obtained by dividing the association rate constant (ka, time-1, M-1) by the dissociation rate constant (kd, time-1). The equilibrium dissociation constant can be measured using any method known in the art. The antibodies and fragments of this disclosure typically have an equilibrium dissociation constant of about 10 -7 or 10 -8 Less than M, for example, about 10 -9 M or 10 -10 Less than M, in some cases about 10 -11 M, 10 -12 M, or 10 -13 It will likely be less than M.

[0061] The term "bioavailability" refers to the systemic availability (i.e., blood / plasma levels) of a given amount of a drug administered to a patient. Bioavailability is an absolute value that indicates both the time (rate) and total amount (amount) of the drug that reaches the systemic circulation from the administered dosage form.

[0062] As used herein, “modification,” “mutation,” or “substitution” of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the initial amino acid sequence (e.g., the wild-type sequence), the change resulting from a sequence alteration involving the amino acid residue / position. Typical modifications include, for example, substitution of a residue (or at the position) by another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more amino acids adjacent to the residue / position, and deletion of the residue / position. An “amino acid mutation” or such mutation refers to the replacement of an existing amino acid residue in a predetermined (initial) amino acid sequence by a different amino acid residue. Generally preferred, modifications result in an alteration of at least one physicobiochemical activity of the mutant polypeptide compared to the polypeptide containing the initial (or “wild-type”) amino acid sequence. For example, in the case of an antibody, the altered physicobiochemical activity may be binding affinity, binding ability, and / or binding effect to a target molecule.

[0063] The term "comprising" encompasses "including" and even "consisting." For example, a composition "containing" X may consist exclusively of X or may include something additional (e.g., X + Y).

[0064] Unless otherwise specified or as is clear from the context, when used herein in relation to numerical values, the term “about” is understood to mean within the normal tolerance range in the art, e.g., within two standard deviations from the mean. Thus, “about” may be within ±10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.05%, or 0.01% of the stated value, preferably within ±10% of the stated value. When used before a numerical range or list of numerical values, the phrase “about” applies sequentially to each numerical value; for example, the phrase “about 1 to 5” should be interpreted as “about 1 to about 5,” or for example, the phrase “about 1, 2, 3, 4” should be interpreted as “about 1, about 2, about 3, about 4, etc.”

[0065] As used herein, “select” and “choose” in relation to patients mean that specific patients are specifically selected from a larger group of patients based on certain patients who meet predetermined criteria. Similarly, “selectively treat patients” means to provide treatment to patients who are specifically selected from a larger group of patients based on certain patients who meet predetermined criteria. Similarly, “selectively administer” means to administer a drug to patients who are specifically selected from a larger group of patients based on certain patients who meet predetermined criteria.

[0066] The word “substantially” does not necessarily exclude “completely”; for example, a composition in which Y is “substantially free” may be one in which Y is completely free. The word “substantially” may be omitted from the definitions in this disclosure as required.

[0067] As used herein, the phrase "essentially consisting of" refers to a genus or species of active agent contained in the method or composition, or any excipient that is inert to the intended purpose of the method or composition. In some embodiments, the phrase "essentially consisting of" expressly excludes the inclusion of one or more additional activators other than the binding molecule of the Disclosure. In some embodiments, the phrase "essentially consisting of" expressly excludes the inclusion of one or more additional activators other than the binding molecule of the Disclosure and a second co-administered agent.

[0068] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, and non-natural amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, carboxyl group, amino group, and R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics refer to compounds that have a different structure from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids.

[0069] The term “conservatively modified variant” is used for both amino acid sequences and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially the same sequence. Due to the degeneracy of genetic coding, many functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at all positions where alanine is identified by a codon, the codon can be changed to one of the corresponding codons described, without changing the coded polypeptide. Such nucleic acid variants are “silent variants,” and these are a type of conservatively modified variant. Furthermore, all nucleic acid sequences in this specification that code for polypeptides describe all possible silent variants of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, usually the sole codon for methionine, and TGG, usually the sole codon for tryptophan) can be modified to produce a functionally identical molecule. Therefore, each silent mutation in the nucleic acid encoding the polypeptide is latent within each described sequence.

[0070] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to the polypeptide sequence that substitute amino acids with chemically similar amino acids. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants also include, but do not exclude, polymorphic variants, interspecific homologs, and alleles. The following eight groups contain amino acids that are conserved substitutions with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term “conservative sequence modification” is used to refer to amino acid modifications that do not significantly affect, or significantly alter, the binding properties of an antibody containing an amino acid sequence.

[0071] As used herein, the term “optimized” refers to a nucleotide sequence modified to encode an amino acid sequence using preferred codons in a producing cell or organism, typically a eukaryotic cell, such as a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese hamster ovary cell (CHO), or a human cell. The optimized nucleotide sequence is manipulated to retain, to the greatest extent possible, the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the “parent” sequence.

[0072] In the context of two or more nucleic acid sequences or polypeptide sequences, the terms “same percentage” or “percent identity” refer to the degree to which two or more sequences or subsequences are identical. Two sequences are “identical” if the sequence of amino acids or nucleotides is the same across the regions being compared. Two sequences are “substantially identical” if, when compared and aligned for maximum match using one of the following sequence comparison algorithms, or by manual alignment and visual inspection, a specific percentage of amino acid residues or nucleotides is the same across a comparison window or a specified region (i.e., 60% identity, and possibly 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity across a specific region, or, if not specified, across the entire sequence). In some cases, identity exists over a region having a length of at least about 30 nucleotides (or 10 amino acids), more preferably a region having a length of 100 to 500 nucleotides or 1000 nucleotides or more (or 20, 50, or 200 amino acids or more).

[0073] In sequence comparison, typically one sequence functions as the reference sequence compared to the test sequence. The test and reference sequences are input into a computer using a sequence comparison algorithm, and, if necessary, the subsequent coordinates and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters.

[0074] As used herein, the “comparison window” includes a reference to one segment of adjacent positions selected from the group consisting of 20 to 600, typically about 50 to about 200, and typically about 100 to about 150, where the sequences can be compared to a reference sequence with the same number of adjacent positions after the two sequences have been optimally aligned. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv.Appl.Math.2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J.Mol.Biol.48:443, by the similarity search method of Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., Current Protocols in Molecular Biology, 2003).

[0075] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST algorithm and the BLAST 2.0 algorithm, described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analysis is generally available from the National Center for Biotechnology Information. This algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with a word of the same length in the database sequence, match or satisfy a certain positive threshold score T. T is called the neighbor word score threshold (Altschul et al., op. cit.). These initial neighbor word hits serve as a basis for initiating a search to find longer HSPs that contain them. Word hits are extended in both directions along each sequence as long as the cumulative alignment score can increase. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction stops if: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or it reaches the end of either sequence. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses, by default, a word length of 11 (W), a prediction of 10 (E), M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program defaults to a word length of 3 and 10 predictions (E), while the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) uses 50 alignments (B), 10 predictions (E), M=5, N=-4, and comparison of both strands.

[0076] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0077] The percentage identicality between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4:11-17 (1988) (which is incorporated into the ALIGN program (version 2.0)), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage identicality between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970 (which is incorporated into the GCG software package (University of South Florida)). The determination can be made using the BLOSUM62 matrix or PAM250 matrix (available from [source], incorporated into the GAP program), and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0078] Another indicator of substantial identity between two nucleic acid sequences or polypeptides, beyond the previously noted percentage of sequence identity, is that the polypeptide encoded by the first nucleic acid may cross-reactive immunologically with an enhanced antibody against the polypeptide encoded by the second nucleic acid, as described below. Therefore, the polypeptide is typically substantially identical to the second polypeptide if, for example, the two peptides differ only by conservative substitutions. Another indicator of substantial identity between two nucleic acid sequences is that the two molecules or their complements may hybridize under stringent conditions, as described below. Yet another indicator of substantial identity is that the same primers may be used to amplify the sequences.

[0079] The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded forms, and polymers thereof. Examples of nucleic acids that are part of this disclosure include cDNA, genomic DNA, recombinant DNA, and RNA (e.g., mRNA). The term encompasses nucleic acids containing known nucleotide analogs or modified skeletal residues or bindings, which include synthetic nucleic acids, naturally occurring nucleic acids, and non-naturally occurring nucleic acids, which have similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs, but not limited to, include phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0080] Unless otherwise specified, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, as detailed below, degenerate codon substitutions can be achieved by producing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

[0081] In the context of nucleic acids, the term "operably bound" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. A typical example is the functional relationship between a transcriptional regulatory sequence and the sequence being transcribed. For example, a promoter or enhancer sequence is operably bound to a coding sequence if it stimulates or modulates the transcription of the coding sequence in a suitable host cell or other expression system. Typically, a promoter transcriptional regulatory sequence operably bound to a transcriptional sequence is physically adjacent to the transcriptional sequence, i.e., cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not need to be physically adjacent to or positioned in close proximity to the coding sequence they enhance transcription of.

[0082] The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms are used for amino acid polymers, which are artificial chemical mimics of corresponding naturally occurring amino acids, and for naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise specified, a particular polypeptide sequence also implicitly includes its conservatively modified variants.

[0083] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" and "subject" are used interchangeably herein.

[0084] As used herein, phrases such as “patients requiring treatment” or “subjects requiring treatment” include subjects such as mammals in which administration of the antibodies or compositions of this disclosure used for detection, diagnostic procedures, and / or treatment is expected to be effective.

[0085] "IC50" (half-inhibitory concentration) refers to the concentration of a specific antibody or its fragment that inhibits the intermediate signal (50%) between the baseline control and the maximum possible signal.

[0086] "EC50" (Circular Effective Concentration) refers to the concentration of a particular antibody or its fragment that induces an intermediate response (50%) between the baseline control and the maximum possible effect after a predetermined exposure or treatment period. For example, EC50 is the concentration of antibody that reduces viral infection by 50%.

[0087] "EC90" refers to the concentration of a specific antibody or its fragment that induces a response corresponding to 90% of the maximum possible effect after a given exposure or treatment time. For example, EC90 is the concentration of an antibody or its fragment that reduces viral infection by 90%.

[0088] The terms “treatment” or “to treat” are defined herein as the application or administration of an antibody or antigen-binding fragment relating to the Disclosure, or a pharmaceutical composition containing such antibody, to or from a subject or tissue or cell line isolated from a subject, wherein the subject has a specific disease (e.g., arthritis), disease-related symptoms, or a predisposition to the development of the disease (if applicable). The objective is to cure the disease (if applicable), delay the onset, reduce the severity, alleviate symptoms, achieve remission of one or more symptoms, improve the disease, or reduce or improve any disease-related symptoms or predisposition to the development of the disease. The terms “treatment” or “to treat” include treating a patient suspected of having the disease, as well as a patient with the disease or a patient diagnosed with the disease or medical condition, and include suppression of clinical relapses. The phrase “reduce prognosis” refers to delaying the onset, development, or progression of a disease, infection, or disorder.

[0089] The terms “therapeutably acceptable amount,” “therapeutably effective amount,” or “therapeutably effective dose” are interchangeable and refer to an amount sufficient to produce the desired outcome (e.g., reduction of disease activity, inhibition of disease progression, etc.). In some embodiments, a therapeutically acceptable amount does not induce or cause any undesirable side effects. A therapeutically acceptable amount may be determined by administering a low dose initially and then gradually increasing the dose until the desired effect is achieved. The “prophylactically effective dose” and “therapeutably effective dose” of the molecules in this disclosure can prevent the onset of symptoms or reduce the severity of symptoms, respectively.

[0090] The term "co-administered" refers to the simultaneous presence of two activators in an individual's blood. Activators (e.g., additional therapeutic agents) co-administered with the antibodies and antigen-binding fragments of this disclosure can be delivered in parallel or sequentially.

