WO2022265331A1 - Fc variants with controlled immune mechanism and increased blood half-life - Google Patents

Abstract

The present invention relates to a polypeptide comprising an Fc variant in which a part of the amino acid sequence of an antibody Fc domain is substituted with another amino acid sequence, or to an antibody comprising same. The Fc variant of the present invention may have uses in a wide range of antibodies and Fc fusion products. According to one aspect, the antibody or Fc fusion of the present invention may be used as a therapeutic, diagnostic, or research reagent, and preferably as a therapeutic medicament. The Fc variant of the present invention may exhibit reduced or silenced effector functions while maximizing in vivo half-life through optimization of some amino acid sequences.

Description

Fc variants with controlled immune mechanism of action and increased blood half-life

The present invention relates to Fc variants with reduced or silenced effector function and/or extended half-life in blood and a method for preparing the same.

Therapeutic antibodies are being developed for the treatment of a variety of diseases, the efficacy of some of which derives in part from the Fc region of the antibody mediating one or more effector functions. These effector functions result from the interaction of antibodies and antibody-antigen complexes with cells of the immune system to stimulate a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Daeron, Annu Rev. Immunol.15:203-234 (1997)).

However, in the treatment of certain diseases, the effector functions mediated by the Fc region may cause undesirable adverse effects, and thus the development of antibodies with reduced or silenced effector functions is required.

The matters described as the above background art are only for improving understanding of the background of the present invention, and should not be taken as an admission that they correspond to prior art already known to those skilled in the art.

The present inventors discover novel Fc variants that have the effect of extending half-life while effector functions of the Fc region, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), are reduced or silenced. made an earnest effort to do so. As a result, by substituting and optimizing some amino acid sequences of the wild-type Fc domain with other amino acid sequences, the present invention identified a method capable of reducing or silencing effector functions without affecting the activity of protein or antibody therapeutics. has been completed.

Accordingly, an object of the present invention is to provide a polypeptide comprising an antibody Fc variant, wherein the Fc variant has reduced binding ability to FcγRIIIa compared to a wild type antibody Fc domain. .

Another object of the present invention is to provide an antibody comprising the polypeptide.

Another object of the present invention is to provide a nucleic acid molecule encoding the polypeptide.

Another object of the present invention is to provide a vector containing the nucleic acid molecule.

Another object of the present invention is to provide a host cell containing the vector.

Another object of the present invention is to provide a method for producing a polypeptide comprising an Fc variant having a reduced effector function compared to a wild-type Fc domain.

Another object of the present invention is to provide a method for preparing an antibody having a reduced effector function compared to a wild type antibody.

Another object of the present invention is an effector function comprising administering a pharmaceutical composition comprising a nucleic acid molecule encoding the polypeptide or a vector containing the nucleic acid molecule to a subject in need thereof. function) to provide a method for delivering an Fc variant with reduced function.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention is a polypeptide comprising an antibody Fc variant, wherein the Fc variant has reduced binding ability to FcγRIIIa compared to a wild type antibody Fc domain, a polypeptide provides

*The present inventors reduce or silence effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), which existing antibodies or Fc regions have, while extending the half-life effect A novel Fc variant with was discovered.

An antibody is a protein that specifically binds to a particular antigen, and natural antibodies are usually heterodimeric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.

There are five main classes of human antibodies used in the present invention: IgA, IgD, IgE, IgG and IgM, preferably IgG. Papain digestion of antibodies forms two Fab fragments and one Fc fragment, and in human IgG molecules, the Fc region is generated by papain digestion of the N-terminus of Cys 226 (Deisenhofer, Biochemistry 20: 2361-2370, 1981) .

The term "Fc region" is used herein to define the C-terminal region of an antibody. The Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody: chain A and chain B. The second and third constant domains are known as CH2 domains and CH3 domains, respectively. The CH2 domain includes the CH2 domain sequence of chain A and the CH2 domain sequence of chain B. The CH3 domain includes the CH3 domain sequence of chain A and the CH3 domain sequence of chain B. As used herein, the Fc region includes the hinge region defined below.

The term “Fc region” or “Fc domain” is used to define the C-terminal region of an immunoglobulin heavy chain. An “Fc region sequence” may be a native Fc region sequence or a variant Fc region sequence. Although the boundaries of the Fc region sequences of immunoglobulin heavy chains can vary, human IgG heavy chain Fc region sequences are usually defined as extending from the amino acid residues at position Cys226 or Pro230 to their carboxyl termini.

The “CH2 domain sequence” (also called “Cy2” domain sequence) of a human IgG Fc region sequence usually extends from about amino acid 231 to about amino acid 340.

A "CH3 domain sequence" includes the extension of the C-terminal residues from the Fc region sequence to the CH2 domain sequence (ie, from about amino acid residue 341 to about amino acid residue 447 of an IgG).

A "functional Fc region" retains the "effector function" or "effector function" of a wild-type Fc region. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (eg B cell receptor; BCR); and the like. Such effector functions generally require an Fc region to be combined with a binding domain (eg an antibody variable domain) and can be assessed using, for example, various assays disclosed herein.

A “wild-type Fc region sequence” includes an amino acid sequence identical to that of an Fc region found in nature. Wild-type Fc regions include wild-type human IgG1 Fc regions (non-A and A allotypes); wild-type human IgG2 Fc region; wild-type human IgG3 Fc region; and wild-type human IgG4 Fc regions as well as naturally occurring variants thereof.

