Effectorless igg1 fc variants

Abstract

The present invention relates to IgG1 Fc variants which do not elicit antibody effector functions, such as CDC, ADCP and ADCC. In particular, the present invention relates to IgG1 Fc variant polypeptides which comprise the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, molecules comprising such IgG1 Fc variant polypeptides and uses of such molecules to reduce antibody effector function.

Description

[0001] EFFECTORLESS IGG1 FC VARIANTS

[0002] The present invention relates to IgGl Fc variants, molecules comprising the same, use of such IgGl variants to decrease antibody effector functions and method to decrease antibody effector functions by introducing mutations into the IgGl Fc.

[0003] 1. BACKGROUND OF THE INVENTION

[0004] Monoclonal antibodies as well as Fc fusion proteins are very promising biopharmaceutical agents and the market for these compounds has increased significantly within the recent years. Reasons for the success of these molecules are their high specificity, safety and long half-lives in the circulation compared to small chemical compounds.

[0005] Antibodies are composed of two different protein chains, a light chain (LC) and a heavy chain (HC). One LC is covalently linked to one HC and the HCs are on their parts covalently linked by disulfide bonds. The LC is composed of a variable domain (VL) and a constant domain (CL), whereas the HC consist of a variable domain (VH) and several constant domains (e.g., CHI, CH2 and CH3 domains for IgG).

[0006] Antibodies are multifunctional proteins. Functionally, an antibody can be divided into two Fab (Fragment antigen binding) regions and one Fc (Fragment crystallizable) region. Each Fab region consists of four domains: VL, CL, VH and CHI. The Fc region comprises the remaining constant domains (CH2 and CH3 domains for IgG). While antigens are bound by Fab regions, effector functions of antibodies are mediated by the Fc region. The effector functions of antibodies include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). These effects are mediated via binding of the antibody Fc region to Fc gamma receptors (FcyRs) and the complement protein Clq, respectively. These antibody effector functions are important for the efficacy of natural antibodies as well as some therapeutic antibodies, e.g., in oncology. On the other hand, the effector functions are undesired for many other engineered therapeutic antibodies, e.g. antibodies with anti-inflammatory effects (such as antibodies blocking pro- inflammatory molecules). Such antibodies are often designed to act mainly or only by blocking their specific targets via binding of the Fab fragment, while any effector function of the Fc region elicits an interfering and potentially harmful immune reaction, at the extreme a “cytokine storm”. On the other hand, the Fc region markedly prolongs half-life of antibodies and Fc fusion proteins in the circulation via a “recycling” mechanism mediated by the neonatal Fc-receptor (FcRn). Moreover, the Fc region contributes to the overall stability of the molecule and another important and desired function of the Fc-domain is its strong and specific binding to protein A. Binding of antibodies and Fc fusion proteins to immobilized protein A is usually the first purification step during the purification of antibodies and Fc fusion proteins. Accordingly, a simple removal of the Fc region from the antibody molecule would have severe drawbacks.

[0007] Therefore, several variants Fc regions have been developed to reduce antibody effector functions while maintaining the positive effects of the Fc region (half-life prolongation, protein A binding, stabilization). These approaches include the aglycosylated variants and variants with point mutations (Wang et al., Protein Cell. 2018;9(l):63-73).

[0008] A common variant with reduced effector function is the so-called IgGl LALA variant, which contains the two mutations L234A and L235A. However, the IgGl LALA variant shows some residual effector functions, in particular residual CDC, which can be undesirable for certain applications. Further mutations have been introduced in the IgGl LALA variant to abolish those residual effector functions, e.g. the additional mutations P329G and P329A (Schlothauer et al., Protein Eng Des Sei. 2016 Oct;29(10):457-466). While it was reported that the introduction of P329G in IgGl LALA abolishes the antibody effector functions completely, P329A was linked to some residual effector functions (Schlothauer et al., Protein Eng Des Sei. 2016 Oct;29(10):457-466).

[0009] As different conditions can require different backbones, e.g. in case of anti-drug-antibodies against particular parts of the antibody backbone, there is the need for IgGl Fc variants which have as least as little residual effector functions as IgGl LALA P329G.

[0010] The present invention meets this need by providing the IgGl LALA GASS variant, which comprises the mutations L234A, L235A, A327G, P329A, A330S, and P331S, and the use of this variant to reduce antibody effector functions.

[0011] 2. SUMMARY OF THE INVENTION

[0012] The present invention provides an IgGl Fc variant polypeptide, wherein said IgGl Fc variant polypeptide comprises the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, wherein the IgGl Fc variant polypeptide does not comprise SEQ ID NO:28.

[0013] In some embodiments, the IgGl Fc variant polypeptide does not consist of SEQ ID NO:31.

[0014] In some embodiments, the IgGl Fc variant polypeptide is a human IgGl Fc variant polypeptide.

[0015] In some embodiments, the IgGl Fc variant polypeptide does not consist of SEQ ID NO:31 and is a human IgGl Fc variant polypeptide

[0016] In some embodiments, the IgGl Fc variant polypeptide comprises SEQ ID NO:31.

[0017] The present invention also provides a molecule comprising two IgGl Fc variant polypeptides according to any of the previous embodiments described before.

[0018] In some embodiments, said two IgGl Fc variant polypeptides are identical or are different.

[0019] In some embodiments, the molecule has not more antibody effector functions compared to the identical molecule comprising, instead of the IgGl Fc variant polypeptides of any of the previous embodiments, two IgGl Fc variant polypeptides comprising the mutations L234A, L235A and P329G.

[0020] In some embodiments, the molecule is an antibody.

[0021] The present invention also provides the use of an IgGl Fc variant polypeptide, comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, to reduce one or more antibody effector functions.

[0022] The present invention also provides the use of a molecule which comprises two IgGl Fc variant polypeptides, comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, to reduce one or more antibody effector functions.

[0023] In some embodiments, one or more antibody effector functions are reduced compared to an identical IgGl Fc variant polypeptide comprising the mutations L234A and L235, but not comprising the mutations A327G, P329A, A330S, and P331S.

[0024] In some embodiments, the reduction of one or more antibody effector functions comprises the reduction of one or more of complement dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC).

[0025] In some embodiments, the reduction of one or more antibody effector functions comprises the reduction of CDC, ADCP, and ADCC. In some embodiments, the IgGl Fc variant polypeptide or the molecule displays no antibody effector function as measured by the CDC, ADCP, and ADCC assays as described in examples 2-5.

[0026] The present invention also provides a method of reducing one or more antibody effector functions of a molecule which comprises an IgGl Fc polypeptide, comprising introducing the mutations L234A, L235A, A327G, P329A, A330S, and P331S into said IgGl Fc polypeptide.

[0027] In some embodiments, said one or more reduced antibody effector functions comprise one or more of CDC, ADCP, and ADCC.

[0028] 3. SHORT DESCRIPTION OF THE FIGURES

[0029] Figure 1: Sequences of the CH2 domains of IgGl, IgG4 and IgGl LALA GASS. The figure shows an alignment of the CH2 domain sequences of IgGl wildtype (wt), IgG4 wt and an IgGl Fc variant of the present invention (named “IgGl LALA GASS”) which comprises the mutations L234A, L235A, A327G, P329A, A330S, and P331S. The numbering is according to the EU numbering system (see, e.g., Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78- 85, 1969). Highlighted are the mutations L234A, L235A (in IgGl LALA GASS) as well as the amino acids forming the CH2-FG-loop (in IgGl wt and IgG4 wt) and the mutations A327G, P329A, A330S, and P331 S (in IgGl LALA GASS).

[0030] Figure 2: CDC assay for the IgGl Fc variants LALA N297A and LALA P329A. CDC was determined by measuring specific lysis conferred by anti-TNF antibodies comprising different IgGl Fc variants. Plotted is the specific lysis (in %) against the employed antibody concentration (ng / mL, logarithmic scale). Tested were Abl (IgGl wt, circle symbols), Ab7.1 (IgGl LALA N297A, square symbols) and Ab7.2 (IgGl LALA P329A, triangle symbols).

