Immunoglobulin variants

Abstract

The present invention provides an IgA antibody with improved efficacy, a pharmaceutical composition, and a method for preparing the antibody. [Solution] The present invention provides an IgA2 antibody comprising an Fc variant having at least 90% identity with the amino acid sequence of a specific Fc polypeptide, wherein the antibody has increased FcαR affinity or increased antibody-dependent cell-mediated cytotoxicity compared to an IgA2 antibody comprising the Fc polypeptide of the specific sequence, and the antibody comprises an amino acid modification at a specific position.

Description

Technical Field

[0001] Sequence Listing This application contains a Sequence Listing that was submitted electronically in ASCII format and is hereby incorporated by reference in its entirety into this specification. The ASCII copy, created on April 19, 2021, is named PAT058743-WO-PCT_SL.txt and is 316 KB in size.

[0002] The present invention relates to Fc mutant polypeptide ptides and antibodies having properties optimized to promote neutrophil recruitment, and engineering methods for preparing them. The Fc mutant polypeptide ptides and antibodies may be useful in the treatment of tumors, particularly solid tumors.

Background Art

[0003] Currently, all clinically approved antibodies include the immunoglobulin IgG isotype. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism for tumor cell killing mediated by IgG antibodies that recognize and bind to Fc gamma receptors (FcγR). However, in patients with high tumor loads, recurrence may occur, and IgG antibody treatment may lose efficacy due to the immunosuppressive environment found in tumors. In the tumor micro environment, FcγR-bearing effector cells such as macrophages and natural killer cells may become refractory to lysis by immunosuppressive factors such as TGFβ. IgA engages the Fc alpha receptor (FcαRI) expressed by bone marrow effector cells, such as neutrophils and tumor resident bone marrow-derived suppressor cells (M DSC). It represents an alternative isotype for antibody therapy. IgA is second only to IgG in human serum. It is the second most abundant immunoglobulin; monomer IgA allotype (IgA1 and Ig Both A2) account for up to 25% of human serum immunoglobulins. Previously, neutrophils were generally They were not considered potential effector cells. However, neutrophils are the most important circulating white blood cells. They are also a large population and have been shown to invade solid tumors (Gregory & H oughton(2011)Cancer Res.,71:2411-16;Vogt Sionov et al.,(2015)Cancer Microenviron .,8(3):125-58;Uribe-Querol & Rosales(201 5)J.Immunol.Res.,Article ID:983698;Rosal es(2018)Front Physiol.,9:113). MDSCs are also in the myeloid lineage. It originates from and is one of the most common immunosuppressive cell types. IgA antibodies mobilize neutrophils and... It has been shown that ADCC can effectively kill tumor cells by improving performance. Unfortunately The use of IgA antibodies as therapeutic agents has several disadvantages and limitations, such as low expression rates. Production is hindered by the large quantities and expensive purification schemes. Furthermore, production is subject to heterogeneous glycosylation. IgA has multiple glycosylation sites that may be susceptible to glycan heterogeneity. The transient expression level for mer IgA is 30-70 μg / L for human IgA1. Reported (Lombana et al., (2019) MABS, 11:112) 2-38; Meyer et al., (2016) MABS, 8:87-98). [Overview of the Initiative]

[0004] Therefore, there is still a need for IgA antibodies that can be developed as therapeutic antibodies. IgA antibodies with improved efficacy mobilize neutrophils, MDSCs, and enhance ADCs. This provides a viable alternative to IgG therapeutic antibodies that have the advantage of effective tumor cell killing by C. It is possible.

[0005] The present invention has improved binding properties to FcαRI and contributes to the recruitment and activation of neutrophils. This provides an Fc variant of the parental Fc polypeptide of an IgA isotype that can be used. The Fc variants of this disclosure independently undergo amino acid insertion, amino acid deletion, and / or amino acid substitution. It may contain amino acid modifications, either by themselves or in combination with other amino acids.

[0006] In one embodiment, the present disclosure provides an Fc variant of a parent Fc polypeptide, where the Fc variant The variant exhibits modified binding to FcαR compared to the parent Fc polypeptide, or modified antibody It exhibits somatic cell-mediated cytotoxicity (ADCC), and the Fc mutant is a derivative of the parent Fc polypeptide. The Fc region contains at least one amino acid modification. In one embodiment, the amino acid modification is:C H2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99 CH3.45, CH3.105, CH3.109, CH3.118 and CH3.124 It is located in a position selected from the group consisting of, and the numbering of amino acid modifications is based on the C-domain. The IMGT numbering follows. In a preferred embodiment, the Fc variant is parent Fc polypeptide The Fc region of the molecule contains at least one amino acid modification, where the amino acid modification is: A_CH 2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2.9 9_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, Q _CH3.118_Y and L_CH3.124_F, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain. In a further embodiment the present disclosure provides an Fc variant comprising at least one amino acid modification in the Fc region, where the amino acid modifications are: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_ D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_C H2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2 .97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y .97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y , M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH 2.97_Y / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3 .105_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_ D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2 .94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, [[ID=3,1]] Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3. 118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M _CH,3.105_Y / Q_CH3.118_Y, A_CH2.10_S, L_CH2. 89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_ CH3.109_D, L_CH3.124_F, L_CH2.89_I / G_CH2.9 1_V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W, selected from the group consisting of, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain. is selected from the group consisting of, and the numbering of the amino acid modifications follows the IMGT numbering for the C-domain. follows.

[0007] In one embodiment, the present disclosure provides an Fc variant of a parental Fc polypeptide comprising amino acid modifications at positions CH2.94, CH2.97, CH3.45, C H3.105 and CH3.118. In one embodiment, the Fc variant of the parental Fc polypeptide comprises an amino acid substitution of Glu at position CH2 .94, Tyr at position CH2.97, Asp at position CH3.45 , Tyr at position CH3.105, or Tyr at position CH3.118. In a preferred embodiment, the Fc variant of the parental Fc polypeptide comprises amino acid substitutions Q_CH2.94_E, L_CH2.97_Y, S_CH3.45_D, M_CH 3.105_Y, and Q_CH3.118_Y. In one embodiment, the present disclosure provides an Fc variant of a parental Fc polypeptide, wherein the parental Fc polypeptide is contained within human IgA1 or the parental Fc polypeptide is contained within human IgA2. In one embodiment of the present disclosure, the Fc variant exhibits altered binding to FcαR compared to the parental Fc polypeptide. An Fc variant of the parental Fc polypeptide is provided herein, where the Fc variant, measured by surface plasmon resonance (SPR), binds to the parental Fc polypeptide

[0008] In one embodiment, the present disclosure provides an Fc variant of a parental Fc polypeptide, where the parental Fc polypeptide is contained within human IgA1 or the parental Fc polypeptide is contained within human IgA2. is contained. <00001l6> In one embodiment of the present disclosure, the Fc variant exhibits altered binding to FcαR compared to the parental Fc polypeptide. An Fc variant of the parental Fc polypeptide is provided herein, where the Fc variant, measured by surface plasmon resonance (SPR), binds to the parental Fc polypeptide and the Fc variant binds to FcαR with a dissociation constant (KD) of less than or equal to 10^−6 M, preferably less than or equal to 10^−7 M, more preferably less than or equal to 10^−8 M, even more preferably less than or equal to 10^−9 M, and most preferably less than or equal to 10^−10 M, as measured by surface plasmon resonance (SPR). and the Fc variant binds to FcαR with a dissociation constant (KD) of less than or equal to 10^−6 M, preferably less than or equal to l0^−7 M, more preferably less than or equal to 10^−8 M, even more preferably less than or equal to 10^−9 M, and most preferably less than or equal to 10^−10 M, as measured by surface plasmon resonance (SPR). It has at least 50 times increased affinity for human FcαRI. In one embodiment, This disclosure provides an Fc variant of a parent Fc polypeptide, wherein the Fc variant is a surface polyp Rasmon resonance was measured to determine at least about 50, about 100, and about 1 of the parent Fc polypeptide. It has increased affinity for human FcαRI by 50, approximately 200, approximately 250, and approximately 300 times. In one embodiment, the present disclosure provides an Fc variant of a parent Fc polypeptide, where F The c mutant, as measured by SPR, has at least approximately 300 times more of the parent Fc polypeptide than the parent Fc polypeptide. It exhibits increased affinity for FcαRI.

[0010] In one embodiment of the present disclosure, the Fc variant is a modified A polypeptide compared to the parent Fc polypeptide. DCC is shown. Fc variants of the parent Fc polypeptide are provided herein, where Fc mutation The body was measured in the MDA-MB-453 cell killing assay, and antibody-dependent cell-mediated Increases cytotoxicity (ADCC) by at least approximately 5 times compared to the parent Fc polypeptide. One embodiment Therefore, this disclosure provides an Fc variant of the parent Fc polypeptide, where the Fc variant is a derivative of the parent F Increased in the Calu-3 cell killing assay, at least approximately twice as much as c polypeptide. It is effective.

[0011] In another aspect, the present disclosure provides an IgA antibody comprising a variant Fc polypeptide, where The antibody showed increased FcαR affinity compared to IgA antibodies containing wild-type Fc polypeptide. or has increased ADCC. In one embodiment, this disclosure is: CH2.10, CH2.8 9, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3 Selected from the group consisting of .105, CH3.109, CH3.118, and CH3.124 We provide an IgA antibody containing amino acid modifications at a specific position, and the numbering of the amino acid modifications is C -Follows IMGT numbering for domains. In preferred embodiments, this disclosure is IAM We provide IgA antibodies including amino acid modifications, where the amino acid modification is: A_CH2.10_S, L _CH2.89_I, G_CH2.91_Q, G_CH2.91_V, Q_CH2.94 _E, N_CH2.97_H, N_CH2.97_Y, G_CH2.99_W, S_CH 3.45_D, M_CH3.105_Y, E_CH3.109_D, Q_CH3.118 Selected from the group consisting of _Y and L_CH3.124_F, the numbering of amino acid modifications is as follows: Follow the IMGT numbering system for the C-domain.

[0012] In further embodiments, the present disclosure provides IgA antibodies comprising amino acid modifications, where a Mino acid modifications are: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_D, M _CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2. 97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_ CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2.97 _Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_ CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.9 7_Y / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_ CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.10 5_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_D, Q _CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94 _E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_C H2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.118 _Y、Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH 3.105_Y / Q_CH3.118_Y, A_CH2.10_S, L_CH2.89_ I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3 .109_D, L_CH3.124_F, L_CH2.89_I / G_CH2.91_V From the group consisting of / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W The selected amino acid modifications are numbered according to the IMGT numbering for the C-domain. .

[0013] In one embodiment, this disclosure relates to positions CH2.94, CH2.97, CH3.45, CH3 This invention provides IgA antibodies including amino acid modifications at .105 and CH3.118. Morphologically, the IgA antibody is Glu at position CH2.94 and at position CH2.97. Tyr, Asp at position CH3.45, Tyr at position CH3.105 or position This includes amino acid substitutions of Tyr at CH3.118. In preferred embodiments, it includes IgA anti The body has amino acid substitutions Q_CH2.94_E, L_CH2.97_Y, S_CH3.45_ Includes D, M_CH3.105_Y, Q, and CH3.118_Y.

[0014] In one embodiment, this disclosure provides an IgA antibody comprising a variant Fc polypeptide, and here The antibody is either a human IgA1 or IgA2 antibody.

[0015] This disclosure provides isolated nucleic acids encoding the Fc variants described herein. This disclosure provides, in some cases, a vector containing nucleic acids operably ligated to a control sequence. This disclosure describes host cells containing a vector, as well as the generation and, optionally, recovery of Fc variants. This disclosure provides a method for doing so. This disclosure provides IgA antibodies containing Fc variants as described herein, and The present invention provides a composition comprising a physiologically or pharmaceutically acceptable carrier or diluent.

[0016] This disclosure relates to the therapeutic and diagnostic use of IgA antibodies containing Fc variants disclosed herein. The intention is to provide for the Fc variants disclosed herein, such as bispecific and multispecific antibodies. It can also be used to construct other binding molecules. The IgA antibodies described herein are used in the proliferation of cancer and other diseases. It can be used to treat a variety of indications, including but not limited to sexually transmitted diseases. [Brief explanation of the drawing]

[0017] [Figure 1] Fc variants with increasing concentrations in the assay described in Example 4: SEQ ID NOs: 3(■), 6(▲), 32(▼), 37(◆) and 42(

number

number

number

[0018] IgA immunoglobulin Fc variants with optimized properties, and these Fc mutations Antibodies containing the parent IgA Fc are disclosed herein. Their optimized properties are those of parent IgA Fc Compared to lipeptides, enhanced binding to FcαR and modified antibody-dependent cells mediated cytotoxicity (ADCC).

[0019] definition To make this disclosure easier to understand, some terms are used throughout the detailed explanation. It is defined specifically. Unless otherwise defined, all technologies used herein and Scientific terms have the same meaning as they are generally understood by those skilled in the art to which this disclosure pertains. ru.

[0020] "Include (comprise)", "Includes (comprises)", or "Include (co Terms such as "sequence" are used in relation to sequences (for example, amino acid sequences). In all cases, the sequence consists of "consist" and "consist" Terms such as "consisting" or "consisting" It should be understood that this can also be limited. When used in this specification, it means "essentially ~ or The phrase "ranal" refers to the genus or species of the active pharmaceutical agent contained in the method or composition, and the method or This refers to any excipient that is inert to the intended purpose of the composition. In some embodiments, this refers to The phrase "qualitatively consisting of ~" means one or more additional activators other than the Fc variants of this disclosure. Explicitly exclude inclusion. In some aspects, the phrase "essentially consists of" means This disclosure explicitly includes the inclusion of one or more additional activators other than the Fc variant and the second co-administered agent. Eliminate them directly.

[0021] As used herein, the term “antibody” means non-covalently bound to the corresponding antigen. This refers to polypeptides of the immunoglobulin family that can bind specifically, reversibly, and in a particular manner. The basic functional unit of each antibody is defined in this specification as "Ig monomer". It is an immunoglobulin monomer containing only one Ig unit. Secretory antibodies contain two Ig units. A dimer with units (e.g., IgA), a tetramer with four Ig units, or five Ig units It can also be a pentamer with units (for example, mammalian IgM). The term "antibody" includes, For example, monoclonal antibodies (for instance, full-length antibodies containing an immunoglobulin Fc region) It contains. The Ig monomer consists of four polypeptide chains; linked by disulfide bonds. It is a Y-type molecule consisting of one identical heavy chain and two identical light chains (Woof & Burt). on(2004)Nature Reviews Immunology,4(2):8 9-99). Each chain is divided into two categories according to its size and function: variable Alternatively, it may contain numerous structural domains containing approximately 70 to 110 amino acids that are classified as constant. The heavy chain consists of one variable domain (abbreviated as VH) and three constant domains (CH1, CH2 It includes CH3 (abbreviated as CH3). Each light chain has one variable domain (abbreviated as VL) and It contains one constant domain (abbreviated as CL). The immunoglobulin domain contains two beta-saturated nucleotides. The conserved cysteine ​​residue is held together by interactions between the cysteine ​​residue and other charged amino acids. It possesses characteristic immunoglobulin folds that create a "sandwich" shape. VH and V The L region is complementary, with scattered areas called the framework region (FR) that are more conserved. It can be further subdivided into highly variable regions called the Decision Region (CDR). VH and VL are three CDRs and four CDRs located from the amino acid terminus to the carboxyl terminus. It consists of three FRs, in the following order: FR1, CDR1, FR2, CDR2, FR 3. CDR3 and FR4. The variable regions of the heavy and light chains are antigen-binding regions that interact with the antigen. Contains a main or antigen-binding site.

[0022] The term "antibody" is not limited to monoclonal antibodies, human antibodies, etc. , humanized antibodies, camel antibodies, chimeric antibodies, and anti-idiotype (anti-Id) antibodies (for example) For example, this includes an anti-Id antibody against the antibody of this disclosure. The antibody is any isotype P / class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or sub Classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) It could be something.

[0023] As used herein, the term “single-specific molecule” means a single specific molecule on a target antigen. This refers to a molecule that binds to a pitope. In some embodiments, the single specific molecule of this disclosure is It is a single-specific antibody-like molecule. In some embodiments, the single-specific molecule of this disclosure is a single It is a monospecific antibody. The term "bispecific molecule" refers to a multi-specific antibody that binds to two different antigens. This refers to a bispecific binding molecule. In some embodiments, the bispecific molecule of this disclosure is a bispecific It is a heteroantibody-like molecule. The term "multi-specific binding molecule" as used herein. This refers to a molecule that binds to two or more different antigens. The recognition of each antigen is generally described as follows: This is achieved via the antigen-binding domain. In some embodiments, the multiple specificity of the present disclosure The binding molecule is a multispecific antibody-like molecule, such as a bispecific antibody-like molecule.

[0024] The term "antigen-binding site" refers to the site that forms the antigen or its epitope-binding interface. This refers to the part of the antibody that contains the determinant. The term "antigen-binding site" is a direct translation of "antigen-binding domain." This term can be used interchangeably with the following term: Regarding proteins (or protein mimes). The antigen-binding site typically forms an interface that binds to the antigen polypeptide (less It contains at least four amino acids or one or more loops of amino acid mimics. Typically, The antigen-binding site of the antibody molecule has at least one or two CDRs and / or hypervariable rings. A lap, or more typically, at least 3, 4, 5 or 6 CDRs and / or Includes highly variable loops.

