Engineered Fc variants

Abstract

The present invention describes engineered immunoglobulin IgG Fc regions by transferring structural elements, namely some CH2 interchain disulfide bonds, from IgA immunoglobulins to IgG immunoglobulins. The engineered Fc variants disclosed exhibit a significant reduction or complete loss of interaction of the engineered Fc with FcγR and C1q while retaining the native ability to interact with FcRn at acidic pH. The disclosed silent Fc molecules have comparable expression and purification yields and improved or maintained thermal stability, thereby reducing the tendency for aggregation, compared to wild-type Fc. Furthermore, the disclosed Fc variant silencing mutations can reduce or compensate for the destabilizing effects of other half-life-extending or chain-pairing-promoting Fc mutations.

Description

[Technical field]

[0001] The present invention relates to molecules such as engineered IgG immunoglobulins, including Fc variants obtained by the transfer of structural elements (e.g., CH2 interchain disulfide bonds) from IgA immunoglobulins to IgG immunoglobulins, which maintain highly stable physicochemical properties while the Fc effector functions are greatly reduced or completely eliminated. These silent Fc variants are particularly advantageous when used in combination with various other, often destabilizing, substitutions in the Fc CH2 domain, such as half-life extension or chain pairing mutations. The molecules according to the invention are useful for the development of therapeutic agents with superior properties such as enhanced stability, development and / or half-life. [Background technology]

[0002] Immunoglobulins (e.g., antibodies) can be functionally separated into variable domains that bind antigens and constant domains that specify effector functions such as activating complement or binding to Fc receptors. There are five major classes of heavy chain constant domains, each of which defines an immunoglobulin isotype (IgM, IgG, IgA, IgD, and IgE). IgG can be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, and IgA can be similarly divided into two subclasses, IgA1 and IgA2. Although the constant domains of immunoglobulin classes G (IgG) and A (IgA) have different amino acid sequences, they show strong structural homology. In fact, both classes are made up of immunoglobulin-like domains and share a very similar protein fold. However, structural differences remain, especially within the CH2 domain of the crystallizable fragments.

[0003] Effector functions attributed to the Fc region of an immunoglobulin (e.g., an antibody) vary depending on the class and subclass of the immunoglobulin (e.g., an antibody) and include the binding of the immunoglobulin (e.g., an antibody) via the Fc region to specific Fc receptors on cells that elicit various biological responses. These receptors are expressed on various immune cells, such as monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells. Formation of an Fc / Fc receptor complex (e.g., an FcγR complex) recruits these effector cells to the site of bound antigen, typically resulting in intracellular signaling events and subsequent important immune responses, such as release of inflammatory mediators, B cell activation, endocytosis, phagocytosis, and / or cytotoxic attack. In addition, overlapping sites on the Fc region of the molecule also control the activation of complement-mediated cell-independent cytotoxicity, also known as complement-dependent cytotoxicity (CDC).

[0004] In some cases, it may be advantageous to reduce or completely eliminate effector functions. This is especially true for antibodies designed to deliver drugs (e.g., toxins and isotopes) to target cells, where Fc / Fc receptor-mediated effector functions bring healthy immune cells into close proximity of the deadly payload, depleting normal lymphoid tissue along with the target cells (Hutchins, et al., 1995; White, et al., 2001). Furthermore, when the mAb is intended to engage cell surface receptors and prevent receptor-ligand interactions (e.g., antagonists, e.g., cytokine antagonists), it may be desirable to reduce or eliminate effector functions, for example, to prevent target cell death or undesirable cytokine secretion. In these cases, the use of antibodies that recruit less complement or effector cells would be highly beneficial. The need to reduce or eliminate effector functions was noted with the first approved mAb, anti-CD3 mAb, muromonab-CD3, intended to prevent T cell activation in tissue transplant patients receiving donor kidneys, lungs, or hearts (Chatenoud and Bluestone, 2007). Many patients receiving muromonab-CD3 had adverse events (e.g., cytokine storm) including induction of proinflammatory cytokines, which were partially attributed to the interaction of muromonab-CD3 with FcγR (Alegre et al., 1992). To reduce this unintended effector function, the human IgG1 variant L234A / L235A was generated (Xu et al., 2000), which reduced inflammatory cytokine release. In particular, the reduced affinity of antibodies for FcγRII receptors favors antibodies that induce platelet activation and aggregation via FcγRII receptor binding, which are important side effects of such antibodies.

[0005] Silent effector functions can be obtained by Fc engineering. Various sets of mutations have been described in the art, such as LALA (L234A, L235A according to EU numbering) (Wines et al., 2000) or DAPA (D265A, P329A according to EU numbering) (Genentech, U.S. Pat. No. 6,737,056). Some researchers have adopted a cross-subclass approach to reduce effector functions. In a further improvement of the cross-subclass approach, an IgG2 variant with point mutations from IgG4 (i.e., H268Q, V309L, A330S, P331S according to EU numbering) was created (An et al., 2009). Another silent IgG1 antibody contains the N297A mutation resulting in an aglycosylated / non-glycosylated antibody (Strohl et al., 2009). Some of the mutation sets used combine previously described techniques to achieve higher levels of silencing, even completely abolishing some or all of the effector functions. DANAPA (D265A, N297A, P329A) is one example (WO 2019 / 068632 Janssen). Other alternative approaches have been reported to engineer or mutate key residues in the Fc region involved in effector function. See, for example, WO 2009 / 100309 (Medimmune), WO 2006 / 076594 (Xencor), US 2006 / 0134709 (Macrogenics), US 6,737,056 (Genentech), US 2010 / 0166740 (Roche).

[0006] Unwanted Fc interactions with Fcγ receptors and the complement receptor C1q can be uncoupled from binding to fetal Fc receptors (FcRn) which may enhance serum persistence. It has been shown that in vivo serum persistence conferred by FcRn is a tunable property that can be modulated by mutations in IgG Fc. Increasing Fc affinity for FcRn in endosomal conditions (acidic pH) by Fc engineering is an effective approach to extend the pharmacokinetics of monoclonal antibodies (Maeda, 2017). The YTE mutation set (M252Y, S254T, T256E according to EU numbering) or the LS mutation set (M428L, N434S according to EU numbering) are examples of such developed mutation sets in the Fc CH2 domain.

[0007] Chain-pairing mutations have been demonstrated to be efficient in driving heavy chain heterodimerization by introducing complementarity at the CH3-CH3 interface of bispecific or multispecific antibodies. Many sets of chain-pairing mutations have been used to generate multispecific antibodies: increasing / decreasing side chain volume (T366W / S354C-T366S / L368A / Y407V / Y349C, knob-into-hole) (Ridgway, 1996), charge reversal (K409D / K392D-D399K / E356K, electrostatic steering) (Gunasekaran, 2010), or multiple IgA substitutions (SEEDbody) (Davis, 2010). However, all these approaches introduce rather large changes at the interface, which destabilize the CH2 and CH3 regions and decrease the melting temperature (Kuglstatter, 2017; Garber, 2007).

[0008] Silent effector functions, extended half-life (enhanced FcRn binding), or Fc chain pairing-promoting mutations achieved by Fc engineering represent great opportunities to improve and enhance current immunotherapies. However, Fc modifications are known to alter the physicochemical properties of engineered antibodies. Modifications of therapeutic antibodies can lead to loss of thermal stability, reduced expression yields, increased aggregation tendency, and reduced solubility (Liu et al., 2013), which may lead to undesirable outcomes in the development of further therapeutics (Yang et al., 2018). In addition, many engineered Fc variants have potential immunogenicity issues, especially when extensive mutagenesis is involved in reducing effector functions, as multiple mutation sites may lead to the formation of new epitopes.

[0009] Thus, there remains a need for effective methods to compensate for the destabilizing effects of the aforementioned sets of mutations, including Fc silencing, half-life extending, and / or linkage pairing promoting mutations, thereby enabling the design of therapeutic antibodies with improved clinical, pharmacokinetic and pharmacodynamic properties, extended half-life, improved manufacturing and formulation behavior, and improved Fc effector functions while retaining IgG-like biophysical properties. Summary of the Invention

[0010] The present invention describes engineered immunoglobulin IgG Fc regions by transferring structural elements, namely several CH2 interchain disulfide bonds, from IgA immunoglobulins to IgG immunoglobulins. The engineered Fc variants show a significant reduction or complete abolition of the engineered Fc's interaction with FcγR and C1q, while retaining the native ability to interact with FcRn at acidic pH. The inventors have found that elimination of antibody effector functions can be achieved by single cysteine ​​substitutions selected from positions 234, 235 or 236, or by combinations thereof, preferably by a single position. The resulting Fc molecules have comparable expression and purification yields and improved or maintained thermal stability, thereby reducing the tendency to aggregate, compared to wild-type Fc. These substitutions can reduce the destabilizing effects of YTE on the thermal stability of engineered antibodies, as well as the KiH (knob-into-hole) chain pairing promoting mutations. Furthermore, single cysteine ​​substitutions that mimic native IgA are unlikely to generate new epitopes and are expected to reduce the risk of immunogenicity. Thus, the present invention provides improved Fc modifications that can significantly reduce Fc effector functions, but still retain the same stable and desirable physicochemical properties as the unmodified Fc in terms of yield, stability, melting temperature, solubility, aggregation tendency, and other behavior in pharmaceutical formulations.

[0011] In one embodiment, provided herein is an engineered immunoglobulin (e.g., engineered antibody) or fragment thereof, comprising an Fc variant of a wild-type human IgG Fc polypeptide and one or more antigen-binding domains, wherein the Fc variant exhibits reduced effector function compared to the wild-type human IgG Fc polypeptide, and wherein the Fc variant comprises one or more cysteine ​​substitutions selected from the group consisting of positions 234, 235, 236, 297 and 299, the amino acid residues being numbered according to EU numbering. Cysteine ​​235 found in IgA can substitute for leucine 235 in IgG CH2, but can also be located instead at the aforementioned leucine at position 234 in IgG, as determined by examining the 3D crystal structures of these molecules. Indeed, some of the neighboring residues (e.g., L234 in particular) were also considered, in particular to form a stable sulfur bridge between both CH2 domains of the paired Fc molecules, since related IgG1 amino acids are not always located exactly in the same spatial position of the equivalent IgA residues.

[0012] In further embodiments, the one or more cysteine ​​substitutions of the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof are selected from positions 234, 235, and 236. In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises a cysteine ​​substitution at position 234. In another embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises a cysteine ​​substitution at position 235. In another embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises a cysteine ​​substitution at position 236.

[0013] In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof further comprises one or more amino acid substitutions in the Fc variant that enhance the half-life of the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof by enhancing FcRn binding, and / or one or more amino acid substitutions that promote correct chain pairing of two different Fc chains.