A “variant Fc region sequence” includes an amino acid sequence that differs from a wild-type Fc region sequence by “one or more amino acid modifications” as defined herein. The variant Fc region sequence has at least one amino acid substitution compared to the wild-type Fc region sequence or the Fc region sequence of the parent polypeptide, for example, from about 1 to about 10 amino acid substitutions in the wild-type Fc region sequence or the Fc region sequence of the parent polypeptide; preferably from about 1 to about 5 amino acid substitutions. In certain embodiments, a variant Fc region sequence herein has at least about 80% identity to, most preferably at least about 90% identity to, more preferably at least about 90% identity to, a wild-type Fc region sequence or an Fc region sequence of a parent polypeptide. have at least about 95% identity with

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are native human FcRs. Moreover, in certain embodiments, an FcR is one that binds an IgG antibody (gamma receptor, FcγR), including allelic variants and alternatively spliced forms of these receptors, FcγRI (CD64), FcγRII (CD32), and receptors of the FcγRIII (CD16) subclass. The term "FcR" also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 1 17:587 (1976)). The term "FcγR" does not include FcRn.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to non-specific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells) that recognize antibodies bound on target cells and subsequently induce lysis of the target cells. cells, neutrophils, and macrophages). NK cells, the main cells mediating ADCC, express FcγRIII and low levels of FcγRIIc, whereas monocytes express FcγRl, FcγRII and FcγRIII.

A “human effector cell” is a leukocyte that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least FcγRIII and performs ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from their natural source, eg blood or PBMCs.

Fc variants with "reduced", "silenced", "negligible" or "negligible" FcγR binding affinity and C1q binding affinity are compared to the parent polypeptide or a polypeptide comprising a wild-type Fc region sequence. It has reduced FcγR binding activity and C1q binding activity. In some embodiments, Fc variants with "reduced", "silenced", "negligible" or "removed" FcγR binding affinity and C1q binding affinity also have a parent polypeptide or wild-type Fc region sequence. An Fc variant that has "reduced", "silenced", "negligible" or "removed" ADCC, ADCP and CDC activity relative to the polypeptide it contains.

According to a preferred embodiment of the present invention, the Fc variant has reduced ADCC compared to the wild-type antibody Fc domain.

According to a preferred embodiment of the present invention, the Fc variant has reduced CDC compared to the wild-type antibody Fc domain.

An Fc variant that “displays reduced or undetectable binding” to an FcγR binds all FcγRs with lower affinity than the parent polypeptide. Such variants that exhibit reduced binding to FcγR may retain little or no appreciable binding to FcγR. In one embodiment, the variant exhibits 0-10% binding to an FcγR relative to a native IgG Fc region, as determined, for example, in the Examples herein or as measured by change in equilibrium constant. In one embodiment, the variant exhibits 0-5% binding to an FcγR compared to a native IgG Fc region. In one embodiment, the variant exhibits 0-3% binding to an FcγR compared to a native IgG Fc region. In one embodiment, the variant exhibits 0-1% binding to an FcγR relative to a native IgG Fc region.

"Parent antibody", "parent polypeptide" or "polypeptide comprising a wild-type Fc region" refers to a construct that does not contain amino acid modifications to the hinge region. In one embodiment, the parent antibody is one that does not contain amino acid modifications to the hinge region and does contain modifications to the CH3 domain that promote formation of a heterodimeric Fc region.

"Amino acid modification" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include amino acid substitutions, insertions and/or deletions. In certain embodiments, an amino acid modification herein is a substitution. For example, an "amino acid modification" at a specified position in an Fc region refers to a substitution or deletion of a specified residue, or insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a specified residue means an insertion within one or both of these residues. In certain embodiments, the insertion is N-terminal or C-terminal to a specified residue.

“Amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different “alternative” amino acid residue. The replacement residue or residues may be “naturally occurring amino acid residues” (ie those encoded by the genetic code) and may be selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); Isoleucine (Ile): Leucine (Leu); Lysine (Lys); methionine (Met); phenylalanine (Phe); Proline (Pro): Serine (Ser); threonine (Thr); tryptophan (Trp); Tyrosine (Tyr); and valine (Val). Preferably, the replacement moiety is not cysteine. Substitutions with one or more non-naturally occurring amino acid residues are also encompassed by the definition of amino acid substitutions herein. A "non-naturally occurring amino acid residue" refers to a residue other than the naturally occurring amino acid residues listed above, which may covalently bind adjacent amino acid residue(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogs such as Ellman et al. Meth. Enzym. 202:301-336 (1991).

An antibody Fc domain can be an Fc domain of an IgA, IgM, IgE, IgD, or IgG antibody, or variants thereof. In one embodiment, the domain is an Fc domain of an IgG antibody (eg, an Fc domain of an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody). In one embodiment, the Fc domain may be an IgG1 Fc domain (eg, trastuzumab or rituximab Fc domain) or an IgG4 Fc domain (eg, nivolumab Fc domain). In the polypeptide comprising the Fc domain of the present invention, some or all of the polypeptide may be non-glycated or glycosylated. In addition, the polypeptide may contain one or more regions from antibodies in addition to the Fc domain. Additionally, the polypeptide may include an antibody-derived antigen binding domain, and a plurality of polypeptides may form an antibody or antibody-like protein.

The amino acid residue numbers of the antibody Fc domain herein follow the Kabat EU numbering system commonly used in the art (Kabat et al., in Sequences of Proteins of Immunological Interest 5th Ed., U.S. Department of EU index number as in Health and Human Services, NIH Publication No. 91-3242, 1991).

According to a preferred embodiment of the present invention, the substituted Fc variant of the present invention comprises an amino acid substitution at one or more positions selected from the group consisting of S228, L234, L235, T299 and P329 according to the Kabat EU numbering system.

The Fc variants of the present invention have reduced binding ability to FcγRIIIa compared to the wild-type antibody Fc domain.

The term "avidity" or "binding affinity" herein generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg antibody) and its binding partner (eg FcγR). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd or KD). Affinity can be measured by common methods known in the art, including those described herein.

The Fc variants of the present invention preferably have a KD of 10 nM or less, more preferably a KD of 5 nM or less, even more preferably a KD of 3 nM or less, and even more preferably a KD of 1 nM or less for FcγRIIIa. , most preferably exhibits a reduced binding force to an unmeasurable level.

