KR20180113904A - Antibody Fc variants for Prolonged Serum 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 a human antibody Fc domain is substituted with another amino acid sequence, or an antibody comprising the same. The Fc variant of the present invention can have use in a wide variety of antibodies and Fc fusions products. In one aspect, the antibodies or Fc fusions of the present invention are therapeutic, diagnostic, or research reagents, preferably therapeutic reagents. The Fc variant of the present invention can maximize half-life in the body through optimization of some amino acid sequences and can be useful for the treatment of cancer. The antibodies and Fc fusions of the present invention are used to kill target cells containing target antigens, e.g., cancer cells. Alternatively, the antibodies and Fc fusions of the present invention are used to block, antagonize, or interfere with the target antigen, for example, to agonize against a cytokine or a cytokine receptor.

Description

Antibody Fc variants for Prolonged Serum Half-Life {Antibody Fc variants for Prolonged Serum Half-Life}

The present invention relates to novel antibody Fc variants with prolonged blood half-life and a method for preparing the same.

With the development of biotechnology such as gene recombination and cell culture around the world, research on the structure and function of proteins has been actively conducted, which is crucial for not only improving understanding of life phenomena, but also identifying the pathogenesis mechanisms of various diseases. By playing a role, it is contributing greatly to improving the quality of life by providing a path for effective disease diagnosis and treatment. In particular, hybridoma technology that produces monoclonal antibodies by fusion of B cells and Myeoloma cells was developed in 1975 (Kohler and Milstein, Nature , 256:495-497, 1975), research and development on immunotherapy using therapeutic antibodies in clinical fields such as cancer, autoimmune diseases, inflammation, cardiovascular diseases, and infections have been actively conducted.

The therapeutic antibody is considered to be one of the most effective cancer treatment methods because it shows very high specificity for the target compared to the existing low-molecular drugs, has low biotoxicity, less side effects, and has an excellent half-life in the blood of about 3 weeks. In fact, large pharmaceutical companies and research institutes around the world are spurring research and development of therapeutic antibodies that specifically bind to and effectively remove cancer cells, including cancer-causing factors. Pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS are dominated by therapeutic antibody drug development companies.In particular, Roche has Herceptin, Avastin, and Rituxan for anticancer treatment. ), as a representative product, is not only generating huge profits by achieving sales of about $19.5 billion in the global market in 2012 with these three therapeutic antibodies, but also leading the global antibody drug market. Johnson & Johnson, who developed Remicade, is also rapidly growing in the global antibody market due to increased sales, and pharmaceutical companies such as Abbott and BMS are also known to possess a number of therapeutic antibodies at the end of development. As a result of this, biopharmaceuticals, including therapeutic antibodies that are specific to disease targets and have low side effects, are rapidly replacing their place in the global pharmaceutical market, where small molecule drugs took the lead.

The Fc region of the antibody serves to recruit immune leukocytes or serum complement molecules so that damaged cells such as cancer cells or infected cells can be removed. The region on the Fc between the Cγ2 and Cγ3 domains mediates the interaction with the neonatal receptor FcRn, and its binding recycles the transfected antibody from the endosome to the bloodstream (Raghavan et al. ., 1996, Annu Rev Cell Dev Biol 12: 181-220; Ghetie et al., 2000, Annu Rev Immunol 18: 739-766). This process, due to the large size of the full length molecule, is associated with the arrest of kidney filtration, and has a favorable antibody serum half-life in the range of 1 to 3 weeks. In addition, binding of Fc to FcRn also plays an important role in antibody transport. Accordingly, the Fc region plays an essential role in maintaining prolonged serum persistence by circulating antibodies through intracellular trafficking and recycling mechanisms.

Administration of an antibody or an Fc fusion protein as a therapeutic agent requires injection at a fixed frequency in consideration of the characteristics of the half-life. Longer half-life in vivo has the obvious advantage of allowing lower frequency injections or lower dosages. Accordingly, in many clinical studies currently underway, the Fc domain in which mutations are introduced to increase the half-life of antibodies, or mutations are introduced to maximize ADCC effect, are widely used in the development of next-generation anti-cancer antibody therapeutics or anti-cancer protein therapeutics. Efforts are being made ( Modified from Cancer Immunol Res . 2015 / Thomson Reuters).

However, the research team is trying to discover some proteins and antibodies with increased FcRn binding affinity and half-life in vivo by introducing some mutations in the Fc domain, but the reality is that the half-life in vivo has not yet been greatly improved. It is a situation in which the development of an antibody introducing a more optimized mutation is urgently required.

Matters described as the above-described background art are only for enhancing an understanding of the background of the present invention, and should not be taken as acknowledging that they correspond to the prior art already known to those of ordinary skill in the art.

The present inventors have made intensive efforts to efficiently increase the half-life in the body of the existing protein or antibody therapeutics. As a result, by substituting some amino acid sequences of the wild-type Fc domain with other amino acid sequences and optimizing them, the present invention was completed by confirming a method capable of maximizing the half-life without almost affecting the activity of the protein or antibody therapeutic agent.

Accordingly, an object of the present invention is to provide a polypeptide comprising an Fc variant in which some of the amino acid sequences of the human antibody Fc domain are substituted with other amino acid sequences.

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 composition comprising the polypeptide, antibody, nucleic acid molecule or vector.

Another object of the present invention is to provide a method for preparing the polypeptide or antibody.

Another object of the present invention is to provide a method for screening the polypeptide.

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

According to one aspect of the present invention, the present invention provides a polypeptide comprising an Fc variant in which a part of the amino acid sequence of a human antibody Fc domain is substituted with another amino acid sequence.

The present inventors have produced an Fc variant by substituting and optimizing some amino acid sequences of the wild-type Fc domain with other amino acid sequences as a method for efficiently increasing the half-life in the body of the existing protein or antibody therapeutic agent, thereby producing a protein or antibody therapeutic agent comprising the same. I tried to find a way to maximize the half-life of.

Antibodies are proteins that exhibit specific binding to specific antigens, and natural antibodies are usually about 150,000 Daltons of heterodimeric glycoprotein, consisting of two identical light chains (L) and two identical heavy chains (H).