[0031] Figure 3: CDC assay for IgGl LALA GASS and other IgGl / IgG4 variants. CDC of the IgGl Fc variant LALA GASS (see also Fig. 1) and other IgGl or IgG4 Fc variants was measured in a cytotoxicity assay. Cell viability (in %) is plotted against antibody concentration (nM, logarithmic scale). Tested were the anti-TNF antibodies Abl (IgGl wt), Ab2 (IgGl LALA), Ab3 (IgG4 PE), Ab4 (IgG4 P-FALA), Ab5 (IgGl NNAS), Ab6 (IgGl LALA P329G), and Ab8 (IgGl LALA GASS), as well as three negative controls of antibodies not binding to TNF (Ab9 (anti-HER2 IgGl wt), Ab 16 (NIST IgGl wt), anti-lysozyme IgGl wt antibody). Dashed lines indicate determination of an EC50 value.

[0032] Figure 4: High-sensitivity ADCP assay for IgGl LALA GASS and other IgGl / IgG4 variants. ADCP of the IgGl Fc variant LALA GASS (see also Fig. 1) and other IgGl or IgG4 Fc variants was measured in two luciferase reporter assays. Determined was activation via FcyRIIa Hl 31 (A) and FcyRI (B), fold luciferase induction is plotted against antibody concentration (nM, logarithmic scale). Tested were the anti-TNF antibodies Abl (IgGl wt), Ab2 (IgGl LALA), Ab3 (IgG4 PE), Ab4 (IgG4 P-FALA), Ab5 (IgGl NNAS), Ab6 (IgGl LALA P329G), and Ab8 (IgGl LALA GASS), as well as three negative controls of antibodies not binding to TNF (Ab9 (anti-HER2 IgGl wt), Ab 16 (NIST IgGl wt), anti-lysozyme IgGl wt antibody). Dashed lines indicate determination of an EC50 value.

[0033] Figure 5: High-sensitivity ADCC assay for IgGl Fc variant LALA GASS and other IgGl / IgG4 Fc variants. ADCC of the IgGl Fc variant LALA GASS (see also Fig. 1) and other IgGl or IgG4 Fc variants was measured in a luciferase reporter assay. Determined was activation via FcyRIIIa VI 58, fold luciferase induction is plotted against antibody concentration (nM, logarithmic scale). Tested were the anti-TNF antibodies Abl (IgGl wt), Ab2 (IgGl LALA), Ab3 (IgG4 PE), Ab4 (IgG4 P-FALA), Ab5 (IgGl NNAS), Ab6 (IgGl LALA P329G), and Ab8 (IgGl LALA GASS), as well as three negative controls of antibodies not binding to TNF (Ab9 (anti-HER2 IgGl wt), Ab 16 (NIST IgGl wt), anti-lysozyme IgGl wt antibody). Dashed lines indicate determination of an EC50 value.

[0034] Figure 6: Pharmacokinetics of IgGl LALA GASS. Pharmacokinetics of antibodies comprising the IgGl Fc variant LALA GASS were compared to the same antibodies comprising instead IgGl wt or the IgGl Fc variant LALA. Antibody concentration (ng / mL) in the blood of mice is plotted against time after injection. Fig. 6A shows the results for the anti-TNF antibodies Abl (IgGl wt, triangle), Ab2 (IgGl LALA, diamonds), and Ab8 (IgGl LALA GASS, circles). Fig. 6B shows the results for the anti-HER2 antibodies Ab9 (IgGl wt, triangle), Ab 10 (IgGl LALA, diamonds), and Abl5 (IgGl LALA GASS, circles). 4. DESCRIPTION OF THE INVENTION

[0035] The present invention provides IgGl Fc variant polypeptides comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S, molecules comprising said IgGl Fc variant polypeptides and their use to reduce antibody effector functions.

[0036] Advantages of the present invention

[0037] Compared to known IgGl Fc variants, like the IgGl Fc variant LAL A, the IgGl Fc variants of the present invention have the advantage that they do completely abolish antibody effector functions, i.e. they do not elicit CDC, ADCP or ADCC.

[0038] This lack of antibody effector functions can be partially contributed to the mutation at position 329. Position 329 is located in the FG-loop of the immunoglobulin fold of the CH2 domain. The proline at position 329 has been shown to be crucial for the interaction of the Fc with FcyRs and for the binding to complement factor Clq which provides an explanation for reduced antibody effector functions for variants which harbor mutations at position 329. However, not all mutations at position 329 have the same effect. While it was reported that the introduction of P329G in IgGl LALA abolishes the antibody effector functions completely, P329A was linked to some residual effector functions (Schlothauer et al., Protein Eng Des Sei. 2016 Oct;29(10):457-466). The present invention overcomes those shortcomings of the P329A Fc variant by introducing the additional mutations A327G, A330S, and P331S in the IgGl Fc variant LALA. The resulting IgGl Fc variant “LALA GASS” showed no residual effector functions.

[0039] The IgGl Fc variants of the present invention provide further advantageous properties. In contrast to IgGl Fc variants which harbor single mutations in the FG loop (like P329G and P329A), IgGl Fc variants of the present invention are considered to be less prone to immunogenicity because their FG-loop resembles the FG-loop of the natural occurring IgG4.

[0040] Furthermore, the IgGl Fc variants of the present invention comprise IgGl Fc regions. Compared to IgG4 Fc regions, IgGl has advantages regarding conformational and colloidal stability, making IgGl the most popular IgGl Fc region for therapeutic antibodies.

[0041] Moreover, it has been shown that molecules comprising the IgGl Fc variants of the present invention show no reduction in pharmacokinetics compared to IgGl wt or the IgGl Fc variant LALA. Thus, the present invention provides novel stable IgGl Fc variants, and molecules comprising the same, with no residual antibody effector functions and good pharmacokinetics.

[0042] 4.1 Definitions

[0043] The term “IgGl Fc” or “IgGl Fc region”, as used herein interchangeably, is the constant region of an IgGl antibody, but excluding CHI and CL domains. Thus, IgGl Fc is a protein complex which is formed by portions of two IgGl heavy chain polypeptides and consists of two CH2 domains, two CH3 domains and the hinge region between CHI and CH2 domains. The hinge region comprises disulfide bridges which link the two IgGl heavy chain polypeptides. Although the exact boundaries of the Fc region may vary, typical boundaries for the human IgGl Fc region are from residue C226 to the C-terminus of the IgGl heavy chain or from P230 to the C-terminus of the IgGl heavy chain, wherein the numbering is according to the EU numbering. “IgGl Fc” or “IgGl Fc region” may refer to the IgGl Fc region in isolation, or in the context of a molecule comprising the Fc, for example an antibody.

[0044] The term “IgGl Fc polypeptide”, as used herein, is a polypeptide which is capable of forming an IgGl Fc, together with another IgGl Fc polypeptide. The two IgGl Fc polypeptides forming an IgGl Fc can be either identical or different. An IgGl Fc polypeptide comprises a CH2 domain, a CH3 domain and an amino acid sequence which can form a hinge region. An IgGl Fc polypeptide can additionally comprise other domains or moieties.

[0045] An “IgGl wildtype Fc polypeptide” or “IgGl wt Fc polypeptide” is a IgGl Fc polypeptide comprising the naturally occurring sequence of SEQ ID NO:30. The term “wildtype” is used correspondingly in other terms, for instance an “IgGl wildtype Fc” is an IgGl Fc formed by two IgGl Fc polypeptides each comprising the sequence of SEQ ID NO:30.

[0046] An “IgGl Fc variant polypeptide” is an IgGl Fc polypeptide which comprises an amino acid sequence which differs from that of the sequence of the IgGl wildtype (wt) Fc polypeptide (SEQ ID NO:30) by virtue of at least one amino acid substitution, also called “mutation”. The terms “amino acid” substitution and “mutation” are used herein interchangeably. Fig. 1 shows how an IgGl Fc variant polypeptide of the present invention (IgGl LALA GASS) is aligned with IgGl wt Fc polypeptide to determine the number of mutations (6 mutations in the case of Fig. 1).

[0047] It should be noted that the IgGl Fc variant polypeptides of the present invention share higher sequence identity with IgGl wt Fc polypeptide than with other IgG wt Fc polypeptide, i.e. the IgGl Fc variant polypeptide of the present invention has higher sequence identity to the IgGl wt Fc polypeptide of SEQ ID NO:30 than to the IgG2 wt Fc polypeptide, the IgG3 wt Fc polypeptide and the IgG4 wt Fc polypeptide. When the IgGl wt Fc polypeptide of the present invention is a human IgGl wt Fc polypeptide, it has higher sequence identity to the IgGl wt Fc polypeptide of SEQ ID NO:30 than to the human IgG2 wt Fc polypeptide, the human IgG3 wt Fc polypeptide and the human IgG4 wt Fc polypeptide.