“Fc-fusion” as used herein refers to a protein in which one or more polypeptides are operably linked to an Fc region or derivative thereof. Fc fusions combine the Fc region of an immunoglobulin with a fusion partner, which generally can be any protein or small molecule. The role of the non-Fc portion of an Fc fusion, i.e. the fusion partner, is to mediate target binding and is therefore functionally similar to the variable region of an antibody. Virtually any protein or small molecule can be linked to Fc to create an Fc fusion. A protein fusion partner may include, but is not limited to, a target-binding region of a receptor, junction attachment, ligand, enzyme, cytokine, chemokine, or some other protein or protein domain. Small molecule fusion partners can include any therapeutic agent that induces Fc fusion to a therapeutic target. Such a target may be any molecule, preferably an extracellular receptor involved in a disease. Two families of surface receptors that are targets of many approved small molecule drugs are G-protein coupled receptors (GPCRs), and ion channels, including K+, Na+, Ca+ channels. Currently, nearly 70% of all drugs marketed worldwide target GPCRs. Thus, an Fc protein described herein may, for example, bind to one or more of GABA receptors, purinergic receptors, adrenergic receptors, histamine receptors, opioid receptors, chemokine receptors, glutamate receptors, nicotinic receptors, 5HT (serotonin) receptors, and estrogen receptors. Can be fused to targeted small molecules. A fusion partner can be a small molecule mimic of a protein that targets a therapeutically useful target. Specific examples of specific drugs that can act as Fc fusion partners are [L. S. Goodman et al, Eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill, New York, ed. 9, 1996). In addition, a variety of linkers can be used to covalently link an Fc to a fusion partner to create an Fc fusion.

According to a preferred embodiment of the present invention, the Fc variant comprises one or more amino acid substitutions selected from the group consisting of S228P, L234A, L235A, L235E, T299L and P329G according to the Kabat EU numbering system in the Fc domain of the wild type antibody.

According to a preferred embodiment of the present invention, the Fc variant comprises the following amino acid substitutions according to the Kabat EU numbering system in the wild-type antibody Fc domain:

(i) L234A and L235A;

(ii) L234A, L235A and P329G;

(iii) T299L;

(iv) L234A, L235A and T299L;

(v) S228P and L235E;

(vi) S228P, L235E and T299L; or

(v) S228P and T299L.

According to a preferred embodiment of the present invention, the Fc variant comprises an additional amino acid mutation for extending half-life compared to the wild-type antibody Fc domain.

For example, the Fc variant may further include an amino acid substitution at one or more positions selected from the group consisting of M252, S254, T256, L309, Q311, M428 and N434 according to the Kabat EU numbering system in the Fc domain of the wild type antibody. there is.

According to a preferred embodiment of the present invention, said additional amino acid substitution may be selected from the group consisting of M252Y, S254T, T256E, L309G, Q311R, M428L and N434S.

According to a preferred embodiment of the present invention, the additional amino acid substitution may include the following amino acid substitution:

(i) M252Y, S254T and T256E;

(ii) M428L and N434S;

(iii) Q311R and M428L; or

(iv) L309G and M428L.

The additional amino acid substitution modulates binding and dissociation to the neonatal Fc receptor (FcRn). In particular, it includes Fc variants, or functional variants thereof, that exhibit increased binding affinity to FcRn at low pH and show no substantially altered binding at high pH.

According to a preferred embodiment of the present invention, the Fc variant has a binding affinity to FcRn at pH 5.6 to 6.2 (preferably pH 5.8 to 6.0) of 10% or more, 20% or more compared to the wild-type Fc domain. ≥, ≥ 30%, ≥ 40%, ≥ 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90%, or ≥ 100%, or at least 2-fold, 3-fold, or 4-fold greater than the wild-type Fc domain. more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 20 times, more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, It can be increased by 80 times or more, 90 times or more or 100 times or more.

According to a preferred embodiment of the present invention, the degree of dissociation of the Fc variant from the neonatal Fc receptor (FcRn) at pH 7.0 to 7.8 (preferably pH 7.2 to 7.6) is equal to or substantially greater than that of the wild-type Fc domain. may not change to

According to one embodiment of the present invention, the substituted Fc variants of the present invention showed significantly improved binding affinity under slightly acidic conditions (e.g., pH 5.8 to 6.0) compared to wild-type Fc or other previously developed Fc variants. , showed the same or substantially equal or higher degree of dissociation under neutral conditions (eg, pH 7.0).

According to a preferred embodiment of the present invention, the substituted Fc variant of the present invention has an increased half-life compared to the wild-type antibody Fc domain.

The half-life of the substituted Fc variants of the present invention is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more compared to the wild-type Fc domain. Increased by at least 100% or more, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold more than the wild-type Fc domain there is.

According to one embodiment of the present invention, the substituted Fc variant of the present invention showed a significantly improved in vivo half-life compared to the wild type.

According to another aspect of the present invention, the present invention provides an antibody comprising the above polypeptide.

The term antibody herein is a polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, antibody mimetic, chimeric antibody, antibody conjugate, human antibody or humanized antibody, or means fragment.

According to a preferred embodiment of the present invention, the half-life of the Fc domain or a polypeptide including the Fc domain is maximized through optimization of the antibody Fc region, while reducing or silencing the effector function.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the polypeptide, a vector containing the nucleic acid molecule, or a host cell containing the vector.

Nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded form, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, cDNA molecule, or oligonucleotide, the nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. Nucleic acids are operably linked when placed into a functional relationship with another nucleic acid sequence. For example, DNA of a full sequence or secretory leader is operably linked to DNA of a polypeptide when the polypeptide is expressed as a preprotein in its pre-secreted form, and a promoter or enhancer is the polypeptide sequence. is operably linked to a coding sequence when it affects transcription of, or when the ribosome binding site is positioned to facilitate translation. In general, operably linked means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

As used herein, the term vector refers to a carrier capable of inserting a nucleic acid sequence into a cell capable of replicating the nucleic acid sequence. A nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, AAV, bacteriophage). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994, etc.).

As used herein, the term expression vector refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.

As used herein, the term host cell includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. The host cell may be transfected or transformed by the vector, which means a process in which an exogenous nucleic acid molecule is delivered or introduced into the host cell.

In this specification, the term "transformation" is used as a meaning including the transfection (Transfected) and transduction (Transduced).