[0091] As used herein, the terms “Fc” or “Fc region” mean a polypeptide containing the CH2-CH3 domain of an IgG molecule, and in some cases, the hinge. In EU numbering for human IgG1, the CH2-CH3 domain contains amino acids 231-447, and the hinge contains amino acids 216-230. Therefore, the definition of “Fc region” includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. In this context, “Fc fragments” may contain fewer amino acids from one or both of the N and C-terminuses, but generally retain the ability to form dimers with other Fc regions so that they are detectable using standard methods based on size (e.g., non-denaturing chromatography, size exclusion chromatography). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0092] A “mutant Fc region” or “modified Fc fragment” contains an amino acid sequence that differs from that of a “natural” or “wild-type” sequence Fc region by at least one “amino acid modification” as defined herein. Preferably, the mutant Fc region has at least one amino acid substitution compared to the natural sequence Fc region or the Fc region of the parent polypeptide, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions. The mutant Fc region herein will preferably have at least about 80% homology to the natural sequence Fc region and / or the Fc region of the parent polypeptide, most preferably at least about 90% homology, and more preferably at least about 95% homology.

[0093] As used herein, the term "Fc variant" refers to a polypeptide containing modifications in the Fc region. The Fc variants of the present invention are defined according to the amino acid modifications that constitute them. For example, P329G is an Fc variant having a proline substitution by glycine at position 329 compared to the parent Fc polypeptide. However, numbering follows the EU index. The identity of the wild-type amino acid may be unspecified, in which case the aforementioned variant is called P329G. For all positions considered in the present invention, numbering follows the EU index. The EU index, or the EU index in Kabat or the EU numbering scheme, refers to the numbering of EU antibodies (Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring and non-naturally occurring amino acids. Variants may include non-natural amino acids. Examples include U.S. Patent No. 6,586,207; International Publication No. 98 / 48032, International Publication No. 03 / 073238, U.S. Patent Application Publication No. 2004 / 0214988A1, International Publication No. 05 / 35727A2; International Publication No. 05 / 74524A2; Chin, JW, et al., Journal of the American Chemical Society 124(2002)9026-9027; Chin, JW, and Schultz, PG, ChemBioChem 11(2002)1135-1137; Chin, JW, et al., PICAS United States of America 99(2002)11020-11024; and Wang, L., and Schultz, PG, Chem. (2002) 1-10 is cited, and the entire work is incorporated by reference.

[0094] The term "Fc region-containing polypeptide" refers to polypeptides that contain an Fc region, such as binding molecules, antibodies, or immunoadhesins.

[0095] The term “Fc receptor” or “FcR” is used to describe receptors that bind to the Fc region of an antibody. The preferred FcR is the natural sequence human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors) and include the FcγRI, FcγRII, and FcγRIII subclass receptors (including allelic variants and alternative splice forms of these receptors). FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See review by Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcR has been reviewed in Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492; Capel, et al., Immunomethods 4 (1994) 25-34; and de Haas, et al., J. Lab. Clin. Med. 126 (1995) 330-41. Other FcRs, including those to be identified in the future, are included in the term “FcR” as used herein.

[0096] As used herein, "IgG Fc ligand" means any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an IgG antibody to form an Fc / Fc ligand complex. Examples of Fc ligands, but not limited to, include FcγR, FcγR, FcγR, FcRn, C1q, C3, mannan-binding lectins, mannose receptors, Staphylococcus protein A, Streptococcus protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), a family of Fc receptors homologous to FcγR (Davis, et al., Immunological Reviews 190 (2002) 123-136 (incorporated in its entirety by reference)). Fc-binding molecules may include undiscovered molecules that bind to Fc. Specific IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, "Fc ligand" means any biologically derived molecule, preferably a polypeptide, that binds to the Fc region of an antibody to form an Fc / Fc ligand complex.

[0097] As used herein, “Fc gamma receptor,” “FcγR,” or “FcgammaR” refers to any member of the family of proteins that bind to the Fc region of an IgG antibody, and is encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI(CD64), including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII(CD32), including isoforms FcγRIIA (including allotypes H131 and R131), FcγRIIB (including FcγRIIB-1 and FcγRIIB-2), and FcγRIIc; and FcγRIII(CD16), including isoforms FcγRIIIA (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIB-NA1 and FcγRIIB-NA2) (Jefferis, et al., Immunol Lett 82(2002) 57-65 (the whole is incorporated by reference)), as well as any undiscovered human FcγR or FcγR isoform or allotype. FcγR may originate from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγR may include, but not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0098] As used herein, “wild-type polypeptide” means an unmodified polypeptide that is subsequently modified to produce mutants. A wild-type polypeptide may be a naturally occurring polypeptide, or a mutant or engineered version of a naturally occurring polypeptide. A wild-type polypeptide may refer to the polypeptide itself, a composition containing the parent polypeptide, or the amino acid sequence encoding it. Therefore, as used herein, “wild-type immunoglobulin” means an unmodified immunoglobulin polypeptide that is modified to produce mutants, and as used herein, “wild-type antibody” means an unmodified antibody that is modified to produce mutant antibodies. It should be noted that “wild-type antibody” includes known commercially available recombinant antibodies outlined below.

[0099] As used herein, the terms “antibody effector function” or “effector function” refer to the function to which the Fc effector domain of IgG (e.g., the Fc region of immunoglobulins) contributes. Such functions can be exerted, for example, by the binding of the Fc effector domain to Fc receptors on phagocytic or lytic immune cells, or by the binding of the Fc effector domain to components of the complement system. Typical effector functions are ADCC, ADCP, and CDC. Effector functions may also include Fc-mediated inflammation and immunomodulation through the induction of cell differentiation and activation.

[0100] As used herein, the term “ADCC” or “antibody-dependent cytotoxicity” activity refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, subsequently causing lysis of those target cells. The primary cells for mediating ADCC (NK cells) express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.

[0101] As used herein, the term “ADCP” or “antibody-dependent cellular phagocytosis” refers to the process by which antibody-coated cells are internalized, either entirely or partially, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc domain.

[0102] As used herein, the term “CDC” or “complement-dependent cytotoxicity” activity refers to a cell death-inducing mechanism by which the Fc effector domain of a target-binding antibody activates a series of enzymatic reactions, resulting in the formation of holes in the target cell membrane. Typically, an antigen-antibody complex, e.g., on an antibody-coated target cell, binds to and activates complement component C1q, thereby activating the complement cascade and leading to target cell death. Complement activation can also lead to the deposition of complement components on the target cell surface, thus promoting ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0103] As used herein, "C1q" is a polypeptide containing a binding site to the Fc region of an immunoglobulin. C1q, together with two serine proteases C1r and C1s, forms the complex C1, the first component of the complement-dependent cytotoxicity (CDC) pathway. Human C1q is commercially available, for example, from Quidel, San Diego, Calif.

[0104] As used herein, “reduced effector function” means a reduction of at least 20% in specific effector function, e.g., ADCC or CDC, compared to a control (e.g., a polypeptide having a wild-type Fc region); “significantly reduced effector function” means a reduction of at least 50% in specific effector function, e.g., ADCC or CDC, compared to a control; and “undetectable effector function” means that the specific effector function, e.g., ADCC or CDC, is reduced to below the detection limit of the assay used.

[0105] As used herein, “human effector cells” are leukocytes that express one or more FcRs to exert effector function. Preferably, the cells express at least FcγRIII to exert ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from their natural sources, for example, from blood or PBMCs as described herein.

[0106] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors can operatively direct the expression of the nucleic acid to which they are ligated. Such vectors are referred to herein as “expression vectors.”

[0107] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” and include primary transformed cells and their offspring, regardless of the number of passages. Offspring may contain mutations, although their nucleic acid content may not be exactly identical to that of the parent cells. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0108] Fc Silent Mutation The present invention provides a binding molecule containing modified IgG1 Fc having mutations in the Fc region, resulting in an “Fc-silent” binding molecule with minimal interaction with effector cells, such as an antibody or its functional fragment, an Fc fusion molecule, or a multispecific antibody format. Generally, the “IgG1 Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including both the native sequence Fc region and the mutant Fc region. The human IgG1 heavy chain Fc region is generally defined as the C-terminal portion of the heavy chain, starting from the amino acid residue at position C226 or from P230 to the carboxyl terminus (K447) of the IgG1 antibody. The numbering of residues in the Fc region is based on the Kabat EU index. The C-terminal lysine (residue K447) of the Fc region may be partially or completely removed during antibody production or purification, for example, based on enzymatic clipping.

[0109] The present invention provides an Fc silent antibody or Fc-containing binding protein or Fc fusion protein containing IgG1 Fc having a combination of amino acid substitutions selected from the group consisting of substitutions: L234A, L235A, G237A (LALAGA), L234A, L235A, S267K, P329A (LALASKPA), D265A, P329A, S267K (DAPASK), G237A, D265A, P329A (GADAPA), G237A, D265A, P329A, S267K (GADAPASK), L234A, L235A, P329G (LALAPG), or L234A, L235A, P329A (LALAPA). However, the amino acid residues are numbered according to the Kabat EU index. The most preferred embodiment is an IgG1 Fc containing either LALASKPA and / or the GADAPASK silencing motif.

[0110] The Fc silent antibodies of this disclosure result in undetectable or severely reduced effector function. For example, the Fc silent antibodies of this disclosure exhibit ADCC of less than 50% specific cell lysis (low ADCC activity), or ADCC of less than 30%, 20%, 10%, 5%, 2%, or 1% specific cell lysis, or ADCC below the detection limit of the assay used (undetectable ADCC activity), compared to wild-type antibodies. At the same time, the Fc silent antibodies and conjugating molecules of this disclosure retain good developable properties; they have a high melting temperature for Fc, are stable, have the ability to be recombinantly produced in high yield, and maintain a stable state for long periods without aggregation when formulated at high concentrations.

[0111] Additional mutations and modifications The binding molecules of this disclosure may further include mutations and / or modifications to improve the properties of the binding molecules.

[0112] In one embodiment, further modifications are carried out to reduce the immunogenicity of the binding molecule.

[0113] For example, one approach involves "reverse mutation" one or more additional framework residues into the corresponding germline sequences. More specifically, somatically mutated antibodies may contain framework residues different from those in the germline sequence from which the antibody originates. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originates. To restore the framework region sequences to their germline configuration, somatic mutations can be "reverse-mutated" into germline sequences, for example, by site-directed mutagenesis. Antibodies with such "reverse mutations" are also intended to be included.

[0114] Another type of framework modification involves reducing the potential immunogenicity of an antibody by mutating one or more residues within a framework region or within one or more CDR regions to remove a T cell epitope. This approach, also known as “deimmunization,” is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al.

[0115] In another embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, for example, increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

[0116] In another embodiment, the Fc hinge region of an antibody or fragment is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of an Fc-hinge fragment so that the antibody has damaged SpA binding compared to the staphylococcal protein A (SpA) binding of the natural Fc-hinge domain. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0117] In another embodiment, one or more amino acid residues are modified to alter the complement-fixing ability of the antibody. This approach is described, for example, in Bodmer et al.'s International Publication No. 94 / 29351. In a specific embodiment, one or more amino acids of the antibody or its antigen-binding fragment of the Disclosure are substituted with one or more allotype amino acid residues for the IgG1 subclass and kappa isotype. Allotype amino acid residues include, but are not limited to, the constant regions of the heavy chains of the IgG1, IgG2, and IgG3 subclasses and the constant regions of the light chains of the kappa isotypes, as described by Jefferis et al., MAbs.1:332-338 (2009).

[0118] In another embodiment, the fragment is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced, as described in Ward's U.S. Patent No. 6,277,375: T252L, T254S, T256F. In addition, to increase the biological half-life, the antibody may be modified within the CH1 or CL region to contain a salvage receptor-binding epitope taken from two loops of the CH2 domain in the Fc region of IgG, as described in Presta et al.'s U.S. Patents No. 5,869,046 and No. 6,121,022. In a preferred embodiment, the modified IgG1-containing binding molecule disclosed herein further includes modification of the Fc to contain a "YTE" mutation (M252Y, S254T, T256E (according to EU numbering)) for half-life extension.

