Antibody FC variants for increasing blood half-life
阅读说明：本技术 用于增加血液半衰期的抗体fc变体 (Antibody FC variants for increasing blood half-life ) 是由 郑相泽 高翔焕 李泰揆 崔昭瑛 李秀汉 孙明湖 金秀珍 朴昭罗 朴钟植 林周铉 于 2018-04-06 设计创作，主要内容包括：本发明涉及包含Fc变体的多肽或包含该多肽的抗体,其中用不同的氨基酸序列置换人抗体Fc结构域的氨基酸序列的一部分。本发明的Fc变体可以用于多种抗体和Fc融合构建体中。在一个方面,本发明的抗体或Fc融合构建体是用于治疗、诊断或研究的试剂,优选为治疗性试剂。本发明的Fc变体可以通过优化氨基酸序列的一部分而使体内半衰期最大化,并且可用于癌症的治疗。本发明的抗体和Fc融合构建体用于杀伤靶抗原,例如含有癌细胞的靶细胞。替代地,本发明的抗体和Fc融合构建体用于阻断、拮抗或干扰靶抗原,以便拮抗例如细胞因子或细胞因子受体。(The present invention relates to a polypeptide comprising an Fc variant in which a portion of the amino acid sequence of the Fc domain of a human antibody is replaced with a different amino acid sequence or an antibody comprising the polypeptide. The Fc variants of the invention can be used in a variety of antibodies and Fc fusion constructs. In one aspect, the antibody or Fc fusion construct of the invention is an agent for therapy, diagnosis or research, preferably a therapeutic agent. The Fc variants of the present invention can maximize half-life in vivo by optimizing a portion of the amino acid sequence, and are useful in the treatment of cancer. The antibodies and Fc fusion constructs of the invention are useful for killing target antigens, such as target cells containing cancer cells. Alternatively, the antibodies and Fc fusion constructs of the invention are used to block, antagonize, or interfere with a target antigen, in order to antagonize, for example, a cytokine or cytokine receptor.)
1. A polypeptide comprising a human antibody Fc variant, wherein said Fc variant comprises M428L and a) Q311R or L309G, b) P228L or c) P230Q or P230S as amino acid substitutions in the Fc domain of a wild-type human antibody according to the Kabat EU numbering system and has increased half-life compared to the wild-type.
2. The polypeptide of claim 1, wherein the Fc variant comprises an additional amino acid substitution at one or more positions selected from the group consisting of position 234, 264, 269, 292, 309, 342, 359, 364, 368, 388, 394, 422, 434, and 445.
3. The polypeptide of claim 2, wherein the additional amino acid substitutions are L309R and N434S.
4. The polypeptide of claim 2 wherein the additional amino acid substitutions are V264M, L368Q, E388D, V422D, and P445S.
5. The polypeptide of claim 2, wherein the additional amino acid substitutions are R292L, T359A, and S364G.
6. The polypeptide of claim 2 wherein the additional amino acid substitutions are L234F, E269D, Q342L, E388D and T394A.
7. The polypeptide of claim 1, wherein the Fc variant comprises additional amino acid substitutions at one or more positions selected from position 243, 246, 295, 320, 356, 361, 384, and 405.
8. The polypeptide of claim 7, wherein the additional amino acid substitutions are F243Y, K246E, N361S, and N384I.
9. The polypeptide of claim 7 wherein the additional amino acid substitutions are Q295L, K320M, D356E, and F405I.
10. The polypeptide of claim 1, wherein the antibody is an IgG antibody.
11. An antibody comprising the polypeptide of claim 1.
12. The antibody of claim 11, 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, a humanized antibody, or a fragment thereof.
13. A nucleic acid molecule encoding the polypeptide of claim 1.
14. A vector comprising the nucleic acid molecule of claim 13.
15. A host cell comprising the vector of claim 14.
16. A composition comprising the polypeptide of claim 1, the antibody of claim 11, the nucleic acid molecule of claim 13, or the vector of claim 14.
17. The composition of claim 16, wherein the composition increases the blood half-life of a therapeutic antibody or protein.
18. The composition according to claim 16, wherein the composition is a pharmaceutical composition for preventing or treating cancer.
19. The composition of claim 18, wherein the composition recognizes a cancer antigen.
20. A method for preventing or treating cancer, comprising administering to a subject the pharmaceutical composition of claim 18.