The (host) cells of the present invention are not limited, but preferably insect cells or mammalian cells, more preferably Sf9 in the case of insect cells, HEK293 cells in the case of mammalian cells, HeLa cells, ARPE-19 cells, RPE- 1 cell, HepG2 cell, Hep3B cell, Huh-7 cell, C8D1a cell, Neuro2A cell, CHO cell, MES13 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell, MCF-7 cell, HC70 cell, HCC1428 cell , BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, MIA PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, NIH3T3 cells and the like can be used.

The host cell of the present invention is preferably an isolated host cell.

According to another aspect of the present invention, the present invention provides a method for producing a polypeptide comprising an Fc variant having a reduced effector function compared to a wild-type Fc domain, comprising the steps of:

a) culturing a host cell containing a vector containing a nucleic acid molecule encoding a polypeptide containing the Fc variant; and

b) recovering the polypeptide expressed by the host cell.

According to another aspect of the present invention, the present invention provides a method for producing an antibody with reduced effector function compared to wild type, comprising the steps of:

a) culturing a host cell expressing an antibody comprising a polypeptide comprising the Fc variant; and

b) purifying the antibody expressed from the host cell.

In the production method of the present invention, antibody purification may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably Protein Affinity chromatography using A can be used.

According to another aspect of the present invention, the present invention provides a composition comprising the polypeptide, the antibody, the nucleic acid molecule or the vector comprising the Fc variant comprising the amino acid substitution.

According to a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising a polypeptide comprising the Fc variant, a nucleic acid molecule encoding the polypeptide, or a vector comprising the nucleic acid molecule, to a subject in need thereof ( It provides a method for delivering an Fc variant with reduced effector function, comprising the step of administering to a subject.

According to one embodiment of the present invention, the Fc variant of the present invention has a low FcγRIIIa binding affinity and / or no ADCC (antibody dependent cellular cytotoxicity) to the degree of undetectable compared to the control group (eg, rituximab or nivolumab) and/or Complement-Dependent Cytotoxicity (CDC) While exhibiting activity, it has remarkable half-life increasing ability at the same time.

The pharmaceutical composition of the present invention comprises (a) the polypeptide, a nucleic acid molecule encoding the polypeptide, or a vector containing the nucleic acid molecule; and (b) a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may be used for prevention or treatment of a specific disease (eg, tumor, viral or bacterial infection, etc.).

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed with, but is likely to have, the disease or condition.

As used herein, the term "treatment" refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition. The composition of the present invention may be a composition for treating viral infection by itself, or may be applied as a therapeutic adjuvant to improve its therapeutic response by being administered together with other pharmacological ingredients. Accordingly, the terms "treatment" or "therapeutic agent" herein include the meaning of "therapeutic aid" or "therapeutic agent".

As used herein, the term "administration" or "administration" refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

In the present invention, the term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the content of the composition contained in a sufficient level to provide a therapeutic or preventive effect of the pharmacological component in the composition to the subject to whom the pharmaceutical composition of the present invention is to be administered. It means, and it is meant to include a "prophylactically effective amount" therein.

As used herein, the term "subject" includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.

The pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions.

The composition of the present invention may contain one or more known active ingredients having anticancer activity, antiviral or antibacterial activity.

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenteral administration, such as intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intravitreal injection, subretinal injection. Subretinal injection, suprachoroidal injection, eye drop administration, intracerebroventricular injection, intrathecal injection, intraamniotic injection, intra-arterial intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisternal injection, intracoronary injection (intracoronary injection), intracranial injection, intrathecal injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection injection), intraperitoneal injection, intrathoracic injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection , intravaginal injection ( It can be administered by intravaginal injection), intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection, local injection and intraperitoneal injection. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type and degree of response to be achieved by administration of the pharmaceutical composition. , various factors, including the type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used simultaneously or at the same time in the subject, and other components of the composition, and the like It can be varied according to similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.

The administration route and administration method of the pharmaceutical composition of the present invention may be each independent, and are not particularly limited in the method, and any administration route and administration method as long as the pharmaceutical composition can reach the target site can follow

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a polypeptide comprising an Fc variant in which part of the amino acid sequence of a human antibody Fc domain is substituted with another amino acid sequence, or an antibody comprising the same.

(ii) In addition, the present invention provides a method for preparing the polypeptide or antibody.

(iii) The Fc variant of the present invention can reduce or silence effector functions and maximize half-life in the body through optimization of some amino acid sequences.

Figure 1 shows the SDS-PAGE results of Rituximab (IgG1) Fc double mutant.

Figure 2 shows the results of SEC-HPLC analysis of Rituximab (IgG1) Fc double mutant.

Figure 3 shows the SDS-PAGE results of Nivolumab (IgG4) Fc double mutant.

Figure 4 shows the results of SEC-HPLC analysis of Nivolumab (IgG4) Fc double mutant.

Figure 5 shows the conditions for measuring the binding force of Rituximab (IgG1) Fc double mutant with FcγRIIIa.

6a, 6b, 6c, 6d, and 6e show binding patterns of Rituximab (IgG1) Fc double mutants to FcγRIIIa.

Figures 7a and 7b show the results of measuring the binding force between human FcRn and Fc double variants at pH 6.0 (Fig. 7a: results of measuring the binding force of four variants (YTE, LS, PFc29, PFc41) compared to Rituximab; Fig. 7b: effector function As a result of introducing additional TL mutatants that decrease).

Figure 8 shows the conditions for measuring the binding force of Nivolumab (IgG4) Fc double mutant with FcγRIIIa.

9a, 9b, and 9c show the results of analysis of binding patterns of Nivolumab (IgG4) Fc Double Variants to FcγRIIIa (FIGS. 9a and 9b: KD values for variants in which SPTL and SPLETL were additionally introduced; FIG. 9c: WT, LS, KD values of PFc29, PFc41).

Figures 10a and 10b show ADCC assay results between Rituximab (IgG1) Fc double mutants (Figure 10a: comparison between original form and LALAPG mutant form; Figure 10b: comparison between original form and TL mutant form).