[0119] Production of antibodies and fragments The conjugation molecules of the present invention may be produced by any means known in the art, but are not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, where the full-length monoclonal antibody may be obtained by hybridoma generation or recombinant generation. Recombinant expression may be derived from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0120] Furthermore, this specification discloses isolated nucleic acid molecules or sets of nucleic acid molecules encoding antibodies or antigen-binding fragments described herein. In some embodiments, the isolated nucleic acid molecules are complementary DNA (cDNA) or messenger RNA (mRNA).

[0121] This disclosure further provides polynucleotides encoding antibodies and binding molecules as described herein, for example, polynucleotides encoding variable regions or segments of heavy or light chains including complementarity-determining regions as described herein.

[0122] The modified IgG1 Fc region of the binding molecule is encoded by the nucleic acid sequences of SEQ ID NOs. 16 and 22 in Table 1. In some embodiments, the polynucleotide encoding the Fc region of the binding molecule has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotide of SEQ ID NOs. 16 or 22 (Table 1).

[0123] Polynucleotide sequences can be generated by de novo solid-phase DNA synthesis of existing sequences or by PCR mutagenesis. Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid-support method of U.S. Patent No. 4,458,066. The introduction of mutations into polynucleotide sequences by PCR can be carried out as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HAErlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[0124] This disclosure also provides expression vectors and host cells for producing the binding molecules described above. This specification discloses cloning and expression vectors comprising one or more nucleic acid molecules or sets of nucleic acid molecules encoding the binding molecules described above. These vectors are suitable for recombinant production of antibodies or their antigen-binding fragments.

[0125] Various expression vectors can be used to express the disclosed binding molecules, such as polynucleotides encoding antibodies. Both virus-based and non-viral expression vectors can be used to generate antibodies in mammalian host cells. Examples of non-viral vectors and non-viral systems include plasmid or episomal vectors (typically having expression cassettes for expressing proteins or RNA) and human artificial chromosomes (see, e.g., Harrington et al., Nat Gen. 15:345, 1997). Examples of non-viral vectors useful for expressing polynucleotides and polypeptides of binding molecules in mammalian (e.g., human) cells include pThioHis A,B & C, pcDNA3.1 / His, pEBVHis A,B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in this technique for expressing other proteins. Useful viral vectors include retrovirus, adenovirus, adeno-associated virus, herpesvirus-based vectors, SV40, papillomavirus, HBP Epstein-Barr virus-based vectors, vaccinia virus vectors, and Semryki Forest virus (SFV). See Brent et al., op. cit.; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[0126] The selection of an expression vector depends on the host cell in which the vector is intended to be expressed. Typically, an expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably bound to a binding molecule, such as a polynucleotide encoding an antibody. In some embodiments, an inducing promoter is used to prevent the expression of the inserted sequence outside of induction conditions. Examples of inducing promoters include arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can grow under non-inducing conditions without biasing the expression product toward a coding sequence better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for the efficient expression of the binding molecule, such as an antibody. Typical examples of such elements include the ATG start codon and adjacent ribosome binding sites or other sequences. In addition, the efficiency of expression can be enhanced by including an enhancer suitable for the cell system being used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, expression in mammalian host cells can be increased using SV40 enhancers or CMV enhancers.

[0127] Expression vectors can also provide secretion signal sequence locations to form fusion proteins with polypeptides encoded by inserted antibody or fragment sequences. Often, the inserted antibody or fragment sequence is bound to the signal sequence before being incorporated into the vector. H and V L The vectors used to accept sequences encoding a constant region or a portion thereof occasionally encode a constant region. Such vectors can produce intact antibodies or fragments thereof by expressing the variable region as a fusion protein with the constant region. Typically, such constant regions are human.

[0128] This specification discloses host cells containing one or more cloning or expression vectors described herein. The host cell for harboring and expressing the binding molecule may be a prokaryote or a eukaryote. Escherichia coli (E. coli) is a useful prokaryotic host for cloning and expressing the polynucleotides of this disclosure. Other suitable microbial hosts include rods, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, those skilled in the art can construct expression vectors, which typically contain expression regulatory sequences (e.g., origins of replication) compatible with the host cell. In addition, there are any number of various well-known promoters, such as lactose promoters, tryptophan (trp) promoters, beta-lactamase promoters, or phage-lambda-derived promoters. Promoters typically control expression, sometimes along with operator sequences, and also contain ribosome binding sites, initiating and completing transcription and translation. Furthermore, other microorganisms, such as yeast, can be used to express the binding molecule. Insect cells combined with baculovirus vectors can also be used.

[0129] In other embodiments, the binding molecules of this disclosure can be expressed and generated using mammalian host cells. For example, the mammalian host cells may be hybridoma cell lines expressing endogenous immunoglobulin genes (e.g., myeloma hybridoma clones) or mammalian cell lines possessing exogenous expression vectors. These include any animal or human cells, whether standard or immortal, that lead to death or are standard or abnormal. For example, several suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293, HEK293T cells, SP2 / 0 cells, NS0 myeloma cell lines, transformed B cells, and hybridomas. CHO cell lines are most preferred. The use of mammalian tissue cell cultures to express polypeptides has generally been discussed, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may contain expression regulatory sequences, such as origins of replication, promoters, and enhancers (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), as well as essential processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Appropriate promoters may be constitutive, cell type-specific, stage-specific, and / or tunable or moduloable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-induced MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-induced CMV promoters (e.g., human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0130] The method for introducing an expression vector containing the target polynucleotide sequence varies depending on the type of cell host. For example, calcium chloride transduction is commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts (generally, see Sambrook et al., op. cit.). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion to the herpesvirus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. In many cases, stable expression is desired for long-term high-yield production of recombinant proteins. For example, cell lines that stably express the binding molecule can be prepared using an expression vector containing a viral replication origin or endogenous expression element and a selection marker gene. After introducing the vector, the cells can be grown in enriched medium for 1-2 days before being switched to a selection medium. The purpose of the selection marker is to confer resistance to selection, and its presence enables the growth of cells that successfully express the introduced sequence in the selection medium. Stable, transfected cells exhibiting resistance can be grown using tissue culture techniques appropriate to the cell type.

[0131] In some embodiments, the binding molecule consists of a single polypeptide chain encoded by a single nucleic acid that can be inserted into a single cloning or expression vector. In other embodiments, the binding molecule consists of two polypeptide chains encoded by two or more nucleic acids, referred herein as a “nucleic acid molecule set.” In some embodiments, the nucleic acid encoding the first chain is inserted into a first cloning or expression vector, and the nucleic acid encoding the second chain is inserted into a second cloning or expression vector. In this situation, the binding molecule is expressed via the cloning or expression vector set. Alternatively, both nucleic acids can be inserted into a single cloning or expression vector.

[0132] This specification discloses a process for producing a binding molecule described herein, comprising culturing host cells described herein under conditions sufficient to express the antibody or its antigen-binding fragment, and then purifying and recovering the antibody or its antigen-binding fragment from the host cell culture as a single polynucleotide chain.

[0133] Isolation of recombinant antibodies and fragments Various methods for screening antibodies and proteins containing their antigen-binding moieties have been described in the art. Such methods can be classified into in vivo systems, such as transgenic mice capable of producing sufficient human antibodies upon antigen immunization, and in vitro systems, which consist of creating an antibody DNA coding library, expressing the DNA library in a system suitable for antibody production, selecting clones that express antibody candidates that bind to a target according to affinity selection criteria, and recovering the corresponding coding sequences of the selected clones. Such in vitro techniques are known as display techniques and include, but are not limited to, phage displays, RNA or DNA displays, ribosome displays, and yeast or mammalian cell displays. These have been well documented in the art (for reviews, see, for example: Nelson et al. 2010, Nature Reviews Drug discovery, “Development trends for human monoclonal antibody therapeutics” (Advance Online Publication) and Hoogenboom et al. 2001, Method in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, NJ). In a particular embodiment, the human recombinant antibody of this disclosure is isolated from a screening library of human recombinant antibody libraries, such as a HuCAL® library, using phage display.

[0134] V H and V LThe repertoire of genes or associated CDR regions can be cloned separately by polymerase chain reaction (PCR) or synthesized by a DNA synthesizer, and then randomly recombined in a phage library and screened for antigen-binding clones. Such phage display methods for isolating human antibodies are established in the art or described in the following examples. For example, see U.S. Patent Nos. 5,223,409, 5,403,484, and 5,571,698 granted to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 granted to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 granted to McCafferty et al.; and U.S. Patent Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081 granted to Griffiths et al.

[0135] In certain embodiments, human antibodies can be identified using transgenic or transchromosomal mice that carry a portion of the human immune system rather than a mouse lineage. These transgenic and transchromosomal mice include mice referred to herein as HUMAB mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice."

[0136] HUMAB mice (registered trademark) (Medarex, Inc.) contain human immunoglobulin gene minilocuses encoding unreconstructed human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg, et al. 1994, Nature 368:856-859). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. 1994; Lonberg, N., 1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. 1995, Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N. 1995, Ann. NYAcad. Sci. 764:536-546). The creation and use of HUMAB mice and the genomic modifications carried by such mice are described in Taylor, L. et al. 1992, Nucleic Acids Research 20:6287-6295; Chen, J. et al. 1993, International Immunology 5:647-656; Tuaillon et al. 1993, Proc. Natl. Acad. Sci. USA 94:3720-3724; Choi et al. 1993, Nature Genetics 4:117-123; Chen, J. et al. 1993, EMBO J. 12:821-830; Tuaillon et al. 1994, J. Immunol. 152:2912-2920; Taylor, L. et al. 1994, International Immunology Further details are found in pp. 579-591 and Fishwild, D. et al. 1996, Nature Biotechnology 14 845-851.Furthermore, U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, all granted to Lonberg and Kay; and U.S. Patent Nos. 5,770,429, all granted to Surani et al. See PCT International Publication No. 5,545,807; PCT International Publication No. 92103918, International Publication No. 93 / 12227, International Publication No. 94 / 25585, International Publication No. 97113852, International Publication No. 98 / 24884 and International Publication No. 99 / 45962, all of which are credited to Lonberg and Kay; and PCT International Publication No. 01 / 14424, credited to Korman et al.

[0137] In another embodiment, the human antibodies of this disclosure can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, for example, mice carrying human heavy chain transgenes and human light chain transchromosomes. Such mice (referred to herein as "KM mice") are described in detail in PCT International Publication No. 02 / 43478, issued to Ishida et al.

[0138] In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used for the production of the antibodies of this disclosure. For example, the alternative transgenic system called Xenomouse from Abgenix, Inc. is available. Such mice are described, for example, in U.S. Patents No. 5,939,598; No. 6,075,181; No. 6,114,598; No. 6,150,584 and No. 6,162,963 granted to Kucherlapati et al. As will be apparent to those skilled in the art, several other mouse models can be used, such as the TRIANNI mouse from Trianni, Inc., the VELOCIMMUNE mouse from Regeneron Pharmaceuticals, Inc., or the KYMOUSE mouse from Kymab Limited.

[0139] Furthermore, alternative transchromosomal animal lines expressing human immunoglobulin genes are available in the art and can be used for the production of the anti-IL-17A antibody of this disclosure. For example, mice carrying both human heavy chain transchromosomes and human light chain transchromosomes (referred to as "TC mice") are available; such mice are described in Tomizuka et al. 2000, Proc. Natl. Acad. Sci. USA 97:722-727.

[0140] The human monoclonal antibodies of this disclosure can also be prepared using SCID mice in which human immune cells have been reconstituted to generate a human antibody response through immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 granted to Wilson et al.

[0141] Production of monoclonal antibodies from rodents Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein 1975, Nature 256:495. Many techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can be employed.