Human antibodies used in the present invention have five main classes: 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 antibody Fc domain may be the Fc domain of an IgA, IgM, IgE, IgD, or IgG antibody, or a modification thereof. In one embodiment, the domain is the Fc domain of an IgG antibody (eg, the Fc domain of an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody). In one embodiment, the Fc domain may be an IgG1 Fc domain (for example, the Fc domain of an anti-HER2 antibody, preferably the Fc domain of trastzumab, more preferably the Fc domain of SEQ ID NO: 28). In the polypeptide comprising the Fc domain of the present invention, some or all of the polypeptide may not be glycosylated or glycosylated. In addition, the polypeptide may contain one or more regions derived from the antibody 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-type protein.

In the present specification, the amino acid residue number of the antibody Fc domain is according to the Kabat numbering system commonly used in the art (Kabat et al., in of Proteins of Immunological Interest5th Ed., US Department of Health and Human Services). , NIH Publication No. 91-3242, EU index number as in 1991).

According to a preferred embodiment of the present invention, the substituted Fc variant of the present invention comprises the following amino acid substitutions according to the Kabat numbering system: a) M428L; And b) Q311R or L309G.

The present invention relates to an Fc variant comprising an amino acid substitution that modulates binding and dissociation to FcRn (neonatal Fc receptor). In particular, it includes an Fc variant, or a functional variant thereof, that exhibits increased binding affinity for FcRn at low pH and does not exhibit substantially altered binding at high pH.

According to a preferred embodiment of the present invention, the Fc variant has a binding affinity for FcRn at pH 5.6 to 6.2 (preferably pH 5.8 to 6.0) compared to the wild-type Fc domain by 10% or more and 20%. Or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more, or more than 2 times, 3 times or more, 4 times than the wild-type Fc domain More than, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 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 Fc variant has the same or substantially the same degree of dissociation from the neonal Fc receptor (FcRn) at pH 7.0 to 7.8 (preferably pH 7.2 to 7.6) compared to the wild-type Fc domain. May not be changed.

According to an embodiment of the present invention, the substituted Fc variant of the present invention exhibits very improved binding affinity under weakly acidic conditions (e.g., pH 5.8 to 6.0) compared to wild-type Fc or other previously developed Fc variants. , In neutral conditions (eg, pH 7.0), the same or substantially equal or higher degree of dissociation was exhibited (Examples 4 and 8).

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.

The half-life of the substituted Fc variant 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% compared to the wild-type Fc domain. It may be increased by more than or equal to 100%, or by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more than the wild-type Fc domain. have.

According to an embodiment of the present invention, the substituted Fc variant of the present invention exhibited a significantly improved half-life in vivo compared to the wild type (Example 11, Table 3).

In the present specification, “Fc gamma receptor” or “FcγR” refers to any member of the protein lineage that binds to the IgG antibody Fc region, and is encoded by the FcγR gene. Examples thereof include FcγRIa, FcγRIb, and FcγRI (CD64), including FcγRIc; FcγRII (CD32) including FcγRIIa, FcγRIIb and FcγRIIc; And FcγRIII (CD16), including FcγRIIIa and FcγRIIIb, and any undiscovered FcγR or FcγR. FcγR includes humans, mice, rats, rabbits and monkeys, but can also be derived from any other organism.

In the present specification, “FcRn” or “neonatal Fc receptor” refers to a protein that binds to the Fc region of an IgG antibody, and is at least partially encoded by the FcRn gene. The FcRn includes humans, mice, rats, rabbits and monkeys, but may be derived from any organism. Functional FcRn proteins comprise two polypeptides referred to as light chain and heavy chain. The light chain is beta-2-microglobulin, and the heavy chain is encoded by the FcRn gene.

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

As used herein, the term antibody is a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimic, a chimeric antibody, an antibody conjugate, a human antibody or a humanized antibody, or Means short.

According to a preferred embodiment of the present invention, the present invention can maximize the half-life of the Fc domain or a polypeptide comprising the same through optimization of the antibody Fc region (M428L and Q311R; or M428L and L309G).

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.

The nucleic acid molecule of the present invention may be isolated or recombinant, and includes single-stranded and double-stranded DNA and RNA as well as corresponding complementary sequences. An isolated nucleic acid is, in the case of a nucleic acid isolated from a naturally occurring source, a nucleic acid isolated from surrounding genetic sequences present in the genome of an individual from which the nucleic acid was isolated. In the case of nucleic acids enzymatically or chemically synthesized from the template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acid resulting from this 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, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when expressed as a preprotein, which is a form before the polypeptide is secreted, and the promoter or enhancer is a polypeptide sequence. Is operably linked to the coding sequence when it affects the transcription of, or the ribosome binding site is operably linked to the coding sequence when arranged to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located adjacent to each other, and in the case of a secretory leader, it means that they are adjacent and exist in the same reading frame. However, enhancers need not be located adjacent to each other. Linkage is achieved by ligation at a convenient restriction enzyme site. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

In the present specification, the term vector refers to a delivery system into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences can be exogenous or heterologous. Examples of the vector include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophage). Those 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 including a nucleic acid sequence encoding at least a portion of the 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 the regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also provide other functions.

In the present specification, 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 exogenous nucleic acid molecules are transferred or introduced into the host cell.

The host cells of the present invention are preferably bacteria cells, CHO cells, HeLa cells, HEK293 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, NIH3T3 cells, etc., but are limited thereto. no.

According to another aspect of the present invention, the present invention provides a method for preparing a polypeptide comprising a human antibody Fc variant comprising the following steps:

a) culturing a host cell comprising a vector containing a nucleic acid molecule encoding the polypeptide of claim 1; 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 preparing an antibody comprising the following steps:

a) culturing a host cell expressing an antibody containing the polypeptide; And

b) purifying the antibody expressed from the host cell.

In the production method of the present invention, purification of the antibody 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 method for screening a polypeptide comprising an Fc variant comprising the following steps:

a) constructing an Fc variant library containing the M428L mutation according to the Kabat numbering system; And

b) selecting an Fc variant having higher affinity for FcRn than wild type at pH 5.6 to 6.2 among the Fc variants containing the M428L mutation.

Fc variants comprising the M428L mutation of the present invention may contain additional amino acid substitutions.

The additional amino acid substitution is not particularly limited, and preferably includes a Q311R or L309G mutation according to the Kabat numbering system.