[0048] The term "antibody effector function" or "effector function" as used herein refers to a function contributed by an Fc region of an antibody, such as IgG. Such function can be effected by, for example, binding of an Fc region to an Fc receptor on an immune cell with phagocytic or cytolytic activity or by binding of an Fc region to components of the complement system. Antibody effector functions are ADCC, ADCP and CDC.

[0049] The term "antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only. FCR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.

[0050] The term "antibody-dependent cellular phagocytosis" (ADCP) refers to a process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region. Unlike antibody-dependent cell-mediated cytotoxicity (ADCC), which is mediated primarily through FcyRIIIa expressed on NK cells, ADCP can be mediated by monocytes, macrophages, neutrophil and dendritic cells mainly via FcyRIIa (CD32a) and FcyRI (CD64).

[0051] The term “complement-dependent cytotoxicity” (CDC) to refers to an immune response in which target cells are lysed through activation and recruitment of the complement cascade to the targeted cell surface. CDC is mediated by binding of the protein Clq to the antibody Fc region. Clq together with two serine proteases, Clr and Cis, forms the complex Cl, the first component of the complement dependent cytotoxicity (CDC) pathway.

[0052] Use of an IgGl Fc variant polypeptide “to reduce” antibody effector functions means that said IgGl Fc variant polypeptide is incorporated in a molecule and the molecule is exposed to a setting where antibody effector functions can potentially occur. The use is achieved if the antibody effector functions are reduced or completely abolished for the molecule comprising said IgGl Fc variant polypeptide, compared to the same molecule comprising instead an IgGl wt Fc polypeptide or an IgGl Fc variant LALA polypeptide.

[0053] Use of a molecule comprising an IgGl Fc variant polypeptide “to reduce” antibody effector functions, as used herein, means that said molecule is exposed to a setting where antibody effector functions can potentially occur. The use is achieved if the antibody effector functions are reduced or completely abolished for the said molecule comprising an IgGl Fc variant polypeptide, compared to the same molecule comprising instead an IgGl wt Fc polypeptide or an IgGl Fc variant LALA polypeptide. Said reduction of antibody effector function can be determined using the assays described in this disclosure as examples 2-5.

[0054] Throughout this application, the numbering of antibodies or antibody domains is according to EU numbering as established in Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85, 1969, unless indicated otherwise. To provide a reference to other numbering systems, the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, are L247A, L248A, A346G, P348A, A349S, P350S, according to Kabat numbering.

[0055] The expression “does not consist of SEQ ID NO:31” means that the IgGl Fc variant polypeptide is not just SEQ ID NO:31, without comprising any other amino acids. This feature does not mean that the IgGl Fc variant polypeptide does not comprise SEQ ID:31 as part of its sequence, provided that the IgGl Fc variant polypeptide further comprise additional amino acids.

[0056] For sake of brevity, the designations “IgGl Fc variant LALA”, “IgGl Fc LALA” and “IgGl LALA” (and analog designations for other IgG Fc variants, e.g. “IgGl Fc variant LALA GASS’TIgGl Fc LALA GASS’TIgGl LALA GASS”) are used interchangeably throughout this application. Similarly, “IgGl wt” is used interchangeably with “IgGl wt Fc region”.

[0057] 4.2 Embodiments of the present invention

[0058] 4.2.1 Mutations in the IgGl Fc variant polypeptides

[0059] The present invention provides IgGl Fc variant polypeptides and molecules comprising said IgGl Fc variant polypeptides. The IgGl Fc variant polypeptides comprise mutations compared to the IgGl wt Fc polypeptide (SEQ ID NO:30). Those mutations comprise the 6 mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering. The IgGl Fc variant polypeptide of the present invention can comprise further mutations. In some embodiments, the IgGl Fc variant polypeptide comprises from 6 to 15 mutations compared to the IgGl wt Fc region polypeptide. In some embodiments, the IgGl Fc variant polypeptide comprises from 6 to 12 mutations compared to the IgGl wt Fc region polypeptide. In some embodiments, the IgGl Fc variant polypeptide comprises from 6 to 10 mutations compared to the IgGl wt Fc region polypeptide. In some embodiments, the IgGl Fc variant polypeptide comprises 6 or 7 mutations compared to the IgGl wt Fc region polypeptide. In some embodiments, the IgGl Fc variant polypeptide comprises 6 mutations compared to the IgGl wt Fc region polypeptide.

[0060] In some embodiments, the IgGl Fc variant polypeptide possess at least about 90% sequence identify, at least about 95% sequence identity or at least about 97% sequence identity with the IgGl wt Fc polypeptide of SEQ ID NO:30.

[0061] The IgGl Fc variant polypeptides of the present invention do not comprise SEQ ID NO:28, which is the heavy chain of a Fab fragment disclosed in W02022003169.

[0062] As SEQ ID NO:28 is a subsequence of SEQ ID NO:29, the IgGl Fc variant polypeptides of the present invention do also not comprise SEQ ID NO:29 which is a heavy chain disclosed in W02022003169.

[0063] In some embodiments, the IgGl Fc variant polypeptide does not consist of SEQ ID NO:31. In some embodiments, the IgGl Fc variant polypeptide comprises SEQ ID NO:31 and comprises one or more additional amino acids.

[0064] In some embodiments, the IgGl Fc variant polypeptide is a human IgGl Fc variant polypeptide, meaning that it comprises a variant of the human IgGl wt Fc sequence (SEQ ID NO:30). In some embodiment, the IgGl Fc variant polypeptide is a rabbit IgGl Fc variant polypeptide, a guinea pig IgGl Fc variant polypeptide, or a monkey IgGl Fc variant polypeptide.

[0065] 4.2.2 Further domains / moieties of the IgGl Fc variant polypeptides

[0066] In some embodiments, the IgGl Fc variant polypeptide further comprises one or more other domains or moieties, in addition to the CH2 domain, the CH3 domain, and the amino acid sequence which can form a hinge region. In some embodiments, the IgGl Fc variant polypeptide further comprises a CHI domain. In some embodiments, the IgGl Fc variant polypeptide further comprises a CHI and a VH domain. In some embodiments, the IgGl Fc variant polypeptide further comprises a CHI and a VH domain linked to a VL and CL domain, forming a single-chain fragment variable (scFv). In some embodiments, the IgGl Fc variant polypeptide further comprises a CHI domain and two VH domains. In some embodiments, the IgGl Fc variant polypeptide further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV. In some embodiments, the IgGl Fc variant polypeptide further comprises an immunoglobulin single variable domain (ISVD). In some embodiments, the IgGl Fc variant polypeptide further comprises a VHH. In some embodiments, the IgGl Fc variant polypeptide further comprises a Nanobody® ISVD. In some embodiments, the IgGl Fc variant polypeptide further comprises 2 Nanobody® ISVDs. In some embodiments, the IgGl Fc variant polypeptide further comprises 3 Nanobody® ISVDs. In some embodiments, the IgGl Fc variant polypeptide further comprises a VNAR.

[0067] The term “immunoglobulin single variable domain” (ISVD), interchangeably used with “single variable domain”, defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab’, F(ab’)2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulfide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

[0068] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0069] As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL- sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). An immunoglobulin single variable domain (ISVD) can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH. In one embodiment, it is a VHH, including a camelized VH or humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.

[0070] For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® ISVD; other single variable domains, or any suitable fragment of any one thereof.

[0071] In particular, the immunoglobulin single variable domain may be a Nanobody® ISVD (such as a VHH, including a humanized VHH or camelized VH) or a suitable fragment thereof. (Note: Nanobody® is a registered trademark of Ablynx N. V.)

[0072] In some embodiments, the IgGl Fc variant polypeptide comprises an antigen-binding moiety on its N-terminus and an antigen-binding moiety on its C-terminus. In some embodiments, both antigen-binding moieties are Fab fragments. In some embodiments, one antigen-binding moiety is a Fab fragment and the other antigen binding moiety is an scFv. In some embodiments, one antigen-binding moiety is a Fab fragment and the other antigen binding moiety is an ISVD. In some embodiments, both antigen binding moieties are scFvs. In some embodiments, one antigen-binding moiety is an scFv and the other antigen binding moiety is an ISVD. In some embodiments, both antigen binding moieties are ISVDs.