[0025] As used herein, the “complementarity determination region” (“CDR”) refers to the supernumerary region of the VL and VH. This refers to the variable region. CDRs are antibody chains that possess specificity for target proteins. These are protein binding sites. Each human VL or VH contains three CDRs (CDR1-3) There are (numbered sequentially from the N-terminus), and approximately 15-20% of the total variable domains. It constitutes. A CD-R can be named according to its area and order. For example, "VH "CDR1" or "HCDR1" both refer to the first CDR in the heavy chain variable region. R is structurally complementary to the epitope of the target protein, and therefore directly contributes to binding specificity. The remaining stretch of VL or VH, the so-called framework region, is the amino acid sequence. This shows a small number of variations (Kuby (2000) Immunology, 4th ed.,Chapter 4.WHFreeman & Co.,New York ). The location of the CDR and framework area is defined by various known definitions in the art, e.g. For example, Kabat, Chothia, IMGT, AbM, and combination definitions (for example, Johnson et al.,(2001)Nucleic Acids Res., 29:205-206;Chothia & Lesk,(1987)J.Mol.Bi ol.,196:901-917;Chothia et al.,(1989)Nat ure,342:877-883;Chothia et al.,(1992)JM ol.Biol.,227:799-817;Lefranc,MP,(2001) Nucleic Acids Res.,29:207-209;Al-Lazikan i et al., (1997) J. Mol. Biol., 273:927-748 and Kabat et al., (1991) Sequences of proteins of immunological interest.5th Edition-U S DHHS, NIH publication n°91-3242, pp 662, This can be determined using (see 680, 689). The definition of the antigen-binding site is as follows: Ruiz et al.,(2000) Nucleic Acids Res.,28: 219-221;MacCallum et al.,(1996)J.Mol.Bio l., 262:732-745; and Martin et al., (1989) Pro. c.Natl.Acad.Sci.USA,86:9268-9272;Martin et al.,(1991)Methods Enzymol.,203:121-15 3; and Rees et al., (1996) In Sternberg MJE (ed.), Protein Structure Prediction, Oxford It is also mentioned in rd University Press, Oxford, pp. 141-172. In combination of Kabat and Chothia numbering schemes, some In some embodiments, the CDR is a Kabat CDR, a Chothia CDR, or a similar CDR. It corresponds to an amino acid residue that is part of both. For example, in some embodiments, CDR is , VH, for example, mammalian VH, for example, amino acid residues 26-35 in human VH (HCDR 1) 50-65 (HCDR2), and 95-102 (HCDR3); and VL, for example. In mammalian VL, for example, amino acid residues 24-34 (LCDR1) and 50-5 in human VL. Compatible with 6 (LCDR2) and 89-97 (LCDR3). Under IMGT, VH The CDR amino acid residues are approximately 26-35 (CDR1), 51-57 (CDR2), and They are numbered 93-102 (CDR3), and there are approximately 27 CDR amino acid residues in VL. The discs are numbered as follows: ~32 (CDR1), 50~52 (CDR2), and 89~97 (CDR3). (Numbering according to "Kabat"). Under IMGT, the CDR region of the antibody is pro This can be determined using Gram IMGT / DomainGap Align. The MGT tool is available on the World Wide Web (www.imgt.org). That is the case.

[0026] In one embodiment, the antibody includes an "antigen-binding fragment" of the antibody. Examples of such fragments are (i) a monovalent fragment consisting of a Fab fragment, VL, VH, CL and CH1 domain; ii) F(ab')2 fragment, linked by disulfide bridges in the hinge region (iii) A bivalent fragment containing two Fab fragments; (iii) An Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) VH domain (vi) a diabody (dAb) fragment consisting of n; (vi) a camel or camelized variable domain; ( vii) Single-chain Fv (scFv), e.g., Bird et al., (1988) Sci ence 242:423-426; and Huston et al., (1988)P See NAS USA 85:5879-5883); (viii) Single-domain antibody; ( ix) Diabody (Dab) (bivalent and bispecific), and (x) total antibody or recombinant D Chimeras can be produced by modifying de novo synthesized materials using NA technology (e.g.) For example, humanized antibodies can be cited. These functional antibody fragments are their respective antigens. Alternatively, they retain the ability to selectively bind to receptors. These antibody fragments are based on methods known to those skilled in the art. Obtained using the technology for which the fragments are screened for usefulness, similar to intact antibodies. It will be done.

[0027] In mammals, there are two types of immunoglobulins called lambda (λ) and kappa (κ). Phosphorus light chains are present. Each antibody always contains the same two light chains; in mammals Only one type of light chain exists for each antibody: either κ or λ. The approximate length of the light chain is 211. It consists of approximately 217 amino acids, and each light chain has two domains, one constant domain and one It has two variable domains.

[0028] There are five types of mammalian Ig heavy chains, denoted as α, δ, ε, γ, and μ, and in antibodies The type of heavy chain present determines the antibody class or isotype: IgM, IgG, IgA, Ig D and IgE are defined respectively. The heavy chain's physicochemical, structural, and immunological properties are variable. However, each heavy chain has two domains: a variable domain and a constant domain. The domain contains a single Ig domain (approximately 110 amino acids long), and its antibody binding specificity is The decision is made. The constant domain is identical in all antibodies of the same isotype, but different. This differs in isotype antibodies. The heavy chains γ, α, and δ consist of three tandem Ig domains. It has a steady region composed of and a hinge region for adding flexibility; heavy chain μ and ε have a constant region composed of four immunoglobulin domains (Woof (& Burton, cited above). The term "immunoglobulin" (Ig) is used herein. It is used interchangeably with the term "antibody."

[0029] IgG is the most abundant antibody in blood (plasma), accounting for 70-75% of human immunoglobulins. It is a somatic isotype. IgG detoxifies harmful substances and is mediated by white blood cells and macrophages. It is important in the recognition of proto-antibody complexes. IgG has four subclasses in humans: IgM is further classified into IgG1, IgG2, IgG3, and IgG4. IgM is usually found in blood. It circulates within the body and accounts for about 10% of human immunoglobulins. IgM is composed of five basic Y-type molecules. They have a pentameric structure that is bound together. B cells respond to microbial infection / antigen invasion as the most It initially produces IgM. IgM has a lower affinity for antigens than IgG, but It has high avidity to antigens due to its pentamer / hexamer structure. IgM is Furthermore, by binding to cell surface receptors, it also activates cellular signaling pathways. IgA is also found in blood It is abundant in human urine, nasal mucosa, saliva, breast milk, and intestinal fluid, and accounts for 25% of human immunoglobulins. IgA forms dimers (i.e., two IgA monomers bound together). IgA in breast milk protects the neonatal gastrointestinal tract from pathogens. IgA is produced in two subclavian cells. IgA1 and IgA2 are classified as follows: IgD is less than 1% of human immunoglobulins. It may be involved in inducing antibody production in B cells, but its exact function remains unknown. IgE is present in trace amounts, accounting for less than 0.001% of human immunoglobulins. Its original role Its purpose is to protect against parasites. In areas where parasitic infections are rare, IgE is primarily used in It is involved in allergies.

[0030] Immune cell activity is determined by the region of the antibody known as the crystallizable fragment region or "Fc region". Modulated. The Fc region consists of two identical polypeptide chains (each referred to as "F" in this specification). It consists of what is called the "c domain," and IgG and IgA have CH2 and CH3 in the heavy chain. Includes the normal domain. The IgM and IgE Fc regions are three in each polypeptide chain. It contains heavy chain constant domains (CH domains 2-4) in the CH2 and CH3 domains. Amino acid residues are numbered according to the EU numbering system (Edelman et al., (1969) PN AS.USA, 63, 78-85), "Kabat" numbering (Kabat et al.) , in accordance with the above, or alternatively using IMGT numbering for the C domain It can be numbered. The IMGT tool is used for the World Wide Web (www.img) It is available on t.org.

[0031] The Fc region is a cell surface receptor that mediates the physiological effects of antibodies, the "Fc receptor," and complement. It binds to proteins. Fc receptors are found in many cells of the immune system, such as B lymphocytes, and filters. Vesicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human It is found on platelets and mast cells. Binding of the antibody Fc region to the Fc receptor is phagocytic or This stimulates cytotoxic cells and, through the mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC), the microorganisms... or destroy infected cells. Several different types of Fc receptors (FcRs) exist, They are classified based on the type of antibody they recognize. For example, those that bind to IgG. The one that binds to IgA is called the Fc-gamma receptor (FcγR), and the one that binds to IgA is called the Fc-alpha receptor. The receptor called the α receptor (FcαRI) is the one that binds to IgE, and the one called the Fc-epsilon receptor (F FcRs are called cεRs. The class of FcRs is defined by the cells that express them (macrophages, granules). The signaling properties of globules, natural killer cells, T and B cells, and their respective receptors. Therefore, they can also be distinguished (Owen J et al., (2009) Immunology (7th ed.).New York:WHFreeman and Compa. (ny.p423). FcαRI is also known as CD89, and its main antibody ligand is I This is gA. This receptor has a low affinity for IgA (Kd>10). -6 M) has, It is found on globules, macrophages, neutrophils, and eosinophils. IgA binding to FcαRI The combination primarily induces phagocytosis and microbial killing.

[0032] In one embodiment, the antibody comprises a full-length antibody or a full-length immunoglobulin chain. In this state, the antibody is either a full-length antibody or an antigen-binding or functional fragment of a full-length immunoglobulin chain. Includes. Antibody preparations may be monoclonal or polyclonal. Antibodies are human, hi This could involve grafting, CDR grafting, or in vitro antibody production.

[0033] In one embodiment, antibodies or immunoglobulins can be recombinantly produced, for example, It can be produced by phage display or by combinatorial methods. Phage display and combinatorial methods for generating bodies are in the field of art. This is publicly known (for example, Ladner et al., U.S. Patent No. 5,223,409). Specification; Kang et al., International Publication No. 92 / 18619 Pamphlet; Dow er et al., International Publication No. 91 / 17271 pamphlet; Winter et al. al., International Publication No. 92 / 20791 pamphlet; Markland et al. International publication no. 92 / 15679; Breitling et al. International Publication No. 93 / 01288 Pamphlet; McCafferty et al., Country International Publication No. 92 / 01047 pamphlet; Garrard et al., International Publication No. Pamphlet No. 92 / 09690; Ladner et al., International Publication No. 90 / 02 Pamphlet No. 809; Fuchs et al., (1991) Bio / Techno logy,9:1370-1372;Hay et al.,(1992)Hum An Tibody Hybridomas,3:81-85;Huse et al.,(1 989)Science 246:1275-1281;Griffths et al. .,(1993)EMBO J.,12:725-734;Hawkins et al. .,(1992)J Mol Biol.,226:889-896;Clackson et al.,(1991)Nature,352:624-628;Gram et al. al.,(1992)PNAS,89:3576-3580;Garrard et al. al.,(1991)Bio / Technology,9:1373-1377;Hoo genboom et al.,(1991)Nuc Acid Res.,19:41 33-4137; and Barbas et al., (1991) PNAS, 88:79 (As described in 78-7982; all of their contents are incorporated herein by reference).

[0034] In one embodiment, the antibody or immunoglobulin is a fully human antibody (e.g., human immunoglobulin) Antibodies produced in genetically engineered mice that produce antibodies from robulin sequences. Antibodies isolated from humans, or non-human antibodies, for example, from rodents (mouse or rat). These are antibodies against goats, primates (e.g., monkeys), and camels. Human monoclonal antibodies are... Instead of using a strain of mice, we used transgenic mice carrying the human immunoglobulin gene. It can be generated from these transgenic mice immunized with the target antigen. Human spleen cells that possess specific affinity for epitopes from human proteins It produces hybridomas that secrete monoclonal antibodies (e.g., Wood et a l., International Publication No. 91 / 00906, Kucherlapati et al., International Publication No. 91 / 10741 pamphlet; Lonberg et al., International Publication No. 92 / 03918 pamphlet; Kay et al., International Publication No. 92 / Pamphlet No. 03917; Lonberg et al., (1994) Nature 368:856-859;Green et al.,(1994)Nature G enet.7:13-21;Morrison et al.,(1994)PNAS USA 81:6851-6855;Bruggeman et al.,(1993) Year Immunol 7:33-40;Tuaillon et al.,(19 93)PNAS 90:3720-3724;Bruggeman et al.,(1 991) Eur J Immunol 21:1323-1326).

[0035] Antibodies or immunoglobulins have a variable region, or part thereof, for example, a CDR in non-human organisms. For example, it may be produced in rats or mice. Chimeras, CDR grafts, And humanized antibodies are within the scope of the present invention. In non-human organisms, for example, rats or mice It is generated and then, for example, a variable framework or to reduce its antigenicity in humans. Antibodies modified within the constant region are within the scope of the present invention. Chimeric antibodies are in the art. It can be produced using known recombinant DNA technology (Robinson et al.) al., International Publication No. 87 / 002671 Pamphlet; Akira et al., Europe State Patent Application Publication No. 184187A1; Taniguchi, European Patent Application Publication No. Specification No. 171496A1; Morrison et al., European Patent Application Publication No. 17 Specification No. 3494A1; Neuberger et al., International Publication No. 86 / 0153 Pamphlet No. 3; Cabilly et al., U.S. Patent No. 4,816,567 Detailed description; Cabilly et al., European Patent Application Publication No. 125023A1; Better et al.,(1988)Science 240:1041-104 3;Liu et al.,(1987)PNAS 84:3439-3443;Liu et al.,(1987),J.Immunol.139:3521-3526;S un et al.,(1987)PNAS 84:214-218;Nishimur a et al.,(1987),Canc.Res.47:999-1005;Woo d et al., (1985) Nature 314:446-449; and Shaw et al., (1988), J. Natl Cancer Inst. 80:155 (See 3-1559).

[0036] Humanized or CDR-grafted antibodies replaced with at least one or It has two recipient CDRs (heavy chain and / or light chain immunoglobulin chains), but generally It has all three recipient CDRs. The antibody replaces at least a portion of the non-human CDR. It is possible to obtain, or to replace only a portion of the CDR with a non-human CDR. Target It is only necessary to replace the number of CDRs required for the binding of humanized antibodies to the antigen. In other words, the donor is a rodent antibody, for example, a rat or mouse antibody, and the recipient is It is a human framework or human consensus framework. Typically, it uses CDR. The immunoglobulins provided are called "donors," and the immunoglobulins that provide the framework are called "donors." The donor immunoglobulin is called an "acceptor." In one embodiment, the donor immunoglobulin is non-Hypertoxic. (For example, rodents). The acceptor framework is naturally occurring (for example, Human-based frameworks or consensus frameworks, or approximately 85% or more of them. Preferably, the sequences are identical for 90%, 95%, or 99% or more of the time.

[0037] As used herein, the term “consensus sequence” refers to the f(x) of the related sequences. A sequence formed from the most frequently occurring amino acids (or nucleotides) in a family. This refers to (for example, Winnaker, From Genes to Clones (Ve rlagsgesellschaft, Weinheim, Germany (1987) (See reference). In a family of proteins, each position in the consensus sequence is: It is occupied by the amino acid that occurs most frequently at that position within its family. If two amino acids occur at equal frequency, both can be included in the consensus sequence. The "consensus framework" is a framework in consensus immunoglobulin sequences. This refers to the area of ​​-.

[0038] Antibodies can be humanized by methods known in the art (for example, M orrison,(1985),Science 229:1202-1207;Oi et al., (1986), BioTechniques 4:214, and Que en et al., U.S. Patent No. 5,585,089, U.S. Patent No. 5,693, See U.S. Patent No. 761 and U.S. Patent No. 5,693,762, all of which are (Incorporated herein by reference). Humanized or CDR-grafted antibodies are immunoglobulins. CDR grafting or CD that can replace 1, 2, or all of the phosphate chain CDRs It can be produced by R substitution. For example, U.S. Patent No. 5,225,539; Jones et al.,(1986)Nature 321:552-525;Ve rhoeyan et al.,(1988)Science 239:1534;Be idler et al.,(1988)J.Immunol.141:4053-40 See U.S. Patent No. 5,225,539 of 60 and Winter, of all of them The contents are expressly incorporated herein by reference. The specified amino acids are substituted, deleted, or added. Humanized antibodies are also within the scope of this invention. Criteria for selecting amino acids from a donor. This refers to U.S. Patent No. 5,585,089, for example, U.S. Patent No. 5,585,089 This is described in columns 12-16 of the specification, and its contents are incorporated herein by reference. Other techniques for humanizing antibodies are described in Padlan et al., European Patent Application Publication. It is described in Specification No. 519596A1.

[0039] Methods for modifying the constant region of an antibody are known in the art. Examples of modified functions include: For example, effector ligands, such as FcR on cells, or the C1 component of complement, can be affected. Antibodies with modified affinity have at least one amino acid residue in the constant portion of the antibody. It can be produced by replacing it with a certain residue (for example, European Patent Application Publication No. 3) U.S. Patent No. 88151A1, U.S. Patent No. 5,624,821 and U.S. Patent No. 5,6 48,260).

[0040] As used herein, "position" refers to the position of an amino acid in a protein sequence. The position is continuous or in an established form, for example, EU in Kabat. They can be numbered according to DEX or IMGT numbering (www.imgt.org). For example, when using IMGT numbering, glutamine 94 (also called Gln94) Q94 (also known as Q94) indicates whether or not it is found in the CH2 or CH3 domain. Therefore, the position in the Fc region is also given. For example, QCH2.94 and SCH3.45 are, The glutamine at position 94 in the CH2 domain of the antibody IgA1, and the CH3 domain This indicates the serine at position 45.

[0041] As used herein, “residue” refers to a protein and its associated amino acid identity. It refers to the position in Q9. For example, glutamine 94 (also called Gln94, Q9) (Also known as 4) is a residue in the human antibody lgA1.

[0042] The “modification” or “mutation” of amino acid residues / positions used herein is a departure from the original. This refers to a change in the primary amino acid sequence compared to the amino acid sequence, and this change is one or more of the above-mentioned changes. It results from sequence changes involving the amino acid residue / position. For example, a typical modification is one Substitution of the above residues (or at the above positions) with another amino acid (e.g., conservative or non-conservative) (Spatial substitution), insertion of one or more amino acids adjacent to the one or more residues / positions, and the Deletion of one or more residues / positions, inversion of the one or more residues / positions, and the one or more remaining Duplication of base / position is one example. Amino acid "substitution" or its variations is a predetermined (out (Derivation) One or more different amino acids of one or more existing amino acid residues in an amino acid sequence. This refers to substitution with an acid residue. Generally and preferably, the modification is made to the starting or parent (or "wild type") At least one variant polypeptide compared to a polypeptide containing an amino acid sequence This results in a change in physicochemical activity. For example, in the case of antibodies or Fc isomers, the substance that is modified... Physiochemical activity can be the binding affinity, binding ability, and / or binding effect to the target molecule. .