11a and 11b show CDC assay results between Rituximab (IgG1) Fc double mutants (FIG. 11a: comparison between original form and LALAPG mutant form; FIG. 11b: comparison between original form and TL mutant form).

12 shows the results of measuring the hFcRn binding affinity between Nivolumab (IgG4) Fc double variants at pH 6.0.

13 shows the results of measuring the hFcRn binding ability between Nivolumab (IgG4) Fc double variants at pH 7.4.

14 shows the results of Effector Silencing Effect confirmation (ADCC Assay) of Nivolumab (IgG4) Fc double variants.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Preparation and production of Rituximab (IgG1) and Nivolumab (IgG4) immune mechanism removal Fc variant construct

go. Construction of Rituximab (IgG1) Fc Double Mutant Constructs

Rituximab Fc variants in which LALA (L234A, L235A), LALAPG (L234A, L235A, P329G), TL (T299L), and LALATL (L234A, L235A, T299L) mutations were introduced into Rituximab wild type, YTE, LS, pFc29, and pFc41 variants, respectively. A total of 25 variants were produced by introducing heavy chain and light chain constructs using the pcDNA3.4 vector (Thermo Fisher Scientific, 14697) (Table 1).

SEQ ID No.	variant name	half-life increasing mutation	Effector-silent mutation
One	Rituximab	WT	-
2	Rituximab-YTE			YTE
3	Rituximab-LS			LS
4	Rituximab-PFc29			PFc29
5	Rituximab-PFc41			PFc41
6	Rituximab-LALA	WT		LALA
7	Rituximab-YTE_LALA				YTE
8	Rituximab-LS_LALA				LS
9	Rituximab-PFc29_LALA				PFc29
10	Rituximab-PFc41_LALA				PFc41
11	Rituximab-LALPG	WT		LALPG
12	Rituximab-YTE_LALPG				YTE
13	Rituximab-LS_LALPG				LS
14	Rituximab-PFc29_LALPG				PFc29
15	Rituximab-PFc41_LALPG				PFc41
16	Rituximab-TL	WT		TL
17	Rituximab-YTE_TL				YTE
18	Rituximab-LS_TL				LS
19	Rituximab-PFc29_TL				PFc29
20	Rituximab-PFc41_TL				PFc41
21	Rituximab-LALATL	WT		LALATL
22	Rituximab-YTE_LALATL				YTE
23	Rituximab-LS_LALATL				LS
24	Rituximab-PFc29_LALATL				PFc29
25	Rituximab-PFc41_LALATL	PFc41

Introduction of additional variants that eliminate the immune mechanism based on half-life increasing variants

me. Rituximab (IgG1) Fc double variant expression purification

Twenty-five Fc variants, including Rituximab wild type, were transiently expressed in animal cells using the Expi293F expression system (Thermo Fisher Scientific, A14527).

The day before transfection, Expi293F cells were subcultured to 2x10 ⁶ cells/mL, and the next day, when the cell density reached 2.9x10 ⁶ cells/mL, the cells were prepared in an amount of 270 mL. In 15 mL of Opti-MEM1 medium (Thermo Fisher Scientific, 31985070), mix a total of 300 ug of plasmid DNA with a heavy chain to light chain transfection ratio of 1:1, and mix ExpiFectamine293 transfection reagent (Thermo Fisher Scientific, A14524) 0.81 mL each was mixed and left for 5 minutes, then the DNA-mixed Opti-MEM1 medium mixed with the ExpiFectamine293 transfection reagent mixed Opti-MEM1 medium was mixed well and left at room temperature for 20 minutes. , in a shaking CO ₂ incubator (INFORS HT, multitron standard) at 37°C, 125 rpm, and 8% CO ₂ conditions. After 20 hours, add 2.5 mL of Enhancer1 and 25 mL of Enhancer2, incubate a total of 300 mL in a shaking CO ₂ incubator for 5 days at 37℃, 125 rpm, 8% CO ₂ conditions, and then centrifuge to collect only the supernatant. did

The expression supernatant was subjected to affinity chromatography using an AKTA prime plus (GE healthcare, 11001313) equipped with a HiTrap MabSelect SuRe column (GE healthcare, #11003495), and 300 mL of the supernatant was flowed at a rate of 3 mL per minute. Elution buffer (100 mM citrate buffer, 5% sucrose, pH3.0) was eluted with 6 fractions of 5 mL each at a rate of 3 mL per minute, and 2 to 4 fractions were collected. Using a 30K Amicon ultra centrifugal filter (Millipore, UFC903024), the protein was finally purified by concentrating while changing the buffer to a pH 7.4 1xPBS buffer (ThermoFisher, 10010031). The produced protein was subjected to SDS-PAGE and SEC-HPLC analysis to secure an antibody with a purity of 92% or more (FIGS. 1 and 2).

all. Fabrication of Nivolumab (IgG4) Fc Double Mutant Constructs

Nivolumab Fc variants in which SPLE (S228P, L235E), SPLETL (S228P, L235E, T299L), and SPTL (S228P, T299L) mutations were introduced into Nivolumab wild type, YTE, LS, pFc29, and pFc41 variants, respectively, were used as pcDNA3.4 vectors. A total of 20 variants were produced by introducing heavy chain and light chain constructs (Table 2).

SEQ ID No.	variant name	half-life increasing mutation	Effector-silent mutation
26	Nivolumab	WT	-
27	Nivolumab-YTE			YTE
28	Nivolumab-LS			LS
29	Nivolumab-PFc29			PFc29
30	Nivolumab-PFc41	PFc41
31	Nivolumab-LALA	WT	SPLE
32	Nivolumab-YTE_SPLE	YTE
33	Nivolumab-LS_SPLE	LS
34	Nivolumab-PFc29_SPLE			PFc29
35	Nivolumab-PFc41_SPLE	PFc41
36	Nivolumab-SPLETL	WT		SPLETL
37	Nivolumab-YTE_SPLETL	YTE
38	Nivolumab-LS_SPLETL	LS
39	Nivolumab-PFc29_SPLETL				PFc29
40	Nivolumab-PFc41_SPLETL				PFc41
41	Nivolumab-SPTL	WT	SPTL
42	Nivolumab-YTE_SPTL	YTE
43	Nivolumab-LS_SPTL	LS
44	Nivolumab-PFc29_SPTL	PFc29
45	Nivolumab-PFc41_SPTL	PFc41

la. Nivolumab (IgG4) Fc double variant expression purification

Twenty Fc variants, including Nivolumab Wild type, were transiently expressed in animal cells using the ExpiCHO expression system (Thermo Fisher Scientific, A29127).