[0142] The animal strain used for hybridoma production is the mouse strain. Hybridoma generation in mice is a well-established procedure. Immunotherapy protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0143] The chimeric or humanized antibodies of this disclosure can be prepared based on the sequences of the murine monoclonal antibodies prepared as described above. The DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and can be manipulated using standard molecular biology techniques to include non-murine (e.g., human) immunoglobulin sequences. For example, to produce a chimeric antibody, the murine variable region can be ligated to the human constant region using methods known in the art (see, for example, U.S. Patent No. 4,816,567, granted to Cabilly et al.). To produce a humanized antibody, the murine CDR region can be inserted into the human framework using methods known in the art. See, for example, U.S. Patent No. 5,225,539, granted to Winter, and U.S. Patents No. 5,530,101, 5,585,089, 5,693,762, and 6,180,370, granted to Queen et al.

[0144] Generation of hybridomas that produce monoclonal antibodies To generate hybridomas that produce the monoclonal antibodies of this disclosure, spleen cells and / or lymph node cells derived from immunized mice can be isolated and fused to suitable immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas can be screened for the production of antigen-specific or epitope-specific antibodies. For example, a single-cell suspension of immunized mouse spleen lymphocytes can be fused to 1 / 6 the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) using 50% PEG. Cells are plated approximately 2 × 145 on flat-bottom microtiter plates and subsequently incubated for 2 weeks in a selective medium containing 20% ​​fetal cloned serum, 18% "653" conditioned medium, 5% Origen (IGEN), 4 mM L-glutamine, 1 mM sodium pirubate, 5 mM HEPES, 0:055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, and 1 × HAT (Sigma; HAT added 24 hours after fusion). After approximately 2 weeks, cells can be cultured in medium in which HAT has been replaced with HT. Individual wells can then be screened for human monoclonal IgM and IgG antibodies by ELISA. Once extensive hybridoma growth has occurred, the medium can usually be observed after 10–14 days. Antibody-secreting hybridomas can be replated and rescreened, and if they remain human IgG-positive, monoclonal antibodies can be subcloned once or twice by limiting dilution. Stable subclones can then be cultured in vitro, and small amounts of antibody can be generated in tissue culture medium for characterization.

[0145] To purify monoclonal antibodies, selected hybridomas can be grown in a 2-liter spinner flask for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography with Protein A Sepharose (Pharmacia, Piscataway, NJ). To ensure purity, eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography. The buffer solution can be replaced with PBS, and OD can be used with an extinction coefficient of 1.43. 280 The concentration can be determined by this method. Monoclonal antibodies can be ali-coated and stored at -80°C.

[0146] Generation of transfectomas that produce monoclonal antibodies The antibodies described herein can be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods, as is well known in the art (e.g., Morrison, S.1985, Science 229:1202).

[0147] For example, to express an antibody or its antibody fragment, it is possible to obtain partial-length or full-length DNA encoding the light and heavy chains by standard molecular biology or biochemical techniques (e.g., DNA chemosynthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector so that the gene is operatively ligated to transcriptional and translational regulatory sequences. In this context, the term “operatively ligated” is intended to mean that the antibody gene is ligated to the vector so that the transcriptional and translational regulatory sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression regulatory sequences are selected to be compatible with the expression host cell in which they are used. The antibody light chain gene and antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or blunt-end ligation if no restriction sites exist). The light and heavy chain variable regions of the antibodies described herein are V H The segment is operationally linked to the CH segment within the vector and V L The full-length antibody gene for any antibody isotype can be generated by inserting the heavy chain constant region and light chain constant region of the desired isotype into an expression vector that already encodes them, so that the segments are operatively linked to the CL segment in the vector. Additionally or alternatively, recombinant expression vectors can encode a signal peptide, also called a leader sequence, which promotes the secretion of the antibody chain from host cells. The antibody chain gene can be cloned into the vector so that the signal peptide is in-frame linked to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0148] In addition to the antibody chain gene, the recombinant expression vectors of this disclosure carry regulatory sequences that control the expression of the antibody chain gene in host cells. The term “regulatory sequence” is intended to include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in Goeddel 1990, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA. Those skilled in the art will see that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, e.g., cytomegalovirus (CMV) Simianvirus 40 (SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyomas. Alternatively, non-viral regulatory sequences such as ubiquitin promoters and P-globin promoters can be used. In addition, the regulatory elements were further composed of an SRa promoter system (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472) containing sequences from different sources, for example, the SV40 initial promoter and sequences derived from the long-terminal repeat of human T-cell leukemia virus type 1.

[0149] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of this disclosure may carry additional sequences such as sequences that regulate vector replication in host cells (e.g., origins of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents No. 4,399,216, 4,634,665, and 5,179,017, all authorized by Axel et al.). Typically, for example, selection marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Selection marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR host cells by methotrexate selection / amplification) and the neo gene (for G418 selection).

[0150] For the expression of the light and heavy chains, host cells were transfected with expression vectors encoding the heavy and light chains using standard techniques. The various forms of the term “transfection” are intended to encompass the wide variety of techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. It is theoretically possible to express the antibodies of this disclosure in either prokaryotic or eukaryotic host cells. We consider antibody expression in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, because such eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies.

[0151] In a particular embodiment, the cloning or expression vector according to the Disclosure comprises at least one nucleic acid coding sequence of the Disclosure operatively ligated to a preferred promoter sequence.

[0152] Mammalian host cells for expressing the recombinant antibodies of this disclosure include Chinese hamster ovary (CHO cells) (including dhfr- CHO cells as described in Urlaub and Chasin 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DH FR selection marker, for example, as described in RJ Kaufman and PA Sharp 1982, Mol. Biol. 159:601-621), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells, and SP2 cells. Specifically, for use with NSO myeloma cells, another expression system is the GS gene expression system as described in PCT International Publication No. 87 / 04462, International Publication No. 89 / 01036, and European Patent No. 0338841. In one embodiment, mammalian host cells for expressing the recombinant antibody of this disclosure include, for example, a mammalian cell line lacking FUT8 gene expression, as described in U.S. Patent No. 6,946,292.

[0153] When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, antibodies are produced by culturing the host cells for a sufficient time to allow antibody expression in the host cells or secretion of antibodies into the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods (see, for example, Abhinav et al. 2007, Journal of Chromatography 848:28-37).

[0154] In one embodiment, the host cell of the Disclosure is a host cell transfected with an expression vector having a nucleic acid encoding the antibody or antigen-binding fragment of the Disclosure.

[0155] These host cells can then be further cultured under conditions suitable for the expression and production of the antibodies or antigen-binding fragments of the present disclosure.

[0156] multispecific molecules In another embodiment, the Disclosure features a bispecific or multispecific molecule comprising a binding molecule, e.g., an antibody, and comprising the modified and silented IgG1 Fc fragment of the Disclosure. The antibody or protein of the Disclosure may be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., a ligand for another antibody or receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody or protein of the Disclosure may be derivatized or linked to two or more other functional molecules to generate a multispecific molecule that binds to three or more different binding sites and / or target molecules, and such multispecific molecules are also intended to be included in the term “bispecific molecule” as used herein. To generate a bispecific molecule of the Disclosure, the antibody or protein of the Disclosure may be functionally linked to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide or binding mimetic, so as to generate a bispecific molecule (e.g., by chemical coupling, genetic fusion, non-covalent association, etc.). Methods for generating bispecific antibodies are well known in the art and are described, for example, in Krah et al, 2017, New Biotechnology, 2017, 39:167-173; Brinkmann and Kontermann, 2017, Mabs, 9(2):182-212; Godar et al., 2018, Expert Opinion on Therapeutic Patents, 28(3):251-256; Spiess et al., 2015, Molecular Immunology 67:95-106; and Kontermann and Brinkmann, 2015, 20(7):838-847.

[0157] In addition, disclosures in which a bispecific molecule is a multispecific molecule may further include a third binding specificity in addition to the first and second target epitopes.

[0158] In one embodiment, the bispecific or multispecific molecule containing the silented IgG1 Fc of this disclosure comprises, as binding specificity, at least one antibody or an antibody fragment thereof (e.g., including Fab, Fab', F(ab')2, Fv, or scFv). The antibody may also be a light chain or heavy chain dimer or any minimal fragment thereof, e.g., Fv or single-chain construct as described in Ladner et al., U.S. Patent No. 4,946,778.

[0159] Other antibodies available for use with the bispecific or multispecific molecules of this disclosure include mouse, chimeric, and humanized monoclonal antibodies.

[0160] Silenced IgG1 Fc containing the bispecific or multispecific molecules of this disclosure can be prepared by conjugating component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificity is a protein or peptide, various coupling agents or crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al. 1984, J. Exp. Med. 160:1686; Liu, MA et al. 1985, Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus 1985, Behring Ins. Mitt. No. 78, 118-132; Brennan et al. 1985, Science 229:81-83), and Glennie et al. 1987, J. Immunol. 139:2367-2375). The conjugating agents are SATA and sulfo-SMCC, both of which are available from Pierce Chemical Co. (Rockford, IL).

[0161] When the binding specificity is an antibody, they can be bound by sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge regions are modified before conjugation to contain an odd number of sulfhydryl residues, for example, one.

[0162] The binding specificity is encodeable in the same vector and expressible and assembleable in the same host cells. This method is particularly useful when the bispecific molecule is an mAb×mAb, mAb×Fab, Fab×F(ab')2, or ligand×Fab fusion protein. The bispecific or multispecific molecules of this disclosure may be a single-chain molecule containing one single-chain antibody and a binding determinant, or a single-chain multispecific molecule containing two binding determinants. The multispecific molecule may contain at least two single-chain molecules. Methods for preparing multispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.

[0163] The binding of multispecific molecules to their specific targets can be confirmed by, for example, enzyme-coupled immunosorbent assays (ELISA), radioimmunoassays (REA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a specific target protein-antibody complex by utilizing a labeling reagent (e.g., antibody) specific to the complex in question.

[0164] knob-in-hole (KIH) (also known as "key-in-hole") A multispecific silencing molecule containing IgG1 Fc, for example, the multispecific antibody or antibody-like molecule of the present invention, may contain one or more mutations, e.g., multiple mutations, on one or more constant domains, e.g., the CH3 domain. In one example, the multispecific binding molecule of the present invention comprises two polypeptides, each containing an antibody heavy chain Fc or constant domain, e.g., a CH2 or CH3 domain. In one example, the two heavy chain constant domains, e.g., the CH2 or CH3 domains of the multispecific binding molecule, contain one or more mutations that enable heterodimeric association between the two chains. In one embodiment, one or more mutations are located on the CH2 domains of the two heavy chains of a multispecific, e.g., a bispecific antibody or antibody-like molecule. In one embodiment, one or more mutations are located on the CH3 domains of at least two polypeptides of the multispecific binding molecule. In one embodiment, one or more mutations in the first polypeptide of the multispecific binding molecule containing a heavy chain constant domain create a "knob" and one or more mutations in the second polypeptide of the multispecific binding molecule containing a heavy chain constant domain create a "hole" and a "knob" such that the heterodimerization of the polypeptide of the multispecific binding molecule containing a heavy chain constant domain causes the "hole" and "knob" to be bounded (for example, so that they interact, for example, so that the CH2 domain of the first polypeptide interacts with the CH2 domain of the second polypeptide, or so that the CH3 domain of the first polypeptide interacts with the CH3 domain of the second polypeptide). When the term is used herein, "knob" means at least one amino acid side chain protruding from the interface of the first polypeptide of the multispecific binding molecule containing a heavy chain constant domain, and is therefore possible to position it in a complementary "hole" at the interface with the second polypeptide of the multispecific binding molecule containing a heavy chain constant domain, for example, to stabilize the heteromultimer and make heteromultimer formation more favorable than homomultimer formation. The knob may be present in the original interface or may be introduced synthetically (for example, by modifying the nucleic acid encoding the interface).Preferred import residues for knob formation are generally naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Most preferred are tryptophan and tyrosine. In preferred embodiments, the original residue for knob formation has a small side chain and is, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

[0165] "Hole" refers to at least one amino acid side chain recessed from the interface of the second polypeptide of the multispecific binding molecule containing a heavy chain constant domain, and thus accommodating the corresponding knob on the adjacent interface surface of the first polypeptide of the multispecific binding molecule containing a heavy chain constant domain. The hole may be present at the original interface or may be introduced synthetically (e.g., by modifying the nucleic acid encoding the interface). Preferred import residues for hole formation are usually naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Most preferred are serine, alanine, or threonine. In preferred embodiments, the original residue for hole formation has a large side chain volume and is, for example, tyrosine, arginine, phenylalanine, or tryptophan.