21. A method for producing a polypeptide comprising a human antibody Fc variant having increased half-life compared to wild-type, the method comprising: a) culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the polypeptide of claim 1; and b) collecting the polypeptide expressed by the host cell.
22. A method for producing an antibody having increased half-life as compared to wild-type, comprising: a) culturing a host cell that expresses an antibody comprising the polypeptide of claim 1; and b) purifying the antibody expressed by the host cell.
23. A method for screening a polypeptide comprising an Fc variant having increased half-life as compared to wild-type, the method comprising: constructing a library comprising M428L and a) Q311R or L309G, b) P228L or c) P230Q or P230S as mutated Fc variants according to the Kabat EU numbering system; and b) selecting from the Fc variants an Fc variant having a higher affinity for FcRn than wild-type at a pH of 5.6 to 6.4.
Technical Field
The present invention relates to novel antibody Fc variants with increased blood half-life and methods for making antibody Fc variants.
Background
With recent advances in biotechnology such as gene recombination and cell culture, a great deal of research has been conducted on the structure and function of proteins worldwide. Biotechnology has promoted a better understanding of important phenomena and has played a crucial role in elucidating the pathogenesis of various diseases to pave the way for effective diagnosis and treatment of diseases, thereby greatly improving quality of life. In particular, since the hybridoma technology for producing monoclonal antibodies by fusing B cells with myeloma cells was developed in 1975 (Kohler and Milstein, Nature, 256: 495-.
Therapeutic antibodies are more specific for targets, less bio-toxic and have fewer side effects than existing small molecule drugs. Another advantage of therapeutic antibodies is their long blood half-life (about 3 weeks). Because of these advantages, therapeutic antibodies are considered to be the most effective cancer treatment. Indeed, large pharmaceutical companies and research institutes have focused their development ability on therapeutic antibodies that specifically bind to and effectively remove cancer cells including carcinogens. Roche, ann, haden, yapei and BMS are major pharmaceutical companies currently developing therapeutic antibody drugs. In particular, roche developed three novel therapeutic antibodies for anti-cancer therapy, namely herceptin, avastin and rituximab, which in 2012 reached us $ 195 million for sales of therapeutic antibodies, obtained considerable profits in the global market, currently leading the global market for antibody drugs. Due to the increase in the sales of the gram-like (Remicade), the niche companies that developed the gram-like are rapidly expanding in the global antibody market. It is well known that other pharmaceutical companies such as yapei and BMS possess many therapeutic antibodies at the final stage of development. As a result, biomedicine, including therapeutic antibodies specific to the target disease and having few side effects, is rapidly replacing small molecule drugs that dominate the global pharmaceutical market.
The Fc region of the antibody recruits immune leukocytes or serum complement molecules, which then triggers the clearance of defective cells, such as tumor cells or infected cells. The Fc interface between the
Administration of an antibody or Fc-fusion protein as a therapeutic agent requires a predetermined injection frequency in consideration of the half-life of the therapeutic agent. Longer in vivo half-life may reduce injection frequency or lower dose. Therefore, in many clinical studies currently in progress, much effort has been focused on developing next-generation anticancer therapeutic antibodies and anticancer therapeutic proteins (Modifiedfrom Cancer Immunol res.2015/Thomson Reuters) by introducing mutations into the Fc domain to increase the half-life of the antibody or variants into the Fc domain to achieve maximum ADCC effect.
However, although research groups have focused on developing some proteins and antibodies with enhanced binding affinity for FcRn and extended in vivo half-life by introducing some mutations into the Fc domain, significant increases in vivo half-life have not been achieved. Under these circumstances, the development of optimally mutated antibodies is urgently required.
The description of the background art is provided only for the purpose of better understanding of the background of the invention and should not be taken as corresponding to the prior art known to those skilled in the art.
Detailed Description
Problems to be solved by the invention
The present inventors earnestly studied to effectively increase the in vivo half-life of existing therapeutic proteins or antibodies, and as a result, found that the therapeutic protein or antibody can be optimized by replacing a part of the amino acid sequence of the wild-type Fc domain with a different amino acid sequence, so that the blood half-life can be maximally extended while maintaining its excellent activity.
It is an object of the present invention to provide a polypeptide comprising an Fc variant produced by replacing a portion of the amino acid sequence of an Fc domain of a human antibody with a different amino acid sequence.