The day before transfection, ExpiCHO cells were subcultured to 3x10 ⁶ cells/mL, and the next day, when the cell density reached 6x10 ⁶ cells/mL, cells were prepared in an amount of 200 mL. In 8 mL of OptiPRO SFM medium (Thermo Fisher Scientific, 12309050), mix a total of 300 ug of plasmid DNA with a heavy chain to light chain transfection ratio of 1:1, and in another 7.4 mL of OptiPRO SFM medium, ExpiFectamineCHO transfection reagent (ThermoFisher, A29129 ) After mixing 0.64 mL each, the two media were mixed well and added to 200 ml of ExpiCHO prepared in advance, and cultured in a shaking CO ₂ incubator at 37°C, 125 rpm, and 8% CO ₂ conditions. After 20 hours, add 1.2 mL of ExpiCHO Enhaner and 32 mL of ExpiCHO Feed, culture at 32°C, 125 rpm, 5% CO ₂ conditions, and after 5 days, add 32 ml of ExpiCHO Feed additionally, incubate for 14 days, and centrifuge Only the supernatant was taken.

The expression supernatant was subjected to affinity chromatography using an AKTA prime plus equipped with a HiTrap MabSelect SuRe column (GE healthcare, 11003495). 300 mL of the supernatant was flowed at a rate of 3 mL per minute. Elution buffer (100 mM citrate buffer, 50 mM NaCl, 5% sucrose, pH3.0) was eluted with 6 fractions of 5 mL each at a rate of 3 mL per minute, and 2-4 fractions were collected. Using a 30K Amicon ultra centrifugal filter (Millipore, UFC903024), the protein was finally purified by concentrating while changing the buffer to a pH 7.4 1xPBS buffer (ThermoFisher, 10010031). The produced protein was subjected to SDS-PAGE and SEC-HPLC analysis to secure an antibody of 95% or more purity (FIGS. 3 and 4).

Example 2: Measurement of FcγRIIIa and FcRn binding affinity of Rituximab (IgG1) and Nivolumab (IgG4) immune mechanism-removed Fc variants

go. Measurement of FcγRIIIa avidity of rituximab (IgG1) Fc double variants

In order to measure the binding force between FcγRIIIa and the Rituximab variant, binding force was measured by capturing FcγRIIIa as a ligand using Biacore T200 equipment (GE healthcare, 28975001) (FIG. 5).

* As a result of measuring binding force with FcγRIIIa, it was confirmed that LALA, TL, LALATL, and LALAPG mutants had significantly reduced binding force to the extent that KD values were not calculated. However, in the case of LALA, the binding pattern was shown, and the binding pattern was not shown in the LALAPG mutant in which the PG mutation was additionally introduced. In the case of the TL variant, since no binding pattern was shown, no additional binding force reduction pattern could be observed in the LALATL variant in which the LALA mutation was additionally introduced (FIG. 6).

me. Measurement of FcRn binding affinity of rituximab (IgG1) Fc double variants

In order to measure the binding force between FcRn and the Rituximab-Fc variant, the binding force was measured by capturing the Rituximab-Fc variant as a ligand on a CM5 chip (GE Healthcare, BR100012) using Biacore T200 equipment (GE Healthcare, 28975001). As a result of measuring the binding force under the condition of endosome pH, pH 6.0, the binding force of 4 variants (YTE, LS, PFc29, PFc41) was improved compared to Rituximab. In addition, even though a TL mutatant that reduces the therapeutic effector function that may exhibit an excessive immune response was introduced, the binding force was maintained compared to before introduction (FIG. 7).

all. Measurement of FcγRIIIa avidity of Nivolumab (IgG4) Fc double variants

In order to measure the binding force of FcγRIIIa and Nivolumab (IgG4) Fc double mutant, binding force was measured by capturing FcγRIIIa as a ligand using Biacore T200 equipment (FIG. 8).

As a result of measuring the binding force with FcγRIIIa, the existing Nivolumab variants showed some remaining binding force to FcγRIIIa, except for YTE, and the variants with additional SPTL and SPLETL were significantly reduced in binding force to such an extent that the KD value was not calculated. In the SPLETL mutant in which the LE mutation was additionally introduced, no additional binding force reduction pattern was observed (FIGS. 9a and 9b). Binding to FcγRIIIa was found in WT, LS, PFc29, and PFc41 except for YTE, and KD values could not be measured due to low binding force (FIG. 9c).

Example 3: Confirmation of difference in antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) reactivity between rituximab (IgG1) Fc double variants

go. ADCC (Antibody-Dependent Cellular Mediated Cytotoxicity) Assay Analysis

In order to compare the ADCC reactivity between the original form of Rituximab (IgG1) Fc variant and the double variant with LALAPG and TL mutations, CD20 is stably expressed Raji cell (ATCC, CCL-86) was selected as the target cell and the experiment was conducted did

On the day of the ADCC assay test, Raji cells are seeded by counting the cell number in the assay buffer so that the cell number of 1.25x10 ⁴ enters each well in a 96 well white plate, and the test substance (original, LALAPG, TL) is assayed The cells were treated in a 96 well white plate seeded so that the buffer had the highest concentration of 1 ug/mL, 1/3 dilution, and 10 points. Take the effector cell out of the liquid nitrogen container, melt it quickly, process it on a 96 well white plate so that the ratio of effector cell to target cell is 25:1, and incubate in a CO ₂ incubator (37℃, 5% CO ₂ conditions) for 6 hours. reacted When the time elapsed, the plate was taken out and the result value was collected by measuring the luminescence fold using EnSpire (Perkin Elmer) equipment. As a result of the measurement, the original form of Rituximab (IgG1) showed an ADCC reactivity of about 50 fold or less, whereas the LALAPG mutant form (FIG. 10a) and the TL mutant form showed no ADCC reactivity (FIG. 10b).

me. CDC (Complement-Dependent Cytotoxicity) Assay Analysis

In order to compare the CDC reactivity of the original form of Rituximab (IgG1) Fc double mutant and LALAPG and TL mutants, Raji cells stably expressing CD20 were selected as target cells and experiments were conducted.