[0166] In one embodiment, the first CH3 domain is mutated at residues 366, 405, or 407 according to Kabat et al.'s EU numbering scheme to produce either a "knob" or a "hole" (as described above) (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)), and the second CH3 domain that heterodimerizes with the first CH3 domain is mutated at residue 407 if residue 366 of the first CH3 domain is mutated, at residue 394 if residue 405 of the first CH3 domain is mutated, or at residue 366 if residue 407 of the first CH3 domain is mutated, in order to produce a "hole" or "knob" complementary to the "knob" or "hole" of the first CH3 domain (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)).

[0167] In another embodiment, the first CH3 domain is mutated at residue 366 according to Kabat et al.'s EU numbering scheme to produce either a "knob" or a "hole" (as described above) (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)), and the second CH3 domain, which heterodimerizes with the first CH3 domain, is mutated at residues 366, 368 and / or 407 according to Kabat et al.'s EU numbering scheme to produce a "hole" or "knob" complementary to the "knob" or "hole" of the first CH3 domain (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)). In one embodiment, a mutation in the first CH3 domain introduces a tyrosine (Y) residue at position 366. In another embodiment, the mutation in the first CH3 is T366Y. In one embodiment, a mutation in the first CH3 domain introduces a tryptophan (W) residue at position 366. In another embodiment, the mutation in the first CH3 is T366W. In embodiments, according to the EU numbering scheme of Kabat et al., mutations in a second CH3 domain that heterodimerizes with a first CH3 domain mutated at position 366 (e.g., having tyrosine (Y) or tryptophan (W) introduced at position 366, e.g., mutant T366Y or T366W) include mutations at position 366, position 368, and position 407 (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)). In embodiments, the mutation at position 366 introduces a serine (S) residue, the mutation at position 368 introduces alanine (A), and the mutation at position 407 introduces valine (V).In one embodiment, the mutations include T366S, L368A, and Y407V. In one embodiment, the first CH3 domain of the multispecific binding molecule contains the mutation T366Y, and the second CH3 domain that heterodimerizes with the first CH3 domain contains the mutations T366S, L368A, and Y407V, or vice versa. In one embodiment, the first CH3 domain of the multispecific binding molecule contains the mutation T366W, and the second CH3 domain that heterodimerizes with the first CH3 domain contains the mutations T366S, L368A, and Y407V, or vice versa.

[0168] Additional knob-in-hole mutant pairs suitable for use in any of the multispecific binding molecules of the present invention are further described, for example, in International Publication No. 1996 / 027011 and Merchant et al., Nat. Biotechnol., 16:677-681 (1998) (their contents are incorporated herein by reference in their entirety).

[0169] In any of the embodiments described herein, the CH3 domain may be additionally mutated to introduce a pair of cysteine ​​residues. Although not theoretically constrained, the introduction of a pair of cysteine ​​residues capable of forming a disulfide bond is thought to provide stability to the heterodimerized multispecific binding molecule. In the embodiments, the first CH3 domain contains cysteine ​​at position 354 according to the EU numbering scheme of Kabat et al. (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)), and the second CH3 domain heterodimerizing with the first CH3 domain contains cysteine ​​at position 349 according to the EU numbering scheme of Kabat et al. (pp. 688-696 in Sequences of proteins of immunological interest, 5th ed., Vol. 1 (1991; NIH, Bethesda, Md.)). In the embodiment, the first CH3 domain of the multispecific binding molecule includes cysteine ​​(e.g., mutant S354C) at position 354 and tyrosine (Y) (e.g., mutant T366Y) at position 366, and the second CH3 domain that heterodimerizes with the first CH3 domain includes cysteine ​​(e.g., mutant Y349C) at position 349, serine (e.g., mutant T366S) at position 366, alanine (e.g., mutant L368A) at position 368, and valine (e.g., mutant Y407V) at position 407. In the embodiment, the first CH3 domain of the multispecific binding molecule comprises cysteine ​​(e.g., mutant S354C) at position 354 and tryptophan (W) (e.g., mutant T366W) at position 366, and the second CH3 domain that heterodimerizes with the first CH3 domain comprises cysteine ​​(e.g., mutant Y349C) at position 349, serine (e.g., mutant T366S) at position 366, alanine (e.g., mutant L368A) at position 368, and valine (e.g., mutant Y407V) at position 407.

[0170] The additional mechanism used for heterodimer formation is sometimes referred to as “electrostatic steering,” as described in Gunasekaran et al., 2010, J. Biol. Chem. 285(25):19637 and Strop et al., 2012, J. Mol. Biol. 420:204-19. This is sometimes referred to herein as “charge pairing.” In this embodiment, electrostatics is used to bias the formation toward heterodimerization. Two positively charged lysines (D339K, E356K) are on one strand and two uncharged aspartates (K409D, K392D) are on the other. In another embodiment, the mutation is introduced only in the CH3 domain (strand A: L368E, strand B: K409D), but also in the hinge region of IgG1 (strand A: D221E and P228E, strand B: D221R and P228R). In another embodiment, the mutations include D221E / P228E / L368E which pairs with D221R / P228R / K409R, and C220E / P228E / 368E which pairs with C220R / E224R / P228R / K409R.

[0171] In certain embodiments, multispecific (e.g., bispecific or trispecific) antibodies or antibody-like molecules can also be generated by Fab arm exchange as described in Labrigin et al., 2011, J.Immunol. 187:3239-46; Labrigin et al., 2013, Proc.Natl.Acad.Sci., 110:5145-50, International Publication No. 08 / 119353, International Publication No. 2011 / 131746, and International Publication No. 2013 / 060867. In another embodiment, the multispecific antibody may include mutations in the CH3 region that promote Fc heterodimerization, as described in Moore et al., 2011, Mabs, 3:546-57: S364H, Y349T, and T394H, and optionally its subtly stability-enhancing mutation T350V.

[0172] In certain embodiments, multispecific (e.g., bispecific or trispecific) antibodies or antibody-like molecules are generated by Strand Exchange Engineered Domains (SEED) heterodimer formation, as described, for example, Davis et al., 2010, Protein Eng. Des. Sel., 23:195-202; Muda et al., 2011, Protein Eng. Des. Sel., 24:447-454; and International Publication No. 07 / 110205. In these embodiments, substantial changes are introduced into the Fc region by manipulating the CH3 domain's substituted IgG and IgA segments, resulting in two non-identical antiparallel chains, referred to as GA and AG, which construct an asymmetric heterodimerization interface.

[0173] In certain embodiments, multispecific (e.g., bispecific or trispecific) antibodies or antibody-like molecules are produced by other techniques well known in the art, for example, biantigen conjugates by antibody crosslinking to generate a bispecific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl-reactive group, as described in U.S. Patent No. 4,433,059; for example, bispecific antibodies or antibody-like molecule determinants produced by recombining half-antibodies (heavy-light chain pairs or Fab) from different antibodies or antibody-like molecules via a reduction and oxidation cycle of the disulfide bond between two heavy chains, as described in U.S. Patent No. 4,444,878; for example, U.S. Trifunctional antibodies such as three Fab' fragments crosslinked via sulfhydryl reactive groups as described in U.S. Patent No. 5273743; biosynthetic binding proteins such as scFv pairs crosslinked via C-terminal tails preferably by disulfide or amine-reactive chemical crosslinking as described in U.S. Patent No. 5534254; bifunctional antibodies such as Fab fragments with different binding specificities dimerized via leucine zippers (e.g., c-fos and c-jun) to replace constant domains as described in U.S. Patent No. 5582996; for example, the CH1 region of one antibody and the V region of the other antibody as described in U.S. Patent No. 5591828 HV of two antibodies (two Fab fragments) linked to a region (typically having associated light chains) via a polypeptide spacer. H -Dual-specific and oligo-specific monovalent and oligovalent receptors such as the CH1 region (Fd region); for example, dual-specific DNA-antibody conjugates such as those in which an antibody or Fab fragment is crosslinked via a double-stranded DNA piece, as described in U.S. Patent No. 5,635,602; for example, dual-specific fusion proteins such as expression constructs containing two scFv via a hydrophilic helical peptide linker between them and the entire constant region, as described in U.S. Patent No. 5,637,481; for example, polyvalent and multi-specific binding proteins such as polypeptide dimers (which can produce higher-order structures for dual-specific, triple-specific, or quadruple-specific molecules) having a first domain with a binding region for the Ig heavy chain variable region and a second domain with a binding region for the Ig light chain variable region, commonly referred to as a diabody, as described in U.S. Patent No. 5,837,242; for example, linked V, which is further connected to the antibody hinge region and the CH3 region by a peptide spacer, as described in U.S. Patent No. 5,837,821 L Chain and V H Mini-body constructs having chains (dimerizable to form bispecific / polyvalent molecules); linked with or without a linker using a short peptide linker (e.g., 5 or 10 amino acids) in either orientation. L and V H Domains (capable of forming dimers to form a bispecific diabody); for example, trimers and tetramers as described in U.S. Patent No. 5,844,094; for example, V to form a series of FVs (or scFvs) as described in U.S. Patent No. 5,864,019 L V is further associated with the domain and linked at the C-terminus by a peptide linkage having a crosslinkable group. H Domain (or V among family members) LA string of domains; for example, as described in International Publication No. 2011 / 028952, one antigen is monovalently bound and the other antigen is divalently bound, and optionally contains a heterodimeric Fc region. L and V H Domains, scFv, or Fab; and, for example, as described in U.S. Patent No. 5,869,620, both scFv or diabody-type formats, linked via peptide linkers to form polyvalent structures via non-covalent or chemical crosslinks to form homodivalent, heterodivalent, trivalent, and tetravalent structures, for example, V L and V HExamples include single-chain binding polypeptides having both domains. Further exemplary multiplespecific and bispecific molecules and methods for producing them are, for example, U.S. Patent Nos. 5910573, 5932448, 5959083, 5989830, 6005079, 6239259, 6294353, 6333396, 6476198, 6511663, 6670453, and 6743896. Detailed specifications, U.S. Patent No. 6809185, U.S. Patent No. 6833441, U.S. Patent No. 7129330, U.S. Patent No. 7183076, U.S. Patent No. 7521056, U.S. Patent No. 7527787, U.S. Patent No. 7534866, U.S. Patent No. 7612181, U.S. Patent Application Publication No. 2002004587A1, U.S. Patent Application Publication No. 2002076406A1, U.S. Patent Application Publication No. 2002103345A1, U.S. Patent Application Publication No. 2003207346A Specification No. 1, U.S. Patent Application Publication No. 2003211078A1, U.S. Patent Application Publication No. 2004219643A1, U.S. Patent Application Publication No. 2004220388A1, U.S. Patent Application Publication No. 2004242847A1, U.S. Patent Application Publication No. 2005003403A1, U.S. Patent Application Publication No. 2005004352A1, U.S. Patent Application Publication No. 2005069552A1, U.S. Patent Application Publication No. 2005079170A1, U.S. Patent Application Publication No. 2005100543A1 Detailed specifications, U.S. Patent Application Publication No. 2005136049A1, U.S. Patent Application Publication No. 2005136051A1, U.S. Patent Application Publication No. 2005163782A1, U.S. Patent Application Publication No. 2005266425A1, U.S. Patent Application Publication No. 2006083747A1, U.S. Patent Application Publication No. 2006120960A1, U.S. Patent Application Publication No. 2006204493A1, U.S. Patent Application Publication No. 2006263367A1, U.S. Patent Application Publication No. 2007004909A1,U.S. Patent Application Publication No. 2007087381A1, U.S. Patent Application Publication No. 2007128150A1, U.S. Patent Application Publication No. 2007141049A1, U.S. Patent Application Publication No. 2007154901A1, U.S. Patent Application Publication No. 2007274985A1, U.S. Patent Application Publication No. 2008050370A1, U.S. Patent Application Publication No. 2008069820A1, U.S. Patent Application Publication No. 2008152645A1, U.S. Patent Application Publication No. 2008171855A1, U.S. Patent Application Publication No. 20082 Specifications 41884A1, U.S. Patent Application Publication No. 2008254512A1, U.S. Patent Application Publication No. 2008260738A1, U.S. Patent Application Publication No. 2009130106A1, U.S. Patent Application Publication No. 2009148905A1, U.S. Patent Application Publication No. 2009155275A1, U.S. Patent Application Publication No. 2009162359A1, U.S. Patent Application Publication No. 2009162360A1, U.S. Patent Application Publication No. 2009175851A1, U.S. Patent Application Publication No. 2009175867A1, United States Patent Application Publication No. 2009232811A1, U.S. Patent Application Publication No. 2009234105A1, U.S. Patent Application Publication No. 2009263392A1, U.S. Patent Application Publication No. 2009274649A1, European Patent Application Publication No. 346087A2, International Publication No. 0006605A2, International Publication No. 02072635A2, International Publication No. 04081051A1, International Publication No. 06020258A2, International Publication No. 2007044887A2, International Publication No. 200 Pamphlet No. 7095338A2, International Publication No. 2007137760A2, International Publication No. 2008119353A1, International Publication No. 2009021754A2, International Publication No. 2009068630A1, International Publication No. 9103493A1, International Publication No. 9323537A1, International Publication No. 9409131A1, International Publication No. 9412625A2, International Publication No. 9509917A1, International Publication No. 9637621A2,The contents of the applications referenced herein are incorporated herein by reference in their entirety.