[0073] In some embodiments, the IgGl Fc variant comprises or is fused to another moiety. In some embodiments, the moiety is a molecule which exerts a biological function by interacting with a biological structure (e.g. a receptor for said molecule). In some embodiments, the moiety is a peptide. In some embodiments, the moiety is a receptor agonist. In some embodiments, the moiety is a receptor antagonist. In some embodiments, the moiety is an enzyme. In some embodiments, the moiety is a cytokine. In some embodiments, the moiety is a chemokine. In some embodiments, the moiety is an interferon. In some embodiments, the moiety is an interleukin. In some embodiments, the moiety is a lymphokine. In some embodiments, the moiety is a tumor necrosis factor. In some embodiments, the moiety is a toxin. In some embodiments, the moiety is hormone. In some embodiments, the moiety is a growth factor. In some embodiments, the moiety is GLP1 or insulin.

[0074] 4.2.3 Molecules of the present invention

[0075] The present invention provides molecules which comprise two IgGl Fc variant polypeptides, at least one of them comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331 S, according to EU numbering, wherein the IgGl Fc variant polypeptide does not comprise SEQ ID NO:28.

[0076] In some embodiments, the molecule comprises one IgGl Fc variant polypeptide and one IgGl wt Fc polypeptide. The IgGl Fc variant polypeptide can be any of the IgGl Fc variant polypeptides peptides described above, i.e. of the IgGl Fc variant polypeptides comprising the 6 mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, wherein the polypeptide, wherein the IgGl Fc variant polypeptide does not comprise SEQ ID NO:28. The IgGl wt Fc polypeptide is a polypeptide comprising SEQ ID NO:30.

[0077] In some embodiments, the molecule comprises two IgGl Fc variant polypeptides. In some embodiments, both IgGl Fc variant polypeptides are IgGl Fc variant polypeptides comprising the 6 mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, wherein the IgGl Fc variant polypeptides do not comprise SEQ ID NO:28. The two IgGl Fc variant polypeptides can be any of the IgGl Fc variant polypeptides peptides described above. The two IgGl Fc variant polypeptides can be identical or can be different. In some embodiments, the two IgGl Fc variant polypeptides are identical. In some embodiments, the two IgGl Fc variant polypeptides are different.

[0078] In some embodiments, the two IgGl Fc variant polypeptides are identical and both further comprise one or more other domains or moieties, in addition to the CH2 domain, the CH3 domain, and the amino acid sequence which can form a hinge region. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a CHI domain. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a CHI and a VH domain. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a CHI and a VH domain linked to a VL and CL domain, forming an scFv. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a CHI domain and two VH domains. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a CHI domain and two VH domains, forming the heavy chain of a CODV. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise an immunoglobulin single variable domain (ISVD). In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a VHH. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a Nanobody® ISVD. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise 2 Nanobody® ISVDs. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise 3 Nanobody® ISVDs. In some embodiments, the two IgGl Fc variant polypeptides are identical and further comprise a VNAR.

[0079] In some embodiments, the two IgGl Fc variant polypeptides are different and one or both of them further comprise one or more other domains or moieties, in addition to the CH2 domain, the CH3 domain, and the amino acid sequence which can form a hinge region.

[0080] In some embodiments, the two IgGl Fc variant polypeptides are different and both further comprise a CHI domain. In some embodiments, the two IgGl Fc variant polypeptides are different and both further comprise a CHI and a VH domain, wherein those two VH domains are different. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other further comprises a CHI and a VH domain linked to a VL and CL domain, forming an scFv. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other further comprises an ISVD. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other further comprises a VHH. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other further comprises an Nanobody® ISVDs. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other further comprises a VNAR. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other does not comprise a antigen-binding moiety. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI and a VH domain while the other consists of SEQ ID NO:31.

[0081] In some embodiments, the two IgGl Fc variant polypeptides are different and both further comprise an scFv, wherein those two scFvs are different. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other further comprises an ISVD. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other further comprises a VHH. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other further comprises an Nanobody® ISVD. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other further comprises a VNAR. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other does not comprise a antigen-binding moiety. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an scFv while the other consists of SEQ ID NO:31.

[0082] In some embodiments, the two IgGl Fc variant polypeptides are different and both further comprise a CHI domain and two VH domains, forming the heavy chain of a CODV, wherein those two CODV heavy chains are different. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV, while the other further comprises an ISVD. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV, while the other further comprises a VHH. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV, while the other further comprises an Nanobody® ISVD. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV, while the other further comprises a VNAR. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV, while the other does not comprise a antigen-binding moiety. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises a CHI domain and two VH domains, forming the heavy chain of a CODV, while the other consists of SEQ ID NO:31.

[0083] In some embodiments, the two IgGl Fc variant polypeptides are different and both further comprise an ISVD, wherein those two ISVDs are different. In some embodiments, one or two of those ISVDs are VHHs. In some embodiment, one or two of those ISVDs are Nanobody® ISVDs. In some embodiments, one or two of those ISVDs are VNARs. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an ISVD while the other does not comprise a antigen-binding moiety. In some embodiments, the two IgGl Fc variant polypeptides are different and one further comprises an ISVD while the other consists of SEQ ID N0:31.

[0084] 4.2.4 Antibodies

[0085] In some embodiments, the molecule of the present invention is an antibody. This means that the molecule comprises at least one antigen-binding moiety, such as a Fab, a scFv or an ISVD, e.g. a VHH, a Nanobody® ISVD or a VNAR. An antibody of the present invention comprises two IgGl Fc variant polypeptide, wherein one or both IgGl Fc variant polypeptide comprises the mutations L234A, L235A, A327G, P329A, A330S, and P33 IS, wherein the IgGl Fc variant polypeptide does not comprise SEQ ID NO:28.

[0086] In some embodiments, an antibody of the present invention has the same polypeptide chain architecture as a wt IgGl, i.e. it consists of two heavy chains and two light chains. The two heavy chains are formed by the two IgGl Fc variant polypeptides, wherein both IgGl Fc variant polypeptides comprise a VH domain and a CHI domain located N-terminally from the portion of the polypeptide which form the Fc region (hinge, CH2, CH3).

[0087] In some embodiments, an antibody of the present invention has the same polypeptide chain architecture as a wt IgGl which lacks one of its Fab fragment, i.e. it consists of two heavy chains and one light chains. The two heavy chains are formed by the two IgGl Fc variant polypeptides, wherein one IgGl Fc variant polypeptide comprises a VH domain and a CHI domain located N-terminally from the portion of the polypeptide which form the Fc region (hinge, CH2, CH3), while the other IgGl Fc variant polypeptide does not comprise a VH domain or CHI domain, but consists e.g. of SEQ ID NO:31.

[0088] 4.2.5 Reduction of antibody effector functions

[0089] The IgGl Fc variants and molecules of the present invention have reduced antibody effector functions compared to molecules comprising IgGl wt Fc or IgGl Fc variant LALA, instead of an IgGl Fc variant of the present inventions. This means that one or more of the antibody effector functions CDC, ADCP and ADCC are reduced. In some embodiments, CDC, ADCP and ADCC are reduced.

[0090] In some embodiments, the molecules of the present invention have also reduced antibody effector functions compared to molecules comprising the IgGl Fc variant LALA P329A, instead of an IgGl Fc variant of the present inventions. This means that one or more of the antibody effector functions CDC, ADCP and ADCC are reduced. In some embodiments, CDC is reduced. In some embodiments, CDC, ADCP and ADCC are reduced.

[0091] In some embodiments, the molecules of the present invention have not more antibody effector functions compared to molecules comprising the IgGl Fc variant LALA P329G, instead of an IgGl Fc variant of the present inventions. This means that all antibody effector functions CDC, ADCP and ADCC are either the same as for the IgGl Fc variant LALA P329G or reduced. In some embodiments, CDC, ADCP and ADCC are the same. In some embodiments, one or more of CDC, ADCP and ADCC are reduced. In some embodiments, CDC, ADCP and ADCC are reduced.

[0092] Whether reduction of one or more antibody effector functions occurs can be determined according to the assays described in example 2 and 3 for CDC, example 4 for ADCP and example 5 for ADCC.