The screening method of the present invention can use fluorescently labeled cell separation (FACS) screening, or other automated flow cytometry techniques. Instruments for carrying out flow cytometry are known to those skilled in the art. Examples of such devices include FACSAria, FACS Star Plus, FACScan and FACSort instruments (Becton Dickinson, Foster City, CA), Epics C (Coulter Epics Division, Hialeah, FL), MOFLO (Cytomation, Colorado Springs, Colo.), MOFLO -XDP (Beckman Coulter, Indianapolis, IN) is mentioned. In general, flow cytometry techniques involve the separation of cells or other particles in a liquid sample. Typically the purpose of a flow cytometer is to analyze the isolated particles for one or more properties of them (eg, the presence of a labeled ligand or other molecule). The particles are passed one by one by the sensor and classified based on size, refraction, light scattering, opacity, illuminance, shape, fluorescence, etc.

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

According to a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition for preventing or treating cancer.

According to an embodiment of the present invention, the Fc variant of the present invention exhibits equivalent or higher ADCC (antibody dependent cellular cytotoxicity) activity compared to a control (e.g., trastzumab), and exhibits high anticancer activity with the remarkable half-life increase. Have (Example 13, Fig. 18).

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

According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising administering the pharmaceutical composition.

The type of cancer to be prevented or treated by the present invention is not limited, and leukemias and acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, and chronic Lymphomas such as chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphomas and multiple myeloma, brain tumors, neuroblastoma , Childhood solid tumors such as retinoblastoma, Wilms Tumor, bone tumors and soft-tissue sarcomas, lung cancer, breast cancer cancer, prostate cancer, urinary cancers, uterine cancers, oral cancers, pancreatic cancer, melanoma and other skin cancers, stomach cancer ( stomach cancer, ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroid cancer, esophageal cancer, and testicular cancer. It can be administered to treat a number of cancers, including common solid tumors.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and may be administered by, for example, intravenous injection, local injection, or intraperitoneal injection.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, Usually the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person skilled in the art. Or it can be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

The pharmaceutical composition of the present invention may be used as a single therapy, but may be used in combination with other conventional chemotherapy or radiation therapy, and when such combination therapy is performed, cancer treatment can be more effectively performed. Chemotherapeutic agents that can be used with the composition of the present invention are cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ipo Spamid, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin , Bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transplatinum, 5-fluoro Uracil (5-fluorouracil), vincristin (vincristin), vinblastin (vinblastin) and methotrexate (methotrexate) and the like. Radiation therapy that can be used with the composition of the present invention is X-ray irradiation and γ-ray irradiation.

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 a 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 producing the polypeptide or antibody.

(Iii) The Fc variant of the present invention can maximize the half-life in the body through optimization of some amino acid sequences, and can be usefully used in the treatment of cancer.

1 shows an expression vector for expression and purification of terameric FcRn and dimeric FcRn, and an SDS-PAGE gel image after purification.
FIG. 2 shows a schematic diagram of a library for making 18 kinds of amino acids in M252 and M428.
3 shows the 2M library search process and selected M428L variants.
4 shows a schematic diagram of an error library and a point library produced based on M428L.
5 shows FACS fluorescence intensity measurement results of variants discovered in the Error library (FIG. 5a) and the point library (FIG. 5b).
6 shows a plasmid for expressing the heavy and light chains of trastzumab in animal cells.
7 shows the expression and purification results of wild-type trastzumab.
8 is a commercial The results of comparing the properties of trastzumab and trastzumab produced in the laboratory are shown (A: CE-cIEF, B: SEC).
9 shows the results of comparison of properties of commercial trastzumab and trastzumab produced in a laboratory by N-Glycan profiling.
10 shows the expression and purification results of 10 kinds of trastzumab Fc variants (A: Affinity Chromatography, B: SDS-PAGE Analysis, C: List of Final Yield).
11 shows the results of SEC characterization of trastzumab Fc variant.
12 shows the results of measuring the FcRn binding ability of the trastzumab Fc variant using ELISA.
13 shows the results of measuring the binding force between the trastzumab Fc variant and hFcRn at pH 6.0 and 7.4 by BiaCore (A: pH 6.0 (capture method) B: pH 7.4 (Avid Format)).
Figure 14 shows the pharmacokinetic comparison results of commercial and laboratory produced trastzumab in normal mice (C57BL/6J (B6)) and human FcRn Tg mice.
Fig. 15 shows the pharmacokinetic analysis results of Fc variants in human FcRn Tg mice (5 mg/kg injection of each variant by intravenous injection, n=5).
Figure 16 shows the results of measuring the binding ability of the FcγRs of trastzumab Fc variant using ELISA.
17 shows the effector function comparison results of the trastzumab Fc variant compared to the normal IgG and the control (trastzumab) (ADCC assay).
Figure 18 shows the effector function (ADCC) comparison results of trastzumab Fc variant.
19 shows the results of measuring the C1q binding ability of the trastzumab Fc variant using ELISA.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill 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 One. Fc Variant For library browsing FcRn (neonatal Fc receptor) expression and purification

In order to search for an Fc variant with improved pH-dependent binding to FcRn, a vector for expression and purification of terameric FcRn and dimeric FcRn was prepared (FIG. 1). To obtain Tetrameric FcRn, a DNA plasmid of pMAZ-β2microglobulin-GSlinker-FcRnα-chain-streptavidin-His was obtained. This DNA was co-transfected into HEK 293F cells and temporarily expressed on a 300 ml scale. After the culture was completed, the culture solution was removed by centrifugation at 7000 rpm for 10 minutes, and the supernatant was taken and equilibrated with 25x PBS. Filtered with a 0.2 μm filter (Merck Millipore) using a bottle top filter. After equilibration with PBS, Ni-NTA resin (Qiagen) was added to allow FcRn and resin to bind at 4°C for 16 hours. After flowing the resin bound to FcRn on the column, 50 ml wash-1 buffer (PBS), 25 ml wash-2 buffer (PBS + 10 mM imidazole), 25 ml wash-3 buffer (PBS + 20 mM imidazole), 200 μl wash Proteins other than tFcRn were removed by flowing -4 buffer (PBS + 250 mM imidazole). And 2.5 ml elution buffer (PBS + 250 mM imidazole) was poured to obtain tFcRn. And centrifugal filter units (Merck Millipore) were used to change the buffer. To obtain a dimeric FcRn, a pcDNA-FcRnα-chain-GST-β2 microglobulin plasmid obtained from Oslo University was obtained. This DNA was co-transfected into HEK 293F cells and temporarily expressed on a 300 ml scale. After the culture was completed, the culture solution was removed by centrifugation at 7000 rpm for 10 minutes, and the supernatant was taken and equilibrated with 25x PBS. Filtered with a 0.2 μm filter (Merck Millipore) using a bottle top filter. After equilibration with PBS, Glutathione agarose 4B (incospharm) resin was added to bind FcRn and the resin at 4°C for 16 hours. Proteins other than dFcRn were removed by flowing the resin bound to the FcRn on the column and flowing 10 ml wash buffer (PBS). Then, 2.5 ml elution buffer (50 mM Tris-HCl + 10 mM GSH pH 8.0) was flowed and centrifugal filter units 3K (Merck Millipore) were used to change the buffer. After purification, the size was confirmed using an SDS-PAGE gel (FIG. 1). The purified terameric FcRn and dimeric FcRn were fluorescently labeled using Alexa 488 so that fluorescent signals could be emitted.