[0174] In another embodiment, the Disclosure provides modified IgG1 Fc-containing polyvalent and multispecific antibodies comprising at least two identical or different antigen-binding moieties of the antibody of the Disclosure. In one embodiment, the polyvalent antibody provides at least two, three, or four antigen-binding moieties of the antibody. The antigen-binding moieties can be linked and integrated via protein fusion or covalent or non-covalent linkage. Alternatively, linkage methods are described for bispecific molecules. A tetravalent compound can be obtained, for example, by crosslinking the antibody of the Disclosure with an antibody that binds to a constant region of the antibody of the Disclosure, e.g., Fc or hinge region.

[0175] Treatment method In one embodiment, the modified IgG1 Fc-containing conjugated molecule according to the present invention is used to treat a disease. In a more specific embodiment, the disease is advantageous in which the effector function of the variant is reduced by at least 50%, 70%, 80%, 90%, 95%, 98%, or 99% compared to a polypeptide containing wild-type IgG Fc polypeptide, or is undetectable.

[0176] In a specific embodiment, the modified IgG1 Fc-containing conjugated molecule according to the present invention is for use as a pharmaceutical. Preferably, the use is advantageous in which the effector function of the polypeptide is significantly reduced compared to the wild-type Fc polypeptide. In a further specific embodiment, the conjugated molecule according to the present invention is for use as a pharmaceutical for the treatment of diseases in which the effector function of the polypeptide is advantageous in which it is reduced by at least 50%, 70%, 80%, 90%, 95%, 98%, or 99% compared to the wild-type Fc polypeptide, or is undetectable.

[0177] A further embodiment is a method for treating an individual with a disease in which it is advantageous that the effector function of the mutant is significantly reduced compared to that of a wild-type IgG1 Fc-containing binding molecule, the method comprising administering an effective amount of the binding molecule according to the present invention to the individual.

[0178] A strong reduction in effector function is defined as a reduction of at least 50%, 70%, 80%, 90%, 95%, 98%, or 99% of effector function compared to the effector function induced by the wild-type polypeptide, or an undetectable effector function.

[0179] Such diseases are, for example, all diseases in which the antigen targeted by a modified IgG1 Fc-containing binding molecule can be present on the cell surface, and the cells should not be destroyed by, for example, ADCC, ADCP and / or CDC, or diseases treatable with therapeutic antibodies or binding molecules designed to deliver drugs (e.g., toxins and radioisotopes) to target cells, where the Fc / FcγR-mediated effector function brings healthy immune cells closer to the lethal payload, leading to the depletion of normal lymphoid tissue along with the target cells (Hutchins, et al, PNAS USA 92(1995)11980-11984; White, et al, Annu Rev Med 52(2001)125-145). In such cases, using antibodies that inadequately recruit complement or effector cells can yield significant benefits (see, for example, Wu, et al., Cell Immunol 200(2000)16-26, Shields, et al., J. Biol Chem 276(9)(2001)6591-6604, U.S. Patent No. 6,194,551, U.S. Patent No. 5,885,573, and PCT International Publication No. 04 / 029207).

[0180] In other cases, for example, in diseases where the therapeutic objective is to block the interaction between widely expressed receptors and their cognitive ligands, it would be advantageous to reduce or eliminate all antibody effector functions to mitigate undesirable toxicity. Furthermore, if a therapeutic antibody exhibits indiscriminate binding across several human tissues and / or cell surfaces, it would be prudent to limit the targeting of effector functions to various groups of tissues and cells to limit toxicity.

[0181] Pharmaceutical composition This specification discloses pharmaceutical compositions comprising a modified IgG1 Fc-containing conjugated molecule described herein in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0182] This specification discloses pharmaceutical compositions comprising the conjugate molecules described herein in combination with one or more additional therapeutic agents.

[0183] The term "pharmaceutical composition" means a mixture of at least one active ingredient (e.g., an antibody or fragment of the present disclosure) and at least one pharmaceutically acceptable excipient, diluent or carrier.

[0184] "Medicine" refers to a substance used in medical procedures.

[0185] The phrase "pharmaceutically acceptable" means that it is approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally accepted pharmacopoeias, for use in animals, or more specifically, in humans.

[0186] A pharmaceutically acceptable carrier includes all physiologically compatible solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents, and absorption retarders. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or dermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for the subcutaneous route. Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific component, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0187] Therapeutic and diagnostic formulations may be prepared by mixing them with physiologically acceptable carriers, excipients, or stabilizers, for example, in the form of lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, 2001; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY, 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; see Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY, 2000).

[0188] The binding molecules of this disclosure may be produced as a lyophilized product in a vial. The lyophilized product can be reconstituted with water or a pharmaceutical carrier suitable for injection. For subsequent intravenous administration, the resulting solution will typically be further diluted in a carrier solution.

[0189] The selection of a therapeutic regimen depends on several factors, including the severity of the infection, the level of symptoms, and the accessibility of target cells within the biological matrix. In certain embodiments, the regimen maximizes the amount of therapeutic agent delivered to the patient while maintaining an acceptable level of side effects. Therefore, the amount of biologic delivered depends in part on the specific entity and the severity of the symptoms being treated. Guidance is available for selecting appropriate doses of antibodies, cytokines, and small molecules (e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY, 1991; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY, 1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al., New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl.J.Med.344:783-792,2001;Beniaminovitz et al.,New Engl.J.Med.342:613-619,2000;Ghosh et al.,New Engl.J.Med.348:24-32,2003;Lipsky et al.,New See Engl.J.Med.343:1594-1602, 2000).

[0190] The determination of the appropriate dose is made by a clinician, for example, using parameters or factors known or presumed in the technique that affect or are expected to affect the procedure. Typically, the dose is started somewhat less than the appropriate dose and then increased in small increments, taking into account any negative side effects, until the desired or optimal effect is achieved. Important diagnostic measures include, for example, measures of symptomatic response to the infusion.

[0191] The actual dosage level of the active ingredient in a pharmaceutical composition containing a binding molecule may vary to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the antibody activity, route of administration, timing of administration, antibody half-life in the patient, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, symptoms, health status, and prior medical history of the patient to be treated, and factors known in the medical technology.

[0192] The dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, depending on the urgency of the treatment situation, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased. It is particularly advantageous to formulate parenteral compositions in the form of dosing units that facilitate administration and ensure uniform dosage. As used herein, unit formulations mean physically individual units appropriate as unit dosing amounts for the subject being treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit formulations of this disclosure are determined by and directly depend on the unique properties of the active compound, the specific therapeutic effect to be achieved, and the inherent limitations in the field of formulation technology for treating the susceptibility of such active compounds in an individual.

[0193] Compositions containing antibodies or fragments thereof may be delivered by continuous infusion, or by administration at intervals of, for example, one day, one week, or one to seven times per week. Doses may be delivered intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. Specific dose protocols should include the maximum dose or dose frequency to avoid undesirable major side effects.

[0194] For antibody or protein administration, the dosage is approximately 0.0001 to 150 mg / kg, for example, 5, 15, and 50 mg / kg subcutaneously, more generally within the range of 0.01 to 5 mg / kg host body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg. Exemplary treatment regimens require administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. The antibody or fragment administration regimens of the present disclosure include intravenous administration of 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg, 10 mg / kg, 20 mg / kg, or 30 mg / kg, and the antibody is administered using one of the following administration schedules: six doses once every four weeks, then once every three months; once every three weeks; once at 3 mg / kg body weight, followed by 1 mg / kg body weight once every three weeks. Administration of the antibody of the present invention may be repeated, and administration may be divided into intervals of at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

[0195] The effective dose for a particular patient may vary depending on factors such as the symptoms being treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects (see, for example, Maynard et al., A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).

[0196] The route of administration may be, for example, topical application or skin application, subcutaneous injection or subcutaneous infusion, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal fluid, intrafocal, or by a continuous release system or implant (e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA (See 77:4030-4034, 1980; U.S. Patent Nos. 6,350,466 and 6,316,024). If necessary, the composition may also contain a solubilizer or a local anesthetic, such as lidocaine to relieve pain at the injection site, or both. In addition, pulmonary administration may also be used, for example, by the use of an inhaler or nebulizer, and by formulation with an aerosolizing agent. For example, see U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and see International Publication Nos. 92 / 19244, 97 / 32572, 97 / 44013, 98 / 31346, and 99 / 66903 (each of these applications is incorporated herein by reference in its entirety).

[0197] The compositions of the present invention, containing the silent modified IgG1 Fc-containing binding molecule and the antibody, can also be administered via one or more routes of administration using one or more of the various methods known in the art. As will be recognized by those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. Routes of administration for selected antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, e.g., by injection or infusion. Parenteral administration may typically refer to methods of administration other than intestinal and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intra-shearing, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions. In addition, the compositions of the present disclosure may be administered via parenteral routes, e.g., topical, epidermal, or mucosal administration routes, e.g., intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the antibody of this disclosure is administered by injection. In another embodiment, the antibody is administered subcutaneously.

[0198] When the modified IgG1 Fc-containing binding molecule or the antibody of the present invention is administered via a controlled-release or sustained-release system, controlled-release or sustained-release can be achieved using a pump (see Langer, op. cit.; Sefton, CRC Crit.Ref Biomed.Eng.14:20,1987; Buchwald et al., Surgery 88:507,1980; Saudek et al., N.Engl.J.Med.321:574,1989). The controlled or sustained release of therapeutic antibodies can be achieved using polymer materials (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; Levy et al., Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howard et al., J. Neurosurg. 7) See also 1:105,1989; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; International Publication No. 99 / 15154; and International Publication No. 99 / 20253.Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of eluting impurities, stable at storage, sterile, and biodegradable. Controlled-release or sustained-release systems may be positioned close to the prophylactic or therapeutic target and therefore require only small systemic doses (see, e.g., Goodson, in Medical Applications of Controlled Release, op. cit., vol. 2, pp. 115-138, 1984).