It is another object of the present invention to provide an antibody comprising the polypeptide.
It is another object of the present invention to provide a nucleic acid molecule encoding said polypeptide.
It is another object of the present invention to provide a vector comprising said nucleic acid molecule.
It is another object of the present invention to provide a host cell comprising said vector.
It is another object of the invention to provide a composition comprising a polypeptide, an antibody, a nucleic acid molecule or a vector.
It is another object of the present invention to provide a method for producing a polypeptide or an antibody.
It is another object of the present invention to provide a method for screening polypeptides.
Other objects and advantages of the present invention will become more fully apparent from the detailed description herein, the appended claims and the accompanying drawings.
Means for solving the problems
One aspect of the present invention provides a polypeptide comprising an Fc variant produced by replacing a portion of the amino acid sequence of an Fc domain of a human antibody with a different amino acid sequence.
Another aspect of the invention provides a composition for increasing the blood half-life of a therapeutic antibody or protein, the composition comprising an Fc variant produced by replacing a portion of the amino acid sequence of a human antibody Fc domain with a different amino acid sequence.
The present inventors have attempted to find a method for effectively increasing the in vivo half-life of existing therapeutic proteins or antibodies and as a result have found that a therapeutic protein or antibody comprising an Fc variant produced by replacing and optimizing a portion of the amino acid sequence of a wild-type Fc domain with a different amino acid sequence can achieve the maximum in vivo half-life.
Antibodies are proteins that specifically bind to a particular antigen. Natural antibodies are heterodimeric glycoproteins with a molecular weight of about 150000 daltons, typically consisting of two identical light chains (L) and two identical heavy chains (H).
Human antibodies for use in the present invention fall into one of five major classes: IgA, IgD, IgE, IgG and IgM. The human antibody is preferably an IgG antibody. The antibody was papain-digested to produce two Fab fragments and an Fc fragment, while the Fc region of the human IgG molecule was produced by papain-digestion of Cys226 at the N-terminus (Deisenhofer, Biochemistry 20: 2361-2370, 1981).
The antibody Fc domain may be an IgA, IgM, IgE, IgD or IgG antibody or a modified Fc domain thereof. In one embodiment, the domain is an Fc domain of an IgG antibody, for example an Fc domain of an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody. In one embodiment, the Fc domain may be an IgG1 Fc domain, e.g. an Fc domain of an anti-HER 2 antibody, preferably an Fc domain of trastuzumab, more preferably a Fc domain having the amino acid sequence of SEQ ID NO: 28, or a Fc domain of the sequence shown in seq id no. The polypeptides of the invention may optionally be partially or fully glycosylated. In addition to the Fc domain, the polypeptide of the invention may also include one or more than one region derived from an antibody. In addition, a polypeptide of the invention may include an antigen binding domain derived from an antibody, and may form an antibody or antibody-like protein with another polypeptide.
Here, the amino acid residues of the Fc domain of an antibody are designated according to the Kabat EU numbering system commonly used in the art, as described in Kabat et al, "Sequences of Proteins of Immunological Interest," 5 th edition, U.S. department of Health and Human Services, NIH Publication No.91-3242, 1991.
According to a preferred embodiment of the invention, the substituted Fc variant comprises M428L as amino acid substitution according to the Kabat EU numbering system.
According to a preferred embodiment of the invention, the substituted Fc variant comprises a) M428L and b) Q311R or L309G as amino acid substitutions according to the Kabat EU numbering system.
According to a preferred embodiment of the invention, the substituted Fc variant comprises P228L and M428L as amino acid substitutions according to the Kabat EU numbering system.
According to a preferred embodiment of the invention, the Fc variant comprising the amino acid substitutions P228L and M428L comprises further amino acid substitutions at one or more positions selected from position 234, 264, 269, 292, 309, 342, 359, 364, 368, 388, 394, 422, 434 and 445, according to the Kabat EU numbering system.
Additional amino acid substitutions may be L309R and N434S.
Additional amino acid substitutions may be V264M, L368Q, E388D, V422D and P445S.
Additional amino acid substitutions may be R292L, T359A, and S364G.
Additional amino acid substitutions may be L234F, E269D, Q342L, E388D and T394A.