[0043] The terms "mutant polypeptide," "polypeptide variant," or "mutation" as used herein refer to the same term. The "body" is a polypeptide that differs from the parent polypeptide sequence by at least one amino acid modification. This refers to the cytoplasmic sequence. The parent polypeptide is either naturally occurring or wild-type (WT) polypeptide. It may be a plutid or a modified version of the WT polypeptide. D refers to the polypeptide itself, a composition containing the polypeptide, or an A that codes for the polypeptide. This may refer to a mino sequence. Preferably, the mutant polypeptide is less than the parent polypeptide. At least one amino acid modification, for example, about 1 to 10 amino acid modifications compared to the parent, preferred The mutant polypeptides described herein have approximately 1 to 5 amino acid modifications. The sequence has at least about 80% homology to the parent polypeptide sequence, preferably at least about It will have 90% homology, more preferably at least about 95% homology. Morphologically, the mutant polypeptide sequences described herein are the parent IgA CH2 polypeptide At least about 85% homology to the cydone sequence, preferably at least about 90% homology, More preferably, it will have at least about 95% homology. In one embodiment, as specified herein The mutant polypeptide sequences described are less different from the parent IgA CH3 polypeptide sequence. Both have approximately 90% homology, preferably at least approximately 95% homology, more preferably less Both will have approximately 97% homology. In preferred embodiments, as described herein The mutant polypeptide sequence is at least approximately 85% different from the parent IgA CH2 polypeptide sequence. homology, preferably at least about 90% homology, more preferably at least about 95% It has homology, and at least about 90% homology with the parent IgA CH3 polypeptide sequence. Preferably, at least about 95% homology, more preferably at least about 97% homology. It will have. Therefore, as used herein, "Fc variant" or "Variant Fc" This means an Fc sequence that is different from the parent Fc sequence due to at least one amino acid modification. Fc variants only encompass the Fc region, or antibodies, Fc fusions, isolated Fc , present in the context of Fc fragments or other polypeptides substantially encoded by Fc It is possible. The Fc variant can be the polypeptide itself, a composition containing the Fc variant polypeptide, or This may refer to the amino acid sequence encoding the Fc variant. A "cydo mutant" or "mutant Fc polypeptide" is formed by modifying at least one amino acid. This refers to an Fc polypeptide that is different from the parent Fc polypeptide. The term "parent Fc polypeptide" refers to the starting Fc polypeptide from which amino acid modifications are performed. The parent Fc polypeptide is either the wild-type Fc polypeptide or the wild-type Fc polypeptide allele. It can be an Fc polypeptide, which is a genetic variation. The parent Fc polypeptide is also It may be an Fc polypeptide that has already undergone amino acid modification. A "protein variant" or "mutant protein" is a protein that has been modified by at least one amino acid. This refers to a protein that is different from the parent protein. The term "antibody variant" as used in this specification also refers to a protein that is different from the parent protein. A "mutant antibody" is an antibody that differs from the parent antibody due to at least one amino acid modification. Taste. The terms "IgA variant" or "IgA variant" used herein refer to at least one This refers to an antibody that is different from parent IgA due to one amino acid modification. Parent IgA is an isotype. It may be IgA1 or IgA2. The term "immunoglobulin mutation" as used herein refers to a mutation. "Body" or "mutant immunoglobulin" is a modified form of the parent immunoglobulin by altering at least one amino acid. This refers to an immunoglobulin sequence that is different from the immunoglobulin sequence.

[0044] In this specification, “wild type” or “WT” refers to naturally occurring varieties, including allelic variations. This refers to the amino acid sequence or nucleotide sequence that is used. WT protein, polypeptide, Antibodies, immunoglobulins, IgA, etc., have amino acid sequences or nucleic acids that have not been intentionally modified. It has a leotide sequence.

[0045] "Conservative amino acid substitution" is when an amino acid residue is replaced with an amino acid residue that has a similar side chain. It is a replaceable substitution. A family of amino acid residues with similar side chains exists in the art. These are defined as follows: basic side chains (e.g., lysine (K) ), arginine (R), histidine (H), acidic side chain (e.g., aspartic acid (D)) , glutamic acid (E), non-charged 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), iso Leucine (I), proline (P), phenylalanine (F), methionine (M), trip Tophan (W), beta branched side chains (e.g., threonine (T), valine (V), isolol isine (I) and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), to Examples include amino acids containing liptophan (W) and histidine (H).

[0046] "Identical percentage" or "identity percentage" for two or more nucleic acid or polypeptide sequences The term "array" refers to two or more identical arrays or subarrays. Two arrays are... Using one of the array comparison algorithms below, or manual alignment and visual inspection. Measured and compared within a comparison window or specified area, and aligned for maximum correspondence. If the two sequences are of the same proportion as specified (i.e., compared to specified regions), or specified If none exists, 60% identity compared to the entire sequence; 65%, 70%, 75%, 80% identity if chosen arbitrarily. amino acid residues or nucleotides that have %, 85%, 90%, 95%, or 99% identity. If it has the same properties, it is "substantially identical." Optionally, identity is defined as having at least approximately 50% of the same properties. On a region that is the length of a creotide (or 10 amino acids), or more preferably 100 to 500 Or a region with a length of 1000 nucleotides or more (or 20, 50, or 200 amino acids or more). It exists in the region. The “percentage of identity” or “percentage of sequence identity” in this disclosure is (i) two Optimally aligned sequences (nucleotides or proteins) are compared in the comparison window. By doing so, (ii) the same nucleic acid base (with respect to the nucleotide sequence) or amino acid Determine the number of positions in both sequences where the residue (for the protein) occurs, and the match positions. By obtaining the number, (iii) the number of matched positions is equal to the total number of positions in the comparison window. By dividing by (iv) this quotient by 100%, the percentage of identity is obtained. It can be calculated by obtaining it. The "identity percentage" is defined in a specific comparison. If calculated with respect to the reference array without a window, the identity percentage is equal to the alignment It is determined by dividing the number of match positions in the range of the entry by the total length of the reference array. For the purposes of this disclosure, the two sequences (query and target) are aligned to When optimally aligned (allowing for gaps), the "identity" of the query array "Percent" represents the number of identical positions between two arrays over its length (or comparison window). It is equal to dividing by the total number of positions in the query array, and then multiplying that by 100%.

[0047] For sequence comparison, typically one sequence is used as the reference sequence against which the test sequence is compared. It is possible. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer. If necessary, the sub-array coordinates are specified, and the array algorithm program parameters are set. It is specified. Default program parameters can be used, or alternative parameters can be used. You can specify the parameter. Then, the array comparison algorithm is set to the program parameters. Based on this, the sequence identity percentage of the test sequence to the reference sequence is calculated.

[0048] As used herein, the term "comparison window" refers to a window between two arrays. 20-6 can be compared to the same number of consecutive reference arrays after optimal alignment. Selected from the groups consisting of 00, typically about 50-200, and more typically about 100-150. The number of consecutive positions includes a reference to one of the segments. The array for comparison The method of alignment is known in the art. Optimal arrangement for comparison Inment is, for example, Smith & Waterman (1970) Adv. App According to the local homology algorithm of l.Math.2:482c, Needleman Homology of allies by & Wunsch (1970) J.Mol.Biol.,48:443 According to the PNAS algorithm, Pearson & Lipman (1988) The similarity search method described in .USA,85:2444 allows these algorithms to compute Execution by (Wisconsin Genetics Software Packa ge,Genetics Computer Group,575 Science D r., Madison, WI GAP, BESTFIT, FASTA, and TFAST A) by or by manual alignment and visual inspection (e.g., Brent et a l.,(2003)Current Protocols in Molecular This can be done according to the Biology section.

[0049] Two examples of algorithms suitable for determining sequence identity percentage and sequence similarity are BL. These are the AST and BLAST 2.0 algorithms, respectively, and are based on Altschul. et al.,(1977)Nuc.Acids Res,.25:3389-340 2; and Altschul et al., (1990) J. Mol. Biol., 21 It is described in 5:403-410. The software for performing BLAST analysis is , National Center for Biotechnology Infor It is publicly available via mation. This algorithm is used in database arrays. When aligned with words of the same length, they match or satisfy a positive threshold score T. High scoring array pairs are achieved by identifying short words of length W in the query array. This involves first identifying HSPs. T is called the adjacent word score threshold (Alt Schul et al., (1990) cited above. These initial adjacent word hits are This serves as a starting point for a search to find longer HSPs that contain these. Word hits are counted as long as the cumulative alignment score can increase for each sequence. It is stretched in both directions along the parameter. The cumulative score is for the nucleotide sequence, parameter M (reward score for a pair of matched residues; always > 0) and N (reward score for mismatched residues) Penalty score; always calculated using <0). For amino acid sequences, cumulative score A scoring matrix is ​​used to calculate the core. The cumulative alignment score is its If the value drops by a factor X from the maximum achieved value; the accumulation of one or more negative score residue alignments If the cumulative score becomes 0 or less due to the product; or if the end of any sequence is reached. The extension of word hits in each direction stops. BLAST algorithm parameters The W, T, and X on the meter determine the alignment sensitivity and speed. BLASTN The program (for nucleotide sequences) defaults to a word length (W) of 11, and the expected value is ( Use E) or 10, M=5, N=-4, and compare both chains. For amino acid sequences, The BLASTP program uses a word length of 3 and an expected value (E) of 10 as defaults. The BLOSUM62 scoring matrix (Henikoff & Henikoff,( (See PNAS.USA, 89:10915, 1989) Alignment (B) 50, The expected value (E) is set to 10, M=5, N=-4, and the comparison of both strands is used as the default.

[0050] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (for example, Karlin & Altschul(1993)PNAS.USA,90:5873- (See 5787). One measure of similarity provided by the BLAST algorithm is the minimum The smallest sum probability (P(N)) is, This is an indicator of the probability that a match between two nucleotide sequences or amino acid sequences occurs by chance. This provides, for example, a minimum summation probability of less than approximately 0.2 in the comparison between the test nucleic acid and the reference nucleic acid. More preferably less than about 0.01, most preferably less than about 0.001, nucleus The acid is considered to be similar to the reference sequence.

[0051] The percentage of identity between two amino acid sequences is calculated using the ALIGN program (version 2). 0) E. Meyers and W. Miller (Comput.Ap Using the algorithm from pl.Biosci.4:11-17(1988), PAM Using a 120-weight residue table, gap length penalty 12, and gap length penalty 4 It can also be determined by this. Furthermore, the percentage of identity between two amino acid sequences is GCG GAP Programming in the software package (available at www.gcg.com) The aforementioned algorithm by Needleman & Wunsch, which is incorporated into the RAM, is used. Then, either the Blossom62 matrix or the PAM250 matrix, and a gapway To 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or It can be determined using 6.

[0052] Aside from the above-mentioned proportion of sequence identity, two nucleic acid sequences or polypeptides are substantially identical. Another indicator is that the polypeptide encoded by the first nucleic acid is compared with the second nucleic acid, as shown below. It is immunologically reactive to antibodies produced against polypeptides encoded by [the specified method]. Therefore, polypeptides are typically, for example, two peptides in a conserved position. It is substantially identical to the second polypeptide, differing only in its substitution. The two nucleic acid sequences are substantially identical. Another indicator is that two molecules or their complements are stringent as follows: This refers to hybridizing with each other under the same conditions. Another indicator is that the same primer can be used to amplify the sequence. .

[0053] The term "nucleic acid" is used herein interchangeably with the term "polynucleotide." Deoxyribonucleotides or ribonucleonucleotides used, which are either single-stranded or double-stranded. This term refers to creotides and their polymers. This term encompasses synthetic, naturally occurring, and unnaturally occurring creotides. It has binding properties similar to the reference nucleic acid and is metabolized in a manner similar to the reference nucleotide. It includes nucleic acids containing known nucleotide analogs or modified skeletal residues or bonds. Examples of analogues like these include, though not limited to, phosphorothioates and phosphothioates. Luamidate, methylphosphonate, chiral-methylphosphonate, 2-O-methylribo Examples include nucleotides and peptide-nucleic acids (PNAs).

[0054] Unless otherwise specified, a particular nucleic acid sequence refers not only to the explicitly stated sequence but also to its conserved sequences. This implicitly includes modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, as detailed below, degenerate codon substitution involves one or more selected codon substitutions. The third position of (or all) codons is substituted with a mixed base and / or a deoxyinosine residue. This can be achieved by generating the sequence (Batzer et al. .,(1991)Nucleic Acid Res.,19:5081;Ohtsuk a et al.,(1985)J Biol Chem.,260:2605-260 8; and Rossolini et al., (1994) Mol Cell Prob (es,8:91-98). When used herein, "optimized nucleotide sequence" The term "produces cells" refers to the cells that produce these cells, in this case, Chinese hamster ovary cells (CHO). The nucleotide sequence has been modified to encode the amino acid sequence using preferred codons. This means that the optimized nucleotide sequence is the starting nucleotide, also known as the "parent" sequence. Artificially engineered to retain the complete amino acid sequence initially encoded by the rheotide sequence. In certain embodiments, the optimized sequence described herein is used in CHO mammalian cells. It has been artificially engineered to have a desirable codon.

[0055] When used herein, "C-terminus" refers to a free carboxyl group (-COOH This refers to the carboxyl-terminal amino acid of a polypeptide chain having ). When this occurs, the "N-terminus" is the amino of the polypeptide chain having a free amine group (-NH2). This refers to terminal amino acids.

[0056] As used herein, “operably coupled” or “functionally coupled” The term refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. This refers to a relationship. Typically, this refers to the functional relationship between a transcriptional regulatory sequence and the sequence being transcribed. For example, a promoter or enhancer sequence may be appropriate for a host cell or other expression system. When stimulating or modulating transcription of the coding sequence within, it binds operably to the coding sequence. Generally, promoter transcriptional regulatory sequences that are operably bound to the sequence being transcribed are They are physically continuous with the sequence being transcribed, meaning they are cis-acting. However However, some transcriptional regulatory sequences, such as enhancers, improve transcription. The elements in the sequence do not need to be physically consecutive or in close proximity.

[0057] The terms "polypeptide" and "protein" refer to polymers of amino acid residues. This term is used interchangeably in this specification. This term corresponds to one or more amino acid residues. Amino acid polymers are artificial chemical mimics of naturally occurring amino acids, as well as naturally occurring amino acids This also applies to amino acid polymers that do not occur naturally and amino acid polymers that do not occur naturally. Unless otherwise specified. To the extent that, a particular polypeptide sequence implicitly includes its conservatively modified variants. do.

[0058] The terms "parent polypeptide," "parent protein," and "precursor polypeptide" as used herein are used in this specification. ", or "precursor protein", is then modified to produce mutants in the unmodified polypeptide. It means "do". The parent polypeptide is a naturally occurring polypeptide, or a naturally occurring polypeptide. It may be a variant or modified version of the lipeptide. The parent polypeptide is a polypeptide The drug itself, a composition containing the parent polypeptide, or an amino acid sequence encoding the parent polypeptide It can refer to. Therefore, as used herein, "parent Fc polypeptide" refers to mutation The term "parental antagonist" as used herein means an Fc polypeptide that has been modified to produce a body. "Antibody" refers to an antibody that has been modified to produce a mutant antibody. In some embodiments, The "parent" protein is the wild-type protein.

[0059] As used herein, the term "in vivo half-life" refers to the half-life of a given mammal in the blood of a given mammal. This refers to the half-life of the molecule or its variant that is intended to circulate.

[0060] The term "subject" includes humans and non-human animals. Non-human animals include all of the following: Vertebrates such as mammals and non-mammals such as non-human primates, sheep, dogs, and cattle Examples include chickens, amphibians, and reptiles. In preferred embodiments, the subject is a human. Unless otherwise noted, the terms “patient” or “subject” are interchangeable in this specification. It is used in a specific way.

[0061] When used in this specification, "patients requiring treatment" or "patients requiring treatment" The phrases such as "targets to be detected" are, for example, used in the detection, diagnostic procedures and / or treatments of this disclosure. Subjects that benefit from the administration of the molecule or pharmaceutical composition include, for example, mammalian subjects.

[0062] When used herein, the terms "treatment" or "to treat" are used in this specification. In the specification, F by the present disclosure to or from isolated tissues or cell lines of the subject. This is defined as the application or administration of a pharmaceutical composition containing the c isomer or the Fc isomer, and the subject is particularly A specific disease (e.g., arthritis), symptoms associated with that disease, or predisposing factors for the onset of that disease (applicable) (if applicable) it has the purpose of curing one or more symptoms of the disease (if applicable) and preventing (If applicable), delay the onset, reduce the severity, alleviate, improve, and treat the disease. The goal is to improve the condition, reduce or improve any associated symptoms of the disease, or reduce predisposing factors for the disease's onset. The terms "treatment" or "to treat" include patients suspected of having a disease and patients with a disease or This includes treating patients diagnosed with a disease or medical condition, and clinical This includes suppressing bed recurrence. The phrase "reducing the likelihood" means reducing the disease, infection, or Refers to delaying the onset, occurrence or progression of a disorder.

[0063] The terms "therapeutically acceptable amount" or "therapeutically effective amount" or "therapeutically effective dose" refer interchangeably to an amount sufficient to produce a desired result ( i.e., reduction of disease activity, reduction of disease progression, reduction of disease signs and / or symptoms, etc.). In some embodiments, a therapeutically acceptable amount does not induce or cause unwanted side effects. A therapeutically acceptable amount can be determined by initially administering a low dose and then gradually increasing the dose until the desired effect is achieved. The "preventive effective dosage" and "therapeutic effective dosage" of the disclosed molecules can each prevent the onset of disease symptoms ( where applicable), or result in a decrease in their severity. When used herein, "selecting" and "selected" with respect to a patient are used to mean specifically selected from a larger group of patients due to a particular patient having a particular criterion. Similarly, "selectively treating a patient"

[0064] refers to providing treatment to a patient specifically selected from a larger group of patients due to a particular patient having a particular criterion. Similarly, "selectively administering" refers to administering a drug to a patient specifically selected from a larger group of patients due to a particular patient having a particular criterion. Unless specifically stated otherwise or otherwise apparent from the context, when used herein, the term "about" with respect to a numerical value is understood to be within the normal acceptable range in the art, e.g., within two standard deviations of the mean. Thus, "about" modifies the description as described.

[0065] Unless specifically stated otherwise or otherwise apparent from the context, when used herein, the term "about" with respect to a numerical value is understood to be within the normal acceptable range in the art, e.g., within two standard deviations of the mean. Thus, "about" modifies the Values ​​+ / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1% , 0.05%, or 0.01%, preferably within a range of + / - 10% of the described value. The term "approximately" when used before a number or a list of numbers refers to each of the numbers in that series. This applies to numbers; for example, the phrase "approximately 1 to 5" should be interpreted as "approximately 1 to approximately 5". It is, or for example, the phrase "approximately 1, 2, 3, 4" is "approximately 1, approximately 2, approximately 3, approximately 4 This should be interpreted as follows.

[0066] The word "substantially" does not exclude "completely"; for example, "substantially does not possess Y." The composition does not have to contain Y entirely. Where necessary, the word “substantially” is used in this disclosure. It can be excluded from the definition.