[0093] 4.2.6 Use for reduction of antibody effector functions

[0094] The present invention provides the use of an IgGl Fc variant polypeptide comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S, or a molecule comprising said Igl Fc variant polypeptide, to reduce one or more antibody effector functions. In such a use of the present invention, antibody effector functions are reduced compared to molecules comprising IgGl wt Fc, instead of an IgGl Fc variant comprising the mutations L234A, L235 A, A327G, P329A, A330S, and P331S. In some embodiments, antibody effector functions are also reduced compared to molecules comprising the IgGl Fc variant LALA, instead of an IgGl Fc variant comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S.

[0095] In some embodiments the use comprises the reduction of one or more of CDC, ADCP, and ADCC. In some embodiments the use comprises the reduction of CDC, ADCP, and ADCC. In some embodiments the use comprises the reduction of CDC and ADCP. In some embodiments the use comprises the reduction of CDC and ADCC. In some embodiments the use comprises the reduction of ADCP, and ADCC. In some embodiments the use comprises the reduction of CDC. In some embodiments the use comprises the reduction of ADCP. In some embodiments the use comprises the reduction of ADCC.

[0096] Whether reduction of one or more antibody effector functions occurs can be determined according to the assays described in example 2 and 3 for CDC, example 4 for ADCP and example 5 for ADCC.

[0097] 4.2. 7 Methods of reducing one or more antibody effector functions The present invention provides methods of reducing one or more antibody effector functions of a molecule which comprises an IgGl Fc polypeptide, comprising introducing the mutations L234A, L235A, A327G, P329A, A330S, and P331S into said IgGl Fc polypeptide. The introduction of the mutations can be done by modifying a nucleic acid encoding the IgGl Fc polypeptide using commonly known methods. Reduction of antibody effector functions means that antibody effector functions are reduced compared to the molecules without the mutations L234A, L235A, A327G, P329A, A330S, and P331S, i.e. having the IgGl wt amino acids at the respective positions.

[0098] In some embodiments the reduction comprises the reduction of one or more of CDC, ADCP, and ADCC. In some embodiments the use comprises the reduction of CDC, ADCP, and ADCC. In some embodiments the use comprises the reduction of CDC and ADCP. In some embodiments the use comprises the reduction of CDC and ADCC. In some embodiments the use comprises the reduction of ADCP, and ADCC. In some embodiments the use comprises the reduction of CDC. In some embodiments the use comprises the reduction of ADCP. In some embodiments the use comprises the reduction of ADCC.

[0099] Whether reduction of one or more antibody effector functions occurs can be determined according to the assays described in example 2 and 3 for CDC, example 4 for ADCP and example 5 for ADCC.

[0100] 4.2.8 Polynucleotides, vectors, cell

[0101] In another aspect, the present invention relates to one or more polynucleotides encoding the IgGl Fc variant polypeptides and molecules of the present invention. This refers to all embodiments described above. The one or more polynucleotides may also encode the one or more additional polypeptides comprised in a complex with the molecule of the present invention. In one embodiment, the one or more polynucleotides encode an antibody or antibody-like structure. In one embodiment, the one or more polynucleotides are isolated.

[0102] In another aspect, the present invention relates to one or more expression vectors comprising the one or more polynucleotides according to the previous aspect of the invention. The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term "vector" includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been fused. One type of vector is a "plasmid," which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes they comprise. Such vectors are referred to herein as "expression vectors".

[0103] In another aspect, the present invention relates to a cell comprising the one or more polynucleotides or the one more expression vectors according to the previous aspects of the invention. A wide variety of cell expression systems can be used to express said polynucleotides including the use of prokaryotic and eukaryotic cells, such as bacterial cells (e.g. E. coli), yeast cells, insect cells or mammalian cells (e.g. mouse cells, rat cells, human cells etc.). For this purpose, a cell is transformed or transfected with said polynucleotide(s) or expression vector(s) such that the polynucleotide(s) of the invention are expressed in the cell and, in one embodiment, secreted into the medium in which the cells are cultured, from where the expression product can be recovered.

[0104] 5. EXAMPLES

[0105] Molecules of the present invention comprise an IgGl Fc variant comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering. Those mutations are depicted in Fig. 1, the respective IgGl Fc variant is named “LALA GASS”. In the following examples, molecules comprising the IgGl Fc variant LALA GASS were produced, characterized and compared to IgGl wt and other IgG Fc variants.

[0106] Example 1: Protein expression., purification and characterization

[0107] Protein expression

[0108] Antibodies were expressed in transiently transfected HEK293 cells. DNA coding for the different variants were cloned into an expression vector under a CMV promoter and a leader sequence directing the proteins into the culture supernatant. For expression in HEK293 cells sequence SEQ ID 001 was used as leader sequence. For large scale expression, cells were grown in non-baffled shake flasks (Corning) at 110 rpm, 37°C and 8% CO2 in Freestyle F17 medium (Gibco) supplemented with 6 mM glutamine and 0.02 % Pluronic F-68 (Gibco). At the time of transfection, the cell density was approximately 1.2 x 106cells / mL. Cell densities were determined with an automated cell counter (Nucleocounter NC-200, Chemometec, Allerod, Denmark). Before transfection, DNA was mixed with linear polyethyleneimine (PEI) at a ratio of 1 :3 in Opti-MEM I-medium (Thermo Fisher Scientific). The transfection mixture was further incubated 10 min at room temperature and then added to the cell cultures. Cultivation was continued for 6 days. For large scale purification, cells were separated from the supernatants by centrifugation and the cell pellets were discarded. The 0.22 pm filtered supernatant was loaded on a protein A column (Mab Select Sure resin, GE Healthcare) equilibrated in phosphate buffered saline (PBS, Gibco). Mabs were eluted with 0.1 M citrate buffer, pH 3.0. Subsequently buffer was exchanged to PBS (Gibco) on a Sephadex G25 desalting column (GE Healthcare). Further purification was done using a Superdex 200 gel filtration column (GE healthcare) equilibrated in 10 mM histidine, 150 mM NaCl, pH 6.0. Corresponding fractions were pooled, concentrated, and stored until usage at 4°C or -80°C.

[0109] Table 1 provides an overview of the generated antibody variants and their respective sequences.

[0110] Table 1 : Produced antibody variants.

[0111]

[0112] Protein characterization

[0113] Analytical size-exclusion chromatography (SEC)

[0114] Analytical SEC was performed using a BioSECcurity instrument (PSS Polymer) with an AdvanceBio 300 column (4.6 mm x 300 mm) and an AdvanceBio 300 guard column (Agilent Technologies). The analysis was performed at 25 °C with a flow rate of 0.5 ml / min using 2x concentrated D-PBS buffer (Thermo Fisher Scientific) with detection at 280 nm after loading lOpl of protein sample (at 1 mg / ml) onto the column. Data evaluation was performed using Chromeleon 7 software (Thermo Fisher Scientific). For estimation of the molecular weight, the SEC column was calibrated with the AdvanceBio SEC 300 A Protein Standard (Agilent Technologies). Purity was calculated by dividing the area of the main peak by the combined area of all peaks.

[0115] Capillary gel electrophoresis (cGE)

[0116] The cGE was conducted according to the Protein Clear HR Assay guide provided by Perkin Elmer. Shortly: To prepare the chip, all assay components were allowed to equilibrate to room temperature. Protein Clear HR Gel matrix was partly mixed with Protein Clear HR Dye solution and filtered before adding to rinsed chip wells according to the manufacturer’s instructions. For assay calibration the provided assay control VeriMAb was diluted in non-reducing sample buffer, denatured (70°C for 10 min) and subsequently diluted with water. The indicated volumes of VeriMAb assay control, Protein Clear HR Ladder and wash buffer were transferred into corresponding tubes and placed in LabChip GXII Touch instrument (Perkin Elmer). Calibration process was finished successfully before sample measurement The samples were normalized to 0.5 mg / mL in water and 5 pL of each sample were added to 18 pL of non-reducing (8.75 rnM fc. lodoacetamide) and reducing sample buffer (50 rnM fc. Dithiothreitol) in a PCR plate. The samples were denatured at 70°C for 10 min on a Thermostat C Thermoblock and subsequently diluted with 35 pL water before it was measured with the LabChip GXII Touch instrument (Perkin Elmer). Data analysis was performed using the LabChip Reviewer Software V5.8.84.0 (Perkin Elmer). All peaks with >0.85% of relative peak area were integrated. Purity was calculated by dividing the area of the main peak by the combined area of all peaks.