According to a preferred embodiment of the invention, the substituted Fc variant comprises a) M428L and b) P230Q or P230S as amino acid substitutions according to the Kabat EU numbering system.
According to a preferred embodiment of the invention, the substituted Fc variant comprising the amino acid substitutions a) M428L and b) P230Q or P230S comprises further amino acid substitutions at one or more than one position selected from
Additional amino acid substitutions may be F243Y, K246E, N361S, and N384I.
Additional amino acid substitutions may be Q295L, K320M, D356E and F405I.
The present invention relates to Fc variants comprising one or more amino acid substitutions that modulate the binding and dissociation of the Fc variant to neonatal Fc receptor (FcRn). In particular, the Fc variants or functional variants thereof of the invention show increased binding affinity for FcRn under acidic conditions (pH below 7) and very low binding to FcRn under neutral pH conditions.
There is no particular limitation on the therapeutic antibody whose half-life is to be increased, and examples thereof include polyclonal antibodies, monoclonal antibodies, miniantibodies, domain antibodies, bispecific antibodies, antibody mimetics, chimeric antibodies, antibody conjugates, human antibodies, humanized antibodies, and fragments thereof.
As the monoclonal antibody, there may be mentioned, for example, human antibodies such as panitumumab (victib), ofatumumab (Arzerra), golimumab (euphonib), ipilimumab (Yervoy), and the like; humanized antibodies such as tositumomab (Jaceostat), trastuzumab (herceptin), bevacizumab (avastin), omalizumab (sorel), meritorizumab (Bosatria), gemtuzumab (Mrituximab), palivizumab (Synagis), ranibizumab (Norada), certuzumab (Cimzia), orelizumab, mogamulizumab (Poteligeo), and eculizumab (suliri); and chimeric antibodies such as rituximab (rituximab), cetuximab (erbitux), infliximab (nixib-like), and basiliximab (sulitu).
There is no particular limitation on the therapeutic protein whose half-life is to be increased, and examples thereof include: hormones, such as insulin; cytokines such as growth factors, interferons, interleukins, erythropoietins, neutrophil growth factors and transforming growth factors; fc fusion proteins such as etanercept (enril), aflibercept (aleya, Zaltrap), aberrance (orihicona), alfasipu (amevivi), belazeprit (Nulojix), and lioxip (Arcalyst); therapeutic peptides such as teriparatide (futaol), exenatide (bacitracin), liraglutide (nord and li), lanreotide (somadurin), pramlintide (Symlin) and enfuvirtide (Fuzeon); and polypeptides including partial or complete VEGF receptors, Her2 receptors, G protein-coupled receptors, and ion channel cell surface receptors.
The half-life of a therapeutic antibody or protein can be extended by binding to or introducing into a vector expressing a polypeptide of the invention or a nucleic acid encoding the polypeptide.
According to a preferred embodiment of the invention, the Fc variant increases the binding affinity to FcRn of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% over the wild-type Fc domain at a pH of 5.6 to 6.4 (preferably 5.8 to 6.2), or 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, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold or at least 100-fold of the binding affinity of the wild-type Fc domain to FcRn.
According to a preferred embodiment of the invention, the extent of dissociation of the Fc variant from the neonatal Fc receptor (FcRn) may be the same as or not substantially altered by the wild-type Fc domain from the neonatal Fc receptor (FcRn) at a pH of 7.0 to 7.8, preferably 7.2 to 7.6.
According to one embodiment of the invention, the substituted Fc variant exhibits a higher binding affinity under mildly acidic conditions (e.g., at a pH of 5.8 to 6.2) than wild-type Fc or other developed Fc variants, and dissociates to the same or substantially the same or higher degree under neutral conditions (e.g., at a pH of 7.4) as wild-type Fc or other developed Fc variants (see example 4 and example 8).
According to a preferred embodiment of the invention, the substituted Fc variant has a long half-life compared to the wild type.
The half-life of a substituted Fc variant according to the present invention may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% longer than that of a wild-type Fc domain, or 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 longer than that of a wild-type Fc domain.
According to one embodiment of the invention, the substituted Fc variant has a significantly improved half-life in vivo compared to the wild type (see example 11 and table 3).