[0014] In one embodiment, the half-life extending / FcRn binding enhancing amino acid substitutions are selected from the group consisting of the set of mutations: M252Y / S254T / T256E (YTE), M428L / N434S (LS), T250Q / M428L (QL), and T307Q / N434A (QA).

[0015] In a further embodiment, the half-life extending / FcRn binding enhancing amino acid substitution is M252Y / S254T / T256E (YTE). In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises L234C and M252Y / S254T / T256E (YTE). In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises L235C and M252Y / S254T / T256E (YTE). In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises G236C and M252Y / S254T / T256E (YTE).

[0016] In another preferred embodiment, the half-life extending / FcRn binding enhancing amino acid substitution is M428L / N434S (LS). In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises L234C and M428L / N434S (LS). In one embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises L235C and M428L / N434S (LS). In another embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof comprises G236C and M428L / N434S (LS).

[0017] In some embodiments, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof is a human IgG1 antibody, a human IgG2 antibody, a human IgG3 antibody, or a human IgG4 antibody. Preferably, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof is a human IgG1 antibody. In another preferred embodiment, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof is a human IgG4 antibody.

[0018] In some embodiments, the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof is part of a multispecific binding molecule (e.g., bispecific or trispecific antibodies or more specific including antibodies) and comprises chain-pairing amino acid substitutions selected from the group consisting of knobs-into-holes (Ridgway, 1996), SEED bodies (Davis, 2010), RF mutations in half-Fc (Eliasson, 1988; Tustian, 2016), DEKK mutations (De, 2017), electrostatic steering mutations (Gunasekaran, 2010), and Fab arm exchange (Labrijn, 2011).

[0019] In one embodiment, the chain-pairing amino acid substitution is a knob-into-hole (KiH) mutation and the multispecific binding molecule comprises a first constant heavy chain having an amino acid substitution of T366W and a second constant heavy chain having an amino acid substitution of Y407T, wherein the amino acid residues are numbered according to EU numbering.

[0020] In another embodiment, the chain-pairing amino acid substitution is a knob-into-hole (KiH) mutation and the multispecific binding molecule comprises a first constant heavy chain with the amino acid substitution T366W and a second constant heavy chain with the amino acid substitutions T366S, L368A and Y407V.

[0021] In a further embodiment, the chain-pairing amino acid substitutions are knobs-into-holes (KiH) mutations and the multispecific binding molecule comprises a first constant heavy chain with the amino acid substitutions S354C and T366W and a second constant heavy chain with the amino acid substitutions Y349C, T366S, L368A and Y407V, wherein the amino acid residues are numbered according to EU numbering.

[0022] In one embodiment, the multispecific binding molecule comprises L234C and T366W / S354C-T366S / L368A / Y407V / Y349C(KiH). In another embodiment, the multispecific binding molecule comprises L235C and T366W / S354C-T366S / L368A / Y407V / Y349C(KiH). In another embodiment, the multispecific binding molecule comprises G236C and T366W / S354C-T366S / L368A / Y407V / Y349C(KiH).

[0023] In one embodiment, the multispecific binding molecule comprises both T366W / S354C-T366S / L368A / Y407V / Y349C(KiH) and M252Y / S254T / T256E(YTE).

[0024] In one embodiment, the multispecific binding molecule comprises L234C, M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH). In one embodiment, the multispecific binding molecule comprises L235C, M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH). In another embodiment, the multispecific binding molecule comprises G236C, M252Y / S254T / T256E (YTE) and T366W / S354C-T366S / L368A / Y407V / Y349C (KiH).

[0025] Also provided herein is an engineered immunoglobulin, or fragment thereof, of the present disclosure for use as a medicament.

[0026] Also provided herein are pharmaceutical compositions comprising the engineered immunoglobulins (e.g., engineered antibodies) or fragments thereof of the present disclosure in combination with one or more pharma- ceutically acceptable excipients, diluents, or carriers.

[0027] In one embodiment, the pharmaceutical composition further comprises one or more additional active agents.

[0028] Also provided herein is an isolated nucleic acid molecule encoding an engineered immunoglobulin (eg, an engineered antibody) of the present disclosure, or a fragment thereof.

[0029] Also provided herein are cloning or expression vectors comprising one or more of the nucleic acid sequences outlined above, where the vector is suitable for the recombinant production of an engineered immunoglobulin (e.g., an engineered antibody) or fragment thereof of the present disclosure.

[0030] Also provided herein are host cells containing one or more of the cloning or expression vectors outlined above.

[0031] Also provided herein is a method of preparing an engineered immunoglobulin (e.g., engineered antibody) or fragment thereof of the present disclosure, comprising culturing a host cell as outlined above, purifying and recovering the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof from the host cell culture, and formulating the engineered immunoglobulin (e.g., engineered antibody) or fragment thereof in a pharmaceutically acceptable composition. [Brief description of the drawings]

[0032] [Figure 1-1] Figure 1: Figure 1 is a schematic of the three-dimensional structure of both IgA2 and IgG1. Figure 1A shows how both CH2 domains of the IgA2 homodimeric Fc contact each other by disulfide bonds and packed loops located spatially on top of the IgA2 Fc (PDB 1OWO). Figure 1B shows how both CH2 domains of the IgG1 homodimeric Fc are separated from each other (PDB 1FC1). Finally, Figure 1C depicts IgG1 Fc and IgA2 Fc (PDB 1FC1 and PDB 1OWO, respectively) superimposed in 3D space, showing the main differences in the region on top of the CH2 domains. [Figure 1-2] (As stated above.) [Figure 1-3](As stated above.) [Figure 2-1] Figure 2: Figure 2 shows some SDS-PAGE protein gels of engineered immunoglobulins expressed in HEK or CHO cells and purified by a two-step purification process. Figures 2A1 and 2A2 show SDS-PAGE analysis of engineered anti-CD3 monospecific IgG1 in non-reducing conditions. Figure 2B shows SDS-PAGE analysis of engineered anti-CD3 x target A x target B trispecific IgG1 in non-reducing conditions. Figure 2C shows SDS-PAGE analysis of engineered IgG1 FC in non-reducing conditions. Figure 2D shows SDS-PAGE analysis of engineered anti-CD3 monospecific IgG4 in non-reducing conditions. Figure 2E shows SDS-PAGE analysis of engineered anti-target C monospecific IgG1 in non-reducing conditions. [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 3-1] Figure 3: Figure 3 shows the overall thermal stability of engineered immunoglobulins compared to the parent immunoglobulin. The data shows that immunoglobulin engineering results in more stable molecules with improved thermal stability over the parent immunoglobulin. Figure 3A shows the results of overall thermal stability measurements performed on engineered anti-CD3 monospecific hIgG1. Figure 3B shows the results of overall thermal stability measurements performed on engineered CD3 x target A x target B trispecific hIgG1. Figure 3C shows the results of overall thermal stability measurements performed on engineered anti-CD3 monospecific hIgG4. Figure 3D shows the results of overall thermal stability measurements performed on engineered anti-target C monospecific hIgG1. [Figure 3-2] (As stated above.) [Figure 3-3] (As stated above.) [Diagram 3-4] (As stated above.) [Figure 3-5] (As stated above.) [Diagram 3-6] (As stated above.) [Diagram 3-7] (As stated above.) [Diagram 3-8] (As stated above.) [Diagram 3-9] (As stated above.) [Figure 3-10] (As stated above.) [Figure 3-11] (As stated above.) [Figure 3-12] (As stated above.) [Figure 3-13] (As stated above.) [Figure 3-14] (As stated above.) [Figure 3-15] (As stated above.) [Figure 3-16] (As stated above.) [Figure 3-17] (As stated above.) [Figure 3-18] (As stated above.) [Figure 3-19] (As stated above.) [Figure 3-20] (As stated above.) [Figure 3-21] (As stated above.) [Figure 3-22] (As stated above.) [Figure 3-23] (As stated above.) [Figure 3-24] (As stated above.) [Figure 3-25] (As stated above.) [Figure 4] FIG. 4 shows the thermal stability of the engineered recombinant Fc, measured independently from the Fab. [Figure 5-1]Figure 5: Figure 5 shows the NFAT activity of engineered anti-CD3 monospecific hIgG1, anti-CD3 monospecific hIgG4 and trispecific anti-CD3 x target A x target B immunoglobulins. Figure 5A shows the results obtained with engineered anti-CD3 monospecific hIgG1 in separate assays (first assay: Figure 5A1, second assay: Figure 5A2, third assay: Figure 5A3). In summary, the parent (CD3_WT) and the corresponding half-life extended variant (CD3_WT_YTE) showed the highest NFAT activity, whereas all engineered immunoglobulins showed a strongly inhibited NFAT activation. Figure 5B shows the results obtained with engineered anti-CD3 x target A x target B trispecific hIgG1. In summary, the parent (CD3 x target A x target B_WT) showed the highest NFAT activity, whereas all engineered immunoglobulins showed a strongly inhibited NFAT activation. Figure 5C shows the results obtained with engineered anti-CD3 monospecific hIgG4. In summary, the parental (IgG4_CD3_WT or IgG4_CD3_S228P) and the corresponding half-life extended variants (IgG4_CD3_WT_YTE or IgG4_CD3_S228P_YTE) displayed maximal NFAT activity, whereas all engineered immunoglobulins displayed greatly suppressed NFAT activation. [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 5-4] (As stated above.) [Figure 5-5] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Disclosed herein are engineered immunoglobulins (e.g., engineered antibodies) or fragments thereof that contain mutated Fc regions such that the engineered immunoglobulins (e.g., engineered antibodies) achieve the elimination of effector functions while still stably retaining physicochemical properties.

[0034] definition In order that the present invention may be more readily understood, certain terms are defined throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

[0036] The terms "comprising" and "including" are used herein in their open-ended, non-limiting sense, unless otherwise specified.

[0037] The term "binding molecule" of the present disclosure encompasses the antigen-binding portion of an antibody, which may also be incorporated into Fc-containing binding molecules, full IgG, including IgG1 and IgG4 antibodies, antibody variants, fragments of antibodies, single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136), or may be a multispecific antibody comprising an Fc domain and two or more binding moieties. In one embodiment, the Fc-containing binding molecules of the present disclosure also include binding moieties such as nanobodies, Fabs, scFvs, Vhhs, DARPins, avimers, affibodies, Sso7d, and anticalins.