Example 2. Fc Variant 2M library production

Trastzumab there is Fc Part of the gene (SEQ ID NO: 29) Sfi Using I restriction enzyme pMopac12 - NlpA - Fc -FLAG Was produced. Crafted Based on vector Fc Within two Met part cys So that it can be substituted with 18 other amino acids except Met pMopac12 - seq - Fw , Fc -M252-1- Rv , Fc -M252-2- Rv , Fc -M252-3-Rv, Fc -M428- Fw , Fc -M428-1- Rv , Fc -M428-2- Rv , Fc -M428-3- Rv , Fc -M428-frg3-Fw, pMopac12 - seq - Rv Primer Library insert using Was produced (Table 1, Fig. 2). Crafted insert Sfi I Restriction enzyme treatment Sfi With I The processed vector and Ligated. That E. coli after Jude1 (( F'[Tn 10( Tet ^r ) proAB ⁺ lacI ^q Δ( lacZ )M15 ] mcrA Δ( mrr - hsdRMS - mcrBC )φ80d lac ZΔM15 Δ lacX74 deoR recA1 araD139 Δ( ara leu )7697 galU galKrpsLendA1nupG ) Transformed into a giant Fc Variant 2M library (library size: 1x10 ⁹ ) Was built.

[Table 1] Primers used for cloning (SEQ ID NO: 1 to 27)

Example 3. Bacterial culture and Flow cytometry Separator (flow flow cytometry ) Using Fc Variant 2M library browsing

To search for the constructed 2M Fc variant library, 1 ml of the transformed Fc variant library cells in E. coli Jude1 cells was added to 2% (w/v) glucose under shaking conditions at 37° C. 250 rpm, and the antibiotic was chloramphenicol (40 μg/mL). It was put into the included Terrific Broth (TB) medium and cultured for 4 hours. Shake-cultured library cells were inoculated in TB medium at 1:100 and then cultured until an _{OD 600 reached 0.5 by shaking at 37°C at 250 rpm.} After incubation at 25° C. for 20 minutes for cooling, 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to induce expression. After the culture was completed, the cells were recovered, and the cells were harvested through OD600 normalization by centrifugation at 14,000 rpm for 1 minute at a uniform amount. Cells were resuspended by adding 1 ml of 10 mM Tris-HCl (pH 8.0), and the washing process of centrifuging for 1 minute was repeated twice. Resuspended in 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] and rotated at 37° C. for 30 minutes to remove the outer cell membrane. After discarding the supernatant by centrifugation, 1 ml of Solution A [0.5 M sucrose, 20 mM MgCl ₂ , 10 mM MOPS pH 6.8] was added, followed by resuspension and centrifugation. 1 ml of a solution of 1 ml of SolutionA and 20 μl of 50 mg/ml lysozyme solution was added and resuspended, and then rotated at 37° C. for 15 minutes to remove the peptidoglycan layer. After centrifugation, the supernatant was removed, resuspended in 1 ml of PBS, 300 μl was taken, 700 μl of PBS and a fluorescently labeled tetrameric FcγRIIIa-Alexa 488 flour probe were added together and rotated at room temperature to label the fluorescent probe on spheroplast. After labeling, after washing once with 1 ml of PBS, sorting was performed to recover the top 3% cells showing high fluorescence using a flow cytometry (S3 sortor (Bio-rad)). To increase the purity of these high cells, the selected cells were re-sorted (resorting). Of the amplified genes by PCR using Taq polymer a material selected samples to pMopac12-seq-Fw and pMopac12-seq-Rv Primer raised (Biosesang) and sorting through the Sfi I restriction enzyme digestion, ligation, transformation procedure cells A sub-library in which the genes were amplified was constructed. After repeating this process for a total of 2 rounds, 40 individual clones were analyzed, respectively, and M428L mutants showing higher affinity with FcRn at pH 5.8 than wild-type Fc were selected (FIG. 3).

Example 3. Fc Variant Error library and point library production

Two additional libraries were produced using the excavated M428L as a template. The first library was prepared by introducing a mutation into Fc using the error prone PCR technique. ^{As for the error rate, a library (library size: 2x10 8} ) was prepared using ep-Fc-Fw and ep-Fc-Rv primers so that a 0.3% error (2.04 bp) could enter Fc (680 bp). The second library uses M428L as a template and selects the Fc and FcRn binding sites, so that mutations are randomly introduced into the corresponding sites, pMopac12-seq-Fw, pMopac12-seq-Rv, Fc-Sub#0-Rv, and Fc-Sub. #1-1-Fw, Fc-Sub#1-2-Fw, Fc-Sub#1-3-Fw, Fc-Sub#1-4-Fw, Fc-Sub#1-5-Fw, Fc-Sub #1-Rv, Fc-Sub#2-1-Fw, Fc-Sub#2-2-Fw, Fc-Sub#2-3-Fw, Fc-Sub#2-Rv, Fc-Sub#3-1 Point libraries were prepared using -Fw, Fc-Sub#3-2-Fw, Fc-Sub#3-3-Fw, and Fc-Sub#3-4-Fw primers (FIG. 4). Then, Jude1 was transformed in the same manner as above to construct an Fc variant library.