[0117] Table 2: Characterization of the antibodies comprising IgGFc variants.

[0118] The antibodies Ab 10, Ab 14, and Ab 15 were further analyzed to evaluate antibody target binding and stability.

[0119] Binding affinity determination

[0120] Binding affinities and kinetics were measured by Surface plasmon resonance (SPR) on a Biacore T200 instrument (Cytiva). For the affinity capture of the anti-HER2 protein samples, an anti-human Fc antibody (human antibody capture kit, Cytiva) was immobilized on a series S CM5 sensor chip (Cytiva) to approximately 10,000 RU. The anti-HER2 antibodies were diluted into HBS-EP+ assay buffer (Cytiva) to 0.15 pg / mL and injected in the sample flow cell (90 seconds, 10 pL / min), whereas the reference flow cell was used without captured antibody. After Fc affinity capture, the human HER2 antigen (Mat no 10004-H08H, Sino Biological) diluted into HBS-EP+ buffer, was injected in a 1 :2 dilution series from 0.19 nM to 100 nM over the reference and sample flow cells. For the antigen inject the flow rate was adjusted to 30 pL / min with an association time of 240 seconds and a dissociation time of 1200 seconds. At the end of each cycle an inject of regeneration solution (3M MgCh) was performed for 1 min at 30 pL / min in order to remove antibody and antigen from the chip surface. Binding kinetics data were evaluated with a 1 : 1 binding model using the Biacore 8K Evaluation Software version (Cytiva).

[0121] Accelerated stress stability

[0122] To monitor changes in the chemical stability of the antibody samples, accelerated stress stability studies were performed. For the accelerated stress stability, the samples were buffer exchanged at room temperature by dialysis into either 10 mM sodium acetate buffer pH 5.0, or 10 mM sodium phosphate buffer pH 8.0 using a robotic system (BRAVO, Agilent). The concentration of the buffer exchanged samples was adjusted to 1 mg / mL and the samples were incubated for up to 21 days at 40 °C in an incubator. Control samples and samples after stress in the respective buffers were frozen at -80 °C before the analyses.

[0123] Determination of active fraction (see below) and SEC analysis (see above) were performed before (DO) and after (D21) exposure of the molecules to accelerated stress conditions.

[0124] Active fraction determination

[0125] Analysis of the active fraction of stressed antibodies was performed by SPR using the same Fc capture assay set up as described above for the affinity determination. Here, the association and dissociation times for human HER2 (Mat no 10004-H08H, Sino Biological) were set to 240 and 300 seconds, respectively. The active fraction of the samples was calculated as the ratio of binding signal for human HER2 and the Fc capture signal of the antibody, normalized to the active fraction of the unstressed control samples. Mean values from three experiments were calculated. The active fraction was determined at before (DO) and after (D21) the exposure of the molecules to accelerated stress conditions.

[0126] Table 3: Binding and stability of antibodies comprising IgG Fc variants.

[0127]

[0128] Example 2: CDC assay of IgGl Fc variants with additional mutations

[0129] It is known that the IgGl Fc variant LALA comprises some residual antibody effector functions. Thus, it was tested in a CDC assay whether the additional mutations N297A and P329A can completely abolish those effector functions. For this purpose, anti-TNF antibodies comprising IgGl Fc variant LALA N297A (Ab7.1) or IgGl Fc variant LALA P329A (Ab7.2) were tested in a complement dependent cytotoxicity assay as described in example 3 below. An anti-TNF antibody with IgGl wt (Abl) served as positive control.

[0130] In the CDC assay, IgGl wt caused lysis of up to around 50%, IgGl Fc variant LALA N297A resulted in lysis of up to 20%, while IgGl Fc variant LALA P329A led to a lysis of around 10% (Fig. 2B). This indicates that the IgGl Fc variants LALA N297A and LALA P329A possess still substantial residual CDC activity.

[0131] Example 3: Complement-dependent cytotoxicity (CPC) assay

[0132] IgG Fc variants were tested to identify Fc variants without residual effector functions. For this purpose, CDC of the IgGl Fc variants of the present invention was evaluated in the following cytotoxicity assay in comparison to other IgG Fc variants. Cytotoxicity assays measure the ability of cytotoxic compounds to cause cell damage or cell death. As in example 2, anti-TNF antibodies comprising the respective IgG Fc variants were used.

[0133] For this CDC assay 3xl03engineered CH0-K1 target cells expressing membrane-bound human TNF per well (CHO-TNF clone MC2-1B10 with mutation resulting in the deletion of amino acids 77 to 88 of wild-type human pro-TNF) were plated in 96-well white / clear flat bottom plates. After an incubation step for 24 hours at 37°C, 5% CO2 and 95% rH the target cells were treated with test antibodies in triplicate wells from 187.5 nM to 0.011 nM concentration in eight serial 1:4 dilution steps and addition of 25 pl / per well Guinea Pig serum complement (Millipore; #234395-5ml) equilibrated with Milli-Q water as described in the manufacturer’s instructions. Incubation was done for 2 hours at 37°C, 5% CO2 and 95% rH followed by an additional 24 hours incubation step at 37°C in 5% CO2 incubator with addition of 7.5 pl per well Cell Proliferation Reagent WST-1 (Roche; #05 015 944 001). The samples were measured in a Tecan Spark luminescence microplate reader for quantification of cellular viability.

[0134] EC50 and the Emax values of the compounds were calculated by applying a Biostat-Speed statistical calculation tool (BIOST@T-SPEED-LTS2.6.1). Results were obtained using the 4- parameter logistic model according to Ratkovsky and Reedy (1986, Biometrics, Vol. 42: 575- 582). The adjustment was obtained by non-linear regression using the Levenberg-Marquardt algorithm in SAS v9.4 under linux software.

[0135] Results

[0136] Abl (IgGl wt) and Ab2 (IgGl Fc variant LALA) were the only active molecules in this CDC assay (Table 7, Fig. 5). Ab 1 showed the highest activity (lower EC50, lower Emax in viability corresponding to higher cell killing). All other tested molecules were fully inactive in this assay.

[0137] Table 4: Results of the CDC assay. Example 4: Antibody-dependent cellular phagocytosis (ADCP) assays

[0138] ADCP of the IgGl Fc variants of the present invention was evaluated in two assays, one based on FcyRIIa binding, the other based on FcyRI binding. ADCP was determined in comparison to other IgG Fc variants. As in example 2 and 3, anti-TNF antibodies comprising the respective IgG Fc variants were used.

[0139] For the high sensitivity ADCP FcyRIIa assay IxlO4engineered CH0-K1 target cells expressing membrane-bound human TNF (CHO-TNF clone MC2-1B10 with mutation resulting in the deletion of amino acids 77 to 88 of wild-type human pro-TNF) per well were plated in 96-well white / clear flat bottom plates. After an incubation step for 24 hours at 37°C, 5% CO2 and 95% rH the target cells were treated with test antibodies in triplicate wells from 125 nM to 0.49 nM concentration in nine serial 1 :2 dilution steps and then co-cultured with 7.5xlO4NFAT-luc2 lurkat effector cells per well overexpressing the human FcyRIIa Hl 31 high sensitivity variant (Promega; #G9871). The ratio of effector versus target cells was 7.5 : 1. An additional incubation step was done for 24 hours at 37°C, 5% CO2 and 95% rH followed by readout using the Bio- Glo Luciferase Assay System (Promega; #G4790) and a Tecan Spark luminescence microplate reader. Assay buffers and medium were applied according to manufacturer’s instructions.

[0140] For the ADCP FcyRI assay NFAT-luc2 urkat effector cells overexpressing the human FcyRI were used (Promega; #GA1341) instead of the overexpressing the human FcyRIIa NFAT-luc2 urkat effector cells. The cells were treated with test antibodies in triplicate wells from 125 nM to 0.02 nM concentration in nine serial 1 :3 dilution steps. Same effector versus target cell ratio, incubation and readout conditions were applied as described for the ADCP FcyRIIa assay.

[0141] EC50 and the Emax values of the compounds were calculated by applying a Biostat-Speed statistical calculation tool (BIOST@T-SPEED-LTS2.6.1). Results were obtained using the 4- parameter logistic model according to Ratkovsky and Reedy (1986, Biometrics, Vol. 42: 575- 582). The adjustment was obtained by non-linear regression using the Levenberg-Marquardt algorithm in SAS v9.4 under linux software.