[0038] As used herein, the term "antibody" refers to a polypeptide of the immunoglobulin family that can bind non-covalently, reversibly, and specifically to a corresponding antigen. The basic functional unit of each antibody is an immunoglobulin monomer containing only one Ig unit, defined herein as an "Ig monomer." Secretory antibodies can also be dimers with two Ig units (e.g., IgA), tetramers with four Ig units, or pentamers with five Ig units (e.g., mammalian IgM). An Ig monomer is a Y-shaped molecule consisting of four polypeptide chains; two identical heavy chains and two identical light chains linked by disulfide bonds (Woof & Burton (2004) Nature Reviews Immunology, 4(2):89-99). Each chain contains multiple structural domains containing about 70-110 amino acids that are classified into two categories according to their size and function: variable or constant. Heavy chains contain one variable domain (variable heavy domain; abbreviated as VH) and three constant domains (abbreviated as CH1, CH2, and CH3). Each light chain contains one variable domain (abbreviated as VL) and one constant domain (abbreviated as CL). Immunoglobulin domains have a characteristic immunoglobulin fold in which two beta sheets create a "sandwich" shape held together by interactions between conserved cysteine ​​residues and other charged amino acids. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino acid terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the antigen-binding domain or site that interacts with an antigen.

[0039] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (e.g., an anti-Id antibody to an antibody of the present disclosure). Antibodies can 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).

[0040] The terms "recognize" or "bind" as used herein refer to a binding molecule, antibody, or antigen-binding fragment thereof that finds and interacts with (e.g., binds to or recognizes) an epitope, regardless of whether the epitope is linear, discontinuous, or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen-binding fragment of the present disclosure specifically binds. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the spatial structure of epitopes include techniques known in the art, such as x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)), or electron microscopy. A "paratope" is the portion of an antibody that recognizes an epitope of an antigen.

[0041] The phrases "specifically bind" or "selectively bind", when used in the context of describing the interaction between an antigen (e.g., a protein) and an antibody, antibody fragment, or antibody-derived binding agent, refer to a binding reaction that determines the presence of the antigen in a heterogeneous population of proteins and other biological products, e.g., a biological sample, such as a blood, serum, plasma, or tissue sample. Thus, under certain specified immunoassay conditions, an antibody or binding agent with a particular binding specificity will bind to a particular antigen at least twice as much as background and will not substantially bind in a significant amount to other antigens present in the sample. In one aspect, under specified immunoassay conditions, an antibody or binding agent with a particular binding specificity will bind to a particular antigen at least 10 times as much as background and will not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or binding agent has been selected for its specificity for a particular protein. If desired or necessary, this selection may be achieved by removing antibodies that cross-react with molecules from other species (e.g., mouse or rat) or other subtypes. Alternatively, in some embodiments, antibodies or antibody fragments are selected that cross-react with a particular desired molecule.

[0042] The term "antigen-binding site" refers to the portion of an antibody that contains determinants that form an interface that binds to an antigen or epitope thereof. The term "antigen-binding site" can be used interchangeably with the term "antigen-binding domain" or antigen-binding portion. For proteins (or protein mimetics), the antigen-binding site typically comprises one or more loops (of at least four amino acids or amino acid mimics) that form an interface that binds to an antigen polypeptide. Typically, the antigen-binding site of an antibody molecule comprises at least one or two CDRs and / or hypervariable loops, or more typically at least three, four, five or six CDRs and / or hypervariable loops.

[0043] "Complementarity determining regions" ("CDRs") as used herein refer to the hypervariable regions of VL and VH. CDRs are the target protein binding sites of the antibody chains that possess the specificity for the target protein. There are three CDRs (CDR1-3, numbered consecutively from the N-terminus) in each human VL or VH, constituting a total of about 15-20% of the variable domain. CDRs can be referred to by their region and order. For example, "VHCDR1" or "HCDR1" both refer to the first CDR of the heavy chain variable region. CDRs are structurally complementary to the epitope of the target protein and are therefore directly responsible for the binding specificity. The remaining stretches of VL or VH, the so-called framework regions, show less variation in amino acid sequence (Kuby (2000) Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York). The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides, including antibodies and antigen-binding fragments, having substantially identical amino acid sequences or derived from the same genetic source. The term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Methods for generating 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).

[0044] The term "human antibody", as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody comprises a constant region, the constant region is also derived from such a human sequence, e.g., an antibody comprising a human germline sequence, or a variant of a human germline sequence, or a consensus framework sequence derived from human framework sequence analysis as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000). In a preferred embodiment, the engineered IgG immunoglobulin or fragment thereof of the present disclosure is a human antibody.

[0045] The human antibodies of the disclosure can include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or conservative substitutions to facilitate stability or manufacturing).

[0046] The antibody or immunoglobulin may be one in which the variable region or a part thereof, such as the CDR, is generated in a non-human organism, such as a rat or a mouse. Chimeric, CDR-grafted, and humanized antibodies are within the scope of the present invention. The antibody may be humanized by methods known in the art (see, for example, Morrison, SL, (1985), Science 229:1202-1207; Oi et al. (1986), BioTechniques 4:214; and Queen et al., U.S. Pat. Nos. 5,585,089, 5,693,761, and 5,693,762, which are incorporated herein by reference). Humanized or CDR-grafted antibodies may be generated by CDR-grafting or CDR-substitution, in which one, two, or all CDRs of an immunoglobulin chain may be replaced. See, for example, U.S. Pat. No. 5,225,539; Jones et al., (1986) Nature 321:552-525; Verhoeyan et al., (1988) Science 239:1534; Beidler et al., (1988) J. Immunol. 141:4053-4060, and Winter U.S. Pat. No. 5,225,539, the entire contents of which are expressly incorporated herein by reference. Humanized antibodies in which specific amino acids have been substituted, deleted, or added are also within the scope of the present invention. Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in European Patent Application Publication No. 519596A1 to Padlan et al.

[0047] In mammals, there are two types of immunoglobulin light chains, called lambda (λ) and kappa (κ). Each antibody always contains two identical light chains; in mammals, only one type of light chain, κ or λ, exists per antibody. The approximate length of a light chain is 211-217 amino acids, and each light chain has two domains, one constant domain and one variable domain.

[0048] There are five types of mammalian Ig heavy chains, designated α, δ, ε, γ, and μ, and the type of heavy chain present in an antibody defines the class or isotype of the antibody: IgM, IgG, IgA, IgD, IgE, respectively. Although heavy chains vary in physicochemical, structural, and immunological properties, each heavy chain has two domains, a variable domain and a constant domain. The variable domain contains a single Ig domain (approximately 110 amino acids long) and determines the antibody binding specificity. The constant domain is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α, and δ have a constant region made up of three tandem Ig domains and a hinge region for added flexibility; heavy chains μ and ε have a constant region made up of four immunoglobulin domains (Woof & Burton, supra). In one embodiment, the "immunoglobulin" can be an antibody. In one embodiment, the "fragment thereof" of an immunoglobulin can be an Fc region or one or more Fc domains.

[0049] The term "Fc region" refers to the crystallizable fragment region of an antibody, which plays an important role in regulating immune cell activity. The Fc region is composed of two polypeptide chains or Fc domains, with IgG containing the CH2 and CH3 constant domains of the heavy chain or "CH2 domain" and "CH3 domain", respectively. IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The amino acid residues in the CH2 and CH3 domains can be numbered according to the EU numbering system (Edelman et al., (1969) PNAS.USA, 63, 78-85), "Kabat" numbering (Kabat et al., supra), or alternatively using the IMGT numbering for C domains. The IMGT tool is available on the World Wide Web (www.imgt.org).

[0050] The Fc region binds to cell surface receptors that mediate the physiological effects of antibodies, the "Fc receptors," and complement proteins. Fc receptors are found on many cells of the immune system, including B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells. Binding of an antibody Fc region to an Fc receptor stimulates phagocytic or cytotoxic cells to destroy microorganisms or infected cells by the mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC). There are several different types of Fc receptors (FcR), which are classified based on the type of antibody they recognize. For example, those that bind IgG are called Fc-gamma receptors (FcγR), those that bind IgA are called Fc-alpha receptors (FcαR), and those that bind IgE are called Fc-epsilon receptors (FcεR). The classes of FcR are also distinguished by the cells that express them (macrophages, granulocytes, natural killer cells, T and B cells) and the signaling properties of each receptor (Owen J et al., (2009) Immunology (7th ed.). New York: WH Freeman and Company. p423). The following table (Table 1) summarizes the different Fc receptors, their ligands, cellular distribution and binding effects.

[0051] [Table 1]

[0052] As used herein, "modification" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, which change results from a sequence alteration involving said one or more amino acid residues / positions. For example, typical modifications include substitution (e.g., conservative or non-conservative substitution) of one or more residues (or at said positions) with another amino acid, insertion of one or more amino acids adjacent to said one or more residues / positions, and deletion of said one or more residues / positions, inversion of said one or more residues / positions, and duplication of said one or more residues / positions.

[0053] "Amino acid substitution" or "substitution" refers to the replacement of one or more existing amino acid residues in a given (starting or parent) amino acid sequence with one or more different amino acid residues. For example, the substitution I332E refers to a variant polypeptide, in this case a constant heavy chain variant, in which the isoleucine at position 332 is replaced with glutamic acid (EU numbering). Alternatively, the position of the substitution in the CH2 or CH3 domain can be mentioned, for example CH2.97 indicates a substitution at position 97 in the CH2 domain, the numbering being according to the IMGT numbering for C-domains. The exact substitution can also be indicated by, for example, L_CH2.97_Y, indicating that the leucine at position 97 in the CH2 domain is replaced with a tyrosine.

[0054] Generally and preferably, the modification results in an alteration of at least one physico-biochemical activity of the variant polypeptide compared to a polypeptide comprising the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody or polyspecific binding molecule, the altered physico-biochemical activity may be binding affinity, binding capacity and / or binding efficacy for the target molecule.

[0055] The term "in vivo half-life," as used herein, refers to the half-life of a molecule of interest or a variant thereof circulating in the blood of a given mammal.

[0056] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), nonpolar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)).

[0057] The term "percent identical" or "percent identity" with respect to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if, when compared over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, and aligned for maximum correspondence, the two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity over a specified region, or, if not specified, over the entire sequence, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity). Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably, over a region that is 100-500 or 1000 nucleotides or more (or 20, 50, 200 amino acids or more) in length. The "percentage of identity" or "percentage of sequence identity" of the present disclosure can be calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a window of comparison, (ii) determining the number of positions where the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to obtain the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percentage of identity" is calculated with respect to a reference sequence without a specific comparison window defined, the percent identity is determined by dividing the number of matched positions on the region of alignment by the total length of the reference sequence. Thus, for the purposes of this disclosure, when two sequences (query and subject) are optimally aligned (allowing for gaps in their alignment), the "percentage of identity" for a query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), then multiplied by 100%.