As used herein, "Fc γ receptor" or "Fc γ R" refers to any member of a family of proteins that bind to the Fc region of an IgG antibody and are encoded by an Fc γ R gene. Examples of such Fc γ receptors or Fc γ rs include, but are not limited to: fc γ RI (CD64), including Fc γ RIa, Fc γ RIb, and Fc γ RIc; fc γ RII (CD32) including Fc γ RIIa, Fc γ RIIb, and Fc γ RIIc; fc γ RIII (CD16), including Fc γ RIIIa and Fc γ RIIIb; and undiscovered Fc γ R. Fc γ R may be derived from mammalian organisms including human, mouse, rat, rabbit and monkey, among others.
As used herein, "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. FcRn may be derived from mammalian organisms including human, mouse, rat, rabbit and monkey, among others. A functional FcRn protein comprises two polypeptides, termed the heavy and light chains. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene.
In another aspect of the invention, an antibody comprising a polypeptide is provided.
The term "antibody" as used herein refers to 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, a humanized antibody, or a fragment thereof (e.g., an antigen-binding antibody fragment).
According to a preferred embodiment of the invention, the half-life of an Fc domain or a polypeptide comprising an Fc domain can be maximized by optimizing the corresponding antibody Fc region (e.g., M428L and Q311R; or M428L and L309G).
In another aspect of the invention there is provided a nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule or a host cell comprising said vector.
The nucleic acid molecules of the invention may be isolated or recombinant nucleic acid molecules. Examples of such nucleic acids include single-and double-stranded DNA and RNA and their corresponding complements. An isolated nucleic acid may be isolated from a natural source. In this case, the isolated nucleic acid is separated from peripheral gene sequences present in the genome of the subject from which the nucleic acid is to be isolated. An isolated nucleic acid is understood to be a nucleic acid, such as a PCR product, a cDNA molecule or an oligonucleotide, which is enzymatically or chemically synthesized from a template. In this case, the nucleic acid produced by the program is understood to be an isolated nucleic acid molecule. An isolated nucleic acid molecule represents a nucleic acid molecule in the form of an isolated fragment or as part of a larger nucleic acid construct. A nucleic acid is "operably linked" when the nucleic acid is arranged in a functional relationship with another nucleic acid sequence. For example, when expressed as a preprotein, the DNA of the prepro sequence or secretory leader is operably linked to the DNA of the polypeptide from which the preprotein is the polypeptide prior to secretion. A promoter or enhancer that affects the transcription of a polypeptide sequence is operably linked to the coding sequence, or a ribosome binding site is operably linked to the coding sequence, and when so arranged, facilitates translation. Generally, the term "operably linked" refers to DNA sequences that are adjacent to each other to be linked. With respect to secretory leaders, the term "operably linked" means that the secretory leaders are present adjacent to each other in the same leader. However, the desired enhancer need not be contiguous. Ligation is performed by ligation at convenient restriction enzyme sites. In the absence of such sites, synthetic oligonucleotide adaptors or linkers are used according to suitable methods known in the art.
As used herein, the term "vector" is used to refer to a vector into which a nucleic acid sequence may be inserted for introduction into a cell so that it may be replicated. The nucleic acid sequence may be "exogenous" or "heterologous". Vectors include plasmids, cosmids, and viruses (e.g., phage). One skilled in the art can construct vectors by standard recombinant techniques, described in Maniatis et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al, In: current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994).
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 capable of being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. The expression vector may contain various "control sequences". In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions.
As used herein, the term "host cell" refers to any transgenic organism capable of replicating a vector or expressing a gene encoded by a vector. Suitable organisms include eukaryotes and prokaryotes. The host cell may be transfected or transformed with a vector. Transfection or transformation refers to the process used to transfer or introduce an exogenous nucleic acid molecule into a host cell.
The host cell of the present invention is preferably a bacterial cell, CHO cell, HeLa cell, HEK293 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell or NIH3T3 cell, but is not limited thereto.
Another aspect of the present invention provides a method for producing a polypeptide comprising an Fc variant of a human antibody, comprising: a) culturing a host cell comprising a vector comprising a nucleic acid molecule encoding the polypeptide; and b) collecting the polypeptide expressed by the host cell.
Another aspect of the present invention provides a method for preparing an antibody, comprising: a) culturing a host cell that expresses an antibody comprising the polypeptide; b) purifying the antibody expressed by the host cell.
In the method of the present invention, the antibody may be purified by filtration, HPLC, anion exchange or cation exchange, High Performance Liquid Chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using protein a.