Example 4. Bacterial culture and Flow cytometry Separator (flow flow cytometry ) Using Fc Variant error and point library search and PFc3 , PFc29 , PFc41 , EFc29 , EFc41 , EFc82 , EFc88, etc. Variant Selection

Based on the previously discovered M428L, the additionally produced error library and point library were selected and re-selected in the same way as above. The error library was repeated through 5 rounds, and the point library was screened once and then 100 individual clones from the groups from the two libraries were analyzed, showing high affinity for FcRn at pH 5.8 and pH 7.4. In, Fc variants showing low affinity were selected. As a result of analyzing each using FACS, EFc6, EFc29, EFc41, EFc46, EFc70, EFc90 EFc82, EFc88 found in the error library showed higher fluorescence intensity at pH 5.8 than wild-type Fc, but Mediimmune discovered in previous studies. YTE of (Gabriel J. Robbie et al., Antimicrob Agents Chemother . 2013 Dec; 57(12): 61476153) or Xencor's LS (U.S. Patent No. 8,324,351) shows higher fluorescence intensity at pH 5.8. It was confirmed that the mutants exhibiting lower fluorescence than LS at pH 7.4 were EFc6, EFc29, EFc41, EFc82, and EFc88. In addition, the variants PFc3, PFc29, and PFc41 discovered in the point library showed higher fluorescence intensity at pH 5.8 than YTE or LS, but PFc30 showed lower fluorescence intensity. And it was confirmed that PFc29 and PFc41 exhibited lower fluorescence than LS at pH 7.4. Finally, EFc6, EFc29, EFc41, EFc82, EFc88, PFc3, PFc29, and PFc41, which are expected to improve blood half-life, were selected (Table 2, Fig. 5).

[Table 2] Point mutations of the discovered variants

Mutation sites were identified by the Kabat EU numbering system (Kabat et al., in “Sequences of Proteins of Immunological Interest”, 5th Ed., US Department of Health and Human Services, NIH Publication No. 91-3242, EU index number as in 1991). According to.

Example 5. Fc Variant Control for introduction Trastzumab Production and refining

In the present invention, a representative trastuzumab (Herceptin ^ⓡ ) among IgG1 therapeutic antibodies was selected, and the discovered Fc variant was introduced and intended to be used as a control in the future.

The wild-type trastzumab gene was synthesized (Genscript) from the amino acid sequence of Drug Bank (http://www.drugbank.ca/) through back-translation and mammalian codon optimization. The synthesized trastzumab heavy chain gene was subcloned into pOptiVEC-Fc vector and the trastzumab light chain gene was subcloned into pDNA3.3 vector (Fig. 6). Plasmids for expression of animal cells encoding trastzumab heavy and light chains were prepared, respectively, and expressed and purified in HEK293 cells.

After incubation in HEK 293F, wild-type trastzumab was purified using Protein A affinity chromatography (AKTA prime plus, cat # 11001313) and gel filtration chromatography (HiTrap MabselectSure, GE, cat # 11-0034-95), 7.7 mg of high-purity wild-type trastzumab was obtained from the 300 ml culture solution (FIG. 7).

Example 6. Lab production ( in-house ) And commercial ( commercial ) Trastzumab Comparative analysis of physical properties

Unlike commercial trastzumab produced by CHO suspension culture, in-house trastzumab produced in the laboratory ( in-house ) was produced in HEK293, so the selected Fc-variant was introduced before analyzing its function. The basic characteristics of the shape were analyzed. Analysis by CE (Capillary Electrophoresis: PA800 Plus, Beckman coulter) was performed using Pharmalyte 3-10 carrier ampholytes (GE Healthcare, 17-0456-01) forming a pH 3-10 gradient. Was analyzed. As a result of the analysis, there was no impurity by SEC (Size Exclusion Chromatography: Tskgel G3000swxl, Tosoh), and the pI value by charge variant by CE (Capillary Electrophoresis: PA800 Plus, Beckman coulter) was 8.27-8.74 in the case of commercial use, and the main peak was The PI was 8.62, the PI of the self-produced trastzumab was 8.29-8.78, and the main peak was 8.65, both of which were almost similar (FIG. 8). However, as a result of cIEF analysis, a slight difference in content was observed in the remaining peaks other than the main peak in trastzumab produced in the laboratory.This is produced by CHO in the case of commercial use, but the production cell line of trastzumab produced in the laboratory is HEK293, which has a different Glycan pattern. There is also a possibility of oxdation due to sialic acid, so Glycan analysis was also performed (FIG. 9).

Glycan analysis is performed by removing N-glycan from protein using PNGase F (NEB, 186007990-1), labeling using RapiFluor-MS reagent (Waters, 186007989-1), and then using UPLC system (Acquity UPLC I class, Waters, FLR detector) was used for analysis. As a result of the glycan analysis, the sugar pattern was similar, but only the difference in the content of each composition was shown, which did not appear to be the cause of oxidation by sialic acid, and it appeared to be caused by the difference in the production cell line. (Data not shown) It was produced by introducing the selected Fc variants.

Example 7. Fc Variant Production, purification and physical property analysis

In addition to the wild type produced in commercial and laboratory, 5 control variants such as LS (XenCor), YTE (MedImmune), 428L, and 5 selected variants such as PFc29, PFc41, EFc29, EFc41 and EFc82 were transfected into HEK 293F animal cells. I did. The day before transfection, HEK293F cells were ^{subcultured at a density of 1×10 6} cells/ml to 300 ml and transfected the next day using Polyethylenimine (PEI, Polyscience, 23966). In 30 ml of Freestyle 293 expression culture medium (Gibco, 12338-018), first mix the heavy and light chain genes of the mutants in a ratio of 2:1, and then mix them in the ratio of PEI: mutant genes = 1:2, and leave for 20 minutes at room temperature and then the day before. After mixing with the subcultured cells, _{incubating for 6 days in a shaking CO 2} incubator under conditions of 37°C, 125 rpm, and 8% CO ₂ , centrifugation was performed to take only the supernatant.

Protein purification from the supernatant was performed by affinity chromatography using AKTA prime plus equipped with a HiTrap MabselectSure column. 300 ml of the supernatant was flowed at a rate of 3 ml per minute, washed with 100 ml of 1xPBS, and then 6 fractions were collected at 5 ml of IgG elution buffer (Thermo scientific, 21009) at a rate of 5 ml per minute, and 1M After neutralizing by mixing 500 ul of Tris (pH9.0), each section was taken to a new tube by confirming the protein with Bradford (BioRad, 5000001) solution. The purified mutants were concentrated using a 30K Amicon ultra centrifugal filter (UFC903096) and then analyzed for physical properties (FIGS. 10 and 11).