[0058] Apart from the percentage of sequence identity described above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by a first nucleic acid is immunologically cross-reactive with an antibody raised against a polypeptide encoded by a second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0059] Various aspects of the disclosure are described in further detail in the following sections and subsections.

[0060] Detailed description of the generation of engineered immunoglobulins (e.g., engineered antibodies) with greatly reduced or eliminated Fc effector functions. To generate IgG immunoglobulins with their stabilizing and silencing properties in the Fc portion, crystal structure analyses of the Fc regions of IgG1 and IgA1 were compared to identify specific amino acid residues in IgA immunoglobulins that are important for binding to FcγR. The basic monomeric unit of IgG or IgA, in common with all antibodies, is arranged in two identical Fab regions linked to the Fc via a hinge region. Both the heavy and light chains fold into globular domains, four in each heavy chain (from the N-terminal VH, CH1, CH2, and CH3) and two in each light chain (VL and CL). Each IgG and IgA domain adopts a characteristic "immunoglobulin fold" that includes a 110-residue β-sheet sandwich of antiparallel strands arranged around a stabilizing internal disulfide bond. There is close pairing of domains between adjacent chains (VH and VL, CH1 and CL, and CH3 and CH3), and interchain disulfide bridges further stabilize the structure. In IgG immunoglobulins, these are found between the heavy chains in the hinge region (C226 and C229 according to EU numbering), whereas in IgA, these interchain disulfide bonds are found between the heavy chains in the CH2 domain. Available X-ray crystal structures (Herr et al., 2003; Ramsland et al., 2007) indicate that four cysteines on each heavy chain (C235, C236, C297, C299 according to EU numbering) may be involved in linking the tops of the CH2 domains. The solved structures of IgA1 Fc complexes with different ligands differ in this exact location, suggesting that some degree of disulfide exchange may be possible (Woof et al., 2011).

[0061] For IgA, both CH2 domains of the homodimeric Fc are in contact and linked by four disulfide bonds at positions C235, C236, C297 and C299 (according to EU numbering). This packed region observed on top of the IgA CH2 region is shown in FIG. 1A. In contrast to IgA, IgG does not have this disulfide bridge. In fact, both CH2 domains of the IgG homodimeric Fc are separated from each other, as shown in FIG. 1B. As a result, the CH2 domains of both the IgG homodimeric Fc and the IgA homodimeric Fc do not share the same 3D position and orientation within the Fc. These observations are shown in FIG. 1C by the 3D superposition of both the IgG Fc and the IgA Fc.

[0062] In the present invention, such IgA structural elements were introduced into IgG Fc by substitution of the relevant positions with IgA amino acids involved in this upper packed region of CH2. Since the relevant IgG1 amino acids are not located exactly in the same spatial positions of the equivalent IgA residues, some of the neighboring residues were also taken into consideration, e.g., especially L234.

[0063] The set of mutations introduced into the IgG FC is listed in Table 2.

[0064] [Table 2]

[0065] As demonstrated in the present disclosure, the transfer of the CH2 interchain disulfide bond from IgA to IgG immunoglobulins has enabled the generation of engineered immunoglobulins (e.g., engineered antibodies) or fragments thereof, including IgG Fc variants with removed Fc effector functions. In one embodiment, the present disclosure provides an engineered IgG immunoglobulin comprising one or more cysteine ​​substitutions selected from the group consisting of positions 234, 235, 236, 297, and 299, where the amino acid residues are numbered according to EU numbering.

[0066] In one embodiment, the present disclosure provides an engineered IgG immunoglobulin (e.g., an engineered antibody) or fragment thereof comprising one or more cysteine ​​substitutions in the Fc domain selected from the group consisting of positions 234, 235, and 236.

[0067] In some embodiments, the engineered IgG immunoglobulin is human IgG1, IgG2, IgG3 or IgG4. In one embodiment, the Fc variant has at least 90% amino acid sequence identity to the Fc domain of wild-type human IgG1. In one embodiment, the Fc variant has at least 95% amino acid sequence identity to the Fc domain of wild-type human IgG1. In one embodiment, the Fc variant has at least 98% amino acid sequence identity to the Fc domain of wild-type human IgG1. In one embodiment, the Fc variant has at least 90% amino acid sequence identity to the Fc domain of wild-type human IgG4. In one embodiment, the Fc variant has at least 95% amino acid sequence identity to the Fc domain of wild-type human IgG4. In one embodiment, the Fc variant has at least 98% amino acid sequence identity to the Fc domain of wild-type human IgG4.

[0068] In one embodiment, the present disclosure provides an engineered IgG immunoglobulin (e.g., engineered antibody) or a fragment thereof further comprising any one of the mutation sets listed in Table 2, where the amino acid residues are numbered according to EU numbering. In one embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or a fragment thereof further comprises one or more amino acid substitutions in the Fc variant that enhance the half-life of the engineered IgG immunoglobulin (e.g., engineered antibody) or a fragment thereof by enhancing FcRn binding, and / or one or more amino acid substitutions that promote correct chain pairing of two different Fc chains. In a preferred embodiment, the half-life extending / FcRn binding enhancing amino acid substitutions are selected from the group consisting of the mutation sets: M252Y / S254T / T256E (YTE), M428L / N434S (LS), and T250Q / M428L (QL), and T307Q / N434A (QA).

[0069] It is known that YTE mutants have lower physical stability than the same mAb without the mutation (Tavakoli-Keshe, 2014). One possibility is that these differences in stability are mediated by changes in the structural dynamics of certain sequences of the mAb due to the YTE mutation. Surprisingly, the present invention shows that cysteine ​​substitutions can reduce the destabilizing effect of the YTE mutation on an engineered IgG immunoglobulin (e.g., engineered antibody) or a fragment thereof. In one embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or a fragment thereof comprises L234C and M252Y / S254T / T256E (YTE). In one embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or a fragment thereof comprises L235C and M252Y / S254T / T256E (YTE). In another embodiment, the engineered IgG immunoglobulin (eg, engineered antibody) or fragment thereof comprises G236C, and M252Y / S254T / T256E (YTE).

[0070] The cysteine ​​substitutions provided by the present invention can also reduce the destabilizing effect of LS mutations. In a preferred embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or fragment thereof comprises L234C and M428L / N434S (LS). In one embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or fragment thereof comprises L235C and M428L / N434S (LS). In another embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or fragment thereof comprises G236C and M428L / N434S (LS).

[0071] In one embodiment, the engineered IgG immunoglobulin (eg, engineered antibody) or fragment thereof comprising any one of the sets of mutations set forth in Table 2 is a monospecific antibody.

[0072] In one embodiment, the monospecific antibody comprises L234C and M252Y / S254T / T256E (YTE). In one embodiment, the monospecific antibody comprises L235C and M252Y / S254T / T256E (YTE). In another embodiment, the monospecific antibody comprises G236C and M252Y / S254T / T256E (YTE).

[0073] In one embodiment, the engineered IgG immunoglobulin (e.g., engineered antibody) or fragment thereof comprising any one of the sets of mutations listed in Table 2 above is a multispecific antibody, in particular a bi- or trispecific antibody.

[0074] The term "monospecific molecule" as used herein refers to an Fc-containing molecule that binds to one epitope on a target antigen. In some embodiments, a monospecific molecule of the present disclosure is a monospecific antibody-like molecule. In some embodiments, a monospecific molecule of the present disclosure is a monospecific antibody. The term "bispecific molecule" refers to a multispecific Fc-containing binding molecule that binds to two different antigens. The term "trispecific molecule" refers to an Fc-containing multispecific binding molecule that binds to three different antigens via three different binding moieties. In some embodiments, a bispecific molecule of the present disclosure is a bispecific antibody-like molecule. In some embodiments, a multispecific binding molecule of the present disclosure is a multispecific antibody-like molecule.

[0075] The term "multispecific antibody" refers to an antibody that can recognize two or more epitopes of an antigen or two or more antigens. The recognition of each antigen is generally achieved by an "antigen-binding domain". In particular, bispecific antibodies recognize two different epitopes on the same or different antigens. All bispecific IgG molecules, i.e. bispecific antibodies that are indistinguishable from natural immunoglobulins in their composition, are bivalent and have an asymmetric structure due to the presence of at least different Fv regions. Depending on the preparation method and source of the heavy and light chains, they may further differ in the constant regions of the heavy or light chains (Brinkmann and Kontermann, 2017).

[0076] In one embodiment, the bispecific antibody further comprises a half-life extending mutation, such as M252Y / S254T / T256E (YTE). In one embodiment, the bispecific antibody comprises L234C and M252Y / S254T / T256E (YTE). In one embodiment, the bispecific antibody comprises L235C and M252Y / S254T / T256E (YTE). In another embodiment, the bispecific antibody comprises G236C and M252Y / S254T / T256E (YTE).

[0077] In a preferred embodiment, the multispecific antibody contains mutations that promote correct HC / HC pairing.

[0078] To ensure proper heterodimerization of the two Fc domains of the Fc region of an engineered immunoglobulin (e.g., engineered antibody) or fragment thereof of the present disclosure, various approaches can be used to enhance dimerization, such as those described in, for example, EP 1870459; U.S. Pat. No. 5,582,996; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,910,573; U.S. Pat. No. 5,932,448; U.S. Pat. No. 6,833,441; U.S. Pat. No. 7,183,076; U.S. Patent Application Publication No. 2006 / 204493 A1; WO 09 / 089004 A1. In one embodiment, one or more mutations in a first Fc domain of an engineered immunoglobulin (e.g., an engineered antibody) or fragment thereof comprising a heavy chain constant domain are generated to create a "knob" and one or more mutations in a second Fc domain of an engineered immunoglobulin (e.g., an engineered antibody) or fragment thereof comprising a heavy chain constant domain are generated to create a "hole", such that the "knob" and the "hole" bind (e.g. interact, e.g. the CH2 domain of the first Fc domain interacts with the CH2 domain of the second Fc domain, or the CH3 domain of the first Fc domain interacts with the CH3 domain of the second Fc domain) upon heterodimerization of the first and second Fc domains.

[0079] "Knob", as the term is used herein, refers to at least one amino acid side chain that protrudes from the interface of a first Fc domain of an engineered immunoglobulin (e.g., engineered antibody) or fragment thereof that comprises a heavy chain constant domain, and thus can be positioned in a compensatory "hole" in the interface with a second Fc domain of an engineered immunoglobulin (e.g., engineered antibody) or fragment thereof that comprises a heavy chain constant domain, such that the heterodimer is stabilized, thereby making it more favorable for heterodimer formation than homodimer formation, for example. The preferred import residues for forming the knob are generally naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Tryptophan and tyrosine are most preferred. In a preferred embodiment, the original residue for forming the protrusion has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.