Another aspect of the present invention provides a method for screening a polypeptide comprising an Fc variant, comprising: constructing a library comprising M428L as a mutated Fc variant according to the Kabat EU numbering system; and b) selecting from the Fc variants comprising the M428L mutation an Fc variant having a higher affinity for FcRn than wild-type at pH 5.6 to 6.4.
An Fc variant including the M428L mutation may include at least one additional amino acid substitution.
According to a preferred embodiment of the invention, the further amino acid substitution comprises Q311R or L309G as a mutation.
According to a preferred embodiment of the invention, the further amino acid substitution comprises P228L as a mutation.
The Fc variant including the P228L mutation may include at least one additional amino acid substitution.
The additional amino acid substitution is not particularly limited, but is preferably an amino acid mutation at least one position selected from the group consisting of positions 234, 264, 269, 292, 309, 342, 359, 364, 368, 388, 394, 422, 434 and 445 according to the Kabat EU numbering system.
According to a preferred embodiment of the invention, the further amino acid substitution comprises P230Q or P230S as a mutation.
The Fc variant including the P230 mutation may include at least one additional amino acid substitution.
The additional amino acid substitution is not particularly limited, but is preferably an amino acid mutation at least one position selected from the group consisting of
The screening method of the present invention may use Fluorescence Activated Cell Sorting (FACS) or automated flow cytometry. Flow cytometry instruments are well known to those skilled in the art. Examples of such instruments include FACSAria, FACS Starplus, FACScan and FACSort (Becton Dickinson, Foster, Calif.), Epics C (Coulter Epics division of Haierria, Florida), MOFLO (Cytomation of Colorado Spprins, Colorado) and MOFLO-XDP (Beckman Coulter of Indianapolis, Indiana). Flow cytometry generally involves the separation of cells or other particles in a liquid sample. Typically, the purpose of flow cytometry is to analyze the isolated particles for one or more characteristics, such as the presence of labeled ligands or other molecules. The particles pass one after another through the sensor and are sorted according to size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc.
Another aspect of the invention provides a composition comprising a polypeptide, antibody, nucleic acid molecule or vector comprising an Fc variant having one or more amino acid substitutions.
According to a preferred embodiment of the present invention, the composition is a pharmaceutical composition for preventing or treating cancer.
According to a preferred embodiment of the invention, the pharmaceutical composition (or polypeptide, antibody, nucleic acid molecule or vector) recognizes a cancer antigen.
According to one embodiment of the present invention, the Fc variant has an antibody-dependent cellular cytotoxicity (ADCC) activity equivalent to or higher than that of a control group (e.g., trastuzumab), thereby achieving a significantly increased half-life and high anti-cancer activity (see example 13 and fig. 18).
The pharmaceutical compositions of the invention may comprise (a) a polypeptide, an antibody, a nucleic acid molecule encoding the polypeptide or a vector comprising the nucleic acid molecule and (b) a pharmaceutically acceptable carrier.
Yet another aspect of the present invention provides a method for preventing or treating cancer, comprising administering a pharmaceutical composition to a subject.
The type of cancer prevented or treated by the method of the present invention is not limited. The pharmaceutical compositions of the invention can be administered to treat a number of cancers, including leukemias and lymphomas such as acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic myelogenous leukemia, hodgkin's disease, non-hodgkin's lymphoma, multiple myeloma, solid tumors of childhood such as brain tumors, neuroblastoma, retinoblastoma, wilms tumors, bone tumors, and soft tissue sarcomas, common solid tumors of adults such as lung, breast, prostate, urinary, uterine, oral, pancreatic, melanoma, and other skin, stomach, ovarian, brain, liver, throat, thyroid, esophageal, and testicular cancers.
The pharmaceutically acceptable carrier of the pharmaceutical composition according to the invention may be any carrier known in the art. Examples of carriers suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may further comprise at least one additive selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's pharmaceutical Sciences (19 th edition, 1995).
The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. For example, the pharmaceutical composition of the present invention may be administered by intravenous, topical or intraperitoneal injection.
The subject is not particularly limited, but is preferably interpreted to include vertebrates, more preferably primates including humans and chimpanzees, domestic pets including dogs and cats, domestic animals including cows, horses, sheep and goats, and rodents including mice and rats.