Laboratory-made wild type trastzumab and other Fc variants were purified with protein A, and purity and molecular weight of 90% or higher were confirmed by SDS-PAGE, and high purity protein samples required for efficacy evaluation and purity analysis were secured (purity 97% or higher). For this, SEC-HPLC (Fig. 11A) experiment was performed. As a result of analysis under isocratic condition (mobile phase 1X PBS pH 7.0, 1 ml/min flow rate), the retention time for the main peak was the same for all of the Fc variants, and the molecular weight prediction result was confirmed as the expected molecular weight (FIG. 11B).

Example 8. Through ELISA Fc Variant FcRn Bonding force measurement

ELISA measurement was performed to measure the binding power of the previously prepared mutants with FcγRs and C1q, which allows them to exhibit the FcRn pH-dependent binding power and effect function. First, to see the pH-dependent binding ability to FcRn, _{50 μl of each IgG Fc variant diluted with 4 μg/ml in 0.05 M Na 2} CO ₃ pH 9.6 was placed in a Flat Bottom Polystyrene High Bind 96 well microplate (costar) at 4℃, 16. After immobilization for an hour, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 5.8/pH 7.4) was blocked at room temperature for 2 hours. After washing 4 times with 180 μl of 0.05% PBST (pH 5.8/pH 7.4), 50 μl of FcRn serially dilution with 1% skim milk (in 0.05% PBST pH 5.8/pH 7.4) was dispensed into each well. It was reacted at room temperature for 1 hour. After the washing process, the antibody reaction was performed for 1 hour at room temperature using 50 μl of anti-GST-HRP conjugate (GE Healthcare), and the washing process was performed. After adding 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) to color development, 50 μl of 2 MH ₂ SO ₄ was added to terminate the reaction, followed by analysis using an Epoch Microplate Spectrophotometer (BioTek). The discovered mutant was found to have a similar avidity to FcRn at pH 5.8 as the LS mutant, and dissociated better than LS at pH 7.4 (FIG. 12 ).

Example 9. At pH 6.0 and pH 7.4 Trastzumab Fc With variants monomeric with hFcRn Cohesion measurement comparative analysis

In this way, in order to compare the difference in binding strength with human FcRn according to the pH of the selected Fc variants other than the commercial and laboratory-made trastzumab identified by the physical property analysis, K _D values were measured and compared using a Biacore T200 (GE Healthcare) equipment. . Under the condition of pH 6.0, human FcRn was used as an analyte in an antigen-mediated antibody capture method according to literature (Yeung YA. et al. , J. Immunol, 2009). The HER2 ECD domain was diluted in running buffer (50 mM Phosphate, pH 6.0, 150 mM NaCl, 0.005% surfactant P20, pH 6.0) as a ligand to the Fc variant on the surface of the CM5 chip fixed at the level of ∼3,000 response units (RU). It was captured by injection at a level of 300 RU. Analyte monomeric FcRn (Sinobiological inc., CT009-H08H) was sequentially diluted in FcRn running buffer from 125 nM, injected for 2 minutes at a flow rate of 30 ul/min, and dissociated for 2 minutes to measure binding force. Regeneration was performed for 30 seconds at a flow rate of 10 mM Glycine (pH1.5) 30 ml/min for each cycle. Sensogram was analyzed by applying a 1:1 binding model in BIAevaluation software (Biacore). As a result, although the Fc variant had better binding power than commercial trastzumab (15 nM) used as a control and laboratory produced trastzumab (16.9 nM) and 428L (9 nM) used as a backbone (PFc 3: 5.6 nM, PFc29: 6.8 nM, PFc 41: 5.9 nM, etc.), and the binding force was somewhat lower than that of YTE (5.7 nM) and LS (4.1 nM), which are known as the world's best, but the difference between the values was confirmed to be almost within the error range. Since the ligand and analytes do not bind well under the condition of pH 7.0, the avid format (Zalevsky J et al . Nat. Biotechnol, 2010) was measured in which monomeric hFcRn was directly immobilized and then Fc variants were injected by concentration. Human FcRn ECD doamin (Sino Biological) was immobilized on the surface of CM5 chip at a level of -1,500 RU. The Fc variants were sequentially diluted in HBS-EP (pH 7.4) from 3000 nM on the surface of the chip on which FcRn was fixed, and injected for 2 minutes at a flow rate of 5 ml/min. The bound Fc variants were dissociated for 2 minutes, and at the end of each cycle, the chip surface was regenerated with 100 mM Tris (pH 9.0) (conatat time 30 seconds; flow rate 30 ul/min). Of these, two Fc variants (PFc29, PFc41) maintain high avidity under pH 6.0 conditions and dissociate faster than YTE and LS under pH 7.4 conditions (consistent with ELISA results), indicating that the effect of increasing the half-life can be expected. It was expected (Fig. 13), and in vivo pharmacokinetic experiments were performed in actual human FcRn Tg mice.

Example 10. With a normal B6 mouse hFcRn Tg Commercial and laboratory-made trastzumab in mice in vivo ( in vivo ) PK experiment comparison analysis

As a result of PK analysis in B6 normal mice (C57BL/6J(B6)) having the same genetic background as human FcRn Tg mice, as reported in the thesis, the affinity between the normal mouse FcRn and the human antibody Fc was human It was confirmed to be higher than FcRn and human antibody Fc. However, PK values in normal mice showed variations between laboratory-made and commercial antibodies, and laboratory-made antibodies appeared unstable, but in Tg mice (B6.Cg-Fcgrttm1Dcr Prkdcscid Tg (Jackson lab, CAGFCGRT) 276Dcr/DcrJ), normal mice Although the AUC was slightly lower than that, it was confirmed that the pharmacokinetic trend between laboratory-made and commercial antibodies was measured similarly. Therefore, it was confirmed that there was no problem in the experimental measurement of the laboratory-derived Fc variants produced by HEK293 in the actual in vivo pharmacokinetic analysis in Tg mice (FIG. 14).