[0080] "Hole" refers to at least one amino acid side chain recessed from the interface of a second Fc domain of an engineered immunoglobulin (e.g. engineered antibody) or fragment thereof comprising a heavy chain constant domain, and thus accommodating a corresponding knob at the adjacent interface of a first Fc domain of an engineered immunoglobulin (e.g. engineered antibody) or fragment thereof comprising a heavy chain constant domain. The preferred import residues for forming the hole are usually naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Serine, alanine or threonine are most preferred. In a preferred embodiment, the original residue for forming the hole has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.

[0081] In one embodiment, the chain-pairing amino acid substitution is a knob-into-hole (KiH) mutation and the multispecific binding molecule comprises a first constant heavy chain having an amino acid substitution of T366W and a second constant heavy chain having an amino acid substitution of Y407T, wherein the amino acid residues are numbered according to EU numbering.

[0082] In another embodiment, the chain-pairing amino acid substitution is a knob-into-hole (KiH) mutation and the multispecific binding molecule comprises a first constant heavy chain with an amino acid substitution of T366W and a second constant heavy chain with amino acid substitutions of T366S, L368A and Y407V, wherein the amino acid residues are numbered according to EU numbering.

[0083] In a further embodiment, the chain-pairing amino acid substitutions are knobs-into-holes (KiH) mutations and the multispecific binding molecule comprises a first constant heavy chain with the amino acid substitutions S354C and T366W and a second constant heavy chain with the amino acid substitutions Y349C, T366S, L368A and Y407V, wherein the amino acid residues are numbered according to EU numbering.

[0084] In one embodiment, the multispecific antibody comprises a first constant heavy chain comprising L234C and the above-mentioned KiH mutations and with amino acid substitutions S354C and T366W, and a second constant heavy chain with amino acid substitutions Y349C, T366S, L368A and Y407V. In one embodiment, the multispecific antibody comprises L235C and a KiH mutation with amino acid substitutions S354C and T366W in the first constant heavy chain and with amino acid substitutions Y349C, T366S, L368A and Y407V in the second constant heavy chain. In another embodiment, the multispecific antibody comprises G236C and a KiH mutation with amino acid substitutions S354C and T366W in the first constant heavy chain and with amino acid substitutions Y349C, T366S, L368A and Y407V in the second constant heavy chain.

[0085] In yet another preferred embodiment, the multispecific antibody comprises the mutations L234C, KiH and YTE. In one embodiment, the bispecific antibody comprises the mutations L235C, KiH and YTE. In another embodiment, the bispecific antibody comprises the mutations G236C, KiH and YTE.

[0086] In some instances, HC / LC pairing was driven by electrosteering to introduce the following sets of mutations onto the HC and LC: Q38K, Q124D, K169 in Kappa LC Q39D, K147, S165D in HC Lambda LC Q38D, E124K, N170D Q39K, K147D, S165R in HC

[0087] In a further embodiment, multispecific antibodies are generated by combining the knobs-into-holes strategy with the electrostatic steering method.

[0088] In one embodiment, the engineered IgG immunoglobulin (eg, engineered antibody) or fragment thereof comprising any one of the sets of mutations set forth in Table 2 is a bispecific antibody.

[0089] In one embodiment, the bispecific antibody further comprises a half-life extending mutation, such as M252Y / S254T / T256E (YTE). In one embodiment, the bispecific antibody comprises L234C and M252Y / S254T / T256E (YTE). In one embodiment, the bispecific antibody comprises L235C and M252Y / S254T / T256E (YTE). In another embodiment, the bispecific antibody comprises G236C and M252Y / S254T / T256E (YTE).

[0090] In a preferred embodiment, the bispecific antibody comprises mutations that promote correct HC / HC pairing, which can be knobs-in-holes or electrostatic steering, or a combination of both.

[0091] In one embodiment, the bispecific antibody comprises L234C and a KiH mutation with amino acid substitutions of S354C and T366W in the first constant heavy chain and with amino acid substitutions of Y349C, T366S, L368A and Y407V in the second constant heavy chain. In one embodiment, the bispecific antibody comprises L235C and a KiH mutation with amino acid substitutions of S354C and T366W in the first constant heavy chain and with amino acid substitutions of Y349C, T366S, L368A and Y407V in the second constant heavy chain. In another embodiment, the bispecific antibody comprises G236C and a KiH mutation with amino acid substitutions of S354C and T366W in the first constant heavy chain and with amino acid substitutions of Y349C, T366S, L368A and Y407V in the second constant heavy chain.

[0092] In a preferred embodiment, the bispecific antibody comprises the mutations L234C, KiH and YTE. In one embodiment, the bispecific antibody comprises the mutations L235C, KiH and YTE. In another embodiment, the bispecific antibody comprises the mutations G236C, KiH and YTE.

[0093] In one embodiment, the engineered IgG immunoglobulin (eg, engineered antibody) or fragment thereof comprising any one of the sets of mutations set forth in Table 2 is a trispecific antibody.

[0094] In one embodiment, the trispecific antibody further comprises a half-life extending mutation, such as M252Y / S254T / T256E (YTE). In one embodiment, the trispecific antibody comprises L234C, and M252Y / S254T / T256E (YTE). In one embodiment, the trispecific antibody comprises L235C, and M252Y / S254T / T256E (YTE). In another embodiment, the trispecific antibody comprises G236C, and M252Y / S254T / T256E (YTE).

[0095] In a preferred embodiment, the trispecific antibody comprises mutations that promote correct HC / HC pairing, which can be knobs-in-holes or electrostatic steering, or a combination of both.

[0096] In one embodiment, the trispecific antibody comprises L234C and a KiH mutation with amino acid substitutions of S354C and T366W in the first constant heavy chain and with amino acid substitutions of Y349C, T366S, L368A and Y407V in the second constant heavy chain. In one embodiment, the trispecific antibody comprises L235C and a KiH mutation with amino acid substitutions of S354C and T366W in the first constant heavy chain and with amino acid substitutions of Y349C, T366S, L368A and Y407V in the second constant heavy chain. In another embodiment, the trispecific antibody comprises G236C and a KiH mutation with amino acid substitutions of S354C and T366W in the first constant heavy chain and with amino acid substitutions of Y349C, T366S, L368A and Y407V in the second constant heavy chain.

[0097] In a preferred embodiment, the trispecific antibody comprises the following mutations: L234C, KiH, and YTE. In one embodiment, the bispecific antibody comprises the following mutations: L235C, KiH, and YTE. In another embodiment, the bispecific antibody comprises the following mutations: G236C, KiH, and YTE.

[0098] Fc fragments of human IgG containing any one of the sets of mutations listed in Table 2 were also generated.

[0099] In one embodiment, the Fc fragment further comprises a half-life extending mutation, such as M252Y / S254T / T256E (YTE). In one embodiment, the Fc fragment comprises L234C and M252Y / S254T / T256E (YTE). In one embodiment, the Fc fragment comprises L235C and M252Y / S254T / T256E (YTE). In another embodiment, the Fc fragment comprises G236C and M252Y / S254T / T256E (YTE).

[0100] In a preferred embodiment, the Fc fragment comprises mutations that promote correct HC / HC pairing, which can be knobs-in-holes or electrostatic steering, or a combination of both.

[0101] In one embodiment, the Fc fragment comprises an L234C and a KiH mutation as described above. In one embodiment, the Fc fragment comprises an L235C and a KiH mutation. In another embodiment, the Fc fragment comprises a G236C and a KiH mutation.

[0102] In a preferred embodiment, the Fc fragment comprises the following mutations: L234C, KiH, and YTE. In one embodiment, the Fc fragment comprises the following mutations: L235C, KiH, and YTE. In another embodiment, the Fc fragment comprises the following mutations: G236C, KiH, and YTE.

[0103] Also provided herein is an engineered immunoglobulin, or fragment thereof, of the present disclosure for use as a medicament.

[0104] Also provided herein is an engineered immunoglobulin, or fragment thereof, of the present disclosure for use in therapy.

[0105] The protein and corresponding nucleotide sequences of the engineered immunoglobulins and Fc fragments are listed in Table 8.

[0106] Nucleic Acids and Expression Systems The present invention also encompasses isolated nucleic acids encoding the polypeptide chains of the engineered immunoglobulins (e.g., engineered antibodies) of the present disclosure or fragments thereof. The nucleic acid molecules of the present disclosure include DNA and RNA in both single-stranded and double-stranded forms, as well as the corresponding complementary sequences. The nucleic acid molecules of the present disclosure include full-length genes or cDNA molecules, as well as combinations of fragments thereof. The nucleic acids of the present disclosure are derived from human sources, but may also include those derived from non-human species.

[0107] An "isolated nucleic acid" is a nucleic acid that, in the case of a nucleic acid isolated from a naturally occurring source, is separated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated. For example, in the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, it is understood that the nucleic acid resulting from such a process is an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of an individual fragment or as a component of a larger nucleic acid construct. In one preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that allows for the identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (e.g., those reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. The sequence of non-translated DNA can be present 5' or 3' from the open reading frame, in which case it does not interfere with manipulation or expression of the coding region.

[0108] Variant sequences can be prepared by site-specific mutagenesis of nucleotides in the DNA encoding the polypeptide using cassette or PCR mutagenesis or other techniques known in the art to produce DNA encoding the variant, as outlined herein, and then expressing the recombinant DNA in cell culture.

[0109] An "optimized nucleotide sequence" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in a production cell, e.g., a Chinese Hamster Ovary cell (CHO). An optimized nucleotide sequence is engineered to completely retain the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence.

[0110] The present disclosure also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes comprising at least one of the above polynucleotides. Additionally, the present disclosure provides host cells comprising such expression systems or constructs. The heavy and light chains of an engineered IgG immunoglobulin or fragment thereof can be encoded by a single nucleic acid (e.g., inserted into a single vector) or can be encoded by multiple nucleic acid molecules, e.g., two nucleic acid molecules (also referred to as a "set"), which can be inserted into multiple vectors (e.g., two vectors, i.e., a set of vectors).

[0111] In one embodiment, a method for preparing an engineered IgG immunoglobulin or fragment thereof comprising an Fc variant as disclosed herein is provided, comprising: (a) culturing a host cell comprising a nucleic acid encoding a heavy chain comprising an engineered Fc domain polypeptide, and a nucleic acid comprising a light chain polypeptide, wherein the cultured host cell expresses the engineered polypeptide, and (b) purifying and recovering the engineered IgG immunoglobulin or fragment thereof from the host cell culture. Optionally, the method may comprise a further step (c) of formulating the IgG immunoglobulin or fragment thereof in a pharma- ceutically acceptable composition.