[0067] The term "simultaneous administration" refers to the simultaneous presence of two activators in an individual's blood. The activator (e.g., additional therapeutic agent) administered simultaneously with the indicated antibody and antigen-binding fragment may also be administered simultaneously. Alternatively, it can be delivered continuously.

[0068] Various aspects of this disclosure are described in further detail in the following sections and subsections. ru.

[0069] Fc variant of the present invention In addition to the ability of antibodies to bind to antigens, a key characteristic of antibodies is the mobilization of immune effector functions. It is the ability to do so. Humoral immune response engagement involves interaction with and complement of C1q. Primarily controlled by the initiation of the body cascade (Meyer et al., (2014) M ABS, 6(5):1133-44). Cellular immune responses are largely composed of antibodies and Fc gamma receptors. This is caused by the interaction with the receptor (FcγR). Intracellular signaling is mediated by the activating receptor. Naring has effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), and anti- This leads to inflammation through somatic phagocytosis (ADCP) and the induction of cytokine secretion. Modulated via phosphorylation of the immune receptor tyrosine-based activation motif (ITAM) It will be done.

[0070] While many antibody-based therapies have shown clinical and commercial success, these treatments are not suitable for patients. It is often effective only in small populations. To date, all commercially available antibodies have been FcγRI IgG classes that mobilize immune effector functions via II (CD16), mainly IgG1 This is the case. A typical reaction is finely tuned by the Fc portion of the IgG antibody that triggers ADCC. Activation of natural killer (NK) cells that display FcγRIII on the cell surface It is accompanied by NK cells. However, NK cells are the only component of the innate immune system, and other forms of white blood cells We were able to improve the IgG-triggered ADCC response by using activation of [specific component / agent]. The IgA class of antibodies engages FcαRI, which is widely expressed on neutrophils. Spheres constitute the largest proportion of innate effector cells found in circulation, and their activation is A It triggers both DCC and ADCP. Furthermore, these can invade solid tumors. This has been shown (Gregory & Houghton (2011), cited above). However, Furthermore, since IgG antibodies do not bind to FcαRI, most commercially available antibody-based therapies are not suitable. It cannot activate medullae. Currently, IgA-based therapeutic antibodies are compared to IgG antibodies. Therefore, due to the recognized drawbacks of this class of antibodies, they have not been commercially developed. However, the present applicants have shown that by making modifications to the Fc region, an IgA antibody having improved binding to FcαRI can be generated. This improved affinity up to 1000-fold was shown to be directly converted to an increase in potency in cell-based assays. Thus, when used therapeutically, fewer antibodies are required and the frequency of antibody administration can be reduced, which is advantageous for the patient. This improved affinity up to 1000-fold was shown to be directly converted to an increase in potency in cell-based assays. This improved affinity up to 1000-fold was shown to be directly converted to an increase in potency in cell-based assays. Thus, when used therapeutically, fewer antibodies are required and the frequency of antibody administration can be reduced, which is advantageous for the patient. Thus, when used therapeutically, fewer antibodies are required and the frequency of antibody administration can be reduced, which is advantageous for the patient.

[0071] Generally, as outlined above, the Fc mutants include amino acid modifications in the CH2 domain and / or CH3 domain of the Fc region. The Fc mutants include one or more amino acid modifications relative to the parental Fc polypeptide, where the amino acid modifications optionally provide one or more optimized properties, but in some cases, the mutants exhibit substantially the same biological properties. properties, but in some cases, the mutants exhibit substantially the same biological properties. Properties that can be optimized include, but are not limited to, enhanced or reduced affinity for FcαRI. In one embodiment, the Fc mutant of the invention is modified to have enhanced affinity for human FcαR I. In a preferred embodiment, the Fc region of the Fc mutant is affinity matured such that the amino acid modifications are made in the CH2 and / or CH3 domains to enhance the binding of the Fc region to its target FcαR I. Such types of modifications can improve the binding and / or dissociation kinetics to the target antigen. These optimized properties are expected to provide Fc mutants with enhanced therapeutic properties in humans, such as enhanced effector function and higher anti-cancer efficacy. function and higher anti-cancer efficacy. These optimized properties are expected to provide Fc mutants with enhanced therapeutic properties in humans, such as enhanced effector function and higher anti-cancer efficacy.

[0072] As used herein, "higher affinity" or "improved affinity" compared to the parental Fc polypeptide "Conformity," "enhanced affinity," or "better affinity" refers to the binding assay with the mutant. If the amount of parent polypeptide is essentially the same, the Fc mutant is greater than the parent Fc polypeptide. Significantly higher equilibrium coupling constant (K A ) or a low equilibrium dissociation constant (K D ) binds to the Fc receptor. This means that, for example, an Fc mutant with improved Fc receptor binding affinity will have a surface Measured by plasmon resonance, it is approximately 10 to 100 times greater than that of the parent Fc polypeptide, for example, a small amount Even without it, it can show about 50 times. For example, Fc mutants of the parent Fc polypeptide have surface plasma Measured by Mon resonance, the parent Fc polypeptide has at least about 50, about 100, and about 150 It may have an increased affinity for human FcαRI, approximately 200, 250, or 300 times. ru.

[0073] The Fc receptor selectivity or specificity of a given Fc variant depends on whether it is an antibody, Fc fusion, or antibody. Depending on whether or not it constitutes an Fc mutant with a combined fusion or conjugate partner Therefore, it will offer different characteristics.

[0074] The Fc variants of the present invention include, but are not limited to, FcγRs and / or FcRn. This may include modifications that regulate interactions with Fc receptors other than FcαRIs.

[0075] The Fc variant of the present invention is obtained by modifying at least one amino acid, thereby altering its parent IgA Fc The region has a different amino acid sequence. Therefore, the Fc variant of the present invention has fewer amino acids compared to the parent. It has at least one amino acid modification. Alternatively, the Fc mutant of the present invention has 1 amino acid modification compared to the parent. More than one amino acid modification, for example, about 1 to 10 amino acid modifications compared to the parent, preferably This includes 1-5 amino acid modifications, 1-4 amino acid modifications, 1-3 amino acid modifications, and 1-2 amino acid modifications. It may have a number of amino acid modifications. Therefore, the sequence of the Fc mutant and the arrangement of the parent Fc polypeptide The sequences are substantially homologous or identical. For example, the mutant Fc mutant sequences described herein are the same as the parent F Approximately 80% homology (including identity) with the c variant sequence, preferably at least about 90% Homology, most preferably having at least about 95, 96, 97, 98 and 99% identity. It will probably happen.

[0076] In one embodiment, one or more amino acid insertions, deletions, or substitutions are made. All of this can be done to IgA molecules, for example, IgA1 or IgA2, especially IgA2. In one embodiment, the amino acid substitution is CH2.10, CH2.89, CH2.91 , CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3 This is performed at locations within the Fc region of .109, CH3.118 and / or CH3.124. Furthermore, the numbering of amino acid modifications follows the IMGT numbering for the C-domain. The amino acid substitutions of these (and any possible combination of substitutions, insertions, and deletions) are: A _CH2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91 _V, Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH 2.99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_ This includes, but is not limited to, D, Q_CH3.118_Y and / or L_CH3.124_F. The numbering of amino acid modifications is not yet established and follows the IMGT numbering for the C-domain. In preferred embodiments, amino acid substitutions or combinations thereof include: Q_CH2.94 _E, N_CH2.97Y, S_CH3.45_D, M_CH3.105_Y, Q_CH 3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D、Q_CH2.94_E / M_CH3.105_Y、N_CH2 .97_Y / S_CH3.45_D, N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3 .118_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y , N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2 .94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_D、Q_CH2.94_E / N_CH2 .97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S _CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / N_CH2.9 7_Y / M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N _CH2.97_Y / S_CH3.45_D / M_CH3.105_Y / Q_CH3.1 18_Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_ CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.12 4_F, L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_C H2.97_Y / G_CH2.99_W may be included, but is not limited to these, amino acid modified The numbering of variants follows the IMGT numbering for the C-domain.

[0077] In one embodiment, the amino acid substitutions are CH2.94, CH2.97, CH3.45, CH This can be done at the locations in the Fc region of 3.105 and CH3.118. One embodiment So, the amino acid substitutions are Glu at position CH2.94 and T at position CH2.97. yr, Asp at position CH3.45, Tyr at position CH3.105, or position C This can be done at the position of Tyr in the Fc region in H3.118. Preferred practice In the application form, the amino acid substitutions are Q_CH2.94_E, L_CH2.97_Y, S_CH In the Fc region of 3.45_D, M_CH3.105_Y, and Q_CH3.118_Y, It is possible.

[0078] Functionally, mutants that result in increased binding to FcαRI are found in several embodiments. It finds specific uses in this. Fc variants may contain more than one protein chain. Fc variants are oligomers containing monomers or mono- or hetero-oligomers. Applications can be found in certain antibodies or Fc fusion products.

[0079] Fc fusions, antibody fusions, and antibody conjugates The Fc polypeptide and antibody of the present invention are antibody fragments, bispecific antibodies, minibodies, and domestically produced antibodies. In antibodies, synthetic antibodies (sometimes referred to as "antibody mimes" in this specification), chimeric antibodies, Humanized antibodies, antibody fusion products (sometimes called "antibody conjugates"), and each The present invention may have a variety of structures, including, but not limited to, these fragments. Protein or polypeptide (or fragment thereof, preferably at least 10, at least 2) 0, at least 30, at least 40, at least 50, at least 60, at least 7 (Polypeptides of 0, at least 80, at least 90, or at least 100 amino acids) Recombinantly fused or chemically conjugated (covalent and non-covalent conjugates) (including both of the following), mutant Fc polypeptides and antibodies that produce fusion proteins (for example) If it contains an antibody or antibody-like molecule) or a fragment thereof, it contains a protein or poly((bacterial, Peptides, or methods for the fusion or conjugation of peptides, are known in the art. Yes. For example, U.S. Patent No. 5,336,603, U.S. Patent No. 5,622,929 U.S. Patent No. 5,359,046, U.S. Patent No. 5,349,053 Detailed Statement, U.S. Patent No. 5,447,851, and U.S. Patent No. 5,112,946 Detailed description; European Patent No. 307434 and European Patent No. 367166; International publication Pamphlet No. 1996 / 04388 and International Publication No. 1991 / 06570 Ashkenazi et al., (1991) PNAS.USA 88:10 535-10539;Zheng et al.,(1995)J.Immunol.1 54:5590-5600; and Vil et al., (1992) PNAS.USA See 89:11337-11341.

[0080] Additional fusion proteins are used for gene shuffling, motif shuffling, and exon shuffling. Shuffling, and / or codon shuffling (collectively, "DNA shuffling") It can be generated through a technology called ''. Using DNA shuffling, The activity of the disclosed molecule or fragment thereof can be modified (e.g., higher affinity and (A molecule or fragment thereof with a very low dissociation rate). Generally, U.S. Patent No. 5,605,793. Specification, U.S. Patent No. 5,811,238, U.S. Patent No. 5,830,721 The Book, U.S. Patent No. 5,834,252, and U.S. Patent No. 5,837,458 Book;Patten et al.,(1997)Curr.Opinion Biote chnol.8:724-33;Harayama(1998)Trends Biot echnol.16(2):76-82;Hansson et al.,(1999) J.Mol.Biol.287:265-76; and Lorenzo & Blasco (1998) Biotechniques, 24(2):308-313 (These patents (Each of these publications is incorporated into this Spec. by reference.) The molecules or fragments thereof described herein were subjected to error-prone PCR and random nucleotides before recombination. It can be altered by insertion or by subjecting it to random mutagenesis by other means. The polynucleotides encoding fragments of this molecule are components of one or more heterogeneous molecules. It can be rearranged into components, motifs, sections, parts, domains, fragments, etc.

[0081] Furthermore, the variant Fc polypeptide and antibody of this disclosure have a marker sequence, for example, peptide It can be fused with the marker to facilitate purification. In a preferred embodiment, the marker The amino acid sequence is a hexa-histidine peptide (SEQ ID NO: 81), for example, pQ E-Vector (QIAGEN, Inc., 9259 Eton Avenue, Chats) These are tags provided during (worth, CA, 91311), and many of them are commercially available. Yes, for example, Gentz ​​et al., (1989) PNAS.USA 86:82 As described in 1-824, hexa-histidine (SEQ ID NO: 81) is used in the fusion protein. It provides a simple purification process. Other peptide tags useful for purification include, but are not limited to, However, the hemagglutinin protein corresponds to an epitope derived from the influenza hemagglutinin protein. Rutinin ("HA") tag (Wilson et al., (1984) Cell 37) Examples include :767), and the "flag" tag.

[0082] In other embodiments, the variant Fc polypeptides and antibodies of this disclosure are used as diagnostic or detection agents. It is conjugated to a disease or disorder as part of a clinical trial procedure. Monitoring of the onset, occurrence, progression and / or severity of the disease, or prognosis, e.g., specific treatment This may be useful in determining the effectiveness of the law. Such diagnosis and detection can detect molecules of substances. For example, various enzymes, for instance, However, horseradish peroxidase, alkaline phosphatase, beta-galactosidase -ase, or acetylcholinesterase; prosthetic group, for example, not limited to However, streptavidin / biotin and avidin / biotin; fluorescent materials, for example, limited It is not something that can be done, but umbelliferone, fluorescein, fluorescein isothiocyanate Fluorescein isothiocynate, rhodamine, dichloro Triazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, e.g. For example, though not limited to, luminol; bioluminescent materials, for example, limited to However, luciferase, luciferin, and aequorin; radioactive materials, for example, limited Although not definitively established, iodine (131I, 125I, 123I, and 121I), carbon Element (14C), sulfur (35S), tritium (3H), indium (115In, 113) In, 112In, and 111In), technetium (99Tc), thallium (201Tc) i) Gallium (68Ga, 67Ga), Palladium (103Pd), Molybdenum (99 Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159 Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186R e, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113S n and 117Tin; and positron-emitting metals using various positron emission tomography techniques, This can be achieved by coupling with non-radioactive paramagnetic metal ions.

[0083] This application relates to the mutant Fc polypeptide of the present disclosure conjugated in the therapeutic portion and anti It further encompasses the use of the body. For example, the therapeutic part is a cytotoxin, e.g., a cell proliferation inhibitor or It may be a cytotoxic agent, a therapeutic agent, or a radioactive metal ion, such as an alpha emitter. Alternatively, cytotoxic agents include any drug that is harmful to cells.

[0084] Furthermore, the mutant Fc polypeptide and antibody are therapeutic molecules that modify a given biological response. Alternatively, it can be conjugated to the drug portion. For example, the drug portion can be conjugated to the desired biological It may be an active protein, peptide, or polypeptide. In terms of quality, for example, toxins, for example, abrin, lysine A, Pseudomonas exotoxin, Diphtheria toxin, or rheumatoid toxin; proteins, e.g., tumor necrosis factor, α-interferon β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen Activators, apoptotic agents, anti-angiogenic agents; or biological response modifiers, for example, Examples include lymphokines.

[0085] Cytotoxins, linker types, and therapeutic agents are conjugated into mutant Fc polypeptides and antibodies. For further consideration of gate methods, see Saito et al., (2003) A dv.Drug Deliv.Rev.55:199-215;Trail et al .,(2003)Cancer Immunol.Immunother.52:328 -337;Payne(2003)Cancer Cell 3:207-212;Al len(2002)Nat.Rev.Cancer,2:750-763;Pastan & Kreitman(2002)Curr.Opin.Investig.Drug s,3:1089-1091;Senter & Springer(2001)Adv See also .Drug Deliv.Rev.53:247-264.

[0086] The variant Fc polypeptide and antibody of this disclosure are conjugated with radioisotopes to produce radioactive material. It can also generate cytotoxic radiopharmaceuticals, also known as immunoconjugates. It can be conjugated into artificially engineered immunoglobulins for therapeutic use. Examples of sex isotopes, though not limited to them, include iodine-131 and indium-111. Examples include yttrium-90 and lutetium-177. Radioactive immunoconjugates are being investigated. The method of production is established in this field. For example, Denardo et al. l.,(1998)Clin Cancer Res.4(10):2483-90;P eterson et al.,(1999)Bioconjug.Chem.10(4 ):553-7; and Zimmerman et al., (1999) Nucl.Me See d.Biol.26(8):943-50, each reference incorporated as a whole. It can be done.

[0087] The therapeutic portion is conjugated to a mutant Fc polypeptide and an antibody, such as an antibody or antibody-like molecule. The techniques for gate are publicly known, for example, Arnon et al., “Monoc lonal Antibodies for Immunotargeting of Drugs in Cancer Therapy”, Monoclonal Anti bodies and cancer therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug De Livery”,Controlled Drug Delivery(2nd Ed. ), Robinson et al.(eds.), pp.623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carri ers of Cytotoxic Agents in Cancer Therapies y:A Review”, Monoclonal Antibodies 84:Bio logical and clinical applications,Pinche ra et al.(eds.),pp.475-506(1985);“Analys is,Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibod y in Cancer Therapy”, Monoclonal Antibodies es for Cancer Detection and Therapy,Bald win et al.(eds.),pp.303-16(Academic Pres. s 1985), and Thorpe et al., (1982) Immunol. Re See v.62:119-58.

[0088] Mutant Fc polypeptides and antibodies are particularly useful for immunoassays or the purification of target antigens. It can also be attached to a solid carrier. Such solid carriers are limited in number. However, glass, cellulose, polyacrylamide, nylon, polystyrene, polysalt Examples include vinyl oxide or polypropylene.

[0089] The Fc variants of the present invention provide one or more Fc variants that offer reduced or enhanced internal translocation. This may include modifications. In one embodiment, the Fc variant of the present invention comprises one or more Fc ligands and It is used to reduce the intracellular translocation of Fc mutants, which occurs through interactions. This can be combined with additional modifications. This characteristic enhances the effector function and the F of the present invention. It is expected that the immunogenicity of the c variant may be potentially reduced. Alternatively, the Fc variant of the present invention Mutations occur within cells of Fc mutants through interactions with one or more Fc ligands. To enhance the transition, it can be used directly or combined with additional modifications.