The appropriate dosage of the pharmaceutical composition according to the present invention depends on various factors such as dosage form, administration mode, age, body weight, sex and pathological condition of the patient, diet, administration time and route, excretion rate and responsiveness. One of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition according to the present invention for the treatment or prevention of the disease or disorder. According to a preferred embodiment of the invention, the daily dose of the pharmaceutical composition according to the invention is between 0.0001mg/kg and 100 mg/kg.
The pharmaceutical compositions of the present invention may be prepared in unit dosage form or dispensed in multi-dose containers with pharmaceutically acceptable carriers and/or excipients by suitable methods readily available to those of ordinary skill in the art. The pharmaceutical compositions of the present invention may be in the form of solutions, suspensions or emulsions in oily or aqueous media. The pharmaceutical composition of the present invention may be in the form of an extract, powder, granule, tablet or capsule. The pharmaceutical composition of the present invention may further comprise a dispersing agent or a stabilizing agent.
The pharmaceutical composition of the present invention may be used in monotherapy. Alternatively, the pharmaceutical composition of the present invention may be used in combination with conventional chemotherapy or radiotherapy. This combination therapy is more effective in cancer treatment. Chemotherapeutic agents that may be used with the compositions of the present invention include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicomycin (plicomycin), mitomycin, etoposide, tamoxifen, paclitaxel, platinatum, 5-fluorouracil, vincristine, vinblastine, methotrexate and the like. Radiation therapy that may be used with the compositions of the present invention includes X-ray radiation and gamma-ray radiation.
Effects of the invention
The features and advantages of the present invention are summarized below.
(i) The present invention provides a polypeptide comprising an Fc variant produced by replacing a portion of the amino acid sequence of an Fc domain of a human antibody with a different amino acid sequence.
(ii) The invention also provides methods of producing the polypeptides or antibodies comprising the polypeptides.
(iii) The Fc variants of the invention are suitable for use in the treatment of cancer because their in vivo half-life can be maximized by optimizing a portion of the amino acid sequence.
Drawings
Figure 1 shows expression vectors for expression and purification of tetrameric and dimeric FcRn, as well as SDS-PAGE gels after purification.
Figure 2 is a schematic of a library constructed to include 18 amino acids at positions M252 and M428.
Figure 3 shows the M428L variant of the 2M library retrieval process and sorting.
Figure 4 schematically shows an error library and a point library constructed based on M428L.
FIG. 5 shows FACS fluorescence intensity of variants sorted from (5a) error library and (5b) spot library.
Figure 6 shows plasmids for expressing trastuzumab heavy and light chains in animal cells.
Figure 7 shows the expression and purification results of wild-type trastuzumab.
FIG. 8 compares the physical properties of commercial trastuzumab and internal trastuzumab (a: CE-cIEF, b: SEC).
Figure 9 compares the physical properties of commercial trastuzumab and internal trastuzumab by N-glycan profiling.
FIG. 10 shows the expression and purification results for 10 trastuzumab Fc variants (a: affinity chromatography, b: SDS-PAGE analysis, c: final yield tabulation).
Figure 11 shows SEC characterization results for trastuzumab Fc variants.
Figure 12 shows the binding of trastuzumab Fc variants to FcRn as measured by ELISA.
Figure 13 shows the binding of trastuzumab Fc variants to hFcRn at pH values of 6.0 and 7.4 as measured using a BiaCore instrument (a: pH6.0 (capture method) b: pH7.4(avid form)).
Figure 14 compares the pharmacokinetics of commercial trastuzumab and internal trastuzumab in conventional mice (C57BL/6J (B6)) and human FcRn Tg mice.
Figure 15 shows the results of pharmacokinetic analysis of Fc variants in human FcRn Tg mice (n-5 after intravenous injection of the variants (5mg/kg each).
Figure 16 shows the binding of trastuzumab Fc variants to Fc γ R, as measured by ELISA.
Figure 17 compares the effector function of trastuzumab Fc variants with that of normal IgG, with trastuzumab as a control (ADCC assay).
Figure 18 compares the effector function (ADCC) of trastuzumab Fc variants.
Figure 19 shows the binding of trastuzumab Fc variant to C1q, measured by ELISA.
Best mode for carrying out the invention
The present invention will be illustrated in more detail with reference to the following examples. 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.
Examples
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