[0199] The controlled-release system is discussed in the overview by Langer (Science 249:1527-1533, 1990). A sustained-release formulation containing one or more antibodies of this disclosure can be produced using any technique known to those skilled in the art. See, for example, U.S. Patent No. 4,526,938, International Publication No. 91 / 05548, International Publication No. 96 / 20698, Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek et al., Pro.Int'l.Symp.Control.Rel.Bioact.Mater. 24:853-854, 1997; and Lam et al., Proc.Int'l.Symp.Control Rel.Bioact.Mater. 24:759-760, 1997 (each of these applications is incorporated herein by reference in its entirety).

[0200] Various means for administering therapeutic compositions are known in the art. For example, in one embodiment, the therapeutic compositions of the present disclosure can be administered by a needle-free subcutaneous injection device, such as the devices shown in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Examples of well-known implants and modules useful for this disclosure include U.S. Patent No. 4,487,603, which shows an implantable microinfusion pump for dispensing pharmaceuticals at a controlled rate; U.S. Patent No. 4,486,194, which shows a therapeutic device for administering pharmaceuticals through the skin; U.S. Patent No. 4,447,233, which shows a pharmaceutical infusion pump for delivering pharmaceuticals at a precise infusion rate; U.S. Patent No. 4,447,224, which shows a variable-flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which shows an osmotic drug delivery system with multiple chamber compartments; and U.S. Patent No. 4,475,196, which shows an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art. In preferred embodiments, means for administering antibodies and fragments are selected from syringes, autoinjectors, injection pens, vials and syringes, infusion pumps, patches, or infusion bags and needles.

[0201] When the silencing IgG1 Fc-containing binding molecule and antibody of the present invention are administered topically, they are formulated as ointments, creams, transdermal patches, lotions, gels, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For topical dosage forms that cannot be sprayed, a viscous semi-solid or solid form containing a carrier or one or more excipients suitable for topical application is typically used, and in some examples, has a dynamic viscosity greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, plasters, etc., which may be sterilized or mixed with excipients (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include, in some examples, sprayable aerosol preparations in which the active ingredient, combined with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile substance (e.g., a gaseous propellant, e.g., freon) or in a squeeze bottle. Moisturizers or humectants may also be added to the pharmaceutical composition and dosage form if desired. Examples of such additional components are well known in the art.

[0202] When a composition comprising a silencing modified IgG1 Fc-containing binding molecule and the antibody of the present invention is administered intranasally, it may be formulated in the form of an aerosol, spray, mist, or drops. In particular, prophylactic or therapeutic agents for use according to this disclosure may be conveniently delivered in the form of aerosol spray dispensers from a pressurized pack or nebulizer by the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for delivering a measured amount. Capsules and cartridges (e.g., composed of gelatin) for use in inhalers or inhalers may be formulated containing the compound and a suitable powder base, e.g., a powder mixture of lactose or starch.

[0203] Methods of co-administration or concurrent treatment with additional therapeutic agents, such as immunosuppressants, cytokines, steroids, chemotherapeutic agents, and antibiotics, are known in the art (see, for example, Hardman et al., (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic agent may reduce symptoms by at least 10%, at least 20%, at least about 30%, at least 40%, or at least 50%.

[0204] Additional treatments (e.g., prophylactic or therapeutic agents) that can be administered in combination with the antibody may be administered at a distance of less than 5 minutes, less than 30 minutes, 1 hour, about 1 hour, about 1 to about 2 hours, about 2 to about 3 hours, about 3 to about 4 hours, about 4 to about 5 hours, about 5 to about 6 hours, about 6 to about 7 hours, about 7 to about 8 hours, about 8 to about 9 hours, about 9 to about 10 hours, about 10 to about 11 hours, about 11 to about 12 hours, about 12 to 18 hours, 18 to 24 hours, 24 to 36 hours, 36 to 48 hours, 48 ​​to 52 hours, 52 to 60 hours, 60 to 72 hours, 72 to 84 hours, 84 to 96 hours, or 96 to 120 hours from the antibody and fragment of the Disclosure. Two or more treatments may be administered during a single visit to the same patient.

[0205] In certain embodiments, the binding molecules of this disclosure may be formulated to ensure appropriate in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the binding molecules traverse the BBB (optionally), they may be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes may contain one or more moieties that are selectively transported into specific cells or organs and thus enhance targeted drug delivery (see, for example, Ranade, (1989) J. Clin. Pharmacol. 29:685). Examples of targeting moieties include phorate or biotin (see, e.g., U.S. Patent No. 5,416,016 by Low et al.); mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibody (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et al., (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al., (1994) J. Biol. Chem. 269:9090); K. Keinanen; M. Laukkanen (1994) FEBS See also Lett.346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0206] This disclosure provides administration protocols for pharmaceutical compositions, comprising a silent modified IgG1 Fc-containing conjugated molecule alone or in combination with other therapies, to a subject requiring such therapy. The combination therapy (preventive or therapeutic) may be administered to the subject simultaneously or sequentially. The combination therapy (preventive or therapeutic) may also be administered cyclically. Cycling therapy involves administering a first therapy (first prophylactic or therapeutic) for a period of time, followed by a second therapy (second prophylactic or therapeutic) for a period of time, and repeating this sequential administration (i.e., a cycle) to reduce the development of resistance to one of the therapies (e.g., agents), to avoid or mitigate the side effects of one of the therapies (e.g., agents), and / or to improve the efficacy of the therapy.

[0207] The combination therapies (preventive or therapeutic) described herein may be administered to the subject simultaneously. The term “simultaneously” is not limited to administering the therapies precisely at the same time, but rather means that the antibody or the pharmaceutical composition containing the antibody or its fragments is administered to the subject in an order and within a time interval such that the antibody acts in conjunction with the other therapy to provide greater benefits than if it were administered normally. For example, each therapy may be administered to the subject simultaneously or sequentially in any order at different time points; however, if not administered simultaneously, they should be administered at times close enough to produce the desired therapeutic or preventive effect. Each therapy may be administered to the subject separately in any appropriate form and by any appropriate route. In various embodiments, the treatment (preventive or therapeutic agent) is administered to the subject at intervals of less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 to 2 hours, about 2 to 3 hours, about 3 to 4 hours, about 4 to 5 hours, about 5 to 6 hours, about 6 to 7 hours, about 7 to 8 hours, about 8 to 9 hours, about 9 to 10 hours, about 10 to 11 hours, about 11 to 12 hours, 24 hours, 48 ​​hours, 72 hours, or 1 week apart. In other embodiments, two or more treatments (preventive or therapeutic agents) are administered during the same patient visit.

[0208] The prophylactic or therapeutic agents in combination therapy may be administered to the subject using the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents in combination therapy may be administered simultaneously to the subject using separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to the subject via the same or different routes of administration.

[0209] Details of one or more embodiments of this disclosure are shown in the accompanying description above. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are listed below. Other features, purposes, and advantages of this disclosure will become apparent from the description and claims. In this specification and the accompanying claims, singular nouns include plural nouns unless specifically provided in context. Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference where applicable unless otherwise indicated. The following examples are provided to more fully illustrate preferred embodiments of this disclosure. These examples should not be construed as limiting the scope of the disclosed subject matter as defined by the accompanying claims. Various embodiments of the present invention are shown below. 1. A binding molecule comprising a human IgG1 Fc variant of the wild-type human IgG1 Fc region and one or more antigen-binding domains, wherein the Fc variant includes a combination of amino acid substitutions selected from the group consisting of substitutions: L234A, L235A, G237A (LALAGA), L234A, L235A, S267K, P329A (LALASKPA), D265A, P329A, S267K (DAPASK), G237A, D265A, P329A (GADAPA), G237A, D265A, P329A, S267K (GADAPASK), L234A, L235A, P329G (LALAPG), or L234A, L235A, P329A (LALAPA), and the amino acid residues are numbered according to the Kabat EU index. 2. The binding molecule according to item 1, wherein the Fc variant includes the sequence of sequence number 15 or 21, or a sequence having at least 95%, 96%, 97%, 98%, or 99% homology thereto. 3. The binding molecule is a human or humanized IgG1 monoclonal antibody, as described in 1 or 2 above. 4. A binding molecule according to any one of 1 to 3 above, wherein the binding molecule has reduced or undetectable binding affinity to the Fc gamma receptor compared to the polypeptide containing the wild-type human IgG1 Fc region (optionally measured by surface plasmon resonance using a Biacore T200 instrument), and the Fc gamma receptor is selected from the group consisting of Fc gamma RIA and Fc gamma RIIIa V158 variants. 5. The binding molecule described in any one of items 1 to 4 above, wherein the antigen is a cell surface antigen. 6. The binding molecule according to any one of 1 to 5 above, wherein the binding molecule has reduced or undetectable effector function compared to the polypeptide containing the wild-type human IgG1 Fc region. 7. The binding molecule described in any of 1 to 6 above, which has the ability to bind to one or more antigens without triggering detectable antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). 8. The binding molecule is a multispecific antibody containing binding domains for two or more antigens, as described in any of items 1 to 7 above. 9. The binding molecule according to 8 above, wherein the binding molecule is a bispecific antibody containing binding domains for two antigens. 10. The binding molecule according to any one of 8 to 9 above, wherein the Fc variant further comprises one or more knob-in-hole mutations. 11. A conjugating molecule according to any one of items 1 to 10 above for use in a method of treating a disease in an individual, wherein the effector function of the conjugating molecule is reduced or undetectable in the individual compared to the effector function induced by the polypeptide comprising the wild-type human IgG1 Fc region, and the method comprises administering the conjugating molecule according to any one of items 1 to 10 above to the individual. 12. The binding molecule described in 11 above, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). 13. The binding molecule described in 11 above, wherein the effector function is antibody-dependent cellular phagocytosis (ADCP). 14. The binding molecule described in 11 above, wherein the effector function is complement-dependent cytotoxicity (CDC). 15. A composition comprising any of the binding molecules described in 1 to 10 above. 16. The composition according to 15, further comprising a pharmaceutically acceptable carrier. 17. An isolated polynucleotide containing a sequence encoding the binding molecule described in any of items 1 to 10 above. 18. A vector containing the polynucleotide described in item 17 above. 19. A host cell containing the vector described in item 18 above or the polynucleotide described in item 17 above.

Example

[0210] Example 1: Selection of Design-Residue Positions The design strategy was tested to produce a set of antibodies with modified Fc regions that exhibit desired properties such as reduced effector function. Early tests defining the major amino acid binding sites on IgG for Fc gamma receptors were performed by mutagenesis analysis, and it was determined that the lower hinge, proximal CH2 region, and glycosylation at N297 were important (Shields et al., 2001). Mutations were introduced into regions that interact with Fc gamma receptors with the goal of reducing residual binding to Fc gamma receptors. For these specific reasons, it was necessary to test various combinations of Fc positions to generate a set of mutations without offsetting antibody drug development potential and immunogenicity risk. Several sets of mutations were generated and compared to wild-type IgG1. LALAPA-IgG1 (L234A / L235A / P329A), LALAGA-IgG1 (L234A / L235A / G237A), LALAPG-IgG1 (L234A / L235A / P329G), DAPA-IgG1 (D265A / P329A), LALASKPA-IgG1 (L234A / L235A / S267K / P329A), DAPASK-IgG1 (D265A / P329A / S267K), GADAPA-IgG1 (G237A / D265A / P329A), GADAPASK-IgG1 (G237A / D265A / P329A / S267K), and DANAPA-IgG1 (D265A / N297A / P329A) were evaluated. The previously described DAPA and DANAPA silencing motifs were included for comparison.