[0090] In preferred embodiments, modifications include stability, solubility, and oligomeric state, but these The invention is made to improve the biophysical properties of Fc variants, not limited to those described herein. For example, to provide greater stability, the Fc variant is preferred. Substitutions that provide better intramolecular interactions, or exposed nonpolar amino acids for greater stability. This may include substitution of ano acids with polar amino acids. To further optimize the Fc variant of the present invention Therefore, several optimization goals and methods available for additional modification operations are provided by reference. The present invention is described in U.S. Patent No. 10 / 379,392, which is incorporated herein by reference. The Fc variant enhances tumor penetration or the in vivo clearance rate as desired. Combine with additional modifications that reduce the oligomeric state or size in order to increase it. It is also possible. Other modifications to the Fc variant of the present invention include specific formation or homodimerization. Examples include those that enable the formation of a single molecule or a homomultimer molecule. Such modifications involve manipulation This may provide a mechanism for generating disulfides and covalent homodimers or homomultimers. This includes, but is not limited to, chemical modification or aggregation methods for such molecules. The operating method and composition are described in Kan et al., (2001) J. lmmunol., 1 66:1320-1326;Stevenson et al.,(2002)Rece nt Results Cancer Res. 159:104-12; US 5, 681,566;Caron et al.,(1992),J.Exp.Med.17 6:1191-1195 and Shapes (1992) J. lmmunol. 148(9 ): As described in 2918-22 (all incorporated herein by reference). Additional modifications to the variant of Akira include specific formations or homodimers, homomultimers, and bifacial structures. Examples include those that enable functional and / or polyfunctional molecules. Such modifications include This includes, but is not limited to, one or more amino acid substitutions in the CH3 domain. Substitutions reduce homodimer formation and increase heterodimer formation. For example, Methods for manipulating molecules and compositions are described in Atwell et al., 1997, J.Mol. Bioi.270(1):26-35 and Carter et al., 2001, J. lmmunol.Methods 248:7-15 (both incorporated herein by reference) (Included) is described. Additional modifications include modifications in the hinge and CH3 domain. This modification involves a change that reduces the tendency to form dimers.

[0091] Preparation of Fc mutant polypeptides The antibodies and fragments thereof containing the variant Fc polypeptide disclosed herein are different from conventional mono Clonal antibody methods, e.g., Kohler & Milstein, (1975) Na Through a variety of techniques, including standard somatic cell hybridization as described in ture 256:495 It can be generated.

[0092] The animal system used to prepare hybridomas is the mouse system. Doma production is a known procedure. Immunization protocol and immunized spleen for fusion. Techniques for isolating cells are known in the art. Fusion partners (e.g., mouse bone) The myeloma cells and fusion procedures are also known.

[0093] The chimeric or humanized antibody is formed by modifying the sequence of the mouse monoclonal antibody prepared as described above. It can be prepared based on the following: DNA encoding heavy chain and light chain immunoglobulins of the desired type Obtained from mouse hybridomas and converted to non-mouse (e.g., human) using standard molecular biology techniques. ) It can be artificially manipulated to contain immunoglobulin sequences. For example, chimeric antibodies. To create this, the mouse variable region was modified to the human steady-state region using a method known in the art. It can be coupled to a region (for example, U.S. Patent No. 4,816,567, Cab). (See illy et al.). To produce humanized antibodies, known in the art The mouse CDR region can be inserted into the human framework using this method. For example For example, U.S. Patent No. 5,225,539, Winter, and U.S. Patent No. 5,530 , Patent No. 101; U.S. Patent No. 5,585,089; U.S. Patent No. 5,693,7 See U.S. Patent No. 62 and U.S. Patent No. 6,180,370, Queen et al. Light.

[0094] In one embodiment, the antibody comprising the antibody or the Fc isomer described herein is The fragment is a human monoclonal antibody. Such human monoclonal antibodies are used in mice. Transgenic or transchromosomal individuals that carry a part of the human immune system rather than the entire system. These can be generated using mice. These transgenic and transchromic In this specification, mosomic mice are referred to as HUMAB mice and KM mice, respectively. Examples of mice referred to as such include the "human Ig mouse," which in this specification are collectively referred to as the "human Ig mouse." .

[0095] HUMAB mice (Medarex, Inc.) are unreorganized human heavy chains (μ Human immunoglobulin gene minigenes encoding γ) and κ light chain immunoglobulin sequences Targeted mutations that inactivate the miniloci locus and the endogenous μ and κ chain gene loci Different (for example, Lonberg, et al., (1994) Nature 368(64 It is contained together with mouse IgM (see 74):856-859). Therefore, mice have mouse IgM or It shows reduced expression of κ, and in response to immunization, introduced human heavy chain and light chain transgenes The offspring undergoes class switching and somatic mutation to produce high-affinity human IgGκ monoclonal. To generate (Lonberg et al., (1994) cited above; Lonberg, (19 94) Handbook of Experimental Pharmacology 113:49-101;Lonberg & Huszar,(1995)Inter n.Rev.Immunol.13:65-93, and Harding & Lonbe An overview can be found in rg, (1995) Ann. NYAcad. Sci. 764:536-546. (It is being done.) Preparation and use of HUMAB mice, and the mice that carry the mice The genome modification described is Taylor et al., (1992) Nucleic Aci ds Research 20:6287-6295;Chen et al.,(19 93)International Immunology 5:647-656;Tu aillon et al., (1993) PNAS USA 94:3720-372 4;Choi et al.,(1993)Nature Genetics 4:11 7-123;Chen et al.,(1993)EMBO J.12:821-83 0;Tuaillon et al.,(1994)J.Immunol.152:29 12-2920;Taylor et al.,(1994)Int.Immun.,5 79-591; and Fishwild et al., (1996) Nature Bi Further details are found in otec., 14:845-851, and all of those contents are referenced. This is specifically incorporated herein as a whole. Furthermore, U.S. 5,545,8 U.S. Patent No. 06; U.S. Patent No. 5,569,825; U.S. Patent No. 5,625,126 U.S. Patent No. 5,633,425; U.S. Patent No. 5,789,650 Detailed document; U.S. Patent No. 5,877,397; U.S. Patent No. 5,661,016 ; U.S. Patent No. 5,814,318; U.S. Patent No. 5,874,299; and U.S. Patent No. 5,770,429; All rights reserved by Lonberg & Kay; U.S. Patent Patent No. 5,545,807, Surani et al.; International Publication No. 92 / 10 Pamphlet No. 3918, International Publication No. 93 / 12227, International Publication No. 94 / Brochure No. 25585, International Publication No. 97113852, International Publication No. Brochure No. 98 / 24884 and International Publication No. 99 / 45962, all Lonberg & Kay; and International Publication No. 01 / 14424, Ko See rman et al.

[0096] In another embodiment, human antibodies are used in the transgene and on the transchromosome, as well as in human immunoglobulins. Mice carrying the chromosome sequence, for example, mice carrying the human heavy chain transgene and the human light chain transchromosome. This can be achieved using mice. Such mice are referred to herein as " It was called the "KM Mouse," and was presented in the International Publication No. 2002 / 43478 pamphlet (Ishida). This is described in detail in (et al).

[0097] Furthermore, alternative transgenic animal systems that express human immunoglobulin genes are being developed for this technology. It is available in the field of technology and can be used to generate human antibodies. Example For example, an alternative transgender person called Xenomouse (Abgenix, Inc.) Nick-type mice can be used. Such mice are, for example, 5,939 in the United States. U.S. Patent No. 598; U.S. Patent No. 6,075,181; U.S. Patent No. 6,114,5 U.S. Patent No. 98; U.S. Patent No. 6,150,584 and U.S. Patent No. 6,162,96 It is described in Specification No. 3 (Kucherlapati et al).

[0098] Furthermore, alternative transchromosomal animal systems that express human immunoglobulin genes This technology is available in this field and can be used to generate human antibodies. For example, both human heavy chain transchromosomes and human light chain transchromosomes, referred to as "TC mice" Mice carrying the gene can be used; such mice were found by Tomizuka et al. This is described in al., (2000) PNAS USA 97:722-727. In this technology, cattle carrying human heavy and light chain transchromosomes have been described ( Kuroiwa et al.,(2002)Nature Biotechnolog (y 20:889-894), and using it to produce useful human antibodies in this application It is possible.

[0099] Human monoclonal antibodies or fragments thereof are used in libraries of human immunoglobulin genes. It can also be prepared using phage display methods for screening. Such phage display methods for isolating antibodies are well established in the art. or as described in the following examples, for example, U.S. Patent No. 5,223,409; U.S. Patent No. 5,403,484; and U.S. Patent No. 5,571,698 ( Ladner et al); U.S. Patent No. 5,427,908 and U.S. Patent No. 5 ,580,717 (Dower et al); U.S. Patent No. 5,969,108 The U.S. Patent No. and U.S. Patent No. 6,172,197 (McCafferty et al. l); and U.S. Patent No. 5,885,793; U.S. Patent No. 6,521,404 Detailed document; U.S. Patent No. 6,544,731; U.S. Patent No. 6,555,313 ; U.S. Patent No. 6,582,915 and U.S. Patent No. 6,593,081 ( See Griffiths et al.

[0100] Human monoclonal antibodies or fragments thereof that are useful in this disclosure are human antibody-responsive during immunization. Using SCID mice in which human immune cells have been reconstituted to produce a response Such mice can also be prepared in the United States, for example, 5,476,996 As described in the U.S. Patent No. 5,698,767 (Wilson et al.) It is listed.

[0101] The human monoclonal antibodies or fragments thereof prepared according to the present invention, as described below, By introducing mutations into amino acid residues within the VH, VL, CH1, CL, CH2, and CH3 domains This determines one or more binding properties of the antibody or fragment thereof against the target receptor (e.g., parent It can be further modified to improve compatibility (a process known as "affinity maturation"). Mutations are introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and receptors The effects on body bonding or other functional properties for a particular purpose are described herein and provided in the examples. It can be evaluated in vitro or in vivo assay. In terms of application, this disclosure relates to affinity-mature antibodies or fragments thereof, particularly affinity-mature Fc regions. Mutations may be amino acid substitutions, additions, or deletions. For example, the Fc mutations of this disclosure. The body is an affinity-mature Fc region, where within the CH2 domain and / or CH3 domain One, two, three, four, or five or more residues have been modified. All of these substitutions are, This can occur in IgA molecules, such as IgA1 or IgA2, and particularly in IgA2. In preferred embodiments, the amino acid substitutions are CH2.10, CH2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, CH3.105, CH3. This may be done at the location in the Fc region of 109, CH3.118 and / or CH3.124. The numbering of amino acid modifications follows the IMGT numbering for the C-domain. Amino acid substitutions include (and any possible combination of substitutions, insertions, and deletions): A_C H2.10_S, L_CH2.89_I, G_CH2.91_Q, G_CH2.91_V , Q_CH2.94_E, N_CH2.97_H, N_CH2.97_Y, G_CH2. 99_W, S_CH3.45_D, M_CH3.105_Y, E_CH3.109_D, This includes, but is not limited to, Q_CH3.118_Y and / or L_CH3.124_F. However, the numbering of amino acid modifications follows the IMGT numbering for the C-domain. In a more appropriate embodiment, amino acid substitutions in affinity-mature Fc mutants of the present invention or their The combinations include: Q_CH2.94_E, N_CH2.97Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2 .97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M _CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2.9 7_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M _CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2. 97_Y / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M _CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.1 05_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.9 4_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_ CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3.11 8_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_C H3.105_Y / Q_CH3.118_Y, A_CH2.10_S, L_CH2.89 _I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH 3.109_D, L_CH3.124_F, L_CH2.89_I / G_CH2.91_ V / Q_CH2.94_E / N_CH2.97_Y / G_CH2.99_W may be included. However, this is not limited to these, and the numbering of amino acid modifications is based on the IMGT number for the C-domain. Follow the numbering system.

[0102] Nucleic acids and expression systems The present invention also includes nucleic acids encoding polypeptide chains of Fc variants described herein. The nucleic acid molecules of this disclosure include both single-stranded and double-stranded DNA and RNA, and Examples of complementary sequences that correspond to each other include the nucleic acid molecule of the present disclosure, such as a full-length gene or cD Examples include NA molecules and combinations thereof. The nucleic acids of this disclosure are derived from human resources. However, this may include species originating from non-human species.

[0103] "Isolated nucleic acid" refers to nucleic acid isolated from naturally occurring resources, or from the organism from which the nucleic acid was isolated. These are nucleic acids isolated from adjacent gene sequences present in the genome. For example, enzymes from a template. nucleic acids synthesized artificially or chemically, such as PCR products, cDNA molecules, or oligonucleotides. In the case of creotides, it is understood that the nucleic acids resulting from such processes are isolated nucleic acids. Isolated nucleic acid molecules exist in the form of individual fragments or as components of larger nucleic acid constructs. This refers to nucleic acid molecules. In one preferred embodiment, the nucleic acid substantially removes contaminating endogenous material. It does not contain. Nucleic acid molecules are preferably in a substantially pure form and by standard biochemical methods. (For example, Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed.,Cold Spring H arbor Laboratory,Cold Spring Harbor,NY(1 Identification, manipulation, and rotation of its constituent nucleotide sequences as outlined in 989) It is derived from DNA or RNA that has been isolated at least once in an amount or concentration that allows for yield. Such sequences are preferably internal untranslated sequences typically found in eukaryotic genes. Provided in the form of an open reading frame that is not interrupted by columns or introns. / or constructed. The sequence of uncoding DNA is taken from the open reading frame at 5' or 3'. It may exist in ', and in that case, it does not interfere with the manipulation or expression of the code area.

[0104] Variant arrays, such as libraries of variant arrays, are outlined herein. and a cassette or PCR mutation to produce the DNA encoding that variant. D, which codes for polypeptides, using heterogeneity or other techniques known in the art. Site-directed mutagenesis of nucleotides in NA, and subsequent recombination in cell cultures. It can be prepared by expressing DNA.

[0105] The "optimized nucleotide sequence" is used in producing cells, such as Chinese hamster ovary cells. (CHO) uses preferred codons to encode the amino acid sequence. This means the nucleotide sequence has been changed. The optimized nucleotide sequence is also the "parent" sequence. It completely preserves the amino acid sequence initially encoded by a known starting nucleotide sequence. Sea urchins are artificially manipulated.

[0106] This disclosure also relates to plasmids containing at least one of the above polynucleotides, expression vectors The present disclosure provides expression systems and constructs in the form of a ter, transcription, or expression cassette. Furthermore, this disclosure provides The present invention provides host cells containing such an expression system or construct.

[0107] In one embodiment, the present invention provides an antibody comprising a variant Fc region as described herein or the The present invention provides a method for preparing fragments, the method of which: (a) mutant heavy chain and light chain polypeptides A step of culturing host cells containing nucleic acids to be mutant, wherein the cultured host cells are mutant (b) expressing a lipeptide; (b) recovering an antibody or fragment thereof from a host cell culture. Includes the process.

[0108] The expression vectors used in this disclosure are derived from a starting vector, for example, a commercially available vector. It is possible to construct a vector and artificially manipulate the polypeptide chain of immunoglobulin. After inserting the nucleic acid molecule to be routed into the appropriate site in the vector, the completed vector is amplified and / or It can be inserted into suitable host cells for polypeptide expression. In selected host cells Transformation of expression vectors into [the target organism] is carried out by known methods, such as transduction, infection, and calcium phosphate. Coprecipitation, electroporation, microinjection, lipofection, D This can be achieved by EAE-dextran-mediated translocation or by other known techniques. The method selected depends in part on the type of host cell to be used. Other suitable methods are known to those skilled in the art, for example, Sambrook et al. As stated above, 2001.

[0109] Typically, expression vectors used in host cells are for plasmid maintenance and It contains sequences for cloning and expression of exogenous nucleotide sequences. The rows are collectively referred to as a "flanking arrangement," and in one embodiment, they are typically, One or more of the following nucleotide sequences: promoter, one or more enhancer sequences, replication A complete intron sequence containing a point, transcription termination sequence, donor and acceptor splice site. Column, sequence encoding the leader sequence for polypeptide secretion, ribosome binding site, A riadenylated sequence, a polymer for inserting nucleic acid encoding the polypeptide to be expressed. This includes marker regions and selection marker elements.

[0110] Host cells, when cultured under appropriate conditions, can be used to express Fc mutants. It can then be removed from the culture medium (if the host cell secretes it into the medium) or It can be recovered directly from the host cell that produces it (if it is not secreted). Careful selection of host cells is crucial, as it involves various factors such as the desired expression level and activity level, and whether they are desirable or necessary. Essential polypeptide modifications (e.g., glycosylation or phosphorylation) and biologically active molecules It depends on the ease of folding. The host cell can be either a eukaryote or a prokaryote. .

[0111] Mammalian cell lines that can be used as expression hosts are known in this field, and However, this is not limited to American Type Culture C Immortalized cell lines available from collection (ATCC) and public in this technology Any cell line used in the knowledge expression system is listed, and using them to perform the artificial manipulations of this disclosure. polypeptides containing immunoglobulins can be produced. Generally, host cells are desired Transformed with a recombinant expression vector containing DNA encoding artificially engineered immunoglobulins Among the host cells that can be used, prokaryotes, yeasts, or higher eukaryotes are present. Prokaryotes include Gram-negative and Gram-positive organisms, such as Escherichia coli (E. coli). Examples include insect cells and established cells of mammalian origin. Cellular cell lines are one example. Suitable mammalian host cell lines include COS-7 cells, L cells, and C cells. L27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, or serum-free medium These derivatives and related cell lines that grow within, HeLa cells, BHK cell lines, CVIIE BNA cell line, human embryonic kidney cells, e.g., 293, 293EBNA or MSR293, human Epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells Cells, cell lines derived from in vitro cultures of primary tissue, primary explants, HL-60, U937 Examples include HaK or Jurkat cells. Optionally, various signaling or repositories are used. When it is desirable to use polypeptides in a transassay, mammalian cell lines, e.g. For example, HepG2 / 3B, KB, NIH3T3, or S49 can be used for polypeptide expression. It can be used in lower eukaryotes, for example, yeast, or in prokaryotes, for example, It is possible to produce polypeptides in bacteria. Suitable yeasts include budding yeast (S .cerevisiae), fission yeast (S. pombe), genus Kluberomyces (Klu Candida strains (yveromyces), Candida genus, or heterologous polypeptides Any yeast strain that may be expressed is listed. Suitable bacterial strains include Escherichia coli (E. coli), Bacillus subtilis, Salmonella typhimurium, and This includes any bacterial strain capable of expressing heterologous polypeptides. Artificially engineered immunoglobulins When prepared in yeast or bacteria, the product produced therein is, for example, phosphorus at the appropriate site. It is sometimes desirable to obtain functional products by modifying them through oxidation or glycosylation. Such covalent bonds can be achieved using known chemical or enzymatic methods.