Example 11. LS , YTE Two other species Variants (PFc29 and PFc41), etc. About 4 types hFcRn Tg Mouse pharmacokinetics

The binding force results measured at pH 6.0 and pH 7.4 with ELISA and BiaCore equipment were consistent, and based on this, two types of PFc29 and PFc41 were selected, which had better binding power as LS in pH 6.0 acidic condition and better dissociation power than LS in pH 7.4. I did. At the same time, XenCor's LS mutant and MedImmune's YTE, which are known to be the best in the world, were used as controls.A total of 4 trastzumab Fc variants were used as 20 human FcRn Tg mice (5 mg/kg of 5 mg/kg in each group as IV (venous vein)). After injection, concentration analysis by ELISA using blood obtained from facial vein blood collection (total 12 times → 0, 30min, 1, 6, 24hr, 3, 7, 14, 21, 28, 35, 42, 50day)) Non-compartmental analysis (NCA) was performed with WinNonlin. As expected from the results of ELISA and BiaCore analysis, both Fc variants (PFc29 and PFc41) showed an effect of increasing the half-life in the body, and in particular, PFc29 showed a higher half-life effect than the LS discovered in previous studies (Fig. 15, Table 3). ).

Table 3 Analysis of non-compartmental pharmacokinetic parameters of trastzumab Fc variant after intravenous administration of mice (5 mg/kg) (Data are expressed as mean ± SD (n = 5))

t _1/2 , terminal half-life; T _max , time at maximal concentration; C ₀ , extrapolated zero time concentration; C _max , maximal concentration; AUC _last , area under the curve from administration to the last measured concentration; AUC _inf , area under the curve from administration to infinity; AUC _%Extrap , percentage of the extrapolated area under the curve at the total area under the curve; V _z , volume of distribution; CL, clearance.

Example 12. Through ELISA Fc Variant FcγRs Bonding force measurement

To measure the binding strength with FcγRs, _{50 μl of each IgG Fc variant diluted with 4 μg/ml in 0.05 M Na 2} CO ₃ pH 9.6 was immobilized in a Flat Bottom Polystyrene High Bind 96 well microplate (costar) at 4°C for 16 hours. Then, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 7.4) was blocked at room temperature for 2 hours. After performing the washing process four times with 180 μl of 0.05% PBST (pH 7.4), FcγRs serially dilution with 1% skim milk (in 0.05% PBST pH 7.4) was dispensed into 50 μl of each well and reacted at room temperature for 1 hour. . After the washing process, the FcRs were subjected to antibody reaction for 1 hour at room temperature using 50 μl of anti-GST-HRP conjugate (GE Healthcare) each, and the washing process was performed. After adding 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) each to develop color, 50 μl of 2 MH ₂ SO ₄ was added to terminate the reaction, and then analyzed using an Epoch Microplate Spectrophotometer (BioTek). Each experiment was conducted in duplicate, and as a result of ELISA, binding strength to each of FcγRs (FcγRI, FcγRIIa(H), FcγRIIa(R), FcγRIIb, FcγRIIIa(V), FcγRIIIa(F)) was confirmed (FIG. 16 ).

Example 13. Fc Variant Antibody dependent cell-mediated cytotoxicity ( ADCC )On by Actuator function ( Effector Function) measurement

Antibody-dependent cellular cytotoxicity (ADCC) activity of the trastzumab Fc variant was evaluated using an ADCC reporter bioassay kit (Promega, G7010). Specifically, SKBR-3, which is used as target cells, is 5X10 ^{3 in each well of a 96-well tissue culture plate.} After dispensing cells/100 μl each, the cells were incubated for 20 hours in a _{CO 2 incubator at 37°C.} After 20 hours, 95 µl of the culture medium was removed from each well of the plate using a multi-pipette, and 25 µl of the ADCC assay buffer provided in the ADCC Reporter Bioassay Kit was dispensed into each well. Normal IgG, trastzumab, and trastzumab Fc variants are prepared by diluting by concentration in ADCC assay buffer, and then dispensed at 25 µl per well of a 96-well tissue culture plate with cells until effector cells are added. Put at room temperature. The effector cells provided by the kit were dissolved in a constant temperature water bath at 37° C. for 2-3 minutes, and then 630 μl was taken and mixed with 3.6 mL of ADCC assay buffer. Effector cells prepared in a plate containing target cells and antibody dilutions were dispensed at a rate of 25 μl per well, and then _{reacted in a 37° C. CO 2} incubator for 6 hours. When the time elapsed, the plate was taken out and allowed to stand at room temperature for 15 minutes, and then ^{75 µl of Bio-Glo TM} Luciferase assay reagent was added per well and reacted at room temperature for 5 minutes. After the reaction, the luminescence of each well was measured using a luminometer (Enspire multimode plate reader). The ADCC activity of each test antibody was expressed as the average value of the experimental results as fold induction, and was determined using the following equation:

Fold induction =

RLU(induced ¹ -background ² )/RLU(noantibodycontrol ³ -background)

1.induced: RLU values obtained from a mixture of target cells, test antibodies and effector cells

2. background: RLU value obtained from ADCC assay buffer

3. no antibody control: RLU value obtained from a sample containing only target cells and effector cells.

The ADCC activities of trastzumab and trastzumab Fc variants (LS, YTE, PFC29 and PFC41) against SKBR-3 were compared with each other (FIG. 17 ). As a result, based on the maximum activity value at the highest concentration, the ADCC activity of each test substance was about 1.5 times higher in LS, 1.18 times in PFc29 and 1.27 times in PFc41 compared to trastzumab, the positive control, but YTE was 4.2. It showed twice as low activity. As a result, the two trastzumab Fc variants (PFc29 and PFc41) and the control variant LS showed 1.18-1.5 times improved ADCC activity compared to the existing control drug (Trastuzumab). However, as shown in the following figure, when looking at the measured EC50 values of each variant, the Fc variants PFc29 (EC50=0.04543 ug/mL) and PFc41 (EC50=) found in the present invention were more than the control LS (EC50=0.05575 ug/mL). 0.05405 ug/mL) showed more stable efficacy (FIG. 18).

Example 14. Through ELISA Fc Variant C1q Bonding force measurement

To measure the binding strength with C1q, _{50 μl of each IgG Fc variant diluted with 4 μg/ml in 0.05 M Na 2} CO ₃ pH 9.6 was immobilized in a Flat Bottom Polystyrene High Bind 96 well microplate (costar) at 4°C for 16 hours. Then, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 7.4) was blocked at room temperature for 2 hours. After washing 4 times with 180 μl of 0.05% PBST (pH 7.4), 50 μl of Complement C1q Human (Millipore) serially dilution with 1% skim milk (in 0.05% PBST pH 7.4) was dispensed into each well at room temperature. It was reacted for 1 hour. After the washing process, the antibody reaction was performed for 1 hour at room temperature using 50 μl of anti-C1qHRP conjugate (Invitrogen), and the washing process was performed. After adding 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) to color development, 50 μl of 2 MH ₂ SO ₄ was added to terminate the reaction, followed by analysis using an Epoch Microplate Spectrophotometer (BioTek). As a result of the analysis, PFc29 discovered in this study showed higher bonding strength than LS and YTE discovered in previous studies (FIG. 19).