[0112] Provided are cloning or expression vectors comprising one or more of the nucleic acid sequences described above, suitable for the recombinant production of engineered immunoglobulins (eg, engineered antibodies) or fragments thereof of the disclosure.

[0113] Expression vectors for use in the present disclosure can be constructed from starting vectors, e.g., commercially available vectors. After constructing the vector and inserting the nucleic acid molecules encoding the polypeptide chains of engineered immunoglobulins into the appropriate sites of the vector, the completed vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the expression vector into the selected host cell can be accomplished by known methods, such as transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell to be used. These methods and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., 2001, supra.

[0114] Typically, expression vectors used in host cells contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" and in certain embodiments typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selection marker element.

[0115] Host cells containing one or more cloning or expression vectors of the disclosure are also provided.

[0116] When cultured under appropriate conditions, host cells can be used to express engineered immunoglobulins (e.g., engineered antibodies) or fragments thereof, which can then be recovered from the culture medium (if the host cells secrete them into the medium) or directly from the host cells that produce it (if not secreted). Selection of an appropriate host cell depends on a variety of factors, such as the desired expression level, modifications of the polypeptide (such as glycosylation or phosphorylation) that are desirable or necessary for activity, and the ease of folding into a biologically active molecule. Host cells can be eukaryotic or prokaryotic.

[0117] Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), and any cell line used in an expression system known in the art can be used to produce polypeptides including engineered immunoglobulins (e.g., engineered antibodies) or fragments thereof of the present disclosure. In general, a host cell is transformed with a recombinant expression vector that includes DNA encoding the desired engineered immunoglobulin. Among the host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include COS-7 cells, L cells, Cl27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells or their derivatives and related cell lines grown in serum-free medium, HeLa cells, BHK cell lines, CVIIEBNA cell lines, human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK, or Jurkat cells.

[0118] Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising an engineered immunoglobulin (e.g., an engineered antibody) or a fragment thereof of the present disclosure. The engineered immunoglobulin may be combined with one or more pharma- ceutically acceptable excipients, diluents, or carriers.

[0119] To prepare a pharmaceutical or sterile composition comprising the artificially engineered immunoglobulin of the present disclosure, the immunoglobulin may be mixed with a pharma- ceutically acceptable excipient, diluent, or carrier. In one embodiment, the pharmaceutical composition of the present disclosure is a combination with one or more pharma- ceutically acceptable excipients, diluents, or carriers. The phrase "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The term "pharmaceutical composition" refers to a mixture of at least one active ingredient (e.g., the artificially engineered immunoglobulin of the present disclosure) and at least one pharma- ceutical agent, diluent, or carrier. A "pharmaceutical product" refers to a substance used in medical treatment.

[0120] Pharmaceutical compositions of therapeutic and diagnostic agents can be prepared, for example, by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Oral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Forms: Disperse Systems, Marcel Dekker, NY; see Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0121] In one embodiment, the pharmaceutical composition of the present disclosure comprises a therapeutically effective amount of an engineered immunoglobulin or fragment thereof of the present disclosure. As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapy (e.g., an engineered antibody) sufficient to reduce and / or ameliorate the severity and / or duration of a given condition, disorder or disease, and / or symptoms associated therewith. These terms also encompass the amount necessary to reduce, slow, slow or ameliorate the progression or progression of a given condition, disorder or disease, reduce, slow or ameliorate the recurrence, occurrence or onset of a given condition, disorder or disease, and / or improve or enhance the prophylactic or therapeutic effect of another therapy.

[0122] The choice of dosing regimen for a therapeutic agent depends on several factors, such as the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Thus, the amount of biologic delivered depends, in part, on the particular entity and the severity of the condition being treated. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available (e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New Engl.J.Med.344:783-792;Beniaminovitz,et al.(2000)New Engl.J.Med.342:613-619;Ghosh,et al.(2003)New Engl.J.Med.348:24-32;Lipsky,et al.(2000)New See Engl.J.Med.343:1594-1602).

[0123] If necessary, the therapeutic agent, including the engineered immunoglobulin of the present disclosure, can be incorporated into a composition that includes a solubilizing agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site. Additionally, pulmonary administration can be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. 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 WO 92 / 19244, WO 97 / 32572, WO 97 / 44013, WO 98 / 31346, and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0124] Therapeutic agents, including engineered immunoglobulins of the present disclosure, can also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and mode of administration will vary depending on the desired outcome. Selected routes of administration of antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. Parenteral administration can generally refer to modes of administration other than intestinal and topical administration by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, compositions of the present disclosure can be administered via parenteral routes, such as topical, epidermal or mucosal routes of administration, such as intranasally, orally, vaginally, rectally, sublingually or topically.

[0125] Therapeutic agents, including engineered immunoglobulins of the present disclosure, can be administered via any of the above routes using, for example, injection devices, injection pens, vials and syringes, prefilled syringes, auto-injectors, infusion pumps, patch pumps, infusion bags and needles, etc. When the molecules or fragments thereof of the present disclosure are administered in a controlled or sustained release system, pumps can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). Polymeric materials can be used to achieve controlled or sustained release of the therapeutic agents of the disclosure (see, e.g., 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 (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., (1985) Science 228:190; During et al., (1989) Ann. Neurol. 25:351; Howard et al., (1989) Ann. Neurol. 25:351; See also, U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; WO 99 / 15154; and WO 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), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymers used in sustained release formulations are inert, free of leachable impurities, storage stable, sterile, and biodegradable. Controlled or sustained release systems can be placed in close proximity to the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0126] Controlled release systems are discussed in the review by Langer (Science (1990) 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations containing one or more molecules of the present application or fragments thereof. See, e.g., U.S. Pat. No. 4,526,938; WO 91 / 05548; WO 96 / 20698; Ning et al., (1996) Radiotherapy & Oncology 39:179-189; Song et al., (1995) PDA Journal of Pharm Sci & Tech., 50:372-397; Cleek et al., (1997) Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; Lam et al., (1997) Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759-760, each of which is incorporated herein by reference in its entirety.

[0127] When the pharmaceutical composition comprising the engineered immunoglobulin of the present disclosure is administered locally, it can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, liquid, emulsion, or other form 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 non-spray topical dosage forms, a viscous to semi-solid or solid form is typically used that contains a carrier or one or more excipients compatible with topical application and, in some cases, has a dynamic viscosity greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like, which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure, if desired. Other suitable topical dosage forms include spray aerosol formulations, in which active ingredient is in some cases combined with solid or liquid inert carrier and mixed with pressurized volatile material (e.g., gaseous propellant, e.g., Freon) or packaged in squeeze bottle.If desired, moisturizer or humectant can also be added to pharmaceutical composition and dosage form.Examples of such additional components are known in the art.

[0128] When the pharmaceutical composition comprising the artificially engineered immunoglobulin of the present disclosure is administered intranasally, it can be formulated in aerosol form, spray, mist, or in the form of droplets. In particular, the prophylactic or therapeutic agent for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray dispensed from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., composed of gelatin) for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0129] Pharmaceutical compositions containing the engineered immunoglobulins of the present disclosure can also be administered periodically to patients.

[0130] In some embodiments, pharmaceutical compositions comprising the engineered immunoglobulins of the present disclosure can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Pat. No. 4,522,811; U.S. Pat. No. 5,374,548; and U.S. Pat. No. 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thus improving targeted drug delivery (see, for example, Ranade VV (1989) J.Clin.Pharmacol.29:685). Exemplary targeting moieties include folates or biotin (see, e.g., U.S. Pat. No. 5,416,016, Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman et al., (1995) FEBS Lett., 357:140; M. 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); Keinaenen & Laukkanen (1994) FEBS Lett., 346:123-6; see also Killion & Fidler (1994) Immunomethods, 4:273.

[0131] In some embodiments, the pharmaceutical compositions of the present disclosure further comprise one or more additional therapeutic agents. EXAMPLES

[0132] The engineered immunoglobulins and Fc fragments were expressed, purified and analyzed, the results of which are shown in Example 1.

[0133] Thermal stability is a crucial pharmaceutical property in the development of therapeutic antibodies. A decrease in the thermal stability of the product leads to a decrease in the stability of the product, which may, for example, increase the degree of aggregation, whereas a high thermal stability of the product will, in principle, decrease the degree of aggregation. The thermal stability of engineered immunoglobulins and their parent IgGs was compared using colorimetric measurements, for example, a differential scanning microcalorimeter (Nano DSC, TA Instrument), which detects the change in heat capacity of a protein solution upon unfolding. The results of the calorimetric measurements are shown in Example 2.

[0134] The binding affinity of engineered immunoglobulins and Fc fragments to human Fc receptors was determined. The technique used to measure binding affinity is surface plasmon resonance (SPR) spectroscopy, which is a label-free technique that allows the measurement of real-time ligand binding affinity and kinetics using relatively small amounts of membrane proteins in a native or native-like environment. Direct binding assays were performed to characterize the binding of engineered immunoglobulins to hFcγR1a, hFcγR2a, hFcγR3a(F158V), hC1q or hFcRn, the results of which are shown in Example 3. Furthermore, direct binding assays were performed to determine the effect of the described manipulations on the binding of engineered anti-CD3 immunoglobulins to the hCD3 epsilon antigen, the results of which are shown in Example 4.

[0135] Activation of Fcγ receptors (FcγR) plays an important role in ADCC. Antibodies bound to cell surface antigens interact with FcγR expressed on effector cells such as natural killer (NK) cells, neutrophils and macrophages, inducing these cells to exert cytotoxicity. To monitor whether Jurkat / FcγR cells were activated by engineered immunoglobulins, Jurkat reporter gene assay (RGA) for nuclear factor of activated T cells (NFAT) pathway was performed using Jurkat NFAT luminescent (JNL) cells and THP-1 cells. The results are shown in Example 5.

[0136] The results regarding the biophysical properties of the engineered immunoglobulins and their binding affinities to human Fc receptors and Fc effector functions are summarized in Table 9.

[0137] Example 1: Expression and purification of engineered immunoglobulins The engineered immunoglobulins that were expressed, purified and analyzed according to the procedures described below are shown in Table 3. The proteins and corresponding nucleotide sequences are listed in Table 8.