[0112] In further embodiments, the Fc variant of the present invention is modified to remove the proteolytic site. These include, for example, protease sites that reduce product yield, and administered tannins. It may contain a protease site that breaks down proteins in vivo.

[0113] In preferred embodiments, the Fc variant is purified or isolated after expression. The protein is It can be isolated or purified by a variety of methods known in the art. A standard purification method is at atmospheric pressure or This involves ion exchange and hydrophobicity, performed under high pressure using systems such as FPLC and HPLC. Chromatographic techniques including interaction, affinity, sizing, or gel filtration, and reversed phase. Includes. Purification methods also include electrophoresis, immunological, precipitation, dialysis, and isoelectric focusing techniques. Ultrafiltration and dialysis filtration techniques, combined with protein concentration, are also useful. In sea urchins, various natural proteins bind to Fc and antibodies, and these proteins are linked to Fc mutations. It can be used in the present invention for the purification of the body. For example, bacterial proteins A and G are F It binds to the c region. Similarly, bacterial protein L, like the target antigen of antibodies, It binds to the Fab region of several antibodies. Purification is often performed by a specific fusion partner. This could be possible. For example, in Fc variants, if a GST fusion is used, glutathione When on resin, His tags are used, Ni +2 affinity chromatography, or flara When gtag is used, it can be purified using immobilized anti-flag antibodies. Appropriate purification techniques are required. For general guidance in this regard, see, for example, the Pro which is incorporated entirely by reference. tein Purification:Principles and Practical e, 3rd Ed., Scopes, Springer-Verlag, NY, 1994. See (which is incorporated into this specification by reference).

[0114] Pharmaceutical composition and administration Pharmaceutical compositions comprising the Fc variant of the present disclosure are provided herein. The Fc variant is an antibody form In the formula, for example, as a monospecific, bispecific, or multispecific antibody, one or more pharmaceutically acceptable antibodies It can be incorporated in combination with excipients, diluents, or carriers that may be used.

[0115] To prepare a pharmaceutical or sterile composition containing the Fc variant of this disclosure, the molecule is pharmaceutically acceptable. Mix with a suitable carrier or excipient. The phrase "pharmaceutically acceptable" is used by federal or state governments. Approved by a government regulatory body, or used in animals, or more specifically, in humans. This means that it is listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "pharmaceutical composition" refers to at least one active ingredient (e.g., Fc as defined in this disclosure). This refers to a mixture of a variant and at least one pharmaceutically acceptable excipient, diluent, or carrier. "Pharmaceuticals" refer to substances used in medical procedures.

[0116] Pharmaceutical compositions for therapeutic and diagnostic agents include, for example, freeze-dried powders, slurries, aqueous solutions, and lotions. Mixed with a physiologically acceptable carrier, excipient, or stabilizer in the form of a nut or suspension. It can be prepared by (for example, Hardman, et al. (2001) Go odman and Gilman's The Pharmacological B asis of Therapeutics,McGraw-Hill,New York k,NY;Gennaro(2000)Remington:The Scienc. e and Practice of Pharmacy, Lippincott, Wi. lliams,and Wilkins,New York,NY;Avis,et al.(eds.)(1993)Pharmaceutical Dosage Fo rms:Oral Medications,Marcel Dekker,NY;Li eberman,et al.(eds.)(1990)Pharmaceutical Dosage Forms:Tablets,Marcel Dekker,NY;L ieberman, et al. (eds.) (1990) Pharmaceutica l Dosage Forms:Disperse Systems,Marcel D ekker,NY;Weiner & Kotkoskie(2000)Excipie nt Toxicity and Safety,Marcel Dekker,Inc (See New York, NY).

[0117] The selection of a drug regimen for therapeutic purposes depends on several factors, such as the substance of the serum or group. Tissue turnover rate, symptom level, immunogenicity of the entity, and biological matrix It depends on the accessibility of the target cells. In one embodiment, the administration regimen is permissible. To maximize the amount of therapeutic drug delivered to the patient in line with Bell's side effects. Therefore, delivery The amount of biopharmaceutical administered depends, in part, on the specific organism and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies, cytokines, and small molecules are available (for example). , Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.Ltd,Oxfordshire,UK;Kresin a(ed.)(1991) Monoclonal Antibodies, Cytoki nes and Arthritis, Marcel Dekker, New York ,NY;Bach(ed.)(1993) Monoclonal Antibodies es and Peptide Therapy in Autoimmune Dis eases,Marcel Dekker,New York,NY;Baert, et al.(2003)New Engl.J.Med.348:601-608;M ilgrom,et al.(1999)New Engl.J.Med.341:19 66-1973;Slamon,et al.(2001)New Engl.J.Me d.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 Engl. J. Med. 343:1594-1602) .

[0118] Determining the appropriate dosage is, for example, a matter known to affect the treatment in the art. Using parameters or factors that are suspected or predicted to affect the treatment This is performed by a clinician. Generally, the dose is started at a level somewhat lower than the optimal dose, and After that, gradually increase it until the desired or optimal effect is achieved for any negative side effects. To cause. Important diagnostic measures include, for example, symptoms of inflammation or the production of inflammatory cells. The level of tocaine can be mentioned.

[0119] The actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein are specific and without toxicity to the patient. Regarding the patient, composition, and method of administration, effective active ingredients are used to achieve the desired therapeutic response. The amount can be changed to obtain the desired dose. The selective dose level depends on various pharmacokinetic factors, e.g. The activity of the specific compositions of this disclosure used, or their esters, salts, or amides, Route of administration, time of administration, excretion rate of the specific compound used, duration of treatment, and usage Other drugs, compounds, and / or materials used in combination with specific compounds, treated The patient's age, sex, weight, medical condition, overall health status, and medical history, as well as information in the medical field. It depends on similar factors that are known.

[0120] The pharmaceutical compositions comprising the Fc variant of this disclosure can be administered by continuous injection, or, for example, daily or weekly. Alternatively, it can be provided in doses administered at intervals of 1 to 7 times per week. The dose is administered intravenously or in a transdermal manner. By lower, locally, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation It can be provided.

[0121] The desired dose of a therapeutic agent containing the Fc variant in this disclosure is based on the amount of antibody or poly(mol / kg body weight). It is almost the same as a lipeptide. The dose administered to the subject is at least 1, 2, 3, 4, 5 It can be 6, 7, 8, 9, 10, 11, or 12 or more.

[0122] For therapeutic agents containing the Fc variants of this disclosure, the dose administered to a patient is approximately 0.0001 The dosage can range from mg / kg to approximately 100 mg / kg of patient body weight.

[0123] When administering a series of doses, for example, administering them almost daily, almost weekly, and almost monthly. The dosage may be adjusted, for example, in the event of disease progression, adverse events, or other circumstances determined by the physician. It can be administered continuously until the end of the course.

[0124] The effective dose for a specific patient depends on factors such as the condition being treated and the patient's overall health. The dosage may vary depending on the condition, method of administration, route and dose, and the severity of side effects (for example, M aynard, et al. (1996) A Handbook of SOPs fo r Good Clinical Practice, Interpharm Pres. s,Boca Raton,Fla.;Dent(2001)Good Laborat ory and Good Clinical Practice,Urch Publ (See London, UK).

[0125] If necessary, therapeutic agents comprising the Fc variants of this disclosure may be used to solubilize and relieve pain at the injection site. A local anesthetic to reduce the effect can be incorporated into a composition containing, for example, lidocaine. Furthermore, for example, the use of inhalers or nebulizers and formulations containing aerosolizing agents. Depending on the application, intrapulmonary administration may also be used. For example, U.S. Patent No. 6,019,968 The document, U.S. Patent No. 5,985,320, U.S. Patent No. 5,985,309, U.S. Patent No. 5,934,272, U.S. Patent No. 5,874,064, United States Japanese Patent No. 5,855,913, U.S. Patent No. 5,290,540, and U.S. Japanese Patent No. 4,880,078; and International Publication No. 92 / 19244 International Publication No. 97 / 32572, International Publication No. 97 / 44013 , International Publication No. 98 / 31346, and International Publication No. 99 / 66903 See the brochure; each of these is incorporated herein by reference as a whole.

[0126] The therapeutic agents comprising the Fc variants of this disclosure may be obtained by one or more of the various methods known in the art. It can also be administered via one or more routes of administration using [this method]. (As recognized by those skilled in the art) As such, the route and method of administration will vary depending on the desired results. This can be administered intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, spinally, or via other parenteral routes, such as injection. Other methods include administration by injection. Parenteral administration usually involves methods other than intestinal and local administration by injection. This can represent the method of administration, and is not limited to intravenous, intramuscular, or arterial administration. Intrathecal, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, capsular Examples include injections and infusions below, subarachnoid, intraspinal, epidural, and intrasternal. Alternatively, the present disclosure The composition is administered via parenteral routes, such as topical, dermal, or mucosal routes, for example, intranasal. It can be administered orally, vaginally, rectally, sublingually, or topically.

[0127] Therapeutic agents comprising the Fc variants of this disclosure include, for example, injection devices, injection pens, vials, and Syringes, pre-filled syringes, auto-injectors, infusion pumps, patch pumps, infusion bags It can also be administered via any of the above routes using a needle or the like. Fc of the present disclosure When administering a therapeutic agent containing a variant in a controlled-release or sustained-release system, use a pump for controlled release. Alternatively, sustained release can be achieved (Langer, op. cit.; Sefton, 1987, CR). C Crit.Ref Biomed.Eng.14:20;Buchwald et al. al.,1980,Surgery 88:507;Saudek et al.,19 See 89, N.Engl.J.Med.321:574). Using polymer materials for the final opening Controlled release or sustained release of the indicated therapeutic agent can be achieved (for example, Medical Ap applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability,D rug Product Design and Performance,Smole n & Ball (eds.), Wiley, New York (1984);Rang er & Peppas(1983)J.Macromol.Sci.Rev.Macr See omol.Chem.23:61; Levy et al., (1985) Scie. nce 228:190;During et al.,(1989)Ann.Neur ol.25:351;Howard et al.,(1989)J.Neurosur g., 7(1):105; U.S. Patent No. 5,679,377; U.S. Patent No. 5,91 U.S. Patent No. 6,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989, U.S. Patent No. 463; U.S. Patent No. 5,128,326; International Publication No. 99 / 15154 See also Pamphlet No. 99 / 20253, International Publication No. 99 / 20253. Sustained-release formulations Examples of polymers used in this process include, but are not limited to, poly(2-hydroxypropyl hydroxide) polymers. Poly(methyl methacrylate), poly(acrylic acid), poly (Ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), Polyanhydrous, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylic Amides, poly(ethylene glycol), polylactide (PLA), poly(lactide-co Examples include (-glycolide) (PLGA) and polyoltoester. In one embodiment, Furthermore, the polymers used in sustained-release formulations are inert, free from leaching impurities, and have storage stability. Furthermore, it is sterile and biodegradable. Controlled-release or sustained-release systems are positioned close to the preventive or therapeutic target. This is possible, and therefore only a small portion of the total systemic dose is required (for example, Goodson,Medical Applications of Controll See ed Release, cited above, vol.2, pp.115-138 (1984).

[0128] The controlled emission system is described in Langer (Science (1990) 249:1527-153). This is discussed in the overview in 3). Using any technology known to those skilled in the art, the present application A sustained-release formulation containing one or more Fc variants can be produced. For example, U.S. Patent No. 4 Specification No. 526,938, International Publication No. 91 / 05548, International Publication No. 9 Pamphlet No. 6 / 20698, Ning et al., (1996) Radio erapy & Oncology 39:179-189;Song et al., (1995)PDA Journal of Pharm Sci & Tech.,5 0:372-397;Cleek et al.,(1997)Pro.Int'l.S ymp.Control.Rel.Bioact.Mater.24:853-854; Lam et al.,(1997)Proc.Int'l.Symp.Control See Rel. Bioact. Mater., 24:759-760, each of them This is incorporated herein by reference in its entirety.

[0129] When a pharmaceutical composition containing the Fc variant of this disclosure is administered topically, it may be an ointment, cream, etc. Transdermal patches, lotions, gels, shampoos, sprays, aerosols, liquids, It can be formulated in the form of an emulsion or in other forms known to those skilled in the art. For example, Rem ington's Pharmaceutical Sciences and Int. production to Pharmaceutical Dosage Forms , 19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-spray topical formulations, it contains a carrier or one or more excipients suitable for topical application, and is partially In the example, a semi-solid or solid form is typically obtained from a viscosity that has a greater dynamic viscosity than water. It is used. Suitable formulations are not limited to liquids, suspensions, and milk. Examples include liquid preparations, creams, ointments, powders, liniments, and ointments, and these are, Depending on the preference, sterilization or auxiliary agents may be used to affect various properties, such as osmotic pressure. It is mixed with (for example, preservatives, stabilizers, humectants, buffers, or salts). Other suitable topical agents In terms of form, the active ingredient is pressurized in combination with a solid or liquid inert carrier in some cases. In a mixture with volatile substances (e.g., gas propellants, e.g., Freon), or squeeze Examples include spray aerosol formulations packaged in bottles. Moisturizers are also available, if desired. Alternatively, humidifiers may be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are This is publicly known in the art.

[0130] When a pharmaceutical composition containing the Fc variant of this disclosure is administered intranasally, it may be administered in aerosol form. It can be formulated in the form of a spray, mist, or droplets. In particular, the use according to this disclosure A preventive or therapeutic agent for this purpose may be a suitable propellant (e.g., dichlorodifluoromethane, trichlorodifluoromethane). (Lolofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases) Conveniently in the form of aerosol spray dispensed from a pressure pack or nebulizer. It can be delivered. In the case of pressurized aerosols, the dose unit is the measured amount delivered. This can be determined by providing a valve for the compound and suitable powder base, for example For use in inhalers or sprayers containing a mixture of lactose or starch powders Capsules and cartridges (for example, made of gelatin) can be formulated for this purpose. can.

[0131] The pharmaceutical compositions comprising the Fc variants of this disclosure may also be administered to patients on a regular basis.

[0132] In one embodiment, a pharmaceutical composition comprising the Fc variant of the present disclosure is appropriately distributed in vivo. It can be formulated to ensure that the blood-brain barrier (BBB) ​​is a high-grade barrier. To eliminate hydrophilic compounds. To ensure that the therapeutic compounds of this disclosure pass through the BBB. (Optionally), these can be formulated, for example, in liposomes. For the manufacturing method, see, for example, U.S. Patent No. 4,522,811; U.S. Patent No. 5 See U.S. Patent Nos. 374,548 and U.S. Patent Nos. 5,399,331. The substance is selectively transported into designated cells or organs, thereby improving targeted drug delivery. It may contain one or more parts (for example, Ranade (1989) J. Clin. Phar (See macol.29:685). Exemplary targeting sites include folate or biotin. For example, see U.S. Patent No. 5,416,016, Low et al.; Mannosi (Umezawa et al., (1988)Biochem.Biophys.R es.Commun.153:1038);Antibodies(Bloeman et al.,(1 995)FEBS Lett.,357:140;Owais et al.(1995) )Antimicrob.Agents Chemother.,39:180);Sur Factant protein A receptor (Briscoe et al., (1995) Am. J.Physiol.1233:134);p120(Schreier et al( (1994) J. Biol. Chem. 269:9090) is cited; Keinaene n & Laukkanen (1994)FEBS Lett.,346:123-6; Killion & Fidler(1994)Immunomethods,4:27 See also 3.

[0133] This application also relates to pharmaceuticals comprising the Fc variant of the Disclosure in combination with other therapeutic agents or therapeutic agents. The protocol provides for the simultaneous administration or treatment of patients using the composition. Additional therapeutic agents For example, simultaneous use of cytokines, steroids, chemotherapy agents, antibiotics, or radiation. The method of administration or treatment is known in the art (e.g., Hardman et al.) al.,(eds.)(2001)Goodman and Gilman's Th e Pharmacological Basis of Therapeutics, 10. sup.th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacoth erapeutics for Advanced Practice:A Pract ical Approach, Lippincott, Williams & Wilk ins,Phila.,Pa.;Chabner and Longo(eds.)(2 001)Cancer Chemotherapy and Biotherapy,L ippincott, Williams & Wilkins, Phila., Pa. (Illuminate). An effective dose of the medication reduces symptoms by at least 10%, at least 20%, or less. It can be reduced by approximately 30%, at least 40%, or at least 50%.

[0134] In some embodiments, the pharmaceutical composition of this disclosure further comprises one or more additional therapeutic agents. nothing.

[0135] In addition to the treatment regimens described above, patients may be subjected to surgery and other forms of physical therapy. .

[0136] therapeutic use Therapeutic or pharmaceutical compositions comprising the Fc variants of this disclosure, but not limited to this specification. A disorder or pathological condition in which abnormal cell proliferation is present, referred to in the book as "cell proliferation disorder or pathological condition". It is useful for treating, preventing, or improving cell proliferation disorders or diseases. In one embodiment, this disclosure is useful for treating, preventing, or improving cell proliferation disorders or diseases. The present invention provides a method for treating a condition. In one embodiment, the subject of treatment is a human being.

[0137] Treatment, prevention, or improvement using therapeutic or pharmaceutical compositions comprising the Fc variant of this disclosure Examples of cell proliferation disorders or pathological conditions that can be caused include, but are not limited to, cancer. In this specification, the term "cancer" refers to a histopathological type. Regardless of the stage of invasion, all types of cancerous growth or carcinogenesis processes, metastatic groups This means that it includes tissue or malignant transformed cells, tissues, or organs.

[0138] In specific embodiments, a therapeutic or pharmaceutical composition comprising the Fc variant of the present disclosure, Administration to subjects according to the method described in this book will achieve one, two, or three or more results: (1) (2) Reduction of tumor or neoplasm growth; (3) Reduction of tumor formation; (4) Primary, localized and / or (4) Eradication, removal, or control of metastatic cancer; (5) Reduction of the spread of metastasis; (6) Reduction of mortality; (6) Increased survival rate; (7) Increased survival time; (8) Increased number of patients in remission; (9) Hospitalization (10) a decrease in the rate; (11) a decrease in the length of hospital stay; and (12) a decrease in tumor size by more than 10%, or Maintenance such that the increase is not more than 8%, or more than 6%, or more than 4%; preferably, tumor Its size does not increase by more than 2%.