[0142] Results

[0143] In the ADCP FcyRIIa assay, Abl (IgGl wt), Ab2 (IgGl Fc variant LALA), Ab3 (IgG4 Fc variant PE), and Ab4 (IgG4 Fc variant P-FALA) showed similar EC50 data (Table 5, Fig. 4A). Emax was highest for Abl and Ab3, and slightly reduced for Ab2 and Ab4. All other tested molecules were fully inactive in this assay.

[0144] Table 5: Results of the high sensitivity ADCP assay for FcyRIIa.

[0145]

[0146] In the ADCP FcyRI assay, Abl (IgGl wt), Ab2 (IgGl Fc variant LALA), Ab3 (IgG4 Fc variant PE), and Ab4 (IgG4 Fc variant P-FALA) and Ab5 (IgGl Fc variant NNAS) showed similar EC50 data (Table 6, Fig. 4B). Compared to Abl, the variants Ab2, Ab3, and Ab4 showed a slightly reduced Emax, while Emax of Ab5 was strongly reduced. All other tested molecules were fully inactive in this assay. This means that only the IgGl Fc variants LALA P329G and LALA GASS were fully silent regarding FcyRI binding in this assay.

[0147] Table 6: Results of the ADCP assay for FcyRI.

[0148] To sum it up, ADCP activity of IgGl Fc variant LALA and the two IgG4 Fc variants were only slightly reduced compared to IgGl wt. IgGl Fc variant NNAS showed weak residual activity in the FcyRI assay and no activity in the FcyRIIa assay. No ADCP activity was detected for the IgGl Fc variants LALA P329G and LALA GASS.

[0149] Example 5: High-sensitivity antibody-dependent cellular cytotoxicity (ADCC) assay

[0150] ADCC of the IgGl Fc variants of the present invention was evaluated in a high-sensitive ADCC assay in comparison to other IgG Fc variants. As in examples 2-4, anti-TNF antibodies comprising the respective IgG Fc variants were used.

[0151] The ADCC Reporter Bioassay was used which is a bioluminescent reporter assay for quantifying biological activity on pathway activation by therapeutic antibody drugs in an ADCC mechanism of action (MOA) assay. For this ADCC assay IxlO4engineered CH0-K1 target cells expressing membrane-bound human TNF (CHO-TNF clone MC2-1B10 with mutation resulting in the deletion of amino acids 77 to 88 of wild-type human pro-TNF) per well were plated in 96-well white / clear flat bottom plates. After an incubation step for 24 hours at 37°C, 5% CO2 and 95% rH the target cells were treated with test antibodies in triplicate wells from 250 nM to 0.04 nM concentration in nine serial 1 :3 dilution steps and then co-cultured with 7.5xl04NFAT-luc2 Jurkat effector cells per well overexpressing the human FcyRIIIa V158 high sensitivity variant (Promega; #G7102). The ratio of effector versus target cells was 7.5:1. An additional incubation step was done for 24 hours at 37°C, 5% CO2 and 95% rH followed by readout using the Bio-Gio Luciferase Assay System (Promega; #G4790) and a Tecan Spark luminescence microplate reader. Assay buffers and medium were applied according to manufacturer’s instructions.

[0152] EC50 and the Emax values of the compounds were calculated by applying a Biostat-Speed statistical calculation tool (BIOST@T-SPEED-LTS2.6.1). Results were obtained using the 4- parameter logistic model according to Ratkovsky and Reedy (1986, Biometrics, Vol. 42: 575- 582). The adjustment was obtained by non-linear regression using the Levenberg-Marquardt algorithm in SAS v9.4 under linux software. Results

[0153] The anti-TNF antibodies with gGl wt and with IgGl Fc variant LALA were the only active molecules in this ADCC assay (Table 4, Fig. 3). Abl (IgGl wt) showed much higher ADCC activity than Ab2 (IgGl Fc variant LALA). All other tested molecules were fully inactive in this assay.

[0154] Table 7: Results of the high sensitivity ADCC assay.

[0155] Example 6: summary of antibody effector functions

[0156] The summarized results of the CDC assay (example 3), the ADCP FcyRIIa assay (example 4), the ADCP FcyRI assay (example 4) and the ADCC FcyRIIIa assay (example 5) are shown in the table below. The compounds are only classified as “active” (+) or “not-active” (-), corresponding to the results in the respective assays.

[0157] It was found that IgGl LALA showed substantial residual ADCC, ADCP, and CDC activity. The IgG4 Fc variants PE and P-FALA display some residual ADCP activity. For IgGl Fc variant NNAS, weak residual activity was detected in the ADCP FcyRI assay. Only IgGl Fc variant LALA P329G and IgGl Fc variant LALA GASS did not confer any antibody effector function in any assay.

[0158] Table 8: Combined results of ADCC, ADCP, and CDC assays.

[0159] Example 7: Pharmacokinetics

[0160] In Vivo Pharmacokinetics

[0161] An in vivo pharmacokinetics (PK) study was performed using treatment naive 8-10 week old male hFcRn Tg32 homozygous mice (The Jackson Laboratory, #014565 MOI). The PK study was conducted at Charles River Labs (CRL) Worcester, MA, USA and was designed and executed within accordance of the Animal Use Protocol (AUP) and adherence to CRLs institutional animal care and use committee (IACUC) regulations. At time=0hrs all treatment groups (groups 1-6, 3 animals per group) were administered with a single intravenous dose of 5 mg / kg (Img / mL solution and dosing volume of 5 mL / kg) of respective molecules. Serial blood samples were collected in tubes containing K2EDTA and processed to plasma at regular timepoints over a period of 504 hrs starting at 0.25 hrs post dose, with an additional terminal sample at 672 hrs for each animal to harvest plasma. Quantitative analysis of hlgG in plasma samples was performed using a targeted LC-MS / MS approach as described below. PK parameter estimates were determined from mean animal data per group using noncompartmental analysis in WinNonlin (Phoenix version 8.2) using the plasma data module with linear up log down interpolation. Concentration values below the limit of quantitation (BLQ) were excluded from PK calculations.

[0162] Bioanalytical methods

[0163] For the quantification of targeted proteins, the samples were first immunoprecipitated using streptavidin beads and biotinylated antibody to capture the target analyte and subsequently analyzed using trypsin digestion-LC-MS / MS method by measuring surrogate peptides. For immunoprecipitation, 25 pL plasma sample was incubated with 10 pL of goat anti-human IgG biotinylated antibody and 10 pL of streptavidin binding magnetic beads in 350 pL of IxPBST for 2 hours. At the end of incubation period the beads were washed twice with IxPBST and once with IX PBS before eluting in 100 pL of 25mM HCL for 15 mins. Digestion was performed overnight at 37 °C and 750 RPM on a Thermo mixer by the addition of 10 pL of trypsin (100 pg / mL) in presence of 25% acetonitrile in TRIS buffer containing 5 mM CaCh. The digestion was stopped by adding 10 pL of 10% formic acid in water.

[0164] Sciex QTRAP 6500+ was used for analysis of surrogate peptides from digestion samples. Chromatographic separation of peptides was carried out on an Ascentis® Express Peptide ES- C18, (75x2.1 mm, 2.7um) column using a gradient elution with the mobile phase A (0.1% formic acid in deionized water) and B (98: 2 Acetonitrile: DMSO) as follows: 0-0.5min 5% B; 0.5-2.5min 5%-40% B; 2.5-2.6 min 40%-90% B; 2.6-3. Omin 90% B; 3.0-3.1 90%-5% B; 3.1- 4.5min 5% B and stop at 4.5 min. The total running time was 4.5 minutes with an injection volume of 75 pL. Mass transitions of 593.8 / 699.4 (Abl (IgGl wt)), 593.8 / 418.2 (Ab2 (IgGl LALA)), 593.8 / 418.2 (Ab8 (IgGl LALA-GASS)), 593.8 / 699.4 (Ab9 (IgGl wt)), 593.8 / 846.5 (AblO (IgGl LALA)), and 593.8 / 418.2 (Abl5 (IgGl LALA-GASS)) were used to monitor surrogate peptides with a linear range 20.0-10240 ng / mL.

[0165] The pharmacokinetics of antibodies comprising the IgGl Fc variant LALA GASS did not differ from antibodies comprising the IgGl wt or the IgGl Fc variant LALA.