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Kookmin University Industry Academy Cooperation Foundation Osong Hi-Tech Medical Industry Promotion Foundation <120> Antibody Fc variants for Prolonged Serum Half-Life <130> HP7178 <160> 29 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pMopac12-seq-Fw <400> 1 ccaggcttta cactttatgc 20 <210> 2 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Fc-M252-1-Rv <400> 2 cctcaggggt ccgggagatg wagagggtgt ccttgggttt tggg 44 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Fc-M252-2-Rv <400> 3 cctcaggggt ccgggagatk nbgagggtgt ccttgggttt tggg 44 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Fc-M252-3-Rv <400> 4 cctcaggggt ccgggagatc cagagggtgt ccttgggttt tggg 44 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fc-M428-Fw <400> 5 atctcccgga cccctgagg 19 <210> 6 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Fc-M428-1-Rv <400> 6 gtagtggttg tgcagagcct catggwacac ggagcatgag aagacgttcc 50 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Fc-M428-2-Rv <400> 7 gtagtggttg tgcagagcct catgknbcac ggagcatgag aagacgttcc 50 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Fc-M428-3-Rv <400> 8 gtagtggttg tgcagagcct catgccacac ggagcatgag aagacgttcc 50 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Fc-M428-frg3-Fw <400> 9 catgaggctc tgcacaacca ctac 24 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> pMopac12-seq-Rv <400> 10 ctgcccatgt tgacgattg 19 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#0-Rv <400> 11 gtccttgggt tttgggggga ag 22 <210> 12 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#1-1-Fw <400> 12 cttcccccca aaacccaagg acnnkctcat gatctcccgg acccctgagg tcacatgcg 59 <210> 13 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#1-2-Fw <400> 13 cttcccccca aaacccaagg acaccnnkat gatctcccgg acccctgagg tcacatgcg 59 <210> 14 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#1-3-Fw <400> 14 cttcccccca aaacccaagg acaccctcat gnnktcccgg acccctgagg tcacatgcg 59 <210> 15 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#1-4-Fw <400> 15 cttcccccca aaacccaagg acaccctcat gatcnnkcgg acccctgagg tcacatgcg 59 <210> 16 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#1-5-Fw <400> 16 cttcccccca aaacccaagg acaccctcat gatctccnnk acccctgagg tcacatgcg 59 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#1-Rv <400> 17 gacggtgagg acgctgacc 19 <210> 18 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#2-1-Fw <400> 18 ggtcagcgtc ctcaccgtcn nkcaccagga ctggctgaat ggcaaggagt acaagtgcaa 60 gg 62 <210> 19 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#2-2-Fw <400> 19 ggtcagcgtc ctcaccgtcc tgcacnnkga ctggctgaat ggcaaggagt acaagtgcaa 60 gg 62 <210> 20 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#2-3-Fw <400> 20 ggtcagcgtc ctcaccgtcc tgcaccagga ctggnnkaat ggcaaggagt acaagtgcaa 60 gg 62 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#2-Rv <400> 21 cacggagcat gagaagacgt tcc 23 <210> 22 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#3-1-Fw <400> 22 ggaacgtctt ctcatgctcc gtgctgcatn nkgctctgca caaccactac acgcagaaga 60 gcctctccct g 71 <210> 23 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#3-2-Fw <400> 23 ggaacgtctt ctcatgctcc gtgctgcatg aggctnnkca caaccactac acgcagaaga 60 gcctctccct g 71 <210> 24 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#3-3-Fw <400> 24 ggaacgtctt ctcatgctcc gtgctgcatg aggctctgca cnnkcactac acgcagaaga 60 gcctctccct g 71 <210> 25 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Fc-Sub#3-4-Fw <400> 25 ggaacgtctt ctcatgctcc gtgctgcatg aggctctgca caaccacnnk acgcagaaga 60 gcctctccct g 71 <210> 26 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> ep-Fc-Fw <400> 26 ccagccggcc atggcg 16 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ep-Fc-Rv <400> 27 gaattcggcc cccgaggccc c 21 <210> 28 <211> 227 <212> PRT <213> Homo sapiens <400> 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 29 <211> 681 <212> DNA <213> Homo sapiens <400> 29 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cacctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ccccgggtaa a 681

Claims (10)

Wherein said Fc variant comprises the amino acid substitution according to the Kabat numbering system in a wild type human antibody Fc domain and has a half-life (&lt; RTI ID = 0.0 &gt; Half-life &lt; / RTI &gt; is increased:
a) M428L; And
b) L309G.
An antibody comprising the polypeptide of claim 1.
The antibody of claim 2, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a chimeric antibody, an antibody conjugate, &Lt; / RTI &gt; or a fragment thereof.
A nucleic acid molecule encoding the polypeptide of claim 1.
A vector comprising the nucleic acid molecule of claim 4.
A host cell comprising the vector of claim 5.
A method for producing a polypeptide comprising a human IgG antibody Fc variant with increased half-life compared to a wild type comprising the steps of:
a) culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the polypeptide of claim 1; And
b) recovering the polypeptide expressed by said host cell.
A method for producing a human IgG antibody having an increased half-life compared to a wild type comprising the steps of:
a) culturing a host cell expressing an antibody comprising the polypeptide of claim 1; And
b) purifying the antibody expressed from said host cell.
A screening method for a polypeptide comprising an Fc variant with increased half-life compared to a wild-type comprising the steps of:
a) constructing an Fc variant library comprising a M428L mutation and an L309G mutation according to a cambator numbering system; And
b) selecting Fc variants with a higher affinity for FcRn than wild type at pH 5.6 to 6.2 among the Fc variants comprising said mutation.
CLAIMS 1. A composition for increasing blood half-life of an antibody or protein therapeutic, said composition comprising a polypeptide comprising a human IgG antibody Fc variant, wherein said Fc variant is selected from the group consisting of Kabat numbering in a wild type human antibody Fc domain, A composition for increasing blood half-life, comprising the following amino acid substitutions according to the Kabat numbering system, wherein the half-life is increased as compared to the wild type:
a) M428L; And
b) L309G.

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