On the day of the CDC assay, Raji cells (ATCC, CCL-86) were placed in a 96-well V-bottom plate (Corning, 3894) by counting the cell numbers in assay buffer so that 1.2x10 ⁴ cells entered each well and counting the cells. After seeding, the test substance (original, LALAPG, TL) is treated in a 96-well V-bottom plate (Corning, 3894) seeded with cells so that the test substance (original, LALAPG, TL) has a maximum concentration of 1 ug/mL, 1/3 dilution, and 10 points in assay buffer and reacted at room temperature for 15 minutes. Take the Complement (Quidel, A112) out of the deepfreezer, melt it quickly, process it in a 96-well V-bottom plate to a 2.5% ratio, and incubate in a CO ₂ incubator (37℃, 5% CO ₂ conditions, Eppendorf, GALAXY 170S) for 1 hour After reacting for a while, the plate was taken out and centrifugation was performed at 300 g for 5 minutes, and 50 uL of the supernatant was transferred to a new 96 well flat-bottom plate (Corning, 3635). Add 50 uL of Cytotox96 reagent (Promega, G1780) to each well, react for 30 minutes, and when the time elapses, add 50 uL of stop reagent to stop the reaction, and use ELISA plate reader (Molecular Device, VersaMAX) Optical density values were measured.

As a result of the measurement, the original form of Rituximab (IgG1) showed cytotoxicity of about 50% or less, whereas the LALAPG mutant form (Fig. 11a) and the TL mutant form (Fig. 11b) showed no CDC reactivity at all.

Example 4: Measurement of pH-dependent dependent hFcRn binding affinity of Nivolumab (IgG4) Fc double variants

* To measure the binding force between hFcRn and Nivolumab-Fc double variants, a Biacore T200 instrument was used. After immobilizing anti-Nivolumab antibody (BIORAD, HCA299) using the Anti-idiotype Ab capture method, Nivolumab-Fc double variants were captured on a CM5 chip at about 160 RU level as a ligand to measure binding force.

The binding force measurement was performed in the range of 31.25 to 1000 nM as a total of 6 points by diluting hFcRn with an analyte at a concentration of 1/2 at a concentration of 1000 nM. As a result of measuring the binding force under the condition of endosome pH, pH 6.0, an Fc duplex introduced with a TL mutatant that reduces the therapeutic effector function that can show an excessive immune response to three variants (LS, PFc29, PFc41) compared to Nivolumab. The binding ability of the variant was greatly improved compared to the control group (FIG. 12). In the case of Xencor's LS double variant currently on the market, the binding affinity with FcRn was the best at pH 6.0 compared to the control group, but the relative dissociation value was the highest at the physical pH of 7.4, so it did not dissociate efficiently (FIG. 13) . According to reports in many literatures, this tendency is expected to decrease the half-life in actual in vivo pharmacokinetics, and it was predicted that it may not be longer than the two variants pFc29 and pFc41. In the case of the control group, efficient dissociation at pH 7.4 is the best, but the binding force of the actual endosome at pH 6.0 is too weak, so it can be predicted that the half-life in the body is not long.

Example 5: Confirmation of effector silencing effect in antibody-dependent cytotoxicity (ADCC) between Nivolumab (IgG4) Fc double variants

In order to compare ADCC (Antibody-Dependent Cellular Mediated Cytotoxicity) reactivity between double mutants in which TL mutations were added to Nivolumab (IgG4) Fc variants, Raji cells overexpressing hPD-1 (Raji-hPD-1 cell, Invivogen, raji- hpd1) was selected as the target cell and the experiment was conducted.

On the day of the ADCC assay, Raji-hPD-1 cells were counted in assay buffer so that the number of cells in each well was 1.25x10 ⁴ in a 96 well white plate (Thermo Fisher Scientific, 136101), and the cells were seeded, The test substances (double mutants) were treated in a 96-well white plate seeded with cells so that the assay buffer had a maximum concentration of 1 ug/mL, 1/3 dilution, and 10 points. Take the effector cell out of the liquid nitrogen container, melt it quickly, process it on a 96 well white plate so that the ratio of effector cell to target cell is 25:1, and incubate in a CO ₂ incubator (37℃, 5% CO ₂ conditions) for 6 hours. reacted When the time elapsed, the plate was taken out and the result value was collected by measuring the luminescence fold using EnSpire (Perkin Elmer) equipment. As a result of the measurement, the ADCC reactivity of each original form variant introduced into Nivolumab (IgG4) was confirmed to have almost no effect as expected, and the ADCC effect, which was insignificant in original form IgG4 (including variants), also showed ADCC in the double variant form introduced with TL mutation. It was confirmed that the reactivity was completely lost (FIG. 14).

Example 6: hFcRn Tg mouse pharmacokinetics of Nivolumab (IgG4) TL variants with increased half-life and ablated mechanisms of action

Human FcRn Tg mice (Jackson Lab., JAX#004919) were used for IgG4-based Nivolumab prepared by introducing half-life increasing mutations LS, PFc29, PFc41 and TL (T299L) mutations reported to inhibit the immune mechanism. The blood half-life improvement effect was compared and verified.

24 human FcRn Tg mice for 4 types of Nivolumab including Fc variants (6 mice per group, 6 mg/kg were injected by I.V (tail vein), and then cross-blooded from the facial vein (total 12 times → 0.5, 2, 8 , 24, 72 hr (0, 1, 4, 12, 72 hr), 7, 14, 21, 28, 35, 42, 49 day) after concentration analysis using ELISA, Phoenix WinNonlin™ (Certara, ver. .8.1.0) and non-compartmental analysis (NCA) was conducted Four pharmacokinetic variables calculated through ELISA analysis were investigated (Table 3).