[0139] In specific embodiments, a therapeutic or pharmaceutical composition comprising the Fc variant of the present disclosure, To a subject with cancer (in some embodiments, an animal model of cancer) using the method described in this book The dose was measured using an assay known in the art and a negative control was administered. Tumor development in subjects with cancer (or, in some embodiments, animal models of the same cancer) The tumor growth relative to the length is at least about twice, preferably at least about 2.5 times, less Approximately 3 times, at least 4 times, at least 5 times, at least 7 times, or at least It inhibits or reduces by approximately 10 times. In another embodiment, the Fc variant of the present disclosure is included A treatment or pharmaceutical composition for a subject having cancer as described herein (some embodiments) In this context, administration to animal models of cancer was performed using assays known in the art. A subject with cancer (in some embodiments, the same cancer) was administered to a negative control and measured using the method. In animal models, tumor growth is reduced by at least approximately 25%, and less than Approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, and at least Approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, and at least 70%, at least 75%, at least about 80%, at least about 85%, at least about It inhibits or reduces by 90%, or at least about 95%.

[0140] Examples of cancer-related complications are not limited to solid tumors, hematological malignancies, and soft tissue tumors. Examples include solid tumors and metastatic lesions. Examples of solid tumors include those of the liver, lungs, breasts, lymphatic system, and stomach. Shadows in the intestines (e.g., colon), urogenital organs (e.g., kidneys, urothelial cells), prostate, and pharynx. Malignant tumors of various organ systems that affect the body, such as sarcomas and carcinomas (adenocarcinomas and squamous carcinomas). Adenocarcinomas include most colorectal cancers, rectal cancers, renal cell carcinomas, and liver cancers. This includes malignant tumors such as non-small cell lung cancer, small intestine cancer, and esophageal cancer. Squamous cell carcinoma includes, for example, For example, malignant tumors of the lungs, esophagus, skin, head and neck region, oral cavity, anus, and cervix. In one embodiment, the cancer is melanoma, for example, advanced melanoma. Metastasis of the aforementioned cancer Sexual lesions can also be treated or prevented using the methods and compositions of this disclosure.

[0141] Growth can be inhibited using therapeutic or pharmaceutical compositions containing the Fc variant of this disclosure. Exemplary cancers include cancers that typically respond to immunotherapy. Preferred cancers for treatment. Non-limiting examples include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., detailed Cellular carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g.) Examples include non-small cell lung cancer and epithelial carcinoma. Furthermore, refractory or recurrent malignant tumors are classified as follows: Treatment can be provided using the combination therapy described in the specification.

[0142] Other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, and cancer of the head or neck. Malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer Cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel's decriminal code Celluloid carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, type A Carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, carcinoma of the soft tissue, carcinoma of the urethra, carcinoma of the penis, acute myeloid leukemia Chronic leukemia, including chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. This includes acute leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, multiple myeloma, and myelodysplasia. Neoplastic syndromes, cancer of the kidney or ureter, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CN S lymphoma, neuroblastoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Positoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and asbestos-induced cancers. Examples include environmentally induced cancers (e.g., mesothelioma) and combinations of such cancers. .

[0143] In specific embodiments, cancers include breast cancer, neuroblastoma, lymphoma, colon cancer, and pancreatic ductal adenocarcinoma. Melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple It is multiple myeloma.

[0144] Combination therapy The phrase "administered in combination" with additional therapeutic agents refers to two (or more) different treatments. This means that it is delivered to the subject during the process of the subject's suffering caused by the disability. In this case, the delivery of one treatment still occurs when the delivery of the second treatment begins. As a result, there is duplication in administration. This is referred to as "simultaneous" or "simultaneous delivery." In another embodiment, the delivery of one treatment is completed before the delivery of another treatment is completed. To begin. This is called "continuous delivery". In some embodiments of any case, The treatment is more effective when administered in combination. Additional treatment of combination therapy as disclosed herein The medication can also be administered regularly. Combination cycling therapy involves a certain period of time. The administration of the first therapeutic agent, followed by the administration of a second therapeutic agent for a certain period, and the repetition of this continuous administration. To be accompanied by.

[0145] Therapeutic or pharmaceutical compositions comprising artificially modified immunoglobulins as described herein include one or more other Treatment methods, for example, administering anticancer drugs, cytokines or antihormone agents together to treat cancer and Treatments including artificially manipulated immunoglobulins as described herein may be administered or managed. Other treatments that can be used in combination with pharmaceutical compositions are limited to the following: However, small molecules, synthetic drugs, peptides (including cyclic peptides), polypeptides, and Proteins, nucleic acids (for example, DNA and RNA nucleotides, as an example, limited to) However, antisense nucleotide sequences, triple helices, RNAi, and biologically Nucleotide sequences encoding active proteins, polypeptides, or peptides), antibodies, Examples include synthetic or natural inorganic molecules, mimics, and synthetic or natural organic molecules.

[0146] Use in addition to therapeutic or pharmaceutical compositions containing the artificially engineered immunoglobulin described herein. One or more other treatments that may be used are, but are not limited to, , chemotherapy, radiotherapy, cytotoxic agents, chemotherapy agents, cytokines, kinase inhibitors, low Examples of dosages include gemcitabine, 5-fluorouracil, and cytokine modulators. In particular, when used in addition to therapeutic or pharmaceutical compositions containing the artificially manipulated immunoglobulins of this disclosure. One or more other possible treatments include, in particular, immuno-oncology that perturbs the tumor microenvironment. Recombinant approaches, for example, recombinant IL-2, recombinant IL-15, recombinant IL-12, recombinant IL-21, anti-IL1β, anti-TGFβ, anti-CD39, anti-CD73, anti-CTLA4, anti-PD (L)1, anti-TIM3, HDAC inhibitors, HIF1a inhibitors, and anti-angiogenic drugs, for example, One example is anti-VEGF.

[0147] kit This disclosure also includes a kit for treating patients with cell proliferation disorders. The kit contains a therapeutically effective amount of a therapeutic or pharmaceutical composition comprising the Fc variant described herein. Furthermore, such kits may contain the Fc variant described herein, or may contain the therapeutic or pharmaceutical composition described herein. Means for administering substances (e.g., auto-injectors, syringes and vials, pre-filled syringes) These kits may include a syringe, pre-filled pen, and instructions for use. It may contain additional therapeutic agents (listed below) to treat the harm. Such kits treat the patient This also includes instructions for administering a therapeutic or pharmaceutical composition containing the Fc variant described herein. Such instructions may be used for therapeutic or pharmaceutical compositions containing the Fc variant described herein. The dosage, route of administration, regimen, and total duration of treatment for use may be provided.

[0148] The phrase "means for administration" refers to any means of administering a drug systemically to a patient. Possible methods, including but not limited to, pre-filled syringes, vials, and Syringes, injection pens, auto-injectors, IV infusions and bags, infusion pumps, patches, infusion bags It is used to indicate the size of the needle and other parts of the device. Using such a tool, patients can self-administer medication. It is possible to administer drugs (i.e., without the assistance of a physician), or a healthcare professional may administer drugs. It can be administered. [Examples]

[0149] The following embodiments are provided to further illustrate the present disclosure, but are not intended to limit its scope. There are none. Other variants of this disclosure are readily apparent to those skilled in the art and are included in the appended claims. It is included by.

[0150] Expression of all constructs derived from the amino acid sequence generated according to Example 1 in a mammalian system The mixture was then purified (Example 4), and human FcαRI was analyzed using surface plasmon resonance (SPR). The binding to rat FcαR was assessed (Example 3). Finally, artificially engineered immunoglobulins were used. The functionality of phosphorus was assessed by a cell-based assay using human fresh isolated PMN (actual Example 4). All examples involve the VH and VL domains that recognize the antigen HER2, and IgA2 Using an Fc variant of an antibody format that includes an artificially modified hinge and Fc region based on [the specified element] The procedure was carried out using the following method. Sequence ID 1 contains the VH domain that binds to HER2 and the hyphen from IgG1. This is the full-length heavy chain sequence of an anti-HER2 binding antibody having a domain and a constant domain. (SEQ ID NO: 2) This involves the VH domain that binds to HER2 and the hinge and determination from the m2 allotype of IgA2. This is the full-length heavy chain sequence of an anti-HER2 binding antibody having a congeneral domain (Lombana et al.). al., (2019) MABS, 11:1122-38). Sequence ID 4 is for HER2. Anti-HER2 binding agent having a binding VL domain and a constant domain (CL) from IgA2. This is the light chain sequence of the antibody.

[0151] Example 1: IgA2 Fc affinity maturation for hFcαRI 1.1 IgA2 Fc Library Design For the IgA1 Fc / hFcαRI complex, the crystal structure PDB 1OW0 was used. An iontophoresis analysis was performed. This is because IgA1 differs from IgA2 only in its hinge region. This is because they have similar structures. All residues located in close proximity to hFcαRI are IgA1 Considering that they are potentially involved in Fc / hFcαRI interactions, they are classified into two categories. (i) Residues from the "core" interface region (LCH2.15, LCH2.15.1, MCH 3.105, ECH3.109, PCH3.113, LCH3.114, ACH3.11 5. FCH3.116, QCH3.118, where the residues are IM for the C-domain. (i) numbered according to GT numbering and (ii) from the “shell” region surrounding the core The residues (QCH2.94, NCH2.97, HCH2.98, RCH3.1, ECH3. 3. RCH3.40, LCH3.42, SCH3.45, ECH3.45.2, here, The residues are numbered according to the IMGT numbering for the C-domain. Otido-specific mutagenesis (TRIM)-based approach (Virnekaes et al.) al,(1994)Nucleic Acids Res.,22:5600-5607 ;Knappik et al.,(2000)J Mol Biol.,296:57 -86) Diversify both groups of residues into "shell" and "core" regions Two libraries, L1 and L2, corresponding to the respective domains, were generated.

[0152] A third library (EP) was created using error-prone PCR of the IgA2 Fc domain. (The library was generated by Gram et al., (1992) PNAS USA, 89:3576-3580).

[0153] 1.2 Library screening using yeast display Yeast display technology (Boder et al., (1997) Nature Bi Using ot.,15:553-557), all three IgA2 Fc libraries were selected. Cleaned. In short, IgA Fc on the yeast cell membrane is α-aglutinin ( Aga1 / Aga2) protein heterodimers convert IgA2 Fc to Aga2 tangent The molecules were fused to the N-terminus of the protein and displayed. Four sorting rounds were performed. :

[0154] (i) During the first sorting round, all three libraries are given 1% of the Raffino In a selective medium containing - and 2% galactose, ferment at 20°C for 2 days with shaking. The cells were grown in stages to induce IgA2 Fc expression on the surface of the yeast cells. Each culture was then processed into pellets. The mixture is then smeared, the supernatant is removed, and the pellet is treated with 1% BSA (bovine serum albumin) and 2 mM Wash once with PBSM (PBS (Gibco, Waltham, MA)) containing EDTA. After resuspending the pellet in 20 ml of PBSM, 100 μl of each strep was added. Toavidin and antibiotin microbeads (Miltenyi Biotec, Berg (Isch Gladbach, Germany) was added. The cells were rotated at room temperature. It was incubated for 1 hour. Then, the MACS LS column (Miltenyi The beads were removed using ). The library was then pelletized and placed in 20 ml of PB. Resuspend in SM+50nM biotinylated FcαRI(CD89)(SEQ ID NO: 5), and The cells were incubated at room temperature for 1 hour while being rotated. Then, the cells were pelleted and placed on top. Remove the clear water, wash once in PBSM, then rinse with 20 ml of PBSM + 100 μl of saturates. The library was resuspended in putavidin microspheres (Miltenyi). Incubate on ice for 5 minutes, shaking occasionally, then pellet the cells and remove the supernatant. Remove, resuspend in 20 ml of PBSM, and then filter on a MACS LS column (Milte The cells were separated on NYI. The column was washed once with 5 ml of PBSM, and then the bound cells were selected. Elute with culture medium, then reduce to a final volume of 10 ml in selective medium and incubate overnight at 30°C with shaking. I made it longer.

[0155] (ii) For the second sorting round, from each of the three libraries One round output containing 1% raffinose and 2% galactose. IgA expression was induced by growing the cells in selective medium at 20°C for 24 hours. Reddened and mixed in PBSF (PBS (Gibco) + 0.1% bovine serum albumin) The libraries were washed and resuspended in PBSF. Each library was divided into two samples. The first sample was converted to 25 nM biotinylated FcαRI in PBSF, and the second sample was converted to We used 10 nM biotinylated FcαRI in PBSF. Each was tagged with rabbit antimyc-tags. Dylight 488 (Rockland, Limerick, PA) at 1:100 Add at the final dilution ratio and incubate at room temperature for 1.5 hours while rotating the sample. The sample was baited. The sample was pelletized, washed once with PBSF, and then rotated while P BSF+1:100 Final streptavidin Dylight 633 (Invitrog The sample was incubated with (en, Waltham, MA) for 5 minutes, then pelleted. After processing, wash once, resuspend in PBSF, filter through a 40 μm strainer, and then... FACS Aria Cell Sorter (Becton Dickinson Bioscal) Analysis was performed using flow cytometry on iences, San Jose, CA. , sorted. For L1 and L2 libraries, 10nM FcαRI samples were used. For the EP library, we sorted the 25nM FcαRI samples. In each case, yeast exhibiting the top 1-2% of signals is gated, harvested, and selected. The plants were grown overnight in culture medium at 30°C.

[0156] (iii) For the third sorting round, the contents from the second sorting round The nutrients were inoculated into a selective medium containing 1% raffinose and 2% galactose, and then left at 20°C. The cells were grown overnight to induce IgA expression. Three libraries were used, as in the second round. Prepare and sort cells from each of the following sources, but use chicken anti-MYC tags as a detection reagent. FITC (Genetex, Irvine, CA) and Neutravidin Dyl I used ight 633 (Invitrogen). Biotinylated FcαRI was used in EP. For the library, 5nM; for the L1 library, 2nM; L2 Ibrary was used at 1nM. In each case, the upper signal was Yeast showing 1-2% concentration was gated, collected, and grown overnight in selective medium at 30°C. Ta.

[0157] (iv) The fourth sorting round is completed only for the EP and L1 libraries. The cultures from the third sorting round were sorted in a selective medium + 1% raffinose and 2 The cells were inoculated into 1% galactose and grown overnight at 20°C to induce IgA expression. Prepare and sort the cells from each of the libraries as done in round 2, however The detection reagent used was mouse anti-myc Dylight 488 (Invitrogen). ) and streptavidin cy5 (Invitrogen) were used. Biotinylated Fc αRI is set to 2nM for the EP library and 1nM for the L1 library. The yeast was used in the following way. In each case, the yeast showing the top 1-2% of the signal was gated. The samples were then collected and grown overnight in a selective medium at 30°C.

[0158] 1.3 Hotspot identification, translation in full-length immunoglobulins, and response to hFcαRI SPR verification From the third (L2 library) and fourth (EP and L1 library) round cultures The plasmid was purified, transformed in E. coli, and then plated on a selective agar plate. Rated, grown overnight at 37°, and Sanger sequencing (Sanger er et al(1975)J Mol Biol.,94(3):441-8;Sa nger et al (1977) PNAS USA.,74(12):5463-7) It was sent to Genewiz (South Plainfield, NJ) for the purpose of [reason]. Clones are selected based on their frequency of occurrence, and they are used for IgA2 / hFcαRI We identified mutations that improve the interaction. The IgA2 residue positions are shown in Table 1.

[0159] [Table 1]

[0160] The identified mutation is a single dot in full-length IgA2 immunoglobulin having sequence number 2. The mutation was incorporated either as a natural mutation or in combination and transiently expressed in HEK293 cells. (As described in Example 2). The same mutation has sequence numbers 3 and 6 and is linked to hFcαRI. In IgG1 isotype immunoglobulins containing artificially modified IgG1 Fc that can be matched, Incorporated. Test mutation sets are shown in Table 2 (based on Sequence ID 2) and Table 4 (Sequence ID 2). This is presented in Table 6 (based on Sequence ID 6), and Table 3 (based on Sequence ID 6).

[0161] The Fc mutant was purified, and surface plasmon resonance (SPR) was measured against hFcαRI. Use this to assess the specific mutations in immunoglobulin affinity for hFcαRI. The effect was evaluated. Interestingly, all mutations were associated with the emission of each immunoglobulin. It had a limited effect on current yield and aggregation tendency, or had no actual effect. PR data and the aggregate content after capture are shown in Tables 3, 5, and 7.

[0162] Finally, lead candidates are selected based on SPR and aggregation data to broaden the range of hFcα SPR experiments were repeated using RI concentrations. The use of the appropriate concentration range was artificially manipulated immunoglobulin. This allowed for more accurate measurement of the interaction between brin and hFcαRI. The results are shown in Table 8. This is shown in 9 and Table 10.

[0163] [Table 2]

[0164] [Table 3]

[0165] [Table 4]

[0166] [Table 5]

[0167] [Table 6]

[0168] [Table 7]

[0169] [Table 8]

[0170] [Table 9]

[0171] [Table 10]

[0172] 1.4 Affinity maturation for improving alpha effector function in in vitro assays conversion Read candidates having homodimer Fc (SEQ ID NOs: 32, 37, and 42) are classified as hFcαR We selected these for their improved binding ability to I. These are then described in Example 4. The PMN killing assay was performed according to the procedure. Efficacy (EC 50 Immunoglobulin (Concentration of immunoglobulin required to produce 50% of the maximum effect) and efficacy (Em The results of ax (the maximum expected effect of immunoglobulin) are shown in Figures 1 to 4.

[0173] All test candidates were active, and in the SK-BR-3 PMN killing assay, parental Ig It was shown to have improved efficacy compared to A2 (Figure 1).

[0174] Furthermore, it is known that Cal has a lower HER2 receptor density than SK-BR-3 cells. Significant improvement in efficacy, up to 2x, was demonstrated in PMN killing assays using u-3 cells. This was done (Figure 2) (described in Example 4). Furthermore, similarly, lower levels of HER2 receptors were produced. In PMN killing assays against MDA-MB-453 cells, which are known to exhibit certain properties A significant improvement in efficacy was shown for the variant with sequence number 42 (Figure 3). The improvement in effectiveness was approximately seven times.

[0175] Finally, the affinity-matured variant with SEQ ID NO: 42 was selected as the minimal HER2-expressing cell line. This was tested in a PMN killing assay using the publicly known MDA-MB-175 cells. It did not show lethality even at extremely high concentrations (Figure 4). This observation was made in the test candidate. Emphasize the secure profile.