[0138] [Table 3]

[0139] [Table 4]

[0140] [Table 5]

[0141] [Table 6]

[0142] Anti-CD3 monospecific IgG1, anti-CD3 monospecific IgG4, and anti-CD3 x target A x target B trispecific IgG1 as shown above were produced in the HEK293T-17SF system. Nucleic acid sequences encoding the heavy and light chains were synthesized at Geneart (LifeTechnologies) and cloned into mammalian expression vectors using a restriction enzyme-ligation based cloning technique. Plasmids encoding the heavy and light chains were co-transfected into HEK293T cells. Briefly, for transient expression of immunoglobulins, equal amounts of light chain and each engineered heavy chain vector were co-transfected into suspension-adapted HEK293T cells using polyethylenimine ((PEI) ref. Cat. No. 24765 Polysciences, Inc.). Typically, cells in 100 ml of suspension at a density of 1-2 Mio cells / ml were transfected with DNA containing 100 μg of the engineered heavy chain-encoding expression vector and the light chain-encoding expression vector (using a HC:LC ratio of 1:1). The constructs were then generated by introducing the recombinant expression vector into the host cells and further culturing the cells for 7 days for secretion into the culture medium (HEK, serum-free medium) supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml antibiotics.

[0143] Anti-Target C monospecific antibodies were produced using Novartis' proprietary Chinese hamster ovary cell line (CHO-C8TD) manufactured expression system. Plasmids encoding the heavy and light chains were incubated at 5.0 × 10 6Live cells were transfected. The transfected cells were seeded in 20 ml of cell culture medium containing low concentration of folic acid in 125 ml shake flasks. The cells were grown in a humidified shaker incubator (50 mm diameter orbital throw) at 150 rpm, 36.5 °C and 10% CO2. Three days after transfection, MTX was added to the culture at a final concentration of 10 nM to start the selection of stable transfectants. The cells went into a selection crisis but recovered within 21 days. A vial of the selected stable pool was then frozen. For the production of engineered anti-target C immunoglobulins, a fed-batch method was used. A vial of frozen cells was thawed. After the cells were recovered from thawing, they were seeded in 100 ml of Novartis proprietary cell culture medium in a 500 ml shake flask. Cultures were grown in a humidified shaker incubator (50 mm diameter orbital throw) at 200 rpm, 36.5° C. and 10% CO2. Five days after seeding of the cultures, the growth temperature was reduced to 33° C. Novartis proprietary feed solution was added on days 3, 4, 5, 6, 7 and 10 after seeding. Cultures were harvested on day 11 after seeding. Cells were separated from the cell culture medium by centrifugation and sterile filtration.

[0144] The produced constructs were then purified from the cell-free supernatant using immunoaffinity chromatography. Protein A resin (CaptivA PrimAb™, Repligen) equilibrated with PBS buffer pH 7.4 was incubated with the filtered conditioned medium using a liquid chromatography system (Aekta pure chromatography system, GE Healthcare Life Sciences). After washing the resin with PBS (pH 7.4), the constructs were eluted with elution buffer (50 mM citrate, 90 mM NaCl, pH 2.7).

[0145] After capture, the eluted proteins were pH neutralized using 1 M Tris solution (pH 10.0) and purified using size-exclusion chromatography technology (HiPrep Superdex 200 16 / 60, GE Healthcare Life Sciences).

[0146] Finally, the engineered immunoglobulins were purified using size-exclusion chromatography technique (HiPrep Superdex 200 16 / 60, GE Healthcare Life Sciences) with PBS (pH 7.4) as equilibration and elution buffer. Finally, the purified proteins were formulated in PBS buffer (pH 7.4).

[0147] Analytical size exclusion chromatography techniques (Superdex 200 Increase 3.2 / 300 GL, GE Healthcare Life Sciences) were used to measure aggregation propensity after capture and pH neutralization steps.

[0148] The purified immunoglobulins were further analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), in which proteins are separated based on their molecular weight. Each protein was mixed with Laemmli buffer and then loaded onto a polyacrylamide gel (Biorad, 4-20% Mini-PROTEAN TGX Stain free). After running for 30 min at 200 V in Tris-glycine-SDS running buffer, the proteins contained in the gel were revealed with a stain-free compatible imager (Biorad, Gel Doc EZ). These gels are shown in Figure 2.

[0149] FIG. 2A shows some of the engineered anti-CD3 monospecific IgG1s generated under non-reducing conditions (FIG. 2A1 and FIG. 2A2): Figure 2A1

[0150] [Table 7]

[0151] Figure 2A2

[0152] [Table 8]

[0153] FIG. 2B shows some of the engineered anti-CD3 x target A x target B trispecific IgG1s generated under non-reducing conditions: Figure 2B

[0154] [Table 9]

[0155] FIG. 2C shows some of the engineered IgG1 FCs produced under non-reducing conditions: Figure 2C

[0156] [Table 10]

[0157] FIG. 2D shows a portion of the produced modified anti-CD3 monospecific IgG4 under non-reducing conditions: Figure 2D

[0158] [Table 11]

[0159] FIG. 2E shows some of the engineered anti-target C monospecific IgG1s produced under non-reducing conditions: Figure 2E

[0160] [Table 12]

[0161] The expression yield results after the two-step purification are shown in Table 4. The aggregate content after the capture step for this set of immunoglobulins is also listed in Table 4.

[0162] [Table 13]

[0163] [Table 14]

[0164] As shown by the results in Table 4, the transfer of IgA upper CH2 structural elements to the hIgG1 FC does not significantly affect either the expression yield or the aggregation tendency of these molecules. Indeed, such engineered molecules maintain both expression yield and aggregation tendency in the same range as observed for the parent hIgG1.

[0165] Furthermore, the described mutation sets are compatible with the YTE mutation sets used for half-life extension. These engineered molecules with the additional YTE mutation sets have expression yields and aggregation propensity in the same range as observed for the parent hIgG1 with the same YTE mutations.

[0166] In addition, the previous results illustrate how the transfer of IgA upper CH2 structural elements to the hIgG1 FC can be applied to different immunoglobulin formats: indeed, such engineering is translatable from monospecific to multispecific IgG1 (i.e., bispecific and trispecific) and is compatible with the techniques used to induce HC / HC pairings (i.e., "knobs-into-holes" mutation sets), allowing the generation of such engineered multispecific antibodies.

[0167] Finally, the data show that it is possible to translate such engineering with or without the YTE mutation set from IgG1 to IgG4 isotypes. Similar to the previous conclusion, such engineered molecules maintain expression yields and aggregation propensity, both in the same range as observed in the parent hIgG4. Furthermore, the described mutation set constitutes an alternative to S228P to prevent IgG4 Fab arm exchange.

[0168] Example 2: Evaluation of thermal stability of engineered immunoglobulins by differential scanning calorimetry (DSC) The thermal stability of the parent and engineered immunoglobulins was measured by calorimetry, as described below.

[0169] Calorimetry was performed in a differential scanning microcalorimeter (Nano DSC, TA Instrument or MicroCal, Malvern). The heating rate was 1° C. / min. All proteins were used at a concentration of 1 mg / ml in PBS (pH 7.4). The heat capacity and molar heat capacity of each protein were estimated by comparison with two samples containing the same buffer minus the protein. Heat capacity, molar heat capacity and melting curves were analyzed using standard procedures. Thermograms were baseline corrected and concentration normalized.

[0170] Figure 3 shows the overall thermal stability of the engineered immunoglobulins compared to the parent immunoglobulin. The data shows that transferring the IgA upper CH2 structural elements to the hIgG1 FC results in a more stable molecule with improved thermal stability over the parent immunoglobulin. Figures 3A and 3B show data obtained using a Nano DSC, TA instrument. Figures 3C and 3D show data obtained on a MicroCal, Malvern instrument.

[0171] Figure 3A shows the results of the overall thermal stability measured for the engineered anti-CD3 monospecific hIgG1. Such improvements brought about by the described engineered manipulations can be clearly observed, for example, when comparing CD3_WT and CD3_1 immunoglobulins. The beneficial effect is even more pronounced when the stabilizing manipulations are combined with the YTE mutation set. As shown when comparing CD3_WT with CD3_WT_YTE, the YTE mutation set destabilizes the immunoglobulin, but this loss in thermal stability is fully compensated by the introduction of the IgA upper CH2 structural element into the IgG1 FC. This can be seen, for example, when comparing CD3_WT_YTE with CD3_1_YTE. Interestingly, the more extensive the manipulations performed, the more the thermal stability is improved. As shown in Figure 3D, a similar CH2 thermal stabilization is observed when applying the described manipulations to the anti-target C monospecific hIgG1.

[0172] Similar observations are made when such engineering is applied to anti-CD3xTargetAxTargetB trispecific hIgG1, as shown in Figure 3B. For example, the improvement in thermal stability brought about by the described engineering can be observed when comparing CD3xTargetAxTargetB_WT and CD3xTargetAxTargetB_1 immunoglobulins.

[0173] FIG. 3C illustrates how thermostability is improved when anti-CD3 monospecific hIgG4 is engineered.

[0174] Taken together, these data show how immunoglobulin CH2 thermostability can be improved by applying Fc engineering as described in this disclosure. When protein engineering is applied to monospecific or multispecific IgG1 Fc, or other isotypes such as IgG4, such stabilizing effects can be observed with or without other mutation sets used for half-life modulation (i.e., YTE mutation set) or Fc heterodimerization (i.e., knobs-into-holes mutation set). Finally, engineered recombinant antibodies show the same CH2 thermostabilization profile regardless of whether they are produced in HEK293 or CHO expression systems.

[0175] Corresponding recombinant Fcs were generated and some of them were used to characterize their thermal stability, independently of the Fabs (Figure 4). The melting temperatures (TM) of the CH2 and CH3 domains could be determined using a MicroCal, Malvern instrument (Table 5). For example, measurements performed on IgG1_FC_1 show how strong the CH2 stabilization is due to the introduction of IgA CH2 structural elements into IgG1. In fact, the CH2 TM is improved by 13°C, increasing from 70.0°C (Ionescu et al, 2007, Contribution of variable domains to stability of humanized IgG1 monoclonal antibodies) to 83°C. Interestingly, the improvement in thermal stability is proportional to the number of IgA residues and the number of additional disulfide bonds introduced into the IgG1 FC. The more extensive the engineering, the more the thermal stability is improved.

[0176] [Table 15]

[0177] Example 3: SPR measurement for human Fc receptors The binding affinity of engineered immunoglobulins or fragments thereof to human Fc receptors was determined using surface plasmon resonance (SPR) spectroscopy. SPR is a technique commonly applied to the affinity and kinetic analysis of protein-protein, protein-peptide, protein-DNA, and protein-small molecule interactions, since it analyzes the interaction between an analyte in solution and a ligand bound to a sensor chip surface, providing a continuous readout of complex formation and dissociation.

[0178] Direct binding assays were performed to characterize the binding of engineered immunoglobulins to hFcγR1a, hFcγR2a, hFcγR3a(F158V), hC1q or hFcRn.