As a result, among variants in which additional TL (T299L) mutations were introduced into blood half-life variants (LS, pFc29 and pFc41) introduced into the IgG4 antibody Nivolumab, XenCore's LS mutants had a more reduced half-life than the control group after introducing the TL additional variant. this has been confirmed This is presumed to be due to the relative instability following the introduction of additional mutations and the dissociation at pH 7.4 (Fig. 11) being relatively less than the other two variants. On the other hand, it was confirmed that the two variants, pFc29 and pFc41, maximized the immune effector silencing effect and stably maintained the half-life under the introduction of additional TL variants when the IgG4 backbone was introduced (Table 3).

Antibody a	Antigen	Animals per Group	Half-Life b (days)			AUC b (d*ug/mL)		Clearance b (mL/d /kg)
			Mean	SD	Fold c	Mean	SD	Mean	SD
WT-TL	PD-1	6	7.4	1.8	1.0	506.7	132.1	12.24	3.36
LS-TL	PD-1	6	6.53	2.8	0.88	418.2	94.7	14.64	3.6
pF29-TL	PD-1	6	10.6	7.5	1.4	552.3	212.2	11.52	2.88
pF41-TL	PD-1	6	8.7	1.8	1.2	513.5	141.8	12.24	3.36

PK analysis of Nivolumab Fc mutants using hFcRn Tg mice

^a The Fc region of anti-PD1 Ab (Nivolumab) for the Fc engineered version. Dose level and route: single ivinfusion at 6 mg/kg.

^b Half-life, area under the curve (AUC), and clearance were computed for individual animals using noncompartment methods and are reported as the mean and standard deviation (SD).

^c Fold half-life=half-life (double variant in Nivo) / half-life (Nivo-single variant (IgG4+TL))

Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (23)

1. A polypeptide comprising an antibody Fc variant, wherein the Fc variant is selected from the group consisting of S228, L234, L235, T299 and P329 according to the Kabat EU numbering system in a wild type antibody Fc domain A polypeptide comprising an amino acid substitution at one or more positions and characterized by reduced binding ability to FcγRIIIa compared to a wild-type antibody Fc domain.
2. The polypeptide according to claim 1, wherein the Fc variant has reduced ADCC (Antibody-Dependent Cellular Mediated Cytotoxicity) compared to the wild-type antibody Fc domain.
3. The polypeptide according to claim 1, wherein the Fc variant has reduced Complement-Dependent Cytotoxicity (CDC) compared to the wild-type antibody Fc domain.
4. The method of claim 1, wherein the Fc variant comprises one or more amino acid substitutions selected from the group consisting of S228P, L234A, L235A, L235E, T299L and P329G according to the Kabat EU numbering system in the Fc domain of the wild type antibody. polypeptide.
5. The polypeptide according to claim 4, wherein the Fc variant comprises the following amino acid substitutions according to the Kabat EU numbering system in the Fc domain of the wild type antibody:

   (i) L234A and L235A;

   (ii) L234A, L235A and P329G;

   (iii) T299L;

   (iv) L234A, L235A and T299L;

   (v) S228P and L235E;

   (vi) S228P, L235E and T299L; or

   (v) S228P and T299L.
6. The method of claim 1, wherein the Fc variant further comprises an amino acid substitution at one or more positions selected from the group consisting of M252, S254, T256, L309, Q311, M428 and N434 according to the Kabat EU numbering system in the wild-type antibody Fc domain. Characterized in that, a polypeptide.
7. 7. The polypeptide of claim 6, wherein the additional amino acid substitution is selected from the group consisting of M252Y, S254T, T256E, L309G, Q311R, M428L and N434S.
8. 8. The polypeptide of claim 7, wherein the additional amino acid substitution comprises the following amino acid substitution:

   (i) M252Y, S254T and T256E;

   (ii) M428L and N434S;

   (iii) Q311R and M428L; or

   (iv) L309G and M428L.
9. The polypeptide according to claim 8, wherein the Fc variant has an increased half-life compared to the wild-type antibody Fc domain.
10. The polypeptide according to claim 1, wherein the antibody is an IgG antibody.
11. 11. The polypeptide according to claim 10, wherein the IgG antibody is an IgG1 or IgG4 antibody.
12. An antibody comprising the polypeptide of claim 1 .
13. 13. The antibody of claim 12, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a chimeric antibody, an antibody conjugate, a human antibody, or a humanized antibody. An antibody characterized in that it is or a fragment thereof.
14. A nucleic acid molecule encoding the polypeptide of claim 1.
15. A vector comprising the nucleic acid molecule of claim 14.
16. A host cell comprising the vector of claim 15.
17. A pharmaceutical composition comprising the polypeptide of claim 1, a nucleic acid molecule encoding the polypeptide, or a vector containing the nucleic acid molecule.
18. 18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition additionally comprises a drug.
19. According to claim 18, wherein the pharmaceutical composition is characterized in that for reducing the effector function (effector function) of the Fc domain, the pharmaceutical composition.
20. The pharmaceutical composition according to claim 19, characterized in that the pharmaceutical composition is for increasing half-life.
21. A method for producing a polypeptide comprising an Fc variant with reduced effector function compared to a wild-type Fc domain comprising the following steps:

    a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the polypeptide of claim 1; and

    b) recovering the polypeptide expressed by the host cell.
22. A method for producing an antibody with reduced effector function compared to wild type comprising the following steps:

    a) culturing a host cell expressing an antibody comprising the polypeptide of claim 1; and

    b) purifying the antibody expressed from the host cell.
23. An effector function comprising administering a pharmaceutical composition comprising the polypeptide of claim 1, a nucleic acid molecule encoding the polypeptide, or a vector containing the nucleic acid molecule to a subject in need thereof. ) Method of delivery of reduced Fc variants.

Images