[0176] The mutation set was applied to heterodimer Fc, resulting in sequence numbers 7-8, 80-8, This resulted in complementation including sequence numbers 7-9 or 80-9 (Figure 5). Tested candidate formulations Column numbers 80-8 and sequence number 80-9 indicate that in the PMN killing assay, their progenitor immunities... Compared to globulin and IgA2 (Figures 5A and 5C), SK-BR-3 cells are more Although it demonstrated good lethal properties, it mediated the γ response in the PBMC killing assay. It did not show any effect (Figures 5B and 5D).

[0177] Example 2: Expression and purification of artificially manipulated proteins Nucleic acid sequences encoding heavy and light chains are generated by Geneart (LifeTechnologi Synthesized in es) using restriction enzyme-ligation-based cloning technology The plasmid was cloned into a mammalian expression vector. The resulting plasmid was then used in HEK293T cells. Simultaneous translocation occurred. In short, immunoglobulins (IgG, IgA and artificially modified immunoglobulins) were introduced. Due to the transient expression of epidemic globulin, equivalent amounts of light chains and their respective artificially engineered heavy chain vectors were used. Polyethyleneimine (PEI) in suspension-compatible HEK293T cells (see catalog number 2) Simultaneous translocation was performed using 4765 Polysciences, Inc. (typical). This includes 100 ml of cells in a suspension with a cell density of 1 to 2 million cells per ml. 50 μg expression vector encoding artificially engineered heavy chain and 50 μg expression vector encoding light chain DNA containing the vector was transfused. Then, the recombinant expression vector was introduced into the host cell. The cells were introduced and further cultured for 7 days to produce constructs with 0.1% plur Cultures supplemented with onic acid, 4 mM glutamine, and 0.25 μg / ml antibiotics. This enabled secretion into culture media (HEK, serum-free medium).

[0178] Next, the produced constructs are obtained from the cell-free supernatant using immunoaffinity chromatography. Purified. Anti-kappa LC resin (KappaSel) equilibrated in PBS buffer pH 7.4. (Etc., GE Healthcare Life Sciences) uses liquid chromatography Graph system (Aekta pure chromatography system, GE Hea Using filtration conditions with medium and incubation (Lthcare Life Sciences) The resin was washed with PBS pH 7.4, and then the construct was eluted with elution buffer (50 mM). The enate was eluted with 90 mM NaCl (pH 2.7).

[0179] After capture, the eluted protein was pH neutralized using a 1M TRIS pH10.0 solution. , size exclusion chromatography technology (HiPrep Superdex 200 16 / 60, GE Healthcare Life Sciences) used poly The mixture was then mixed with purified protein in PBS buffer at pH 7.4.

[0180] Aggregation tendency is determined by analytical size exclusion chromatography after capture and pH neutralization steps. Superdex 200 Increase 3.2 / 300GL,GE Healt Measurements were taken using (hcare Life Sciences).

[0181] Example 3: Human Fcα receptor (hFcαRI) and rat Fcα receptor (rFcαR) SPR measurement Fc variant antibodies with the light chain of SEQ ID NO: 4 against human FcαRI or rat FcαR A direct binding assay was performed to characterize the formal binding.

[0182] The dynamic binding affinity constant (KD) is measured using the BIAcore® T200 instrument (GE He Measured at room temperature in Althcare, Glattbrugg, Switzerland. The protein is in a running buffer of 10 mM NaP, 150 mM NaCl, and 0.05% Diluted with Tween 20 at pH 7.6. Biotinylated anti-κ light chain scFv was immobilized. Streptoavidin Sensor Chip (Sensor Chip SA, GE Health) Using Care Life Sciences, we supplemented the manipulated immunoglobulins. Recombinant human hFcαRI or recombinant rat FcαR was used as the analyte.

[0183] To function as a reference, one flow cell does not capture any immunoglobulins. The combined data was obtained from subsequent injections and flow cells of the analyte dilution series on the reference. Data was collected by measurement. Data evaluation included zero-concentration samples (running buffer only). This enabled dual referencing between the two. For data evaluation, the dual-referencing sensorgram was connected in a 1:1 relationship. The equilibrium dissociation constant (KD) was generated by applying a composite model analysis. rFCα The results for R are summarized in Table 11.

[0184] This experiment demonstrates that the candidate Fc variant is cross-reactive with human / rat FcαRI. This is a desirable characteristic for candidate trials in in vivo disease models.

[0185] [Table 11]

[0186] Example 4: Antibody-dependent cytotoxicity (ADCC) assay method Blood samples from healthy donors are collected using the Swiss Human Research Protocol (Swiss human re In accordance with the Search Act (Basel Organizational Donor Program - Prevoted) It was recovered from freshly drawn peripheral blood. After lysing of red blood cells with ACK lysis buffer, Formulae and peripheral blood mononuclear cells (PMNs and PBMCs) are subjected to a ficoll-paque gradient. Further isolation was achieved. The alpha-effector function of artificially manipulated immunoglobulins was then utilized with PMN. While characterizing the PBMC, we used it to characterize the gamma effector function.

[0187] Effector cells (freshly isolated PMN or PBMC cells) are used to target HER2 expression. Cells (SK-BR-3, Calu-3, MDA-MB-453 or MDA-MB-175 cells) American Type Culture Collection,Rockv (Purchased from ille MD) Added to the effector at a ratio of 20:1 to the target. K-BR-3 is a breast cancer cell lineage that overexpresses HER2. Calu-3 and MDA- MB-453, compared to SK-BR-3, exceeds HER2 at lower levels. It is an overexpression of lung and breast cancer cell lines (Cheung et al., 2019). MDA -MD-175 is a breast cancer cell lineage that expresses the lowest amount of HER2 (Crocker e (t al., 2005). PMN cell killing was not observed in any of the candidate Fc mutants, low It has shown a favorable safety profile for HER2-expressing cell lines.

[0188] Add the immunoglobulin construct at the indicated concentration and gently mix the combination. Next, the target and effector cells were centrifuged at 260×g for 4 minutes without destruction. This promoted localization. Subsequently, the assay product was placed in a standard tissue culture incubator with 5% C The cells were incubated in O2 at 37°C for 18 hours. After 18 hours, Cytotox96 Use the reagent (Promega) according to the manufacturer's instructions and use the supernatant for LDH emission measurement. The absorbance at 490 nm was measured using a Biotek Synergy HT platelet. The data was read on the device. The data was analyzed using GraphPad Prism 6.0. , and graphed it.

[0189] Example 5: Improvement of pharmacokinetic (PK) properties of artificially modified immunoglobulins compared to IgA 5.1 Material production of artificially engineered immunoglobulins Anti-HER2 individuals with sequence numbers 1, 2, 7, 8, 40, 80, 82, 83, and 84 Geneart(LifeTe) generates nucleic acids encoding immunoglobulin heavy chain variants. Synthesized in (technologies), restriction enzyme-ligation based clonin The selected N-glycosylation site was cloned into a mammalian expression vector using a special technique. This was removed by substitution of a specific Asp residue with an Ala residue. The resulting heavy chain encoding was obtained. Plasmids that encode light chains (SEQ ID NO: 4) and mammalian expression systems simultaneously produce traits. The cells were transferred. For the HEK293T-expressing cell line, expression was carried out according to the procedure described in Example 2. The procedure was carried out. For the CHO-S expressing cell line (Thermo), the following procedure was used. To put it simply, for transient protein expression, the expression vector is adapted to the suspension of CHO-S cells. Translocation was performed using the expifectamine CHO translocation agent (Thermo) inside. Typically, 400 ml of suspension at a cell density of 6 million cells per ml The cells were subjected to DNA containing a 400 μg expression vector encoding an artificially manipulated protein. Transfusion was performed. Subsequently, the recombinant expression vector was cultured in host cells for further secretion. Culture medium (ExpiCHO expression culture medium supplemented with ExpiCHO feed and enhancer reagent) It was introduced in a thermostatic environment for 7 days. Then, the expression construct was introduced in Example 2. The immunoglobulin was purified from the cell-free supernatant according to the procedure described. The measured immunoglobulin concentration in serum was measured over time. The results were plotted as a function of , as shown in Figures 6 and 7. The generated materials are shown in Tables 12 and 13. It is stated.

[0190] [Table 12]

[0191] [Table 13]

[0192] In accordance with the construct design, the operations have sequence numbers 7-8 and 8-80. The immunoglobulin, while retaining its binding to FcRn, binds to CD89, as shown in Figure 6. Thus, it exhibits improved PK properties and an improved half-life compared to IgA immunoglobulin. Furthermore, as shown in Figure 6, affinity-mature mutants having sequence numbers 8-80 have sequence numbers. This shows the same PK as the parent construct with numbers 7-8. This is the parent for CD89. This study demonstrates that sexual maturation impairs the manipulated immunoglobulin PK properties.

[0193] The data shown in Figure 7 illustrates how the N-glycosylation pattern influences immunoglobulin PK. This shows the impact. The PK properties of the manipulated immunoglobulins are improved compared to IgA. Therefore, when individual N-glycosylation sites CH2.84.4 were removed, the results were improved (sequence number No. 83).

[0194] 5.1 Mouse Test Male CD1 mice were obtained from Charles River Laboratories. Upon arrival, all mice are placed in a pathogen-free animal facility under standard 12-hour light / 12-hour dark conditions. The mice were reared under an electric current at a room temperature of 21°C with continuous feeding and watering. All mice were raised under the conditions described above. IgG or IgA or artificially engineered immunoglobulin (3 mg / kg) produced and purified as described above. Each of the compounds was administered as a single intravenous (IV) injection into three mice. Blood samples were collected at various time points after injection into serum separation tubes via the saphenous vein. The blood was allowed to coagulate at ambient temperature for at least 20 minutes. The coagulated sample was centrifuged. The samples were kept at room temperature until the collection time was started within one hour. The mixture was centrifuged at 2-8°C for 5 minutes under a relative centrifugal force of 1500-2000 × g. Within 20 minutes after separation, the serum was separated from the blood sample and labeled into 2.0 mL of polypropylene The samples were transferred to conical-bottom microcentrifuge tubes. The animals were clearly healthy and clearly Only animals without any abnormalities were used in the tests. All animal work performed was conducted by Novart. is'Institutional Animal Care and Use Com Reviewed and approved by mittee.

[0195] 5.2 Immunoglobulin ELISA for pharmacokinetic studies Immunoglobulin levels were measured by serial sandwich ELISA. Regarding IgA medication... Therefore, the wells of the Nunc Maxisorp microtiter plate contain goat anti-human I gA (Southern Biotech, catalog number 2053-01) at 4°C odor It was coated overnight. For IgG and artificially engineered immunoglobulin administration, Roch Biotinylated SB goat antibody wells of StreptaWell microtiter plate Human IgG (Southern Biotech, catalog number 2049-08) at room temperature The mixture was coated for 1 hour in blocking buffer (PBS, 0.5% bovine serum). After 1 hour incubation with lubumin (BSA), in the same blocking buffer... The diluted sample was added to a blocked plate and left at room temperature for 2 hours. Incubated. After incubation, horseradish peroxidase conjugate Goat anti-human IgA (SouthernBiotech, catalog number 2053-05) This is horseradish peroxidase conjugate goat anti-human IgG (Southern Add Biotech (catalog number 2049-05) and incubate at room temperature for 1 hour. The plate was then coated with substrate solution (BM Blue POD substrate TMB, Roc He (catalog number 11484281001) was incubated and refracted in 0.5M sulfuric acid. The response was stopped. The absorbance was measured at 450 nm using a plate reader, and 65 Subtraction was performed at 0 nm. Between steps, the plate was washed with washing buffer (0.05% Twe in PBS). It was washed three times with en-20.

[0196] Sequence information Table 14 shows the variant Fc region described in the examples, which is used to generate complete antibodies. The amino acid sequences (sequence numbers) of the full-length heavy chain and light chain, including Fc, are described herein. Mutants, full-length heavy chains, light chains, or complete antibodies are obtained through conventional recombinant protein production and purification processes. It can be produced using

[0197] All sequences (sequence numbers) mentioned herein can be found in Table 14. Reference numbers The number refers to the internal sequence reference number. Throughout the text of this application, the text of this specification (for example, In the event of any inconsistency between Table 14) and the sequence listing, the text of this specification shall prevail.

[0198] [Table 14]

[0199] [Table 15]

[0200] Table 16

[0201] Table 17

[0202] Table 18

[0203] Table 19

[0204] Table 20

[0205] Table 21

[0206] Table 22

[0207] Table 23

Claims

1. An Fc variant of the parent Fc polypeptide, wherein the Fc variant is the parent Fc polypeptide Binding to modified FcαR compared to D, or modified antibody-dependent cell-mediated cells The Fc variant exhibits toxicity (ADCC), and the Fc variant has the Fc region of the parent Fc polypeptide. The amino acid modification comprises at least one amino acid modification, wherein the amino acid modification is: CH2,10, CH 2.89, CH2.91, CH2.94, CH2.97, CH2.99, CH3.45, Select from the group consisting of CH3.105, CH3.109, CH3.118, and CH3.

124. Located in a selected position, the numbering of the amino acid modification is IMGT for the C-domain Fc variants that follow the numbering system.

2. The aforementioned at least one amino acid modification is: A_CH2.10_S, L_CH2.89_I , G_CH2.91_Q, G_CH2.91_V, Q_CH2.94_E, N_CH2. 97_H, N_CH2.97_Y, G_CH2.99_W, S_CH3.45_D, M_ CH3.105_Y, E_CH3.109_D, Q_CH3.118_Y and L_CH3 Selected from the group consisting of 124_F, the numbering of the amino acid modification is in the C-domain The Fc variant according to claim 1, wherein the IMGT numbering is applied accordingly.

3. The aforementioned at least one amino acid modification is: Q_CH2.94_E, N_CH2.97_Y, S_CH3.45_D, M_CH3.105_Y, Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y, Q_CH2.94_E / S_CH3.45_D, Q_CH2.94_E / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D, N_CH2.97_Y / M_CH3.105_Y, S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y, N_CH2.97_Y / S_CH3.45_D / M_CH3.105_Y, Q_CH2.94_E / S_CH3.45_D / M_CH3.105_Y, M_CH3.105_Y / Q_CH3.118_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.1 05_Y, Q_CH2.94_E / N_CH2.97_Y / M_CH3.105_Y / Q_CH3. 118_Y, Q_CH2.94_E / N_CH2.97_Y / S_CH3.45_D / M_CH3.1 05_Y / Q_CH3.118_Y, A_CH2.10_S, L_CH2.89_I, G_CH2.91_V, N_CH2.97_H, G_CH2.99_W, E_CH3.109_D, L_CH3.124_F, and L_CH2.89_I / G_CH2.91_V / Q_CH2.94_E / N_CH2.9 7_Y / G_CH2.99_W Selected from the group consisting of, the numbering of the amino acid modification is IM for the C-domain An Fc variant according to claim 1 or claim 2, in accordance with GT numbering.

4. The parent Fc polypeptide is contained within human IgA, as described in any one of claims 1 to 3. The Fc variant shown.

5. The parent Fc polypeptide is contained within human IgA2, according to any one of claims 1 to 4. The Fc variant described.

6. The Fc mutant was measured by surface plasmon resonance and showed a decrease in the parent Fc polypeptide. Claims 1 to 5, which have an increased affinity for human FcαRI of at least approximately 50 times. An Fc variant described in any one of the items.

7. The parent Fc polypeptide comprises human IgG1, according to any one of claims 1 to 3. Fc mutant.

8. The Fc mutant was measured by surface plasmon resonance and showed a decrease in the parent Fc polypeptide. At the very least, it has an increased affinity for human FcαRI of approximately 300 times, as described in claim 7. The Fc variant shown.

9. The Fc variant was measured in the MDA-MB-453 cell killing assay and was antibody-dependent. Claim to increase the persistence of cell-mediated cytotoxicity by at least about five times that of the parent Fc polypeptide. An Fc variant as described in any one of items 1 to 8.

10. The Fc mutant is at least about twice as numerous as the parent Fc polypeptide in Calu-3 cells. F according to any one of claims 1 to 9, which has increased efficacy in a lethal assay. c variant.

11. An IgA antibody containing an Fc variant, wherein the antibody contains a parent Fc polypeptide. Increased FcαR affinity compared to the antibody, or increased antibody-dependent cell-mediated cytotoxicity Possesses IgA antibodies.

12. The antibodies mentioned above are: CH2.10, CH2.89, CH2.91, CH2.94, CH2.9 7, CH2.99, CH3.45, CH3.105, CH3.109, CH3.118 and The amino acid modification includes a position selected from the group consisting of CH3, 124, and the aforementioned amino acid The numbering of the no-acid modifications follows the IMGT numbering for the C-domain, as in claim 11. The IgA antibody described.

13. The IgA antibody according to claim 12, wherein the antibody is a human IgA1 or IgA2 antibody.

14. The IgA antibody according to any one of claims 11 to 13, wherein the antibody binds to the tumor antigen. 。

15. The Fc variant according to any one of claims 1 to 10 or any one of claims 11 to 14 The antibodies described in the section are combined with one or more pharmaceutically acceptable excipients, diluents, or carriers. A pharmaceutical composition containing [the specified ingredient].

16. The pharmaceutical composition according to claim 15, further comprising one or more additional activators.

17. Any one of claims 1 to 10 for use in the treatment of cell proliferation disorders or conditions The Fc variant described in the original or the antibody described in any one of claims 11 to 14.

18. The aforementioned cell proliferative disorders or conditions include: breast cancer, neuroblastoma, lymphoma, pancreatic ductal adenocarcinoma, melanoma, kidney cancer. Cellular carcinoma, bladder cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, and multiple myeloma An Fc variant or antibody for use according to claim 17, selected from the group comprising the following:

19. The Fc variant according to any one of claims 1 to 10 or any one of claims 11 to 14 An isolated nucleic acid molecule encoding the antibody described in the section.

20. A cloning or expression vector comprising one or more nucleic acid sequences according to claim 19, , the Fc variant according to any one of claims 1 to 10 or any one of claims 11 to 14 A vector suitable for recombinant production of the antibodies described in the section.

21. A host cell comprising one or more cloning or expression vectors according to claim 20.

22. The Fc variant according to any one of claims 1 to 10 or any one of claims 11 to 13 A method for preparing the antibody described in the section, comprising culturing the host cells described in claim 21. , Purifying the Fc mutant or antibody from the host cell culture, and the host cell culture A method comprising recovering the Fc variant or antibody from a substance.

Images