[0211] Example 2: Expression and Purification of Modified Antibodies For the experiment, antibodies against CD3 (SEQ ID NOs: 1-24) listed below, containing the indicator amino acid substitutions and expressed according to the indicator nucleotide sequence, were used. IgG1 molecules were expressed in HEK293 mammalian cells and purified using protein A and size exclusion chromatography. Briefly, the heavy and light chain DNA of anti-CD3, WT IgG1 was synthesized at GeneArt (Regensburg, Germany) and cloned into mammalian expression vectors using restriction enzyme ligation-based cloning techniques. Then, all the mutants described herein were generated using PCR-based mutagenesis. The resulting plasmids were cotransfected into HEK293 T cells. For transient antibody expression, equal amounts of the vector for each chain were cotransfected into suspension-compatible HEK293 T cells using polyethyleneimine (PEI; Cat#24765 Polysciences, Inc.). Typically, 100 ml of suspended cells were transfected at a density of 1–2 mio cells / ml with 50 μg of expression vector encoding the heavy chain containing DNA and 50 μg of expression vector encoding the light chain. The recombinant expression vectors were then introduced into host cells, and the cells were further cultured for 7 days to produce constructs by secretion into culture medium (HEK, serum-free medium) supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml antibiotic.

[0212] Next, the produced construct was purified from the cell-free supernatant using immunoaffinity chromatography. MabSelect Sure resin (GE Healthcare Life Sciences), equilibrated in PBS buffer pH 7.4, was incubated with filter-conditioned medium using a liquid chromatography system (Aekta Pure Chromatography System, GE Healthcare Life Sciences). The resin was washed with PBS pH 7.4, and the construct was then eluted with elution buffer (50 mM citrate, 90 mM NaCl, pH 2.7). After capture, the eluted protein was pH-neutralized using 1 M TRIS pH 10.0 solution, and then finished using size exclusion chromatography (HiPrep Superdex200 16 / 60, GE Healthcare Life Sciences). Finally, the purified protein was combined with PBS buffer pH 7.4.

[0213] [Table 1]

[0214] [Table 2]

[0215] [Table 3]

[0216] [Table 4]

[0217] [Table 5]

[0218] [Table 6]

[0219]

Table 7

[0220]

Table 8

[0221] [[ID=*19]]

Table 9

[0222] Example 3: Biophysical Properties of the Modified Antibody Binding of the Modified Antibody to SPR-Human Fc Gamma Receptor and Human C1q To analyze the interactions of human activating receptors FcγR1A, FcγR3A (V158) and human C1q with IgG1 WT and antibody Fc variants, surface plasmon resonance (SPR) experiments were performed. Binding kinetics and their relative binding affinities were examined. Binding affinity is an important property of the interaction between an antibody and an antigen. Equilibrium dissociation constant (K D Note: There seems to be a formatting issue in the original text where the superscript in "Equilibrium dissociation constant (K " is not properly closed. I've left it as is in the translation. If this is an error in the original, it should be corrected before translation for a more accurate result. Also, the asterisk on line 19 is just for highlighting the potential formatting issue in the original text and has no meaning in the translation.) determines the strength of the interaction and, therefore, the degree to which antibody-antigen complexes are formed in equilibrium. Knowledge of antibody properties is essential not only for selecting the best therapeutic antibody candidates but also for understanding in vivo behavior and predicting cellular immune responses. The goal is to generate antibody variants with little or no binding to the Fc gamma receptor in order to reduce or eliminate effector function, aiming to improve the safety of monoclonal antibody therapy. Binding to human C1q was evaluated. All SPR buffers were prepared using deionized water. Samples were prepared in running buffer PBS pH 7.4 containing 0.005% Tween-20. SPR measurements were performed on a Biacore T200 (GE-Healthcare Life Sciences) controlled by Biacore T200 control software version 2.0.1. Surface plasmon resonance (SPL) was performed using Biacore T200 to evaluate the binding affinity of IgG1 WT and variant antibodies to human Fc receptors, including FcγR1A, FcγR3A(V158), and human C1q.

[0223] Antibodies were covalently immobilized on the CM5 sensor chip, while Fc gamma receptor or human C1q acted as an analyte in solution (Figure 1). For Fc gamma receptor binding evaluation (Method 1), a standard amine coupling procedure was applied, diluting the antibody in 10 mM sodium acetate pH 4 and immobilizing it on the CM5 sensor chip at a density of approximately 950 resonance units (RU). Flow cell 1 was immobilized as a blank and served as a reference. Kinetic binding data were collected by subsequent injection of 1:2 serially diluted human Fc gamma receptor onto all flow cells at a flow rate of 30 μl / min and a temperature of 25°C. Fc gamma receptors were diluted in running buffer at concentrations ranging from 0.2 nM to 1000 nM (e.g., FcγR1A: 0.2 to 100 nM, FcγR3A V158: 1.95 to 1000 nM). The chip surface was regenerated after each measurement cycle using a 20 mM glycine pH 2.0 solution. For human C1q binding evaluation (Method 2), the antibody was diluted with 10 mM sodium acetate pH 4 and immobilized on the CM5 sensor chip at a density of approximately 7000 resonance units (RU) using a standard amine coupling procedure. Flow cell 1 was immobilized as a blank and served as a reference. Kinetic binding data was collected by subsequent injection of 1:2 serially diluted human C1q onto all flow cells at a flow rate of 30 μl / min and a temperature of 25°C. Human C1q was diluted with running buffer to concentrations ranging from 0.49 nM to 250 nM. The chip surface was regenerated after each measurement cycle using a 50 mM NaOH solution. Zero-concentration samples (blank runs) were measured by both methods to allow for dual reference during data evaluation.

[0224] The data were evaluated using Biacore T200 evaluation software. Raw data were double-referenced; that is, the response of the measurement flow cell was corrected for the response of the reference flow cell, and in the second step, the response of the blank injection was subtracted. The sensorgrams were then fitted by applying a 1:1 kinetic binding model to calculate the dissociation equilibrium constant. In addition, the maximum response was reached during experimental monitoring. The maximum response describes the ability to bind to the surface based on the response at saturation. The maximum response values ​​summarizing these interactions are shown in Table 2. The SPR Biacore binding sensorgrams of each mutant for each receptor are plotted in Figure 2 in the concentration ranges: 0.2 nM to 100 nM for FcgR1, and 7.8 nM to 4000 nm for FcgR2A R131 and FcγR3A (V158 and F158). Figure 2A shows representative sensorograms and response plots for WT and mutants against FcgammaR1A (concentration range: 0.2nM-100nM for human FcγR1A). Figure 2B shows representative sensorograms and response plots for WT and mutants against FcgammaR3A V158 (concentration range: 1.95nM-1000nM for human FcγR3A V158). Figure 2C shows representative sensorograms and response plots for WT and mutants against human C1q (concentration range: 0.49nM-250nM for human C1q). All IgG1 antibody Fc mutants inhibited binding to the Fc gamma receptor compared to WT (SEQ ID NOs: 1 and 3), and no residual binding was measured. All IgG1 antibody Fc mutants inhibited binding to human C1q compared to WT (SEQ ID NOs: 1 and 3), and low residual binding was measured.

[0225] [Table 10]

[0226] Example 4: Differential scanning calorimetry of modified antibodies - melting temperature As shown in Table 3, the thermal stability of the manipulated antibody CH2 domain was compared using calorimetry. Calorimetric measurements were performed using a differential scanning microcalorimeter (Nano DSC, TA instruments). The cell volume was 0.5 ml, and the heating rate was 1 °C / min. All proteins were used at a concentration of 1 mg / ml in PBS (pH 7.4). The molar heat capacity of each protein was estimated by comparison with duplicate samples containing the same buffer but without the protein. Partial molar heat capacity and melting curves were analyzed using standard procedures. Thermograms were baseline-corrected and normalized at the specified concentrations. The silent version LALASKPA (70 °C) showed significantly better Tm compared to DANAPA (62 °C).

[0227] [Table 11]

[0228] Aggregation tendency after capture of IgG1 anti-CD3 antibody and Fc mutant Size exclusion chromatography was performed to evaluate the aggregation tendency (%HMW) of IgG1 antibody and Fc-modified derivatives. The produced and purified anti-CD3 antibody was applied to an analytical size exclusion chromatography column (SEC 200, GE Healthcare) equilibrated with PBS buffer pH 7.4. The results are summarized in Table 4.

[0229] [Table 12]

[0230] Example 5: Anti-CD3 NFAT signaling assay Jurkat NFAT luciferase-mediated (JNL) cells and THP1 cells (ATCC, TIB202) were used to perform a Jurkat reporter gene assay (RGA) of the activated T cell nuclear factor (NFAT) pathway. THP1 cells express FcγRI, FcγRII, and FcγRIII. Cells were co-incubated at 37°C and 5% CO2 for 6 hours with each sample at various concentrations as depicted, in an effector-to-tumor ratio of 5:1. An equal volume of ONE-Glo® reagent (Promega, E6110) was added relative to the culture volume. The plate was shaken for 2 minutes, then protected from light and incubated for a further 8 minutes. For the JNL+THP+IFNg experiment, THP-1 cells were pre-treated with 100 u / mL IFNg at 37°C and 5% CO2 for 48 hours prior to co-culture. IFNg stimulation increases FcγRI expression. Luciferase activity was quantified using an EnVision plate reader (PerkinElmer). The data were analyzed and fitted to a 5-parameter logistic curve using GraphPad Prism.

[0231] In both treatments, the WT cells exhibited maximum NFAT activity. All silent mutation sets showed significantly suppressed NFAT activation overall. In RGA, when performed without IFNg (Figure 3A), all silent mutation sets showed similar levels of T cell activation, except for DAPA. When THP1 cells were incubated with IFNg (Figure 3B), the mutation sets exhibited lower activity, demonstrating strong Fc silencing, although some activity remained with DAPA, LALAPA, and GADAPA.

Claims

1. A binding molecule comprising a human IgG1 Fc variant of the wild-type human IgG1 Fc region and one or more antigen-binding domains, The Fc variant includes a combination of amino acid substitutions L234A, L235A, S267K, and P329A (LALASKPA), and the Fc variant includes the sequence of Sequence ID No. 15, or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto. The amino acid substitution residues are numbered according to the EU index of Kabat. The aforementioned binding molecule.

2. The binding molecule according to claim 1, wherein the binding molecule is a human or humanized IgG1 monoclonal antibody.

3. A binding molecule according to claim 1 or 2, wherein the binding molecule has a reduced or undetectable binding affinity to the Fc gamma receptor compared to the polypeptide comprising the wild-type human IgG1 Fc region (optionally measured by surface plasmon resonance using a Biacore T200 instrument), and the Fc gamma receptor is selected from the group consisting of Fc gamma RIA and Fc gamma RIIIa V158 variants.

4. The binding molecule according to any one of claims 1 to 3, wherein the antigen is a cell surface antigen.

5. The binding molecule according to any one of claims 1 to 4, wherein the binding molecule has reduced or undetectable effector function compared to the polypeptide containing the wild-type human IgG1 Fc region.

6. The binding molecule according to any one of claims 1 to 5, wherein the binding molecule has the ability to bind to one or more antigens without triggering detectable antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC).

7. The binding molecule according to any one of claims 1 to 6, wherein the binding molecule is a multispecific antibody comprising binding domains for two or more antigens.

8. The binding molecule according to claim 7, wherein the binding molecule is a bispecific antibody containing binding domains for two antigens.

9. The binding molecule according to claim 7 or 8, wherein the Fc variant further comprises one or more knob-in-hole mutations.

10. A binding molecule according to any one of claims 1 to 9 for use in a method of treating a disease in an individual, wherein the effector function of the binding molecule is reduced or undetectable in the individual compared to the effector function induced by a polypeptide comprising the wild-type human IgG1 Fc region, and the method comprises administering the binding molecule according to any one of claims 1 to 9 to the individual.

11. The binding molecule according to claim 10, wherein the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

12. The binding molecule according to claim 10, wherein the effector function is antibody-dependent cellular phagocytosis (ADCP).

13. The binding molecule according to claim 10, wherein the effector function is complement-dependent cytotoxicity (CDC).

14. A composition comprising the binding molecule described in any one of claims 1 to 9.

15. The composition according to claim 14, further comprising a pharmaceutically acceptable carrier.

16. An isolated polynucleotide comprising a sequence encoding the binding molecule according to any one of claims 1 to 9.

17. A vector comprising the polynucleotide described in claim 16.

18. A host cell comprising the vector according to claim 17 or the polynucleotide according to claim 16.

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