[0166] References

[0167] Wang et al., Protein Cell. 2018;9(l):63-73

[0168] Schlothauer et al., Protein Eng Des Sei. 2016 Oct;29(10):457-466

[0169] Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85, 1969

[0170] Ravetch and Kinet Annu. Rev. Immunol 9 (1991) 457-492 Sequences

[0171] SEQ ID NO:1 (Signal sequence, UniProt P01749, Ighvl-61, HVM05 MOUSE, Aa 1-19)

[0172] MGWSCIILFLVATATGVHS

[0173] SEQ ID NO:2 (anti-TNFa, light chain)

[0174] DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGV

[0175] PSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFI

[0176] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

[0177] LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0178] SEQ ID NO:3(anti-Her2, light chain)

[0179] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG

[0180] VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFI

[0181] FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS

[0182] LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0183] SEQ ID NO:4 (NIST, light chain)

[0184] DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGV

[0185] PSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIF

[0186] PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

[0187] S STLTLSKADYEKHKVYACEVTHQGLS SP VTKSFNRGEC

[0188] SEQ ID NO:5 (anti-TNFa-huIgGl, heavy chain)

[0189] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0190] GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0191] SEQ ID NO: 6 (anti-TNFa-huIgGl (LAL A), heavy chain)

[0192] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS

[0193] GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0194] GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TI

[0195] SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

[0196] TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0197] SEQ ID NO: 7 (anti-TNFa-huIgG4(PE), heavy chain)

[0198] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0199] GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0200] SEQ ID NO:8 (anti-TNFa-huIgG4(P-FALA), heavy chain)

[0201] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0202] GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0203] SEQ ID NO: 9 (anti-TNFa-huIgGl(NNAS), heavy chain)

[0204] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0205] GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0206] SEQ ID NO: 10 (anti-TNFa-huIgGl(LALA) P329G, heavy chain)

[0207] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0208] GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0209] GVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<ALGAPIEI<TI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT

[0210] TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0211] SEQ ID NO: 11 (anti-TNFa-huIgGl(LALA-GASS), heavy chain)

[0212] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0213] GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0214] SEQ ID NO: 12 (anti-HER2-huIgGl, heavy chain)

[0215] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

[0216] KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0217] SEQ ID NO: 13 (anti-HER2-huIgGl(LALA), heavy chain)

[0218] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

[0219] AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0220] SEQ ID NO: 14 (anti-HER2-huIgG4(PE), heavy chain)

[0221] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0222] DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO: 15 (anti-HER2-huIgG4(P-FALA), heavy chain)

[0223] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0224] DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0225] SEQ ID NO: 16 (anti-HER2-huIgGl(NNAS), heavy chain)

[0226] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

[0227] KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0228] SEQ ID NO: 17 (anti-HER2-huIgGl(LALA) P329G, heavy chain)

[0229] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALGAPIEI<TISI<

[0230] AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0231] SEQ ID NO: 18 (anti-HER2-huIgGl(LALA-GASS), heavy chain)

[0232] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISK

[0233] AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 19 ((NIST8671)-huIgGl, heavy chain)

[0234] QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDD KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ

[0235] GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0236] SEQ ID NO:20 ((NIST8671)-huIgGl(LALA), heavy chain)

[0237] QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDD KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ

[0238] GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0239] SEQ ID NO:21 ((NIST8671)-huIgG4(PE), heavy chain)

[0240] Q VTLRESGPALVKPTQTLTLTCTF SGF SLSTAGMS VGWIRQPPGKALEWLADIWWDD KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0241] SEQ ID NO:22 ((NIST8671)-huIgG4(P-FALA), heavy chain)

[0242] QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDD

[0243] KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ

[0244] GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0245] HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP

[0246] CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH

[0247] NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ

[0248] PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0249] DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0250] SEQ ID NO:23 ((NIST8671)-huIgGl(NNAS), heavy chain) QVTLRESGPALVKPTQTLTLTCTF SGF SLSTAGMS VGWIRQPPGKALEWLADIWWDD KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0251] SEQ ID NO:24 ((NIST8671)-huIgGl(LALA) P329G, heavy chain)

[0252] Q VTLRESGPALVKPTQTLTLTCTF SGF SLSTAGMS VGWIRQPPGKALEWLADIWWDD KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0253] SEQ ID NO:25 ((NIST8671)-huIgGl(LALA-GASS), heavy chain)

[0254] Q VTLRESGPALVKPTQTLTLTCTF SGF SLSTAGMS VGWIRQPPGKALEWLADIWWDD KKHYNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP AVLQS SGLYSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0255] SEQ ID NO: 26 (anti-TNFa-huIgGl(LALA) N297A, heavy chain)

[0256] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW

[0257] GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0258] SEQ ID NO: 27 (anti-TNFa-huIgGl(LALA) P329A, heavy chain)

[0259] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNS

[0260] GHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFP AVLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALAAPIEI<TI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0261] SEQ ID NO:28 (anti-FGFGl-16H7 VH-CH1)

[0262] QVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMGVSWIRQPPGKALEWLAHIFSND EKSYSTSLKSRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGGYYYDGMDV

[0263] WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV

[0264] SEQ ID NO: 29 (anti-FGFRl-16H7 huIgGl(LALA) GASS, heavy chain)

[0265] QVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMGVSWIRQPPGKALEWLAHIFSND EKSYSTSLKSRLTISKDTSKSQVVLIMTNMDPVDTATYYCARSVVTGGYYYDGMDV

[0266] WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

[0267] DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0268] SEQ ID NO: 30 (IgGl wt Fc)

[0269] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK

[0270] FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G

[0271] SEQ ID NO: 31 (huIgGl(LALA) GASS Fc)

[0272] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

[0273] I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CT<VSNI<G

[0274] LASSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ

[0275] PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG

Claims

CLAIMS1. An IgGl Fc variant polypeptide, wherein said IgGl Fc variant polypeptide comprises the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, wherein the IgGl Fc variant polypeptide does not comprise SEQ ID NO:28.

2. The IgGl Fc variant polypeptide of claim 1, wherein the IgGl Fc variant polypeptide does not consist of SEQ ID NO:31.

3. The IgGl Fc variant polypeptide of claim 1 or 2, wherein the IgGl Fc variant polypeptide is a human IgGl Fc variant polypeptide.

4. The IgGl Fc variant polypeptide of any of claims 1-3, wherein the IgGl Fc variant polypeptide comprises SEQ ID NO:31.

5. A molecule comprising two IgGl Fc variant polypeptides according to any of claims 1- 4.

6. The molecule of claim 5, wherein said two IgGl Fc variant polypeptides are identical or are different.

7. The molecule of claim 5 or 6, wherein the molecule has not more antibody effector functions compared to the identical molecule comprising, instead of the IgGl Fc variant polypeptides of any of claims 1-4, two IgGl Fc variant polypeptides comprising the mutations L234A, L235A and P329G.

8. The molecule of any of claims 5-7, wherein the molecule is an antibody.

9. Use of an IgGl Fc variant polypeptide, comprising the mutations L234A, L235A, A327G, P329A, A330S, and P33 IS, according to EU numbering, to reduce one or more antibody effector functions.

10. Use of a molecule which comprises two IgGl Fc variant polypeptides, comprising the mutations L234A, L235A, A327G, P329A, A330S, and P331S, according to EU numbering, to reduce one or more antibody effector functions.

11. The use of claim 9 or 10, wherein one or more antibody effector functions are reduced compared to an identical IgGl Fc variant polypeptide comprising the mutations L234A and L235, but not comprising the mutations A327G, P329A, A330S, and P331S.

12. The use of any of claims 9-11, wherein the reduction of one or more antibody effector functions comprises the reduction of one or more of complement dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC).

13. The use of any of claims 9-12, wherein the reduction of one or more antibody effector functions comprises the reduction of CDC, ADCP, and ADCC.

14. The use of any of claims 9-13, wherein the IgGl Fc variant polypeptide or the molecule displays no antibody effector function as measured by the CDC, ADCP, and ADCC assays as described in examples 2-5.

15. A method of reducing one or more antibody effector functions of a molecule which comprises an IgGl Fc polypeptide, comprising introducing the mutations L234A, L235A, A327G, P329A, A330S, and P331S into said IgGl Fc polypeptide.

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