[0179] Kinetics and binding capacity were measured on a BIAcore® T200 instrument (GE Healthcare, Glattbrugg, Switzerland) at room temperature using proteins diluted in running buffer: 10 mM NaP, 150 mM NaCl, 0.05% Tween 20 (pH 7.6). Engineered immunoglobulins were immobilized by amine coupling using a CM5 sensor chip (Sensor Chip SA, GE Healthcare Life Sciences). Recombinant human hFcγR1a, or recombinant human hFcγR3a (F158V), or recombinant human hFcRn, or hC1q were then used as analytes.

[0180] To serve as a reference, one flow cell did not capture any immunoglobulin and was inactivated using ethanolamine. Binding data was obtained by injecting a series of analyte dilutions into the reference and measurement flow cells. A zero concentration sample (running buffer only) was included to allow double referencing in the data evaluation. For data evaluation, the double-referenced sensorgrams were analyzed and the maximum response reached during the experiment was monitored. The maximum response represents the binding capacity of the surface in terms of the response at saturation. Finally, the measured maximum response was normalized to the maximum response measured with the parent immunoglobulin (not engineered). The affinity (KD) for hFcRn at pH 5.8 was determined for the anti-target C monospecific antibody. The results are shown in Table 6.

[0181] [Table 16]

[0182] The data show that introduction of IgA upper CH2 structural elements into IgG1 FC (monospecific or multispecific) reduces binding to gamma receptors (e.g., FCγR1a, FCγR2a, FCγR3a, C1q) while retaining adequate binding to FcRn.

[0183] The use of the YTE mutation set for half-life extension remains compatible with the stabilization procedures described, and indeed, increased levels of binding to FcRn could be demonstrated with immunoglobulins carrying the IgA upper CH2 structural element in combination with the YTE mutation set.

[0184] Example 4: SPR measurements for human CD3 epsilon To determine the effect of the described manipulations on the binding of engineered anti-CD3 immunoglobulins to the hCD3 epsilon antigen, a direct binding assay was performed.

[0185] Kinetic binding affinity constants (KD) were measured on a BIAcore® T200 instrument (GE Healthcare, Glattbrugg, Switzerland) at room temperature using proteins diluted in running buffer: 10 mM NaP, 150 mM NaCl, 0.05% Tween 20 (pH 7.6). A CM5 sensor chip (Sensor Chip SA, GE Healthcare Life Sciences) was used to immobilize the hCD3 epsilon-FC antigen by amine coupling. Engineered anti-CD3 hIgG1 was then used as the analyte.

[0186] To serve as a reference, one flow cell did not capture any antigen and was inactivated using ethanolamine. Binding data were obtained by injecting a series of analyte dilutions into the reference and measurement flow cells. A zero concentration sample (running buffer only) was included to allow double referencing in the data evaluation. For data evaluation, the double-referenced sensorgrams were analyzed by applying a 1:1 binding model analysis to generate equilibrium dissociation constants (KD). Since the immunoglobulins used as analytes are bivalent with respect to the immobilized antigen, the binding constants listed in Table 7 are considered as apparent KDs. In addition, the maximum response reached during the experiment was monitored. The maximum response represents the binding capacity of the surface with respect to the response at saturation. Finally, the measured maximum response was normalized to the maximum response measured with the parent immunoglobulin (not engineered). The results shown in Table 7 show that all engineered immunoglobulins bind to the hCD3 epsilon antigen similarly to their parent anti-CD3 IgG1 (CD3_WT).

[0187] [Table 17]

[0188] These results indicate that introduction of IgA upper CH2 structural elements into IgG1 Fc does not affect its antigen recognition by Fab.

[0189] Example 5: Anti-CD3 NFAT signaling assay Jurkat reporter gene assay (RGA) for nuclear factor of activated T cells (NFAT) pathway was performed using Jurkat NFAT luminescent (JNL) cells and THP-1 cells (ATCC, TIB202). THP-1 cells expressing gamma receptors FcγRI, FcγRII and FcγRIII were pretreated with 100u / mL IFNg at 37°C, 5% CO2 for 48 hours before co-culture. Cells were co-incubated with each sample at various concentrations as indicated at 37°C, 5% CO2, target to effector cell ratio of 10:1 for 6 hours. An equal volume of ONE-Glo™ reagent (Promega, E6120) was added to the culture medium. Plates were shaken for 2 minutes and then incubated for an additional 8 minutes protected from light. Luciferase activity was quantified on a Biotek Synergy HT plate reader. Data were analyzed and fitted to a four-parameter logistic curve using GraphPad Prism. NFAT activity translates directly to the ability of the tested immunoglobulins to crosslink Jurkat and THP-1 cells. Finally, such activity correlates with the ability of the tested immunoglobulins to bind to gamma receptors exposed on the THP-1 cell membrane. The stronger the activity, the higher the affinity.

[0190] Figures 5A1, 5A2 and 5A3 show the results obtained using engineered anti-CD3 monospecific hIgG1 in separate assays (first assay: Figure 5A1, second assay: Figure 5A2, third assay: Figure 5A3). In summary, the parent (CD3_WT) and the corresponding half-life extended variant (CD3_WT_YTE) showed maximal NFAT activity, whereas all engineered immunoglobulins showed a strongly suppressed NFAT activation.

[0191] Figure 5B shows the results obtained with engineered anti-CD3 x target A x target B trispecific hIgG1. In summary, the parent (CD3 x target A x target B_WT) showed maximal NFAT activity, whereas all engineered immunoglobulins showed greatly suppressed NFAT activation.

[0192] Figure 5C shows the results obtained with engineered anti-CD3 monospecific hIgG4. In summary, the parent (IgG4_WT or IgG4_CD3_S228P) and the corresponding half-life extended variants (IgG4_CD3_WT_YTE or IgG4_CD3_S228P_YTE) showed maximal NFAT activity, whereas all engineered immunoglobulins showed a strongly suppressed NFAT activation.

[0193] Taken together, these results show that stabilization of IgG1 immunoglobulins by introduction of IgA upper CH2 structural elements significantly reduces interaction with gamma receptors. In agreement with previous SPR measurements (against FcγRI, FcγRII, and FcγRIII) shown in Example 4, this cell-based assay confirms the surprising silencing effect of such stabilization manipulations. Interestingly, some variants are measured to have a silencing effect at least as strong as that observed by introduction of the DANAPA mutation set used as a benchmark.

[0194] [Table 18]

[0195] [Table 19]

[0196] [Table 20]

[0197] [Table 21]

[0198]

Table 22

[0199]

Table 23

[0200]

Table 24

[0201]

Table 25

[0202]

Table 26

[0203]

Table 27

[0204]

Table 28

[0205]

Table 29

[0206]

Table 30

[0207]

Table 31

[0208]

Table 32

[0209]

Table 33

[0210]

Table 34

[0211]

Table 35

[0212]

Table 36

[0213]

Table 37

[0214]

Table 38

[0215]

Table 39

[0216]

Table 40

[0217]

Table 41

[0218]

Table 42

[0219]

Table 43

[0220]

Table 44

[0221]

Table 45

[0222]

Table 46

[0223]

Table 47

[0224]

Table 48

[0225]

Table 49

[0226]

Table 50

[0227]

Table 51

[0228]

Table 52

[0229]

Table 53

[0230]

Table 54

Claims

1. 1. An engineered immunoglobulin or fragment thereof comprising an Fc variant of a wild-type human IgG Fc polypeptide and one or more antigen-binding domains, wherein said Fc variant exhibits reduced effector function compared to said wild-type human IgG Fc polypeptide, and wherein said Fc variant comprises a cysteine ​​substitution at position 235, and wherein amino acid residues are numbered according to EU numbering.

2. 2. The engineered immunoglobulin or fragment thereof of claim 1, further comprising one or more amino acid substitutions in the Fc variant that enhance the half-life of the engineered immunoglobulin or fragment thereof by enhancing FcRn binding, and / or one or more amino acid substitutions that promote correct chain pairing of two different Fc chains.

3. 3. The engineered immunoglobulin or fragment thereof of claim 2, wherein the half-life extending / FcRn binding enhancing amino acid substitutions are selected from the group consisting of the following mutation sets: M252Y / S254T / T256E (YTE), M428L / N434S (LS), and T250Q / M428L (QL), and T307Q / N434A (QA).

4. 4. The engineered immunoglobulin or fragment thereof of claim 3, wherein the half-life extending / FcRn binding enhancing amino acid substitutions are M252Y / S254T / T256E (YTE).

5. 2. The artificially engineered immunoglobulin or fragment thereof of claim 1, which is a human IgG1 antibody, a human IgG2 antibody, a human IgG3 antibody, or a human IgG4 antibody.

6. 2. The artificially engineered immunoglobulin or fragment thereof of claim 1, which is a human IgG1 antibody.

7. 2. The artificially engineered immunoglobulin or fragment thereof of claim 1, which is a multispecific binding molecule comprising chain-pairing amino acid substitutions selected from the group consisting of knob-into-hole (KiH), SEED body, RF mutation, DEKK mutation, electrostatic steering mutation, and Fab arm exchange.

8. 8. The engineered immunoglobulin or fragment thereof of claim 7, wherein the chain-pairing amino acid substitutions are knob-into-hole (KiH) mutations, and the multispecific binding molecule comprises a first constant heavy chain with the amino acid substitutions S354C and T366W and a second constant heavy chain with the amino acid substitutions Y349C, T366S, L368A and Y407V, wherein the amino acid residues are numbered according to EU numbering.

9. 9. The engineered immunoglobulin or fragment thereof of claim 8, wherein the multispecific binding molecule further comprises M252Y / S254T / T256E (YTE).

10. 10. A pharmaceutical composition comprising the engineered immunoglobulin or fragment thereof of claim 1 in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

11. 11. The pharmaceutical composition of claim 10, further comprising one or more additional active agents.

12. 2. An isolated nucleic acid molecule encoding the engineered immunoglobulin or fragment thereof of claim 1.

13. 13. A cloning or expression vector comprising one or more nucleic acid sequences according to claim 12, said cloning or expression vector being suitable for the recombinant production of an engineered immunoglobulin or fragment thereof according to claim 1.

14. 14. A recombinant host cell comprising one or more cloning or expression vectors according to claim 13.

15. 15. A method for preparing an artificially engineered immunoglobulin or fragment thereof of claim 1, comprising culturing the host cell of claim 14; purifying and recovering the artificially engineered immunoglobulin or fragment thereof from the host cell culture; and formulating the artificially engineered immunoglobulin or fragment thereof into a pharmaceutically acceptable composition.

16. 10. The artificially engineered immunoglobulin or fragment thereof of claim 1 for use as a medicament.

Images